Sample records for bumetanide

  1. Bumetanide (United States)

    ... fluid retention; excess fluid held in body tissues) caused by various medical problems, including heart, kidney, and liver disease. Bumetanide is in a class of medications called diuretics ('water pills'). It works by causing the kidneys to ...

  2. Structure-activity relationships of bumetanide derivatives

    DEFF Research Database (Denmark)

    Pedersen, Kasper Lykke; Töllner, Kathrin; Römermann, Kerstin;


    of diuretics such as bumetanide. Bumetanide was discovered by screening ∼5000 3-amino-5-sulfamoylbenzoic acid derivatives, long before NKCC2 was identified in the kidney. Therefore, structure-activity studies on effects of bumetanide derivatives on NKCC2 are not available. EXPERIMENTAL APPROACH: In this study...

  3. Bumetanide to Treat Parkinson Disease: A Report of 4 Cases. (United States)

    Damier, Philippe; Hammond, Constance; Ben-Ari, Yeheskel


    Relying on recent experimental data in 2 animal models of Parkinson disease (PD), we have tested the effects of the loop diuretic bumetanide as an add-on treatment to dopaminergic drugs in 4 volunteered patients with PD using the Unified Parkinson's Disease Rating Scale. Bumetanide is a specific antagonist of the chloride importer NKCC1 (sodium/potassium/chloride cotransporter isoform 1) that ameliorates neuronal inhibition by reducing intracellular chloride levels in a variety of pathological conditions. Bumetanide is however not labeled for use in PD. We report an improvement of PD motor symptoms in the 4 patients treated with bumetanide (5 mg/d for 2 months). Bumetanide also improved gait and freezing in 2 of these patients. Our results suggest that bumetanide is well tolerated and call for double-blind, placebo-controlled, randomized trials to confirm the therapeutic efficacy of bumetanide.





    Objective: Formulation and Development of oral dissolving Films of Bumetanide. Materials and methods: BUMETANIDE, HPMC E-50, HPMC E-5, HPMC E-3, PE, 4000 (Flakes) and Aspartame. Citric Acid could be formulated with low viscosity film formers viz. HPMC E50 in combination with HPMC E5, E15. Bumetanide could be successfully incorporated in FDFs with of the above polymers and polyethylene glycol 4000 is used as a plasticizer. PEG 4000 itself has a solubulizing affect and result in faster dissolut...

  5. Paradoxical Benzodiazepine Response : A Rationale for Bumetanide in Neurodevelopmental Disorders?

    NARCIS (Netherlands)

    Bruining, Hilgo; Passtoors, Laurien; Goriounova, Natalia; Jansen, Floor; Hakvoort, Britt; de Jonge, Maretha; Poil, Simon-Shlomo


    The diuretic agent bumetanide has recently been put forward as a novel, promising treatment of behavioral symptoms in autism spectrum disorder (ASD) and related conditions. Bumetanide can decrease neuronal chloride concentrations and may thereby reinstate γ-aminobutyric acid (GABA)-ergic inhibition

  6. Treating Schizophrenia With the Diuretic Bumetanide: A Case Report. (United States)

    Lemonnier, Eric; Lazartigues, Alain; Ben-Ari, Yehezkel


    Administration of the diuretic and NKCC1 chloride cotransporter antagonist bumetanide reduces the severity of autism spectrum disorders in children, and this effect is mediated by a reduction of the elevated intracellular chloride concentrations and a reinforcement of GABAergic inhibition (Lemonnier et al Transl Psychiatry. 2012;2:e202; Tyzio et al Science. 2014;343:675-679). Here, we report that this treatment also reduces the severity of symptoms in an adolescent with schizophrenia. Long-term treatment reduced hallucinations significantly, suggesting that this treatment may also be useful to treat schizophrenia. Further clinical trials and experimental studies are warranted to test this hypothesis.

  7. A retrospective evaluation of the efficacy of intravenous bumetanide and comparison of potency with furosemide

    Directory of Open Access Journals (Sweden)

    Nappi JM


    Full Text Available Background: The potency of intravenous bumetanide to furosemide using a ratio of 1:40 has been suggested; however, there are little data supporting this ratio. Recent drug shortages required the use of bumetanide in a large patient population, enabling further characterization of the efficacy of IV bumetanide.Objective: The primary objective of this study was to estimate a dose-response effect of IV bumetanide on urine output (UOP in all patients that received 48 hours of therapy as well as in a subgroup of patients with heart failure (HF. This subgroup was used to compare the potency of bumetanide with furosemide. A secondary safety objective described electrolyte replacement required during therapy. Methods: This was a single-center retrospective study examining the dose-response effect of IV bumetanide in patients receiving at least 48 hours of intermittent (iIV or continuous (cIV dosing, measured by UOP per mg of drug received (mL/mg. The potency of IV bumetanide was compared with furosemide in a subset of patients with HF using pre-existing data. The safety of IV bumetanide was analyzed by quantifying electrolyte replacement received during the study period.Results: The primary outcome was higher in the iIV group (n=93 at 1273 ± 844 mL/mg compared with the cIV group (n=16 at 749 ± 370 mL/mg (P=0.002. Among patients with HF who received furosemide (iIV n=30, cIV n=26 or bumetanide (iIV n=30, cIV n=3, a potency ratio of 41:1 was found for the iIV group and 34:1 for all patients with HF. There was no significant difference in electrolyte replacement between groups.Conclusion: A greater response was seen with intermittent bumetanide compared with continuous infusion bumetanide. This study supports the 40:1 dose equivalence ratio (furosemide:bumetanide in patients with HF receiving at least 48 hours of intravenous intermittent bumetanide.

  8. The effect of bumetanide treatment on the sensory behaviours of a young girl with Asperger syndrome. (United States)

    Grandgeorge, Marine; Lemonnier, Eric; Degrez, Céline; Jallot, Nelle


    Sensory behaviours were not considered as core features of autism spectrum disorders until recently. However, they constitute an important part of the observed symptoms that result in social maladjustment and are currently quite difficult to treat. One promising strategy for the treatment of these behaviours is the use of bumetanide, which was previously shown to reduce the severity of autism spectrum disorders. In this study, we proposed to evaluate sensory behaviours using Dunn's Sensory Profile after 18 months of bumetanide treatment in a 10-year-old girl with Asperger syndrome. Reported improvements covered a large range of sensory behaviours, including auditory, vestibular, tactile, multisensory and oral sensory processing. Although our results were limited to a single case report, we believe that our clinical observations warrant clinical trials to test the long-term efficacy of bumetanide to manage the sensory behaviours of people with autism spectrum disorders.

  9. A randomised controlled trial of bumetanide in the treatment of autism in children. (United States)

    Lemonnier, E; Degrez, C; Phelep, M; Tyzio, R; Josse, F; Grandgeorge, M; Hadjikhani, N; Ben-Ari, Y


    Gamma aminobutyric acid (GABA)-mediated synapses and the oscillations they orchestrate are altered in autism. GABA-acting benzodiazepines exert in some patients with autism paradoxical effects, raising the possibility that like in epilepsies, GABA excites neurons because of elevated intracellular concentrations of chloride. Following a successful pilot study,(1) we have now performed a double-blind clinical trial using the diuretic, chloride-importer antagonist bumetanide that reduces intracellular chloride reinforcing GABAergic inhibition. Sixty children with autism or Asperger syndrome (3-11 years old) received for 3 months placebo or bumetanide (1 mg daily), followed by 1-month wash out. Determination of the severity of autism was made with video films at day 0 (D0) and D90 by blind, independent evaluators. Bumetanide reduced significantly the Childhood Autism Rating Scale (CARS) (D90-D0; PAutism Diagnostic Observation Schedule values when the most severe cases (CARS values above the mean ± s.d.; n=9) were removed (Wilcoxon test: P-value=0.031; Student's t-test: P-value=0.017). Side effects were restricted to an occasional mild hypokalaemia (3.0-3.5 mM l(-1) K(+)) that was treated with supplemental potassium. In a companion study, chronic bumetanide treatment significantly improved accuracy in facial emotional labelling, and increased brain activation in areas involved in social and emotional perception (Hadjikhani et al., submitted). Therefore, bumetanide is a promising novel therapeutic agent to treat autism. Larger trials are warranted to better determine the population best suited for this treatment.

  10. The effect on serum enzymes of intramuscular injections of digoxin, bumetanide, pentazocine and isotonic sodium chloride

    DEFF Research Database (Denmark)

    Andersen, Klaus Ejner; Damsgaard, T


    Intramuscular injections of digoxin, bumetanide, pentazocine or isotonic sodium chloride have been given to 39 patients. We followed the serum concentrations of creatine kinase (CK), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH) and LDH isoenzymes for 4 days. Ten patients receiving...... 500 mug digoxin showed a significant rise in CK, which lasted for 48 hours, and 6 of them had CK values exceeding the upper normal limit. Pentazocine in a dose of 30 mg given to 9 patients caused a significant rise in CK and LDH isoenzyme 1, but in no case did the level exceed the upper normal limit....... No rise in ASAT or total LDH was found after digoxin and pentazocine injections. No changes in enzymes were discovered after bumetanide or isotonic sodium chloride. In the diagnostic evaluation of acute myocardial infarction, a moderate rise in CK must be assessed with caution when the patients have...

  11. Bumetanide for the treatment of seizures in newborn babies with hypoxic ischaemic encephalopathy (NEMO) : an open-label, dose finding, and feasibility phase 1/2 trial

    NARCIS (Netherlands)

    Pressler, Ronit M.; Boylan, Geraldine B.; Marlow, Neil; Blennow, Mats; Chiron, Catherine; Cross, J. Helen; de Vries, Linda S.; Hallberg, Boubou; Hellstrom-Westas, Lena; Jullien, Vincent; Livingstone, Vicki; Mangum, Barry; Murphy, Brendan; Murray, Deirdre; Pons, Gerard; Rennie, Janet; Swarte, Renate; Toet, Mona C.; Vanhatalo, Sampsa; Zohar, Sarah


    Background Predinical data suggest that the loop-diuretic bumetanide might be an effective treatment for neonatal seizures. We aimed to assess dose and feasibility of intravenous bumetanide as an add-on to phenobarbital for treatment of neonatal seizures. Methods In this open-label, dose finding, an

  12. Bumetanide promotes neural precursor cell regeneration and dendritic development in the hippocampal dentate gyrus in the chronic stage of cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Wang-shu Xu; Xuan Sun; Cheng-guang Song; Xiao-peng Mu; Wen-ping Ma; Xing-hu Zhang; Chuan-sheng Zhao


    Bumetanide has been shown to lessen cerebral edema and reduce the infarct area in the acute stage of cerebral ischemia. Few studies focus on the effects of bumetanide on neuroprotection and neurogenesis in the chronic stage of cerebral ischemia. We established a rat model of cerebral ischemia by injecting endothelin-1 in the left cortical motor area and left corpus striatum. Seven days later, bumeta-nide 200 µg/kg/day was injected into the lateral ventricle for 21 consecutive days with a mini-osmotic pump. Results demonstrated that the number of neuroblasts cells and the total length of dendrites increased, escape latency reduced, and the number of platform crossings increased in the rat hippocampal dentate gyrus in the chronic stage of cerebral ischemia. These ifndings suggest that bumetanide promoted neural precursor cell regeneration, dendritic development and the recovery of cognitive function, and protected brain tissue in the chronic stage of ischemia.

  13. Bumetanide, an NKCC1 antagonist, does not prevent formation of epileptogenic focus but blocks epileptic focus seizures in immature rat hippocampus. (United States)

    Nardou, Romain; Ben-Ari, Yehezkel; Khalilov, Ilgam


    Excitatory GABA action induced by high [Cl(-)](i) is thought to contribute to seizure generation in neonatal neurons although the mechanism of this effect remains unclear. We report that bumetanide, a NKCC1 antagonist, reduces driving force of GABA-mediated currents (DF(GABA)) in neonatal hippocampal neurons and blocks the giant depolarizing potentials (GDPs), a spontaneous pattern of network activity. In the preparation composed of two intact interconnected hippocampi, bumetanide did not prevent generation of kainate-induced seizures, their propagation to the contralateral hippocampus, and formation of an epileptogenic mirror focus. However, in the isolated mirror focus, bumetanide effectively blocked spontaneous epileptiform activity transforming it to the GDP-like activity pattern. Bumetanide partially reduced DF(GABA) and therefore the excitatory action of GABA in epileptic neurons. Therefore bumetanide is a potent anticonvulsive agent although it cannot prevent formation of the epileptogenic mirror focus. We suggest that an additional mechanism other than NKCC1-mediated contributes to the persistent increase of DF(GABA) in epileptic neurons.

  14. Failure of the Nemo trial: bumetanide is a promising agent to treat many brain disorders but not newborn seizures

    Directory of Open Access Journals (Sweden)

    Yehezkel eBen-Ari


    Full Text Available The diuretic bumetanide failed to treat acute seizures due to hypoxic ischemic encephalopathy (HIE in newborn babies and was associated with hearing loss (NEMO trial; 1. On the other hand, clinical and experimental observations suggest that the diuretic might provide novel therapy for many brain disorders including autistic spectrum disorder, schizophrenia, Rett syndrome and Parkinson disease. Here, we discuss the differences between the pathophysiology of severe recurrent seizures in the neonates and neurological and psychiatric disorders stressing the uniqueness of severe seizures in newborn in comparison to other disorders.

  15. Failure of the Nemo Trial: Bumetanide Is a Promising Agent to Treat Many Brain Disorders but Not Newborn Seizures. (United States)

    Ben-Ari, Yehezkel; Damier, Philippe; Lemonnier, Eric


    The diuretic bumetanide failed to treat acute seizures due to hypoxic ischemic encephalopathy (HIE) in newborn babies and was associated with hearing loss (NEMO trial, Pressler et al., 2015). On the other hand, clinical and experimental observations suggest that the diuretic might provide novel therapy for many brain disorders including Autism Spectrum Disorders (ASD), schizophrenia, Rett syndrome, and Parkinson disease. Here, we discuss the differences between the pathophysiology of severe recurrent seizures in the neonates and neurological and psychiatric disorders stressing the uniqueness of severe seizures in newborn in comparison to other disorders.

  16. A randomized, placebo-controlled study of loop diuretics in patients with essential hypertension : The bumetanide and furosemide on lipid profile (BUFUL) clinical study report

    NARCIS (Netherlands)

    Donders, SH; Cleophas, TJ; Niemeyer, MG; van der Meulen, J; Bernink, PJ; de Planque, BA; van der Wall, EE


    This study was conducted to determine whether loop diuretics are more effective than placebo in reducing blood pressure without raising serum lipid levels, and whether bumetanide is more effective than furosemide in this respect In a double-blind, 24-week placebo-controlled crossover study, 27 patie

  17. Improving Emotional Face Perception in Autism with Diuretic Bumetanide: A Proof-of-Concept Behavioral and Functional Brain Imaging Pilot Study (United States)

    Hadjikhani, Nouchine; Zürcher, Nicole R; Rogier, Ophelie; Ruest, Torsten; Hippolyte, Loyse; Ben-Ari, Yehezkel; Lemonnier, Eric


    Clinical observations have shown that GABA-acting benzodiazepines exert paradoxical excitatory effects in autism, suggesting elevated intracellular chloride (Cl-)[subscript i] and excitatory action of GABA. In a previous double-blind randomized study, we have shown that the diuretic NKCC1 chloride importer antagonist bumetanide, that decreases…

  18. Bumetanide reduce the seizure susceptibility induced by pentylenetetrazol via inhibition of aberrant hippocampal neurogenesis in neonatal rats after hypoxia-ischemia. (United States)

    Hu, Jiang-Jian; Yang, Xing-Liang; Luo, Wen-Di; Han, Song; Yin, Jun; Liu, Wan-Hong; He, Xiao-Hua; Peng, Bi-Wen


    Hypoxia-ischemia brain damage (HIBD) is one of prevalent causes of neonatal mortality and morbidity. Our data demonstrated that hypoxia-ischemia (HI) induced Na(+)-K(+)-Cl(-)-co-transporter 1 (NKCC1) increasing in hippocampus. Previous studies demonstrated that NKCC1 regulates various stages of neurogenesis. In this study, we studied the role of increased NKCC1 in regulating of HI-induced neurogenesis. HIBD model was established in 7days old Sprague-Dawley rat pup, and the expression of NKCC1 was detected by western blot and qPCR. Brain electrical activity in freely rats was monitored by electroencephalography (EEG) recordings. HI-induced neurogenesis was detected by immunofluorescence staining. Neurobehavioral test was to investigate the neuro-protective role of bumetanide, an inhibitor of NKCC1, on neonatal rats after HI. The results showed that bumetanide treatment significantly reduced brain electrical activity and the seizure stage of epilepsy induced by pentylenetetrazol (PTZ) in vivo after HI. In addition, bumetanide restored aberrant hippocampal neurogenesis and associated cognitive function. Our data demonstrated that bumetanide reduces the susceptibility of epilepsy induced by PTZ in rats suffering from HI injury during neonatal period via restoring the ectopic newborn neurons in dentate gyrus (DG) and cognitive function.

  19. Analgesic effect of intrathecal bumetanide is accompanied by changes in spinal sodium-potassium-chloride co-transporter 1 and potassium-chloride co-transporter 2 expression in a rat model of incisional pain

    Institute of Scientific and Technical Information of China (English)

    Yanbing He; Shiyuan Xu; Junjie Huang; Qingjuan Gong


    Accumulating evidence has demonstrated that the sodium-potassium-chloride co-transporter 1 and potassium-chloride co-transporter 2 have a role in the modulation of pain transmission at the spinal level through chloride regulation in the pain pathway and by effecting neuronal excitability and pain sensitization. The present study aimed to investigate the analgesic effect of the speciifc sodium-potassium-chloride co-transporter 1 inhibitor bumetanide, and the change in spinal sodium-potassium-chloride co-transporter 1 and potassium-chloride co-transporter 2 expression in a rat model of incisional pain. Results showed that intrathecal bumetanide could decrease cumulative pain scores, and could increase thermal and mechanical pain thresholds in a rat model of incisional pain. Sodium-potassium-chloride co-transporter 1 expression in-creased in neurons from dorsal root ganglion and the deep laminae of the ipsilateral dorsal horn following incision. By contrast, potassium-chloride co-transporter 2 expression decreased in neurons of the deep laminae from the ipsilateral dorsal horn. These ifndings suggest that spinal sodium-potassium-chloride co-transporter 1 expression was up-regulated and spinal potassi-um-chloride co-transporter 2 expression was down-regulated following incision. Intrathecal bumetanide has analgesic effects on incisional pain through inhibition of sodium-potassi-um-chloride co-transporter 1.

  20. Altered expression of renal bumetanide-sensitive sodium-pota-ssium-2 chloride cotransporter and Cl- channel -K2 gene in angiotensin Ⅱ-infused hypertensive rats

    Institute of Scientific and Technical Information of China (English)

    YE Tao; LIU Zhi-quan; SUN Chao-feng; ZHENG Yong; MA Ai-qun; FANG Yuan


    Background Little information is available regarding the effect of angiotensin Ⅱ (Ang Ⅱ) on the bumetanide-sensitive sodium-potassium-2 chloride cotransporter (NKCC2), the thiazide-sensitive sodium-chloride cotransporter (NCC), and the Cl- channel (CLC)-K2 at both mRNA and protein expression level in Ang Ⅱ-induced hypertensive rats. This study was conducted to investigate the influence of Ang Ⅱ with chronic subpressor infusion on nephron-specific gene expression of NKCC2, NCC and CLC-K2. Results Ang Ⅱ significantly increased blood pressure and up-regulated NKCC2 mRNA and protein expression in the kidney. Expression of CLC-K2 mRNA in the kidney increased 1.6 fold (P<0.05).There were no changes in NCC mRNA or protein expression in AngII-treated rats versus control. Conclusions Chronic subpressor Ang Ⅱ infusion can significantly alter NKCC2 and CLC-K2 mRNA expression in the kidney, and protein abundance of NKCC2 in kidney is positively regulated by Ang Ⅱ. These effects may contribute to enhanced renal Na+ and Cl- reabsorption in response to Ang Ⅱ.

  1. Cellular distribution of the renal bumetanide-sensitive Na-K-2Cl cotransporter BSC-1 in the inner stripe of the outer medulla during the development of hypertension in the spontaneously hypertensive rat. (United States)

    Sonalker, Prajakta A; Tofovic, Stevan P; Jackson, Edwin K


    1. The renal bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1) is expressed only in the thick ascending limb and selectively traffics from intracellular vesicles (IVs) to apical plasma membranes (PMs), where BSC-1 regulates sodium reabsorption. We showed previously that in kidneys from adult spontaneously hypertensive rats (SHR; model of essential hypertension) total protein expression of BSC-1 was higher compared with kidneys from normotensive Wistar-Kyoto (WKY) rats. However, whether this change is associated with an increased trafficking of BSC-1 from IVs to PMs is unknown. The goal of the present study was to test the hypothesis that the increase in total renal BSC-1 protein expression in SHR is accompanied by an augmented distribution of BSC-1 from IVs to PMs. 2. To test the hypothesis, we obtained renal tissue from the inner stripe of the outer medulla (ISOM; enriched in thick ascending limbs) and isolated IVs and PMs from this tissue by differential centrifugation. Total BSC-1 protein expression in ISOM and BSC-1 protein expression in ISOM IVs and PMs were measured by semiquantitative western blotting in SHR and aged-matched WKY rats at different ages and stages of hypertension. 3. At 5 weeks of age, SHR were prehypertensive (mean arterial blood pressure (MABP) 97 mmHg). At this age, both the total abundance and cellular distribution of BSC-1 were similar in ISOM from SHR and WKY rats. 4. As SHR aged, their hypertension progressed (MABP 137 and 195 mmHg at 8 and 14 weeks of age, respectively). Associated with the increase in MABP was an increase in both steady state protein levels of ISOM BSC-1 and the distribution of ISOM BSC-1 to PMs (four- and sixfold increases at 8 and 14 weeks of age, respectively, compared with age-matched WKY rats; P BSC-1 mRNA was measured and was found not to differ between SHR and WKY rat ISOM at any age or level of MABP. 6. We conclude that as SHR transition from prehypertensive to established hypertension, there is a marked

  2. Bumetanide, an Inhibitor of NKCC1 (Na-K-2Cl Cotransporter Isoform 1), Enhances Propofol-Induced Loss of Righting Reflex but Not Its Immobilizing Actions in Neonatal Rats


    Koyama, Yukihide; Andoh, Tomio; Kamiya, Yoshinori; Miyazaki, Tomoyuki; Maruyama, Koichi; Kariya, Takayuki; Goto, Takahisa


    Gamma-aminobutyric acid (GABA) has been shown to induce excitation on immature neurons due to increased expression of Na+-K+-2Cl- co-transporter isoform 1 (NKCC1), and the transition of GABAergic signaling from excitatory to inhibitory occurs before birth in the rat spinal cord and spreads rostrally according to the developmental changes in cation-chloride co-transporter expression. We previously showed that midazolam activates the hippocampal CA3 area and induces less sedation in neonatal ra...

