Changes of muscarinic cholinergic receptors during aging process of primary cultured neutrons

International Nuclear Information System (INIS)

Fan Guohuang; Yi Ningyu; Xia Zongqin

1996-01-01

The dynamic changes of muscarinic receptor density and its reactivity during aging process in primary cultured neutrons were studied. Muscarinic receptor density was measured by 3 H-QNB binding assay, and muscarinic receptor reactivity was assessed by carbachol stimulation of cGMP formation, the latter was measured by RIA. After 2 weeks' incubation of neonatal rat brain cells, the nutrients began to rupture and the cell bodies shrank markedly showing senescent feature. The muscarinic receptor density reached peak at the 12th day in vitro (12 DIV), but the muscarinic receptor reactivity reached peak at 9 DIV and declined significantly at 12 DIV. The results demonstrated that during aging process of primary cultured neutrons, the decline of muscarinic receptor reactivity is likely prior to the decrease of receptor density

Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice.

Science.gov (United States)

Joseph, Lauren; Thomsen, Morgane

2017-06-30

Muscarinic M 1 /M 4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (S D ) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M 1 , M 2 , or M 4 receptors (M 1 -/- , M 2 -/- , M 4 -/- ), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M 1 , M 2 , or M 4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M 1 /M 4 receptors partially substituted for cocaine and increased the S D effect of cocaine, while M 2 -preferring antagonists did not substitute, and reduced the S D effect of cocaine. The cocaine-like effects of scopolamine were absent in M 1 -/- mice. The cocaine S D attenuating effects of methoctramine were absent in M 2 -/- mice and almost absent in M 1 -/- mice. The findings indicate that the cocaine-like S D effects of muscarinic antagonists are primarily mediated through M 1 receptors, with a minor contribution of M 4 receptors. The data also support our previous findings that stimulation of M 1 receptors and M 4 receptors can each attenuate the S D effect of cocaine, and show that this can also be achieved by blocking M 2 autoreceptors, likely via increased acetylcholine release. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of muscarinic receptor subtypes in human tissues

International Nuclear Information System (INIS)

Giraldo, E.; Martos, F.; Gomez, A.; Garcia, A.; Vigano, M.A.; Ladinsky, H.; Sanchez de La Cuesta, F.

1988-01-01

The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with [ 3 H]Pirenzepine and [ 3 H]N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M 1 neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M 1 , the cardiac M 2 and the glandular M 3

Characterization of muscarinic receptor subtypes in primary cultures of cerebellar granule cells using specific muscarinic receptor antagonists

International Nuclear Information System (INIS)

McLeskey, S.W.

1989-01-01

In cerebellar granule cell cultures, two muscarinic receptor mediated responses were observed: inhibition of adenylate cyclase (M-AC) and stimulation of phosphoinositide hydrolysis (M-PI). These responses were antagonized by three purported specific muscarinic antagonists: pirenzipine and (-)QNX (specific for M-PI) and methoctramine (specific for M-AC). However, the specificity for the three antagonists in blocking these responses is not comparable to the specificity observed in binding studies on these cells or to that quoted in the literature. Two peaks of molecular sizes were found in these cells corresponding to the two molecular sizes of muscarinic receptive proteins reported in the literature. Muscarinic receptive proteins were alkylated with 3 H-propylbenzilylcholine mustard followed by sodium dodecylsulfate polyacrylamide gel electrophoresis. Pirenzipine and (-)QNX were able to block alkylation of the high molecular size peak, which corresponds to the receptive protein m 3 reported in the literature. Methoctramine was able to block alkylation of a portion of the lower molecular size peak, possibly corresponding to the m 2 and/or m 4 receptive proteins reported in the literature. Studies attempting to show the presence of receptor reserve for either of the two biochemical responses present in these cells by alkylation of the receptive protein with nonradiolabeled propylbenzilylcholine mustard (PBCM) were confounded by specificity of this agent for the lower molecular weight peak of muscarinic receptive protein. Thus the muscarinic receptive proteins coupled to M-AC were alkylated preferentially over the ones coupled to M-PI

Effect of aging on airway remodeling and muscarinic receptors in a murine acute asthma model

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Kang JY

2013-10-01

Full Text Available Ji Young Kang, Sook Young Lee, Chin Kook Rhee, Seung Joon Kim, Soon Seog Kwon, Young Kyoon KimDepartment of Internal Medicine, College of Medicine, Catholic University of Korea, Seoul, KoreaBackground and objectives: The influence of aging on the development of asthma has not been studied thoroughly. The aim of this study was to investigate age-related airway responses involving lung histology and expression of muscarinic receptors in a murine model of acute asthma. Methods: Female BALB/c mice at the ages of 6 weeks and 6, 9, and 12 months were sensitized and challenged with ovalbumin (OVA for 1 month (n = 8–12 per group. We analyzed inflammatory cells and T-helper (Th2 cytokines in bronchoalveolar lavage (BAL fluid and parameters of airway remodeling and expression of muscarinic receptors in lung tissue. Results: Among the OVA groups, total cell and eosinophil numbers in BAL fluid were significantly higher in the older (6-, 9-, and 12-month-old mice than in the young (6-week-old mice. Interleukin (IL 4 (IL-4 concentration increased, but IL-5 and IL-13 concentrations showed a decreased tendency, with age. IL-17 concentration tended to increase with age, which did not reach statistical significance. periodic acid-Schiff (PAS staining area, peribronchial collagen deposition, and area of α-smooth muscle staining were significantly higher in the 6-month older OVA group than in the young OVA group. The expression of the M3 and M2 muscarinic receptors tended to increase and decrease, respectively, with age. Conclusion: The aged mice showed an active and unique pattern not only on airway inflammation, but also on airway remodeling and expression of the muscarinic receptors during the development of acute asthma compared with the young mice. These findings suggest that the aging process affects the pathogenesis of acute asthma and age-specific approach might be more appropriate for better asthma control in a clinical practice.Keywords: aging, asthma

Convulsant bicuculline modifies CNS muscarinic receptor affinity

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Rodríguez de Lores Arnaiz Georgina

2006-04-01

Full Text Available Abstract Background Previous work from this laboratory has shown that the administration of the convulsant drug 3-mercaptopropionic acid (MP, a GAD inhibitor, modifies not only GABA synthesis but also binding of the antagonist [3H]-quinuclidinyl benzilate ([3H]-QNB to central muscarinic receptors, an effect due to an increase in affinity without modifications in binding site number. The cholinergic system has been implicated in several experimental epilepsy models and the ability of acetylcholine to regulate neuronal excitability in the neocortex is well known. To study the potential relationship between GABAergic and cholinergic systems with seizure activity, we analyzed the muscarinic receptor after inducing seizure by bicuculline (BIC, known to antagonize the GABA-A postsynaptic receptor subtype. Results We analyzed binding of muscarinic antagonist [3H]-QNB to rat CNS membranes after i.p. administration of BIC at subconvulsant (1.0 mg/kg and convulsant (7.5 mg/kg doses. Subconvulsant BIC dose failed to develop seizures but produced binding alteration in the cerebellum and hippocampus with roughly 40% increase and 10% decrease, respectively. After convulsant BIC dose, which invariably led to generalized tonic-clonic seizures, binding increased 36% and 15% to cerebellar and striatal membranes respectively, but decreased 12% to hippocampal membranes. Kd value was accordingly modified: with the subconvulsant dose it decreased 27% in cerebellum whereas it increased 61% in hippocampus; with the convulsant dose, Kd value decreased 33% in cerebellum but increased 85% in hippocampus. No change in receptor number site was found, and Hill number was invariably close to unity. Conclusion Results indicate dissimilar central nervous system area susceptibility of muscarinic receptor to BIC. Ligand binding was modified not only by a convulsant BIC dose but also by a subconvulsant dose, indicating that changes are not attributable to the seizure process

M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

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Zhao, Lan-Xue; Ge, Yan-Hui; Xiong, Cai-Hong; Tang, Ling; Yan, Ying-Hui; Law, Ping-Yee; Qiu, Yu; Chen, Hong-Zhuan

2018-03-06

M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

Muscarinic M4 Receptors on Cholinergic and Dopamine D1 Receptor-Expressing Neurons Have Opposing Functionality for Positive Reinforcement and Influence Impulsivity

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Anna M. Klawonn

2018-04-01

Full Text Available The neurotransmitter acetylcholine has been implicated in reward learning and drug addiction. However, the roles of the various cholinergic receptor subtypes on different neuron populations remain elusive. Here we study the function of muscarinic M4 receptors (M4Rs in dopamine D1 receptor (D1R expressing neurons and cholinergic neurons (expressing choline acetyltransferase; ChAT, during various reward-enforced behaviors and in a “waiting”-impulsivity test. We applied cell-type-specific gene deletions targeting M4Rs in D1RCre or ChATCre mice. Mice lacking M4Rs in D1R-neurons displayed greater cocaine seeking and drug-primed reinstatement than their littermate controls in a Pavlovian conditioned place preference (CPP paradigm. Furthermore, the M4R-D1RCre mice initiated significantly more premature responses (PRs in the 5-choice-serial-reaction-time-task (5CSRTT than their littermate controls, indicating impaired waiting impulse control. In contrast, mice lacking M4Rs in cholinergic neurons did not acquire cocaine Pavlovian conditioning. The M4R-ChATCre mice were also unable to learn positive reinforcement to either natural reward or cocaine in an operant runway paradigm. Immediate early gene (IEG expression (cFos and FosB induced by repeated cocaine injections was significantly increased in the forebrain of M4R-D1RCre mice, whereas it remained normal in the M4R-ChATCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality. Furthermore, we found that neurons expressing both M4Rs and D1Rs are important for signaling impulse control.

Muscarinic M4 Receptors on Cholinergic and Dopamine D1 Receptor-Expressing Neurons Have Opposing Functionality for Positive Reinforcement and Influence Impulsivity.

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Klawonn, Anna M; Wilhelms, Daniel B; Lindström, Sarah H; Singh, Anand Kumar; Jaarola, Maarit; Wess, Jürgen; Fritz, Michael; Engblom, David

2018-01-01

The neurotransmitter acetylcholine has been implicated in reward learning and drug addiction. However, the roles of the various cholinergic receptor subtypes on different neuron populations remain elusive. Here we study the function of muscarinic M4 receptors (M4Rs) in dopamine D1 receptor (D1R) expressing neurons and cholinergic neurons (expressing choline acetyltransferase; ChAT), during various reward-enforced behaviors and in a "waiting"-impulsivity test. We applied cell-type-specific gene deletions targeting M4Rs in D1RCre or ChATCre mice. Mice lacking M4Rs in D1R-neurons displayed greater cocaine seeking and drug-primed reinstatement than their littermate controls in a Pavlovian conditioned place preference (CPP) paradigm. Furthermore, the M4R-D1RCre mice initiated significantly more premature responses (PRs) in the 5-choice-serial-reaction-time-task (5CSRTT) than their littermate controls, indicating impaired waiting impulse control. In contrast, mice lacking M4Rs in cholinergic neurons did not acquire cocaine Pavlovian conditioning. The M4R-ChATCre mice were also unable to learn positive reinforcement to either natural reward or cocaine in an operant runway paradigm. Immediate early gene (IEG) expression ( cFos and FosB ) induced by repeated cocaine injections was significantly increased in the forebrain of M4R-D1RCre mice, whereas it remained normal in the M4R-ChATCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality. Furthermore, we found that neurons expressing both M4Rs and D1Rs are important for signaling impulse control.

Muscarinic acetylcholine receptor expression in aganglionic bowel.

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Oue, T; Yoneda, A; Shima, H; Puri, P

2000-01-01

In Hirschsprung's disease (HD) there exists an overabundance of acetylcholine (ACh), which in turn stimulates excessive production of the enzyme acetylcholinesterase. Muscarinic ACh receptors (mAChRs) play an important role in smooth-muscle contraction. Recent studies have indicated five different subtypes of mAChRs encoded by five different genes, ml to m5. The purpose of this study was to investigate the expression of each mAChR subtype in aganglionic (AG) colon to further understand the pathophysiology of HD. Entire colon resected at the time of pull-through operation for HD was obtained from 14 patients. Specimens obtained at autopsy from 8 age-matched patients without gastrointestinal disease acted as controls. Frozen sections were used for indirect immunohistochemistry as well as in-situ hybridization. Immunohistochemistry was performed using specific antiserum against each mAChR subtype and in-situ hybridization was performed using specific oligonucleotide probes against ml to m5 subtypes. Messenger RNA (mRNA) was extracted from normoganglionic (NG) and AG bowel of HD patients and normal control bowel. Reverse transcription-polymerase chain reaction was performed to evaluate mRNA levels of each mAChR subtype. To adjust the levels of mRNA expression, a housekeeping gene G3PDH, known to be expressed normally, was used as an internal control. Strong m2 and m3 immunoreactivity was observed in the mucosal layer, smooth-muscle layers, and myenteric plexus of NG bowel, whereas ml immunoreactivity was only detected in the mucosal layer. The most striking finding was the abundance of m3-immunoreactive fibers in muscle layers of NG bowel while there was a total lack of m3 fibers in smooth-muscle of AG bowel. Intense mRNA signals encoding m2 and m3 and to a lesser degree ml were detected in NG bowel, and these signals were weak in AG bowel. Immunoreactivity and mRNA expression of m4 and m5 was not detected in NG or AG bowel. The lack of m3-immunoreactive fibers in the

Biochemical and immunological studies of the Muscarinic acetylcholine receptor

International Nuclear Information System (INIS)

Gainer, M.W.

1985-01-01

Muscarinic acetylcholine receptors were solubilized from bovine brain membranes with 3[3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS). A combination of 10 mM CHAPS and 1 M NaCl solubilized 15-40% of the specific receptor binding sites from these membranes. The solubilized receptors displayed high affinity binding of the muscarinic antagonist, [ 3 H]quinuclidinyl benzilate with a K/sub D/ = 300 pM. In addition, the solubilized and retained guanyl nucleotide regulation of agonist binding characteristic of membrane bound receptors. Gel filtration experiments showed that solubilized receptors from cortex and cerebellum had different elution profiles. Analysis by sucrose density gradient centrifugation showed that receptors in the lower molecular weight peak sedimented with a coefficient of 5S. Receptors in the larger molecular weight peak sedimented to the bottom of the gradient. Attempts to purify receptors by chromatography on propylbenzilycholine Sepharose were unsuccessful. The technique used to attach the ligand to the solid support, however, was used to synthesize a PrBCM-BSA conjugate and the conjugate used as an antigen in the production of anti-ligand antibodies. Two anti-PrBCM monoclonal antibodies were isolated that recognize muscarinic but not nicotinic cholinergic ligands. The abilities of the antibodies to recognize other muscarinic ligands indicated the antibodies recognized a portion of PrBCM involved in binding to the receptor. Construction of an antibody affinity resin resulted in the purification of this fragment a minimum of 170 fold

Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

International Nuclear Information System (INIS)

Large, T.H.; Cho, N.J.; De Mello, F.G.; Klein, W.L.

1985-01-01

Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed

Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation.

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Romero-Fernandez, Wilber; Borroto-Escuela, Dasiel O; Alea, Mileidys Perez; Garcia-Mesa, Yoelvis; Garriga, Pere

2011-12-01

The human M(3) muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M(3) muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [(3)H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity. © The Author 2011. Published by Oxford University Press. All rights reserved.

Investigation of the presence and antinociceptive function of muscarinic acetylcholine receptors in the African naked mole-rat (Heterocephalus glaber).

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Jørgensen, Kristine B; Krogh-Jensen, Karen; Pickering, Darryl S; Kanui, Titus I; Abelson, Klas S P

2016-01-01

The present study investigated the cholinergic system in the African naked mole-rat (Heterocephalus glaber) with focus on the muscarinic acetylcholine receptor subtypes M1 and M4. The protein sequences for the subtypes m 1-5 of the naked mole-rat were compared to that of the house mouse (Mus musculus) using basic local alignment search tool (BLAST). The presence and function of M1 and M4 was investigated in vivo, using the formalin test with the muscarinic receptor agonists xanomeline and VU0152100. Spinal cord tissue from the naked mole-rat was used for receptor saturation binding studies with [(3)H]-N-methylscopolamine. The BLAST test revealed 95 % protein sequence homology showing the naked mole-rat to have the genetic potential to express all five muscarinic acetylcholine receptor subtypes. A significant reduction in pain behavior was demonstrated after administration of 8.4 mg/kg in the formalin test. Administration of 50 mg/kg VU0152100 resulted in a non-significant tendency towards antinociception. The antinociceptive effects were reversed by the muscarinic acetylcholine receptor antagonist atropine. Binding studies indicated presence of muscarinic acetylcholine receptors with a radioligand affinity comparable to that reported in mice. In conclusion, muscarinic acetylcholine receptor subtypes are present in the naked mole-rat and contribute to antinociception in the naked mole-rat.

New pharmacological approaches to the cholinergic system: an overview on muscarinic receptor ligands and cholinesterase inhibitors.

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Greig, Nigel H; Reale, Marcella; Tata, Ada M

2013-08-01

The cholinergic system is expressed in neuronal and in non-neuronal tissues. Acetylcholine (ACh), synthesized in and out of the nervous system can locally contribute to modulation of various cell functions (e.g. survival, proliferation). Considering that the cholinergic system and its functions are impaired in a number of disorders, the identification of new pharmacological approaches to regulate cholinergic system components appears of great relevance. The present review focuses on recent pharmacological drugs able to modulate the activity of cholinergic receptors and thereby, cholinergic function, with an emphasis on the muscarinic receptor subtype, and additionally covers the cholinesterases, the main enzymes involved in ACh hydrolysis. The presence and function of muscarinic receptor subtypes both in neuronal and non-neuronal cells has been demonstrated using extensive pharmacological data emerging from studies on transgenic mice. The possible involvement of ACh in different pathologies has been proposed in recent years and is becoming an important area of study. Although the lack of selective muscarinic receptor ligands has for a long time limited the definition of therapeutic treatment based on muscarinic receptors as targets, some muscarinic ligands such as cevimeline (patents US4855290; US5571918) or xanomeline (patent, US5980933) have been developed and used in pre-clinical or in clinical studies for the treatment of nervous system diseases (Alzheimer' and Sjogren's diseases). The present review focuses on the potential implications of muscarinic receptors in different pathologies, including tumors. Moreover, the future use of muscarinic ligands in therapeutic protocols in cancer therapy will be discussed, considering that some muscarinic antagonists currently used in the treatment of genitourinary disease (e.g. darifenacin, patent, US5096890; US6106864) have also been demonstrated to arrest tumor progression in nude mice. The involvement of muscarinic

Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M₁, M₂, and M₄ receptor knockout mice

DEFF Research Database (Denmark)

Joseph, Lauren; Thomsen, Morgane

2017-01-01

Muscarinic M1/M4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (SD) effects, but the receptor subtypes mediating those...

Muscarinic receptor compensation in hippocampus of alzheimer patients. [Autoradiography

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Nordberg, A; Larsson, C; Adolfsson, R; Alafuzoff, I; Winblad, B [Uppsala Univ. (Sweden)

1983-01-01

The activity of the acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) (presynaptic marker) and number of muscarine-like receptor binding sites have been measured in the hippocampus from eight individuals with senile dementia of Alzheimer type (SDAT) and ten controls. A negative correlation (r=0.80; p<0.05) was found between the ChAT activity and the number of muscarine-like receptors in the SDAT group but not in the controls. The findings might indicate an ongoing compensatory receptor mechanism as a response to changes in presynaptic cholinergic activity.

Differential expression of muscarinic acetylcholine receptor subtypes in Jurkat cells and their signaling.

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Alea, Mileidys Perez; Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Fuxe, Kjell; Garriga, Pere

2011-08-15

Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were investigated. Semiquantitative real-time PCR and radioligand binding studies, using a wide set of antagonist compounds, showed the co-existence of M(3), M(4), and M(5) subtypes. Stimulation of these subpopulations caused a concentration and time- dependent activation of second messengers and ERK signaling pathways, with a major contribution of the M(3) subtype in a G(q/11)-mediated response. In addition, we found that T-cell stimulation leads to increased expression of M(3) and M(5) both at transcriptional and protein levels in a PLC/PKCθ dependent manner. Our data clarifies the functional role of AChR subtypes in Jurkat cells and pave the way to future studies on the potential cross-talk among these subpopulations and their regulation of T lymphocytes immune function. Copyright © 2011 Elsevier B.V. All rights reserved.

Autoradiographic visualization of muscarinic receptor subtypes in human and guinea pig lung

International Nuclear Information System (INIS)

Mak, J.C.; Barnes, P.J.

1990-01-01

Muscarinic receptor subtypes have been localized in human and guinea pig lung sections by an autoradiographic technique, using [3H](-)quinuclidinyl benzilate [( 3H]QNB) and selective muscarinic antagonists. [3H]QNB was incubated with tissue sections for 90 min at 25 degrees C, and nonspecific binding was determined by incubating adjacent serial sections in the presence of 1 microM atropine. Binding to lung sections had the characterization expected for muscarinic receptors. Autoradiography revealed that muscarinic receptors were widely distributed in human lung, with dense labeling over submucosal glands and airway ganglia, and moderate labeling over nerves in intrapulmonary bronchi and of airway smooth muscle of large and small airways. In addition, alveolar walls were uniformly labeled. In guinea pig lung, labeling of airway smooth muscle was similar, but in contrast to human airways, epithelium was labeled but alveolar walls were not. The muscarinic receptors of human airway smooth muscle from large to small airways were entirely of the M3-subtype, whereas in guinea pig airway smooth muscle, the majority were the M3-subtype with a very small population of the M2-subtype present. In human bronchial submucosal glands, M1- and M3-subtypes appeared to coexist in the proportions of 36 and 64%, respectively. In human alveolar walls the muscarinic receptors were entirely of the M1-subtype, which is absent from the guinea pig lung. No M2-receptors were demonstrated in human lung. The localization of M1-receptors was confirmed by direct labeling with [3H]pirenzepine. With the exception of the alveolar walls in human lung, the localization of muscarinic receptor subtypes on structures in the lung is consistent with known functional studies

Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods.

Science.gov (United States)

Collin, Caitlin; Hauser, Frank; Gonzalez de Valdivia, Ernesto; de Valdivia, Ernesto Gonzalez; Li, Shizhong; Reisenberger, Julia; Carlsen, Eva M M; Khan, Zaid; Hansen, Niels O; Puhm, Florian; Søndergaard, Leif; Niemiec, Justyna; Heninger, Magdalena; Ren, Guilin R; Grimmelikhuijzen, Cornelis J P

2013-09-01

Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5). In this study, we cloned two structurally related GPCRs from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine (EC50, 5 × 10(-8) M) and muscarine (EC50, 6 × 10(-8) M) and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked by the antagonists. A- and B-type mAChRs were also cloned and functionally characterized from the red flour beetle Tribolium castaneum. Recently, Haga et al. (Nature 2012, 482: 547-551) published the crystal structure of the human m2 mAChR, revealing 14 amino acid residues forming the binding pocket for QNB. These residues are identical between the human m2 and the D. melanogaster and T. castaneum A-type mAChRs, while many of them are different between the human m2 and the B-type receptors. Using bioinformatics, one orthologue of the A-type and one of the B-type mAChRs could also be found in all other arthropods with a sequenced genome. Protostomes, such as arthropods, and deuterostomes, such as mammals and other vertebrates, belong to two evolutionarily distinct lineages of animal evolution that split about 700 million years ago. We found that animals that originated before this split, such as cnidarians (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs.

Hydrostatic pressure and muscarinic receptors are involved in the release of inflammatory cytokines in human bladder smooth muscle cells.

Science.gov (United States)

Liang, Zhou; Xin, Wei; Qiang, Liu; Xiang, Cai; Bang-Hua, Liao; Jin, Yang; De-Yi, Luo; Hong, Li; Kun-Jie, Wang

2017-06-01

Abnormal intravesical pressure results in a series of pathological changes. We investigated the effects of hydrostatic pressure and muscarinic receptors on the release of inflammatory cytokines in rat and human bladder smooth muscle cells (HBSMCs). Animal model of bladder outlet obstruction was induced by urethra ligation. HBSMCs were subjected to elevated hydrostatic pressure and/or acetylcholine (Ach). Macrophage infiltration in the bladder wall was determined by immunohistochemical staining. The expression of inflammatory genes was measured by RT-PCR, ELISA and immunofluorescence. In obstructed bladder, inflammatory genes and macrophage infiltration were remarkably induced. When HBSMCs were subjected to 200-300 cm H 2 O pressure for 2-24 h in vitro, the expressions of IL-6 and RANTES were significantly increased. Hydrostatic pressure promoted the protein levels of phospho-NFκB p65 and phospho-ERK1/2 as well as muscarinic receptors. Moreover, NFκB or ERK1/2 inhibitors suppressed pressure-induced inflammatory genes mRNA. When cells were treated with 1 μM acetylcholine for 6 h, a significant increase in IL-6 mRNA expression was detected. Acetylcholine also enhanced pressure-induced phospho-NFκB p65 and IL-6 protein expression. Additionally, pressure-induced IL-6 was partially suppressed by muscarinic receptors antagonists. Hydrostatic pressure and muscarinic receptors were involved in the secretion of inflammatory cytokines in HBSMCs, indicating a pro-inflammatory effect of the two factors in the pathological process of BOO. © 2016 Wiley Periodicals, Inc.

Loss of muscarinic receptors and of stimulated phospholipid labeling in ibotenate-treated hippocampus

International Nuclear Information System (INIS)

Fisher, S.K.; Frey, K.A.; Agranoff, B.W.

1981-01-01

The stimulation of phospholipid labeling by muscarinic agonists has been examined in nerve ending preparations from lesioned hippocampus in order to investigate the synaptic locus of the effect. Unilateral injections of the neurotoxin, ibotenic acid, into the hippocampus resulted in an extensive loss of nerve cells from both the dentate gyrus and hippocampus on the lesioned side and a parallel loss of muscarinic receptors as revealed by [ 3 H]quinuclidinyl benzilate autoradiography. Homogenates and nerve ending fractions prepared from the lesioned side of the hippocampus possessed a reduced specific activity (expressed per milligram of protein) of glutamic acid decarboxylase as well as a reduced number of muscarinic receptors compared with the control side. By contrast, choline acetyltransferase activity was either unchanged or slightly increased on the lesioned side. Although there was a reduced yield (25%) of nerve endings from the lesioned side, the specific activity of 32 Pi incorporation into phospholipids in the absence of added carbachol was comparable to that of the control side. There was, however, a marked reduction in the carbachol stimulation of phosphatidic acid and phosphatidylinositol labeling in nerve ending fractions obtained from he lesioned hippocampus. These results indicate that the muscarinic receptors present in nerve ending fractions from hippocampus and implicated in stimulated phospholipid turnover are derived from cholinoceptive intrinsic neurons

MUSCARINIC ACETYLCHOLINE RECEPTOR-EXPRESSION IN ASTROCYTES IN THE CORTEX OF YOUNG AND AGED RATS

NARCIS (Netherlands)

VANDERZEE, EA; DEJONG, GI; STROSBERG, AD; LUITEN, PGM

The present report describes the cellular and subcellular distribution pattern of immunoreactivity to M35, a monoclonal antibody raised against purified muscarinic acetylcholine receptor protein, in astrocytes in the cerebral cortex of young and aged rats. Most M35-positive astrocytes were localized

Antipsychotic-like effect of the muscarinic acetylcholine receptor agonist BuTAC in non-human primates

DEFF Research Database (Denmark)

Andersen, Maibritt B; Croy, Carrie Hughes; Dencker, Ditte

2015-01-01

Cholinergic, muscarinic receptor agonists exhibit functional dopamine antagonism and muscarinic receptors have been suggested as possible future targets for the treatment of schizophrenia and drug abuse. The muscarinic ligand (5R,6R)-6-(3-butylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane...... (BuTAC) exhibits high affinity for muscarinic receptors with no or substantially less affinity for a large number of other receptors and binding sites, including the dopamine receptors and the dopamine transporter. In the present study, we wanted to examine the possible antipsychotic-like effects...

The Cu-Zn superoxide dismutase (SOD1) inhibits ERK phosphorylation by muscarinic receptor modulation in rat pituitary GH3 cells

International Nuclear Information System (INIS)

Secondo, Agnese; De Mizio, Mariarosaria; Zirpoli, Laura; Santillo, Mariarosaria; Mondola, Paolo

2008-01-01

The Cu-Zn superoxide dismutase (SOD1) belongs to a family of isoenzymes that are able to dismutate the oxygen superoxide in hydrogen peroxide and molecular oxygen. This enzyme is secreted by many cellular lines and it is also released trough a calcium-dependent depolarization mechanism involving SNARE protein SNAP 25. Using rat pituitary GH3 cells that express muscarinic receptors we found that SOD1 inhibits P-ERK1/2 pathway trough an interaction with muscarinic M1 receptor. This effect is strengthened by oxotremorine, a muscarinic M agonist and partially reverted by pyrenzepine, an antagonist of M1 receptor; moreover this effect is independent from increased intracellular calcium concentration induced by SOD1. Finally, P-ERK1/2 inhibition was accompanied by the reduction of GH3 cell proliferation. These data indicate that SOD1 beside the well studied antioxidant properties can be considered as a neuromodulator able to affect mitogen-activated protein kinase in rat pituitary cells trough a M1 muscarinic receptor

Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

Directory of Open Access Journals (Sweden)

Meyyammai Swaminathan

2014-06-01

Full Text Available Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM, comparable to that of acetylcholine (Ki = 59 µM. Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

Allosteric Modulation of Muscarinic Acetylcholine Receptors

Czech Academy of Sciences Publication Activity Database

Jakubík, Jan; El-Fakahany, E. E.

2010-01-01

Roč. 3, č. 9 (2010), s. 2838-2860 ISSN 1424-8247 R&D Projects: GA ČR GA305/09/0681 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic acetylcholine receptors * allosteric modulation * Alzheimer´s disease Subject RIV: CE - Biochemistry

New advances in pharmacological approaches to the cholinergic system: an overview on muscarinic receptor ligands and cholinesterase inhibitors

Science.gov (United States)

Greig, Nigel H.; Reale, Marcella; Tata, Ada Maria

2016-01-01

The cholinergic system is expressed in neuronal and in non-neuronal tissues. Acetylcholine (ACh), synthesized in and out of the nervous system can locally contribute to modulation of various cell functions (e.g. survival, proliferation). Considering that the cholinergic system and its functions are impaired in a number of disorders, the identification of new pharmacological approaches to regulate cholinergic system components appears of great relevance. The present review focuses on recent pharmacological drugs able to modulate the activity of cholinergic receptors and thereby, cholinergic function, with an emphasis on the muscarinic receptor subtype, and additionally covers the cholinesterases, the main enzymes involved in ACh hydrolysis. The presence and function of muscarinic receptor subtypes both in neuronal and non-neuronal cells has been demonstrated using extensive pharmacological data emerging from studies on transgenic mice. The possible involvement of ACh in different pathologies has been proposed in recent years and is becoming an important area of study. Although the lack of selective muscarinic receptor ligands has for a long time limited the definition of therapeutic treatment based on muscarinic receptors as targets, some muscarinic ligands such as cevimeline (patents US4855290; US5571918) or xanomeline (patent, US5980933) have been developed and used in pre-clinical or in clinical studies for the treatment of nervous system diseases (Alzheimer’ and Sjogren’s diseases). The present review focuses on the potential implications of muscarinic receptors in different pathologies, including tumors. Moreover, the future use of muscarinic ligands in therapeutic protocols in cancer therapy will be discussed, considering that some muscarinic antagonists currently used in the treatment of genitourinary disease (e.g. darifenacin, patent, US5096890; US6106864) have also been demonstrated to arrest tumor progression in nude mice. The involvement of muscarinic

G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

International Nuclear Information System (INIS)

Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

1988-01-01

The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na + channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [ 3 H] acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [ 3 H]batrachotoxin to Na + channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22 Na + uptake in the presence and absence of tetrodotoxin, which blocks Na + channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na + channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na + channel-is such that at resting potential the muscarinic receptor induces opening of Na + channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues

Muscarinic receptor subtypes in porcine detrusor: comparison with humans and regulation by bladder augmentation

NARCIS (Netherlands)

Goepel, M.; Gronewald, A.; Krege, S.; Michel, M. C.

1998-01-01

The properties of muscarinic acetylcholine receptors of porcine and human bladder detrusor were compared in radioligand binding studies using [3H]quinuclidinylbenzylate as the radioligand. The receptor affinity for the radioligand and the density of muscarinic receptors was similar in male and

Role of dopamine receptor and muscarinic acetylcholine receptor blockade in the antiapomorphine action of neuroleptics

Energy Technology Data Exchange (ETDEWEB)

Zharkovskii, A.M.; Langel, Yu.L.; Chereshka, K.S.; Zharkovskaya, T.A.

1987-08-01

The authors analyze the role of dopamine and muscarinic acetylcholine receptor blocking components in the antistereotypic action of neuroleptics with different chemical structure. To determine dopamine-blocking activity in vitro, binding of /sup 3/H-spiperone with membranes of the rat striatum was measured. To study the blocking action of the substances on muscarinic acetylcholine receptors, binding of /sup 3/H-quinuclidinyl benzylate with brain membranes was chosen.

Antipsychotic-like effect of the muscarinic acetylcholine receptor agonist BuTAC in non-human primates.

Directory of Open Access Journals (Sweden)

Maibritt B Andersen

Full Text Available Cholinergic, muscarinic receptor agonists exhibit functional dopamine antagonism and muscarinic receptors have been suggested as possible future targets for the treatment of schizophrenia and drug abuse. The muscarinic ligand (5R,6R-6-(3-butylthio-1,2,5-thiadiazol-4-yl-1-azabicyclo[3.2.1]octane (BuTAC exhibits high affinity for muscarinic receptors with no or substantially less affinity for a large number of other receptors and binding sites, including the dopamine receptors and the dopamine transporter. In the present study, we wanted to examine the possible antipsychotic-like effects of BuTAC in primates. To this end, we investigated the effects of BuTAC on d-amphetamine-induced behaviour in antipsychotic-naive Cebus paella monkeys. Possible adverse events of BuTAC, were evaluated in the same monkeys as well as in monkeys sensitized to antipsychotic-induced extrapyramidal side effects. The present data suggests that, the muscarinic receptor ligand BuTAC exhibits antipsychotic-like behaviour in primates. The behavioural data of BuTAC as well as the new biochemical data further substantiate the rationale for the use of muscarinic M1/M2/M4-preferring receptor agonists as novel pharmacological tools in the treatment of schizophrenia.

Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice.

Science.gov (United States)

Gainetdinov, R R; Bohn, L M; Walker, J K; Laporte, S A; Macrae, A D; Caron, M G; Lefkowitz, R J; Premont, R T

1999-12-01

G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.

Muscarinic receptor M₄ positive allosteric modulators attenuate central effects of cocaine

DEFF Research Database (Denmark)

Dall, Camilla; Weikop, Pia; Dencker, Ditte

2017-01-01

BACKGROUND: Cocaine addiction is a chronic brain disease affecting neurotransmission. Muscarinic cholinergic receptors modulate dopaminergic signaling in the reward system, and muscarinic receptor stimulation can block direct reinforcing effects of cocaine. Here, we tested the hypothesis...... that specific muscarinic M4receptor stimulation can attenuate the discriminative stimulus effects and conditioned rewarding effects of cocaine, measures believed to predict the ability of cocaine and cocaine-associated cues to elicit relapse to drug taking. METHODS: We tested the M4-selective positive...

Activation of muscarinic receptors protects against retinal neurons damage and optic nerve degeneration in vitro and in vivo models.

Science.gov (United States)

Tan, Pan-Pan; Yuan, Hai-Hong; Zhu, Xu; Cui, Yong-Yao; Li, Hui; Feng, Xue-Mei; Qiu, Yu; Chen, Hong-Zhuan; Zhou, Wei

2014-03-01

Muscarinic acetylcholine receptor agonist pilocarpine reduces intraocular pressure (IOP) of glaucoma mainly by stimulating ciliary muscle contraction and then increasing aqueous outflow. It is of our great interest to know whether pilocarpine has the additional properties of retinal neuroprotection independent of IOP lowering in vitro and in vivo models. In rat primary retinal cultures, cell viability was measured using an MTT assay and the trypan blue exclusion method, respectively. Retinal ganglion cells (RGCs) were identified by immunofluorescence and quantified by flow cytometry. For the in vivo study, the retinal damage after retinal ischemia/reperfusion injury in rats was evaluated by histopathological study using hematoxylin and eosin staining, transmission electron microscopy, and immunohistochemical study on cleaved caspase-3, caspase-3, and ChAT. Pretreatment of pilocarpine attenuated glutamate-induced neurotoxicity of primary retinal neurons in a dose-dependent manner. Protection of pilocarpine in both retinal neurons and RGCs was largely abolished by the nonselective muscarinic receptor antagonist atropine and the M1-selective muscarinic receptor antagonist pirenzepine. After ischemia/reperfusion injury in retina, the inner retinal degeneration occurred including ganglion cell layer thinning and neuron lost, and the optic nerve underwent vacuolar changes. These degenerative changes were significantly lessened by topical application of 2% pilocarpine. In addition, the protective effect of pilocarpine on the ischemic rat retina was favorably reflected by downregulating the expression of activated apoptosis marker cleaved caspase-3 and caspase-3 and upregulating the expression of cholinergic cell marker ChAT. Taken together, this highlights pilocarpine through the activation of muscarinic receptors appear to afford significant protection against retinal neurons damage and optic nerve degeneration at clinically relevant concentrations. These data also

Multiple promoters drive tissue-specific expression of the human M2 muscarinic acetylcholine receptor gene

Czech Academy of Sciences Publication Activity Database

Krejčí, Alena; Bruce, A. W.; Doležal, Vladimír; Tuček, Stanislav; Buckley, N. J.

2004-01-01

Roč. 91, č. 1 (2004), s. 88-98 ISSN 0022-3042 R&D Projects: GA AV ČR IAA5011306 Institutional research plan: CEZ:AV0Z5011922 Keywords : M2 muscarinic receptor * neuron-restrictive silence factor * promoter Subject RIV: ED - Physiology Impact factor: 4.824, year: 2004

Muscarinic receptors mediate cold stress-induced detrusor overactivity in type 2 diabetes mellitus rats.

Science.gov (United States)

Imamura, Tetsuya; Ishizuka, Osamu; Ogawa, Teruyuki; Yamagishi, Takahiro; Yokoyama, Hitoshi; Minagawa, Tomonori; Nakazawa, Masaki; Gautam, Sudha Silwal; Nishizawa, Osamu

2014-10-01

This study determined if muscarinic receptors could mediate the cold stress-induced detrusor overactivity induced in type 2 diabetes mellitus rats. Ten-week-old female Goto-Kakizaki diabetic rats (n = 12) and Wister Kyoto non-diabetic rats (n = 12) were maintained on a high-fat diet for 4 weeks. Cystometric investigations of the unanesthetized rats were carried out at room temperature (27 ± 2°C) for 20 min. They were intravenously administered imidafenacin (0.3 mg/kg, n = 6) or vehicle (n = 6). After 5 min, the rats were transferred to a low temperature (4 ± 2°C) for 40 min where the cystometry was continued. The rats were then returned to room temperature for the final cystometric measurements. Afterwards, expressions of bladder muscarinic receptor M3 and M2 messenger ribonucleic acids and proteins were assessed by reverse transcription polymerase chain reaction and immunohistochemistry. In non-diabetic Wister Kyoto rats, imidafenacin did not reduce cold stress-induced detrusor overactivity. In diabetic Goto-Kakizaki rats, just after transfer to a low temperature, the cold stress-induced detrusor overactivity in imidafenacin-treated rats was reduced compared with vehicle-treated rats. Within the urinary bladders, the ratio of M3 to M2 receptor messenger ribonucleic acid in the diabetic Goto-Kakizaki rats was significantly higher than that of the non-diabetic Wister Kyoto rats. The proportion of muscarinic M3 receptor-positive area within the detrusor in diabetic Goto-Kakizaki rats was also significantly higher than that in non-diabetic Wister Kyoto rats. Imidafenacin partially inhibits cold stress-induced detrusor overactivity in diabetic Goto-Kakizaki rats. In this animal model, muscarinic M3 receptors partially mediate cold stress-induced detrusor overactivity. © 2014 The Japanese Urological Association.

Decrease in the number of rat brain dopamine and muscarinic receptors after chronic alcohol intake

International Nuclear Information System (INIS)

Syvaelahti, E.K.G.; Hietala, J.; Roeyttae, M.; Groenroos, J.

1988-01-01

The effect of 32 weeks' alcohol treatment on the number and affinity of dopamine and muscarinic receptor sites in rat striatum were measured using 3 H-spiperone and 3 H-quinuclidinylbenzilate ( 3 H-QNB) as radioligans. The number of dopamine receptor sites was 38 per cent and the number of muscarinic receptor sites 36 per cent lower in the alcohol group than in control rats. The differences in receptor affinities were less marked. In conclusion, a long-term alcohol intake with rather moderate doses seems to induce a pronounced down-regulation in dopamine and muscarinic receptor systems in rat striatum. (author)

Eosinophils express muscarinic receptors and corticotropin-releasing factor to disrupt the mucosal barrier in ulcerative colitis.

Science.gov (United States)

Wallon, Conny; Persborn, Mats; Jönsson, Maria; Wang, Arthur; Phan, Van; Lampinen, Maria; Vicario, Maria; Santos, Javier; Sherman, Philip M; Carlson, Marie; Ericson, Ann-Charlott; McKay, Derek M; Söderholm, Johan D

2011-05-01

Altered intestinal barrier function has been implicated in the pathophysiology of ulcerative colitis (UC) in genetic, functional, and epidemiological studies. Mast cells and corticotropin-releasing factor (CRF) regulate the mucosal barrier in human colon. Because eosinophils are often increased in colon tissues of patients with UC, we assessed interactions among mast cells, CRF, and eosinophils in the mucosal barrier of these patients. Transmucosal fluxes of protein antigens (horseradish peroxidase) and paracellular markers ((51)Cr-EDTA, fluorescein isothiocyanate-dextran 4000) were studied in noninflamed, colonic mucosal biopsy samples collected from 26 patients with UC and 53 healthy volunteers (controls); samples were mounted in Ussing chambers. We also performed fluorescence and electron microscopy of human tissue samples, assessed isolated eosinophils, and performed mechanistic studies using in vitro cocultured eosinophils (15HL-60), mast cells (HMC-1), and a colonic epithelial cell line (T84). Colon tissues from patients with UC had significant increases in permeability to protein antigens compared with controls. Permeability was blocked by atropine (a muscarinic receptor antagonist), α-helical CRF(9-41) (a CRF receptor antagonist), and lodoxamide (a mast-cell stabilizer). Eosinophils were increased in number in UC tissues (compared with controls), expressed the most M2 and M3 muscarinic receptors of any mucosal cell type, and had immunoreactivity to CRF. In coculture studies, carbachol activation of eosinophils caused production of CRF and activation of mast cells, which increased permeability of T84 epithelial cells to macromolecules. We identified a neuroimmune intercellular circuit (from cholinergic nerves, via eosinophils to mast cells) that mediates colonic mucosal barrier dysfunction in patients with UC. This circuit might exacerbate mucosal inflammation. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

Alkylating derivative of oxotremorine interacts irreversibly with the muscarinic receptor

International Nuclear Information System (INIS)

Ehlert, F.J.; Jenden, D.J.; Ringdahl, B.

1984-01-01

A 2-chloroethylamine derivative of oxotremorine was studied in pharmacological experiments and muscarinic receptor binding assays. The compound, N-[4-(2-chloroethylmethylamino)-2-butynyl]-2-pyrrolidone (BM 123), forms an aziridinium ion in aqueous solution at neutral pH that stimulates contractions of guinea pig ileum with a potency similar to that of oxotremorine. Following the initial stimulation, there is a long lasting period of lack of sensitivity of the guinea pig ileum to muscarinic agonists. BM 123 also produces muscarinic effects in vivo. When homogenates of the rat cerebral cortex were incubated with BM 123 and assayed subsequently in muscarinic receptor binding assays, a loss of binding capacity for the muscarinic antagonist, [ 3 H]N-methylscopolamine ([ 3 H]NMS), was noted without a change in affinity. Similar observations were made in [ 3 H]1-3-quinuclidinyl benzilate ([ 3 H]-QNB) binding assays on the forebrains of mice that had been injected with BM 123 24 hr earlier. The loss in receptor capacity for both [ 3 H]NMS and [ 3 H]-QNB was prevented by atropine treatment. Kinetic studies of the interaction of BM 123 with homogenates of the rat cerebral cortex in vitro showed that the half-time for the loss of [ 3 H]-QNB binding sites increased from 10 to 45 min as the concentration of BM 123 decreased from 10 to 1 μM. In contrast to the aziridinium ion, the parent 2-chloroethylamine compound and the alcoholic hydrolysis product were largely devoid of pharmacological and binding activity

Muscarinic receptor blockade in ventral hippocampus and prelimbic cortex impairs memory for socially transmitted food preference.

Science.gov (United States)

Carballo-Márquez, Anna; Vale-Martínez, Anna; Guillazo-Blanch, Gemma; Martí-Nicolovius, Margarita

2009-05-01

Acetylcholine is involved in learning and memory and, particularly, in olfactory tasks, but reports on its specific role in consolidation processes are somewhat controversial. The present experiment sought to determine the effects of blocking muscarinic cholinergic receptors in the ventral hippocampus (vHPC) and the prelimbic cortex (PLC) on the consolidation of social transmission of food preference, an odor-guided relational task that depends on such brain areas. Adult male Wistar rats were bilaterally infused with scopolamine (20 microg/site) immediately after social training and showed impairment, relative to vehicle-injected controls, in the expression of the task measured 24 h after learning. Results indicated that scopolamine in the PLC completely abolished memory, suggesting that muscarinic transmission in this cortical region is crucial for consolidation of recent socially acquired information. Muscarinic receptors in the vHPC contribute in some way to task consolidation, as the rats injected with scopolamine in the vHPC showed significantly lower trained food preference than control rats, but higher than both chance level and that of the PLC-injected rats. Behavioral measures such as social interaction, motivation to eat, neophobia, or exploration did not differ between rats infused with scopolamine or vehicle. Such data suggest a possible differential role of muscarinic receptors in the PLC and the vHPC in the initial consolidation of a naturalistic form of nonspatial relational memory. Copyright 2008 Wiley-Liss, Inc.

