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Sample records for carbachol

  1. The cholinomimetic agent carbachol induces headache in healthy subjects

    DEFF Research Database (Denmark)

    Schytz, H W; Wienecke, Troels; Oturai, P S;

    2009-01-01

    The parasympathetic nervous system is likely to be involved in migraine pathogenesis. We hypothesized that the cholinomimetic agonist carbachol would induce headache and vasodilation of cephalic and radial arteries. Carbachol (3 microg/kg) or placebo was randomly infused into 12 healthy subjects ...

  2. Carbachol induces headache, but not migraine-like attacks, in patients with migraine without aura

    DEFF Research Database (Denmark)

    Schytz, H W; Wienecke, T; Olesen, J;

    2009-01-01

    Schytz HW, Wienecke T, Olesen J & Ashina M. Carbachol induces headache, but not migraine-like attacks, in patients with migraine without aura. Cephalalgia 2009. London. ISSN 0333-1024Carbachol induces headache in healthy subjects, but the migraine eliciting effect of carbachol has not previously...... been studied. We hypothesized that the cholinomimetic agonist carbachol would induce headache and migraine-like attacks in migraineurs. Carbachol (3 microg/kg) or placebo was randomly infused into 18 patients with migraine without aura in a double-blind crossover study. Headache was scored on a verbal...

  3. Effects of aerosolized histamine and carbachol in the conscious horse.

    OpenAIRE

    Mirbahar, K B; McDonell, W. N.; Bignell, W; Eyre, P.

    1985-01-01

    Pulmonary function tests were performed in seven conscious, standing horses. Changes in pulmonary mechanics and ventilation volumes were measured after inhalation challenge with saline (baseline), histamine (1% w/v solution for 5 min) and carbachol (0.5% w/v solution for 3 min). Comparisons between baseline and posthistamine values revealed a significant (P less than 0.05) increase in nonelastic work of breathing (Wb), maximum change in transpulmonary pressure (max delta Ppl), and pulmonary r...

  4. Renshaw cells are inactive during motor inhibition elicited by the pontine microinjection of carbachol.

    Science.gov (United States)

    Morales, F R; Engelhardt, J K; Pereda, A E; Yamuy, J; Chase, M H

    1988-04-12

    The present study was undertaken to determine whether the postsynaptic inhibition of motoneurons that occurs following the pontine microinjection of carbachol in the decerebrate cat is due to the activity of Renshaw cells. Thirty-two out of 37 Renshaw cells (86%) were spontaneously active prior to the administration of carbachol, whereas only 2 out of 13 Renshaw cells (15%) discharged during carbachol-induced motor inhibition. In addition, discrete inhibitory synaptic potentials were observed in 33% of the Renshaw cells from which intracellular recordings were obtained after carbachol administration, indicating that these cells were actively inhibited. The finding that a population of Renshaw cells, which inhibit motoneurons, were themselves inhibited during a period of profound motoneuron inhibition was quite unexpected. These results support the conclusion that Renshaw cells are not the inhibitory interneurons that are responsible for the powerful inhibition of motoneurons that occurs following the pontine microinjection of carbachol. PMID:3380320

  5. Carbachol as miotic agent in intra-ocular lens implant surgery

    Directory of Open Access Journals (Sweden)

    Agarwal Jitendra

    1988-01-01

    Full Text Available The intra cameral use of 0.025% carbachol as a miotic agent in anterior chamber intraocular lens im-plant surgery us reported in 15 cases. Carbachol produced prompt and effective moisis and was found to be harmless and non-irritating to the anterior chamber structures. A rebound dialatation of the pipit was noticed in the post operative period in 3 of our first 5 cases where only carbachol was used. For prolonged miosis instillation of a more powerful miotic like pilocarpine is recommended at the completion of surgery:

  6. Carbachol-induced rhythmic slow activity (theta) in cat hippocampal formation slices.

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    Konopacki, J; Gołebiewski, H; Eckersdorf, B

    1992-04-24

    Application of the cholinergic agonist, carbachol, produced theta-like rhythmical waveforms, recorded in the stratum moleculare of the dentate gyrus in the cat hippocampal formation slices. This effect of carbachol was antagonized by atropine but not D-tubocurarine. These results provide first direct evidence that the hippocampal formation neuronal network in the cat is capable of producing synchronized slow wave activity when isolated from pulsed rhythmic inputs of the medial septum. PMID:1511270

  7. Changes in electrophysiological properties of cat hypoglossal motoneurons during carbachol-induced motor inhibition.

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    Fung, S J; Yamuy, J; Xi, M C; Engelhardt, J K; Morales, F R; Chase, M H

    2000-12-01

    The control of hypoglossal motoneurons during sleep is important from a basic science perspective as well as to understand the bases for pharyngeal occlusion which results in the obstructive sleep apnea syndrome. In the present work, we used intracellular recording techniques to determine changes in membrane properties in adult cats in which atonia was produced by the injection of carbachol into the pontine tegmentum (AS-carbachol). During AS-carbachol, 86% of the recorded hypoglossal motoneurons were found to be postsynaptically inhibited on the basis of analyses of their electrical properties; the electrical properties of the remaining 14% were similar to motoneurons recorded during control conditions. Those cells that exhibited changes in their electrical properties during AS-carbachol also displayed large-amplitude inhibitory synaptic potentials. Following sciatic nerve stimulation, hypoglossal motoneurons which responded with a depolarizing potential during control conditions exhibited a hyperpolarizing potential during AS-carbachol. Both spontaneous and evoked inhibitory potentials recorded during AS-carbachol were comparable to those that have been previously observed in trigeminal and spinal cord motoneurons under similar experimental conditions as well as during naturally occurring active sleep. Calculations based on modeling the changes that we found in input resistance and membrane time constant with a three-compartment neuron model suggest that shunts are present in all three compartments of the hypoglossal motoneuron model. Taken together, these data indicate that postsynaptic inhibitory drives are widely distributed on the soma-dendritic tree of hypoglossal motoneurons during AS-carbachol. These postsynaptic inhibitory actions are likely to be involved in the pathophysiology of obstructive sleep apnea. PMID:11102580

  8. Nonlinear dynamical analysis of carbachol induced hippocampal oscillations in mice

    Institute of Scientific and Technical Information of China (English)

    Metin AKAY; Kui WANG; Yasemin M AKAY; Andrei DRAGOMIR; Jie WU

    2009-01-01

    Aim: Hippocampal neuronal network and synaptic impairment underlie learning and memory deficit in Alzheimer's disease (AD) patients and animal models. In this paper, we analyzed the dynamics and complexity of hippocampal neuronal network synchronization induced by acute exposure to carbachol, a nicotinic and muscarinic receptor co-agonist, using the nonlinear dynamical model based on the Lempel-Ziv estimator. We compared the dynamics of hippocampal oscillations between wild-type (WT) and triple-transgenic (3xTg) mice, as an AD animal model. We also compared these dynamic alterations between different age groups (5 and 10 months). We hypothesize that there is an impairment of complexity of CCh-induced hippocampal oscillations in 3xTg AD mice compared to WT mice, and that this impairment is age-dependent. Methods: To test this hypothesis, we used electrophysiological recordings (field potential) in hippocampal slices. Results: Acute exposure to 100 nmol/L CCh induced field potential oscillations in hippocampal CA1 region, which exhibited three distinct patterns: (1) continuous neural firing, (2) repeated burst neural firing and (3) the mixed (continuous and burst) pattern in both WT and 3xTg AD mice. Based on Lempel-Ziv estimator, pattern (2) was significantly lower than patterns (1) and (3) in 3xTg AD mice compared to WT mice (P<0.001), and also in 10-month old WT mice compared to those in 5-month old WT mice (P<0.01).Conclusion: These results suggest that the burst pattern (theta oscillation) of hippocampal network is selectively impaired in 3xTg AD mouse model, which may reflect a learning and memory deficit in the AD patients.

  9. Intracerebral implantation of carbachol in the rat: Its effect on water intake and body temperature

    NARCIS (Netherlands)

    Hulst, S.G.Th.

    1972-01-01

    Intracerebral carbachol produces a fall in body temperature as well as drinking in the rat when implanted in various subcortical structures, related to the emotion-motivation limbic circuit. These effects are due to a central cholinergic stimulation since they can be prevented by the systemic admini

  10. Regional haemodynamic effects of carbachol injected into the hypothalamic paraventricular nuclei of conscious, unrestrained rats.

    Science.gov (United States)

    Bachelard, H; Gardiner, S M; Kemp, P A; Bennett, T

    1994-06-01

    Carbachol was injected into the hypothalamic paraventricular nuclei (PVN) of conscious, unrestrained Long Evans rats, chronically instrumented with intravascular catheters and pulsed Doppler probes to assess changes in regional haemodynamics. Bilateral microinjections of carbachol (1 ng-1 microgram) produced increases in blood pressure, bradycardias and vasoconstrictions in renal, superior mesenteric and hindquarters vascular beds. In the presence of phentolamine, the bradycardic and hindquarters vasoconstrictor responses to carbachol were unchanged while the pressor response was smaller due to a reduction in the renal and the superior mesenteric vasoconstriction. In the presence of propranolol, the bradycardic response was reduced, but the pressor and renal vasoconstrictor responses were potentiated, whereas the superior mesenteric and hindquarter vasoconstrictions were not changed significantly. In the presence of phentolamine and propranolol, the heart rate and pressor responses, as well as the renal vasoconstriction, were unchanged, whereas the superior mesenteric vasoconstriction was reduced and the hindquarters vasoconstriction was potentiated. Together these results are consistent with an involvement of the sympathoadrenal system in the pressor response to carbachol injected into the PVN of untreated animals. They indicate that alpha-adrenoceptor-mediated vasoconstriction in the superior mesenteric vascular bed is a particularly important component in that regard. In the presence of the vasopressin antagonist, d(CH2)5(Tyr(Et))DAVP, alone or in combination with phentolamine and propranolol, the pressor response to carbachol was substantially reduced, while the renal and superior mesenteric vasoconstrictor effects were completely abolished; the bradycardia was not significantly affected by this treatment. These results indicate an important involvement of vasopressin in the cardiovascular responses to carbachol injected into the PVN of untreated animals

  11. The effect of M & B 22948 on carbachol-induced inositol trisphosphate accumulation and contraction in iris sphincter smooth muscle.

    Science.gov (United States)

    Akhtar, R A; Abdel-Latif, A A

    1991-04-25

    The effect of a cyclic GMP phosphodiesterase inhibitor, M & B 22948, on carbachol-induced phosphatidylinositol 4,5-bis-phosphate (PIP2) breakdown and phosphatidic acid labeling, 1,4,5-inositol trisphosphate (IP3) accumulation and muscle contraction was studied in bovine iris sphincter smooth muscle. Addition of carbachol (10 microM) to 32P-labeled tissue resulted in increased labeling of phosphatidic acid and hydrolysis of PIP2. In myo[3H]inositol labeled tissue, carbachol caused rapid accumulation of IP3 which reached its maximum at about 2 min. Under identical experimental conditions, carbachol initiated a rapid increase in muscle contraction (phasic component) which was followed by a slightly lower contractile response (tonic component) that lasted for several minutes. Pretreatment of the iris sphincter with M & B 22948 did not alter carbachol-stimulated PIP2 breakdown and phosphatidic acid labeling, IP3 accumulation, or phasic component of the contractile response. However, the tonic component of the contractile response was increasingly attenuated by increasing concentrations of the drug. In conclusion, the data presented demonstrate a close correlation between carbachol-induced IP3 accumulation and muscle contraction, and that M & B 22948 does not inhibit carbachol-induced responses in the iris sphincter.

  12. Proteomic response analysis of endothelial cells of human coronary artery to stimulation with carbachol

    Institute of Scientific and Technical Information of China (English)

    Min YU; Dong-mei CHEN; Gang HU; Hai WANG

    2004-01-01

    AIM: To identify the molecular basis of the endothelial target for acetylcholine (ETA). METHODS: Proteomic methods were used to monitor changes in protein expression in the first 10 h following the stimulation of human coronary endothelial cells with carbachol 100 μmol/L. Thirty proteins showing the largest changes were identified by mass spectrometry. RESULTS: Based on analysis with Image Master 2-D Elite software, about 623 protein spots were detected in control cells and 825 protein spots in carbachol-treated cells, the matching rate was 68.1%.Among all the detected spots, 39 were up-regulated and 29 were down-regulated, showing detectable changes varied from 1.7-3.8 folds. Forty one spots in the peptide mass fingerprints were successfully obtained. The most interesting feature was that all the four newly synthesized proteins belonged to the heat shock protein family.CONCLUSION: These identified proteins played key roles in the molecular mechanism of ETA.

  13. Effects of carbachol matching oral fluid resuscitation on intestinal mucosa blood flow and absorption rate of dogs suffered hemorrhagic shock

    Directory of Open Access Journals (Sweden)

    Lin LI

    2011-04-01

    Full Text Available Objective To investigate the effects of carbachol matching oral fluid resuscitation on intestinal mucosa blood flow(IMBF and intestinal absorption rate of dogs suffered hemorrhagic shock.Methods Twenty-four hours after a preliminary intubation of carotid artery,jugular vein and jejunum by asepsis,twelve Beagle dogs were subjected to a loss of 40% total blood volume to establish animal model of hemorrhagic shock.Animals were then divided into oral resuscitation group and carbachol group(6 each.Dogs in oral resuscitation group were given by gastric tube the glucose-electrolyte solution(GES,which was 3 times volume of blood loss,within 24h after bleeding;while dogs in carbachol group were given GES added carbachol(0.25μg/kg.The IMBF and intestinal absorption rate of water before hemorrhage(0h and 2,4 and 8h after hemorrhage were measured.All the animals were sacrificed at 8h after hemorrhage to record the intestinal GES volume.Results The intestinal absorption rate of water remarkably decreased after hemorrhage in both groups,while in carbachol,group it was obviously higher than that in oral resuscitation group(P < 0.05.The GES volume absorbed by intestine in carbachol group was high than that in oral resuscitation group 8h after hemorrhage(P < 0.05.The IMBF decreased significantly in the both groups after hemorrhage,and then increased gradually 2h after hemorrhage.The IMBF in carbachol group was obviously higher than that in oral resuscitation group(P < 0.05.Conclusion Carbachol in oral resuscitation with GES can improve intestinal absorption rate of water and GES,and increase IMBF in dogs with 40% blood loss.

  14. Carbachol can be released at a cholinergic ganglionic synapse as a false transmitter.

    OpenAIRE

    Baux, G; Tauc, L

    1983-01-01

    Carbachol was injected into a presynaptic cholinergic neuron in the buccal ganglion of Aplysia and the quantal aspects of the Cl- -dependent postsynaptic response to a prolonged stimulation were analyzed by a statistical fluctuation method. The calculated amplitude of the miniature postsynaptic current was increased with respect to control. Statistical fluctuation analysis was also used to analyze the postsynaptic response obtained during ionophoretic application of acetylcholine and carbacho...

  15. Carbachol-Induced Reduction in the Activity of Adult Male Zebra Finch RA Projection Neurons.

    Science.gov (United States)

    Meng, Wei; Wang, Song-Hua; Li, Dong-Feng

    2016-01-01

    Cholinergic mechanism is involved in motor behavior. In songbirds, the robust nucleus of the arcopallium (RA) is a song premotor nucleus in the pallium and receives cholinergic inputs from the basal forebrain. The activity of projection neurons in RA determines song motor behavior. Although many evidences suggest that cholinergic system is implicated in song production, the cholinergic modulation of RA is not clear until now. In the present study, the electrophysiological effects of carbachol, a nonselective cholinergic receptor agonist, were investigated on the RA projection neurons of adult male zebra finches through whole-cell patch-clamp techniques in vitro. Our results show that carbachol produced a significant decrease in the spontaneous and evoked action potential (AP) firing frequency of RA projection neurons, accompanying a hyperpolarization of the membrane potential, an increase in the evoked AP latency, afterhyperpolarization (AHP) peak amplitude, and AHP time to peak, and a decrease in the membrane input resistance, membrane time constant, and membrane capacitance. These results indicate that carbachol reduces the activity of RA projection neurons by hyperpolarizing the resting membrane potential and increasing the AHP and the membrane conductance, suggesting that the cholinergic modulation of RA may play an important role in song production. PMID:26904300

  16. Carbachol ameliorates lipopolysaccharide-induced intestinal epithelial tight junction damage by down-regulating NF-{kappa}{beta} and myosin light-chain kinase pathways

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ying [Department of Anesthesia, Critical Care Medicine and Emergency Medicine Center, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, People' s Republic of China (China); Li, Jianguo, E-mail: 2010lijianguo@sina.cn [Department of Anesthesia, Critical Care Medicine and Emergency Medicine Center, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, People' s Republic of China (China)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Carbachol reduced the lipopolysaccharide-induced intestinal barrier breakdown. Black-Right-Pointing-Pointer Carbachol ameliorated the lipopolysaccharide-induced ileal tight junction damage. Black-Right-Pointing-Pointer Carbachol prevented the LPS-induced NF-{kappa}{beta} and myosin light-chain kinase activation. Black-Right-Pointing-Pointer Carbachol exerted its beneficial effects in an {alpha}7 nicotinic receptor-dependent manner. -- Abstract: Carbachol is a cholinergic agonist that protects the intestines after trauma or burn injury. The present study determines the beneficial effects of carbachol and the mechanisms by which it ameliorates the lipopolysaccharide (LPS)-induced intestinal barrier breakdown. Rats were injected intraperitoneally with 10 mg/kg LPS. Results showed that the gut barrier permeability was reduced, the ultrastructural disruption of tight junctions (TJs) was prevented, the redistribution of zonula occludens-1 and claudin-2 proteins was partially reversed, and the nuclear factor-kappa beta (NF-{kappa}{beta}) and myosin light-chain kinase (MLCK) activation in the intestinal epithelium were suppressed after carbachol administration in LPS-exposed rats. Pretreatment with the {alpha}7 nicotinic acetylcholine receptor ({alpha}7nAchR) antagonist {alpha}-bungarotoxin blocked the protective action of carbachol. These results suggested that carbachol treatment can protect LPS-induced intestinal barrier dysfunction. Carbachol exerts its beneficial effect on the amelioration of the TJ damage by inhibiting the NF-{kappa}{beta} and MLCK pathways in an {alpha}7nAchR-dependent manner.

  17. Basolateral K channel activated by carbachol in the epithelial cell line T84.

    Science.gov (United States)

    Tabcharani, J A; Harris, R A; Boucher, A; Eng, J W; Hanrahan, J W

    1994-11-01

    Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na > Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba

  18. Motoneuron properties during motor inhibition produced by microinjection of carbachol into the pontine reticular formation of the decerebrate cat.

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    Morales, F R; Engelhardt, J K; Soja, P J; Pereda, A E; Chase, M H

    1987-04-01

    It is well established that cholinergic agonists, when injected into the pontine reticular formation in cats, produce a generalized suppression of motor activity (1, 3, 6, 14, 18, 27, 33, 50). The responsible neuronal mechanisms were explored by measuring ventral root activity, the amplitude of the Ia-monosynaptic reflex, and the basic electrophysiological properties of hindlimb motoneurons before and after carbachol was microinjected into the pontine reticular formation of decerebrate cats. Intrapontine microinjections of carbachol (0.25-1.0 microliter, 16 mg/ml) resulted in the tonic suppression of ventral root activity and a decrease in the amplitude of the Ia-monosynaptic reflex. An analysis of intracellular records from lumbar motoneurons during the suppression of motor activity induced by carbachol revealed a considerable decrease in input resistance and membrane time constant as well as a reduction in motoneuron excitability, as evidenced by a nearly twofold increase in rheobase. Discrete inhibitory postsynaptic potentials were also observed following carbachol administration. The changes in motoneuron properties (rheobase, input resistance, and membrane time constant), as well as the development of discrete inhibitory postsynaptic potentials, indicate that spinal cord motoneurons were postsynaptically inhibited following the pontine administration of carbachol. In addition, the inhibitory processes that arose after carbachol administration in the decerebrate cat were remarkably similar to those that are present during active sleep in the chronic cat. These findings suggest that the microinjection of carbachol into the pontine reticular formation activates the same brain stem-spinal cord system that is responsible for the postsynaptic inhibition of alpha-motoneurons that occurs during active sleep. PMID:3585456

  19. C-fos expression in the pons and medulla of the cat during carbachol-induced active sleep.

    Science.gov (United States)

    Yamuy, J; Mancillas, J R; Morales, F R; Chase, M H

    1993-06-01

    Microinjection of carbachol into the rostral pontine tegmentum of the cat induces a state that is comparable to naturally occurring active (REM, rapid eye movement) sleep. We sought to determine, during this pharmacologically induced behavioral state, which we refer to as active sleep-carbachol, the distribution of activated neuron within the pons and medulla using c-fos immunocytochemistry as a functional marker. Compared with control cats, which were injected with saline, active sleep-carbachol cats exhibited higher numbers of c-fos-expressing neurons in (1) the medial and portions of the lateral reticular formation of the pons and medulla, (2) nuclei in the dorsolateral rostral pons, (3) various raphe nuclei, including the dorsal, central superior, magnus, pallidus, and obscurus, (4) the medial and lateral vestibular, prepositus hypoglossi, and intercalatus nuclei, and (5) the abducens nuclei. On the other hand, the mean number of c-fos-expressing neurons found in the masseter, facial, and hypoglossal nuclei was lower in carbachol-injected than in control cats. The data indicate that c-fos expression can be employed as a marker of state-dependent neuronal activity. The specific sites in which there were greater numbers of c-fos-expressing neurons during active sleep-carbachol are discussed in relation to the state of active sleep, as well as the functional role that these sites play in generating the various physiological patterns of activity that occur during this state. PMID:8501533

  20. Neocortical 40 Hz oscillations during carbachol-induced rapid eye movement sleep and cataplexy.

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    Torterolo, Pablo; Castro-Zaballa, Santiago; Cavelli, Matías; Chase, Michael H; Falconi, Atilio

    2016-02-01

    Higher cognitive functions require the integration and coordination of large populations of neurons in cortical and subcortical regions. Oscillations in the gamma band (30-45 Hz) of the electroencephalogram (EEG) have been involved in these cognitive functions. In previous studies, we analysed the extent of functional connectivity between cortical areas employing the 'mean squared coherence' analysis of the EEG gamma band. We demonstrated that gamma coherence is maximal during alert wakefulness and is almost absent during rapid eye movement (REM) sleep. The nucleus pontis oralis (NPO) is critical for REM sleep generation. The NPO is considered to exert executive control over the initiation and maintenance of REM sleep. In the cat, depending on the previous state of the animal, a single microinjection of carbachol (a cholinergic agonist) into the NPO can produce either REM sleep [REM sleep induced by carbachol (REMc)] or a waking state with muscle atonia, i.e. cataplexy [cataplexy induced by carbachol (CA)]. In the present study, in cats that were implanted with electrodes in different cortical areas to record polysomnographic activity, we compared the degree of gamma (30-45 Hz) coherence during REMc, CA and naturally-occurring behavioural states. Gamma coherence was maximal during CA and alert wakefulness. In contrast, gamma coherence was almost absent during REMc as in naturally-occurring REM sleep. We conclude that, in spite of the presence of somatic muscle paralysis, there are remarkable differences in cortical activity between REMc and CA, which confirm that EEG gamma (≈40 Hz) coherence is a trait that differentiates wakefulness from REM sleep. PMID:26670051

  1. GABAergic neurons of the cat dorsal raphe nucleus express c-fos during carbachol-induced active sleep.

    Science.gov (United States)

    Torterolo, P; Yamuy, J; Sampogna, S; Morales, F R; Chase, M H

    2000-11-24

    Serotonergic neurons of the dorsal raphe nucleus (DRN) cease firing during active sleep (AS, also called rapid-eye-movement sleep). This cessation of electrical activity is believed to play a 'permissive' role in the generation of AS. In the present study we explored the possibility that GABAergic cells in the DRN are involved in the suppression of serotonergic activity during AS. Accordingly, we examined whether immunocytochemically identified GABAergic neurons in the DRN were activated, as indicated by their expression of c-fos, during carbachol-induced AS (AS-carbachol). Three chronically-prepared cats were euthanized after prolonged episodes of AS that was induced by microinjections of carbachol into the nucleus pontis oralis. Another four cats (controls) were maintained 2 h in quiet wakefulness before being euthanized. Thereafter, immunocytochemical studies were performed on brainstem sections utilizing antibodies against Fos, GABA and serotonin. When compared with identically prepared tissue from awake cats, the number of Fos+ neurons was larger in the DRN during AS-carbachol (35.9+/-5.6 vs. 13.9+/-4.4, P<0.05). Furthermore, a larger number of GABA+ Fos+ neurons were observed during AS-carbachol than during wakefulness (24.8+/-3.3 vs. 4.0+/-1.0, P<0.001). These GABA+ Fos+ neurons were distributed asymmetrically with a larger number located ipsilaterally to the site of injection. There was no significant difference between control and experimental animals in the number of non-GABAergic neurons that expressed c-fos in the DRN. We therefore suggest that activated GABAergic neurons of the DRN are responsible for the inhibition of serotonergic neurons that occurs during natural AS. PMID:11082488

  2. Population synaptic potentials evoked in lumbar motoneurons following stimulation of the nucleus reticularis gigantocellularis during carbachol-induced atonia.

    Science.gov (United States)

    Yamuy, J; Jiménez, I; Morales, F; Rudomin, P; Chase, M

    1994-03-14

    The effect of electrical stimulation of the medullary nucleus reticularis gigantocellularis (NRGc) on lumbar spinal cord motoneurons was studied in the decerebrate cat using sucrose-gap recordings from ventral roots. The NRGc was stimulated ipsi- and contralaterally before and during atonia elicited by the microinjection of carbachol into the pontine reticular formation. Prior to carbachol administration, the NRGc-induced response recorded from the sucrose-gap consisted of two consecutive excitatory population synaptic potentials followed by a long-lasting, small amplitude inhibitory population synaptic potential. Following carbachol injection, the same NRGc stimulus evoked a distinct, large amplitude inhibitory population synaptic potential, whereas the excitatory population synaptic potentials decreased in amplitude. In addition, after carbachol administration, the amplitude of the monosynaptic excitatory population synaptic potential, which was evoked by stimulation of group Ia afferents in hindlimb nerves, was reduced by 18 to 43%. When evoked at the peak of the NRGc-induced inhibitory response, this potential was further decreased in amplitude. Systemic strychnine administration (0.07-0.1 mg/kg, i.v.) blocked the NRGc-induced inhibitory population synaptic potential and promoted an increase in the amplitude of the excitatory population synaptic potentials induced by stimulation of the NRGc and group Ia afferents. These data indicate that during the state of carbachol-induced atonia, the NRGc effects on ipsi- and contralateral spinal cord motoneurons are predominantly inhibitory and that glycine is likely to be involved in this inhibitory process. These results support the hypothesis that the nucleus reticularis gigantocellularis is part of the system responsible for state-dependent somatomotor inhibition that occurs during active sleep. PMID:8205484

  3. Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid

    Energy Technology Data Exchange (ETDEWEB)

    Takuwa, Y.; Takuwa, N.; Rasmussen, H.

    1986-11-05

    The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of (/sup 3/H)inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in (/sup 3/H)inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of (/sup 3/H)inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca/sup 2 +/ and does not appear to be secondary to an increase in intracellular Ca/sup 2 +/. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.

  4. Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid.

    Science.gov (United States)

    Takuwa, Y; Takuwa, N; Rasmussen, H

    1986-11-01

    The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of [3H]inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in [3H]inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of [3H]inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca2+ and does not appear to be secondary to an increase in intracellular Ca2+. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.

  5. Electrophysiological properties of lumbar motoneurons in the alpha-chloralose-anesthetized cat during carbachol-induced motor inhibition.

    Science.gov (United States)

    Xi, M C; Liu, R H; Yamuy, J; Morales, F R; Chase, M H

    1997-07-01

    The present study was undertaken 1) to examine the neuronal mechanisms responsible for the inhibition of spinal cord motoneurons that occurs in alpha-chloralose-anesthetized cats following the microinjection of carbachol into the nucleus pontis oralis (NPO), and 2) to determine whether the inhibitory mechanisms are the same as those that are responsible for the postsynaptic inhibition of motoneurons that is present during naturally occurring active sleep. Accordingly, the basic electrophysiological properties of lumbar motoneurons were examined, with the use of intracellular recording techniques, in cats anesthetized with alpha-chloralose and compared with those present during naturally occurring active sleep. The intrapontine administration of carbachol resulted in a sustained reduction in the amplitude of the spinal cord Ia monosynaptic reflex. Discrete large-amplitude inhibitory postsynaptic potentials (IPSPs), which are only present during the state of active sleep in the chronic cat, were also observed in high-gain recordings from lumbar motoneurons after the injection of carbachol. During carbachol-induced motor inhibition, lumbar motoneurons exhibited a statistically significant decrease in input resistance, membrane time constant and a reduction in the amplitude of the action potential's afterhyperpolarization. In addition, there was a statistically significant increase in rheobase and in the delay between the initial-segment (IS) and somadendritic (SD) portions of the action potential (IS-SD delay). There was a significant increase in the mean motoneuron resting membrane potential (i.e., hyperpolarization). The preceding changes in the electrophysiological properties of motoneurons, as well as the development of discrete IPSPs, indicate that lumbar motoneurons are postsynaptically inhibited after the intrapontine administration of carbachol in cats that are anesthetized with alpha-chloralose. These changes in the electrophysiological properties of lumbar

  6. c-fos Expression in mesopontine noradrenergic and cholinergic neurons of the cat during carbachol-induced active sleep: a double-labeling study.

    Science.gov (United States)

    Yamuy, J; Sampogna, S; Morales, F R; Chase, M H

    1998-01-01

    The interaction of cholinergic and catecholaminergic mechanisms in the mesopontine region has been hypothesized as being critical for the generation and maintenance of active (REM) sleep. To further examine this hypothesis, we sought to determine the pattern of neuronal activation (via c-fos expression) of catecholaminergic and cholinergic neurons in this region during active sleep induced by the pontine microapplication of carbachol (designated as active sleep-carbachol). Accordingly, we used two sets of double-labeling techniques; the first to identify tyrosine hydroxylase-containing neurons (putative catecholaminergic cells) which also express the c-fos protein product Fos, and the second to reveal choline acetyltransferase-containing neurons (putative cholinergic cells) which also express Fos. Compared to control cats, active sleep-carbachol cats exhibited a significantly greater number of Fos-expressing neurons in the dorsolateral region of the pons, which encompasses the locus coeruleus, the lateral pontine reticular formation, the peribrachial nuclei and the latero-dorsal and pedunculo-pontine tegmental nuclei. However, both control and active sleep-carbachol cats exhibited a similar number of catecholaminergic and cholinergic neurons in those regions that expressed Fos (i.e., double-labeled cells). A large number of c-fos-expressing neurons in the active sleep-carbachol cats whose neurotransmitter phenotype was not identified suggests that non-catecholaminergic, non-cholinergic neuronal populations in mesopontine regions are involved in the generation and maintenance of active sleep. The lack of increased c-fos expression in catecholaminergic neurons during active sleep-carbachol confirms and extends previous data that indicate that these cells are silent during active sleep-carbachol and naturally-occurring active sleep. The finding that cholinergic neurons of the dorsolateral pons were not activated either during wakefulness or active sleep-carbachol

  7. Carbachol increases basolateral K+ conductance in T84 cells. Simultaneous measurements of cell [Ca] and gK explore calcium's role.

    Science.gov (United States)

    Wong, S M; Tesfaye, A; DeBell, M C; Chase, H S

    1990-12-01

    To explore the role of calcium in mediating the action of carbachol in chloride-secreting epithelia, we simultaneously measured intracellular free [Ca] ([Ca]i) and the potassium conductance (gK) of the basolateral membrane in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value ([Ca]'i) normalized to control. To assess changes in basolateral gK, we measured the short circuit current (Isc) in the presence of luminal amphotericin and a transepithelial mucosa-to-serosa K+ gradient (Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson. 1986. J. Gen. Physiol. 88:237-251). Treatment of the monolayers with carbachol resulted in a parallel increase and then decrease in [Ca]'i and gK. The carbachol-induced changes in gK appeared to be dependent on the increase in [Ca]i because stimulation of gK was significantly diminished when the hormone-induced increase in [Ca]'i was blunted, either by loading the cells with BAPTA or by reducing the extracellular [Ca]. The carbachol-stimulated increase in gK appeared to be the direct result of the increase in steady-state [Ca]'i. The changes in gK and [Ca]'i after stimulation with carbachol were correlated and ionomycin also increased gK and [Ca]'i in a parallel manner. The carbachol-induced delta gK per delta[Ca]'i, however, was greater than that after ionomycin. Because ionomycin and carbachol appear to open the same channel, a conclusion based on inhibitor and selectivity experiments, carbachol may have a second action that amplifies the effect of calcium on gK. PMID:2126802

  8. Age-dependent contribution of Rho kinase in carbachol-induced contraction of human detrusor smooth muscle in vitro

    OpenAIRE

    Kirschstein, Timo; Protzel, Chris; Porath, Katrin; Sellmann, Tina; Köhling, Rüdiger; Hakenberg, Oliver W

    2013-01-01

    Aim: Activation of muscarinic receptors on the detrusor smooth muscle is followed by contraction, which involves both myosin light chain kinase (MLCK) and Rho kinase (ROCK). The aim of this study was to determine the relative contributions of MLCK and ROCK to carbachol-induced contraction of human detrusor smooth muscle in vitro. Methods: Detrusor smooth muscle strips were prepared from the macroscopically unaffected bladder wall of patients underwent cystectomy. The strips were fixed in an o...

  9. Naloxone reduces the amplitude of IPSPs evoked in lumbar motoneurons by reticular stimulation during carbachol-induced motor inhibition.

    Science.gov (United States)

    Xi, M C; Liu, R H; Yamuy, J; Morales, F R; Chase, M H

    1999-02-20

    During active sleep or carbachol-induced motor inhibition, electrical stimulation of the medullary nucleus reticularis gigantocellularis (NRGc) evoked large amplitude, glycinergic inhibitory postsynaptic potentials (IPSPs) in cat motoneurons. The present study was directed to determine whether these IPSPs, that are specific to the state of active sleep, are modulated by opioid peptides. Accordingly, intracellular recordings were obtained from lumbar motoneurons of acute decerebrate cats during carbachol-induced motor inhibition while an opiate receptor antagonist, naloxone, was microiontophoretically released next to the recorded cells. Naloxone reversibly reduced by 26% the mean amplitude of NRGc-evoked IPSPs (1.9+/-0.2 mV (S.E.M.) vs. 1.4+/-0.2 mV; n=11, control and naloxone, respectively, p<0.05), but had no effect on the other waveform parameters of these IPSPs (e.g., latency-to-onset, latency-to-peak, duration, etc.). The mean resting membrane potential, input resistance and membrane time constant of motoneurons following naloxone ejection were not statistically different from those of the control. These data indicate that opioid peptides have a modulatory effect on NRGc-evoked IPSPs during carbachol-induced motor inhibition. We therefore suggest that endogenous opioid peptides may act as neuromodulators to regulate inhibitory glycinergic synaptic transmission at motoneurons during active sleep. PMID:10082872

  10. Heart rate variability during carbachol-induced REM sleep and cataplexy.

    Science.gov (United States)

    Torterolo, Pablo; Castro-Zaballa, Santiago; Cavelli, Matías; Velasquez, Noelia; Brando, Victoria; Falconi, Atilio; Chase, Michael H; Migliaro, Eduardo R

    2015-09-15

    The nucleus pontis oralis (NPO) exerts an executive control over REM sleep. Cholinergic input to the NPO is critical for REM sleep generation. In the cat, a single microinjection of carbachol (a cholinergic agonist) into the NPO produces either REM sleep (REMc) or wakefulness with muscle atonia (cataplexy, CA). In order to study the central control of the heart rate variability (HRV) during sleep, we conducted polysomnographic and electrocardiogram recordings from chronically prepared cats during REMc, CA as well as during sleep and wakefulness. Subsequently, we performed statistical and spectral analyses of the HRV. The heart rate was greater during CA compared to REMc, NREM or REM sleep. Spectral analysis revealed that the low frequency band (LF) power was significantly higher during REM sleep in comparison to REMc and CA. Furthermore, we found that during CA there was a decrease in coupling between the RR intervals plot (tachogram) and respiratory activity. In contrast, compared to natural behavioral states, during REMc and CA there were no significant differences in the HRV based upon the standard deviation of normal RR intervals (SDNN) and the mean squared difference of successive intervals (rMSSD). In conclusion, there were differences in the HRV during naturally-occurring REM sleep compared to REMc. In addition, in spite of the same muscle atonia, the HRV was different during REMc and CA. Therefore, the neuronal network that controls the HRV during REM sleep can be dissociated from the one that generates the muscle atonia during this state. PMID:25997581

  11. Heart rate variability during carbachol-induced REM sleep and cataplexy.

    Science.gov (United States)

    Torterolo, Pablo; Castro-Zaballa, Santiago; Cavelli, Matías; Velasquez, Noelia; Brando, Victoria; Falconi, Atilio; Chase, Michael H; Migliaro, Eduardo R

    2015-09-15

    The nucleus pontis oralis (NPO) exerts an executive control over REM sleep. Cholinergic input to the NPO is critical for REM sleep generation. In the cat, a single microinjection of carbachol (a cholinergic agonist) into the NPO produces either REM sleep (REMc) or wakefulness with muscle atonia (cataplexy, CA). In order to study the central control of the heart rate variability (HRV) during sleep, we conducted polysomnographic and electrocardiogram recordings from chronically prepared cats during REMc, CA as well as during sleep and wakefulness. Subsequently, we performed statistical and spectral analyses of the HRV. The heart rate was greater during CA compared to REMc, NREM or REM sleep. Spectral analysis revealed that the low frequency band (LF) power was significantly higher during REM sleep in comparison to REMc and CA. Furthermore, we found that during CA there was a decrease in coupling between the RR intervals plot (tachogram) and respiratory activity. In contrast, compared to natural behavioral states, during REMc and CA there were no significant differences in the HRV based upon the standard deviation of normal RR intervals (SDNN) and the mean squared difference of successive intervals (rMSSD). In conclusion, there were differences in the HRV during naturally-occurring REM sleep compared to REMc. In addition, in spite of the same muscle atonia, the HRV was different during REMc and CA. Therefore, the neuronal network that controls the HRV during REM sleep can be dissociated from the one that generates the muscle atonia during this state.

  12. Modeling carbachol-induced hippocampal network synchronization using hidden Markov models

    Science.gov (United States)

    Dragomir, Andrei; Akay, Yasemin M.; Akay, Metin

    2010-10-01

    In this work we studied the neural state transitions undergone by the hippocampal neural network using a hidden Markov model (HMM) framework. We first employed a measure based on the Lempel-Ziv (LZ) estimator to characterize the changes in the hippocampal oscillation patterns in terms of their complexity. These oscillations correspond to different modes of hippocampal network synchronization induced by the cholinergic agonist carbachol in the CA1 region of mice hippocampus. HMMs are then used to model the dynamics of the LZ-derived complexity signals as first-order Markov chains. Consequently, the signals corresponding to our oscillation recordings can be segmented into a sequence of statistically discriminated hidden states. The segmentation is used for detecting transitions in neural synchronization modes in data recorded from wild-type and triple transgenic mice models (3xTG) of Alzheimer's disease (AD). Our data suggest that transition from low-frequency (delta range) continuous oscillation mode into high-frequency (theta range) oscillation, exhibiting repeated burst-type patterns, occurs always through a mode resembling a mixture of the two patterns, continuous with burst. The relatively random patterns of oscillation during this mode may reflect the fact that the neuronal network undergoes re-organization. Further insight into the time durations of these modes (retrieved via the HMM segmentation of the LZ-derived signals) reveals that the mixed mode lasts significantly longer (p < 10-4) in 3xTG AD mice. These findings, coupled with the documented cholinergic neurotransmission deficits in the 3xTG mice model, may be highly relevant for the case of AD.

  13. Involvement of inward rectifier and M-type currents in carbachol-induced epileptiform synchronization.

    Science.gov (United States)

    Cataldi, Mauro; Panuccio, Gabriella; Cavaccini, Anna; D'Antuono, Margherita; Taglialatela, Maurizio; Avoli, Massimo

    2011-03-01

    Exposure to cholinergic agonists is a widely used paradigm to induce epileptogenesis in vivo and synchronous activity in brain slices maintained in vitro. However, the mechanisms underlying these effects remain unclear. Here, we used field potential recordings from the lateral entorhinal cortex in horizontal rat brain slices to explore whether two different K(+) currents regulated by muscarinic receptor activation, the inward rectifier (K(IR)) and the M-type (K(M)) currents, have a role in carbachol (CCh)-induced field activity, a prototypical model of cholinergic-dependent epileptiform synchronization. To establish whether K(IR) or K(M) blockade could replicate CCh effects, we exposed slices to blockers of these currents in the absence of CCh. K(IR) channel blockade with micromolar Ba(2+) concentrations induced interictal-like events with duration and frequency that were lower than those observed with CCh; by contrast, the K(M) blocker linopirdine was ineffective. Pre-treatment with Ba(2+) or linopirdine increased the duration of epileptiform discharges induced by subsequent application of CCh. Baclofen, a GABA(B) receptor agonist that activates K(IR), abolished CCh-induced field oscillations, an effect that was abrogated by the GABA(B) receptor antagonist CGP 55845, and prevented by Ba(2+). Finally, when applied after CCh, the K(M) activators flupirtine and retigabine shifted leftward the cumulative distribution of CCh-induced event duration; this effect was opposite to what seen during linopirdine application under similar experimental conditions. Overall, our findings suggest that K(IR) rather than K(M) plays a major regulatory role in controlling CCh-induced epileptiform synchronization. PMID:21144855

  14. Fos and serotonin immunoreactivity in the raphe nuclei of the cat during carbachol-induced active sleep: a double-labeling study.

    Science.gov (United States)

    Yamuy, J; Sampogna, S; López-Rodríguez, F; Luppi, P H; Morales, F R; Chase, M H

    1995-07-01

    The microinjection of carbachol into the nucleus pontis oralis produces a state which is polygraphically and behaviorally similar to active sleep (rapid eye movement sleep). In the present study, using double-labeling techniques for serotonin and the protein product of c-fos (Fos), we sought to examine whether immunocytochemically identified serotonergic neurons of the raphe nuclei of the cat were activated, as indicated by their expression of c-fos, during this pharmacologically-induced behavioral state (active sleep-carbachol). Compared with control cats, which were injected with saline, active sleep-carbachol cats exhibited a significantly greater number of c-fos-expressing neurons in the raphe dorsalis, magnus and pallidus. Whereas most of the c-fos-expressing neurons in the raphe dorsalis were small, those in the raphe magnus were medium-sized and in the raphe pallidus they were small and medium-sized. The mean number of serotonergic neurons that expressed c-fos (i.e. double-labeled cells) was similar in control and active sleep-carbachol cats. These data indicate that there is an increased number of non-serotonergic, c-fos-expressing neurons in the raphe dorsalis, magnus and pallidus during the carbachol-induced state.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7477901

  15. GABAergic neurons of the laterodorsal and pedunculopontine tegmental nuclei of the cat express c-fos during carbachol-induced active sleep.

    Science.gov (United States)

    Torterolo, P; Yamuy, J; Sampogna, S; Morales, F R; Chase, M H

    2001-02-23

    The laterodorsal and pedunculopontine tegmental nuclei (LDT-PPT) are involved in the generation of active sleep (AS; also called REM or rapid eye movement sleep). Although the LDT-PPT are composed principally of cholinergic neurons that participate in the control of sleep and waking states, the function of the large number of GABAergic neurons that are also located in the LDT-PPT is unknown. Consequently, we sought to determine if these neurons are activated (as indicated by their c-fos expression) during active sleep induced by the microinjection of carbachol into the rostro-dorsal pons (AS-carbachol). Accordingly, immunocytochemical double-labeling techniques were used to identify GABA and Fos protein, as well as choline acetyltransferase (ChAT), in histological sections of the LDT-PPT. Compared to control awake cats, there was a larger number of GABAergic neurons that expressed c-fos during AS-carbachol (31.5+/-6.1 vs. 112+/-15.2, P<0.005). This increase in the number of GABA+Fos+ neurons occurred on the ipsilateral side relative to the injection site; there was a small decrease in GABA+Fos+ cells in the contralateral LDT-PPT. However, the LDT-PPT neurons that exhibited the largest increase in c-fos expression during AS-carbachol were neither GABA+ nor ChAT+ (47+/-22.5 vs. 228.7+/-14.0, P<0.0005). The number of cholinergic neurons that expressed c-fos during AS-carbachol was not significantly different compared to wakefulness. These data demonstrate that, during AS-carbachol, GABAergic as well as an unidentified population of neurons are activated in the LDT-PPT. We propose that these non-cholinergic LDT-PPT neurons may participate in the regulation of active sleep. PMID:11172778

  16. Carbachol- and elevated Ca(2+)-induced translocation of functionally active protein kinase C to the brush border of rabbit ileal Na+ absorbing cells.

    OpenAIRE

    Cohen, M E; Wesolek, J; McCullen, J; Rys-Sikora, K; Pandol, S; Rood, R P; Sharp, G W; Donowitz, M

    1991-01-01

    Protein kinase C is involved in mediating the effects of elevated Ca2+ in ileal villus Na+ absorbing cells to inhibit NaCl absorption. The present studies were undertaken to understand the mechanism by which this occurs. The effects of carbachol and the calcium ionophore A23187, agents which elevate intracellular Ca2+ and inhibit NaCl absorption in ileal villus cells, were studied. Carbachol treatment of villus cells caused a rapid decrease in protein kinase C activity in cytosol, with an acc...

  17. The novel β3-adrenoceptor agonist mirabegron reduces carbachol-induced contractile activity in detrusor tissue from patients with bladder outflow obstruction with or without detrusor overactivity

    DEFF Research Database (Denmark)

    Nordling, Jørgen; Bouchelouche, Kirsten; Andersson, Karl-Erik;

    2013-01-01

    prostatic hyperplasia undergoing cystoscopy and from patients undergoing radical prostatectomy/cystectomy (in total 33 donors). Detrusor contractility was evaluated by organ bath studies and strips were incubated with carbachol (1μM) to induce and enhance tension. Both mirabegron and isoprenaline reduced...... preparations from patients with bladder outflow obstruction (BOO) with and without detrusor overactivity (DO), and from patients with normal bladder function. We compared the effects to those of isoprenaline, a non-selective β-adrenoceptor agonist. Detrusor specimens were obtained from patients with benign...

  18. Carbachol increases basolateral K+ conductance in T84 cells. Simultaneous measurements of cell [Ca] and gK explore calcium's role

    OpenAIRE

    1990-01-01

    To explore the role of calcium in mediating the action of carbachol in chloride-secreting epithelia, we simultaneously measured intracellular free [Ca] ([Ca]i) and the potassium conductance (gK) of the basolateral membrane in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value ([Ca]'i) normalized to control. To assess changes in basolateral gK, we measured the short circuit current (Isc) in the presence of lu...

  19. Biphasic increase of apical Cl- conductance by muscarinic stimulation of ht-29cl.19a human colon carcinoma cell line: Evidence for activation of different cl- conductances by carbachol and forskolin

    NARCIS (Netherlands)

    R.B. Bajnath (R.); K. Dekker (K.); A.B. Vaandrager (Arie); H.R. de Jonge (Hugo); J.A. Groot (J.)

    1992-01-01

    textabstractThe modulation of ion transport pathways in filtergrown monolayers of the Cl--secreting subclone (19A) of the human colon carcinoma cell line HT-29 by muscarinic stimulation was studied by combined Ussing chamber and microimpalement experiments. Basolateral addition of 10-4m carbachol in

  20. Role of orexin-2 receptors in the nucleus accumbens in antinociception induced by carbachol stimulation of the lateral hypothalamus in formalin test.

    Science.gov (United States)

    Yazdi, Fatemeh; Jahangirvand, Mahboubeh; Ezzatpanah, Somayeh; Haghparast, Abbas

    2016-08-01

    Orexins, which are mainly produced by orexin-expressing neurons in the lateral hypothalamus (LH), play an important role in pain modulation. Previously, it has been established that the nucleus accumbens (NAc) is involved in the modulation of formalin-induced nociceptive responses, a model of tonic pain. In this study, the role of intra-accumbal orexin-2 receptors (OX2rs) in the mediation of formalin-induced pain was investigated. A volume of 0.5 μl of 10, 20, and 40 nmol/l solutions of TCS OX2 29, an OX2r antagonist, were unilaterally microinjected into the NAc 5 min before an intra-LH carbachol microinjection (0.5 μl of 250 nmol/l solution). After 5 min, animals received a subcutaneous injection of formalin 2.5% (50 μl) into the hind paw. Pain-related behaviors were assessed at 5 min intervals during a 60-min test period. The findings showed that TCS OX2 29 administration dose dependently blocked carbachol-induced antinociception during both phases of formalin-induced pain. The antianalgesic effect of TCS OX2 29 was greater during the late phase compared with the early phase. These observations suggest that the NAc, as a part of a descending pain-modulatory circuitry, partially mediates LH-induced analgesia in the formalin test through recruitment of OX2rs. This makes the orexinergic system a good potential therapeutic target in the control of persistent inflammatory pain. PMID:26871404

  1. Phase dependency of long-term potentiation induction during the intermittent bursts of carbachol-induced β oscillation in rat hippocampal slices.

    Science.gov (United States)

    Nishimura, Motoshi; Nakatsuka, Hiroki; Natsume, Kiyohisa

    2012-01-01

    The rodent hippocampus possesses theta (θ) and beta (β) rhythms, which occur intermittently as bursts. Both rhythms are related to spatial memory processing in a novel environment. θ rhythm is related to spatial memory encoding process. β rhythm is related to the match/mismatch process. In the match/mismatch process, rodent hippocampus detects a representation matching sensory inputs of the current place among the retrieved internal representations of places. Long-term synaptic potentiation (LTP) is induced in both processes. The cholinergic agent carbachol induces intermittent θ and β oscillations in in vitro slices similar to in vivo bursts. LTP is facilitated during the generation of θ oscillation, suggesting that the facilitation of LTP is dependent upon the phases of intermittent burst (burst phases) of the oscillation. However, whether this is the case for β oscillation has not yet been studied. In the present study, LTP-inducing θ-burst stimulation was administered at the different burst phases of carbachol-induced β oscillations (CIBO), and the synaptic changes were measured at CA3-CA3 pyramidal cell synapses (CA3 synapse) and at CA3-CA1 pyramidal cell synapses (CA1 synapse). At the CA3 synapse, the largest magnitude of LTP was induced at the late burst phases of CIBO. At the CA1 synapse, LTP was induced only at the late burst phases. Modulation of LTP was suppressed when CIBO was blocked by the application of atropine at both synapses. The results suggest that the bursts of hippocampal β rhythm can determine the optimal temporal period for completing with the match/mismatch process.

  2. PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells.

    Science.gov (United States)

    Zachos, Nicholas C; Lee, Luke J; Kovbasnjuk, Olga; Li, Xuhang; Donowitz, Mark

    2013-08-01

    Elevated levels of intracellular Ca(2+) ([Ca(2+)]i) inhibit Na(+)/H(+) exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca(2+)]i inhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca(2+) signaling proteins necessary for regulation of NHE3 activity. [Ca(2+)]i regulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca(2+)]i inhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y(416) phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca(2+)]i inhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca(2+)]i conditions, and 4) does not directly bind NHE3. Under elevated [Ca(2+)]i conditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3.

  3. 卡巴胆碱对休克延迟复苏大鼠脏器功能的影响%EFFECTS OF CARBACHOL ON ORGAN FUNCTION OF BURN SHOCK DELAYED RESUSCITATION IN RATS

    Institute of Scientific and Technical Information of China (English)

    程爱斌; 谢宇曦; 王瑞刚; 贺瑞新; 罗永伟; 高素玲; 王林; 余亮科

    2014-01-01

    目的:研究卡巴胆碱对烫伤休克大鼠延迟复苏时脏器功能的影响。方法 Wistar大鼠随即分假烧伤组(A组),标准组(B组),对照组(C组),卡巴胆碱组(D组),制备30%体表面积Ⅲ度烫伤模型。C组经损伤处理0.5h后管饲卡巴胆碱。损伤处理后大鼠经外科游离颈静脉及颈动脉并分别置入无菌导管,以微蠕动泵控制输入乳酸钠林格液,对照组和卡巴胆碱组给药剂量及输注速度为标准组药量或输注速度的1/2。A组仅行备皮及动、静脉导管置入,无任何损伤处理及后续措施。数据采纳输注相关液体后0,1,2,4h二组的血细胞比容(Hct)、平均动脉压(MAP)及心率(HR)等,同时检测大鼠肝、肾、肠组织血量。结果 D组经处理2h及4h后可明显降低心率,提升血压,改善烧伤休克大鼠早期脏器的功能。结论卡巴胆碱能够维护烧伤大鼠休克早期的脏器功能,同时减少机体液量需求,对休克及并发症的处理具有明显积极意义。%Objective To study the effects of Carbachol on organ function of burn shock delayed resuscita-tion in rats .Methods Wistar rats were divided into false burn group ( group A) ,standard group ( group B ) ,control group ( group C) and Carbachol group (group D) ,then 30% BSA third degree burn model was prepared .Group C was received Carbachol at half an hour after oral administration of .The scalded rats first exposed jugular vein ,artery incision and inserted into a sterile silicone tube ,with a micro peristal-tic pump control input of lactated Ringer's solution .The control group and the Kaba group were transfused drugs according to the standard dose of choline or velocity of the given 1/2 ,while group A only shearing , jugular vein ,artery intubation ,but not scald and rehydration .Recording fluid infusion was started after 0 , 1 ,2 ,4h in two groups in mean arterial pressure (MAP) ,heart rate (HR

  4. The protection of carbachol on intestinal barrier function in patients with trauma%卡巴胆碱对创伤患者肠屏障功能的保护作用

    Institute of Scientific and Technical Information of China (English)

    王印华; 尤丕聪; 唐明贵; 王宝华; 柴海霞; 浦践一

    2014-01-01

    Objective To observe the protection of carbachol on intestinal barrier function in patients with trauma. Methods A prospective randomized controlled trial was conducted. Seventy patients after trauma with a definite diagnosis of multiple organ dysfunction syndrome(MODS)from Department of Critical Care Medicine in Hebei United University Affiliated Hospital were included. According to random number table,the patients were divided into a carbachol treatment group(37 cases)and a mosapride citrate treatment group(33 cases),and all the patients in the two groups were treated by antibacterial drugs,supportive agents for organ function,surgery, etc symptomatic treatment. Based on the conventional treatment,in the carbachol treatment group,carbachol was administered through a stomach tube at the dose of 0.2 mg/kg,twice a day,and the dose was doubled if no exhaust or defecation persisted for 3 days after treatment,while in the mosapride group,mosapride citrate was given at the dose of 5 mg once and thrice a day,the therapeutic course of both groups being 7 days. On the 1st,3rd,5th, 7th day after admission,peripheral venous fasting blood in early morning was collected,the activity of diamine oxidase(DAO),expression rates of CD11b+and CD18+in polymorphonuclear neutrophil(PMN),contents of tumour necrosis factor-α(TNF-α)and interleukin-10(IL-10) were detected,and the clinical curative effects were observed. Results Compared to the mosapride citrate treatment group,the total effective rate was significantly higher in the carbachol treatment group on the 7th day after treatment〔70.3%(26/37)vs. 45.5%(15/33),P<0.05〕. The activity of DAO,expression rates of CD11b+and CD18+in PMN,contents of TNF-αand IL-10 in the carbachol treatment group were decreased with the extension of time,and reached valley values on the 7th day,the differences were statistically significant in the comparisons with those in mosapride citrate treatment group at the same time point〔DAO(mg/L):3.21±0

  5. 卡巴胆碱对大鼠烫伤休克期肠内补液时肠道的影响%Carbachol alleviates intestinal inflammation during enteral fluid resuscitation of rats with burn shock

    Institute of Scientific and Technical Information of China (English)

    车晋伟; 胡森; 耿世佳; 吴静; 王磊; 杜颖; 田易军; 盛志勇

    2008-01-01

    Objective To investigate the effect of carbachol on local gut inflammation during entetal resuscitation of rats with bum shock. Method Thirty-eight Wistar rats were subjected to 35%TBSA full thickness scald injury, and enteral fluid was infused into animal intestines via duodenal stomas 30 minutes post bum. The animals were randomly divided into four groups: no resuscitation (Control, n = 8), enteral resuscitation using either a glucose electrolyte solution (GES, n = 10) or GES plus carbachol (60 μg·kg-1,GES/CAR, n = 10), or carbachol alone (CAR, n = 10) .The volumeof GES infusion was based on the Parkland formula (4 ml· 1% TB-SA-1·Kg-1) - All animals were sacrificed 4 hours post bum, and specimens of jejunal tissue were collected to determine the levels of tumor necrosis factor (TNF)-α, nitric oxide (NO), nitric oxide synthase (NOS) and myeloperoxidase (MPO). Serum assays for plasma diamine oxidase (DAO) activities were also performed. Results There were no statistical differences in the intestinal levels of NOS, NO, TNF-α and MPO, and plasma OAO activities, between the GES group and the control group. Compared to the GES group, the GES/CAR group showed significantly lowered levels of intestinal NOS (1.276 ±0.391 vs. 1.818 ±0.436, P<0.05), NO (0.925 ±0.402 vs. 1.561 ±0.190, P < 0.05, TNF-α (0.87±0.13 vs. 1.94±0.47, P <0.01) and MPO (0.465 ±0.092 vs. 0.832±0.214, P<0.05),and reduction in plasma DAO activites (0.732±0.192 vs. 1.381 ±0.564, P <0.05). The CAR group also showed significantly lowered levels of intestinal NOS, NO, TNF-α and MPO and reduced plasma DAO activites, compared to the GES group. Conclusions Theses results suggest that carbachol significantly inhibits the release of proinflammatory mediator and attenuates local inflammation in gut during enteral fluid resuscitation of rats in rats with bum shock. We postulate that carbachol may exert its and-inflammatory effects via the cholinergic anti-inflammatory pathway.%目的

  6. 单纯卡巴胆碱或联合膳食纤维治疗对弥漫性脑损伤后肠屏障的影响%Effect of pure carbachol or combined with dietary fiber on intestinal mucosal barrier of rats after diffuse brain injury

    Institute of Scientific and Technical Information of China (English)

    王印华; 王宝华; 唐明贵; 浦践一; 柴海霞

    2015-01-01

    Objective To study the protective effect of pure carbachol or combined with dietary fiber on intestinal mucosal barrier of rats after diffuse brain injury (DBI).Methods An adult male Wistar rat model of DBI was reproduced by gravitational shock method. The rats injured and survived after resuscitation were divided into three groups: model group (n = 40), carbachol group (n = 40) and carbachol combined with dietary fiber group (combined group,n = 32). In addition, a control group was established by simply an incision performed on the scalp, and the rats could drink freely (n = 5). In the experimental groups, 2 hours after resuscitation the rats began to receive gavage, 6 hours once, the liquid amount 15 mL/kg should be assured in every 6 hours, and if insufficient, normal saline was supplemented. In model group, normal saline 90 mL/kg was given, in carbachol group, carbachol 300μg/kg was administered and in combined group, carbachol 300μg/kg combined with dietary fiber 60 mL/kg was supplied. At 3 (combined group being excluded), 6, 12, 24 and 48 hours after resuscitation, the rats were anesthetized to collect samples and detect the plasma levels of D-lactate and activity of diamine oxidase (DAO) respectively, and the changes of villus height of small intestine were examined by a light microscope.Results The plasma D-lactate levels and the activities of DAO at any time point in the experimental groups were significant higher than those in control group (allP < 0.01). Along with the prolongation of time, the levels of plasma D-lactate and DAO activities in carbachol and carbachol plus diatary fiber groups were gradually lower than those of the model group, and at 48 hours after injury they reached their valley values [D-lactate (ng/L): 6.32±0.79, 7.46±1.67 vs. 17.65±1.53, DAO activity (kU/L): 0.76±0.01, 0.86±0.01 vs. 2.23±0.15]. Under light microscopy, compared with control group, the villus height of small intestinal mucosa at any time point in any

  7. Carbachol阻断亚硒酸钠对离体皮质神经元的致凋亡作用%Carbachol blocks apoptosis induced by sodium selenite in cultured cortical neurons

    Institute of Scientific and Technical Information of China (English)

    肖荣; 闫秀珍; 王瑞; 乔中东; 乔健天

    2001-01-01

    在早期工作发现小剂量亚硒酸钠(Sodium selenite)对离体皮质神经元的致凋亡作用的基础上,观察了在亚硒酸钠致凋亡模型中加入M型胆碱能受体激动剂Carbachol(Carb)能否阻止凋亡过程和相伴随的有关基因表达的改变.结果显示:①用不同剂量(0.004,0.02,0.1,0.5,2.5和12.5 mmol/L)的Carb和0.5μmol/L亚硒酸钠共同处理培养的新生小鼠皮质神经元时,用琼脂糖凝胶电泳DNA梯形检测和流式细胞仪亚二倍体峰检测,都证明Carb表现出对亚硒酸钠致凋亡作用的剂量依赖性保护效应;②用RT-PCR技术证明,在与Carb共同孵育的实验中,不再出现或减弱了亚硒酸钠单独处理时出现的bcl-2基因表达的下调和bax,p53,c-fos以及AChE基因表达的上调.上述结果表明,亚硒酸钠对皮质神经元的致凋亡作用和与之相伴随的基因表达的改变,可被一定浓度的Carb所阻断,提示亚硒酸钠致凋亡作用可能同本室发现的β-淀粉样蛋白的C末端5肽片段的致凋亡作用具有类似的途径,以类似的基因表达改变为基础,并对Carb的抗凋亡机制进行了讨论.

  8. Effect of propofol on carbachol-induced intestinal mucosa secretion%异丙酚对卡巴胆碱引起的肠黏膜分泌的影响

    Institute of Scientific and Technical Information of China (English)

    孙华; 汤树海; 王颢诒; 安宁; 许翠萍; 赵东波

    2016-01-01

    目的:观察异丙酚对卡巴胆碱(CCh)引起的肠黏膜分泌的影响。方法利用 Ussing chamber 技术监测黏膜短路电流(Isc )的变化。结果CCh 引起大鼠回肠黏膜 Isc升高;异丙酚明显抑制 CCh 引起的大鼠回肠黏膜 Isc的变化,且呈现明显的浓度依赖效应(10-2~10-6 mol/L),这一抑制效应仅发生在浆膜层。钠离子通道阻断剂河豚毒素及 N 型胆碱能受体阻断剂六甲溴铵都不能影响异丙酚引起的抑制作用。结论静脉麻醉药异丙酚可抑制 CCh引起的肠黏膜分泌增加,其作用是通过抑制肠黏膜上皮基底侧的 M 型胆碱能受体信号途径实现。%Objective To explore the effect of propofol on carbachol(CCh)-induced chloride secretion in rat ileum mu-cosa.Methods Cch-induced chloride secretion was measured by Ussing chamber technique.Results Propofol atten-uated the CCh-stimulated increase in short-circuit current (Isc )of rat ileum mucosa in a dose-dependent manner (10 -2-10 -6 mol/L).The propofol-evoked inhibitory effect was serosal side-dependent.Furthermore,both TTX,a Na + channel blocker,and hexamethonium bromide,a nicotinic acetylcholine receptor antagonist,had no effect on propofol-evoked in-hibitory effect.Conclusion Propofol inhibites CCh-induced intestinal secretion via basolateral mAChR pathway.