  3. In vitro studies of theophylline-induced changes in Na, K and Cl transport in hen (Gallus domesticus) colon suggesting bidirectional, basolateral NaK2Cl cotransport

    DEFF Research Database (Denmark)

    Andersen, Vibeke; Munck, B G; Munck, L K;


    1. In isolated mucosa from a NaCl-loaded hen theophylline stimulates both unidirectional chloride fluxes (JmsCl and JsmCl). Conductive and electroneutral exchange processes, besides a bumetanide-sensitive, rheogenic process contribute. 2. The bumetanide-sensitive fraction of the theophylline......-induced delta JcmCl is sodium-dependent. 3. Incubation in nominally K(+)-free solutions reduces the bumetanide-sensitive fraction delta JsmCl more than treatment with ouabain. 4. With respect to chloride the bumetanide-sensitive fraction of delta JsmCl has a Hill coefficient of 1.93 +/- 0.03, a Jmax of 12...

  4. Dosage dependent hormonal counter regulation to combination therapy in patients with left ventricular dysfunction

    DEFF Research Database (Denmark)

    Galløe, A.M.; Skagen, K.; Christensen, Niels Juel


    rate and plasma noradrenaline in a dose dependent manner. Doses of bumetanide of more than 0.5 mg, given twice daily significantly decreased the quality of life and increased diuresis. Weight loss was maximal on 0.5 mg bumetanide twice daily. Trandolapril significantly reduced systolic blood pressure...

  5. Roles of the cytoskeleton and of Protein Phosphorylation Events in the Osmotic Stress Response in EEL Intestinal Epithelium

    DEFF Research Database (Denmark)

    Lionetto, Maria G; Pedersen, Stine F; Hoffmann, Else K;


    phase is bumetanide-insensitive, the second, sustained phase is bumetanide-sensitive, reflecting activation of the apically located Na(+)-K(+)-2Cl(-) (NKCC) cotransporter, which correlates with the cellular RVI response. Here, we investigated the involvement of the cytoskeleton and of serine...... 15%, detectable morphologically mainly as a decrease in the intensity of the apical brush border F-actin labeling.The bumetanide-sensitive response of V(te) and Isc to hypertonicity was potently inhibited by treatment with either cytochalasin, latrunculin A, colchicine, the protein kinase C (PKC...

  6. Is it safe to use a diuretic to treat seizures early in development ? (United States)

    Ben-Ari, Y; Tyzio, R


    There has been considerable interest in using bumetanide, a diuretic chloride importer NKCC1 antagonist, to reduce intracellular chloride ([Cl(-)](i)) in epileptic neurons, thereby shifting the polarity of GABA from excitatory to inhibitory and ameliorating the actions of GABA-acting antiepileptic drugs. However, a recent study raises the important issue of potential deleterious actions of bumetanide on immature neurons, because reduction of (Cl(-))(i) also alleviates a major source of excitation in developing neurons, upon which GABA exerts a trophic action. This review considers the importance of separating intrauterine from postnatal effects of bumetanide in normal versus pathologic neurons.

  7. Cisplatin Injection (United States)

    ... Dilantin), bumetanide (Bumex), ethacrynic acid (Edecrin), furosemide (Lasix), pyridoxine (Vitamin B-6). Your doctor may need to ... looks like coffee grounds Cisplatin may increase the risk that you will develop other cancers. Talk to ...

  8. Is It Safe to Use a Diuretic to Treat Seizures Early in Development ?


    Ben-Ari, Y.; Tyzio, R


    There has been considerable interest in using bumetanide, a diuretic chloride importer NKCC1 antagonist, to reduce intracellular chloride ([Cl−]i) in epileptic neurons, thereby shifting the polarity of GABA from excitatory to inhibitory and ameliorating the actions of GABA-acting antiepileptic drugs. However, a recent study raises the important issue of potential deleterious actions of bumetanide on immature neurons, because reduction of (Cl−)i also alleviates a major source of excitation in ...

  9. Loop diuretics have anxiolytic effects in rat models of conditioned anxiety.

    Directory of Open Access Journals (Sweden)

    Andrew D Krystal

    Full Text Available A number of antiepileptic medications that modulate GABA(A mediated synaptic transmission are anxiolytic. The loop diuretics furosemide (Lasix and bumetanide (Bumex are thought to have antiepileptic properties. These drugs also modulate GABA(A mediated signalling through their antagonism of cation-chloride cotransporters. Given that loop diuretics may act as antiepileptic drugs that modulate GABAergic signalling, we sought to investigate whether they also mediate anxiolytic effects. Here we report the first investigation of the anxiolytic effects of these drugs in rat models of anxiety. Furosemide and bumetanide were tested in adult rats for their anxiolytic effects using four standard anxiety models: 1 contextual fear conditioning; 2 fear-potentiated startle; 3 elevated plus maze, and 4 open-field test. Furosemide and bumetanide significantly reduced conditioned anxiety in the contextual fear-conditioning and fear-potentiated startle models. At the tested doses, neither compound had significant anxiolytic effects on unconditioned anxiety in the elevated plus maze and open-field test models. These observations suggest that loop diuretics elicit significant anxiolytic effects in rat models of conditioned anxiety. Since loop diuretics are antagonists of the NKCC1 and KCC2 cotransporters, these results implicate the cation-chloride cotransport system as possible molecular mechanism involved in anxiety, and as novel pharmacological target for the development of anxiolytics. In view of these findings, and since furosemide and bumetanide are safe and well tolerated drugs, the clinical potential of loop diuretics for treating some types of anxiety disorders deserves further investigation.

  10. Loop Diuretics Have Anxiolytic Effects in Rat Models of Conditioned Anxiety (United States)

    Krystal, Andrew D.; Sutherland, Janice; Hochman, Daryl W.


    A number of antiepileptic medications that modulate GABAA mediated synaptic transmission are anxiolytic. The loop diuretics furosemide (Lasix) and bumetanide (Bumex) are thought to have antiepileptic properties. These drugs also modulate GABAA mediated signalling through their antagonism of cation-chloride cotransporters. Given that loop diuretics may act as antiepileptic drugs that modulate GABAergic signalling, we sought to investigate whether they also mediate anxiolytic effects. Here we report the first investigation of the anxiolytic effects of these drugs in rat models of anxiety. Furosemide and bumetanide were tested in adult rats for their anxiolytic effects using four standard anxiety models: 1) contextual fear conditioning; 2) fear-potentiated startle; 3) elevated plus maze, and 4) open-field test. Furosemide and bumetanide significantly reduced conditioned anxiety in the contextual fear-conditioning and fear-potentiated startle models. At the tested doses, neither compound had significant anxiolytic effects on unconditioned anxiety in the elevated plus maze and open-field test models. These observations suggest that loop diuretics elicit significant anxiolytic effects in rat models of conditioned anxiety. Since loop diuretics are antagonists of the NKCC1 and KCC2 cotransporters, these results implicate the cation-chloride cotransport system as possible molecular mechanism involved in anxiety, and as novel pharmacological target for the development of anxiolytics. In view of these findings, and since furosemide and bumetanide are safe and well tolerated drugs, the clinical potential of loop diuretics for treating some types of anxiety disorders deserves further investigation. PMID:22514741

  11. Choroid plexus potassium cotransport: modulation by osmotic stress and external potassium. (United States)

    Keep, R F; Xiang, J


    The choroid plexuses are involved in CSF secretion and CSF K homeostasis. This study examines the potential role of K cotransport in these two processes using isolated rat lateral ventricle choroid plexuses. Bumetanide-sensitive 86Rb influx and efflux were measured to assess the response of K cotransport to changes in media osmolality and K concentration. Alterations in osmolality had no effect on K uptake (in the presence or absence of bumetanide). However, the efflux rate constant for K was 0.29 +/- 0.02, 0.44 +/- 0.04, and 0.84 +/- 0.06 min-1 in 240, 300, and 424 mOsm/kg solutions, respectively (p brain shrinkage during hyperosmotic stress if the cotransporter is present on the apical membrane. The rate of bumetanide-sensitive efflux was unaffected by changes in external [K]. However, the rate of K uptake (measured on return to normal [K] media) was reduced gradually by exposure to low [K]. It was 21 +/- 1, 19 +/- 3, 13 +/- 2, and 6 +/- 1 nmol/mg/min after 0, 10, 30, and 60-min exposure to 1 mM K. Sixty minutes of exposure to 1 mM [K] abolished the bumetanide-sensitive K uptake present in plexuses exposed continually to normal media. This modulation of K cotransport by external [K] may be important in CSF K homeostasis by limiting K loss from the CSF if CSF [K] is low.


    Directory of Open Access Journals (Sweden)

    A. V. Skvortsov


    Full Text Available The aim of this study was to investigate the role of the Na+, K+, 2Cl– -transport and potassium conductance, in the implementation of the effects of carbon monoxide in the SMC taenia coli and ureter of guinea pig.Materials and methods. The study was conducted by the method of double saccharose bridge. Was studied the effects of carbon monoxide donor CORM2 in ureter and smooth muscle of guinea pig of taenia coli, in normal Krebs solution, against inhibitor-bumetanide and potassium channel blocker tetraethylammonium chloride (TEA. Studied the effects of bumetanide in normal Krebs solution and the background of TEA.Main results. CORM2 causes inhibition of contractile activity of SMC taenia coli and guinea pig ureter. Its effect is weakened by bumetanide and TEA. Bumetanide causes inhibition of contractile activity of SMC taenia coli, and its effect is weakened by TEA.Conclusions. In this way, it shows that there are tissue-specific mechanisms of interrelation effects CO and ion conductivity.

  13. Response to Comment on "Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring". (United States)

    Eftekhari, Sanaz; Shahrokhi, Amene; Tsintsadze, Vera; Nardou, Romain; Brouchoud, Corinne; Conesa, Magali; Burnashev, Nail; Ferrari, Diana C; Ben-Ari, Yehezkel


    Bambini-Junior et al. questioned whether our treatment in two rodent models of autism has a long-lasting effect into adulthood. In response, we show that bumetanide treatment around delivery attenuates autistic behavioral features in adult offspring. Therefore, the polarity of γ-aminobutyric acid (GABA) actions during delivery exerts long-lasting priming actions after birth.

  14. Dosage dependent hormonal counter regulation to combination therapy in patients with left ventricular dysfunction

    DEFF Research Database (Denmark)

    Galløe, A.M.; Skagen, K.; Christensen, N.J.


    The present study attempts to assess the efficacy combination therapy for heart failure. Genuine dose-response studies on combination therapy are not available and published studies involved adding one drug on top of 'usual treatment'. Sixteen different dosage combinations of trandolapril...... rate and plasma noradrenaline in a dose dependent manner. Doses of bumetanide of more than 0.5 mg, given twice daily significantly decreased the quality of life and increased diuresis. Weight loss was maximal on 0.5 mg bumetanide twice daily. Trandolapril significantly reduced systolic blood pressure...... of life and weight loss. Estimated by the reduction in systolic blood pressure the optimal dosage of Trandolapril appeared to be 0.5 mg once daily. CONCLUSIONS: It appears that patients should be given less than the usually recommended dosages. Patients may be treated with a low dose loop diuretic...

  15. Loop-acting diuretics do not bind to Tamm-Horsfall urinary glycoprotein. (United States)

    Brunisholz, M C; Lynn, K L; Hunt, J S


    1. Binding between the radiolabelled loop-acting diuretics ([14C]frusemide, [14C]ethacrynic acid and [3H]bumetanide) and human Tamm-Horsfall glycoprotein or human serum albumin in vitro was evaluated by equilibrium dialysis. 2. The diuretic action and binding to urinary Tamm-Horsfall glycoprotein of the radiolabelled diuretics in vivo, after intravenous administration, were examined in rabbits. 3. In vitro, all three radiolabelled diuretics bound strongly to human serum albumin, but not to Tamm-Horsfall glycoprotein. 4. Radiolabelled frusemide and bumetanide, but not ethacrynic acid, caused a diuresis in rabbits, but no binding between the drugs and Tamm-Horsfall glycoprotein was seen in vivo. 5. Binding to Tamm-Horsfall glycoprotein does not appear to be an important mechanism in the action of loop diuretics.

  16. Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring. (United States)

    Tyzio, Roman; Nardou, Romain; Ferrari, Diana C; Tsintsadze, Timur; Shahrokhi, Amene; Eftekhari, Sanaz; Khalilov, Ilgam; Tsintsadze, Vera; Brouchoud, Corinne; Chazal, Genevieve; Lemonnier, Eric; Lozovaya, Natalia; Burnashev, Nail; Ben-Ari, Yehezkel


    We report that the oxytocin-mediated neuroprotective γ-aminobutyric acid (GABA) excitatory-inhibitory shift during delivery is abolished in the valproate and fragile X rodent models of autism. During delivery and subsequently, hippocampal neurons in these models have elevated intracellular chloride levels, increased excitatory GABA, enhanced glutamatergic activity, and elevated gamma oscillations. Maternal pretreatment with bumetanide restored in offspring control electrophysiological and behavioral phenotypes. Conversely, blocking oxytocin signaling in naïve mothers produced offspring having electrophysiological and behavioral autistic-like features. Our results suggest a chronic deficient chloride regulation in these rodent models of autism and stress the importance of oxytocin-mediated GABAergic inhibition during the delivery process. Our data validate the amelioration observed with bumetanide and oxytocin and point to common pathways in a drug-induced and a genetic rodent model of autism.

  17. Water permeability of Na+-K+-2C1- cotransporters in mammalian epithelial cells

    DEFF Research Database (Denmark)

    Hammann, Steffen; Herrera-Perez, J.J.; Bundgaard, Magnus


    Water transport properties of the Na+-K+-2Cl- cotransporter (NKCC) were studied in cultures of pigmented epithelial cells (PE) from the ciliary body of the eye. Here, the membrane that faces upwards contains NKCCs and can be subjected to rapid changes in bathing solution composition and osmolarity...... changes of the cotransporter and interaction with Na+, K+ and Cl-. Similar measurements were performed on immortalized cell cultures from the thick ascending limb of the loop of Henle (TALH). Given similar overall transport rates of bumetanide-sensitive 86Rb+, the NKCCs of this tissue did not contribute...... any bumetanide-sensitive Lp. This suggests that the cotransporters of the two tissues are either different isoforms or the same cotransporter but in two different transport modes....

  18. Mechanisms of guanylin action on water and ion absorption at different regions of seawater eel intestine. (United States)

    Ando, Masaaki; Wong, Marty K S; Takei, Yoshio


    Guanylin (GN) inhibited water absorption and short-circuit current (Isc) in seawater eel intestine. Similar inhibition was observed after bumetanide, and the effect of bumetanide was abolished by GN or vice versa, suggesting that both act on the same target, Na(+)-K(+)-2Cl(-) cotransporter (NKCC), which is a key player for the Na(+)-K(+)-Cl(-) transport system responsible for water absorption in marine teleost intestine. However, effect of GN was always greater than that of bumetanide: 10% greater in middle intestine (MI) and 40% in posterior intestine (PI) for Isc, and 25% greater in MI and 34% in PI for water absorption. After treatment with GN, Isc decreased to zero, but 20-30% water absorption still remained. The remainder may be due to the Cl(-)/HCO3 (-) exchanger and Na(+)-Cl(-) cotransporter (NCC), since inhibitors for these transporters almost nullified the remaining water absorption. Quantitative PCR analysis revealed the presence of major proteins involved in water absorption; the NKCC2β and AQP1 genes whose expression was markedly upregulated after seawater acclimation. The SLC26A6 (anion exchanger) and NCCβ genes were also expressed in small amounts. Consistent with the inhibitors' effect, expression of NKCC2β was MI > PI, and that of NCCβ was MI intestine, and its role may be minor, as indicated by the small effect of its inhibitors.

  19. Potassium channel and NKCC cotransporter involvement in ocular refractive control mechanisms.

    Directory of Open Access Journals (Sweden)

    Sheila G Crewther

    Full Text Available Myopia affects well over 30% of adult humans globally. However, the underlying physiological mechanism is little understood. This study tested the hypothesis that ocular growth and refractive compensation to optical defocus can be controlled by manipulation of potassium and chloride ion-driven transretinal fluid movements to the choroid. Chicks were raised with +/-10D or zero power optical defocus rendering the focal plane of the eye in front of, behind, or at the level of the retinal photoreceptors respectively. Intravitreal injections of barium chloride, a non-specific inhibitor of potassium channels in the retina and RPE or bumetanide, a selective inhibitor of the sodium-potassium-chloride cotransporter were made, targeting fluid control mechanisms. Comparison of refractive compensation to 5 mM Ba(2+ and 10(-5 M bumetanide compared with control saline injected eyes shows significant change for both positive and negative lens defocus for Ba(2+ but significant change only for negative lens defocus with bumetanide (Rx(SAL(-10D = -8.6 +/- .9 D; Rx(Ba2+(-10D = -2.9 +/- .9 D; Rx(Bum(-10D = -2.9 +/- .9 D; Rx(SAL(+10D = +8.2 +/- .9 D; Rx(Ba2+(+10D = +2.8 +/- 1.3 D; Rx(Bum(+10D = +8.0 +/- .7 D. Vitreous chamber depths showed a main effect for drug conditions with less depth change in response to defocus shown for Ba(2+ relative to Saline, while bumetanide injected eyes showed a trend to increased depth without a significant interaction with applied defocus. The results indicate that both K channels and the NKCC cotransporter play a role in refractive compensation with NKCC blockade showing far more specificity for negative, compared with positive, lens defocus. Probable sites of action relevant to refractive control include the apical retinal pigment epithelium membrane and the photoreceptor/ON bipolar synapse. The similarities between the biometric effects of NKCC inhibition and biometric reports of the blockade of the retinal ON response, suggest a

  20. Characterization of slow waves generated by myenteric interstitial cells of Cajal of the rabbit small intestine. (United States)

    Kito, Yoshihiko; Mitsui, Retsu; Ward, Sean M; Sanders, Kenton M


    Slow waves (slow wavesICC) were recorded from myenteric interstitial cells of Cajal (ICC-MY) in situ in the rabbit small intestine, and their properties were compared with those of mouse small intestine. Rabbit slow wavesICC consisted of an upstroke depolarization followed by a distinct plateau component. Ni(2+) and nominally Ca(2+)-free solutions reduced the rate-of-rise and amplitude of the upstroke depolarization. Replacement of Ca(2+) with Sr(2+) enhanced the upstroke component but decreased the plateau component of rabbit slow wavesICC. In contrast, replacing Ca(2+) with Sr(2+) decreased both components of mouse slow wavesICC. The plateau component of rabbit slow wavesICC was inhibited in low-extracellular-Cl(-)-concentration (low-[Cl(-)]o) solutions and by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of Cl(-) channels, cyclopiazonic acid (CPA), an inhibitor of internal Ca(2+) pumps, or bumetanide, an inhibitor of Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). Bumetanide also inhibited the plateau component of mouse slow wavesICC. NKCC1-like immunoreactivity was observed mainly in ICC-MY in the rabbit small intestine. Membrane depolarization with a high-K(+) solution reduced the upstroke component of rabbit slow wavesICC. In cells depolarized with elevated external K(+), DIDS, CPA, and bumetanide blocked slow wavesICC. These results suggest that the upstroke component of rabbit slow wavesICC is partially mediated by voltage-dependent Ca(2+) influx, whereas the plateau component is dependent on Ca(2+)-activated Cl(-) efflux. NKCC1 is likely to be responsible for Cl(-) accumulation in ICC-MY. The results also suggest that the mechanism of the upstroke component differs in rabbit and mouse slow wavesICC in the small intestine.

  1. Effects of cyclooxygenase and lipoxygenase inhibition on basal- and serotonin-induced ion transport in rat colon

    DEFF Research Database (Denmark)

    Engelmann, Bodil Elisabeth; Bindslev, Niels; Poulsen, Steen Seier;


    . 5-HT and PGE2 both induced a concentration-dependent increase in SCC by activation of multiple receptors. The response to 5-HT was bumetanide-sensitive. Neither the non-selective COX inhibitor piroxicam, nor the selective COX-2 inhibitor SC-'236, altered basal- SCC or 5-HT-induced SCC. Indomethacin...... reduced both basal- and 5-HT-induced SCC in both segments. Nordihydroguaiaretic acid reduced the 5-HT-induced increase in SCC, but did not change basal SCC. 5-HT-induced a concentration-dependent release of PGE2. Only high concentrations of piroxicam and indomethacin reduced basal PGE2 release and 5-HT...

  2. Osmoregulation Requires Brain Expression of the Renal Na-K-2Cl Cotransporter NKCC2 (United States)

    Konopacka, Agnieszka; Qiu, Jing; Yao, Song T.; Greenwood, Michael P.; Greenwood, Mingkwan; Lancaster, Thomas; Inoue, Wataru; de Souza Mecawi, Andre; Vechiato, Fernanda M.V.; de Lima, Juliana B.M.; Coletti, Ricardo; Hoe, See Ziau; Martin, Andrew; Lee, Justina; Joseph, Marina; Hindmarch, Charles; Paton, Julian; Antunes-Rodrigues, Jose; Bains, Jaideep


    The Na-K-2Cl cotransporter 2 (NKCC2) was thought to be kidney specific. Here we show expression in the brain hypothalamo-neurohypophyseal system (HNS), wherein upregulation follows osmotic stress. The HNS controls osmotic stability through the synthesis and release of the neuropeptide hormone, arginine vasopressin (AVP). AVP travels through the bloodstream to the kidney, where it promotes water conservation. Knockdown of HNS NKCC2 elicited profound effects on fluid balance following ingestion of a high-salt solution—rats produced significantly more urine, concomitant with increases in fluid intake and plasma osmolality. Since NKCC2 is the molecular target of the loop diuretics bumetanide and furosemide, we asked about their effects on HNS function following disturbed water balance. Dehydration-evoked GABA-mediated excitation of AVP neurons was reversed by bumetanide, and furosemide blocked AVP release, both in vivo and in hypothalamic explants. Thus, NKCC2-dependent brain mechanisms that regulate osmotic stability are disrupted by loop diuretics in rats. PMID:25834041

  3. NKCC1 and NHE1 are abundantly expressed in the basolateral plasma membrane of secretory coil cells in rat, mouse, and human sweat glands

    DEFF Research Database (Denmark)

    Nejsum, Lene Niemann; Prætorius, Jeppe; Nielsen, Søren


    In isolated sweat glands, bumetanide inhibits sweat secretion. The mRNA encoding bumetanide-sensitive Na(+)-K(+)-Cl(-) cotransporter (NKCC) isoform 1 (NKCC1) has been detected in sweat glands; however, the cellular and subcellular protein localization is unknown. Na(+)/H(+) exchanger (NHE) isoform...... 1 (NHE1) protein has been localized to both the duct and secretory coil of human sweat duct; however, the NHE1 abundance in the duct was not compared with that in the secretory coil. The aim of this study was to test whether mRNA encoding NKCC1, NKCC2, and Na(+)-coupled acid-base transporters...... and the corresponding proteins are expressed in rodent sweat glands and, if expressed, to determine the cellular and subcellular localization in rat, mouse, and human eccrine sweat glands. NKCC1 mRNA was demonstrated in rat palmar tissue, including sweat glands, using RT-PCR, whereas NKCC2 mRNA was absent. Also, NHE1 m...

  4. Loop Diuretics in the Treatment of Hypertension. (United States)

    Malha, Line; Mann, Samuel J


    Loop diuretics are not recommended in current hypertension guidelines largely due to the lack of outcome data. Nevertheless, they have been shown to lower blood pressure and to offer potential advantages over thiazide-type diuretics. Torsemide offers advantages of longer duration of action and once daily dosing (vs. furosemide and bumetanide) and more reliable bioavailability (vs. furosemide). Studies show that the previously employed high doses of thiazide-type diuretics lower BP more than furosemide. Loop diuretics appear to have a preferable side effect profile (less hyponatremia, hypokalemia, and possibly less glucose intolerance). Studies comparing efficacy and side effect profiles of loop diuretics with the lower, currently widely prescribed, thiazide doses are needed. Research is needed to fill gaps in knowledge and common misconceptions about loop diuretic use in hypertension and to determine their rightful place in the antihypertensive arsenal.