Pet measurements of postsynaptic muscarinic and beta adrenergic receptors in the heart

International Nuclear Information System (INIS)

Syrota, A.

1991-01-01

There is ample evidence from both experimental and clinical studies that changes in β-adrenergic and muscarinic receptor density can be associated with such cardiac diseases as congestive heart failure, myocardial ischemia and infarction, cardiomyopathy, diabetes, or thyroid-induced muscle disease. Changes in B-adrenergic density also have been shown in the denervated transplanted heart. These alterations of cardiac receptors have been demonstrated in vitro on homogenates from samples collected mainly during surgery or post mortem. Recent developments of Positron Emission Tomography (PET) techniques and of radioligands suitable for cardiac receptor binding studies in vivo have made possible both the imaging and the measurement of receptor density. From these studies, important information is now available concerning physiologic and pathologic conditions, as well as alterations induced by treatment. For the investigation of myocardial B-adrenergic receptors we have used [ 11 C] CGP 12177, a potent hydrophilic antagonist of the 3-adrenergic receptor. The quantification of myocardial muscarinic receptors in vivo has been obtained with [ 11 C] MQNB, a nonmetabolized hydrophilic antagonist of the muscarinic receptor. Receptor density and affinity have been measured by a kinetic, nonequilibrium approach in an experimental protocol that provides sufficient data to determine values for all parameters from a single experiment

Antipsychotic-induced catalepsy is attenuated in mice lacking the M4 muscarinic acetylcholine receptor

DEFF Research Database (Denmark)

Fink-Jensen, Anders; Schmidt, Lene S; Dencker, Ditte

2011-01-01

of the striatum, suggesting a role for muscarinic M4 receptors in the motor side effects of antipsychotics, and in the alleviation of these side effects by anticholinergics. Here we investigated the potential role of the muscarinic M4 receptor in catalepsy induced by antipsychotics (haloperidol and risperidone...

Attenuation of cocaine's reinforcing and discriminative stimulus effects via muscarinic M1 acetylcholine receptor stimulation

DEFF Research Database (Denmark)

Thomsen, Morgane; Conn, P Jeffrey; Lindsley, Craig

2010-01-01

Muscarinic cholinergic receptors modulate dopaminergic function in brain pathways thought to mediate cocaine's abuse-related effects. Here, we sought to confirm and extend in the mouse species findings that nonselective muscarinic receptor antagonists can enhance cocaine's discriminative stimulus...... for cocaine addiction....

Hypoxia increases exercise heart rate despite combined inhibition of β-adrenergic and muscarinic receptors

DEFF Research Database (Denmark)

Siebenmann, Christoph; Rasmussen, Peter; Sørensen, Henrik

2015-01-01

Hypoxia increases the heart rate (HR) response to exercise but the mechanism(s) remain unclear. We tested the hypothesis that the tachycardic effect of hypoxia persists during separate but not combined inhibition of β-adrenergic and muscarinic receptors. Nine subjects performed incremental exercise...... combined β-adrenergic and muscarinic receptor inhibition....

Decreased ipsilateral [123I]iododexetimide binding to cortical muscarinic receptors in unilaterally 6-hydroxydopamine lesioned rats

International Nuclear Information System (INIS)

Knol, Remco J.J.; Bruin, Kora de; Opmeer, Brent; Voorn, Pieter; Jonker, Allert J.; Eck-Smit, Berthe L.F. van; Booij, Jan

2014-01-01

Introduction: Dysfunction of the cholinergic neurotransmitter system is present in Parkinson’s disease, Parkinson’s disease related dementia and dementia with Lewy bodies, and is thought to contribute to cognitive deficits in these patients. In vivo imaging of the cholinergic system in these diseases may be of value to monitor central cholinergic disturbances and to select cases in which treatment with cholinesterase inhibitors could be beneficial. The muscarinic receptor tracer [ 123 I]iododexetimide, predominantly reflecting M 1 receptor binding, may be an appropriate tool for imaging of the cholinergic system by means of SPECT. In this study, we used [ 123 I]iododexetimide to study the effects of a 6-hydroxydopamine lesion (an animal model of Parkinson’s disease) on the muscarinic receptor availability in the rat brain. Methods: Rats (n = 5) were injected in vivo at 10–13 days after a confirmed unilateral 6-hydroxydopamine lesion. Muscarinic receptor availability was measured bilaterally in multiple brain areas on storage phosphor images by region of interest analysis. Results: Autoradiography revealed a consistent and statistically significant lower [ 123 I]iododexetimide binding in all examined neocortical areas on the ipsilateral side of the lesion as compared to the contralateral side. In hippocampal and subcortical areas, such asymmetry was not detected. Conclusions: This study suggests that evaluation of muscarinic receptor availability in dopamine depleted brains using [ 123 I]iododexetimide is feasible. We conclude that 6-hydroxydopamine lesions induce a decrease of neocortical muscarinic receptor availability. We hypothesize that this arises from down regulation of muscarinic postsynaptic M 1 receptors due to hyperactivation of the cortical cholinergic system in response to dopamine depletion. Advances in knowledge: In rats, dopamine depletion provokes a decrease in neocortical muscarinic receptor availability, which is evaluable by [ 123 I

Muscarinic Receptor Agonists and Antagonists

Directory of Open Access Journals (Sweden)

David R. Kelly

2001-02-01

Full Text Available A comprehensive review of pharmacological and medical aspects of the muscarinic class of acetylcholine agonists and antagonists is presented. The therapeutic benefits of achieving receptor subtype selectivity are outlined and applications in the treatment of Alzheimer’s disease are discussed. A selection of chemical routes are described, which illustrate contemporary methodology for the synthesis of chiral medicinal compounds (asymmetric synthesis, chiral pool, enzymes. Routes to bicyclic intrannular amines and intramolecular Diels-Alder reactions are highlighted.

Quantitative autoradiographic analysis of muscarinic receptor subtypes and their role in representational memory

International Nuclear Information System (INIS)

Messer, W.S.

1986-01-01

Autoradiographic techniques were used to examine the distribution of muscarinic receptors in rat brain slices. Agonist and selective antagonist binding were examined by measuring the ability for unlabeled ligands to inhibit [ 3 H]-1-QNB labeling of muscarinic receptors. The distribution of high affinity pirenzepine binding sites (M 1 subtype) was distinct from the distribution of high affinity carbamylcholine sites, which corresponded to the M 2 subtype. In a separate assay, the binding profile for pirenzepine was shown to differ from the profile for scopolamine, a classical muscarinic antagonist. Muscarinic antagonists, when injected into the Hippocampus, impaired performance of a representational memory task. Pirenzepine, the M 1 selective antagonist, produced representational memory deficits. Scopolamine, a less selective muscarinic antagonist, caused increases in running times in some animals which prevented a definitive interpretation of the nature of the impairment. Pirenzepine displayed a higher affinity for the hippocampus and was more effective in producing a selective impairment of representational memory than scopolamine. The data indicated that cholinergic activity in the hippocampus was necessary for representation memory function

Hyperfunction of muscarinic receptor maintains long-term memory in 5-HT4 receptor knock-out mice.

Directory of Open Access Journals (Sweden)

Luis Segu

Full Text Available Patients suffering from dementia of Alzheimer's type express less serotonin 4 receptors (5-HTR(4, but whether an absence of these receptors modifies learning and memory is unexplored. In the spatial version of the Morris water maze, we show that 5-HTR(4 knock-out (KO and wild-type (WT mice performed similarly for spatial learning, short- and long-term retention. Since 5-HTR(4 control mnesic abilities, we tested whether cholinergic system had circumvented the absence of 5-HTR(4. Inactivating muscarinic receptor with scopolamine, at an ineffective dose (0.8 mg/kg to alter memory in WT mice, decreased long-term but not short-term memory of 5-HTR(4 KO mice. Other changes included decreases in the activity of choline acetyltransferase (ChAT, the required enzyme for acetylcholine synthesis, in the septum and the dorsal hippocampus in 5-HTR(4 KO under baseline conditions. Training- and scopolamine-induced increase and decrease, respectively in ChAT activity in the septum in WT mice were not detected in the 5-HTR(4 KO animals. Findings suggest that adaptive changes in cholinergic systems may circumvent the absence of 5-HTR(4 to maintain long-term memory under baseline conditions. In contrast, despite adaptive mechanisms, the absence of 5-HTR(4 aggravates scopolamine-induced memory impairments. The mechanisms whereby 5-HTR(4 mediate a tonic influence on ChAT activity and muscarinic receptors remain to be determined.

Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.

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Eva Šantrůčková

Full Text Available Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

The binding of [3H]AF-DX 384 to rat ileal smooth muscle muscarinic receptors

International Nuclear Information System (INIS)

Entzeroth, M.; Mayer, N.

1991-01-01

The tritiated cardioselective muscarinic antagonist AF-DX 384 (5,11-dihydro-11-[2-[-(8-dipropylamino)methyl]-1-piperidinyl-ethyl-amino-carbonyl]-6H-pyrido [2,3-b] [1,4]benzodiazepin-6-one) was used to label muscarinic receptors in the rat ileum. Saturation binding to membrane suspensions revealed a high affinity binding site with a Kd of 9.2 nM. The maximal number of binding sites labeled in this tissue (Bmax) is 237 fmol/mg protein. The association and dissociation kinetics were well represented by single exponential reactions, and the dissociation constant obtained from the ratio of rate constants was in agreement with that derived from saturation experiments. Specific binding was inhibited by muscarinic antagonists with a rank order of potencies of atropine (pKi: 8.80) greater than 4-DAMP (pKi: 8.23) = AF-DX 384 (pKi: 8.20) greater than AF-DX 116 (pKi: 7.09) = hexahydro-sila-difenidol (pKi: 6.97) greater than pirenzepine (pKi: 6.49) and is consistent with the interaction of [3H]AF-DX 384 with muscarinic receptors of the M2 subtype. It can be concluded that [3H]AF-DX 384 can be used to selectively label M2 muscarinic receptors in heterogeneous receptor populations

COLOCALIZATION OF MUSCARINIC AND NICOTINIC RECEPTORS IN CHOLINOCEPTIVE NEURONS OF THE SUPRACHIASMATIC REGION IN YOUNG AND AGED RATS

NARCIS (Netherlands)

VANDERZEE, EA; STREEFLAND, C; STROSBERG, AD; SCHRODER, H; LUITEN, PGM; Schröder, H.

1991-01-01

In the present study muscarinic and nicotinic cholinergic receptors in the SCN region were demonstrated and analyzed, employing monoclonal antibodies to purified muscarinic and nicotinic cholinergic receptor proteins. A near-total colocalization of the two acetylcholine receptor subclasses in

Organophosphorus pesticides decrease M2 muscarinic receptor function in guinea pig airway nerves via indirect mechanisms.

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Becky J Proskocil

Full Text Available BACKGROUND: Epidemiological studies link organophosphorus pesticide (OP exposures to asthma, and we have shown that the OPs chlorpyrifos, diazinon and parathion cause airway hyperreactivity in guinea pigs 24 hr after a single subcutaneous injection. OP-induced airway hyperreactivity involves M2 muscarinic receptor dysfunction on airway nerves independent of acetylcholinesterase (AChE inhibition, but how OPs inhibit neuronal M2 receptors in airways is not known. In the central nervous system, OPs interact directly with neurons to alter muscarinic receptor function or expression; therefore, in this study we tested whether the OP parathion or its oxon metabolite, paraoxon, might decrease M2 receptor function on peripheral neurons via similar direct mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Intravenous administration of paraoxon, but not parathion, caused acute frequency-dependent potentiation of vagally-induced bronchoconstriction and increased electrical field stimulation (EFS-induced contractions in isolated trachea independent of AChE inhibition. However, paraoxon had no effect on vagally-induced bradycardia in intact guinea pigs or EFS-induced contractions in isolated ileum, suggesting mechanisms other than pharmacologic antagonism of M2 receptors. Paraoxon did not alter M2 receptor expression in cultured cells at the mRNA or protein level as determined by quantitative RT-PCR and radio-ligand binding assays, respectively. Additionally, a biotin-labeled fluorophosphonate, which was used as a probe to identify molecular targets phosphorylated by OPs, did not phosphorylate proteins in guinea pig cardiac membranes that were recognized by M2 receptor antibodies. CONCLUSIONS/SIGNIFICANCE: These data indicate that neither direct pharmacologic antagonism nor downregulated expression of M2 receptors contributes to OP inhibition of M2 function in airway nerves, adding to the growing evidence of non-cholinergic mechanisms of OP neurotoxicity.

SPET imaging of central muscarinic receptors with (R,R)[123I]-I-QNB: methodological considerations

International Nuclear Information System (INIS)

Norbury, R.; Travis, M.J.; Erlandsson, K.; Waddington, W.; Owens, J.; Ell, P.J.; Murphy, D.G.

2004-01-01

Investigations on the effect of normal healthy ageing on the muscarinic system have shown conflicting results. Also, in vivo determination of muscarinic receptor binding has been hampered by a lack of subtype selective ligands and differences in methods used for quantification of receptor densities. Recent in vitro and in vivo work with the muscarinic antagonist (R,R)-I-QNB indicates this ligand has selectivity for m 1 and m 4 muscarinic receptor subtypes. Therefore, we used (R,R)[ 123 I]-I-QNB and single photon emission tomography to study brain m 1 and m 4 muscarinic receptors in 25 healthy female subjects (11 younger subjects, age range 26-32 years and 14 older subjects, age range 57-82 years). Our aims were to ascertain the viability of tracer administration and imaging within the same day, and to evaluate whether normalization to whole brain, compared to normalization to cerebellum, could alter the clinical interpretation of results. Images were analyzed using the simplified reference tissue model and by two ratio methods: normalization to whole brain and normalization to cerebellum. Significant correlations were observed between kinetic analysis and normalization to cerebellum, but not to whole brain. Both the kinetic analysis and normalization to cerebellum showed age-related reductions in muscarinic binding in frontal, orbitofrontal, and parietal regions. Normalization to whole brain, however, failed to detect age-related changes in any region. Here we show that, for this radiotracer, normalizing to a region of negligible specific binding (cerebellum) significantly improves sensitivity when compared to global normalization

Role of muscarinic receptor antagonists in urgency and nocturia

NARCIS (Netherlands)

Michel, Martin C.; de La Rosette, Jean J. M. C. H.

2005-01-01

The overactive bladder (OAB) syndrome is defined as urgency, with or without urgency incontinence, usually accompanied by frequency and nocturia. Muscarinic receptor antagonists are the most established form of treatment for OAB, but until recently their effectiveness was only confirmed for symptoms

Highly specific detection of muscarinic M3 receptor, G protein interaction and intracellular trafficking in human detrusor using Proximity Ligation Assay (PLA).

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Berndt-Paetz, Mandy; Herbst, Luise; Weimann, Annett; Gonsior, Andreas; Stolzenburg, Jens-Uwe; Neuhaus, Jochen

2018-05-01

Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis - BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples. Paraffin sections of formalin-fixed bladder tissue (FFPE) of BPS/IC patients receiving transurethral biopsy were examined by Cy3-PLA for M3 expression, coupling of M3 to GPs (G αq/11 , G αs , G αi ) and interaction of M3 with endocytic regulator proteins. Membranes were labeled with wheat germ agglutinin-Alexa Fluor ® 488, nuclei were stained with DAPI. Object density and co-localization were analyzed in 3D-reconstruction of high resolution confocal z-stacks. Confocal image stack processing resulted in well demarcated objects. Calculated receptor densities correlated significantly with existing confocal expression data, while significantly improved specificity of M3 detection by PLA was verified using bladder tissue samples from transgenic mice. 50-60% of the M3 receptor complexes were plasma membrane associated in human bladder detrusor. Application of PLA for M3 and GPs allowed visualization of M3-GP interactions and revealed individual GP

Current status of muscarinic M1 and M4 receptors as drug targets for neurodegenerative diseases.

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Felder, Christian C; Goldsmith, Paul J; Jackson, Kimberley; Sanger, Helen E; Evans, David A; Mogg, Adrian J; Broad, Lisa M

2018-01-25

The cholinergic signalling system has been an attractive pathway to seek targets for modulation of arousal, cognition, and attention which are compromised in neurodegenerative and neuropsychiatric diseases. The acetylcholine muscarinic receptor M1 and M4 subtypes which are highly expressed in the central nervous system, in cortex, hippocampus and striatum, key areas of cognitive and neuropsychiatric control, have received particular attention. Historical muscarinic drug development yielded first generation agonists with modest selectivity for these two receptor targets over M2 and M3 receptors, the major peripheral sub-types hypothesised to underlie the dose-limiting clinical side effects. More recent compound screening and medicinal chemistry optimization of orthosteric and allosteric agonists, and positive allosteric modulators binding to sites distinct from the highly homologous acetylcholine binding pocket have yielded a collection of highly selective tool compounds for preclinical validation studies. Several M1 selective ligands have progressed to early clinical development and in time will hopefully lead to useful therapeutics for treating symptoms of Alzheimer's disease and related disorders. Copyright © 2018. Published by Elsevier Ltd.

Effects of agonist efficacy on desensitization of phosphoinositide hydrolysis mediated by m1 and m3 muscarinic receptors expressed in Chinese hamster ovary cells

International Nuclear Information System (INIS)

Hu, J.; Wang, S.Z.; el-Fakahany, E.E.

1991-01-01

Muscarinic receptor agonist-induced desensitization of phosphoinositide (PI) hydrolysis and loss of receptors were studied in Chinese hamster ovary (CHO) cells transfected with the m1 and m3 muscarinic receptor genes. Long-term exposure to the full agonist carbamylcholine (CBC) resulted in a time-dependent attenuation of the maximal PI response and a decrease in agonist potency. This desensitization was accompanied by a parallel loss of maximal ligand binding without an alteration of the binding affinity. The time course of both receptor desensitization and down-regulation was similar in m1 and m3 CHO cells. The PI response to the partial agonist McN-A-343 (McN) in m1 cells was more sensitive to desensitization by CBC than the response to the latter agonist, and this desensitization was faster than receptor down-regulation. Desensitization of the PI response to McN was reflected as a decrease in the maximal response without a marked change in potency. McN induced slow desensitization of the PI response to CBC but a much faster desensitization of its own response. Our data provide evidence that although muscarinic agonist-induced desensitization of PI hydrolysis in CHO cells is due mainly to loss of receptors, there are other important factors which play a role in this process, e.g., receptor-effector uncoupling. The relative contribution of these different mechanisms depends on the efficacy of the agonists used for the receptor desensitization and activation steps

Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

International Nuclear Information System (INIS)

Gil, D.W.; Wolfe, B.B.

1986-01-01

Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [ 3 H]quinuclidinyl benzilate or [ 3 H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [ 3 H]quinuclidinyl benzilate in a biphasic manner

Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging

International Nuclear Information System (INIS)

Gurwitz, D.; Egozi, Y.; Henis, Y.I.; Kloog, Y.; Sokolovsky, M.

1987-01-01

The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of [ 3 H]-antagonist as well as of the labeled natural neurotransmitter, [ 3 H]-acetylcholine [( 3 H]-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of [ 3 H]-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity [ 3 H]-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with [ 3 H]-antagonist represent a loss of low-affinity agonist binding sites. In contrast, [ 3 H]-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms

Muscarinic Acetylcholine Receptor Subtypes as Potential Drug Targets for the Treatment of Schizophrenia, Drug Abuse and Parkinson's Disease

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Dencker, Ditte; Thomsen, Morgane; Wörtwein, Gitta

2011-01-01

's disease and drug abuse. Dopaminergic systems are regulated by cholinergic, especially muscarinic, input. Not surprisingly, increasing evidence implicates muscarinic acetylcholine receptor-mediated pathways as potential targets for the treatment of these disorders classically viewed as "dopamine based...... site. Such agents may lead to the development of novel classes of drugs useful for the treatment of psychosis, drug abuse and Parkinson's disease. The present review highlights recent studies carried out using muscarinic receptor knock-out mice and new subtype-selective allosteric ligands to assess...... the roles of M(1), M(4), and M(5) receptors in various central processes that are under strong dopaminergic control. The outcome of these studies opens new perspectives for the use of novel muscarinic drugs for several severe disorders of the CNS....

Urtica dioica leaves modulates muscarinic cholinergic system in the hippocampus of streptozotocin-induced diabetic mice.

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Patel, Sita Sharan; Parashar, Arun; Udayabanu, Malairaman

2015-06-01

Diabetes mellitus is a chronic metabolic disorder and has been associated with cognitive dysfunction. In our earlier study, chronic Urtica dioica (UD) treatment significantly ameliorated diabetes induced associative and spatial memory deficit in mice. The present study was designed to explore the effect of UD leaves extract on muscarinic cholinergic system, which has long been known to be involved in cognition. Streptozotocin (STZ) (50 mg/kg, i.p., consecutively for 5 days) was used to induce diabetes followed by treatment with UD extract (50 mg/kg, oral) or rosiglitazone (5 mg/kg, oral) for 8 weeks. STZ-induced diabetic mice showed significant reduction in hippocampal muscarinic acetylcholine receptor-1 and choline acetyltransferase expressions. Chronic diabetes significantly up-regulated the protein expression of acetylcholinesterase associated with oxidative stress in hippocampus. Besides, STZ-induced diabetic mice showed hypolocomotion with up-regulation of muscarinic acetylcholine receptor-4 expression in striatum. Chronic UD treatment significantly attenuated the cholinergic dysfunction and oxidative stress in the hippocampus of diabetic mice. UD had no effect on locomotor activity and muscarinic acetylcholine receptor-4 expression in striatum. In conclusion, UD leaves extract has potential to reverse diabetes mediated alteration in muscarinic cholinergic system in hippocampus and thereby improve memory functions.

Muscarinic acetylcholine receptor subtypes: localization and structure/function

DEFF Research Database (Denmark)

Brann, M R; Ellis, J; Jørgensen, H

1993-01-01

Based on the sequence of the five cloned muscarinic receptor subtypes (m1-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues...... are described, as well as the implied structures of these functional domains....

Comparative study of muscarinic acetylcholine receptors of human and rat cortical glial cells

International Nuclear Information System (INIS)

Demushkin, V.P.; Burbaeva, G.S.; Dzhaliashvili, T.A.; Plyashkevich, Y.G.

1985-01-01

The aim of the present investigation was a comparative studyof muscarinic acetylcholine receptors in human and rat glial cells. ( 3 H)Quinuclidinyl-benzylate (( 3 H)-QB), atropine, platiphylline, decamethonium, carbamylcholine, tubocurarine, and nicotine were used. The glial cell fraction was obtained from the cerebral cortex of rats weighing 130-140 g and from the frontal pole of the postmortem brain from men aged 60-70 years. The use of the method of radioimmune binding of ( 3 H)-QB with human and rat glial cell membranes demonstrated the presence of a muscarinic acetylcholine receptor in the glial cells

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

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González, Alfredo; Crittenden, Elizabeth L; García, Dana M

2004-07-13

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

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

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Crittenden Elizabeth L

2004-07-01

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

Muscarinic Acetylcholine Receptor M3 Mutation Causes Urinary Bladder Disease and a Prune-Belly-like Syndrome.

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Weber, Stefanie; Thiele, Holger; Mir, Sevgi; Toliat, Mohammad Reza; Sozeri, Betül; Reutter, Heiko; Draaken, Markus; Ludwig, Michael; Altmüller, Janine; Frommolt, Peter; Stuart, Helen M; Ranjzad, Parisa; Hanley, Neil A; Jennings, Rachel; Newman, William G; Wilcox, Duncan T; Thiel, Uwe; Schlingmann, Karl Peter; Beetz, Rolf; Hoyer, Peter F; Konrad, Martin; Schaefer, Franz; Nürnberg, Peter; Woolf, Adrian S

2011-11-11

Urinary bladder malformations associated with bladder outlet obstruction are a frequent cause of progressive renal failure in children. We here describe a muscarinic acetylcholine receptor M3 (CHRM3) (1q41-q44) homozygous frameshift mutation in familial congenital bladder malformation associated with a prune-belly-like syndrome, defining an isolated gene defect underlying this sometimes devastating disease. CHRM3 encodes the M3 muscarinic acetylcholine receptor, which we show is present in developing renal epithelia and bladder muscle. These observations may imply that M3 has a role beyond its known contribution to detrusor contractions. This Mendelian disease caused by a muscarinic acetylcholine receptor mutation strikingly phenocopies Chrm3 null mutant mice. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Adenosine receptors and muscarinic receptors cooperate in acetylcholine release modulation in the neuromuscular synapse.

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Santafe, M M; Priego, M; Obis, T; Garcia, N; Tomàs, M; Lanuza, M A; Tomàs, J

2015-07-01

Adenosine receptors (ARs) are present in the motor terminals at the mouse neuromuscular junction. ARs and the presynaptic muscarinic acetylcholine receptors (mAChRs) share the functional control of the neuromuscular junction. We analysed their mutual interaction in transmitter release modulation. In electrophysiological experiments with unaltered synaptic transmission (muscles paralysed by blocking the voltage-dependent sodium channel of the muscle cells with μ-conotoxin GIIIB), we found that: (i) a collaborative action between different AR subtypes reduced synaptic depression at a moderate activity level (40 Hz); (ii) at high activity levels (100 Hz), endogenous adenosine production in the synaptic cleft was sufficient to reduce depression through A1 -type receptors (A1 Rs) and A2 A-type receptors (A2 A Rs); (iii) when the non-metabolizable 2-chloroadenosine (CADO) agonist was used, both the quantal content and depression were reduced; (iv) the protective effect of CADO on depression was mediated by A1 Rs, whereas A2 A Rs seemed to modulate A1 Rs; (v) ARs and mAChRs absolutely depended upon each other for the modulation of evoked and spontaneous acetylcholine release in basal conditions and in experimental conditions with CADO stimulation; (vi) the purinergic and muscarinic mechanisms cooperated in the control of depression by sharing a common pathway although the purinergic control was more powerful than the muscarinic control; and (vii) the imbalance of the ARs created by using subtype-selective and non-selective inhibitory and stimulatory agents uncoupled protein kinase C from evoked transmitter release. In summary, ARs (A1 Rs, A2 A Rs) and mAChRs (M1 , M2 ) cooperated in the control of activity-dependent synaptic depression and may share a common protein kinase C pathway. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Muscarinic receptor antagonists for overactive bladder treatment: does one fit all?

NARCIS (Netherlands)

Witte, Lambertus P. W.; Mulder, Wilhelmina M. C.; de La Rosette, Jean J. M. C. H.; Michel, Martin C.

2009-01-01

Purpose of review To review evidence and regulatory dosing recommendations for muscarinic receptor antagonists used in the treatment of overactive bladder symptom complex (darifenacin, fesoterodine oxybutynin propiverine solifenacin tolterodine trospium) in special patient populations. Recent

Muscarinic receptors as targets for anti-inflammatory therapy.

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Sales, María Elena

2010-11-01

ACh, the main neurotransmitter in the neuronal cholinergic system, is synthesized by pre-ganglionic fibers of the sympathetic and parasympathetic autonomic nervous system and by post-ganglionic parasympathetic fibers. There is increasing experimental evidence that ACh is widely expressed in prokaryotic and eukaryotic non-neuronal cells. The neuronal and non-neuronal cholinergic systems comprise ACh, choline acetyltransferase and cholinesterase, enzymes that synthesize and catabolize ACh, and the nicotinic and muscarinic ACh receptors (nAChRs and mAChRs, respectively), which are the targets for ACh action. This review analyzes the participation of the cholinergic system, particularly through mAChRs, in inflammation, and discusses the role of the different mAChR antagonists that have been used to treat skin inflammatory disorders, asthma and COPD, as well as intestinal inflammation and systemic inflammatory diseases, to assess the potential application of these compounds as therapeutic tools.

Quantitative autoradiography of muscarinic and benzodiazepine receptors in the forebrain of the turtle, Pseudemys scripta

International Nuclear Information System (INIS)

Schlegel, J.R.; Kriegstein, A.R.

1987-01-01

The distribution of muscarinic and benzodiazepine receptors was investigated in the turtle forebrain by the technique of in vitro receptor autoradiography. Muscarinic binding sites were labeled with 1 nM 3 H-quinuclidinyl benzilate ( 3 H-QNB), and benzodiazepine sites were demonstrated with the aid of 1 nM 3 H-flunitrazepam ( 3 H-FLU). Autoradiograms generated on 3 H-Ultrofilm apposed to tissue slices revealed regionally specific distributions of muscarinic and benzodiazepine binding sites that are comparable with those for mammalian brain. Dense benzodiazepine binding was found in the anterior olfactory nucleus, the lateral and dorsal cortices, and the dorsal ventricular ridge (DVR), a structure with no clear mammalian homologue. Muscarinic binding sites were most dense in the striatum, accumbens, DVR, lateral geniculate, and the anterior olfactory nucleus. Cortical binding sites were studied in greater detail by quantitative analysis of autoradiograms generated by using emulsion-coated coverslips. Laminar gradients of binding were observed that were specific for each radioligand; 3 H-QNB sites were most dense in the inner molecular layer in all cortical regions, whereas 3 H-FLU binding was generally most concentrated in the outer molecular layer and was least dense through all layers in the dorsomedial cortex. Because pyramidal cells are arranged in register in turtle cortex, the laminar patterns of receptor binding may reflect different receptor density gradients along pyramidal cell dendrites

M3 muscarinic receptor interaction with phospholipase C beta3 determines its signaling efficiency

NARCIS (Netherlands)

Kan, W.; Adjobo-Hermans, M.J.; Burroughs, M.; Faibis, G.; Malik, S.; Tall, G.G.; Smrcka, A.V.

2014-01-01

Phospholipase Cbeta (PLCbeta) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on Galphabetagamma heterotrimers containing Gq family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and

Attenuation of cocaine's reinforcing and discriminative stimulus effects via muscarinic M1 acetylcholine receptor stimulation

DEFF Research Database (Denmark)

Thomsen, Morgane; Conn, P Jeffrey; Lindsley, Craig

2010-01-01

substituted for cocaine and enhanced its discriminative stimulus. Conversely, muscarinic agonists blunted cocaine discrimination and abolished cocaine self-administration with varying effects on food-maintained behavior. Specifically, increasing selectivity for the M(1) subtype (oxotremorine ...'s abuse-related effects, whereas non-M(1)/M(4) receptors probably contribute to undesirable effects of muscarinic stimulation. These data provide the first demonstration of anticocaine effects of systemically applied, M(1) receptor agonists and suggest the possibility of a new approach to pharmacotherapy...

Perirhinal Cortex Muscarinic Receptor Blockade Impairs Taste Recognition Memory Formation

Science.gov (United States)

Gutierrez, Ranier; De la Cruz, Vanesa; Rodriguez-Ortiz, Carlos J.; Bermudez-Rattoni, Federico

2004-01-01

The relevance of perirhinal cortical cholinergic and glutamatergic neurotransmission for taste recognition memory and learned taste aversion was assessed by microinfusions of muscarinic (scopolamine), NMDA (AP-5), and AMPA (NBQX) receptor antagonists. Infusions of scopolamine, but not AP5 or NBQX, prevented the consolidation of taste recognition…

The pharmacological rationale for combining muscarinic receptor antagonists and beta-adrenoceptor agonists in the treatment of airway and bladder disease

NARCIS (Netherlands)

Dale, Philippa R.; Cernecka, Hana; Schmidt, Martina; Dowling, Mark R.; Charlton, Steven J.; Pieper, Michael P.; Michel, Martin C.

Muscarinic receptor antagonists and beta-adrenoceptor agonists are used in the treatment of obstructive airway disease and overactive bladder syndrome. Here we review the pharmacological rationale for their combination. Muscarinic receptors and beta-adrenoceptors are physiological antagonists for

Connexins and M3 Muscarinic Receptors Contribute to Heterogeneous Ca2+ Signaling in Mouse Aortic Endothelium

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François-Xavier Boittin

2013-02-01

Full Text Available Background/Aims: Smooth muscle tone is controlled by Ca2+ signaling in the endothelial layer. Mouse endothelial cells are interconnected by gap junctions made of Connexin40 (Cx40 and Cx37, which allow the exchange of signaling molecules to coordinate their activity. Here, we investigated the role of Cx40 in the endothelial Ca2+ signaling of the mouse aorta. Methods: Ca2+ imaging was performed on intact aortic endothelium from both wild type (Cx40+/+ and Connexin40-deficient (Cx40 -/- mice. Results: Acetylcholine (ACh induced early fast and high amplitude Ca2+ transients in a fraction of endothelial cells expressing the M3 muscarinic receptors. Inhibition of intercellular communication using carbenoxolone or octanol fully blocked the propagation of ACh-induced Ca2+ transients toward adjacent cells in WT and Cx40-/- mice. As compared to WT, Cx40-/- mice displayed a reduced propagation of ACh-induced Ca2+ waves, indicating that Cx40 contributes to the spreading of Ca2+ signals. The propagation of those Ca2+ responses was not blocked by suramin, a blocker of purinergic ATP receptors, indicating that there is no paracrine effect of ATP release on the Ca2+ waves. Conclusions: Altogether our data show that Cx40 and Cx37 contribute to the propagation and amplification of the Ca2+ signaling triggered by ACh in endothelial cells expressing the M3 muscarinic receptors.

Cholinergic nicotinic and muscarinic receptors in dementia of Alzheimer, Parkinson and Lewy body types.

Science.gov (United States)

Perry, E K; Smith, C J; Court, J A; Perry, R H

1990-01-01

Cholinergic nicotinic and muscarinic receptor binding were measured in post mortem human brain tissue, using low (nM) concentrations of (3H)-nicotine to detect predominately the high affinity nicotinic site and (3H)-N-methylscopolamine in the presence and absence of 3 x 10(-4) M carbachol to measure both the low and high affinity agonist subtypes of the muscarinic receptor group. Consistent with most previous reports, the nicotinic but not muscarinic binding was reduced in the different forms of dementia associated with cortical cholinergic deficits, including Alzheimer's and Parkinson's disease, senile dementia of Lewy body type (SDLT) and Down's syndrome (over 50 years). Analysis of (3H)-nicotine binding displaced by a range of carbachol concentrations (10(-9)-10(-3) M) indicated 2 binding sites for nicotine and that the high affinity rather than low affinity site was reduced in Alzheimer's disease. In all 3 cortical areas investigated (temporal, parietal and occipital) there were increases in the low affinity muscarinic site in Parkinson's disease and SDLT but not Alzheimer's disease or middle-aged Down's syndrome. This observation raised the question of whether the presence of neurofibrillary tangles (evident in the latter but not former 2 disorders) is incompatible with denervation-induced muscarinic supersensitivity in cholinoceptive neurons which include cortical pyramids generally affeted by tangle formation.

Comparative Effects of Oral Chlorpyrifos Exposure on Cholinesterase Activity and Muscarinic Receptor Binding in Neonatal and Adult Rat Heart

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Howard, Marcia D.; Mirajkar, Nikita; Karanth, Subramanya; Pope, Carey N.

2010-01-01

Organophosphorus (OP) pesticides elicit acute toxicity by inhibiting acetylcholinesterase (AChE), the enzyme responsible for inactivating acetylcholine (ACh) at cholinergic synapses. A number of OP toxicants have also been reported to interact directly with muscarinic receptors, in particular the M2 muscarinic subtype. Parasympathetic innervation to the heart primarily regulates cardiac function by activating M2 receptors in the sinus node, atrial-ventricular node and conducting tissues. Thus, OP insecticides can potentially influence cardiac function in a receptor–mediated manner indirectly by inhibiting acetylcholinesterase and directly by binding to muscarinic M2 receptors. Young animals are generally more sensitive than adults to the acute toxicity of OP insecticides and age related differences in potency of direct binding to muscarinic receptors by some OP toxicants have been reported. We thus compared the effects of the common OP insecticide chlorpyrifos (CPF) on functional signs of toxicity and cardiac ChE activity and muscarinic receptor binding in neonatal and adult rats. Dosages were based on acute lethality (i.e., 0.5 and 1 × LD10: neonates, 7.5 and 15 mg/kg; adults, 68 and 136 mg/kg). Dose- and time-related changes in body weight and cholinergic signs of toxicity (involuntary movements) were noted in both age groups. With 1 × LD10, relatively similar maximal reductions in ChE activity (95%) and muscarinic receptor binding (≈ 30%) were noted, but receptor binding reductions appeared earlier in adults and were more prolonged in neonates. In vitro inhibition studies indicated that ChE in neonatal tissues was markedly more sensitive to inhibition by the active metabolite of chlorpyrifos (i.e., chlorpyrifos oxon, CPO) than enzyme in adult tissues (IC50 values: neonates, 17 nM; adults, 200 nM). Chelation of free calcium with EDTA had relatively little effect on in vitro cholinesterase inhibition, suggesting that differential A-esterase activity was not

Involvement of Striatal Cholinergic Interneurons and M1 and M4 Muscarinic Receptors in Motor Symptoms of Parkinson's Disease.

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Ztaou, Samira; Maurice, Nicolas; Camon, Jeremy; Guiraudie-Capraz, Gaëlle; Kerkerian-Le Goff, Lydia; Beurrier, Corinne; Liberge, Martine; Amalric, Marianne

2016-08-31

Over the last decade, striatal cholinergic interneurons (ChIs) have reemerged as key actors in the pathophysiology of basal-ganglia-related movement disorders. However, the mechanisms involved are still unclear. In this study, we address the role of ChI activity in the expression of parkinsonian-like motor deficits in a unilateral nigrostriatal 6-hydroxydopamine (6-OHDA) lesion model using optogenetic and pharmacological approaches. Dorsal striatal photoinhibition of ChIs in lesioned ChAT(cre/cre) mice expressing halorhodopsin in ChIs reduces akinesia, bradykinesia, and sensorimotor neglect. Muscarinic acetylcholine receptor (mAChR) blockade by scopolamine produces similar anti-parkinsonian effects. To decipher which of the mAChR subtypes provides these beneficial effects, systemic and intrastriatal administration of the selective M1 and M4 mAChR antagonists telenzepine and tropicamide, respectively, were tested in the same model of Parkinson's disease. The two compounds alleviate 6-OHDA lesion-induced motor deficits. Telenzepine produces its beneficial effects by blocking postsynaptic M1 mAChRs expressed on medium spiny neurons (MSNs) at the origin of the indirect striatopallidal and direct striatonigral pathways. The anti-parkinsonian effects of tropicamide were almost completely abolished in mutant lesioned mice that lack M4 mAChRs specifically in dopamine D1-receptor-expressing neurons, suggesting that postsynaptic M4 mAChRs expressed on direct MSNs mediate the antiakinetic action of tropicamide. The present results show that altered cholinergic transmission via M1 and M4 mAChRs of the dorsal striatum plays a pivotal role in the occurrence of motor symptoms in Parkinson's disease. The striatum, where dopaminergic and cholinergic systems interact, is the pivotal structure of basal ganglia involved in pathophysiological changes underlying Parkinson's disease. Here, using optogenetic and pharmacological approaches, we investigated the involvement of striatal

Outline of therapeutic interventions with muscarinic receptor-mediated transmission

Czech Academy of Sciences Publication Activity Database

Jakubík, Jan; Šantrůčková, Eva; Randáková, Alena; Janíčková, Helena; Zimčík, Pavel; Rudajev, Vladimír; Michal, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

2014-01-01

Roč. 63, Suppl.1 (2014), S177-S189 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA305/09/0681; GA ČR(CZ) GAP304/12/0259; GA MŠk(CZ) 7E10060 Institutional support: RVO:67985823 Keywords : cholinergic transmission * muscarinic receptors * therapy * Alzheimer's disease, * schizophrenia Subject RIV: ED - Physiology Impact factor: 1.293, year: 2014

Visualization of cholinoceptive neurons in the rat neocortex : colocalization of muscarinic and nicotinic acetylcholine receptors

NARCIS (Netherlands)

Zee, E.A. van der; Streefland, C.; Strosberg, A.D.; Schröder, H.; Luiten, P.G.M.

The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of

Vitamin C Deficiency Reduces Muscarinic Receptor Coronary Artery Vasoconstriction and Plasma Tetrahydrobiopterin Concentration in Guinea Pigs

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Gry Freja Skovsted

2017-07-01

Full Text Available Vitamin C (vitC deficiency is associated with increased cardiovascular disease risk, but its specific interplay with arteriolar function is unclear. This study investigates the effect of vitC deficiency in guinea pigs on plasma biopterin status and the vasomotor responses in coronary arteries exposed to vasoconstrictor/-dilator agents. Dunkin Hartley female guinea pigs (n = 32 were randomized to high (1500 mg/kg diet or low (0 to 50 mg/kg diet vitC for 10–12 weeks. At euthanasia, coronary artery segments were dissected and mounted in a wire-myograph. Vasomotor responses to potassium, carbachol, sodium nitroprusside (SNP, U46619, sarafotoxin 6c (S6c and endothelin-1 (ET-1 were recorded. Plasma vitC and tetrahydrobiopterin were measured by HPLC. Plasma vitC status reflected the diets with deficient animals displaying reduced tetrahydrobiopterin. Vasoconstrictor responses to carbachol were significantly decreased in vitC deficient coronary arteries independent of their general vasoconstrictor/vasodilator capacity (p < 0.001. Moreover, in vitC deficient animals, carbachol-induced vasodilator responses correlated with coronary artery diameter (p < 0.001. Inhibition of cyclooxygenases with indomethacin increased carbachol-induced vasoconstriction, suggesting an augmented carbachol-induced release of vasodilator prostanoids. Atropine abolished carbachol-induced vasomotion, supporting a specific muscarinic receptor effect. Arterial responses to SNP, potassium, S6c, U46619 and ET-1 were unaffected by vitC status. The study shows that vitC deficiency decreases tetrahydrobiopterin concentrations and muscarinic receptor mediated contraction in coronary arteries. This attenuated vasoconstrictor response may be linked to altered production of vasoactive arachidonic acid metabolites and reduced muscarinic receptor expression/signaling.

Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

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Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria Angel; Cilleros, Victor; Tomàs, Josep Maria

2016-01-01

The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M1, M2, and M4 subtypes) and adenosine receptors (A1 and A2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A2A, M1, M4, and A1 receptors (in this order of ability) delayed axonal elimination at P7. M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway, which suggests that they cooperate. M2 receptors may modulate (by allowing a permissive action) the other receptors, mainly M4 and A1. The continued action of these receptors (now including M2 but not M4) finally promotes axonal loss at P9. All 4 receptors (M2, M1, A1, and A2A, in this order of ability) are necessary. The M4 receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M1, A1, and A2A receptors, which show an additive effect, whereas the potent M2 effect is largely independent of the other receptors (though can be modulated by M4). At P9, there is a full mutual dependence between the A1 and A2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes. �

The A- and B-type muscarinic acetylcholine receptors from Drosophila melanogaster couple to different second messenger pathways

DEFF Research Database (Denmark)

Ren, Guilin Robin; Folke, Jonas; Hauser, Frank

2015-01-01

Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors (GPCRs) that are activated by the agonists acetylcholine and muscarine and blocked by several antagonists, among them atropine. In mammals five mAChRs (m1-m5) exist of which m1, m3, and m5 are coupled to members of the Gq...

Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

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Marlet Martinez-Archundia

2012-01-01

Full Text Available The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS. Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

Muscarinic Acetylcholine Receptors Act in Synergy to Facilitate Learning and Memory

Science.gov (United States)

Leaderbrand, Katherine; Chen, Helen J.; Corcoran, Kevin A.; Guedea, Anita L.; Jovasevic, Vladimir; Wess, Jurgen; Radulovic, Jelena

2016-01-01

Understanding how episodic memories are formed and retrieved is necessary if we are to treat disorders in which they malfunction. Muscarinic acetylcholine receptors (mAChR) in the hippocampus and cortex underlie memory formation, but there is conflicting evidence regarding their role in memory retrieval. Additionally, there is no consensus on…

Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ.

Science.gov (United States)

Słoniecka, Marta; Le Roux, Sandrine; Boman, Peter; Byström, Berit; Zhou, Qingjun; Danielson, Patrik

2015-01-01

Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and β2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides

Increased cocaine self-administration in M4 muscarinic acetylcholine receptor knockout mice

DEFF Research Database (Denmark)

Schmidt, Lene Sørensen; Thomsen, Morgane; Weikop, Pia

2011-01-01

Rationale The reinforcing effects of cocaine are mediated by the mesolimbic dopamine system. Behavioral and neurochemical studies have shown that the cholinergic muscarinic M4 receptor subtype plays an important role in regulation of dopaminergic neurotransmission. Objectives Here we investigated...... of drug addiction...

Iodine-123 N-methyl-4-iododexetimide: a new radioligand for single-photon emission tomographic imaging of myocardial muscarinic receptors

International Nuclear Information System (INIS)

Hicks, R.J.; Kassiou, M.; Eu, P.; Katsifis, A.G.; Garra, M.; Power, J.; Najdovski, L.; Lambrecht, R.M.

1995-01-01

Cardiac muscarinic receptor ligands suitable for positron emission tomography have previously been characterised. Attempts to develop radioligands of these receptors suitable for single-photon emission tomographic (SPET) imaging have not been successful due to high lung retention and high non-specific binding of previously investigated potential tracers. The purpose of this study was to evaluate the biodistribution and in vivo imaging characteristics of a new radiopharmaceutical, [ 123 I]N-methyl-4-iododexetimide. Biodistribution studies performed in rats showed high cardiac uptake (2.4% ID/g) 10 min after injection with a heart to lung activity ratio of 5:1. Specificity and stereoselectivity of cardiac binding were demonstrated using blocking experiments in rats. Dynamic imaging studies in anaesthetised greyhounds demonstrated rapid and high myocardial uptake and low lung binding with stable heart to lung activity ratios of >2.5:1 between 10 and 30 min, making SPET imaging feasible. Administration of an excess of an unlabelled muscarinic antagonist, methyl-quinuclidinyl benzylate rapidly displaced myocardial activity to background levels and the pharmacologically inactive enantiomer, [ 123 I]N-methyl-4-iodolevetimide, had no detectable cardiac uptake, indicating specific and stereoselective muscarinic receptor binding. SPET revealed higher activity in the inferior than in the anterior wall, this being consistent with previously described regional variation of cardiac parasympathetic innervation. [ 123 I]N-methyl-4-iododexetimide shows promise as an imaging agent for muscarinic receptor distribution in the heart and may be helpful in evaluating diverse cardiac diseases associated with altered muscarinic receptor function, including heart failure and diabetic heart disease. (orig.)