  9. Influence of carbachol on myocardium in patients with intestinal barrier dysfunction induced by emergency trauma%卡巴胆碱对急诊创伤后肠屏障功能障碍者心肌损伤的影响

    Institute of Scientific and Technical Information of China (English)

    王印华; 王宝华; 唐明贵; 柴海霞; 浦践一

    2014-01-01

    目的 探讨卡巴胆碱对急诊创伤后肠屏障功能障碍患者心肌的保护作用.方法 选择70例急诊创伤后肠屏障功能障碍综合征的患者完全随机分为卡巴胆碱组(37例)和莫沙必利组(33例),监测患者第1、7天外周血二胺氧化酶活性、嗜中性多形核粒细胞CD11b+/CD18的表达率、左心室射血分数及肌酸激酶同工酶峰值,观察临床疗效.结果 治疗后7d,卡巴胆碱组有效26例(70.3%),死亡7例(18.9%);莫沙必利组有效15例(45.5%),死亡13例(39.4%).卡巴胆碱组有效率优于莫沙必利组,差异有统计学意义(x2=4.43,P< 0.05).卡巴胆碱组治疗第1天血二胺氧化酶、CD11b+和CD18分别为(4.8±0.5)kU/L、(30.6±3.6)%、(75.8±3.6)%,第7天分别为(3.2±0.5) kU/L、(14.9±2.2)%、(53.7±2.4)%;莫沙必利组治疗第1天血二胺氧化酶、CD11b+和CD18分别为(4.7±0.3)kU/L、(31.1±2.9)%、(74.6±2.7)%,第7天分别为(3.9±0.5)kU/L、(28.9±1.6)%、(72.5±4.1)%,治疗第7天卡巴胆碱组患者血二胺氧化酶水平、嗜中性多形核粒细胞CD11b及CD18阳性表达率均低于入组第1天及莫沙必利组,差异均有统计学意义(均P<0.05).2组患者入组时肌酸激酶同工酶差异无统计学意义,而卡巴胆碱组肌酸激酶同工酶峰值明显低于莫沙必利组[(60±19) U/L比(79±18) U/L],差异有统计学意义(P<0.05);2组患者入组时左心室射血分数差异无统计学意义,卡巴胆碱组左心室射血分数峰值高于莫沙必利组[(60±7)%比(55±7)%],差异有统计学意义(P<0.05).结论 卡巴胆碱通过降低急诊创伤后肠屏障功能障碍患者肠道缺血再灌注损伤、减少活性炎症介质释放、促进肠蠕动,从而减轻心肌损伤,保护远隔脏器-心脏的功能.%Objective To explore protection of carbachol for myocardium in patients with intestinal barrier dysfunction induced by emergency trauma.Methods Seventy patients after trauma with a definite

  10. 早期肠内给予膳食纤维复合卡巴胆碱对弥漫性颅脑损伤大鼠肠黏膜屏障功能的影响%Impact of Early Enteral-intestinal Dietary Fiber Combined with Carbachol on Intestinal Mucosal Barrier Function of Rats with Diffuse Craniocerebral Injury

    Institute of Scientific and Technical Information of China (English)

    唐明贵; 邱涵坤; 王宝华; 王印华; 柴海霞; 浦践一; 邱方

    2015-01-01

    目的:研究早期肠内给予膳食纤维复合卡巴胆碱对弥漫性颅脑损伤大鼠肠黏膜屏障功能的影响。方法从126只清洁级Wistar大鼠中不规则抽取20只大鼠作为假手术组(对照组),其余106只大鼠采用Marmarou模型制备法制备弥漫性颅脑损伤模型,最终建模成功64只,按随机数字表法分为0.9%氯化钠溶液组( NS组,n=32)和膳食纤维复合卡巴胆碱组(DF+CAR组,n=32)。对照组大鼠自由饮水,NS组大鼠建模成功后采用0.9%氯化钠溶液灌胃,DF+CAR组大鼠建模成功后采用膳食纤维+卡巴胆碱灌胃,灌胃量为15 ml·kg-1·(6 h)-1,不足时加0.9%氯化钠溶液。3组大鼠分别于建模后6 h、12 h、24 h、48 h处死(对照组各时间点5只,NS组和DF+CAR组各时间点8只)。比较各组大鼠不同时间点血浆二胺氧化酶( DAO)、D-乳酸水平及肠绒毛高度。结果 NS组及DF+CAR组大鼠建模后6 h、12 h、24 h、48 h血浆DAO水平均高于对照组, DF+CAR组大鼠建模后6 h、12 h、24 h、48 h血浆DAO水平均低于NS组(P﹤0.05)。NS组及DF+CAR组大鼠建模后6 h、12 h、24 h、48 h血浆D-乳酸水平均高于对照组,DF+CAR组大鼠建模后12 h、24 h、48 h血浆D-乳酸水平均低于NS组(P﹤0.05)。NS组及DF+CAR组大鼠建模后6 h、12 h、24 h肠绒毛高度低于对照组,NS组大鼠建模后48 h肠绒毛高度低于对照组,DF+CAR组大鼠建模后24 h、48 h肠绒毛高度高于NS组(P﹤0.05)。结论早期肠内给予膳食纤维复合卡巴胆碱对弥漫性颅脑损伤大鼠肠黏膜屏障功能有一定的保护作用。%Objective To investigate the impact of early enteral-intestinal dietary fiber combined with carbachol on intestinal mucosal barrier function of rats with diffuse craniocerebral injury. Methods A total of 126 cleaning Wistar rats were enrolled,and 20 rats were irregularly selected as control group and were given free drinking only

  11. Die Carbachol-induzierte Chloridsekretion am proximalen Kolon des Pferdes

    OpenAIRE

    Lamberti, Nicole

    2005-01-01

    Akute Durchfallerkrankungen kommen weltweit bei Pferden vor. Ihre besondere Bedeutung liegt zum einen in ihrem häufig letalen Ausgang, zum anderen kann ihre Therapie sehr zeit- und kostenaufwendig sein. Eine beim adulten Pferd wichtige Erkrankung des Dickdarms ist die Colitis X. Hierbei handelt es sich um eine hämorrhagisch-nekrotisierende Typhlocolitis, welche auch bei intensiver Therapie durch die massiven Flüssigkeitsverluste in 75-95 % der Fälle zu einem letal endenden hypovolämischen Sch...

  12. Investigation of carbachol and PACAP38 in a human model of migraine

    DEFF Research Database (Denmark)

    Schytz, Henrik Winther

    2011-01-01

    The parasympathetic signalling molecules acetylcholine, pituitary adenylate cyclase activating peptide-38 (PACAP38) and vasoactive intestinal peptide (VIP) may be released from parasympathetic fibres and activate sensory nerve fibres during migraine attacks. Recently, it was shown that VIP does...... in migraine patients as well as sustained dilatation of cephalic vessels. In study IV VIP and PACAP38 evoked skin pain, central sensitization, neurogenic inflammation and mast cell degranulation, but VIP showed to be more potent than PACAP38 in inducing neurogenic inflammation and mast cell degranulation...

  13. Differences in time to peak carbachol-induced contractions between circular and longitudinal smooth muscles of mouse ileum.

    Science.gov (United States)

    Azuma, Yasu-Taka; Samezawa, Nanako; Nishiyama, Kazuhiro; Nakajima, Hidemitsu; Takeuchi, Tadayoshi

    2016-01-01

    The muscular layer in the GI tract consists of an inner circular muscular layer and an outer longitudinal muscular layer. Acetylcholine (ACh) is the representative neurotransmitter that causes contractions in the gastrointestinal tracts of most animal species. There are many reports of muscarinic receptor-mediated contraction of longitudinal muscles, but few studies discuss circular muscles. The present study detailed the contractile response in the circular smooth muscles of the mouse ileum. We used small muscle strips (0.2 mm × 1 mm) and large muscle strips (4 × 4 mm) isolated from the circular and longitudinal muscle layers of the mouse ileum to compare contraction responses in circular and longitudinal smooth muscles. The time to peak contractile responses to carbamylcholine (CCh) were later in the small muscle strips (0.2 × 1 mm) of circular muscle (5.7 min) than longitudinal muscles (0.4 min). The time to peak contractile responses to CCh in the large muscle strips (4 × 4 mm) were also later in the circular muscle (3.1 min) than the longitudinal muscle (1.4 min). Furthermore, a muscarinic M2 receptor antagonist and gap junction inhibitor significantly delayed the time to peak contraction of the large muscle strips (4 × 4 mm) from the circular muscular layer. Our findings indicate that muscarinic M2 receptors in the circular muscular layer of mouse ileum exert a previously undocumented function in gut motility via the regulation of gap junctions.

  14. Electrophysiological characterization of neurons in the dorsolateral pontine REM sleep induction zone of the rat: intrinsic membrane properties and responses to carbachol and orexins

    OpenAIRE

    Brown§, Ritchie E.; Winston, Stuart; Basheer, Radhika; Thakkar, Mahesh M.; McCarley, Robert W.

    2006-01-01

    Pharmacological, lesion and single-unit recording techniques in several animal species have identified a region of the pontine reticular formation (Subcoeruleus, SubC) just ventral to the locus coeruleus as critically involved in the generation of rapid-eye-movement (REM) sleep. However, the intrinsic membrane properties and responses of SubC neurons to neurotransmitters important ...

  15. Dissociated insulinotropic sensitivity to glucose and carbachol in high-fat diet-induced insulin resistance in C57BL/6J mice

    NARCIS (Netherlands)

    Ahren, B; Simonsson, E; Scheurink, AJW; Mulder, H; Myrsen, U; Sundler, F

    1997-01-01

    To study islet function following reduced insulin sensitivity, we examined mice of the C57BL/6J strain, the genotype of which carries an increased propensity to develop insulin resistance when metabolically challenged. The mice received either a high-fat diet (58% fat on an energy basis) or a contro

  16. Membrane potential and conductance of frog skin gland acinar cells in resting conditions and during stimulation with agonists of macroscopic secretion

    DEFF Research Database (Denmark)

    Sørensen, Jakob B.; Larsen, Erik Hviid

    1999-01-01

    Adrenaline; carbachol; Cl- secretion; exocrine gland; isoproterenol; noradrenaline; prostaglandin E*U2......Adrenaline; carbachol; Cl- secretion; exocrine gland; isoproterenol; noradrenaline; prostaglandin E*U2...

  17. Effects of carbachol on proliferation of microglia participating in inflammatory reaction of central nervous system%卡巴胆碱对参与中枢炎症反应的小胶质细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    关艳中; 高天明

    2007-01-01

    目的:观察卡巴胆碱是否对小胶质细胞增殖产生影响.方法:培养的小胶质细胞系,加入不同浓度的卡巴胆碱或卡巴胆碱与粒细胞巨噬细胞集落刺激因子(Granulocyte-Macrophage Colony-Stimulating Factor,GM-CSF),用MTT方法检测细胞活性,观察对小胶质细胞数量的影响.结果:我们在培养的小胶质细胞系发现,卡巴胆碱显著减少小胶质细胞的数量,并能有效抑制粒细胞巨噬细胞集落刺激因子诱导的小胶质细胞数量增多,并存在剂量依赖效应,10μ M浓度效果最为显著.结论:卡巴胆碱可抑制小胶质细胞炎性反应.

  18. Augmentation of cholinergic-mediated amylase release by forskolin in mouse parotid gland

    International Nuclear Information System (INIS)

    Cholinergic-mediated amylase release in mouse parotid acini was augmented by forskolin; the potency but not the maximal response to carbachol was altered. Amylase released by carbachol plus forskolin was dependent on extracellular calcium and was mimicked by the calcium ionophore, A23187 plus forskolin. Forskolin was also shown to enhance carbachol-stimulated 45Ca2+ uptake into isolated acini. Hydroxylamine, nitroprusside, and 8-bromo-c-GMP each in combination with forskolin mimicked the effects of carbachol plus forskolin on amylase release. In the presence of carbachol (10-8M) forskolin did not augment c-AMP levels. However, in the presence of carbachol (5 x 10-7 M) or hydroxylamine (50 μM) forskolin did significantly augment c-AMP accumulation. These results suggest that calcium and c-GMP may mediate the augmentation of cholinergic-mediated amylase release by effects on c-AMP metabolism. 21 references, 1 figure, 3 tables

  19. Interactions between chemical and electrical kindling of the rat amygdala.

    Science.gov (United States)

    Wasterlain, C G; Morin, A M; Jonec, V

    1982-09-16

    Holtzman rats were implanted with a chemitrode into the left basolateral amygdala, which could then be stimulated electrically (400 microA, 1 s, AC) or chemically by injection of carbachol (1 microliter, 2.7 nmoles, sterile, isotonic). Group A received a daily injection of carbachol and developed kindled seizures. Group B received carbachol mixed with equimolar atropine, which blocked seizures and kindling. After 20 injections, both groups were stimulated electrically once a day and kindled at similar rates. Two additional groups received electrical or sham stimulation, followed by carbachol kindling. No transfer effects were observed. Four additional groups received 27 nmoles of atropine through the chemitrode, followed 15 min later by electrical stimulation, sham stimulation, carbachol injection or saline injection, respectively. Atropine completely blocked carbachol kindling but did not alter the rate of electrical kindling. No different in the number of QNB binding sites was observed in the amygdala of rats sacrificed two weeks after full electrical kindling. The lack of interaction between electrical and carbachol kindling and the failure of atropine to block electrical kindling of the amygdala suggest that the activation of local muscarinic synapses, while essential for carbachol kindling, is not required for electrical kindling of the rat amygdala.

  20. Ethanol impairs muscarinic receptor-induced neuritogenesis in rat hippocampal slices: Role of astrocytes and extracellular matrix proteins.

    Science.gov (United States)

    Giordano, Gennaro; Guizzetti, Marina; Dao, Khoi; Mattison, Hayley A; Costa, Lucio G

    2011-12-01

    In an in vitro co-culture system of astrocytes and neurons, stimulation of cholinergic muscarinic receptors in astrocytes had been shown to cause neuritogenesis in hippocampal neurons, and this effect was inhibited by ethanol. The present study sought to confirm these earlier findings in a more complex system, in vitro rat hippocampal slices in culture. Exposure of hippocampal slices to the cholinergic agonist carbachol (1mM for 24h) induced neurite outgrowth in hippocampal pyramidal neurons, which was mediated by activation of muscarinic M3 receptors. Specifically, carbachol induced a >4-fold increase in the length of the longest neurite, and a 4-fold increase in the length of minor neurites and in the number of branches. Co-incubation of carbachol with ethanol (50mM) resulted in significant inhibition of the effects induced by carbachol on all parameters measured. Neurite outgrowth in CNS neurons is dependent on various permissive factors that are produced and released by glial cells. In hippocampal slices carbachol increased the levels of two extracellular matrix protein, fibronectin and laminin-1, by 1.6-fold, as measured by Western blot. Co-incubation of carbachol with ethanol significantly inhibited these increases. Carbachol-induced increases in levels of extracellular matrix proteins were antagonized by a M3 muscarinic receptor antagonist. Furthermore, function-blocking fibronectin or laminin-1 antibodies antagonized the effect of carbachol on neurite outgrowth. These results indicate that in hippocampal slices stimulation of muscarinic M3 receptors induces neurite outgrowth, which is mediated by fibronectin and laminin-1, two extracellular matrix proteins released by astrocytes. By decreasing fibronectin and laminin levels ethanol prevents carbachol-induced neuritogenesis. These findings highlight the importance of glial-neuronal interactions as important targets in the developmental neurotoxicity of alcohol.

  1. Experimental testing of Mackay's model for functional antagonism in the isolated costo-uterus of the rat.

    OpenAIRE

    Henry, P. J.; Lulich, K M; Paterson, J W

    1985-01-01

    Several key predictions of a recently developed model for functional antagonism (Mackay, 1981) were experimentally tested using the rat isolated costo-uterine preparation. In the presence of the functional antagonist fenoterol (Fen), the functional constants (KAF) for carbachol and oxotremorine (Oxo) were respectively 9.9 and 3.4 fold greater than their corresponding affinity constants (KA). According to Mackay's model for functional antagonism, the higher KAF/KA ratio for carbachol indicates...

  2. Rapid recycling of Ca2+ between IP3-sensitive stores and lysosomes.

    Directory of Open Access Journals (Sweden)

    Cristina I López Sanjurjo

    Full Text Available Inositol 1,4,5-trisphosphate (IP3 evokes release of Ca2+ from the endoplasmic reticulum (ER, but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

  3. Barrier Qualities of the Mouse Eye to Topically Applied Drugs

    OpenAIRE

    Wang, Zhao; Do, Chi Wai; Avila, Marcel Y.; Stone, Richard A.; Jacobson, Kenneth A.; Civan, Mortimer M.

    2007-01-01

    The mouse eye displays unusually rapid intraocular pressure (IOP) responses to topically applied drugs as measured by the invasive servo-null micropipette system (SNMS). To learn if the time course reflected rapid drug transfer across the thin mouse cornea and sclera, we monitored a different parameter, pupillary size, following topical application of droplets containing 40 μM (0.073μg) carbachol. No miosis developed from this low carbachol concentration unless the cornea was impaled with an ...

  4. The control of chick myoblast fusion by ion channels operated by prostaglandins and acetylcholine

    OpenAIRE

    1988-01-01

    Chick myoblast fusion in culture was investigated using prostanoid synthesis inhibitors to delay spontaneous fusion. During this delay myoblast fusion could be induced by prostaglandin E1 (PGE1), by raising extracellular potassium and by addition of carbachol. Carbachol-induced fusion, but not PGE-induced fusion, was prevented by the acetylcholine receptor blocker alpha-bungarotoxin. Fusion induced by any of these agents was prevented by the Ca channel blockers lanthanum and D600. The thresho...

  5. Cuneiform neurons activated during cholinergically induced active sleep in the cat.

    Science.gov (United States)

    Pose, I; Sampogna, S; Chase, M H; Morales, F R

    2000-05-01

    In the present study, we report that the cuneiform (Cun) nucleus, a brainstem structure that before now has not been implicated in sleep processes, exhibits a large number of neurons that express c-fos during carbachol-induced active sleep (AS-carbachol). Compared with control (awake) cats, during AS-carbachol, there was a 671% increase in the number of neurons that expressed c-fos in this structure. Within the Cun nucleus, three immunocytochemically distinct populations of neurons were observed. One group consisted of GABAergic neurons, which predominantly did not express c-fos during AS-carbachol. Two other different populations expressed c-fos during this state. One of the Fos-positive (Fos(+)) populations consisted of a distinct group of nitric oxide synthase (NOS)-NADPH-diaphorase (NADPH-d)-containing neurons; the neurotransmitter of the other Fos(+) population remains unknown. The Cun nucleus did not contain cholinergic, catecholaminergic, serotonergic, or glycinergic neurons. On the basis of neuronal activation during AS-carbachol, as indicated by c-fos expression, we suggest that the Cun nucleus is involved, in an as yet unknown manner, in the physiological expression of active sleep. The finding of a population of NOS-NADPH-d containing neurons, which were activated during AS-carbachol, suggests that nitrergic modulation of their target cell groups is likely to play a role in active sleep-related physiological processes. PMID:10777795

  6. The Cholinergic Signaling Responsible for the Expression of a Memory-Related Protein in Primary Rat Cortical Neurons.

    Science.gov (United States)

    Chen, Tsan-Ju; Chen, Shun-Sheng; Wang, Dean-Chuan; Hung, Hui-Shan

    2016-11-01

    Cholinergic dysfunction in the brain is closely related to cognitive impairment including memory loss. In addition to the degeneration of basal forebrain cholinergic neurons, deficits in the cholinergic receptor signaling may also play an important role. In the present study, to examine the cholinergic signaling pathways responsible for the induction of a memory-related postsynaptic protein, a cholinergic agonist carbachol was used to induce the expression of activity-regulated cytoskeleton associated protein (Arc) in primary rat cortical neurons. After pretreating neurons with various antagonists or inhibitors, the levels of carbachol-induced Arc protein expression were detected by Western blot analysis. The results show that carbachol induces Arc protein expression mainly through activating M1 acetylcholine receptors and the downstream phospholipase C pathway, which may lead to the activation of the MAPK/ERK signaling pathway. Importantly, carbachol-mediated M2 receptor activation exerts negative effects on Arc protein expression and thus counteracts the enhanced effects of M1 activation. Furthermore, it is suggested for the first time that M1-mediated enhancement of N-methyl-D-aspartate receptor (NMDAR) responses, leading to Ca(2+) entry through NMDARs, contributes to carbachol-induced Arc protein expression. These findings reveal a more complete cholinergic signaling that is responsible for carbachol-induced Arc protein expression, and thus provide more information for developing treatments that can modulate cholinergic signaling and consequently alleviate cognitive impairment. J. Cell. Physiol. 231: 2428-2438, 2016. © 2016 Wiley Periodicals, Inc. PMID:26895748

  7. Relationship between PI decomposition and M3R expression in human cultured detrusor muscle cells

    Institute of Scientific and Technical Information of China (English)

    卢根生; 宋波; 金锡御; 熊恩庆

    2003-01-01

    Objective: To study the relationship between the expression of muscarinic receptor subtype M3R and the decomposition of phosphatidylinositol (PI). Methods: The [3H]-IP contents were detected with [3H] incorporation in human cultured detrusor muscle cells after being stimulated by carbachol, atropine, methoctramine, and 4-DAMP, respectively. Results: The [3H]-IP contents were increased with the elevation of carbachol concentration. Both atropine and 4-DAMP significantly inhibited the effect of carbachol on PI decomposition (P<0.01), but methoctramine did not. No significant difference was seen in the effect of atropine and 4-DAMP on PI decomposition. Conclusion: M3R is closely related to the decomposition of PI, the second messenger molecule IP3.

  8. Role of M1 receptor in regulation of gastric fundus smooth muscle contraction

    Directory of Open Access Journals (Sweden)

    Marta Gajdus

    2011-09-01

    Full Text Available Background:The subject of this study is determination of the influence of drugs on gastric fundus smooth muscle contraction induced by activation of muscarinic receptors M1. Experiments tested interactions between a receptor agonist, carbachol and muscarinic receptor antagonists, atropine and pirenzepine.Material/Methods:Testing was conducted on tissues isolated from rat’s stomach. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg. The stomach was dissected, and later the gastric fundus was isolated. Tissue was placed in a dish for insulated organs with 20 ml in capacity, filled with Krebs fluid. Results contained in the study are average values ± SE. In order to determine statistical significance, the principles of receptor theory were used (Kenakin modification.Results:According to tests, carbachol, in concentrations ranging between 10–8 M to 10–4 M, in a dosage-dependent way induces gastric fundus smooth muscle contraction. Presented results indicate that carbachol meets the conditions posed to full agonists. On the other hand, atropine, a non-selective muscarinic receptor antagonist, causes a concentration-dependent shift of concentration-effect curve (for carbachol to the right, maintaining maximum reaction. According to analysis of the curve determined, we can deduce that atropine meets the conditions posed to competitive antagonists. The use of pirenzepine, a competitive receptor agonist M1, causes shift of concentration-effect curve (for carbachol to the right, maintaining maximum reaction.Conclusions:From the testing conducted on the preparation of the gastric fundus we can deduce that atropine causes shift of concentration-effect curves for carbachol to the right. A similar effect is released by pirenzepine, selectively blocking muscarinic receptors of M1 type. The results indicate that in the preparation of the gastric fundus smooth muscle, M1 type

  9. The subpopulation of microglia expressing functional muscarinic acetylcholine receptors expands in stroke and Alzheimer's disease.

    Science.gov (United States)

    Pannell, Maria; Meier, Maria Almut; Szulzewsky, Frank; Matyash, Vitali; Endres, Matthias; Kronenberg, Golo; Prinz, Vincent; Waiczies, Sonia; Wolf, Susanne A; Kettenmann, Helmut

    2016-03-01

    Microglia undergo a process of activation in pathology which is controlled by many factors including neurotransmitters. We found that a subpopulation (11 %) of freshly isolated adult microglia respond to the muscarinic acetylcholine receptor agonist carbachol with a Ca(2+) increase and a subpopulation of similar size (16 %) was observed by FACS analysis using an antibody against the M3 receptor subtype. The carbachol-sensitive population increased in microglia/brain macrophages isolated from tissue of mouse models for stroke (60 %) and Alzheimer's disease (25 %), but not for glioma and multiple sclerosis. Microglia cultured from adult and neonatal brain contained a carbachol-sensitive subpopulation (8 and 9 %), which was increased by treatment with interferon-γ to around 60 %. This increase was sensitive to blockers of protein synthesis and correlated with an upregulation of the M3 receptor subtype and with an increased expression of MHC-I and MHC-II. Carbachol was a chemoattractant for microglia and decreased their phagocytic activity. PMID:25523105

  10. Functional subtyping of muscarinic receptors on canine esophageal mucosa.

    Science.gov (United States)

    Lad, R; Donoff, B; Rangachari, P K

    1991-09-01

    Serosal addition of muscarinic agonists elicited rapid changes in electrical parameters across the isolated canine esophageal epithelium set up in vitro. Both carbachol and the M1-selective agonist, McNeil A343 (McN), increased transmucosal potential differences (PDs), decreased transmucosal resistances (R), and increased short-circuit currents (Isc). Carbachol was more potent and more effective than McN. Muscarinic antagonists were used to define the muscarinic receptor involved. The pA2 values obtained with Schild plots were as follows: atropine 9.14, 4-DAMP 8.98, AFDX-116 6.71, and pirenzepine 7.12. Low concentrations of pirenzepine (10(-8) M), produced a rightward shift in the dose-response curve to McN, without inhibiting responses to carbachol. Thus the receptor subtype is clearly not an M2. As in other glandular systems, M3 receptors are present. Whether M1 receptors also exist requires better definition of receptor densities-reserves in this tissue. Carbachol induced net secretion of Na and Cl and converted a predominantly absorptive tissue to a secretory one. PMID:1716057

  11. Relationship between stimulated phosphatidic acid production and inositol lipid hydrolysis in intestinal longitudinal smooth muscle from guinea pig.

    Science.gov (United States)

    Mallows, R S; Bolton, T B

    1987-06-15

    Accumulation of [32P]phosphatidic acid (PA) and total [3H]inositol phosphates (IPs) was measured in the longitudinal smooth-muscle layer from guinea-pig small intestine. Stimulation with carbachol, histamine and substance P produced increases in accumulation of both [3H]IPs and [32P]PA over the same concentration range. The increase in [32P]PA accumulation in response to carbachol (1 microM-0.1 mM) was inhibited in the presence of atropine (0.5 microM). Buffering the external free [Ca2+] to 10 nM did not prevent the carbachol-stimulated increase in [32P]PA accumulation. Carbachol and Ca2+ appear to act synergistically to increase accumulation of [32P]PA. In contrast, although incubation with noradrenaline also increased accumulation of [3H]IPs, no increase in accumulation of [32P]PA could be detected. These results suggest that an increase in formation of IPs is not necessarily accompanied by an increase in PA formation, and imply the existence of receptor-modulated pathways regulating PA concentrations other than by phospholipase-C-catalysed inositol phospholipid hydrolysis.

  12. Antisecretory Action of the Extract of the Aerial Parts of Eremomastax speciosa (Acanthaceae) Occurs through Antihistaminic and Anticholinergic Pathways.

    Science.gov (United States)

    André Perfusion, Amang; Tan, Paul V; Ernestine, Nkwengoua; Barthélemy, Nyasse

    2014-01-01

    Objective. The objective of this study was to find out the possible antiulcer mechanism of action of Eremomastax speciosa. Method. Carbachol- and histamine-induced hypersecretion, associated with the pylorus ligation technique, were used in rats. Gastric mucosal ulceration, mucus production, pH, gastric volume, and acidity were measured. Results. Histamine and carbachol raised gastric acidity to 86.50 and 84.80 mEq/L, respectively, in the control rats, and the extracts (200 mg/kg) reduced gastric acidity to 34.60 and 39.00 mEq/L, respectively. Intraduodenal aqueous extract (400 mg/kg) in histamine- and carbachol-treated rats produced significant (P mEq/L, respectively, and 100 percent inhibition of gastric ulceration. Augmented histamine-induced gastric acid secretion (90.20 mEq/L) was significantly reduced to 52.60 and 27.50 mEq/L by the 200 and 400 mg/kg doses of the aqueous extract, respectively. The extract significantly reduced (P < 0.001) the volume of gastric secretion and significantly increased mucus production. The ulcer inhibition potential of the extract significantly dropped to 25-44% (oral extract) and to 29-37% (duodenal extract) in carbachol/indomethacin-treated rats. Conclusion. The aqueous extract of E. speciosa has both cytoprotective and antisecretory effects. The antisecretory effect may involve a mechanism common to both cholinergic and histaminergic pathways. PMID:24695819

  13. Further evidence for the role of the caudate nucleus in programming motor and nonmotor behavior in Java monkeys.

    Science.gov (United States)

    Vrijmoed-de Vries, M C; Cools, A R

    1985-01-01

    This study describes the short-term effects of intracaudate microinjections of carbachol in temporarily isolated and restrained Java monkeys. The monkeys were found to display a series of motor disturbances including blepharoptosis, facial twitches, tongue protrusions, ear flattening, torticollis, and compulsive alternations of rapid flexions and extensions of the extremities. In general, carbachol was found to produce consistent effects as far as it concerns its ability to elicit motor disturbances. Three of the five tested monkeys had previously received another series of carbachol injections when they were freely moving and living in a stabilized social group. Accordingly, the present study enabled us to compare the effectiveness of threshold doses of carbachol in the same monkey in two distinct situations. We concluded first, that motor disturbances and disturbances in social communication were closely coupled in relation to the involvement of a particular cholinoceptive substrate within the caudate nucleus of Java monkeys. Second, the motor disturbances under study appeared to require a larger degree of dysfunctioning of this substrate than did subtle disturbances in the social communication of these monkeys. And, finally, stress inherent to restraint increased the susceptibility of the cholinoceptive substrate within the caudate nucleus. The clinical impact of our findings is discussed in view of differences between the premorbid and manifest phases of Parkinson's disease.

  14. Effects of cholinergic and noradrenergic agents on locomotion in the mudpuppy (Necturus maculatus).

    Science.gov (United States)

    Fok, M; Stein, R B

    2002-08-01

    Some neurotransmitters act consistently on the central pattern generator (CPG) for locomotion in a wide range of vertebrates. In contrast, acetylcholine (ACh) and noradrenaline (NA) have various effects on locomotion in different preparations. The roles of ACh and NA have not been studied in amphibian walking, so we examined their effects in an isolated spinal cord preparation of the mudpuppy ( Necturus maculatus). This preparation contains a CPG that produces locomotor activity when N-methyl- D-aspartic acid (NMDA), an excitatory amino acid agonist, is added to the bath. The addition of carbachol, a long acting ACh agonist, to the bath disrupted the walking rhythm induced by NMDA, while not changing the level of activity in flexor and extensor motoneurons. Adding clonidine, an alpha(2)-noradrenergic agonist, had no effect on the NMDA-induced walking rhythm. Physostigmine, an ACh-esterase inhibitor, disrupted the walking rhythm, presumably by potentiating the effects of endogenously released ACh. Atropine, an ACh antagonist that binds to muscarinic ACh receptors, blocked the effects of carbachol, indicating that the action is mediated, at least in part, by muscarinic receptors. In the absence of carbachol, atropine had no effect. Locomotion was not induced by carbachol, atropine or clonidine in a resting spinal cord preparation. Cholinergic actions do not seem to be essential to the CPG for walking in the mudpuppy, but ACh may convert a rhythmic walking state to a more tonic state with occasional bursts of EMG activity for postural adjustments.

  15. Functional partial agonism at cloned human muscarinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Ebert, B; Brann, M R;

    1996-01-01

    of maximal response, depending on the molar ratio of agonist and antagonist used. Using recombinant human muscarinic acetylcholine receptors (m1 and m5) and the functional assay, receptor selection and amplification technology (R-SAT), we have now shown that co-administration of the full agonist, carbachol...

  16. Lithium dampens neurotransmitter response in smooth muscle: relevance to action in affective illness.

    OpenAIRE

    Menkes, H A; Baraban, J M; Freed, A N; Snyder, S H

    1986-01-01

    Lithium, by inhibiting inositol phosphate metabolism, interferes with the phosphatidylinositol ("phosphoinositide") cycle, which is stimulated by numerous hormones and neurotransmitters. To examine the relevance of this action to neurotransmission, we evaluated effects of lithium treatment on smooth muscle responses to transmitters. In lithium-pretreated tracheal muscle, the relaxation following carbachol or histamine contractions is retarded. Lithium does not affect relaxation following cont...

  17. Supramaximal stimuli do not evoke a maximal contraction in urinary bladder smooth muscle fibers

    NARCIS (Netherlands)

    J. Minekus (Joanne); A.J. Visser (Anna); R. van Mastrigt (Ron)

    2001-01-01

    textabstractBACKGROUND: Smooth muscle fibers can be stimulated with an electrical field, high potassium or carbachol. We studied the effect of combined, supramaximal stimulation on the isometric force and the maximum shortening velocity of the pig urinary bladder. MATERIALS AND METHODS: After determ

  18. Changes of phasic and tonic smooth muscle function of jejunum in type 2 diabetic Goto-Kakizaki rats

    DEFF Research Database (Denmark)

    Zhao, Jing-Bo; Chen, Peng-Min; Gregersen, Hans;

    2013-01-01

    ) were included in the study. Jejunal segments were distended up to a pressure of 10 cm H2O in an organ bath containing 37 °C Krebs solution with addition of carbachol (CA). The pressure and outer diameter changes were synchronously recorded. Passive conditions were obtained using calcium-free Krebs...

  19. NSC23766, a widely used inhibitor of Rac1 activation, additionally acts as a competitive antagonist at muscarinic acetylcholine receptors.

    Science.gov (United States)

    Levay, Magdolna; Krobert, Kurt Allen; Wittig, Karola; Voigt, Niels; Bermudez, Marcel; Wolber, Gerhard; Dobrev, Dobromir; Levy, Finn Olav; Wieland, Thomas

    2013-10-01

    Small molecules interfering with Rac1 activation are considered as potential drugs and are already studied in animal models. A widely used inhibitor without reported attenuation of RhoA activity is NSC23766 [(N(6)-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride]. We found that NSC23766 inhibits the M2 muscarinic acetylcholine receptor (M2 mAChR)-induced Rac1 activation in neonatal rat cardiac myocytes. Surprisingly, NSC27366 concomitantly suppressed the carbachol-induced RhoA activation and a M2 mAChR-induced inotropic response in isolated neonatal rat hearts requiring the activation of Rho-dependent kinases. We therefore aimed to identify the mechanisms by which NSC23766 interferes with the differentially mediated, M2 mAChR-induced responses. Interestingly, NSC23766 caused a rightward shift of the carbachol concentration response curve for the positive inotropic response without modifying carbachol efficacy. To analyze the specificity of NSC23766, we compared the carbachol and the similarly Giβγ-mediated, adenosine-induced activation of Gi protein-regulated potassium channel (GIRK) channels in human atrial myocytes. Application of NSC23766 blocked the carbachol-induced K(+) current but had no effect on the adenosine-induced GIRK current. Similarly, an adenosine A1 receptor-induced positive inotropic response in neonatal rat hearts was not attenuated by NSC23766. To investigate its specificity toward the different mAChR types, we studied the carbachol-induced elevation of intracellular Ca(2+) concentrations in human embryonic kidney 293 (HEK-293) cells expressing M1, M2, or M3 mAChRs. NSC23766 caused a concentration-dependent rightward shift of the carbachol concentration response curves at all mAChRs. Thus, NSC23766 is not only an inhibitor of Rac1 activation, but it is within the same concentration range a competitive antagonist at mAChRs. Molecular docking analysis at M2 and M3 mAChR crystal

  20. Involvement of M3 Cholinergic Receptor Signal Transduction Pathway in Regulation of the Expression of Chemokine MOB-1, MCP-1 Genes in Pancreatic Acinar Cells

    Institute of Scientific and Technical Information of China (English)

    郑海; 陈道达; 张景輝; 田原

    2004-01-01

    Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-κB and the expression of chemokine MOB-1, MCP-1genes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-κB was monitored by using electrophoretic mobility shift assay.The results showed that as compared with control group, M3 cholinergic receptor agonist (103mol/L, 104-4ol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10 -3mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-κB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10-3 mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10-5 mol/L atropine) or NF-κB inhibitor (10-2 mol/L PDTC) could obviously inhibit the activation of NF-κB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P <0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-1genes in pancreatic acinar cells in vitro through the activation of NF-κB.

  1. Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium.

    Science.gov (United States)

    Suchard, S J; Lattanzio, F A; Rubin, R W; Pressman, B C

    1982-09-01

    The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+-sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation. PMID:7130269

  2. Muscarinic receptors participation in angiogenic response induced by macrophages from mammary adenocarcinoma-bearing mice

    International Nuclear Information System (INIS)

    The role of macrophages in tumor progression has generated contradictory evidence. We had previously demonstrated the ability of peritoneal macrophages from LMM3 murine mammary adenocarcinoma-bearing mice (TMps) to increase the angiogenicity of LMM3 tumor cells, mainly through polyamine synthesis. Here we investigate the ability of the parasympathetic nervous system to modulate angiogenesis induced by TMps through the activation of the muscarinic acetylcholine receptor (mAchR). Peritoneal macrophages from female BALB/c mice bearing a 7-day LMM3 tumor were inoculated intradermally (3 × 105 cells per site) into syngeneic mice. Before inoculation, TMps were stimulated with the muscarinic agonist carbachol in the absence or presence of different muscarinic antagonists or enzyme inhibitors. Angiogenesis was evaluated by counting vessels per square millimeter of skin. The expression of mAchR, arginase and cyclo-oxygenase (COX) isoforms was analyzed by Western blotting. Arginase and COX activities were evaluated by urea and prostaglandin E2 (PGE2) production, respectively. TMps, which stimulate neovascularization, express functional mAchR, because carbachol-treated TMps potently increased new blood vessels formation. This response was completely blocked by preincubating TMps with pirenzepine and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), M1 and M3 receptor antagonists, and partly by the M2 receptor antagonist methoctramine. M1 receptor activation by carbachol in TMps triggers neovascularization through arginase products because Nω-hydroxy-L-arginine reversed the agonist action. Preincubation of TMps with methoctramine partly prevented carbachol-stimulated urea formation. In addition, COX-derived liberation of PGE2 is responsible for the promotion of TMps angiogenic activity by M3 receptor. We also detected a higher expression of vascular endothelial growth factor (VEGF) in TMps than in macrophages from normal mice. Carbachol significantly increased VEGF expression

  3. Regulation of the Na+/Ca2+ exchanger in rat pancreatic ducts

    DEFF Research Database (Denmark)

    Ankorina-Stark, I; Amstrup, J; Novak, I

    2002-01-01

    hormones/agonists affecting pancreatic secretion. Whole pancreas, pure pancreatic acini and ducts were obtained from rats and used for RT-PCR and Western blot analysis, immunohistochemistry and intracellular Ca2+ measurements using Fura-2. RT-PCR analysis indicated Na+/Ca2+-exchanger isoforms NCX1.3 and...... intralobular/extralobular ducts the exchanger was predominantly on the luminal membrane. Na+/Ca2+ exchange in ducts was monitored by changes in intracellular Ca2+ activity upon reversal of the Na+ gradient. Secretin (1 nM) and carbachol (1 mM) reduced Na+/Ca2+ exchange by 40% and 51%, respectively. Insulin (1...... nM) increased Na+/Ca2+ exchange by 230% within 5 min. The present study shows that pancreatic ducts express the Na+/Ca2+ exchanger. Its distinct localization along the ductal tree and regulation by secretin, carbachol and insulin indicate that ducts might be involved in regulation of Ca2...

  4. Lateral Hypothalamic Stimulation Reduces Hyperalgesia Through Spinally Descending Orexin-A Neurons in Neuropathic Pain.

    Science.gov (United States)

    Wardach, Jacob; Wagner, Monica; Jeong, Younhee; Holden, Janean E

    2016-03-01

    No evidence to date shows that lateral hypothalamic (LH) stimulation produces orexin-A-mediated antinociception in the spinal cord dorsal horn (SCDH) in a model of neuropathic pain. We conducted experiments to examine the effect of orexin-A-mediated LH stimulation in female rats with chronic constriction injury (CCI) on thermal hyperalgesia. Rats receiving carbachol into the LH demonstrated antinociception on both the left CCI and right nonligated paws (p < .05). Rats were given carbachol in the LH followed by intrathecal injection of the orexin-1 (OX1) receptor antagonist SB-334867, which blocked LH-induced antinociception compared with control groups (p < .05) in the left paw, but not in the right paw. These findings support the hypothesis that LH stimulation produces antinociception in rats with thermal hyperalgesia from neuropathic pain via an orexin-A connection between the LH and the SCDH. Identification of this pathway may lead to studies using orexins to manage clinical pain.

  5. Effects of phenylalaninol on centrally induced gastric acid secretion.

    Science.gov (United States)

    Hashizume, H; Miyamae, T; Morikawa, T; Hagiwara, M

    1992-11-01

    The effects of phenylalaninol (D-isomer) on gastric acid secretion and gastric ulcer were studied in rats. The compound reduced the gastric acid secretion stimulated by intracisternal thyrotropin releasing hormone and intravenous 2-deoxy-D-glucose, but not that stimulated by subcutaneous carbachol or histamine. Phenylalaninol prevented stress- and indomethacin-induced gastric ulcers. We conclude that phenylalaninol inhibits ulcer formation mainly by central inhibition of gastric acid secretion. PMID:1477931

  6. State-dependent Control of Lumbar Motoneurons by the Hypocretinergic System

    OpenAIRE

    Yamuy, Jack; Fung, Simon J.; Xi, MingChu; Chase, Michael H.

    2009-01-01

    Neurons in the lateral hypothalamus (LH) that synthesize hypocretins (Hcrt-1 and 2) are active during wakefulness and excite lumbar motoneurons. Because hypocretinergic cells also discharge during phasic periods of rapid eye movement (REM) sleep, we sought to examine their action on the activity of motoneurons during this state. Accordingly, cat lumbar motoneurons were intracellularly recorded, under α-chloralose anesthesia, prior to (control) and during the carbachol-induced REM sleep-like a...

  7. State-dependent phenomena in cat masseter motoneurons.

    Science.gov (United States)

    Kohlmeier, K A; López-Rodríguez, F; Liu, R H; Morales, F R; Chase, M H

    1996-05-25

    In the present study we explored the mechanisms of carbachol-induced muscle atonia in the alpha-chloralose-anesthetized animal. We compared our findings to those that have been previously obtained in unanesthetized cats during muscle atonia occurring during natural active sleep. Accordingly, in cats anesthetized with alpha-chloralose, intracellular records were obtained from masseter motoneurons before and after carbachol-induced motor atonia. Following the induction of atonia, the membrane potential activity was dominated by high-frequency, discrete, hyperpolarizing potentials. These hyperpolarizing potentials were reversed in polarity by the intracellular injection of chloride ions and abolished by the application of strychnine. These findings indicate that they were inhibitory postsynaptic potentials (IPSPs) mediated by glycine. These IPSPs appeared exclusively during muscle atonia. In addition, masseter motoneurons were significantly hyperpolarized and their rheobase increased. There was a decrease in input resistance and membrane time constant. In the alpha-chloralose-anesthetized preparation, stimulation of the nucleus pontis oralis (NPO) induced IPSPs in masseter motoneurons following, but never prior to, the pontine injection of carbachol. Thus, this is the first demonstration that "reticular response-reversal' may be elicited in an anesthetized preparation. Another state-dependent phenomenon of active sleep, the occurrence of IPSPs in motoneurons that are temporally correlated with ponto-geniculo-occipital (PGO) waves, was also observed in this preparation only after carbachol administration. Based on the data in this report, we conclude that the inhibitory system that mediates atonia during the state of active sleep can be activated in an animal that is anesthetized with alpha-chloralose. Specifically, the neuronal groups that generate spontaneous IPSPs, those that mediate the phenomenon of reticular response-reversal, and those involved in the generation

  8. Menthol inhibits detrusor contractility independently of TRPM8 activation.

    Directory of Open Access Journals (Sweden)

    Antonio Celso Saragossa Ramos-Filho

    Full Text Available Agonists such as icilin and menthol can activate the cool temperature-sensitive ion channel TRPM8. However, biological responses to menthol may occur independently of TRPM8 activation. In the rodent urinary bladder, menthol facilitates the micturition reflex but inhibits muscarinic contractions of the detrusor smooth muscle. The site(s of TRPM8 expression in the bladder are controversial. In this study we investigated the regulation of bladder contractility in vitro by menthol. Bladder strips from wild type and TRPM8 knockout male mice (25-30 g were dissected free and mounted in organ baths. Isometric contractions to carbachol (1 nM-30 µM, CaCl2 (1 µM to 100 mM and electrical field stimulation (EFS; 8, 16, 32 Hz were measured. Strips from both groups contracted similarly in response to both carbachol and EFS. Menthol (300 µM or nifedipine (1 µM inhibited carbachol and EFS-induced contractions in both wild type and TRPM8 knockout bladder strips. Incubation with the sodium channel blocker tetrodotoxin (1 µM, replacement of extracellular sodium with the impermeant cation N-Methyl-D-Glucamine, incubation with a cocktail of potassium channel inhibitors (100 nM charybdotoxin, 1 µM apamin, 10 µM glibenclamide and 1 µM tetraethylammonium or removal of the urothelium did not affect the inhibitory actions of menthol. Contraction to CaCl2 was markedly inhibited by either menthol or nifedipine. In cultured bladder smooth muscle cells, menthol or nifedipine abrogated the carbachol or KCl-induced increases in [Ca2+]i. Intravesical administration of menthol increased voiding frequency while decreasing peak voiding pressure. We conclude that menthol inhibits muscarinic bladder contractions through blockade of L-type calcium channels, independently of TRPM8 activation.

  9. [The formation of motor programs during pharmacological kindling].

    Science.gov (United States)

    Shandra, A A; Godlevskiĭ, L S; Mazarati, A M; Lobenko, A A

    1990-10-01

    In Wistar rats with the kindling phenomenon, the number of animals with three or more not stimulus-directed forms of swimming was increased. Obvious changes of swimming behaviour in these animals were also observed after intrastriatal injections of carbachol, apomorphine, haloperidol, muscimol and picrotoxin. These findings are discussed from the standpoint of a latent generator of pathologically enhanced excitation in the striatum which changed the ability of these rats to develop behavioral programs.

  10. Antisecretory Action of the Extract of the Aerial Parts of Eremomastax speciosa (Acanthaceae Occurs through Antihistaminic and Anticholinergic Pathways

    Directory of Open Access Journals (Sweden)

    Amang André Perfusion

    2014-01-01

    Full Text Available Objective. The objective of this study was to find out the possible antiulcer mechanism of action of Eremomastax speciosa. Method. Carbachol- and histamine-induced hypersecretion, associated with the pylorus ligation technique, were used in rats. Gastric mucosal ulceration, mucus production, pH, gastric volume, and acidity were measured. Results. Histamine and carbachol raised gastric acidity to 86.50 and 84.80 mEq/L, respectively, in the control rats, and the extracts (200 mg/kg reduced gastric acidity to 34.60 and 39.00 mEq/L, respectively. Intraduodenal aqueous extract (400 mg/kg in histamine- and carbachol-treated rats produced significant (P<0.001 decreases in acid secretion to 28.50 and 28.80 mEq/L, respectively, and 100 percent inhibition of gastric ulceration. Augmented histamine-induced gastric acid secretion (90.20 mEq/L was significantly reduced to 52.60 and 27.50 mEq/L by the 200 and 400 mg/kg doses of the aqueous extract, respectively. The extract significantly reduced (P<0.001 the volume of gastric secretion and significantly increased mucus production. The ulcer inhibition potential of the extract significantly dropped to 25–44% (oral extract and to 29–37% (duodenal extract in carbachol/indomethacin-treated rats. Conclusion. The aqueous extract of E. speciosa has both cytoprotective and antisecretory effects. The antisecretory effect may involve a mechanism common to both cholinergic and histaminergic pathways.

  11. In vitro assessment of gastric mucosal transfer of anti-Helicobacter therapeutic agents.

    OpenAIRE

    Goddard, A. F.; Spiller, R C

    1997-01-01

    A novel animal model for studying antibiotic transfer across gastric mucosa was developed by using adult rats. Gastric corpus mucosa was mounted in an Ussing chamber system and bathed in oxygenated Krebs solution. Metronidazole flux from serosa to mucosa (J(S-->M)) was measured over 60 min under basal conditions and compared with mucosa-to-serosa flux (J(M-->S)). The effects of varying the chamber cross-sectional diameter and of stimulation by histamine and carbachol were assessed. Metronidaz...

  12. Intestinal Ioidide Secretion and its Dependence Upon Mucosal I Permeability

    OpenAIRE

    Ilundáin Larrañeta, María Anunciación; Ondarra, A.; Larralde, J.

    1985-01-01

    Iodide secretion across different regions of rat small inlestine ha.s been investigated in vitro using the standard Wilson- Wisema technique. Net I secretion was observed along the entire small intestine, being significantly higher in the central region. Anaerobic conditions, ouabain {2 mM) and Na• free Ringer solution prevented net I· secretion, whilts, both theophyilline (1 mM) and carbachol {0,1 mM) enhanced the observed basal intestinal I· secretion. Furthermore, Caº'-deprived bathing...

  13. Fluid and amylase secretion by perfused parotid gland: physio-morphological approach.

    OpenAIRE

    Murakami, M.; Yoshimura, K.; H. Sugiya; Segawa, A; Loffredo, F; Testa-Riva, F; Riva, A.

    2000-01-01

    Whole gland perfusion technique was applied to rat parotid glands to assess whether amylase affects fluid secretion. Control perfusion without any secretagogue evoked no spontaneous secretion. Carbachol (CCh 1 microM) induced both amylase and fluid secretion with distinctive kinetics. Fluid secretion occurred constantly around 60 microL/g-min, whereas amylase secretion exhibited an initial peak, followed by a rapid decrease to reach a plateau. Isoproterenol (Isop 1 microM) alone did not induc...

  14. Are there valid reasons for using anti-muscarinic drugs in the management of renal colic?

    Science.gov (United States)

    Tomiak, R H; Barlow, R B; Smith, P J

    1985-10-01

    Experiments have been carried out with isolated ring preparations of human ureter. The tissue displayed spontaneous activity and contracted when exposed to barium chloride (0.5-4 mM) but no responses were obtained with carbachol (0.1 micromolar-0.1 mM). This raises questions about the value of treating ureteric colic with anti-muscarinic drugs. PMID:4063728

  15. Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill

    Directory of Open Access Journals (Sweden)

    Crittenden Elizabeth L

    2004-07-01

    Full Text Available Abstract Background In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. Results The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Conclusions Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

  16. Cholinergic signal activated renin angiotensin system associated with cardiovascular changes in the ovine fetus

    OpenAIRE

    Geng, Chunsong; Mao, Caiping; Wu, Lei; Cheng, Yu; Liu, Rulu; Chen, Bingxin; Chen, Ling; Zhang, Lubo; Xu, Zhice

    2010-01-01

    Aim: Cholinergic regulation is important in the control of cardiovascular and endocrine responses. The mechanisms behind cardiovascular responses induced by cholinergic activation are explored by studying hormonal systems, including renin-angiotensin and vasopressin (VP). Results: In chronically prepared fetal sheep, intravenous infusion of the cholinergic agonist carbachol increased fetal systolic, diastolic, and mean arterial pressure accompanied with bradycardia at near-term. Although int...

  17. Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium

    OpenAIRE

    1982-01-01

    The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secr...

  18. Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Martinson, E.A.; Goldstein, D.; Brown, J.H. (Univ. of California, San Diego, La Jolla (USA))

    1989-09-05

    We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of (3H)choline and (3H)phosphorylcholine ((3H)Pchol) from cells containing (3H)choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of (3H)phosphatidic acid ((3H)PA) in cells containing (3H)myristate-labeled PC. (3H)Diacylglycerol ((3H)DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with (3H)myristate and (14C)arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol.

  19. Force-inhibiting effect of Ser/Thr protein phosphatase 2A inhibitors on bovine ciliary muscle.

    OpenAIRE

    石田, 美織

    2015-01-01

    Ciliary muscle is a smooth muscle characterized by a rapid response to muscarinic receptor stimulation and sustained contraction. Although it is evident that these contractions are Ca(2+)-dependent, detailed molecular mechanisms are still unknown. In order to elucidate the role of Ser/Thr protein phosphatase 2A (PP2A) in ciliary muscle contraction, we examined the effects of okadaic acid and other PP2A inhibitors on contractions induced by carbachol (CCh) and ionomycin in bovine ciliary muscl...

  20. Salmeterol, formoterol, and salbutamol in the isolated guinea pig trachea: differences in maximum relaxant effect and potency but not in functional antagonism.

    OpenAIRE

    Lindén, A.; Bergendal, A; Ullman, A.; Skoogh, B. E.; Löfdahl, C G

    1993-01-01

    BACKGROUND--Formoterol and salmeterol are new long acting beta 2 adrenoceptor agonists. The maximum relaxant effect, potency and functional antagonism against carbachol induced contraction for salmeterol, formoterol and salbutamol have been compared in the guinea pig isolated trachea. In addition, the possibility of inducing a non-beta adrenoceptor mediated relaxation by salmeterol was studied. METHODS--Concentration response experiments were conducted with isolated tracheal preparations (n =...

  1. Aclidinium bromide combined with formoterol inhibits remodeling parameters in lung epithelial cells through cAMP.

    Science.gov (United States)

    Lambers, Christopher; Costa, Luigi; Ying, Qi; Zhong, Jun; Lardinois, Didier; Dekan, Gerhard; Schuller, Elisabeth; Roth, Michael

    2015-12-01

    Combined muscarinic receptor antagonists and long acting β2-agonists improve symptom control in chronic obstructive pulmonary disease (COPD) significantly. In clinical studies aclidinium bromide achieved better beneficial effects than other bronchodilators; however, the underlying molecular mechanisms are unknown. This study assessed the effect of aclidinium bromide combined with formoterol on COPD lung (n=20) and non-COPD lung (n=10) derived epithelial cells stimulated with TGF-β1+carbachol on: (i) the generation of mesenchymal cells in relation to epithelial cells, (II) extracellular matrix (ECM) deposition, and (iii) the interaction of ECM on the generation of epithelial and mesenchymal cells. TGF-β1+carbachol enhanced the generation of mesenchymal cells, which was significantly reduced by aclidinium bromide or formoterol. The effect of combined drugs was additive. Inhibition of p38 MAP kinase and Smad by specific inhibitors or aclidinium bromide reduced the generation of mesenchymal cells. In mesenchymal cells, TGF-β1+carbachol induced the deposition of collagen-I and fibronectin which was prevented by both drugs dose-dependently. Formoterol alone reduced collagen-I deposition via cAMP, this however, was overruled by TGF-β1+carbachol and rescued by aclidinium bromide. Inhibition of fibronectin was cAMP independent, but involved p38 MAP kinase and Smad. Seeding epithelial cells on ECM collagen-I and fibronectin induced mesenchymal cell generation, which was reduced by aclidinium bromide and formoterol. Our results suggest that the beneficial effect of aclidinium bromide and formoterol involves cAMP affecting both, the accumulation of mesenchymal cells and ECM remodeling, which may explain the beneficial effect of the drugs on lung function in COPD. PMID:26546746

  2. Luminal DMSO: Effects on Detrusor and Urothelial/Lamina Propria Function

    Directory of Open Access Journals (Sweden)

    Katrina J. Smith

    2014-01-01

    Full Text Available DMSO is used as a treatment for interstitial cystitis and this study examined the effects of luminal DMSO treatment on bladder function and histology. Porcine bladder was incubated without (controls or with DMSO (50% applied to the luminal surface and the release of ATP, acetylcholine, and LDH assessed during incubation and in tissues strips after DMSO incubation. Luminally applied DMSO caused ATP, Ach, and LDH release from the urothelial surface during treatment, with loss of urothelial layers also evident histologically. In strips of urothelium/lamina propria from DMSO pretreated bladders the release of both ATP and Ach was depressed, while contractile responses to carbachol were enhanced. Detrusor muscle contractile responses to carbachol were not affected by DMSO pretreatment, but neurogenic responses to electrical field stimulation were enhanced. The presence of an intact urothelium/lamina propria inhibited detrusor contraction to carbachol by 53% and this inhibition was significantly reduced in DMSO pretreated tissues. Detection of LDH in the treatment medium suggests that DMSO permeabilised urothelial membranes causing leakage of cytosolic contents including ATP and Ach rather than enhancing release of these mediators. The increase in contractile response and high levels of ATP are consistent with initial flare up in IC/PBS symptoms after DMSO treatment.

  3. (-)-Carvone: antispasmodic effect and mode of action.

    Science.gov (United States)

    Souza, Fábia Valéria M; da Rocha, Marcelly Barbosa; de Souza, Damião P; Marçal, Rosilene Moretti

    2013-03-01

    (-)-Carvone is a monoterpene ketone found in spearmint (Mentha spicata var. crispa) essential oil that is widely used as an odor and flavor additive. An intestinal antispasmodic effect was recently reported for (-)-carvone, and it has been shown to be more potent than its (+)-antipode. The mechanism of (-)-carvone action in the intestines has not been investigated. To gain a better understanding of the (-)-carvone antispasmodic effect, we investigated its pharmacological effects in the guinea pig ileum. Terminal portions of the ileum were mounted for isotonic contraction recordings. The effect of (-)-carvone was compared with that of the classical calcium channel blocker (CCB) verapamil. In isolated ileal smooth muscle, (-)-carvone did not produce direct contractile or relaxation responses and did not modify electrically elicited contractions or low K(+)-evoked contractions. The submaximal contractions induced by histamine (p<0.001), BaCl2 (p<0.05), and carbachol (p<0.01) were significantly reduced by (-)-carvone. The contractile response elicited by high concentrations of carbachol was reduced but not abolished by (-)-carvone. No additive action was detected with co-incubation of (-)-carvone and verapamil on carbachol-induced contraction. (-)-Carvone reduced the contraction induced by high K(+) and was almost 100 times more potent than verapamil. Thus, (-)-carvone showed a typical and potent CCB-like action. Many effects described for both (-)-carvone and spearmint oil can be explained as a CCB-like mode of action. PMID:23103297

  4. Fatal asthma in a subject sensitized to toluene diisocyanate

    Energy Technology Data Exchange (ETDEWEB)

    Fabbri, L.M.; Danieli, D.; Crescioli, S.; Bevilacqua, P.; Meli, S.; Saetta, M.; Mapp, C.E.

    1988-06-01

    We report the case of a 43-yr-old car painter who died within 1 h of exposure to a polyurethane paint in the workplace. A diagnosis of asthma induced by toluene diisocyanate (TDI) had been established 6 yr before, when he underwent inhalation challenges with carbachol and with TDI. The subject had airway hyperresponsiveness to carbachol (PD20FEV1 carbachol = 0.32 mg; normal value greater than 1.0 mg) and developed an early and long-lasting asthmatic reaction after exposure to TDI in the laboratory. Although it was recommended that he change his job or stop using paints containing isocyanates, he continued to work as a car painter, taking antiasthmatic drugs both at work and at home to control asthma symptoms. On Monday, October 6, 1986, at 11:30 A. M., he developed a severe attack of asthma while he was mixing the 2 components of a polyurethane paint. Taken to hospital, he was dead on arrival. Autopsy showed no evidence of cardiac or brain disease; lungs were overinflated, the cut surface showed grey glistening mucous plugs in in the airways. Histologic examination showed denudation of airway epithelium and thickening of the basement membrane with infiltration of the lamina propria by polymorphonuclear leukocytes, mainly eosinophils, and diffuse mucous plugging of bronchioles. Bronchial smooth muscle appeared hyperplastic and disarrayed, and lung parenchyma showed focal areas of alveolar destruction adjacent to areas of perfectly intact alveolar walls.

  5. Regulation of pulmonary surfactant secretion in the developing lizard, Pogona vitticeps.

    Science.gov (United States)

    Sullivan, Lucy C; Orgeig, Sandra; Daniels, Christopher B

    2002-11-01

    Pulmonary surfactant is a mixture of lipids and proteins that is secreted by alveolar type II cells in the lungs of all air-breathing vertebrates. Pulmonary surfactant functions to reduce the surface tension in the lungs and, therefore, reduce the work of breathing. In mammals, the embryonic maturation of the surfactant system is controlled by a host of factors, including glucocorticoids, thyroid hormones and autonomic neurotransmitters. We have used a co-culture system of embryonic type II cells and lung fibroblasts to investigate the ability of dexamethasone, tri-iodothyronine (T(3)), adrenaline and carbamylcholine (carbachol) to stimulate the cellular secretion of phosphatidylcholine in the bearded dragon (Pogona vitticeps) at day 55 (approx. 92%) of incubation and following hatching. Adrenaline stimulated surfactant secretion both before and after hatching, whereas carbachol stimulated secretion only at day 55. Glucocorticoids and triiodothyronine together stimulated secretion at day 55 but did not after hatching. Therefore, adrenaline, carbachol, dexamethasone and T(3), are all involved in the development of the surfactant system in the bearded dragon. However, the efficacy of the hormones is attenuated during the developmental process. These differences probably relate to the changes in the cellular environment during development and the specific biology of the bearded dragon. PMID:12443912

  6. Laxative and anti-diarrheal activity of polycarbophil in mice and rats.

    Science.gov (United States)

    Saito, Takaharu; Mizutani, Fujie; Iwanaga, Yuji; Morikawa, Kouji; Kato, Hideo

    2002-06-01

    We investigated the laxative and anti-diarrheal activity of polycarbophil, an insoluble hydrophilic polymer, in comparison with other agents used for treating functional bowel disorder (FBD). In naive rats, polycarbophil (500 mg/kg) increased fecal weight and water contents without producing diarrhea. Carboxymethylcellulose (CMC) did not produce evident changes in bowel movement. Picosulfate markedly produced diarrhea. Loperamide, trimebutine and granisetron decreased stool output dose-dependently. Constipation, indicated by decrease in fecal weight, was produced by loperamide and clonidine in rats. Polycarbophil (500 mg/kg) and CMC increased fecal weight without diarrhea. Conversely trimebutine further decreased fecal weight in constipated rats. Polycarbophil (500 mg/kg) suppressed diarrhea induced by castor oil, and at 250-500 mg/kg, it produced shaped stools in animals with stools loosened by prostaglandin E2, serotonin or carbachol in mice. Polycarbophil (500 mg/kg) also reduced stools in rats with stool output increased by wrap restraint stress (WRS). CMC had no effect in the diarrhea models, except for carbachol-induced diarrhea, and WRS-induced evacuation. Loperamide, trimebutine and granisetron inhibited diarrhea production and WRS-induced evacuation, except for carbachol-induced diarrhea. The results show that polycarbophil prevents constipation and diarrhea without inducing diarrhea or constipation, which is different from the other agents. Hydrophilic polymers such as polycarbophil will be promising agents for the treatment of FBD.