  5. Unexpected Extra-renal Effects of Loop Diuretics in the Preterm Neonate (United States)

    Cotton, Robert; Suarez, Sandra; Reese, Jeff


    The loop diuretics furosemide and bumetanide are commonly used in neonatal intensive care units (NICUs). Furosemide, due to its actions on the ubiquitous NKCC1 co-transporter and its promotion of prostanoid production and release, also has non-diuretic effects on vascular smooth muscle, airways, the ductus arteriosus, and theoretically the gastrointestinal tract. Loop diuretics also affect the central nervous system through the inhibitory neurotransmitter, GABA. Conclusion The loop diuretics have a variety of biological effects that are potentially harmful as well as beneficial. Care should be taken with the use of these agents since the range of their effects may be broader than the single action sought by the prescribing physician. PMID:22536874

  6. Localization and functional characterization of the human NKCC2 isoforms

    DEFF Research Database (Denmark)

    Carota, I; Theilig, F; Oppermann, M;


    AIM: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2...... isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were...... performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant...

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

    Directory of Open Access Journals (Sweden)

    Joo NS


    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

  8. Phototoxicity to sulphonamide-derived oral antidiabetics and diuretics: comparative in-vitro and in-vivo investigations (United States)

    Selvaag, Edgar; Anholt, Helle; Moan, Johan; Thune, Per


    Seven oral antidiabetics (chlorpropamide, glibenclamide, glipizide, gliquidone, glymidine, tolazamide, and tolbutamide), and 14 diuretics (bemetizide, bendroflumethiazide, benzylhydrochlorothiazide, bumetanide, butizide, chlortalidone, furosemide, hydrochlorothiazide, hydroflumethiazide, indapamide, piretanide, polythiazide, trichlormethiazide, and xipamide) were investigated for potential phototoxicity in vitro using a cell culture model and in vivo in hairless mice. After exposure to broad band UVA, the majority of the substances tested in vitro yielded phototoxic action leading to loss of culture forming ability. In vivo, all tested substances induced edema or ulceration, and lead to a significant increase in skin fold thickness of the mouse skin. In all a number of substances not described to induce clinical photosensitivity nor phototoxicity in vitro or in vivo were detected in our testing. In determining potential photosensitizers, it seems important to utilize different test methods, as not all substances will exhibit action in a given assay.

  9. Functional characterization of serotonin receptor subtypes in human duodenal secretion

    DEFF Research Database (Denmark)

    Engelmann, Bodil Elisabeth; Bindslev, Niels; Poulsen, Steen Seier;


    of dyspeptic patients with or without Helicobacter pylori infection, and to determine the 5-HT receptor subtypes functionally involved. Biopsies from the second part of duodenum were obtained from 43 dyspeptic patients during routine endoscopy. Biopsies were mounted in modified Ussing chambers with air suction......: ketanserin, ondansetron, or SB-204070 (1-butyl-4 piperidinmethyl-8-amino-7-chloro-2,3-dihydro-1,4-benzodioxin-5-carboxylate HCl). Histological examination was performed on duodenal biopsies. Helicobacter urease testing and histological examination determined Helicobacter pylori infection. 5-HT induced a dose......-dependent and bumetanide-sensitive short-circuit current, which was independent of the presence of Helicobacter pylori infection. All the three 5-HT receptor antagonists failed to significantly effect basal and 5-HT-induced short-circuit current. Our results indicate that in human duodenum 1) 5-HT is a potent stimulator...

  10. Regulation of electrolyte transport with IL-1β in rabbit distal colon

    Directory of Open Access Journals (Sweden)

    F. R. Homaidan


    Full Text Available Interletrkin-1β levels are elevated in inflammatory bowel disease. In this study the mechanism by which interleukin-1β affects electrolyte transport in the rabbit distal colon, was investigated. Interleukin-1β caused a delayed increase in short-circuit current (Isc which was attributed to protein synthesis since the effect was inhibited by cycloheximide. The interleukin-1β induced increase in Isc was not affected by amiloride treatment but was completely inhibited by bumetanide or in chloride-free buffer and by indomethacin. Prostaglandin E2 levels increased in tissue treated with interleukin-1β, but this increase was reversed by cycloheximide. These data suggest that interleukin-1β causes its effect via a yet to be identified second messenger, by increasing chloride secretion through a prostaglandin E2 mediated mechanism.

  11. Comparison of ion transport by cultured secretory and absorptive canine airway epithelia

    DEFF Research Database (Denmark)

    Boucher, R C; Larsen, Erik Hviid


    The use of primary cell culture techniques to predict the function of native respiratory epithelia was tested in studies of dog airway epithelia. Epithelial cells from Cl- secretory (tracheal) and Na+ absorptive (bronchial) airway regions were isolated by enzymatic digestion, plated on collagen...... sensitive to amiloride but insensitive to bumetanide. As compared with the trachea, the bronchial (absorptive) epithelium is characterized by 1) a large amiloride-sensitive cellular conductance and 2) a relatively depolarized basolateral membrane. We conclude that this primary cell culture technique...... matrices, and maintained in serum-free, hormone-supplemented media. Transepithelial and intracellular studies showed that both the tracheal and bronchial culture preparations exhibited bioelectric parameters quantitatively similar to those of intact tissues. Similar to the native tissue, the tracheal...

  12. Comparison of Cannabidiol, Antioxidants, and Diuretics in Reversing Binge Ethanol-Induced Neurotoxicity (United States)

    Hamelink, Carol; Hampson, Aidan; Wink, David A.; Eiden, Lee E.; Eskay, Robert L.


    Binge alcohol consumption in the rat induces substantial neurodegeneration in the hippocampus and entorhinal cortex. Oxidative stress and cytotoxic edema have both been shown to be involved in such neurotoxicity, whereas N-methyl-D-aspartate (NMDA) receptor activity has been implicated in alcohol withdrawal and excitoxic injury. Because the nonpsychoactive cannabinoid cannabidiol (CBD) was previously shown in vitro to prevent glutamate toxicity through its ability to reduce oxidative stress, we evaluated CBD as a neuroprotectant in a rat binge ethanol model. When administered concurrently with binge ethanol exposure, CBD protected against hippocampal and entorhinal cortical neurodegeneration in a dose-dependent manner. Similarly, the common antioxidants butylated hydroxytoluene and α-tocopherol also afforded significant protection. In contrast, the NMDA receptor antagonists dizocilpine (MK-801) and memantine did not prevent cell death. Of the diuretics tested, furosemide was protective, whereas the other two anion exchanger inhibitors, L-644,711 [(R)-(+)-(5,6-dichloro2,3,9,9a-tetrahydro 3-oxo-9a-propyl-1H-fluoren-7-yl)oxy acetic acid] and bumetanide, were ineffective. In vitro comparison of these diuretics indicated that furosemide is also a potent antioxidant, whereas the nonprotective diuretics are not. The lack of efficacy of L-644,711 and bumetanide suggests that the antioxidant rather than the diuretic properties of furosemide contribute most critically to its efficacy in reversing ethanol-induced neurotoxicity in vitro, in our model. This study provides the first demonstration of CBD as an in vivo neuroprotectant and shows the efficacy of lipophilic antioxidants in preventing binge ethanol-induced brain injury. PMID:15878999

  13. Loop diuretics are open-channel blockers of the cystic fibrosis transmembrane conductance regulator with distinct kinetics (United States)

    Ju, Min; Scott-Ward, Toby S; Liu, Jia; Khuituan, Pissared; Li, Hongyu; Cai, Zhiwei; Husbands, Stephen M; Sheppard, David N


    BACKGROUND AND PURPOSE Loop diuretics are widely used to inhibit the Na+, K+, 2Cl− co-transporter, but they also inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Here, we investigated the mechanism of CFTR inhibition by loop diuretics and explored the effects of chemical structure on channel blockade. EXPERIMENTAL APPROACH Using the patch-clamp technique, we tested the effects of bumetanide, furosemide, piretanide and xipamide on recombinant wild-type human CFTR. KEY RESULTS When added to the intracellular solution, loop diuretics inhibited CFTR Cl− currents with potency approaching that of glibenclamide, a widely used CFTR blocker with some structural similarity to loop diuretics. To begin to study the kinetics of channel blockade, we examined the time dependence of macroscopic current inhibition following a hyperpolarizing voltage step. Like glibenclamide, piretanide blockade of CFTR was time and voltage dependent. By contrast, furosemide blockade was voltage dependent, but time independent. Consistent with these data, furosemide blocked individual CFTR Cl− channels with ‘very fast’ speed and drug-induced blocking events overlapped brief channel closures, whereas piretanide inhibited individual channels with ‘intermediate’ speed and drug-induced blocking events were distinct from channel closures. CONCLUSIONS AND IMPLICATIONS Structure–activity analysis of the loop diuretics suggests that the phenoxy group present in bumetanide and piretanide, but absent in furosemide and xipamide, might account for the different kinetics of channel block by locking loop diuretics within the intracellular vestibule of the CFTR pore. We conclude that loop diuretics are open-channel blockers of CFTR with distinct kinetics, affected by molecular dimensions and lipophilicity. PMID:24117047

  14. Site-specific regulation of ion transport by prolactin in rat colon epithelium. (United States)

    Deachapunya, Chatsri; Poonyachoti, Sutthasinee; Krishnamra, Nateetip


    The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 μg/ml) induced a sustained decrease in short-circuit current (I(sc)) in the distal colon with an EC(50) value of 100 ng/ml and increased I(sc) in the proximal colon with an EC(50) value of 49 ng/ml. In the distal colon, the PRL-induced decrease in I(sc) was not affected by Na(+) channel blocker amiloride or Cl(-) channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K(+) channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K(+) channel blockers, Ba(2+), tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRL-induced decrease in I(sc) was also inhibited by Na(+)-K(+)-2Cl(-) transporter inhibitor bumetanide, Ba(2+), and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in I(sc) was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in I(sc) in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca(2+) chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K(+) secretion in the distal colon and activation of Cl(-) secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca(2+) mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon.

  15. Cell-attached recordings of responses evoked by photorelease of GABA in the immature cortical neurons

    Directory of Open Access Journals (Sweden)

    Marat eMinlebaev


    Full Text Available We present a novel non-invasive technique to measure the polarity of GABAergic responses based on cell-attached recordings of currents activated by laser-uncaging of GABA. For these recordings, a patch pipette was filled with a solution containing RuBi-GABA, and GABA was released from this complex by a laser beam conducted to the tip of the patch pipette via an optic fiber. In cell-attached recordings from neocortical and hippocampal neurons in postnatal days P2-5 rat brain slices in vitro, we found that laser-uncaging of GABA activates integral cell-attached currents mediated by tens of GABA(A channels. The initial response was inwardly directed, indicating a depolarizing response to GABA. The direction of the initial response was dependent on the pipette potential and analysis of its slope-voltage relationships revealed a depolarizing driving force of +11 mV for the currents through GABA channels. Initial depolarizing responses to GABA uncaging were inverted to hyperpolarizing in the presence of the NKCC1 blocker bumetanide. Current-voltage relationships of the currents evoked by Rubi-GABA uncaging using voltage-ramps at the peak of responses not only revealed a bumetanide-sensitive depolarizing reversal potential of the GABA(A receptor mediated responses, but also showed a strong voltage-dependent hysteresis. Upon desensitization of the uncaged-GABA response, current-voltage relationships of the currents through single GABA(A channels revealed depolarizing responses with the driving force values similar to those obtained for the initial response. Thus, cell-attached recordings of the responses evoked by local intrapipette GABA uncaging are suitable to assess the polarity of the GABA(A-Rs mediated signals in small cell compartments.

  16. Modulation of NaCl absorption by [HCO(3)(-)] in the marine teleost intestine is mediated by soluble adenylyl cyclase. (United States)

    Tresguerres, Martin; Levin, Lonny R; Buck, Jochen; Grosell, Martin


    Intestinal HCO(3)(-) secretion and NaCl absorption are essential for counteracting dehydration in marine teleost fish. We investigated how these two processes are coordinated in toadfish. HCO(3)(-) stimulated a luminal positive short-circuit current (I(sc)) in intestine mounted in Ussing chamber, bathed with the same saline solution on the external and internal sides of the epithelium. The I(sc) increased proportionally to the [HCO(3)(-)] in the bath up to 80 mM NaHCO(3), and it did not occur when NaHCO(3) was replaced with Na(+)-gluconate or with NaHCO(3) in Cl(-)-free saline. HCO(3)(-) (20 mM) induced a approximately 2.5-fold stimulation of I(sc), and this [HCO(3)(-)] was used in all subsequent experiments. The HCO(3)(-)-stimulated I(sc) was prevented or abolished by apical application of 10 muM bumetanide (a specific inhibitor of NKCC) and by 30 microM 4-catechol estrogen [CE; an inhibitor of soluble adenylyl cyclase (sAC)]. The inhibitory effects of bumetanide and CE were not additive. The HCO(3)(-)-stimulated I(sc) was prevented by apical bafilomycin (1 microM) and etoxolamide (1 mM), indicating involvement of V-H(+)-ATPase and carbonic anhydrases, respectively. Immunohistochemistry and Western blot analysis confirmed the presence of an NKCC2-like protein in the apical membrane and subapical area of epithelial intestinal cells, of Na(+)/K(+)-ATPase in basolateral membranes, and of an sAC-like protein in the cytoplasm. We propose that sAC regulates NKCC activity in response to luminal HCO(3)(-), and that V-H(+)-ATPase and intracellular carbonic anhydrase are essential for transducing luminal HCO(3)(-) into the cell by CO(2)/HCO(3)(-) hydration/dehydration. This mechanism putatively coordinates HCO(3)(-) secretion with NaCl and water absorption in toadfish intestine.

  17. Estradiol reduces activity of the blood-brain barrier Na-K-Cl cotransporter and decreases edema formation in permanent middle cerebral artery occlusion. (United States)

    O'Donnell, Martha E; Lam, Tina I; Tran, Lien Q; Foroutan, Shahin; Anderson, Steven E


    Estrogen has been shown to protect against stroke-induced brain damage, yet the mechanism is unknown. During the early hours of stroke, cerebral edema forms as increased transport of Na and Cl from blood into brain occurs across an intact blood-brain barrier (BBB). We showed previously that a luminal BBB Na-K-Cl cotransporter is stimulated by hypoxia and arginine vasopressin (AVP), factors present during cerebral ischemia, and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema in rats subjected to permanent middle cerebral artery occlusion (MCAO). The present study was conducted to determine whether estrogen protects in stroke at least in part by reducing activity of the BBB cotransporter, thereby decreasing edema formation. Ovariectomized rats were subjected to 210 mins of permanent MCAO after 7-day or 30-min pretreatment with 17beta-estradiol and then brain swelling and 2,3,5-triphenyltetrazolium chloride staining were assessed as measures of brain edema and lesion volume, respectively. Diffusion-weighed imaging was used to monitor permanent MCAO-induced decreases in apparent diffusion coefficient (ADC) values, an index of changes in brain water distribution and mobility. Na-K-Cl cotransporter activity of cerebral microvascular endothelial cells (CMECs) was assessed as bumetanide-sensitive K influx and cotransporter abundance by Western blot analysis after estradiol treatment. Estradiol significantly decreased brain swelling and lesion volume and attenuated the decrease in ADC values during permanent MCAO. Estradiol also abolished CMEC cotransporter stimulation by chemical hypoxia or AVP and decreased cotransporter abundance. These findings support the hypothesis that estrogen attenuates stimulation of BBB Na-K-Cl cotransporter activity, reducing edema formation during stroke.

  18. Inward flux of lactate⁻ through monocarboxylate transporters contributes to regulatory volume increase in mouse muscle fibres.

    Directory of Open Access Journals (Sweden)

    Michael I Lindinger

    Full Text Available Mouse and rat skeletal muscles are capable of a regulatory volume increase (RVI after they shrink (volume loss resultant from exposure to solutions of increased osmolarity and that this RVI occurs mainly by a Na-K-Cl-Cotransporter (NKCC-dependent mechanism. With high-intensity exercise, increased extracellular osmolarity is accompanied by large increases in extracellular [lactate⁻]. We hypothesized that large increases in [lactate⁻] and osmolarity augment the NKCC-dependent RVI response observed with a NaCl (or sucrose-induced increase in osmolarity alone; a response that is dependent on lactate⁻ influx through monocarboxylate transporters (MCTs. Single mouse muscle fibres were isolated and visualized under light microscopy under varying osmolar conditions. When solution osmolarity was increased by adding NaLac by 30 or 60 mM, fibres lost significantly less volume and regained volume sooner compared to when NaCl was used. Phloretin (MCT1 inhibitor accentuated the volume loss compared to both NaLac controls, supporting a role for MCT1 in the RVI response in the presence of elevated [lactate⁻]. Inhibition of MCT4 (with pCMBS resulted in a volume loss, intermediate to that seen with phloretin and NaLac controls. Bumetanide (NKCC inhibitor, in combination with pCMBS, reduced the magnitude of volume loss, but volume recovery was complete. While combined phloretin-bumetanide also reduced the magnitude of the volume loss, it also largely abolished the cell volume recovery. In conclusion, RVI in skeletal muscle exposed to raised tonicity and [lactate⁻] is facilitated by inward flux of solute by NKCC- and MCT1-dependent mechanisms. This work demonstrates evidence of a RVI response in skeletal muscle that is facilitated by inward flux of solute by MCT-dependent mechanisms. These findings further expand our understanding of the capacities for skeletal muscle to volume regulate, particularly in instances of raised tonicity and lactate

  19. Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium. (United States)

    Nickell, William T; Kleene, Nancy K; Kleene, Steven J


    When olfactory receptor neurons respond to odours, a depolarizing Cl(-) efflux is a substantial part of the response. This requires that the resting neuron accumulate Cl(-) against an electrochemical gradient. In isolated olfactory receptor neurons, the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 is essential for Cl(-) accumulation. However, in intact epithelium, a robust electrical olfactory response persists in mice lacking NKCC1. This response is largely due to a neuronal Cl(-) efflux. It thus appears that NKCC1 is an important part of a more complex system of Cl(-) accumulation. To identify the remaining transport proteins, we first screened by RT-PCR for 21 Cl(-) transporters in mouse nasal tissue containing olfactory mucosa. For most of the Cl(-) transporters, the presence of mRNA was demonstrated. We also investigated the effects of pharmacological block or genetic ablation of Cl(-) transporters on the olfactory field potential, the electroolfactogram (EOG). Mice lacking the common Cl(-)/HCO(3)(-) exchanger AE2 had normal EOGs. Block of NKCC cotransport with bumetanide reduced the EOG in epithelia from wild-type mice but had no effect in mice lacking NKCC1. Hydrochlorothiazide, a blocker of the Na(+)-Cl(-) cotransporter, had only a small effect. DIDS, a blocker of some KCC cotransporters and Cl(-)/HCO(3)(-) exchangers, reduced the EOG in epithelia from both wild-type and NKCC1 knockout mice. A combination of bumetanide and DIDS decreased the response more than either drug alone. However, no combination of drugs completely abolished the Cl(-) component of the response. These results support the involvement of both NKCC1 and one or more DIDS-sensitive transporters in Cl(-) accumulation in olfactory receptor neurons.

  20. Interaction between {sup 99}Tc{sup m}-hydroxmethylene diphosphonate and loop-diuretics in an experimental mouse system

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    Cronhjort, M. [Dept. of Diagnostic Radiology, Karolinska Hospital, Stockholm (Sweden); Sjoeberg, H.E. [Dept. of Endocrinology, Karolinska Hospital, Stockholm (Sweden); Schnell, P.O. [Dept. of Hospital Physics, Karolinska Hospital, Stockholm (Sweden); Jacobsson, H. [Dept. of Diagnostic Radiology, Karolinska Hospital, Stockholm (Sweden)


    The image quality at bone scintigraphy depends largely on the bone/soft-tissue activity ratio. This varies considerably between different patients and may sometimes be strongly reduced. The ratio increases with time due to urinary excretion of extracelluar activity. The possibility to utilize the phosphaturic effect of loop-diuretics to enhance the excretion of the soft tissue activity caused by radiolabeled phosphonate compounds at bone scintigraphy has been studied. Three loop-diuretics (Bumetanide, Ethacrynic acid and Furosemide) were injected at different times in relation to {sup 99}Tc{sup m}-Hydroxymethylene diphosphonate (HDP) in mice. By assessing the activity of different organs as well as of peripheral blood by a gamma-counter, the activity distribution in the animals was established. Administration of diuretics together with, or after HDP has a negative influence on the quality of the potential HDP-image. Administration of diuretics prior to the radiopharmaceutical slightly improves the image quality, but not to an extent justifying it use for this purpose in practice. The complex effects on HDP may be explained by the influence of Furosemide on calcium, pH and on blood plasma volume and their hormonal consequences. (orig.) [Deutsch] Bei der Knochenszintigraphie haengt die Bildqualitaet hauptsaechlich von der Quote der Isotopenaktivitaet in den Knochen und in den Weichteilen ab. Die extrazellulaere Weichteilaktivitaet wird durch die Nieren ausgeschieden, zur Steigerung der Quote fuehrend. Die Moeglichkeit die Diuretica wegen ihrer Wirkung die extrazellulaeren Isotopen aus dem Koerper auszuschneiden zu benuetzen ist hier untersucht worden. Diesbezueglich hat man insgesamt drei Typen von Loop-Diuretica (Bumetanid, Ethacrynsaeure und Furosemid) verwendet. Die Diuretica hat man den Maeusen bei verschiedenen Zeitpunkten im Verhaeltnis zu dem {sup 99}Tc{sup m}-HDP (HDP) zugefuehrt. Um die Verteilung des Isotops in den Tierkoerpern festzustellen, ist die

  1. Na+,2Cl-,K+ cotransport system as a marker of antihypertensive activity of new torasemide derivatives. (United States)

    Masereel, B; Ferrari, P; Ferrandi, M; Pirotte, B; Schynts, M; Parenti, P; Delarge, J


    A series of compounds related to torasemide, a loop diuretic, were synthesized and examined for their diuretic potency and inhibitory activity on the erythrocyte and renal medullary thick ascending limb vesicle Na+,2Cl-,K+ cotransport in Milan hypertensive (MHS) and normotensive (MNS) rat strains, where previous studies had demonstrated an alteration of the cotransport system genetically related to hypertension. From the results of the screening, structure-activity relationships were drawn and two compounds, JDL 961 and C 2921 were selected. Their IC50 on renal vesicle cotransport were similar in the two strains (JDL 961: MHS = 1.8 microM; MNS = 1.2 microM; C 2921: MHS = 4 microM; MNS = 3.8 microM), and were 4-8 times lower than those of torasemide (MHS = 13 microM; MNS = 31 microM, P less than 0.01) and 50-60 times lower than those of bumetanide (MHS = 145 microM; MNS = 206 microM, P less than 0.05) taken as reference compounds. Their ability to reduce the development rate of hypertension was tested both in MHS and in Okamoto spontaneously hypertensive rats (SHR) strain, in which cotransport alterations are opposite to those of MHS. Both torasemide derivatives (7.5 os per day) prevented development of hypertension in the two strains. The time course of this hypotensive activity was faster and the percentage of blood pressure fall greater in MHS (20-25%) than in SHR rats (12-15%), even though the absolute value of blood pressure fall was similar in MHS (JDL 961 = -17 mm Hg; C 2921 = -30 mm Hg) and SHR (JDL 961 = -25 mm Hg; C 2921 = -20 mm Hg). A superimposable effect of bumetanide was observed in the two strains, but at 8 times higher daily dose (60 These results suggest that new loop diuretics can be selected for their antihypertensive activity on the basis of their in vitro potency in inhibiting the Na+,2Cl-,K+.