A novel muscarinic receptor ligand which penetrates the blood brain barrier and displays in vivo selectivity for the m2 subtype

International Nuclear Information System (INIS)

Gitler, M.S.; Cohen, V.I.; De La Cruz, R.; Boulay, S.F.; Jin, B.; Zeeberg, B.R.; Reba, R.C.

1993-01-01

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in the posterior parietal cortex of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood-brain barrier. In our efforts to prepare such a radioligand, the authors have used competition studies against currently existing muscarinic receptor radioligands to infer the in vitro and in vivo properties of a novel muscarinic receptor ligand, 5-[[4-[4-(diisobutylamino)butyl]-1-phenyl]acetyl]-10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-11-one (DIBD). In vitro competition studies against [ 3 H](R)-3-quinuclidinylbenzilate ([ 3 H]QNB) and [ 3 H]N-methylscopolamine ([ 3 H]NMS), using membranes derived from transfected cells expressing only m1, m2, m3, or m4 receptor subtypes, indicate that DIBD is selective for m2/m4 over m1/m3. In vivo competition studies against (R,R)-[ 125 I]IQNB indicate that DIBD crosses the blood brain barrier (BBB). The relationship of the regional percentage decrease in (R,R)-[ 125 I]IQNB versus the percentage of each of the receptor subtypes indicates that DIBD competes more effectively in those brain regions which are known to be enriched in the m2, relative to the m1, m3, and m4, receptor subtype; however, analysis of the data using a mathematical model shows that caution is required when interpreting the in vivo results. The authors conclude that a suitably radiolabeled derivative of DIBD may be of potential use in emission tomographic study of changes in m2 receptors in the central nervous system

Effects of dopamine D1-like and D2-like antagonists on cocaine discrimination in muscarinic receptor knockout mice.

Science.gov (United States)

Thomsen, Morgane; Caine, Simon Barak

2016-04-05

Muscarinic and dopamine brain systems interact intimately, and muscarinic receptor ligands, like dopamine ligands, can modulate the reinforcing and discriminative stimulus (S(D)) effects of cocaine. To enlighten the dopamine/muscarinic interactions as they pertain to the S(D) effects of cocaine, we evaluated whether muscarinic M1, M2 or M4 receptors are necessary for dopamine D1 and/or D2 antagonist mediated modulation of the S(D) effects of cocaine. Knockout mice lacking M1, M2, or M4 receptors, as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline in a food-reinforced drug discrimination procedure. Effects of pretreatments with the dopamine D1 antagonist SCH 23390 and the dopamine D2 antagonist eticlopride were evaluated. In intact mice, both SCH 23390 and eticlopride attenuated the cocaine discriminative stimulus effect, as expected. SCH 23390 similarly attenuated the cocaine discriminative stimulus effect in M1 knockout mice, but not in mice lacking M2 or M4 receptors. The effects of eticlopride were comparable in each knockout strain. These findings demonstrate differences in the way that D1 and D2 antagonists modulate the S(D) effects of cocaine, D1 modulation being at least partially dependent upon activity at the inhibitory M2/M4 muscarinic subtypes, while D2 modulation appeared independent of these systems. Copyright © 2016 Elsevier B.V. All rights reserved.

Muscarinic and alpha 1-adrenergic receptor binding characteristics of saw palmetto extract in rat lower urinary tract.

Science.gov (United States)

Suzuki, Mayumi; Oki, Tomomi; Sugiyama, Tomomi; Umegaki, Keizo; Uchida, Shinya; Yamada, Shizuo

2007-06-01

To elucidate the in vitro and ex vivo effects of saw palmetto extract (SPE) on autonomic receptors in the rat lower urinary tract. The in vitro binding affinities for alpha 1-adrenergic, muscarinic, and purinergic receptors in the rat prostate and bladder were measured by radioligand binding assays. Rats received vehicle or SPE (0.6 to 60 mg/kg/day) orally for 4 weeks, and alpha 1-adrenergic and muscarinic receptor binding in tissues of these rats were measured. Saw palmetto extract inhibited specific binding of [3H]prazosin and [N-methyl-3H]scopolamine methyl chloride (NMS) but not alpha, beta-methylene adenosine triphosphate [2,8-(3)H]tetrasodium salt in the rat prostate and bladder. The binding activity of SPE for muscarinic receptors was four times greater than that for alpha 1-adrenergic receptors. Scatchard analysis revealed that SPE significantly reduced the maximal number of binding sites (Bmax) for each radioligand in the prostate and bladder under in vitro condition. Repeated oral administration of SPE to rats brought about significant alteration in Bmax for prostatic [3H]prazosin binding and for bladder [3H]NMS binding. Such alteration by SPE was selective to the receptors in the lower urinary tract. Saw palmetto extract exerts significant binding activity on autonomic receptors in the lower urinary tract under in vitro and in vivo conditions.

Autoradiography of H-3-pirenzepine and H-3-AFDX-384 in Mouse Brain Regions: Possible Insights into M-1, M-2, and M-4 Muscarinic Receptors Distribution

Czech Academy of Sciences Publication Activity Database

Valuskova, P.; Farar, V.; Forczek, Sándor; Křížová, I.; Mysliveček, J.

2018-01-01

Roč. 9, FEB 20 (2018), č. článku 124. ISSN 1663-9812 Institutional support: RVO:61389030 Keywords : 3 h-afdx-384 * 3 H-pirenzepine * 3 h-qnb * Autoradiography * M muscarinic receptor 1 * M muscarinic receptor 2 * M muscarinic receptor 4 Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 4.400, year: 2016

Effects of selective activation of M1 and M4 muscarinic receptors on object recognition memory performance in rats.

Science.gov (United States)

Galloway, Claire R; Lebois, Evan P; Shagarabi, Shezza L; Hernandez, Norma A; Manns, Joseph R

2014-01-01

Acetylcholine signaling through muscarinic receptors has been shown to benefit memory performance in some conditions, but pan-muscarinic activation also frequently leads to peripheral side effects. Drug therapies that selectively target M1 or M4 muscarinic receptors could potentially improve memory while minimizing side effects mediated by the other muscarinic receptor subtypes. The ability of three recently developed drugs that selectively activate M1 or M4 receptors to improve recognition memory was tested by giving Long-Evans rats subcutaneous injections of three different doses of the M1 agonist VU0364572, the M1 positive allosteric modulator BQCA or the M4 positive allosteric modulator VU0152100 before performing an object recognition memory task. VU0364572 at 0.1 mg/kg, BQCA at 1.0 mg/kg and VU0152100 at 3.0 and 30.0 mg/kg improved the memory performance of rats that performed poorly at baseline, yet the improvements in memory performance were the most statistically robust for VU0152100 at 3.0 mg/kg. The results suggested that selective M1 and M4 receptor activation each improved memory but that the likelihood of obtaining behavioral efficacy at a given dose might vary between subjects even in healthy groups depending on baseline performance. These results also highlighted the potential of drug therapies that selectively target M1 or M4 receptors to improve memory performance in individuals with impaired memory.

Ventricular, but not atrial, M2-muscarinic receptors increase in the canine pacing-overdrive model of heart failure.

Science.gov (United States)

Wilkinson, M; Giles, A; Armour, J A; Cardinal, R

1996-01-01

To investigate the effects of heart failure induced by chronic rapid ventricular pacing (six weeks) on canine atrial and ventricular muscarinic receptors. Dogs (n = 4) were fitted with a bipolar pacing electrode connected to a Medtronic pacemaker set at 240 stimuli/min. Pacing was maintained for six weeks. Tissue samples obtained from the left atrium and ventral wall of the left ventricle were frozen at -70 degrees C. Control tissue was obtained from normal dogs (n = 6) following anesthesia and thoracotomy. M2-muscarinic receptors were characterized and quantified in tissue micropunches using the hydrophilic ligand [3H] N-methyl-scopolamine (NMS). Cardiac tissue bound [3H] NMS with the specificity of an M2 subtype. Tachycardia-induced heart failure did not affect atrial muscarinic receptors but signify left ventricular myocytes (control 160.0 +/- 10.0 fmol/mg protein versus heart failure 245.0 +/- 25.0 fmol/mg protein; P failure was accompanied by an increase (+ 53%) in ventricular, but not atrial, M2 receptors compared with normal dogs.

Cholinergic regulation of VIP gene expression in human neuroblastoma cells

DEFF Research Database (Denmark)

Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

1997-01-01

Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

Muscarinic receptors in separate populations of noradrenaline- and adrenaline-containing chromaffin cells

International Nuclear Information System (INIS)

Michelena, P.; Moro, M.A.; Castillo, C.J.; Garcia, A.G.

1991-01-01

We have performed binding experiments of (a)[3H]quinuclidinyl benzilate to partially purified membranes from noradrenaline- and adrenaline-containing chromaffin cells and (b) [3H]N-methyl-quinuclidinyl benzilate to acutely isolated, or 48-h cultured, chromaffin cells subpopulations. Using this approach, we obtained enough evidence to conclude (1st) that muscarinic receptors are present in both noradrenaline- and adrenaline containing cells; (2nd) that noradrenaline cells contain in fact 2-3 fold higher density of those receptors; and (3rd) that those receptors undergo plastic changes upon chronic culturing of the cells

Perirhinal Cortex Muscarinic Receptor Blockade Impairs Taste Recognition Memory Formation

OpenAIRE

Gutiérrez, Ranier; De la Cruz, Vanesa; Rodriguez-Ortiz, Carlos J.; Bermudez-Rattoni, Federico

2004-01-01

The relevance of perirhinal cortical cholinergic and glutamatergic neurotransmission for taste recognition memory and learned taste aversion was assessed by microinfusions of muscarinic (scopolamine), NMDA (AP-5), and AMPA (NBQX) receptor antagonists. Infusions of scopolamine, but not AP5 or NBQX, prevented the consolidation of taste recognition memory using attenuation of neophobia as an index. In addition, learned taste aversion in both short- and long-term memory tests was exclusively impa...

Investigation of the presence and antinociceptive function of muscarinic acetylcholine receptors in the African naked mole-rat (Heterocephalus glaber)

DEFF Research Database (Denmark)

Jørgensen, Kristine B.; Krogh-Jensen, Karen; Pickering, Darryl S

2016-01-01

The present study investigated the cholinergic system in the African naked mole-rat (Heterocephalus glaber) with focus on the muscarinic acetylcholine receptor subtypes M1 and M4. The protein sequences for the subtypes m 1–5 of the naked mole-rat were compared to that of the house mouse (Mus...... musculus) using basic local alignment search tool (BLAST). The presence and function of M1 and M4 was investigated in vivo, using the formalin test with the muscarinic receptor agonists xanomeline and VU0152100. Spinal cord tissue from the naked mole-rat was used for receptor saturation binding studies...

Biological sex influences learning strategy preference and muscarinic receptor binding in specific brain regions of prepubertal rats.

Science.gov (United States)

Grissom, Elin M; Hawley, Wayne R; Hodges, Kelly S; Fawcett-Patel, Jessica M; Dohanich, Gary P

2013-04-01

According to the theory of multiple memory systems, specific brain regions interact to determine how the locations of goals are learned when rodents navigate a spatial environment. A number of factors influence the type of strategy used by rodents to remember the location of a given goal in space, including the biological sex of the learner. We recently found that prior to puberty male rats preferred a striatum-dependent stimulus-response strategy over a hippocampus-dependent place strategy when solving a dual-solution task, while age-matched females showed no strategy preference. Because the cholinergic system has been implicated in learning strategy and is known to be sexually dimorphic prior to puberty, we explored the relationship between learning strategy and muscarinic receptor binding in specific brain regions of prepubertal males and female rats. We confirmed our previous finding that at 28 days of age a significantly higher proportion of prepubertal males preferred a stimulus-response learning strategy than a place strategy to solve a dual-solution visible platform water maze task. Equal proportions of prepubertal females preferred stimulus-response or place strategies. Profiles of muscarinic receptor binding as assessed by autoradiography varied according to strategy preference. Regardless of biological sex, prepubertal rats that preferred stimulus-response strategy exhibited lower ratios of muscarinic receptor binding in the hippocampus relative to the dorsolateral striatum compared to rats that preferred place strategy. Importantly, much of the variance in this ratio was related to differences in the ventral hippocampus to a greater extent than the dorsal hippocampus. The ratios of muscarinic receptors in the hippocampus relative to the basolateral amygdala also were lower in rats that preferred stimulus-response strategy over place strategy. Results confirm that learning strategy preference varies with biological sex in prepubertal rats with males

Why does the agonist [18F]FP-TZTP bind preferentially to the M2 muscarinic receptor?

International Nuclear Information System (INIS)

Ravasi, L.; Kiesewetter, D.O.; Shimoji, K.; Lucignani, G.; Eckelman, W.C.

2006-01-01

Preferential binding of FP-TZTP at the M 2 receptor in vivo led to investigation of [ 18 F]FP-TZTP as a potential PET tracer for Alzheimer's disease, in which a substantial reduction of M 2 receptors has been observed in autopsy studies. We hereby investigated in vitro the FP-TZTP behavior to further elucidate the properties of FP-TZTP that lead to its M 2 selectivity. Chinese hamster ovarian cells expressing the five subtypes of human muscarinic receptor as well as the wild type were harvested in culture to assess equilibrium binding. Specific binding was calculated by subtraction of non-specific binding from total binding. Internal specific binding was calculated by subtraction of external specific binding from the total specific binding. Saturation assays were also performed to calculate B max , K i , and IC 50 . In addition, equilibrium binding and dissociation kinetic studies were performed on rat brain tissue. Selected regions of interest were drawn on the digital autoradiograms and [ 18 F]FP-TZTP off-rates were determined by measurement of the rate of release into a buffer solution of [ 18 F]FP-TZTP from slide-bound cells that had been preincubated with [ 18 F]FP-TZTP. At equilibrium in vitro, M 2 subtype selectivity of [ 18 F]FP-TZTP was not evident. We demonstrated that ATP-dependent mechanisms are not responsible for FP-TZTP M 2 selectivity. In vitro off-rate studies from rat brain tissue showed that the off-rate of FP-TZTP varied with the percentage of M 2 subtype in the tissue region. The slower dissociation kinetics of FP-TZTP from M 2 receptors compared with the four other muscarinic receptor subtypes may be a factor in its M 2 selectivity. (orig.)

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

Czech Academy of Sciences Publication Activity Database

Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

2017-01-01

Roč. 7, Jan 16 (2017), č. článku 40381. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptors * N-methylscopolamine * ligand binding * molecular dynamics Subject RIV: ED - Physiology OBOR OECD: Physiology (including cytology) Impact factor: 4.259, year: 2016

Role of ventrolateral orbital cortex muscarinic and nicotinic receptors in modulation of capsaicin-induced orofacial pain-related behaviors in rats.

Science.gov (United States)

Tamaddonfard, Esmaeal; Erfanparast, Amir; Abbas Farshid, Amir; Delkhosh-Kasmaie, Fatmeh

2017-11-15

Acetylcholine, as a major neurotransmitter, mediates many brain functions such as pain. This study was aimed to investigate the effects of microinjection of muscarinic and nicotinic acetylcholine receptor antagonists and agonists into the ventrolateral orbital cortex (VLOC) on capsaicin-induced orofacial nociception and subsequent hyperalgesia. The right side of VLOC was surgically implanted with a guide cannula in anaesthetized rats. Orofacial pain-related behaviors were induced by subcutaneous injection of a capsaicin solution (1.5µg/20µl) into the left vibrissa pad. The time spent face rubbing with ipsilateral forepaw and general behavior were recorded for 10min, and then mechanical hyperalgesia was determined using von Frey filaments at 15, 30, 45 and 60min post-capsaicin injection. Alone intra-VLOC microinjection of atropine (a muscarinic acetylcholine receptor antagonist) and mecamylamine (a nicotinic acetylcholine receptor antagonist) at a similar dose of 200ng/site did not alter nocifensive behavior and hyperalgesia. Microinjection of oxotremorine (a muscarinic acetylcholine receptor agonist) at doses of 50 and 100ng/site and epibatidine (a nicotinic acetylcholine receptor agonist) at doses of 12.5, 25, 50 and 100ng/site into the VLOC suppressed pain-related behaviors. Prior microinjections of 200ng/site atropine and mecamylamine (200ng/site) prevented oxotremorine (100ng/site)-, and epibatidine (100ng/site)-induced antinociception, respectively. None of the above-mentioned chemicals changed general behavior. These results showed that the VLOC muscarinic and nicotinic acetylcholine receptors might be involved in modulation of orofacial nociception and hypersensitivity. Copyright © 2017 Elsevier B.V. All rights reserved.

The muscarinic M1/M4 receptor agonist xanomeline exhibits antipsychotic-like activity in Cebus apella monkeys

DEFF Research Database (Denmark)

Andersen, Maibritt B; Fink-Jensen, Anders; Peacock, Linda

2003-01-01

Xanomeline is a muscarinic M(1)/M(4) preferring receptor agonist with little or no affinity for dopamine receptors. The compound reduces psychotic-like symptoms in patients with Alzheimer's disease and exhibits an antipsychotic-like profile in rodents without inducing extrapyramidal side effects ...

An allosteric enhancer of M4muscarinic acetylcholine receptor function inhibits behavioral and neurochemical effects of cocaine

DEFF Research Database (Denmark)

Dencker, Ditte; Weikop, Pia; Sørensen, Gunnar

2012-01-01

The mesostriatal dopamine system plays a key role in mediating the reinforcing effects of psychostimulant drugs like cocaine. The muscarinic M4 acetylcholine receptor subtype is centrally involved in the regulation of dopamine release in striatal areas. Consequently, striatal M4 receptors could...

Characterization of muscarinic and P2X receptors in the urothelium and detrusor muscle of the rat bladder

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Masaki Ogoda

2016-05-01

Full Text Available Muscarinic and purinergic (P2X receptors play critical roles in bladder urothelium under physiological and pathological conditions. Aim of present study was to characterize these receptors in rat bladder urothelium and detrusor muscle using selective radioligands of [N-methyl-3H]scopolamine methyl chloride ([3H]NMS and αβ-methylene ATP [2,8-3H]tetrasodium salt ([3H]αβ-MeATP. Similar binding parameters for each radioligand were observed in urothelium and detrusor muscle. Pretreatment with N-(2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP mustard mustard revealed co-existence of M2 and M3 receptors, with the number of M2 receptors being larger in the urothelium and detrusor muscle. Intravesical administration of imidafenacin and Dpr-P-4 (N → O (active metabolite of propiverine displayed significant binding of muscarinic receptors in the urothelium and detrusor muscle. The treatment with cyclophosphamide (CYP or resiniferatoxin (RTX resulted in a significant decrease in maximal number of binding sites (Bmax for [3H]NMS and/or [3H]αβ-MeATP in the urothelium and detrusor muscle. These results demonstrated that 1 pharmacological characteristics of muscarinic and P2X receptors in rat bladder urothelium were similar to those in the detrusor muscle, 2 that densities of these receptors were significantly altered by pretreatments with CYP and RTX, and 3 that these receptors may be pharmacologically affected by imidafenacin and Dpr-P-4 (N → O which are excreted in the urine.

Blocking muscarinic receptors in the olfactory bulb impairs performance on an olfactory short term memory task

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Sasha eDevore

2012-09-01

Full Text Available Cholinergic inputs to cortical processing networks have long been associated with attentional and top-down processing. Experimental and theoretical studies suggest that cholinergic inputs to the main olfactory bulb (OB can modulate both neural and behavioral odor discrimination. Previous experiments from our laboratory and others demonstrate that blockade of nicotinic receptors directly impairs olfactory discrimination, whereas blockade of muscarinic receptors only measurably impairs olfactory perception when task demands are made more challenging, such as when very low-concentration odors are used or rats are required to maintain sensory memory over long durations. To further investigate the role of muscarinic signaling in the OB, we developed an olfactory delayed match-to-sample task using a digging-based behavioral paradigm. We find that rats are able to maintain robust short-term odor memory for tens to hundreds of seconds. To investigate the role of muscarinic signaling in task performance, we bilaterally infused scopolamine into the OB. We find that high dosages of scopolamine (38 mM impair performance on the task across all delays tested, including the baseline condition with no delay, whereas lower dosages (7.6 mM and 22.8 mM had no measureable effects. These results indicate that general execution of the match-to-sample task, even with no delay, is at least partially dependent on muscarinic signaling in the OB.

Blocking muscarinic receptors in the olfactory bulb impairs performance on an olfactory short-term memory task.

Science.gov (United States)

Devore, Sasha; Manella, Laura C; Linster, Christiane

2012-01-01

Cholinergic inputs to cortical processing networks have long been associated with attentional and top-down processing. Experimental and theoretical studies suggest that cholinergic inputs to the main olfactory bulb (OB) can modulate both neural and behavioral odor discrimination. Previous experiments from our laboratory and others demonstrate that blockade of nicotinic receptors directly impairs olfactory discrimination, whereas blockade of muscarinic receptors only measurably impairs olfactory perception when task demands are made more challenging, such as when very low-concentration odors are used or rats are required to maintain sensory memory over long durations. To further investigate the role of muscarinic signaling in the OB, we developed an olfactory delayed match-to-sample task using a digging-based behavioral paradigm. We find that rats are able to maintain robust short-term odor memory for 10-100 s. To investigate the role of muscarinic signaling in task performance, we bilaterally infused scopolamine into the OB. We find that high dosages of scopolamine (38 mM) impair performance on the task across all delays tested, including the baseline condition with no delay, whereas lower dosages (7.6 mM and 22.8 mM) had no measureable effects. These results indicate that general execution of the match-to-sample task, even with no delay, is at least partially dependent on muscarinic signaling in the OB.

Role of membrane cholesterol in differential sensitivity of muscarinic receptor subtypes to persistently bound xanomeline

Czech Academy of Sciences Publication Activity Database

Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, E. E.; Jakubík, Jan

2018-01-01

Roč. 133, May 1 (2018), s. 129-144 ISSN 0028-3908 R&D Projects: GA ČR(CZ) GA14-05696S; GA ČR(CZ) GA17-16182S Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptors * membrane cholesterol * xanomeline * receptor activation * molecular dynamics Subject RIV: ED - Physiology OBOR OECD: Physiology (including cytology) Impact factor: 5.012, year: 2016

Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors

Czech Academy of Sciences Publication Activity Database

Jakubík, Jan; Randáková, Alena; El-Fakahany, E. E.; Doležal, Vladimír

2009-01-01

Roč. 9, č. 15 (2009), s. 1-20 ISSN 1471-2210 R&D Projects: GA ČR GA305/09/0681; GA MŠk(CZ) LC554; GA AV ČR(CZ) IAA500110703 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * allosteric modulation * rapacuronium Subject RIV: ED - Physiology

Drugs Interfering with Muscarinic Acetylcholine Receptors and Their Effects on Place Navigation

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Jan Svoboda

2017-11-01

Full Text Available Muscarinic acetylcholine receptors (mAChRs have been found to regulate many diverse functions, ranging from motivation and feeding to spatial navigation, an important and widely studied type of cognitive behavior. Systemic administration of non-selective antagonists of mAChRs, such as scopolamine or atropine, have been found to have adverse effects on a vast majority of place navigation tasks. However, many of these results may be potentially confounded by disruptions of functions other than spatial learning and memory. Although studies with selective antimuscarinics point to mutually opposite effects of M1 and M2 receptors, their particular contribution to spatial cognition is still poorly understood, partly due to a lack of truly selective agents. Furthermore, constitutive knock-outs do not always support results from selective antagonists. For modeling impaired spatial cognition, the scopolamine-induced amnesia model still maintains some limited validity, but there is an apparent need for more targeted approaches such as local intracerebral administration of antagonists, as well as novel techniques such as optogenetics focused on cholinergic neurons and chemogenetics aimed at cells expressing metabotropic mAChRs.

Selective expression of muscarinic acetylcholine receptor subtype M3 by mouse type III taste bud cells.

Science.gov (United States)

Mori, Yusuke; Eguchi, Kohgaku; Yoshii, Kiyonori; Ohtubo, Yoshitaka

2016-11-01

Each taste bud cell (TBC) type responds to a different taste. Previously, we showed that an unidentified cell type(s) functionally expresses a muscarinic acetylcholine (ACh) receptor subtype, M3, and we suggested the ACh-dependent modification of its taste responsiveness. In this study, we found that M3 is expressed by type III TBCs, which is the only cell type that possesses synaptic contacts with taste nerve fibers in taste buds. The application of ACh to the basolateral membrane of mouse fungiform TBCs in situ increased the intracellular Ca 2+ concentration in 2.4 ± 1.4 cells per taste bud (mean ± SD, n = 14). After Ca 2+ imaging, we supravitally labeled type II cells (phospholipase C β2 [PLCβ2]-immunoreactive cells) with Lucifer yellow CH (LY), a fluorescent dye and investigated the positional relationship between ACh-responding cells and LY-labeled cells. After fixation, the TBCs were immunohistostained to investigate the positional relationships between immunohistochemically classified cells and LY-labeled cells. The overlay of the two positional relationships obtained by superimposing the LY-labeled cells showed that all of the ACh-responding cells were type III cells (synaptosomal-associated protein 25 [SNAP-25]-immunoreactive cells). The ACh responses required no added Ca 2+ in the bathing solution. The addition of 1 μM U73122, a phospholipase C inhibitor, decreased the magnitude of the ACh response, whereas that of 1 μM U73343, a negative control, had no effect. These results suggest that type III cells respond to ACh and release Ca 2+ from intracellular stores. We also discuss the underlying mechanism of the Ca 2+ response and the role of M3 in type III cells.

Muscarinic receptors modulate dendrodendritic inhibitory synapses to sculpt glomerular output.

Science.gov (United States)

Liu, Shaolin; Shao, Zuoyi; Puche, Adam; Wachowiak, Matt; Rothermel, Markus; Shipley, Michael T

2015-04-08

Cholinergic [acetylcholine (ACh)] axons from the basal forebrain innervate olfactory bulb glomeruli, the initial site of synaptic integration in the olfactory system. Both nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs) are expressed in glomeruli. The activation of nAChRs directly excites both mitral/tufted cells (MTCs) and external tufted cells (ETCs), the two major excitatory neurons that transmit glomerular output. The functional roles of mAChRs in glomerular circuits are unknown. We show that the restricted glomerular application of ACh causes rapid, brief nAChR-mediated excitation of both MTCs and ETCs in the mouse olfactory bulb. This excitation is followed by mAChR-mediated inhibition, which is blocked by GABAA receptor antagonists, indicating the engagement of periglomerular cells (PGCs) and/or short axon cells (SACs), the two major glomerular inhibitory neurons. Indeed, selective activation of glomerular mAChRs, with ionotropic GluRs and nAChRs blocked, increased IPSCs in MTCs and ETCs, indicating that mAChRs recruit glomerular inhibitory circuits. Selective activation of glomerular mAChRs in the presence of tetrodotoxin increased IPSCs in all glomerular neurons, indicating action potential-independent enhancement of GABA release from PGC and/or SAC dendrodendritic synapses. mAChR-mediated enhancement of GABA release also presynaptically suppressed the first synapse of the olfactory system via GABAB receptors on sensory terminals. Together, these results indicate that cholinergic modulation of glomerular circuits is biphasic, involving an initial excitation of MTC/ETCs mediated by nAChRs followed by inhibition mediated directly by mAChRs on PGCs/SACs. This may phasically enhance the sensitivity of glomerular outputs to odorants, an action that is consistent with recent in vivo findings. Copyright © 2015 the authors 0270-6474/15/355680-13$15.00/0.

Regional distribution of muscarinic acetylcholine receptors in the telencephalon of the pigeon (Columba livia f. domestica)

International Nuclear Information System (INIS)

Waechtler, K.

1985-01-01

The distribution of muscarinic acetylcholine receptors was studied autoradiographically in croystat sections of the pigeon telencephalon using 3 H-quinuclidinylbenzylate as a ligand. Highest receptor density was observed in the hyperstriatum ventrale, palaeostriatum augmentatum, septum, and parts of the archistriatum. In sites of known sensory input of neostriatum (field L) and ectostriatum low receptor binding was observed. Acetylcholinesterase distribution is in good agreement with the receptor picture only in the basal telencephalon. In the pallium differences in the pattern of these two components can be seen. (author)

Presynaptic muscarinic receptors: Change of sensitivity during long-term drug treatment

International Nuclear Information System (INIS)

Marchi, M.; Raiteri, M.

1986-01-01

The authors investigate some of the characteristics of auto- and heteroreceptors from different brain areas in male rats; their alteration in sensitivity following chronic drug treatment is monitored. The synaptosomes were prelabeled with tritium-choline or tritium-dopamine and the release of tritium-acetylcholine and tritium-DA was studied in superfusion. It is shown that the difference in susceptibility between auto- and heteroreceptors with respect to changes of sensitivity may represent a further criterion to discriminate between muscarinic receptor subtypes

Hook-up of GluA2, GRIP and liprin-α for cholinergic muscarinic receptor-dependent LTD in the hippocampus

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Wu Long-Jun

2009-06-01

Full Text Available Abstract The molecular mechanism underlying muscarinic acetylcholine receptor-dependent LTD (mAChR-LTD in the hippocampus is less studied. In a recent study, a novel mechanism is described. The induction of mAChR-LTD required the activation of protein tyrosine phosphatase (PTP, and the expression was mediated by AMPA receptor endocytosis via interactions between GluA2, GRIP and liprin-α. The hook-up of these proteins may result in the recruitment of leukocyte common antigen-related receptor (LAR, a PTP that is known to be involved in AMPA receptor trafficking. Interestingly, the similar molecular interaction cannot be applied to mGluR-LTD, despite the fact that the same G-protein involved in LTD is activated by both mAChR and mGluR. This discovery provides key molecular insights for cholinergic dependent cognitive function, and mAChR-LTD can serve as a useful cellular model for studying the roles of cholinergic mechanism in learning and memory.

Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms

Czech Academy of Sciences Publication Activity Database

Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, E. E.; Jakubík, Jan

2015-01-01

Roč. 97, Jul 2015 (2015), s. 27-39 ISSN 1043-6618 R&D Projects: GA ČR(CZ) GA305/09/0681; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptors * atypical agonists * xanomeline * activation mechanism Subject RIV: ED - Physiology Impact factor: 4.816, year: 2015

In vivo and in vitro studies on the potentiation of muscarinic receptor stimulation by alaproclate, a selective 5-HT uptake blocker

International Nuclear Information System (INIS)

Oegren, S.O.; Nordstroem, Oe.; Danielsson, E.; Peterson, L.-L.; Bartfai, T.

1985-01-01

Alaproclate (10-60 mg/kg) injected i.p. into male mice potentiated and prolonged the oxotremorine and physostigmine-induced tremor in a dosedependent manner. Atropine completely blocked the tremor caused by oxotremorine or physostigmine both in the presence and absence of alaproclate. Pretreatment with the 5-HT receptor antagonist metitepine completely blocked the enhancement of oxotremorine-induced tremor caused by alaproclate. Biochemical studies indicated that the above effects cannot be explained by assuming that alaproclate a) acts as a cholinergic agonist, b) inhibits the acetylcholine esterase, c) interferes with choline uptake or acetylcholine synthesis, or d) directly potentiates the release of acetylcholine. In ligand binding studies alaproclate was found to be a weak competitive inhibitor of muscarinic antagonist binding to membranes from the rat cerebral cortex, rat striatum, human cerebral cortex and human striatum. (Ksub(i) approximately 28-40 μM in all four tissues). The present results suggest that alaproclate may potentiate muscarinic responses by a mechanism involving serotonergic receptor mechanisms rather than by a direct interaction with the muscarinic cholinergic receptors. (Author)

In vivo and in vitro studies on the potentiation of muscarinic receptor stimulation by alaproclate, a selective 5-HT uptake blocker

Energy Technology Data Exchange (ETDEWEB)

Oegren, S.O. (Astra Pharmaceuticals AB, Soedertaelje (Sweden)); Nordstroem, Oe.; Danielsson, E.; Peterson, L.L.; Bartfai, T.

1985-01-01

Alaproclate (10-60 mg/kg) injected i.p. into male mice potentiated and prolonged the oxotremorine and physostigmine-induced tremor in a dose dependent manner. Atropine completely blocked the tremor caused by oxotremorine or physostigmine both in the presence and absence of alaproclate. Pretreatment with the 5-HT receptor antagonist metitepine completely blocked the enhancement of oxotremorine-induced tremor caused by alaproclate. Biochemical studies indicated that the above effects cannot be explained by assuming that alaproclate a) acts as a cholinergic agonist, b) inhibits the acetylcholine esterase, c) interferes with choline uptake or acetylcholine synthesis, or d) directly potentiates the release of acetylcholine. In ligand binding studies alaproclate was found to be a weak competitive inhibitor of muscarinic antagonist binding to membranes from the rat cerebral cortex, rat striatum, human cerebral cortex and human striatum. (Ksub(i) approximately 28-40 ..mu..M in all four tissues). The present results suggest that alaproclate may potentiate muscarinic responses by a mechanism involving serotonergic receptor mechanisms rather than by a direct interaction with the muscarinic cholinergic receptors.

Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

International Nuclear Information System (INIS)

Sanborn, B.B.; Schneider, A.S.

1990-01-01

Inositol trisphosphate (IP 3 ), a product of the phosphoinositide cycle, mobilizes intracellular Ca 2+ in many cell types. New evidence suggests that inositol tetrakisphosphate (IP 4 ), an IP 3 derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP 4 are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP 4 in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine stimulated an increase in [ 3 H]IP 4 and [ 3 H]IP 3 accumulation in chromaffin cells and this effect was completely blocked by atropine. [ 3 H]IP 4 accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP 3 and IP 4 hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP 4 and calcium homeostasis

Costimulation of N-methyl-d-aspartate and muscarinic neuronal receptors modulates gap junctional communication in striatal astrocytes

OpenAIRE

Rouach, N.; Tencé, M.; Glowinski, J.; Giaume, C.

2002-01-01

Cocultures of neurons and astrocytes from the rat striatum were used to determine whether the stimulation of neuronal receptors could affect the level of intercellular communication mediated by gap junctions in astrocytes. The costimulation of N-methyl-D-asparte (NMDA) and muscarinic receptors led to a prominent reduction of astrocyte gap junctional communication (GJC) in coculture. This treatment was not effective in astrocyte cultures, these cells being devoid of NMDA receptors. Both types ...

Identification of four areas each enriched in a unique muscarinic receptor subtype

International Nuclear Information System (INIS)

Hoss, W.; Ellerbrock, B.R.; Goldman, P.S.; Collins, D.A.; Messer, W.S. Jr.

1990-01-01

The affinities of muscarinic agonists and antagonists were determined by autoradiography and image analysis in selected areas of the rat brain. IC 50 values and Hill coefficients for the inhibition of the binding of 0.2 nM [ 3 H]-QNB to dentate gyrus, superior colliculus, rhomboid thalamus and substantia nigra were measured in coronal sections. Pirenzepine displayed a high affinity for receptors in the dentate gyrus and AF-DX 116, the superior colliculus. Both pirenzepine and AF-DX 116 had high affinities for the substantia nigra and low affinities for the rhomboid thalamus. Gallamine displayed a 50-fold preference for superior colliculus over dentate gyrus receptors. Amitriptyline was less selective, showing a modest preference for substantia nigra receptors and 4-DAMP was essentially nonselective. Carbachol was the most selective agonist with a 4000-fold preference for superior colliculus over dentate gyrus receptors. Other agonists except RS 86 were also selective for superior colliculus receptors in the order carbachol >> arecoline > bethanechol > McN A343 = oxotremorine = pilocarpine

Neuronal M3 muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth.

Science.gov (United States)

Gautam, Dinesh; Jeon, Jongrye; Starost, Matthew F; Han, Sung-Jun; Hamdan, Fadi F; Cui, Yinghong; Parlow, Albert F; Gavrilova, Oksana; Szalayova, Ildiko; Mezey, Eva; Wess, Jürgen

2009-04-14

The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the M(3) muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from M(3) receptor/GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) M(3) receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central M(3) receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells.

Effect of paraoxon on muscarinic, dopamine and γ-aminobutyric acid receptors of brain and sensitivity to muscarinic antagonists

International Nuclear Information System (INIS)

Fernando, J.C.R.; Hoskins, B.; Ho, I.K.

1986-01-01

Several acetylcholinesterase (AChE) inhibitors decrease muscarinic cholinergic (mACh) receptors in the brain, alteration of dopamine (DA) and γ-aminobutyric acid (GABA) receptors after AChE inhibition was also reported. In view of the important interactions among DA, GABA and ACh systems, whether this is a common effect of AChE inhibitors should be established. They report the effect of the AChE inhibitor, paraoxon, on DA, GABA and mACh receptors in the rat. The binding of 3 H-QNB (for mACh), 3 H-spiperone (for DA) and 3 H-muscimol (for GABA) to striatal and hippocampal membranes was analyzed. Also, behavioral sensitivity to atropine was studied. Twenty-four hr after a single dose (0.75 mg/kg, s.c.) of paraoxon, the density of mACh receptors in the striatum was decreased but, at 3 days, no change was seen. In the hippocampus, the mACh receptors were not affected. Repeated treatment with paraoxon (0.3 mg/kg, 48 hourly) for 2 weeks reduced the mACh receptor density in both regions. Neither single nor repeated paraoxon treatment had an effect on DA or GABA receptors. After single or repeated dosing with paraoxon, myoclonus induced by atropine (10 mg/kg, i.p.) was enhanced. The results show rapid downregulation of mACh receptors by paraoxon. DA or GABA, however, appear not to be affected under these treatment regimens

Dual effects of muscarinic M2 acetylcholine receptors on the synthesis of cyclic AMP in CHO cells: dependence on time, receptor density and receptor agonists

Czech Academy of Sciences Publication Activity Database

Michal, Pavel; Lysíková, Michaela; Tuček, Stanislav

2001-01-01

Roč. 132, č. 6 (2001), s. 1217-1228 ISSN 0007-1188 R&D Projects: GA ČR GA309/99/0214; GA AV ČR IAA7011910 Institutional research plan: CEZ:AV0Z5011922 Keywords : cyclic AMP * muscarinic receptors * CHO cells Subject RIV: ED - Physiology Impact factor: 3.502, year: 2001

The effects of nicotinic and muscarinic receptor activation on patch-clamped cells in the optic tectum of Rana pipiens.

Science.gov (United States)

Yu, C-J; Debski, E A

2003-01-01

Both nicotinic and muscarinic cholinergic receptors are present in the optic tectum. To begin to understand how the activation of these receptors affects visual activity patterns, we have determined the types of physiological responses induced by their activation. Using tectal brain slices from the leopard frog, we found that application of nicotine (100 microM) evoked long-lasting responses in 60% of patch-clamped tectal cells. Thirty percent of these responses consisted of an increase in spontaneous postsynaptic currents (sPSCs) and had both a glutamatergic and GABAergic component as determined by the use of 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM) and bicuculline (25 microM), respectively. Remaining response types consisted of an inward membrane current (16%) and an increase in sPSCs combined with an inward membrane current (14%). All responses could be elicited in the presence of tetrodotoxin (0.5 microM). Muscarinic receptor-mediated responses, induced by carbachol (100 microM) application after nicotinic receptor desensitization, produced responses in 70% of tectal cells. In contrast to responses elicited by nicotine, carbachol-induced responses could be evoked multiple times without significant decrement. Responses consisted of either an outward current (57%), a decrease in sPSCs (5%) or an increase in sPSCs, with (almost 6%) or without (almost 3%) an outward current. The response elicited by carbachol was not predicted by the response of the cell to nicotine. Our results suggest that nicotinic receptors are found predominantly at presynaptic locations in the optic tectum while muscarinic receptors are most often present at postsynaptic sites. We conclude that both of these receptor types could substantially modulate visual activity by changing either the input to tectal neurons or the level of their response to that input.

Permanent alterations in muscarinic receptors and pupil size produced by chronic atropinization in kittens

International Nuclear Information System (INIS)

Smith, E.L.; Redburn, D.A.; Harwerth, R.S.; Maguire, G.W.

1984-01-01

Chronic mydriasis was induced in six kittens (four monocular, two binocular) and two adult cats (both monocular) by the daily topical application of atropine. Both the kittens and the adult cats were atropinized for a 13-week period with the treatment regimen beginning at the time of eye opening for the kittens. Pupil size measurements, obtained 1 year after the atropinization were discontinued, revealed that, although the pupils of the adult cats were normal, the pupils of the kittens' treated eyes were consistently smaller than pupils in control eyes. The status of the muscarinic receptors in the kittens' irides was investigated using 3 H-QNB binding assays. In comparison with iris muscle homogenates from the control eyes, those from the treated eyes demonstrated an eightfold increase in the number of receptor binding sites. The results indicate that pupil size can be altered permanently by chronic mydriasis initiated early in the life of a kitten and that the permanent change in pupil size may result, in part, from a type of permanent supersensitivity response in the muscle following chronic blockade of muscarinic transmission by atropine

Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

Science.gov (United States)

Redka, Dar'ya S; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V; Ellis, John; Ernst, Oliver P; Wells, James W

2014-08-29

G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[(3)H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Determinants of positive cooperativity between strychnine-like allosteric modulators and N-methylscopolamine at muscarinic receptors

Czech Academy of Sciences Publication Activity Database

Jakubík, Jan; Doležal, Vladimír

2006-01-01

Roč. 30, č. 1-2 (2006), s. 111-112 ISSN 0895-8696 R&D Projects: GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * strychnine -like allosteric modulators * cooperativity Subject RIV: ED - Physiology Impact factor: 2.965, year: 2006

Distinct interneuron types express m2 muscarinic receptor immunoreactivity on their dendrites or axon terminals in the hippocampus.

Science.gov (United States)

Hájos, N; Papp, E C; Acsády, L; Levey, A I; Freund, T F

1998-01-01

In previous studies m2 muscarinic acetylcholine receptor-immunoreactive interneurons and various types of m2-positive axon terminals have been described in the hippocampal formation. The aim of the present study was to identify the types of interneurons expressing m2 receptor and to examine whether the somadendritic and axonal m2 immunostaining labels the same or distinct cell populations. In the CA1 subfield, neurons immunoreactive for m2 have horizontal dendrites, they are located at the stratum oriens/alveus border and have an axon that project to the dendritic region of pyramidal cells. In the CA3 subfield and the hilus, m2-positive neurons are multipolar and are scattered in all layers except stratum lacunosum-moleculare. In stratum pyramidale of the CA1 and CA3 regions, striking axon terminal staining for m2 was observed, surrounding the somata and axon initial segments of pyramidal cells in a basket-like manner. The co-localization of m2 with neurochemical markers and GABA was studied using the "mirror" technique and fluorescent double-immunostaining at the light microscopic level and with double-labelling using colloidal gold-conjugated antisera and immunoperoxidase reaction (diaminobenzidine) at the electron microscopic level. GABA was shown to be present in the somata of most m2-immunoreactive interneurons, as well as in the majority of m2-positive terminals in all layers. The calcium-binding protein parvalbumin was absent from practically all m2-immunoreactive cell bodies and dendrites. In contrast, many of the terminals synapsing on pyramidal cell somata and axon initial segments co-localized parvalbumin and m2, suggesting a differential distribution of m2 receptor immunoreactivity on the axonal and somadendritic membrane of parvalbumin-containing basket and axo-axonic cells. The co-existence of m2 receptors with the calcium-binding protein calbindin and the neuropeptides cholecystokinin and vasoactive intestinal polypeptide was rare throughout the

Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

Science.gov (United States)

Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

2017-06-05

Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The distribution of cerebral muscarinic acetylcholine receptors in vivo in patients with dementia. A controlled study with 123IQNB and single photon emission computed tomography

International Nuclear Information System (INIS)

Weinberger, D.R.; Gibson, R.; Coppola, R.; Jones, D.W.; Molchan, S.; Sunderland, T.; Berman, K.F.; Reba, R.C.

1991-01-01

A high-affinity muscarinic receptor antagonist, 123IQNB (3-quinuclidinyl-4-iodobenzilate labeled with iodine 123), was used with single photon emission computed tomography to image muscarinic acetylcholine receptors in 14 patients with dementia and in 11 healthy controls. High-resolution single photon emission computed tomographic scanning was performed 21 hours after the intravenous administration of approximately 5 mCi of IQNB. In normal subjects, the images of retained ligand showed a consistent regional pattern that correlated with postmortem studies of the relative distribution of muscarinic receptors in the normal human brain, having high radioactivity counts in the basal ganglia, occipital cortex, and insular cortex, low counts in the thalamus, and virtually no counts in the cerebellum. Eight of 12 patients with a clinical diagnosis of Alzheimer's disease had obvious focal cortical defects in either frontal or posterior temporal cortex. Both patients with a clinical diagnosis of Pick's disease had obvious frontal and anterior temporal defects. A region of interest statistical analysis of relative regional activity revealed a significant reduction bilaterally in the posterior temporal cortex of the patients with Alzheimer's disease compared with controls. This study demonstrates the practicability of acetylcholine receptor imaging with 123IQNB and single photon emission computed tomography. The data suggest that focal abnormalities in muscarinic binding in vivo may characterize some patients with Alzheimer's disease and Pick's disease, but further studies are needed to address questions about partial volume artifacts and receptor quantification

Hypoglycemia induced changes in cholinergic receptor expression in the cerebellum of diabetic rats

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Anju TR

2010-02-01

Full Text Available Abstract Glucose homeostasis in humans is an important factor for the functioning of nervous system. Hypoglycemia and hyperglycemia is found to be associated with central and peripheral nerve system dysfunction. Changes in acetylcholine receptors have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS. In the present study we showed the effects of insulin induced hypoglycemia and streptozotocin induced diabetes on the cerebellar cholinergic receptors, GLUT3 and muscle cholinergic activity. Results showed enhanced binding parameters and gene expression of Muscarinic M1, M3 receptor subtypes in cerebellum of diabetic (D and hypoglycemic group (D + IIH and C + IIH. α7nAchR gene expression showed a significant upregulation in diabetic group and showed further upregulated expression in both D + IIH and C + IIH group. AchE expression significantly upregulated in hypoglycemic and diabetic group. ChAT showed downregulation and GLUT3 expression showed a significant upregulation in D + IIH and C + IIH and diabetic group. AchE activity enhanced in the muscle of hypoglycemic and diabetic rats. Our studies demonstrated a functional disturbance in the neuronal glucose transporter GLUT3 in the cerebellum during insulin induced hypoglycemia in diabetic rats. Altered expression of muscarinic M1, M3 and α7nAchR and increased muscle AchE activity in hypoglycemic rats in cerebellum is suggested to cause cognitive and motor dysfunction. Hypoglycemia induced changes in ChAT and AchE gene expression is suggested to cause impaired acetycholine metabolism in the cerebellum. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. The results shows that cerebellar cholinergic neurotransmission is impaired during hyperglycemia and hypoglycemia and the hypoglycemia is causing more prominent imbalance in cholinergic neurotransmission which is suggested to be a cause of cerebellar

Activation of multiple G-proteins by muscarinic M1 and M2 receptors

Czech Academy of Sciences Publication Activity Database

Michal, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

2006-01-01

Roč. 27, č. S1 (2006), s. 404-404 ISSN 1671-4083. [World Congress of Pharmacology /15./. 02.07.2006-07.07.2006, Beijing] R&D Projects: GA ČR(CZ) GP305/05/P209; GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * multiple G-protein coupling Subject RIV: ED - Physiology

Experiment K-6-18. Study of muscarinic and gaba (benzodiazepine) receptors in the sensory-motor cortex, hippcampus and spinal code

Science.gov (United States)

Daunton, N.; Damelio, F.; Krasnov, I.