  7. Fatal asthma in a subject sensitized to toluene diisocyanate.

    Science.gov (United States)

    Fabbri, L M; Danieli, D; Crescioli, S; Bevilacqua, P; Meli, S; Saetta, M; Mapp, C E

    1988-06-01

    We report the case of a 43-yr-old car painter who died within 1 h of exposure to a polyurethane paint in the workplace. A diagnosis of asthma induced by toluene diisocyanate (TDI) had been established 6 yr before, when he underwent inhalation challenges with carbachol and with TDI. The subject had airway hyperresponsiveness to carbachol (PD20FEV1 carbachol = 0.32 mg; normal value greater than 1.0 mg) and developed an early and long-lasting asthmatic reaction after exposure to TDI in the laboratory. Although it was recommended that he change his job or stop using paints containing isocyanates, he continued to work as a car painter, taking antiasthmatic drugs both at work and at home to control asthma symptoms. On Monday, October 6, 1986, at 11:30 A. M., he developed a severe attack of asthma while he was mixing the 2 components of a polyurethane paint. Taken to hospital, he was dead on arrival. Autopsy showed no evidence of cardiac or brain disease; lungs were overinflated, the cut surface showed grey glistening mucous plugs in in the airways. Histologic examination showed denudation of airway epithelium and thickening of the basement membrane with infiltration of the lamina propria by polymorphonuclear leukocytes, mainly eosinophils, and diffuse mucous plugging of bronchioles. Bronchial smooth muscle appeared hyperplastic and disarrayed, and lung parenchyma showed focal areas of alveolar destruction adjacent to areas of perfectly intact alveolar walls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2849334

  8. (-)-Carvone: antispasmodic effect and mode of action.

    Science.gov (United States)

    Souza, Fábia Valéria M; da Rocha, Marcelly Barbosa; de Souza, Damião P; Marçal, Rosilene Moretti

    2013-03-01

    (-)-Carvone is a monoterpene ketone found in spearmint (Mentha spicata var. crispa) essential oil that is widely used as an odor and flavor additive. An intestinal antispasmodic effect was recently reported for (-)-carvone, and it has been shown to be more potent than its (+)-antipode. The mechanism of (-)-carvone action in the intestines has not been investigated. To gain a better understanding of the (-)-carvone antispasmodic effect, we investigated its pharmacological effects in the guinea pig ileum. Terminal portions of the ileum were mounted for isotonic contraction recordings. The effect of (-)-carvone was compared with that of the classical calcium channel blocker (CCB) verapamil. In isolated ileal smooth muscle, (-)-carvone did not produce direct contractile or relaxation responses and did not modify electrically elicited contractions or low K(+)-evoked contractions. The submaximal contractions induced by histamine (p<0.001), BaCl2 (p<0.05), and carbachol (p<0.01) were significantly reduced by (-)-carvone. The contractile response elicited by high concentrations of carbachol was reduced but not abolished by (-)-carvone. No additive action was detected with co-incubation of (-)-carvone and verapamil on carbachol-induced contraction. (-)-Carvone reduced the contraction induced by high K(+) and was almost 100 times more potent than verapamil. Thus, (-)-carvone showed a typical and potent CCB-like action. Many effects described for both (-)-carvone and spearmint oil can be explained as a CCB-like mode of action.

  9. Protein kinase C mediates cholinergically regulated protein phosphorylation in a Cl(-)-secreting epithelium.

    Science.gov (United States)

    Cohn, J A

    1990-02-01

    T84 cell monolayers were used to study the cholinergic regulation of protein phosphorylation in epithelial cells. When T84 cell monolayers are labeled with 32Pi and stimulated with carbachol, six proteins exhibit altered phosphorylation. The most prominent response is a fivefold increase in labeling of p83, an acidic protein of Mr 83,000. Increasing labeling of p83 parallels stimulated secretion with respect to the onset of agonist action, agonist potency, and antagonism by atropine. However, the p83 and secretory responses differ in that the p83 response is more sustained. When T84 cell fractions are incubated with [gamma-32P]ATP, Ca2(+)-phospholipid stimulates p83 labeling. Phosphorylation of p83 also occurs when a T84 cell extract is incubated with purified protein kinase C and when intact cells are exposed to phorbol myristate acetate. p83 does not become phosphorylated in cell fractions incubated with adenosine 3',5'-cyclic monophosphate (cAMP) or in monolayers stimulated with agonists acting via cAMP. Thus carbachol stimulates the phosphorylation of an endogenous substrate for protein kinase C in T84 cells. The duration of this phosphorylation response suggests that protein kinase C may mediate a sustained response to carbachol, possibly acting to limit the duration of stimulated secretion.

  10. Extremely-low-frequency magnetic fields disrupt rhythmic slow activity in rat hippocampal slices.

    Science.gov (United States)

    Bawin, S M; Satmary, W M; Jones, R A; Adey, W R; Zimmerman, G

    1996-01-01

    Several studies have indicated that weak, extremely-low-frequency (ELF; 1-100 Hz) magnetic fields affect brain electrical activity and memory processes in man and laboratory animals. Our studies sought to determine whether ELF magnetic fields could couple directly with brain tissue and affect neuronal activity in vitro. We used rat hippocampal slices to study field effects on a specific brain activity known as rhythmic slow activity (RSA), or theta rhythm, which occurs in 7-15 s bursts in the hippocampus during memory functions. RSA, which, in vivo, is a cholinergic activity, is induced in hippocampal slices by perfusion of the tissue with carbachol, a stable analog of acetylcholine. We previously demonstrated that the free radical nitric oxide (NO), synthesized in carbachol-treated hippocampal slices, lengthened and destabilized the intervals between successive RSA episodes. Here, we investigate the possibility that sinusoidal ELF magnetic fields could trigger the NO-dependent perturbation of the rate of occurrence of the RSA episodes. Carbachol-treated slices were exposed for 10 min epochs to 1 or 60 Hz magnetic fields with field intensities of 5.6, 56, or 560 microT (rms), or they were sham exposed. All exposures took place in the presence of an ambient DC field of 45 microT, with an angle of -66 degrees from the horizontal plane. Sinusoidal 1 Hz fields at 56 and 560 microT, but not at 5.6 microT, triggered the irreversible destabilization of RSA intervals. Fields at 60 Hz resulted in similar, but not statistically significant, trends. Fields had no effects on RSA when NO synthesis was pharmacologically inhibited. However, field effects could take place when extracellular NO, diffusing from its cell of origin to the extracellular space,was chelated by hemoglobin. These results suggest that ELF magnetic fields exert a strong influence on NO systems in the brain; therefore, they could modulate the functional state of a variety of neuronal ensembles. PMID:8915548

  11. MCH-containing neurons in the hypothalamus of the cat: searching for a role in the control of sleep and wakefulness.

    Science.gov (United States)

    Torterolo, Pablo; Sampogna, Sharon; Morales, Francisco R; Chase, Michael H

    2006-11-13

    Neurons that utilize melanin-concentrating hormone (MCH) and others that employ hypocretin as neurotransmitter are located in the hypothalamus and project diffusely throughout the CNS, including areas that participate in the generation and maintenance of the states of sleep and wakefulness. In the present report, immunohistochemical methods were employed to examine the distribution of MCHergic and hypocretinergic neurons. In order to test the hypothesis that the MCHergic system is capable of influencing specific behavioral states, we studied Fos immunoreactivity in MCH-containing neurons during (1) quiet wakefulness, (2) active wakefulness with motor activity, (3) active wakefulness without motor activity, (4) quiet sleep and (5) active sleep induced by carbachol (AS-carbachol). We determined that MCHergic neuronal somata in the cat are intermingled with hypocretinergic neurons in the dorsal and lateral hypothalamus, principally in the tuberal and tuberomammillary regions; however, hypocretinergic neurons extended more in the anterior-posterior axis than MCHergic neurons. Axosomatic and axodendritic contacts were common between these neurons. In contrast to hypocretinergic neurons, which are known to be active during motor activity and AS-carbachol, Fos immunoreactivity was not observed in MCH-containing neurons in conjunction with any of the preceding behavioral conditions. Non-MCHergic, non-hypocretinergic neurons that expressed c-fos during active wakefulness with motor activity were intermingled with MCH and hypocretin-containing neurons, suggesting that these neurons are related to some aspect of motor function. Further studies are required to elucidate the functional sequela of the interactions between MCHergic and hypocretinergic neurons and the phenotype of the other neurons that were active during motor activity. PMID:17027934

  12. Modulation of presynaptic nAChRs on postsynaptic GABA receptor in optictectum of juvenile Xenopus

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Using the blind patch-clamp technique with the whole-cell mode, we have studied the modulation of presynaptic receptor on postsynaptic γ-aminobutyric acid (GABA) receptor measuring miniature inhibitory postsynaptic currents (mIPSCs) in optic tectum of Xenopus during critical peroid. It was demonstrated that compared with mature neurons, mIPSCs recorded from immature neurons had smaller amplitude and longer decay time. mIPSCs are mediated by GABAa receptor. The nicotinic acetylcholine receptor agonists (carbachol, cytisine, nicotine, DMPP and so on) could increase the frequency of mIPSCs. The enhancement of mIPSCs frequency induced by nAChR agonists was calcium-dependent. However, the choline, a product of hydrolyzed acetylcholine, could not increase the frequency of mIPSCs. DH-β-E, a competitive antagonist of nAChR, blocked the increase of mIPSCs frequency induced by carbachol. Mecamyllamine, an α3β4 subtype of nAChR antagonist, also blocked the carbachol-induced enhancement of mIPSCs. On the other hand, MLA, α7 subtype of nAChR antagonist, had no effect on it. Thus, it seems that nAChR could presynaptically modulate the mIPSCs and α3β4 subtype of nAChR might be involved. But α7 nAChR subtype of nAChR would not be involved. The modulation is calcium- dependent. Meanwhile, we found that Ca2+-free solution could elicit giant PSCs. The frequency of mIPSCs also is related with the level of HP.

  13. Food allergy alters jejunal circular muscle contractility and induces local inflammatory cytokine expression in a mouse model

    Directory of Open Access Journals (Sweden)

    Kovanen Petri T

    2009-05-01

    Full Text Available Abstract Background We hypothesized that food allergy causes a state of non-specific jejunal dysmotility. This was tested in a mouse model. Methods Balb/c mice were epicutaneously sensitized with ovalbumin and challenged with 10 intragastric ovalbumin administrations every second day. Smooth muscle contractility of isolated circular jejunal sections was studied in organ bath with increasing concentrations of carbamylcholine chloride (carbachol. Smooth muscle layer thickness and mast cell protease-1 (MMCP-1 positive cell density were assayed histologically. Serum MMCP-1 and immunoglobulins were quantified by ELISA, and mRNA expressions of IFN-γ, IL-4, IL-6 and TGFβ-1 from jejunal and ileal tissue segments were analyzed with quantitative real-time PCR. Results Ovalbumin-specific serum IgE correlated with jejunal MMCP-1+ cell density. In the allergic mice, higher concentrations of carbachol were required to reach submaximal muscular stimulation, particularly in preparations derived from mice with diarrhoea. Decreased sensitivity to carbachol was associated with increased expression of IL-4 and IL-6 mRNA in jejunum. Smooth muscle layer thickness, as well as mRNA of IFN-γ and TGF-β1 remained unchanged. Conclusion In this mouse model of food allergy, we demonstrated a decreased response to a muscarinic agonist, and increased levels of proinflammatory IL-6 and Th2-related IL-4, but not Th1-related IFN-γ mRNAs in jejunum. IgE levels in serum correlated with the number of jejunal MMCP-1+ cells, and predicted diarrhoea. Overall, these changes may reflect a protective mechanism of the gut in food allergy.

  14. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

    Science.gov (United States)

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

    2013-01-01

    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  15. Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells.

    Science.gov (United States)

    Hoffmann, Carsten; Nuber, Susanne; Zabel, Ulrike; Ziegler, Nicole; Winkler, Christiane; Hein, Peter; Berlot, Catherine H; Bünemann, Moritz; Lohse, Martin J

    2012-08-01

    Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3

  16. Increased cholinergic contractions of jejunal smooth muscle caused by a high cholesterol diet are prevented by the 5-HT4 agonist – tegaserod

    Directory of Open Access Journals (Sweden)

    Shaffer Eldon

    2006-02-01

    Full Text Available Abstract Background Excess cholesterol in bile and in blood is a major risk factor for the respective development of gallbladder disease and atherosclerosis. This lipid in excess negatively impacts the functioning of other smooth muscles, including the intestine. Serotonin is an important mediator of the contractile responses of the small intestine. Drugs targeting the serotonin receptor are used as prokinetic agents to manage intestinal motor disorders, in particular irritable bowel syndrome. Thus, tegaserod, acting on 5-HT4 receptor, ideally should obviate detrimental effects of excessive cholesterol on gastrointestinal smooth muscle. In this study we examined the effect of tegaserod on cholesterol-induced changes in the contractile responses of intestinal smooth muscle. Methods The effects of a high cholesterol (1% diet on the in vitro contractile responses of jejunal longitudinal smooth muscle from Richardson ground squirrels to the cholinergic agonist carbachol were examined in the presence or absence of tetrodrodotoxin (TTX. Two groups of animals, fed either low (0.03% or high cholesterol rat chow diet, were further divided into two subgroups and treated for 28 days with either vehicle or tegaserod. Results The high cholesterol diet increased, by nearly 2-fold, contractions of the jejunal longitudinal smooth muscle elicited by carbachol. These cholinergic contractions were mediated by muscarinic receptors since they were blocked by scopolamine, a muscarinic receptor antagonist, but not by the nicotinic receptor antagonist, hexamethonium. Tegaserod treatment, which did not affect cholinergic contractions of tissues from low cholesterol fed animals, abrogated the increase caused by the high cholesterol diet. With low cholesterol diet TTX enhanced carbachol-evoked contractions, whereas this action potential blocker did not affect the augmented cholinergic contractions seen with tissues from animals on the high cholesterol diet. Tegaserod

  17. Gestational alterations in phospholipase c coupled muscarinic response

    Energy Technology Data Exchange (ETDEWEB)

    Varol, F.G.; Hadjiconstantinou, M.; Zuspan, F.P.; Neff, N.H. (Ohio State Univ. college of Medicine, Columbus (USA))

    1989-01-01

    In the pregnant rat, carbachol-induced phosphoinositol hydrolysis by myometrium at the placental attachment region progressively decreased toward term, whereas hydrolysis was relatively stable in the myometrium of the non-attachment region. Tritium-quinuclidinyl benzilate binding increased in the myometrium of non-attachment regions as pregnancy progressed. At placental attachment sites binding remained relatively stable until parturition when it increased. Apparently the myometrium associated with the placental attachment site is less sensitive to cholinergic influence during pregnancy compared with the non-attachment site when evaluated by muscarinic activation of phospholipase C or ligand binding.

  18. Activation of the nitric oxide-cGMP pathway reduces phasic contractions in neonatal rat bladder strips via protein kinase G

    OpenAIRE

    Artim, Debra E.; Kullmann, F. Aura; Daugherty, Stephanie L.; Wu, Hsi-Yang; de Groat, William C.

    2009-01-01

    Nitric oxide (NO), a neurotransmitter in the lower urinary tract, stimulates soluble guanylyl cyclase (sGC) and in turn cGMP-dependent protein kinase G (PKG) to modulate a number of downstream targets. NO donors reduce bladder hyperactivity in some pathological models but do not affect normal bladder activity in the adult rat. In this study, the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP; 100 μM) decreased the amplitude and frequency of spontaneous and carbachol-enhanced contractions ...

  19. The activity of phosphorothioate analogues of ATP in various smooth muscle systems.

    OpenAIRE

    Stone, T.W.

    1985-01-01

    Phosphorothioate analogues of adenosine 5'-triphosphate (ATP) have been tested on the rat and guinea-pig vas deferens, the guinea-pig taenia coli and urinary bladder. Adenosine 5'0-(2-thiotriphosphate) (ATP beta S) was more active than adenosine 5'0(1-thiotriphosphate) (ATP alpha S) and ATP in producing contractile responses on the vas deferens of rat and guinea-pig, and guinea-pig bladder, though the difference of potency was less marked for producing relaxation of the carbachol-contracted t...

  20. The biphasic response of rat vesical smooth muscle to ATP.

    OpenAIRE

    Bolego, C; Pinna, C.; Abbracchio, M. P.; Cattabeni, F.; Puglisi, L.

    1995-01-01

    1. Adenosine-5'-triphosphate (ATP) is known to exert a variety of biological effects via the activation of either ionotropic P2x- or G-protein coupled P2Y-purinoceptor subtypes. In this study the effects induced by ATP and ATP analogues on rat bladder strips were characterized at resting tone and in carbachol-prestimulated tissues. 2. ATP exerted a clear concentration-dependent biphasic response, which was maximal at 1 mM concentration and was characterized by an immediate and transient contr...

  1. Defective fluid secretion from submucosal glands of nasal turbinates from CFTR-/- and CFTR (ΔF508/ΔF508 pigs.

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    Hyung-Ju Cho

    Full Text Available BACKGROUND: Cystic fibrosis (CF, caused by reduced CFTR function, includes severe sinonasal disease which may predispose to lung disease. Newly developed CF pigs provide models to study the onset of CF pathophysiology. We asked if glands from pig nasal turbinates have secretory responses similar to those of tracheal glands and if CF nasal glands show reduced fluid secretion. METHODOLOGY/PRINCIPAL FINDINGS: Unexpectedly, we found that nasal glands differed from tracheal glands in five ways, being smaller, more numerous (density per airway surface area, more sensitive to carbachol, more sensitive to forskolin, and nonresponsive to Substance P (a potent agonist for pig tracheal glands. Nasal gland fluid secretion from newborn piglets (12 CF and 12 controls in response to agonists was measured using digital imaging of mucus bubbles formed under oil. Secretion rates were significantly reduced in all conditions tested. Fluid secretory rates (Controls vs. CF, in pl/min/gland were as follows: 3 µM forskolin: 9.2±2.2 vs. 0.6±0.3; 1 µM carbachol: 143.5±35.5 vs. 52.2±10.3; 3 µM forskolin + 0.1 µM carbachol: 25.8±5.8 vs. CF 4.5±0.9. We also compared CF(ΔF508/ΔF508 with CFTR(-/- piglets and found significantly greater forskolin-stimulated secretion rates in the ΔF508 vs. the null piglets (1.4±0.8, n = 4 vs. 0.2±0.1, n = 7. An unexpected age effect was also discovered: the ratio of secretion to 3 µM forskolin vs. 1 µM carbachol was ∼4 times greater in adult than in neonatal nasal glands. CONCLUSIONS/SIGNIFICANCE: These findings reveal differences between nasal and tracheal glands, show defective fluid secretion in nasal glands of CF pigs, reveal some spared function in the ΔF508 vs. null piglets, and show unexpected age-dependent differences. Reduced nasal gland fluid secretion may predispose to sinonasal and lung infections.

  2. Correlation between oxytocin neuronal sensitivity and oxytocin receptor binding: An electrophysiological and autoradiographical study comparing rat and guinea pig hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Raggenbass, M.; Tribollet, E.; Dubois-Dauphin, M.; Dreifuss, J.J. (Univ. Medical Center, Geneva (Switzerland))

    1989-01-01

    In transverse hippocampal slices from rat and guinea pig brains, the authors obtained unitary extracellular recordings from nonpyramidal neurones located in or near the stratum pyramidale in the CA1 field and in the transition region between the CA1 and the subiculum. In rats, these neurones responded to oxytocin at 50-1,000 nM by a reversible increase in firing rate. The oxytocin-induced excitation was suppressed by a synthetic structural analogue that acts as a potent, selective antioxytocic on peripheral receptors. Nonpyramidal neurones were also excited by carbachol at 0.5-10 {mu}M. The effect of this compound was postsynaptic and was blocked by the muscarinic antagonist atropine. In guinea pigs, by contrast, nonpyramidal neurones were unaffected by oxytocin, although they were excited by carbachol. Light microscopic autoradiography, carried out using a radioiodinated selective antioxytocic as a ligand, revealed labeling in the subiculum and in the CA1 area of the hippocampus of rats, whereas no oxytocin-binding sites were detected in the hippocampus of guinea pigs. The results indicate (i) that a hippocampal action of oxytocin is species-dependent and (ii) that a positive correlation exists between neuronal responsiveness to oxytocin and the presence in the hippocampus of high-affinity binding sites for this peptide.

  3. Neuronal network analysis based on arrival times of active-sleep specific inhibitory postsynaptic potentials in spinal cord motoneurons of the cat.

    Science.gov (United States)

    Engelhardt, J K; Chase, M H

    2001-07-20

    The neuronal network responsible for motoneuron inhibition and loss of muscle tone during active (REM) sleep can be activated by the injection of the cholinergic agonist carbachol into a circumscribed region of the brainstem reticular formation. In the present report, we studied the arrival times of inhibitory postsynaptic potentials (IPSPs) observed in intracellular recordings from cat spinal cord motoneurons. These recordings were obtained during episodes of motor inhibition induced by carbachol or during motor inhibition associated with naturally occurring active sleep. When the observed IPSP arrival times were analyzed as a superposition of renewal processes occurring in a pool of pre-motor inhibitory interneurons, it was possible to estimate the following parameters: (1) the number of independent sources of the IPSPs; (2) the rate at which each source was bombarded with excitatory postsynaptic potentials (EPSPs); and (3) the number of EPSPs required to bring each source to threshold. From the data based upon the preceding parameters and the unusually large amplitudes of the active sleep-specific IPSPs, we suggest that each source is a cluster of synchronously discharging pre-motor inhibitory interneurons. The analysis of IPSP arrival times as a superposition of renewal processes, therefore, provides quantitative information regarding neuronal activity that is as far as two synapses upstream from the site of the recording electrode. Consequently, we suggest that a study of the temporal evolution of these parameters could provide a basis for dynamic analyses of this neuronal network and, in the future, for other neuronal networks as well. PMID:11457433

  4. Noncholinergic penile erection in mice lacking the gene for endothelial nitric oxide synthase.

    Science.gov (United States)

    Burnett, Arthur L; Chang, Alex G; Crone, Julie K; Huang, Paul L; Sezen, Sena E

    2002-01-01

    With the current understanding that nitric oxide (NO) mediates penile erection, the endothelial isoform of NO synthase (eNOS) has been implicated in this function. We undertook this study applying transgenic mice with targeted deletion of the eNOS gene (eNOS-/- mice) as an experimental approach to evaluate the importance of eNOS in cholinergically stimulated erectile function in vivo. Combined pharmacostimulation with intracavernosal carbachol (3 ng) administration and submaximal cavernous nerve (CN) electrical stimulation (16 Hz, 5 millisecond, 1 V) simultaneous with intracavernosal pressure (ICP) monitoring, and both biochemical assay of NO synthase activity and Western blot analysis of eNOS protein content in penile tissue, were performed on eNOS-/- mice and wild-type controls. Combined intracavernosal carbachol administration and submaximal CN electrical stimulation raised the recorded ICP, elicited by CN electrical stimulation alone in wild-type mice (from 35.7 +/- 2.7 to 48.1 +/- 5.5 mm Hg, P penes of eNOS-/- mice, approximately 60% NO synthase activity of wild-type penis levels was retained (NS), and eNOS protein was absent. We concluded that eNOS-/- mice preserve erectile function on the basis of a noncholinergic but NO-dependent mechanism and that eNOS physiologically mediates penile erection under cholinergic stimulation. PMID:11780929

  5. A Simplified In vitro Experimental Model Encompasses the Essential Features of Sleep

    Science.gov (United States)

    Colombi, Ilaria; Tinarelli, Federico; Pasquale, Valentina; Tucci, Valter; Chiappalone, Michela

    2016-01-01

    In this paper, we show that neuronal assemblies plated on Micro Electrode Arrays present synchronized, low frequency firing patterns similar to in vivo slow wave oscillations, which are a key parameter of sleep-like state. Although neuronal cultures lack the characteristic high-frequency waves of wakefulness, it is possible to modulate their spontaneous firing pattern through the administration of specific neurotransmitters such as acetylcholine. We thus stimulated the cortical cultures with an agonist of acetylcholine receptor, Carbachol, which caused a desynchronization of the spontaneous firing of the cultures. We recorded and monitored the cultures for a period of over 31 h. We analyzed the electrophysiological signals by exploiting novel methodological approaches, taking into account the different temporal scales of the recorded signals, and considering both spikes and local field potentials. Supporting the electrophysiological analysis results, gene expressions of targeted genes showed the activation of specific markers involved in sleep-wake rhythms. Our results demonstrate that the Carbachol treatment induces desynchronization of neuronal activity, altering sleep-like properties in an in vitro model. PMID:27458335

  6. Activation of muscarinic receptors inhibits glutamate-induced GSK-3β overactivation in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Ke MA; Li-min YANG; Hong-zhuan CHEN; Yang LU

    2013-01-01

    Aim:To investigate the actions of the muscarinic agonist carbachol on glutamate-induced neurotoxicity in PC12 cells,and the underlying mechanisms.Methods:PC12 cells were treated with different concentrations of glutamate for 24 or 48 h.The cell viability was measured using MTT assay,and the expression and activation of GSK-3β were detected with Western blot.β-Catenin translocation was detected using immunofluorescence.Luciferase reporter assay and real-time PCR were used to analyze the transcriptional activity of β-catenin.Results:Glutamate (1,3,and 10 mmol/L) induced PC12 cell death in a dose-dependent manner.Moreover,treatment of the cells with glutamate (1 mmol/L) caused significant overactivation of GSK-3β and prevented β-catenin translocation to the nucleus.Pretreatment with carbachol (0.01 μmol/L) blocked glutamate-induced cell death and GSK-3β overactivation,and markedly enhanced β-catenin transcriptional activity.Conclusion:Activation of muscarinic receptors exerts neuroprotection in PC12 cells by attenuating glutamate-induced GSK-3β overactivation,suggesting potential benefits of muscarinic agonists for Alzheimer's disease.

  7. Hyposmotic membrane stretch potentiated muscarinic receptor agonist-induced depolarization of membrane potential in guinea-pig gastric myocytes

    Institute of Scientific and Technical Information of China (English)

    Lin Li; Nan-Ge Jin; Lin Piao; Ming-Yu Hong; Zheng-Yuan Jin; Ying Li; Wen-Xie Xu

    2002-01-01

    AIM: To investigate the relationship betweenhyposmotic membrane stretch and muscarinic receptoragonist-induced depolarization of membrane potentialin antral gastric circular myocytes of guinea-pig.METHODS: Using whole cell patch-clamp techniquerecorded membrane potential and current in singlegastric myocytes isolated by collagena se.RESULTS: Hyposmotic membrane stretch hyperpolarizedmembrane potential from -60.0mV±1.0mV to -67.9mV±1.0mV. TEA (10mmol/L), a nonselective potassiumchannel blocker significantly inhibited hyposmoticmembrane stretch-induced hyperpolarization. After KCIin the pipette and NaCI in the external solution werereplaced by CsCI to block the potassium current,hyposmotic membrane stretch depolarized the membranepotential from -60.0 mV±-1.0mV to -44.8 mV±2.3mV(P<0.05), and atropine (1 pmol/L) inhibited thedepolarization of the membrane potential. Muscarinicreceptor agonist Carbachol depolarized membranepotential from -60.0mV±1.0mV to -50.3 mV±0.3mV(P<0.05) and hyposmotic membrane stretchpotentiated the depolarization. Carbachol inducedmuscarinic current (Icch) was greatly increased byhyposmotic membrane stretch.CONCLUSION: Hyposmotic membrane stretchpotentiated muscarinic receptor agonist-induceddepolarization of membrane potential, which is relatedto hyposmotic membrane stretch-induced increase ofmuscarinic current.

  8. Amyloid Beta Peptides Differentially Affect Hippocampal Theta Rhythms In Vitro

    Directory of Open Access Journals (Sweden)

    Armando I. Gutiérrez-Lerma

    2013-01-01

    Full Text Available Soluble amyloid beta peptide (Aβ is responsible for the early cognitive dysfunction observed in Alzheimer's disease. Both cholinergically and glutamatergically induced hippocampal theta rhythms are related to learning and memory, spatial navigation, and spatial memory. However, these two types of theta rhythms are not identical; they are associated with different behaviors and can be differentially modulated by diverse experimental conditions. Therefore, in this study, we aimed to investigate whether or not application of soluble Aβ alters the two types of theta frequency oscillatory network activity generated in rat hippocampal slices by application of the cholinergic and glutamatergic agonists carbachol or DHPG, respectively. Due to previous evidence that oscillatory activity can be differentially affected by different Aβ peptides, we also compared Aβ25−35 and Aβ1−42 for their effects on theta rhythms in vitro at similar concentrations (0.5 to 1.0 μM. We found that Aβ25−35 reduces, with less potency than Aβ1−42, carbachol-induced population theta oscillatory activity. In contrast, DHPG-induced oscillatory activity was not affected by a high concentration of Aβ25−35 but was reduced by Aβ1−42. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of exerting a generalized inhibitory effect on neuronal network function.

  9. Inositol phospholipid hydrolysis in cultured astrocytes and oligodendrocytes

    International Nuclear Information System (INIS)

    Cultures of astrocytes and oligodendrocytes were prelabeled with 3H-inositol and the accumulation of 3H-inositol phosphates was determined following stimulation with a number of neuroactive substances. In astrocytes, norepinephrine (NE) produced the greatest stimulation with significant increase also observed with bradykinin. In oligodendrocytes, the greatest stimulation was produced by carbachol with significant increase also produced by bradykinin, histamine and NE. Carbachol was found to be ineffective in producing stimulation in astrocytes. The accumulation of 3H-inositol phosphates in astrocytes in response to NE was found to be dependent on the presence of Li+. The NE stimulation in astrocytes was dose-dependent and had an EC50 of 1.2 μM. This stimulation was blocked by the low concentration of the α1-adrenergic antagonist prazosin but not by the α2-adrenergic antagonist yohimbine. The NE-stimulated accumulation of 3H-inositol phosphates in astrocytes was inhibited by the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine as well as by the cAMP analog dibutyryl cAMP. 34 references, 4 figures, 4 tables

  10. Comparison of normal and asthmatic subjects' responses to sulfate pollutant aerosols

    Energy Technology Data Exchange (ETDEWEB)

    Utell, M.J.; Morrow, P.E.; Hyde, R.W.

    1980-01-01

    Epidemiological studies support an association between elevated levels of sulfates and acute respiratory disease. To determine if these pollutants produce airway hyperreactivity, 16 normal and 17 asthmatic subjects inhaled a control NaCl aerosol and the following sulfates: ammonium sulfate, sodium bisulfate, ammonium bisulfate, and sulfuric acid. A Lovelace generator produced particles with an average MMAD of approx. 1.0 ..mu..m (sigma/sub g/ approx. = 2.0) and concentrations of 0.1 and 1.0 mg/m/sup 3/. By double-blind randomization, all subjects breathed these aerosols for a 16-minute period. To determine if sulfate inhalation caused increased reactivity to a known bronchoconstrictor, all subjects inhaled carbachol following each 16-minute exposure. Before, during, and after exposure, pulmonary function studies were performed. When compared to NaCl, sulfate (1 mg/m/sup 3/) produced significant reductions in airway conductance and flow rates in asthmatics. The two most sensitive asthmatics demonstrated changes even at 0.1 mg/m/sup 3/ sulfate. To a far more significant degree, the bronchoconstrictor action of carbachol was potentiated by sulfates more or less in relation to their acidity in normals and asthmatics.

  11. Physical exercise and pancreatic islets: acute and chronic actions on insulin secretion.

    Science.gov (United States)

    Almeida, Felipe N; Proença, André R G; Chimin, Patrícia; Marçal, Anderson C; Bessa-Lima, Fábio; Carvalho, Carla R O

    2012-01-01

    Diabetes mellitus (DM) is a great public health problem, which attacks part of the world population, being characterized by an imbalance in body glucose homeostasis. Physical exercise is pointed as a protective agent and is also recommended to people with DM. As pancreatic islets present an important role in glucose homeostasis, we aim to study the role of physical exercise (chronic adaptations and acute responses) in pancreatic islets functionality in Wistar male rats. First, animals were divided into two groups: sedentary (S) and aerobic trained (T). At the end of 8 weeks, half of them (S and T) were submitted to an acute exercise session (exercise until exhaustion), being subdivided as acute sedentary (AS) and acute trained (AT). After the experimental period, periepididymal, retroperitoneal and subcutaneous fat pads, blood, soleus muscle and pancreatic islets were collected and prepared for further analysis. From the pancreatic islets, total insulin content, insulin secretion stimulated by glucose, leucine, arginine and carbachol were analyzed. Our results pointed that body adiposity and glucose homeostasis improved with chronic physical exercise. In addition, total insulin content was reduced in group AT, insulin secretion stimulated by glucose was reduced in trained groups (T and AT) and insulin secretion stimulated by carbachol was increased in group AT. There were no significant differences in insulin secretion stimulated by arginine and leucine. We identified a possible modulating action on insulin secretion, probably related to the association of chronic adaptation with an acute response on cholinergic activity in pancreatic islets. PMID:22868676

  12. Tolerance to the carbamate insecticide propoxur.

    Science.gov (United States)

    Costa, L G; Hand, H; Schwab, B W; Murphy, S D

    1981-01-01

    Male mice were given the carbamate insecticide propoxur (2-isopropoxy phenyl methylcarbamate; Baygon) in the drinking water at weekly increasing concentrations (from 50 to 2000 ppm), for a period of 6 weeks. At the end of the treatment the LD50 for propoxur was significantly higher in the treated animals as compared with controls. Propoxur-treated animals were also resistant to the hypothermic effect of an acute administration of the same compound. Groups of mice were challenged with the cholinergic agonist carbachol at intervals during the drinking water dosing and at its end. No differences in sensitivity to carbachol acute toxicity were found between control and treated animals. Propoxur-tolerant animals were also not resistant to the hypothermic effect of oxotremorine, another cholinergic agonist. [3H]Quinuclidinyl benzilate ([3H]QNB) binding (a measure of muscarinic receptor density and affinity) in forebrain, hindbrain and ileum never differed in control and treated mice. The possibility that repeated administrations of propoxur induced increased metabolic inactivation was tested by measuring hexobarbital sleeping time and carboxylesterase activity in treated and control mice. No changes in tissue carboxylesterase activities occurred but hexobarbital sleeping time was significantly reduced in propoxur treated animals suggesting an induction of hepatic microsomal enzymes. These results suggest that tolerance to propoxur is not mediated by a decrease of cholinergic receptors, as reported for other acetylcholinesterase inhibitors, but possibly by an enhancement of its metabolism.

  13. SENSITIVE EFFECTS OF POTASSIUM AND CALCIUM CHANNEL BLOCKING AND ATP-SENSITIVE POTASSIUM CHANNEL ACTIVATORS ON SEMINAL VESICLE SMOOTH MUSCLE CONTRACTIONS

    Directory of Open Access Journals (Sweden)

    H SADRAEI

    2000-12-01

    Full Text Available Background. Seminal vesicle smooth muscle contraction is mediated through sympathetic and parasympathetic neurons activity. Although seminal vesicle plays an important role in male fertility, but little attention is given to mechanism involved in contraction of this organ.
    Methods. In this study effects of drugs which activate ATP - sensitive K channels and blockers of K and Ca channels were examined on contraction of guinea - pig isolated seminal vesicle due to electrical filled stimulation (EFS, noradrenaline, carbachol and KCI.
    Results. The K channel blocker tetraethyl ammonium potentate the EFS responses at all frequencies, while, the ATP - sensitive K channel inhibitor glibenclamide and the K channel opener levcromakalim, diazoxide, minoxidil and Ca channel blocker nifedipine all had relaxant effect on guinea - pig seminal vesicle.
    Discussion. This study indicate that activities of K and Ca channels is important in regulation of seminal vesicle contraction due to nerve stimulation, noradrenaline or carbachol.

  14. Cholinergic pairing with visual activation results in long-term enhancement of visual evoked potentials.

    Directory of Open Access Journals (Sweden)

    Jun Il Kang

    Full Text Available Acetylcholine (ACh contributes to learning processes by modulating cortical plasticity in terms of intensity of neuronal activity and selectivity properties of cortical neurons. However, it is not known if ACh induces long term effects within the primary visual cortex (V1 that could sustain visual learning mechanisms. In the present study we analyzed visual evoked potentials (VEPs in V1 of rats during a 4-8 h period after coupling visual stimulation to an intracortical injection of ACh analog carbachol or stimulation of basal forebrain. To clarify the action of ACh on VEP activity in V1, we individually pre-injected muscarinic (scopolamine, nicotinic (mecamylamine, alpha7 (methyllycaconitine, and NMDA (CPP receptor antagonists before carbachol infusion. Stimulation of the cholinergic system paired with visual stimulation significantly increased VEP amplitude (56% during a 6 h period. Pre-treatment with scopolamine, mecamylamine and CPP completely abolished this long-term enhancement, while alpha7 inhibition induced an instant increase of VEP amplitude. This suggests a role of ACh in facilitating visual stimuli responsiveness through mechanisms comparable to LTP which involve nicotinic and muscarinic receptors with an interaction of NMDA transmission in the visual cortex.

  15. Relaxation of rat distal colon by activation of muscarinic, neuronal receptors: possible involvement of P(2y)-purinoceptors.

    Science.gov (United States)

    Börjesson, L; Ali, A; Nordgren, S; Delbro, D S

    2000-07-01

    McN-A-343, which is a ligand at muscarinic receptors on myenteric ganglia, was found to concentration-dependently (1-44 microM) elicit non-adrenergic relaxation of the longitudinal muscle of rat distal colon, having been precontracted with carbachol (1 microM). This effect was partly antagonized by the muscarinic receptor antagonist, pirenzepine (0.3 microM), the nerve blocker, tetrodotoxin (1 microM), or by drugs which interfere with purinergic neurotransmission (apamin [0.5 microM], reactive blue 2 [50 microM]). Blockade of nitric oxide synthase (L-NNA [100 microM]), or of the cAMP (H-89 [1 microM]), or cGMP (ODQ [10 microM]) second messenger pathways did not affect the relaxatory response to McN-A-343 (14 microM). An additional, non-neurogenic component of the relaxation to this compound on carbachol induced tone is suggested to reflect a partial antagonism of the muscarinic receptors on the gut muscle by McN-A-343.

  16. Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors.

    Science.gov (United States)

    Sodhi, Puneet; Hartwick, Andrew T E

    2016-09-01

    Melanopsin-dependent phototransduction in intrinsically photosensitive retinal ganglion cells (ipRGCs) involves a Gq-coupled phospholipase C (PLC) signaling cascade. Acetylcholine, released in the mammalian retina by starburst amacrine cells, can also activate Gq-PLC pathways through certain muscarinic acetylcholine receptors (mAChRs). Using multielectrode array recordings of rat retinas, we demonstrate that robust spiking responses can be evoked in neonatal and adult ipRGCs after bath application of the muscarinic agonist carbachol. The stimulatory action of carbachol on ipRGCs was a direct effect, as confirmed through calcium imaging experiments on isolated ipRGCs in purified cultures. Using flickering (6 Hz) yellow light stimuli at irradiances below the threshold for melanopsin activation, spiking responses could be elicited in ipRGCs that were suppressed by mAChR antagonism. Therefore, this work identified a novel melanopsin-independent pathway for stimulating sustained spiking in ganglion cell photoreceptors. This mAChR-mediated pathway could enhance ipRGC spiking responses in conditions known to evoke retinal acetylcholine release, such as those involving flickering or moving visual stimuli. Furthermore, this work identifies a pharmacological approach for light-independent ipRGC stimulation that could be targeted by mAChR agonists. PMID:27055770

  17. Effects of Ammi visnaga (Bisnaga) Extract on the Volume and Acidity of Stimulated Gastric Secretion in Fasting Rabbits

    International Nuclear Information System (INIS)

    Objective: To determine the efficacy of extract of Ammi visnaga on volume and acidity of stimulated gastric secretion in rabbits and also its safety on liver and kidney function. Study Design: Quasi experimental study. Place and Duration of Study: Pharmacology Department, Saidu Medical College, Swat, in the years 2008-9. Methodology: Thirty rabbits weighing 1 - 1.5 kg were divided into groups A, B and C each having 10 animals. After fasting for 48 hours, pylorus of animals of group A and B was ligated. Group A was administered Carbachol and group B was given extracts of Ammi visnaga followed by Carbachol after 15 minutes intraperitoneally. After 4 hours, stomach contents were measured for volume and then centrifuged and estimated for acidity. The extract was also administered to group C animals for 45 days to observe its effects on liver and kidney function. Results: In group B, reduction in volume, free and total acidity of gastric juice was highly significant when the mean values were compared with group A. In group C, mean values of liver and kidney function test compared with pre-treated values, were found statistically non-significant. Conclusion: Ammi visnaga extract can be used effectively and safely in the treatment of hyper acidity conditions and peptic ulcer after evaluation of its effects in human being. (author)

  18. Reductions of {sup 56}Fe heavy-particle irradiation-induced deficits in striatal muscarinic receptor sensitivity by selective cross-activation/inhibition of second-messenger systems

    Energy Technology Data Exchange (ETDEWEB)

    Joseph, J.A.; Villalobos-Molina, R.; Rabin, B.M.; Dalton, T.K.; Harris, A.; Kandasamy, S. [Armed Forces Radiobiology Research Institute, Bethesda, MD (United States)

    1994-07-01

    Recent experiments have revealed radiation-induced losses of sensitivity of rodent neostriatal muscarinic receptors to stimulation by cholinergic agonists that appears as reduction in oxotremorine enhancement of K{sup +}-evoked dopamine release. These losses were postulated to be the result of radiation-induced alterations early in phosphoinositide-mediated signal transduction. Additional findings indicated that if the ligand-receptor-G protein interface was bypassed no radiation deficits were seen. In the present study, radiation-induced deficits in K{sup +}-evoked dopamine release were examined in perifused striatal tissue obtained from rats exposed to 0,0.1 or 1.0 Gy of {sup 56}Fe particles. Results showed that these deficits could be reduced by co-applying combinations of various pharmacological agents that were known to have differential effects on various second messengers such as 1,4,5-inositoltrisphosphate (IP{sub 3}). Combinations included oxotremorine-carbachol, and either oxotremorine or carbachol with arginine vasopressin or arachidonic acid. These results are discussed in terms of putative radiation-induced changes in receptor-containing membranes which alter receptor-G protein coupling/uncoupling. 49 refs., 4 figs.

  19. Absence of force suppression in rabbit bladder correlates with low expression of heat shock protein 20

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    Murphy Richard A

    2005-11-01

    Full Text Available Abstract Background Nitroglycerin can induce relaxation of swine carotid artery without sustained reductions in [Ca2+]i or myosin regulatory light chain (MRLC phosphorylation. This has been termed force suppression and been found to correlate with ser16-phosphorylation of heat shock protein 20 (HSP20. We tested for the existence of this mechanism in a smooth muscle that is not responsive to nitric oxide. Methods Isometrically mounted mucosa free rabbit bladder strips were contracted with carbachol and relaxed with 8-Br-cGMP, forskolin, or isoprenaline. Results Contraction was associated with a highly cooperative relation between MRLC phosphorylation and force such that very small increases in MRLC phosphorylation induced large increases in force. Relaxation induced by 8-Br-cGMP, forskolin, or isoprenaline did not shift the MRLC phosphorylation-force relation from that observed with carbachol alone, i.e. there was no force suppression. HSP20 content was negligible (approximately two hundred-fold less than swine carotid. Conclusion The lack of force suppression in the absence of HSP20 is consistent with the hypothesized role for HSP20 in the force suppression observed in tonic smooth muscles.

  20. Aluminium inhibits muscarinic agonist-induced inositol 1,4,5-trisphosphate production and calcium mobilization in permeabilized SH-SY5Y human neuroblastoma cells.

    Science.gov (United States)

    Wood, P C; Wojcikiewicz, R J; Burgess, J; Castleden, C M; Nahorski, S R

    1994-06-01

    The effects of aluminium (as Al3+) on carbachol-induced inositol 1,4,5-trisphosphate (InsP3) production and Ca2+ mobilisation were assessed in electropermeabilised human SH-SY5Y neuroblastoma cells. Al3+ had no effect on InsP3-induced Ca2+ release but appreciably reduced carbachol-induced Ca2+ release (IC50 of approximately 90 microM). Al3+ also inhibited InsP3 production (IC50 of approximately 15 microM). Dimethyl hydroxypyridin-4-one, a potent Al3+ chelator (Ks = 31), at 100 microM was able to abort and reverse the effects of Al3+ on both Ca2+ release and InsP3 production. These data suggest that, in permeabilised cells, the effect of Al3+ on the phosphoinositide-mediated signalling pathway is at the level of phosphatidylinositol 4,5-bisphosphate hydrolysis. This may reflect interference with receptor-G protein-phospholipase C coupling or an interaction with phosphatidylinositol 4,5-bisphosphate. PMID:8189229

  1. Distribution of airway narrowing responses across generations and at branching points, assessed in vitro by anatomical optical coherence tomography

    Directory of Open Access Journals (Sweden)

    Eastwood Peter R

    2010-01-01

    Full Text Available Abstract Background Previous histological and imaging studies have shown the presence of variability in the degree of bronchoconstriction of airways sampled at different locations in the lung (i.e., heterogeneity. Heterogeneity can occur at different airway generations and at branching points in the bronchial tree. Whilst heterogeneity has been detected by previous experimental approaches, its spatial relationship either within or between airways is unknown. Methods In this study, distribution of airway narrowing responses across a portion of the porcine bronchial tree was determined in vitro. The portion comprised contiguous airways spanning bronchial generations (#3-11, including the associated side branches. We used a recent optical imaging technique, anatomical optical coherence tomography, to image the bronchial tree in three dimensions. Bronchoconstriction was produced by carbachol administered to either the adventitial or luminal surface of the airway. Luminal cross sectional area was measured before and at different time points after constriction to carbachol and airway narrowing calculated from the percent decrease in luminal cross sectional area. Results When administered to the adventitial surface, the degree of airway narrowing was progressively increased from proximal to distal generations (r = 0.80 to 0.98, P Conclusions Our findings demonstrate that the bronchial tree expresses intrinsic serial heterogeneity, such that narrowing increases from proximal to distal airways, a relationship that is influenced by the route of drug administration but not by structural variations accompanying branching sites.

  2. Muscarinic Receptor Agonists Protect Cultured Bovine Trabecular Meshwork Cells against Apoptosis Induced by Dexamethasone

    Institute of Scientific and Technical Information of China (English)

    Yajuan Gu; Shujun Zeng; PengXin Qiu; Yuping Wu; Dawei Peng; Guangmei Yan1

    2004-01-01

    Purpose:To study whether muscarinic receptor agonists can protect cultured bovine trabecular meshwork cells against apoptosis induced by dexamethasone.Methods:The third to fifth passags of bovine trabecular meshwork cells were grown to confluence and incubated for 1~14 days in growth media with dexamethasone or pretreatment of pilocarpine or carbachol.The cultures were evaluated for apoptosis by phase-contrast microscopy, fluorescence microscopy, DNA laddering and flow cytometric analysis.Results :Dexamethasone (0.24~0.96 mmol·L-1) induced apoptosis of trabecular meshwork cells in a dose and time-dependent manner. Before 0.48 mmol·L-1 dexamethasone-treatment, 1.84 mmol· L-1 of pilocarpine or 2.74 mmol· L-1 of carbachol added could significantly reduce apoptotic percentage. Conclusion: Muscarinie receptor agonists can protect cultured bovine trabecular meshwork cells against apoptosis induced by dexamethasone. Eye Science 2004;20:42-47.

  3. The effects of cannabidiolic acid and cannabidiol on contractility of the gastrointestinal tract of Suncus murinus.

    Science.gov (United States)

    Cluny, Nina L; Naylor, Robert J; Whittle, Brian A; Javid, Farideh A

    2011-09-01

    Cannabidiol (CBD) has been shown to inhibit gastrointestinal (GI) transit in pathophysiologic in vivo models, while having no effect in physiologic controls. The actions of the precursor of CBD, cannabidiolic acid (CBDA), have not been investigated in the GI tract. The actions of these phytocannabinoids on the contractility of the GI tract of Suncus murinus were investigated in the current study. The effects of CBDA and CBD in resting state and pre-contracted isolated intestinal segments, and on the contractile effects of carbachol and electrical field stimulation (EFS) on the intestines of S. murinus were examined. CBDA and CBD induced a reduction in resting tissue tension of isolated intestinal segments which was not blocked by the cannabinoid CB1 receptor antagonist, AM251, the CB(2) receptor antagonist AM630, or tetrodotoxin. CBDA and CBD reduced the magnitude of contractions induced by carbachol and the tension of intestinal segments that were pre-contracted with potassium chloride. In tissues stimulated by EFS, CBDA inhibited contractions induced by lower frequencies (0.1-4.0 Hz) of EFS, while CBD inhibited contractions induced by higher frequencies (4.0-20.0 Hz) of EFS. The data suggest that CBDA and CBD have inhibitory actions on the intestines of S. murinus that are not neuronallymediated or mediated via CB(1) or CB(2) receptors. PMID:21975813

  4. Barrier qualities of the mouse eye to topically applied drugs.

    Science.gov (United States)

    Wang, Zhao; Do, Chi Wai; Avila, Marcel Y; Stone, Richard A; Jacobson, Kenneth A; Civan, Mortimer M

    2007-07-01

    The mouse eye displays unusually rapid intraocular pressure (IOP) responses to topically applied drugs as measured by the invasive servo-null micropipette system (SNMS). To learn if the time course reflected rapid drug transfer across the thin mouse cornea and sclera, we monitored a different parameter, pupillary size, following topical application of droplets containing 40 microM (0.073 microg) carbachol. No miosis developed from this low carbachol concentration unless the cornea was impaled with an exploring micropipette as used in the SNMS. We also compared the mouse IOP response to several purinergic drugs, measured by the invasive SNMS and non-invasive pneumotonometry. Responses to the previously studied non-selective adenosine-receptor (AR) agonist adenosine, the A(3)-selective agonist Cl-IB-MECA and the A(3)-selective antagonist MRS 1191 were all enhanced to varying degrees, in time and magnitude, by corneal impalement. We conclude that the thin ocular coats of the mouse eye actually present a substantial barrier to drug penetration. Corneal impalement with even fine-tipped micropipettes can significantly enhance entry of topically-applied drugs into the mouse aqueous humor, reflecting either direct diffusion around the tip or a more complex impalement-triggered change in ocular barrier properties. Comparison of invasive and non-invasive measurement methods can document drug efficacy at intraocular target sites even if topical drug penetration is too slow to manifest convincing physiologic effects in intact eyes. PMID:17490649

  5. Effects of ghrelin and motilin on smooth muscle contractility of the isolated gastrointestinal tract from the bullfrog and Japanese fire belly newt.

    Science.gov (United States)

    Kitazawa, Takio; Shimazaki, Misato; Kikuta, Ayumi; Yaosaka, Noriko; Teraoka, Hiroki; Kaiya, Hiroyuki

    2016-06-01

    Ghrelin has been identified in some amphibians and is known to stimulate growth hormone release and food intake as seen in mammals. Ghrelin regulates gastrointestinal motility in mammals and birds. The aim of this study was to determine whether ghrelin affects gastrointestinal smooth muscle contractility in bullfrogs (anuran) and Japanese fire belly newts (urodelian) in vitro. Neither bullfrog ghrelin nor rat ghrelin affected longitudinal smooth muscle contractility of gastrointestinal strips from the bullfrog. Expression of growth hormone secretagogue receptor 1a (GHS-R1a) mRNA was confirmed in the bullfrog gastrointestinal tract, and the expression level in the gastric mucosa was lower than that in the intestinal mucosa. In contrast, some gastrointestinal peptides, including substance P, neurotensin and motilin, and the muscarinic receptor agonist carbachol showed marked contraction, indicating normality of the smooth muscle preparations. Similar results were obtained in another amphibian, the Japanese fire belly newt. Newt ghrelin and rat ghrelin did not cause any contraction in gastrointestinal longitudinal muscle, whereas substance P and carbachol were effective causing contraction. In conclusion, ghrelin does not affect contractility of the gastrointestinal smooth muscle in anuran and urodelian amphibians, similar to results for rainbow trout and goldfish (fish) but different from results for rats and chickens. The results suggest diversity of ghrelin actions on the gastrointestinal tract across animals. This study also showed for the first time that motilin induces gastrointestinal contraction in amphibians. PMID:26704852

  6. Fatty acid-induced changes in vascular reactivity in healthy adult rats.

    Science.gov (United States)

    Christon, Raymond; Marette, André; Badeau, Mylène; Bourgoin, Frédéric; Mélançon, Sébastien; Bachelard, Hélène

    2005-12-01

    Dietary fatty acids (FAs) are known to modulate endothelial dysfunction, which is the first stage of atherosclerosis. However, their exact role in this initial phase is still unclear. The effects of isolated or combined (by 2) purified FAs from the main FA families were studied on the vascular response of isolated thoracic aorta in healthy rats to get a better understanding of the mechanisms of action of dietary FAs in regulating vascular endothelial function. Cumulative contraction curves to phenylephrine and relaxation curves to carbachol and then to sodium nitroprusside were obtained in the absence or presence of the FAs studied allowing endothelium-dependent and endothelium-independent ability of the smooth muscle to relax to be assessed in each experimental group. The endothelium-dependent vasodilator response to carbachol was lowered by eicosapentaenoic acid, whereas it was not altered either by docosahexaenoic acid alone or by combined eicosapentaenoic acid-docosahexaenoic acid, oleic acid, or stearic acid, and it was increased by linoleic acid (LA). A decreased phenylephrine-induced contraction was observed after incubation with arachidonic acid and with stearic acid. On the other hand, the endothelium-dependent relaxation was reduced by the addition of combined LA-arachidonic acid and LA-oleic acid. In conclusion, these data point out the differential effects of different types of FAs and of FAs alone vs combined on vascular reactivity. The complex nature of these effects could be partially linked to metabolic specificities of endothelial cells and to interactions between some FAs.

  7. Brainstem glycinergic neurons and their activation during active (rapid eye movement) sleep in the cat.

    Science.gov (United States)

    Morales, F R; Sampogna, S; Rampon, C; Luppi, P H; Chase, M H

    2006-09-29

    It is well established that, during rapid eye movement (REM) sleep, somatic motoneurons are subjected to a barrage of inhibitory synaptic potentials that are mediated by glycine. However, the source of this inhibition, which is crucial for the maintenance and preservation of REM sleep, has not been identified. Consequently, the present study was undertaken to determine in cats the location of the glycinergic neurons, that are activated during active sleep, and are responsible for the postsynaptic inhibition of motoneurons that occurs during this state. For this purpose, a pharmacologically-induced state of active sleep (AS-carbachol) was employed. Antibodies against glycine-conjugated proteins were used to identify glycinergic neurons and immunocytochemical techniques to label the Fos protein were employed to identify activated neurons. Two distinct populations of glycinergic neurons that expressed c-fos were distinguished. One population was situated within the nucleus reticularis gigantocellularis (NRGc) and nucleus magnocellularis (Mc) in the rostro-ventral medulla; this group of neurons extended caudally to the ventral portion of the nucleus paramedianus reticularis (nPR). Forty percent of the glycinergic neurons in the NRGc and Mc and 25% in the nPR expressed c-fos during AS-carbachol. A second population was located in the caudal medulla adjacent to the nucleus ambiguus (nAmb), wherein 40% of the glycinergic cells expressed c-fos during AS-carbachol. Neither population of glycinergic cells expressed c-fos during quiet wakefulness or quiet (non-rapid eye movement) sleep. We suggest that the population of glycinergic neurons in the NRGc, Mc, and nPR participates in the inhibition of somatic brainstem motoneurons during active sleep. These neurons may also be responsible for the inhibition of sensory and other processes during this state. It is likely that the group of glycinergic neurons adjacent to the nucleus ambiguus (nAmb) is responsible for the active

  8. Activation of protein kinase C accelerates contraction kinetics of airway smooth muscle.

    Science.gov (United States)

    Peiper, U; Knipp, S C; Thies, B; Henke, R

    1996-01-01

    Contraction kinetics of isolated rat tracheal smooth muscle were studied by analysing the increase of force subsequent to force-inhibiting passive length changes lasting 1 s (100 Hz, sinus, 5% of muscle length). Compared with carbachol activation, phorboldibutyrate (PDBu)-induced stimulation of protein kinase C (PKC) demonstrated no significant difference in the extent of force development in the polarized preparation [mean peak force 9.16 +/- 0.37 mN (carbachol) vs. 9.12 +/- 0.37 mN (PDBu)]. However, the time constant calculated for the slow component of post-vibration force recovery was 6.40 +/- 0.29 s after addition of PDBu vs. 22.39 +/- 1.40 s during carbachol activation, indicating a significant phorbol ester-induced acceleration of the cross-bridge cycling rate. In the K-depolarized preparation, treatment with 26.4 microM indolactam (IL) to activate PKC produced muscle relaxation (9.94 +/- 0.16 mN measured 0-30 min after the onset of depolarization vs. 4.13 +/- 0.05 mN measured during 30-60 min of IL treatment). Again, even in the presence of high sarcoplasmic Ca2+ resulting from tonic depolarization, PKC activation was associated with a distinct diminution of the time constant (25.99 +/- 0.79 s during the first 30 min of depolarization vs. 10.32 +/- 0.21 s during 30-60 min of IL treatment). In contrast, addition of 0.035 microM verapamil, 1.5 microM isoproterenol, and 32 microM dibutyryl-cAMP to the bathing medium induced relaxation without affecting the rate of post-vibration force recovery. The results suggest that the calcium-dependent signal cascade (agonist receptor/inositol trisphosphate/ Ca(2+)-calmodulin/myosin light chain kinase) hardly affects the regulation of contraction kinetics in the tonically activated intact smooth muscle preparation. PKC stimulation, however, accelerates actin/myosin interaction kinetics, possibly by inhibition of phosphatase(s).

  9. HCO3- Transport in Relation to Mucus Secretion from Submucosal Glands

    Directory of Open Access Journals (Sweden)

    Joo NS

    2001-07-01

    Full Text Available The role of HCO(3(- transport in relation to fluid secretion by submucosal glands is being studied in sheep, pigs, cats and humans. Optical methods have been developed to measure secretion rates of mucus volume from single glands with sufficient temporal resolution to detect differences in minute-by-minute secretion rates among glands. The ionic composition and viscoelastic properties of the uncontaminated gland mucus are measured with a combination of ratiometric fluorescent indicators, ion-selective microelectrodes, FRAP, and a miniaturized, magnetic force viscometer. Sheep glands secreted basally at low rates, showed small, transient responses to alpha- and beta-adrenergic agonists, and large responses to a cholinergic agonist, carbachol. Peak rates and temporal patterns of responses to carbachol differed markedly among glands. To assess the contribution of HCO(3(- transport to gland secretion, we either inhibited Na(+/K(+/2Cl(- cotransporter (NKCC with bumetanide or replaced HCO(3(- with HEPES and gassed with O(2. Bumetanide caused a small, non-significant inhibition of basal secretion, but removal of HCO(3(-/CO(2 significantly reduced basal secretion almost by half. Both bumetanide and removal of HCO(3(-/CO(2 reduced carbachol-stimulated secretion significantly, with HCO(3(- removal having the larger effect: a reduction to 33% of control (P less than 0.01. The remaining secretory response to carbachol was nearly eliminated by bumetanide. Sheep mucus pH measured with ion selective electrodes was about 0.4 log more acidic than the bath. In humans, we observed the same pattern of responses to agonists and antagonists as in sheep, and observed a mucus pH of 7.0 using 2',7'-bis(carboxyethyl-5,6-carboxyfluorescein (BCECF. We hypothesize that HCO(3(- transport is important in the formation of mucus secretion, but that most HCO(3(- is scavenged before the final mucus appears at the duct opening. Cystic fibrosis transmembrane conductance regulator

  10. Antioxidant, larvicidal and antiacetylcholinesterase activities of cashew nut shell liquid constituents.

    Science.gov (United States)

    Oliveira, Micheline Soares Costa; Morais, Selene Maia de; Magalhães, Davi Varela; Batista, Williams Pereira; Vieira, Icaro Gusmão Pinto; Craveiro, Afrânio Aragão; de Manezes, Jane Eire Silva Alencar; Carvalho, Ana Fontenelle Urano; de Lima, Glauber Pacelli Gomes

    2011-03-01

    Anacardic acid, cardanol and cardol, the main constituents of natural cashew nut shell liquid (CNSL), were obtained by solvent extraction and assayed for antioxidant, larvicidal and antiacetylcholinesterase activity. Their relative percent composition was obtained by HPLC analysis. Antioxidant activity was assessed using the DPPH and ABTS(+) tests, which showed cardanol as the most active, followed by cardol and anacardic acid. The three CNSL components demonstrated good larvicidal activity against Aedes aegypti (LC(50)=12.40 for anacardic acid, 10.22 for cardol and 14.45 for cardanol) and exhibited inhibition zones for acetylcholinesterase enzymes in the TLC test similar to carbachol, which was used as standard. Based on the results, these multipotent compounds represent promising agents in the control of Ae. aegypti, the main dengue vector in Brazil. PMID:20707981

  11. Impaired Sympathoadrenal Axis Function Contributes to Enhanced Insulin Secretion in Prediabetic Obese Rats

    Directory of Open Access Journals (Sweden)

    Ana Eliza Andreazzi

    2011-01-01

    Full Text Available The involvement of sympathoadrenal axis activity in obesity onset was investigated using the experimental model of treating neonatal rats with monosodium L-glutamate. To access general sympathetic nervous system activity, we recorded the firing rates of sympathetic superior cervical ganglion nerves in animals. Catecholamine content and secretion from isolated adrenal medulla were measured. Intravenous glucose tolerance test was performed, and isolated pancreatic islets were stimulated with glucose and adrenergic agonists. The nerve firing rate of obese rats was decreased compared to the rate for lean rats. Basal catecholamine secretion decreased whereas catecholamine secretion induced by carbachol, elevated extracellular potassium, and caffeine in the isolated adrenal medulla were all increased in obese rats compared to control. Both glucose intolerance and hyperinsulinaemia were observed in obese rats. Adrenaline strongly inhibited glucose-induced insulin secretion in obese animals. These findings suggest that low sympathoadrenal activity contributes to impaired glycaemic control in prediabetic obese rats.

  12. Effects of thapsigargin in isolated rat thoracic aorta

    DEFF Research Database (Denmark)

    Mikkelsen, E O; Thastrup, Ole; Christensen, S B

    1988-01-01

    The effect of thapsigargin (Tg) was studied in rat thoracic aorta. Tg (10(-8)-10(-5) M) had a dual effect on rat aorta. Thus, Tg induced a concentration dependent increase in basal tone in normal physiological salt solution (PSS), while Tg in potassium (K+) precontracted aortic rings caused...... a concentration related relaxation and shifted the K+-concentration response curve to the right and depressed the maximal response to K+. Removal of vascular endothelium abolished the relaxant response to Tg and increased the sensitivity of the preparations to the contractile effect of Tg. The contractile......, but had no effect on the Tg or on the calcium ionophore A 23187 evoked relaxation. Ultraviolet radiation decreased the relaxant effect of Tg and A 23187 without affecting the carbachol induced relaxations. The results showed that vascular endothelium depressed the contractile effect of Tg and that Tg like...

  13. Antinociceptive and antispasmodic effects of the essential oil of Ocimum micranthum: potential anti-inflammatory properties.