  2. Improvement of barrier function and stimulation of colonic epithelial anion secretion by Menoease Pills

    Institute of Scientific and Technical Information of China (English)

    Jin-Xia Zhu; Ning Yang; Gui-Hong Zhang; Lai-Ling Tsang; Yu-Lin Gou; Hau-Yan Connie Wong; Yiu-Wa Chung; Hsiao-Chang Chan


    AIM: Menoease Pills (MP), a Chinese medicine-based new formula for postmenopausal women, has been shown to modulate the endocrine and immune systems[1]. The present study investigated the effects of MP and one of its active ingredients, ligustrazine, on epithelial barrier and ion transport function in a human colonic cell line, T84.METHODS: Colonic transepithelial electrophysiological characteristics and colonic anion secretion were studied using the short circuit current (ISC) technique. RT-PCR was used to examine the expression of cytoplasmic proteins associated with the tight junctions, ZO-1(zonula occludens-1) and ZO-2 (zonula occludens-2).RESULTS: Pretreatment of T84 cells with MP (15 μg/mL) for 72 h significantly increased basal potential difference,transepithelial resistance and basal ISC. RT-PCR results showed that the expressions of ZO-1 and ZO-2 were significantly increased after MP treatment, consistent with improved epithelial barrier function. Results of acute stimulation showed that apical addition of MP produced a concentrationdependent (10-5 000 μg/mL, EC50 = 293.9 μg/mL) increase in ISC. MP-induced ISC was inhibited by basolateral treatment with bumetanide (100 μmol/L), an inhibitor of the Na+-K+-2Cl- cotransporter, apical addition of Cl-channel blockers, diphenylamine-2, 2'-dicarboxylic acid (1 mmol/L) or glibenclamide (1 mmol/L), but not 4, 4'-diisothiocyanostilbene2, 2'-disulfonic acid or epithelial Na+ channel blocker,amiloride. The effect of MP on ZO-1 and ZO-2 was mimicked by Ligustrazine and the ligustrazine-induced ISC was also blocked by basolateral application of bumetanide and apical addition of diphenylamine-2, 2'-dicarboxylic acid or glibenclamide, and reduced by a removal of extracellular Cl-.CONCLUSION: The results of the present study suggest that MP and lligustrazine may improve epithelial barrier function and exert a stimulatory effect on colonic anion secretion, indicating the potential use of MP and its active ingredients

  3. Neonatal Propofol and Etomidate Exposure Enhance Inhibitory Synaptic Transmission in Hippocampal Cornus Ammonis 1 Pyramidal Neurons

    Institute of Scientific and Technical Information of China (English)

    Jia-Qiang Zhang; Wan-Ying Xu; Chang-Qing Xu


    Background:Propofol and etomidate are the most important intravenous general anesthetics in the current clinical use and that mediate gamma-aminobutyric acid's (GABAergic) synaptic transmission.However,their long-term effects on GABAergic synaptic transmission induced by neonatal propofol or etomidate exposure remain unclear.We investigated the long-term GABAergic neurotransmission alterations,following neonatal propofol and etomidate administration.Methods:Sprague-Dawley rat pups at postnatal days 4 6 were underwent 6-h-long propofol-induced or 5-h-long etomidate-induced anesthesia.We performed whole-cell patch-clamp recording from pyramidal cells in the cornus ammonis 1 area of acute hippocampal slices of postnatal 80-90 days.Spontaneous and miniature inhibitory GABAergic currents (spontaneous inhibitory postsynaptic currents [sIPSCs] and miniature inhibitory postsynaptic currents [mIPSCs]) and their kinetic characters were measured.The glutamatergic tonic effect on inhibitory transmission and the effect of bumetanide on neonatal propofol exposure were also examined.Results:Neonatal propofol exposure significantly increased the frequency of mIPSCs (from 1.87 ± 0.35 Hz to 3.43 ± 0.51 Hz,P < 0.05) and did not affect the amplitude of mIPSCs and sIPSCs.Both propofol and etomidate slowed the decay time of mIPSCs kinetics (168.39 ± 27.91 ms and 267.02 ± 100.08 ms vs.68.18 ± 12.43 ms;P < 0.05).Bumetanide significantly blocked the frequency increase and reversed the kinetic alteration of mIPSCs induced by neonatal propofol exposure (3.01 ± 0.45 Hz and 94.30 ± 32.56 ms).Conclusions:Neonatal propofol and etomidate exposure has long-term effects on inhibitory GABAergic transmission.Propofol might act at pre-and post-synaptic GABA receptor A (GABAA) receptors within GABAergic synapses and impairs the glutamatergic tonic input to GABAergic synapses;etomidate might act at the postsynaptic site.

  4. Diuretics Prime Plant Immunity in Arabidopsis thaliana (United States)

    Noutoshi, Yoshiteru; Ikeda, Mika; Shirasu, Ken


    Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application. PMID:23144763

  5. Seizure-induced disinhibition of the HPA axis increases seizure susceptibility. (United States)

    O'Toole, Kate K; Hooper, Andrew; Wakefield, Seth; Maguire, Jamie


    Stress is the most commonly reported precipitating factor for seizures. The proconvulsant actions of stress hormones are thought to mediate the effects of stress on seizure susceptibility. Interestingly, epileptic patients have increased basal levels of stress hormones, including corticotropin-releasing hormone (CRH) and corticosterone, which are further increased following seizures. Given the proconvulsant actions of stress hormones, we proposed that seizure-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis may contribute to future seizure susceptibility. Consistent with this hypothesis, our data demonstrate that pharmacological induction of seizures in mice with kainic acid or pilocarpine increases circulating levels of the stress hormone, corticosterone, and exogenous corticosterone administration is sufficient to increase seizure susceptibility. However, the mechanism(s) whereby seizures activate the HPA axis remain unknown. Here we demonstrate that seizure-induced activation of the HPA axis involves compromised GABAergic control of CRH neurons, which govern HPA axis function. Following seizure activity, there is a collapse of the chloride gradient due to changes in NKCC1 and KCC2 expression, resulting in reduced amplitude of sIPSPs and even depolarizing effects of GABA on CRH neurons. Seizure-induced activation of the HPA axis results in future seizure susceptibility which can be blocked by treatment with an NKCC1 inhibitor, bumetanide, or blocking the CRH signaling with Antalarmin. These data suggest that compromised GABAergic control of CRH neurons following an initial seizure event may cause hyperexcitability of the HPA axis and increase future seizure susceptibility.

  6. Altered expression of renal NHE3, TSC, BSC-1, and ENaC subunits in potassium-depleted rats. (United States)

    Elkjaer, Marie-Louise; Kwon, Tae-Hwan; Wang, Weidong; Nielsen, Jakob; Knepper, Mark A; Frøkiaer, Jørgen; Nielsen, Søren


    The purpose of this study was to examine whether hypokalemia is associated with altered abundance of major renal Na+ transporters that may contribute to the development of urinary concentrating defects. We examined the changes in the abundance of the type 3 Na+/H+ exchanger (NHE3), Na+ - K+-ATPase, the bumetanide-sensitive Na+ - K+ - 2Cl- cotransporter (BSC-1), the thiazide-sensitive Na+ - Cl- cotransporter (TSC), and epithelial sodium channel (ENaC) subunits in kidneys of hypokalemic rats. Semiquantitative immunoblotting revealed that the abundance of BSC-1 (57%) and TSC (46%) were profoundly decreased in the inner stripe of the outer medulla (ISOM) and cortex/outer stripe of the outer medulla (OSOM), respectively. These findings were confirmed by immunohistochemistry. Moreover, total kidney abundance of all ENaC subunits was significantly reduced in response to the hypokalemia: alpha-subunit (61%), beta-subunit (41%), and gamma-subunit (60%), and this was confirmed by immunohistochemistry. In contrast, the renal abundance of NHE3 in hypokalemic rats was dramatically increased in cortex/OSOM (736%) and ISOM (210%). Downregulation of BSC-1, TSC, and ENaC may contribute to the urinary concentrating defect, whereas upregulation of NHE3 may be compensatory to prevent urinary Na+ loss and/or to maintain intracellular pH levels.

  7. Participation of central GABAA receptors in the trigeminal processing of mechanical allodynia in rats (United States)

    Kim, Min Ji; Park, Young Hong; Yang, Kui Ye; Ju, Jin Sook; Bae, Yong Chul


    Here we investigated the central processing mechanisms of mechanical allodynia and found a direct excitatory link with low-threshold input to nociceptive neurons. Experiments were performed on male Sprague-Dawley rats weighing 230-280 g. Subcutaneous injection of interleukin 1 beta (IL-1β) (1 ng/10 µL) was used to produce mechanical allodynia and thermal hyperalgesia. Intracisternal administration of bicuculline, a gamma aminobutyric acid A (GABAA) receptor antagonist, produced mechanical allodynia in the orofacial area under normal conditions. However, intracisternal administration of bicuculline (50 ng) produced a paradoxical anti-allodynic effect under inflammatory pain conditions. Pretreatment with resiniferatoxin (RTX), which depletes capsaicin receptor protein in primary afferent fibers, did not alter the paradoxical anti-allodynic effects produced by the intracisternal injection of bicuculline. Intracisternal injection of bumetanide, an Na-K-Cl cotransporter (NKCC 1) inhibitor, reversed the IL-1β-induced mechanical allodynia. In the control group, application of GABA (100 µM) or muscimol (3 µM) led to membrane hyperpolarization in gramicidin perforated current clamp mode. However, in some neurons, application of GABA or muscimol led to membrane depolarization in the IL-1β-treated rats. These results suggest that some large myelinated Aβ fibers gain access to the nociceptive system and elicit pain sensation via GABAA receptors under inflammatory pain conditions. PMID:28066142

  8. Effect of brain-derived neurotropic factor released from hypoxic astrocytes on gamma-aminobutyric acid type A receptor function in normal hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Hongliang Liu; Tijun Dai


    Astrocytes can release increased levels of brain-derived neurotrophic factor during cerebral ischemia, but it is unclear whether brain-derived neurotrophic factor affects γ-aminobutyric acid type A receptor function in normal neurons. Results from this study demonstrated that γ-aminobutyric acid at 100 μmol/L concentration raised the intracellular calcium level in neurons treated with medium from cultured hypoxic astrocytes, and the rise in calcium level could be inhibited by γ-aminobutyric acid type A receptor antagonist bicuculline or brain-derived neurotrophic factor receptor antagonist k252a. Γ-aminobutyric acid type A-gated current induced by 100 μmol/L γ-aminobutyric acid was in an inward direction in physiological conditions, but shifted to the outward direction in neurons when treated with the medium from cultured hypoxic astrocytes, and this effect could be inhibited by k252a. The reverse potential was shifted leftward to -93 Mv, which could be inhibited by k252a and Na+-K+-Cl- cotransporter inhibitor bumetanide. Brain-derived neurotrophic factor was released from hypoxic astrocytes at a high level. It shifted the reverse potential of γ-aminobutyric acid type A-gated currents leftward in normal neurons by enhancing the function of Na+-K+-Cl- cotransporter, and caused γ-aminobutyric acid to exert an excitatory effect by activating γ-aminobutyric acid type A receptor.

  9. Primary afferent terminals acting as excitatory interneurons contribute to spontaneous motor activities in the immature spinal cord. (United States)

    Bos, Rémi; Brocard, Frédéric; Vinay, Laurent


    Patterned, spontaneous activity plays a critical role in the development of neuronal networks. A robust spontaneous activity is observed in vitro in spinal cord preparations isolated from immature rats. The rhythmic ventral root discharges rely mainly on the depolarizing/excitatory action of GABA and glycine early during development, whereas at later stages glutamate drive is primarily responsible for the rhythmic activity and GABA/glycine are thought to play an inhibitory role. However, rhythmic discharges mediated by the activation of GABA(A) receptors are recorded from dorsal roots (DRs). In the present study, we used the in vitro spinal cord preparation of neonatal rats to identify the relationship between discharges that are conducted antidromically along DRs and the spontaneous activity recorded from lumbar motoneurons. We show that discharges in DRs precede those in ventral roots and that primary afferent depolarizations (PADs) start earlier than EPSPs in motoneurons. EPSP-triggered averaging revealed that the action potentials propagate not only antidromically in the DR but also centrally and trigger EPSPs in motoneurons. Potentiating GABAergic antidromic discharges by diazepam increased the EPSPs recorded from motoneurons; conversely, blocking DR bursts markedly reduced these EPSPs. High intracellular concentrations of chloride are maintained in primary afferent terminals by the sodium-potassium-chloride cotransporter NKCC1. Blocking these cotransporters by bumetanide decreased both dorsal and ventral root discharges. We conclude that primary afferent fibers act as excitatory interneurons and that GABA, through PADs reaching firing threshold, is still playing a key role in promoting spontaneous activity in neonates.

  10. The role of cation-dependent chloride transporters in neuropathic pain following spinal cord injury

    Directory of Open Access Journals (Sweden)

    Rajpal Sharad


    Full Text Available Abstract Background Altered Cl- homeostasis and GABAergic function are associated with nociceptive input hypersensitivity. This study investigated the role of two major intracellular Cl- regulatory proteins, Na+-K+-Cl- cotransporter 1 (NKCC1 and K+-Cl- cotransporter 2 (KCC2, in neuropathic pain following spinal cord injury (SCI. Results Sprague-Dawley rats underwent a contusive SCI at T9 using the MASCIS impactor. The rats developed hyperalgesia between days 21 and 42 post-SCI. Thermal hyperalgesia (TH was determined by a decrease in hindpaw thermal withdrawal latency time (WLT between days 21 and 42 post-SCI. Rats with TH were then treated with either vehicle (saline containing 0.25% NaOH or NKCC1 inhibitor bumetanide (BU, 30 mg/kg, i.p. in vehicle. TH was then re-measured at 1 h post-injection. Administration of BU significantly increased the mean WLT in rats (p Conclusion Taken together, expression of NKCC1 and KCC2 proteins was differentially altered following SCI. The anti-hyperalgesic effect of NKCC1 inhibition suggests that normal or elevated NKCC1 function and loss of KCC2 function play a role in the development and maintenance of SCI-induced neuropathic pain.

  11. Posttraumatic GABA(A)-mediated [Ca2+]i increase is essential for the induction of brain-derived neurotrophic factor-dependent survival of mature central neurons. (United States)

    Shulga, Anastasia; Thomas-Crusells, Judith; Sigl, Thomas; Blaesse, Anne; Mestres, Pedro; Meyer, Michael; Yan, Qiao; Kaila, Kai; Saarma, Mart; Rivera, Claudio; Giehl, Klaus M


    A shift of GABA(A)-mediated responses from hyperpolarizing to depolarizing after neuronal injury leads to GABA(A)-mediated increase in [Ca2+](i). In addition, central neurons become dependent on BDNF for survival. Whether these two mechanisms are causally interrelated is an open question. Here, we show in lesioned CA3 hippocampal neurons in vitro and in axotomized corticospinal neurons in vivo that posttraumatic downregulation of the neuron-specific K-Cl cotransporter KCC2 leads to intracellular chloride accumulation by the Na-K-2Cl cotransporter NKCC1, resulting in GABA-induced [Ca2+](i) transients. This mechanism is required by a population of neurons to survive in a BDNF-dependent manner after injury, because blocking GABA(A)-depolarization with the NKCC1 inhibitor bumetanide prevents the loss of neurons on BDNF withdrawal. The resurgence of KCC2 expression during recovery coincides with loss of BDNF dependency for survival. This is likely mediated through BDNF itself, because injured neurons reverse their response to this neurotrophin by switching the BDNF-induced downregulation of KCC2 to upregulation.

  12. Intravenous HOE-642 reduces brain edema and Na uptake in the rat permanent middle cerebral artery occlusion model of stroke: evidence for participation of the blood-brain barrier Na/H exchanger. (United States)

    O'Donnell, Martha E; Chen, Yi-Je; Lam, Tina I; Taylor, Kelleen C; Walton, Jeffrey H; Anderson, Steven E


    Cerebral edema forms in the early hours of ischemic stroke by processes involving increased transport of Na and Cl from blood into brain across an intact blood-brain barrier (BBB). Our previous studies provided evidence that the BBB Na-K-Cl cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema and infarct in rats subjected to permanent middle cerebral artery occlusion (pMCAO). More recently, we showed that BBB Na/H exchanger activity is also stimulated by hypoxia, aglycemia, and AVP. The present study was conducted to further investigate the possibility that a BBB Na/H exchanger also participates in edema formation during ischemic stroke. Sprague-Dawley rats were subjected to pMCAO and then brain edema and Na content assessed by magnetic resonance imaging diffusion-weighed imaging and magnetic resonance spectroscopy Na spectroscopy, respectively, for up to 210 minutes. We found that intravenous administration of the specific Na/H exchange inhibitor HOE-642 significantly decreased brain Na uptake and reduced cerebral edema, brain swelling, and infarct volume. These findings support the hypothesis that edema formation and brain Na uptake during the early hours of cerebral ischemia involve BBB Na/H exchanger activity as well as Na-K-Cl cotransporter activity.

  13. Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

    Directory of Open Access Journals (Sweden)

    Florian Wegner

    Full Text Available BACKGROUND: Human fetal midbrain-derived neural progenitor cells (NPCs may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+-K(+-Cl(- co-transporter 1 (NKCC1-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. CONCLUSIONS/SIGNIFICANCE: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

  14. Regulation of renal peripheral benzodiazepine receptors by anion transport inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Basile, A.S.; Lueddens, W.M.; Skolnick, P.


    The in vitro and in vivo regulation of (/sup 3/H)Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) by ion transport/exchange inhibitors was studied in the kidney. The potencies of 9-anthroic acid, furosemide, bumetanide, hydrochlorothiazide and SITS as inhibitors of (/sup 3/H)Ro 5-4864 binding to renal membranes were consistent with their actions as anion transport inhibitors (Ki approx. = 30 - 130 In contrast, spironolactone, amiloride, acetazolamide, and ouabain were less potent (Ki=100-1000 Administration of furosemide to rats for five days resulted in a profound diuresis accompanied by a significant increase in PBR density (43%) that was apparent by the fifth day of treatment. Administration of hydrochlorothiazide or Ro 5-4864 for five days also caused diuresis and increased renal PBR density. Both the diuresis and increased density of PBR produced by Ro 5-4864 were blocked by coadministration of PK 11195, which alone had no effect on either PBR density or urine volume. The equilibrium binding constants of (/sup 3/H)Ro 5-4864 to cardiac membranes were unaffected by administration of any of these drugs. These findings suggest that renal PBR may be selectively modulated in vivo and in vitro by administration of ion transport/exchange inhibitors. 36 references, 4 tables.

  15. [Glomerulo-tubular balance in diabetes mellitus: molecular evidence and clinical consequences]. (United States)

    Evangelista, C; Rizzo, M; Cantone, A; Corbo, G; Di Donato, L; Trocino, C; Zacchia, M; Capasso, G


    Diabetes mellitus is fast becoming a world epidemic. About one-third of individuals with diabetes, after 10 yrs, develop diabetic nephropathy, the first cause of end-stage kidney disease. The evolution of diabetic nephropathy can be considered in three stages: glomerular hyperfiltration, microalbuminuria (30-300 mg/24 hr) and proteinuria (>300 mg/24 hr). This study was designed to investigate the tubular basis of glomerular hyperfiltration in early diabetes mellitus. Diabetes was inducted in rats with i.p. streptozotocin (65 mg/kg bw) for 6 days. At the end of the treatment, the glomerular filtration rate (GFR), measured by inulin clearance, had substantially increased in diabetic rats compared with controls. Quantitative polymerase chain reaction (PCR) and Western blot analysis reveal that in diabetic rats compared with controls, mRNA and protein abundance was higher for type 3 sodium/hydrogen exchanger (NHE3) in proximal tubule and ascending limbs of Henle's loop, and higher for bumetanide-sensitive sodium-potassium-2 chloride cotransporter (NKCC2) in ascending limbs of Henle's loop. Western blot analysis confirmed the PCR results. Finally, the abundance of á -ENaC protein was unchanged in diabetic rats compared to controls. These results show that the primary sodium reabsorption increase in proximal tubule reduces salt concentrations at the macula densa. This elicits a tubuloglomerular feedback-dependent increase in single nephron GFR.

  16. Diuretics prime plant immunity in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Yoshiteru Noutoshi

    Full Text Available Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application.

  17. Abnormal expression of cerebrospinal fluid cation chloride cotransporters in patients with Rett syndrome.

    Directory of Open Access Journals (Sweden)

    Sofia Temudo Duarte

    Full Text Available OBJECTIVE: Rett Syndrome is a progressive neurodevelopmental disorder caused mainly by mutations in the gene encoding methyl-CpG-binding protein 2. The relevance of MeCP2 for GABAergic function was previously documented in animal models. In these models, animals show deficits in brain-derived neurotrophic factor, which is thought to contribute to the pathogenesis of this disease. Neuronal Cation Chloride Cotransporters (CCCs play a key role in GABAergic neuronal maturation, and brain-derived neurotrophic factor is implicated in the regulation of CCCs expression during development. Our aim was to analyse the expression of two relevant CCCs, NKCC1 and KCC2, in the cerebrospinal fluid of Rett syndrome patients and compare it with a normal control group. METHODS: The presence of bumetanide sensitive NKCC1 and KCC2 was analysed in cerebrospinal fluid samples from a control pediatric population (1 day to 14 years of life and from Rett syndrome patients (2 to 19 years of life, by immunoblot analysis. RESULTS: Both proteins were detected in the cerebrospinal fluid and their levels are higher in the early postnatal period. However, Rett syndrome patients showed significantly reduced levels of KCC2 and KCC2/NKCC1 ratio when compared to the control group. CONCLUSIONS: Reduced KCC2/NKCC1 ratio in the cerebrospinal fluid of Rett Syndrome patients suggests a disturbed process of GABAergic neuronal maturation and open up a new therapeutic perspective.

  18. GABA(A) receptor-mediated presynaptic inhibition on glutamatergic transmission. (United States)

    Yamamoto, Sokatsu; Yoshimura, Megumu; Shin, Min-Chul; Wakita, Masahito; Nonaka, Kiku; Akaike, Norio


    We investigated the functional roles of presynaptic GABA(A) receptors on excitatory nerve terminals in contributing to spontaneous and action potential-evoked glutamatergic transmission to rat hippocampal CA3 pyramidal neurons. Single CA3 neurons were mechanically isolated with adherent nerve terminals, namely the 'synaptic bouton preparation', and spontaneous glutamatergic excitatory synaptic potentials (sEPSCs) and EPSCs evoked by focal electrical stimuli of a single presynaptic glutamatergic boutons (eEPSCs) were recorded using conventional whole-cell patch recordings. Selective activation of presynaptic GABA(A) receptors on these excitatory nerve terminals by muscimol, markedly facilitated sEPSCs frequency but inhibited eEPSC amplitude. The facilitation of sEPSC frequency was completely occluded by GABA(A) receptor-Cl⁻ channel blockers bicuculline or penicillin (PN). PN itself concentration-dependently inhibited the GABA(A) receptor response induced by bath application of muscimol, but had no effect on the glutamate receptor response. In addition, pretreatment with a blocker of the Na(+), K(+), 2Cl⁻ co-transporter type 1 (NKCC-1), bumetanide, prevented the muscimol-induced inhibition of eEPSCs. The results indicate that activation of presynaptic GABA(A) receptors directly depolarizes glutamatergic excitatory nerve terminals and thereby differentially modulates sEPSCs and eEPSCs.