1990-01-01

Frontal lobe samples of rat brains flown aboard Cosmos 1887 were processed for the study of muscarinic (cholinergic) and GABA (benzodiazepine) receptors and for immunocytochemical localization of the neurotransmitter gamma-aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP). Although radioactive labeling of both muscarinic cholinergic and GABA (benzodiazepine) receptors proved to be successful with the techniques employed, distinct receptor localization of individual laminae of the frontal neocortex was not possible since the sampling of the area was different in the various groups of animals. In spite of efforts made for proper orientation and regional identification of laminae, it was found that a densitometric (quantitation of autoradiograms) analysis of the tissue did not contribute to the final interpretation of the effects of weightlessness on these receptors. As to the immunocytochemical studies the use of both markers, GFAP and GABA antiserum, confirmed the suitability of the techniques for use in frozen material. However, similar problems to those encountered in the receptor studies prevented an adequate interpretation of the effects of micro-G exposure on the localization and distribution of GABA and GFAP. This study did, however, confirm the feasibility of investigating neurotransmitters and their receptors in future space flight experiments.

Uncoupling of M1 muscarinic receptor/G-protein interaction by amyloid beta(1-42)

Czech Academy of Sciences Publication Activity Database

Janíčková, Helena; Rudajev, Vladimír; Zimčík, Pavel; Jakubík, Jan; Tanila, H.; El-Fakahany, E. E.; Doležal, Vladimír

2013-01-01

Roč. 67, April (2013), s. 272-283 ISSN 0028-3908 R&D Projects: GA ČR(CZ) GA305/09/0681; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) 7E10060 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : Alzheimer ´s Disease * muscarinic receptors * G-proteins Subject RIV: ED - Physiology Impact factor: 4.819, year: 2013

Nucleus accumbens neurotransmission and effort-related choice behavior in food motivation: effects of drugs acting on dopamine, adenosine, and muscarinic acetylcholine receptors.

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Nunes, Eric J; Randall, Patrick A; Podurgiel, Samantha; Correa, Mercè; Salamone, John D

2013-11-01

Mesolimbic dopamine (DA) is a critical component of the brain circuitry regulating behavioral activation and effort-related processes. Although nucleus accumbens (NAc) DA depletions or antagonism leave aspects of appetite and primary food motivation intact, rats with impaired DA transmission reallocate their instrumental behavior away from food-reinforced tasks with high response requirements, and instead select less effortful food-seeking behaviors. Previous work showed that adenosine A2A antagonists can reverse the effects of DA D2 antagonists on effort-related choice, and that stimulation of adenosine A2A receptors produces behavioral effects that are similar to those induced by DA antagonism. The present review summarizes the literature on the role of NAc DA and adenosine in effort-related processes, and also presents original data on the effects of local stimulation of muscarinic acetylcholine receptors in NAc core. Local injections of the muscarinic agonist pilocarpine directly into NAc core produces shifts in effort-related choice behavior similar to those induced by DA antagonism or A2A receptor stimulation, decreasing lever pressing but increasing chow intake in rats responding on a concurrent fixed ratio/chow feeding choice task. In contrast, injections into a neostriatal control site dorsal to the NAc were ineffective. The actions of pilocarpine on this task were attenuated by co-administration of the muscarinic antagonist scopolamine. Thus, drugs that act on DA, adenosine A2A, and muscarinic receptors regulate effort-related choice behavior, which may have implications for the treatment of psychiatric symptoms such as psychomotor slowing, fatigue or anergia that can be observed in depression and other disorders. Copyright © 2013 Elsevier Ltd. All rights reserved.

Topographical distribution of decrements and recovery in muscarinic receptors from rat brains repeatedly exposed to sublethal doses of soman

International Nuclear Information System (INIS)

Churchill, L.; Pazdernik, T.L.; Jackson, J.L.; Nelson, S.R.; Samson, F.E.; McDonough, J.H. Jr.

1984-01-01

[3H]Quinuclidinyl benzilate binding to rat brain muscarinic receptors decreased after repeated exposure to soman, a potent organophosphorus cholinesterase inhibitor. The topographical distribution of this decrement was analyzed by quantitative receptor autoradiography. After 4 weeks of soman, three times a week, quinuclidinyl benzilate binding decreased to 67 to 80% of control in frontal and parietal cortex, caudate-putamen, lateral septum, hippocampal body, dentate gyrus, superior colliculus, nucleus of the fifth nerve, and central grey. Minor or no decreases were observed in thalamic or hypothalamic nuclei, reticular formation, pontine nuclei, inferior colliculus, nucleus of the seventh nerve, and cerebellum. Scatchard analyses of saturation curves using frontal cortex sections from soman-treated rats revealed a decrease in maximal quinuclidinyl benzilate binding from that in control rats and a return toward control levels by 24 days without any significant change in affinity. These brain areas showing significant decrements in muscarinic receptors recovered with a similar time course. An estimate of the time for 50% recovery for some of the brain areas was 14 days for superior colliculus, 16 days for cortex, and 19 days for hippocampal body. The application of quantitative receptor autoradiography to analyze receptor alterations has been valuable in localizing the telencephalon as a region more susceptible to change in receptor concentration

Topographical distribution of decrements and recovery in muscarinic receptors from rat brains repeatedly exposed to sublethal doses of soman

Energy Technology Data Exchange (ETDEWEB)

Churchill, L.; Pazdernik, T.L.; Jackson, J.L.; Nelson, S.R.; Samson, F.E.; McDonough, J.H. Jr.

1984-08-01

(3H)Quinuclidinyl benzilate binding to rat brain muscarinic receptors decreased after repeated exposure to soman, a potent organophosphorus cholinesterase inhibitor. The topographical distribution of this decrement was analyzed by quantitative receptor autoradiography. After 4 weeks of soman, three times a week, quinuclidinyl benzilate binding decreased to 67 to 80% of control in frontal and parietal cortex, caudate-putamen, lateral septum, hippocampal body, dentate gyrus, superior colliculus, nucleus of the fifth nerve, and central grey. Minor or no decreases were observed in thalamic or hypothalamic nuclei, reticular formation, pontine nuclei, inferior colliculus, nucleus of the seventh nerve, and cerebellum. Scatchard analyses of saturation curves using frontal cortex sections from soman-treated rats revealed a decrease in maximal quinuclidinyl benzilate binding from that in control rats and a return toward control levels by 24 days without any significant change in affinity. These brain areas showing significant decrements in muscarinic receptors recovered with a similar time course. An estimate of the time for 50% recovery for some of the brain areas was 14 days for superior colliculus, 16 days for cortex, and 19 days for hippocampal body. The application of quantitative receptor autoradiography to analyze receptor alterations has been valuable in localizing the telencephalon as a region more susceptible to change in receptor concentration.

The modulatory role of M2 muscarinic receptor on apomorphine-induced yawning and genital grooming.

Science.gov (United States)

Gamberini, Maria Thereza; Bolognesi, Maria Laura; Nasello, Antonia Gladys

2012-12-07

The interaction between dopaminergic and cholinergic pathways in the induction of behavioral responses has been previously established. In the brain, M2 receptors are found predominantly in presynaptic cholinergic neurons as autoreceptors, and in dopaminergic neurons as heteroceptors, suggesting a control role of acetylcholine and dopamine release, respectively. Our aim was to investigate the role of M2 receptors on the yawning and genital grooming of rats induced by apomorphine, a dopaminergic receptor agonist, focusing on the interaction between cholinergic and dopaminergic pathways. Initially, the effect of atropine, a non-selective muscarinic antagonist, on yawning and genital grooming induced by apomorphine (100 μg/kg s.c.) was analyzed. Atropine doses of 0.5, 1 and 2 mg/kg i.p. were administered to Wistar rats 30 min before induction of the behavioral responses by apomorphine. Number of yawns and time spent genital grooming were quantified over a 60 min period. Apomorphine-induced yawning was increased by low dose (0.5 mg/kg i.p.) but not by high doses (1 and 2 mg/kg, i.p.) of atropine. Genital grooming was antagonized by 2 mg/kg i.p. of atropine and showed no changes at the other doses tested. Tripitramine, a selective M2 cholinergic antagonist, was used as a tool for distinguishing between M2 and all other muscarinic receptor subtypes in yawning and genital grooming. Tripitramine doses of 0.01, 0.02 and 0.04 μmol/kg i.p. were administered to Wistar rats 30 min before apomorphine (100 μg/kg s.c.). Number of yawns and time spent genital grooming were also quantified over a 60 min period. Tripitramine 0.01 μmol/kg increased all parameters. Higher doses, which possibly block all subtypes of muscarinic receptor, did not modify the response of apomorphine, suggesting a non-selective effect of tripitramine at these doses. Given that low doses of tripitramine increased the behavioral responses induced by apomorphine and that the main distribution of the M2

The M3 muscarinic receptor is required for optimal adaptive immunity to helminth and bacterial infection.

Directory of Open Access Journals (Sweden)

Matthew Darby

2015-01-01

Full Text Available Innate immunity is regulated by cholinergic signalling through nicotinic acetylcholine receptors. We show here that signalling through the M3 muscarinic acetylcholine receptor (M3R plays an important role in adaptive immunity to both Nippostrongylus brasiliensis and Salmonella enterica serovar Typhimurium, as M3R-/- mice were impaired in their ability to resolve infection with either pathogen. CD4 T cell activation and cytokine production were reduced in M3R-/- mice. Immunity to secondary infection with N. brasiliensis was severely impaired, with reduced cytokine responses in M3R-/- mice accompanied by lower numbers of mucus-producing goblet cells and alternatively activated macrophages in the lungs. Ex vivo lymphocyte stimulation of cells from intact BALB/c mice infected with N. brasiliensis and S. typhimurium with muscarinic agonists resulted in enhanced production of IL-13 and IFN-γ respectively, which was blocked by an M3R-selective antagonist. Our data therefore indicate that cholinergic signalling via the M3R is essential for optimal Th1 and Th2 adaptive immunity to infection.

The allosteric site regulates the voltage sensitivity of muscarinic receptors.

Science.gov (United States)

Hoppe, Anika; Marti-Solano, Maria; Drabek, Matthäus; Bünemann, Moritz; Kolb, Peter; Rinne, Andreas

2018-01-01

Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein-coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M 1 -Rs and M 3 -Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the G q protein cycle. In the presence of ACh, M 1 -R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M 3 -R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed "allosteric site" M 3 /M 1 -R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M 3 -Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects. Copyright © 2017 Elsevier Inc. All rights reserved.

Differentiation-associated decrease in muscarinic receptor sensitivity in human neuroblastoma cells

International Nuclear Information System (INIS)

Heikkilae, J.E.; Scott, J.G.; Suominen, L.A.; Akerman, K.E.O.

1987-01-01

Muscarinic receptor-linked increases in intracellular free Ca 2+ as measured with quin-2 and Ca 2+ release from monolayers of cells have been measured in the human neuroblastoma cell line SH-SY5Y. Induction of differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca 2+ and stimulation of Ca 2+ efflux. No decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, 3 H-NMS, by a non-labelled agonist was studied

Differential involvement of cortical muscarinic and NMDA receptors in short- and long-term taste aversion memory.

Science.gov (United States)

Ferreira, G; Gutiérrez, R; De La Cruz, V; Bermúdez-Rattoni, F

2002-09-01

In conditioned taste aversion, an animal avoids a taste previously associated with toxic effects, and this aversive memory formation requires an intact insular cortex. In this paper, we investigated the possible differential involvement of cholinergic and glutamatergic receptors in the insular cortex in short-term memory (STM) and long-term memory (LTM) of taste aversion in rats. Taste aversion was induced by intraperitoneal administration of lithium chloride (a malaise-inducing drug) 15 min after experience with an unfamiliar taste. In order to test STM and LTM of taste aversion, taste stimulus was again presented 4 h and 72 h after lithium injection, respectively. During the acquisition, microinjection of the muscarinic antagonist, scopolamine, in the insular cortex before, but not after, the presentation of the new taste, abolished STM as well as LTM. Blockade of the NMDA receptor, in the insular cortex, by AP5 before, but not after, the presentation of the taste stimulus, impaired LTM but left STM intact. Moreover, when injected 1 h after malaise induction (i.e., during taste-illness association), AP5 disrupted both STM and LTM. These results suggest that activation of muscarinic receptors in the insular cortex is involved in the acquisition of taste memory, whereas NMDA receptors participate in taste memory consolidation. These data demonstrate that different neurochemical mechanisms subserve different memory phases. NMDA receptors are also probably involved in processing the visceral input, thus allowing subsequent taste-illness association. This indicates that in the same cortical area the same neurotransmitter system can be involved in distinct processes: taste memory consolidation vs. taste-illness association.

Angiotensin II potentiates adrenergic and muscarinic modulation of guinea pig intracardiac neurons.

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Girasole, Allison E; Palmer, Christopher P; Corrado, Samantha L; Marie Southerland, E; Ardell, Jeffrey L; Hardwick, Jean C

2011-11-01

The intrinsic cardiac plexus represents a major peripheral integration site for neuronal, hormonal, and locally produced neuromodulators controlling efferent neuronal output to the heart. This study examined the interdependence of norepinephrine, muscarinic agonists, and ANG II, to modulate intrinsic cardiac neuronal activity. Intracellular voltage recordings from whole-mount preparations of the guinea pig cardiac plexus were used to determine changes in active and passive electrical properties of individual intrinsic cardiac neurons. Application of either adrenergic or muscarinic agonists induced changes in neuronal resting membrane potentials, decreased afterhyperpolarization duration of single action potentials, and increased neuronal excitability. Adrenergic responses were inhibited by removal of extracellular calcium ions, while muscarinic responses were inhibited by application of TEA. The adrenergic responses were heterogeneous, responding to a variety of receptor-specific agonists (phenylephrine, clonidine, dobutamine, and terbutaline), although α-receptor agonists produced the most frequent responses. Application of ANG II alone produced a significant increase in excitability, while application of ANG II in combination with either adrenergic or muscarinic agonists produced a much larger potentiation of excitability. The ANG II-induced modulation of firing was blocked by the angiotensin type 2 (AT(2)) receptor inhibitor PD 123319 and was mimicked by the AT(2) receptor agonist CGP-42112A. AT(1) receptor blockade with telmasartin did not alter neuronal responses to ANG II. These data demonstrate that ANG II potentiates both muscarinically and adrenergically mediated activation of intrinsic cardiac neurons, doing so primarily via AT(2) receptor-dependent mechanisms. These neurohumoral interactions may be fundamental to regulation of neuronal excitability within the intrinsic cardiac nervous system.

Characterization of the effect of penehyclidine hydrochloride on muscarinic receptor subtypes mediating the contraction of guinea-pig isolated gastrointestinal smooth muscle.

Science.gov (United States)

Xiao, Hong-Tao; Liao, Zhi; Meng, Xian-Min; Yan, Xiao-Yan; Chen, Shu-Jie; Mo, Zheng-Ji

2009-07-01

The aim was to characterize the effect of penehyclidine hydrochloride, which mediates the relaxation of guinea-pig isolated gastrointestinal smooth muscle, on muscarinic receptor subtypes. Radioimmune assay was used to determine cAMP levels in isolated guinea-pig gastrointestinal smooth muscle to compare the selective effects of penehyclidine hydrochloride on muscarinic receptor subtypes. The results indicated that the relaxing effect of penehyclidine hydrochloride on isolated gastrointestinal smooth muscle contraction induced by acetylcholine was stronger than that of atropine (based on PA2 values). In the radioimmune assay, penehyclidine hydrochloride increased the cAMP content in isolated guinea-pig stomach smooth muscle and decreased the cAMP content in isolated guinea-pig intestinal smooth muscle, but the difference was not statistically significant at a dose of 10 mumol/l. The results suggest that penehyclidine hydrochloride has little or no effect on M2 receptor subtypes in guinea-pig gastrointestinal smooth muscle.

The detection of the non-M2 muscarinic receptor subtype in the rat heart atria and ventricles

Czech Academy of Sciences Publication Activity Database

Mysliveček, J.; Klein, M.; Nováková, M.; Říčný, Jan

2008-01-01

Roč. 378, č. 1 (2008), s. 103-116 ISSN 0028-1298 R&D Projects: GA MŠk(CZ) LC554; GA ČR(CZ) GA304/08/0256 Institutional research plan: CEZ:AV0Z50110509 Keywords : heart * muscarinic receptors * PLC activity Subject RIV: FH - Neuro logy Impact factor: 2.830, year: 2008

Pre- and postynaptic effects of muscarinic antagonists in the isolated guinea pig ileum

International Nuclear Information System (INIS)

Kilbinger, H.; Weiler, W.; Wessler, I.

1986-01-01

The authors have studied in the guinea-pig ileum whether the presynaptic muscarinic receptors of he cholinergic nerves differ from the postsynaptic muscarinic receptors of tthe longitudinal muscle in their affinities for several muscarinic antagonists. The method of measuring the release of tritium-ACh from the myenteric plexus-longitudinal muscle preparation in tthe guinea-pig ileum in the absence of a chlinesterase inhibitor is described in which two longitudinal muscle strips were incubated in a 2 ml organ bath with tritium-choline are subsequently superfused with Tyrode solution

Synthesis of dibenzodioxazocines and their effects on cholinesterases and muscarinic cholinergic receptors.

Science.gov (United States)

Gaál, J; Batke, J; Borsodi, A; Rózsa, L; Somogyi, G

1989-01-01

A new family of tricyclic compounds, the dibenzodioxazocines were synthesized. These compounds were the following: 2-chloro-12-(2-piperidino-ethyl)-dibenzo d,g 1,3,6 dioxazocine hydrochloride: EGYT-2347, 2-chloro-12-(3-dimethylamino-2-methyl-propyl)-dibenzo [d,g] [1,3,6]-dibenzodioxazocine hydrochloride: EGYT-2509, 2-chloro-12-(3-dimethylamino-propyl)-dibenzo [d,g] [1,3,6] dioxazocine-maleate: EGYT-2474 and 2-chloro-12-2-(4-methyl-piperazino)-ethyl-dibenzo [d,g] [1,3,6]-dioxazocine-dihydrochloride: EGYT-2541. These compounds are inhibitors of both butyryl- and acetylcholinesterase to and they exhibited relatively good anticholinergic properties in receptor binding experiments. The most selective inhibitor of butyrylcholinesterase is the compound EGYT-2347 (Ki = 1.5 x 10(-7) M) which strongly binds to rat brain muscarinic cholinergic receptor (KD = 4.1 x 10(-8) M).

Effect of partial volume correction on muscarinic cholinergic receptor imaging with single-photon emission tomography in patients with temporal lobe epilepsy

International Nuclear Information System (INIS)

Weckesser, M.; Ziemons, K.; Griessmeier, M.; Sonnenberg, F.; Langen, K.J.; Mueller-Gaertner, H.W.; Hufnagel, A.; Elger, C.E.; Hacklaender, T.; Holschbach, M.

1997-01-01

Animal experiments and preliminary results in humans have indicated alterations of hippocampal muscarinic acetylcholine receptors (mAChR) in temporal lobe epilepsy. Patients with temporal lobe epilepsy often present with a reduction in hippocampal volume. The aim of this study was to investigate the influence of hippocampal atrophy on the quantification of mAChR with single photon emission tomography (SPET) in patients with temporal lobe epilepsy. Cerebral uptake of the muscarinic cholinergic antagonist [ 123 I]4-iododexetimide (IDex) was investigated by SPET in patients suffering from temporal lobe epilepsy of unilateral (n=6) or predominantly unilateral (n=1) onset. Regions of interest were drawn on co-registered magnetic resonance images. Hippocampal volume was determined in these regions and was used to correct the SPET results for partial volume effects. A ratio of hippocampal IDex binding on the affected side to that on the unaffected side was used to detect changes in muscarinic cholinergic receptor density. Before partial volume correction a decrease in hippocampal IDex binding on the focus side was found in each patient. After partial volume no convincing differences remained. Our results indicate that the reduction in hippocampal IDex binding in patients with epilepsy is due to a decrease in hippocampal volume rather than to a decrease in receptor concentration. (orig.). With 2 figs., 2 tabs

Bovine pancreatic polypeptide as an antagonist of muscarinic cholinergic receptors

International Nuclear Information System (INIS)

Pan, G.Z.; Lu, L.; Qian, J.; Xue, B.G.

1987-01-01

In dispersed acini from rat pancreas, it was found that bovine pancreatic polypeptide (BPP) and its C-fragment hexapeptide amide (PP-6), at concentrations of 0.1 and 30 μM, respectively, could significantly inhibit amylase secretion stimulated by carbachol, and this inhibition by BPP was dose dependent. 45 Ca outflux induced by carbachol was also inhibited by BPP or PP-6, but they had no effect on cholecystokinin octapeptide- (CCK-8) or A23187-stimulated 45 Ca outflux. BPP was also capable of displacing the specific binding of [ 3 H]-quinuclidinyl benzilate to its receptors, and it possessed a higher affinity (K/sub i/35nM) than carbachol (K/sub i/ 1.8 μM) in binding with M-receptors. It is concluded from this study that BPP acts as an antagonist of muscarinic cholinergic receptors in rat pancreatic acini. In addition, BPP inhibited the potentiation of amylase secretion caused by the combination of carbachol plus secretin or vasoactive intestinal peptide. This may be a possible explanation of the inhibitory effect of BPP on secretin-induced pancreatic enzyme secretion shown in vivo, since pancreatic enzyme secretion stimulated by secretin under experimental conditions may be the result of potentiation of enzyme release produced by the peptide in combination with a cholinergic stimulant

Regional distribution of muscarinic acetylcholine receptors in the telencephalon of the pigeon (Columba livia f. domestica). [Use of TH-quinuclidinylbenzylate

Energy Technology Data Exchange (ETDEWEB)

Waechtler, K.

1985-01-01

The distribution of muscarinic acetylcholine receptors was studied autoradiographically in croystat sections of the pigeon telencephalon using TH-quinuclidinylbenzylate as a ligand. Highest receptor density was observed in the hyperstriatum ventrale, palaeostriatum augmentatum, septum, and parts of the archistriatum. In sites of known sensory input of neostriatum (field L) and ectostriatum low receptor binding was observed. Acetylcholinesterase distribution is in good agreement with the receptor picture only in the basal telencephalon. In the pallium differences in the pattern of these two components can be seen.

ZMS regulation of M2 muscarinic receptor mRNA stability requires protein factor

International Nuclear Information System (INIS)

Zhang Yongfang; Xia Zongqin; Hu Ya'er

2010-01-01

Aim The aim of this work is to study the elevation mechanism of ZMS on muscarinic M2 receptor mRNA expression. Methods Actinomycin D was added to cultured CHOm2 cells to stop the de novo synthesis of M2 receptor mRNA and samples were taken at various times to determine the time course of mRNA of M2 receptor with real-time quantitative RT-PCR. Half-life of M2 receptor mRNA and the effect of ZMS on the half-life was obtained from the slope of the exponential curves. Cycloheximide was added at 4 h prior to and 24 h after the addition of ZMS to examine the effect of de novo protein synthesis on the action of ZMS. Results The half-life of m2 mRNA was prolonged by ZMS treatment without cycloheximide (4.75±0.54 h and 2.13 h±0.23 h for ZMS and vehicle treated groups, respectively, P<0.05). When cycloheximide was added to the culture medium 4h prior to the addition of ZMS, the effect of ZMS in prolonging the half-life of m2 mRNA disappeared (3.06 h±0.23 h and 3.00 h±l.20 h for cells with and without ZMS, respectively). However, when the ZMS was added to the medium 24h prior to the addition of cycloheximide, the action of ZMS was not abolished by cycloheximide (half-life was 5.43 h±1.13 h and 2.46 h±0.09 h for cells with and without ZMS, respectively). Conclusion These data suggest that de novo protein synthesis was required for the increase in M2 mRNA stability induced by ZMS. (authors)

Z-(-,-)-[76Br]BrQNP: a high affinity PET radiotracer for central and cardiac muscarinic receptors

International Nuclear Information System (INIS)

Strijckmans, V.; Coulon, C.; Loc'h, C.; Maziere, B.; Luo, H.; McPherson, D.W.; Knapp, F.F.

1996-01-01

Racemic E-1-azabicyclo[2.2.2]oct-3-yl α-(1-bromo-1-1-propen-3-yl)-α -hydroxy-α-phenylacetate (BrQNP) was prepared and evaluated in vivo as a potential candidate for imaging muscarinic acetylcholinergic receptors by Positron Emission Tomography. Initial in vivo blocking studies utilizing Z-(-,-)-[ 125 I]IQNP as a radiolabelled muscarinic probe demonstrated that a preinjection of cold E-BrQNP effectively blocks the uptake of the radiolabelled probe in the brain and heart, by 71% and 86% respectively. Z-(-,-)-[ 76 Br]BrQNP was prepared by electrophilic substitution from a tributylstannyl precursor. Peracetic acid and chloramine T was evaluated as oxidizing agents. After purification by SPE and RP-HPLC, radiolabelling yields of 85% and 95% were obtained with peracetic acid and chloramine T, respectively. The final radiochemical yield was 70% for both oxidizing agents. (author)

Muscarinic acetylcholine receptors: location of the ligand binding site

International Nuclear Information System (INIS)

Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

1987-01-01

The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, 3 H-propylbenzilycholine mustard aziridinium ion ( 3 H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that 3 H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin

The muscarinic M1/M4 receptor agonist xanomeline exhibits antipsychotic-like activity in Cebus apella monkeys

DEFF Research Database (Denmark)

Andersen, Maibritt B; Fink-Jensen, Anders; Peacock, Linda

2003-01-01

(EPS) at therapeutically relevant doses. In the present study, we examined whether the xanomeline-induced functional dopamine antagonism found in rodent studies could also be observed in nonhuman primates. In addition, we studied whether the lack of EPS observed in rodents also applies to primates......Xanomeline is a muscarinic M(1)/M(4) preferring receptor agonist with little or no affinity for dopamine receptors. The compound reduces psychotic-like symptoms in patients with Alzheimer's disease and exhibits an antipsychotic-like profile in rodents without inducing extrapyramidal side effects...

Myotropic Effects of Cholinergic Muscarinic Agonists and Antagonists in the Beetle Tenebrio molitor L.

Science.gov (United States)

Chowanski, Szymon; Rosinski, Grzegorz

2017-01-01

In mammals, the cholinergic nervous system plays a crucial role in neuronal regulation of physiological processes. It acts on cells by two types of receptors - nicotinic and muscarinic receptors. Both signal transmission pathways also operate in the central and peripheral cholinergic nervous system of insects. In our pharmacological experiments, we studied the effects of two muscarinic agonists (carbachol, pilocarpine) and two muscarinic antagonists (atropine, scopolamine) on the muscle contractile activity of visceral organs in the beetle, Tenebrio molitor. Both antagonists, when injected to haemolymph at concentration 10-5 M, caused delayed and prolonged cardioinhibitory effects on heart contractility in ortho- and antidromic phases of heart activity in T. molitor pupa what was observed as negative chrono- and inotropic effects. Agonist of muscarinic receptors - carbachol evoked opposite effect and increased contraction rate but only in antidromic phase. Pilocarpine, the second agonist induced weak negative chronotropic effects in the antiand orthodromic phases of heart activity. However, neither agonists had an effect on semi-isolated beetle heart in vitro. Only atropine at the highest tested concentrations slightly decreased the frequency of myocardial contractions. These suggest the regulation of heart activity by muscarinic system indirectly. The tested compounds also affected the contractility of the oviduct and hindgut, but the responses of these organs were varied and depended on the concentration of the applied compounds. These pharmacological experiments suggest the possible modulation of insect visceral muscle contractility by the cholinergic nervous system and indirectly indicate the presence of muscarinic receptor(s) in the visceral organs of the beetle T. molitor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Immunocytochemical demonstration of M1 muscarinic acetylcholine receptors at the presynaptic and postsynaptic membranes of rat diaphragm endplates

Czech Academy of Sciences Publication Activity Database

Malomouzh, A. I.; Arkhipova, S. S.; Nikolsky, E. E.; Vyskočil, František

2011-01-01

Roč. 60, č. 1 (2011), s. 185-188 ISSN 0862-8408 R&D Projects: GA AV ČR(CZ) IAA500110905; GA ČR GA202/09/0806 Institutional research plan: CEZ:AV0Z50110509 Keywords : skeletal muscle * M1 muscarinic receptor Subject RIV: ED - Physiology Impact factor: 1.555, year: 2011

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

Science.gov (United States)

Jakubík, J; Janíčková, H; El-Fakahany, E E; Doležal, V

2011-03-01

Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

The Effects of Nicotinic and Muscarinic Receptor Activation on Patch-Clamped Cells in the Optic Tectum of Rana Pipiens

OpenAIRE

Yu, C.-J.; Debski, E. A.

2003-01-01

Both nicotinic and muscarinic cholinergic receptors are present in the optic tectum. To begin to understand how the activation of these receptors affects visual activity patterns, we have determined the types of physiological responses induced by their activation. Using tectal brain slices from the leopard frog, we found that application of nicotine (100 μM) evoked long-lasting responses in 60% of patch-clamped tectal cells. Thirty percent of these responses consisted of an increase in sponta...

Inhibition of allergen-induced basophil activation by ASM-024, a nicotinic receptor ligand.

Science.gov (United States)

Watson, Brittany M; Oliveria, John Paul; Nusca, Graeme M; Smith, Steven G; Beaudin, Sue; Dua, Benny; Watson, Rick M; Assayag, Evelynne Israël; Cormier, Yvon F; Sehmi, Roma; Gauvreau, Gail M

2014-01-01

Nicotinic acetylcholine receptors (nAChRs) were identified on eosinophils and shown to regulate inflammatory responses, but nAChR expression on basophils has not been explored yet. We investigated surface receptor expression of nAChR α4, α7 and α1/α3/α5 subunits on basophils. Furthermore, we examined the effects of ASM-024, a synthetic nicotinic ligand, on in vitro anti-IgE and in vivo allergen-induced basophil activation. Basophils were enriched from the peripheral blood of allergic donors and the expression of nAChR subunits and muscarinic receptors was determined. Purified basophils were stimulated with anti-IgE in the presence of ASM-024 with or without muscarinic or nicotinic antagonists for the measurement of CD203c expression and histamine release. The effect of 9 days of treatment with 50 and 200 mg ASM-024 on basophil CD203c expression was examined in the blood of mild allergic asthmatics before and after allergen inhalation challenge. nAChR α4, α7 and α1/α3/α5 receptor subunit expression was detected on basophils. Stimulation of basophils with anti-IgE increased CD203c expression and histamine release, which was inhibited by ASM-024 (10(-5) to 10(-)(3) M, p ASM-024 was reversed in the presence of muscarinic and nicotinic antagonists. In subjects with mild asthma, ASM-024 inhalation significantly inhibited basophil CD203c expression measured 24 h after allergen challenge (p = 0.03). This study shows that ASM-024 inhibits IgE- and allergen-induced basophil activation through both nicotinic and muscarinic receptors, and suggests that ASM-024 may be an efficacious agent for modulating allergic asthma responses. © 2015 S. Karger AG, Basel.

The selective positive allosteric M1 muscarinic receptor modulator PQCA attenuates learning and memory deficits in the Tg2576 Alzheimer's disease mouse model.

Science.gov (United States)

Puri, Vanita; Wang, Xiaohai; Vardigan, Joshua D; Kuduk, Scott D; Uslaner, Jason M

2015-01-01

We have recently shown that the M1 muscarinic receptor positive allosteric modulator, PQCA, improves cognitive performance in rodents and non-human primates administered the muscarinic receptor antagonist scopolamine. The purpose of the present experiments was to characterize the effects of PQCA in a model more relevant to the disease pathology of Alzheimer's disease. Tg2576 transgenic mice that have elevated Aβ were tested in the novel object recognition task to characterize recognition memory as a function of age and treatment with the PQCA. The effects of PQCA were compared to the acetylcholinesterase inhibitor donepezil, the standard of care for Alzheimer's disease. In addition, the effect of co-administering PQCA and donepezil was evaluated. Aged Tg2576 mice demonstrated a deficit in recognition memory that was significantly attenuated by PQCA. The positive control donepezil also reversed the deficit. Furthermore, doses of PQCA and donepezil that were inactive on their own were found to improve recognition memory when given together. These studies suggest that M1 muscarinic receptor positive allosteric modulation can ameliorate memory deficits in disease relevant models of Alzheimer's disease. These data, combined with our previous findings demonstrating PQCA improves scopolamine-induced cognitive deficits in both rodents and non-human primates, suggest that M1 positive allosteric modulators have therapeutic potential for the treatment of Alzheimer's disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Noninvasive quantification of muscarinic receptors in vivo with positron emission tomography in the dog heart

International Nuclear Information System (INIS)

Delforge, J.; Janier, M.; Syrota, A.; Crouzel, C.; Vallois, J.M.; Cayla, J.; Lancon, J.P.; Mazoyer, B.M.

1990-01-01

The in vivo quantification of myocardial muscarinic receptors has been obtained in six closed-chest dogs by using positron emission tomography. The dogs were injected with a trace amount of 11C-labeled methylquinuclidinyl benzilate (MQNB), a nonmetabolized antagonist of the muscarinic receptor. This was followed 30 minutes later by an injection of an excess of unlabeled MQNB (displacement experiment). Two additional injections of unlabeled MQNB with [11C]MQNB and without [11C]MQNB (second displacement experiment) were administered after 70 and 120 minutes, respectively. This protocol allowed a separate evaluation of the quantity of available receptors (B'max) as well as the association and dissociation rate constants (k+1 and k-1) in each dog. The parameters were calculated by using a nonlinear mathematical model in regions of interest over the left ventricle and the interventricular septum. The average value of B'max was 42 +/- 11 pmol/ml tissue, the rate constants k+1, k-1, and Kd were 0.6 +/- 0.1 ml.pmol-1.min-1, 0.27 +/- 0.03 ml.pmol-1.min-1, and 0.49 +/- 0.14 pmol.ml-1, respectively, taking into account the MQNB reaction volume estimated to 0.15 ml/ml tissue. Although [11C]MQNB binding would appear irreversible, our findings indicate that the association of the antagonist is very rapid and that the dissociation is far from negligible. The dissociated ligand, however, has a high probability of rebinding to a free receptor site instead of escaping into the microcirculation. We deduce that the positron emission tomographic images obtained after injecting a trace amount of [11C]MQNB are more representative of blood flow than of receptor density or affinity. We also suggest a simplified protocol consisting of a tracer injection of [11C]MQNB and a second injection of an excess of cold MQNB, which is sufficient to measure B'max and Kd in humans

Molecular basis of the functional heterogeneity of the muscarinic acetylcholine receptor

International Nuclear Information System (INIS)

Numa, S.; Fukuda, K.; Kubo, T.; Maeda, A.; Akiba, I.; Bujo, H.; Nakai, J.; Mishina, M.; Higashida, H.

1988-01-01

The muscarinic acetylcholine receptor (mAChR) mediates a variety of cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositides, and modulation of potassium channels, through the action of guanine-nucleotide-binding regulatory proteins (G proteins). The question then arises as to whether multiple mAChR species exist that are responsible for the various biochemical and physiological effects. In fact, pharmacologically distinguishable forms of the mAChR occur in different tissues and have been provisionally classified into M 1 (I), M 2 cardiac (II), and M 2 glandular (III) subtypes on the basis of their difference in apparent affinity for antagonists. Here, the authors have made attempts to understand the molecular basis of the functional heterogeneity of the mAChR, using recombinant DNA technology

Muscarinic receptor plasticity in the brain of senescent rats: down-regulation after repeated administration of diisopropyl fluorophosphate

International Nuclear Information System (INIS)

Pintor, A.; Fortuna, S.; Volpe, M.T.; Michalek, H.

1988-01-01

Potential age-related differences in the response of Fischer 344 rats to subchronic treatment with diisopropylfluorophosphate (DFP) were evaluated in terms of brain cholinesterase (ChE) inhibition and muscarinic receptor sites. Male 3- and 24-month old rats were sc injected with sublethal doses of DFP for 2 weeks and killed 48 hrs after the last treatment. In the cerebral cortex, hippocampus and striatum of control rats a significant age-related reduction of ChE and of maximum number of 3 H-QNB binding sites (Bmax) was observed. The administration of DFP to senescent rats resulted in more pronounced and longer lasting syndrome of cholinergic stimulation, with marked body weight loss and 60% mortality. The percentage inhibition of brain ChE induced by DFP did not differ between young and senescent rats. As expected, in young rats DFP caused a significant decrease of Bmax, which in the cerebral cortex reached about 40%. In the surviving senescent rats, the percentage decrease of Bmax due to DFP with respect to age-matched controls was very similar to that of young animals, especially in the cerebral cortex. Thus there is great variability in the response of aged rats to DFP treatment, from total failure of adaptive mechanisms resulting in death to considerable muscarinic receptor plasticity

Muscarinic receptor plasticity in the brain of senescent rats: down-regulation after repeated administration of diisopropyl fluorophosphate

Energy Technology Data Exchange (ETDEWEB)

Pintor, A.; Fortuna, S.; Volpe, M.T.; Michalek, H.

1988-01-01

Potential age-related differences in the response of Fischer 344 rats to subchronic treatment with diisopropylfluorophosphate (DFP) were evaluated in terms of brain cholinesterase (ChE) inhibition and muscarinic receptor sites. Male 3- and 24-month old rats were sc injected with sublethal doses of DFP for 2 weeks and killed 48 hrs after the last treatment. In the cerebral cortex, hippocampus and striatum of control rats a significant age-related reduction of ChE and of maximum number of /sup 3/H-QNB binding sites (Bmax) was observed. The administration of DFP to senescent rats resulted in more pronounced and longer lasting syndrome of cholinergic stimulation, with marked body weight loss and 60% mortality. The percentage inhibition of brain ChE induced by DFP did not differ between young and senescent rats. As expected, in young rats DFP caused a significant decrease of Bmax, which in the cerebral cortex reached about 40%. In the surviving senescent rats, the percentage decrease of Bmax due to DFP with respect to age-matched controls was very similar to that of young animals, especially in the cerebral cortex. Thus there is great variability in the response of aged rats to DFP treatment, from total failure of adaptive mechanisms resulting in death to considerable muscarinic receptor plasticity.

Time-course of 5-HT(6) receptor mRNA expression during memory consolidation and amnesia.

Science.gov (United States)

Huerta-Rivas, A; Pérez-García, G; González-Espinosa, C; Meneses, A

2010-01-01

Growing evidence indicates that antagonists of the 5-hydroxytryptamine (serotonin) receptor(6) (5-HT(6)) improve memory and reverse amnesia although the mechanisms involved are poorly understood. Hence, in this paper RT-PCR was used to evaluate changes in mRNA expression of 5-HT(6) receptor in trained and untrained rats treated with the 5-HT(6) receptor antagonist SB-399885 and amnesic drugs scopolamine or dizocilpine. Changes in mRNA expression of 5-HT(6) receptor were investigated at different times in prefrontal cortex, hippocampus and striatum. Data indicated that memory in the Pavlovian/instrumental autoshaping task was a progressive process associated to reduced mRNA expression of 5-HT(6) receptor in the three structures examined. SB-399885 improved long-term memory at 48h, while the muscarinic receptor antagonist scopolamine or the non-competitive NMDA receptor antagonist dizocilpine impaired it at 24h. Autoshaping training and treatment with SB-399885 increased 5-HT(6) receptor mRNA expression in (maximum increase) prefrontal cortex and striatum, 24 or 48h. The scopolamine-induced amnesia suppressed 5-HT(6) receptor mRNA expression while the dizocilpine-induced amnesia did not modify 5-HT(6) receptor mRNA expression. SB-399885 and scopolamine or dizocilpine were able to reestablish memory and 5-HT(6) receptor mRNA expression. These data confirmed previous memory evidence and of more interest is the observation that training, SB-399885 and amnesic drugs modulated 5-HT(6) receptor mRNA expression in prefrontal cortex, hippocampus and striatum. Further investigation in different memory tasks, times and amnesia models together with more complex control groups might provide further clues. Copyright 2009 Elsevier Inc. All rights reserved.

Presynaptic Muscarinic Acetylcholine Receptors and TrkB Receptor Cooperate in the Elimination of Redundant Motor Nerve Terminals during Development.

Science.gov (United States)

Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Cilleros, Victor; Tomàs, Josep

2017-01-01

The development of the nervous system involves the overproduction of synapses but connectivity is refined by Hebbian activity-dependent axonal competition. The newborn skeletal muscle fibers are polyinnervated but, at the end of the competition process, some days later, become innervated by a single axon. We used quantitative confocal imaging of the autofluorescent axons from transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice to investigate the possible cooperation of the muscarinic autoreceptors (mAChR, M 1 -, M 2 - and M 4 -subtypes) and the tyrosine kinase B (TrkB) receptor in the control of axonal elimination after the mice Levator auris longus (LAL) muscle had been exposed to several selective antagonist of the corresponding receptor pathways in vivo . Our previous results show that M 1 , M 2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M 1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M 2 receptor is largely independent of both M 1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination.

Allosteric modulation by persistent binding of xanomeline of the interaction of competitive ligands with the M1 muscarinic acetylcholine receptor

Czech Academy of Sciences Publication Activity Database

Jakubík, Jan; Tuček, Stanislav; El-Fakahany, E. E.

2002-01-01

Roč. 301, č. 3 (2002), s. 1033-1041 ISSN 0022-3565 R&D Projects: GA ČR GP305/01/D119 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z5011922 Keywords : xanomeline * M(1) muscarinic acetylcholine receptor Subject RIV: FH - Neurology Impact factor: 3.991, year: 2002

SELECTIVITY PROFILE OF SOME RECENT MUSCARINIC ANTAGONISTS IN BOVINE AND GUINEA-PIG TRACHEA AND HEART

NARCIS (Netherlands)

ROFFEL, AF; HAMSTRA, JJ; ELZINGA, CRS; ZAAGSMA, J

1994-01-01

The functional affinities of some recently developed subtype-selective muscarinic antagonists towards bovine tracheal smooth muscle muscarinic M(3) receptors were established and compared to binding affinities for bovine cardiac M(2) and functional affinities for guinea-pig tracheal smooth muscle

PASSIVE-AVOIDANCE TRAINING INDUCES ENHANCED LEVELS OF IMMUNOREACTIVITY FOR MUSCARINIC ACETYLCHOLINE-RECEPTOR AND COEXPRESSED PKC-GAMMA AND MAP-2 IN RAT CORTICAL-NEURONS

NARCIS (Netherlands)

VANDERZEE, EA; DOUMA, BRK; BOHUS, B; LUITEN, PGM

1994-01-01

Changes in neocortical immunoreactivity (ir) for muscarinic acetylcholine receptors (mAChRs), protein kinase C gamma (PKC gamma), microtubule-associated protein 2 (MAP-2), and the calcium-binding protein parvalbumin (PARV) induced by the performance of a one-trial passive shock avoidance (PSA) task

Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M(3) receptors

Czech Academy of Sciences Publication Activity Database

Krejčí, Alena; Tuček, Stanislav

2001-01-01

Roč. 60, č. 4 (2001), s. 761-767 ISSN 0026-895X R&D Projects: GA ČR GA309/99/0214 Institutional research plan: CEZ:AV0Z5011922 Keywords : muscarinic receptors * allosteric modulators Subject RIV: FH - Neurology Impact factor: 5.297, year: 2001

Muscarinic M1 acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism

Czech Academy of Sciences Publication Activity Database

Malomouzh, A. I.; Mukhtarov, M. R.; Nikolsky, E. E.; Vyskočil, František

2007-01-01

Roč. 102, č. 6 (2007), s. 2110-2117 ISSN 0022-3042 R&D Projects: GA AV ČR(CZ) IAA5011411; GA MŠk(CZ) LC554 Grant - others:-(RU) 112.0/001/481 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * NO synthese Subject RIV: ED - Physiology Impact factor: 4.451, year: 2007

Demonstration of a reduction in muscarinic receptor binding in early Alzheimer's disease using iodine-123 dexetimide single-photon emission tomography

International Nuclear Information System (INIS)

Claus, J.J.; Dubois, E.A.; Booij, J.; Habraken, J.; Munck, J.C. van; Herk, M. van; Verbeeten, B. Jr.; Royen, E.A. van

1997-01-01

Decreased muscarinic receptor binding has been suggested in single-photon emission tomography (SPET) studies of Alzheimer's disease. However, it remains unclear whether these changes are present in mildly demented patients, and the role of cortical atrophy in receptor binding assessment has not been investigated. We studied muscarinic receptor binding normalized to neostriatum with SPET using [ 123 I[4-iododexetimide in five mildly affected patients with probable Alzheimer's disease and in five age-matched control subjects. Region of interest (ROI) analysis was performed in a consensus procedure blind to clinical diagnosis using matched magnetic resonance (MRI) images. Cortical atrophy was assessed by calculating percentages of cerebrospinal fluid in each ROI. An observer study with three observers was conducted to validate this method. Alzheimer patients showed statistically significantly less [ 123 I[4-iododexetimide binding in left temporal and right temporo-parietal cortex compared with controls, independent of age, sex and cortical atrophy. Mean intra-observer variability was 3.6% and inter-observer results showed consistent differences in [ 123 I[4-iododexetimide binding between observers. However, differences between patients and controls were comparable among observers and statistically significant in the same regions as in the consensus procedure. Using an MRI-SPET matching technique, we conclude that [ 123 I[4-iododexetimide binding is reduced in patients with mild probable Alzheimer's disease in areas of temporal and temporo-parietal cortex. (orig.). With 1 fig., 4 tabs

Muscarinic responses of gastric parietal cells

International Nuclear Information System (INIS)

Wilkes, J.M.; Kajimura, M.; Scott, D.R.; Hersey, S.J.; Sachs, G.