    Science.gov (United States)

    Pinho, João Paulo de; Silva, Andressa Santa; Pinheiro, Bruno Gonçalves; Sombra, Igor; Bayma, Joaquim de Carvalho; Lahlou, Saad; Sousa, Pergentino José da Cunha; Magalhães, Pedro Jorge

    2012-05-01

    The antinociceptive and antispasmodic properties of the essential oil of OCIMUM micranthum (EOOM) were characterized. In mice, EOOM (15-100 mg · kg (-1), p. o.) reduced both the writhing responses induced by acetic acid and the licking-time induced by formalin, being inert on the hot plate test. In rat trachea, EOOM relaxed sustained contractions induced by KCl or carbachol (CCh). Its constituents, ( E)- [( E)-MC] and ( Z)-methyl cinnamate [( Z)-MC], reproduced several effects of EOOM. Inhaled as aerosol, EOOM prevented tracheal hyperresponsiveness to KCl or CCh in ovalbumin-sensitized animals after antigen challenge. Thus, EOOM exerts peripheral analgesia in nociception of inflammatory origin and has antispasmodic actions on rat airways under an inflammatory environment. Its effects are mainly due to ( E)-MC, which makes this substance potentially interesting for studies involving conjunctly smooth muscle cells, nociception, and inflammation. Other EOOM constituents also appear to be involved in its pharmacological actions.

  14. Ethanol inhibits the motility of rabbit sphincter of Oddi in vitro

    Institute of Scientific and Technical Information of China (English)

    Réka Sári; Attila Pálv(o)lgyi; Zoltán Rakonczay Jr; Tamás Takács; János Lonovics; László Czakó; Zoltán Szilvássy; Péter Hegyi

    2004-01-01

    AIM: The role of the sphincter of Oddi (SO) in ethanol (ETOH)-induced pancreatitis is controversial. Our aim was to characterise the effect of ETOH on basal and stimulated SO motility.METHODS: SOs removed from white rabbits were placed in an organ bath (Krebs solution, pH7.4, 37 ℃). The effects of 2 mL/L, 4 mL/L, 6 mL/L and 8 mL/L of ETOH on the contractile responses of the sphincter were determined.SOs were stimulated with either 0.1 μmol/L carbachol, 1 μmol/L erythromycin or 0.1 μmol/L cholecystokinin (CCK).RESULTS: ETOH at a dose of 4 mL/L significantly decreased the baseline contractile amplitude from 11.98±0.05 mN to 11.19±0.07 mN. However, no significant changes in the contractile frequency were observed. ETOH (0.6%)significantly decreased both the baseline amplitude and the frequency compared to the control group (10.50±0.01 mN,12.13±0.10 mN and 3.53±0.13 c/min, 5.5±0.13 cycles(c)/min,respectively). Moreover, 0.8% of ETOH resulted in complete relaxation of the SO. Carbachol (0.1 μmol/L) or erythromycin (1 μmol/L) stimulated the baseline amplitudes (by 82%and 75%, respectively) and the contractile frequencies (by 150% and 106%, respectively). In the carbachol or erythromycin-stimulated groups 2-6 mL/L of ETOH significantly inhibited both the amplitude and the frequency. Interestingly,a 4-5 min administration of 6 mL/L ETOH suddenly and completely relaxed the SO. CCK (0.1 μmol/L) stimulated the baseline amplitude from 12.37±0.05 mN to 27.40±1.82mN within 1.60±0.24 min. After this peak, the amplitude decreased to 17.17±0.22 mN and remained constant during the experiment. The frequency peaked at 12.8±0.2 c/min,after which the constant frequency was 9.43±0.24 c/minthroughout the rest of the experiment. ETOH at a dose of 4 mL/L significantly decreased the amplitude from 16.13±0.23 mN to 14.93±0.19 mN. However, no significant changes in the contractile frequency were observed. ETOH at a dose of 6 mL/L inhibited both the amplitudes and the

  15. Pretreatment with adenosine and adenosine A1 receptor agonist protects against intestinal ischemia-reperfusion injury in rat

    Institute of Scientific and Technical Information of China (English)

    V Haktan Ozacmak; Hale Sayan

    2007-01-01

    AIM: To examine the effects of adenosine and A1 receptor activation on reperfusion-induced small intestinal injury.METHODS: Rats were randomized into groups with sham operation, ischemia and reperfusion, and systemic treatments with either adenosine or 2-chloro-N6-cyclopentyladenosine, A1 receptor agonist or 8-cyclopentyl-1,3-dipropylxanthine, A1 receptor antagonist, plus adenosine before ischemia. Following reperfusion, contractions of ileum segments in response to KCl, carbachol and substance P were recorded. Tissue myeloperoxidase,malondialdehyde, and reduced glutathione levels were measured.RESULTS: Ischemia significantly decreased both contraction and reduced glutathione level which were ameliorated by adenosine and agonist administration. Treatment also decreased neutrophil infiltration and membrane lipid peroxidation. Beneficial effects of adenosine were abolished by pretreatment with A1 receptor antagonist.CONCLUSION: The data suggest that adenosine and A1 receptor stimulation attenuate ischemic intestinal injury via decreasing oxidative stress, lowering neutrophil infiltration, and increasing reduced glutathione content.

  16. Vasoactive intestinal polypeptide excites medial pontine reticular formation neurons in the brainstem rapid eye movement sleep-induction zone

    DEFF Research Database (Denmark)

    Kohlmeier, Kristi Anne; Reiner, P B

    1999-01-01

    Although it has long been known that microinjection of the cholinergic agonist carbachol into the medial pontine reticular formation (mPRF) induces a state that resembles rapid eye movement (REM) sleep, it is likely that other transmitters contribute to mPRF regulation of behavioral states. A key...... candidate is the peptide vasoactive intestinal polypeptide (VIP), which innervates the mPRF and induces REM sleep when injected into this region of the brainstem. To begin understanding the cellular mechanisms underlying this phenomenon, we examined the effects of VIP on mPRF cells using whole-cell patch...... conclude that VIP excites mPRF neurons by activation of a sodium current. This effect is mediated at least in part by G-protein stimulation of adenylyl cyclase, cAMP, and protein kinase A. These data suggest that VIP may play a physiological role in REM induction by its actions on mPRF neurons....

  17. Isolation and Pharmacological Characterization of α-Elapitoxin-Ot1a, a Short-Chain Postsynaptic Neurotoxin from the Venom of the Western Desert Taipan, Oxyuranus temporalis

    Science.gov (United States)

    Barber, Carmel M.; Ahmad Rusmili, Muhamad Rusdi; Hodgson, Wayne C.

    2016-01-01

    Taipans (Oxyuranus spp.) are elapids with highly potent venoms containing presynaptic (β) and postsynaptic (α) neurotoxins. O. temporalis (Western Desert taipan), a newly discovered member of this genus, has been shown to possess venom which displays marked in vitro neurotoxicity. No components have been isolated from this venom. We describe the characterization of α-elapitoxin-Ot1a (α-EPTX-Ot1a; 6712 Da), a short-chain postsynaptic neurotoxin, which accounts for approximately 30% of O. temporalis venom. α-Elapitoxin-Ot1a (0.1–1 µM) produced concentration-dependent inhibition of indirect-twitches, and abolished contractile responses to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The inhibition of indirect twitches by α-elapitoxin-Ot1a (1 µM) was not reversed by washing the tissue. Prior addition of taipan antivenom (10 U/mL) delayed the neurotoxic effects of α-elapitoxin-Ot1a (1 µM) and markedly attenuated the neurotoxic effects of α-elapitoxin-Ot1a (0.1 µM). α-Elapitoxin-Ot1a displayed pseudo-irreversible antagonism of concentration-response curves to carbachol with a pA2 value of 8.02 ± 0.05. De novo sequencing revealed the main sequence of the short-chain postsynaptic neurotoxin (i.e., α-elapitoxin-Ot1a) as well as three other isoforms found in O. temporalis venom. α-Elapitoxin-Ot1a shows high sequence similarity (i.e., >87%) with other taipan short-chain postsynaptic neurotoxins. PMID:26938558

  18. Mechanisms underlying probucol-induced hERG-channel deficiency

    Directory of Open Access Journals (Sweden)

    Shi YQ

    2015-07-01

    Full Text Available Yuan-Qi Shi,1,* Cai-Chuan Yan,1,* Xiao Zhang,1 Meng Yan,1 Li-Rong Liu,1 Huai-Ze Geng,1 Lin Lv,1 Bao-Xin Li1,21Department of Pharmacology, Harbin Medical University, 2State-Province Key Laboratory of Biopharmaceutical Engineering, Harbin, Heilongjiang, People’s Republic of China*These authors contributed equally to this workAbstract: The hERG gene encodes the pore-forming α-subunit of the rapidly activating delayed rectifier potassium channel (IKr, which is important for cardiac repolarization. Reduction of IhERG due to genetic mutations or drug interferences causes long QT syndrome, leading to life-threatening cardiac arrhythmias (torsades de pointes or sudden death. Probucol is a cholesterol-lowering drug that could reduce hERG current by decreasing plasma membrane hERG protein expression and eventually cause long QT syndrome. Here, we investigated the mechanisms of probucol effects on IhERG and hERG-channel expression. Our data demonstrated that probucol reduces SGK1 expression, known as SGK isoform, in a concentration-dependent manner, resulting in downregulation of phosphorylated E3 ubiquitin ligase Nedd4-2 expression, but not the total level of Nedd4-2. As a result, the hERG protein reduces, due to the enhanced ubiquitination level. On the contrary, carbachol could enhance the phosphorylation level of Nedd4-2 as an alternative to SGK1, and thus rescue the ubiquitin-mediated degradation of hERG channels caused by probucol. These discoveries provide a novel mechanism of probucol-induced hERG-channel deficiency, and imply that carbachol or its analog may serve as potential therapeutic compounds for the handling of probucol cardiotoxicity.Keywords: long QT, hERG potassium channels, probucol, SGK1, Nedd4-2

  19. TRPC1 protects human SH-SY5Y cells against salsolinol-induced cytotoxicity by inhibiting apoptosis.

    Science.gov (United States)

    Bollimuntha, Sunitha; Ebadi, Manuchair; Singh, Brij B

    2006-07-12

    Salsolinol, an endogenous neurotoxin, may be involved in the pathogenesis of Parkinson's disease. In this study, we sought to determine whether salsolinol-induced cytotoxicity in SH-SY5Y human neuroblastoma cells, a cloned cell line which expresses dopaminergic activity, could be prevented by overexpressing a Ca(2+) channel, transient receptor potential (TRPC1) protein. Exposure of SH-SY5Y cells to 500 microM salsolinol for 12 h resulted in a significant decrease in thapsigargin or carbachol-mediated Ca(2+) influx. Consistent with these results, SH-SY5Y cells treated with salsolinol showed approximately 60% reduction in TRPC1 protein levels. Confocal microscopy also showed that SH-SY5Y cells treated with salsolinol had a significant decrease in the plasma membrane staining of the TRPC1 protein. Interestingly, overexpression of TRPC1 increases TRPC1 protein levels and also protected SH-SY5Y neuroblastoma cells against salsolinol-mediated cytotoxicity as determined by 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The protective effect of TRPC1 was blocked by the addition of TRPC1 blockers lanthanum, or 2APB. Activation of TRPC1 protein by either thapsigargin or carbachol further protected SH-SY5Y cells from salsolinol treatments. Staining of SH-SY5Y cells with an apoptotic marker (YO-PRO-1) showed that TRPC1 protein protects against apoptosis. Furthermore, TRPC1 overexpression also inhibited cytochrome c release and decreased BAX protein levels required for apoptosis. Taken together, these findings suggest that the reduction in cell surface TRPC1 protein expression in response to salsolinol may be a contributory factor in cellular toxicity of the dopaminergic neurons. Furthermore, overexpression of TRPC1 could inhibit apoptotic complex thereby increasing neuronal cell survivability in Parkinson's disease.

  20. Activation of angiotensin-converting enzyme 2 (ACE2) attenuates allergic airway inflammation in rat asthma model.

    Science.gov (United States)

    Dhawale, Vaibhav Shrirang; Amara, Venkateswara Rao; Karpe, Pinakin Arun; Malek, Vajir; Patel, Deep; Tikoo, Kulbhushan

    2016-09-01

    Angiotensin-I converting enzyme (ACE) is positively correlated to asthma, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS) and is highly expressed in lungs. ACE2, the counteracting enzyme of ACE, was proven to be protective in pulmonary, cardiovascular diseases. In the present study we checked the effect of ACE2 activation in animal model of asthma. Asthma was induced in male wistar rats by sensitization and challenge with ovalbumin and then treated with ACE2 activator, diminazene aceturate (DIZE) for 2weeks. 48h after last allergen challenge, animals were anesthetized, blood, BALF, femoral bone marrow lavage were collected for leucocyte count; trachea for measuring airway responsiveness to carbachol; lungs and heart were isolated for histological studies and western blotting. In our animal model, the characteristic features of asthma such as altered airway responsiveness to carbachol, eosinophilia and neutrophilia were observed. Western blotting revealed the increased pulmonary expression of ACE1, IL-1β, IL-4, NF-κB, BCL2, p-AKT, p-p38 and decreased expression of ACE2 and IκB. DIZE treatment prevented these alterations. Intraalveolar interstitial thickening, inflammatory cell infiltration, interstitial fibrosis, oxidative stress and right ventricular hypertrophy in asthma control animals were also reversed by DIZE treatment. Activation of ACE2 by DIZE conferred protection against asthma as evident from biochemical, functional, histological and molecular parameters. To the best of our knowledge, we report for the first time that activation of ACE2 by DIZE prevents asthma progression by altering AKT, p38, NF-κB and other inflammatory markers. PMID:27343405

  1. Autocrine Signaling Underlies Fast Repetitive Plasma Membrane Translocation of Conventional and Novel Protein Kinase C Isoforms in β Cells.

    Science.gov (United States)

    Wuttke, Anne; Yu, Qian; Tengholm, Anders

    2016-07-15

    PKC signaling has been implicated in the regulation of many cell functions, including metabolism, cell death, proliferation, and secretion. Activation of conventional and novel PKC isoforms is associated with their Ca(2+)- and/or diacylglycerol (DAG)-dependent translocation to the plasma membrane. In β cells, exocytosis of insulin granules evokes brief (<10 s) local DAG elevations ("spiking") at the plasma membrane because of autocrine activation of P2Y1 purinoceptors by ATP co-released with insulin. Using total internal reflection microscopy, fluorescent protein-tagged PKCs, and signaling biosensors, we investigated whether DAG spiking causes membrane recruitment of PKCs and whether different classes of PKCs show characteristic responses. Glucose stimulation of MIN6 cells triggered DAG spiking with concomitant repetitive translocation of the novel isoforms PKCδ, PKCϵ, and PKCη. The conventional PKCα, PKCβI, and PKCβII isoforms showed a more complex pattern with both rapid and slow translocation. K(+) depolarization-induced PKCϵ translocation entirely mirrored DAG spiking, whereas PKCβI translocation showed a sustained component, reflecting the subplasma membrane Ca(2+) concentration ([Ca(2+)]pm), with additional effect during DAG spikes. Interference with DAG spiking by purinoceptor inhibition prevented intermittent translocation of PKCs and reduced insulin secretion but did not affect [Ca(2+)]pm elevation or sustained PKCβI translocation. The muscarinic agonist carbachol induced pronounced transient PKCβI translocation and sustained recruitment of PKCϵ. When rise of [Ca(2+)]pm was prevented, the carbachol-induced DAG and PKCϵ responses were somewhat reduced, but PKCβI translocation was completely abolished. We conclude that exocytosis-induced DAG spikes efficiently recruit both conventional and novel PKCs to the β cell plasma membrane. PKC signaling is thus implicated in autocrine regulation of β cell function.

  2. Autocrine Signaling Underlies Fast Repetitive Plasma Membrane Translocation of Conventional and Novel Protein Kinase C Isoforms in β Cells*

    Science.gov (United States)

    Wuttke, Anne; Yu, Qian; Tengholm, Anders

    2016-01-01

    PKC signaling has been implicated in the regulation of many cell functions, including metabolism, cell death, proliferation, and secretion. Activation of conventional and novel PKC isoforms is associated with their Ca2+- and/or diacylglycerol (DAG)-dependent translocation to the plasma membrane. In β cells, exocytosis of insulin granules evokes brief (<10 s) local DAG elevations (“spiking”) at the plasma membrane because of autocrine activation of P2Y1 purinoceptors by ATP co-released with insulin. Using total internal reflection microscopy, fluorescent protein-tagged PKCs, and signaling biosensors, we investigated whether DAG spiking causes membrane recruitment of PKCs and whether different classes of PKCs show characteristic responses. Glucose stimulation of MIN6 cells triggered DAG spiking with concomitant repetitive translocation of the novel isoforms PKCδ, PKCϵ, and PKCη. The conventional PKCα, PKCβI, and PKCβII isoforms showed a more complex pattern with both rapid and slow translocation. K+ depolarization-induced PKCϵ translocation entirely mirrored DAG spiking, whereas PKCβI translocation showed a sustained component, reflecting the subplasma membrane Ca2+ concentration ([Ca2+]pm), with additional effect during DAG spikes. Interference with DAG spiking by purinoceptor inhibition prevented intermittent translocation of PKCs and reduced insulin secretion but did not affect [Ca2+]pm elevation or sustained PKCβI translocation. The muscarinic agonist carbachol induced pronounced transient PKCβI translocation and sustained recruitment of PKCϵ. When rise of [Ca2+]pm was prevented, the carbachol-induced DAG and PKCϵ responses were somewhat reduced, but PKCβI translocation was completely abolished. We conclude that exocytosis-induced DAG spikes efficiently recruit both conventional and novel PKCs to the β cell plasma membrane. PKC signaling is thus implicated in autocrine regulation of β cell function. PMID:27226533

  3. Electrophysiological Studies into the Safety of the Anti-diarrheal Drug Clotrimazole during Oral Rehydration Therapy.

    Directory of Open Access Journals (Sweden)

    Willem S Lexmond

    2015-09-01

    Full Text Available Morbidity and mortality from acute diarrheal disease remains high, particularly in developing countries and in cases of natural or man-made disasters. Previous work has shown that the small molecule clotrimazole inhibits intestinal Cl- secretion by blocking both cyclic nucleotide- and Ca(2+-gated K(+ channels, implicating its use in the treatment of diarrhea of diverse etiologies. Clotrimazole, however, might also inhibit transporters that mediate the inwardly directed electrochemical potential for Na(+-dependent solute absorption, which would undermine its clinical application. Here we test this possibility by examining the effects of clotrimazole on Na(+-coupled glucose uptake.Short-circuit currents (Isc following administration of glucose and secretagogues were studied in clotrimazole-treated jejunal sections of mouse intestine mounted in Ussing chambers.Treatment of small intestinal tissue with clotrimazole inhibited the Cl- secretory currents that resulted from challenge with the cAMP-agonist vasoactive intestinal peptide (VIP or Ca(2+-agonist carbachol in a dose-dependent fashion. A dose of 30 μM was effective in significantly reducing the Isc response to VIP and carbachol by 50% and 72%, respectively. At this dose, uptake of glucose was only marginally affected (decreased by 14%, p = 0.37. There was no measurable effect on SGLT1-mediated sugar transport, as uptake of SGLT1-restricted 3-O-methyl glucose was equivalent between clotrimazole-treated and untreated tissue (98% vs. 100%, p = 0.90.Treatment of intestinal tissue with clotrimazole significantly reduced secretory responses caused by both cAMP- and Ca(2+-dependent agonists as expected, but did not affect Na(+-coupled glucose absorption. Clotrimazole could thus be used in conjunction with oral rehydration solution as a low-cost, auxiliary treatment of acute secretory diarrheas.

  4. Functional antidopaminergic and anticholinergic effects of thioridazine and its metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Niedzwiecki, D.

    1986-01-01

    The relative potency of thioridazine and two of its clinically active metabolites, mesoridazine and sulforidazine was studied. In each of three separate methods, mesoridazine and sulforidazine exhibited greater potency than did thioridazine. Both metabolites showed greater affinities for striatal DA receptors as estimated by their competition for (/sup 3/H)spiperone binding sites in crude striatal membrane preparations. On a more functional level, both metabolites more potently antagonized the inhibitory effects of either the direct acting DA agonist apomorphine, or of endogenous DA, on the electrically evoked release of radiolabeled DA and ACh from perfused striatal slices. While thioridazine effectively blocked the agonist-induced inhibition at those striatal DA receptors which control DA release, it showed significantly lower potency at the striatal DA receptors which modulate ACh release. Thioridazine exhibited moderate affinity for striatal muscarinic cholinergic receptors. It was only five times less potent than atropine in competing for (/sup 3/H) quinuclidinylbenzilate binding sites in striatal membrane preparations. Unlike dopamine receptors, thioridazine showed greater antimuscarinic potency than did its metabolites. Despite this significant affinity for muscarinic binding sites in striatal homogenates, neither thioridezine nor its metabolites could block the inhibitory effects of the full muscarinic agonist carbachol, on the evoked release of ACh from striatal slices. This lack of effect contrasted with the antagonism of the carbachol-induced inhibition by such classical muscarinic blockers as quinuclindinylbenzilate or atropine. In combination with available pharmacokinetic data, these studies have demonstrated that the metabolites of thioridazine probably contribute to the antidopaminergic effects of this drug within the CNS.

  5. Functional differences in the acinar cells of the murine major salivary glands.

    Science.gov (United States)

    Kondo, Y; Nakamoto, T; Jaramillo, Y; Choi, S; Catalan, M A; Melvin, J E

    2015-05-01

    In humans, approximately 90% of saliva is secreted by the 3 major salivary glands: the parotid (PG), the submandibular (SMG), and the sublingual glands (SLG). Even though it is known that all 3 major salivary glands secrete saliva by a Cl(-)-dependent mechanism, salivary secretion rates differ greatly among these glands. The goal of this study was to gain insight into the properties of the ion-transporting pathways in acinar cells that might account for the differences among the major salivary glands. Pilocarpine-induced saliva was simultaneously collected in vivo from the 3 major salivary glands of mice. When normalized by gland weight, the amount of saliva secreted by the PG was more than 2-fold larger than that obtained from the SMG and SLG. At the cellular level, carbachol induced an increase in the intracellular [Ca(2+)] that was more than 2-fold larger in PG and SMG than in SLG acinar cells. Carbachol-stimulated Cl(-) efflux and the protein levels of the Ca(2+)-activated Cl(-) channel TMEM16A, the major apical Cl(-) efflux pathway in salivary acinar cells, were significantly greater in PG compared with SMG and SLG. In addition, we evaluated the transporter activity of the Na(+)-K(+)-2Cl(-) cotransporters (NKCC1) and anion exchangers (AE), the 2 primary basolateral Cl(-) uptake mechanisms in acinar cells. The SMG NKCC1 activity was about twice that of the PG and more than 12-fold greater than that of the SLG. AE activity was similar in PG and SLG, and both PG and SLG AE activity was about 2-fold larger than that of SMG. In summary, the salivation kinetics of the 3 major glands are distinct, and these differences can be explained by the unique functional properties of each gland related to Cl(-) movement, including the transporter activities of the Cl(-) uptake and efflux pathways, and intracellular Ca(2+) mobilization.

  6. Calorie restriction: A new therapeutic intervention for age-related dry eye disease in rats

    International Nuclear Information System (INIS)

    A decrease in lacrimal gland secretory function is closely related to aging and leads to an increased prevalence of dry eye syndrome. Since calorie restriction (CR) is considered to prevent functional decline of various organs due to aging, we hypothesized that CR could prevent age-related lacrimal dysfunction. Six-month-old male Fischer 344 rats were randomly divided into ad libitum (AL) and CR (-35%) groups. After 6 months of CR, tear function was examined under conscious state. After euthanasia, lacrimal glands were subjected to histological examination, tear protein secretion stimulation test with Carbachol, and assessment of oxidative stress with 8-hydroxy-2 deoxyguanosine (8-OHdG) and 4-hydroxynonenal (HNE) antibodies. CR significantly improved tear volume and tended to increase tear protein secretion volume after stimulation with Carbachol compared to AL. The acinar unit density was significantly higher in the CR rats compared to AL rats. Lacrimal glands in the CR rats showed a lesser degree of interstitial fibrosis. CR reduced the concentration of 8-OHdG and the extent of staining with HNE in the lacrimal gland, compared to AL. Furthermore, our electron microscopic observations showed that mitochondrial structure of the lacrimal gland obtained from the middle-aged CR rats was preserved in comparison to the AL rats. Collectively, these results demonstrate for the first time that CR may attenuate oxidative stress related damage in the lacrimal gland with preservation of lacrimal gland functions. Although molecular mechanism(s) by which CR maintains lacrimal gland function remains to be resolved, CR might provide a novel therapeutic strategy for treating dry eye syndrome.

  7. Histamine H1 receptor induces cytosolic calcium increase and aquaporin translocation in human salivary gland cells.

    Science.gov (United States)

    Kim, Ji-Hyun; Park, Seong-Hae; Moon, Young Wha; Hwang, Sungmin; Kim, Donghoon; Jo, Su-Hyun; Oh, Seog Bae; Kim, Joong Soo; Jahng, Jeong Won; Lee, Jong-Ho; Lee, Sung Joong; Choi, Se-Young; Park, Kyungpyo

    2009-08-01

    One of the common side effects of antihistamine medicines is xerostomia (dry mouth). The current consensus is that antihistamine-induced xerostomia comes from an antimuscarinic effect. Although the effect of antihistamines on salivary secretion is both obvious and significant, the cellular mechanism whereby this happens is still unclear because of the lack of knowledge of histamine signaling in human salivary glands. Here, we have studied histamine receptors and the effect of antihistamines on human submandibular acinar cells. In primary cultured human submandibular gland and a HSG cell line, histamine increased the intracellular Ca(2+) concentration. The histamine-induced cytosolic free Ca(2+) concentration ([Ca(2+)](i)) increase was inhibited by histamine H1 receptor-specific antagonists, and the expression of the functional histamine H1 receptor was confirmed by reverse transcription-polymerase chain reaction. Interestingly, histamine pretreatment did not inhibit a subsequent carbachol-induced [Ca(2+)](i) rise without "heterologous desensitization." Chlorpheniramine inhibited a carbachol-induced [Ca(2+)](i) increase at a 100-fold greater concentration than histamine receptor antagonism, whereas astemizole and cetrizine showed more than 1000-fold difference, which in part explains the xerostomia-inducing potency among the antihistamines. Notably, histamine resulted in translocation of aquaporin-5 to the plasma membrane in human submandibular gland cells and green fluorescent protein-tagged aquaporin-5 expressing HSG cells. We found that histidine decarboxylase and the histamine H1 receptor are broadly distributed in submandibular gland cells, whereas choline acetyltransferase is localized only at the parasympathetic terminals. Our results suggest that human salivary gland cells express histamine H1 receptors and histamine-synthesizing enzymes, revealing the cellular mechanism of antihistamine-induced xerostomia. PMID:19443731

  8. Pretreatment with remifentanil protects against the reduced-intestinal contractility related to the ischemia and reperfusion injury in rat

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    Hale Sayan-Ozacmak

    2015-12-01

    Full Text Available BACKGROUND AND OBJECTIVES: Serious functional and structural alterations of gastrointestinal tract are observed in failure of blood supply, leading to gastrointestinal dismotility. Activation of opioid receptors provides cardioprotective effect against ischemia-reperfusion (I/R injury. The aim of the present study was to determine whether or not remifentanil could reduce I/R injury of small intestine. METHODS: Male Wistar Albino rats were subjected to mesenteric ischemia (30 min followed by reperfusion (3 h. Four groups were designed: sham control; remifentanil alone; I/R control; and remifentanil + I/R. Animals in remifentanil + I/R group were subjected to infusion of remifentanil (2 ug kg-1 min-1 for 60 min, half of which started before inducing ischemia. Collecting the ileum tissues, evaluation of damage was based on contractile responses to carbachol, levels of lipid peroxidation and neutrophil infiltration, and observation of histopathological features in intestinal tissue. RESULTS: Following reperfusion, a significant decrease in carbachol-induced contractile response, a remarkable increase in both lipid peroxidation and neutrophil infiltration, and a significant injury in mucosa were observed. An average contractile response of remifentanil + I/R group was significantly different from that of the I/R group. Lipid peroxidation and neutrophil infiltration were also significantly suppressed by the treatment. The tissue samples of the I/R group were grade 4 in histopathological evaluation. In remifentanil + I/R group, on the other hand, the mucosal damage was moderate, staging as grade 1. CONCLUSIONS: The pretreatment with remifentanil can attenuate the intestinal I/R injury at a remarkable degree possibly by lowering lipid peroxidation and leukocyte infiltration.

  9. Effects of age and zinc supplementation on transport properties in the jejunum of piglets.

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    Gefeller, E M; Martens, H; Aschenbach, J R; Klingspor, S; Twardziok, S; Wrede, P; Pieper, R; Lodemann, U

    2015-06-01

    Zinc is effective in the prevention and treatment of post-weaning diarrhoea and in promoting piglet growth. Its effects on the absorption of nutrients and the secretory capacity of the intestinal epithelium are controversial. We investigated the effects of age, dietary pharmacological zinc supplementation and acute zinc exposure in vitro on small-intestinal transport properties of weaned piglets. We further examined whether the effect of zinc on secretory responses depended on the pathway by which chloride secretion is activated. A total of 96 piglets were weaned at 26 days of age and allocated to diets containing three different levels of zinc oxide (50, 150 and 2500 ppm). At the age of 32, 39, 46 and 53 days, piglets were killed, and isolated epithelia from the mid-jejunum were used for intestinal transport studies in conventional Ussing chambers, with 23 μm ZnSO4 being added to the serosal side for testing acute effects. Absorptive transport was stimulated by mucosal addition of d-glucose or l-glutamine. Secretion was activated by serosal addition of prostaglandin E2 , carbachol or by mucosal application of Escherichia coli heat-stable enterotoxin (Stp ). Jejunal transport properties showed significant age-dependent alterations (p < 0.03). Both absorptive and secretory responses were highest in the youngest piglets (32 d). The dietary zinc supplementation had no significant influence on jejunal absorptive and secretory responses. However, the pre-treatment of epithelia with ZnSO4 in vitro led to a small but significant decrease in both absorptive and secretory capacities (p < 0.05), with an exception for carbachol (p = 0.07). The results showed that, in piglets, chronic supplementation with zinc did not sustainably influence the jejunal transport properties in the post-weaning phase. Because transport properties are influenced by the addition of zinc in vitro, we suggest that possible epithelial effects of zinc depend on the acute presence of this ion. PMID

  10. Feed supplemented with organic acids does not affect starch digestibility, nor intestinal absorptive or secretory function in broiler chickens.

    Science.gov (United States)

    Ruhnke, I; Röhe, I; Goodarzi Boroojeni, F; Knorr, F; Mader, A; Hafeez, A; Zentek, J

    2015-04-01

    The current study aimed to determine the impact of acidified feed on apparent ileal starch digestibility, intestinal transport and barrier function and intestinal glucose transporter expression. The experiment included a control group and a treatment group with broilers fed a standard diet without or with 1.5% of a commercial organic acid product (64% formic acid, 25% propionic acid, 11% water). Broilers were fed with the experimental diets from hatching until days 32-35. Starch digestibility was determined using 0.2% titanium dioxide as ingestible marker. Gene expressions of the intestinal sodium glucose transporter 1 (SGLT-1) and glucose transporter 2 (GLUT-2) were analysed using qPCR analysis. Additionally, SGLT-1 function and chloride secretion were analysed in Ussing chamber experiments. Jejunal samples were sequentially exposed to 10 mm glucose, 100 μm phloridzin, 100 μm histamine and 100 μm carbachol. Apparent ileal starch digestibility (±SEM) of the control group (97.5 ± 0.35%) and the acid-treated group (97.0 ± 0.59%) did not differ (p = 0.674). The mean tissue conductance of intestinal samples obtained from the control group and the treatment group was similar [10.6 mS/cm(2) (±0.68) and 9.4 mS/cm(2) (±0.80) respectively (p = 0.147)]. The mean short-circuit currents (ΔIsc ) of the samples exposed to glucose, phloridzin, histamine and carbachol did not differ (p > 0.05). Additionally, no differences in the expression of SGLT-1 and GLUT-2 could be observed (p = 0.942, p = 0.413). Based on this study, the consumption of feed supplemented with organic acids was not associated with effects on ileal starch digestibility and functional traits of jejunal tissues, indicating that these additives have no major impact on the small intestinal function in broilers. PMID:25865420

  11. Electrophysiological Studies into the Safety of the Anti-diarrheal Drug Clotrimazole during Oral Rehydration Therapy

    Science.gov (United States)

    Lexmond, Willem S.; Rufo, Paul A.; Fiebiger, Edda; Lencer, Wayne I.

    2015-01-01

    Background and Aims Morbidity and mortality from acute diarrheal disease remains high, particularly in developing countries and in cases of natural or man-made disasters. Previous work has shown that the small molecule clotrimazole inhibits intestinal Cl- secretion by blocking both cyclic nucleotide- and Ca2+-gated K+ channels, implicating its use in the treatment of diarrhea of diverse etiologies. Clotrimazole, however, might also inhibit transporters that mediate the inwardly directed electrochemical potential for Na+-dependent solute absorption, which would undermine its clinical application. Here we test this possibility by examining the effects of clotrimazole on Na+-coupled glucose uptake. Materials and Methods Short-circuit currents (Isc) following administration of glucose and secretagogues were studied in clotrimazole-treated jejunal sections of mouse intestine mounted in Ussing chambers. Results Treatment of small intestinal tissue with clotrimazole inhibited the Cl- secretory currents that resulted from challenge with the cAMP-agonist vasoactive intestinal peptide (VIP) or Ca2+-agonist carbachol in a dose-dependent fashion. A dose of 30 μM was effective in significantly reducing the Isc response to VIP and carbachol by 50% and 72%, respectively. At this dose, uptake of glucose was only marginally affected (decreased by 14%, p = 0.37). There was no measurable effect on SGLT1-mediated sugar transport, as uptake of SGLT1-restricted 3-O-methyl glucose was equivalent between clotrimazole-treated and untreated tissue (98% vs. 100%, p = 0.90). Conclusion Treatment of intestinal tissue with clotrimazole significantly reduced secretory responses caused by both cAMP- and Ca2+-dependent agonists as expected, but did not affect Na+-coupled glucose absorption. Clotrimazole could thus be used in conjunction with oral rehydration solution as a low-cost, auxiliary treatment of acute secretory diarrheas. PMID:26405813

  12. Mechanism of antiulcerogenic activity of semi-synthetic crotonin obtained from Croton cajucara Benth.

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    Ana Beatriz A. Almeida

    2002-01-01

    Full Text Available The bark of Croton cajucara Benth. is used in Brazilian folk medicine to treat gastrointestinal disorders. Transdehydrocrotonin (DHC isolated from the bark of Croton cajucara has antiulcerogenic activity25. The presence of similar activity in semi-synthetic crotonin obtained from dehydrocrotonin from Croton cajucara was observed in gastric ulcer-induced models (HCl/ethanol, ethanol, indomethacin, stress and pylorus ligature. The aim of the present study was to assess the mechanisms involved in the antiulcerogenic activity of semi-synthetic crotonin. We investigated the effects of semi-synthetic crotonin on the response to histamine of right atria isolated from guinea pigs and on the response to carbachol of stomach fundus strips from rats. Semi-synthetic crotonin (3, 10 or 30 mM induced a shift to the right in the concentrationresponse curves to carbachol in the isolated rat stomach at the pD2 level (pD2: 5.42±0.05, 5.76±0.061, 5.77±0.076, 6.48±0.012, respectively, without any alteration in the maximum response. Semi-synthetic crotonin also induced a shift to the right in the concentration-response curves to histamine in guinea pig right atria, pD2 (5.54±0.06, 6.01±0.06, 5.89±0.06, 5.92±0.03 and (% maximum response (80±6.18, 118±6.18, 114±6.18, 122±1.4, respectively. Thus, the protective effect of semi-synthetic crotonin on induced gastric lesions could be due to antagonism of histaminergic and cholinergic effects on gastric secretion.

  13. Oscillatory pressure transients after flow interruption during bronchial challenge test in children.

    Science.gov (United States)

    Frey, U; Kraemer, R

    1997-01-01

    The measurement of the conventional interrupter resistance (Rint) is dependent on pressure equilibration between alveolar and airway opening pressure, which is often not achieved in the presence of severe airways obstruction or in small children. The damping properties (d) of postocclusional oscillatory pressure transients after rapid flow interrruption can be assessed independent of complete pressure equilibration, and we have previously shown them to be correlated to resistive properties of the respiratory system. We wanted to determine whether these transients were an expression of acoustic properties of the air in the airways, or whether they were caused by an interaction of gas and lung tissue, and whether d was more sensitive than Rint to changes in airway mechanics. Bronchial challenge tests were carried out with cumulative doses of inhaled carbachol in 10 healthy children (aged 7-14 yrs) and 50 asthmatic children (aged 5-15 yrs). The airflow interruptions were performed with a combined nebulizer-shutter head, allowing resistance measurements with each breath. The frequency, and the damping factor of the postocclusional pressure transients changed significantly during carbachol challenge in both groups of children. The provocation dose (PD) at which the damping factor (d) of the oscillatory pressure transients increased more than 2 SD above the baseline mean ("variance-based", PDvb) was lower than the PDvb of the end-interruption resistance (Rint, EI). These changes in frequency and damping factor were reversible after inhaling salbutamol. These findings suggest that the damping properties of the postocclusional pressure transients after flow interruption can be used as a sensitive parameter to assess changes in airway mechanics during bronchial challenge test in children in whom pressure equilibration is frequently not achieved during airflow interruption due to airways obstruction. PMID:9032496

  14. Calorie restriction: A new therapeutic intervention for age-related dry eye disease in rats

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    Kawashima, Motoko; Kawakita, Tetsuya; Okada, Naoko; Ogawa, Yoko [Department of Ophthalmology, Keio University School of Medicine, Tokyo (Japan); Murat, Dogru [Department of Ocular Surface and Visual Optics, Keio University School of Medicine, Tokyo (Japan); Nakamura, Shigeru; Nakashima, Hideo [Research Center, Ophtecs Corporation, Hyogo (Japan); Shimmura, Shigeto [Department of Ophthalmology, Keio University School of Medicine, Tokyo (Japan); Shinmura, Ken [Division of Geriatric Medicine, Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Tsubota, Kazuo, E-mail: tsubota@sc.itc.keio.ac.jp [Department of Ophthalmology, Keio University School of Medicine, Tokyo (Japan)

    2010-07-09

    A decrease in lacrimal gland secretory function is closely related to aging and leads to an increased prevalence of dry eye syndrome. Since calorie restriction (CR) is considered to prevent functional decline of various organs due to aging, we hypothesized that CR could prevent age-related lacrimal dysfunction. Six-month-old male Fischer 344 rats were randomly divided into ad libitum (AL) and CR (-35%) groups. After 6 months of CR, tear function was examined under conscious state. After euthanasia, lacrimal glands were subjected to histological examination, tear protein secretion stimulation test with Carbachol, and assessment of oxidative stress with 8-hydroxy-2 deoxyguanosine (8-OHdG) and 4-hydroxynonenal (HNE) antibodies. CR significantly improved tear volume and tended to increase tear protein secretion volume after stimulation with Carbachol compared to AL. The acinar unit density was significantly higher in the CR rats compared to AL rats. Lacrimal glands in the CR rats showed a lesser degree of interstitial fibrosis. CR reduced the concentration of 8-OHdG and the extent of staining with HNE in the lacrimal gland, compared to AL. Furthermore, our electron microscopic observations showed that mitochondrial structure of the lacrimal gland obtained from the middle-aged CR rats was preserved in comparison to the AL rats. Collectively, these results demonstrate for the first time that CR may attenuate oxidative stress related damage in the lacrimal gland with preservation of lacrimal gland functions. Although molecular mechanism(s) by which CR maintains lacrimal gland function remains to be resolved, CR might provide a novel therapeutic strategy for treating dry eye syndrome.

  15. Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion.

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    Yanan Hou

    Full Text Available The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1. Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.

  16. Tracheal relaxation of five medicinal plants used in Mexico for the treatment of several diseases

    Institute of Scientific and Technical Information of China (English)

    Amanda Snchez-Recillas; Paul Mantecn-Reyes; Patricia Castillo-Espaa; Rafael Villalobos-Molina; Maximiliano Ibarra-Barajas; Samuel Estrada-Soto

    2014-01-01

    Objective:To assess the relaxant effect of several organic extracts obtained fromAgastache mexicana(A. mexicana),Cochlospermum vitifolium(C. vitifolium),Cordia morelosana(C. morelosana),Lepechinia caulescens(L. caulescens)andTalauma mexicana(T. mexicana) used inMexican traditional medicine for the treatment of several diseases.Methods:Extracts were obtained by maceration at room temperature using hexane, dichloromethane and methanol for each plant material.The organic extracts were evaluatedex vivo to determine their relaxant activity on the contractions induced by carbachol(cholinergic receptor agonist,1 μmol/L) in isolated rat tracheal rings.Results:A total of15 extracts were evaluated(three for each species).All test samples showed significant relaxant effect, in a concentration-dependent manner, on the contractions induced by1 μmol/L carbachol, with exception of extracts fromC. morelosana.Active extracts were less potent than theophylline [phosphodiesterase inhibitor,EC50:(28.79±0.82) μg/mL] that was used as positive control.Concentration-response curves revealed that the extracts with more significant effects were dichloromethanic extracts ofT. mexicana [Emax:(103.03±3.32)% andEC50:(159.39±3.72) μg/mL) andC. vitifolium [Emax:(106.58±2.42)% andEC50:(219.54±7.61) μg/mL].Finally, hexanic and dichloromethanic extracts fromA. mexicana were fully effective but less potent than T. mexicana andC. vitifolium.Conclusions:Less polar extracts obtained fromA. mexicana,T. mexicana and C. vitifolium exhibited greater relaxant effect on tracheal rat rings, which allows us to suggest them as sources for the isolation of bioactive molecules with potential therapeutic value in the treatment of asthma.

  17. Inhibition of intermediary metabolism by amiodarone in dog thyroid slices

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    Pasquali, D.; Tseng, F.Y.; Rani, C.S.; Field, J.B. (Baylor College of Medicine, Houston, TX (USA))

    1990-10-01

    Amiodarone, an iodine-containing antiarrhythmic drug, has been reported to interfere with thyroid function and thyroid hormone metabolism. We studied the effects of amiodarone on basal and agonist (thyroid-stimulating hormone (TSH), phorbol ester, or carbachol)-stimulated glucose oxidation, 32PO4 incorporation into phospholipids, and adenosine 3',5'-cyclic monophosphate (cAMP) concentration in dog thyroid slices. Slices were preincubated with amiodarone at 37 degrees C for 1 h before the addition of agonist and the appropriate radioisotope. cAMP stimulation was measured after 20 min, glucose oxidation for 45 min, and 32PO4 incorporation into phospholipids for 2 h. Amiodarone (0.5 mM) had no effect on basal 14CO2 formation or 32PO4 incorporation into phospholipids but significantly inhibited TSH, phorbol ester, and carbachol stimulation of these parameters. It also inhibited cAMP stimulation by TSH. Inhibition of TSH-stimulated (14C)glucose oxidation was also obtained with another iodide-containing compound, iopanoic acid (0.5 mM), but not with iothalamate (up to 10 mM). Inhibition by amiodarone was still present, but to a lesser extent, when it was added at the same time as the agonist. Inhibition of stimulated (14C)glucose oxidation persisted even after the slices were incubated without amiodarone for 6 h. Inhibition by amiodarone, in contrast to that by inorganic iodide, was not prevented by 1 mM methimazole added at the same time as amiodarone. These results indicate that the inhibitory effects of amiodarone on thyroid function are not due to dissociation of iodide from the molecule.

  18. Association of overactive bladder and stress urinary incontinence in rats with pudendal nerve ligation injury.

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    Furuta, Akira; Kita, Masafumi; Suzuki, Yasuyuki; Egawa, Shin; Chancellor, Michael B; de Groat, William C; Yoshimura, Naoki

    2008-05-01

    Approximately one-third of patients with stress urinary incontinence (SUI) also suffer from urgency incontinence, which is one of the major symptoms of overactive bladder (OAB) syndrome. Pudendal nerve injury has been recognized as a possible cause for both SUI and OAB. Therefore, we investigated the effects of pudendal nerve ligation (PNL) on bladder function and urinary continence in female Sprague-Dawley rats. Conscious cystometry with or without capsaicin pretreatment (125 mg/kg sc), leak point pressures (LPPs), contractile responses of bladder muscle strips to carbachol or phenylephrine, and levels of nerve growth factor (NGF) protein and mRNA in the bladder were compared in sham and PNL rats 4 wk after the injury. Urinary frequency detected by a reduction in intercontraction intervals and voided volume was observed in PNL rats compared with sham rats, but it was not seen in PNL rats with capsaicin pretreatment that desensitizes C-fiber-afferent pathways. LPPs in PNL rats were significantly decreased compared with sham rats. The contractile responses of detrusor muscle strips to phenylephrine, but not to carbachol, were significantly increased in PNL rats. The levels of NGF protein and mRNA in the bladder of PNL rats were significantly increased compared with sham rats. These results suggest that pudendal nerve neuropathy induced by PNL may be one of the potential risk factors for OAB, as well as SUI. Somato-visceral cross sensitization between somatic (pudendal) and visceral (bladder) sensory pathways that increases NGF expression and alpha(1)-adrenoceptor-mediated contractility in the bladder may be involved in this pathophysiological mechanism.

  19. Isolation and Pharmacological Characterization of α-Elapitoxin-Ot1a, a Short-Chain Postsynaptic Neurotoxin from the Venom of the Western Desert Taipan, Oxyuranus temporalis

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    Carmel M. Barber

    2016-02-01

    Full Text Available Taipans (Oxyuranus spp. are elapids with highly potent venoms containing presynaptic (β and postsynaptic (α neurotoxins. O. temporalis (Western Desert taipan, a newly discovered member of this genus, has been shown to possess venom which displays marked in vitro neurotoxicity. No components have been isolated from this venom. We describe the characterization of α-elapitoxin-Ot1a (α-EPTX-Ot1a; 6712 Da, a short-chain postsynaptic neurotoxin, which accounts for approximately 30% of O. temporalis venom. α-Elapitoxin-Ot1a (0.1–1 µM produced concentration-dependent inhibition of indirect-twitches, and abolished contractile responses to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The inhibition of indirect twitches by α-elapitoxin-Ot1a (1 µM was not reversed by washing the tissue. Prior addition of taipan antivenom (10 U/mL delayed the neurotoxic effects of α-elapitoxin-Ot1a (1 µM and markedly attenuated the neurotoxic effects of α-elapitoxin-Ot1a (0.1 µM. α-Elapitoxin-Ot1a displayed pseudo-irreversible antagonism of concentration-response curves to carbachol with a pA2 value of 8.02 ± 0.05. De novo sequencing revealed the main sequence of the short-chain postsynaptic neurotoxin (i.e., α-elapitoxin-Ot1a as well as three other isoforms found in O. temporalis venom. α-Elapitoxin-Ot1a shows high sequence similarity (i.e., >87% with other taipan short-chain postsynaptic neurotoxins.

  20. Inhibition of allergic airway responses by heparin derived oligosaccharides: identification of a tetrasaccharide sequence

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    Ahmed Tahir

    2012-01-01

    Full Text Available Abstract Background Previous studies showed that heparin's anti-allergic activity is molecular weight dependent and resides in oligosaccharide fractions of Objective To investigate the structural sequence of heparin's anti-allergic domain, we used nitrous acid depolymerization of porcine heparin to prepare an oligosaccharide, and then fractionated it into disaccharide, tetrasaccharide, hexasaccharide, and octasaccharide fractions. The anti-allergic activity of each oligosaccharide fraction was tested in allergic sheep. Methods Allergic sheep without (acute responder and with late airway responses (LAR; dual responder were challenged with Ascaris suum antigen with and without inhaled oligosaccharide pretreatment and the effects on specific lung resistance and airway hyperresponsiveness (AHR to carbachol determined. Additional inflammatory cell recruitment studies were performed in immunized ovalbumin-challenged BALB/C mice with and without treatment. Results The inhaled tetrasaccharide fraction was the minimal effective chain length to show anti-allergic activity. This fraction showed activity in both groups of sheep; it was also effective in inhibiting LAR and AHR, when administered after the antigen challenge. Tetrasaccharide failed to modify the bronchoconstrictor responses to airway smooth muscle agonists (histamine, carbachol and LTD4, and had no effect on antigen-induced histamine release in bronchoalveolar lavage fluid in sheep. In mice, inhaled tetrasaccharide also attenuated the ovalbumin-induced peribronchial inflammatory response and eosinophil influx in the bronchoalveolar lavage fluid. Chemical analysis identified the active structure to be a pentasulfated tetrasaccharide ([IdoU2S (1→4GlcNS6S (1→4 IdoU2S (1→4 AMan-6S] which lacked anti-coagulant activity. Conclusions These results demonstrate that heparin tetrasaccharide possesses potent anti-allergic and anti-inflammatory properties, and that the domains responsible for anti

  1. Rapid effects of phytoestrogens on human colonic smooth muscle are mediated by oestrogen receptor beta.

    LENUS (Irish Health Repository)

    Hogan, A M

    2012-02-01

    Epidemiological studies have correlated consumption of dietary phytoestrogens with beneficial effects on colon, breast and prostate cancers. Genomic and non-genomic mechanisms are responsible for anti-carcinogenic effects but, until now, the effect on human colon was assumed to be passive and remote. No direct effect on human colonic smooth muscle has previously been described. Institutional research board approval was granted. Histologically normal colon was obtained from the proximal resection margin of colorectal carcinoma specimens. Circular smooth muscle strips were microdissected and suspended under 1g of tension in organ baths containing oxygenated Krebs solution at 37 degrees C. After an equilibration period, tissues were exposed to diarylpropionitrile (DPN) (ER beta agonist) and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) (ER alpha agonist) or to the synthetic phytoestrogen compounds genistein (n=8), daidzein (n=8), fisetin (n=8) and quercetin (n=8) in the presence or absence of fulvestrant (oestrogen receptor antagonist). Mechanism of action was investigated by inhibition of downstream pathways. The cholinergic agonist carbachol was used to induce contractile activity. Tension was recorded isometrically. Phytoestrogens inhibit carbachol-induced colonic contractility. In keeping with a non-genomic, rapid onset direct action, the effect was within minutes, reversible and similar to previously described actions of 17 beta oestradiol. No effect was seen in the presence of fulvestrant indicating receptor modulation. While the DPN exerted inhibitory effects, PPT did not. The effect appears to be reliant on a p38\\/mitogen activated protein kinase mediated induction of nitric oxide production in colonic smooth muscle. The present data set provides the first description of a direct effect of genistein, daidzein, fisetin and quercetin on human colonic smooth muscle. The presence of ER in colonic smooth muscle has been functionally proven and the beta

  2. Role of PKC and CaV1.2 in detrusor overactivity in a model of obesity associated with insulin resistance in mice.

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    Luiz O Leiria

    Full Text Available Obesity/metabolic syndrome are common risk factors for overactive bladder. This study aimed to investigate the functional and molecular changes of detrusor smooth muscle (DSM in high-fat insulin resistant obese mice, focusing on the role of protein kinase C (PKC and Ca(v1.2 in causing bladder dysfunction. Male C57BL/6 mice were fed with high-fat diet for 10 weeks. In vitro functional responses and cystometry, as well as PKC and Ca(v1.2 expression in bladder were evaluated. Obese mice exhibited higher body weight, epididymal fat mass, fasting glucose and insulin resistance. Carbachol (0.001-100 µM, α,β-methylene ATP (1-10 µM, KCl (1-300 mM, extracellular Ca(2+ (0.01-100 mM and phorbol-12,13-dibutyrate (PDBu; 0.001-3 µM all produced greater DSM contractions in obese mice, which were fully reversed by the Ca(v1.2 blocker amlodipine. Cystometry evidenced augmented frequency, non-void contractions and post-void pressure in obese mice that were also prevented by amlodipine. Metformin treatment improved the insulin sensitivity, and normalized the in vitro bladder hypercontractility and cystometric dysfunction in obese mice. The PKC inhibitor GF109203X (1 µM also reduced the carbachol induced contractions. PKC protein expression was markedly higher in bladder tissues from obese mice, which was normalized by metformin treatment. The Ca(v1.2 channel protein expression was not modified in any experimental group. Our findings show that Ca(v1.2 blockade and improvement of insulin sensitization restores the enhanced PKC protein expression in bladder tissues and normalizes the overactive detrusor. It is likely that insulin resistance importantly contributes for the pathophysiology of this urological disorder in obese mice.

  3. Gastric motor effects of ghrelin and growth hormone releasing peptide 6 in diabetic mice with gastroparesis

    Institute of Scientific and Technical Information of China (English)

    Wen-Cai Qiu; Zhi-Gang Wang; Wei-Gang Wang; Jun Yan; Qi Zheng

    2008-01-01

    AIM:To investigate the potential therapeutic significance of ghrelin and growth hormone releasing peptide 6(GHRP-6) in diabetic mice with gastric motility disorders.METHODS:A diabetic mouse model was established by intraperitoneal (ip) injection of alloxan.Diabetic mice were injected ip with ghrelin or GHRP-6 (20-200 μg/kg),and the effects on gastric emptying were measured after intragastric application of phenol red.The effect of atropine,NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) or D-Lys3-GHRP-6 (a growth hormone secretagogue receptor (GHS-R) antagonist) on the gastroprokinetic effect of ghrelin or GHRP-6 (100 μg/kg)was also investigated.The effects of ghrelin or GHRP-6(0.01-10 μmol/L) on spontaneous or carbachol-induced contractile amplitude were also investigated in vitro,in gastric fundic circular strips taken from diabetic mice.The presence of growth hormone secretagogue receptor la transcripts in the fundic strips of diabetic mice was detected by reverse transcriptase polymerase chain reaction (RT-PCR).RESULTS:We established a diabetic mouse model with delayed gastric emptying.Ghrelin and GHRP-6accelerated gastric emptying in diabetic mice with gastroparesis.In the presence of atropine or L-NAME,which delayed gastric emptying,ghrelin and GHRP-6(100 μg/kg) failed to accelerate gastric emptying.D-Lys3-GHRP-6 also delayed gastric emptying induced by the GHS-R agonist.Ghrelin and GHRP-6 increased the carbachol-induced contractile amplitude in gastric fundic strips taken from diabetic mice.RT-PCR confirmed the presence of GHS-R mRNA in the strip preparations.CONCLUSION:Ghrelin and GHRP-6 increase gastric emptying in diabetic mice with gastroparesis,perhaps by activating peripheral cholinergic pathways in the enteric nervous system.

  4. Effect of different bronchodilators on airway smooth muscle responsiveness to contractile agents.

    Science.gov (United States)

    Gustafsson, B; Persson, C G

    1991-05-01

    "Functional antagonism" is often used to describe the general relaxant effect of beta 2 agonists and xanthines and their ability to protect the airways against bronchoconstrictor stimuli. This study in guinea pig isolated trachea addresses the question of whether the capacity of these drugs to protect against constrictor stimuli is related to smooth muscle relaxation. Three antimuscarinic drugs were also examined to determine whether antagonism of mediators other than muscarinic agonists might contribute to bronchodilatation by these antimuscarinic drugs. Terbutaline (1.1 x 10(-7), 2.2 x 10(-7) M), theophylline (2.2 x 10(-4), 4.4 x 10(-4) M), and enprofylline (5.2 x 10(-5), 1.0 x 10(-4) M) relaxed the tracheal tension that remained after indomethacin treatment. They did not, however, alter the carbachol concentration-response curve significantly. In addition, neither theophylline (2.2 x 10(-4) M) nor terbutaline (1.1 x 10(-7) M) altered histamine induced contraction. Atropine sulphate, glycopyrrolate, and ipratropium bromide had EC50 values of 10(-9) - 10(-8) M for relaxation of carbachol induced contractions, whereas concentrations of 10(-6) - 10(-3) M or greater were required to relax contractions induced by allergen and nine other non-muscarinic mediators. It is suggested that bronchodilatation by antimuscarinic drugs in vivo is due to inhibition of acetylcholine induced bronchoconstriction alone and that beta 2 agonists and xanthines have poor ability to protect airway smooth muscle against constrictor stimuli. Hence mechanisms other than bronchodilatation and "functional antagonism" should be considered to explain the protection against constrictor stimuli in asthma seen with beta 2 agonists and xanthines. PMID:2068693

  5. Activity of sap from Croton lechleri on rat vascular and gastric smooth muscles.

    Science.gov (United States)

    Froldi, G; Zagotto, G; Filippini, R; Montopoli, M; Dorigo, P; Caparrotta, L

    2009-08-01

    The effects of red sap from Croton lechleri (SdD), Euphorbiaceae, on vascular and gastric smooth muscles were investigated. SdD, from 10 to 1000 microg/ml, induced concentration-dependent vasoconstriction in rat caudal arteries, which was endothelium-independent. In arterial preparations pre-constricted by phenylephrine (0.1 microM) or KCl (30 mM), SdD also produced concentration-dependent vasoconstriction. To study the mechanisms implicated in this effect we used selective inhibitors such as prazosin (0.1 microM), an antagonist of alpha(1)-adrenoceptors, atropine (0.1 microM), an antagonist of muscarinic receptors, and ritanserin (50 nM), a 5-HT(2A) antagonist; none of these influenced vasoconstriction caused by SdD. Likewise, nifedipine (50 nM), an inhibitor of L-type calcium channels, did not modify the action of SdD. Capsaicin (100 nM), an agonist of vanilloid receptors, also did not affect vasoconstriction by SdD. We also investigated the action of SdD (10-1000 microg/ml) on rat gastric fundus; per se the sap slightly increased contractile tension. When the gastric fundus was pre-treated with SdD (100 microg/ml) the contraction induced by carbachol (1 microM) was increased, whereas that by KCl (60mM) or capsaicin (100 nM) were unchanged. The data shows that SdD increased contractile tension in a concentration-dependent way, both on vascular and gastric smooth muscles. The vasoconstriction is unrelated to alpha(1), M, 5-HT(2A) and vanilloid receptors as well as L-type calcium channels. SdD increased also contraction by carbachol on rat gastric fundus. Thus for the first time, experimental data provides evidence that sap from C. lechleri owns constricting activity on smooth muscles. PMID:19406630

  6. 内毒素耐受和拟胆碱药物对巨噬细胞分泌肿瘤坏死因子-α的影响%Effects of endotoxin tolerance;and;cholinergic drug on macrophages in secreting tumor necrosis factor-α

    Institute of Scientific and Technical Information of China (English)

    吕艺; 周国勇; 胡森

    2011-01-01

    目的:探讨内毒素耐受和拟胆碱药物卡巴胆碱对巨噬细胞肿瘤坏死因子-α(TNF-α)分泌的调节作用.方法:①分离小鼠腹腔巨噬细胞,将其与内毒素(LPS)作用不同时间(0、2、4、8 h);②小鼠腹腔巨噬细胞分为3组:空白对照组(RPMI1640常规培养),LPS耐受组(LPS预刺激20 h,再用LPS刺激4 h)和LPS不耐受组(RPMI1640常规培养20 h后,再加LPS刺激4 h);③分离大鼠腹腔巨噬细胞,分为3组:空白对照组、LPS刺激组及卡巴胆碱+LPS刺激组(卡巴胆碱20 μmol/L预处理后LPS刺激).各组LPS的剂量均为100 ng/ml.收集各组细胞培养上清液及细胞裂解液,用酶联免疫分析(ELISA)方法检测TNF-α浓度.结果:LPS预刺激巨噬细胞20 h后再次用LPS刺激时,其TNF-α分泌量不增加,与空白对照组接近,同时细胞内TNF-α含量也不增加.未经LPS预刺激的巨噬细胞在受到LPS刺激后TNF-α分泌量大幅增加,同时细胞内TNF-α含量也伴随增加,即细胞分泌和贮存的TNF-α量的变化一致.卡巴胆碱预处理巨噬细胞受内毒素刺激后TNF-α分泌量低于单纯LPS刺激组细胞,但细胞内的TNF-α含量明显高于空白对照组(P<001),与单纯LPS刺激组接近.结论:巨噬细胞建立LPS耐受机制或受到拟胆碱药物作用后,再次受到LPS刺激后均表现为TNF-α分泌的减少,但细胞内TNF-α量变化规律不一致.提示内毒素耐受和胆碱能抗炎所致巨噬细胞炎症反应特性改变的机制不同.%Objective:To investigate the difference in regulating tumor necrosis factor-α (TNF-α) secretion between endotoxin tolerance and cholinergic drug-carbachol pre-treated macrophages. Methods: ①The peritoneal macrophages were harvested from male mice, and they were exposed to endotoxin (LPS) for 0. 2, 4, and 8 hours. ②The isolated peritoneal macrophages of mice were divided into 3 groups: control group (RPMI1640 routine culture), LPS pre-treatment group (pre-exposure to LPS followed by LPS

  7. In vitro Tracheobronchial Relaxation of Fritillaria Alkaloids%贝母生物碱的体外气管松弛作用研究

    Institute of Scientific and Technical Information of China (English)

    陈舜宏; 李萍; 关耀华; 林鸽

    2011-01-01

    AIM:Beimu(Bulbus Fritillariae)is a commonly used anti-tussive traditional Chinese herbal medicine and the major Fritillaria alkaloids in the herb are believed to be responsible for its antitussive activity.However,the detailed underlying mechanism and potency of individual Fritillaria alkaloids are still not very clear.METHODS:In the present study,we investigated and compared the relaxant effect of five major Fritillaria alkaloids[imperialine(IMP),verticine(VER),verticinone(VERN),ebeiedine(EBE)and puqietinone(PUQ)]using rat isolated tracheal and bronchial preparations pre-contracted with carbachol.RESULTS:All five Fritillaria alkaloids caused a concentration-dependent relaxation of both tracheal and bronchial preparations with the relative relaxing potency of IMP>VER≌VERN>EBE>PUQ.Amongst all five alkaloids tested,IMP(an isosteroidal Fritillaria alkaloid with a cis-configuration in rings D and E)was found to be the most potent;whereas PUQ(a steroidal Fritillaria alkaloid)was the least effective one.Furthermore,three Fritillaria alkaloids,namely IMP,VER and VERN(≥1 μmol-L-1)were capable of eliciting parallel rightward shifts of the carbachol concentration-response curve,demonstrating a competitive antagonism in muscarinic pathway.In addition,the suppression of carbachol-induced contraction caused by all five isosteroidal Fritillaria alkaloids was also partly due to the inhibition of influx of calcium ions.CONCLUSION:The results demonstrated that the major isosteroidal Fritillaria alkaloids might be the most active constituents responsible for the anti-tussive activity of herbal Beimu with the mechanisms of competitive antagonism of muscarinic pathway and also the inhibition of influx of calcium ions.%目的:贝母是一种常用的传统止咳中药,贝母生物碱被认为是其镇咳的主要活性成分,但其作用机理和各种生物碱的作用强度仍然不是很明确.方法:选用离体大鼠气管和支气管作为体外模型并用卡巴胆

  8. Effects of excitation of sensory pathways on the membrane potential of cat masseter motoneurons before and during cholinergically induced motor atonia.