  19. Basolateral Cl- uptake mechanisms in Xenopus laevis lung epithelium. (United States)

    Berger, Jens; Hardt, Martin; Clauss, Wolfgang G; Fronius, Martin


    A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl(-) uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na(+)/K(+)/2 Cl(-) cotransporter (NKCC) and HCO(3)(-)/Cl(-) anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl(-)-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl(-) gradient. Further, we were interested in the involvement of the HCO(3)(-)/Cl(-) anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (I(SC)). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO(3)(-) concentrations. Cl(-) secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO(3)(-). These experiments indicate that the AE at least partially contributes to Cl(-) secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl(-) uptake, which contrasts with the common model for Cl(-) secretion in pulmonary epithelia.

  20. Newborn Analgesia Mediated by Oxytocin during Delivery. (United States)

    Mazzuca, Michel; Minlebaev, Marat; Shakirzyanova, Anastasia; Tyzio, Roman; Taccola, Giuliano; Janackova, Sona; Gataullina, Svetlana; Ben-Ari, Yehezkel; Giniatullin, Rashid; Khazipov, Rustem


    The mechanisms controlling pain in newborns during delivery are poorly understood. We explored the hypothesis that oxytocin, an essential hormone for labor and a powerful neuromodulator, exerts analgesic actions on newborns during delivery. Using a thermal tail-flick assay, we report that pain sensitivity is two-fold lower in rat pups immediately after birth than 2 days later. Oxytocin receptor antagonists strongly enhanced pain sensitivity in newborn, but not in 2-day-old rats, whereas oxytocin reduced pain at both ages suggesting an endogenous analgesia by oxytocin during delivery. Similar analgesic effects of oxytocin, measured as attenuation of pain-vocalization induced by electrical whisker pad stimulation, were also observed in decerebrated newborns. Oxytocin reduced GABA-evoked calcium responses and depolarizing GABA driving force in isolated neonatal trigeminal neurons suggesting that oxytocin effects are mediated by alterations of intracellular chloride. Unlike GABA signaling, oxytocin did not affect responses mediated by P2X3 and TRPV1 receptors. In keeping with a GABAergic mechanism, reduction of intracellular chloride by the diuretic NKCC1 chloride co-transporter antagonist bumetanide mimicked the analgesic actions of oxytocin and its effects on GABA responses in nociceptive neurons. Therefore, endogenous oxytocin exerts an analgesic action in newborn pups that involves a reduction of the depolarizing action of GABA on nociceptive neurons. Therefore, the same hormone that triggers delivery also acts as a natural pain killer revealing a novel facet of the protective actions of oxytocin in the fetus at birth.

  1. Effects of oxytocin on GABA signalling in the foetal brain during delivery. (United States)

    Khazipov, Rustem; Tyzio, Roman; Ben-Ari, Yehezkel


    Oxytocin (OXT) exerts multiple effects in the adult central nervous system. However, little is known about the effects of OXT on foetal neurons during delivery, at the time when a surge of OXT occurs. In a recent study, the effects of OXT on gamma-aminobutyric acid (GABA) signalling have been reported in foetal and newborn rats. In the immature rat hippocampal and neocortical neurons at birth, endogenous OXT induced a switch in the action of GABA from excitatory to inhibitory. This excitatory-to-inhibitory switch was caused by a switch in the polarity of the GABAergic responses from depolarizing to hyperpolarizing, reflecting a decrease in the intracellular chloride concentration. The effects of OXT were mimicked and occluded by bumetanide, a selective blocker of the chloride co-transporter NKCC1, suggesting that the effects of OXT involve inhibition of NKCC1. Neuronal death caused by anoxic-aglycaemic episodes was substantially delayed in the foetal hippocampus by endogenous OXT. These findings suggest that OXT plays important role in the preparation of the foetal brain to delivery.

  2. Beta-adrenergic activation of solute coupled water uptake by toad skin epithelium results in near-isosmotic transport

    DEFF Research Database (Denmark)

    Nielsen, Robert; Larsen, Erik Hviid


    (V) decreased to 0.50+/-0.15 nL cm(-2) x s(-1), which is significantly different from zero. Isoproterenol decreased the osmotic concentration of the transported fluid, C(osm) approximately 2 x I(SC)(Eqv)/J(V), from 351+/-72 to 227+/-28 mOsm (Ringer's solution: 252.8 mOsm). J(V) depicted a saturating function......(V) with a [Na+] of the transported fluid of 130+/-24 mM ([Na+]Ringer's solution = 117.4 mM). Addition of bumetanide to the inside solution reduced J(V). Water was transported uphill and J(V) reversed at an excess outside osmotic concentration, deltaC(S,rev) = 28.9+/-3.9 mOsm, amiloride decreased delta......Transepithelial potential (V(T)), conductance (G(T)), and water flow (J(V)) were measured simultaneously with good time resolution (min) in isolated toad (Bufo bufo) skin epithelium with Ringer on both sides. Inside application of 5 microM isoproterenol resulted in the fast increase in G(T) from 1...

  3. Propofol facilitated excitatory postsynaptic currents frequency on nucleus tractus solitarii (NTS) neurons. (United States)

    Jin, Zhenhua; Choi, Myung-Jin; Park, Cheung-Seog; Park, Young Seek; Jin, Young-Ho


    Propofol, an intravenous anesthetic, is broadly used for general anesthesia and diagnostic sedations due to its fast onset and recovery. Propofol depresses respiratory and cardiovascular reflex responses, however, their underlying mechanisms are not well known. Cardiorespiratory information from visceral afferent vagus nerves is integrated in the nucleus tractus solitarii (NTS). Cardiac and respiratory signals transducing vagal afferent neurons release the excitatory neurotransmitter glutamate onto NTS neurons in an activity dependent manner and trigger negative feedback reflex responses. In this experiment, the effects of propofol on glutamatergic synaptic responses at NTS neurons was tested using patch clamp methods. Glutamatergic excitatory postsynaptic currents (EPSC) were recorded at chloride reversal potential (-49mV) without γ-aminobutyric acid type A (GABA(A)) receptor antagonists. Propofol (≥3μM) facilitated frequency of the spontaneous EPSCs in a concentration dependent manner without altering amplitude and decay time. The GABA(A) receptor selective antagonist, gabazine (6μM), attenuated propofol effects on glutamate release. Propofol (10μM) evoked glutamate release was also blocked in the presence of the voltage dependent Na(+) and Ca(2+) channel blockers TTX (0.3μM) and Cd(2+) (0.2mM), respectively. In addition, the Na(+)-K(+)-Cl(-) cotransporter type 1 antagonist bumetanide (10μM) also inhibited propofol evoked increase in sEPSC frequency. These results suggest that propofol evoked glutamate release onto NTS neurons by GABA(A) receptor-mediated depolarization of the presynaptic excitatory terminals.

  4. Combined data mining strategy for the systematic identification of sport drug metabolites in urine by liquid chromatography time-of-flight mass spectrometry. (United States)

    Domínguez-Romero, Juan C; García-Reyes, Juan F; Martínez-Romero, Rubén; Berton, Paula; Martínez-Lara, Esther; Del Moral-Leal, María L; Molina-Díaz, Antonio


    The development of comprehensive methods able to tackle with the systematic identification of drug metabolites in an automated fashion is of great interest. In this article, a strategy based on the combined use of two complementary data mining tools is proposed for the screening and systematic detection and identification of urinary drug metabolites by liquid chromatography full-scan high resolution mass spectrometry. The proposed methodology is based on the use of accurate mass extraction of diagnostic ions (compound-dependent information) from in-source CID fragmentation without precursor ion isolation along with the use of automated mass extraction of accurate-mass shifts corresponding to typical biotransformations (non compound-dependent information) that xenobiotics usually undergo when metabolized. The combined strategy was evaluated using LC-TOFMS with a suite of nine sport drugs representative from different classes (propranolol, bumetanide, clenbuterol, ephedrine, finasteride, methoxyphenamine, methylephedrine, salbutamol and terbutaline), after single doses administered to rats. The metabolite identification coverage rate obtained with the systematic method (compared to existing literature) was satisfactory, and provided the identification of several non-previously reported metabolites. In addition, the combined information obtained helps to minimize the number of false positives. As an example, the systematic identification of urinary metabolites of propranolol enabled the identification of up to 24 metabolites, 15 of them non previously described in literature, which is a valuable indicator of the usefulness of the proposed systematic procedure.

  5. Gamma-aminobutyric acid and autism spectrum disorders%γ-氨基丁酸与孤独症谱系障碍

    Institute of Scientific and Technical Information of China (English)

    王刚; 单玲; 杜琳; 贾飞勇; 王伟


    孤独症谱系障碍是一组以社会交往、交流障碍和重复刻板性行为为主要特征的疾病,病因尚不明确。近年研究发现在孤独症谱系障碍动物模型中分娩时及生后早期Na+-K+-2Cl-共转运体1(NKCC1)与K+-2Cl-共转运体2(KCC2)的表达时相发生改变,造成海马神经元细胞内氯离子堆积,致使γ-氨基丁酸介导的神经兴奋性与抑制性的转换被破坏。应用 NKCC1抑制剂布美他尼对模型动物母体进行干预后纠正了它们的神经发育顺序,改善了其孤独症样行为,并且已有研究应用布美他尼改善了孤独症患者的症状。故而γ-氨基丁酸信号通路为孤独症谱系障碍发病机制的研究提供了新的方向,可能成为治疗孤独症谱系障碍新的靶点。本文对γ-氨基丁酸与孤独症谱系障碍关系的研究现状作一综述。%Autism spectrum disorder (ASD) is a group of diseases characterized by social interaction and communication defects and repeat stereotyped behaviors, and the etiology is not clear now. In recent years, the study found that in the ASD animal model of childbirth and early postnatal the time of expression of Na+-K+-2Cl-transporter 1 (NKCC1) and K+-2Cl- (KCC2) had changed, and the accumulation of chlorine ion neurons within the hippocampus, lead to the conversion of excitatory and inhibitory mediated by gamma-aminobutyric acid (GABA) was damaged. Application of bumetanide, as a NKCC1 inhibitor, in animal models’ matrix may correct their neurological development order, improve autistic behaviors. And the existing research by applying bumetanide improved symptoms in patients with ASD. So GABA signaling pathway provides a new direction of the pathogenesis of ASD, may become a new target in the treatment of ASD. In this paper, the research was about the relationship of GABA and ASD.

  6. Glucose stimulates calcium-activated chloride secretion in small intestinal cells. (United States)

    Yin, Liangjie; Vijaygopal, Pooja; MacGregor, Gordon G; Menon, Rejeesh; Ranganathan, Perungavur; Prabhakaran, Sreekala; Zhang, Lurong; Zhang, Mei; Binder, Henry J; Okunieff, Paul; Vidyasagar, Sadasivan


    The sodium-coupled glucose transporter-1 (SGLT1)-based oral rehydration solution (ORS) used in the management of acute diarrhea does not substantially reduce stool output, despite the fact that glucose stimulates the absorption of sodium and water. To explain this phenomenon, we investigated the possibility that glucose might also stimulate anion secretion. Transepithelial electrical measurements and isotope flux measurements in Ussing chambers were used to study the effect of glucose on active chloride and fluid secretion in mouse small intestinal cells and human Caco-2 cells. Confocal fluorescence laser microscopy and immunohistochemistry measured intracellular changes in calcium, sodium-glucose linked transporter, and calcium-activated chloride channel (anoctamin 1) expression. In addition to enhancing active sodium absorption, glucose increased intracellular calcium and stimulated electrogenic chloride secretion. Calcium imaging studies showed increased intracellular calcium when intestinal cells were exposed to glucose. Niflumic acid, but not glibenclamide, inhibited glucose-stimulated chloride secretion in mouse small intestines and in Caco-2 cells. Glucose-stimulated chloride secretion was not seen in ileal tissues incubated with the intracellular calcium chelater BAPTA-AM and the sodium-potassium-2 chloride cotransporter 1 (NKCC1) blocker bumetanide. These observations establish that glucose not only stimulates active Na absorption, a well-established phenomenon, but also induces a Ca-activated chloride secretion. This may explain the failure of glucose-based ORS to markedly reduce stool output in acute diarrhea. These results have immediate potential to improve the treatment outcomes for acute and/or chronic diarrheal diseases by replacing glucose with compounds that do not stimulate chloride secretion.

  7. Subcellular localization and displacement by diuretics of the peripheral benzodiazepine binding site (PBS) from rat kidney

    Energy Technology Data Exchange (ETDEWEB)

    Lukeman, S.; Fanestil, D.


    Although the PBS has been identified in many organs, its function and cellular location are speculative. Using rapid filtration, binding of (/sup 3/H)RO 5-4864 (*RO) (.75 nM) was assessed in four subcellular fractions (.3 mg/ml) derived from depapillated rat kidney by differential centrifugation: N (450g x 2 min), O (13,000 x 10), P (105,000 x 30), and S. The binding distribution was: N-18%, O-74%, P-6%, and S-2%. Marker enzyme analysis revealed that O was enriched in mitochondria (M), lysosomes (L), peroxisomes (P), and endoplasmic reticulum (ER), but not plasma membrane, and that N contained small amounts (10-15%) of markers for the above. Repeated washing of O removed ER enzymes but preserved *RO binding. O was further fractionated with centrifugation (57,000g x 4 hr) on a linear sucrose gradient (18-65%); *RO binding then comigrated with M but not P and L markers. Centrifugation of isolated M (5500 x 10 min) on another linear sucrose gradient (37-65%) gave low and high density bands, which contained 65% and 35% of *RO binding activity, resp. *RO binding in O was specific, saturable, reversible, and inhibited by diuretics. Inhibitors with the highest potency were indacrinone (K/sub d/ = 35, hydrochlorothiazide (100, and ethacrynic acid (325 Low potency inhibitors (K/sub d/ greater than or equal to 1 mM) included amiloride, triamterene, furosemide, bumetanide, and ozolinone.

  8. Direct block of the cystic fibrosis transmembrane conductance regulator Cl(-) channel by niflumic acid. (United States)

    Scott-Ward, T S; Li, H; Schmidt, A; Cai, Z; Sheppard, D N


    Niflumic acid is widely used to inhibit Ca(2+) -activated Cl(-) channels. However, the chemical structure of niflumic acid resembles that of diphenylamine-2-carboxylate, a drug that inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. To investigate how niflumic acid inhibits CFTR Cl(-) channel, we studied recombinant wild-type human CFTR in excised inside-out membrane patches. When added to the intracellular solution, niflumic acid caused a concentration- and voltage-dependent decrease of CFTR Cl(-) current with half-maximal inhibitory concentration (K(i)) of 253 microM and Hill co-efficient of approximately 1, at -50 mV. Niflumic acid inhibition of single CFTR Cl(-) channels was characterized by a very fast, flickery block that decreased dramatically current amplitude without altering open-probability. Consistent with these data, spectral analysis of CFTR Cl(-) currents suggested that channel block by niflumic acid was described by the closed open blocked kinetic scheme with blocker on rate (k(on)) = 13.9 x 10(6) M(-1)s(-1), off rate (k(off))=3348 s(-1) and dissociation constant (K(d)) = 241 microM, at -50 mV. Based on these data, we tested the effects of niflumic acid on transepithelial Cl(-) secretion and cyst growth using type I MDCK epithelial cells. Niflumic acid (200 microM) inhibited cAMP-stimulated, bumetanide-sensitive short-circuit current by 55%. Moreover, the drug potently retarded cyst growth. We conclude that niflumic acid is an open-channel blocker of CFTR that inhibits Cl(-) permeation by plugging the channel pore. It or related agents might be of value in the development of new therapies for autosomal dominant polycystic kidney disease.

  9. GABAergic signaling as therapeutic target for Autism Spectrum Disorders

    Directory of Open Access Journals (Sweden)

    Giada eCellot


    Full Text Available GABA, the main inhibitory neurotransmitter in the adult brain, early in postnatal life exerts a depolarizing and excitatory action. This depends on accumulation of chloride inside the cell via the cation-chloride importer NKCC1, being the expression of the chloride exporter KCC2 very low at birth. The developmentally regulated expression of KCC2 results in extrusion of chloride with age and a shift of GABA from the depolarizing to the hyperpolarizing direction. The depolarizing action of GABA leads to intracellular calcium rise through voltage-dependent calcium channels and/or NMDA receptors. GABA-mediated calcium signals regulate a variety of developmental processes from cell proliferation migration, differentiation, synapse maturation and neuronal wiring. Therefore, it is not surprising that some forms of neuro-developmental disorders such as Autism Spectrum Disorders (ASDs are associated with alterations of GABAergic signaling and impairment of the excitatory/inhibitory balance in selective neuronal circuits. In this review we will discuss how changes of GABAA-mediated neurotransmission affect several forms of ASDs including the Fragile X, the Angelman and Rett syndromes. Then, we will describe various animal models of ASDs with GABAergic dysfunctions, highlighting their behavioral deficits and the possibility to rescue them by targeting selective components of the GABAergic synapse. In particular, we will discuss how in some cases, reverting the polarity of GABA responses from the depolarizing to the hyperpolarizing direction with the diuretic bumetanide, a selective blocker of NKCC1, may have beneficial effects on ASDs, thus opening new therapeutic perspectives for the treatment of these devastating disorders.

  10. CFTR mediated chloride secretion in the avian renal proximal tubule. (United States)

    Laverty, Gary; Anttila, Ashley; Carty, Jenava; Reddy, Varudhini; Yum, Jamie; Arnason, Sighvatur S


    In primary cell cultures of the avian (Gallus gallus) renal proximal tubule parathyroid hormone and cAMP activation generate a Cl(-)-dependent short circuit current (I(SC)) response, consistent with net transepithelial Cl(-) secretion. In this study we investigated the expression and physiological function of the Na-K-2Cl (NKCC) transporter and CFTR chloride channel, both associated with Cl(-) secretion in a variety of tissues, in these proximal tubule cells. Using both RT-PCR and immunoblotting approaches, we showed that NKCC and CFTR are expressed, both in proximal tubule primary cultures and in a proximal tubule fraction of non-cultured (native tissue) fragments. We also used electrophysiological methods to assess the functional contribution of NKCC and CFTR to forskolin-activated I(SC) responses in filter grown cultured monolayers. Bumetanide (10 μM), a specific blocker of NKCC, inhibited forskolin activated I(SC) by about 40%, suggesting that basolateral uptake of Cl(-) is partially mediated by NKCC transport. In monolayers permeabilized on the basolateral side with nystatin, forskolin activated an apical Cl(-) conductance, manifested as bidirectional diffusion currents in the presence of oppositely directed Cl(-) gradients. Under these conditions the apical conductance appeared to show some bias towards apical-to-basolateral Cl(-) current. Two selective CFTR blockers, CFTR Inhibitor 172 and GlyH-101 (both at 20 μM) inhibited the forskolin activated diffusion currents by 38-68%, with GlyH-101 having a greater effect. These data support the conclusion that avian renal proximal tubules utilize an apical CFTR Cl(-) channel to mediate cAMP-activated Cl(-) secretion.

  11. At immature mossy fibers-CA3 connections, activation of presynaptic GABAB receptors by endogenously released GABA contributes to synapses silencing

    Directory of Open Access Journals (Sweden)

    Victoria F Safiulina


    Full Text Available Early in postnatal life correlated GABAergic activity in the hippocampus is thought to play a crucial role in synaptogenesis and in the development of adult neuronal networks. Unlike adulthood, at this developmental stage, mossy fibers (MF which are the axons of granule cells, release GABA into CA3 principal cells and interneurons. Here, we tested the hypothesis that at MF-CA3 connections, tonic activation of GABAB autoreceptors by GABA is responsible for the low probability of release and synapse silencing. Blocking GABAB receptors with CGP55845 enhanced the probability of GABA release and switched on silent synapses while the opposite was observed with baclofen. Both these effects were presynaptic and were associated with changes in paired-pulse ratio and coefficient of variation. In addition, enhancing the extracellular GABA concentration by repetitive stimulation of MF or by blocking the GABA transporter GAT-1, switched off active synapses, an effect that was prevented by CGP55845. In the presence of CGP55845, stimulation of MF induced synaptic potentiation. The shift of EGABA from the depolarizing to the hyperpolarizing direction with bumetanide, a blocker of the cation-chloride co-transporter NKCC1, prevented synaptic potentiation and caused synaptic depression, suggesting that the depolarizing action of GABA observed in the presence of CGP55845 is responsible for the potentiating effect. It is proposed that, activation of GABAB receptors by spillover of GABA from MF terminals reduces the probability of release and contributes to synapses silencing. This would act as a filter to prevent excessive activation of the auto-associative CA3 network and the emergence of seizures.

  12. Regulatory volume increase in astrocytes exposed to hypertonic medium requires β1 -adrenergic Na(+) /K(+) -ATPase stimulation and glycogenolysis. (United States)

    Song, Dan; Xu, Junnan; Hertz, Leif; Peng, Liang


    The cotransporter of Na(+) , K(+) , 2Cl(-) , and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K(+) concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1-mediated transport occurs as secondary active transport driven by Na(+) /K(+) -ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na(+) , K(+) , and Cl(-) × Cl(-) . In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na(+) /K(+) -ATPase activity is stimulated by elevated K(+) or the β-adrenergic agonist isoproterenol. Extracellular K(+) concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 µM in well-differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the β1 -adrenergic antagonist betaxolol, but not by ICI118551, a β2 -adrenergic antagonist. A large part of the β1 -adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na(+) /K(+) -ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post-excitatory undershoot.

  13. Acute study of interaction among cadmium, calcium, and zinc transport along the rat nephron in vivo. (United States)

    Barbier, O; Jacquillet, G; Tauc, M; Poujeol, P; Cougnon, M


    This study investigates the effect in rats of acute CdCl(2) (5 microM) intoxication on renal function and characterizes the transport of Ca(2+), Cd(2+), and Zn(2+) in the proximal tubule (PT), loop of Henle (LH), and terminal segments of the nephron (DT) using whole kidney clearance and nephron microinjection techniques. Acute Cd(2+) injection resulted in renal losses of Na(+), K(+), Ca(2+), Mg(2+), PO(4)(-2), and water, but the glomerular filtration rate remained stable. (45)Ca microinjections showed that Ca(2+) permeability in the DT was strongly inhibited by Cd(2+) (20 microM), Gd(3+) (100 microM), and La(3+) (1 mM), whereas nifedipine (20 microM) had no effect. (109)Cd and (65)Zn(2+) microinjections showed that each segment of nephron was permeable to these metals. In the PT, 95% of injected amounts of (109)Cd were taken up. (109)Cd fluxes were inhibited by Gd(3+) (90 microM), Co(2+) (100 microM), and Fe(2+) (100 microM) in all nephron segments. Bumetanide (50 microM) only inhibited (109)Cd fluxes in LH; Zn(2+) (50 and 500 microM) inhibited transport of (109)Cd in DT. In conclusion, these results indicate that 1) the renal effects of acute Cd(2+) intoxication are suggestive of proximal tubulopathy; 2) Cd(2+) inhibits Ca(2+) reabsorption possibly through the epithelial Ca(2+) channel in the DT, and this blockade could account for the hypercalciuria associated with Cd(2+) intoxication; 3) the PT is the major site of Cd(2+) reabsorption; 4) the paracellular pathway and DMT1 could be involved in Cd(2+) reabsorption along the LH; 5) DMT1 may be one of the major transporters of Cd(2+) in the DT; and 6) Zn(2+) is taken up along each part of the nephron and its transport in the terminal segments could occur via DMT1.