1991-01-01

Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC50 of 13 microM; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC50 of 110 microM; and by the M1/M3 antagonist, diphenyl-acetoxy-4-methylpiperidinemethiodide, with an IC50 of 35 nM. The three antagonists displayed equivalent IC50 values for the inhibition of carbachol-stimulated production of 14CO2 from radiolabeled glucose, which is a measure of the turnover of the H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 microM La3+. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC50 of 1.7 nM, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations, suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the [Ca]i transient

In vivo biodistribution of two [18F]-labelled muscarinic cholinergic receptor ligands: 2-[18F]- and 4-[18F]-fluorodexetimide

International Nuclear Information System (INIS)

Wilson, A.A.; Scheffel, U.A.; Dannals, R.F.; Stathis, M.; Ravert, H.T.; Wagner, H.N. Jr.

1991-01-01

Two [ 18 F]-labelled analogues of the potent muscarinic cholinergic receptor (m-AChR) antagonist, dexetimide, were evaluated as potential ligands for imaging m-AChR by positron emission tomography (PET). Intravenous administration of both 2-[ 18 F]- or 4-[ 18 F]-fluorodexetimide resulted in high brain uptake of radioactivity in mice. High binding levels were observed in m-AChR rich areas, such as cortex and striatum, with low levels in the receptor-poor cerebellum. Uptake of radioactivity was saturable and could be blocked by pre-administration of dexetimide or atropine. Drugs with different sites of action were ineffective at blocking receptor binding. The results indicate that both radiotracers are promising candidates for use in PET studies

Membrane cholesterol content influences binding properties of muscarinic M2 receptors and differentially impacts activation of second messenger pathways

Czech Academy of Sciences Publication Activity Database

Michal, Pavel; Rudajev, Vladimír; El-Fakahany, E. E.; Doležal, Vladimír

2009-01-01

Roč. 606, 1-3 (2009), s. 50-60 ISSN 0014-2999 R&D Projects: GA ČR(CZ) GA305/05/0452; GA AV ČR(CZ) IAA500110703; GA MŠk(CZ) LC554 Grant - others:National Institutes of Health(US) NS25743; EC(XE) LipiDiDiet Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic * cholesterol * receptors Subject RIV: ED - Physiology Impact factor: 2.585, year: 2009

Muscarinic receptors in amygdala control trace fear conditioning.

Directory of Open Access Journals (Sweden)

Amber N Baysinger

Full Text Available Intelligent behavior requires transient memory, which entails the ability to retain information over short time periods. A newly-emerging hypothesis posits that endogenous persistent firing (EPF is the neurophysiological foundation for aspects or types of transient memory. EPF is enabled by the activation of muscarinic acetylcholine receptors (mAChRs and is triggered by suprathreshold stimulation. EPF occurs in several brain regions, including the lateral amygdala (LA. The present study examined the role of amygdalar mAChRs in trace fear conditioning, a paradigm that requires transient memory. If mAChR-dependent EPF selectively supports transient memory, then blocking amygdalar mAChRs should impair trace conditioning, while sparing delay and context conditioning, which presumably do not rely upon transient memory. To test the EPF hypothesis, LA was bilaterally infused, prior to trace or delay conditioning, with either a mAChR antagonist (scopolamine or saline. Computerized video analysis quantified the amount of freezing elicited by the cue and by the training context. Scopolamine infusion profoundly reduced freezing in the trace conditioning group but had no significant effect on delay or context conditioning. This pattern of results was uniquely anticipated by the EPF hypothesis. The present findings are discussed in terms of a systems-level theory of how EPF in LA and several other brain regions might help support trace fear conditioning.

Muscarinic receptors in amygdala control trace fear conditioning.

Science.gov (United States)

Baysinger, Amber N; Kent, Brianne A; Brown, Thomas H

2012-01-01

Intelligent behavior requires transient memory, which entails the ability to retain information over short time periods. A newly-emerging hypothesis posits that endogenous persistent firing (EPF) is the neurophysiological foundation for aspects or types of transient memory. EPF is enabled by the activation of muscarinic acetylcholine receptors (mAChRs) and is triggered by suprathreshold stimulation. EPF occurs in several brain regions, including the lateral amygdala (LA). The present study examined the role of amygdalar mAChRs in trace fear conditioning, a paradigm that requires transient memory. If mAChR-dependent EPF selectively supports transient memory, then blocking amygdalar mAChRs should impair trace conditioning, while sparing delay and context conditioning, which presumably do not rely upon transient memory. To test the EPF hypothesis, LA was bilaterally infused, prior to trace or delay conditioning, with either a mAChR antagonist (scopolamine) or saline. Computerized video analysis quantified the amount of freezing elicited by the cue and by the training context. Scopolamine infusion profoundly reduced freezing in the trace conditioning group but had no significant effect on delay or context conditioning. This pattern of results was uniquely anticipated by the EPF hypothesis. The present findings are discussed in terms of a systems-level theory of how EPF in LA and several other brain regions might help support trace fear conditioning.

Wash-Resistantly Bound Xanomeline Inhibits Acetylcholine Release by Persistent Activation of Presynaptic M2 and M4 Muscarinic Receptors in Rat Brain

Czech Academy of Sciences Publication Activity Database

Machová, Eva; Jakubík, Jan; El-Fakahany, E. E.; Doležal, Vladimír

2007-01-01

Roč. 322, č. 1 (2007), s. 316-323 ISSN 0022-3565 R&D Projects: GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:-(US) NS25743 Institutional research plan: CEZ:AV0Z50110509 Keywords : acetylcholine * xanomeline * muscarinic receptor Subject RIV: ED - Physiology Impact factor: 4.003, year: 2007

3-(2-Benzofuranyl)quinuclidin-2-ene derivatives: novel muscarinic antagonists.

Science.gov (United States)

Nordvall, G; Sundquist, S; Johansson, G; Glas, G; Nilvebrant, L; Hacksell, U

1996-08-16

A series of 26 derivatives of the novel muscarinic antagonist 3-(2-benzofuranyl)quinuclidin-2-ene (1) has been synthesized and evaluated for muscarinic and antimuscarinic properties. The affinity of the compounds was determined by competition experiments in homogenates of cerebral cortex, heart, parotid gland, and urinary bladder from guinea pigs using (-)-[3H]-3-quinuclidinyl benzilate as the radioligand, and the antimuscarinic-potency was determined in a functional assay on isolated guinea pig urinary bladder using carbachol as the agonist. The 5-fluorobenzofuranyl derivative was slightly more potent than 1. The 7-bromo-substituted 8 displayed a 14-fold tissue selectivity ratio for muscarinic receptors in the cortex versus the parotid gland. Comparative molecular field analysis and quantitative structure-activity relationship models were developed for this series of substituted benzofuranyl derivatives.

Functional Characterization of CCHamide and Muscarinic Acetylcholine Receptor Signalling in Drosophila melanogaster

DEFF Research Database (Denmark)

Ren, Guilin Robin

G-protein coupled receptors (GPCRs) constitute a large and ancient superfamily of membraneproteins responsible for the transduction of extracellular signals to the inside of the cells. In thisPh.D. thesis, Drosophila melanogaster (Dm) was used as a model organism to investigate a numberof topics...... is a newly discovered insect peptide hormone. The function of this novel peptide hasnot been well characterised. In this Ph.D. thesis, I identified CCHamide-2 peptides in endocrinecells of the gut and neurones of the brain of larvae and endocrine cells of the gut of adultDrosophila. Behavioural assays...... little is known about muscarinic acetylcholine receptorsignalling in insects. In this study, I found that two types of mAChRs occur in D. melanogaster, onecoupling to Gq (A-type) and the other to Gi (B-type). Both A- and B-type Dm-mAChRs can beactivated by acetylcholine (ACh), but the classical...

Concomitant alteration in number and affinity of P2X and muscarinic receptors are associated with bladder dysfunction in early stage of diabetic rats.

Science.gov (United States)

Yoshizawa, Tsuyoshi; Hayashi, Yukio; Yoshida, Akira; Yoshida, Shohei; Ito, Yoshihiko; Yamaguchi, Kenya; Yamada, Shizuo; Takahashi, Satoru

2018-03-01

To investigate time course of bladder dysfunction and concurrent changes in number and affinity of the muscarinic and P 2 X receptor in the early stage of streptozotocin (STZ)-induced diabetic rats. Diabetic rats were prepared by the intraperitoneal injection of 50 mg/kg of STZ to 7-week-old female Wistar rats. We performed recording of 24-h voiding behavior and cystometry at 1, 4, 8, and 12 weeks after the induction of diabetes. A muscle strip experiments with electrical field stimulation (EFS), carbachol, and α,β-methylene adenosine 5'-triphosphate (α,β-MeATP) were also performed at the same time-points. Additionally, concurrent changes in number and affinity of bladder muscarinic and P 2 X receptor were measured by a radioreceptor assay using [N-methyl- 3 H] scopolamine methyl chloride ([ 3 H]NMS) and α,β-methylene-ATP (2,8- 3 H) tetrasodium salt ([ 3 H]α,β-MeATP). In STZ-induced diabetic rats, polydipsic polyuric pollakiuria were noted on recording of 24-h voiding behavior from early stage. Also, the residual urine volume markedly increased in diabetic rats on cystometry. In the muscle strip experiment, the detrusor contractions induced by EFS, carbachol, and α,β-MeATP were enhanced in STZ-induced diabetic rats. Based on the radioreceptor assay, the maximum number of sites (Bmax) for the specific binding of [ 3 H]NMS and [ 3 H]α,β-MeATP was concurrently increased in the bladder from diabetic rats. Increased bladder contractility is found in early stage of diabetic rats. Then, bladder dysfunction is associated with increased number of muscarinic and P 2 X receptors in STZ-induced diabetic rats.

Changes in Membrane Cholesterol Differentially Influence Preferential and Non-preferential Signaling of the M1 and M3 Muscarinic Acetylcholine Receptors

Czech Academy of Sciences Publication Activity Database

Michal, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

2015-01-01

Roč. 40, č. 10 (2015), s. 2068-2070 ISSN 0364-3190 R&D Projects: GA ČR(CZ) GA14-05696S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) 7E10060 Institutional support: RVO:67985823 Keywords : muscarinic receptors * agonist binding * cholesterol * G-proteins * signal transduction * cAMP synthesis Subject RIV: FH - Neurology Impact factor: 2.472, year: 2015

Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

Science.gov (United States)

Miao, Yinglong; McCammon, J Andrew

2018-03-20

Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M(1) and M(2) muscarinic acetylcholine receptors

Czech Academy of Sciences Publication Activity Database

Jakubík, Jan; El-Fakahany, E. E.; Doležal, Vladimír

2006-01-01

Roč. 70, č. 2 (2006), s. 656-666 ISSN 0026-895X R&D Projects: GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z5011922 Keywords : xanomeline * muscarinic receptors * G-protein activation Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 4.469, year: 2006

An Anti-Nicotinic Cognitive Challenge Model using Mecamylamine in Comparison with the Anti-Muscarinic Cognitive Challenge using Scopolamine

NARCIS (Netherlands)

Baakman, A. C.; Alvarez-jimenez, R.; Rissmann, R.; Klaassen, E. S.; Stevens, J.; Goulooze, S. C.; Burger, J.; Swart, E. L.; Van Gerven, J. M. A.; Groeneveld, G. J.

Aims The muscarinic acetylcholine receptor antagonist scopolamine is often used for proof-of-pharmacology studies with pro-cognitive compounds. From a pharmacological point of view, it would seem more rational to use a nicotinic rather than a muscarinic anticholinergic challenge to prove

Nicotinic and muscarinic cholinergic receptors are recruited by acetylcholine-mediated neurotransmission within the locus coeruleus during the organisation of post-ictal antinociception.

Science.gov (United States)

de Oliveira, Rithiele Cristina; de Oliveira, Ricardo; Biagioni, Audrey Franceschi; Falconi-Sobrinho, Luiz Luciano; Dos Anjos-Garcia, Tayllon; Coimbra, Norberto Cysne

2016-10-01

Post-ictal antinociception is characterised by an increase in the nociceptive threshold that accompanies tonic and tonic-clonic seizures (TCS). The locus coeruleus (LC) receives profuse cholinergic inputs from the pedunculopontine tegmental nucleus. Different concentrations (1μg, 3μg and 5μg/0.2μL) of the muscarinic cholinergic receptor antagonist atropine and the nicotinic cholinergic receptor antagonist mecamylamine were microinjected into the LC of Wistar rats to investigate the role of cholinergic mechanisms in the severity of TCS and the post-ictal antinociceptive response. Five minutes later, TCS were induced by systemic administration of pentylenetetrazole (PTZ) (64mg/kg). Seizures were recorded inside the open field apparatus for an average of 10min. Immediately after seizures, the nociceptive threshold was recorded for 130min using the tail-flick test. Pre-treatment of the LC with 1μg, 3μg and 5μg/0.2μL concentrations of both atropine and mecamylamine did not cause a significant effect on seizure severity. However, the same treatments decreased the post-ictal antinociceptive phenomenon. In addition, mecamylamine caused an earlier decrease in the post-ictal antinociception compared to atropine. These results suggest that muscarinic and mainly nicotinic cholinergic receptors of the LC are recruited to organise tonic-clonic seizure-induced antinociception. Copyright © 2016 Elsevier Inc. All rights reserved.

A novel mechanism of hippocampal LTD involving muscarinic receptor-triggered interactions between AMPARs, GRIP and liprin-α

Directory of Open Access Journals (Sweden)

Dickinson Bryony A

2009-06-01

Full Text Available Abstract Background Long-term depression (LTD in the hippocampus can be induced by activation of different types of G-protein coupled receptors, in particular metabotropic glutamate receptors (mGluRs and muscarinic acethycholine receptors (mAChRs. Since mGluRs and mAChRs activate the same G-proteins and isoforms of phospholipase C (PLC, it would be expected that these two forms of LTD utilise the same molecular mechanisms. However, we find a distinct mechanism of LTD involving GRIP and liprin-α. Results Whilst both forms of LTD require activation of tyrosine phosphatases and involve internalisation of AMPARs, they use different molecular interactions. Specifically, mAChR-LTD, but not mGluR-LTD, is blocked by peptides that inhibit the binding of GRIP to the AMPA receptor subunit GluA2 and the binding of GRIP to liprin-α. Thus, different receptors that utilise the same G-proteins can regulate AMPAR trafficking and synaptic efficacy via distinct molecular mechanisms. Conclusion Our results suggest that mAChR-LTD selectively involves interactions between GRIP and liprin-α. These data indicate a novel mechanism of synaptic plasticity in which activation of M1 receptors results in AMPAR endocytosis, via a mechanism involving interactions between GluA2, GRIP and liprin-α.

Mutations in the third extracellular loop of M3 muscarinic receptor induce positive cooperativity between N-Methylscopolamine and Wieland-Gumlich aldehyde

Czech Academy of Sciences Publication Activity Database

Jakubík, Jan; Doležal, Vladimír

2005-01-01

Roč. 272, č. S1 (2005), s. 221-221 ISSN 1474-3833. [FEBS Congress /30./ and IUBMB Conference /9./. 02.07.2005-07.07.2005, Budapest] R&D Projects: GA AV ČR(CZ) IAA5011306; GA ČR(CZ) GP305/02/D090 Institutional research plan: CEZ:AV0Z5011922 Keywords : muscarinic receptors * allosteric interaction * strychnine -like modulators * mutations * extracellular loop Subject RIV: ED - Physiology

Muscarinic contribution to the acute cortical effects of vagus nerve stimulation

Science.gov (United States)

Nichols, Justin A.

2011-12-01

Electrical stimulation of the vagus nerve (VNS) has been used to treat more than 60,000 patients with drug-resistant epilepsy and is under investigation as a treatment for several other neurological disorders and conditions. Among these, VNS increases memory performance and enhances recovery of motor and cognitive function in animal models of traumatic brain injury. Recent research indicates that pairing brief VNS with tones multiple-times a day for several weeks induces long-term, input specific cortical plasticity, which can be used to re-normalize the pathological cortical reorganization and eliminate a behavioral correlate of chronic tinnitus in noise exposed rats. Despite the therapeutic potential, the mechanisms of action of VNS remain speculative. In chapter 2 of this dissertation, the acute effects of VNS on cortical synchrony, excitability, and temporal processing are examined. In anesthetized rats implanted with multi-electrode arrays, VNS increased and decorrelated spontaneous multi-unit activity, and suppressed entrainment to repetitive noise burst stimulation at 6 to 8 Hz, but not after systemic administration of the muscarinic antagonist scopolamine. Chapter 3 focuses on VNS-tone pairing induced cortical plasticity. Pairing VNS with a tone one hundred times in anesthetized rats resulted in frequency specific plasticity in 31% of the auditory cortex sites. Half of these sites exhibited a frequency specific increase in firing rate and half exhibited a frequency specific decrease. Muscarinic receptor blockade with scopolamine almost entirely prevented the frequency specific increases, but not decreases. Collectively, these experiments demonstrate the capacity for VNS to not only acutely influence cortical synchrony, and excitability, but to also influence temporal and spectral tuning via muscarinic receptor activation. These results strengthen the hypothesis that acetylcholine and muscarinic receptors are involved in the mechanisms of action of VNS and

Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

International Nuclear Information System (INIS)

Scherer, N.M.; Nathanson, N.M.

1990-01-01

Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA - ). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA + or PKA - cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA - cells. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA + and PKA - cells was accompanied by desensitization of functional responses. Exposure of PKA + cells to 10 -7 M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 ± 9% decrease in the number of m1 receptors on the cell surface. The m2 receptor was not internalized following treatment of either PKA + or PKA - cells with PMA. Thus, the m1 and m2 receptors show differential sensitivity to internalization by PMA. Agonist-dependent internalization of the m1 receptor appeared to be independent of activation of PKC because (1) agonist-dependent internalization of m1 was not attenuated in PKA - cells, (2) the rate and extent of internalization of m1 in cells exposed to PMA were less than those in cells exposed to agonist, and (3) treatment of cells with concanavalin A selectivity blocked internalization of m1 in cells exposed to PMA, but not to agonist. The effects of agonist and PMA on receptor internalization were not additive. Exposure of PKA + or PKA - cells to PMA reduced the magnitude of pilocarpine-stimulated PI hydrolysis by about 25%

Preparation of an imaging agent for cerebral muscarinic acetylcholine receptor, (R,S)131I-QNB

International Nuclear Information System (INIS)

Ding Shiyu; Chen Zhengping; Ji Shuren; Lu Chunxiong; Zhou Xiang; Fang Ping; Wu Chunying; Wang Bocheng; Xiang Jingde; Lin Yansong

2003-01-01

The method to synthesize a high affinity muscarinic receptor antagonist (R,S)I-QNB[(R)-(-)-l-azabicyclo [2,2,2]oct-3-yl-(S)-(+)-α-hydroxy-α-(4-[127I]iodophenyl)-α-phenyl acetate] from 4-nitrobenzophenone with improvement compared to literatures was reported in this article. IR, MS and 1 HNMR characterized the final product. (R,S) 131 I-QNB was prepared using Cu(I) assisted iodine exchange labeling, and showed by TLC that the radiolabeling yield (RLY) was over 80%, and radiochemical purity (RCP) was over 95%. Stability of the labelled compound was also determined. It was found that (R,S) 131 I-QNB dried by nitrogen blowing can stay at 4-10 degree C for a week without change of RCP

The M1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

International Nuclear Information System (INIS)

Mei, Lin.

1989-01-01

The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated [ 3 H]IP 1 accumulation in the SH-SY5Y cells was decreased in the presence of 1μg/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M 1 mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m 1 gene. The transfected B82 cells (cTB10) showed specific [ 3 H](-)QNB binding activity. The mAChRs in these cells are of the M 1 type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M 1 mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M 1 mAChR densities in these cells characterized by [ 3 H](-)MQNB binding ranged from 12 fmol/10 6 cells in LK3-1 cells to 260 fmol/10 6 cells in the LK3-8 cells

Immunocytochemical localization of choline acetyltransferase and muscarinic ACh receptors in the antenna during development of the sphinx moth Manduca sexta.

Science.gov (United States)

Clark, Julie; Meisner, Shannon; Torkkeli, Päivi H

2005-04-01

Immunocytochemistry with monoclonal antibodies was used to investigate the locations of muscarinic acetylcholine receptors (mAChR) and choline acetyltransferase (ChAT) in sections of the developing antennae of the moth Manduca sexta. The results were correlated with a previous morphological investigation in the developing antennae which allowed us to locate different cell types at various stages of development. Our findings indicated that the muscarinic cholinergic system was not restricted to the sensory neurons but was also present in glial and epidermal cells. By day 4-5 of adult development, immunoreactivity against both antibodies was present in the axons of the antennal nerve, and more intense labeling was present in sections from older pupae. At days 4-9, the cell bodies of the sensory neurons in the basal part of the epidermis were also intensely immunolabeled by the anti-mAChR antibody. In mature flagella, large numbers of cells, some with processes into hairs, were strongly labeled by both antibodies. Antennal glial cells were intensely immunolabeled with both antibodies by days 4-5, but in later stages, it was not possible to discriminate between glial and neural staining. At days 4-9, we observed a distinctly labeled layer of epidermal cells close to the developing cuticle. The expression of both ChAT and mAChRs by neurons in moth antennae may allow the regulation of excitability by endogenous ACh. Cholinergic communication between neurons and glia may be part of the system that guides axon elongation during development. The cholinergic system in the apical part of the developing epidermis could be involved in cuticle formation.

Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors

Science.gov (United States)

Moreau, Christophe J.; Revilloud, Jean; Caro, Lydia N.; Dupuis, Julien P.; Trouchet, Amandine; Estrada-Mondragón, Argel; Nieścierowicz, Katarzyna; Sapay, Nicolas; Crouzy, Serge; Vivaudou, Michel

2017-01-01

Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications. PMID:28145461

Effects of muscarinic blockade in perirhinal cortex during visual recognition

Science.gov (United States)

Tang, Yi; Mishkin, Mortimer; Aigner, Thomas G.

1997-01-01

Stimulus recognition in monkeys is severely impaired by destruction or dysfunction of the perirhinal cortex and also by systemic administration of the cholinergic-muscarinic receptor blocker, scopolamine. These two effects are shown here to be linked: Stimulus recognition was found to be significantly impaired after bilateral microinjection of scopolamine directly into the perirhinal cortex, but not after equivalent injections into the laterally adjacent visual area TE or into the dentate gyrus of the overlying hippocampal formation. The results suggest that the formation of stimulus memories depends critically on cholinergic-muscarinic activation of the perirhinal area, providing a new clue to how stimulus representations are stored. PMID:9356507

Changes in acetylcholine content, release and muscarinic receptors in rat hippocampus under cold stress

International Nuclear Information System (INIS)

Fatranska, M.; Budai, D.; Gulya, K; Kvetnansky, R.

1989-01-01

The aim was to study the mechanism of the previously established decrease in acetylcholine (ACh) concentration in the rat hippocampus under cold stress. Male rats were exposed for 14 days to cold (5 degree C) or kept (controls) at room temperature (24 degree C). Acetylcholine content, release and muscarinic receptor binding were investigated in the hippocampus. Cold exposure resulted in a decrease of ACh concentration in the dorsal hippocampus. Moreover, the potassium-evoked release of ACh from hippocampal slices was increased and an increase of maximal binding capacity of [ 3 H](-) quinuclidinyl benzilate in the dorsal hippocampus of cold exposed animals was also observed. Thus the decrease of hippocampal ACh concentration under cold exposure is probably due to its increased release. On balance then, our results demonstrate that cold stress in the rat induces significant activation of the hippocampal cholinergic system

Involvement of a subpopulation of neuronal M4 muscarinic acetylcholine receptors in the antipsychotic-like effects of the M1/M4 preferring muscarinic receptor agonist xanomeline

DEFF Research Database (Denmark)

Dencker, Ditte; Wörtwein, Gitta; Weikop, Pia

2011-01-01

Disturbances in central dopaminergic neurotransmission are believed to be centrally involved in the pathogenesis of schizophrenia. Central dopaminergic and cholinergic systems interact and the cholinergic muscarinic agonist xanomeline has shown antipsychotic effects in clinical studies. Preclinic...

The interaction between tropomyosin-related kinase B receptors and presynaptic muscarinic receptors modulates transmitter release in adult rodent motor nerve terminals.

Science.gov (United States)

Garcia, Neus; Tomàs, Marta; Santafé, Manel M; Besalduch, Nuria; Lanuza, Maria A; Tomàs, Josep

2010-12-08

The neurotrophin brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase B (trkB) and p75(NTR) are present in the nerve terminals on the neuromuscular junctions (NMJs) of the levator auris longus muscle of the adult mouse. Exogenously added BDNF or NT-4 increased evoked ACh release after 3 h. This presynaptic effect (the size of the spontaneous potentials is not affected) is specific because it is not produced by neurotrophin-3 (NT-3) and is prevented by preincubation with trkB-IgG chimera or by pharmacological block of trkB [K-252a (C₂₇H₂₁N₃O₅)] or p75(NTR) [Pep5 (C₈₆H₁₁₁N₂₅O₁₉S₂] signaling. The effect of BDNF depends on the M₁ and M₂ muscarinic acetylcholine autoreceptors (mAChRs) because it is prevented by atropine, pirenzepine and methoctramine. We found that K-252a incubation reduces ACh release (~50%) in a short time (1 h), but the p75(NTR) signaling inhibitor Pep5 does not have this effect. The specificity of the K-252a blocking effect on trkB was confirmed with the anti-trkB antibody 47/trkB, which reduces evoked ACh release, like K-252a, whereas the nonpermeant tyrosine kinase blocker K-252b does not. Neither does incubation with the fusion protein trkB-IgG (to chelate endogenous BDNF/NT-4), anti-BDNF or anti-NT-4 change ACh release. Thus, the trkB receptor normally seems to be coupled to ACh release when there is no short-term local effect of neurotrophins at the NMJ. The normal function of the mAChR mechanism is a permissive prerequisite for the trkB pathway to couple to ACh release. Reciprocally, the normal function of trkB modulates M₁- and M₂-subtype muscarinic pathways.

Involvement of Ca2+ Signaling in the Synergistic Effects between Muscarinic Receptor Antagonists and β2-Adrenoceptor Agonists in Airway Smooth Muscle

Directory of Open Access Journals (Sweden)

Kentaro Fukunaga

2016-09-01

Full Text Available Long-acting muscarinic antagonists (LAMAs and short-acting β2-adrenoceptor agonists (SABAs play important roles in remedy for COPD. To propel a translational research for development of bronchodilator therapy, synergistic effects between SABAs with LAMAs were examined focused on Ca2+ signaling using simultaneous records of isometric tension and F340/F380 in fura-2-loaded tracheal smooth muscle. Glycopyrronium (3 nM, a LAMA, modestly reduced methacholine (1 μM-induced contraction. When procaterol, salbutamol and SABAs were applied in the presence of glycopyrronium, relaxant effects of these SABAs are markedly enhanced, and percent inhibition of tension was much greater than the sum of those for each agent and those expected from the BI theory. In contrast, percent inhibition of F340/F380 was not greater than those values. Bisindolylmaleimide, an inhibitor of protein kinase C (PKC, significantly increased the relaxant effect of LAMA without reducing F340/F380. Iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ (KCa channels, significantly suppressed the effects of these combined agents with reducing F340/F380. In conclusion, combination of SABAs with LAMAs synergistically enhances inhibition of muscarinic contraction via decreasing both Ca2+ sensitization mediated by PKC and Ca2+ dynamics mediated by KCa channels. PKC and KCa channels may be molecular targets for cross talk between β2-adrenoceptors and muscarinic receptors.

Effect of aqueous extract of mango kernel (Mangifera Indica on basal activity of virgin rat uterine smooth muscle: role of muscarinic receptors

Directory of Open Access Journals (Sweden)

Mehdi Noureddini

2017-04-01

Full Text Available Background: Mango belongs to the Anacardiaceae and the extracts from its stems, leaves, fruit and kernel are reported to affect smooth muscle contractility. We studied the role of cholinergic muscarinic receptors for the effects of aqueous extract of mango kernel (Mangifera indica on the basal activity of virgin rat uterine smooth muscle. Materials and Methods: In this experimental study, mid-sections (n=24 of the uterine of healthy virgin rats were placed in an organ bath containing carbonated Tyrode’s solution under 1 g tension. The cumulative effects of the aqueous extracts of mango kernel (0.002, 0.02, 0.2, 2, 20, 200, and 2000 μg/mL or extract vehicle (Tyrode’s solution in the presence or absence of atropine were examined by isometric method using the strength, frequency and contractile activity of uterine smooth muscle. Results: The cumulative concentrations (0.002-20 µg/ml of mango kernel aqueous extract was significantly decreased the strength, frequency and contractile activity of uterine smooth muscle, but the contractile activity was returned to the basal level at the concentrations of 200 and 2000 µg/ml. Atropine (1 µM could not significantly change the effects of cumulative use of extract on the strength and contractile activity of uterine smooth muscle, but it significantly enhanced the contractile frequency at low concentrations. Conclusion: The effects of aqueous extract of mango kernel on the activity of the uterine smooth muscle might not be through cholinergic muscarinic receptors and atropine could enhance the effects of the extract on frequency through other receptors.

Acetylcholine receptors in the human retina

International Nuclear Information System (INIS)

Hutchins, J.B.; Hollyfield, J.G.

1985-01-01

Evidence for a population of acetylcholine (ACh) receptors in the human retina is presented. The authors have used the irreversible ligand 3 H-propylbenzilylcholine mustard ( 3 H-PrBCM) to label muscarinic receptors. 3 H- or 125 I-alpha-bungarotoxin (alpha-BTx) was used to label putative nicotinic receptors. Muscarinic receptors are apparently present in the inner plexiform layer of the retina. Autoradiographic grain densities are reduced in the presence of saturating concentrations of atropine, quinuclidinyl benzilate or scopolamine; this indicates that 3 H-PrBCM binding is specific for a population of muscarinic receptors in the human retina. Binding sites for radiolabeled alpha-BTx are found predominantly in the inner plexiform layer of the retina. Grain densities are reduced in the presence of d-tubocurarine, indicating that alpha-BTx may bind to a pharmacologically relevant nicotinic ACh receptor. This study provides evidence for cholinergic neurotransmission in the human retina

Muscarinic M1 receptor inhibition reduces gastroduodenal bicarbonate secretion and promotes gastric prostaglandin E2 synthesis in healthy volunteers

DEFF Research Database (Denmark)

Mertz-Nielsen, A; Hillingsø, Jens; Eskerod, O

1995-01-01

stimulated gastric and basal duodenal bicarbonate secretion by about 50% (p basal and vagally stimulated PGE2 output increased significantly (p ...The selective muscarinic M1 receptor antagonist, pirenzepine, considerably stimulates duodenal mucosal bicarbonate secretion in the rat and increases gastric luminal release of prostaglandin E2 (PGE2) in humans. This study, therefore, looked at the effect of pirenzepine on bicarbonate secretion...... sham feeding and acid exposure (HCl 0.1 M; 20 ml; 5 min) of the duodenal bulb increased mucosal bicarbonate secretion from 191 (14) mumol/cm x h to 266 (27) mumol/cm x h (p basal and vagally...

Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor

Science.gov (United States)

Wan, Min; Zhang, Wenhua; Tian, Yangli; Xu, Chanjuan; Xu, Tao; Liu, Jianfeng; Zhang, Rongying

2015-01-01

Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence 374KKKPPPS380 servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching 374KKKPPPS380 to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, 361VARKIVKMTKQPA373, which is normally masked in the presence of the downstream sequence 374KKKPPPS380. Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent. PMID:26094760

An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

International Nuclear Information System (INIS)

Basu, Niladri; Stamler, Christopher J.; Loua, Kovana Marcel; Chan, H.M.

2005-01-01

Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl 2 ) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl 2 and MeHg on [ 3 H]-quinuclidinyl benzilate ([ 3 H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B max ) and ligand affinity (K d ). Subsequently, samples were exposed to HgCl 2 or MeHg to derive IC50 values and inhibition constants (K i ). Results demonstrate that HgCl 2 is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [ 3 H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies

Effects of Saw Palmetto Extract on Urodynamic Parameters, Bladder Muscarinic and Purinergic Receptors and Urinary Cytokines in Rats with Cyclophosphamide-Induced Cystitis.

Science.gov (United States)

Nasrin, Sweety; Masuda, Eiji; Kugaya, Haruna; Osano, Ayaka; Ito, Yoshihiko; Yamada, Shizuo

2014-01-01

To clarify the effect of saw palmetto extract (SPE), a phytotherapeutic agent, on urodynamic parameters, bladder muscarinic and purinergic receptors, and urinary cytokines in rats with cystitis induced by cyclophosphamide (CYP). Saw palmetto extract (60 mg/kg per day) was administered orally twice a day for 7 days to rats. The urodynamic parameters in CYP (150 mg/kg i.p.)-treated rats were monitored by a cystometric method under anesthesia. The muscarinic and purinergic receptors in the bladder and submaxillary gland were measured by radioreceptor assays using [N-methyl-(3) H] scopolamine chloride([(3) H]NMS) and αβ-methylene-ATP [2,8-(3) H] tetrasodium salt ([(3) H]αβ-MeATP), respectively. Urinary cytokines (interleukin-1β [IL-1β], IL-6 and L-17) were measured with enzyme linked immunosorbent assay kits. Micturition interval and micturition volume were significantly decreased and the frequency of micturition and basal pressure were significantly increased in the CYP-treated rats compared with sham-operated rats. Orally administered SPE significantly increased the micturition interval and micturition volume and decreased the frequency of micturition and basal pressure. The maximal number of sites (Bmax ) for the specific binding of [(3) H]NMS and [(3) H]αβ-MeATP was significantly decreased in the bladder. The decrease in receptors was attenuated by repeated treatment with SPE. An elevation in urinary cytokine (IL-1β and IL-17) levels were seen, and this increase was effectively suppressed by SPE treatment. Saw palmetto extract attenuates the alteration of urodynamic parameters, pharmacologically relevant receptors, and urinary cytokines in CYP-treated rats. Therefore, SPE may be a potential therapeutic agent for improving the clinical symptoms of cystitis. © 2013 Wiley Publishing Asia Pty Ltd.

Acute food deprivation reverses morphine-induced locomotion deficits in M5 muscarinic receptor knockout mice.

Science.gov (United States)

Steidl, Stephan; Lee, Esther; Wasserman, David; Yeomans, John S

2013-09-01

Lesions of the pedunculopontine tegmental nucleus (PPT), one of two sources of cholinergic input to the ventral tegmental area (VTA), block conditioned place preference (CPP) for morphine in drug-naïve rats. M5 muscarinic cholinergic receptors, expressed by midbrain dopamine neurons, are critical for the ability of morphine to increase nucleus accumbens dopamine levels and locomotion, and for morphine CPP. This suggests that M5-mediated PPT cholinergic inputs to VTA dopamine neurons critically contribute to morphine-induced dopamine activation, reward and locomotion. In the current study we tested whether food deprivation, which reduces PPT contribution to morphine CPP in rats, could also reduce M5 contributions to morphine-induced locomotion in mice. Acute 18-h food deprivation reversed the phenotypic differences usually seen between non-deprived wild-type and M5 knockout mice. That is, food deprivation increased morphine-induced locomotion in M5 knockout mice but reduced morphine-induced locomotion in wild-type mice. Food deprivation increased saline-induced locomotion equally in wild-type and M5 knockout mice. Based on these findings, we suggest that food deprivation reduces the contribution of M5-mediated PPT cholinergic inputs to the VTA in morphine-induced locomotion and increases the contribution of a PPT-independent pathway. The contributions of cholinergic, dopaminergic and GABAergic neurons to the effects of acute food deprivation are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Asparagine, valine, and threonine in the third extracellular loop of muscarinic receptor have essential roles in the positive cooperativity of strychnine-like allosteric modulators

Czech Academy of Sciences Publication Activity Database

Jakubík, Jan; Krejčí, Alena; Doležal, Vladimír

2005-01-01

Roč. 313, č. 2 (2005), s. 688-696 ISSN 0022-3565 R&D Projects: GA ČR(CZ) GA309/02/1331; GA ČR(CZ) GP305/02/D090; GA AV ČR(CZ) IAA5011306; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * positive cooperativity * mutation Subject RIV: ED - Physiology Impact factor: 4.098, year: 2005

Individual receptor profiling as a novel tool to support diagnosis of bladder pain syndrome/interstitial cystitis (BPS/IC).

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Neuhaus, Jochen; Schulte-Baukloh, Heinrich; Stolzenburg, Jens-Uwe; Speroni di Fenizio, Pietro; Horn, Lars-Christian; Rüffert, Henrik; Hartenstein, Siegurd; Burger, Maximilian; Schulze, Matthias; Schwalenberg, Thilo

2012-10-01

Dysregulation of neurotransmitter receptors may contribute to bladder overactivity (OAB) symptoms. To address the question whether specific receptor expression patterns are associated with bladder pain syndrome/interstitial cystitis (BPS/IC), we examined the expression of muscarinic, purinergic and histamine receptors in the detrusor. Detrusor receptor expression was investigated in bladder biopsies of female BPS/IC patients (n = 44; age 60.64 ± 13.78, mean ± SD) and carcinoma patients (n = 11; age 58.91 ± 12.72) undergoing cystectomy. Protein expression of muscarinic (M2, M3), purinergic (P2X1-3) and histamine receptors (H1, H2) was analysed by confocal immunofluorescence, and gene expression was quantified by real-time polymerase chain reaction (qPCR). M2, P2X1, P2X2 and H1 receptor immunoreactivity (-IR) was significantly enhanced in BPS/IC compared to the control group, while there was no difference for M3-, P2X3- and H2-IR. We calculated a score, which separated BPS/IC from control patients with an AUC of 89.46%, showing 84.09% sensitivity and 90.91% specificity. Patients had a 9.25 times enhanced calculated risk for BPS/IC. In addition, two patient subgroups (M2 > M3 and M3 > M2) were observed, which differed in associated purinergic and histamine receptor expression. M2, P2X1, P2X2 and H1 were significantly upregulated in BPS/IC patients, and H2 was occasionally highly overexpressed. There was no significant correlation between receptor protein and gene expression, implying posttranslational mechanisms being responsible for the altered receptor expressions. On the basis of individual receptor profiles, upregulated receptors could be targeted by monotherapy or combination therapy with already approved receptor inhibitors, thereby promoting tailored therapy for patients suffering from BPS/IC-like symptoms.

Vitamin C deficiency reduces muscarinic receptor coronary artery vasoconstriction and plasma tetrahydrobiopterin concentration in guinea pigs

DEFF Research Database (Denmark)

Skovsted, Gry Freja; Tveden-Nyborg, Pernille; Lindblad, Maiken Marie

2017-01-01

Vitamin C (vitC) deficiency is associated with increased cardiovascular disease risk, but its specific interplay with arteriolar function is unclear. This study investigates the effect of vitC deficiency in guinea pigs on plasma biopterin status and the vasomotor responses in coronary arteries...... exposed to vasoconstrictor/-dilator agents. Dunkin Hartley female guinea pigs (n = 32) were randomized to high (1500 mg/kg diet) or low (0 to 50 mg/kg diet) vitC for 10-12 weeks. At euthanasia, coronary artery segments were dissected and mounted in a wire-myograph. Vasomotor responses to potassium......-1 were unaffected by vitC status. The study shows that vitC deficiency decreases tetrahydrobiopterin concentrations and muscarinic receptor mediated contraction in coronary arteries. This attenuated vasoconstrictor response may be linked to altered production of vasoactive arachidonic acid...

Synthesis of novel and functionally selective non‐competitive muscarinic antagonists as chemical probes

Czech Academy of Sciences Publication Activity Database

Boulos, J. F.; Jakubík, Jan; Boulos, J. M.; Randáková, Alena; Momirov, J.

2018-01-01

Roč. 91, č. 1 (2018), s. 93-104 ISSN 1747-0277 R&D Projects: GA ČR(CZ) GA14-05696S; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : G- protein -coupled receptor * muscarinic acetylcholine receptor * N-methylscopolamine * Parkinson's disease * positive allosteric modulator Subject RIV: ED - Physiology OBOR OECD: Physiology (including cytology) Impact factor: 2.396, year: 2016

Evidence for inhibitory nicotinic and facilitatory muscarinic receptors in cholinergic nerve terminals of the rat urinary bladder.

Science.gov (United States)

Somogyi, G T; de Groat, W C

1992-02-01

Cholinergic prejunctional modulatory receptors on parasympathetic nerves in the rat urinary bladder were studied by measuring 3H-acetylcholine (ACh) release in muscle strips from the bladder body. Electrical field stimulation markedly increased 3H-ACh overflow in strips preloaded with 3H-choline. Oxotremorine (1 microM), an M2 receptor agonist and DMPP (10 microM) a nicotinic (N) receptor agonist decreased the release of ACh (50% and 55% respectively); whereas McN-A 343 (50 microM) an M1 receptor agonist increased the release (33%), indicating the presence of three types of modulatory receptors. The anticholinesterase agent, physostigmine in concentrations of 1, 5 and 25 microM and neostigmine (5 microM) increased ACh release (44-710%). However a low concentration of physostigmine (0.05 microM) decreased release. Pirenzepine, an M1 muscarinic antagonist or atropine blocked the increased ACh release in physostigmine-treated strips, but in normal strips pirenzepine did not change release and atropine increased release. McN-A 343 or prolonged application (15 min) of DMPP increased ACh release (376% and 391% respectively) in physostigmine-treated strips. The response to McN-A 343 was blocked by pirenzepine. d-Tubocurarine (DTC), a nicotinic receptor blocker, enhanced ACh release in the presence of physostigmine but proved to be ineffective in normal preparations. These findings suggest that all three cholinergic receptors (M1 facilitatory, N inhibitory and M2 inhibitory) are activated by endogenous ACh in physostigmine treated preparations whereas only M2-inhibitory receptors are activated in normal preparations. It will be important in future studies to determine whether M1 and M2 mechanisms can also be activated under more physiological conditions in the bladder and whether they are present at other cholinergic synapses.

Nucleus Accumbens Acetylcholine Receptors Modulate Dopamine and Motivation.

Science.gov (United States)

Collins, Anne L; Aitken, Tara J; Greenfield, Venuz Y; Ostlund, Sean B; Wassum, Kate M

2016-11-01

Environmental reward-predictive cues can motivate reward-seeking behaviors. Although this influence is normally adaptive, it can become maladaptive in disordered states, such as addiction. Dopamine release in the nucleus accumbens core (NAc) is known to mediate the motivational impact of reward-predictive cues, but little is known about how other neuromodulatory systems contribute to cue-motivated behavior. Here, we examined the role of the NAc cholinergic receptor system in cue-motivated behavior using a Pavlovian-to-instrumental transfer task designed to assess the motivating influence of a reward-predictive cue over an independently-trained instrumental action. Disruption of NAc muscarinic acetylcholine receptor activity attenuated, whereas blockade of nicotinic receptors augmented cue-induced invigoration of reward seeking. We next examined a potential dopaminergic mechanism for this behavioral effect by combining fast-scan cyclic voltammetry with local pharmacological acetylcholine receptor manipulation. The data show evidence of opposing modulation of cue-evoked dopamine release, with muscarinic and nicotinic receptor antagonists causing suppression and augmentation, respectively, consistent with the behavioral effects of these manipulations. In addition to demonstrating cholinergic modulation of naturally-evoked and behaviorally-relevant dopamine signaling, these data suggest that NAc cholinergic receptors may gate the expression of cue-motivated behavior through modulation of phasic dopamine release.

Guanosine 5'-triphosphate binding protein (G/sub i/) and two additional pertussis toxin substrates associated with muscarinic receptors in rat heart myocytes: characterization and age dependency

International Nuclear Information System (INIS)

Moscona-Amir, E.; Henis, Y.I.; Sokolovsky, M.

1988-01-01

The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. The coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completely different effects of 5'-guanylyl imidodiphosphate [Gpp(NH)p] on muscarinic agonist binding to homogenates from young vs aged cultures. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no such effect was observed in homogenates from young cultures. IAP-catalyzed [ 32 P]ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-α/sub i/ antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-α/sub i/ or anti-α 0 antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. The parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures. The role of the membrane lipid composition in these phenomena is discussed

Laminar pattern of cholinergic and adrenergic receptors in rat visual cortex using quantitative receptor autoradiography

International Nuclear Information System (INIS)

Schliebs, R.; Walch, C.