    Science.gov (United States)

    Kohlmeier, K A; López-Rodríguez, F; Morales, F R; Chase, M H

    1998-09-01

    Electrical stimulation of the nucleus pontis oralis during wakefulness enhances somatic reflex activity; identical stimuli during the motor atonia of active (rapid eye movement) sleep induces reflex suppression. This phenomenon, which is called reticular response-reversal, is based upon the generation of excitatory postsynaptic potential activity in motoneurons during wakefulness and inhibitory postsynaptic potential activity during the motor atonia of active sleep. In the present study, instead of utilizing artificial electrical stimulation to directly excite brainstem structures, we sought to examine the effects on motoneurons of activation of sensory pathways by exogenously applied stimuli (auditory) and by stimulation of a peripheral (sciatic) nerve. Accordingly, we examined the synaptic response of masseter motoneurons prior to and during cholinergically induced motor atonia in a pharmacological model of active sleep-specific motor atonia, the alpha-chloralose-anesthetized cat, to two different types of afferent input, one of which has been previously demonstrated to elicit excitatory motor responses during wakefulness. Following the pontine injection of carbachol, auditory stimuli (95 dB clicks) elicited a hyperpolarizing potential in masseter motoneurons. Similar responses were obtained upon stimulation of the sciatic nerve. Responses of this nature were never seen prior to the injection of carbachol. Thus, stimulation of two different afferent pathways (auditory and somatosensory) that produce excitatory motor responses during wakefulness instead, during motor atonia, results in the inhibition of masseter motoneurons. The switching of the net result of the synaptic response from one of potential motor excitation to primarily inhibition in response to the activation of sensory pathways was comparable to the phenomenon of reticular response-reversal. This is the first report to examine the synaptic mechanisms whereby exogenously or peripherally applied

  9. Evaluation of gut modulatory and bronchodilator activities of Amaranthus spinosus Linn.

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    Chaudhary Mueen

    2012-10-01

    Full Text Available Abstract Background The aqueous-methanolic extract of Amaranthus spinosus (A. spinosus Linn., whole plant, was studied for its laxative, spasmolytic and bronchodilator activities to validate some of its medicinal uses. Methods The crude extract of A. spinosus was studied in-vivo for bronchodilator and laxative activities and in-vitro using isolated tissue preparations which were mounted in tissue baths assembly containing physiological salt solutions, maintained at 37°C and aerated with carbogen, to assess the spasmolytic effect and to find out the possible underlying mechanisms. Results In the in-vivo experiments in mice, the administration of A. spinosus increased fecal output at doses of 100 and 300 mg/kg showing laxative activity. It also inhibited carbachol-induced bronchospasm in anesthetized rats at 1, 3, 10 and 30 mg/kg indicative of bronchodilator activity. When tested on isolated gut preparations, the plant extract showed a concentration-dependent (0.01-10.0 mg/ml spasmogenic effect in spontaneously contracting rabbit jejunum and guinea-pig ileum. The spasmogenic effect was partially blocked in tissues pretreated with atropine (0.1 μM. When tested on K+ (80 mM-induced sustained contractions in isolated rabbit jejunum, the plant extract caused complete relaxation and also produced a shift in the Ca++ concentration-response curves (CRCs towards right, similar to diltiazem. In rabbit trachea, the plant extract completely inhibited K+ (80 mM and carbachol (CCh, 1 μM-induced contractions at 1 mg/ml but pretreatment of tissue with propranolol (1 μM, caused around 10 fold shift in the inhibitory CRCs of the plant extract constructed against CCh-induced contraction. The plant extract (up to 0.3 mg/ml also increased both force and rate of spontaneous contractions of isolated guinea-pig atria, followed by relaxation at higher concentration (1.0-5.0 mg/ml. The cardio-stimulant effect was abolished in the presence of propranolol, similar to

  10. The role of extrinsic and intrinsic factors in the evolution of the control of pulmonary surfactant maturation during development in the amniotes.

    Science.gov (United States)

    Sullivan, Lucy C; Orgeig, Sandra; Daniels, Christopher B

    2003-01-01

    Pulmonary surfactant is a mixture of lipids and proteins that is secreted by alveolar Type II cells. It reduces alveolar surface tension and hence the work of breathing. Despite the tremendous diversity of lung structures amongst the vertebrates, the composition of surfactant is highly conserved. Conserved elements of the surfactant system amongst distantly related species are likely to be crucial factors for successful lung development. Understanding the mechanisms by which the surfactant system becomes operational in animals with dramatically different birthing strategies and in distantly related species will provide important information about the role of the surfactant system in the commencement of air breathing and the processes regulating surfactant maturation and secretion. In mammals, the embryonic maturation of the surfactant system is controlled by a host of factors, including glucocorticoids, thyroid hormones, and autonomic neurotransmitters. Here we review the mechanisms controlling the maturation of surfactant production, including birthing strategy, phylogeny, lung structure, and posthatching environment. Using four species of egg-laying amniote (chicken, dragon lizard, sea turtle, and crocodile) previously described in detail and the large amount of information available for mammals, we examine the hypothesis that the control of surfactant production is dependent on glucocorticoids (dexamethasone [Dex]), thyroid hormones (T3), and autonomic neurotransmitters (epinephrine and carbachol). We also examine whether the overall intrinsic pattern of the control of surfactant maturation is conserved throughout the vertebrate radiation and then how the environment (extrinsic factors) may account for the observed differences in the patterns of development. We also discuss the utility of a coculture system of embryonic Type II cells and fibroblasts to determine the evolutionary pattern behind the control of surfactant and to demonstrate that the surfactant

  11. K(Ca3.1 channel-blockade attenuates airway pathophysiology in a sheep model of chronic asthma.

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    Joanne Van Der Velden

    Full Text Available BACKGROUND: The Ca(2+-activated K(+ channel K(Ca3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of K(Ca3.1 modifies experimental asthma in sheep. METHODOLOGY AND PRINCIPAL FINDINGS: Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073, a selective inhibitor of the K(Ca3.1-channel, or vehicle alone (0.5% methylcellulose twice daily (orally. Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147. The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288. Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340. Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling. CONCLUSIONS: These findings suggest that K(Ca3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of K(Ca3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.

  12. Urinary Bladder Dysfunction in Transgenic Sickle Cell Disease Mice.

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    Mário Angelo Claudino

    Full Text Available Urological complications associated with sickle cell disease (SCD, include nocturia, enuresis, urinary infections and urinary incontinence. However, scientific evidence to ascertain the underlying cause of the lower urinary tract symptoms in SCD is lacking.Thus, the aim of this study was to evaluate urinary function, in vivo and ex vivo, in the Berkeley SCD murine model (SS.Urine output was measured in metabolic cage for both wild type and SS mice (25-30 g. Bladder strips and urethra rings were dissected free and mounted in organ baths. In isolated detrusor smooth muscle (DSM, relaxant response to mirabegron and isoproterenol (1nM-10μM and contractile response to (carbachol (CCh; 1 nM-100μM, KCl (1 mM-300mM, CaCl2 (1μM-100mM, α,β-methylene ATP (1, 3 and 10 μM and electrical field stimulation (EFS; 1-32 Hz were measured. Phenylephrine (Phe; 10nM-100μM was used to evaluate the contraction mechanism in the urethra rings. Cystometry and histomorphometry were also performed in the urinary bladder.SS mice present a reduced urine output and incapacity to produce typical bladder contractions and bladder emptying (ex vivo, compared to control animals. In DSM, relaxation in response to a selective β3-adrenergic agonist (mirabegron and to a non-selective β-adrenergic (isoproterenol agonist were lower in SS mice. Additionally, carbachol, α, β-methylene ATP, KCl, extracellular Ca2+ and electrical-field stimulation promoted smaller bladder contractions in SS group. Urethra contraction induced by phenylephrine was markedly reduced in SS mice. Histological analyses of SS mice bladder revealed severe structural abnormalities, such as reductions in detrusor thickness and bladder volume, and cell infiltration.Taken together, our data demonstrate, for the first time, that SS mice display features of urinary bladder dysfunction, leading to impairment in urinary continence, which may have an important role in the pathogenesis of the enuresis and infections

  13. Pharmacological profiles of an anticholinergic agent, phencynonate hydrochloride, and its optical isomers

    Institute of Scientific and Technical Information of China (English)

    Li-yun WANG; Yun WANG; Jian-quan ZHENG; Bo-hua ZHONG; He LIU; Si-jian DONG; Jin-xiu RUAN; Ke-liang LIU

    2005-01-01

    Aim: To comparatively study the pharmacological profiles of 3-methyl-3-azabicyclo (3,3,1)nonanyl-9-α-yl-α-cyclopentyl-α-phenyl-α-glycolate (phencynonate hydrochloride, CPG), an anticholinergic agent, and its enantiomers [R(-)- and S(+)-CPG]. Methods: The affinity and relative efficacy were tested using radioligand-binding assay with muscarinic acetylcholine receptors from rat cerebral cortex. The pharmacological activities were assessed in three individual experiments: (1) potentiating the effect of subthreshold hypnotic dose of sodium pentobarbital; (2) inhibiting oxotremorine-induced salivation; and (3) inhibiting the contractile response to carbachol. Results: The order of potency of phencynonate hydrochloride and its optical isomers to inhibit the binding of [3H]quinuclidinyl benzilate ([3H] QNB) was R(-)-CPG (Ki=46.49± 1.27 nmol/L)>CPG (Ki=271.37±72.30 nmol/L)>S(+)-CPG (Ki=1263.12±131.64 nmol/L). The results showed that R(-)-CPG had the highest affinity to central muscarinic receptors among the three compounds, but did not show any central depressant effects at dose from 10.00 to 29.15 mg/kg. CPG increased the effects of subthreshold hypnotic dose of sodium pentobarbital induced-sleeping [the ED50±95 % LC value was 21.06±3.04 mg/kg]. CPG and R(-)-CPG displayed nearly equipotent effect in depressing oxotremorineinduced salivation [the ED50±95% LC for R(-) and CPG were 1.10±0.28 and 1.07±0.15 mg/kg, respectively], and the contractile response to carbachol (pA2 values for R(-) and CPG were 6.84 and 6.80, respectively). S(+)-CPG presented the lowest anticholinergic profiles, but could potentate effects of its enantiomers in some manner. Conclusions: These data suggested that R(-)-CPG acted as an eutomer in racemate and a competitive antagonist to acetylcholine muscarinic receptors,but S(+)-CPG was less active in comparison to R(-)-CPG and its racemate. The central depressant effects of R(-)-CPG and S(+)-CPG were lower in comparison to its racemate.

  14. Comparative study on pharmacological effects of DM-phencynonate hydrochloride and its optical isomers

    Institute of Scientific and Technical Information of China (English)

    Li-yun WANG; Jian-quan ZHENG; Yun WANG; Bo-hua ZHONG; Jin-xiu RUAN; Ke-liang LIU

    2005-01-01

    Aim: The 3 -azabicyclo(3,3, 1)nonanyl-9-α-yl-α-cyclopentyl-α-phenyl-α-glycolate (DM-phencynonate hydrochloride, DMCPG) is a demethylated metabolite of 3-methyl-3-azabicyclo(3,3,1)nonanyl-9-α-yl-α-cyclopentyl-α-phenyl-α-glycolate (phencynonate hydrochloride, CPG). (±)DMCPG had one chiral center and two enantiomers [R(-) and S(+)DMCPG]. Here we carried out a comparative study of the pharmacological profiles of these optical isomers. Methods: Affinity and relative efficacy were tested using a radioligand-binding assay with muscarinic acetylcholine receptors from the rat cerebral cortex. Pharmacological activity was assessed in three individual experiments: (1) potentiating the effect of a subthreshold hypnotic dose of sodium pentobarbital; (2) inhibiting oxotremorineinduced salivation; and (3) inhibiting the contractile response to carbachol.Results: In the competitive binding assay, R(-)DMCPG (Ki=763.75 nmol/L) was 4-and 2-fold more potent than (±)DMCPG (Ki=3186 nmol/L) and S(+)DMCPG (Ki= 1699 nmol/L) in inhibiting the binding of [3H]QNB. The R(-) and S (+) configurations showed positive cooperation (nH>1) with the muscarinic receptor, whereas (±)DMCPG had a negative cooperation (nH<1) relationship with the muscarinic receptor in a radio-binding assay. Both the R(-) and S(+) configurations could potentiate the effect of sub-threshold hypnotic dose of sodium pentobarbital in a dose-dependent manner (the ED50 values were 2.53 and 18.65 mg/kg, respectively),but (±)DMCPG did not display significant central depressant effects at doses from 10 to 29.15 mg/kg (P>0.05). (±)DMCPG and its optical isomers suppressed the guinea pig ileum contractile response to carbachol. The IC50 values were 7.78×10-9,1.88×10-7, and 1.03× 10-7 nmol/L, respectively. In the anti-salivation study,(±)DMCPG and its enantiomers depressed oxotremorine- induced salivation in a dose-dependent manner, and the order of potency was R(-)DMCPG (ED50=0.44 mg/kg)>(±)DMCPG (ED50

  15. Activation of ERK1/2 by store-operated calcium entry in rat parotid acinar cells.

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    Stephen P Soltoff

    Full Text Available The regulation of intracellular Ca(2+ concentration ([Ca(2+]i plays a critical role in a variety of cellular processes, including transcription, protein activation, vesicle trafficking, and ion movement across epithelial cells. In many cells, the activation of phospholipase C-coupled receptors hydrolyzes membrane phosphoinositides and produces the depletion of endoplasmic reticulum Ca(2+ stores, followed by the sustained elevation of [Ca(2+]i from Ca(2+ entry across the plasma membrane via store-operated Ca(2+ entry (SOCE. Ca(2+ entry is also increased in a store-independent manner by arachidonate-regulated Ca(2+ (ARC channels. Using rat parotid salivary gland cells, we examined multiple pathways of Ca(2+ entry/elevation to determine if they activated cell signaling proteins and whether this occurred in a pathway-dependent manner. We observed that SOCE activates extracellular signal-related kinases 1 and 2 (ERK1/2 to ∼3-times basal levels via a receptor-independent mechanism when SOCE was initiated by depleting Ca(2+ stores using the endoplasmic reticulum Ca(2+-ATPase inhibitor thapsigargin (TG. TG-initiated ERK1/2 phosphorylation increased as rapidly as that initiated by the muscarinic receptor agonist carbachol, which promoted an increase to ∼5-times basal levels. Notably, ERK1/2 phosphorylation was not increased by the global elevation of [Ca(2+]i by Ca(2+ ionophore or by Ca(2+ entry via ARC channels in native cells, although ERK1/2 phosphorylation was increased by Ca(2+ ionophore in Par-C10 and HSY salivary cell lines. Agents and conditions that blocked SOCE in native cells, including 2-aminoethyldiphenyl borate (2-APB, SKF96363, and removal of extracellular Ca(2+, also reduced TG- and carbachol-stimulated ERK1/2 phosphorylation. TG-promoted ERK1/2 phosphorylation was blocked when SRC and Protein Kinases C (PKC were inhibited, and it was blocked in cells pretreated with β-adrenergic agonist isoproterenol. These observations demonstrate

  16. Effect of L-arginine on neuromuscular transmission of the chick biventer cervicis muscle

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    B. Esfandiar

    2008-01-01

    Full Text Available biventer cervicis muscleD. Effect of L-arginine on neuromuscular transmission of the chick EsfandiarAbstractBackground and Purpose: NO is a short-lived gas molecule generated by degradation of L-arg to citrulline and by the activation of enzyme NOS Ca2+/calmodulin-dependent. There are multiple NOS isoforms that strongly are expressed in skeletal muscle, suggesting the crucial role of NO in regulating muscular metabolism and function. In this study, the effect of L-arginine was examined at the neuromuscular junction of the chick biventer cervicis muscle.Materials and Methods: Biventer cervicis muscle preparations from chick’s age of 3 weeks were set up in the organ bath. The organ bath had a vessel with volume of about 70 ml; it contained Tyrode solution aerated with oxygen and was kept at 37º C. NO levels was also measured in the chick biventer cervicis muscle homogenates, using spectrophotometer method for the direct detection of NO, nitrite and nitrate. Total nitrite (nitrite+nitrate was measured by a spectrophotometer at 540 nm after the conversion of nitrate to nitrite by copperized cadmium granules.Results: L-Arginine at 500 µg/ml, decreased twitch response to electrical stimulation, and produced rightward shift of the dose-response curve for acetylcholine or carbachol. L-arginine at 1000 µg/ml produced a strong shift to the right of the dose-response curve for acetylcholine or carbachol with a reduction in efficacy. The inhibitory effect of L-arginine on the twitch response was blocked by caffeine (200 µg/ml. NO levels were found to be significantly increased in concentrations 500 and 1000 µg/ml of L-arginine in comparison with the control group (p < 0.001.Conclusion: These findings indicate a possible role of increased NO levels in the suppressive action of L-arginie on the twitch response. In addition, the results indicate that the post-junctional antagonistic action of L-arginine is probably the result of impaired sarcoplasmic

  17. Therapeutic effects of ghrelin and growth hormone releasing peptide 6 on gastroparesis in streptozotocin-induced diabetic guinea pigs in vivo and in vitro

    Institute of Scientific and Technical Information of China (English)

    QIU Wen-cai; WANG Zhi-gang; WANG Wei-gang; YAN Jun; ZHENG Qi

    2008-01-01

    Background Diabetic gastroparesis is a disabling condition with no consistently effective treatment.In normal animals,both ghrelin and its synthetic peptide,growth hormone releasing peptide 6(GHRP-6),increase gastric emptying.Thus,we investigated the potential therapeutic significance of ghrelin and GHRP-6 in diabetic guinea pigs with gastric motility disorders.Methods A diabetic guinea pig model was produced by intraperitoneal(i.p.)injection of streptozotocin(STZ,280 mg/kg).Diabetic guinea pigs were injected i.p.with ghrelin or GHRP-6(10-100 pg/kg),and the effects on gastric emptying were measured after intragastric application of phenol red.The effect of atropine or a growth hormone secretagogue receptor(GHS-R)antagonist,D-Lys3-GHRP-6,on the gastroprokinetic effects of ghrelin or GHRP-6(100 μg/kg)was also investigated.Further,the in vitro effects of ghrelin or GHRP-6(0.01-10 μmol/L)on spontaneous or carbachol-induced contractile amplitude in gastric fundic circular strips taken from diabetic guinea pigs were examined.Growth hormone secretagogue receptor transcripts in the fundic strips of diabetic guinea pigs were detected by reverse transcriptase polymerase chain reaction(RT-PCR).Results We established a guinea pig model of delayed gastric emptying.Ghrelin(20,50,or 100 μg/kg)and GHRP-6 (20,50,or 1 00 μg/kg)accelerated gastric emptying in diabetic guinea pigs with gastroparesis(n=-6,P<0.05).In the presence of atropine,which delayed gastric emptying,ghrelin and GHRP-6(100 μg/kg)failed to accelerate gastric emptying(n=6,P<0.05).D-Lys3-GHRP-6 also delayed gastric emptying induced by the GHS-R agonist(n=6,P<0.05).Ghrelin and GHRP-6 increased the carbachol-induced contractile amplitude in gastric fundic strips taken from diabetic guinea pigs(n=6,P<0.05).RT-PCR confirmed the presence of GHS-R mRNA in the strip preparations.Conclusions Ghrelin and GHRP-6 increased gastric emptying in diabetic guinea pigs with gastroparesis,potentially,by activating the

  18. Transcutaneous electrical nerve stimulation (TENS) improves the diabetic cytopathy (DCP) via up-regulation of CGRP and cAMP.

    Science.gov (United States)

    Ding, Liucheng; Song, Tao; Yi, Chaoran; Huang, Yi; Yu, Wen; Ling, Lin; Dai, Yutian; Wei, Zhongqing

    2013-01-01

    The objective of this study was to investigate the effects and mechanism of Transcutaneous Electrical Nerve Stimulation (TENS) on the diabetic cytopathy (DCP) in the diabetic bladder. A total of 45 rats were randomly divided into diabetes mellitus (DM)/TENS group (n=15), DM group (n=15) and control group (n=15). The rats in the DM/TENS and TENS groups were electronically stimulated (stimulating parameters: intensity-31 V, frequency-31 Hz, and duration of stimulation of 15 min) for three weeks. Bladder histology, urodynamics and contractile responses to field stimulation and carbachol were determined. The expression of calcitonin gene-related peptide (CGRP) was analyzed by RT-PCR and Western blotting. The results showed that contractile responses of the DM rats were ameliorated after 3 weeks of TENS. Furthermore, TENS significantly increased bladder wet weight, volume threshold for micturition and reduced PVR, V% and cAMP content of the bladder. The mRNA and protein levels of CGRP in dorsal root ganglion (DRG) in the DM/TENS group were higher than those in the DM group. TENS also significantly up-regulated the cAMP content in the bladder body and base compared with diabetic rats. We conclude that TENS can significantly improve the urine contractility and ameliorate the feeling of bladder fullness in DM rats possibly via up-regulation of cAMP and CGRP in DRG. PMID:23468996

  19. Hormonal and cholinergic influences on pancreatic lysosomal and digestive enzymes in rats.

    Science.gov (United States)

    Evander, A; Ihse, I; Lundquist, I

    1983-01-01

    Hormonal and cholinergic influences on lysosomal and digestive enzyme activities in pancreatic tissue were studied in normal adult rats. Hormonal stimulation by the cholecystokinin analogue, caerulein, induced a marked enhancement of the activities of cathepsin D and N-acetyl-beta-D-glucosaminidase in pancreatic tissue, whereas the activities of amylase and lipase tended to decrease. Acid phosphatase activity was not affected. Further, caerulein was found to induce a significant increase of cathepsin D output in bile-pancreatic juice. This output largely parallelled that of amylase. Cholinergic stimulation by the muscarinic agonist carbachol, at a dose level giving the same output of amylase as caerulein, did not affect pancreatic activities of cathepsin D and N-acetyl-beta-D-glucosaminidase. Further, cholinergic stimulation induced an increase of amylase activity and a slight decrease of acid phosphatase activity in pancreatic tissue. Lipase activity was not affected. No apparent effect on cathepsin D output in bile-pancreatic juice was encountered after cholinergic stimulation. The activities of neither the digestive nor the lysosomal enzymes were influenced by the administration of secretin. The results suggest a possible lysosomal involvement in caerulein-induced secretion and/or inactivation of pancreatic digestive enzymes, whereas cholinergic stimulation seems to act through different mechanisms.

  20. Method for the preparation of mucosal flaps from the jejunum of laying hens for transporter studies in Ussing chambers.

    Science.gov (United States)

    Ruhnke, Isabelle; Röhe, Ilen; Meyer, Wilfried; Kröger, Susan; Neumann, Konrad; Zentek, Jürgen

    2013-04-01

    Ussing chambers are frequently used for in vitro evaluation of intestinal transport physiology. The current study describes investigating the jejunal tissue from laying hens using a specific preparation method and evaluates the effect of glutamine in the maintenance buffer. Tunica mucosa was stripped from 104 jejunal samples from 10 hens and stabilised by a net device. Fifty samples were maintained with modified Krebs-Henseleit buffer (Control), 54 samples with additional 5 mM glutamine (Group Gln). The percentage of responding samples varied between 87 and 100%. Mean short circuit current (ΔI sc,) [µA/cm(2)] of samples exposed to 10 mM glucose in the Control group and Group Gln was 17.0 and 14.6 (p = 0.836), respectively, of samples exposed to 100 µM phloridzin -13.3 and -11.8 (p = 0.712), respectively, and of samples exposed to 100 µM carbachol 4.7 and 3.7 (p = 0.450), respectively. In conclusion, the net-supported method enabled a reliable investigation of jejunum from laying hens. Glutamine in the maintenance buffer was of no significant benefit.

  1. Argan (Argania spinosa) oil lowers blood pressure and improves endothelial dysfunction in spontaneously hypertensive rats.

    Science.gov (United States)

    Berrougui, Hicham; Alvarez de Sotomayor, Maria; Pérez-Guerrero, Concepción; Ettaib, Abdelkader; Hmamouchi, Mohamed; Marhuenda, Elisa; Herrera, Maria Dolores

    2004-12-01

    Traditionally hand-pressed argan oil, obtained from Argania spinosa seeds, is eaten raw in south-west Morocco; its rich composition of tocopherols, MUFA and PUFA make a study of its actions on risk factors for CVD, such as hypertension, interesting. The effects of 7 weeks of treatment with argan oil (10 ml/kg) on the blood pressure and endothelial function of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats were investigated. Systolic blood pressure and heart rate were measured every week by the tail-cuff method and endothelial function was assessed by carbachol (10(-8) to 10(-4) M)-induced relaxations of aortic rings and small mesenteric arteries pre-contracted with phenylephrine. Argan-oil administration reduced the mean blood pressure of SHR after the fifth week of treatment (Pargan-oil treatment of SHR. Experiments in the presence of the thromboxane A2-prostaglandin H2 receptor antagonist ICI 192,605 (10(-5) M) confirmed this result. Results after incubation with the antioxidants superoxide dismutase and catalase suggested that a decreased oxidative stress might contribute to explain the beneficial effects of argan-oil treatment.

  2. Fast oscillatory activity in the anterior cingulate cortex: dopaminergic modulation and efect of perineuronal net loss

    Directory of Open Access Journals (Sweden)

    Pascal eSteullet

    2014-08-01

    Full Text Available Dopamine release in the prefrontal cortex plays a critical role in cognitive function such as working memory, attention and planning. Dopamine exerts complex modulation on excitability of pyramidal neurons and interneurons, and regulates excitatory and inhibitory synaptic transmission. Because of the complexity of this modulation, it is difficult to fully comprehend the effect of dopamine on neuronal network activity. In this study, we investigated the effect of dopamine on local high-frequency oscillatory neuronal activity (in  band in slices of the mouse anterior cingulate cortex (ACC. We found that dopamine enhanced the power of these oscillations induced by kainate and carbachol, but did not affect their peak frequency. Activation of D2R and in a lesser degree D1R increased the oscillation power, while activation of D4R had no effect. These high-frequency oscillations in the ACC relied on both phasic inhibitory and excitatory transmission and functional gap junctions. Thus, dopamine released in the ACC promotes high-frequency synchronized local cortical activity which is known to favor information transfer, fast selection and binding of distributed neuronal responses. Finally, the power of these oscillations was significantly enhanced after degradation of the perineuronal nets enwrapping most parvalbumin interneurons. This study provides new insights for a better understanding of the abnormal prefrontal gamma activity in schizophrenia patients who display prefrontal anomalies of both the dopaminergic system and the perineuronal nets.

  3. An Exploratory Study on the Development of an Animal Model of Acute Pancreatitis Following Nicotine Exposure

    Directory of Open Access Journals (Sweden)

    Chowdhury P

    2003-09-01

    Full Text Available Abstract Cigarette smoking is known to be a major risk factor for pancreatic cancer and pancreatitis is believed to be a predisposed condition for pancreatic cancer. As of this date, there is no established experimental animal model to conduct detailed studies on these two deadly diseases. Our aim is to establish a rodent model by which we can systematically study the pathogenesis of pancreatitis and pancreatic cancer. Methods Adult Male Sprague Dawley rats were exposed to graded doses of nicotine by various routes for periods of three to 16 weeks. Blood samples were measured for hormonal and metabolic parameters. The pancreas was evaluated for histopathological changes and its function was assessed in isolated pancreatic acini upon stimulation with cholecystokinin (CCK or carbachol (Cch. The pancreatic tissue was evaluated further for oncogene expression. Results Body weight, food and fluid intakes, plasma glucose and insulin levels were significantly reduced in animals with nicotine exposure when compared to control. However, CCK and gastrin levels in the blood were significantly elevated. Pancreatic function was decreased significantly with no alteration in CCK receptor binding. Pancreatic histology revealed vacuolation, swelling, cellular pyknosis and karyorrhexis. Mutant oncogene, H-ras, was overexpressed in nicotine-treated pancreatic tissue. Summary and conclusion The results suggest that alterations in metabolic, hormonal and pathologic parameters following nicotine-treatment appear consistent with diagnostic criteria of human pancreatitis. It is proposed that rats could be considered as a potential animal model to study the pathogenesis of pancreatitis.

  4. c-fos expression in brainstem premotor interneurons during cholinergically induced active sleep in the cat.

    Science.gov (United States)

    Morales, F R; Sampogna, S; Yamuy, J; Chase, M H

    1999-11-01

    The present study was undertaken to identify trigeminal premotor interneurons that become activated during carbachol-induced active sleep (c-AS). Their identification is a critical step in determining the neural circuits responsible for the atonia of active sleep. Accordingly, the retrograde tracer cholera toxin subunit B (CTb) was injected into the trigeminal motor nuclei complex to label trigeminal interneurons. To identify retrograde-labeled activated neurons, immunocytochemical techniques, designed to label the Fos protein, were used. Double-labeled (i.e., CTb(+), Fos(+)) neurons were found exclusively in the ventral portion of the medullary reticular formation, medial to the facial motor nucleus and lateral to the inferior olive. This region, which encompasses the ventral portion of the nucleus reticularis gigantocellularis and the nucleus magnocellularis, corresponds to the rostral portion of the classic inhibitory region of. This region contained a mean of 606 +/- 41.5 ipsilateral and 90 +/- 32.0 contralateral, CTb-labeled neurons. These cells were of medium-size with an average soma diameter of 20-35 micrometer. Approximately 55% of the retrogradely labeled cells expressed c-fos during a prolonged episode of c-AS. We propose that these neurons are the interneurons responsible for the nonreciprocal postsynaptic inhibition of trigeminal motoneurons that occurs during active sleep. PMID:10531453

  5. Pharmacology of JB-9315, a new selective histamine H2-receptor antagonist.

    Science.gov (United States)

    Palacios, B; Montero, M J; Sevilla, M A; San Román, L

    1998-02-01

    1. The histamine H2-receptor antagonistic activity and antisecretory and antiulcer effects of JB-9315 were studied in comparison with the standard H2 blocker ranitidine. 2. In vitro, JB-9315 is a competitive antagonist of histamine H2 receptors in the isolated, spontaneously beating guinea-pig right atrium, with a pA2 value of 7.30 relative to a value of 7.36 for ranitidine. JB-9315 was specific for the histamine H2 receptor because, at high concentration, it did not affect histamine- or acetylcholine-induced contractions in guinea-pig isolated ileum or rat isolated duodenum, respectively. 3. JB-9315 dose dependently inhibited histamine-, pentagastrin- or carbachol-stimulated acid secretion and basal secretion in the perfused stomach preparation of the anesthetized rat. In the pylorus-ligated rat after intraperitoneal administration, total acid output over 4 h was inhibited by JB-9315 with an ID50 of 32.8 mg/kg, confirming its H2-receptor antagonist properties. 4. JB-9315 showed antiulcer activity against cold stress plus indomethacin-induced lesions with an ID50 of 6.8 mg/kg. 5. JB-9315, 50 and 100 mg/kg, inhibited macroscopic gastric hemorrhagic lesions induced by ethanol. In contrast, ranitidine (50 mg/kg) failed to reduce these lesions. 6. These results indicate that JB-9315 is a new antiulcer drug that exerts a cytoprotective effect in addition to its gastric antisecretory activity.

  6. JB-9322, a new selective histamine H2-receptor antagonist with potent gastric mucosal protective properties.

    Science.gov (United States)

    Palacios, B; Montero, M J; Sevilla, M A; Román, L S

    1995-05-01

    1. JB-9322 is a selective histamine H2-receptor antagonist with gastric antisecretory activity and mucosal protective properties. 2. The affinity of JB-9322 for the guinea-pig atria histamine H2-receptor was approximately 2 times greater than that of ranitidine. 3. In vivo, the ID50 value for the inhibition of gastric acid secretion in pylorus-ligated rats was 5.28 mg kg-1 intraperitoneally. JB-9322 also dose-dependently inhibited gastric juice volume and pepsin secretion. In gastric lumen-perfused rats, intravenous injection of JB-9322 dose-dependently reduced histamine-, pentagastrin- and carbachol-stimulated gastric acid secretion. 4. JB-9322 showed antiulcer activity against aspirin and indomethacin-induced gastric lesions and was more potent than ranitidine. 5. JB-9322 effectively inhibited macroscopic gastric haemorrhagic lesions induced by ethanol. Intraperitoneal injection was effective in preventing the lesions as well as oral treatment. The oral ID50 value for these lesions was 1.33 mg kg-1. By contrast, ranitidine (50 mg kg-1) failed to reduce these lesions. In addition, the protective effect of JB-9322 was independent of prostaglandin synthesis. 6. These results indicate that JB-9322 is a new antiulcer drug that exerts a potent cytoprotective effect in addition to its gastric antisecretory activity.

  7. Smooth muscle length-dependent PI(4,5)P2 synthesis and paxillin tyrosine phosphorylation.

    Science.gov (United States)

    Sul, D; Baron, C B; Broome, R; Coburn, R F

    2001-07-01

    We studied effects of increasing the length of porcine trachealis muscle on 5.5 microM carbachol (CCh)-evoked phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] synthesis and other parameters of phosphatidylinositol (PI) turnover. PI(4,5)P2 resynthesis rates in muscle held at 1.0 optimal length (L(o)), measured over the first 6 min of CCh stimulation, were 140 +/- 12 and 227 +/- 14% of values found in muscle held at 0.5 L(o) and in free-floating muscle, respectively. Time-dependent changes in cellular masses of PI(4,5)P2, PI, and phosphatidic acid, and PI resynthesis rates, were also altered by the muscle length at which contraction occurred. In free-floating muscle, CCh did not evoke increases in tyrosine-phosphorylated paxillin (PTyr-paxillin), an index of beta1-integrin signaling; however, there were progressive increases in PTyr-paxillin in muscle held at 0.5 and 1.0 L(o) during contraction, which correlated with increases in PI(4,5)P2 synthesis rates. These data indicate that PI(4,5)P2 synthesis rates and other parameters of CCh-stimulated inositol phospholipid turnover are muscle length-dependent and provide evidence that supports the hypothesis that length-dependent beta1-integrin signals may exert control on CCh-activated PI(4,5)P2 synthesis.

  8. Determination of the vectorelectrogram in isolated rat atria: application to the study of arrhythmias

    International Nuclear Information System (INIS)

    Atrial tachyarrhythmias, the most frequent type of cardiac arrhythmia, are associated with increased stroke risk. Reentry and focal activity are considered as the main mechanisms underlying this dysfunction. In this study, we describe determination of the vectorelectrogram in isolated rat atria as a means to distinguish different patterns of electrical propagation. In all studied right atria beating at sinus rhythm, the mean electric vector (MEV) trajectory was clockwise, and each cycle was preceded by electric diastole (null MEV), either in the absence or presence of muscarinic cholinergic or β-adrenergic receptor stimulation. During cholinergic tachyarrhythmia (induced by high-rate electric stimulation in both atria, plus exposure to carbachol in left atria), vector loops were ellipsoidal and stable, with variable direction, and did not cross the origin, which is consistent with reentrant activation and with findings obtained in vivo by other authors. In contrast, during spontaneous activity induced by rapid pacing in isoproterenol-exposed left atria, vector loops were similar to those in right atria at sinus rhythm, thus suggestive of focal activity. It is concluded that the vectorelectrogram approach allows discrimination of different patterns of propagation during arrhythmia in isolated atria and may be useful for high-output tests of pro- and anti-arrhythmic compounds

  9. The discovery of potent blockers of the canonical transient receptor channels, TRPC3 and TRPC6, based on an anilino-thiazole pharmacophore.

    Science.gov (United States)

    Washburn, David G; Holt, Dennis A; Dodson, Jason; McAtee, Jeff J; Terrell, Lamont R; Barton, Linda; Manns, Sharada; Waszkiewicz, Anna; Pritchard, Christina; Gillie, Dan J; Morrow, Dwight M; Davenport, Elizabeth A; Lozinskaya, Irina M; Guss, Jeffrey; Basilla, Jonathan B; Negron, Lorena Kallal; Klein, Michael; Willette, Robert N; Fries, Rusty E; Jensen, Timothy C; Xu, Xiaoping; Schnackenberg, Christine G; Marino, Joseph P

    2013-09-01

    Lead optimization of piperidine amide HTS hits, based on an anilino-thiazole core, led to the identification of analogs which displayed low nanomolar blocking activity at the canonical transient receptor channels 3 and 6 (TRPC3 & 6) based on FLIPR (carbachol stimulated) and electrophysiology (OAG stimulated) assays. In addition, the anilino-thiazole amides displayed good selectivity over other TRP channels (TRPA1, TRPV1, and TRPV4), as well as against cardiac ion channels (CaV1.2, hERG, and NaV1.5). The high oxidation potential of the aliphatic piperidine and aniline groups, as well as the lability of the thiazole amide group contributed to the high clearance observed for this class of compounds. Conversion of an isoquinoline amide to a naphthyridine amide markedly reduced clearance for the bicyclic piperidines, and improved oral bioavailability for this compound series, however TRPC3 and TRPC6 blocking activity was reduced substantially. Although the most potent anilino-thiazole amides ultimately lacked oral exposure in rodents and were not suitable for chronic dosing, analogs such as 14-19, 22, and 23 are potentially valuable in vitro tool compounds for investigating the role of TRPC3 and TRPC6 in cardiovascular disease.

  10. Munc18/SNARE proteins' regulation of exocytosis in guinea pig duodenal Brunner's gland acini

    Institute of Scientific and Technical Information of China (English)

    Laura I Cosen-Binker; Gerry P Morris; Stephen Vanner; Herbert Y Gaisano

    2008-01-01

    AIM: To examine the molecular mechanism of exocytosis in the Brunner's gland acinar cell.METHODS: We used a submucosal preparation of guinea pig duodenal Brunner's gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy.RESULTS: One and 6 μmol/L carbachol evoked a dosedependent dilation of Brunner's gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact.CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner's gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.

  11. Antidiarrheal Activity of 19-Deoxyicetexone Isolated from Salvia ballotiflora Benth in Mice and Rats

    Directory of Open Access Journals (Sweden)

    Ernesto Sánchez-Mendoza

    2013-07-01

    Full Text Available The antidiarrheal properties of 19-deoxyicetexone, a diterpenoid isolated from Salvia ballotiflora were evaluated on castor oil-, arachidonic acid (AA- and prostaglandin (PGE2-induced diarrhea in rodent models. The structure of 19-deoxyicetexone was determined by X-ray crystallography, mass spectrometry (EI-MS, as well as ultraviolet (UV-Vis, infrared (FT-IR and nuclear magnetic resonance (NMR spectroscopies. This compound significantly and dose-dependently reduced frequency of stooling in castor oil-induced diarrhea, and at dose of 25 mg/kg it also inhibited diarrhea induced with AA, while it had no effect on PGE2-induced diarrhea. This compound at doses of 25 mg/kg also diminished castor oil-induced enteropooling and intestinal motility, and inhibited the contraction of the rats’ ileum induced by carbachol chloride at a concentration of 100 µg/mL. 19-Deoxyicetexone did not present acute toxicity at doses of 625 mg/kg. Its antidiarrheal activity may be due to increased reabsorption of NaCl and water and inhibition of the release of prostaglandins, gastrointestinal motility and fluid accumulation in the intestinal tracts of rats. These findings suggest that 19-deoxyicetexone may be used in the treatment of diarrhea, although more studies must be carried out to confirm this.

  12. Anti-diarrheal constituents of Alpinia oxyphylla.

    Science.gov (United States)

    Zhang, Junqing; Wang, Sheng; Li, Yonghui; Xu, Peng; Chen, Feng; Tan, Yinfeng; Duan, Jinao

    2013-09-01

    Isolation, screening and in vivo assays have been used for evaluating anti-diarrhea bioactive of Alpinia oxyphylla. Preliminary experimental results showed that 95% ethanol extract and 90% ethanol elution significantly extended the onset time of diarrhea and reduced the wet feces proportion, however 50% ethanol election had no effect on diarrhea. Chemical analysis results displayed that Nootkatone, Tectochrysin and yakuchinone A may be bioactive ingredients for curing diarrhea. Duodenum in vitro experiment showed that Tectochrysin 50, 100 μM reduces carbachol-induced contraction, while yakuchinone A and Nootkatone had no effect. Bioinformatic computational method as molecular docking has been complementary to experimentally work to explore the potential mechanism. The study of pathogenesis of diarrhea in humans and animal models suggested that Na(+)/H(+) exchanger3 (NHE3) and aquaporin4 (AQP4) are causative agents of diarrhea. The analysis was done on the basis of scoring and binding ability and the docking analysis showed that Tectochrysin has maximum potential against NHE3 (PDB ID: 2OCS) and AQP4 (PDB ID: 3GD8). Tectochrysin indicated minimum energy score and the highest number of interactions with active site residues. These results suggested that A. oxyphylla might exhibit its anti-diarrhea effect partially by affecting the proteins of NHE3 and AQP4 with its active ingredient Tectochrysin. PMID:23583435

  13. Phase-locked signals elucidate circuit architecture of an oscillatory pathway.

    Science.gov (United States)

    Jovic, Andreja; Howell, Bryan; Cote, Michelle; Wade, Susan M; Mehta, Khamir; Miyawaki, Atsushi; Neubig, Richard R; Linderman, Jennifer J; Takayama, Shuichi

    2010-12-23

    This paper introduces the concept of phase-locking analysis of oscillatory cellular signaling systems to elucidate biochemical circuit architecture. Phase-locking is a physical phenomenon that refers to a response mode in which system output is synchronized to a periodic stimulus; in some instances, the number of responses can be fewer than the number of inputs, indicative of skipped beats. While the observation of phase-locking alone is largely independent of detailed mechanism, we find that the properties of phase-locking are useful for discriminating circuit architectures because they reflect not only the activation but also the recovery characteristics of biochemical circuits. Here, this principle is demonstrated for analysis of a G-protein coupled receptor system, the M3 muscarinic receptor-calcium signaling pathway, using microfluidic-mediated periodic chemical stimulation of the M3 receptor with carbachol and real-time imaging of resulting calcium transients. Using this approach we uncovered the potential importance of basal IP3 production, a finding that has important implications on calcium response fidelity to periodic stimulation. Based upon our analysis, we also negated the notion that the Gq-PLC interaction is switch-like, which has a strong influence upon how extracellular signals are filtered and interpreted downstream. Phase-locking analysis is a new and useful tool for model revision and mechanism elucidation; the method complements conventional genetic and chemical tools for analysis of cellular signaling circuitry and should be broadly applicable to other oscillatory pathways.

  14. Pyocyanin inhibits both nitric oxide-dependent and -independent relaxation in porcine coronary arteries.

    Science.gov (United States)

    Hempenstall, Allison; Grant, Gary D; Anoopkumar-Dukie, Shailendra; Johnson, Peter J

    2015-02-01

    The effects of the Pseudomonas aeruginosa virulence factor pyocyanin (PCN) on the contractile function of porcine coronary arteries was investigated in vitro. Artery rings (5 mm) were suspended in organ baths containing Krebs' solution for the measurement of isometric tension. The effect of PCN on resting and precontracted coronary arteries was initially investigated with various agents. Arteries were precontracted with prostaglandin (PG) F2α or potassium chloride and endothelium-dependent relaxations were induced by various agents in the presence of PCN. Pyocyanin (0.1-10 μmol/L) evoked small-amplitude, dose-dependent contractions in resting porcine coronary arteries. In addition, PCN amplified the contractile response to PGF2α , but did not alter responses to carbachol. Pyocyanin (0.1-10 μmol/L) significantly inhibited endothelium-dependent relaxations evoked by neurokinin A. Pyocyanin also inhibited relaxations evoked by diethylamine nitric oxide (a nitric oxide donor), forskolin (an adenylate cyclase activator), dibuytyryl-cAMP (a cAMP analogue), 8-bromo-cGMP (a cGMP analogue) and P1075 (a KATP channel activator), but not isoprenaline (β-adrenoceceptor agonist). These results indicate that physiological concentrations of PCN interfere with multiple intracellular processes involved in vascular smooth muscle relaxation, in particular pathways downstream of nitric oxide release. Thus, PCN may alter normal vascular function in patients infected with P. aeruginosa.

  15. Effect of long-term partial bladder outlet obstruction on caldesmon isoforms and their correlation with contractile function

    Institute of Scientific and Technical Information of China (English)

    Lin YANG; Da-lin Hei; Shu WANG; He-peng CHENG; Xin-yang WANG

    2008-01-01

    Aim: In the present study, we investigate the expression of caldesmon (CAD) isoforms in rabbit detrusor smooth muscles (DSM) during the progression of partial bladder outlet obstruction and relate them with the time course of obstruction. Methods: Detrusor samples were obtained from the bladders of rabbits with partial bladder outlet obstruction and sham-operated control rabbits after 1, 2, 4, and 8 weeks of obstruction. Contractile responses to field stimulation and carbachol were determined in the isolated bladder strips. Western blotting was used to determine the relative levels of CaD isoform expression at the protein levels. Results: The contractile responses decreased progressively over the course of obstruction. The expression of 1-CaD increased significantly to approximately the same extent as the 1-4-week obstructed groups and further in the 8-week ob-structed group. The expression of h-CaD increased in all of the obstructed bladders, but at significantly higher levels in the 1-2-week obstructed bladders compared to the control and 4-8-week obstructed bladders. Conclusions: The changes in the isoforms of CaD may be part of the molecular mechanism for bladder compensa-tion following partial bladder outlet obstruction. The overexpression of 1-CaD and the h-CaD/1-CaD ratio could be markers for the status of DSM remodeling and dysfunction.

  16. The Regulatory Role of Rolipram on Inflammatory Mediators and Cholinergic/Adrenergic Stimulation-Induced Signals in Isolated Primary Mouse Submandibular Gland Cells

    Directory of Open Access Journals (Sweden)

    Dong Un Lee

    2016-01-01

    Full Text Available Exposure to bacterial lipopolysaccharides (LPS induces inflammatory signals in salivary glands. We investigated the regulatory role of phosphodiesterase 4 (PDE4 inhibitor rolipram on inflammatory mediators and cholinergic/adrenergic stimulation-induced intracellular Ca2+ signaling in salivary acinar and ductal cells. Submandibular gland (SMG expressed PDE4A through 4D mRNA and PDE4 was localized in the luminal membrane of SMG. LPS induced Ca2+ signaling and ROS production in SMG. Treatment with rolipram blocked LPS-induced Ca2+ increase and ROS production. The application of histamine evoked Ca2+ signals and ROS production, which were attenuated by rolipram in SMG cells. Moreover, LPS-induced NLRP3 inflammasome and cleaved caspase-1 were inhibited by rolipram. The inhibitory role of rolipram in ROS-induced Ca2+ signaling was mainly observed in acinar cells and not in ductal cells. Rolipram also protected SMG acinar but not ductal cells from LPS-induced cell membrane damage. In the case of cholinergic/adrenergic stimulation, carbachol/isoproterenol-induced Ca2+ signals were upregulated by the treatment of rolipram in SMG. In the case of cAMP-dependent ductal bicarbonate secretion by rolipram, no effect was observed on the modulation of ductal chloride/bicarbonate exchange activity. Rolipram could suppress the inflammatory signals and could be a potential therapeutic strategy against LPS-induced inflammation to protect the salivary gland cells.

  17. Actions of ionomycin in rat parotid gland

    Energy Technology Data Exchange (ETDEWEB)

    Poggioli, J.; Leslie, B.A.; McKinney, J.S.; Weiss, S.J.; Putney, J.W. Jr.

    1982-04-01

    The effects of ionomycin (SQ 23,377), a carboxylic acid Ca-ionophore, on the rat parotid acinar cell were investigated. Ionomycin stimulated 86Rb efflux from parotid slices and was substantially more potent and efficacious than the Ca-ionophore, A-23187. The release of 86Rb was dependent on the concentration of ionomycin and of Ca. Ionomycin also stimulated 22Na uptake and 3H-protein secretion, but did not stimulate the incorporation of 32PO4 into phosphatidylinositol. These observations are consistent with an action of ionomycin in increasing cytosolic Ca by acting as an ionophore and not involving endogenous receptors. Pretreatment with ionomycin inhibited the transient, Ca-independent responses to carbachol or physalaemin. When ionomycin was added to parotid cells pre-equilibrated with 45Ca, a net loss of radiocalcium was observed. These observations suggest that ionomycin can release the receptor-regulated cellular Ca pool. Morphological studies did not reveal any nonspecific deleterious effects in the cells after incubation with 2.67 microM ionomycin.

  18. The Regulatory Role of Rolipram on Inflammatory Mediators and Cholinergic/Adrenergic Stimulation-Induced Signals in Isolated Primary Mouse Submandibular Gland Cells

    Science.gov (United States)

    Lee, Dong Un; Shin, Dong Min; Hong, Jeong Hee

    2016-01-01

    Exposure to bacterial lipopolysaccharides (LPS) induces inflammatory signals in salivary glands. We investigated the regulatory role of phosphodiesterase 4 (PDE4) inhibitor rolipram on inflammatory mediators and cholinergic/adrenergic stimulation-induced intracellular Ca2+ signaling in salivary acinar and ductal cells. Submandibular gland (SMG) expressed PDE4A through 4D mRNA and PDE4 was localized in the luminal membrane of SMG. LPS induced Ca2+ signaling and ROS production in SMG. Treatment with rolipram blocked LPS-induced Ca2+ increase and ROS production. The application of histamine evoked Ca2+ signals and ROS production, which were attenuated by rolipram in SMG cells. Moreover, LPS-induced NLRP3 inflammasome and cleaved caspase-1 were inhibited by rolipram. The inhibitory role of rolipram in ROS-induced Ca2+ signaling was mainly observed in acinar cells and not in ductal cells. Rolipram also protected SMG acinar but not ductal cells from LPS-induced cell membrane damage. In the case of cholinergic/adrenergic stimulation, carbachol/isoproterenol-induced Ca2+ signals were upregulated by the treatment of rolipram in SMG. In the case of cAMP-dependent ductal bicarbonate secretion by rolipram, no effect was observed on the modulation of ductal chloride/bicarbonate exchange activity. Rolipram could suppress the inflammatory signals and could be a potential therapeutic strategy against LPS-induced inflammation to protect the salivary gland cells. PMID:27143817

  19. Antiulcerogenic effect of Securigera securidaca L. seed extract on various experimental gastric ulcer models in rats.

    Science.gov (United States)

    Mard, S A; Bahari, Z; Eshaghi, N; Farbood, Y

    2008-12-01

    Securigera securidaca belongs to the family Fabaceae is used in Iranian folk medicine to treat gastric disturbances. The present study was undertaken to evaluate the Securigera securidaca seed hydroalcoholic extract (SSE) and its subfractions for their gastroprotective effect in rat. Acute gastric ulceration in rats was produced by oral administration of ethanol (100%; 1 mL/200 g of body weight) or water immersion restraint-stress (5 h, water immersion restraint stress at 20-22 degrees C). Ranitidine (100 mg kg(-1), p.o.) was used as the reference antiulcer drug. After ethanol administration, the gastric wall mucus was examined. Chronic gastric ulceration was produced by injection of acetic acid in rat gastric subserosa. The antisecretory effect of the extract and its subfractions (ethyl acetate, chloroform and aqueous fractions) were investigated in pylorus-ligated rats. Administration of SSE significantly inhibited gastric mucosa damage induced by ethanol, water immersion restraint-stress and acetic acid in a dose-dependent manner. In pylorus ligature rats, SSE and its subfractions significantly reduced the basal gastric acid secretion and total acidity; moreover, it inhibited the increase in total acidity induced by carbachol. However, the antisecretory effect of the chloroform fraction was more potent than two other fractions. Administration of SSE did not affect the gastric mucus production. The results obtained in the present study indicate that the SSE has gastroprotective and antisecretory effects on gastric mucosa in rats. PMID:19630213

  20. Effects of acetylcholine and electrical stimulation on glial cell line-derived neurotrophic factor production in skeletal muscle cells.

    Science.gov (United States)

    Vianney, John-Mary; Miller, Damon A; Spitsbergen, John M

    2014-11-01

    Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor required for survival of neurons in the central and peripheral nervous system. Specifically, GDNF has been characterized as a survival factor for spinal motor neurons. GDNF is synthesized and secreted by neuronal target tissues, including skeletal muscle in the peripheral nervous system; however, the mechanisms by which GDNF is synthesized and released by skeletal muscle are not fully understood. Previous results suggested that cholinergic neurons regulate secretion of GDNF by skeletal muscle. In the current study, GDNF production by skeletal muscle myotubes following treatment with acetylcholine was examined. Acetylcholine receptors on myotubes were identified with labeled alpha-bungarotoxin and were blocked using unlabeled alpha-bungarotoxin. The question of whether electrical stimulation has a similar effect to that of acetylcholine was also investigated. Cells were stimulated with voltage pulses; at 1 and 5 Hz frequencies for times ranging from 30 min to 48 h. GDNF content in myotubes and GDNF in conditioned culture medium were quantified by enzyme-linked immunosorbant assay. Results suggest that acetylcholine and short-term electrical stimulation reduce GDNF secretion, while treatment with carbachol or long-term electrical stimulation enhances GDNF production by skeletal muscle.

  1. The effect of cannabinoids on dinitrofluorobenzene-induced experimental asthma in mice.

    Science.gov (United States)

    Bozkurt, Turgut Emrah; Kaya, Yesim; Durlu-Kandilci, Nezahat Tugba; Onder, Sevgen; Sahin-Erdemli, Inci

    2016-09-01

    Cannabinoids have anti-inflammatory effects and can produce bronchodilation in the airways. We have investigated the effects of cannabinoids on tracheal hyperreactivity and airway inflammation in dinitrofluorobenzene (DNFB)-induced experimental non-atopic asthma in mice. 5-hydroxytryptamine (5-HT)-induced contraction response was enhanced while carbachol- and electrical field stimulation-induced contractions, and isoprenaline-induced relaxation responses were remained unchanged in DNFB group. The increased 5-HT-induced contractions were inhibited by incubation with either atropine or tetrodotoxin. DNFB application resulted in increased macrophage number in the bronchoalveolar lavage fluid (BALF). In vivo ACEA (CB1 agonist) treatment prevented the increase in 5-HT contractions, while JWH133 (CB2 agonist) had no effect. However, neither ACEA nor JWH133 prevented the increase in macrophage number in BALF. In vitro ACEA incubation also inhibited the increase in 5-HT contraction in DNFB group. These results show that cannabinoid CB1 receptor agonist can prevent tracheal hyperreactivity to 5-HT in DNFB-induced non-atopic asthma in mice. PMID:27216000

  2. Pharmacological Evaluation of Prosopis cineraria (L. Druce in Gastrointestinal, Respiratory, and Vascular Disorders

    Directory of Open Access Journals (Sweden)

    Khalid Hussain Janbaz

    2012-01-01

    Full Text Available In this paper, a crude methanolic extract from the stem bark of Prosopis cineraria, a plant native of Pakistan, was tested for its possible presence of spasmolytic, bronchodilator, and vasodilator activities in an attempt to validate some of its folkloric uses. Moreover, attempts were made to provide plausible explanations of the observed biological activities. The extract caused relaxation of the spontaneous as well as K+ (80 mM-induced contractions at tissue bath concentrations of 3–10 mg/mL in isolated rabbit jejunum preparations, probably mediated through blockade of Ca+2 channels. This finding was further confirmed by the shifting of the Ca+2 concentration response curves to the rightward in a manner similar to verapamil used as a standard Ca+2 channel blocker. The extract also exhibited nonspecific relaxant effect on carbachol (1 μM- and K+ (80 mM-induced contractions in isolated rabbit tracheal preparations. The same effect was recorded for phenylephrine (11 μM and K+ (80 mM-induced contractions in isolated rabbit aortic preparations in a manner similar to verapamil. These observations confirm that observed bronchodilator and vasodilator activities were possibly mediated through blockade of Ca+2 channels. The above-mentioned observations validate the traditional use of the plant in the treatment of respiratory and gastrointestinal ailments.

  3. Smooth-muscle-like cells derived from human embryonic stem cells support and augment cord-like structures in vitro.

    Science.gov (United States)

    Vo, Elaine; Hanjaya-Putra, Donny; Zha, Yuanting; Kusuma, Sravanti; Gerecht, Sharon

    2010-06-01

    Engineering vascularized tissue is crucial for its successful implantation, survival, and integration with the host tissue. Vascular smooth muscle cells (v-SMCs) provide physical support to the vasculature and aid in maintaining endothelial viability. In this study, we show an efficient derivation of v-SMCs from human embryonic stem cells (hESCs), and demonstrate their functionality and ability to support the vasculature in vitro. Human ESCs were differentiated in monolayers and supplemented with platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-beta1). Human ESC-derived smooth-muscle-like cells (SMLCs) were found to highly express specific smooth muscle cell (SMC) markers--including alpha-smooth muscle actin, calponin, SM22, and smooth muscle myosin heavy chain--to produce and secrete fibronectin and collagen, and to contract in response to carbachol. In vitro tubulogenesis assays revealed that these hESC-derived SMLCs interacted with human endothelial progenitor cell (EPCs) to form longer and thicker cord-like structures in vitro. We have demonstrated a simple protocol for the efficient derivation of highly purified SMLCs from hESCs. These in vitro functional SMLCs interacted with EPCs to support and augment capillary-like structures (CLSs), demonstrating the potential of hESCs as a cell source for therapeutic vascular tissue engineering.

  4. Zinc sulphate attenuates chloride secretion in human colonic mucosae in vitro.

    Science.gov (United States)

    Medani, Mekki; Bzik, Victoria A; Rogers, Ailin; Collins, Danielle; Kennelly, Rory; Winter, Des C; Brayden, David J; Baird, Alan W

    2012-12-01

    Zinc's usefulness in the treatment of diarrhoea is well established as an addition to oral rehydration. Mechanisms of action of zinc have been explored in intestinal epithelia from rodents and in cell lines. The aim was to examine how zinc alters ion transport and signal transduction in human colon in vitro. Voltage clamped colonic sheets obtained at the time of surgical resection were used to quantify ion transport responses to established secretagogues. Nystatin permeabilisation was used to study basolaterally-sited ion channels. Direct actions of zinc were determined using preparations of colonic crypts isolated from human mucosal sheets. Electrophysiological measurements revealed zinc to be an inhibitor of electrogenic ion transport stimulated by forskolin, PGE(2), histamine and carbachol in isolated human colonic epithelium. Basolateral addition of zinc sulphate had no direct effect on the epithelium. To further outline the mechanism of action, levels of secondary intracellular messengers (3', 5'-cyclic adenosine monophosphate; cAMP) were determined in isolated colonic crypts, and were found to be reduced by zinc sulphate. Finally, indirect evidence from nystatin-permeabilised mucosae further suggested that zinc inhibits basolateral K(+) channels, which are critical for transepithelial Cl(-) secretion linked to water flux. Anti-secretory, and therefore anti-diarrhoeal, actions of exogenous zinc are due, at least in part, to direct basolateral epithelial K(+) channel inhibition. PMID:23022335

  5. Cell discharge correlates of posterior hypothalamic theta rhythm. Recipe for success in recording stable field potential.

    Science.gov (United States)

    Bocian, Renata; Kłos-Wojtczak, Paulina; Konopacki, Jan

    2016-09-01

    The theta rhythm discovered in the posterior hypothalamus area (PHa) differs from theta observed in the hippocampal formation. In comparison to hippocampal spontaneous theta, the theta recorded in the PHa is rarely registered, has lower amplitude, often disappears, and sometimes returns after a few minutes. These features indicate that spontaneous theta recorded in the PHa is not an appropriate experimental model to search for the correlation between PHa cell discharges and local field potential. In this paper we present standard experimental conditions necessary to record theta-related cells in the PHa in anesthetized rats. Three pharmacological agents were used in the experiments to induce PHa theta rhythm in urethanized rats: carbachol (CCH), carbenoxolone and kainic acid, which are potent enough to induce well-synchronized PHa theta. However, CCH was found to be the best pharmacological tool to induce PHa theta oscillations, due to its longest duration of action and lack of preliminary epileptogenic effects. It seems that CCH-induced theta can be the most suitable pharmacological model for experiments with the use of protocol of long-lasting recordings of PHa theta-related cell discharges. PMID:27353451

  6. Participation of citral in the bronchodilatory effect of ginger oil and possible mechanism of action.

    Science.gov (United States)

    Mangprayool, Thitiya; Kupittayanant, Sajeera; Chudapongse, Nuannoi

    2013-09-01

    The extract of ginger, the rhizomes of Zingiber officinale Roscoe (Zingiberaceae), has been reported to possess anti-hyperactivity and anti-inflammation on airway. The present study described brochodilatory activity of ginger oil and identified its active compound. Ginger oil was extracted by hydro-distillation. The compositions of ginger oil were analyzed by gas chromatography and mass spectrometer. Citral, eucalyptol and camphene were found to be the major components. Ginger oil and citral, but not camphene, suppressed rat tracheal contraction induced by carbachol (CCh). Consistent with previous report, eucalyptol showed a relaxing effect on rat airway. Since the content of eucalyptol in ginger oil was relatively low, the contribution of eucalyptol to the bronchodilatory effect of ginger oil was small. To elucidate the mechanisms responsible for the myorelaxing effect, propranolol (a β-adrenergic receptor antagonist), indomethacin (a COX inhibitor) and L-NAME (a NOS inhibitor) were used to block the inhibitory effects of ginger oil and citral. It was found that propranolol, but not indomethacin and L-NAME, reversed bronchodilatory effects of both ginger oil and citral, suggesting that a possible mechanism involved β-adrenergic receptor. This study provides the pharmacological basis supporting the therapeutic potential of Z. officinale rhizomes as a bronchodilator. PMID:23685048

  7. Distinct regulation of vasoactive intestinal peptide (VIP) expression at mRNA and peptide levels in human neuroblastoma cells.

    Science.gov (United States)

    Agoston, D V; Colburn, S; Krajniak, K G; Waschek, J A

    1992-05-25

    Neuronal differentiation was induced in cultures of the human neuroblastoma cell line subclone SH-SY5Y by 14-day treatment with dibutyryl cAMP (dBcAMP), retinoic acid, and phorbol 12-myristate 13-acetate (PMA). An approximate 4-fold increase in vasoactive intestinal peptide (VIP) mRNA concentration was observed after differentiation with retinoic acid, whereas no change in VIP mRNA concentration was observed after differentiation with dBcAMP or PMA. A short-term treatment of cells with PMA did however result in a 5-fold transient increase in VIP mRNA; prior differentiation with retinoic acid or dBcAMP diminished this effect. Observed increases in VIP mRNA were in all cases accompanied by increases in VIP immunoreactivity. Remarkably, however, long-term treatment of cells with dBcAMP, which caused no change in mRNA levels, resulted in a six-fold increase in VIP immunoreactivity. Acute (36-h) treatment with carbachol also caused an increase in VIP immunoreactivity (about 2-fold, and blocked by atropine) without an increase in VIP mRNA level. Thus, a quantitative change in gene transcription or mRNA stability appears not to be a prerequisite for increased VIP expression, indicating that regulation can occur at translational or post-translational steps.

  8. Differential activation of nitric oxide synthase through muscarinic acetylcholine receptors in rat salivary glands.

    Science.gov (United States)

    Leirós, C P; Rosignoli, F; Genaro, A M; Sales, M E; Sterin-Borda, L; Santiago BordaE

    2000-03-15

    Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) appears to be one of the multiple effectors coupled to muscarinic receptors in both submandibular and sublingual glands although some differences have been found depending on the gland studied. First, submandibular glands had a lower basal activity of nitric oxide synthase than sublingual glands and the concentration-response curve for carbachol was bell-shaped in the former but not in sublingual glands. Second, cGMP levels displayed a similar profile to that observed for NOS activity in both glands. Third, protein kinase C also coupled to muscarinic receptor activation in the glands might have a regulatory effect on nitric oxide production since its activity was higher in basal conditions in submandibular than sublingual glands and it also increased in the presence of the agonist at a concentration that inhibited NOS activity in submandibular glands. The effects appear to be partly related to the expression of a minor population of M(1) receptors in submandibular glands absent in sublingual as determined in binding and signaling experiments with the muscarinic receptor antagonist pirenzepine.