  14. Ion transport mechanisms linked to bicarbonate secretion in the esophageal submucosal glands. (United States)

    Abdulnour-Nakhoul, Solange; Nakhoul, Hani N; Kalliny, Medhat I; Gyftopoulos, Alex; Rabon, Edd; Doetjes, Rienk; Brown, Karen; Nakhoul, Nazih L


    The esophageal submucosal glands (SMG) secrete HCO(3)(-) and mucus into the esophageal lumen, where they contribute to acid clearance and epithelial protection. This study characterized the ion transport mechanisms linked to HCO(3)(-) secretion in SMG. We localized ion transporters using immunofluorescence, and we examined their expression by RT-PCR and in situ hybridization. We measured HCO(3)(-) secretion by using pH stat and the isolated perfused esophagus. Using double labeling with Na(+)-K(+)-ATPase as a marker, we localized Na(+)-coupled bicarbonate transporter (NBCe1) and Cl(-)-HCO(3)(-) exchanger (SLC4A2/AE2) to the basolateral membrane of duct cells. Expression of cystic fibrosis transmembrane regulator channel (CFTR) was confirmed by immunofluorescence, RT-PCR, and in situ hybridization. We identified anion exchanger SLC26A6 at the ducts' luminal membrane and Na(+)-K(+)-2Cl(-) (NKCC1) at the basolateral membrane of mucous and duct cells. pH stat experiments showed that elevations in cAMP induced by forskolin or IBMX increased HCO(3)(-) secretion. Genistein, an activator of CFTR, which does not increase intracellular cAMP, also stimulated HCO(3)(-) secretion, whereas glibenclamide, a Cl(-) channel blocker, and bumetanide, a Na(+)-K(+)-2Cl(-) blocker, decreased it. CFTR(inh)-172, a specific CFTR channel blocker, inhibited basal HCO(3)(-) secretion as well as stimulation of HCO(3)(-) secretion by IBMX. This is the first report on the presence of CFTR channels in the esophagus. The role of CFTR in manifestations of esophageal disease in cystic fibrosis patients remains to be determined.

  15. At immature mossy-fiber-CA3 synapses, correlated presynaptic and postsynaptic activity persistently enhances GABA release and network excitability via BDNF and cAMP-dependent PKA. (United States)

    Sivakumaran, Sudhir; Mohajerani, Majid H; Cherubini, Enrico


    In the adult rat hippocampus, the axons of granule cells in the dentate gyrus, the mossy fibers (MF), form excitatory glutamatergic synapses with CA3 principal cells. In neonates, MF release into their targets mainly GABA, which at this developmental stage is depolarizing. Here we tested the hypothesis that, at immature MF-CA3 synapses, correlated presynaptic [single fiber-evoked GABA(A)-mediated postsynaptic potentials (GPSPs)] and postsynaptic activity (back propagating action potentials) may exert a critical control on synaptic efficacy. This form of plasticity, called spike-timing-dependent plasticity (STDP), is a Hebbian type form of learning extensively studied at the level of glutamatergic synapses. Depending on the relative timing, pairing postsynaptic spiking and single MF-GPSPs induced bidirectional changes in synaptic efficacy. In case of positive pairing, spike-timing-dependent-long-term potentiation (STD-LTP) was associated with a persistent increase in GPSP slope and in the probability of cell firing. The transduction pathway involved a rise of calcium in the postsynaptic cell and the combined activity of cAMP-dependent PKA (protein kinase A) and brain-derived neurotrophic factor (BDNF). Retrograde signaling via BDNF and presynaptic TrkB receptors led to a persistent increase in GABA release. In "presynaptically" silent neurons, the enhanced probability of GABA release induced by the pairing protocol, unsilenced these synapses. Shifting E(GABA) from the depolarizing to the hyperpolarizing direction with bumetanide failed to modify synaptic strength. Thus, STD-LTP of GPSPs provides a reliable way to convey information from granule cells to the CA3 associative network at a time when glutamatergic synapses are still poorly developed.

  16. At immature mossy fibers-CA3 connections, activation of presynaptic GABA(B) receptors by endogenously released GABA contributes to synapses silencing. (United States)

    Safiulina, Victoria F; Cherubini, Enrico


    Early in postnatal life correlated GABAergic activity in the hippocampus is thought to play a crucial role in synaptogenesis and in the development of adult neuronal networks. Unlike adulthood, at this developmental stage, mossy fibers (MF) which are the axons of granule cells, release GABA into CA3 principal cells and interneurons. Here, we tested the hypothesis that at MF-CA3 connections, tonic activation of GABA(B) autoreceptors by GABA is responsible for the low probability of release and synapse silencing. Blocking GABA(B) receptors with CGP55845 enhanced the probability of GABA release and switched on silent synapses while the opposite was observed with baclofen. Both these effects were presynaptic and were associated with changes in paired-pulse ratio and coefficient of variation. In addition, enhancing the extracellular GABA concentration by repetitive stimulation of MF or by blocking the GABA transporter GAT-1, switched off active synapses, an effect that was prevented by CGP55845. In the presence of CGP55845, stimulation of MF-induced synaptic potentiation. The shift of E(GABA) from the depolarizing to the hyperpolarizing direction with bumetanide, a blocker of the cation-chloride co-transporter NKCC1, prevented synaptic potentiation and caused synaptic depression, suggesting that the depolarizing action of GABA observed in the presence of CGP55845 is responsible for the potentiating effect. It is proposed that, activation of GABA(B) receptors by spillover of GABA from MF terminals reduces the probability of release and contributes to synapses silencing. This would act as a filter to prevent excessive activation of the auto-associative CA3 network and the emergence of seizures.

  17. Hippocampus and epilepsy: Findings from human tissues. (United States)

    Huberfeld, G; Blauwblomme, T; Miles, R


    Surgical removal of the epileptogenic zone provides an effective therapy for several focal epileptic syndromes. This surgery offers the opportunity to study pathological activity in living human tissue for pharmacoresistant partial epilepsy syndromes including temporal lobe epilepsies with hippocampal sclerosis, cortical dysplasias, epilepsies associated with tumors and developmental malformations. Slices of tissue from patients with these syndromes retain functional neuronal networks and may generate epileptic activities. The properties of cells in this tissue may not be greatly changed, but excitatory synaptic transmission is often enhanced and GABAergic inhibition is preserved. Typically epileptic activity is not generated spontaneously by the neocortex, whether dysplastic or not, but can be induced by convulsants. The initiation of ictal discharges in the neocortex depends on both GABAergic signaling and increased extracellular potassium. In contrast, a spontaneous interictal-like activity is generated by tissues from patients with temporal lobe epilepsies associated with hippocampal sclerosis. This activity is initiated, not in the hippocampus but in the subiculum, an output region, which projects to the entorhinal cortex. Interictal events seem to be triggered by GABAergic cells, which paradoxically excite about 20% of subicular pyramidal cells while simultaneously inhibiting the majority. Interictal discharges thus depend on both GABAergic and glutamatergic signaling. The depolarizing effects of GABA depend on a pathological elevation in levels of chloride in some subicular cells, similar to those of developmentally immature cells. Such defect is caused by a perturbed expression of the cotransporters regulating intracellular chloride concentration, the importer NKCC1 and the extruder KCC2. Blockade of NKCC1 actions by the diuretic bumetanide restores intracellular chloride and thus hyperpolarizing GABAergic actions and consequently suppressing interictal


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    I. V. Kovalev


    Full Text Available Aim. The effect of hypoxia on the electrical and contractile activities of smooth muscles cells (SMCs of the guinea pig ureter was studied by the method of the double sucrose bridge.Materials and methods. This method allows registering simultaneously parameters of the action potential (AP and the contraction of SMCs, caused by an electrical stimulus.Results. It was found that lowering the oxygen content in the perfusion solution for 10 min resulted to an increase of electrical and contractile activity of ureteral SMCs. Addition of tetraethylammonium chloride (TEA, 5 mM – nonselective blocker of potassium membrane conductance – in hypoxic conditions causing an additional increase in the amplitude of the AP, duration of the AP plateau and the contractile responses of smooth muscles. Thus, the hypoxia decreased the potassium membrane conductance of ureteral SMCs. Inhibition of the effect of the α1 -adrenergic receptors agonist phenylephrine (PE, 10 mM on the electrical and contractile properties of SMCs in hypoxic condition indicate the involvement of the protein kinase C-dependent signaling system in effects of hypoxia. Pretreatment of ureteral smooth muscles with bumetanide (100 mM – selective inhibitor of Na+,K+,2Cl- - cotransporter (NKCC – caused a decrease of the activating effect of hypoxia on the SMCs of guinea pig ureter.Conclusion.Thus, the impact of hypoxia on the regulation of electrical activity and contractions of smooth muscles of guinea pig ureter may be due to changes in ion permeability of membranes SMCs and operation of ion-transporting systems. 

  19. Transepithelial electrical potential of nonsensory region of gerbil utricle in vitro. (United States)

    Marcus, D C


    Transepithelial electrical potential difference (VT) was measured across the vestibular labyrinth of the inner ear in vitro by puncturing the epithelial wall of the utricle with a glass microelectrode. A region of nonsensory cells of the utricle was isolated from the sensory regions by introducing columns of liquid Sylgard 184. Under control conditions, the VT of this region was +7.5 +/- 0.3 mV (means +/- SE), lumen positive. This potential difference was rapidly reduced by either 1 mM ouabain, 10-100 microM bumetanide, 0.5-5.0 mM Ba (in the bathing solution), or cooling, but not by the disulfonic stilbene, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Changes in VT due to reductions of Cl or Na or to increases of K in the bathing solution in exchange for presumably impermeant ions were observed in this region and were compared with those in a preparation in which the insulating seals were absent. The K-induced voltage change was significantly higher in the unblocked preparation, a finding consistent with a high K permeability of the sensory cells. The voltage change due to reduction of Cl was not inhibited by Cl channel blockers (9-anthracenecarboxylate and diphenylamine-2-carboxylate) in the bathing solution. These results represent the first direct demonstration that the nonsensory cells of the utricle produce a lumen-positive active-transport potential and characterize some of the properties of the cell membranes in terms of their pharmacological sensitivities and net voltage responses to changes in the bathing medium ions Na, K, and Cl.

  20. Evidence against luminal one-for-one Cl -HCO3 exchange in urodele small intestine

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    White, J.F.


    The ratio of Cl absorbed to HCO3 secreted by the in vitro small intestine of Amphiuma was measured using TWCl and titration. The aim was to estimate the stoichiometry and thereby elucidate the underlying transport mechanisms. For every mole of HCO3 secreted 1.8 mol of Cl underwent net absorption. Indirect measures of net Cl absorption and HCO3 secretion were validated. Several known and putative Cl transport inhibitors were examined for their ability to inhibit the anion transport events. Disulfonic stilbenes (DIDS) and the diuretics piretanide and furosemide inhibited the Cl absorptive flux (J/sub m s/sup Cl/) and simultaneously the HCO3 secretory flux (J/sup HCO3 /). The diuretics acetazolamide and bumetanide also reduced J/sup HCO3 and J/sub m s/sup Cl/, although the latter effect was not statistically significant. The ratio of inhibition, J/sub m s/sup Cl// J/sup HCO3 /, varied from 1.2 to 1.8 for the different inhibitors. The presence of Cl -HCO3 exchange at the serosal membrane was deduced from 1) the reduction of J/sub m s/sup Cl/ and J/sup HCO3 / by serosally added stilbenes, 2) the reduction of Cl absorption when serosal Cl was replaced, 3) inhibition of the secretory-to-mucosal Cl flux by serosal stilbenes, and 4) enhancement of J/sup HCO3 when serosal medium HCO3 was elevated. The observations are not consistent with one-for-one exchange of Cl for HCO3 at the mucosal membrane. The observed coupling ratio is compatible with a one-for-one exchange of Cl for HCO3 at the serosal membrane.

  1. Low salt concentrations activate AMP-activated protein kinase in mouse macula densa cells. (United States)

    Cook, Natasha; Fraser, Scott A; Katerelos, Marina; Katsis, Frosa; Gleich, Kurt; Mount, Peter F; Steinberg, Gregory R; Levidiotis, Vicki; Kemp, Bruce E; Power, David A


    The energy-sensing kinase AMP-activated protein kinase (AMPK) is associated with the sodium-potassium-chloride cotransporter NKCC2 in the kidney and phosphorylates it on a regulatory site in vitro. To identify a potential role for AMPK in salt sensing at the macula densa, we have used the murine macula densa cell line MMDD1. In this cell line, AMPK was rapidly activated by isosmolar low-salt conditions. In contrast to the known salt-sensing pathway in the macula densa, AMPK activation occurred in the presence of either low sodium or low chloride and was unaffected by inhibition of NKCC2 with bumetanide. Assays using recombinant AMPK demonstrated activation of an upstream kinase by isosmolar low salt. The specific calcium/calmodulin-dependent kinase kinase inhibitor STO-609 failed to suppress AMPK activation, suggesting that it was not part of the signal pathway. AMPK activation was associated with increased phosphorylation of the specific substrate acetyl-CoA carboxylase (ACC) at Ser(79), as well as increased NKCC2 phosphorylation at Ser(126). AMPK activation due to low salt concentrations was inhibited by an adenovirus construct encoding a kinase dead mutant of AMPK, leading to reduced ACC Ser(79) and NKCC2 Ser(126) phosphorylation. This work demonstrates that AMPK activation in macula densa-like cells occurs via isosmolar changes in sodium or chloride concentration, leading to phosphorylation of ACC and NKCC2. Phosphorylation of these substrates in vivo is predicted to increase intracellular chloride and so reduce the effect of salt restriction on tubuloglomerular feedback and renin secretion.

  2. Possible role of GABAergic depolarization in neocortical neurons in generating hyperexcitatory behaviors during emergence from sevoflurane anesthesia in the rat

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    Byung‑Gun Lim


    Full Text Available Hyperexcitatory behaviors occurring after sevoflurane anesthesia are of serious clinical concern, but the underlying mechanism is unknown. These behaviors may result from the potentiation by sevoflurane of GABAergic depolarization/excitation in neocortical neurons, cells implicated in the genesis of consciousness and arousal. The current study sought to provide evidence for this hypothesis with rats, the neocortical neurons of which are known to respond to GABA (γ-aminobutyric acid with depolarization/excitation at early stages of development (i.e., until the second postnatal week and with hyperpolarization/inhibition during adulthood. Employing behavioral tests and electrophysiological recordings in neocortical slice preparations, we found: (1 sevoflurane produced PAHBs (post-anesthetic hyperexcitatory behaviors in postnatal day (P1–15 rats, whereas it failed to elicit PAHBs in P16 or older rats; (2 GABAergic PSPs (postsynaptic potentials were depolarizing/excitatory in the neocortical neurons of P5 and P10 rats, whereas mostly hyperpolarizing/inhibitory in the cells of adult rats; (3 at P14–15, <50% of rats had PAHBs and, in general, the cells of the animals with PAHBs exhibited strongly depolarizing GABAergic PSPs, whereas those without PAHBs showed hyperpolarizing or weakly depolarizing GABAergic PSPs; (4 bumetanide [inhibitor of the Cl− importer NKCC (Na+–K+–2Cl− cotransporter] treatment at P5 suppressed PAHBs and depolarizing GABAergic responses; and (5 sevoflurane at 1% (i.e., concentration <1 minimum alveolar concentration potentiated depolarizing GABAergic PSPs in the neurons of P5 and P10 rats and of P14–15 animals with PAHBs, evoking action potentials in ≥50% of these cells. On the basis of these results, we conclude that sevoflurane may produce PAHBs by potentiating GABAergic depolarization/excitation in neocortical neurons.

  3. Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation.

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    Carol Taylor-Burds

    Full Text Available A developmental "switch" in chloride transporters occurs in most neurons resulting in GABAA mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABAA receptor activation. Among this group are gonadotropin-releasing hormone-1 (GnRH neurons, which control puberty and reproduction. NKCC1 is the primary chloride accumulator in neurons, expressed at high levels early in development and contributes to depolarization after GABAA receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABAA receptor activation. Anion exchangers (AEs are also potential modulators of responses to GABAA activation since they accumulate chloride and extrude bicarbonate. To evaluate the mechanism(s underlying GABAA mediated depolarization, GnRH neurons were analyzed for 1 expression of chloride transporters and AEs in embryonic, pre-pubertal, and adult mice 2 responses to GABAA receptor activation in NKCC1-/- mice and 3 function of AEs in these responses. At all ages, GnRH neurons were immunopositive for NKCC1 and AE2 but not KCC2 or AE3. Using explants, calcium imaging and gramicidin perforated patch clamp techniques we found that GnRH neurons from NKCC1-/- mice retained relatively normal responses to the GABAA agonist muscimol. However, acute pharmacological inhibition of NKCC1 with bumetanide eliminated the depolarization/calcium response to muscimol in 40% of GnRH neurons from WT mice. In the remaining GnRH neurons, HCO3- mediated mechanisms accounted for the remaining calcium responses to muscimol. Collectively these data reveal mechanisms responsible for maintaining depolarizing GABAA mediated transmission in GnRH neurons.

  4. Cell Volume Regulation and Apoptotic Volume Decrease in Rat Distal Colon Superficial Enterocytes

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    Stefania Antico


    Full Text Available Background: The colon epithelium is physiologically exposed to osmotic stress, and the activation of cell volume regulation mechanisms is essential in colonocyte physiology. Moreover, colon is characterized by a high apoptotic rate of mature cells balancing the high division rate of stem cells. Aim: The aim of the present work was to investigate the main cell volume regulation mechanisms in rat colon surface colonocytes and their role in apoptosis. Methods: Cell volume changes were measured by light microscopy and video imaging on colon explants; apoptosis sign appearance was monitored by confocal microscopy on annexin V/propidium iodide labeled explants. Results: Superficial colonocytes showed a dynamic regulation of their cell volume during anisosmotic conditions with a Regulatory Volume Increase (RVI response following hypertonic shrinkage and Regulatory Volume Decrease (RVD response following hypotonic swelling. RVI was completely inhibited by bumetanide, while RVD was completely abolished by high K+ or iberiotoxin treatment and by extracellular Ca2+ removal. DIDS incubation was also able to affect the RVD response. When colon explants were exposed to H2O2 as apoptotic inducer, colonocytes underwent an isotonic volume decrease ascribable to Apoptotic Volume Decrease (AVD within about four hours of exposure. AVD was shown to precede annexin V positivity. It was also inhibited by high K+ or iberiotoxin treatment. Interestingly, treatment with iberiotoxin significantly inhibited apoptosis progression. Conclusions: In rat superficial colonocytes K+ efflux through high conductance Ca2+-activated K+ channels (BK channels was demonstrated to be the main mechanism of RVD and to plays also a crucial role in the AVD process and in the progression of apoptosis.

  5. Characterization of Na+ uptake in the endangered desert pupfish, Cyprinodon macularius (Baird and Girard) (United States)

    Brix, Kevin V.; Grosell, Martin


    This study provided an initial characterization of Na+ uptake in saline freshwater by the endangered pupfish, Cyprinodon macularius. This species occurs only in several saline water systems in the southwestern USA and northern Mexico, where salinity is largely controlled by water-management practices. Consequently, understanding the osmoregulatory capacity of this species is important for their conservation. The lower acclimation limit of C. macularius in freshwater was found to be 2 mM Na+. Fish acclimated to 2 or 7 mM Na+ displayed similar Na+ uptake kinetics, with Km values of 4321 and 3672 μM and Vmax values of 4771 and 3602 nmol g−1 h−1, respectively. A series of experiments using pharmacological inhibitors indicated that Na+ uptake in C. macularius was not sensitive to bumetanide, metolazone, or phenamil. These results indicate the Na+–K+–2Cl− cotransporter, Na+–Cl− cotransporter, and the Na+ channel–H+-ATPase system are likely not to be involved in Na+ uptake at the apical membrane of fish gill ionocytes in fish acclimated to 2 or 7 mM Na+. However, Na+ uptake was sensitive to 1 × 10−3 M amiloride (not 1 × 10−4 or 1 × 10−5 M), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and ethoxzolamide. These data suggest that C. macularius relies on a low-affinity Na+–H+ exchanger for apical Na+ uptake and that H+ ions generated via carbonic anhydrase-mediated CO2 hydration are important for the function of this protein. PMID:27293589

  6. Characterization of Na(+) uptake in the endangered desert pupfish, Cyprinodon macularius (Baird and Girard). (United States)

    Brix, Kevin V; Grosell, Martin


    This study provided an initial characterization of Na(+) uptake in saline freshwater by the endangered pupfish, Cyprinodon macularius. This species occurs only in several saline water systems in the southwestern USA and northern Mexico, where salinity is largely controlled by water-management practices. Consequently, understanding the osmoregulatory capacity of this species is important for their conservation. The lower acclimation limit of C. macularius in freshwater was found to be 2 mM Na(+). Fish acclimated to 2 or 7 mM Na(+) displayed similar Na(+) uptake kinetics, with K m values of 4321 and 3672 μM and V max values of 4771 and 3602 nmol g(-1) h(-1), respectively. A series of experiments using pharmacological inhibitors indicated that Na(+) uptake in C. macularius was not sensitive to bumetanide, metolazone, or phenamil. These results indicate the Na(+)-K(+)-2Cl(-) cotransporter, Na(+)-Cl(-) cotransporter, and the Na(+) channel-H(+)-ATPase system are likely not to be involved in Na(+) uptake at the apical membrane of fish gill ionocytes in fish acclimated to 2 or 7 mM Na(+). However, Na(+) uptake was sensitive to 1 × 10(-3) M amiloride (not 1 × 10(-4) or 1 × 10(-5) M), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and ethoxzolamide. These data suggest that C. macularius relies on a low-affinity Na(+)-H(+) exchanger for apical Na(+) uptake and that H(+) ions generated via carbonic anhydrase-mediated CO2 hydration are important for the function of this protein.

  7. Age-dependent susceptibility to phenobarbital-resistant neonatal seizures: role of chloride co-transporters

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    Seok Kyu eKang


    Full Text Available Ischemia in the immature brain is an important cause of neonatal seizures. Temporal evolution of acquired neonatal seizures and their response to anticonvulsants are of great interest, given the unreliability of the clinical correlates and poor efficacy of first-line anti-seizure drugs. The expression and function of the electroneutral chloride co-transporters KCC2 and NKCC1 influence the anti-seizure efficacy of GABAA-agonists. To investigate ischemia-induced seizure susceptibility and efficacy of the GABAA-agonist phenobarbital (PB, with NKCC1 antagonist bumetanide (BTN as an adjunct treatment, we utilized permanent unilateral carotid-ligation to produce acute ischemic-seizures in postnatal day 7, 10 and 12 CD1 mice. Immediate post-ligation video-electroencephalograms (EEGs quantitatively evaluated baseline and post-treatment seizure burdens. Brains were examined for stroke-injury and western blot analyses to evaluate the expression of KCC2 and NKCC1. Severity of acute ischemic seizures post-ligation was highest at P7. PB was an efficacious anti-seizure agent at P10 and P12, but not at P7. BTN failed as an adjunct, at all ages tested and significantly blunted PB-efficacy at P10. Significant acute post-ischemic downregulation of KCC2 was detected at all ages. At P7, males displayed higher age-dependent seizure susceptibility, associated with a significant developmental lag in their KCC2 expression. This study established a novel neonatal mouse model of PB-resistant seizures that demonstrates age/sex-dependent susceptibility. The age-dependent profile of KCC2 expression and its post-insult downregulation may underlie the PB-resistance reported in this model. Blocking NKCC1 with low-dose BTN following PB treatment failed to improve PB-efficacy.