1989-01-01

The laminar distribution of muscarinic acetylcholine receptors, including the M1-receptor subtype, of beta-adrenergic receptors, and noradrenaline uptake sites, was studied in the adult rat visual, frontal, somatosensory and motor cortex, using quantitative receptor autoradiography. In the visual cortex, the highest density of muscarinic acetylcholine receptors was found in layer I. From layer II/III to layer V binding decreases continueously reaching a constant binding level in layers V and VI. This laminar pattern of muscarinic receptor density differs somewhat from that observed in the non-visual cortical regions examined: layer II/III contained the highest receptor density followed by layer I and IV: lowest density was found in layer V and VI. The binding profile of the muscarinic cholinergic M1-subtype through the visual cortex shows a peak in cortical layer II and in the upper part of layer VI, whereas in the non-visual cortical regions cited the binding level was high in layer II/III, moderate in layer I and IV, and low in layer VI. Layers I to IV of the visual cortex contained the highest beta-adrenergic receptor densities, whereas only low binding levels were observed in the deeper layers. A similar laminar distribution was found also in the frontal, somatosensory and motor cortex. The density of noradrenaline uptake sites was high in all layers of the cortical regions studied, but with noradrenaline uptake sites somewhat more concentrated in the superficial layers than in deeper ones. The distinct laminar pattern of cholinergic and noradrenergic receptor sites indicates a different role for acetylcholine and noradrenaline in the functional anatomy of the cerebral cortex, and in particular, the visual cortex. (author)

Laminar pattern of cholinergic and adrenergic receptors in rat visual cortex using quantitative receptor autoradiography

Energy Technology Data Exchange (ETDEWEB)

Schliebs, R; Walch, C [Leipzig Univ. (German Democratic Republic). Bereich Medizin; Stewart, M G [Open Univ., Milton Keynes (UK)

1989-01-01

The laminar distribution of muscarinic acetylcholine receptors, including the M1-receptor subtype, of beta-adrenergic receptors, and noradrenaline uptake sites, was studied in the adult rat visual, frontal, somatosensory and motor cortex, using quantitative receptor autoradiography. In the visual cortex, the highest density of muscarinic acetylcholine receptors was found in layer I. From layer II/III to layer V binding decreases continueously reaching a constant binding level in layers V and VI. This laminar pattern of muscarinic receptor density differs somewhat from that observed in the non-visual cortical regions examined: layer II/III contained the highest receptor density followed by layer I and IV: lowest density was found in layer V and VI. The binding profile of the muscarinic cholinergic M1-subtype through the visual cortex shows a peak in cortical layer II and in the upper part of layer VI, whereas in the non-visual cortical regions cited the binding level was high in layer II/III, moderate in layer I and IV, and low in layer VI. Layers I to IV of the visual cortex contained the highest beta-adrenergic receptor densities, whereas only low binding levels were observed in the deeper layers. A similar laminar distribution was found also in the frontal, somatosensory and motor cortex. The density of noradrenaline uptake sites was high in all layers of the cortical regions studied, but with noradrenaline uptake sites somewhat more concentrated in the superficial layers than in deeper ones. The distinct laminar pattern of cholinergic and noradrenergic receptor sites indicates a different role for acetylcholine and noradrenaline in the functional anatomy of the cerebral cortex, and in particular, the visual cortex. (author).

Stimulation of Na+ -K+ -pump currents by epithelial nicotinic receptors in rat colon.

Science.gov (United States)

Bader, Sandra; Lottig, Lena; Diener, Martin

2017-05-01

Acetylcholine-induced epithelial Cl - secretion is generally thought to be mediated by epithelial muscarinic receptors and nicotinic receptors on secretomotor neurons. However, recent data have shown expression of nicotinic receptors by intestinal epithelium and the stimulation of Cl - secretion by nicotine, in the presence of the neurotoxin, tetrodotoxin. Here, we aimed to identify the transporters activated by epithelial nicotinic receptors and to clarify their role in cholinergic regulation of intestinal ion transport. Ussing chamber experiments were performed, using rat distal colon with intact epithelia. Epithelia were basolaterally depolarized to measure currents across the apical membrane. Apically permeabilized tissue was also used to measure currents across the basolateral membrane in the presence of tetrodotoxin. Nicotine had no effect on currents through Cl - channels in the apical membrane or on currents through K + channels in the apical or the basolateral membrane. Instead, nicotine stimulated the Na + -K + -pump as indicated by Na + -dependency and sensitivity of the nicotine-induced current across the basolateral membrane to cardiac steroids. Effects of nicotine were inhibited by nicotinic receptor antagonists such as hexamethonium and mimicked by dimethyl-4-phenylpiperazinium, a chemically different nicotinic agonist. Simultaneous stimulation of epithelial muscarinic and nicotinic receptors led to a strong potentiation of transepithelial Cl - secretion. These results suggest a novel concept for the cholinergic regulation of transepithelial ion transport by costimulation of muscarinic and nicotinic epithelial receptors and a unique role of nicotinic receptors controlling the activity of the Na + -K + -ATPase. © 2017 The British Pharmacological Society.

Triazolam-induced modulation of muscarinic acetylcholine receptor in living brain slices as revealed by a new positron-based imaging technique

International Nuclear Information System (INIS)

Murata, T.; Matsumura, K.; Onoe, H.; Watanabe, Y.; Sihver, S.; Sihver, W.; Langstroem, B.; Bergstroem, M.; Yonekura, Y.

1997-01-01

The effect of triazolam, a potent benzodiazepine (BZ) agonist, on muscarinic acetylcholinergic receptor (mAChR) binding was investigated in living brain slices by use of a novel positron-based imaging technique. Fresh rat brain slices were incubated with [ 11 C]N-methyl-4-piperidylbenzilate ([ 11 C]NMPB), a mAChR antagonist, in oxygenated Krebs-Ringer solution at 37 degree C. During incubation, time-resolved imaging of [ 11 C]NMPB binding in the slices was constructed on the storage phosphor screens. Addition of triazolam (1 μM) plus muscimol (30 μM), a GABA A receptor agonist, to the incubation mixture decreased the specific binding of [ 11 C]NMPB. Ro15-1788, a BZ receptor antagonist, prevented this effect, indicating that the effect was exerted through the GABA A /BZ receptor complex. These results demonstrated that stimulation of the GABA A /BZ receptor lowers the affinity of the mAChR for its ligand, which may underlie the BZ-induced amnesia, a serious clinical side effect of BZ. No such effect in the P2-fraction instead implies that the integrity of the neuronal cells and/or their environment is prerequisite for the modulation of mAChR by GABA A /BZ stimulation. (author)

Combined treatment with a β3 -adrenergic receptor agonist and a muscarinic receptor antagonist inhibits detrusor overactivity induced by cold stress in spontaneously hypertensive rats.

Science.gov (United States)

Imamura, Tetsuya; Ogawa, Teruyuki; Minagawa, Tomonori; Nagai, Takashi; Suzuki, Toshiro; Saito, Tetsuichi; Yokoyama, Hitoshi; Nakazawa, Masaki; Ishizuka, Osamu

2017-04-01

This study determined if combined treatment with the muscarinic receptor (MR) antagonist solifenacin and the β 3 -adrenergic receptor (AR) agonist mirabegron could inhibit detrusor overactivity induced by cold stress in spontaneously hypertensive rats (SHRs). Thirty-two female 10-week-old SHRs were fed an 8% NaCl-supplemented diet for 4 weeks. Cystometric measurements of the unanesthetized, unrestricted rats were performed at room temperature (RT, 27 ± 2°C) for 20 min. The rats were then intravenously administered vehicle, 0.1 mg/kg solifenacin alone, 0.1 mg/kg mirabegron alone, or the combination of 0.1 mg/kg mirabegron and 0.1 mg/kg solifenacin (n = 8 each group). Five minutes later, the treated rats were exposed to low temperature (LT, 4 ± 2°C) for 40 min. Finally, the rats were returned to RT. After the cystometric investigations, the β 3 -ARs and M 3 -MRs expressed within the urinary bladders were analyzed. Just after transfer from RT to LT, vehicle-, solifenacin-, and mirabegron-treated SHRs exhibited detrusor overactivity that significantly decreased voiding interval and bladder capacity. However, treatment with the combination of solifenacin and mirabegron partially inhibited the cold stress-induced detrusor overactivity patterns. The decreases of voiding interval and bladder capacity in the combination-treated rats were significantly inhibited compared to other groups. Within the urinary bladders, there were no differences between expression levels of M 3 -MR and β 3 -AR mRNA. The tissue distribution of M 3 -MRs was similar to that of the β 3 -ARs. This study suggested that the combination of solifenacin and mirabegron act synergistically to inhibit the cold stress-induced detrusor overactivity in SHRs. Neurourol. Urodynam. 36:1026-1033, 2017. © 2016 The Authors. Neurourology and Urodynamics Published by Wiley Periodicals, Inc. © 2016 The Authors. Neurourology and Urodynamics Published by Wiley Periodicals, Inc.

A comparison of β-adrenoceptors and muscarinic cholinergic receptors in tissues of brown bullhead catfish (Ameiurus nebulosus) from the black river and old woman creek, Ohio

Science.gov (United States)

Steevens, Jeffery A.; Baumann, Paul C.; Jones, Susan B.

1996-01-01

β-Adrenoceptors (βARs) and muscarinic cholinergic receptors were measured in brain, gill, and heart tissues of brown bullhead catfish exposed to polycyclic aromatic hydrocarbons in the Black River, Ohio, USA, and were compared to values from Old Woman Creek, Ohio, a reference site. A decreased number of βARs were found in the gill from Black River fish, possibly indicating a compensatory response subsequent to chemical stress.

Synthesis of N-Substituted Piperidine Salts as Potential Muscarinic Ligands for Alzheimer's Applications

Czech Academy of Sciences Publication Activity Database

Boulos, J.; Jakubík, Jan; Randáková, Alena; Avila, C.

2013-01-01

Roč. 50, č. 6 (2013), s. 1363-1367 ISSN 0022-152X R&D Projects: GA ČR(CZ) GA305/09/0681 Institutional support: RVO:67985823 Keywords : N-piperidine substituted salts * muscarinic receptor antagonists * selectivity Subject RIV: ED - Physiology Impact factor: 0.873, year: 2013

Interactions between allosteric modulators and 4-DAMP and other antagonists at muscarinic receptors: potential significance of the distance between the N and Carboxyl C atoms in the molecules of antagonists

Czech Academy of Sciences Publication Activity Database

Lysíková, Michaela; Havlas, Zdeněk; Tuček, Stanislav

2001-01-01

Roč. 26, č. 4 (2001), s. 383-394 ISSN 0364-3190 R&D Projects: GA ČR GA309/99/0214; GA MŠk LN00A032 Institutional research plan: CEZ:AV0Z5011922 Keywords : muscarinic receptors * allosteric modulation * 4-DAMP Subject RIV: ED - Physiology Impact factor: 1.638, year: 2001

Agonist actions of clothianidin on synaptic and extrasynaptic nicotinic acetylcholine receptors expressed on cockroach sixth abdominal ganglion.

Science.gov (United States)

Thany, Steeve H

2009-11-01

Clothianidin is new neonicotinoid insecticide acting selectively on insect nicotinic acetylcholine receptors (nAChRs). Its effects on nAChRs expressed on cercal afferent/giant interneuron synapses and DUM neurons have been studied using mannitol-gap and whole-cell patch-clamp techniques, respectively. Bath-application of clothianidin-induced dose-dependent depolarizations of cockroach cercal afferent/giant interneuron synapses which were not reversed after wash-out suggesting a strong desensitization of postsynaptic interneurons at the 6th abdominal ganglion (A6). Clothinidin activity on the nerve preparation was characterized by an increased firing rate of action potentials which then ceased when the depolarization reached a peak. Clothianidin responses were insensitive to all muscarinic antagonists tested but were blocked by co-application of specific nicotinic antagonists methyllicaconitine, alpha-bungarotoxin and d-tubocurarine. In a second round of experiment, clothianidin actions were tested on DUM neurons isolated from the A6. There was a strong desensitization of nAChRs which was not affected by muscarinic antagonists, pirenzepine and atropine, but was reduced with nicotinic antagonist alpha-bungarotoxin. In addition, clothianidin-induced currents were completely blocked by methyllicaconitine suggesting that (1) clothianidin acted as a specific agonist of nAChR subtypes and (2) a small proportion of receptors blocked by MLA was insensitive to alpha-bungarotoxin. Moreover, because clothianidin currents were blocked by d-tubocurarine and mecamylamine, we provided that clothianidin was an agonist of both nAChRs: imidacloprid-sensitive nAChR1 and -insensitive nAChR2 subtypes.

Muscarinic cholinergic and alpha 2-adrenergic receptors in the epithelium and muscularis of the human ileum

International Nuclear Information System (INIS)

Lepor, H.; Rigaud, G.; Shapiro, E.; Baumann, M.; Kodner, I.J.; Fleshman, J.W.

1990-01-01

The aim of this study was to characterize the binding and functional properties of muscarinic cholinergic (MCh) and alpha 2-adrenergic receptors in the human ileum to provide insight into pharmacologic strategies for managing urinary and fecal incontinence after bladder and rectal replacement with intestinal segments. MCh and alpha 2-adrenergic binding sites were characterized in the epithelium and muscularis of eight human ileal segments with 3H-N-methylscopolamine and 3H-rauwolscine, respectively. The dissociation constant for 3H-N-methylscopolamine in the epithelium and muscularis was 0.32 +/- 0.07 nmol/L and 0.45 +/- 0.10 nmol/L, respectively (p = 0.32). The MCh receptor content was approximately eightfold greater in the muscularis compared with the epithelium (p = 0.008). The dissociation constant for 3H-rauwolscine in the muscularis and epithelium was 2.55 +/- 0.42 nmol/L and 2.03 +/- 0.19 nmol/L, respectively (p = 0.29). The alpha 2-adrenoceptor density was twofold greater in the epithelium compared with the muscularis (p = 0.05). Noncumulative concentration-response experiments were performed with carbachol, an MCh agonist, and UK-14304, a selective alpha 2-adrenergic agonist. The epithelium did not contract in the presence of high concentrations of carbachol and UK-14304. The muscularis preparations were responsive only to carbachol. The muscularis contains primarily MCh receptors mediating smooth muscle contraction. The alpha 2-adrenoceptors are localized primarily to the epithelium and may regulate water secretion in the intestine. The distribution and functional properties of ileal MCh and alpha 2-adrenergic receptors provide a theoretic basis for the treatment of incontinence after bladder and rectal replacement with intestinal segments

Beta3 adrenoceptors substitute the role of M(2) muscarinic receptor in coping with cold stress in the heart: evidence from M(2)KO mice.

Science.gov (United States)

Benes, Jan; Novakova, Martina; Rotkova, Jana; Farar, Vladimir; Kvetnansky, Richard; Riljak, Vladimir; Myslivecek, Jaromir

2012-07-01

We investigated the role of beta3-adrenoceptors (AR) in cold stress (1 or 7 days in cold) in animals lacking main cardioinhibitive receptors-M2 muscarinic receptors (M(2)KO). There was no change in receptor number in the right ventricles. In the left ventricles, there was decrease in binding to all cardiostimulative receptors (beta1-, and beta2-AR) and increase in cardiodepressive receptors (beta3-AR) in unstressed KO in comparison to WT. The cold stress in WT animals resulted in decrease in binding to beta1- and beta2-AR (to 37%/35% after 1 day in cold and to 27%/28% after 7 days in cold) while beta3-AR were increased (to 216% of control) when 7 days cold was applied. MR were reduced to 46% and 58%, respectively. Gene expression of M2 MR in WT was not changed due to stress, while M3 was changed. The reaction of beta1- and beta2-AR (binding) to cold was similar in KO and WT animals, and beta3-AR in stressed KO animals did not change. Adenylyl cyclase activity was affected by beta3-agonist CL316243 in cold stressed WT animals but CL316243 had almost no effects on adenylyl cyclase activity in stressed KO. Nitric oxide activity (NOS) was not affected by BRL37344 (beta3-agonist) both in WT and KO animals. Similarly, the stress had no effects on NOS activity in WT animals and in KO animals. We conclude that the function of M2 MR is substituted by beta3-AR and that these effects are mediated via adenylyl cyclase rather than NOS.

Effects of trihexyphenidyl and L-dopa on brain muscarinic cholinergic receptor binding measured by positron emission tomography

Energy Technology Data Exchange (ETDEWEB)

Shinotoh, H; Asahina, M; Hirayama, K [Dept. of Neurology, School of Medicine, Chiba Univ., Chiba (Japan); Inoue, O; Suhara, T; Tateno, Y [Division of Clinical Research, National Inst. of Radiological Sciences, Chiba (Japan)

1994-01-01

The effects of pharmacological intervention on brain muscarinic cholinergic receptor (mAChR) binding were assessed in seven patients with Parkinson's disease by positron emission tomography and carbon-11 labelled N-methyl-4-piperidyl benzilate ([[sup 11]C]NMPB). [[sup 11]C]NMPB was injected twice, approximately 2 hours apart, in each patient, to assess the effect of single doses of 4 mg of trihexyphenidyl (n=5) or 400 mg of L-dopa with 57 mg of benserazide (n=2) on the binding parameter of mAChRs (K[sub 3]). There was a mean 28% inhibition of K[sub 3] values in the brain in the presence of trihexyphenidyl, which was assumed to reflect mAChR occupancy. No significant change in K[sub 3] was observed in the presence of L-dopa. This study demonstrates the feasibility of measuring mAChR occupancy by an anticholinergic medication with PET.

Nitric oxide/cGMP/PKG signaling pathway activated by M1-type muscarinic acetylcholine receptor cascade inhibits Na+-activated K+ currents in Kenyon cells

Science.gov (United States)

Hasebe, Masaharu

2016-01-01

The interneurons of the mushroom body, known as Kenyon cells, are essential for the long-term memory of olfactory associative learning in some insects. Some studies have reported that nitric oxide (NO) is strongly related to this long-term memory in Kenyon cells. However, the target molecules and upstream and downstream NO signaling cascades are not completely understood. Here we analyzed the effect of the NO signaling cascade on Na+-activated K+ (KNa) channel activity in Kenyon cells of crickets (Gryllus bimaculatus). We found that two different NO donors, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-dl-penicillamine (SNAP), strongly suppressed KNa channel currents. Additionally, this inhibitory effect of GSNO on KNa channel activity was diminished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), and KT5823, an inhibitor of protein kinase G (PKG). Next, we analyzed the role of ACh in the NO signaling cascade. ACh strongly suppressed KNa channel currents, similar to NO donors. Furthermore, this inhibitory effect of ACh was blocked by pirenzepine, an M1 muscarinic ACh receptor antagonist, but not by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) and mecamylamine, an M3 muscarinic ACh receptor antagonist and a nicotinic ACh receptor antagonist, respectively. The ACh-induced inhibition of KNa channel currents was also diminished by the PLC inhibitor U73122 and the calmodulin antagonist W-7. Finally, we found that ACh inhibition was blocked by the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME). These results suggested that the ACh signaling cascade promotes NO production by activating NOS and NO inhibits KNa channel currents via the sGC/cGMP/PKG signaling cascade in Kenyon cells. PMID:26984419

Muscarinic supersensibility of anterior pituitary ACTH and beta-endorphin release in major depressive illness

International Nuclear Information System (INIS)

Risch, S.C.; Gillin, J.C.; Janowsky, D.S.

1986-01-01

Since numerious physiological systems display muscarinic receptor supersensitivity in major depressive illnesses, the authors have hypothesize that anterior pituitary release of ACTH and beta-Endorphin immunoreactivity may also be muscarinically supersensitive in depression. The studies were conducted under FDA-approved IND and with local human subjects committee approval. Plasma ACTH concentrations were determined as follows: samples were assayed in duplicate using equilibrium radioimmunoassay utilizing a rabbit anti-porcine ACTH antibody. Significance ofchanges in plasma cortisol, ACTH and beta-endorphin immunoreactivity after physostigmine and saline were determined by repeated measures analysis of variance. In all subject groups, physostigmine (relative to placebo) caused significant increases in plasma concentrations of cortisol, ACTH, and beta-endorphin immonoreactivity

Fluoxetine induces vasodilatation of cerebral arterioles by co-modulating NO/muscarinic signalling

Science.gov (United States)

Ofek, Keren; Schoknecht, Karl; Melamed-Book, Naomi; Heinemann, Uwe; Friedman, Alon; Soreq, Hermona

2012-01-01

Ischaemic stroke patients treated with Selective Serotonin Reuptake Inhibitors (SSRI) show improved motor, cognitive and executive functions, but the underlying mechanism(s) are incompletely understood. Here, we report that cerebral arterioles in the rat brain superfused with therapeutically effective doses of the SSRI fluoxetine showed consistent, dose-dependent vasodilatation (by 1.2 to 1.6-fold), suppressible by muscarinic and nitric oxide synthase (NOS) antagonists [atropine, NG-nitro-l-arginine methyl ester (l-NAME)] but resistant to nicotinic and serotoninergic antagonists (mecamylamine, methylsergide). Fluoxetine administered 10–30 min. following experimental vascular photo-thrombosis increased arterial diameter (1.3–1.6), inducing partial, but lasting reperfusion of the ischaemic brain. In brain endothelial b.End.3 cells, fluoxetine induced rapid muscarinic receptor-dependent increases in intracellular [Ca2+] and promoted albumin- and eNOS-dependent nitric oxide (NO) production and HSP90 interaction. In vitro, fluoxetine suppressed recombinant human acetylcholinesterase (rhAChE) activity only in the presence of albumin. That fluoxetine induces vasodilatation of cerebral arterioles suggests co-promotion of endothelial muscarinic and nitric oxide signalling, facilitated by albumin-dependent inhibition of serum AChE. PMID:22697296

Differential anti-ischaemic effects of muscarinic receptor blockade in patients with obstructive coronary artery disease; impaired vs normal left ventricular function.

NARCIS (Netherlands)

A.F. van den Heuvel; D.J. van Veldhuisen (Dirk); G.L. Bartels; M. van der Ent (Martin); W.J. Remme (Willem)

1999-01-01

textabstractAIMS: In patients with coronary artery disease acetylcholine (a muscarinic agonist) causes vasoconstriction. The effect of atropine (a muscarinic antagonist) on coronary vasotone in patients with normal or impaired left ventricular function is unknown.

Activation of muscarinic receptors by a hydroalcoholic extract of Dicksonia sellowiana Presl. HooK (Dicksoniaceae) induces vascular relaxation and hypotension in rats.

Science.gov (United States)

Rattmann, Yanna D; Crestani, Sandra; Lapa, Fernanda R; Miguel, Obdúlio G; Marques, Maria C A; da Silva-Santos, J Eduardo; Santos, Adair R S

2009-01-01

Dicksonia sellowiana (Presl.) Hook is a native plant from the Central and South Americas that contain high levels of polyphenols, antioxidant compounds involved in protection against inflammation, cancer and cardiovascular risk. A phytomedicinal preparation obtained from aerial parts of D. sellowiana is currently under clinical evaluation in Brazil against asthma, and has been associated with several other beneficial effects. This study demonstrates that a hydroalcoholic extract obtained from D. sellowiana leaves (HEDS) fully relax, in a concentration-dependent manner, rat aortic rings precontracted with phenylephrine. Moreover, administration of HEDS (10, 20 and 40 mg/kg, i.v.) in anaesthetized rats resulted in a strong but reversible hypotension. Aortic relaxation induced by HEDS was abolished by endothelium removal, by incubation of the nitric oxide synthase inhibitor L-NAME, or the soluble guanylate cyclase inhibitor ODQ. In addition, this effect was partially inhibited by indomethacin (a cyclooxygenase inhibitor) and KT 5730 (a PKA inhibitor). The potassium channels blockade by either tetraethylammonium or charybdotoxin also resulted in a potent inhibition of HEDS-induced aortic relaxation, whereas apamine only slightly reduced it. In addition HEDS-induced relaxation was unchanged by 4-amynopiridine and glibenclamide. The selective muscarinic receptor antagonist atropine counteracted both aortic relaxation and blood pressure reduction generated by HEDS. Experiments using HPLC revealed the presence of high amounts of phenolic compounds in this extract. Taken together, our results reveal that the D. sellowiana possess substances with both in vivo and in vitro activities and that the vascular effect of HEDS involves activation of muscarinic receptors, stimulation of the nitric oxide pathway and opening of calcium-activated potassium channels.

Muscarinic M2 receptors directly activate Gq/11 and Gs G-proteins

Czech Academy of Sciences Publication Activity Database

Michal, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

2007-01-01

Roč. 320, č. 2 (2007), s. 607-614 ISSN 0022-3565 R&D Projects: GA ČR GP305/05/P209; GA ČR GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic * siRNA * G-proteins Subject RIV: CE - Biochemistry Impact factor: 4.003, year: 2007

Relations between immune and mediator receptors of mouse lymphocytes

International Nuclear Information System (INIS)

Ado, A.D.; Alekseeva, T.A.; Kravchenko, S.A.

1985-01-01

This paper examines the action of the specific muscarinic antogonist tritium-quinuclidinyl benzilate (tritium-QNB) on immune rosette formation in mice. It is shown that since the specific muscarini antagonist tritium-QNB inhibits immune rosette formation, this process must be regarded as interconnected with muscarinic receptors of lymphocytes. Interaction of immune (antigen-binding) and mediator receptors, however, is an important factor maintaining immune homeostasis at a certain level

The role of muscarinic cholinergic signaling in cost-benefit decision making

Science.gov (United States)

Fobbs, Wambura

Animals regularly face decisions that affect both their immediate success and long term survival. Such decisions typically involve some form of cost-benefit analysis and engage a number of high level cognitive processes, including learning, memory and motivational influences. While decision making has been a focus of study for over a century, it's only in the last 20 years that researchers have begun to identify functional neural circuits that subserve different forms of cost-benefit decision making. Even though the cholinergic system is both functionally and anatomically positioned to modulate cost-benefit decision circuits, the contribution of the cholinergic system to decision making has been little studied. In this thesis, I investigated the cognitive and neural contribution of muscarinic cholinergic signaling to cost-benefit decision making. I, first, re-examined the effects of systemic administration of 0.3 mg/kg atropine on delay and probability discounting tasks and found that blockade of muscarinic acetylcholine receptors by atropine induced suboptimal choices (impulsive and risky) in both tasks. Since the effect on delay discounting was restricted to the No Cue version of the delay discounting task, I concluded that muscarinic cholinergic signaling mediates both forms of cost-benefit decision making and is selectively engaged when decisions require valuation of reward options whose costs are not externally signified. Second, I assessed the impact of inactivating the nucleus basalis (NBM) on both forms decision making and the effect of injecting atropine locally into the orbitofrontal cortex (OFC), basolateral amygdala (BLA), or nucleus accumbens (NAc) core during the No Cue version of the delay discounting task. I discovered that although NBM inactivation failed to affect delay discounting, it induced risk aversion in the probability discounting task; and blockade of intra- NAc core, but not intra-OFC or intra-BLA, muscarinic cholinergic signaling lead to

Serotonin-S2 and dopamine-D2 receptors are the same size in membranes

International Nuclear Information System (INIS)

Brann, M.R.

1985-01-01

Target size analysis was used to compare the sizes of serotonin-S2 and dopamine-D2 receptors in rat brain membranes. The sizes of these receptors were standardized by comparison with the muscarinic receptor, a receptor of known size. The number of serotonin-S2 receptors labeled with (3H)ketanserin or (3H)spiperone in frontal cortex decreased as an exponential function of radiation dose, and receptor affinity was not affected. The number of dopamine-D2 receptors labeled with (3H)spiperone in striatum also decreased as an exponential function of radiation dose, and D2 and S2 receptors were equally sensitive to radiation. In both striatum and frontal cortex, the number of muscarinic receptors labeled with (3H)QNB decreased as an exponential function of radiation dose, and were much less sensitive to radiation than S2 and D2 receptors. These data indicate that in rat brain membranes, S2 and D2 receptors are of similar size, and both molecules are much larger than the muscarinic receptor

Brain regional acetylcholinesterase activity and muscarinic acetylcholine receptors in rats after repeated administration of cholinesterase inhibitors and its withdrawal

International Nuclear Information System (INIS)

Kobayashi, Haruo; Suzuki, Tadahiko; Sakamoto, Maki; Hashimoto, Wataru; Kashiwada, Keiko; Sato, Itaru; Akahori, Fumiaki; Satoh, Tetsuo

2007-01-01

Activity of acetylcholinesterase (AChE) and specific binding of [ 3 H]quinuclidinyl benzilate (QNB), [ 3 H]pirenzepine (PZP) and [ 3 H]AF-DX 384 to muscarinic acetylcholine receptor (mAChR) preparations in the striatum, hippocampus and cortex of rats were determined 1, 6 and 11 days after the last treatment with an organophosphate DDVP, a carbamate propoxur or a muscarinic agonist oxotremorine as a reference for 7 and 14 days. AChE activity was markedly decreased in the three regions 1 day after the treatment with DDVP for 7 and 14 days with a gradual recovery 6 to 11 days, and much less decreased 1, 6 and 11 days after the treatment with propoxur for 7 days but not for 14 days in the hippocampus and cortex. The binding of [ 3 H]-QNB, PZP and AF-DX 384 in the three regions was generally decreased by the treatment with DDVP for 7 and 14 days. Such down-regulations were generally restored 6 or 11 days after the treatment for 7 but not for 14 days. The down-regulation or up-regulation as measured by [ 3 H]-QNB, PZP and AF-DX 384 was observed 1, 6 or 11 days after treatment with propoxur for 7 days and/or 14 days. Repeated treatment with oxotremorine produced similar effects except AChE activity to DDVP. These results suggest that repeated inhibition of AChE activity may usually cause down-regulation of mAChRs with some exception in the hippocampus when a reversible antiChE propoxur is injected

Sex-related differences in the muscarinic acetylcholinergic receptor in the healthy human brain. A positron emission tomography study

International Nuclear Information System (INIS)

Yoshida, Tsuyoshi; Kuwabara, Yasuo; Sasaki, Masayuki; Ichimiya, Atsushi; Takita, Masashi; Ogomori, Koji; Masuda, Kouji; Fukumura, Toshimitsu; Ichiya, Yuichi

2000-01-01

We evaluated the sex-related differences in the decline of the cerebral muscarinic acetylcholinergic receptor (mACh-R) due to aging by using 11 C-N-methyl-4-piperidyl benzilate ( 11 C-NMPB) and positron emission tomography (PET). The subjects consisted of 37 (20 males and 17 females) healthy volunteers. The 11 C-NMPB uptake was evaluated by the ratio method (regional 11 C-NMPB uptake/Cerebellar 11 C-NMPB uptake; rNMPB ratio). The correlation between sex, aging, and the rNMPB ratio in normal aging was evaluated by a multiple regression analysis. The rNMPB ratio was higher in females than in males throughout the entire cerebral region (p<0.01-p<0.0001) and the rNMPB ratio might thus possibly decline with age more rapidly in females. Our study therefore revealed the existence of sex-related differences in the cerebral mACh-R. (author)

No changes in lymphocyte muscarinic receptors and platelet monoamine oxidase-B examined as surrogate central nervous system biomarkers in a Faroese children cohort prenatally exposed to methylmercury and polychlorinated biphenyls

DEFF Research Database (Denmark)

Coccini, Teresa; Manzo, Luigi; Debes, Frodi

2009-01-01

Experimental evidence suggests that monoamine oxidase B (MAO-B) and muscarinic cholinergic receptors (mAChRs) are involved in the pathogenesis of neurotoxicity caused by methylmercury and polychlorinated biphenyls (PCBs). Blood samples from 7-year-old exposed children were analyzed for platelet M....../or PCB exposure, whereas these markers are significantly altered in sustained exposure scenarios, as shown by clinical studies in drug addicts or patients treated with psychopharmacological agents....

Impairment of muscarinic transmission in transgenic APPswe/PS1dE9 mice

Czech Academy of Sciences Publication Activity Database

Machová, Eva; Jakubík, Jan; Michal, Pavel; Oksman, M.; Iivonen, H.; Tanila, H.; Doležal, Vladimír

2008-01-01

Roč. 29, č. 3 (2008), s. 368-378 ISSN 0197-4580 R&D Projects: GA MŠk(CZ) LC554; GA AV ČR(CZ) IAA5011206 Grant - others:EC(XE) QLK1-CT-2002-00172 Institutional research plan: CEZ:AV0Z50110509 Keywords : Alzheimer ´s disease * muscarinic receptors * cholinergic neurotransmission Subject RIV: FH - Neurology Impact factor: 5.959, year: 2008

Expression of GABAergic receptors in mouse taste receptor cells.

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Margaret R Starostik

Full Text Available BACKGROUND: Multiple excitatory neurotransmitters have been identified in the mammalian taste transduction, with few studies focused on inhibitory neurotransmitters. Since the synthetic enzyme glutamate decarboxylase (GAD for gamma-aminobutyric acid (GABA is expressed in a subset of mouse taste cells, we hypothesized that other components of the GABA signaling pathway are likely expressed in this system. GABA signaling is initiated by the activation of either ionotropic receptors (GABA(A and GABA(C or metabotropic receptors (GABA(B while it is terminated by the re-uptake of GABA through transporters (GATs. METHODOLOGY/PRINCIPAL FINDINGS: Using reverse transcriptase-PCR (RT-PCR analysis, we investigated the expression of different GABA signaling molecules in the mouse taste system. Taste receptor cells (TRCs in the circumvallate papillae express multiple subunits of the GABA(A and GABA(B receptors as well as multiple GATs. Immunocytochemical analyses examined the distribution of the GABA machinery in the circumvallate papillae. Both GABA(A-and GABA(B- immunoreactivity were detected in the peripheral taste receptor cells. We also used transgenic mice that express green fluorescent protein (GFP in either the Type II taste cells, which can respond to bitter, sweet or umami taste stimuli, or in the Type III GAD67 expressing taste cells. Thus, we were able to identify that GABAergic receptors are expressed in some Type II and Type III taste cells. Mouse GAT4 labeling was concentrated in the cells surrounding the taste buds with a few positively labeled TRCs at the margins of the taste buds. CONCLUSIONS/SIGNIFICANCE: The presence of GABAergic receptors localized on Type II and Type III taste cells suggests that GABA is likely modulating evoked taste responses in the mouse taste bud.

Evidence for cholinergic participation in the control of bird song; acetylcholinesterase distribution and muscarinic receptor autoradiography in the zebra finch brain

International Nuclear Information System (INIS)

Ryan, S.M.; Arnold, A.P.

1981-01-01

Brain regions thought to be involved in the control of song in the zebra finch (Poephila guttata), were examined histochemically using the Karnovsky and Roots direct-coloring method for the detection of acetylcholinesterase (AChE) and the autoradiographic method for the localization of muscarinic cholinergic receptors following injection of tritiated quinuclidinyl benzilate (3H QNB). All presently identified vocal control nuclei in both males and females contain AChE. These nuclei include Area X, magnocellular nucleus of the anterior neostriatum (MAN), nucleus interface (NIF), caudal nucleus of the hyperstriatum ventrale (HVc), intercollicular nucleus (ICo), nucleus uva, robust nucleus of the archistriatum (RA), and tracheosyringeal portion of the hypoglossal nerve nucleus (nXIIts). All nuclei except Area X contain mostly AChE-synthesizing cell bodies. All of these nuclei contain some AChE in the neuropil, with particularly intense staining in Area X, the surrounding LPO, and the dorsomedial portion of ICo. In agreement with this description are very high concentrations of 3H QNB in both Area X and the dorsomedial ICo. HVc also appears specifically labeled. Evidence from these two histological technique suggests that efferent projections of most vocal control area may utilize acetylcholine, and that several of the vocal control nuclei may themselves receive muscarinic cholinergic projection. In Area X, there are sex differences of AChE neuropil staining. This evidence suggesting that sexually dimorphic projections to or within Area X are cholinergic or cholinoceptive

Development of radiohalogenated muscarinic ligands for the in vivo imaging of m-AChR by nuclear medicine techniques

Energy Technology Data Exchange (ETDEWEB)

McPherson, D.W.; Luo, H.; Knapp, F.F. Jr.

1994-06-01

Alterations in the density of acetylcholinergic muscarinic receptors (m-AChR) have been observed in various dementias. This has spurred interest in the development of radiohalogenated ligands which can be used for the non-invasive in vivo detection of m-AChR by nuclear medicine techniques. We have developed a new ligand 1-azabicyclo[2.2.2]oct-3-yl ({alpha}-hydroxy-{alpha}-(1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (IQNP,12) which demonstrates high affinity for the muscarinic receptor. When labeled with radioiodine it has been shown to be selective and specific for m-ACHR. Initial studies on the separation and in vivo evaluation of the various isomers of IQNP have shown that the stereochemistry of the chiral centers and the configuration around the double bond play an important role in m-AChR subtype specificity. In vivo evaluation of these stereoisomers demonstrate that E-(R,R)-IQNP has a high affinity for the M{sub 1} muscarinic subtype while Z-(R,R)-IQNP demonstrate a high affinity for M{sub 1} and M{sub 2} receptor subtypes. These data demonstrate IQNP (12) has potential for use in the non-evasive in vivo detection of m-AChR by single photon emission computed tomography (SPECT). A brominated analogue, ``BrQNP,`` in which the iodine has been replaced by a bromine atom, has also been prepared and was shown to block the in vivo uptake of IQNP in the brain and heart and therefore has potential for positron emission tomographic (PET) studies of m-AChR.

Sex-related differences in the muscarinic acetylcholinergic receptor in the healthy human brain. A positron emission tomography study

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Yoshida, Tsuyoshi; Kuwabara, Yasuo; Sasaki, Masayuki; Ichimiya, Atsushi; Takita, Masashi; Ogomori, Koji; Masuda, Kouji [Kyushu Univ., Fukuoka (Japan). Graduate School of Medical Sciences; Fukumura, Toshimitsu; Ichiya, Yuichi

2000-04-01

We evaluated the sex-related differences in the decline of the cerebral muscarinic acetylcholinergic receptor (mACh-R) due to aging by using {sup 11}C-N-methyl-4-piperidyl benzilate ({sup 11}C-NMPB) and positron emission tomography (PET). The subjects consisted of 37 (20 males and 17 females) healthy volunteers. The {sup 11}C-NMPB uptake was evaluated by the ratio method (regional {sup 11}C-NMPB uptake/Cerebellar {sup 11}C-NMPB uptake; rNMPB ratio). The correlation between sex, aging, and the rNMPB ratio in normal aging was evaluated by a multiple regression analysis. The rNMPB ratio was higher in females than in males throughout the entire cerebral region (p<0.01-p<0.0001) and the rNMPB ratio might thus possibly decline with age more rapidly in females. Our study therefore revealed the existence of sex-related differences in the cerebral mACh-R. (author)

Exposure to Gulf War Illness chemicals induces functional muscarinic receptor maladaptations in muscle nociceptors.

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Cooper, B Y; Johnson, R D; Nutter, T J

2016-05-01

Chronic pain is a component of the multisymptom disease known as Gulf War Illness (GWI). There is evidence that pain symptoms could have been a consequence of prolonged and/or excessive exposure to anticholinesterases and other GW chemicals. We previously reported that rats exposed, for 8 weeks, to a mixture of anticholinesterases (pyridostigmine bromide, chlorpyrifos) and a Nav (voltage activated Na(+) channel) deactivation-inhibiting pyrethroid, permethrin, exhibited a behavior pattern that was consistent with a delayed myalgia. This myalgia-like behavior was accompanied by persistent changes to Kv (voltage activated K(+)) channel physiology in muscle nociceptors (Kv7, KDR). In the present study, we examined how exposure to the above agents altered the reactivity of Kv channels to a muscarinic receptor (mAChR) agonist (oxotremorine-M). Comparisons between muscle nociceptors harvested from vehicle and GW chemical-exposed rats revealed that mAChR suppression of Kv7 activity was enhanced in exposed rats. Yet in these same muscle nociceptors, a Stromatoxin-insensitive component of the KDR (voltage activated delayed rectifier K(+) channel) exhibited decreased sensitivity to activation of mAChR. We have previously shown that a unique mAChR-induced depolarization and burst discharge (MDBD) was exaggerated in muscle nociceptors of rats exposed to GW chemicals. We now provide evidence that both muscle and vascular nociceptors of naïve rats exhibit MDBD. Examination of the molecular basis of the MDBD in naïve animals revealed that while the mAChR depolarization was independent of Kv7, the action potential burst was modulated by Kv7 status. mAChR depolarizations were shown to be dependent, in part, on TRPA1. We argue that dysfunction of the MDBD could be a functional convergence point for maladapted ion channels and receptors consequent to exposure to GW chemicals. Copyright © 2016 Elsevier Inc. All rights reserved.

The Role of Muscarinic Receptors on spasmolytic Activity of Hydroalcohlic Extract of Parsley(petroselinum crispumSeed in Adult Male Rat's Ileum

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Raede Tavalaee

2016-08-01

Full Text Available Abstract Background: because of the side effects of chemical drugs, using plant drugs was considered during recent decades. Based on previous studies, various concentrations of plant extracts can reduce ileum contraction caused by KCl and carbachol. Therefore, in this study, the role of muscarinic receptors in anti-spasmolytic activity of hydroalcohlic extract of parsley(petroselinum crispumseed in adult male rat's ileum was investigated. Materials and Methods: The distal part of Wistar rat’s ileum was separated and its contractions were recorded under 1 gr stretching and 37oC temperature in the bathroom containing the solution of Tyrode by Isotonic method. The ileum tissue was inserted in separately in first group affected by KCl and parsley seed, in the second group affected by KCl and carbachol with concentration 10-2 mg/ml and after that parsley seed, the third group affected by carbachol with concentration 10-2 mg/ml and then the extraction of parsley seed and the fourth group affected by scopolamine with concentration 10-3 M and after that KCl and then the extraction of parsley seed. Results: Non-cumulative concentrations of extract reduced the ileum contraction caused by KCl (60mM in a concentration-dependent form (p<0.001. There was a significant difference between the effect of extract in the presence of carbachol and extract concentration 0.2 mg/ml (p<0.01. There was no significant difference between the effect of extract in the presence of carbachol without stimulating tissue by KCl (60mM with extract concentration 0.2mg/ml (p<0.05. There was no significant difference between the effect of extract in the presence of scopolamine with extract concentration 0.2mg/ml(p<0.05. Conclusion: This study showed that the hydroalcholic of parsley seed extract has a relaxatory effect on rat’s ileum. Carbachol with KCl has a synergistic effect on smooth muscle contraction of the ileum wall and muscarinic receptors do not involve in the

Autoantibodies against Muscarinic Type 3 Receptor in Sjögren's Syndrome Inhibit Aquaporin 5 Trafficking

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Lee, Byung Ha; Gauna, Adrienne E.; Perez, Geidys; Park, Yun-jong; Pauley, Kaleb M.; Kawai, Toshihisa; Cha, Seunghee

2013-01-01

Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS. PMID:23382834

Comparison of the butyrate effects on neurotransmitter receptors in neurohybrids NG108-15 and NCB-20 cells

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Zhu, X.Z.; Chuang, D.M.

1987-01-01

The authors previous study demonstrated that long term treatment of NCB-20 cells with sodium butyrate resulted in a marked increase in the density of delta-opioid receptors with a much lesser effect on muscarinic cholinergic and no effect on alpha 2 -adrenergic receptors. In the present study the authors investigated the effect of sodium butyrate on these three types of receptors in NG108-15 cells whose neuroblastoma parent is the same as that of NCB-20 cells. Long term treatment of NG108-15 cells with sodium butyrate (0.5 mM) induced a 2-fold increase in the density of the specific binding of 3 H-clonidine. A comparable increase in the number of binding sites was detected when 3 H-yohimbine was used as the receptor ligand. The butyrate-induced increase in the alpha 2 -adrenergic receptor binding could be totally abolished by treatment with a protein synthesis inhibitor, cycloheximide, suggesting that synthesis of receptor protein is involved. The same butyrate treatment had no significant effect on opioid and muscarinic cholinergic receptor bindings. Thus, butyrate effects on the expression of these three types of receptors in NG108-15 and NCB-20 cells are dramatically different. These data suggest that induction by butyrate of neurotransmitter receptors requires concerted action of genetic factors of both parents of the neurohybrids. 22 references, 2 figures, 2 tables

Blockade of muscarinic receptors impairs the retrieval of well-trained memory

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Shogo eSoma

2014-04-01

Full Text Available Acetylcholine (ACh is known to play an important role in memory functions, and its deficit has been proposed to cause the cognitive decline associated with advanced age and Alzheimer’s disease (the cholinergic hypothesis. Although many studies have tested the cholinergic hypothesis for recently acquired memory, only a few have investigated the role of ACh in the retrieval process of well-trained cognitive memory, which describes the memory established from repetition and daily routine. To examine this point, we trained rats to perform a two-alternative forced-choice visual detection task. Each trial was started by having the rats pull upward a central-lever, which triggered the presentation of a visual stimulus to the right or left side of the display monitor, and then pulling upward a stimulus-relevant choice-lever located on both sides. Rats learned the task within 10 days, and the task training was continued for a month. Task performance was measured with or without systemic administration of a muscarinic ACh receptor (mAChR antagonist, scopolamine (SCOP, prior to the test. After 30 min of SCOP administration, rats stopped manipulating any lever even though they explored the lever and surrounding environment, suggesting a loss of the task-related associative memory. Three hours later, rats were recovered to complete the trial, but the rats selected the levers irrespective of the visual stimulus, suggesting they remembered a series of lever-manipulations in association with a reward, but not association between the reward and visual stimulation. Furthermore, an m1-AChR, but not nicotinic AChR antagonist caused a similar deficit in the task execution. SCOP neither interfered with locomotor activity nor drinking behavior, while it influenced anxiety. These results suggest that the activation of mAChRs at basal ACh levels is essential for the recall of well-trained cognitive memory.

Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

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Bencherif, M.; Lukas, R.J.

1991-01-01

Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate [ 3 H-QNB]. The rank order potency of selective antagonists that inhibit specific 3 HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2[2-[(diethylamino)methyl]-1-[piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10 microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of 3 HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA

A crosstalk between muscarinic and CRF2 receptors regulates cellular adhesion properties of human colon cancer cells.