  9. The effects of cyclophosphamide on neurotransmission in the urinary bladder of Suncus murinus, the house musk shrew.

    Science.gov (United States)

    Mok, M H; Knight, G E; Andrews, P L; Hoyle, C H; Burnstock, G

    2000-05-12

    This study has shown that cyclophosphamide treatment of the insectivore Suncus murinus, causes a down regulation in both muscarinic and P2X receptors, together with a reduced responsiveness to exogenous histamine (0.3 mM) in the urinary bladder. Electrical field stimulation (70 V, 0.3 ms, 0.5-16 Hz, 10 s every 5 min) of bladders from both control and cyclophosphamide-treated animals showed identical responses. Since post-junctional alterations have been revealed by the reduced responsiveness to exogenous carbachol (0.1 microM-3 mM) and beta,gamma-methylene ATP (0.3-300 microM), it would appear that in the bladders of cyclophosphamide-treated animals there is also a pre-junctional effect, increased transmitter release compensating for the down regulation of the receptors. As the pattern of neurotransmission of the bladder of suncus more closely resembles that of human detrusor than other commonly studied laboratory animals, this insectivore appears to be a useful animal model for the study of bladder neurotransmission in pathophysiological conditions.

  10. Antispasmodic Effects and Action Mechanism of Essential Oil of Chrysactinia mexicana A. Gray on Rabbit Ileum

    Directory of Open Access Journals (Sweden)

    Daniel Zavala-Mendoza

    2016-06-01

    Full Text Available The Chrysactinia mexicana A. Gray (C. mexicana plant is used in folk medicine to treat fever and rheumatism; it is used as a diuretic, antispasmodic; and it is used for its aphrodisiac properties. This study investigates the effects of the essential oil of C. mexicana (EOCM on the contractility of rabbit ileum and the mechanisms of action involved. Muscle contractility studies in vitro in an organ bath to evaluate the response to EOCM were performed in the rabbit ileum. EOCM (1–100 µg·mL−1 reduced the amplitude and area under the curve of spontaneous contractions of the ileum. The contractions induced by carbachol 1 µM, potassium chloride (KCl 60 mM or Bay K8644 1 µM were reduced by EOCM (30 µg·mL−1. Apamin 1 µM and charybdotoxin 0.01 µM decreased the inhibition induced by EOCM. The d-cAMP 1 µM decreased the inhibition induced by EOCM. l-NNA 10 µM, Rp-8-Br-PET-cGMPS 1 µM, d,l-propargylglycine 2 mM, or aminooxyacetic acid hemihydrochloride 2 mM did not modify the EOCM effect. In conclusion, EOCM induces an antispasmodic effect and could be used in the treatment of intestinal spasms or diarrhea processes. This effect would be mediated by Ca2+, Ca2+-activated K+ channels and cAMP.

  11. Inhibition of the mechanical activity of mouse ileum by cactus pear (Opuntia Ficus Indica, L, Mill.) fruit extract and its pigment indicaxanthin.

    Science.gov (United States)

    Baldassano, S; Tesoriere, L; Rotondo, A; Serio, R; Livrea, M A; Mulè, F

    2010-07-14

    We investigated, using an organ bath technique, the effects of a hydrophilic extract from Opuntia ficus indica fruit pulp (cactus fruit extract, CFE) on the motility of mouse ileum, and researched the extract component(s) responsible for the observed responses. CFE (10-320 mg of fresh fruit pulp equivalents/mL of organ bath) reduced dose-dependently the spontaneous contractions. This effect was unaffected by tetrodotoxin, a neuronal blocker, N(omega)-nitro-l-arginine methyl ester, a nitric oxide synthase blocker, tetraethylammonium, a potassium channel blocker, or atropine, a muscarinic receptor antagonist. CFE also reduced the contractions evoked by carbachol, without affecting the contractions evoked by high extracellular potassium. Indicaxanthin, but not ascorbic acid, assayed at concentrations comparable with their content in CFE, mimicked the CFE effects. The data show that CFE is able to exert direct antispasmodic effects on the intestinal motility. The CFE inhibitory effects do not involve potassium channels or voltage-dependent calcium channels but rather pathways of calcium intracellular release. The fruit pigment indicaxanthin appears to be the main component responsible for the CFE-induced effects.

  12. Effects of changes in frequency on guinea pig ventricular action potential duration and on QT interval under different experimental conditions.

    Science.gov (United States)

    von Savigny, L; Hohnloser, S; Antoni, H

    1981-01-01

    Isolated perfused guinea pig hearts (Langendorff preparation) were arrested by carbachol (0.1-0.2 mg/l) and electrically stimulated in the region of the av-conducting system. The QT interval was determined by means of extracellular electrodes at different driving frequencies. Separate experiments were performed on papillary muscles from the right ventricle to measure the duration of the transmembrane action potential under comparable conditions. At 35 degrees C (Ke+ 5.4 mmol/l) increasing the frequency of stimulation (range 12-120/min) caused the action potential duration (APD) to decrease to a greater extent than the QT interval. Stepwise rising of the external K+ concentration up to 16.2 mmol/l produced a nearly parallel shift to the APD-frequency relation to lower values. Again, the QT interval was less affected by increasing the external K+ concentration than the APD. Stepwise reduction of the temperature down to 20 degrees C prolonged the APD as well as the QT interval, the effects being more pronounced at lower than at higher stimulation frequencies. Under all examined experimental conditions, the APD proved to be markedly shorter than the QT interval even when the latter is diminished by the duration of QRS. The results suggest that no close relation exists between the APD and the QT interval. The observed divergencies may be due to functional differences among various parts of the ventricles.

  13. 5-Hydroxytryptamine-induced bladder hyperactivity via the 5-HT2A receptor in partial bladder outlet obstruction in rats.

    Science.gov (United States)

    Sakai, Takumi; Kasahara, Ken-ichi; Tomita, Ken-ichi; Ikegaki, Ichiro; Kuriyama, Hiroshi

    2013-04-01

    We investigated the effects of partial bladder outlet obstruction (BOO) on the function and gene expression of 5-hydroxytryptamine (5-HT) receptor subtypes in rat bladder. Isometric contractions of the isolated bladders from sham-operated control and BOO rats were examined. The contractile responses to 5-HT were significantly increased in BOO rat bladder strips, while the responses to KCl, carbachol, or phenylephrine were not different from the control. The 5-HT-induced hypercontraction in BOO rat bladder strips was inhibited by ketanserin, a 5-HT(2A) receptor antagonist. The contractile responses to 5-HT in bladder strips were not affected by urothelium removal from the intact bladder. The gene expression of 5-HT receptor subtypes in the bladders was analyzed by RT-PCR. The mRNA expression of the 5-HT(2A), 5-HT(2B), 5-HT(2C), 5-HT(4), and 5-HT(7) receptors was detected in both the control and BOO rat bladders. Quantitative RT-PCR analysis showed there was a significant increase of 5-HT(2A) receptor mRNA in the BOO rat bladder compared with the control bladder. On the other hand, the gene expression of the 5-HT(4) receptor was not changed in the BOO rat bladder. These results suggest that the increased contractile responses to 5-HT in BOO rat bladder may be partly caused by 5-HT(2A) receptor upregulation in the detrusor smooth muscles. PMID:23344575

  14. Pharmaceutical evaluation of cultivated Glycyrrhiza uralensis roots in comparison of their antispasmodic activity and glycycoumarin contents with those of licorice.

    Science.gov (United States)

    Nagai, Hidemasa; Yamamoto, Yutaka; Sato, Yuji; Akao, Teruaki; Tani, Tadato

    2006-12-01

    In China, the collection of wild Glycyrrhiza uralensis, one of the raw materials of Chinese licorice, has been restricted to prevent desertification. To compensate for the reduced supply of wild Glycyrrhiza plants, cultivation programs of G. uralensis have been initiated in eastern Inner Mongolia. The goal of the present study was to compare the chemical and pharmacological properties of cultivated G. uralensis roots to those of licorice prepared from wild Glycyrrhiza plants. The antispasmodic effect of boiled water extract of 4-year-old cultivated G. uralensis roots and licorice on carbachol-induced contraction in mice jejunum was similar (ED(50): 134+/-21 microg/ml vs. 134+/-16 microg/ml). In addition, glycycoumarin content, which is an antispasmodic and species-specific ingredient of G. uralensis, was similar when comparing the boiled water extracts of 4-year-old cultivated roots and licorice (0.10+/-0.02% vs. 0.10+/-0.06%). These data suggest that cultivated G. uralensis roots may be an adequate replacement for the generation of licorice in the context of the restriction of wild Glycyrrhiza plant collection. PMID:17142979

  15. Essential oil from the leaves of Xylopia langsdorfiana (Annonaceae) as a possible spasmolytic agent.

    Science.gov (United States)

    Correia, Ana Carolina de C; Ferreira, Tamyris F; Martins, Italo Rossi R; Macêdo, Cibério L; Monteiro, Fabio de S; Costa, Vicente Carlos O; Tavares, Josean F; Silva, Marcelo S; Paredes-Gamero, Edgar J; Buri, Marcus V; Rigoni, Vera L S; Nouailhetas, Viviane L A; Da Silva, Bagnólia A

    2015-01-01

    Xylopia langsdorfiana A. St.-Hil. &Tul. (Annonaceae) is popularly known in the northeast of Brazil as 'pimenteira da terra', and an essential oil (XL-OE) was extracted from its leaves. Since Xylopia species are cited in folk medicine and diterpenes from X. langsdorfiana have spasmolytic activity, this study aimed to investigate a possible spasmolytic action of XL-OE on smooth muscle models. XL-OE (243 and 729 μg/mL) showed low pharmacologic efficacy on guinea pig trachea and rat aorta and uterus. However, in guinea pig ileum, XL-OE (27-729 μg/mL) inhibited carbachol or histamine-induced phasic contractions (1 μM) in a significant and concentration-dependent manner. In addition, XL-OE (81 μg/mL) reduced fluorescence intensity in ileal myocytes stimulated by histamine, indicating a decrease in cytosolic calcium concentration, which could explain the spasmolytic activity. Thus, XL-OE proved to be a promising natural product to be used in gastrointestinal diseases acting by modulating the cytosolic calcium concentration.

  16. Effects of hypoxia and glucose-removal condition on muscle contraction of the smooth muscles of porcine urinary bladder.

    Science.gov (United States)

    Nagai, Yuta; Kaneda, Takeharu; Miyamoto, Yasuyuki; Nuruki, Takaomi; Kanda, Hidenori; Urakawa, Norimoto; Shimizu, Kazumasa

    2016-01-01

    To elucidate the dependence of aerobic energy metabolism and utilization of glucose in contraction of urinary bladder smooth muscle, we investigated the changes in the reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity, and determined the phosphocreatine (PCr) and ATP contents of the porcine urinary bladder during contractions induced by high K(+) or carbachol (CCh) and with and without hypoxia (achieved by bubbling N2 instead of O2) or in a glucose-free condition. Hyperosmotic addition of 65 mM KCl (H-65K(+)) and 1 µM CCh induced a phasic contraction followed by a tonic contraction. A glucose-free physiological salt solution (PSS) did not change the subsequent contractile responses to H-65K(+) and CCh. However, hypoxia significantly attenuated H-65K(+)- and CCh-induced contraction. H-65K(+) and CCh induced a sustained increase in PNred fluorescence, representing glycolysis activity. Hypoxia enhanced H-65K(+)- and CCh-induced increases in PNred fluorescence, whereas glucose-free PSS decreased these increases, significantly. In the presence of H-65K(+), hypoxia decreased the PCr and ATP contents; however, the glucose-free PSS did not change the PCr contents. In conclusion, we demonstrated that high K(+)- and CCh-induced contractions depend on aerobic metabolism and that an endogenous substrate may be utilized to maintain muscle contraction in a glucose-free PSS in the porcine urinary bladder.

  17. Oestrogen inhibits human colonic motility by a non-genomic cell membrane receptor-dependent mechanism.

    LENUS (Irish Health Repository)

    Hogan, A M

    2012-02-01

    BACKGROUND: Classical effects of oestrogen involve activation of target genes after binding nuclear receptors. Oestrogenic effects too rapid for DNA transcription (non-genomic) are known to occur. The effect of oestrogen on colonic motility is unknown despite the prevalence of gastrointestinal symptoms in pregnant and premenopausal women. METHODS: Histologically normal colon was obtained from proximal resection margins of colorectal carcinoma specimens. Circular smooth muscle strips were microdissected and suspended in organ baths under 1 g of tension. After equilibration, they were exposed to 17beta-oestradiol (n = 8) or bovine serum albumin (BSA)-conjugated 17beta-oestradiol (n = 8). Fulvestrant, an oestrogen receptor antagonist, was added to some baths (n = 8). Other strips were exposed to calphostin C or cycloheximide. Carbachol was added in increasing concentrations and contractile activity was recorded isometrically. RESULTS: Oestrogen inhibited colonic contractility (mean difference 19.7 per cent; n = 8, P < 0.001). In keeping with non-genomic, rapid-onset steroid action, the effect was apparent within minutes and reversible. It was observed with both 17beta-oestradiol and BSA-conjugated oestrogen, and was not altered by cycloheximide. Effects were inhibited by fulvestrant, suggesting receptor mediation. CONCLUSION: Oestrogen decreases contractility in human colonic smooth muscle by a non-genomic mechanism involving cell membrane coupling.

  18. Denatonium and 6-n-Propyl-2-thiouracil, Agonists of Bitter Taste Receptor, Inhibit Contraction of Various Types of Smooth Muscles in the Rat and Mouse.

    Science.gov (United States)

    Sakai, Hiroyasu; Sato, Ken; Kai, Yuki; Chiba, Yoshihiko; Narita, Minoru

    2016-01-01

    Recently the global expression of taste 2 receptors (TAS2Rs) on smooth muscle cells in human airways was demonstrated. Here, the effects of agonists of taste receptor, type 2, denatonium and 6-n-propyl-2-thiouracil, on smooth-muscle contraction were examined in the rat and mouse. Contractions induced by carbachol (CCh), high K(+), and sodium fluoride, but not calyculin-A, were inhibited significantly in the presence of a TAS2R agonist in the bronchial smooth muscle of mice. The contraction induced by CCh was inhibited by TAS2R agonists in ileal smooth muscle. Phenylephrine-induced contraction was also inhibited by TAS2R agonists in aortic smooth muscle. Gastrointestinal motility and blood pressure were attenuated by administration of TAS2R agonists in vivo. These findings suggest that TAS2R may be receptor for endogenous biologically active substances as well as for bitter tastes on the tongue. TAS2R signaling could be employed in the development of anti-asthmatic, anti-spasmodic, and anti-hypertensive drugs.

  19. Transcutaneous electrical nerve stimulation (TENS improves the diabetic cytopathy (DCP via up-regulation of CGRP and cAMP.

    Directory of Open Access Journals (Sweden)

    Liucheng Ding

    Full Text Available The objective of this study was to investigate the effects and mechanism of Transcutaneous Electrical Nerve Stimulation (TENS on the diabetic cytopathy (DCP in the diabetic bladder. A total of 45 rats were randomly divided into diabetes mellitus (DM/TENS group (n=15, DM group (n=15 and control group (n=15. The rats in the DM/TENS and TENS groups were electronically stimulated (stimulating parameters: intensity-31 V, frequency-31 Hz, and duration of stimulation of 15 min for three weeks. Bladder histology, urodynamics and contractile responses to field stimulation and carbachol were determined. The expression of calcitonin gene-related peptide (CGRP was analyzed by RT-PCR and Western blotting. The results showed that contractile responses of the DM rats were ameliorated after 3 weeks of TENS. Furthermore, TENS significantly increased bladder wet weight, volume threshold for micturition and reduced PVR, V% and cAMP content of the bladder. The mRNA and protein levels of CGRP in dorsal root ganglion (DRG in the DM/TENS group were higher than those in the DM group. TENS also significantly up-regulated the cAMP content in the bladder body and base compared with diabetic rats. We conclude that TENS can significantly improve the urine contractility and ameliorate the feeling of bladder fullness in DM rats possibly via up-regulation of cAMP and CGRP in DRG.

  20. Transcutaneous electrical nerve stimulation (TENS) improves the diabetic cytopathy (DCP) via up-regulation of CGRP and cAMP.

    Science.gov (United States)

    Ding, Liucheng; Song, Tao; Yi, Chaoran; Huang, Yi; Yu, Wen; Ling, Lin; Dai, Yutian; Wei, Zhongqing

    2013-01-01

    The objective of this study was to investigate the effects and mechanism of Transcutaneous Electrical Nerve Stimulation (TENS) on the diabetic cytopathy (DCP) in the diabetic bladder. A total of 45 rats were randomly divided into diabetes mellitus (DM)/TENS group (n=15), DM group (n=15) and control group (n=15). The rats in the DM/TENS and TENS groups were electronically stimulated (stimulating parameters: intensity-31 V, frequency-31 Hz, and duration of stimulation of 15 min) for three weeks. Bladder histology, urodynamics and contractile responses to field stimulation and carbachol were determined. The expression of calcitonin gene-related peptide (CGRP) was analyzed by RT-PCR and Western blotting. The results showed that contractile responses of the DM rats were ameliorated after 3 weeks of TENS. Furthermore, TENS significantly increased bladder wet weight, volume threshold for micturition and reduced PVR, V% and cAMP content of the bladder. The mRNA and protein levels of CGRP in dorsal root ganglion (DRG) in the DM/TENS group were higher than those in the DM group. TENS also significantly up-regulated the cAMP content in the bladder body and base compared with diabetic rats. We conclude that TENS can significantly improve the urine contractility and ameliorate the feeling of bladder fullness in DM rats possibly via up-regulation of cAMP and CGRP in DRG.

  1. Muscle-wide secretion of a miniaturized form of neural agrin rescues focal neuromuscular innervation in agrin mutant mice

    Science.gov (United States)

    Lin, Shuo; Maj, Marcin; Bezakova, Gabriela; Magyar, Josef P.; Brenner, Hans Rudolf; Ruegg, Markus A.

    2008-01-01

    Agrin and its receptor MuSK are required for the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). In the current model the local deposition of agrin by the nerve and the resulting local activation of MuSK are responsible for creating and maintaining the postsynaptic apparatus including clusters of acetylcholine receptors (AChRs). Concomitantly, the release of acetylcholine (ACh) and the resulting depolarization disperses those postsynaptic structures that are not apposed by the nerve and thus not stabilized by agrin-MuSK signaling. Here we show that a miniaturized form of agrin, consisting of the laminin-binding and MuSK-activating domains, is sufficient to fully restore NMJs in agrin mutant mice when expressed by developing muscle. Although miniagrin is expressed uniformly throughout muscle fibers and induces ectopic AChR clusters, the size and the number of those AChR clusters contacted by the motor nerve increase during development. We provide experimental evidence that this is due to ACh, because the AChR agonist carbachol stabilizes AChR clusters in organotypic cultures of embryonic diaphragms. In summary, our results show that agrin function in NMJ development requires only two small domains, and that this function does not depend on the local deposition of agrin at synapses. Finally, they suggest a novel local function of ACh in stabilizing postsynaptic structures. PMID:18685098

  2. Comparative Studies of the Venom of a New Taipan Species, Oxyuranus temporalis, with Other Members of Its Genus

    Directory of Open Access Journals (Sweden)

    Carmel M. Barber

    2014-07-01

    Full Text Available Taipans are highly venomous Australo-Papuan elapids. A new species of taipan, the Western Desert Taipan (Oxyuranus temporalis, has been discovered with two specimens housed in captivity at the Adelaide Zoo. This study is the first investigation of O. temporalis venom and seeks to characterise and compare the neurotoxicity, lethality and biochemical properties of O. temporalis venom with other taipan venoms. Analysis of O. temporalis venom using size-exclusion and reverse-phase HPLC indicated a markedly simplified “profile” compared to other taipan venoms. SDS-PAGE and agarose gel electrophoresis analysis also indicated a relatively simple composition. Murine LD50 studies showed that O. temporalis venom is less lethal than O. microlepidotus venom. Venoms were tested in vitro, using the chick biventer cervicis nerve-muscle preparation. Based on t90 values, O. temporalis venom is highly neurotoxic abolishing indirect twitches far more rapidly than other taipan venoms. O. temporalis venom also abolished responses to exogenous acetylcholine and carbachol, indicating the presence of postsynaptic neurotoxins. Prior administration of CSL Taipan antivenom (CSL Limited neutralised the inhibitory effects of all taipan venoms. The results of this study suggest that the venom of the O. temporalis is highly neurotoxic in vitro and may contain procoagulant toxins, making this snake potentially dangerous to humans.

  3. Maternal Fructose Exposure Programs Metabolic Syndrome-Associated Bladder Overactivity in Young Adult Offspring

    Science.gov (United States)

    Lee, Wei-Chia; Tain, You-Lin; Wu, Kay L. H.; Leu, Steve; Chan, Julie Y. H.

    2016-01-01

    Maternal fructose exposure (MFE) programs the development of metabolic syndrome (MetS) in young adult offspring. Epidemiological data indicate that MetS may increase the risks of overactive bladder (OAB) symptoms. However, it remains unknown whether MFE programs MetS-associated bladder dysfunction in adult offspring. Using Sprague-Dawley rats, we investigated the effects of MFE during pregnancy and lactation on developmental programming of MetS-associated bladder dysfunction. In addition, next generation sequencing technology was used to identify potential transcripts involved in the programmed bladder dysfunction in adult male offspring to MFE. We found that MFE programmed the MetS-associated OAB symptoms (i.e., an increase in micturition frequency and a shortened mean inter-contractile interval) in young adult male offspring, alongside significant alterations in bladder transcripts, including Chrm2, Chrm3, P2rx1, Trpv4, and Vipr2 gene expression. At protein level, the expressions of M2-, M3-muscarinic and P2X1 receptor proteins were upregulated in the MFE bladder. Functionally, the carbachol-induced detrusor contractility was reduced in the MFE offspring. These data suggest that alterations in the bladder transcripts and impairment of the bladder cholinergic pathways may underlie the pathophysiology of programmed bladder dysfunction in adult offspring to MFE. PMID:27703194

  4. Profiles of secreted neuropeptides and catecholamines illustrate similarities and differences in response to stimulation by distinct secretagogues.

    Science.gov (United States)

    Podvin, Sonia; Bundey, Richard; Toneff, Thomas; Ziegler, Michael; Hook, Vivian

    2015-09-01

    The goal of this study was to define profiles of secreted neuropeptide and catecholamine neurotransmitters that undergo co-release from sympathoadrenal chromaffin cells upon stimulation by distinct secretagogues. Chromaffin cells of the adrenal medulla participate in the dynamic responses to stress, especially that of 'fight and flight', and, thus, analyses of the co-release of multiple neurotransmitters is necessary to gain knowledge of how the stress response regulates cell-cell communication among physiological systems. Results of this study demonstrated that six different secretagogues stimulated the co-release of the neuropeptides Met-enkephalin, galanin, NPY, and VIP with the catecholamines dopamine, norepinephrine, and epinephrine. Importantly, the quantitative profiles of the secreted neurotransmitters showed similarities and differences upon stimulation by the different secretagogues evaluated, composed of KCl depolarization, nicotine, carbachol, PACAP, bradykinin, and histamine. The rank-orders of the secreted profiles of the neurotransmitters were generally similar among these secretagogues, but differences in the secreted amounts of each neurotransmitter occurred with different secretagogues. Epinephrine among the catecholamines showed the highest level of secretion. (Met)enkephalin showed the largest levels of secretion compared to the other neuropeptides examined. Levels of secreted catecholamines were greater than that of the neuropeptides. These data support the hypothesis that profiles of secreted neuropeptide and catecholamine neurotransmitters show similarities and differences upon stimulation by distinct secretagogues. These results illustrate the co-release of concerted neurotransmitter profiles that participate in the stress response of the sympathoadrenal nervous system.

  5. Stereoselectivity of satropane, a novel tropane analog, on iris muscarinic receptor activation and intraocular hypotension

    Institute of Scientific and Technical Information of China (English)

    Liang ZHU; Hong-zhuan CHEN; Li-min YANG; Yong-yao CUI; Pei-li ZHENG; Yin-yao NIU; Hao WANG; Yang LU; Qiu-shi REN; Pi-jing WEI

    2008-01-01

    Aim: To study the stereoselectivity of satropane (3-paramethylbenzene sulfonyloxy-6-acetoxy tropane), a novel tropane analog, on iris muscarinic receptor activation and intraocular hypotension. Methods: The assays for radioligand-receptor binding, the contractile responses of isolated iris muscle, the miosis response, and the intraocular hypotension of the enantiomers of satropane were investigated. Results: In the binding analysis, S(-)satropane (lesatropane) completely com-peted against the [3H]quinuclydinyl benzilate-labeled ligand at muscarinic recep-tors in the iris muscle, whereas R(+)satropane failed to completely compete. In an isolated iris contractile assay, R,S(±)satropane and S(-)satropane produced a concentration-dependent contractile response with similar efficacy and potency to that of carbachol. R(+)satropane did not induce any contractile response. In the pupil diameter measurement assay in vivo, S(-)satropane induced miosis much more effectively than pilocarpine, while R(+)satropane failed to produce any miosis. In the water loading-induced and methylcellulose-induced ocular hypertensive models, S(-)satropane, but not R(+)satropane, significantly suppressed intraocu-lar pressure at a much lower concentration than pilocarpine. Conclusion: The ago-nistic and hypotensive properties of satropane on rabbit eyes are stereoselective, with the S(-)isomer being its active form.

  6. Rho kinase acts as a downstream molecule to participate in protein kinase Cε regulation of vascular reactivity after hemorrhagic shock in rats.

    Science.gov (United States)

    Li, Tao; Zhu, Yu; Zang, Jia-tao; Peng, Xiao-yong; Lan, Dan; Yang, Guang-ming; Xu, Jing; Liu, Liang-ming

    2014-09-01

    Our previous study demonstrated that Rho kinase and protein kinase C (PKC) played important parts in the regulation of vascular reactivity after shock. Using superior mesenteric arteries (SMAs) from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs), relationship of PKCε regulation of vascular reactivity to Rho kinase, as well as the signal transduction after shock, was investigated. The results showed that inhibition of Rho kinase with the Rho kinase-specific inhibitor Y-27632 antagonized the PKCε-specific agonist carbachol and highly expressed PKCε-induced increase of vascular reactivity in SMAs and VSMCs, whereas inhibition of PKCε with its specific inhibitory peptide did not antagonize the Rho kinase agonist (U-46619)-induced increase of vascular reactivity in SMAs and VSMCs. Activation of PKCε or highly expressed PKCε upregulated the activity of Rho kinase and the phosphorylation of PKC-dependent phosphatase inhibitor 17 (CPI-17), zipper interacting protein kinase (ZIPK), and integrin-linked kinase (ILK), whereas activation of Rho kinase increased only CPI-17 phosphorylation. The specific neutralization antibodies of ZIPK and ILK antagonized PKCε-induced increases in the activity of Rho kinase, but CPI-17 neutralization antibody did not antagonize this effect. These results suggested that Rho kinase takes part in the regulation of PKCε on vascular reactivity after shock. Rho kinase is downstream of PKCε. Protein kinase Cε activates Rho kinase via ZIPK and ILK; CPI-17 is downstream of Rho kinase.

  7. Genetic dissection of theta rhythm heterogeneity in mice.

    Science.gov (United States)

    Shin, Jonghan; Kim, Daesoo; Bianchi, Riccardo; Wong, Robert K S; Shin, Hee-Sup

    2005-12-13

    Rhythmic oscillatory activities at the theta frequency (4-12 Hz) in the hippocampus have long-attracted attention because they have been implicated in diverse brain functions, including spatial cognition. Although studies based on pharmacology and lesion experiments suggested heterogeneity of these rhythms and their behavioral correlates, controversies are abundant on these issues. Here we show that mice harboring a phospholipase C (PLC)-beta1(-/-) mutation (PLC-beta1(-/-) mice) lack one subset of theta rhythms normally observed during urethane anesthesia, alert immobility, and passive whole-body rotation. In contrast, the other subset of theta rhythms observed during walking or running was intact in these mutant mice. PLC-beta1(-/-) mice also have somewhat disrupted theta activity during paradoxical sleep but do have an atropine-resistant component of theta rhythm. In addition, carbachol-induced oscillations were obliterated in hippocampal slices of PLC-beta1(-/-) mice. Interestingly, PLC-beta1(-/-) mice showed deficits in a hidden platform version of the Morris water maze yet performed well in motor coordination tests and a visual platform version of the Morris water maze. The results genetically define the existence of at least two subtypes of theta rhythms and reveal their association with different behaviors.

  8. A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Venglarik, C.J.; Bridges, R.J.; Frizzell, R.A. (Univ. of Alabama, Birmingham (USA))

    1990-08-01

    We developed a convenient flux assay that permits simultaneous measurement of Cl and K conductance pathways in Cl-secreting epithelial cells. Monolayers of the colonic tumor cell line T84 were preloaded with 125I and 86Rb, and isotope effluxes were monitored by a sample-replace procedure. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agonists forskolin and prostaglandin E2 increased I efflux with little effect on Rb efflux, whereas the Ca-mediated agonists ionomycin, A23187, and carbachol increased both I and Rb effluxes. Simultaneous determinations of I and Cl or Rb and K effluxes indicated that I and Rb provide good measures of the effluxes of Cl and K, respectively. Forskolin- and ionomycin-stimulated I effluxes were inhibited by the Cl-channel blockers diphenylamine-2-dicarboxylate (DPC), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), and 2-(cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H- inden-5-yl-oxy)acetic acid (IAA-94) and by high external K. The Rb efflux evoked by ionomycin was inhibited by the K-channel blockers Ba and charybdotoxin. These findings suggest that I and Rb effluxes provide qualitative estimates of agonist-stimulated Cl and K conductance pathways. Thus this method can provide a simple and relatively inexpensive screening assay for Cl and K conductances in cultured cells to assess the effects of agonist, blockers, or genetic manipulations.

  9. Antidiarrheal activity of 19-deoxyicetexone isolated from Salvia ballotiflora Benth in mice and rats.

    Science.gov (United States)

    Pérez-Gutiérrez, Salud; Zavala-Mendoza, Daniel; Hernández-Munive, Abigail; Mendoza-Martínez, Angel; Pérez-González, Cuauhtemoc; Sánchez-Mendoza, Ernesto

    2013-01-01

    The antidiarrheal properties of 19-deoxyicetexone, a diterpenoid isolated from Salvia ballotiflora were evaluated on castor oil-, arachidonic acid (AA)- and prostaglandin (PGE₂)-induced diarrhea in rodent models. The structure of 19-deoxyicetexone was determined by X-ray crystallography, mass spectrometry (EI-MS), as well as ultraviolet (UV-Vis), infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopies. This compound significantly and dose-dependently reduced frequency of stooling in castor oil-induced diarrhea, and at dose of 25 mg/kg it also inhibited diarrhea induced with AA, while it had no effect on PGE₂-induced diarrhea. This compound at doses of 25 mg/kg also diminished castor oil-induced enteropooling and intestinal motility, and inhibited the contraction of the rats' ileum induced by carbachol chloride at a concentration of 100 µg/mL. 19-Deoxyicetexone did not present acute toxicity at doses of 625 mg/kg. Its antidiarrheal activity may be due to increased reabsorption of NaCl and water and inhibition of the release of prostaglandins, gastrointestinal motility and fluid accumulation in the intestinal tracts of rats. These findings suggest that 19-deoxyicetexone may be used in the treatment of diarrhea, although more studies must be carried out to confirm this. PMID:23896615

  10. The Raf-1 inhibitor GW5074 and dexamethasone suppress sidestream smoke-induced airway hyperresponsiveness in mice

    Directory of Open Access Journals (Sweden)

    Xu Cang-Bao

    2008-11-01

    Full Text Available Abstract Background Sidestream smoke is closely associated with airway inflammation and hyperreactivity. The present study was designed to investigate if the Raf-1 inhibitor GW5074 and the anti-inflammatory drug dexamethasone suppress airway hyperreactivity in a mouse model of sidestream smoke exposure. Methods Mice were repeatedly exposed to smoke from four cigarettes each day for four weeks. After the first week of the smoke exposure, the mice received either dexamethasone intraperitoneally every other day or GW5074 intraperitoneally every day for three weeks. The tone of the tracheal ring segments was recorded with a myograph system and concentration-response curves were obtained by cumulative administration of agonists. Histopathology was examined by light microscopy. Results Four weeks of exposure to cigarette smoke significantly increased the mouse airway contractile response to carbachol, endothelin-1 and potassium. Intraperitoneal administration of GW5074 or dexamethasone significantly suppressed the enhanced airway contractile responses, while airway epithelium-dependent relaxation was not affected. In addition, the smoke-induced infiltration of inflammatory cells and mucous gland hypertrophy were attenuated by the administration of GW5074 or dexamethasone. Conclusion Sidestream smoke induces airway contractile hyperresponsiveness. Inhibition of Raf-1 activity and airway inflammation suppresses smoking-associated airway hyperresponsiveness.

  11. Vestibular-evoked myogenic potentials in miniature pigs

    Institute of Scientific and Technical Information of China (English)

    Xi Shi; Yan Zhang; Ya Li; Shiwei Qiu; Shili Zhang; Yaohan Li; Na Yuan; Yuehua Qiao; Shiming Yang

    2016-01-01

    Objective:To report detection of vestibular-evoked myogenic potentials (VEMPs) in the miniature pig. Methods:Potentials evoked by 1000 Hz tone bursts were recorded from neck extensor muscles and the masseter muscles in normal adult Bama miniature pigs anesthetized with 3%pentobarbital sodium and Carbachol II. Results:The latency of the first positive wave P from neck extensor muscles was 7.65 ± 0.64 ms, with an amplitude of 1.66 ± 0.34 uv and a rate of successful induction of 75%at 80 dB SPL. The latency of potentials evoked from the masseter muscles was 7.60 ± 0.78 ms, with an amplitude of 1.31 ± 0.28 uv and a rate successful induction of 66%at 80 dB SPL. Conclusion:The latencies and thresholds of VEMPs recorded from the neck extensor muscle and the masseter muscle appear to be comparable in normal adult Bama miniature pigs, although the amplitude recorded from the neck extensor muscle seems to be higher than that from the masseter muscle. However, because of their usually relatively superficial and easily accessible location, as well as their large volume and strong contractions, masseter muscles may be better target muscles for recording myogenic potentials.

  12. IP3R deficit underlies loss of salivary fluid secretion in Sjögren’s Syndrome

    Science.gov (United States)

    Teos, Leyla Y.; Zhang, Yu; Cotrim, Ana P.; Swaim, William; Won, Jon H.; Ambrus, Julian; Shen, Long; Bebris, Lolita; Grisius, Margaret; Jang, Shyh-Ing; Yule, David I.; Ambudkar, Indu S.; Alevizos, Ilias

    2015-01-01

    The autoimmune exocrinopathy, Sjögren’s syndrome (SS), is associated with secretory defects in patients, including individuals with mild lymphocytic infiltration and minimal glandular damage. The mechanism(s) underlying the secretory dysfunction is not known. We have used minor salivary gland biopsies from SS patients and healthy individuals to assess acinar cell function in morphologically intact glandular areas. We report that agonist-regulated intracellular Ca2+ release, critically required for Ca2+ entry and fluid secretion, is defective in acini from SS patients. Importantly, these acini displayed reduction in IP3R2 and IP3R3, but not AQP5 or STIM1. Similar decreases in IP3R and carbachol (CCh)-stimulated [Ca2+]i elevation were detected in acinar cells from lymphotoxin-alpha (LTα) transgenic (TG) mice, a model for (SS). Treatment of salivary glands from healthy individuals with LT α, a cytokine linked to disease progression in SS and IL14α mice, reduced Ca2+ signaling. Together, our findings reveal novel IP3R deficits in acinar cells that underlie secretory dysfunction in SS patients. PMID:26365984

  13. CaMK Ⅱ在收缩促进骨骼肌细胞GLUT4转位中的作用%The Role of CaMKⅡ in Contraction-Stimulated GLUT4 Translocation in Skeletal Muscle Cells

    Institute of Scientific and Technical Information of China (English)

    于俊娜; 牛文彦

    2011-01-01

    Objective: To study the role of calcium-/calmodulin- dependent protein kinase Ⅱ (CaMK Ⅱ) in the mechanism of contraction-stimulated translocation of glucose transporter 4(GLUT4) in skeletal muscle cells. Methods:C2C12GLUT4myc myotubes were divided into two groups treated with or without carbachol (Cch). CaMK Ⅱ inhibitor KN93or KN62 was added to the medium prior to treatment, respectively. Then cell surface levels of GLUT4myc were measured by enzyme linked immunosorbent assay(ELISA). The phosphorylation of CaMK Ⅱ was detected by immunoblotting after pre-incubation with KN93. Results: Carbachol increased phosphorylation of CaMK Ⅱ which was reduced by KN93. KN93 and KN62 inhibited the gain of cell surface GLUT4myc induced by Cch. KN93 did not affect the phosphorylation of CaMK Ⅱ but it inhibited the CaMK Ⅱ phosphorylation stimulated by Cch. Conclusion: CaMK Ⅱ is the downstream signal of CaZ+ and mediates contraction-stimulated GLUT4myc traffic in skeletal muscle cells.%目的:探讨钙一钙调蛋白依赖性蛋白激酶Ⅱ(CaMK Ⅱ)在收缩促进骨骼肌细胞葡萄糖转运子4(GLUT4)转位机制中的作用.方法:将接种在培养板中的C2C12 GLUT4myc小鼠肌管随机分为对照组和氨乙酰胆碱(Cch)组,各组分为抑制剂亚组和对照亚组.分别用10 μmol/L CaMK Ⅱ的特异性抑制剂KN93或KN62预孵育,用酶联免疫吸附法(ELISA)测定细胞膜上GLUT4myc的含量(转位);用KN93预孵育后,免疫印迹法测定蛋白激酶CaMKⅡ的磷酸化.结果:氨乙酰胆碱使细胞膜上GLUT4myc的水平显著增加.KN93和KN62抑制氨乙酰胆碱刺激的GLUT4myc转位.氨乙酰胆碱可增加CaMKⅡ的磷酸化,KN93不影响对照组CaMK Ⅱ的磷酸化水平,但可以抑制Cch刺激的CaMK Ⅱ磷酸化.结论:CaMK Ⅱ位于Ca2+下游并有介导收缩促进骨骼肌细胞GLUT4myc转位的作用.

  14. Incontinência urinária após gonadectomia em fêmeas caninas Urinary incontinence after gonadectomy in female dogs

    Directory of Open Access Journals (Sweden)

    Fabiana Azevedo Voorwald

    2010-03-01

    Full Text Available A incontinência urinária adquirida é uma condição debilitante e, muitas vezes, incurável que acomete fêmeas castradas e raramente fêmeas inteiras ou machos. A manifestação clínica pode ocorrer em qualquer momento após a gonadectomia e resulta em graves problemas no manejo do paciente. Os mecanismos que desencadeiam a incontinência após ovariectomia envolvem decréscimo na pressão de fechamento uretral, alterações hormonais, aumento na deposição de colágeno na musculatura lisa da bexiga, diminuição na contratilidade do músculo detrusor e redução na resposta aos estímulos elétricos e ao carbachol. O diagnóstico é realizado pelo histórico do animal, pelo exame físico, pelos exames laboratoriais, pelo perfil de pressão uretral, pela ultrassonografia e pelas radiografias abdominais. O tratamento clínico envolve utilização de fármacos -adrenérgicos, estrógenos, análogos de GnRH e agentes antidepressivos. As técnicas cirúrgicas recomendadas correspondem à uretropexia, cistouretropexia, aplicação de colágeno na uretra e colpossuspensão. Melhor compreensão da etiologia, da fisiopatologia, dos métodos de diagnóstico e tratamentos é fundamental em razão do pouco conhecimento e da identificação dessa condição no Brasil.Acquired urinary incontinence is a debilitating, incurable condition, prevalent in spayed bitches and rarely seen in entire bitches or males. In bitches, acquired urinary incontinence can occur anytime from one week after neutering and is associated with severe management problems. Incontinence in neutered bitches can be associated with a decrease in maximal urethral closure pressure, hormonal changes and increase in the percentage of collagen in the bladder wall. It's also associated to a reduced contractility of the detrusor muscle, decrease in the magnitude of response of the bladder wall to both carbachol and eletrical field stimulation, and there are risk factors involved. The

  15. Connectivity, excitability and activity patterns in neuronal networks

    Science.gov (United States)

    le Feber, Joost; Stoyanova, Irina I.; Chiappalone, Michela

    2014-06-01

    Extremely synchronized firing patterns such as those observed in brain diseases like epilepsy may result from excessive network excitability. Although network excitability is closely related to (excitatory) connectivity, a direct measure for network excitability remains unavailable. Several methods currently exist for estimating network connectivity, most of which are related to cross-correlation. An example is the conditional firing probability (CFP) analysis which calculates the pairwise probability (CFPi,j) that electrode j records an action potential at time t = τ, given that electrode i recorded a spike at t = 0. However, electrode i often records multiple spikes within the analysis interval, and CFP values are biased by the on-going dynamic state of the network. Here we show that in a linear approximation this bias may be removed by deconvoluting CFPi,j with the autocorrelation of i (i.e. CFPi,i), to obtain the single pulse response (SPRi,j)—the average response at electrode j to a single spike at electrode i. Thus, in a linear system SPRs would be independent of the dynamic network state. Nonlinear components of synaptic transmission, such as facilitation and short term depression, will however still affect SPRs. Therefore SPRs provide a clean measure of network excitability. We used carbachol and ghrelin to moderately activate cultured cortical networks to affect their dynamic state. Both neuromodulators transformed the bursting firing patterns of the isolated networks into more dispersed firing. We show that the influence of the dynamic state on SPRs is much smaller than the effect on CFPs, but not zero. The remaining difference reflects the alteration in network excitability. We conclude that SPRs are less contaminated by the dynamic network state and that mild excitation may decrease network excitability, possibly through short term synaptic depression.

  16. Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors.

    Directory of Open Access Journals (Sweden)

    Paola Llanos

    Full Text Available Glucose-stimulated insulin secretion (GSIS from pancreatic β-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]. Glucose uptake into β-cells promotes Ca2+ influx and reactive oxygen species (ROS generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC, which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated β-cells with H2O2. Addition of stimulatory glucose or H2O2 (in basal glucose to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS.

  17. Synergistic actions of diacylglycerol and inositol 1,4,5 trisphosphate for Ca2+-dependent inactivation of TRPC7 channel1

    Institute of Scientific and Technical Information of China (English)

    Hua ZHANG; Ryuji INOUE; Juan SHI; Xiao-hang JIN; Yun-qing LI

    2008-01-01

    Aim: The aim of the present study was to explore the mechanism for the Ca2+-dependent inactivation of the canonical transient receptor potential (TRPC) 7 channel expressed in human embryonic kidney 293 cells. Method: The whole-cell patch-clamp technique was used in the study. Results: With Ca2+-free external solution, the perfusion of 100 μmol/L carbachol to, or dialysis of the cell with 100 μnol/L guanosine 5'-3-O-(thio)triphosphate (GTPγS), induced large inward currents, respectively. These currents were rapidly inhibited by the addition of 1 mmol/L Ca2+ into the bath, and recovery from this inhibition was only partial after the Ca2+ removal, unless vigorous intracellular Ca2+ buffering with 10 mmol/L 1,2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (plus 4 mmol/L Ca2+) was employed. In contrast, the current induced by a membrane-permeable analog of diacylglycerol (DAG), 1-oleoyl-2-acetyl-sn-glycerol (OAG; 100 μnol/L) did not undergo the inhibition persisting after Ca2+ removal. Interestingly, the inclusion of inositol 1,4,5 trisphosphate (IP3; 100 μmol/L) in the patch pipette rendered the OAG-induced current susceptible to the persistent Ca2+-mediated inhibition inde-pendent of the IP3 receptor in the majority of the tested cells, as evidenced by the inability of heparin and thapsigargin in reversing the effect of IP3. Conclusion: The present results suggest that Ca2+ entry via the activated TRPC7 channel plays a critical role in inactivating the channel where the cooperative actions of DAG and IP3 are essentially involved.

  18. Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction.

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    Saghir Akhtar

    Full Text Available Diabetes mellitus leads to vascular complications but the underlying signalling mechanisms are not fully understood. Here, we examined the role of ErbB2 (HER2/Neu, a transmembrane receptor tyrosine kinase of the ErbB/EGFR (epidermal growth factor receptor family, in mediating diabetes-induced vascular dysfunction in an experimental model of type 1 diabetes. Chronic treatment of streptozotocin-induced diabetic rats (1 mg/kg/alt diem or acute, ex-vivo (10(-6, 10(-5 M administration of AG825, a specific inhibitor of ErbB2, significantly corrected the diabetes-induced hyper-reactivity of the perfused mesenteric vascular bed (MVB to the vasoconstrictor, norephinephrine (NE and the attenuated responsiveness to the vasodilator, carbachol. Diabetes led to enhanced phosphorylation of ErbB2 at multiple tyrosine (Y residues (Y1221/1222, Y1248 and Y877 in the MVB that could be attenuated by chronic AG825 treatment. Diabetes- or high glucose-mediated upregulation of ErbB2 phosphorylation was coupled with activation of Rho kinases (ROCKs and ERK1/2 in MVB and in cultured vascular smooth muscle cells (VSMC that were attenuated upon treatment with either chronic or acute AG825 or with anti-ErbB2 siRNA. ErbB2 likley heterodimerizes with EGFR, as evidenced by increased co-association in diabetic MVB, and further supported by our finding that ERK1/2 and ROCKs are common downstream effectors since their activation could also be blocked by AG1478. Our results show for the first time that ErbB2 is an upstream effector of ROCKs and ERK1/2 in mediating diabetes-induced vascular dysfunction. Thus, potential strategies aimed at modifying actions of signal transduction pathways involving ErbB2 pathway may prove to be beneficial in treatment of diabetes-induced vascular complications.

  19. Novel role for the transient potential receptor melastatin 4 channel in guinea pig detrusor smooth muscle physiology.

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    Smith, Amy C; Hristov, Kiril L; Cheng, Qiuping; Xin, Wenkuan; Parajuli, Shankar P; Earley, Scott; Malysz, John; Petkov, Georgi V

    2013-03-01

    Members of the transient receptor potential (TRP) channel superfamily, including the Ca(2+)-activated monovalent cation-selective TRP melastatin 4 (TRPM4) channel, have been recently identified in the urinary bladder. However, their expression and function at the level of detrusor smooth muscle (DSM) remain largely unexplored. In this study, for the first time we investigated the role of TRPM4 channels in guinea pig DSM excitation-contraction coupling using a multidisciplinary approach encompassing protein detection, electrophysiology, live-cell Ca(2+) imaging, DSM contractility, and 9-phenanthrol, a recently characterized selective inhibitor of the TRPM4 channel. Western blot and immunocytochemistry experiments demonstrated the expression of the TRPM4 channel in whole DSM tissue and freshly isolated DSM cells with specific localization on the plasma membrane. Perforated whole cell patch-clamp recordings and real-time Ca(2+) imaging experiments with fura 2-AM, both using freshly isolated DSM cells, revealed that 9-phenanthrol (30 μM) significantly reduced the cation current and decreased intracellular Ca(2+) levels. 9-Phenanthrol (0.1-30 μM) significantly inhibited spontaneous, 0.1 μM carbachol-induced, 20 mM KCl-induced, and nerve-evoked contractions in guinea pig DSM-isolated strips with IC50 values of 1-7 μM and 70-80% maximum inhibition. 9-Phenanthrol also reduced nerve-evoked contraction amplitude induced by continuous repetitive electrical field stimulation of 10-Hz frequency and shifted the frequency-response curve (0.5-50 Hz) relative to the control. Collectively, our data demonstrate the novel finding that TRPM4 channels are expressed in guinea pig DSM and reveal their critical role in the regulation of guinea pig DSM excitation-contraction coupling.

  20. Muscarinic cholinergic regulation of L-type calcium channel in heart of embryonic mice at different developmental stages

    Institute of Scientific and Technical Information of China (English)

    Hua-minLIANG; MingTANG; Chang-jinLIU; Hong-yanLUO; Yuan-longSONG; Xin-wuHU; Jiao-yaXI; Lin-linGAO; BinNIE; Su-yunLI; Ling-lingLAI; JuergenHESCHELER

    2004-01-01

    AIM: To investigate the muscarinic regulation of L-type calcium current (ICa-L) during development. METHODS:The whole cell patch-clamp technique was used to record ICa-L in mice embryonic cardiomyocytes at different stages (the early developmental stage, EDS; the intermediate developmental stage, IDS; and the late developmental stage, LDS). Carbachol (CCh) was used to stimulate M-receptor in the embryonic cardiomyocytes of mice.RESULTS: The expression of lCa.L density did not change in different developmental stages (P>0.05). There was no difference in the sensitivity of ICa-L to CCh during development (P>0.05). This inhibitory action of CCh was mediated by inhibition of cyclic AMP since 8-bromo-cAMP completely reversed the muscarinic inhibitory action. IBMX, a non-selective inhibitor of phosphodiesterase (PDE), reversed the inhibitory action of M-receptor on ICa-L current by 71.2 %±9.2% (n=8) and 11.3%±2.5% (n=9) in EDS and LDS respectively. However forskolin, an agonist of adenylyl cyclase (AC), reversed the action of CCh by 14.5%±3.5% (n=5) and 82.7%± 10.4% (n=7) in EDS and LDS respectively. CONCLUSION: The inhibitory action of CCh on lca.L current was mediated in different pathways: in EDS, the inhibitory action of M-receptor on ICa-L channel mainly depended on the stimulation of PDE. However, in LDS, the regulation by M-receptor on lCa.L channel mainly depended on the inactivation of AC.

  1. Evidence that CFTR is expressed in rat tracheal smooth muscle cells and contributes to bronchodilation

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    Mettey Yvette

    2006-08-01

    Full Text Available Abstract Background The airway functions are profoundly affected in many diseases including asthma, chronic obstructive pulmonary disease (COPD and cystic fibrosis (CF. CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR gene, which normally encodes a multifunctional and integral membrane protein, the CF transmembrane conductance regulator (CFTR expressed in airway epithelial cells. Methods To demonstrate that CFTR is also expressed in tracheal smooth muscle cells (TSMC, we used iodide efflux assay to analyse the chloride transports in organ culture of rat TSMC, immunofluorescence study to localize CFTR proteins and isometric contraction measurement on isolated tracheal rings to observe the implication of CFTR in the bronchodilation. Results We characterized three different pathways stimulated by the cAMP agonist forskolin and the isoflavone agent genistein, by the calcium ionophore A23187 and by hypo-osmotic challenge. The pharmacology of the cAMP-dependent iodide efflux was investigated in detail. We demonstrated in rat TSMC that it is remarkably similar to that of the epithelial CFTR, both for activation (using three benzo [c]quinolizinium derivatives and for inhibition (glibenclamide, DPC and CFTRinh-172. Using rat tracheal rings, we observed that the activation of CFTR by benzoquinolizinium derivatives in TSMC leads to CFTRinh-172-sensitive bronchodilation after constriction with carbachol. An immunolocalisation study confirmed expression of CFTR in tracheal myocytes. Conclusion Altogether, these observations revealed that CFTR in the airways of rat is expressed not only in the epithelial cells but also in tracheal smooth muscle cells leading to the hypothesis that this ionic channel could contribute to bronchodilation.

  2. In-vitro effects of PDE5 inhibitor and statin treatment on the contractile responses of experimental MetS rabbit's cavernous smooth muscle

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    Yasin Erden

    2014-03-01

    Full Text Available Objective: Hypercholesterolaemia promotes erectile dysfunction through increased superoxide formation and decreased nitric oxide bioactivity in cavernosal tissue. The role of nitric oxide on erectile function is well known. Statins have lipid lowering properties and can modulate endothelial nitric oxide bioavailability. Sildenafil, enhances smooth muscle relaxation in corpus cavernosum. We invastigated in-vitro effects of sildenafil and rosuvastatin on nonadrenergic, non-cholinergic and nitric oxide mediated cavernosal smooth musle relaxation in metabolic syndrome rabbits, since alterations in this pathway are recognised in diabetic and hypercholesterolemic erectile dysfunction. Methods: Ten male rabbits were fed a standart diet as control group, fourty male rabbits were fed a hypercholesterolemic diet for 12 weeks. Hypercholesterolemic group were divided for without treatment, rosuvastatin treatment, sildenafil teratment, and rosuvastatin + sildenafil treatment (N = 10 per groups. Results: Serum levels of cholesterol and glucose were significantly higher in the experimental group than in the control group (p < 0.05. After theraphy no differences were found among the groups in relaxation responses to sodium nitroprusside. The relaxation responses to carbachol and EFS were significantly reduced in metabolic syndrome group to control group (p < 0.05, but there were no differences between the other groups and control group. There was a significantly lower in-vitro relaxation response in the metabolic syndrome rabbits than in controls and the others (p < 0.05. Conclusion: Both agents improve in-vitro relaxation responses of erectile tissue from metabolic syndrome rabbits to endothelial non-adrenergic, non-cholinergic and nitric oxide. This finding supports to the results of other clinical studies with these drugs.

  3. Muscarinic depolarization of layer II neurons of the parasubiculum.

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    Stephen D Glasgow

    Full Text Available The parasubiculum (PaS is a component of the hippocampal formation that sends its major output to layer II of the entorhinal cortex. The PaS receives strong cholinergic innervation from the basal forebrain that is likely to modulate neuronal excitability and contribute to theta-frequency network activity. The present study used whole cell current- and voltage-clamp recordings to determine the effects of cholinergic receptor activation on layer II PaS neurons. Bath application of carbachol (CCh; 10-50 µM resulted in a dose-dependent depolarization of morphologically-identified layer II stellate and pyramidal cells that was not prevented by blockade of excitatory and inhibitory synaptic inputs. Bath application of the M1 receptor antagonist pirenzepine (1 µM, but not the M2-preferring antagonist methoctramine (1 µM, blocked the depolarization, suggesting that it is dependent on M1 receptors. Voltage-clamp experiments using ramped voltage commands showed that CCh resulted in the gradual development of an inward current that was partially blocked by concurrent application of the selective Kv7.2/3 channel antagonist XE-991, which inhibits the muscarine-dependent K(+ current I M. The remaining inward current also reversed near EK and was inhibited by the K(+ channel blocker Ba(2+, suggesting that M1 receptor activation attenuates both I M as well as an additional K(+ current. The additional K(+ current showed rectification at depolarized voltages, similar to K(+ conductances mediated by Kir 2.3 channels. The cholinergic depolarization of layer II PaS neurons therefore appears to occur through M1-mediated effects on I M as well as an additional K(+ conductance.

  4. How do the rotavirus NSP4 and bacterial enterotoxins lead differently to diarrhea?

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    Vasseur Monique

    2007-03-01

    Full Text Available Abstract Rotavirus is the major cause of infantile gastroenteritis and each year causes 611 000 deaths worldwide. The virus infects the mature enterocytes of the villus tip of the small intestine and induces a watery diarrhea. Diarrhea can occur with no visible tissue damage and, conversely, the histological lesions can be asymptomatic. Rotavirus impairs activities of intestinal disaccharidases and Na+-solute symports coupled with water transport. Maldigestion of carbohydrates and their accumulation in the intestinal lumen as well as malabsorption of nutrients and a concomitant inhibition of water reabsorption can lead to a malabsorption component of diarrhea. Since the discovery of the NSP4 enterotoxin, diverse hypotheses have been proposed in favor of an additional secretion component in the pathogenesis of diarrhea. Rotavirus induces a moderate net chloride secretion at the onset of diarrhea, but the mechanisms appear to be quite different from those used by bacterial enterotoxins that cause pure secretory diarrhea. Rotavirus failed to stimulate Cl- secretion in crypt, whereas it stimulated Cl- reabsorption in villi, questioning, therefore, the origin of net Cl- secretion. A solution to this riddle was that intestinal villi do in fact secrete chloride as a result of rotavirus infection. Also, the overall chloride secretory response is regulated by a phospholipase C-dependent calcium signaling pathway induced by NSP4. However, the overall response is weak, suggesting that NSP4 may exert both secretory and subsequent anti-secretory actions, as did carbachol, hence limiting Cl- secretion. All these characteristics provide the means to make the necessary functional distinction between viral NSP4 and bacterial enterotoxins.

  5. Inhibition of Release of Vasoactive and Inflammatory Mediators in Airway and Vascular Tissues and Macrophages by a Chinese Herbal Medicine Formula for Allergic Rhinitis

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    George Binh Lenon

    2007-01-01

    Full Text Available Herbal therapies are being used increasingly for the treatment of allergic rhinitis. The aim of this study was to investigate the possible pharmacological actions and cellular targets of a Chinese herbal formula (RCM-101, which was previously shown to be effective in reducing seasonal allergic rhinitis symptoms in a randomized, placebo-controlled clinical trial. Rat and guinea pig isolated tissues (trachea and aorta were used to study the effects of RCM-101 on responses to various mediators. Production of leukotriene B4 in porcine neutrophils and of prostaglandin E2 and nitric oxide (NO in Raw 264.7 cells were also measured. In rat and guinea pig tracheal preparations, RCM-101 inhibited contractile responses to compound 48/80 but not those to histamine (guinea pig preparations or serotonin (rat preparations. Contractile responses of guinea pig tracheal preparations to carbachol and leukotriene C4, and relaxant responses to substance P and prostaglandin E2 were not affected by RCM-101. In rat aortic preparations, precontracted with phenylephrine, endothelium-dependent relaxant responses to acetylcholine and endothelium-independent relaxant responses to sodium nitroprusside were not affected by RCM-101. However, RCM-101 inhibited relaxations to l-arginine in endothelium-denuded rat aortic preparations, which had been pre-incubated with lipopolysaccharide. RCM-101 did not affect leukotriene B4 formation in isolated porcine neutrophils, induced by the calcium ionophore A23187; however, it inhibited prostaglandin E2 and NO production in lipopolysaccharide-stimulated murine macrophages (Raw 264.7 cells.The findings indicate that RCM-101 may have multiple inhibitory actions on the release and/or synthesis of inflammatory mediators involved in allergic rhinitis.

  6. Chronic Treatment with Ang-(1-7) Reverses Abnormal Reactivity in the Corpus Cavernosum and Normalizes Diabetes-Induced Changes in the Protein Levels of ACE, ACE2, ROCK1, ROCK2 and Omega-Hydroxylase in a Rat Model of Type 1 Diabetes

    Science.gov (United States)

    Yousif, Mariam H. M.; Makki, Batoul; El-Hashim, Ahmed Z.; Benter, Ibrahim F.

    2014-01-01

    Angiotensin-(1-7) [Ang-(1-7)] may have beneficial effects in diabetes mellitus-induced erectile dysfunction (DMIED) but its molecular actions in the diabetic corpus cavernosum (CC) are not known. We characterized the effects of diabetes and/or chronic in vivo administration of Ang-(1-7) on vascular reactivity in the rat corpus cavernosum (CC) and on protein expression levels of potential downstream effectors of the renin-angiotensin-aldosterone system (RAAS) such as angiotensin-converting enzyme (ACE), ACE2, Rho kinases 1 and 2 (ROCK1 and ROCK2), and omega-hydroxylase, the cytochrome-P450 enzyme that metabolizes arachidonic acid to form the vasoconstrictor, 20-hydroxyeicosatetraenoic acid. Streptozotocin-treated rats were chronicically administered Ang-(1-7) with or without A779, a Mas receptor antagonist, during weeks 4 to 6 of diabetes. Ang-(1-7) reversed diabetes-induced abnormal reactivity to vasoactive agents (endothelin-1, phenylepherine, and carbachol) in the CC without correcting hyperglycemia. Six weeks of diabetes led to elevated ACE, ROCK1, ROCK 2, and omega-hydroxylase and a concomitant decrease in ACE2 protein expression levels that were normalized by Ang-(1-7) treatment but not upon coadministration of A779. These data are supportive of the notion that the beneficial effects of Ang-(1-7) in DMIED involve counterregulation of diabetes-induced changes in ACE, ACE2, Rho kinases, and omega-hydroxylase proteins in the diabetic CC via a Mas receptor-dependent mechanism. PMID:25309930

  7. Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine.

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    Schreiber, Rainer; Faria, Diana; Skryabin, Boris V; Wanitchakool, Podchanart; Rock, Jason R; Kunzelmann, Karl

    2015-06-01

    Intestinal epithelial electrolyte secretion is activated by increase in intracellular cAMP or Ca(2+) and opening of apical Cl(-) channels. In infants and young animals, but not in adults, Ca(2+)-activated chloride channels may cause secretory diarrhea during rotavirus infection. While detailed knowledge exists concerning the contribution of cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) channels, analysis of the role of Ca(2+)-dependent Cl(-) channels became possible through identification of the anoctamin (TMEM16) family of proteins. We demonstrate expression of several anoctamin paralogues in mouse small and large intestines. Using intestinal-specific mouse knockout models for anoctamin 1 (Ano1) and anoctamin 10 (Ano10) and a conventional knockout model for anoctamin 6 (Ano6), we demonstrate the role of anoctamins for Ca(2+)-dependent Cl(-) secretion induced by the muscarinic agonist carbachol (CCH). Ano1 is preferentially expressed in the ileum and large intestine, where it supports Ca(2+)-activated Cl(-) secretion. In contrast, Ano10 is essential for Ca(2+)-dependent Cl(-) secretion in jejunum, where expression of Ano1 was not detected. Although broadly expressed, Ano6 has no role in intestinal cholinergic Cl(-) secretion. Ano1 is located in a basolateral compartment/membrane rather than in the apical membrane, where it supports CCH-induced Ca(2+) increase, while the essential and possibly only apical Cl(-) channel is CFTR. These results define a new role of Ano1 for intestinal Ca(2+)-dependent Cl(-) secretion and demonstrate for the first time a contribution of Ano10 to intestinal transport.

  8. Contractile profile of esophageal and gastric fundus strips in experimental doxorubicin-induced esophageal atresia

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    F.A. Capeto

    2015-05-01

    Full Text Available Esophageal atresia (EA is characterized by esophageal and gastric motility changes secondary to developmental and postsurgical damage. This study evaluated the in vitro contractile profile of the distal esophagus and gastric fundus in an experimental model of EA induced by doxorubicin (DOXO. Wistar pregnant rats received DOXO 2.2 mg/kg on the 8th and 9th gestational days. On day 21.5, fetuses were collected, sacrificed, and divided into groups: control, DOXO without EA (DOXO-EA, and DOXO with EA (DOXO+EA. Strips from the distal esophagus and gastric fundus were mounted on a wire myograph and isolated organ-bath system, respectively, and subjected to increasing concentrations of carbamylcholine chloride (carbachol, CCh. The isolated esophagus was also stimulated with increasing concentrations of KCl. In esophagus, the concentration-effect curves were reduced in response to CCh in the DOXO+EA and DOXO-EA groups compared to the control group (P0.05. In response to KCl, the distal esophagus samples in the three groups were not statistically different with regard to Emax or EC50 values (P>0.05. No significant difference was noted for EC50 or Emax values in fundic strips stimulated with CCh (P>0.05. In conclusion, exposure of dams to DOXO during gestation inhibited the contractile behavior of esophageal strips from offspring in response to CCh but not KCl, regardless of EA induction. The gastric fundus of DOXO-exposed offspring did not have altered contractile responsiveness to cholinergic stimulation.