  8. Prevention of NKCC1 phosphorylation avoids downregulation of KCC2 in central sensory pathways and reduces neuropathic pain after peripheral nerve injury. (United States)

    Mòdol, Laura; Cobianchi, Stefano; Navarro, Xavier


    Neuropathic pain after peripheral nerve injury is characterized by loss of inhibition in both peripheral and central pain pathways. In the adult nervous system, the Na(+)-K(+)-2Cl(-) (NKCC1) and neuron-specific K(+)-Cl(-) (KCC2) cotransporters are involved in setting the strength and polarity of GABAergic/glycinergic transmission. After nerve injury, the balance between these cotransporters changes, leading to a decrease in the inhibitory tone. However, the role that NKCC1 and KCC2 play in pain-processing brain areas is unknown. Our goal was to study the effects of peripheral nerve injury on NKCC1 and KCC2 expression in dorsal root ganglia (DRG), spinal cord, ventral posterolateral (VPL) nucleus of the thalamus, and primary somatosensory (S1) cortex. After sciatic nerve section and suture in adult rats, assessment of mechanical and thermal pain thresholds showed evidence of hyperalgesia during the following 2 months. We also found an increase in NKCC1 expression in the DRG and a downregulation of KCC2 in spinal cord after injury, accompanied by later decrease of KCC2 levels in higher projection areas (VPL and S1) from 2 weeks postinjury, correlating with neuropathic pain signs. Administration of bumetanide (30 mg/kg) during 2 weeks following sciatic nerve lesion prevented the previously observed changes in the spinothalamic tract projecting areas and the appearance of hyperalgesia. In conclusion, the present results indicate that changes in NKCC1 and KCC2 in DRG, spinal cord, and central pain areas may contribute to development of neuropathic pain.

  9. Modulation of ion transport across rat distal colon by cysteine

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    Martin eDiener


    Full Text Available The aim of this study was to identify the actions of stimulation of endogenous production of H2S by cysteine, the substrate for the two H2S-producing enzymes, cystathionin-beta-synthase and cystathionin-gamma-lyase, on ion transport across rat distal colon. Changes in short-circuit current (Isc induced by cysteine were measured in Ussing chambers. Free cysteine caused a concentration-dependent, transient fall in Isc, which was sensitive to amino-oxyacetate and beta-cyano-L-alanine, i.e. inhibitors of H2S-producing enzymes. In contrast, Na cysteinate evoked a biphasic change in Isc, i.e. an initial fall followed by a secondary increase, which was also reduced by these enzyme inhibitors. All responses were dependent on the presence of Cl- and inhibited by bumetanide, suggesting that free cysteine induces an inhibition of transcellular Cl- secretion, whereas Na cysteinate – after a transient inhibitory phase – activates anion secretion. The assumed reason for this discrepancy is a fall in the cytosolic pH induced by free cysteine, but not by Na cysteinate, as observed in isolated colonic crypts loaded with the pH-sensitive dye, BCECF. Intracellular acidification is known to inhibit epithelial K+ channels. Indeed, after preinhibition of basolateral K+ channels with tetrapentylammonium or Ba2+, the negative Isc induced by free cysteine was reduced significantly. In consequence, stimulation of endogenous H2S production by Na cysteinate causes, after a short inhibitory response, a delayed activation of anion secretion, which is missing in the case of free cysteine, probably due to the cytosolic acidification. In contrast, diallyl trisulfide, which is intracellularly converted to H2S, only evoked a monophasic increase in Isc without the initial fall observed with Na cysteinate. Consequently, time course and amount of produced H2S seem to strongly influence the functional response of the colonic epithelium evoked by this gasotransmitter.

  10. Mechanisms underlying spontaneous constrictions of postcapillary venules in the rat stomach. (United States)

    Mitsui, Retsu; Hashitani, Hikaru


    Postcapillary venules (PCVs) play a critical role in regulating capillary hydrostatic pressure, but their contractile mechanisms are not well understood. We examined the properties of spontaneous vasomotion and corresponding Ca(2+) transients in gastric PCV. In the rat gastric submucosa, changes in PCV diameter and intracellular Ca(2+) dynamics were visualised by video tracking system and fluorescent Ca(2+) imaging, respectively, while PCV morphology was examined by immunohistochemistry. Stellate-shaped PCV mural cells expressing α-smooth muscle actin exhibited synchronised spontaneous Ca(2+) transients to develop vasomotion which was abolished by nifedipine (1 μM), cyclopiazonic acid (10 μM), or Ca(2+)-activated Cl(-) channel inhibitors (100 μM niflumic acid, 1 μM T16Ainh-A01). A gap junction blocker (3 μM carbenoxolone) disrupted the synchrony of spontaneous Ca(2+) transients amongst PCV mural cells and attenuated spontaneous vasomotion. Low chloride solution ([Cl(-)]0 = 12.4 mM) also disrupted the synchrony of spontaneous Ca(2+) transients and abolished vasomotion. Na(+)-K(+)-Cl(-) co-transporter inhibitors (10 μM bumetanide, 30 μM furosemide) suppressed spontaneous Ca(2+) transients and vasoconstrictions. A phosphodiesterase type 5 (PDE5) inhibitor (1 μM tadalafil) disrupted the spontaneous Ca(2+) transient synchrony and abolished vasomotion in a nitric oxide (NO)-dependent manner. Thus, gastric PCVs exhibit spontaneous vasomotion, resulting from synchronised spontaneous Ca(2+) transients within a network of stellate-shaped PCV mural cells. An active Cl(-) accumulation partly via Na(+)-K(+)-Cl(-) co-transport appears to be fundamental in maintaining depolarisation upon the opening of Ca(2+)-activated Cl(-) channels that triggers Ca(2+) influx via voltage-dependent L-type Ca(2+) channels. Basal PDE5 activity may continuously counteract vaso-relaxing effects of endothelial NO to maintain spontaneous vasomotion.

  11. Temporal coding at the immature depolarizing GABAergic synapse

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    Guzel Valeeva


    Full Text Available In the developing hippocampus, GABA exerts depolarizing and excitatory actions and contributes to the generation of neuronal network driven Giant Depolarizing Potentials (GDPs. Here, we studied spike time coding at immature GABAergic synapses and its impact on synchronization of the neuronal network during GDPs in the neonatal (postnatal days P2-6 rat hippocampal slices. Using extracellular recordings, we found that the delays of action potentials (APs evoked by synaptic activation of GABA(A receptors are long (mean, 65 ms and variable (within a time window of 10-200 ms. During patch-clamp recordings, depolarizing GABAergic responses were mainly subthreshold and their amplification by persistent sodium conductance was required to trigger APs. AP delays at GABAergic synapses shortened and their variability reduced with an increase in intracellular chloride concentration during whole-cell recordings. Negative shift of the GABA reversal potential (EGABA with low concentrations of bumetanide, or potentiation of GABA(A receptors with diazepam reduced GDPs amplitude, desynchronized neuronal firing during GDPs and slowed down GDPs propagation. Partial blockade of GABA(A receptors with bicuculline increased neuronal synchronization and accelerated GDPs propagation. We propose that spike-timing at depolarizing GABA synapses is determined by intracellular chloride concentration. At physiological levels of intracellular chloride GABAergic depolarization does not reach the action potential threshold and amplification of GABAergic responses by non-inactivating sodium conductance is required for postsynaptic AP initiation. Slow and variable excitation at GABAergic synapse determines the level of neuronal synchrony and the rate of GDPs propagation in the developing hippocampus.

  12. Layer-specific generation and propagation of seizures in slices of developing neocortex: role of excitatory GABAergic synapses. (United States)

    Rheims, Sylvain; Represa, Alfonso; Ben-Ari, Yehezkel; Zilberter, Yuri


    The neonatal period is critical for seizure susceptibility, and neocortical networks are central in infantile epilepsies. We report that application of 4-aminopyridine (4-AP) to immature (P6-P9) neocortical slices generates layer-specific interictal seizures (IISs) that transform after recurrent seizures to ictal seizures (ISs). During IISs, cell-attached recordings show action potentials in interneurons and pyramidal cells in L5/6 and interneurons but not pyramidal neurons in L2/3. However, L2/3 pyramidal neurons also fire during ISs. Using single N-methyl-d-aspartate (NMDA) channel recordings for measuring the cell resting potential (Em), we show that transition from IISs to ISs is associated with a gradual Em depolarization of L2/3 and L5/6 pyramidal neurons that enhances their excitability. Bumetanide, a NKCC1 co-transporter antagonist, inhibits generation of IISs and prevents their transformation to ISs, indicating the role excitatory GABA in epilepsies. Therefore deep layer neurons are more susceptible to seizures than superficial ones. The initiating phase of seizures is characterized by IISs generated in L5/6 and supported by activation of both L5/6 interneurons and pyramidal cells. IISs propagate to L2/3 via activation of L2/3 interneurons but not pyramidal cells, which are mostly quiescent at this phase. In superficial layers, a persistent increase in excitability of pyramidal neurons caused by Em depolarization is associated with a transition from largely confined GABAergic IIS to ictal events that entrain the entire neocortex.

  13. Temporal coding at the immature depolarizing GABAergic synapse. (United States)

    Valeeva, Guzel; Abdullin, Azat; Tyzio, Roman; Skorinkin, Andrei; Nikolski, Evgeny; Ben-Ari, Yehezkiel; Khazipov, Rustem


    In the developing hippocampus, GABA exerts depolarizing and excitatory actions and contributes to the generation of neuronal network driven giant depolarizing potentials (GDPs). Here, we studied spike time coding at immature GABAergic synapses and its impact on synchronization of the neuronal network during GDPs in the neonatal (postnatal days P2-6) rat hippocampal slices. Using extracellular recordings, we found that the delays of action potentials (APs) evoked by synaptic activation of GABA(A) receptors are long (mean, 65 ms) and variable (within a time window of 10-200 ms). During patch-clamp recordings, depolarizing GABAergic responses were mainly subthreshold and their amplification by persistent sodium conductance was required to trigger APs. AP delays at GABAergic synapses shortened and their variability reduced with an increase in intracellular chloride concentration during whole-cell recordings. Negative shift of the GABA reversal potential (E(GABA)) with low concentrations of bumetanide, or potentiation of GABA(A) receptors with diazepam reduced GDPs amplitude, desynchronized neuronal firing during GDPs and slowed down GDPs propagation. Partial blockade of GABA(A) receptors with bicuculline increased neuronal synchronization and accelerated GDPs propagation. We propose that spike timing at depolarizing GABA synapses is determined by intracellular chloride concentration. At physiological levels of intracellular chloride GABAergic depolarization does not reach the action potential threshold and amplification of GABAergic responses by non-inactivating sodium conductance is required for postsynaptic AP initiation. Slow and variable excitation at GABAergic synapse determines the level of neuronal synchrony and the rate of GDPs propagation in the developing hippocampus.

  14. Excitatory GABA in rodent developing neocortex in vitro. (United States)

    Rheims, Sylvain; Minlebaev, Marat; Ivanov, Anton; Represa, Alfonso; Khazipov, Rustem; Holmes, Gregory L; Ben-Ari, Yehezkel; Zilberter, Yuri


    GABA depolarizes immature cortical neurons. However, whether GABA excites immature neocortical neurons and drives network oscillations as in other brain structures remains controversial. Excitatory actions of GABA depend on three fundamental parameters: the resting membrane potential (Em), reversal potential of GABA (E(GABA)), and threshold of action potential generation (Vthr). We have shown recently that conventional invasive recording techniques provide an erroneous estimation of these parameters in immature neurons. In this study, we used noninvasive single N-methyl-d-aspartate and GABA channel recordings in rodent brain slices to measure both Em and E(GABA) in the same neuron. We show that GABA strongly depolarizes pyramidal neurons and interneurons in both deep and superficial layers of the immature neocortex (P2-P10). However, GABA generates action potentials in layer 5/6 (L5/6) but not L2/3 pyramidal cells, since L5/6 pyramidal cells have more depolarized resting potentials and more hyperpolarized Vthr. The excitatory GABA transiently drives oscillations generated by L5/6 pyramidal cells and interneurons during development (P5-P12). The NKCC1 co-transporter antagonist bumetanide strongly reduces [Cl(-)]i, GABA-induced depolarization, and network oscillations, confirming the importance of GABA signaling. Thus a strong GABA excitatory drive coupled with high intrinsic excitability of L5/6 pyramidal neurons and interneurons provide a powerful mechanism of synapse-driven oscillatory activity in the rodent neocortex in vitro. In the companion paper, we show that the excitatory GABA drives layer-specific seizures in the immature neocortex.

  15. The chemokine CXCL12 and the HIV-1 envelope protein gp120 regulate spontaneous activity of Cajal-Retzius cells in opposite directions. (United States)

    Marchionni, Ivan; Beaumont, Michael; Maccaferri, Gianmaria


    Activation of the CXC chemokine receptor 4 (CXCR4) in Cajal–Retzius cells by CXC chemokine ligand 12 (CXCL12) is important for controlling their excitability. CXCR4 is also a co-receptor for the glycoprotein 120 (gp120) of the envelope of the human immunodeficiency virus type 1 (HIV-1), and binding of gp120 to CXCR4 may produce pathological effects. In order to study CXCR4-dependent modulation of membrane excitability, we recorded in cell-attached configuration spontaneous action currents from hippocampal stratum lacunosum-moleculare Cajal–Retzius cells of the CXCR4-EGFP mouse. CXCL12 (50 nM) powerfully inhibited firing independently of synaptic transmission, suggesting that CXCR4 regulates an intrinsic conductance. This effect was prevented by conditioning slices with BAPTA-AM (200 μM), and by blockers of the BK calcium-dependent potassium channels (TEA (1 mM), paxilline (10 μM) and iberiotoxin (100 nM)). In contrast, exposure to gp120 (pico- to nanomolar range, alone or in combination with soluble cluster of differentiation 4 (CD4)), enhanced spontaneous firing frequency. This effect was prevented by the CXCR4 antagonist AMD3100 (1 μM) and was absent in EGFP-negative stratum lacunosum-moleculare interneurons. Increased excitability was prevented by treating slices with BAPTA-AM or bumetanide, suggesting that gp120 activates a mechanism that is both calcium- and chloride-dependent. In conclusion, our results demonstrate that CXCL12 and gp120 modulate the excitability of Cajal–Retzius cells in opposite directions. We propose that CXCL12 and gp120 either generate calcium responses of different strength or activate distinct pools of intracellular calcium, leading to agonist-specific responses, mediated by BK channels in the case of CXCL12, and by a chloride-dependent mechanism in the case of gp120.

  16. Impaired intestinal sodium and chloride transport in the blind loop syndrome of the rat. (United States)

    Schulzke, J D; Fromm, M; Menge, H; Riecken, E O


    Self-filling blind loops of rat jejunum were used as a model for the blind loop syndrome in humans. Electrical resistance, short circuit current, and unidirectional sodium and chloride fluxes were measured using the Ussing technique. Whereas net fluxes for sodium and chloride did not differ significantly from zero in the blind loop or in the control, unidirectional fluxes of either direction were decreased and electrical resistance was increased, indicating an increase in the tightness of the intestinal wall. Measurements of alternating current impedance and micropuncture experiments revealed that this was due to an increase in epithelial resistance from 9 +/- 1 omega X cm2 (n = 15, results of both methods) to 27 +/- 4 omega X cm2 (n = 15) and in subepithelial resistance from 40 +/- 2 omega X cm2 (n = 15) to 76 +/- 7 omega X cm2 (n = 15). As the ratio of epithelial to subepithelial resistance was similar in the blind loop and in the control, lower transport rates in the blind loop are indicative of impaired epithelial transport function. Subsequently, two different transport systems were characterized. First, the 3-o-methyl-glucose-induced, phlorizin-reversible increase in short circuit current, representing glucose-coupled sodium absorption, showed a 77% decrease in maximum velocity in the blind loop and no change in Km. Second, the chloride-induced, bumetanide-reversible increase in short circuit current in tissues stimulated simultaneously by prostaglandin E1 and theophylline, representing rheogenic chloride secretion, also showed a decrease in maximum velocity (of 83%) and no change in Km. A morphometric analysis revealed that the crypt surface area increased by 100% in the blind loop, whereas the villous surface area was not significantly different between blind loops and controls. We conclude that the jejunal self-filling blind loop is characterized by impaired active ion transport processes and an increase in epithelial and subepithelial resistance.

  17. Lipoxin A4 stimulates calcium-activated chloride currents and increases airway surface liquid height in normal and cystic fibrosis airway epithelia.

    LENUS (Irish Health Repository)


    Cystic Fibrosis (CF) is a genetic disease characterised by a deficit in epithelial Cl(-) secretion which in the lung leads to airway dehydration and a reduced Airway Surface Liquid (ASL) height. The endogenous lipoxin LXA(4) is a member of the newly identified eicosanoids playing a key role in ending the inflammatory process. Levels of LXA(4) are reported to be decreased in the airways of patients with CF. We have previously shown that in normal human bronchial epithelial cells, LXA(4) produced a rapid and transient increase in intracellular Ca(2+). We have investigated, the effect of LXA(4) on Cl(-) secretion and the functional consequences on ASL generation in bronchial epithelial cells obtained from CF and non-CF patient biopsies and in bronchial epithelial cell lines. We found that LXA(4) stimulated a rapid intracellular Ca(2+) increase in all of the different CF bronchial epithelial cells tested. In non-CF and CF bronchial epithelia, LXA(4) stimulated whole-cell Cl(-) currents which were inhibited by NPPB (calcium-activated Cl(-) channel inhibitor), BAPTA-AM (chelator of intracellular Ca(2+)) but not by CFTRinh-172 (CFTR inhibitor). We found, using confocal imaging, that LXA(4) increased the ASL height in non-CF and in CF airway bronchial epithelia. The LXA(4) effect on ASL height was sensitive to bumetanide, an inhibitor of transepithelial Cl(-) secretion. The LXA(4) stimulation of intracellular Ca(2+), whole-cell Cl(-) currents, conductances and ASL height were inhibited by Boc-2, a specific antagonist of the ALX\\/FPR2 receptor. Our results provide, for the first time, evidence for a novel role of LXA(4) in the stimulation of intracellular Ca(2+) signalling leading to Ca(2+)-activated Cl(-) secretion and enhanced ASL height in non-CF and CF bronchial epithelia.

  18. In situ measurement of airway surface liquid [K+] using a ratioable K+-sensitive fluorescent dye. (United States)

    Namkung, Wan; Song, Yuanlin; Mills, Aaron D; Padmawar, Prashant; Finkbeiner, Walter E; Verkman, A S


    The airway surface liquid (ASL) is the thin fluid layer lining airway surface epithelial cells, whose volume and composition are tightly regulated and may be abnormal in cystic fibrosis (CF). We synthesized a two-color fluorescent dextran to measure ASL [K(+)], TAC-Lime-dextran-TMR, consisting of a green-fluorescing triazacryptand K(+) ionophore-Bodipy conjugate, coupled to dextran, together with a red fluorescing tetramethylrhodamine reference chromophore. TAC-Lime-dextran-TMR fluorescence was K(+)-selective, increasing >4-fold with increasing [K(+)] from 0 to 40 mm. In well differentiated human airway epithelial cells, ASL [K(+)] was 20.8 +/- 0.3 mm and decreased by inhibition of the Na(+)/K(+) pump (ouabain), ENaC (amiloride), CF transmembrane conductance regulator (CFTR(inh)-172), or K(+) channels (TEA or XE991). ASL [K(+)] was increased by forskolin but not affected by Na(+)/K(+)/2Cl(-) cotransporter inhibition (bumetanide). Functional and expression studies indicated the involvement of [K(+)] channels KCNQ1, KCNQ3, and KCNQ5 as determinants of ASL [K(+)]. [K(+)] in CF cultures was similar to that in non-CF cultures, suggesting that abnormal ASL [K(+)] is not a factor in CF lung disease. In intact airways, ASL [K(+)] was also well above extracellular [K(+)]: 22 +/- 1 mm in pig trachea ex vivo and 16 +/- 1 mm in mouse trachea in vivo. Our results provide the first noninvasive measurements of [K(+)] in the ASL and indicate the involvement of apical and basolateral membrane ion transporters in maintaining a high ASL [K(+)].

  19. Cellular mechanisms underlying the laxative effect of flavonol naringenin on rat constipation model.

    Directory of Open Access Journals (Sweden)

    Zi-Huan Yang

    Full Text Available BACKGROUND & AIMS: Symptoms of constipation are extremely common, especially in the elderly. The present study aim to identify an efficacious treatment strategy for constipation by evaluating the secretion-promoting and laxative effect of a herbal compound, naringenin, on intestinal epithelial anion secretion and a rat constipation model, respectively. METHODS/PRINCIPAL FINDINGS: In isolated rat colonic crypts, mucosal addition of naringenin (100 microM elicited a concentration-dependent and sustained increase in the short-circuit current (I(SC, which could be inhibited in Cl- free solution or by bumetanide and DPC (diphenylamine-2-carboxylic acid, but not by DIDS (4, 4'- diisothiocyanatostilbene-2, 2'-disulfonic acid. Naringenin could increase intracellular cAMP content and PKA activity, consisted with that MDL-12330A (N-(Cis-2-phenyl-cyclopentyl azacyclotridecan-2-imine-hydrochloride pretreatment reduced the naringenin-induced I(SC. In addition, significant inhibition of the naringenin-induced I(SC by quinidine indicated that basolateral K+ channels were involved in maintaining this cAMP-dependent Cl- secretion. Naringenin-evoked whole cell current which exhibited a linear I-V relationship and time-and voltage- independent characteristics was inhibited by DPC, indicating that the cAMP activated Cl- conductance most likely CFTR (cystic fibrosis transmembrane conductance regulator was involved. In rat constipation model, administration of naringenin restored the level of fecal output, water content and mucus secretion compared to loperamide-administrated group. CONCLUSIONS: Taken together, our data suggest that naringenin could stimulate Cl- secretion in colonic epithelium via a signaling pathway involving cAMP and PKA, hence provide an osmotic force for subsequent colonic fluid secretion by which the laxative effect observed in the rat constipation model. Naringenin appears to be a novel alternative treatment strategy for constipation.