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Pelissier-Rota, M; Chartier, N T; Bonaz, B; Jacquier-Sarlin, M R

2017-07-01

Patients with inflammatory bowel disease often suffer from chronic and relapsing intestinal inflammation that favor the development of colitis associated cancer. An alteration of the epithelial intestinal barrier function observed in IBD is supposed to be a consequence of stress. It has been proposed that corticotrophin-releasing factor receptor (CRF2), one of the two receptors of CRF, the principal neuromediator of stress, acts on cholinergic nerves to induce stress-mediated epithelial barrier dysfunction. Non-neuronal acetylcholine (Ach) and muscarinic receptors (mAchR) also contribute to alterations of epithelial cell functions. In this study, we investigated the mechanisms through which stress and Ach modulate epithelial cell adhesive properties. We show that Ach-induced activation of mAchR in HT-29 cells results in cell dissociation together with changes in cell-matrix contacts, which correlates with the acquisition of invasive potential consistent with a matrix metalloproteinase (MMP) mode of invasion. These processes result from mAchR subsequent stimulation of the cascade of src/Erk and FAK activation. Ach-induced secretion of laminin 332 leads to α3β1 integrin activation and RhoA-dependent reorganization of the actin cytoskeleton. We show that Ach-mediated effects on cell adhesion are blocked by astressin 2b, a CRF2 antagonist, suggesting that Ach action depends partly on CRF2 signaling. This is reinforced by the fact that Ach-mediated activation of mAchR stimulates both the synthesis and the release of CRF2 ligands in HT-29 cells (effects blocked by atropine). In summary, our data provides evidence for a novel intracellular circuit involving mAchR acting on CRF2-signaling that could mediate colonic mucosal barrier dysfunction and exacerbate mucosal inflammation. Copyright © 2017. Published by Elsevier B.V.

Endosulfan and cholinergic (muscarinic) transmission: effect on electroencephalograms and [3H]quinuclidinyl benzilate in pigeon brain

International Nuclear Information System (INIS)

Anand, M.; Agrawal, A.K.; Gopal, K.; Sur, R.N.; Seth, P.K.

1986-01-01

Single exposure of endosulfan (5 mg/kg) to pigeons (Columbia livia) caused neuronal hyperexcitability as evidence by spike discharges of 200-500 μV in the electroencephalograms (EEG) from the telencephalon and hyperstriatum, but there was not effect on the ectostriatal area. Cholinergic (muscarinic) receptor binding study using [ 3 H]quinuclidinyl benzilate ([ 3 H]QNB) as a specific ligand indicated that a single exposure to 5 mg/kg of endosulfan caused a significant increase in [ 3 H]QNB binding to the striatal membrane. Behavior study further indicated that a single dose of 200 μg/kg of oxotremorine produced a significant induction in the tremor in endosulfan-pretreated pigeons. The results of this behavioral and biochemical study indicate the involvement of a cholinergic (muscarinic) transmitter system in endosulfan-induced neurotoxicity

Beta amyloid differently modulate nicotinic and muscarinic receptor subtypes which regulate in vitro and in vivo the release of glycine in the rat hippocampus

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Stefania eZappettini

2012-07-01

Full Text Available Using both in vitro (hippocampal synaptosomes in superfusion and in vivo (microdialysis approaches we investigated whether and to what extent β amyloid peptide 1-40 (Aβ 1-40 interferes with the cholinergic modulation of the release of glycine (GLY in the rat hippocampus. The nicotine-evoked overflow of endogenous GLY in hippocampal synaptosomes in superfusion was significantly inhibited by Aβ 1-40 (10 nM while increasing the concentration to 100 nM the inhibitory effect did not further increase. Both the Choline (Ch (α7 agonist; 1 mM and the 5-Iodo-A-85380 dihydrochloride (5IA85380, α4β2 agonist; 10 nM-evoked GLY overflow were inhibited by Aβ1-40 at 100 nM but not at 10nM concentrations. The KCl evoked [3H]GLY and [3H]Acetylcholine (ACh overflow were strongly inhibited in presence of oxotremorine; however this inhibitory muscarinic effect was not affected by Aβ1-40. The effects of Aβ1-40 on the administration of nicotine, veratridine, 5IA85380 and PHA 543613 hydrochloride (PHA543613 (a selective agonist of α7 subtypes on hippocampal endogenous GLY release in vivo were also studied. Aβ 1-40 significantly reduced (at 10 μM but not at 1 μM the nicotine evoked in vivo release of GLY. Aβ 1-40 (at 10 μM but not at 1 μM significantly inhibited the PHA543613 (1 mM-elicited GLY overflow while was ineffective on the GLY overflow evoked by 5IA85380 (1 mM. Aβ 40-1 (10 μM did not produce any inhibitory effect on nicotine evoked GLY overflow both in the in vitro and in vivo experiments. Our results indicate that a the cholinergic modulation of the release of GLY occurs by the activation of both α7 and α4β2 nicotinic ACh receptors (nAChRs as well as by the activation of inhibitory muscarinic ACh receptors (mAChRs and b Aβ 1-40 can modulate cholinergic evoked GLY release exclusively through the interaction with α7 and the α4β2 nAChR nicotinic receptors but not through mAChR subtypes.

Mercury Vapour Long-Lasting Exposure: Lymphocyte Muscarinic Receptors as Neurochemical Markers of Accidental Intoxication

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E. Roda

2016-01-01

Full Text Available Introduction. Chronic poisoning may result in home setting after mercury (Hg vapours inhalation from damaged devices. We report a chronic, nonoccupational Hg poisoning due to 10-year indoor exposure to mercury spillage. Case Report. A 72-year-old man with polyneuropathy of suspected toxic origin. At hospitalization, toxicological clinical evaluations confirmed the altered neurological picture documented across the last decade. Periodic blood and urine Hg levels (BHg, UHg monitoring were performed from admission (t0, until 1 year later (t2, paralleled by blood neurochemical markers assessment, that is, lymphocytes muscarinic receptors (l-MRs. At t0: BHg and UHg were 27 and 1.4 microg/L, respectively (normal values: BHg 1–4.5; UHg 0.1–4.5, associated with l-MRs increase, 185.82 femtomoL/million lymphocytes (normal range: 8.0–16.0. At t1 (two days after DMSA-mobilization test, BHg weak reduction, paralleled by UHg 3.7-fold increase, was measured together with further l-MRs enhancement (205.43 femtomoL/million lymphocytes. At t2 (eight months after two cycles of DMSA chelating therapy ending, gradual improving of clinical manifestations was accompanied by progressive decrease of BHg and UHg (4.0 and 2.8 microg/L, resp. and peripheral l-MRs neurochemical marker (24.89 femtomoL/million lymphocytes. Conclusion. l-MRs modulatory effect supports their use as peripheral neurochemical marker in Hg poisoning diagnosis and chelation therapy monitoring.

Interactions of alaproclate, a selective 5HT-uptake blocker, with muscarinic receptors: in vivo and in vitro studies

International Nuclear Information System (INIS)

Danielsson, E.; Bartfai, T.; Nordstrom, O.; Ogren, S.O.; Unden, A.

1986-01-01

Cholinergic mechanisms play an important role in higher brain functions such as learning or memory. It is hoped that drugs which improve cholinergic transmission would be therapeutically effective in senile dementia. The results of biochemical studies on the interaction of alaproclate with the muscarinic system are summarized. Tritium-4-N-methylpiperidinylbenzilate and methyl tritium-choline chloride were used in the studies. Salivation and hypothermia were studied in adult male mice following injection of alaproclate preceding the injection of oxotremorine by 30 min. Salivation, tremor and hypothermia caused by oxotremorine were found to be centrally mediated muscarinic responses. Tritium-ACh release was studied from synaptosomes. The metabolites of alaproclate, alanine and 2(4 chlorophenyl) 1-1 dimethylethanol, do not produce tremor

Autonomic receptors in urinary tract: Sex and age differences

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Latifpour, J.; Kondo, S.; O'Hollaren, B.; Morita, T.; Weiss, R.M.

1990-01-01

As age and sex affect the function of the lower urinary tract, we studied the characteristics of adrenergic and cholinergic receptors in various parts of lower urinary tract smooth muscle of young (6 months) and old (4 1/2-5 years) male and female rabbits. Saturation experiments performed with [3H]prazosin, [3H]yohimbine, [3H]dihydroalprenolol and [3H]quinuclidinyl benzylate in rabbit bladder base, bladder dome and urethra indicate the presence of regional, sex- and age-related differences in the density of alpha-1, alpha-2, and beta adrenergic and muscarinic cholinergic receptors. Alpha-2 adrenergic receptor density is considerably higher in the female than in the male urethra of both age groups, whereas the higher density of beta adrenergic receptors in the female than in the male bladder base is observed only in the younger animals. The density of muscarinic receptors is higher in bladder dome than in bladder base or urethra in young rabbits of both sexes. In the old animals, the density of muscarinic receptors in bladder base increases to the level observed in bladder dome. Inhibition experiments with selective adrenergic agonists and antagonists indicate that the pharmacological profiles of alpha-2 adrenergic receptors in the urethra and beta adrenergic receptors in the bladder dome and bladder base are similar in both sexes and at both ages. Beta-2 adrenergic receptors are shown to be predominant in bladder base and bladder dome of rabbits. Parallel studies in rabbit urethra, adult rat cortex and neonatal rat lung show that the urethral alpha-2 adrenergic receptors are of the alpha-2A subtype

Impairment of the vascular relaxation and differential expression of caveolin-1 of the aorta of diabetic +db/+db mice.

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Lam, Tze Yan; Seto, Sai Wang; Lau, Yee Man; Au, Lai Shan; Kwan, Yiu Wa; Ngai, Sai Ming; Tsui, Kwong Wing

2006-09-28

In this study, we compared the endothelium-dependent and -independent relaxation of the isolated thoracic aorta of control (+db/+m) and diabetic (+db/+db) (C57BL/KsJ) mice. The gene expression (mRNA and protein) level of the muscarinic M(3) receptors, endothelial nitric oxide synthase (eNOS) and caveolin-1 of the aorta was also evaluated. Acetylcholine caused a concentration-dependent, N(G)-nitro-L-arginine methyl-ester (20 microM)-sensitive relaxation, with approximately 100% relaxation at 10 microM, in +db/+m mice. In +db/+db mice, the acetylcholine-induced relaxation was significantly smaller (maximum relaxation: approximately 80%). The sodium nitroprusside-mediated relaxation was slightly diminished in +db/+db mice, compared to +db/+m mice. However, there was no significant difference in the isoprenaline- and cromakalim-induced relaxation observed in both species. The mRNA and protein expression levels of caveolin-1 were significantly higher in the aorta of +db/+db mice. In contrast, there was no difference in the mRNA and protein expression levels of eNOS and muscarinic M(3) receptors between these mice. Our results demonstrate that the impairment of the acetylcholine-induced, endothelium-dependent aortic relaxation observed in +db/+db mice was probably associated with an enhanced expression of caveolin-1 mRNA and protein.

Distinct muscarinic acetylcholine receptor subtypes mediate pre- and postsynaptic effects in rat neocortex

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Gigout Sylvain

2012-04-01

Full Text Available Abstract Background Cholinergic transmission has been implicated in learning, memory and cognition. However, the cellular effects induced by muscarinic acetylcholine receptors (mAChRs activation are poorly understood in the neocortex. We investigated the effects of the cholinergic agonist carbachol (CCh and various agonists and antagonists on neuronal activity in rat neocortical slices using intracellular (sharp microelectrode and field potential recordings. Results CCh increased neuronal firing but reduced synaptic transmission. The increase of neuronal firing was antagonized by pirenzepine (M1/M4 mAChRs antagonist but not by AF-DX 116 (M2/M4 mAChRs antagonist. Pirenzepine reversed the depressant effect of CCh on excitatory postsynaptic potential (EPSP but had marginal effects when applied before CCh. AF-DX 116 antagonized the depression of EPSP when applied before or during CCh. CCh also decreased the paired-pulse inhibition of field potentials and the inhibitory conductances mediated by GABAA and GABAB receptors. The depression of paired-pulse inhibition was antagonized or prevented by AF-DX 116 or atropine but only marginally by pirenzepine. The inhibitory conductances were unaltered by xanomeline (M1/M4 mAChRs agonist, yet the CCh-induced depression was antagonized by AF-DX 116. Linopirdine, a selective M-current blocker, mimicked the effect of CCh on neuronal firing. However, linopirdine had no effect on the amplitude of EPSP or on the paired-pulse inhibition, indicating that M-current is involved in the increase of neuronal excitability but neither in the depression of EPSP nor paired-pulse inhibition. Conclusions These data indicate that the three effects are mediated by different mAChRs, the increase in firing being mediated by M1 mAChR, decrease of inhibition by M2 mAChR and depression of excitatory transmission by M4 mAChR. The depression of EPSP and increase of neuronal firing might enhance the signal-to-noise ratio, whereas the

SPET imaging of central muscarinic acetylcholine receptors with iodine-123 labelled E-IQNP and Z-IQNP

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Nobuhara, K.; Farde, L.; Halldin, C.; Karlsson, P.; Swahn, C.G.; Olsson, H.; Sedvall, G. [Dept. of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, Stockholm (Sweden); Bergstroem, K.A. [Dept. of Clinical Physiology, Kuopio University Hospital, Kuopio (Finland); Larsson, S.A.; Schnell, P.-O. [Dept. of Radiation Physics, Karolinska Hospital, Stockholm (Sweden); McPherson, D.W. [Oak Ridge National Laboratory (ORNL), Nuclear Medicine Group, TN (United States); Savonen, A.; Hiltunen, J. [MAP Medical Technologies Oy, Tikkakoski (Finland)

2001-01-01

1-Azabicyclo[2.2.2]oct-3-yl {alpha}-hydroxy-{alpha}-(1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (IQNP) is a muscarinic acetylcholine receptor (mAChR) antagonist and the racemic ligand contains eight stereoisomers. In a single-photon emission tomography (SPET) study in monkeys we recently confirmed that [{sup 123}I]E-(R,R)-IQNP ([{sup 123}I]E-IQNP) is a radioligand with modest selectivity for the M{sub 1} and M{sub 4} subtypes, whereas [{sup 123}I]Z-(R,R)-IQNP ([{sup 123}I]Z-IQNP) is non-subtype selective. In the present SPET study, E- and Z-IQNP were examined in human subjects. SPET examination was performed on three male subjects after i.v. injection of [{sup 123}I]E-IQNP and in another three after i.v. injection of [{sup 123}I]Z-IQNP. The binding potential (BP) for [{sup 123}I]E-IQNP was calculated using several quantitative approaches with the cerebellum as a reference region. High-performance liquid chromatography was used to measure radioligand metabolism in plasma. Following [{sup 123}I]E-IQNP, the radioactivity was high in the neocortex and striatum, intermediate in the thalamus and low in the pons and cerebellum, which is consistent with the rank order for the regional density of M{sub 1} and M{sub 4} subtypes in vitro. For all regions, peak equilibrium was identified within the 48-h data acquisition. The simplified reference tissue approach using SPET data from 0 to 48 h was the most reliable in this limited series of subjects. Following injection of [{sup 123}I]Z-IQNP, radioactivity was high in the neocortex and striatum, intermediate in the thalamus and pons and low in the cerebellum, which is in agreement with the density of M{sub 1}, M{sub 2} and M{sub 4} subtypes as measured in vitro. Quantitative analyses provided indirect support for specific M{sub 2} binding of Z-IQNP in the cerebellum. The high selectivity of [{sup 123}I]E-IQNP for M{sub 1} and M{sub 4} receptors allowed the use of cerebellum as a reference region devoid of specific binding, and

Androgen Receptor Expression in Thai Breast Cancer Patients

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Suthat Chottanapund

2016-09-01

Full Text Available The aim of this study was to investigate prevalence and related factors of androgen receptor (AR expression in Thai breast cancer patients. A descriptive study was done in 95 patients, who were admitted to Charoenkrung Pracharak Hospital, Bangkok (2011–2013. Statistical relationships were examined between AR protein expression, tumor status, and patient characteristics. Compared with those from Western countries, ethnic Thai patients were younger at age of diagnosis and had a higher proliferative index (high Ki-67 expression, which indicates unfavorable prognosis. In addition, 91% of the Thai breast tumors that were positive for any of the following receptors, estrogen receptor (ER, progesterone receptor (PR, and human epidermal growth factor receptor 2 (HER2 also expressed the AR protein, while in triple negative breast tumors only 33% were AR positive. ER and PR expression was positively related with AR expression, while AR expression was inversely correlated to Ki-67 expression. AR status was strongly correlated with ER and PR status in Thai patients. There is an inverse relationship between Ki-67 and AR, which suggests that AR may be a prognostic factor for breast cancer.

Evolution of the toxins muscarine and psilocybin in a family of mushroom-forming fungi.

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Pawel Kosentka

Full Text Available Mushroom-forming fungi produce a wide array of toxic alkaloids. However, evolutionary analyses aimed at exploring the evolution of muscarine, a toxin that stimulates the parasympathetic nervous system, and psilocybin, a hallucinogen, have never been performed. The known taxonomic distribution of muscarine within the Inocybaceae is limited, based only on assays of species from temperate regions of the northern hemisphere. Here, we present a review of muscarine and psilocybin assays performed on species of Inocybaceae during the last fifty years. To supplement these results, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS to determine whether muscarine was present in 30 new samples of Inocybaceae, the majority of which have not been previously assayed or that originated from either the tropics or temperate regions of the southern hemisphere. Our main objective is to test the hypothesis that the presence of muscarine is a shared ancestral feature of the Inocybaceae. In addition, we also test whether species of Inocyabceae that produce psilocybin are monophyletic. Our findings suggest otherwise. Muscarine has evolved independently on several occasions, together with several losses. We also detect at least two independent transitions of muscarine-free lineages to psilocybin-producing states. Although not ancestral for the family as a whole, muscarine is a shared derived trait for an inclusive clade containing three of the seven major lineages of Inocybaceae (the Inocybe, Nothocybe, and Pseudosperma clades, the common ancestor of which may have evolved ca. 60 million years ago. Thus, muscarine represents a conserved trait followed by several recent losses. Transitions to psilocybin from muscarine-producing ancestors occurred more recently between 10-20 million years ago after muscarine loss in two separate lineages. Statistical analyses firmly reject a single origin of muscarine-producing taxa.

Evolution of the toxins muscarine and psilocybin in a family of mushroom-forming fungi.

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Kosentka, Pawel; Sprague, Sarah L; Ryberg, Martin; Gartz, Jochen; May, Amanda L; Campagna, Shawn R; Matheny, P Brandon

2013-01-01

Mushroom-forming fungi produce a wide array of toxic alkaloids. However, evolutionary analyses aimed at exploring the evolution of muscarine, a toxin that stimulates the parasympathetic nervous system, and psilocybin, a hallucinogen, have never been performed. The known taxonomic distribution of muscarine within the Inocybaceae is limited, based only on assays of species from temperate regions of the northern hemisphere. Here, we present a review of muscarine and psilocybin assays performed on species of Inocybaceae during the last fifty years. To supplement these results, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine whether muscarine was present in 30 new samples of Inocybaceae, the majority of which have not been previously assayed or that originated from either the tropics or temperate regions of the southern hemisphere. Our main objective is to test the hypothesis that the presence of muscarine is a shared ancestral feature of the Inocybaceae. In addition, we also test whether species of Inocyabceae that produce psilocybin are monophyletic. Our findings suggest otherwise. Muscarine has evolved independently on several occasions, together with several losses. We also detect at least two independent transitions of muscarine-free lineages to psilocybin-producing states. Although not ancestral for the family as a whole, muscarine is a shared derived trait for an inclusive clade containing three of the seven major lineages of Inocybaceae (the Inocybe, Nothocybe, and Pseudosperma clades), the common ancestor of which may have evolved ca. 60 million years ago. Thus, muscarine represents a conserved trait followed by several recent losses. Transitions to psilocybin from muscarine-producing ancestors occurred more recently between 10-20 million years ago after muscarine loss in two separate lineages. Statistical analyses firmly reject a single origin of muscarine-producing taxa.

Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

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Bencherif, M.; Lukas, R.J. (Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (USA))

1991-06-01

Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate ({sup 3}H-QNB). The rank order potency of selective antagonists that inhibit specific {sup 3}HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2(2-((diethylamino)methyl)-1-(piperidinyl) acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10 microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of {sup 3}HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.

The expression, localization and function of α7 nicotinic acetylcholine receptor in rat corpus cavernosum.

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Faghir-Ghanesefat, Hedyeh; Rahimi, Nastaran; Yarmohammadi, Fatemeh; Mokhtari, Tahmineh; Abdollahi, Ali Reza; Ejtemaei Mehr, Shahram; Dehpour, Ahmad R

2017-12-01

Alpha7 nicotinic acetylcholine receptor (α7-nAChR), an emerging pharmacological target for a variety of medical conditions, is expressed in the most mammalian tissues with different effects. So, this study was designed to investigate the expression, localization and effect of α7-nAChR in rat corpus cavernosum (CC). Reverse transcription polymerase chain reaction (RT-PCR) revealed that α7-nAChR was expressed in rat CC and double immunofluorescence studies demonstrated the presence of α7-nAChR in corporal neurons. The rat CC segments were mounted in organ bath chambers and contracted with phenylephrine (0.1 μm -300 μm) to investigate the relaxation effect of electrical field stimulation (EFS,10 Hz) assessed in the presence of guanethidine (adrenergic blocker, 5 μm) and atropine (muscarinic cholinergic blocker, 1 μm) to obtain non-adrenergic non-cholinergic (NANC) response. Cumulative administration of nicotine significantly potentiated the EFS-induced NANC relaxation (-log EC50 = 7.5 ± 0.057). Whereas, the potentiated NANC relaxation of nicotine was significantly inhibited with different concentrations of methyllycaconitine citrate (α7-nAChR antagonist, P < 0.05) in preincubated strips. L-NAME (non-specific nitric oxide synthase inhibitor, 1 μm) completely blocked the neurogenic relaxation induced by EFS plus nicotine. To conclude α7-nAChR is expressed in rat CC and modulates the neurogenic relaxation response to nicotine. © 2017 Royal Pharmaceutical Society.

Expressing exogenous functional odorant receptors in cultured olfactory sensory neurons

Directory of Open Access Journals (Sweden)

Fomina Alla F

2008-09-01

Full Text Available Abstract Background Olfactory discrimination depends on the large numbers of odorant receptor genes and differential ligand-receptor signaling among neurons expressing different receptors. In this study, we describe an in vitro system that enables the expression of exogenous odorant receptors in cultured olfactory sensory neurons. Olfactory sensory neurons in the culture express characteristic signaling molecules and, therefore, provide a system to study receptor function within its intrinsic cellular environment. Results We demonstrate that cultured olfactory sensory neurons express endogenous odorant receptors. Lentiviral vector-mediated gene transfer enables successful ectopic expression of odorant receptors. We show that the ectopically expressed mouse I7 is functional in the cultured olfactory sensory neurons. When two different odorant receptors are ectopically expressed simultaneously, both receptor proteins co-localized in the same olfactory sensory neurons up to 10 days in vitro. Conclusion This culture technique provided an efficient method to culture olfactory sensory neurons whose morphology, molecular characteristics and maturation progression resembled those observed in vivo. Using this system, regulation of odorant receptor expression and its ligand specificity can be studied in its intrinsic cellular environment.

Studies for transitional changes of the muscarinic acetylcholine receptor and mRNA distribution by focal ischemia using nuclear medicine

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Kuji, Ichiei [Kanazawa Univ. (Japan). School of Medicine

1994-04-01

Assessing stress-induced brain receptor responses is important in understanding clinical brain receptor images for nuclear medicine. It is known that cholinergic neurons are decreased by Alzheimer`s disease and that there is a close relationship between cholinergic neurons and muscarinic acetylcholine receptors (mAchR). Thus, this study assessed the response of mAchR to focal ischemia using infarction model rats (prepared by middle cerebral artery occlusion) and sham-operated rats. In the same rats, three kinds of images -- ex vivo regional cerebral blood flow (rCBF) images with {sup 99m}Tc-hexametyl-propyleneamine oxime ({sup 99m}Tc-HMPAO), in vitro mAchR binding images with [{sup 3}H] quinuclidinyl benzilate ({sup 3}H-QNB), and mAchR-mRNA images by in situ hybridization method using {sup 35}S-labeled-oligonucleotide probes specific for mAchR gene subtypes of m1 to m5 -- were obtained in acute and chronic phases. Each image datum was digitalized and assessed semi-quantitatively. There were significant changes in global distribution among rCBF, mAchR and mAchR-mRNAs. In the acute phase, there was no significant change in mAchR in the infarcted area, although rCBF markedly decreased. In the chronic phase, there was a significant decrease in mAchR in the infarct-sided thalamus, although there was no change in rCBF; and there was a significant decrease in mAchR of the infarct-sided substantia nigra in spite of increase in rCBF. In the acute phase, mAchR-mRNAs of the infarct-sided caudate-putamen was decreased, suggesting that the ability of cholinergic neuron to synthesize receptor protein had decreased in the acute phase. Because mAchR was not decreased in the acute phase, some viable neurons with no normal function may be preserved in the acute phase. These results were encouraging in understanding mAchR brain images of patients with memory disturbances such as cerebrovascular dementia and Alzheimer`s disease. (N.K.).

Effects of muscarinic M1 and M4 acetylcholine receptor stimulation on extinction and reinstatement of cocaine seeking in male mice, independent of extinction learning.

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Stoll, Kevin; Hart, Rachel; Lindsley, Craig W; Thomsen, Morgane

2018-03-01

Stimulating muscarinic M 1 /M 4 receptors can blunt reinforcing and other effects of cocaine. A hallmark of addiction is continued drug seeking/craving after abstinence and relapse. We tested whether stimulating M 1 and/or M 4 receptors could facilitate extinction of cocaine seeking, and whether this was mediated via memory consolidation. Experimentally naïve C57BL/6J mice were allowed to acquire self-administration of intravenous cocaine (1 mg/kg/infusion) under a fixed-ratio 1 schedule of reinforcement. Then, saline was substituted for cocaine until responding extinguished to ≤30% of cocaine-reinforced responding. Immediately after each extinction session, mice received saline, the M 1 /M 4 receptor-preferring agonist xanomeline, the M 1 receptor-selective allosteric agonist VU0357017, the M 4 receptor-selective positive allosteric modulator VU0152100, or VU0357017 + VU0152100. In additional experiments, xanomeline was administered delayed after the session or in the home cage before extinction training began. In the latter group, reinstatement of responding by a 10-mg/kg cocaine injection was also tested. Stimulating M 1  + M 4 receptors significantly expedited extinction from 17.2 sessions to 8.3 using xanomeline or 7.8 using VU0357017 + VU0152100. VU0357017 alone and VU0152100 alone did not significantly modify rates of extinction (12.6 and 14.6 sessions). The effect of xanomeline was fully preserved when administered delayed after or unpaired from extinction sessions (7.5 and 6.4 sessions). Xanomeline-treated mice showed no cocaine-induced reinstatement. These findings show that M 1 /M 4 receptor stimulation can decrease cocaine seeking in mice. The effect lasted beyond treatment duration and was not dependent upon extinction learning. This suggests that M 1 /M 4 receptor stimulation modulated or reversed some neurochemical effects of cocaine exposure.

Expression of prostanoid receptors in human ductus arteriosus

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Leonhardt, Andreas; Glaser, Alexander; Wegmann, Markus; Schranz, Dietmar; Seyberth, Hannsjörg; Nüsing, Rolf

2003-01-01

Prostaglandins play a major role in maintaining ductal patency in utero. Ductal tone is regulated by both locally released and circulating vasodilatory prostaglandins. In infants with ductus arteriosus-dependent congenital heart disease, ductal patency is maintained by intravenous administration of prostaglandin (PG) E1. Little information is available regarding the expression of prostaglandin receptors in man. By means of RT–PCR and immunohistochemistry we studied the expression of the PGI2 receptor (IP), the four different PGE2 receptors (EP1, EP2, EP3 and EP4), and the receptors for thromboxane (Tx) A2 (TP), PGD2 (DP) and PGF2α (FP) in the ductus arteriosus of three newborn infants with ductus arteriosus-dependent congenital heart disease and intravenous infusion of PGE1 and of one 8 month old child with a patent ductus arteriosus. The EP3, EP4, FP, IP and TP receptor were markedly expressed at the mRNA and protein level, whereas the EP2 receptor was weakly expressed and the EP1 receptor was detected in two out of four tissue specimens only. The DP receptor was not detected in any of the samples. The most pronounced expression, which was located in the media of the ductus arteriosus, was observed for the EP4 and TP receptors followed by IP and FP receptor protein. These data indicate that ductal patency during the infusion of PGE1 in infants with ductus arteriosus-dependent congenital heart disease might be mediated by the EP4 and IP receptor. The data further suggest that a heterogeneous population of prostanoid receptors may contribute to the regulation of ductus arteriosus tone in humans. PMID:12598419

Pharmacodynamic Study of Interaction of Aqueous Leaf Extract of Psidium Guajava Linn. (Myrtaceae) with Receptor Systems Using Isolated Tissue Preparations.

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Mahaseth, R K; Kumar, S; Dutta, Shagun; Sehgal, Ratika; Rajora, Preety; Mathur, Rajani

2015-01-01

The present study investigates the interaction of aqueous leaf extract of Psidium guajava with muscarinic, serotonergic and adrenergic receptor system using isolated rat ileum, gastric fundus and trachea, respectively. The concentration-dependent contractile response of aqueous leaf extract of Psidium guajava was parallel and rightward of standard agonists, ACh and 5-HT indicating agonistic activity on muscarinic and serotonergic receptor systems. The inhibition of aqueous leaf extract of Psidium guajava mediated contractions in presence of atropine (10(-7) M) and ketanserin (10(-6) M) confirmed the activity. Relaxant effect of PG (0.2 mg/ml) on carbachol induced pre-contracted rat tracheal chain indicated its agonistic action on adrenergic receptor system. Inhibition (P<0.05) of the action in the presence of propranolol (1 ng/ml) confirmed the activity. It may be concluded that PG possesses agonistic action on muscarinic, serotonergic and adrenergic receptor systems.

Characterization of cholinergic muscarinic receptor-stimulated phosphoinositide metabolism in brain from immature rats

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Balduini, W.; Murphy, S.D.; Costa, L.G.

1990-01-01

Hydrolysis of phosphoinositides elicited by stimulation of cholinergic muscarinic receptors has been studied in brain from neonatal (7-day-old) rats in order to determine: (1) whether the neonatal rat could provide a good model system to study this signal-transduction pathway; and (2) whether potential differences with adult nerve tissue would explain the differential, age-related effects of cholinergic agonists. Accumulation of [3H] inositol phosphates in [3H]inositol prelabeled slices from neonatal and adult rats was measured as an index of phosphoinositide metabolism. Full (acetylcholine, methacholine, carbachol) and partial (oxotremorine, bethanechol) agonists had qualitatively similar, albeit quantitatively different, effects in neonatal and adult rats. Atropine and pirenzepine effectively blocked the carbachol-induced response with inhibition constants of 1.2 and 20.7 nM, respectively. In all brain areas, response to all agonists was higher in neonatal than adult rats, and in hippocampus and cerebral cortex the response was higher than in cerebellum or brainstem. The relative intrinsic activity of partial agonists was higher in the latter two areas (0.6-0.7) than in the former two (0.3-0.4). Carbachol-stimulated phosphoinositide metabolism in brain areas correlated well with the binding of [3H]QNB (r2 = 0.627) and, particularly, with [3H]pirenzepine (r2 = 0.911). In cerebral cortex the effect of carbachol was additive to that of norepinephrine and glutamate. The presence of calcium (250-500 microM) was necessary for maximal response to carbachol to be elicited; the EC50 value for Ca2+ was 65.4 microM. Addition of EDTA completely abolished the response. Removal of sodium ions from the incubation medium reduced the response to carbachol by 50%

Differential effects of m1 and m2 receptor antagonists in perirhinal cortex on visual recognition memory in monkeys.

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Wu, Wei; Saunders, Richard C; Mishkin, Mortimer; Turchi, Janita

2012-07-01

Microinfusions of the nonselective muscarinic antagonist scopolamine into perirhinal cortex impairs performance on visual recognition tasks, indicating that muscarinic receptors in this region play a pivotal role in recognition memory. To assess the mnemonic effects of selective blockade in perirhinal cortex of muscarinic receptor subtypes, we locally infused either the m1-selective antagonist pirenzepine or the m2-selective antagonist methoctramine in animals performing one-trial visual recognition, and compared these scores with those following infusions of equivalent volumes of saline. Compared to these control infusions, injections of pirenzepine, but not of methoctramine, significantly impaired recognition accuracy. Further, similar doses of scopolamine and pirenzepine yielded similar deficits, suggesting that the deficits obtained earlier with scopolamine were due mainly, if not exclusively, to blockade of m1 receptors. The present findings indicate that m1 and m2 receptors have functionally dissociable roles, and that the formation of new visual memories is critically dependent on the cholinergic activation of m1 receptors located on perirhinal cells. Published by Elsevier Inc.

Muscarinic receptor binding increases in anterior thalamus and cingulate cortex during discriminative avoidance learning

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Vogt, B.A.; Gabriel, M.; Vogt, L.J.; Poremba, A.; Jensen, E.L.; Kubota, Y.; Kang, E.

1991-01-01

Training-induced neuronal activity develops in the mammalian limbic system during discriminative avoidance conditioning. This study explores behaviorally relevant changes in muscarinic ACh receptor binding in 52 rabbits that were trained to one of five stages of conditioned response acquisition. Sixteen naive and 10 animals yoked to criterion performance served as control cases. Upon reaching a particular stage of training, the brains were removed and autoradiographically assayed for 3H-oxotremorine-M binding with 50 nM pirenzepine (OxO-M/PZ) or for 3H-pirenzepine binding in nine limbic thalamic nuclei and cingulate cortex. Specific OxO-M/PZ binding increased in the parvocellular division of the anterodorsal nucleus early in training when the animals were first exposed to pairing of the conditional and unconditional stimuli. Elevated binding in this nucleus was maintained throughout subsequent training. In the parvocellular division of the anteroventral nucleus (AVp), OxO-M/PZ binding progressively increased throughout training, reached a peak at the criterion stage of performance, and returned to control values during extinction sessions. Peak OxO-M/PZ binding in AVp was significantly elevated over that for cases yoked to criterion performance. In the magnocellular division of the anteroventral nucleus (AVm), OxO-M/PZ binding was elevated only during criterion performance of the task, and it was unaltered in any other limbic thalamic nuclei. Specific OxO-M/PZ binding was also elevated in most layers in rostral area 29c when subjects first performed a significant behavioral discrimination. Training-induced alterations in OxO-M/PZ binding in AVp and layer Ia of area 29c were similar and highly correlated

Acetylcholine muscarinic receptors and response to anti-cholinesterase therapy in patients with Alzheimer's disease

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Brown, Derek; Chisholm, Jennifer A.; Patterson, Jim; Wyper, David; Owens, Jonathan; Pimlott, Sally

2003-01-01

An acetylcholine deficit remains the most consistent neurotransmitter abnormality found in Alzheimer's disease and various therapeutic agents have been targeted at this. In this study we investigated the action of Donepezil, a cholinesterase inhibitor that has few side-effects. In particular we set out to investigate whether muscarinic acetylcholine receptor (mAChR) availability influences the response to this therapy. We used the novel single-photon emission tomography (SPET) tracer (R,R)[ 123 I]I-quinuclidinyl benzilate (R,R[ 123 I]I-QNB), which has high affinity for the M1 subtype of mAChR. Regional cerebral perfusion was also assessed using technetium-99m hexamethylpropylene amine oxime. We investigated 20 patients on Donepezil treatment and ten age-matched controls. The results showed a reduction in (R,R)[ 123 I]I-QNB binding in the caudal anterior cingulate in patients compared with controls and relatively high binding in the putamen and rostral anterior cingulate, suggesting a relative sparing of mAChR in these regions. The main finding of the study was that mAChR availability as assessed by (R,R)[ 123 I]I-QNB binding did not distinguish responders from non-responders. Interestingly, we found that the extent of cognitive improvement showed no positive correlation with (R,R)[ 123 I]I-QNB binding in any brain region but was inversely related to binding in the insular cortex. This suggests that, within the advised cognitive performance band for use of Donepezil, response is greater in those patients with evidence of a more marked cholinergic deficit. A larger study should investigate this. (orig.)

Selective Activation of M4 Muscarinic Acetylcholine Receptors Reverses MK-801-Induced Behavioral Impairments and Enhances Associative Learning in Rodents

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2015-01-01

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a novel approach for the treatment of psychotic symptoms associated with schizophrenia and other neuropsychiatric disorders. We recently reported that the selective M4 PAM VU0152100 produced an antipsychotic drug-like profile in rodents after amphetamine challenge. Previous studies suggest that enhanced cholinergic activity may also improve cognitive function and reverse deficits observed with reduced signaling through the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) in the central nervous system. Prior to this study, the M1 mAChR subtype was viewed as the primary candidate for these actions relative to the other mAChR subtypes. Here we describe the discovery of a novel M4 PAM, VU0467154, with enhanced in vitro potency and improved pharmacokinetic properties relative to other M4 PAMs, enabling a more extensive characterization of M4 actions in rodent models. We used VU0467154 to test the hypothesis that selective potentiation of M4 receptor signaling could ameliorate the behavioral, cognitive, and neurochemical impairments induced by the noncompetitive NMDAR antagonist MK-801. VU0467154 produced a robust dose-dependent reversal of MK-801-induced hyperlocomotion and deficits in preclinical models of associative learning and memory functions, including the touchscreen pairwise visual discrimination task in wild-type mice, but failed to reverse these stimulant-induced deficits in M4 KO mice. VU0467154 also enhanced the acquisition of both contextual and cue-mediated fear conditioning when administered alone in wild-type mice. These novel findings suggest that M4 PAMs may provide a strategy for addressing the more complex affective and cognitive disruptions associated with schizophrenia and other neuropsychiatric disorders. PMID:25137629

Neuropeptide S ameliorates olfactory spatial memory impairment induced by scopolamine and MK801 through activation of cognate receptor-expressing neurons in the subiculum complex.

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Shao, Yu-Feng; Wang, Can; Xie, Jun-Fan; Kong, Xiang-Pan; Xin, Le; Dong, Chao-Yu; Li, Jing; Ren, Wen-Ting; Hou, Yi-Ping

2016-07-01

Our previous studies have demonstrated that neuropeptide S (NPS), via selective activation of the neurons bearing NPS receptor (NPSR) in the olfactory cortex, facilitates olfactory function. High level expression of NPSR mRNA in the subiculum complex of hippocampal formation suggests that NPS-NPSR system might be involved in the regulation of olfactory spatial memory. The present study was undertaken to investigate effects of NPS on the scopolamine- or MK801-induced impairment of olfactory spatial memory using computer-assisted 4-hole-board spatial memory test, and by monitoring Fos expression in the subiculum complex in mice. In addition, dual-immunofluorescence microscopy was employed to identify NPS-induced Fos-immunereactive (-ir) neurons that also bear NPSR. Intracerebroventricular administration of NPS (0.5 nmol) significantly increased the number of visits to switched odorants in recall trial in mice suffering from odor-discriminating inability induced by scopolamine, a selective muscarinic cholinergic receptor antagonist, or MK801, a N-methyl-D-aspartate receptor antagonist, after training trials. The improvement of olfactory spatial memory by NPS was abolished by the NPSR antagonist [D-Val(5)]NPS (40 nmol). Ex vivo c-Fos and NPSR immunohistochemistry revealed that, as compared with vehicle-treated mice, NPS markedly enhanced Fos expression in the subiculum complex encompassing the subiculum (S), presubiculum (PrS) and parasubiculum (PaS). The percentages of Fos-ir neurons that also express NPSR were 91.3, 86.5 and 90.0 % in the S, PrS and PaS, respectively. The present findings demonstrate that NPS, via selective activation of the neurons bearing NPSR in the subiculum complex, ameliorates olfactory spatial memory impairment induced by scopolamine and MK801 in mice.

Distribution of cellular HSV-1 receptor expression in human brain.

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Lathe, Richard; Haas, Juergen G

2017-06-01

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus linked to a range of acute and chronic neurological disorders affecting distinct regions of the brain. Unusually, HSV-1 entry into cells requires the interaction of viral proteins glycoprotein D (gD) and glycoprotein B (gB) with distinct cellular receptor proteins. Several different gD and gB receptors have been identified, including TNFRSF14/HVEM and PVRL1/nectin 1 as gD receptors and PILRA, MAG, and MYH9 as gB receptors. We investigated the expression of these receptor molecules in different areas of the adult and developing human brain using online transcriptome databases. Whereas all HSV-1 receptors showed distinct expression patterns in different brain areas, the Allan Brain Atlas (ABA) reported increased expression of both gD and gB receptors in the hippocampus. Specifically, for PVRL1, TNFRFS14, and MYH9, the differential z scores for hippocampal expression, a measure of relative levels of increased expression, rose to 2.9, 2.9, and 2.5, respectively, comparable to the z score for the archetypical hippocampus-enriched mineralocorticoid receptor (NR3C2, z = 3.1). These data were confirmed at the Human Brain Transcriptome (HBT) database, but HBT data indicate that MAG expression is also enriched in hippocampus. The HBT database allowed the developmental pattern of expression to be investigated; we report that all HSV1 receptors markedly increase in expression levels between gestation and the postnatal/adult periods. These results suggest that differential receptor expression levels of several HSV-1 gD and gB receptors in the adult hippocampus are likely to underlie the susceptibility of this brain region to HSV-1 infection.

The vestibulo- and preposito-cerebellar cholinergic neurons of a ChAT-tdTomato transgenic rat exhibit heterogeneous firing properties and the expression of various neurotransmitter receptors.

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Zhang, Yue; Kaneko, Ryosuke; Yanagawa, Yuchio; Saito, Yasuhiko

2014-04-01

Cerebellar function is regulated by cholinergic mossy fiber inputs that are primarily derived from the medial vestibular nucleus (MVN) and prepositus hypoglossi nucleus (PHN). In contrast to the growing evidence surrounding cholinergic transmission and its functional significance in the cerebellum, the intrinsic and synaptic properties of cholinergic projection neurons (ChPNs) have not been clarified. In this study, we generated choline acetyltransferase (ChAT)-tdTomato transgenic rats, which specifically express the fluorescent protein tdTomato in cholinergic neurons, and used them to investigate the response properties of ChPNs identified via retrograde labeling using whole-cell recordings in brainstem slices. In response to current pulses, ChPNs exhibited two afterhyperpolarisation (AHP) profiles and three firing patterns; the predominant AHP and firing properties differed between the MVN and PHN. Morphologically, the ChPNs were separated into two types based on their soma size and dendritic extensions. Analyses of the firing responses to time-varying sinusoidal current stimuli revealed that ChPNs exhibited different firing modes depending on the input frequencies. The maximum frequencies in which each firing mode was observed were different between the neurons that exhibited distinct firing patterns. Analyses of the current responses to the application of neurotransmitter receptor agonists revealed that the ChPNs expressed (i) AMPA- and NMDA-type glutamate receptors, (ii) GABAA and glycine receptors, and (iii) muscarinic and nicotinic acetylcholine receptors. The current responses mediated by these receptors of MVN ChPNs were not different from those of PHN ChPNs. These findings suggest that ChPNs receive various synaptic inputs and encode those inputs appropriately across different frequencies. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Highly selective and sensitive detection of neurotransmitters using receptor-modified single-walled carbon nanotube sensors

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Kim, Byeongju; Song, Hyun Seok; Jin, Hye Jun; Park, Eun Jin; Lee, Sang Hun; Lee, Byung Yang; Park, Tai Hyun; Hong, Seunghun

2013-07-01

We present receptor-modified carbon nanotube sensors for the highly selective and sensitive detection of acetylcholine (ACh), one kind of neurotransmitter. Here, we successfully expressed the M1 muscarinic acetylcholine receptor (M1 mAChR), a family of G protein-coupled receptors (GPCRs), in E. coli and coated single-walled carbon nanotube (swCNT)-field effect transistors (FETs) with lipid membrane including the receptor, enabling highly selective and sensitive ACh detection. Using this sensor, we could detect ACh at 100 pM concentration. Moreover, we showed that this sensor could selectively detect ACh among other neurotransmitters. This is the first demonstration of the real-time detection of ACh using specific binding between ACh and M1 mAChR, and it may lead to breakthroughs for various applications such as disease diagnosis and drug screening.

Highly selective and sensitive detection of neurotransmitters using receptor-modified single-walled carbon nanotube sensors

International Nuclear Information System (INIS)

Kim, Byeongju; Jin, Hye Jun; Park, Eun Jin; Hong, Seunghun; Song, Hyun Seok; Lee, Sang Hun; Park, Tai Hyun; Lee, Byung Yang

2013-01-01

We present receptor-modified carbon nanotube sensors for the highly selective and sensitive detection of acetylcholine (ACh), one kind of neurotransmitter. Here, we successfully expressed the M1 muscarinic acetylcholine receptor (M1 mAChR), a family of G protein-coupled receptors (GPCRs), in E. coli and coated single-walled carbon nanotube (swCNT)-field effect transistors (FETs) with lipid membrane including the receptor, enabling highly selective and sensitive ACh detection. Using this sensor, we could detect ACh at 100 pM concentration. Moreover, we showed that this sensor could selectively detect ACh among other neurotransmitters. This is the first demonstration of the real-time detection of ACh using specific binding between ACh and M1 mAChR, and it may lead to breakthroughs for various applications such as disease diagnosis and drug screening. (paper)

An anti-nicotinic cognitive challenge model using mecamylamine in comparison with the anti-muscarinic cognitive challenge using scopolamine.