  9. Matrine Inhibits Pacing Induced Atrial Fibrillation by Modulating IKM3 and ICa-L

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    Yuhong Zhou, Wei Xu, Ruyi Han, Jiaying Zhou, Zhenwei Pan, Huo Rong, Junnan Li, Changqing Xu, Guofen Qiao, Yanjie Lu

    2012-01-01

    Full Text Available Aim: To elucidate the protective effects of Matrine on atrial fibrillation (AF induced by electric pacing in mice and underlying molecular and ion channel mechanisms.Methods: AF was introduced by electric pacing in mice and the incidence and duration of AF were evaluated. Functional expression of M3 receptor (M3-R and Cav1.2 were explored by western and Real-time PCR, action potential (AP and the density of (IKM3 L-type calcium channel (ICa-L were both recorded using whole-cell patch in isolated atrial cardiomyocytes.Results: In control group, incidence and duration of AF induced by electric pacing were 50 ± 17% and 3.68 ± 1.84 s, respectively; after application of carbachol 50 µg/kg both incidence and duration of AF were significantly increased to 86 ± 24% and 65.2 ± 29.0 s. Compared with control group, pretreatment of Matrine for 15 days significantly reduced AF incidence and duration in dose-dependent manner. Atrial membrane-protein expression of M3-R was decreased and membrane Cav1.2 expression was up-regulated. In single Matrine-treated atrial cardiomyocyte the density of IKM3 was significantly decreased by 39% as well compared with control group, P Ca-L density of atrium was increased by 40%.Conclusion: These data demonstrated at the first time that the anti-AF effects of Matrine may due, at least in part, to down-regulation of IKM3 density and M3-R expression and up-regulation of ICa-L density and α1C/Cav1.2 expression.

  10. Effects of fetal and neonatal exposure to nicotine on blood pressure and perivascular adipose tissue function in adult life.

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    Gao, Yu-Jing; Holloway, Alison C; Su, Li-Ying; Takemori, Kumiko; Lu, Chao; Lee, Robert M K W

    2008-08-20

    In Wistar rats, maternal exposure to nicotine was shown to impair the inhibitory function of perivascular adipose tissue on vascular contractility in the aorta of the offspring. It is not known whether an impairment of perivascular adipose tissue function occurs in smaller arteries, and whether the control of blood pressure is affected. Here we studied the blood pressure effects and the alteration of perivascular adipose tissue function in mesenteric arteries of the offspring born to Wistar-Kyoto rat (WKY) dams exposed to nicotine. Nulliparous female WKY rats were given either nicotine bitartrate (1 mg/kg/day) or saline (vehicle) by subcutaneous injection 2 weeks prior to mating, during pregnancy and until weaning. Blood pressure of the offspring and functional studies with mesenteric arteries were conducted. Tissue samples (thoracic aorta, mesenteric arteries, and kidneys) were collected for morphological and immunohistochemical examinations. Blood pressure increased from 14 weeks of age onwards in the offspring born to nicotine-exposed dams. Nicotine-exposed offspring showed a significant increase in the number of brown adipocytes in aortic perivascular adipose tissue relative to control offspring. In mesenteric arteries from control offspring, contractile responses induced by phenylephrine, serotonin, and 9,11-dideoxy-11alpha, 9alpha-epoxymethanoprostaglandin F(2)alpha (U44619) were significantly attenuated in the presence of perivascular adipose tissue, an effect not observed in the nicotine-exposed tissues. Endothelium-dependent relaxation responses to carbachol, kidney weight, the total number of nephrons and glomerulus' size were comparable in nicotine and saline groups. We conclude that fetal and neonatal exposure to nicotine caused blood pressure elevation. Alterations in perivascular adipose tissue composition and modulatory function are some of the mechanisms associated with this blood pressure increase. PMID:18647709

  11. Opiatergic participation in the thirst-inhibiting effect of acute third ventricle injections of cadmium (Cd2+ and lead (Pb2+

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    E. De-Castro-e-Silva

    1998-06-01

    Full Text Available We have previously demonstrated that acute third ventricle injections of both lead and cadmium prevent the dipsogenic response elicited by dehydration or by central injections of dipsogenic agents such as angiotensin II, carbachol and isoproterenol in rats. We have also shown that the antidipsogenic action of cadmium may be due, at least in part, to activation of thirst-inhibitory central serotonergic pathways. In the present paper we show that in Wistar male rats the antidipsogenic effect of both lead acetate (3.0 nmol/rat and cadmium chloride (3.0 nmol/rat may be partially dependent on the activation of brain opiatergic pathways since central injections of naloxone (82.5 nmol/rat, a non-selective opioid antagonist, blunt the thirst-inhibiting effect of these metals. One hundred and twenty minutes after the second third ventricle injections, dehydrated animals (14 h overnight receiving saline + sodium acetate displayed a high water intake (7.90 ± 0.47 ml/100 g body weight whereas animals receiving saline + lead acetate drank 3.24 ± 0.47 ml/100 g body weight. Animals receiving naloxone + lead acetate drank 6.94 ± 0.60 ml/100 g body weight. Animals receiving saline + saline drank 8.16 ± 0.66 ml/100 g body weight whilst animals receiving saline + cadmium chloride drank 1.63 ± 0.37 ml/100 g body weight. Animals receiving naloxone + cadmium chloride drank 8.01 ± 0.94 ml/100 g body weight. It is suggested that acute third ventricle injections of both lead and cadmium exert their antidipsogenic effect by activating thirst-inhibiting opioid pathways in the brain.

  12. Vagotomy ameliorates islet morphofunction and body metabolic homeostasis in MSG-obese rats

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    Lubaczeuski, C.; Balbo, S.L. [Laboratório de Fisiologia Endócrina e Metabolismo, Centro de Ciências Biológicas e da Saúde, Universidade Estadual do Oeste do Paraná, Cascavel, PR (Brazil); Ribeiro, R.A. [Universidade Federal do Rio de Janeiro, Macaé, RJ (Brazil); Vettorazzi, J.F.; Santos-Silva, J.C.; Carneiro, E.M. [Laboratório de Pâncreas Endócrino e Metabolismo, Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP (Brazil); Bonfleur, M.L. [Laboratório de Fisiologia Endócrina e Metabolismo, Centro de Ciências Biológicas e da Saúde, Universidade Estadual do Oeste do Paraná, Cascavel, PR (Brazil)

    2015-02-24

    The parasympathetic nervous system is important for β-cell secretion and mass regulation. Here, we characterized involvement of the vagus nerve in pancreatic β-cell morphofunctional regulation and body nutrient homeostasis in 90-day-old monosodium glutamate (MSG)-obese rats. Male newborn Wistar rats received MSG (4 g/kg body weight) or saline [control (CTL) group] during the first 5 days of life. At 30 days of age, both groups of rats were submitted to sham-surgery (CTL and MSG groups) or subdiaphragmatic vagotomy (Cvag and Mvag groups). The 90-day-old MSG rats presented obesity, hyperinsulinemia, insulin resistance, and hypertriglyceridemia. Their pancreatic islets hypersecreted insulin in response to glucose but did not increase insulin release upon carbachol (Cch) stimulus, despite a higher intracellular Ca{sup 2+} mobilization. Furthermore, while the pancreas weight was 34% lower in MSG rats, no alteration in islet and β-cell mass was observed. However, in the MSG pancreas, increases of 51% and 55% were observed in the total islet and β-cell area/pancreas section, respectively. Also, the β-cell number per β-cell area was 19% higher in MSG rat pancreas than in CTL pancreas. Vagotomy prevented obesity, reducing 25% of body fat stores and ameliorated glucose homeostasis in Mvag rats. Mvag islets demonstrated partially reduced insulin secretion in response to 11.1 mM glucose and presented normalization of Cch-induced Ca{sup 2+} mobilization and insulin release. All morphometric parameters were similar among Mvag and CTL rat pancreases. Therefore, the higher insulin release in MSG rats was associated with greater β-cell/islet numbers and not due to hypertrophy. Vagotomy improved whole body nutrient homeostasis and endocrine pancreatic morphofunction in Mvag rats.

  13. Vagotomy ameliorates islet morphofunction and body metabolic homeostasis in MSG-obese rats

    International Nuclear Information System (INIS)

    The parasympathetic nervous system is important for β-cell secretion and mass regulation. Here, we characterized involvement of the vagus nerve in pancreatic β-cell morphofunctional regulation and body nutrient homeostasis in 90-day-old monosodium glutamate (MSG)-obese rats. Male newborn Wistar rats received MSG (4 g/kg body weight) or saline [control (CTL) group] during the first 5 days of life. At 30 days of age, both groups of rats were submitted to sham-surgery (CTL and MSG groups) or subdiaphragmatic vagotomy (Cvag and Mvag groups). The 90-day-old MSG rats presented obesity, hyperinsulinemia, insulin resistance, and hypertriglyceridemia. Their pancreatic islets hypersecreted insulin in response to glucose but did not increase insulin release upon carbachol (Cch) stimulus, despite a higher intracellular Ca2+ mobilization. Furthermore, while the pancreas weight was 34% lower in MSG rats, no alteration in islet and β-cell mass was observed. However, in the MSG pancreas, increases of 51% and 55% were observed in the total islet and β-cell area/pancreas section, respectively. Also, the β-cell number per β-cell area was 19% higher in MSG rat pancreas than in CTL pancreas. Vagotomy prevented obesity, reducing 25% of body fat stores and ameliorated glucose homeostasis in Mvag rats. Mvag islets demonstrated partially reduced insulin secretion in response to 11.1 mM glucose and presented normalization of Cch-induced Ca2+ mobilization and insulin release. All morphometric parameters were similar among Mvag and CTL rat pancreases. Therefore, the higher insulin release in MSG rats was associated with greater β-cell/islet numbers and not due to hypertrophy. Vagotomy improved whole body nutrient homeostasis and endocrine pancreatic morphofunction in Mvag rats

  14. Hypocretinergic and non-hypocretinergic projections from the hypothalamus to the REM sleep executive area of the pons.

    Science.gov (United States)

    Torterolo, Pablo; Sampogna, Sharon; Chase, Michael H

    2013-01-23

    Within the postero-lateral hypothalamus neurons that utilize hypocretin or melanin-concentrating hormone (MCH) as neuromodulators are co-distributed. These neurons have been involved in the control of behavioral states, and a deficit in the hypocretinergic system is the pathogenic basis of narcolepsy with cataplexy. In this report, utilizing immunohistochemistry and retrograde tracing techniques, we examined the hypocretinergic innervation of the nucleus pontis oralis (NPO), which is the executive site that is responsible for the generation of REM sleep in the cat. The retrograde tracer cholera toxin subunit b (CTb) was administered in pontine regions where carbachol microinjections induced REM sleep. Utilizing immunohistochemical techniques, we found that approximately 1% of hypocretinergic neurons in the tuberal area of the hypothalamus project to the NPO. In addition, approximately 6% of all CTb+ neurons in this region were hypocretinergic. The hypocretinergic innervation of the NPO was also compared with the innervation of the same site by MCH-containing neurons. More than three times as many MCHergic neurons were found to project to the NPO compared with hypocretinergic cells; both neuronal types exhibited bilateral projections. We also identified a group of non-hypocretinergic non-MCHergic neuronal group of neurons that were intermingled with both hypocretinergic and MCHergic neurons that also projected to this same brainstem region. These neurons were grater in number that either hypocretin or MCH-containing neurons; their soma size was also smaller and their projections were mainly ipsilateral. The present anatomical data suggest that hypocretinergic, MCHergic and an unidentified companion group of neurons of the postero-lateral hypothalamus participate in the regulation of the neuronal activity of NPO neurons, and therefore, are likely to participate in the control of wakefulness and REM sleep. PMID:23122879

  15. State-dependent control of lumbar motoneurons by the hypocretinergic system.

    Science.gov (United States)

    Yamuy, Jack; Fung, Simon J; Xi, Mingchu; Chase, Michael H

    2010-02-01

    Neurons in the lateral hypothalamus (LH) that synthesize hypocretins (Hcrt-1 and Hcrt-2) are active during wakefulness and excite lumbar motoneurons. Because hypocretinergic cells also discharge during phasic periods of rapid eye movement (REM) sleep, we sought to examine their action on the activity of motoneurons during this state. Accordingly, cat lumbar motoneurons were intracellularly recorded, under alpha-chloralose anesthesia, prior to (control) and during the carbachol-induced REM sleep-like atonia (REMc). During control conditions, LH stimulation induced excitatory postsynaptic potentials (composite EPSP) in motoneurons. In contrast, during REMc, identical LH stimulation induced inhibitory PSPs in motoneurons. We then tested the effects of LH stimulation on motoneuron responses following the stimulation of the nucleus reticularis gigantocellularis (NRGc) which is part of a brainstem-spinal cord system that controls motoneuron excitability in a state-dependent manner. LH stimulation facilitated NRGc stimulation-induced composite EPSP during control conditions whereas it enhanced NRGc stimulation-induced IPSPs during REMc. These intriguing data indicate that the LH exerts a state-dependent control of motor activity. As a first step to understand these results, we examined whether hypocretinergic synaptic mechanisms in the spinal cord were state dependent. We found that the juxtacellular application of Hcrt-1 induced motoneuron excitation during control conditions whereas motoneuron inhibition was enhanced during REMc. These data indicate that the hypocretinergic system acts on motoneurons in a state-dependent manner via spinal synaptic mechanisms. Thus, deficits in Hcrt-1 may cause the coexistence of incongruous motor signs in cataplectic patients, such as motor suppression during wakefulness and movement disorders during REM sleep. PMID:19962375

  16. Nitrergic ventro-medial medullary neurons activated during cholinergically induced active (rapid eye movement) sleep in the cat.

    Science.gov (United States)

    Pose, I; Sampogna, S; Chase, M H; Morales, F R

    2011-01-13

    The rostral ventro-medial medullary reticular formation is a complex structure that is involved with a variety of motor functions. It contains glycinergic neurons that are activated during active (rapid eye movement (REM)) sleep (AS); these neurons appear to be responsible for the postsynaptic inhibition of motoneurons that occurs during this state. We have reported that neurons in this same region contain nitric oxide (NO) synthase and that they innervate brainstem motor pools. In the present study we examined the c-fos expression of these neurons after carbachol-induced active sleep (C-AS). Three control and four experimental cats were employed to identify c-fos expressing nitrergic neurons using immunocytochemical techniques to detect the Fos protein together with neuronal nitric oxide synthase (nNOS) or nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity. The classical neurotransmitter content of the nitrergic cells in this region was examined through the combination of immunocytochemical techniques for the detection of glutamate, glycine, choline acetyltransferase (ChAT), tyrosine hydroxilase (TH) or GABA together with nNOS. During C-AS, there was a 1074% increase in the number of nitrergic neurons that expressed c-fos. These neurons did not contain glycine, ChAT, TH or GABA, but a subpopulation (15%) of them displayed glutamate-like immunoreactivity. Therefore, some of these neurons contain both an excitatory neurotransmitter (glutamate) and an excitatory neuromodulator (NO); the neurotransmitter content of the rest of them remains to be determined. Because some of the nitrergic neurons innervate brainstem motoneurons it is possible that they participate in the generation of tonic and excitatory phasic motor events that occur during AS. We also suggest that these nitrergic neurons may be involved in autonomic regulation during this state. In addition, because NO has trophic effects on target neurons, the present findings represent the

  17. Identification of ML204, a Novel Potent Antagonist That Selectively Modulates Native TRPC4/C5 Ion Channels*

    Science.gov (United States)

    Miller, Melissa; Shi, Jie; Zhu, Yingmin; Kustov, Maksym; Tian, Jin-bin; Stevens, Amy; Wu, Meng; Xu, Jia; Long, Shunyou; Yang, Pu; Zholos, Alexander V.; Salovich, James M.; Weaver, C. David; Hopkins, Corey R.; Lindsley, Craig W.; McManus, Owen; Li, Min; Zhu, Michael X.

    2011-01-01

    Transient receptor potential canonical (TRPC) channels are Ca2+-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca2+ rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca2+ rise with an IC50 value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5′-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10–20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4. PMID:21795696

  18. Transient and sustained afterdepolarizations in accessory olfactory bulb mitral cells are mediated by distinct mechanisms that are differentially regulated by neuromodulators

    Directory of Open Access Journals (Sweden)

    Guy eShpak

    2015-01-01

    Full Text Available Social interactions between mammalian conspecifics rely heavily on molecular communication via the main and accessory olfactory systems. These two chemosensory systems show high similarity in the organization of information flow along their early stages: social chemical cues are detected by the sensory neurons of the main olfactory epithelium and the vomeronasal organ. These neurons then convey sensory information to the main (MOB and accessory (AOB olfactory bulbs, respectively, where they synapse upon mitral cells that project to higher brain areas. Yet, the functional difference between these two chemosensory systems remains unclear. We have previously shown that MOB and AOB mitral cells exhibit very distinct intrinsic biophysical properties leading to different types of information processing. Specifically, we found that unlike MOB mitral cells, AOB neurons display persistent firing responses to strong stimuli. These prolonged responses are mediated by long-lasting calcium-activated non-selective cationic current (Ican. In the current study we further examined the firing characteristics of these cells and their modulation by several neuromodulators. We found that AOB mitral cells display transient depolarizing afterpotentials (DAPs following moderate firing. These DAPs are not found in MOB mitral cells that show instead robust hyperpolarizing afterpotentials. Unlike Ican, the DAPs of AOB mitral cells are activated by low levels of intracellular calcium and are relatively insensitive to flufenamic acid. Moreover, the cholinergic agonist carbachol exerts opposite effects on the persistent firing and DAPs of AOB mitral cells. We conclude that these phenomena are mediated by distinct biophysical mechanisms that may serve to mediate different types of information processing in the AOB at distinct brain states.

  19. Transient and sustained afterdepolarizations in accessory olfactory bulb mitral cells are mediated by distinct mechanisms that are differentially regulated by neuromodulators.

    Science.gov (United States)

    Shpak, Guy; Zylbertal, Asaph; Wagner, Shlomo

    2014-01-01

    Social interactions between mammalian conspecifics rely heavily on molecular communication via the main and accessory olfactory systems. These two chemosensory systems show high similarity in the organization of information flow along their early stages: social chemical cues are detected by the sensory neurons of the main olfactory epithelium and the vomeronasal organ. These neurons then convey sensory information to the main (MOB) and accessory (AOB) olfactory bulbs, respectively, where they synapse upon mitral cells that project to higher brain areas. Yet, the functional difference between these two chemosensory systems remains unclear. We have previously shown that MOB and AOB mitral cells exhibit very distinct intrinsic biophysical properties leading to different types of information processing. Specifically, we found that unlike MOB mitral cells, AOB neurons display persistent firing responses to strong stimuli. These prolonged responses are mediated by long-lasting calcium-activated non-selective cationic current (Ican). In the current study we further examined the firing characteristics of these cells and their modulation by several neuromodulators. We found that AOB mitral cells display transient depolarizing afterpotentials (DAPs) following moderate firing. These DAPs are not found in MOB mitral cells that show instead robust hyperpolarizing afterpotentials. Unlike Ican, the DAPs of AOB mitral cells are activated by low levels of intracellular calcium and are relatively insensitive to flufenamic acid. Moreover, the cholinergic agonist carbachol exerts opposite effects on the persistent firing and DAPs of AOB mitral cells. We conclude that these phenomena are mediated by distinct biophysical mechanisms that may serve to mediate different types of information processing in the AOB at distinct brain states. PMID:25642164

  20. Vascular progenitor cells isolated from human embryonic stem cells give rise to endothelial and smooth muscle like cells and form vascular networks in vivo.

    Science.gov (United States)

    Ferreira, Lino S; Gerecht, Sharon; Shieh, Hester F; Watson, Nicki; Rupnick, Maria A; Dallabrida, Susan M; Vunjak-Novakovic, Gordana; Langer, Robert

    2007-08-01

    We report that human embryonic stem cells contain a population of vascular progenitor cells that have the ability to differentiate into endothelial-like and smooth muscle (SM)-like cells. Vascular progenitor cells were isolated from EBs grown in suspension for 10 days and were characterized by expression of the endothelial/hematopoietic marker CD34 (CD34+ cells). When these cells are subsequently cultured in EGM-2 (endothelial growth medium) supplemented with vascular endothelial growth factor-165 (50 ng/mL), they give rise to endothelial-like cells characterized by a cobblestone cell morphology, expression of endothelial markers (platelet endothelial cell-adhesion molecule-1, CD34, KDR/Flk-1, vascular endothelial cadherin, von Willebrand factor), incorporation of acetylated low-density lipoprotein, and formation of capillary-like structures when placed in Matrigel. In contrast, when CD34+ cells are cultured in EGM-2 supplemented with platelet-derived growth factor-BB (50 ng/mL), they give rise to SM-like cells characterized by spindle-shape morphology, expression of SM cell markers (alpha-SM actin, SM myosin heavy chain, calponin, caldesmon, SM alpha-22), and the ability to contract and relax in response to common pharmacological agents such as carbachol and atropine but rarely form capillary-like structures when placed in Matrigel. Implantation studies in nude mice show that both cell types contribute to the formation of human microvasculature. Some microvessels contained mouse blood cells, which indicates functional integration with host vasculature. Therefore, the vascular progenitors isolated from human embryonic stem cells using methods established in the present study could provide a means to examine the mechanisms of endothelial and SM cell development, and they could also provide a potential source of cells for vascular tissue engineering.

  1. Evaluation of intestinal absorption enhancement and local mucosal toxicity of two promoters. I. Studies in isolated rat and human colonic mucosae.

    Science.gov (United States)

    Maher, Sam; Kennelly, Rory; Bzik, Victoria A; Baird, Alan W; Wang, Xuexuan; Winter, Desmond; Brayden, David J

    2009-11-01

    The effects of two absorption promoters, (sodium caprate (C(10)) and melittin), on intestinal permeability and viability were measured in intact rat and human colonic epithelia mounted in Ussing chambers. Apical-side addition of C(10) (10 mM) and melittin (10-50 microM) rapidly reduced the transepithelial electrical resistance (TEER) and increased the apparent permeability coefficient (Papp) of [(14)C]-mannitol and FITC-dextran-4 kDa (FD4) across colonic mucosae from both species. Effects of C(10) on flux were greater than those of melittin at the concentrations selected. C(10) irreversibly decreased TEER, but the effects of melittin were partially reversible. Enhanced permeability of polar sugars (0.18-70 kDa) in colonic mucosae with C(10) was accompanied by significant release of lactate dehydrogenase (LDH) from the luminal surface as well as by inhibition of electrogenic chloride secretion induced by the muscarinic agonist, carbachol (0.1-10 microM). Although melittin did not alter electrogenic chloride secretion in rat or human colonic mucosae, it caused leakage of LDH from rat tissue. Gross histology and electron microscopy of rat and human colonic mucosae demonstrated that each permeation enhancer can induce colonic epithelial damage at concentrations required to increase marker fluxes. C(10) led to more significant mucosal damage than melittin, characterised by sloughing and mucosal erosion. Overall, these results indicate that while C(10) and melittin increase transport of paracellular flux markers across isolated human and rat colonic mucosae in vitro, these effects are associated with some cytotoxicity. PMID:19737613

  2. Muscarinic acetylcholine receptor subtype 4 is essential for cholinergic stimulation of duodenal bicarbonate secretion in mice - relationship to D cell/somatostatin.

    Science.gov (United States)

    Takeuchi, K; Kita, K; Takahashi, K; Aihara, E; Hayashi, S

    2015-06-01

    We investigated the roles of muscarinic (M) acetylcholine receptor subtype in the cholinergic stimulation of duodenal HCO3(-) secretion using knockout (KO) mice. Wild-type and M1-M5 KO C57BL/6J mice were used. The duodenal mucosa was mounted on an Ussing chamber, and HCO3(-) secretion was measured at pH 7.0 using a pH-stat method in vitro. Carbachol (CCh) or other agents were added to the serosal side. CCh dose-dependently stimulated HCO3(-) secretion in wild-type mice, and this effect was completely inhibited in the presence of atropine. The HCO3(-) response to CCh in wild-type mice was also inhibited by pirenzepine (M1 antagonist), 4DAMP (M3 antagonist), and tropicamide (M4 antagonist), but not by methoctramine (M2 antagonist). CCh stimulated HCO3(-) secretion in M2 and M5 KO animals as effectively as in WT mice; however, this stimulatory effect was significantly attenuated in M1, M3, and M4 KO mice. The decrease observed in the CCh-stimulated HCO3(-) response in M4 KO mice was reversed by the co-application of CYN154806, a somatostatin receptor type 2 (SST2) antagonist. Octreotide (a somatostatin analogue) decreased the basal and CCh-stimulated secretion of HCO3(-) in wild-type mice. The co-localized expression of somatostatin and M4 receptors was confirmed immunohistologically in the duodenum. We concluded that the duodenal HCO3(-) response to CCh was directly mediated by M1/M3 receptors and indirectly modified by M4 receptors. The activation of M4 receptors was assumed to inhibit the release of somatostatin from D cells and potentiate the HCO3(-) response by removing the negative influence of somatostatin via the activation of SST2 receptors. PMID:26084221

  3. Cholinergic signalling-regulated KV7.5 currents are expressed in colonic ICC-IM but not ICC-MP.

    Science.gov (United States)

    Wright, George W J; Parsons, Sean P; Loera-Valencia, Raúl; Wang, Xuan-Yu; Barajas-López, Carlos; Huizinga, Jan D

    2014-09-01

    Interstitial cells of Cajal (ICC) and the enteric nervous system orchestrate the various rhythmic motor patterns of the colon. Excitation of ICC may evoke stimulus-dependent pacemaker activity and will therefore have a profound effect on colonic motility. The objective of the present study was to evaluate the potential role of K(+) channels in the regulation of ICC excitability. We employed the cell-attached patch clamp technique to assess single channel activity from mouse colon ICC, immunohistochemistry to determine ICC K(+) channel expression and single cell RT-PCR to identify K(+) channel RNA. Single channel activity revealed voltage-sensitive K(+) channels, which were blocked by the KV7 blocker XE991 (n = 8), which also evoked inward maxi channel activity. Muscarinic acetylcholine receptor stimulation with carbachol inhibited K(+) channel activity (n = 8). The single channel conductance was 3.4 ± 0.1 pS (n = 8), but with high extracellular K(+), it was 18.1 ± 0.6 pS (n = 22). Single cell RT-PCR revealed Ano1-positive ICC that were positive for KV7.5. Double immunohistochemical staining of colons for c-Kit and KV7.5 in situ revealed that intramuscular ICC (ICC-IM), but not ICC associated with the myenteric plexus (ICC-MP), were positive for KV7.5. It also revealed dense cholinergic innervation of ICC-IM. ICC-IM and ICC-MP networks were found to be connected. We propose that the pacemaker network in the colon consists of both ICC-MP and ICC-IM and that one way of exciting this network is via cholinergic KV7.5 channel inhibition in ICC-IM.

  4. Argan (Argania spinosa) oil lowers blood pressure and improves endothelial dysfunction in spontaneously hypertensive rats.

    Science.gov (United States)

    Berrougui, Hicham; Alvarez de Sotomayor, Maria; Pérez-Guerrero, Concepción; Ettaib, Abdelkader; Hmamouchi, Mohamed; Marhuenda, Elisa; Herrera, Maria Dolores

    2004-12-01

    Traditionally hand-pressed argan oil, obtained from Argania spinosa seeds, is eaten raw in south-west Morocco; its rich composition of tocopherols, MUFA and PUFA make a study of its actions on risk factors for CVD, such as hypertension, interesting. The effects of 7 weeks of treatment with argan oil (10 ml/kg) on the blood pressure and endothelial function of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats were investigated. Systolic blood pressure and heart rate were measured every week by the tail-cuff method and endothelial function was assessed by carbachol (10(-8) to 10(-4) M)-induced relaxations of aortic rings and small mesenteric arteries pre-contracted with phenylephrine. Argan-oil administration reduced the mean blood pressure of SHR after the fifth week of treatment (P<0.05) and increased (P<0.01) the endothelial responses of arteries from SHR. The NO synthase inhibitor, L-N-omega-nitroarginine (3 x 10(-5) M) revealed a greater participation of NO in the relaxant effect after the treatment. When cyclooxygenase (COX) was blocked with indomethacin (10(-5) M), an involvement of COX products in the endothelium-dependent response was characterized. Enzyme immunoassay of thromboxane B2 showed a significant decrease (P<0.05) in the release of thromboxane A2 in both aorta and small mesenteric artery after argan-oil treatment of SHR. Experiments in the presence of the thromboxane A2-prostaglandin H2 receptor antagonist ICI 192,605 (10(-5) M) confirmed this result. Results after incubation with the antioxidants superoxide dismutase and catalase suggested that a decreased oxidative stress might contribute to explain the beneficial effects of argan-oil treatment. PMID:15613254

  5. NANC relaxation of the circular smooth muscle of the oesophagus of the Agama lizard involves the L-arginine-nitric oxide synthase pathway.

    Science.gov (United States)

    Knight, G E; Burnstock, G

    1999-02-01

    On carbachol (CCh; 10-30 microM) pre-contracted circular muscle strips of the Agama lizard oesophagus, electrical field stimulation evoked frequency-dependent relaxations in the presence of guanethidine (1 microM) and indomethacin (1 microM). These non-adrenergic inhibitory responses were concentration-dependently inhibited by the nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) within a concentration range of 30-300 microM but not D-NAME (up to 300 microM), although a component remained at 4-16 Hz even with 300 microM L-NAME. The inhibition by L-NAME (300 microM) was completely prevented when L-arginine (L-Arg; 15 mM) but not D-Arg (up to 15 mM) was applied simultaneously with L-NAME (300 microM). Increasing the L-NAME concentration to 1 mM had no additional inhibitory effect. Sodium nitroprusside (SNP) concentration-dependently relaxed pre-contracted oesophageal strips, L-NAME (up to 300 microM) had no effect. Neither adenosine 5'-triphosphate (up to 0.1 mM) nor vasoactive intestinal polypeptide (up to 0.1 microM) caused the pre-contracted oesophagus to relax. This study has shown that the NANC inhibitory response of the Agama lizard oesophagus circular muscle largely involves the L-Arg-NOS pathway as seen by the effect of L-NAME, L-Arg and SNP. The identity of the L-NAME-resistant component(s) and the lack of effect of tetrodotoxin (up to 3 microM) and omega-conotoxin GVIA (up to 0.1 microM) in relation to the nature of the inhibitory response are discussed. PMID:10190041

  6. Effects of ginger constituents on the gastrointestinal tract: role of cholinergic M3 and serotonergic 5-HT3 and 5-HT4 receptors.

    Science.gov (United States)

    Pertz, Heinz H; Lehmann, Jochen; Roth-Ehrang, René; Elz, Sigurd

    2011-07-01

    The herbal drug ginger (Zingiber officinale Roscoe) may be effective for treating nausea, vomiting, and gastric hypomotility. In these conditions, cholinergic M (3) receptors and serotonergic 5-HT (3) and 5-HT (4) receptors are involved. The major chemical constituents of ginger are [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol. We studied the interaction of [6]-gingerol, [8]-gingerol, [10]-gingerol (racemates), and [6]-shogaol with guinea pig M (3) receptors, guinea pig 5-HT (3) receptors, and rat 5-HT (4) receptors. In whole segments of guinea pig ileum (bioassay for contractile M (3) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol slightly but significantly depressed the maximal carbachol response at an antagonist concentration of 10 µM. In the guinea pig myenteric plexus preparation (bioassay for contractile 5-HT (3) receptors), 5-HT maximal responses were depressed by [10]-gingerol from 93 ± 3 % to 65 ± 6 % at an antagonist concentration of 3 µM and to 48 ± 3 % at an antagonist concentration of 5 µM following desensitization of 5-HT (4) receptors and blockade of 5-HT (1) and 5-HT (2) receptors. [6]-Shogaol (3 µM) induced depression to 61 ± 3 %. In rat esophageal tunica muscularis mucosae (bioassay for relaxant 5-HT (4) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol (2-6.3 µM) showed no agonist effects. The maximal 5-HT response remained unaffected in the presence of the compounds. It is concluded that the efficiency of ginger in reducing nausea and vomiting may be based on a weak inhibitory effect of gingerols and shogaols at M (3) and 5-HT (3) receptors. 5-HT (4) receptors, which play a role in gastroduodenal motility, appear not to be involved in the action of these compounds. PMID:21305447

  7. Remodeling of Aorta Extracellular Matrix as a Result of Transient High Oxygen Exposure in Newborn Rats: Implication for Arterial Rigidity and Hypertension Risk

    Science.gov (United States)

    Castro, Michele M.; Cloutier, Anik; Bertagnolli, Mariane; Sartelet, Hervé; Germain, Nathalie; Comte, Blandine; Schulz, Richard; DeBlois, Denis; Nuyt, Anne Monique

    2014-01-01

    Neonatal high-oxygen exposure leads to elevated blood pressure, microvascular rarefaction, vascular dysfunction and arterial (aorta) rigidity in adult rats. Whether structural changes are present in the matrix of aorta wall is unknown. Considering that elastin synthesis peaks in late fetal life in humans, and early postnatal life in rodents, we postulated that transient neonatal high-oxygen exposure can trigger premature vascular remodelling. Sprague Dawley rat pups were exposed from days 3 to 10 after birth to 80% oxygen (vs. room air control) and were studied at 4 weeks. Blood pressure and vasomotor response of the aorta to angiotensin II and to the acetylcholine analogue carbachol were not different between groups. Vascular superoxide anion production was similar between groups. There was no difference between groups in aortic cross sectional area, smooth muscle cell number or media/lumen ratio. In oxygen-exposed rats, aorta elastin/collagen content ratio was significantly decreased, the expression of elastinolytic cathepsin S was increased whereas collagenolytic cathepsin K was decreased. By immunofluorescence we observed an increase in MMP-2 and TIMP-1 staining in aortas of oxygen-exposed rats whereas TIMP-2 staining was reduced, indicating a shift in the balance towards degradation of the extra-cellular matrix and increased deposition of collagen. There was no significant difference in MMP-2 activity between groups as determined by gelatin zymography. Overall, these findings indicate that transient neonatal high oxygen exposure leads to vascular wall alterations (decreased elastin/collagen ratio and a shift in the balance towards increased deposition of collagen) which are associated with increased rigidity. Importantly, these changes are present prior to the elevation of blood pressure and vascular dysfunction in this model, and may therefore be contributory. PMID:24743169

  8. Remodeling of aorta extracellular matrix as a result of transient high oxygen exposure in newborn rats: implication for arterial rigidity and hypertension risk.

    Directory of Open Access Journals (Sweden)

    Fanny Huyard

    Full Text Available Neonatal high-oxygen exposure leads to elevated blood pressure, microvascular rarefaction, vascular dysfunction and arterial (aorta rigidity in adult rats. Whether structural changes are present in the matrix of aorta wall is unknown. Considering that elastin synthesis peaks in late fetal life in humans, and early postnatal life in rodents, we postulated that transient neonatal high-oxygen exposure can trigger premature vascular remodelling. Sprague Dawley rat pups were exposed from days 3 to 10 after birth to 80% oxygen (vs. room air control and were studied at 4 weeks. Blood pressure and vasomotor response of the aorta to angiotensin II and to the acetylcholine analogue carbachol were not different between groups. Vascular superoxide anion production was similar between groups. There was no difference between groups in aortic cross sectional area, smooth muscle cell number or media/lumen ratio. In oxygen-exposed rats, aorta elastin/collagen content ratio was significantly decreased, the expression of elastinolytic cathepsin S was increased whereas collagenolytic cathepsin K was decreased. By immunofluorescence we observed an increase in MMP-2 and TIMP-1 staining in aortas of oxygen-exposed rats whereas TIMP-2 staining was reduced, indicating a shift in the balance towards degradation of the extra-cellular matrix and increased deposition of collagen. There was no significant difference in MMP-2 activity between groups as determined by gelatin zymography. Overall, these findings indicate that transient neonatal high oxygen exposure leads to vascular wall alterations (decreased elastin/collagen ratio and a shift in the balance towards increased deposition of collagen which are associated with increased rigidity. Importantly, these changes are present prior to the elevation of blood pressure and vascular dysfunction in this model, and may therefore be contributory.

  9. VdTX-1, a reversible nicotinic receptor antagonist isolated from venom of the spider Vitalius dubius (Theraphosidae).

    Science.gov (United States)

    Rocha-E-Silva, Thomaz A A; Rostelato-Ferreira, Sandro; Leite, Gildo B; da Silva, Pedro Ismael; Hyslop, Stephen; Rodrigues-Simioni, Léa

    2013-08-01

    Theraphosid spider venoms can block neurotransmission in vertebrate nerve-muscle preparations in vitro, but few of the components involved have been characterized. In this work, we describe the neuromuscular activity of venom from the Brazilian theraphosid Vitalius dubius and report the purification and pharmacological characterization of VdTX-1, a 728 Da toxin that blocks nicotinic receptors. Neuromuscular activity was assayed in chick biventer cervicis preparations and muscle responses to exogenous ACh and KCl were determined before and after incubation with venom or toxin. Changes in membrane resting potential were studied in mouse diaphragm muscle. The toxin was purified by a combination of filtration through Amicon® filters, cation exchange HPLC and RP-HPLC; toxin purity and mass were confirmed by mass spectrometry. Venom caused progressive neuromuscular blockade and muscle contracture; the blockade but not the contracture was reversible by washing. Venom attenuated contractures to exogenous ACh and KCl. Filtration yielded low (LM, 5 kDa) fractions, with the latter reproducing the contracture seen in venom but with a slight and progressive twitch blockade. The LM fraction caused reversible blockade and attenuated contractures to ACh, but had no effect on contractures to KCl. VdTX-1 (728 Da) purified from the LM fraction was photosensitive and reduced the E(max) to ACh in biventer cervicis muscle without affecting the EC₅₀; VdTX-1 also abolished carbachol-induced depolarizations. V. dubius venom contains at least two components that affect vertebrate neurotransmission. One component, VdTX-1, blocks nicotinic receptors non-competitively to produce reversible blockade without muscle contracture.

  10. Effects of mild bladder outlet obstruction on rabbit bladder structure and function.

    Science.gov (United States)

    Kuo, H C

    1995-09-01

    Mild bladder outlet obstruction was created in 42 New Zealand white rabbits by placing a 5-mm inner diameter polyethylene tube around the urethra. Periods of obstruction lasted for 1 day (7 rabbits), 3 days (7 rabbits), 1 week (7 rabbits), 2 weeks (6 rabbits), 4 weeks (7 rabbits), 6 weeks (4 rabbits) and 8 weeks (4 rabbits). Whole bladder functional study was performed in control (5 rabbits) and the obstructed bladders. The obstructed bladders increased in weight two-to threefold. There was no significant difference in bladder weight after different periods of obstruction. Electric stimulation showed a diminished response after 3 days of obstruction. Pharmacologic stimulation with 500 microM carbachol revealed a decreased response after obstruction, but intravesical pressure returned to the control level at 6 and 8 weeks of obstruction. However, the bladder weight and the expelling function of the obstructed bladders remained the same as on day 1 of obstruction. Histologic study of control and obstructed bladders revealed that: 1) dense submucosal fibrous tissue developed after 3 days of obstruction, 2) detrusor muscle hypertrophy and hyperplasia were noted with intermuscular collagenous fiber deposition after 2 weeks of obstruction, 3) trabeculation of the bladder wall with hyperplasia of muscular bundles was noted after 2 weeks of obstruction, 4) acute edematous and inflammatory reaction were apparent after 3 days and 1 week of obstruction, but resolved later and 5) degenerative changes of muscular cells were noted at 8 weeks of obstruction. The results show that despite apparent morphologic changes after outlet obstruction, no significant difference was observed in bladder expelling between short-term and long-term infravesical mild outlet obstruction. The bladder contractile apparatus appears to have a compensatory ability after mild outlet obstruction. PMID:8696170

  11. Differences in airway reactivity in normal and allergic sheep after exposure to sulfur dioxide

    Energy Technology Data Exchange (ETDEWEB)

    Abraham, W.M.; Oliver, W. Jr.; Welker, M.J.; King, M.M.; Wanner, A.; Sackner, M.A.

    1981-12-01

    The effect of breathing 5 ppm sulfur dioxide (SO/sub 2/) on airway reactivity was studied in both normal and allergic conscious sheep. Allergic sheep were defined as animals in which inhalation of Axcaris suum extract resulted in bronchospasm as evidenced by an increase in mean pulmonary flow resistance (RL), hyperinflation, and a fall in dynamic compliance. Airway reactivity was assessed by measuring the increase of RL after 18 breaths of 0.25% carbachol (c), from an initial RL value obtained after 18 breaths of buffered saline (s) (RL(c-s)). RL and RL(c-s) were determined prior to, immediately after, and 24 h after exposure to 5 ppm SO/sub 2/ for 4 h. In both groups RL remained unchanged after SO/sub 2/ exposure. Prior to exposure, RL(c-s) was not significantly different in seven normal (0.3 +/- 0.1) and seven allergic sheep (0.4 +/- 0.2 (SD) cmH/sub 2/O.l/sup -1/.s), and there was no significant change in RL (c-s) immediately after SO/sub 2/ exposure in either group. Twenty-four h later, RL(c-s) RL(c-s) increased to 0.7 +/- 0.8 (P < 0.2) in normal and to 1.8 +/- 0.9 cmH/sub 2/O.l/sup -1/.s (P < 0.01) in allergic sheep. Because the increase in RL(c-s) after 24 h was greater (P < 0.01) in allergic than in normal sheep, we conclude that SO/sub 2/ exposure increased airway reactivity more in the former than in the latter.

  12. Chronic Treatment with Ang-(1-7 Reverses Abnormal Reactivity in the Corpus Cavernosum and Normalizes Diabetes-Induced Changes in the Protein Levels of ACE, ACE2, ROCK1, ROCK2 and Omega-Hydroxylase in a Rat Model of Type 1 Diabetes

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    Mariam H. M. Yousif

    2014-01-01

    Full Text Available Angiotensin-(1-7 [Ang-(1-7] may have beneficial effects in diabetes mellitus-induced erectile dysfunction (DMIED but its molecular actions in the diabetic corpus cavernosum (CC are not known. We characterized the effects of diabetes and/or chronic in vivo administration of Ang-(1-7 on vascular reactivity in the rat corpus cavernosum (CC and on protein expression levels of potential downstream effectors of the renin-angiotensin-aldosterone system (RAAS such as angiotensin-converting enzyme (ACE, ACE2, Rho kinases 1 and 2 (ROCK1 and ROCK2, and omega-hydroxylase, the cytochrome-P450 enzyme that metabolizes arachidonic acid to form the vasoconstrictor, 20-hydroxyeicosatetraenoic acid. Streptozotocin-treated rats were chronicically administered Ang-(1-7 with or without A779, a Mas receptor antagonist, during weeks 4 to 6 of diabetes. Ang-(1-7 reversed diabetes-induced abnormal reactivity to vasoactive agents (endothelin-1, phenylepherine, and carbachol in the CC without correcting hyperglycemia. Six weeks of diabetes led to elevated ACE, ROCK1, ROCK 2, and omega-hydroxylase and a concomitant decrease in ACE2 protein expression levels that were normalized by Ang-(1-7 treatment but not upon coadministration of A779. These data are supportive of the notion that the beneficial effects of Ang-(1-7 in DMIED involve counterregulation of diabetes-induced changes in ACE, ACE2, Rho kinases, and omega-hydroxylase proteins in the diabetic CC via a Mas receptor-dependent mechanism.

  13. Mesenchymal Stem Cells Induce Suppressive Macrophages Through Phagocytosis in a Mouse Model of Asthma.

    Science.gov (United States)

    Braza, Faouzi; Dirou, Stéphanie; Forest, Virginie; Sauzeau, Vincent; Hassoun, Dorian; Chesné, Julie; Cheminant-Muller, Marie-Aude; Sagan, Christine; Magnan, Antoine; Lemarchand, Patricia

    2016-07-01

    Mesenchymal stem cell (MSC) immunosuppressive functions make them attractive candidates for anti-inflammatory therapy in allergic asthma. However, the mechanisms by which they ensure therapeutic effects remain to be elucidated. In an acute mouse model of house dust mite (Der f)-induced asthma, one i.v. MSC injection was sufficient to normalize and stabilize lung function in Der f-sensitized mice as compared to control mice. MSC injection decreased in vivo airway responsiveness and decreased ex vivo carbachol-induced bronchial contraction, maintaining bronchial expression of the inhibitory type 2 muscarinic receptor. To evaluate in vivo MSC survival, MSCs were labeled with PKH26 fluorescent marker prior to i.v. injection, and 1 to 10 days later total lungs were digested to obtain single-cell suspensions. 91.5 ± 2.3% and 86.6 ± 6.3% of the recovered PKH26(+) lung cells expressed specific macrophage markers in control and Der f mice, respectively, suggesting that macrophages had phagocyted in vivo the injected MSCs. Interestingly, only PKH26(+) macrophages expressed M2 phenotype, while the innate PKH26(-) macrophages expressed M1 phenotype. Finally, the remaining 0.5% PKH26(+) MSCs expressed 10- to 100-fold more COX-2 than before injection, suggesting in vivo MSC phenotype modification. Together, the results of this study indicate that MSCs attenuate asthma by being phagocyted by lung macrophages, which in turn acquire a M2 suppressive phenotype. Stem Cells 2016;34:1836-1845. PMID:26891455

  14. GABAergic synapse properties may explain genetic variation in hippocampal network oscillations in mice

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    Tim S Heistek

    2010-06-01

    Full Text Available Cognitive ability and the properties of brain oscillation are highly heritable in humans. Genetic variation underlying oscillatory activity might give rise to differences in cognition and behavior. How genetic diversity translates into altered properties of oscillations and synchronization of neuronal activity is unknown. To address this issue, we investigated cellular and synaptic mechanisms of hippocampal fast network oscillations in eight genetically distinct inbred mouse strains. The frequency of carbachol-induced oscillations differed substantially between mouse strains. Since GABAergic inhibition sets oscillation frequency, we studied the properties of inhibitory synaptic inputs (IPSCs received by CA3 and CA1 pyramidal cells of three mouse strains that showed the highest, lowest and intermediate frequencies of oscillations. In CA3 pyramidal cells, the frequency of rhythmic IPSC input showed the same strain differences as the frequency of field oscillations. Furthermore, IPSC decay times in both CA1 and CA3 pyramidal cells were faster in mouse strains with higher oscillation frequencies than in mouse strains with lower oscillation frequency, suggesting that differences in GABAA-receptor subunit composition exist between these strains. Indeed, gene expression of GABAA-receptor β2 (Gabrb2 and β3 (Gabrb2 subunits was higher in mouse strains with faster decay kinetics compared with mouse strains with slower decay kinetics. Hippocampal pyramidal neurons in mouse strains with higher oscillation frequencies and faster decay kinetics fired action potential at higher frequencies. These data indicate that differences in genetic background may result in different GABAA-receptor subunit expression, which affects the rhythm of pyramidal neuron firing and fast network activity through GABA synapse kinetics.

  15. Avaliação dos efeitos miorelaxante, antiespasmódico e antinociceptivo do extrato aquoso da Phoradendron piperoides (Kunt. Trel. (Viscaceae Investigation on miorelaxant, antispasmodic and analgesic effects of the aqueous extract of Phoradendron piperoides

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    Kellyane S. Dias

    2007-09-01

    Full Text Available Foram investigados os efeitos miorelaxante, antiespasmódico e antinociceptivo do extrato aquoso liofilizado das folhas da Phoradendron piperoides. A toxicidade aguda também foi avaliada. No íleo isolado de cobaio, o extrato aquoso da P. piperoides (0,05 - 2,0 mg/mL produziu relaxamento de forma concentração-dependente (IC50 = 0,114 mg/mL e, na concentração de 1,5 mg/mL, reduziu a amplitude das contrações induzidas por carbacol (2 µM, histamina (2 µM e BaCl2 (0,03 M em 46,6; 38,6 e 55,3% (p The present work evaluated the antinociceptive, miorelaxant and antispasmodic effects as well as the acute toxicity of the aqueous extract from leaves of Phoradendron piperoides. In guinea pig ileum, the plant extract (0.05 - 2.0 mg/kg decreased the preparations basal tone in a dose-dependent manner (IC50 = 0.114 mg/mL and it (1.5 mg/mL reduced (p < 0.001 the contractions induced by carbachol (2 µM, histamine (2 µM and BaCl2 (0.03M. The extract, at oral doses of 100, 200, and 400 mg/kg, did not manifest a significant antinociceptive effect in the writhing, formalin and hot-plate tests. Moreover, no animal deaths were observed in doses up to 5 g/kg. In conclusion, the aqueous extract of Phoradendron piperoides showed no antinociceptive effect and no acute toxicity in mice. Indeed, it revealed miorelaxant and antispasmodic activities that are probably miogenic and not specific for neurotransmitters.

  16. Pinaverium acts as L-type calcium channel blocker on smooth muscle of colon.

    Science.gov (United States)

    Malysz, J; Farraway, L A; Christen, M O; Huizinga, J D

    1997-08-01

    The effect of pinaverium was electrophysiologically characterized and compared with the established L-type calcium channel blockers diltiazem, D600, and nitrendipine on canine colonic circular smooth muscle. Effects were studied on the electrical activity of the smooth muscle cells, in particular the spontaneously occurring slow wave. In addition, effects were examined on spontaneous contraction patterns and contractile activities generated by stimulation of cholinergic nerves or directly by stimulating muscarinic receptors. Effects were also examined on excitation of NO-releasing intrinsic nerves. Pinaverium bromide affected the slow wave by selectively inhibiting the plateau potential that is associated with generation of contractile activity. Pinaverium, similar to diltiazem and D600, produced reductions in cholinergic responses as well as spontaneous contractions. The IC50 values for inhibition of cholinergic responses for pinaverium, diltiazem, and D600 were 1.0 x 10(-6), 4.1 x 10(-7), and 5.3 x 10(-7) M, respectively. The IC50 values for inhibition of spontaneous contractile activity for pinaverium, diltiazem, and D600 were 3.8 x 10(-6), 9.7 x 10(-7), and 8.0 x 10(-7) M, respectively. Increases in contractility by carbachol were abolished by pretreatment with either pinaverium or D600. In addition, neither pinaverium nor D600 had any effects on the inhibitory NO-mediated relaxations. These data provide a rationale for the use of pinaverium in the treatment of colonic motor disorders where excessive contraction has to be suppressed. PMID:9360010

  17. Effects of Kampo medicine, Keishi-ka Shakuyaku-to (TJ-60) on alteration of diacylglycerol metabolism in gastrointestinal smooth muscle of diabetic rats

    Institute of Scientific and Technical Information of China (English)

    NOBE Koji; MOMOSE Kazutaka; SAKAI Yasushi

    2002-01-01

    AIM: To examine the effects of Kampo medicine, Keishi-ka-Shakuyaku-to (TJ-60) on the signal transduction in diabetic gastrointestinal dysfunction. METHODS: Experimental diabetic models were prepared using streptozotocin (STZ)-treated Wistar rats. Randomly selected STZ rats were treated with insulin (12 U@kg-1@d-1) or TJ-60 (1% of food intake). Diacylglycerol (DG) and DG kinase activities were quantified in isolated aortic smooth muscle tissue.RESULTS: One of the key element of the PI-turnover, DG kinase activity in resting state in gastric smooth muscle was significantly increased compared to the control value, and carbachol (CCh)-induced response was not detectable,but it was detected in the control rats. On the other hand resting activity in ileum did not differ from the control, but the CCh-induced responses were suppressed. Treatment with TJ-60 indicated resistant effects for the alteration of DG kinase activities in diabetic intestinal tissues. In order to reveal the mechanism of the effects, total content of DG was measured, because the DG plays important role in the PI-turnover and the DG converted from not only PI but also incorporated glucose under high glucose condition. Patterns of the change in DG levels were similar to those in DG kinase. These results indicate that the effect of TJ-60 occurs at the cellular level of DG. CONCLUSION:Dysfunction of gastrointestinal smooth muscle in diabetes is mediated by an alternation of DG and DG kinase. TJ-60 influences the alteration and relief the dysfunction.

  18. Electrophysiological, vasoactive, and gastromodulatory effects of stevia in healthy Wistar rats.

    Science.gov (United States)

    Yesmine, Saquiba; Connolly, Kylie; Hill, Nicholas; Coulson, Fiona R; Fenning, Andrew S

    2013-07-01

    Antihypertensive and antidiabetic effects of stevia, Stevia rebaudiana (Asteraceae), have been demonstrated in several human and animal models. The current study aims to define stevia's role in modifying the electrophysiological and mechanical properties of cardiomyocytes, blood vessels, and gastrointestinal smooth muscle. Tissues from thoracic aorta, mesenteric arteries, ileum, and left ventricular papillary muscles were excised from 8-week-old healthy Wistar rats. The effects of stevia (1 × 10-9 M to 1 × 10-4 M) were measured on these tissues. Stevia's effects in the presence of verapamil, 4-AP, and L-NAME were also assessed. In cardiomyocytes, stevia attenuated the force of contraction, decreased the average peak amplitude, and shortened the repolarisation phase of action potential - repolarisation phase of action potential20 by 25 %, repolarisation phase of action potential50 by 34 %, and repolarisation phase of action potential90 by 36 %. Stevia caused relaxation of aortic tissues which was significantly potentiated in the presence of verapamil. In mesenteric arteries, incubation with L-NAME failed to block stevia-induced relaxation indicating the mechanism of action may not be fully via nitric oxide-dependent pathways. Stevia concentration-dependently reduced electrical field stimulated and carbachol-induced contractions in the isolated ileum. This study is the first to show the effectiveness of stevia in reducing cardiac action potential duration at 20 %, 50 %, and 90 % of repolarisation. Stevia also showed beneficial modulatory effects on cardiovascular and gastrointestinal tissues via calcium channel antagonism, activation of the M2 muscarinic receptor function, and enhanced nitric oxide release.

  19. Stimulation of large-conductance calcium-activated potassium channels inhibits neurogenic contraction of human bladder from patients with urinary symptoms and reverses acetic acid-induced bladder hyperactivity in rats.

    Science.gov (United States)

    La Fuente, José M; Fernández, Argentina; Cuevas, Pedro; González-Corrochano, Rocío; Chen, Mao Xiang; Angulo, Javier

    2014-07-15

    We have analysed the effects of large-conductance calcium-activated potassium channel (BK) stimulation on neurogenic and myogenic contraction of human bladder from healthy subjects and patients with urinary symptoms and evaluated the efficacy of activating BK to relief bladder hyperactivity in rats. Bladder specimens were obtained from organ donors and from men with benign prostatic hyperplasia (BPH). Contractions elicited by electrical field stimulation (EFS) and carbachol (CCh) were evaluated in isolated bladder strips. in vivo cystometric recordings were obtained in anesthetized rats under control and acetic acid-induced hyperactive conditions. Neurogenic contractions of human bladder were potentiated by blockade of BK and small-conductance calcium-activated potassium channels (SK) but were unaffected by the blockade of intermediate calcium-activated potassium channels (IK). EFS-induced contractions were inhibited by BK stimulation with NS-8 or NS1619 or by SK/IK stimulation with NS309 (3µM). CCh-induced contractions were not modified by blockade or stimulation of BK, IK or SK. The anti-cholinergic agent, oxybutynin (0.3µM) inhibited either neurogenic or CCh-induced contractions. Neurogenic contractions of bladders from BPH patients were less sensitive to BK inhibition and more sensitive to BK activation than healthy bladders. The BK activator, NS-8 (5mg/kg; i.v.), reversed bladder hyperactivity induced by acetic acid in rats, while oxybutynin was ineffective. NS-8 did not significantly impact blood pressure or heart rate. BK stimulation specifically inhibits neurogenic contractions in patients with urinary symptoms and relieves bladder hyperactivity in vivo without compromising bladder contractile capacity or cardiovascular safety, supporting its potential therapeutic use for relieving bladder overactivity. PMID:24747752

  20. Protease-activated receptor 2 mediates mucus secretion in the airway submucosal gland.

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    Hyun Jae Lee

    Full Text Available Protease-activated receptor 2 (PAR2, a G protein-coupled receptor expressed in airway epithelia and smooth muscle, plays an important role in airway inflammation. In this study, we demonstrated that activation of PAR2 induces mucus secretion from the human airway gland and examined the underlying mechanism using the porcine and murine airway glands. The mucosa with underlying submucosal glands were dissected from the cartilage of tissues, pinned with the mucosal side up at the gas/bath solution interface of a physiological chamber, and covered with oil so that secretions from individual glands could be visualized as spherical bubbles in the oil. Secretion rates were determined by optical monitoring of the bubble diameter. The Ca(2+-sensitive dye Fura2-AM was used to determine intracellular Ca(2+ concentration ([Ca(2+](i by means of spectrofluorometry. Stimulation of human tracheal mucosa with PAR2-activating peptide (PAR2-AP elevated intracellular Ca(2+ and induced glandular secretion equal to approximately 30% of the carbachol response in the human airway. Porcine gland tissue was more sensitive to PAR2-AP, and this response was dependent on Ca(2+ and anion secretion. When the mouse trachea were exposed to PAR2-AP, large amounts of secretion were observed in both wild type and ΔF508 cystic fibrosis transmembrane conductance regulator mutant mice but there is no secretion from PAR-2 knock out mice. In conclusion, PAR2-AP is an agonist for mucus secretion from the airway gland that is Ca(2+-dependent and cystic fibrosis transmembrane conductance regulator-independent.

  1. Estudo da hiper-responsividade brônquica em pacientes portadores de refluxo gastroesofágico Bronchial hyperreactivity in patients with gastroesophageal reflux disease

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    Mônica Silveira Lapa

    2005-08-01

    Full Text Available OBJETIVO: O intuito deste trabalho foi evidenciar a existência desse reflexo vagal, através de uma broncoprovocação, em pacientes portadores de refluxo gastroesofágico. MÉTODOS: Onze pacientes com endoscopia sem evidências de refluxo gastroesofágico ou hérnia hiatal (grupo controle e dez pacientes com hérnia hiatal ou refluxo gastroesofágico foram submetidos à broncoprovocação com carbacol. RESULTADOS: O teste foi positivo em 5 dos pacientes com hérnia hiatal ou refluxo gastroesofágico (50%, e em 3 do grupo controle (27% (p = 0,64. CONCLUSÃO: A hipótese de que as vias aéreas de pacientes com refluxo gastroesofágico sem sintomas asmatiformes anteriores possam ser mais responsivas do que as de pacientes sem refluxo gastroesofágico permanece não comprovada.OBJECTIVE: To identify this vagal reflex using bronchial provocation tests in patients with gastroesophageal reflux disease. METHODS: The study group was composed of 10 patients presenting endoscopic evidence of hiatal hernia or gastroesophageal reflux disease, and the control group consisted of 11 patients presenting no evidence of either condition. All subjects were submitted to bronchial provocation with carbachol. RESULTS: The provocation test was positive in 5 (50% of the study group patients and 3 (27% of the control group patients (p = 0.64. CONCLUSION: The hypothesis that the airways of patients with gastroesophageal reflux disease (and no history of asthma-like respiratory symptoms might be more responsive than those of individuals without the disease remains unproven.

  2. Activation of CFTR by ASBT-mediated bile salt absorption.

    Science.gov (United States)

    Bijvelds, Marcel J C; Jorna, Huub; Verkade, Henkjan J; Bot, Alice G M; Hofmann, Franz; Agellon, Luis B; Sinaasappel, Maarten; de Jonge, Hugo R

    2005-11-01

    In cholangiocytes, bile salt (BS) uptake via the apical sodium-dependent bile acid transporter (ASBT) may evoke ductular flow by enhancing cAMP-mediated signaling to the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. We considered that ASBT-mediated BS uptake in the distal ileum might also modulate intestinal fluid secretion. Taurocholate (TC) induced a biphasic rise in the short circuit current across ileal tissue, reflecting transepithelial electrogenic ion transport. This response was sensitive to bumetanide and largely abrogated in Cftr-null mice, indicating that it predominantly reflects CFTR-mediated Cl- secretion. The residual response in Cftr-null mice could be attributed to electrogenic ASBT activity, as it matched the TC-coupled absorptive Na+ flux. TC-evoked Cl- secretion required ASBT-mediated TC uptake, because it was blocked by a selective ASBT inhibitor and was restricted to the distal ileum. Suppression of neurotransmitter or prostaglandin release, blocking of the histamine H1 receptor, or pretreatment with 5-hydroxytryptamine did not abrogate the TC response, suggesting that neurocrine or immune mediators of Cl- secretion are not involved. Responses to TC were retained after carbachol treatment and after permeabilization of the basolateral membrane with nystatin, indicating that BS modulate CFTR channel gating rather than the driving force for Cl- exit. TC-induced Cl- secretion was maintained in cGMP-dependent protein kinase II-deficient mice and only partially inhibited by the cAMP-dependent protein kinase inhibitor H89, suggesting a mechanism of CFTR activation different from cAMP or cGMP signaling. We conclude that active BS absorption in the ileum triggers CFTR activation and, consequently, local salt and water secretion, which may serve to prevent intestinal obstruction in the postprandial state. PMID:16037545

  3. Nicotinic acetylcholine receptors containing the α7-like subunit mediate contractions of muscles responsible for space positioning of the snail, Helix pomatia L. tentacle.

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    Tibor Kiss

    Full Text Available Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh responses in muscles to determine the properties of the ACh receptor (AChR, the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium, methyllycaconitine, α-Bungarotoxin (αBgTx and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na+ and Ca2+ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery.

  4. A novel muscarinic antagonist R2HBJJ inhibits non-small cell lung cancer cell growth and arrests the cell cycle in G0/G1.

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    Nan Hua

    Full Text Available Lung cancers express the cholinergic autocrine loop, which facilitates the progression of cancer cells. The antagonists of mAChRs have been demonstrated to depress the growth of small cell lung cancers (SCLCs. In this study we intended to investigate the growth inhibitory effect of R2HBJJ, a novel muscarinic antagonist, on non-small cell lung cancer (NSCLC cells and the possible mechanisms. The competitive binding assay revealed that R2HBJJ had a high affinity to M3 and M1 AChRs. R2HBJJ presented a strong anticholinergic activity on carbachol-induced contraction of guinea-pig trachea. R2HBJJ markedly suppressed the growth of NSCLC cells, such as H1299, H460 and H157. In H1299 cells, both R2HBJJ and its leading compound R2-PHC displayed significant anti-proliferative activity as M3 receptor antagonist darifenacin. Exogenous replenish of ACh could attenuate R2HBJJ-induced growth inhibition. Silencing M3 receptor or ChAT by specific-siRNAs resulted in a growth inhibition of 55.5% and 37.9% on H1299 cells 96 h post transfection, respectively. Further studies revealed that treatment with R2HBJJ arrested the cell cycle in G0/G1 by down-regulation of cyclin D1-CDK4/6-Rb. Therefore, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, as a novel mAChRs antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth, which suggest that M3 receptor antagonist might be a potential chemotherapeutic regimen for NSCLC.

  5. A novel muscarinic antagonist R2HBJJ inhibits non-small cell lung cancer cell growth and arrests the cell cycle in G0/G1.