  20. Activity-dependent endogenous taurine release facilitates excitatory neurotransmission in the neocortical marginal zone of neonatal rats

    Directory of Open Access Journals (Sweden)

    Taizhe eQian


    Full Text Available In the developing cerebral cortex, the marginal zone (MZ, consisting of early-generated neurons such as Cajal-Retzius cells, plays an important role in cell migration and lamination. There is accumulating evidence of widespread excitatory neurotransmission mediated by γ-aminobutyric acid (GABA in the MZ. Cajal-Retzius cells express not only GABAA receptors but also α2/β subunits of glycine receptors, and exhibit glycine receptor-mediated depolarization due to high [Cl−]i. However, the physiological roles of glycine receptors and their endogenous agonists during neurotransmission in the MZ are yet to be elucidated. To address this question, we performed optical imaging from the MZ using the voltage-sensitive dye JPW1114 on tangential neocortical slices of neonatal rats. A single electrical stimulus evoked an action-potential-dependent optical signal that spread radially over the MZ. The amplitude of the signal was not affected by glutamate receptor blockers, but was suppressed by either GABAA or glycine receptor antagonists. Combined application of both antagonists nearly abolished the signal. Inhibition of Na+, K+-2Cl− cotransporter by 20 µM bumetanide reduced the signal, indicating that this transporter contributes to excitation. Analysis of the interstitial fluid obtained by microdialysis from tangential neocortical slices with high-performance liquid chromatography revealed that GABA and taurine, but not glycine or glutamate, were released in the MZ in response to the electrical stimulation. The ambient release of taurine was reduced by the addition of a voltage-sensitive Na+ channel blocker. Immunohistochemistry and immunoelectron microscopy indicated that taurine was stored both in Cajal-Retzius and non-Cajal-Retzius cells in the MZ, but was not localized in presynaptic structures. Our results suggest that activity-dependent non-synaptic release of endogenous taurine facilitates excitatory neurotransmission through activation of

  1. Prostaglandin E2-induced colonic secretion in patients with and without colorectal neoplasia

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    Poulsen Steen S


    Full Text Available Abstract Background The pathogenesis for colorectal cancer remains unresolved. A growing body of evidence suggests a direct correlation between cyclooxygenase enzyme expression, prostaglandin E2 metabolism and neoplastic development. Thus further understanding of the regulation of epithelial functions by prostaglandin E2 is needed. We hypothesized that patients with colonic neoplasia have altered colonic epithelial ion transport and express functionally different prostanoid receptor levels in this respect. Methods Patients referred for colonoscopy were included and grouped into patients with and without colorectal neoplasia. Patients without endoscopic findings of neoplasia served as controls. Biopsy specimens were obtained from normally appearing mucosa in the sigmoid part of colon. Biopsies were mounted in miniaturized modified Ussing air-suction chambers. Indomethacin (10 μM, various stimulators and inhibitors of prostanoid receptors and ion transport were subsequently added to the chamber solutions. Electrogenic ion transport parameters (short circuit current and slope conductance were recorded. Tissue pathology and tissue damage before and after experiments was assessed by histology. Results Baseline short circuit current and slope conductance did not differ between the two groups. Patients with neoplasia were significantly more sensitive to indomethacin with a decrease in short circuit current of 15.1 ± 2.6 μA·cm-2 compared to controls, who showed a decrease of 10.5 ± 2.1 μA·cm-2 (p = 0.027. Stimulation or inhibition with theophylline, ouabain, bumetanide, forskolin or the EP receptor agonists prostaglandin E2, butaprost, sulprostone and prostaglandin E1 (OH did not differ significantly between the two groups. Histology was with normal findings in both groups. Conclusions Epithelial electrogenic transport is more sensitive to indomethacin in normal colonic mucosa from patients with previous or present colorectal neoplasia compared

  2. Colonic epithelial ion transport is not affected in patients with diverticulosis

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    Tilotta Maria C


    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.

  3. Chronic noradrenaline increases renal expression of NHE-3, NBC-1, BSC-1 and aquaporin-2. (United States)

    Sonalker, Prajakta A; Tofovic, Stevan P; Bastacky, Sheldon I; Jackson, Edwin K


    1. Because chronic activation of the renal sympathetic nervous system promotes sodium and water retention, it is conceivable that long-term exposure of the kidney to the sympathetic neurotransmitter noradrenaline upregulates the expression of key renal epithelial transport systems. 2. To test this hypothesis, we used immunoblotting of renal cortical and medullary tissue to investigate the abundance of major transport systems expressed along the renal tubule in response to long-term (15 days) infusions of noradrenaline (600 ng/min) in rats. 3. Mean arterial blood pressure and heart rate were significantly elevated in rats receiving chronic infusions of noradrenaline (128 +/- 10 mmHg and 492 +/- 16 b.p.m., respectively) compared with animals treated with saline only (89 +/- 3 mmHg and 376 +/- 14 b.p.m., respectively). 4. Chronic infusions of noradrenaline also increased the protein abundance of the cortical Na(+)/H(+) exchanger isoform 3 (NHE-3; 2.5-fold; P = 0.0142), the cortical sodium-bicarbonate cotransporter NBC-1 (2.5-fold; P = 0.0067), the bumetanide-sensitive sodium-potassium-chloride cotransporter BSC-1/NKCC2 in the inner stripe of outer medulla (threefold; P = 0.0020) and aquaporin-2 in the inner medulla (twofold; P = 0.0039). 5. In contrast, noradrenaline did not significantly affect expression of the thiazide-sensitive Na(+)-Cl(-) cotransporter in the cortex, Na(+)/K(+)-ATPase-alpha(1) in the cortex and inner stripe of the outer or inner medulla, the inwardly rectifying K(+) channel (ROMK-1) in the inner stripe of the outer medulla or aquaporin-1 in the cortex or inner medulla. Noradrenaline did significantly, but modestly (less than twofold), increase aquaporin-1 in the inner stripe of the outer medulla. 6. We conclude that noradrenaline-induced increases in the expression of NHE-3, NBC-1, BSC-1 and aquaporin-2 are likely to play an important role in the regulation of salt and water transport by noradrenaline in the kidney and may explain, at least in

  4. Volume regulation in mammalian skeletal muscle: the role of sodium-potassium-chloride cotransporters during exposure to hypertonic solutions. (United States)

    Lindinger, Michael I; Leung, Matthew; Trajcevski, Karin E; Hawke, Thomas J


    Controversy exists as to whether mammalian skeletal muscle is capable of volume regulation in response to changes in extracellular osmolarity despite evidence that muscle fibres have the required ion transport mechanisms to transport solute and water in situ. We addressed this issue by studying the ability of skeletal muscle to regulate volume during periods of induced hyperosmotic stress using single, mouse extensor digitorum longus (EDL) muscle fibres and intact muscle (soleus and EDL). Fibres and intact muscles were loaded with the fluorophore, calcein, and the change in muscle fluorescence and width (single fibres only) used as a metric of volume change. We hypothesized that skeletal muscle exposed to increased extracellular osmolarity would elicit initial cellular shrinkage followed by a regulatory volume increase (RVI) with the RVI dependent on the sodium–potassium–chloride cotransporter (NKCC). We found that single fibres exposed to a 35% increase in extracellular osmolarity demonstrated a rapid, initial 27–32% decrease in cell volume followed by a RVI which took 10-20 min and returned cell volume to 90–110% of pre-stimulus values. Within intact muscle, exposure to increased extracellular osmolarity of varying degrees also induced a rapid, initial shrinkage followed by a gradual RVI, with a greater rate of initial cell shrinkage and a longer time for RVI to occur with increasing extracellular tonicities. Furthermore, RVI was significantly faster in slow-twitch soleus than fast-twitch EDL. Pre-treatment of muscle with bumetanide (NKCC inhibitor) or ouabain (Na+,K+-ATPase inhibitor), increased the initial volume loss and impaired the RVI response to increased extracellular osmolarity indicating that the NKCC is a primary contributor to volume regulation in skeletal muscle. It is concluded that mouse skeletal muscle initially loses volume then exhibits a RVI when exposed to increases in extracellular osmolarity. The rate of RVI is dependent on the

  5. Factors affecting ammonium uptake by C11 clone of MDCK cells. (United States)

    Tararthuch, A L; Fernandez, R; Ramirez, M A; Malnic, G


    In several tissues ammonium ions are able to use the transport pathways of other ions, particularly of K+. We investigated this possibility in the C11 clone of MDCK cells, thought to represent intercalated cells, in control and 0 Cl- conditions. Cell pH was measured by ratiometric fluorescence microscopy using the pH indicator BCECF. After preincubating the cells for 10 min in control or 0 Cl- (substituted by gluconate) Ringer, an ammonium pulse was applied to induce cell acidification. The magnitude of the initial alkalinization (DeltapH) was 0.24+/-0.03 ( n=28) pH units in controls, which fell to 0.023+/-0.01 ( n=12) in 0 Cl-, suggesting uptake of NH4+ balancing the alkalinization by NH3. Addition of 10(-3) M bumetanide or furosemide to the 0 Cl- medium, or 10(-4 )M hexamethylene amiloride, did not alter DeltapH. However, with 5 mM Ba+, DeltapH increased to 38% of control. When 2.5x10(-4) M ouabain, an inhibitor of Na+-K+ ATPase, was used, DeltapH increased to 46% of control. Inhibition of H+-K+ ATPase by SCH28080 or by omeprazol caused significant increase in DeltapH. In 0 Cl- solution, these cells underwent a mean volume reduction (-d V) of -10.24+/-1.96% per 10 min as measured by confocal microscopy. To investigate if NH4+ influx was regulated by cell volume or by cell Cl-, volume reduction was avoided by two procedures. When preincubating with NPPB, a Cl- channel blocker, in 0 Cl-, volume reduction was inhibited (d V=-2.12% per 10 min), and DeltapH was 0.24+/-0.04 ( n=5). When the cells were preincubated in hypotonic 0 Cl- (260 mosmol/l), cell volume reduction was abolished (d V=+2.6% per 10 min) and DeltapH was 0.52+/-0.07 ( n=7). Thus, activation of NH4+ influx by several transporters was due to volume reduction rather than to [Cl-] alteration.

  6. Effect of genistein on basal jejunal chloride secretion in R117H CF mice is sex and route specific

    Directory of Open Access Journals (Sweden)

    Rayyan E


    Full Text Available Esa Rayyan,1 Sarah Polito,1 Lana Leung,1 Ashesh Bhakta,1 Jonathan Kang,1 Justin Willey,1 Wasim Mansour,1 Mitchell L Drumm,2 Layla Al-Nakkash11Department of Physiology, Arizona College of Osteopathic Medicine, Midwestern University, Glendale, AZ, USA; 2Pediatric Pulmonology Division, Case Western Reserve University, Cleveland, OH, USAAbstract: Cystic fibrosis (CF results from the loss or reduction in function of the CFTR (cystic fibrosis transmembrane conductance regulatory protein chloride channel. The third most common CFTR mutation seen clinically is R117H. Genistein, a naturally occurring phytoestrogen, is known to stimulate CFTR function in vitro. We aimed to determine whether route of administration of genistein could mediate differential effects in R117H male and female CF mice. Mice were fed (4 weeks or injected subcutaneously (1 week with the following: genistein 600 mg/kg diet (600Gd; genistein-free diet (0Gd; genistein injection 600 mg/kg body weight (600Gi; dimethyl sulfoxide control (0Gi. In male R117H mice fed 600Gd, basal short circuit current (Isc was unchanged. In 600Gd-fed female mice, there was a subgroup that demonstrated a significant increase in basal Isc (53.14±7.92 µA/cm2, n=6, P<0.05 and a subgroup of nonresponders (12.05±6.59 µA/cm2, n=4, compared to 0Gd controls (29.3±6.5 µA/cm2, n=7. In R117H mice injected with 600Gi, basal Isc was unchanged in both male and female mice compared to 0Gi controls. Isc was measured in response to the following: the adenylate cyclase activator forskolin (10 µM, bilateral, bumetanide (100 µM, basolateral to indicate the Cl- secretory component, and acetazolamide (100 µM, bilateral to indicate the HCO3- secretory component; however, there was no effect of genistein (diet or injection on any of these parameters. Jejunal morphology (ie, villi length, number of goblet cells per villus, crypt depth, and number of goblet cells per crypt in R117H mice suggested no genistein

  7. Research advances in the management of autism spectrum disorders in children%儿童孤独症谱系障碍的治疗研究进展

    Institute of Scientific and Technical Information of China (English)

    李洪华; 单玲; 杜琳; 贾飞勇


    孤独症谱系障碍(ASD)为一类广泛性神经系统发育障碍,以社会交往及交流障碍、兴趣狭窄、刻板与重复行为为主要特点。目前ASD的发病率呈显著上升趋势,早期合理的个性化综合干预治疗可明显改善患儿预后。由于ASD的病因不明,目前尚无特效药,主要以行为与教育干预为主;对ASD的伴发症状,如易激惹、自伤行为、注意缺陷多动障碍、睡眠问题等,合理应用一些药物,可改善ASD患儿的行为干预效果。随着ASD发病机制的深入研究,布美他尼、催产素、维生素D及高压氧治疗,可有望改善ASD核心症状。该文对目前针对ASD的行为与发展干预及治疗方法进行了综述。%Autism spectrum disorders (ASD) are a group of developmental dysfuntion of nervous system characterized by social interaction and communication disorders, restricted interests and repetitive stereotyped behaviors. The incidence of ASD has been increasing through the world. Some studies have shown that early reasonable individualized comprehensive intervention can obviously improve the prognosis of children with ASD. The etiology of ASD is unclear now, and behavioral and developmental intervention is the main therapy for ASD. The reasonable application of some drugs can improve the efifcacy of the behavioral intervention for concomitant symptoms in ASD. With the in-depth study of the pathogenesis of ASD, bumetanide, oxytocin, vitamin D and hyperbaric oxygen therapy have been found to be promising for the improvement of core symptoms of ASD. This article reviews the research advances in the behavioral and developmental intervention and drug therapy for ASD.

  8. Inhibition by mercuric chloride of Na-K-2Cl cotransport activity in rectal gland plasma membrane vesicles isolated from Squalus acanthias. (United States)

    Kinne-Saffran, E; Kinne, R K


    The rectal gland of the dogfish shark is a model system for active transepithelial transport of chloride. It has been shown previously that mercuric chloride, one of the toxic environmental pollutants, inhibits chloride secretion in this organ. In order to investigate the mechanism of action of HgCl(2) at a membrane-molecular level, plasma membrane vesicles were isolated from the rectal gland and the effect of mercury on the activity of the Na-K-2Cl cotransporter was investigated in isotope flux studies. During a 30 s exposure HgCl(2) inhibited cotransport activity in a dose-dependent manner with an apparent K(i) of approx. 50 microM. The inhibition was complete after 15 s, partly reversible by dilution of the incubation medium and completely attenuated upon addition of reduced glutathione. The extent of inhibition by mercury depended on the ionic composition of the medium. The sensitivity of the cotransporter was highest when only the high affinity binding sites for sodium and chloride were saturated. Organic mercurials such as p-chloromercuribenzoic acid and p-chloromercuriphenylsulfonic acid at 100 microM did not inhibit the cotransporter, similarly exposure of the vesicles to 10 mM H(2)O(2) or 1 mM dithiothreitol for 30 min at 15 degrees C did not change cotransport activity. Transport activity was, however, reduced by 45.9+/-2.5% after an incubation with 3 mM N-ethylmaleimide for 20 min. Blocking free amino groups by N-hydroxysuccinimide or biotinamidocapronate-N-hydroxysulfosuccinimide had no effect. Investigations on the sidedness of the plasma membrane vesicles, employing the asymmetry of the (Na+K)-ATPase, demonstrated a right-side-out orientation in which the former extracellular face of the membrane is exposed to the incubation medium. In addition, extracellular mercury (5x10(-5) M) inhibited bumetanide-sensitive rubidium uptake into T84 cells by 48.5+/-7.1% after a 2 min incubation period. This inhibition was reversible in a manner similar to that

  9. Multicenter intestinal current measurements in rectal biopsies from CF and non-CF subjects to monitor CFTR function.

    Directory of Open Access Journals (Sweden)

    John P Clancy

    Full Text Available Intestinal current measurements (ICM from rectal biopsies are a sensitive means to detect cystic fibrosis transmembrane conductance regulator (CFTR function, but have not been optimized for multicenter use. We piloted multicenter standard operating procedures (SOPs to detect CFTR activity by ICM and examined key questions for use in clinical trials. SOPs for ICM using human rectal biopsies were developed across three centers and used to characterize ion transport from non-CF and CF subjects (two severe CFTR mutations. All data were centrally evaluated by a blinded interpreter. SOPs were then used across four centers to examine the effect of cold storage on CFTR currents and compare CFTR currents in biopsies from one subject studied simultaneously either at two sites (24 hours post-biopsy or when biopsies were obtained by either forceps or suction. Rectal biopsies from 44 non-CF and 17 CF subjects were analyzed. Mean differences (µA/cm(2; 95% confidence intervals between CF and non-CF were forskolin/IBMX=102.6(128.0 to 81.1, carbachol=96.3(118.7 to 73.9, forskolin/IBMX+carbachol=200.9(243.1 to 158.6, and bumetanide=-44.6 (-33.7 to -55.6 (P<0.005, CF vs non-CF for all parameters. Receiver Operating Characteristic curves indicated that each parameter discriminated CF from non-CF subjects (area under the curve of 0.94-0.98. CFTR dependent currents following 18-24 hours of cold storage for forskolin/IBMX, carbachol, and forskolin/IBMX+carbachol stimulation (n=17 non-CF subjects were 44%, 47.5%, and 47.3%, respectively of those in fresh biopsies. CFTR-dependent currents from biopsies studied after cold storage at two sites simultaneously demonstrated moderate correlation (n=14 non-CF subjects, Pearson correlation coefficients 0.389, 0.484, and 0.533. Similar CFTR dependent currents were detected from fresh biopsies obtained by either forceps or suction (within-subject comparisons, n=22 biopsies from three non-CF subjects. Multicenter ICM is a

  10. Neuronal chloride accumulation and excitatory GABA underlie aggravation of neonatal epileptiform activities by phenobarbital. (United States)

    Nardou, Romain; Yamamoto, Sumii; Chazal, Geneviève; Bhar, Asma; Ferrand, Nadine; Dulac, Olivier; Ben-Ari, Yehezkel; Khalilov, Ilgam


    Phenobarbital produces its anti-epileptic actions by increasing the inhibitory drive of γ-aminobutyric acid. However, following recurrent seizures, γ-aminobutyric acid excites neurons because of a persistent increase of chloride raising the important issue of whether phenobarbital could aggravate persistent seizures. Here we compared the actions of phenobarbital on initial and established ictal-like events in an in vitro model of mirror focus. Using the in vitro three-compartment chamber preparation with the two hippocampi and their commissural fibres placed in three different chambers, kainate was applied to one hippocampus and phenobarbital contralaterally, either after one ictal-like event or after many recurrent ictal-like events that produce an epileptogenic mirror focus. Field, perforated patch and single-channel recordings were used to determine the effects of γ-aminobutyric acid and their modulation by phenobarbital, and alterations of the chloride cotransporters were investigated using sodium-potassium-chloride cotransporter 1 and potassium chloride cotransporter 2 antagonists, potassium chloride cotransporter 2 immunocytochemistry and sodium-potassium-chloride cotransporter 1 knockouts. Phenobarbital reduced initial ictal-like events and prevented the formation of a mirror focus when applied from the start. In contrast, phenobarbital aggravated epileptiform activities when applied after many ictal-like events by enhancing the excitatory actions of γ-aminobutyric acid due to increased chloride. The accumulation of chloride and the excitatory actions of γ-aminobutyric acid in mirror foci neurons are mediated by the sodium-potassium-chloride cotransporter 1 chloride importer and by downregulation and internalization of the chloride-exporter potassium-chloride cotransporter 2. Finally, concomitant applications of the sodium-potassium-chloride cotransporter 1 antagonist bumetanide and phenobarbital decreased excitatory actions of γ-aminobutyric acid and

  11. Simultaneous Determination of 20 Anti-Obesity Drugs Illegally Added in Weight-loss Functional Foods by Matrix Solid Phase Dispersion-High Performance Liquid Chromatography Tandem Mass Spectrometry%基质分散固相萃取-高效液相色谱-串联质谱法同时测定减肥保健食品中20种违禁添加药物

    Institute of Scientific and Technical Information of China (English)

    马微; 程丽; 张兰威; 张英春; 王海波; 焦月华; 代汉慧; 唐英章


    建立了测定减肥保健食品中20种违禁添加药物(芬氟拉明、苯丙醇胺、西布曲明、舍曲林、利莫那班、安非他酮、西酞普兰、氟西汀、苯氟雷司、托吡酯、唑尼沙胺、咖啡因、酚酞、大黄素、吲达帕胺、布美他尼、托拉塞米、三氨喋啶、奥利司他、苯乙双胍)的基质分散固相萃取-高效液相色谱-电喷雾串联质谱分析方法。不同类型的样品经乙醇-丙酮(7:3, V/V)超声提取后,提取液经N-丙基乙二胺( PSA)、十八烷基硅烷( ODS)净化,以Waters Atlantis T3柱(150 mm ×2.1 mm,3μm)分离,采用HPLC/MS/MS电喷雾电离,多反应监测模式检测,以保留时间和子离子比进行定性分析,外标法进行定量分析。20种减肥药物的检出限为0.05~3.0 mg/kg,在不同添加水平范围内的回收率为67.1%~101.4%,日内精密度均小于10%,日间精密度均小于15%。本方法分析速度快,适用于减肥保健食品的实际检验工作。%An analytical method based on high performance liquid chromatography tandem mass spectrometry has been developed for the simultaneous determination of 20 anti-obesity drugs ( fenfluramine, phenylpropanolamine, sibutramine, sertraline, rimonabant, bupropion, citalopram, fluoxetine, benfluorex, topiramate, zonisamide, caffeine, phenolphthalein, emodin, indapamide, bumetanide, torasemide, triamterene, orlistat, phenformin). that were extracted from various weight-loss functional foods by ethanol-acetone(7:3, V/V)and purified by primary secondary amine (PSA) and octadecyltrimethoxysilane(ODS) under ultrasonication. The analysis was carried out on HPLC-MS /MS by electrospray ionization using multiple reaction monitoring after the chromatographic separation on Waters Atlantis T3 (3 μm, 150 mm × 2. 1 mm) column. Identification was achieved by the retention time and the ion ratio, quantification was done by the external standard method. The limits of detection for the appetite suppressants were 0. 05


    Oh, Vernon Min Sen


    reduction in the daily dietary salt intake between the age group 40-49 years (9000 mg) and those aged 60-69 years (7700 mg). The Indian and Chinese respondents in the Survey consumed more salt, 8700 and 8500 mg/day respectively, than the Malays (7600 mg/day).A study led by Associate Professor Rob Martinus Van Dam in the School of Public Health, National University of Singapore, is addressing the validation and calibration of a technique to estimate the 24-hour urinary content of sodium from measurements of sodium concentration in a spot urine sample. The technique is intended to raise the accuracy of spot urine sampling.It is difficult to interpret the significance of the decreased daily intake of salt in older persons, aged 50 to 69, in the face of the greater prevalence of hypertension in these age bands (31.9% in those aged 50-59 years, and 53.4% in those aged 60-69). For instance, some older persons might ingest less dietary sodium, or the net renal tubular excretion of sodium in the urine might decrease between age 50 and 69 years without any change in dietary sodium intake.Moreover, the treatment of hypertension in persons aged 50 to 69 years is likely to include diuretic agents, both thiazide drugs, and loop diuretics such as furosemide and bumetanide. In most hypertensive patients, long-term treatment with diuretics exposes older persons to a risk of hyponatraemia, hypokalemia, or both. The latter conditions result from a net increase in the urinary excretion of sodium and potassium (apart from increased free water excretion). Such diuretic-related loss of sodium in the urine also needs to be factored into the estimation of dietary sodium intake by means of 24-hour urinary sodium output.The increased prescription of renin-angiotensin-aldosterone blocking drugs to control HTn further confounds the estimation of dietary sodium intake from 24-hour urine collections. Angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers, both reduce the