    Science.gov (United States)

    Hua, Nan; Wei, Xiaoli; Liu, Xiaoyan; Ma, Xiaoyun; He, Xinhua; Zhuo, Rengong; Zhao, Zhe; Wang, Liyun; Yan, Haitao; Zhong, Bohua; Zheng, Jianquan

    2012-01-01

    Lung cancers express the cholinergic autocrine loop, which facilitates the progression of cancer cells. The antagonists of mAChRs have been demonstrated to depress the growth of small cell lung cancers (SCLCs). In this study we intended to investigate the growth inhibitory effect of R2HBJJ, a novel muscarinic antagonist, on non-small cell lung cancer (NSCLC) cells and the possible mechanisms. The competitive binding assay revealed that R2HBJJ had a high affinity to M3 and M1 AChRs. R2HBJJ presented a strong anticholinergic activity on carbachol-induced contraction of guinea-pig trachea. R2HBJJ markedly suppressed the growth of NSCLC cells, such as H1299, H460 and H157. In H1299 cells, both R2HBJJ and its leading compound R2-PHC displayed significant anti-proliferative activity as M3 receptor antagonist darifenacin. Exogenous replenish of ACh could attenuate R2HBJJ-induced growth inhibition. Silencing M3 receptor or ChAT by specific-siRNAs resulted in a growth inhibition of 55.5% and 37.9% on H1299 cells 96 h post transfection, respectively. Further studies revealed that treatment with R2HBJJ arrested the cell cycle in G0/G1 by down-regulation of cyclin D1-CDK4/6-Rb. Therefore, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, as a novel mAChRs antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth, which suggest that M3 receptor antagonist might be a potential chemotherapeutic regimen for NSCLC. PMID:23285263

  6. Regulation of Ca²⁺ release through inositol 1,4,5-trisphosphate receptors by adenine nucleotides in parotid acinar cells.

    Science.gov (United States)

    Park, Hyung Seo; Betzenhauser, Matthew J; Zhang, Yu; Yule, David I

    2012-01-01

    Secretagogue-stimulated intracellular Ca(2+) signals are fundamentally important for initiating the secretion of the fluid and ion component of saliva from parotid acinar cells. The Ca(2+) signals have characteristic spatial and temporal characteristics, which are defined by the specific properties of Ca(2+) release mediated by inositol 1,4,5-trisphosphate receptors (InsP(3)R). In this study we have investigated the role of adenine nucleotides in modulating Ca(2+) release in mouse parotid acinar cells. In permeabilized cells, the Ca(2+) release rate induced by submaximal [InsP(3)] was increased by 5 mM ATP. Enhanced Ca(2+) release was not observed at saturating [InsP(3)]. The EC(50) for the augmented Ca(2+) release was ∼8 μM ATP. The effect was mimicked by nonhydrolysable ATP analogs. ADP and AMP also potentiated Ca(2+) release but were less potent than ATP. In acini isolated from InsP(3)R-2-null transgenic animals, the rate of Ca(2+) release was decreased under all conditions but now enhanced by ATP at all [InsP(3)]. In addition the EC(50) for ATP potentiation increased to ∼500 μM. These characteristics are consistent with the properties of the InsP(3)R-2 dominating the overall features of InsP(3)R-induced Ca(2+) release despite the expression of all isoforms. Finally, Ca(2+) signals were measured in intact parotid lobules by multiphoton microscopy. Consistent with the release data, carbachol-stimulated Ca(2+) signals were reduced in lobules exposed to experimental hypoxia compared with control lobules only at submaximal concentrations. Adenine nucleotide modulation of InsP(3)R in parotid acinar cells likely contributes to the properties of Ca(2+) signals in physiological and pathological conditions.

  7. JNK mitogen-activated protein kinase limits calcium-dependent chloride secretion across colonic epithelial cells.

    LENUS (Irish Health Repository)

    Donnellan, Fergal

    2010-01-01

    Neuroimmune agonists induce epithelial Cl(-) secretion through elevations in intracellular Ca2+ or cAMP. Previously, we demonstrated that epidermal growth factor receptor (EGFR) transactivation and subsequent ERK MAPK activation limits secretory responses to Ca2+-dependent, but not cAMP-dependent, agonists. Although JNK MAPKs are also expressed in epithelial cells, their role in regulating transport function is unknown. Here, we investigated the potential role for JNK in regulating Cl(-) secretion in T(84) colonic epithelial cells. Western blot analysis revealed that a prototypical Ca2+-dependent secretagogue, carbachol (CCh; 100 microM), induced phosphorylation of both the 46-kDa and 54-kDa isoforms of JNK. This effect was mimicked by thapsigargin (TG), which specifically elevates intracellular Ca2+, but not by forskolin (FSK; 10 microM), which elevates cAMP. CCh-induced JNK phosphorylation was attenuated by the EGFR inhibitor, tyrphostin-AG1478 (1 microM). Pretreatment of voltage-clamped T(84) cells with SP600125 (2 microM), a specific JNK inhibitor, potentiated secretory responses to both CCh and TG but not to FSK. The effects of SP600125 on CCh-induced secretion were not additive with those of the ERK inhibitor, PD98059. Finally, in apically permeabilized T(84) cell monolayers, SP600125 potentiated CCh-induced K+ conductances but not Na+\\/K+ATPase activity. These data demonstrate a novel role for JNK MAPK in regulating Ca2+ but not cAMP-dependent epithelial Cl(-) secretion. JNK activation is mediated by EGFR transactivation and exerts its antisecretory effects through inhibition of basolateral K+ channels. These data further our understanding of mechanisms regulating epithelial secretion and underscore the potential for exploitation of MAPK-dependent signaling in treatment of intestinal transport disorders.

  8. Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells.

    Science.gov (United States)

    Devor, D C; Frizzell, R A

    1993-11-01

    Single-channel recording techniques were used to identify and characterize the K+ channel activated by Ca(2+)-mediated secretory agonists in T84 cells. Carbachol (CCh; 100 microM) and taurodeoxycholate (TDC; 0.75 mM) stimulated oscillatory outward K+ currents. With K gluconate in bath and pipette, cell-attached single-channel K+ currents stimulated by CCh and ionomycin (2 microM) were inwardly rectified and reversed at 0 mV. The single-channel chord conductance was 32 pS at -90 mV and 14 pS at +90 mV. Similar properties were observed in excised inside-out patches in symmetric K+, permitting further characterization of channel properties. Partial substitution of bath or pipette K+ with Na+ gave a K(+)-to-Na+ selectivity ratio of 5.5:1. Channel activity increased with increasing bath Ca2+ concentration in the physiological range of 50-800 nM. Maximal channel activity occurred at intracellular pH 7.2 and decreased at more acidic or alkaline pH values. Extracellular charybdotoxin (CTX; 50 nM) blocked inward but not outward currents. Extracellular tetraethylammonium (TEA; 10 mM) reduced single-channel amplitude at all voltages. No apparent block of the channel was observed with extracellular Ba2+ (1 mM), apamin (1 microM), 4-aminopyridine (4-AP; 4 mM), quinine (500 microM), or glyburide (10 microM). Cytosolic quinine and 4-AP blocked both inward and outward currents, whereas Ba2+ blocked only outward currents. Apamin, CTX, TEA, and glyburide did not affect channel activity. The agonist activation and pharmacological profile of this inwardly rectified K+ channel indicate that it is responsible for the increase in basolateral K+ conductance stimulated by Ca(2+)-mediated agonists in T84 cells. PMID:7694492

  9. Methodologic aspects of acetylcholine-evoked relaxation of rabbit aorta.

    Science.gov (United States)

    Hansen, K; Nedergaard, O A

    1999-08-01

    The acetylcholine-evoked relaxation of rabbit isolated thoracic aorta precontracted by phenylephrine was studied. Phenylephrine caused a steady contraction that was maintained for 6 h. In the presence of calcium disodium ethylenediaminetetraacetate (EDTA) and ascorbic acid the contraction decreased with time. N(G)-Nitro-L-arginine abolished the inhibitory effect of EDTA and ascorbic acid. Acetylcholine evoked a rapid concentration-dependent relaxation that recovered spontaneously and slowly, but fully, with time. Relaxation evoked by equieffective concentrations of carbachol and acetylcholine had the same time course. Cumulative addition of acetylcholine (10(-7)-3 x 10(-5) M) caused a marked relaxation that was reverted slightly at high concentrations. The relaxation was the same with rings derived from the upper, middle, and lower part of the thoracic aorta. Two consecutive concentration-response curves for acetylcholine obtained at a 2-h interval demonstrated a slight development of tachyphylaxis. The relaxation was inversely related to precontractile tension evoked by phenylephrine when expressed as a percentage, but independent when expressed as g tension. Storage of aorta in cold salt solution for 24 h did not alter the relaxation. EDTA and ascorbic acid did not alter the relaxation. It is concluded that (1) EDTA and ascorbic acid can not be used with impunity to stabilize catecholamines used as preconstriction agents; (2) the reversal of the acetylcholine-evoked relaxation is not due to hydrolysis of acetylcholine; (3) the relaxation is uniform in all segments of thoracic aorta; (4) cold storage of aorta does not alter the relaxation; and (5) acetylcholine releases the same amount of relaxing factor, irrespective of the precontractile tension. PMID:10691020

  10. Antispasmodic effect of Tecoma stans (L. Juss leaf extract on rat ileum

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    Gharib Naseri M.K.

    2007-06-01

    Full Text Available Tecoma stans (L. Juss or Yellow bells from Bignoniaceae is a ornamental tropical shrub or small tree predominantly found in central, and south America and in Latin America is used traditionally for reducing blood glucose. However, its other pharmacological effects have not been yet elucidated. The aim of present study was to investigate the effect of its leaves extract on rat ileum contractility and involved mechanism(s. Tecoma stans Juss hydroalcoholic leaf extract (TLE was prepared by macerated method using 70% alcohol. Distal segment of ileum (2 cm from male Wistar rat was mounted in an organ bath containing Tyrode solution (10 ml, pH 7, 37 °C and pre-contracted by carbachol (CCh, 10 µM or by KCl (60 mM. The antispasmodic effects of TLE (0.125-2 mg/ml were studied prior and after 20-30 min incubation of ileum with propranolol (1µM, naloxone (1µM, L-NAME (100 µM, or 5 min incubation with glibenclamide (10 µM and tetraethylammonium (TEA, 1mM. The effect of TLE on CaCl2-induced contraction in Ca2+-free with high K+ Tyrode solution was also studied. The CCh- and KCl-induced ileal contractions were reduced by TLE (P<0.0001. This effect was not attenuated by propranolol, naloxone, L-NAME, glibenclamide and TEA. In Ca2+-free Tyrode solution with high K+, cumulative concentrations of CaCl2 induced contractions which were inhibited by TLE dose-dependently. Our results indicate that the Tecoma stans (L. Juss leaf extract induces its antispasmodic effects without involvement β-adrenoceptors, opioid receptors, potassium channels and NO production. It seems that, the calcium channels are involved in this spasmolytic effect.

  11. Mechanism underlying the reversal of contractility dysfunction in experimental colitis by cyclooxygenase-2 inhibition.

    Science.gov (United States)

    Khan, I; Oriowo, M A

    2006-03-01

    Inflammatory bowel diseases are associated with reduced colonic contractility and induction of cyclooxygenase-2. In this study a possible role of cyclooxygenase-2 in and the underlying mechanism of the reduced contractility were investigated in experimental colitis. The effects of meloxicam, a cyclooxygenase-2 selective inhibitor were examined on colonic contractility and MAP kinase p38 and ERK(1/2) expression. Colitis was induced in Sprague-Dawley male rats by intra-colonic instillation of trinitrobenzenesulphonic acid (TNBS; 40 mg/rat in 50 ethanol). The animals were divided into three groups. Group 1 (n=9) received meloxicam (3 mg/kg-day) gavage 1 h before and 1 day (Group 2) after induction of colitis. Group 3 (n=9) received phosphate buffered saline (PBS) in a similar manner and served as colitic control. The non colitic control animals received meloxicam in a similar manner. The animals were sacrificed after 5 days of treatment, colon was cleaned with PBS and colonic smooth muscle was obtained which was used in this study. Meloxicam treatment given 1 h before or 1 day after administration of colitis restored the reduced colonic contractility without affecting the sensitivity to carbachol. The levels of colonic smooth muscle IL-1beta mRNA, PGE(2), ERK(1/2), p38, malondialdehyde, myeloperoxidase activity and colonic mass were increased, whereas the body weight was decreased due to TNBS. The changes except colonic muscle mass and p38 expression were reversed by meloxicam treatment. These findings indicate that restoration of reduced colonic contractility by meloxicam is mediated by ERK(1/2), and that ERK(1/2) may serve as an important anti inflammatory target for treatment of colitis. PMID:16835710

  12. Functional and molecular evidence for Kv7 channel subtypes in human detrusor from patients with and without bladder outflow obstruction.

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    Julie Svalø

    Full Text Available The aim of the study was to investigate whether Kv7 channels and their ancillary β-subunits, KCNE, are functionally expressed in the human urinary bladder. Kv7 channels were examined at the molecular level and by functional studies using RT-qPCR and myography, respectively. We found mRNA expression of KCNQ1, KCNQ3-KCNQ5 and KCNE1-5 in the human urinary bladder from patients with normal bladder function (n = 7 and in patients with bladder outflow obstruction (n = 3. Interestingly, a 3.4-fold up-regulation of KCNQ1 was observed in the latter. The Kv7 channel subtype selective modulators, ML277 (activator of Kv7.1 channels, 10 μM and ML213 (activator of Kv7.2, Kv7.4, Kv7.4/7.5 and Kv7.5 channels, 10 μM, reduced the tone of 1 μM carbachol pre-constricted bladder strips. XE991 (blocker of Kv7.1-7.5 channels, 10 μM had opposing effects as it increased contractions achieved with 20 mM KPSS. Furthermore, we investigated if there is interplay between Kv7 channels and β-adrenoceptors. Using cumulative additions of isoprenaline (β-adrenoceptor agonist and forskolin (adenylyl cyclase activator in combination with the Kv7 channel activator and blocker, retigabine and XE991, we did not find interplay between Kv7 channels and β-adrenoceptors in the human urinary bladder. The performed gene expression analysis combined with the organ bath studies imply that compounds that activate Kv7 channels could be useful for treatment of overactive bladder syndrome.

  13. Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

    Science.gov (United States)

    Phelan, K. D.; Gallagher, J. P.

    1992-01-01

    We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca2+/high Mg2+ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinic-receptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the gamma-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.

  14. Non-adrenergic non-cholinergic (NANC) excitatory response of the channel catfish intestine.

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    Venugopalan, C S; Holmes, E P; Jarboe, H H; Kleinow, K M

    1994-06-01

    1. Optimal parameters for electrical field stimulation (EFS) of catfish pyloric and middle intestinal segments were determined (15 Hz, 60 V) from a range of frequencies (5-45 Hz) and voltages (40-120 V) using a modified Magnus' method. Contractile responses were produced by EFS which were reproducible and showed no significant difference between the tissues. 2. The contractile cholinergic responses of the tissues to carbachol and acetylcholine (ACh) were blocked by atropine on an equimolar concentration, whereas, these responses were enhanced in the presence of neostigmine, and acetylcholinesterase inhibitor. 3. Adrenergic responses were examined with noradrenaline (NA). NA produced contraction of the segments only, at a concentration of 10(-4) M. Among the various adrenoceptors, beta-adrenoceptor stimulation produced a weak relaxation whereas, both alpha 1- and alpha 2-adrenoceptor stimulation produced contractions, of which alpha 2-induced contraction was of greater magnitude. The beta, alpha 1 and alpha 2 responses were blocked by their respective blocking agents propranolol, prazosin and yohimbine. 4. The autonomic components of the response to EFS were determined by using selected cholinergic and adrenergic antagonists separately or collectively. Cholinergic blockade with atropine did not produce a significant blockade of the EFS-induced response. Similarly, blockade of beta-adrenoceptors with propranolol did not modulate the contractile response to EFS to any significant level. Blockade by prazosin or yohimbine did not significantly change the contractile response to EFS. After a complete blockade of the adrenergic and cholinergic divisions, the intestinal segments still showed a contractile response to EFS which was not significantly different from the control response. This indicated the presence of a non-adrenergic non-cholinergic (NANC) response. 5. Tetrodotoxin, at 10(-6) M, significantly blocked the EFS-induced NANC response suggesting a neurogenic

  15. C(8) substituted 1-azabicyclo[3.3.1]non-3-enes and C(8) substituted 1-azabicyclo[3.3.1]nonan-4-ones: novel muscarinic receptor antagonists.

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    Kim, Myoung Goo; Bodor, Erik T; Wang, Chen; Harden, T Kendall; Kohn, Harold

    2003-05-22

    Expedient syntheses of C(8) substituted 1-azabicyclo[3.3.1]non-3-enes and C(8) substituted 1-azabicyclo[3.3.1]nonan-4-ones are reported to begin with 2,5-disubstituted pyridines. Catalytic reduction of the pyridine to the piperidine followed by treatment with ethyl acrylate and Dieckmann cyclization gave diastereomeric mixtures of C(8) substituted 3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]non-3-enes, which were separable by chromatography. We found that the catalytic reduction (PtO2, H2) procedure provided the cis-substituted piperidine but that pyridine reduction was accompanied by competitive cleavage of the C(2) pyridyl substituent. Accordingly, an alternative route was devised that afforded a diastereomeric mixture of the cis- and trans-2,5-disubstituted piperidine. Treatment of the substituted pyridine with m-CPBA gave the pyridine N-oxide, which was reduced to the piperidine by sequential reduction with ammonium formate in the presence of Pd-C followed by NaBH3CN. Addition of ethyl acrylate completed the synthesis of the substituted piperidine. The overall four-step reaction gave higher yields (57%) than the two-step procedure (13%) with little cleavage of the C(2) pyridyl substituent. Acid decarboxylation of the bicyclo[3.3.1]non-3-enes provided the C(8) substituted 1-azabicyclo[3.3.1]nonan-4-ones. Structural studies revealed diagnostic 13C NMR signals that permit assignment of the orientation of the C(8) substituent. Pharmacological investigations documented that 3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]non-3-enes efficiently bind to the human M1-M5 muscarinic receptors and function as antagonists. We observed that exo-8-benzyloxymethyl-3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]non-3-ene (3) displayed the highest affinity, exhibiting Ki values at all five muscarinic receptors that were approximately 10-50 times lower than carbachol and approximately 30-230 times lower than arecoline. Receptor selectivity was observed for 3. Compound 3 contained

  16. Comparison of soluble guanylate cyclase stimulators and activators in models of cardiovascular disease associated with oxidative stress

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    Melissa H Costell

    2012-07-01

    Full Text Available Soluble guanylate cyclase (sGC, the primary mediator of nitric oxide (NO bioactivity, exists as reduced (NO-sensitive and oxidized (NO-insensitive forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A (a non-depressor dose and a higher dose which lowered mean arterial pressure [MAP] by 5-10mmHg and equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R and spontaneously hypertensive stroke prone rats (SHR-SP on a high salt/fat diet (HSFD. In I/R, neither compound reduced infarct size. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria and mortality, caused left ventricular hypertrophy and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552 but not GSK2181236A decreased urine output and mortality. Conversely, the low dose of GSK2181236A attenuated cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and mortality. In addition, the high dose of BAY 60-4552 reduced urine output, microalbuminuria and MAP. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the sGC oxidative state is responsible for the vascular dysfunction. In summary, GSK2181236A and BAY 60-4552 provide partial benefit against hypertension-induced end organ damage. The differential beneficial effects observed between these compounds could reflect tissue-specific changes in the s

  17. Comparison of soluble guanylate cyclase stimulators and activators in models of cardiovascular disease associated with oxidative stress.

    Science.gov (United States)

    Costell, Melissa H; Ancellin, Nicolas; Bernard, Roberta E; Zhao, Shufang; Upson, John J; Morgan, Lisa A; Maniscalco, Kristeen; Olzinski, Alan R; Ballard, Victoria L T; Herry, Kenny; Grondin, Pascal; Dodic, Nerina; Mirguet, Olivier; Bouillot, Anne; Gellibert, Francoise; Coatney, Robert W; Lepore, John J; Jucker, Beat M; Jolivette, Larry J; Willette, Robert N; Schnackenberg, Christine G; Behm, David J

    2012-01-01

    Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5-10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of GSK2181236A, decreased urine output, and improved survival. Conversely, the low dose of GSK2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did GSK2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible

  18. Acellular bi-layer silk fibroin scaffolds support functional tissue regeneration in a rat model of onlay esophagoplasty.

    Science.gov (United States)

    Algarrahi, Khalid; Franck, Debra; Ghezzi, Chiara E; Cristofaro, Vivian; Yang, Xuehui; Sullivan, Maryrose P; Chung, Yeun Goo; Affas, Saif; Jennings, Russell; Kaplan, David L; Estrada, Carlos R; Mauney, Joshua R

    2015-06-01

    Surgical management of long-gap esophageal defects with autologous gastrointestinal tissues is frequently associated with adverse complications including organ dysmotility, dysphagia, and donor site morbidity. In order to develop alternative graft options, bi-layer silk fibroin (SF) scaffolds were investigated for their potential to support functional tissue regeneration in a rodent model of esophageal repair. Onlay esophagoplasty was performed with SF matrices (N = 40) in adult rats for up to 2 m of implantation. Parallel groups consisted of animals implanted with small intestinal submucosa (SIS) scaffolds (N = 22) or sham controls receiving esophagotomy alone (N = 20). Sham controls exhibited a 100% survival rate while rats implanted with SF and SIS scaffolds displayed respective survival rates of 93% and 91% prior to scheduled euthanasia. Animals in each experimental group were capable of solid food consumption following a 3 d post-op liquid diet and demonstrated similar degrees of weight gain throughout the study period. End-point μ-computed tomography at 2 m post-op revealed no evidence of contrast extravasation, fistulas, strictures, or diverticula in any of the implant groups. Ex vivo tissue bath studies demonstrated that reconstructed esophageal conduits supported by both SF and SIS scaffolds displayed contractile responses to carbachol, KCl and electrical field stimulation while isoproterenol produced tissue relaxation. Histological (Masson's trichrome and hematoxylin and eosin) and immunohistochemical (IHC) evaluations demonstrated both implant groups produced de novo formation of skeletal and smooth muscle bundles positive for contractile protein expression [fast myosin heavy chain (MY32) and α-smooth muscle actin (α-SMA)] within the graft site. However, SF matrices promoted a significant 4-fold increase in MY32+ skeletal muscle and a 2-fold gain in α-SMA+ smooth muscle in comparison to the SIS cohort as determined by histomorphometric

  19. Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

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    Thiel Gerald

    2009-05-01

    Full Text Available Abstract Background The serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts. Results Stimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca2+, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK. Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M1 muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M

  20. Activation of Muscarinic Acetylcholine Receptor Subtype 4 is Essential for Cholinergic Stimulation of Gastric Acid Secretion - Relation To D Cell/Somatostatin -

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    Koji Takeuchi

    2016-08-01

    Full Text Available AbstractBackground/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1~M5, and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1~M5 KO mice, the importance of M4 receptors in carbachol (CCh stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice of wild-type (WT and M1-M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 µg/kg was given s.c. to stimulate acid secretion. Atropine or octreotide (a somatostatin analogue was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist was given i.p. 20 min before the administration of octreotide or CCh. Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analogue, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh. Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect

  1. Adenovirus-mediated hAQP1 expression in irradiated mouse salivary glands causes recovery of saliva secretion by enhancing acinar cell volume decrease.

    Science.gov (United States)

    Teos, L Y; Zheng, C-Y; Liu, X; Swaim, W D; Goldsmith, C M; Cotrim, A P; Baum, B J; Ambudkar, I S

    2016-07-01

    Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands

  2. State-dependent pattern of Fos protein expression in regionally-specific sites within the preoptic area of the cat.

    Science.gov (United States)

    Torterolo, Pablo; Benedetto, Luciana; Lagos, Patricia; Sampogna, Sharon; Chase, Michael H

    2009-04-24

    Clinical and experimental data have shown that the preoptic area of the hypothalamus (POA) is involved in the generation and maintenance of NREM sleep. However, the activity of specific populations of POA neurons during REM sleep, NREM sleep and different waking conditions is still not firmly established. Consequently, we performed a quantitative, regionally-specific analysis of the Fos immunoreactivity of neurons in the POA of the cat during NREM sleep and REM sleep induced by microinjections of carbachol into the nucleus pontis oralis (REMc), as well as during quiet and alert wakefulness. We observed that while the total number of Fos immunoreactive neurons in the POA did not change as a function of these behavioral states, state-specific differences in neuronal activity were detected in restricted regions of the POA. An increase in the number of Fos+ neurons was observed in the rostral tip of the suprachiasmatic nucleus (SCN) during NREM (83.4+/-25.6) compared to quiet wakefulness (5.1+/-1.3, p<0.05) but not with the other behavioral states. In the median preoptic nucleus (MnPN), the number of Fos immunoreactive neurons was greater during NREM sleep (39.5+/-6.1) compared with quiet wakefulness (13.5+/-1.4, p<0.05) and REMc (16.2+/-2.0, p<0.05). State-specific Fos immunoreactive neurons were not observed in the ventro-lateral preoptic nucleus (VLPO). Finally, there was no significant increase in the number of Fos+ neurons during REMc in any of the subregions of the POA. In conclusion, within the POA, a selective neuronal activation during NREM sleep was found only in the MnPN. In addition, our data suggest a potential role of the SCN in NREM sleep. Finally, based on the distribution of Fos+ neurons in the entire POA, we conclude that the neuronal network involved in the regulation of NREM sleep is dispersed and intermingled with waking-related neurons. PMID:19269274

  3. The role of tropomyosin-related kinase receptors in neurotrophin-induced rapid eye movement sleep in the cat.

    Science.gov (United States)

    Yamuy, J; Ramos, O; Torterolo, P; Sampogna, S; Chase, M H

    2005-01-01

    The microinjection of nerve growth factor and neurotrophin-3 into the rostro-dorsal pontine tegmentum of the cat evokes a state that is comparable to naturally-occurring rapid eye movement sleep. Using two experimental paradigms, we tested the hypothesis that neurotrophin high-affinity receptors (trkA and trkC, tropomyosin-related kinase A and C, respectively) mediate this effect. First, trk and fos immunohistochemistry were combined to determine whether tyrosine kinase receptor-containing neurons in the dorsal pontine tegmentum are active in cats that exhibit long-lasting periods of rapid eye movement sleep following the local microinjection of nerve growth factor. During approximately two hours of recording, nerve growth factor-treated cats spent 59.8% of the time in a rapid eye movement sleep-like state; vehicle-injected (control) animals remained in quiet wakefulness and non-rapid eye movement sleep. Whereas control and nerve growth factor-treated cats exhibited a similar mean number of trkA- and trkC-immunoreactive neurons in the dorsal pontine tegmentum, the number of trkA- and trkC-immunoreactive neurons that expressed Fos, i.e. double-labeled cells that are presumably activated, was significantly larger in cats that were injected with nerve growth factor. Axon terminals contained tyrosine kinase receptor immunoreactivity in this region; many were apposed to Fos-immunoreactive neurons. In addition, patterns of tyrosine kinase receptor and Fos immunoreactivity similar to those observed in nerve growth factor-injected cats were present, in conjunction with long-lasting rapid eye movement sleep, following the microinjection of carbachol into the dorsal pons. In a second series of studies, nerve growth factor or neurotrophin-3 was injected alone or after K-252a, a blocker of tyrosine kinase receptors, into the rostro-dorsal pontine tegmentum. Nerve growth factor or neurotrophin-3 alone produced, with a mean latency of 4 min, a rapid eye movement sleep-like state

  4. Evidence for Active Electrolyte Transport by Two-Dimensional Monolayers of Human Salivary Epithelial Cells.

    Science.gov (United States)

    Hegyesi, Orsolya; Földes, Anna; Bori, Erzsébet; Németh, Zsolt; Barabás, József; Steward, Martin C; Varga, Gábor

    2015-12-01

    Functional reconstruction of lost tissue by regenerative therapy of salivary glands would be of immense benefit following radiotherapy or in the treatment of Sjogren's syndrome. The purpose of this study was to develop primary cultures of human salivary gland cells as potential regenerative resources and to characterize their acinar/ductal phenotype using electrophysiological measurements of ion transport. Human salivary gland cultures were prepared either from adherent submandibular gland cells (huSMG) or from mixed adherent and nonadherent cells (PTHSG) and were cultivated in Hepato-STIM or minimum essential medium (MEM). Expression of key epithelial marker proteins was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). Transepithelial electrical resistance (TER) was monitored following seeding the cells on Transwell membranes. Transepithelial ion transport was estimated by short-circuit current (Isc) measurements in an Ussing chamber. Both huSMG and PTHSG cells showed epithelial characteristics when cultivated in Hepato-STIM, while fibroblast-like elements dominated in MEM. Compared to intact tissue, cultivation of the cells resulted in substantial decreases in AQP5 and NKCC1 expression and moderate increases in claudin-1 and ENaC expression. Both cultures achieved high TER and transepithelial electrolyte movement in Hepato-STIM, but not in MEM. The Isc was substantially reduced by basolateral Cl(-) and bicarbonate withdrawal, indicating the involvement of basolateral-to-apical anion transport, and by the blockade of apical ENaC by amiloride, indicating the involvement of apical-to-basolateral Na(+) transport. An almost complete inhibition was observed following simultaneous ENaC block and withdrawal of the two anions. Isc was enhanced by either apical adenosine triphosphate (ATP) or basolateral carbachol application, but not by forskolin, confirming the expected role of Ca(2+)-activated regulatory pathways in electrolyte

  5. Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells.

    Science.gov (United States)

    Martinez, Namyr A; Ayala, Alondra M; Martinez, Magdiel; Martinez-Rivera, Freddyson J; Miranda, Jorge D; Silva, Walter I

    2016-06-01

    Damage to the CNS can cause a differential spatio-temporal release of multiple factors, such as nucleotides, ATP and UTP. The latter interact with neuronal and glial nucleotide receptors. The P2Y2 nucleotide receptor (P2Y2R) has gained prominence as a modulator of gliotic responses after CNS injury. Still, the molecular mechanisms underlying these responses in glia are not fully understood. Membrane-raft microdomains, such as caveolae, and their constituent caveolins, modulate receptor signaling in astrocytes; yet, their role in P2Y2R signaling has not been adequately explored. Hence, this study evaluated the role of caveolin-1 (Cav-1) in modulating P2Y2R subcellular distribution and signaling in human 1321N1 astrocytoma cells. Recombinant hP2Y2R expressed in 1321N1 cells and Cav-1 were found to co-fractionate in light-density membrane-raft fractions, co-localize via confocal microscopy, and co-immunoprecipitate. Raft localization was dependent on ATP stimulation and Cav-1 expression. This hP2Y2R/Cav-1 distribution and interaction was confirmed with various cell model systems differing in the expression of both P2Y2R and Cav-1, and shRNA knockdown of Cav-1 expression. Furthermore, shRNA knockdown of Cav-1 expression decreased nucleotide-induced increases in the intracellular Ca(2+) concentration in 1321N1 and C6 glioma cells without altering TRAP-6 and carbachol Ca(2+) responses. In addition, Cav-1 shRNA knockdown also decreased AKT phosphorylation and altered the kinetics of ERK1/2 activation in 1321N1 cells. Our findings strongly suggest that P2Y2R interaction with Cav-1 in membrane-raft caveolae of 1321N1 cells modulates receptor coupling to its downstream signaling machinery. Thus, P2Y2R/Cav-1 interactions represent a novel target for controlling P2Y2R function after CNS injury. PMID:27129210

  6. CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors

    Science.gov (United States)

    Mamedova, Liaman; Capra, Valérie; Accomazzo, Maria Rosa; Gao, Zhan-Guo; Ferrario, Silvia; Fumagalli, Marta; Abbracchio, Maria P.; Rovati, G. Enrico; Jacobson, Kenneth A.

    2016-01-01

    Montelukast and pranlukast are orally active leukotriene receptor antagonists selective for the CysLT1 receptor. Conversely, the hP2Y1,2,4,6,11,12,13,14 receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937). Montelukast and pranlukast inhibited the effects of UTP with IC50 values of 7.7 and 4.3 μM, respectively, and inhibited the effects of UDP with IC50 values of 4.5 and 1.6 μM, respectively, in an insurmountable manner. Furthermore, ligand binding studies using [3H]LTD4 excluded the possibility of orthosteric nucleotide binding to the CysLT1 receptor. dU937 cells were shown to express P2Y2, P2Y4, P2Y6, P2Y11, P2Y13 and P2Y14 receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing human P2Y1,2,4,6 receptors, CysLT1 antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization. IC50 values at P2Y1 and P2Y6 receptors were astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10 μM montelukast had no effect on the carbachol-induced rise in intracellular Ca2+. These data demonstrated that CysLT1 receptor antagonists interact functionally with signaling pathways of P2Y receptors, and this should foster the study of possible implications for the clinical use of these compounds in asthma or in other inflammatory conditions. PMID:16280122

  7. Influence of statins on distinct circulating microRNAs during prolonged aerobic exercise.

    Science.gov (United States)

    Min, Pil-Ki; Park, Joseph; Isaacs, Stephanie; Taylor, Beth A; Thompson, Paul D; Troyanos, Chris; D'Hemecourt, Pierre; Dyer, Sophia; Chan, Stephen Y; Baggish, Aaron L

    2016-03-15

    Statins exacerbate exercise-induced skeletal muscle injury. Muscle-specific microRNAs (myomiRs) increase in plasma after prolonged exercise, but the patterns of myomiRs release after statin-associated muscle injury have not been examined. We examined the relationships between statin exposure, in vitro and in vivo muscle contraction, and expression of candidate circulating myomiRs. We measured plasma levels of myomiRs, circulating microRNA-1 (c-miR-1), c-miR-133a, c-miR-206, and c-miR-499-5p from 28 statin-using and 28 nonstatin-using runners before (PRE), immediately after (FINISH), and 24 h after they ran a 42-km footrace (the 2011 Boston marathon) (POST-24). To examine these cellular-regulation myomiRs, we used contracting mouse C2C12 myotubes in culture with and without statin exposure to compare intracellular and extracellular expression of these molecules. In marathoners, c-miR-1, c-miR-133a, and c-miR-206 increased at FINISH, returned to baseline at POST-24, and were unaffected by statin use. In contrast, c-miR-499-5p was unchanged at FINISH but increased at POST-24 among statin users compared with PRE and runners who did not take statins. In cultured C2C12 myotubes, extracellular c-miR-1, c-miR-133a, and c-miR-206 were significantly increased by muscle contraction regardless of statin use. In contrast, extracellular miR-499-5p was unaffected by either isolated statin exposure or isolated carbachol exposure but it was increased when muscle contraction was combined with statin exposure. In summary, we found that statin-potentiated muscle injury during exercise is accompanied by augmented extracellular release of miR-499-5p. Thus c-miR-499-5p may serve as a biomarker of statin-potentiated muscle damage. PMID:26472872

  8. Adenovirus-mediated hAQP1 expression in irradiated mouse salivary glands causes recovery of saliva secretion by enhancing acinar cell volume decrease.

    Science.gov (United States)

    Teos, L Y; Zheng, C-Y; Liu, X; Swaim, W D; Goldsmith, C M; Cotrim, A P; Baum, B J; Ambudkar, I S

    2016-07-01

    Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands

  9. The relaxant action of osthole isolated from Angelica pubescens in guinea-pig trachea.

    Science.gov (United States)

    Teng, C M; Lin, C H; Ko, F N; Wu, T S; Huang, T F

    1994-02-01

    The effect of osthole, isolated from Angelica pubescens, on the contraction of guinea-pig trachea was studied. Osthole (25-100 mumol/l), theophylline (10-1000 mumol/l) and higher concentrations of nifedipine (0.1-100 mumol/l) suppressed the contraction response curves of tracheal smooth muscle caused by carbachol, prostaglandin F2 alpha (PGF2 alpha), U46619 (thromboxane A2 analogue) and leukotriene C4 (LTC4) in a concentration-dependent manner. The contraction caused by high K+ (120 mmol/l) and cumulative concentrations of CaCl2 (0.03-3 mmol/l) was also inhibited concentration-dependently by osthole (25-100 mumol/l), theophylline (10-1000 mumol/l) and lower concentrations of nifedipine (0.01-0.1 mumol/l). The relaxant actions of osthole were not affected by propranolol (1 mumol/l), glibenclamide (10 mumol/l) or removal of tracheal epithelium. Osthole (100 mumol/l) was still effective in causing tracheal relaxation in the presence of nifedipine (1 mumol/l). In Ca(2+)-free- and EGTA (0.2 mmol/l)-containing medium, the relaxing effect of osthole was more potent than in normal Krebs solution. Osthole (25 and 50 mumol/l) caused 2.9 and 6.5, or 3.0 and 5.6 fold, respectively, increase in potency of forskolin or sodium nitroprusside in causing tracheal relaxation but did not affect that by cromakalim. Osthole (50 mumol/l) enhanced the increase in tissue cAMP and cGMP levels induced by forskolin and sodium nitroprusside, respectively, and in higher concentrations (100 and 250 mumol/l), itself increased markedly tissue cAMP and cGMP contents. Osthole (10-250 mol/l) inhibited the activity of cAMP and cGMP phosphodiesterases in a concentration-dependent manner. It is concluded that osthole exerts a non-specific relaxant effect on the trachealis by inhibiting the cAMP and cGMP phosphodiesterases. PMID:8170504

  10. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yuan, E-mail: yuan.xu@ki.se; Cardell, Lars-Olaf

    2014-02-15

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin- (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in

  11. Blebbistain, a myosin II inhibitor, as a novel strategy to regulate detrusor contractility in a rat model of partial bladder outlet obstruction.

    Directory of Open Access Journals (Sweden)

    Xinhua Zhang

    Full Text Available Partial bladder outlet obstruction (PBOO, a common urologic pathology mostly caused by benign prostatic hyperplasia, can coexist in 40-45% of patients with overactive bladder (OAB and is associated with detrusor overactivity (DO. PBOO that induces DO results in alteration in bladder myosin II type and isoform composition. Blebbistatin (BLEB is a myosin II inhibitor we recently demonstrated potently relaxed normal detrusor smooth muscle (SM and reports suggest varied BLEB efficacy for different SM myosin (SMM isoforms and/or SMM vs nonmuscle myosin (NMM. We hypothesize BLEB inhibition of myosin II as a novel contraction protein targeted strategy to regulate DO. Using a surgically-induced male rat PBOO model, organ bath contractility, competitive and Real-Time-RT-PCR were performed. It was found that obstructed-bladder weight significantly increased 2.74-fold while in vitro contractility of detrusor to various stimuli was impaired ∼50% along with decreased shortening velocity. Obstruction also altered detrusor spontaneous activities with significantly increased amplitude but depressed frequency. PBOO switched bladder from a phasic-type to a more tonic-type SM. Expression of 5' myosin heavy chain (MHC alternatively spliced isoform SM-A (associated with tonic-type SM increased 3-fold while 3' MHC SM1 and essential light chain isoform MLC(17b also exhibited increased relative expression. Total SMMHC expression was decreased by 25% while the expression of NMM IIB (SMemb was greatly increased by 4.5-fold. BLEB was found to completely relax detrusor strips from both sham-operated and PBOO rats pre-contracted with KCl, carbachol or electrical field stimulation although sensitivity was slightly decreased (20% only at lower doses for PBOO. Thus we provide the first thorough characterization of the response of rat bladder myosin to PBOO and demonstrate complete BLEB-induced PBOO bladder SM relaxation. Furthermore, the present study provides valuable

  12. Testosterone and farnesoid X receptor agonist INT-747 counteract high fat diet-induced bladder alterations in a rabbit model of metabolic syndrome.

    Science.gov (United States)

    Morelli, Annamaria; Comeglio, Paolo; Filippi, Sandra; Sarchielli, Erica; Cellai, Ilaria; Vignozzi, Linda; Yehiely-Cohen, Ravit; Maneschi, Elena; Gacci, Mauro; Carini, Marco; Adorini, Luciano; Vannelli, Gabriella B; Maggi, Mario

    2012-10-01

    In the male, metabolic syndrome (MetS) is associated to an increased risk of benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS). A recently established rabbit model of high fat diet (HFD)-induced MetS showed hypogonadism and the presence of prostate gland alterations, including inflammation, hypoxia and fibrosis. The present study investigated whether HFD-induced MetS might also alter bladder structure and function. Testosterone and the farnesoid X receptor (FXR) agonist INT-747, were evaluated for possible effects on HFD bladder. MetS rabbits develop bladder alterations, including fibrosis (reduced muscle/fiber ratio), hypoxia [2-fold increase as compared to regular diet (RD) group], low-grade inflammation (increased leukocyte infiltration and inflammatory markers) and RhoA/ROCK hyperactivity. Bladder strips from HFD rabbits, pre-contracted with carbachol, showed an overactive response to the selective ROCK inhibitor Y-27632. All these HFD-induced bladder alterations were partially blunted by testosterone and almost completely reverted by INT-747. Both treatments prevented some MetS features (glucose intolerance and visceral fat increase), thus suggesting that their effects on bladder could be ascribed to an improvement of the metabolic and/or hypogonadal state. However, a pathogenetic role for hypogonadism has been ruled out as GnRH analog-induced hypogonadal rabbits, fed a regular diet, did not show any detectable bladder alterations. In addition, INT-747 did not revert the MetS-induced hypogonadal state. FXR mRNA was highly expressed in rabbit bladder and positively associated with visceral fat increase. A direct effect of INT-747 on bladder smooth muscle was further suggested by inhibition of RhoA/ROCK-mediated activity by in vitro experiments on isolated cells. In conclusion, HFD-related MetS features are associated to bladder derangements, which are ameliorated by testosterone or INT-747 administration. INT-747 showed the most marked

  13. Colonic epithelial ion transport is not affected in patients with diverticulosis

    Directory of Open Access Journals (Sweden)

    Tilotta Maria C

    2007-09-01

    Full Text Available Abstract Background Colonic diverticular disease is a bothersome condition with an unresolved pathogenesis. It is unknown whether a neuroepithelial dysfunction is present. The aim of the study was two-fold; (1 to investigate colonic epithelial ion transport in patients with diverticulosis and (2 to adapt a miniaturized Modified Ussing Air-Suction (MUAS chamber for colonic endoscopic biopsies. Methods Biopsies were obtained from the sigmoid part of the colon. 86 patients were included. All patients were referred for colonoscopy on suspicion of neoplasia and they were without pathological findings at colonoscopy (controls except for diverticulosis in 22 (D-patients. Biopsies were mounted in MUAS chambers with an exposed area of 5 mm2. Electrical responses to various stimulators and inhibitors of ion transport were investigated together with histological examination. The MUAS chamber was easy to use and reproducible data were obtained. Results Median basal short circuit current (SCC was 43.8 μA·cm-2 (0.8 – 199 for controls and 59.3 μA·cm-2 (3.0 – 177.2 for D-patients. Slope conductance was 77.0 mS·cm-2 (18.6 – 204.0 equal to 13 Ω·cm2 for controls and 96.6 mS·cm-2 (8.4 – 191.4 equal to 10.3 Ω·cm2 for D-patients. Stimulation with serotonin, theophylline, forskolin and carbachol induced increases in SCC in a range of 4.9 – 18.6 μA·cm-2, while inhibition with indomethacin, bumetanide, ouabain and amiloride decreased SCC in a range of 6.5 – 27.4 μA·cm-2, and all with no significant differences between controls and D-patients. Histological examinations showed intact epithelium and lamina propria before and after mounting for both types of patients. Conclusion We conclude that epithelial ion transport is not significantly altered in patients with diverticulosis and that the MUAS chamber can be adapted for studies of human colonic endoscopic biopsies.

  14. HSP20 phosphorylation and airway smooth muscle relaxation

    Directory of Open Access Journals (Sweden)

    Mariam Ba

    2009-06-01

    Full Text Available Mariam Ba1, Cherie A Singer1, Manoj Tyagi2, Colleen Brophy3, Josh E Baker4, Christine Cremo4, Andrew Halayko5, William T Gerthoffer21Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA; 2Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA; 3Harrington Department of Biochemistry, Arizona State University, Tempe, AZ, USA; 4Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA; 5Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, CanadaAbstract: HSP20 (HSPB6 is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1, and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200–500 kDa to smaller complexes (<60 kDa. The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway

  15. Toxicological, Antidiarrheal and Spasmolytic Activities of Solanum paniculatum.

    Science.gov (United States)

    Clementino-Neto, José; Pereira, Joedna C; Vasconcelos, Luiz H C; de Souza, Iara L L; Silva, Anne D S; Silva, Telma M G; Ramos, Natalia S M; Pessôa, Hilzeth L F; Silva, Tania M S; da Silva, Bagnólia A; de Cavalcante, Fabiana A

    2016-01-01

    Solanum paniculatum is popularly known as "jurubeba-verdadeira". In folk medicine, its roots, stems, and leaves are used as tonics, anti-inflammatories, carminatives, diuretics, and for gastrointestinal disorders. This species is listed in the Brazilian Pharmacopoeia and belongs to the "Relação Nacional de Plantas Medicinais de Interesse ao SUS". Based on folk medicine data of the Solanum genus, we decided to investigate whether the crude ethanol extract from S. paniculatum aerial parts presents toxicological, antidiarrheal, and spasmolytic activities. The crude ethanol extract from S. paniculatum aerial parts did not produce in vitro or in vivo toxicity and showed dose-dependent antidiarrheal activity, inhibiting equipotently both the defecation frequency (ED50 = 340.3 ± 35.1 mg/kg) and liquid stool formation (ED50 = 370.1 ± 19.4 mg/kg) in mice. Conversely, the crude ethanol extract from S. paniculatum aerial parts did not inhibit normal intestinal transit, even though it has shown a dose-dependent reduction of both the castor oil-induced intestinal transit (Emax = 36.9 ± 1.3 %, ED50 = 242.0 ± 8.6 mg/kg) and intestinal fluid content (Emax = 74.8 ± 2.4 %, ED50 = 328.9 ± 15.9 mg/kg). Additionally, the crude ethanol extract from S. paniculatum aerial parts was approximately 2-fold more potent in antagonizing the phasic contractions induced with histamine (IC50 = 63.7 ± 3.5 µg/mL) than carbachol 10(-6) M (IC50 = 129.3 ± 14.1 µg/mL). Therefore, we concluded that the crude ethanol extract from S. paniculatum aerial parts presents antidiarrheal activity in mice related to the inhibition of small intestinal motility and secretion as well as nonselective spasmolytic activity on the guinea pig ileum. PMID:26366752

  16. Separation of four stereoisomers of anisodamine and comparison of their anticholinergic activity%山莨菪碱4个异构体的分离及其抗胆碱活性比较

    Institute of Scientific and Technical Information of China (English)

    孙凯; 谢一凡; 崔永耀; 陈红专; 陆阳; 杨丽敏

    2011-01-01

    目的 探讨山莨菪碱立体异构与生物活性的关系.方法 采用MDS-5型反相制备色谱柱直接分离山莨菪碱得到外消旋体Ⅰ和Ⅱ,用手性半制备高效液相色谱柱分离Ⅰ和Ⅱ得到山莨菪碱的4个单一异构体,通过抗卡巴胆碱致大鼠气管收缩效应实验考察4个异构体的抗胆碱活性.结果 由H NMR谱和比旋光度值确定了山莨菪碱4个异构体的绝对构型,不同的立体构型对卡巴胆碱诱导的大鼠气管平滑肌收缩表现出不同程度的抑制作用,效应强度为(6S,2'S)>(6S,2'R)>(6R,2'S)>(6R,2'R).结论 山莨菪碱莨菪烷母核上的S构型在抗胆碱活性上发挥更重要的作用.%Objective To investigate the relationship between stereoisomerism and pharmacological activity of anisodamine.Methods Two racemates of anisodamine ( Ⅰ and Ⅱ ) were separated from synthetic anisodamine using MDS-5 column, and four stereoisomers of anisodamine were separated from Ⅰ and Ⅱ by high performance liquid chromatography using CHIRALPAK AD-H.The anticholinergic activities of four stereoisomers were compared by their inhibitory effects on carbachol-induced contractions of isolated rat tracheal smooth muscle.Results The absolute configurations of four stereoiscmers were assigned using 1H NMR chemical shifts and specific rotation values.The difference of anticholinergic activities was observed among the four isomers of anisodamine.The order of potency was (6S, 2'S) > (6S, 2'R) > (6R, 2'S) > (6R, 2'R).Conclusion (S) configuration at the tropane ring plays more important role in the anticholinergic activity of anisodamine.

  17. Effects of anti-peptide antibody against human M2 muscarinic receptors on the cAMP content in rat ventricles%心肌M2胆碱受体抗体对大鼠心房及心室肌cAMP含量的影响

    Institute of Scientific and Technical Information of China (English)

    朱琳; 李香珍; 冯羡菊; 王文泽; 张琳; 陈爱莲

    2001-01-01

    目的:了解心肌M2胆碱受体抗体(M2-Ab)对大鼠心房和心室肌cAMP含量的影响,并与M2胆碱受体激动剂卡巴可(Carb)的作用进行了对比观察。方法:采用离体生化放射免疫分析法测定M2-Ab及M2胆碱受体激动剂Carb对心肌cAMP含量的影响。结果:①M2-Ab及Carb两者均可剂量依赖性抑制异丙肾上腺素(Isoproterenol,Iso)所刺激的大鼠心房及心室肌cAMP的增加。卡巴可浓度为2 μmol/L,10 μmol/L,50 μmol/L时可分别抑制Iso所刺激的cAMP含量(8.5±1.2)%,(16.2±1.4)%,(29.5±2.1)%,而M2-Ab浓度为50 nmol/L,100 nmol/L,400 nmol/L时,可分别抑制(6.1±0.6)%,(17.3±1.8)%,(31.7±3.1)%(P<0.01)。②Carb(10 μmol/L)及M2-Ab(100 nmol/L)两者可分别抑制基础cAMP(49.2±4.3)%和(64.3±5.1)%。③M受体阻断剂阿托品(Atr)(1.5 μmol/L)不但可阻断Carb对Iso的抑制反应,亦能阻断M2-Ab的这种反应。而相应的抗原性肽段也能阻断M2-Ab的这种反应。结论:M2-Ab抑制Iso引起的心室肌细胞cAMP生成量的增加反应,类似于M受体激动剂Carb,两者效应均通过作用于M2受体途径实现。%Aim:To study the effects of anti-peptide antibodies(M2-Ab) against the second extracellular loop of human muscarinic receptor 2 on the cAMP content in rat atria and ventricles.These effects were compared with those of the muscarinic receptor agonist carbachol (Carb).Methods:Radioimmunoassay technique,was used for determining the effects of anti-pepitide antibodies and carb on cAMP content in rat ventricles.Result:①both Carb and M2-Ab were able to inhibit the isoproterenol (Iso) stimulated cAMP production in rat atria and ventricles.Carb at 2μmol/L,10μmol/L and 50μmol/L decreased Iso-stimulated cAMP production by (8.5±1.2)%,(16.2±1.4)% and (29.5±2.1)%,respectively;whereas M2-Ab at 50nmol/L,100nmol/L and 400nmol/L decreased it by (6.1±0.6)%,(17.3±1.8)% and (31.7±3.1)% (P<0.01),respectively.②Both Carb and M2-Ab

  18. The effects of neuropeptide Y and its fragments upon basal and electrically stimulated ion secretion in rat jejunum mucosa.

    Science.gov (United States)

    Cox, H M; Cuthbert, A W

    1990-10-01

    , by definition be Y2-like. NPY (11-36) and NPY (13-36) at 300nm and 1 microM did not significantly attenuate secretory responses to either carbachol (CCh) or substance P (SP). A 1 microM concentration of either fragment was equivalent in effect to 30nm NPY upon basal current, but NPY at this concentration significantly reduced both CCh- and SP-induced secretion. The reduced spectrum of fragment activity together with the different order and potency ratios obtained with these three peptides indicates a presynaptic action for NPY and the fragments. PMID:2257432

  19. O Ácido (817,12E,14-labdatrieno-18-óico (labdano302, diterpeno tipo labdano isolado de Xylopia langsdorffiana St. Hil. & Tul. (Annonaceae relaxa a traquéia isolada de cobaia The (817,12E,14-labdatrien-18-oic acid (labdane302, labdane-type diterpene isolated from Xylopia langsdorffiana St. Hil. & Tul. (Annonaceae relaxes the guinea-pig trachea

    Directory of Open Access Journals (Sweden)

    Luciano A. A. Ribeiro

    2007-06-01

    Full Text Available A espécie Xylopia langsdorffiana St. Hil.. & Tul. é popularmente conhecida como "pimenteira-da-terra" no Sudeste do Brasil. A partir do fracionamento do extrato etanólico, obtido das cascas do caule desta espécie, foi isolado um diterpeno tipo labdano, identificado como sendo o ácido 8(17,12E,14-labdatrieno-18-óico, e que neste trabalho é codificado como labdano302. O labdano302 relaxou o tônus basal dos anéis de traquéia isolada de cobaia com um valor de CE50 de 6,7 ± 0,5 x 10-8 M. O diterpeno labdano302 relaxou de maneira dependente de concentração os anéis pré-contraídos com carbacol (10-6 M, tanto na presença (CE50 = 1,4 ± 0,7 x 10-5 M como na ausência de epitélio funcional (CE50 = 1,5 ± 0,3 x 10-5 M, bem como anéis pré-contraídos com 18 ou 60 mM de KCl, apresentando valores de CE50 de 2,3 ± 0,4 x 10(7 M e 1,8 ± 0,8 x 10-5 M, respectivamente. Este efeito relaxante, sobre as contrações induzidas por 18 mM de KCl, tanto foi significantemente mais potente quanto mais eficaz quando comparado ao efeito sobre as contrações induzidas por 60 mM de KCl. Assim, labdano302 mostra um efeito relaxante em traquéia isolada de cobaia, tanto em seu tônus basal como sob estímulo contrátil, aparentemente sem a participação dos fatores relaxantes derivados do epitélio, contudo com possível participação dos canais de K+.Xylopia langsdorfiana St. Hil. & Tul. is popularly known as "pimenteira-da-terra" in Southeast of Brazil. The fractionation of the ethanol extract obtained from the stem-bark of this species yielded a labdane-type diterpene identified as 8(17,12E,14-labdatrien-18-oic acid, referred here as labdane302. In this study, we investigated the effect of labdane302 in guinea-pig trachea. labdane302 relaxed the basal tonus of trachea rings with EC50 value of 6.7 ± 0.5 x 10-8 M. The diterpene labdane302 relaxed the pre-contracted rings by carbachol 10-6 M both in the presence (EC50 = 1.4 ± 0.7 x 10-5 M and absence

  20. 脑源性神经营养因子在小鼠肠神经-平滑肌重构中的作用及其对肠动力的影响%Role of brain-derived neurotrophic factor in the plasticity of enteric nervous-smooth muscle and its effects on gastrointestinal motility in mice

    Institute of Scientific and Technical Information of China (English)

    陈飞雪; 于岩波; 王鹏; 左秀丽; 李延青

    2016-01-01

    接头和平滑肌存在显著的超微结构改变.结论 BDNF可引起小鼠肠神经-平滑肌形态及功能重构,此重构作用在影响小鼠肠动力过程中发挥重要作用.%Objective To observe the role of brain-derived neurotrophic factor (BDNF) in the plasticity of enteric nervous-smooth muscle system,and to investigate the effects of BDNF induced plasticity on gastrointestinal motility in mice.Methods Male hybrid BDNF knockout (BDNF+/) mice and wild type (BDNF+/+) mice were selected,eight in each group.Gastrointestinal motility of BDNF+/+ mice and BDNF+/ mice were tested and compared.Longitudinal muscle strips of mice colon smooth muscle were prepared.The effects of carbachol (1 × 10 5 mol/L) and BDNF (1 × 10 7 mol/L) on contractile function of muscle strips were observed.And the effects of tetrodotoxin (TTX,1 × 10-6 mol/L) on BDNF induced contractile function of muscle strips were also studied.The changes of the density of mice intestinal myenteric plexus and the expression of smooth muscle α-actin (α-SMA) in colon smooth muscle were detected by immunohistochemical techniqne.The ultrastruetural alterations of myenteric plexus,neuromuscular junction (NMJ) and smooth muscle cells were detected by transmission electron microscope (TEM).T-test or Rank sum test was performed for comparison between groups.Results Number of feces particles and water content in feces of BDNF+/-mice ((3.80±0.75) and (39.60±1.47)%) were both lower than those of BDNF+/+ mice ((6.30± 1.03) and (51.00± 1.61) %),and the differences were statistically significant (t=4.792,12.827;both P<0.05).Carbachol (1 × 10-5 mol/L) could significantly increase contraction activity of smooth muscle of BDNF+/+ mice (R =3.26 ± 0.43) and BDNF+/-mice (R=2.15 ± 0.36),and the difference was statistically significant (t=15.754,9.632;both P<0.05).The effects on contraction exciting of smooth muscle strips of BDNF+/+ mice were more significant than BDNF+/ mice,and the difference was

  1. 抗M2受体的抗肽段抗体对cAMP产生量和钙电流的影响%Effects of anti-peptide antibodies against the second extrace llular loop of human M2 muscarinic receptors on the cAMP production and calci um currents

    Institute of Scientific and Technical Information of China (English)

    王文泽; 赵荣瑞; 吴博威; 郭光伟; 唐进; 李家成; Hjalmarson; Ake; Michael; Fu; LX

    2001-01-01

    Effects of anti-peptide antibodies aga inst the second extracellular loop of human M2 muscarinic receptors on the cAMP production and calcium currents(ICa) in guinea pig ventricular m yocytes we re studied by using radioimmunoassay and whole-cell patch clamp technique an d a comparison was also made with those of the muscarinic receptor agonist. Both muscarinic receptor agonist carbachol (Carb 10 μmol/L) and anti-pepti de antibodies (Abs 100 nmol/L) could decrease the isoprenaline (Iso 0.8 μm ol/L) -induced increases of cAMP〔from (108.2±7.0) to (88.4±7.2) pmol/(mg* min)for Carb and (88.6±5.1) pmol/(mg*min) for Abs respectively〕 production and i ncreases of ICa〔from (53.5± 7.0)% to (13.0±2.0)% for Carb and (66.9± 10.5)% to (34.5±8.0)% for Abs res pectively〕. The muscarinic receptor antagonist atropine (Atr) was able to prevent these effects of Carb and Abs.These results indicated that the anti-p eptide antibodies against an epitope located in the second extracellular loop o f human M2 muscarinic receptors, similar to muscarinic receptor agonist , could decrease the β-receptor agonist stimulated incre ase of ICa by decreasing the β -receptor agonist stimulated increa se of cAMP productions and therefore could have an effect on their target receptor. These results further suggest that autoimmunity may participate in the pathogenesis of human idiopathic dilated cardiomyopathy(DCM) and the seco nd extracellular loop of human M2 musarinic receptor could be the ma in immunodominant region.%应用放射免疫分析方法及全细胞膜片钳技术研究了抗M2受体细胞外第二环上的抗肽段 抗体(Abs )对豚鼠心室肌细胞cAMP产生量和内向钙电流(ICa)的影响并同M受体激 动剂的 效应进行了比较。结果如下:M受体激动剂carbachol(Carb 10 μmol/L)和抗肽段抗体( Abs 100 nmol/L)两者均可抑制异丙肾上腺素(Iso 0.8 μmol/L)所引起的cAMP量的增 加〔Carb可使组织中cAMP从(108

  2. Effects of Berberine on Contractile Activity of Circular Muscle in Rat Gastric Body Under High Glucose and Insulin Conditions%高糖高胰岛素环境下小檗碱对大鼠胃体环形肌收缩活动的影响

    Institute of Scientific and Technical Information of China (English)

    王霄; 马建红; 郝璋森; 赵丁

    2012-01-01

    Objective To observe the effects of berberine on the contractile responses in circular muscle strips of rat gastric body at high glucose or/and high insulin conditions. Methods By measuring muscular tone, we observed the contractions induced by adenosine triphosphate (ATP) or carbachol ( CCh ) in circular muscle strips of the rat gastric body, and the effects of berberine on the contractions in normal rats or rats with high glucose or/and high insulin. Results (1 )Berberine inhibited the contractile responses to CCh(0.3 μmo·L-1) or ATP(3 μmol·L-1) in circular muscle strips of the rat gastric body with dose-dependent manner. (2)Compared to the normal condition, the contractions of gastric circular muscle to CCh and ATP were not changed significantly under high glucose (15.6, 31.2 mmol·L-1) or high insulin( 50-200 mU · L-1 ) conditions(P > 0.05). ( 3 )The inhibited effect of berberine on the contractile responses to CCh was enhanced (P < 0.05 or P < 0.01), but on that to ATP was decreased significantly under high glu cose (31.2 mmol·L-1) and high insulin (200 mU ·L-1) conditions (P < 0.05) when compared to the normal condition. Conclusion Berberine inhibits the contractile responses to CCh and ATP significantly in circular muscle strips of the rat gastric body. Under high glucose or high insulin condition, the inhibited effect of berberine on the contractile activity in circular muscle of the rat gastric body are different, and the inhibition of berberine on the contractile re sponses to CCh is enhanced, which may aggravate the diabetic gastrointestinal dysfunction in rats.%目的 探讨小檗碱在高糖高胰岛素环境下对大鼠胃体环形肌收缩活动的影响.方法 采用张力测定法,在正常、高糖或/和高胰岛素环境下观察小檗碱对三磷酸腺苷( ATP)、卡巴胆碱(CCh)诱导大鼠胃体环形肌收缩反应的影响.结果 (1)小檗碱对ATP(3μmol· L-1)、CCh(0.3 μmol·L-1)引起的大鼠胃体环形肌收缩有明显

  3. Effects of samarium chloride on nicotinic transmission in superior cervical ganglia of rats%氯化钐对大鼠颈上神经节烟碱传递的影响

    Institute of Scientific and Technical Information of China (English)

    何萍; 莫宁

    2006-01-01

    , Guangxi Medical University. MATERIALS: Totally 40 adult Wistar rats (weighing 250-300 g) of either sex, provided by the Experimental Animal Center of Guangxi Medical University, were used in this study. SmCl3 was made by the chlorination of Samarium Oxide with purity 99.5% and relative molecule mass 348.7, presented by Professor Liu Da-yuan, Guangxi Medical University. Acetylcholine chloride (Ach) and carbachol (Carb) were purchased from Sigma.METHODS: The experiment was completed at the neuropharmacology lab of the experimental center of Guangxi Medical University from September 2001 to December 2002. After sacrificing animals by acute exsanguination,SCG together with their preganglionic nerve trunks were isolated rapidly,then transferred to the recording chamber, the preganglionic nerve trunk was drawn into a suction electrode for orthodromic stimulation. The ganglia were superfused continuously with a Krebs solution, saturated with 950 mL/L 02 and 5mL/L CO2, pH 7.4±0.05, (34±0.5) ℃.The fiber containing glass microelectrodes filled with 3 mol/L KC1 (30-60 MΩ tip resistance) were used to impaled cells and do intracellular recording. The fast excitatory postsynaptic potentials (FEPSPs) were evoked in SCG neurons by single pulse stimulations (0.2-0.5 Hz, 0.5-1.0 ms, 2-10 V)on preganglionic nerve trunk. The remarkable membrane depolarization would be recorded in SCG neurons by superfusing ganglia with exogenous Ach (0.1 mmol/L) or Carb(0.1 mmol/L) for 30-60 s. The effects of 1×(10-7-10-4) mol/L SmCl3 on FEPSPs, membrane potentials, membrane resistance, exogenous Ach and Carb-induced membrane depolarization of SCG neurons were investigated in this experiment.The effects of SmCl3 on the facilitation of high Ca2+ (10 mmol/L ) on FEPSPs were also be observed, namely, first superfusing the ganglia with high Ca2+ (10 mmol/L)to facilitate FEPSPs, then superfusing the ganglia with Ca2+(10 mmol/L)contained SmCl3. All the drugs were solved in Krebs solution or improved Krebs