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Baakman, Anne Catrien; Alvarez-Jimenez, Ricardo; Rissmann, Robert; Klaassen, Erica S; Stevens, Jasper; Goulooze, Sebastiaan C; den Burger, Jeroen C G; Swart, Eleonora L; van Gerven, Joop M A; Groeneveld, Geert Jan

2017-08-01

The muscarinic acetylcholine receptor antagonist scopolamine is often used for proof-of-pharmacology studies with pro-cognitive compounds. From a pharmacological point of view, it would seem more rational to use a nicotinic rather than a muscarinic anticholinergic challenge to prove pharmacology of a nicotinic acetylcholine receptor agonist. This study aims to characterize a nicotinic anticholinergic challenge model using mecamylamine and to compare it to the scopolamine model. In this double-blind, placebo-controlled, four-way cross-over trial, 12 healthy male subjects received oral mecamylamine 10 and 20 mg, intravenous scopolamine 0.5 mg and placebo. Pharmacokinetics were analysed using non-compartmental analysis. Pharmacodynamic effects were measured with a multidimensional test battery that includes neurophysiological, subjective, (visuo)motor and cognitive measurements. All treatments were safe and well tolerated. Mecamylamine had a t max of 2.5 h and a C max of 64.5 ng ml -1 for the 20 mg dose. Mecamylamine had a dose-dependent effect decreasing the adaptive tracking performance and VAS alertness, and increasing the finger tapping and visual verbal learning task performance time and errors. Scopolamine significantly affected almost all pharmacodynamic tests. This study demonstrated that mecamylamine causes nicotinic receptor specific temporary decline in cognitive functioning. Compared with the scopolamine model, pharmacodynamic effects were less pronounced at the dose levels tested; however, mecamylamine caused less sedation. The cognitive effects of scopolamine might at least partly be caused by sedation. Whether the mecamylamine model can be used for proof-of-pharmacology of nicotinic acetylcholine receptor agonists remains to be established. © 2017 The British Pharmacological Society.

A regulatory code for neuron-specific odor receptor expression.

Directory of Open Access Journals (Sweden)

Anandasankar Ray

2008-05-01

Full Text Available Olfactory receptor neurons (ORNs must select-from a large repertoire-which odor receptors to express. In Drosophila, most ORNs express one of 60 Or genes, and most Or genes are expressed in a single ORN class in a process that produces a stereotyped receptor-to-neuron map. The construction of this map poses a problem of receptor gene regulation that is remarkable in its dimension and about which little is known. By using a phylogenetic approach and the genome sequences of 12 Drosophila species, we systematically identified regulatory elements that are evolutionarily conserved and specific for individual Or genes of the maxillary palp. Genetic analysis of these elements supports a model in which each receptor gene contains a zip code, consisting of elements that act positively to promote expression in a subset of ORN classes, and elements that restrict expression to a single ORN class. We identified a transcription factor, Scalloped, that mediates repression. Some elements are used in other chemosensory organs, and some are conserved upstream of axon-guidance genes. Surprisingly, the odor response spectra and organization of maxillary palp ORNs have been extremely well-conserved for tens of millions of years, even though the amino acid sequences of the receptors are not highly conserved. These results, taken together, define the logic by which individual ORNs in the maxillary palp select which odor receptors to express.

Reduced muscarinic receptors in the cingulate cortex in mild Alzheimer's disease demonstrated with 123I iodo-dexetamide SPECT

International Nuclear Information System (INIS)

Rowe, C.C.; Barnden, L.R.; Nicholas, C.; Nowakowski, K.; Boundy, K.

2000-01-01

Full text: Parietal hypoperfusion/hypometabolism is a feature of Alzheimer's disease (AD). In early AD this may be preceded by changes in the posterior cingulate cortex, part of the cortico-limbic circuit with connections to the medial temporal lobes. Because cholinergic function is affected in early AD, we aimed to investigate the binding of the muscarinic receptor label, I-123 iodo-dexetamide (IDEX). We recruited 11 mild (MiniMental State Examination 27-24) and 11 moderate (MMSE 23-16) Alzheimer's patients and 10 age and sex-matched normal subjects. SPECT was performed six hours after injection of 185 MBq IDEX. Sections were reconstructed with attenuation correction using an iterative algorithm (OSEM). Statistical Parametric Mapping (SPM 99) was used to analyse the data. Because there is very little IDEX uptake in the cerebellum and thalamus it was necessary to edit them from the SPM PET template. Facial and scalp activity was also edited. Global scaling relative to the basal ganglia was used. Significant areas of decreased IDEX binding were found in the mild Alzheimer's group in the cingulate cortex with pvoxel = .08 and pcluster < 0.001, (particularly the posterior cingulate), left parietotemporal junction (pcluster = 0.01) and posteromedial left temporal lobe (pcluster = 0.03). In moderate AD extensive areas of decreased binding were found in the posterior cingulate, parietal and temporal lobes. The difference between the group-means at the posterior cingulate was 14% (mild AD) and 22% (moderate AD). Hypoperfusion, hypometabolism and now reduced cholinergic receptors have been demonstrated in the posterior cingulate in mild AD. Greater attention to this area may enhance the diagnostic value of functional imaging in early AD. Copyright (2000) The Australian and New Zealand Society of Nuclear Medicine Inc

Behavioral analysis of Drosophila transformants expressing human taste receptor genes in the gustatory receptor neurons.

Science.gov (United States)

Adachi, Ryota; Sasaki, Yuko; Morita, Hiromi; Komai, Michio; Shirakawa, Hitoshi; Goto, Tomoko; Furuyama, Akira; Isono, Kunio

2012-06-01

Transgenic Drosophila expressing human T2R4 and T2R38 bitter-taste receptors or PKD2L1 sour-taste receptor in the fly gustatory receptor neurons and other tissues were prepared using conventional Gal4/UAS binary system. Molecular analysis showed that the transgene mRNAs are expressed according to the tissue specificity of the Gal4 drivers. Transformants expressing the transgene taste receptors in the fly taste neurons were then studied by a behavioral assay to analyze whether transgene chemoreceptors are functional and coupled to the cell response. Since wild-type flies show strong aversion against the T2R ligands as in mammals, the authors analyzed the transformants where the transgenes are expressed in the fly sugar receptor neurons so that they promote feeding ligand-dependently if they are functional and activate the neurons. Although the feeding preference varied considerably among different strains and individuals, statistical analysis using large numbers of transformants indicated that transformants expressing T2R4 showed a small but significant increase in the preference for denatonium and quinine, the T2R4 ligands, as compared to the control flies, whereas transformants expressing T2R38 did not. Similarly, transformants expressing T2R38 and PKD2L1 also showed a similar preference increase for T2R38-specific ligand phenylthiocarbamide (PTC) and a sour-taste ligand, citric acid, respectively. Taken together, the transformants expressing mammalian taste receptors showed a small but significant increase in the feeding preference that is taste receptor and also ligand dependent. Although future improvements are required to attain performance comparable to the endogenous robust response, Drosophila taste neurons may serve as a potential in vivo heterologous expression system for analyzing chemoreceptor function.

Muscarinic Long-Term Enhancement of Tonic and Phasic GABAA Inhibition in Rat CA1 Pyramidal Neurons

Science.gov (United States)

Domínguez, Soledad; Fernández de Sevilla, David; Buño, Washington

2016-01-01

Acetylcholine (ACh) regulates network operation in the hippocampus by controlling excitation and inhibition in rat CA1 pyramidal neurons (PCs), the latter through gamma-aminobutyric acid type-A receptors (GABAARs). Although, the enhancing effects of ACh on GABAARs have been reported (Dominguez et al., 2014, 2015), its role in regulating tonic GABAA inhibition has not been explored in depth. Therefore, we aimed at determining the effects of the activation of ACh receptors on responses mediated by synaptic and extrasynaptic GABAARs. Here, we show that under blockade of ionotropic glutamate receptors ACh, acting through muscarinic type 1 receptors, paired with post-synaptic depolarization induced a long-term enhancement of tonic GABAA currents (tGABAA) and puff-evoked GABAA currents (pGABAA). ACh combined with depolarization also potentiated IPSCs (i.e., phasic inhibition) in the same PCs, without signs of interactions of synaptic responses with pGABAA and tGABAA, suggesting the contribution of two different GABAA receptor pools. The long-term enhancement of GABAA currents and IPSCs reduced the excitability of PCs, possibly regulating plasticity and learning in behaving animals. PMID:27833531

Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

International Nuclear Information System (INIS)

Waser, Beatrice; Reubi, Jean Claude

2014-01-01

Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the 125 iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer 125 I-GLP-1(7-36)amide. Receptor autoradiography studies with 125 I-GLP-1(7-36)amide agonist or 125 I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist 125 I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer 125 I-GLP-1(7-36)amide. For comparison, 125 I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with 125 I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

A search for presynaptic inhibitory histamine receptors in guinea-pig tissues: Further H3 receptors but no evidence for H4 receptors.

Science.gov (United States)

Petri, Doris; Schlicker, Eberhard

2016-07-01

The histamine H4 receptor is coupled to Gi/o proteins and expressed on inflammatory cells and lymphoid tissues; it was suggested that this receptor also occurs in the brain or on peripheral neurones. Since many Gi/o protein-coupled receptors, including the H3 receptor, serve as presynaptic inhibitory receptors, we studied whether the sympathetic neurones supplying four peripheral tissues and the cholinergic neurones in the hippocampus from the guinea-pig are equipped with release-modulating H4 and H3 receptors. For this purpose, we preincubated tissue pieces from the aorta, atrium, renal cortex and vas deferens with (3)H-noradrenaline and hippocampal slices with (3)H-choline and determined the electrically evoked tritium overflow. The stimulation-evoked overflow in the five superfused tissues was inhibited by the muscarinic receptor agonist oxotremorine, which served as a positive control, but not affected by the H4 receptor agonist 4-methylhistamine. The H3 receptor agonist R-α-methylhistamine inhibited noradrenaline release in the peripheral tissues without affecting acetylcholine release in the hippocampal slices. Thioperamide shifted the concentration-response curve of histamine in the aorta and the renal cortex to the right, yielding apparent pA2 values of 8.0 and 8.1, respectively, which are close to its affinity at other H3 receptors but higher by one log unit than its pKi at the H4 receptor of the guinea-pig. In conclusion, histamine H4 receptors could not be identified in five experimental models of the guinea-pig that are suited for the detection of presynaptic inhibitory receptors whereas H3 receptors could be shown in the peripheral tissues but not in the hippocampus. This article is part of the Special Issue entitled 'Histamine Receptors'. Copyright © 2015 Elsevier Ltd. All rights reserved.

A complex pattern of chemokine receptor expression is seen in osteosarcoma

International Nuclear Information System (INIS)

Luettichau, Irene von; Huss, Ralf; Nelson, Peter J; Segerer, Stephan; Wechselberger, Alexandra; Notohamiprodjo, Mike; Nathrath, Michaela; Kremer, Markus; Henger, Anna; Djafarzadeh, Roghieh; Burdach, Stefan

2008-01-01

Osteosarcoma is the most frequent bone tumor in childhood and adolescence. Patients with primary metastatic disease have a poor prognosis. It is therefore important to better characterize the biology of this tumor to define new prognostic markers or therapeutic targets for tailored therapy. Chemokines and their receptors have been shown to be involved in the development and progression of malignant tumors. They are thought to be active participants in the biology of osteosarcoma. The function of specific chemokines and their receptors is strongly associated with the biological context and microenvironment of their expression. In this report we characterized the expression of a series of chemokine receptors in the complex environment that defines osteosarcoma. The overall level of chemokine receptor mRNA expression was determined using TaqMan RT-PCR of microdissected archival patient biopsy samples. Expression was then verified at the protein level by immunohistochemistry using a series of receptor specific antibody reagents to elucidate the cellular association of expression. Expression at the RNA level was found for most of the tested receptors. CCR1 expression was found on infiltrating mononuclear and polynuclear giant cells in the tumor. Cells associated with the lining of intratumoral vessels were shown to express CCR4. Infiltrating mononuclear cells and tumor cells both showed expression of the receptor CCR5, while CCR7 was predominantly expressed by the mononuclear infiltrate. CCR10 was only very rarely detected in few scattered infiltrating cells. Our data elucidate for the first time the cellular context of chemokine receptor expression in osteosarcoma. This is an important issue for better understanding potential chemokine/chemokine receptor function in the complex biologic processes that underlie the development and progression of osteosarcoma. Our data support the suggested involvement of chemokines and their receptors in diverse aspects of the biology

Differences in muscarinic acetylcholine receptor subtypes in the central nervous system of long sleep and short sleep mice

International Nuclear Information System (INIS)

Watson, M.; Ming, X.; McArdle, J.J.

1989-01-01

Differences in voluntary ethanol consumption have been noted in various inbred strains of mice and pharmacogenetic approaches have been used to study the mechanisms of action of many drugs such as ethanol. Long-sleep (LS) and short-sleep (SS) mice, selectively bred for differences in ethanol induced narcosis, provide a method by which a relationship between the differential responsiveness of these geno-types and muscarinic acetylcholine receptors (mAChR) may be evaluated. Sleep times after injection of 3ml ethanol/kg (i.p.) verified the higher sensitivity of LS vs. SS. Mean body weights of LS (26.5g) vs. SS (22g) were also significantly (p 3 H](-) quinuclidinylbenzilate ([ 3 H](-)QNB), a specific but nonsubtype selective mAChR antagonist, [ 3 H]pirenzepine ([ 3 H]PZ), a specific M1 mAChR antagonist and [ 3 H]11-2-[[2-[(diethylamino) methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido (2,3-b) (1,4) benzodiazepine-6-one, ([ 3 H]AF-DX 116), an M2 selective antagonist were performed to determine mAChR affinity (K d ) and density (B max ) in CNS regions such as the cerebral cortex, hippocampus, corpus striatum and other areas. Significantly lower (30-40%) [ 3 H](-)QNB binding suggests that SS have fewer mAChR's than LS in many areas. These differences may relate to their differential ethanol sensitivity

Sexual dimorphism in the volume of song control nuclei in European starlings: assessment by a Nissl stain and autoradiography for muscarinic cholinergic receptors.

Science.gov (United States)

Bernard, D J; Casto, J M; Ball, G F

1993-08-22

Previous studies have found that the volume of several song control nuclei is larger in male songbirds than in female songbirds. The degree of this volumetric sex difference within a given species appears to be systematically related to the degree of the behavioral sex difference. The largest volumetric differences have been reported in species in which the male sings and the female sings little, if at all, and the smallest sex differences in volume have been reported in species in which males and females both sing in nearly equal amounts. We compared the volume of three song control nuclei in male and female European starlings (Sturnus vulgaris), a species in which females are known to sing, though at a much lower rate than males. We investigated the volume of hyperstriatum ventrale, pars caudale, nucleus robustus archistriatalis, and area X of the lobus parolfactorius as defined with the use of a Nissl stain. In addition, we measured the volume of area X as defined by the density of muscarinic cholinergic receptors visualized by in vitro receptor autoradiographic methods. The volumes of all three of the song nuclei, as defined by Nissl staining, are significantly larger in males than in females. For area X, Nissl staining and receptor autoradiography indicate the same significant volumetric sex difference. The three nuclei are approximately one and one half to two times larger in males than in females, a degree of dimorphism that is intermediate to those reported for other species. Previous investigations of sex differences in the avian vocal control system have used only Nissl stains to define nuclear volumes. We demonstrate in this paper that receptor autoradiography can be used to assess dimorphisms in nuclear volume. Broad application of this approach to a number of neurotransmitter receptor systems will better characterize the dimorphisms in the song system, and therefore will provide greater insight into the neuroanatomical and neurochemical control of

Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

Energy Technology Data Exchange (ETDEWEB)

Waser, Beatrice; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, PO Box 62, Berne (Switzerland)

2014-06-15

Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the {sup 125}iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer {sup 125}I-GLP-1(7-36)amide. Receptor autoradiography studies with {sup 125}I-GLP-1(7-36)amide agonist or {sup 125}I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist {sup 125}I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer {sup 125}I-GLP-1(7-36)amide. For comparison, {sup 125}I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with {sup 125}I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

Hormone-receptor expression and ovarian cancer survival

DEFF Research Database (Denmark)

Sieh, Weiva; Köbel, Martin; Longacre, Teri A

2013-01-01

Few biomarkers of ovarian cancer prognosis have been established, partly because subtype-specific associations might be obscured in studies combining all histopathological subtypes. We examined whether tumour expression of the progesterone receptor (PR) and oestrogen receptor (ER) was associated ...

Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography

International Nuclear Information System (INIS)

Gillberg, P.-G.; Aquilonius, S.-M.

1985-01-01

Binding sites for the receptor ligands 3 H-quinuclidinylbenzilate, 3 H-alpha-bungarotoxin ( 3 H-alpha-Btx), 3 H-etorphine and 3 H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3 H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distribute within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged. (author)

The expression of the ACTH receptor

Directory of Open Access Journals (Sweden)

L.L.K. Elias

2000-10-01

Full Text Available Adrenal glucocorticoid secretion is regulated by adrenocorticotropic hormone (ACTH acting through a specific cell membrane receptor (ACTH-R. The ACTH-R is a member of the G protein superfamily-coupled receptors and belongs to the subfamily of melanocortin receptors. The ACTH-R is mainly expressed in the adrenocortical cells showing a restricted tissue specificity, although ACTH is recognized by the other four melanocortin receptors. The cloning of the ACTH-R was followed by the study of this gene in human diseases such as familial glucocorticoid deficiency (FGD and adrenocortical tumors. FGD is a rare autosomal recessive disease characterized by glucocorticoid deficiency, elevated plasma ACTH levels and preserved renin/aldosterone secretion. This disorder has been ascribed to an impaired adrenal responsiveness to ACTH due to a defective ACTH-R, a defect in intracellular signal transduction or an abnormality in adrenal cortical development. Mutations of the ACTH-R have been described in patients with FGD in segregation with the disease. The functional characterization of these mutations has been prevented by difficulties in expressing human ACTH-R in cells that lack endogenous melanocortin receptor activity. To overcome these difficulties we used Y6 cells, a mutant variant of the Y1 cell line, which possesses a non-expressed ACTH-R gene allowing the functional study without any background activity. Our results demonstrated that the several mutations of the ACTH-R found in FGD result in an impaired cAMP response or loss of sensitivity to ACTH stimulation. An ACTH-binding study showed an impairment of ligand binding with loss of the high affinity site in most of the mutations studied.

Hormonal control of spermatogenesis: expression of FSJH receptor and androgen receptor genes

NARCIS (Netherlands)

L.J. Blok (Leen)

1992-01-01

textabstractFSH and testosterone are the main hormonal regulators of spermatogenesis. The actions of androgens and FSH are mediated by their respective receptors. Receptor gene expression (mRNA and protein). is an important determinant of hormone action. Biochemical aspects of the regulation of

Imaging of muscarinic acetylcholine receptors using (+)N-[11C]methyl-3-piperidyl benzilate (11C-3NMPB) in vascular dementia and Alzheimer's disease

International Nuclear Information System (INIS)

Saito, Hirohiko

2006-01-01

In order to clarify the integrity of muscarinic acetylcholine receptor (mAChR) in vascular dementia (VaD) and Alzheimer's disease (AD), PET imaging with (+) N-[ 11 C]methyl-3-piperidyl benzilate ( 11 C-3NMPB) was performed in 12 patients with VaD, 11 patients with AD, and 7 normal controls (NC group). The mAChR binding was compared by the ratios compared with the cerebellum which were calculated from the regions of interest (ROI), and by three-dimensional statistic analysis. Compared with the NC group, mAChR was not significantly reduced in any ROI in AD patients. In those with VaD due to cortical lesions, mAChR was reduced in the infarcted areas. On the other hand, mAChR was significantly reduced in the thalamus and anterior cingulated gyrus, but not in other cerebral cortices in patients with VaD due to subcortical lesions. Accordingly, it is suggested that the mAChR is preserved in the cerebral cortices in patients with VaD due to subcortical lesions as well as in AD patients. (author)

Profiling neurotransmitter receptor expression in the Ambystoma mexicanum brain.

Science.gov (United States)

Reyes-Ruiz, Jorge Mauricio; Limon, Agenor; Korn, Matthew J; Nakamura, Paul A; Shirkey, Nicole J; Wong, Jamie K; Miledi, Ricardo

2013-03-22

Ability to regenerate limbs and central nervous system (CNS) is unique to few vertebrates, most notably the axolotl (Ambystoma sp.). However, despite the fact the neurotransmitter receptors are involved in axonal regeneration, little is known regarding its expression profile. In this project, RT-PCR and qPCR were performed to gain insight into the neurotransmitter receptors present in Ambystoma. Its functional ability was studied by expressing axolotl receptors in Xenopus laevis oocytes by either injection of mRNA or by direct microtransplantation of brain membranes. Oocytes injected with axolotl mRNA expressed ionotropic receptors activated by GABA, aspartate+glycine and kainate, as well as metabotropic receptors activated by acetylcholine and glutamate. Interestingly, we did not see responses following the application of serotonin. Membranes from the axolotl brain were efficiently microtransplanted into Xenopus oocytes and two types of native GABA receptors that differed in the temporal course of their responses and affinities to GABA were observed. Results of this study are necessary for further characterization of axolotl neurotransmitter receptors and may be useful for guiding experiments aimed at understanding activity-dependant limb and CNS regeneration. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Expression of Plant Receptor Kinases in Tobacco BY-2 Cells.

Science.gov (United States)

Shinohara, Hidefumi; Matsubayashi, Yoshikatsu

2017-01-01

Although more than 600 single-transmembrane receptor kinase genes have been found in the Arabidopsis genome, only a few of them have known physiological functions, and even fewer plant receptor kinases have known specific ligands. Ligand-binding analysis must be operated using the functionally expressed receptor form. However, the relative abundance of native receptor kinase molecules in the plasma membrane is often quite low. Here, we present a method for stable and functional expression of plant receptor kinases in tobacco BY-2 cells that allows preparation of microsomal fractions containing the receptor. This procedure provides a sufficient amount of receptor proteins while maintaining its ligand-binding activities.

Functional importance of GLP-1 receptor species and expression levels in cell lines.

Science.gov (United States)

Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan

2012-04-10

Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.

Transitional cell carcinoma express vitamin D receptors

DEFF Research Database (Denmark)

Hermann, G G; Andersen, C B

1997-01-01

Recently, vitamin D analogues have shown antineoplastic effect in several diseases. Vitamin D analogues exert its effect by interacting with the vitamin D receptor (VDR). Studies of VDR in transitional cell carcinoma (TCC) have not been reported. The purpose of the present study was therefore.......05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...... studies of vitamin D's effect on TCC cells in vitro are necessary before the efficacy of treatment with vitamin D analogues in TCC can be evaluated in patients....

Odor memories regulate olfactory receptor expression in the sensory periphery.

Science.gov (United States)

Claudianos, Charles; Lim, Julianne; Young, Melanie; Yan, Shanzhi; Cristino, Alexandre S; Newcomb, Richard D; Gunasekaran, Nivetha; Reinhard, Judith

2014-05-01

Odor learning induces structural and functional modifications throughout the olfactory system, but it is currently unknown whether this plasticity extends to the olfactory receptors (Or) in the sensory periphery. Here, we demonstrate that odor learning induces plasticity in olfactory receptor expression in the honeybee, Apis mellifera. Using quantitative RT-PCR analysis, we show that six putative floral scent receptors were differentially expressed in the bee antennae depending on the scent environment that the bees experienced. Or151, which we characterized using an in vitro cell expression system as a broadly tuned receptor binding floral odorants such as linalool, and Or11, the specific receptor for the queen pheromone 9-oxo-decenoic acid, were significantly down-regulated after honeybees were conditioned with the respective odorants in an olfactory learning paradigm. Electroantennogram recordings showed that the neural response of the antenna was similarly reduced after odor learning. Long-term odor memory was essential for inducing these changes, suggesting that the molecular mechanisms involved in olfactory memory also regulate olfactory receptor expression. Our study demonstrates for the first time that olfactory receptor expression is experience-dependent and modulated by scent conditioning, providing novel insight into how molecular regulation at the periphery contributes to plasticity in the olfactory system. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

The Expression Profiles of Lysophospholipid Receptors (LPLRs in Different Endothelial Cells

Directory of Open Access Journals (Sweden)

Yu-Wei Lee

2006-03-01

Full Text Available Sphingosine-1-phosphate (S1P and lysophosphatidic acid (LPA are two bioactive lysophospholipids (LPLs, stored primarily in platelets and released during platelet activation. Both LPLs are capable of regulating endothelial cell functions. The physiological functions of S1P and LPA are mediated by interacting with eight different G-protein coupled receptors: S1P1 through 5 and LPA1 through 3, which activate three different heterotrimeric GTP proteins－including Gi、Gq and G(12/13. The expression of LPL receptors in endothelial cells would affect the responses of S1P and LPA to these cells. There is no previous report discussing the expression profiles of LPL receptors in different endothelial cells from various species. In this study, we aim to investigate the expression profiles of S1P and LPA receptors in different endothelial cells isolated from human, rat, mouse and bovine origin. We used RT-PCR to determine LPLs receptors expression profiles in different endothelial cells. Our results indicated that endothelial cells from various species express different LPL receptors. Endothelial cells isolated from the same source of different species also had different LPLs receptors expression profiles. Therefore, different endothelial cells should respond to LPLs in different manners.

Expression profile and prognostic role of sex hormone receptors in gastric cancer

International Nuclear Information System (INIS)

Gan, Lu; He, Jian; Zhang, Xia; Zhang, Yong-Jie; Yu, Guan-Zhen; Chen, Ying; Pan, Jun; Wang, Jie-Jun; Wang, Xi

2012-01-01

Increasing interest has been devoted to the expression and possible role of sex hormone receptors in gastric cancer, but most of these findings are controversial. In the present study, the expression profile of sex hormone receptors in gastric cancer and their clinicopathological and prognostic value were determined in a large Chinese cohort. The mRNA and protein expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), progesterone receptor (PR), and androgen receptor (AR) in primary gastric tumors and corresponding adjacent normal tissues from 60 and 866 Chinese gastric cancer patients was detected by real-time quantitative PCR and immunohistochemistry method, respectively. The expression profile of the four receptors was compared and their associations with clinicopathological characteristics were assessed by using Chi-square test. The prognostic value of the four receptors in gastric cancer was evaluated by using univariate and multivariate Cox regression analysis. The presence of ERα, ERβ, PR, and AR in both gastric tumors and normal tissues was confirmed but their expression levels were extremely low except for the predominance of ERβ. The four receptors were expressed independently and showed a decreased expression pattern in gastric tumors compared to adjacent normal tissues. The positive expression of the four receptors all correlated with high tumor grade and intestinal type, and ERα and AR were also associated with early TNM stage and thereby a favorable outcome. However, ERα and AR were not independent prognostic factors for gastric cancer when multivariate survival analysis was performed. Our findings indicate that the sex hormone receptors may be partly involved in gastric carcinogenesis but their clinicopathological and prognostic significance in gastric cancer appears to be limited

Autoantibodies Enhance Agonist Action and Binding to Cardiac Muscarinic Receptors in Chronic Chagas’ Disease

Science.gov (United States)

Hernández, Ciria C.; Nascimento, José H.; Chaves, Elen A.; Costa, Patrícia C.; Masuda, Masako O.; Kurtenbach, Eleonora; Campos de Carvalho, Antônio C.; Giménez, Luis E.

2009-01-01

Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M2-muscarinic acetylcholine receptors (M2AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M2AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M2AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [3H]-N-methyl scopolamine ([3H]-NMS) in allosterism binding assays. A peptide corresponding to the M2AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [3H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [3H]-NMS dissociation right shifted from an IC50 of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 × 10−8, 1.33 × 10−7, and 2.0 × 10−7 mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M2AChRs as a positive cooperativity effect on acetylcholine action. PMID:18702010

Local anesthetic inhibition of G protein-coupled receptor signaling by interference with Galpha(q) protein function

NARCIS (Netherlands)

Hollmann, M. W.; Wieczorek, K. S.; Berger, A.; Durieux, M. E.

2001-01-01

Although local anesthetics are considered primarily Na(+) channel blockers, previous studies suggest a common intracellular site of action on different G protein-coupled receptors. In the present study, we characterized this site for the LPA, m1 muscarinic, and trypsin receptor. Xenopus laevis

Point mutation of a conserved aspartate, D69, in the muscarinic M2 receptor does not modify voltage-sensitive agonist potency.

Science.gov (United States)

Ågren, Richard; Sahlholm, Kristoffer; Nilsson, Johanna; Århem, Peter

2018-01-29

The muscarinic M 2 receptor (M 2 R) has been shown to display voltage-sensitive agonist binding, based on G protein-activated inward rectifier potassium channel (GIRK) opening and radioligand binding at different membrane voltages. A conserved aspartate in transmembrane segment (TM) II of M 2 R, D69, has been proposed as the voltage sensor. While a recent paper instead presented evidence of tyrosines in TMs III, VI, and VII acting as voltage sensors, these authors were not able to record GIRK channel activation by a D69N mutant M 2 R. In the present study, we succeeded in recording ACh-induced GIRK channel activation by this mutant at -80 and 0 mV. The acetylcholine EC 50 was about 2.5-fold higher at 0 mV, a potency shift very similar to that observed at wild-type M 2 R, indicating that voltage sensitivity persists at the D69N mutant. Thus, our present observations corroborate the notion that D69 is not responsible for voltage sensitivity of the M 2 R. Copyright © 2018 Elsevier Inc. All rights reserved.

Steroidal Hormone Receptor Expression in Male Breast Cancer

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Fatemeh Homaei-Shandiz

2014-01-01

Full Text Available Introduction: The etiology of male breast cancer is unclear, but hormonal levels may play a role in development of this disease. It seems that the risk of male breast cancer related to increased lifelong exposure to estrogen or reduced androgen. The aim of this study was to investigate the expression of the steroid hormone receptors including estrogen receptor (ER and progesterone receptor (PR in Iranian cases with male breast cancer. Methods: This is a prospective review of 18 cases of male breast cancer in in Omid Hospital, Mashhad, North East of Iran, between October 2001 and October 2006. ER and PR were measured by immunohistochemistry. Clinicopathologic features and family history were obtained by interview. Data were analyzed with SPSS 13 using descriptive statistics.  Results: The median age was 63.2 year. All the cases were infiltrating ductal carcinoma. A high rate of expression of ER (88.8% and PR (66.6% was found in the studied cases. Conclusion: Cancers of the male breast are significantly more likely than cancers of the female breast to express hormonal receptors.

Integrated olfactory receptor and microarray gene expression databases

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Crasto Chiquito J

2007-06-01

Full Text Available Abstract Background Gene expression patterns of olfactory receptors (ORs are an important component of the signal encoding mechanism in the olfactory system since they determine the interactions between odorant ligands and sensory neurons. We have developed the Olfactory Receptor Microarray Database (ORMD to house OR gene expression data. ORMD is integrated with the Olfactory Receptor Database (ORDB, which is a key repository of OR gene information. Both databases aim to aid experimental research related to olfaction. Description ORMD is a Web-accessible database that provides a secure data repository for OR microarray experiments. It contains both publicly available and private data; accessing the latter requires authenticated login. The ORMD is designed to allow users to not only deposit gene expression data but also manage their projects/experiments. For example, contributors can choose whether to make their datasets public. For each experiment, users can download the raw data files and view and export the gene expression data. For each OR gene being probed in a microarray experiment, a hyperlink to that gene in ORDB provides access to genomic and proteomic information related to the corresponding olfactory receptor. Individual ORs archived in ORDB are also linked to ORMD, allowing users access to the related microarray gene expression data. Conclusion ORMD serves as a data repository and project management system. It facilitates the study of microarray experiments of gene expression in the olfactory system. In conjunction with ORDB, ORMD integrates gene expression data with the genomic and functional data of ORs, and is thus a useful resource for both olfactory researchers and the public.

A new nosology of psychosis and the pharmacological basis of affective and negative symptom dimensions in schizophrenia

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Costa Vakalopoulos

2010-01-01

Full Text Available Although first rank symptoms focus on positive symptoms of psychosis they are shared by a number of psychiatric conditions. The difficulty in differentiating bipolar disorder from schizophrenia with affective features has led to a third category of patients often loosely labeled as schizoaffective. Research in schizophrenia has attempted to render the presence or absence of negative symptoms and their relation to etiology and prognosis more explicit. A dichotomous population is a recurring theme in experimental paradigms. Thus, schizophrenia is defined as process or reactive, deficit or non-deficit and by the presence or absence of affective symptoms. Laboratory tests confirm the clinical impression showing conflicting responses to dexamethasone suppression and clearly defined differences in autonomic responsiveness, but their pathophysiological significance eludes mainstream theory. Added to this is the difficulty in agreeing to what exactly constitutes useful clinical features differentiating, for example, negative symptoms of a true deficit syndrome from features of depression. Two recent papers proposed that the general and specific cognitive features of schizophrenia and major depression result from a monoamine-cholinergic imbalance, the former due to a relative muscarinic receptor hypofunction and the latter, in contrast, to a muscarinic hypersensitivity exacerbated by monoamine depletion. Further development of these ideas will provide pharmacological principles for what is currently an incomplete and largely, descriptive nosology of psychosis. It will propose a dimensional view of affective and negative symptoms based on relative muscarinic integrity and is supported by several exciting intracellular signaling and gene expression studies. Bipolar disorder manifests both muscarinic and dopaminergic hypersensitivity. The greater the imbalance between these two receptor signaling systems, the more the clinical picture will resemble

Sex Steroid Hormone Receptor Expression Affects Ovarian Cancer Survival

DEFF Research Database (Denmark)

Jönsson, Jenny-Maria; Skovbjerg Arildsen, Nicolai; Malander, Susanne

2015-01-01

BACKGROUND AND AIMS: Although most ovarian cancers express estrogen (ER), progesterone (PR), and androgen (AR) receptors, they are currently not applied in clinical decision making. We explored the prognostic impact of sex steroid hormone receptor protein and mRNA expression on survival...... in epithelial ovarian cancer. METHODS: Immunohistochemical stainings for ERα, ERβ, PR, and AR were assessed in relation to survival in 118 serous and endometrioid ovarian cancers. Expression of the genes encoding the four receptors was studied in relation to prognosis in the molecular subtypes of ovarian cancer...... in ovarian cancer and support that tumors should be stratified based on molecular as well as histological subtypes in future studies investigating the role of endocrine treatment in ovarian cancer....

Prognostic value of sex-hormone receptor expression in non-muscle-invasive bladder cancer.

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Nam, Jong Kil; Park, Sung Woo; Lee, Sang Don; Chung, Moon Kee

2014-09-01

We investigated sex-hormone receptor expression as predicting factor of recurrence and progression in patients with non-muscle invasive bladder cancer. We retrospectively evaluated tumor specimens from patients treated for transitional cell carcinoma of the bladder at our institution between January 2006 and January 2011. Performing immunohistochemistry using a monoclonal androgen receptor antibody and monoclonal estrogen receptor-beta antibody on paraffin-embedded tissue sections, we assessed the relationship of immunohistochemistry results and prognostic factors such as recurrence and progression. A total of 169 patients with bladder cancer were evaluated in this study. Sixty-threepatients had expressed androgen receptors and 52 patients had estrogen receptor beta. On univariable analysis, androgen receptor expression was significant lower in recurrence rates (p=0.001), and estrogen receptor beta expression was significant higher in progression rates (p=0.004). On multivariable analysis, significant association was found between androgen receptor expression and lower recurrence rates (hazard ratio=0.500; 95% confidence interval, 0.294 to 0.852; p=0.011), but estrogen receptor beta expression was not significantly associated with progression rates. We concluded that the possibility of recurrence was low when the androgen receptor was expressed in the bladder cancer specimen and it could be the predicting factor of the stage, number of tumors, carcinoma in situ lesion and recurrence.

Expression of sulfonylurea receptors in rat taste buds.

Science.gov (United States)

Liu, Dian-Xin; Liu, Xiao-Min; Zhou, Li-Hong; Feng, Xiao-Hong; Zhang, Xiao-Juan

2011-07-01

To test the possibility that a fast-onset promoting agent repaglinide may initiate prandial insulin secretion through the mechanism of cephalic-phase insulin release, we explored the expression and distribution character of sulfonylurea receptors in rat taste buds. Twenty male Wistar rats aged 10 weeks old were killed after general anesthesia. The circumvallate papillae, fungiform papillae and pancreas tissues were separately collected. Immunohistochemical staining was used to detect the expression and distribution of sulfonylurea receptor 1 (SUR1) or sulfonylurea receptor 2 (SUR2) in rat taste buds. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the expression of SUR1 or SUR2 mRNA. The pancreatic tissues from the same rat were used as positive control. This is the first study to report that SUR1 is uniquely expressed in the taste buds of fungiform papillae of each rat tongue, while the expression of SUR1 or SUR2 was not detected in the taste buds of circumvallate papillae. SUR1 is selectively expressed in rat taste buds, and its distribution pattern may be functionally relevant, suggesting that the rapid insulin secretion-promoting effect of repaglinide may be exerted through the cephalic-phase secretion pathway mediated by taste buds. Copyright © 2010 Elsevier GmbH. All rights reserved.

Oleocanthal Modulates Estradiol-Induced Gene Expression Involving Estrogen Receptor α.

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Keiler, Annekathrin Martina; Djiogue, Sefirin; Ehrhardt, Tino; Zierau, Oliver; Skaltsounis, Leandros; Halabalaki, Maria; Vollmer, Günter

2015-09-01

Oleocanthal is a bioactive compound from olive oil. It has attracted considerable attention as it is anti-inflammatory, antiproliferative, and has been shown to possess neuroprotective properties in vitro and in vivo. Delineated from its polyphenolic structure, the aim of this study was to characterize oleocanthal towards estrogenic properties. This might contribute to partly explain the beneficial effects described for the Mediterranean diet. Estrogenic properties of oleocanthal were assessed by different methods: a) stimulation of reporter gene activity in MVLN or RNDA cells either expressing estrogen receptor α or β, b) stimulation of luciferase reporter gene activity in U2OS osteosarcoma cells expressing estrogen receptor α or β, and c) elucidation of the impact on estradiol-induced gene expression in U2OS cells transduced with both estrogen receptors. Depending on the cell line origin, oleocanthal inhibited luciferase activity (MVLN, U2OS-estrogen receptor β) or weakly induced reporter gene activity at 10 µM in U2OS-estrogen receptor α cells. However, oleocanthal inhibited stimulation of luciferase activity by estradiol from both estrogen receptors. Oleocanthal, if given alone, did not stimulate gene expression in U2OS cells, but it significantly modulated the response of estradiol. Oleocanthal enhanced the effect of estradiol on the regulation of those genes, which are believed to be regulated through heterodimeric estrogen receptors. As the estrogenic response pattern of oleocanthal is rather unique, we compared the results obtained with oleacein. Oleocanthal binds to both estrogen receptors inducing estradiol-agonistic or antiagonistic effects depending on the cell line. Regarding regulation of gene expression in U2OS-estrogen receptor α/β cells, oleocanthal and oleacein enhanced estradiol-mediated regulation of heterodimer-regulated genes. Georg Thieme Verlag KG Stuttgart · New York.

Role of a Ubiquitously Expressed Receptor in the Vertebrate Olfactory System

OpenAIRE

DeMaria, Shannon; Berke, Allison P.; Van Name, Eric; Heravian, Anisa; Ferreira, Todd; Ngai, John

2013-01-01

Odorant cues are recognized by receptors expressed on olfactory sensory neurons, the primary sensory neurons of the olfactory epithelium. Odorant receptors typically obey the “one receptor, one neuron” rule, in which the receptive field of the olfactory neuron is determined by the singular odorant receptor that it expresses. Odor-evoked receptor activity across the population of olfactory neurons is then interpreted by the brain to identify the molecular nature of the odorant stimulus. In the...

Presynaptic muscarinic receptors, calcium channels, and protein kinase C modulate the functional disconnection of weak inputs at polyinnervated neonatal neuromuscular synapses.

Science.gov (United States)

Santafe, M M; Garcia, N; Lanuza, M A; Tomàs, M; Besalduch, N; Tomàs, J

2009-04-01

We studied the relation among calcium inflows, voltage-dependent calcium channels (VDCC), presynaptic muscarinic acetylcholine receptors (mAChRs), and protein kinase C (PKC) activity in the modulation of synapse elimination. We used intracellular recording to determine the synaptic efficacy in dually innervated endplates of the levator auris longus muscle of newborn rats during axonal competition in the postnatal synaptic elimination period. In these dual junctions, the weak nerve terminal was potentiated by partially reducing calcium entry (P/Q-, N-, or L-type VDCC-specific block or 500 muM magnesium ions), M1- or M4-type selective mAChR block, or PKC block. Moreover, reducing calcium entry or blocking PKC or mAChRs results in unmasking functionally silent nerve endings that now recover neurotransmitter release. Our results show interactions between these molecules and indicate that there is a release inhibition mechanism based on an mAChR-PKC-VDCC intracellular cascade. When it is fully active in certain weak motor axons, it can depress ACh release and even disconnect synapses. We suggest that this mechanism plays a central role in the elimination of redundant neonatal synapses, because functional axonal withdrawal can indeed be reversed by mAChRs, VDCCs, or PKC block.

Functional expression of rat VPAC1 receptor in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Hansen, M.K.; Tams, J.W.; Fahrenkrug, Jan

1999-01-01

G protein-coupled receptor; heterologous expression; membrane protein; Saccharomyces cerevisiae, vasoactive intestinal polypeptide; yeast mating factor-pre-pro *Ga-leader peptide......G protein-coupled receptor; heterologous expression; membrane protein; Saccharomyces cerevisiae, vasoactive intestinal polypeptide; yeast mating factor-pre-pro *Ga-leader peptide...

Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in Vibrio vulnificus.

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Kim, Choon-Mee; Kim, Seong-Jung; Shin, Sung-Heui

2012-04-01

The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.

Sex steroid receptor expression in idiopathic pulmonary fibrosis.

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Mehrad, Mitra; Trejo Bittar, Humberto E; Yousem, Samuel A

2017-08-01

Usual interstitial pneumonia (UIP) is characterized by progressive scarring of the lungs and is associated with high morbidity and mortality despite therapeutic interventions. Sex steroid receptors have been demonstrated to play an important role in chronic lung conditions; however, their significance is unknown in patients with UIP. We retrospectively reviewed 40 idiopathic UIP cases for the expression of hormonal receptors. Forty cases including 10 normal lung, 10 cryptogenic organizing pneumonia, 10 idiopathic organizing diffuse alveolar damage, 7 hypersensitivity pneumonitis, and 3 nonspecific interstitial pneumonitis served as controls. Immunohistochemistry for estrogen receptor α, progesterone receptor (PR), and androgen receptor was performed in all groups. Expression of these receptors was assessed in 4 anatomic/pathologic compartments: alveolar and bronchiolar epithelium, arteries/veins, fibroblastic foci/airspace organization, and old scar. All UIPs (100%) stained positive for PR in myofibroblasts in the scarred areas, whereas among the control cases, only 1 nonspecific interstitial pneumonitis case stained focally positive and the rest were negative. PR was positive in myocytes of the large-sized arteries within the fibrotic areas in 31 cases (77.5%). PR was negative within the alveolar and bronchial epithelium, airspace organization, and center of fibroblastic foci; however, weak PR positivity was noted in the peripheral fibroblasts of the fibroblastic foci where they merged with dense fibrous connective tissue scar. All UIP and control cases were negative for androgen receptor and estrogen receptor α. This is the first study to show the expression of PR within the established fibrotic areas of UIP, indicating that progesterone may have profibrotic effects in UIP patients. Hormonal therapy by targeting PR could be of potential benefit in patients with UIP/IPF. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of somatotropin receptor messenger ribonucleic acid in bovine tissues

International Nuclear Information System (INIS)

Lucy, M.C.; Boyd, C.K.; Koenigsfeld, A.T.; Okamura, C.S.

1998-01-01

The somatotropin receptor mRNA is controlled by at least two different gene promoters that generate 2 two variants with different exon 1 sequences (1A and 1B). The location of 1A and 1B somatotropin receptor mRNA within cattle tissues and, hence, the tissue specificity of the 1A and 1B promoters are unknown. In addition, the cDNA sequence of the 1B somatotropin receptor has not been determined. Our objective, therefore, was to sequence a cDNA for the 1B somatotropin receptor and to analyze bovine tissues for expression of 1A and 1B somatotropin receptor mRNA. Twenty adult tissues and six fetal tissues were collected at slaughter from each of four cows and two fetuses. Messenger RNA was analyzed using ribonuclease protection assays. The adult liver expressed both 1A and 1B mRNA. All other adult tissues expressed 1B mRNA but not 1A mRNA. The greatest amount of 1B mRNA was detected in liver and adipose (abdominal and subcutaneous) tissues. Other tissues had approximately one-half to one-tenth of the amount of 1B mRNA in the liver or adipose tissue. Fetal tissues (including fetal liver) expressed 1B mRNA and not 1A mRNA. Based on cDNA sequencing, the protein encoded by the 1A and 1B mRNA was nearly identical. We concluded that 1A somatotropin receptor mRNA is specific to adult bovine liver. Other adult and fetal bovine tissues expressed 1B somatotropin receptor mRNA with a predicted protein sequence that was similar to the 1A somatotropin receptor

Prolactin receptor, growth hormone receptor, and putative somatolactin receptor in Mozambique tilapia: tissue specific expression and differential regulation by salinity and fasting.

Science.gov (United States)

Pierce, A L; Fox, B K; Davis, L K; Visitacion, N; Kitahashi, T; Hirano, T; Grau, E G

2007-01-01

In fish, pituitary growth hormone family peptide hormones (growth hormone, GH; prolactin, PRL; somatolactin, SL) regulate essential physiological functions including osmoregulation, growth, and metabolism. Teleost GH family hormones have both differential and overlapping effects, which are mediated by plasma membrane receptors. A PRL receptor (PRLR) and two putative GH receptors (GHR1 and GHR2) have been identified in several teleost species. Recent phylogenetic analyses and binding studies suggest that GHR1 is a receptor for SL. However, no studies have compared the tissue distribution and physiological regulation of all three receptors. We sequenced GHR2 from the liver of the Mozambique tilapia (Oreochromis mossambicus), developed quantitative real-time PCR assays for the three receptors, and assessed their tissue distribution and regulation by salinity and fasting. PRLR was highly expressed in the gill, kidney, and intestine, consistent with the osmoregulatory functions of PRL. PRLR expression was very low in the liver. GHR2 was most highly expressed in the muscle, followed by heart, testis, and liver, consistent with this being a GH receptor with functions in growth and metabolism. GHR1 was most highly expressed in fat, liver, and muscle, suggesting a metabolic function. GHR1 expression was also high in skin, consistent with a function of SL in chromatophore regulation. These findings support the hypothesis that GHR1 is a receptor for SL. In a comparison of freshwater (FW)- and seawater (SW)-adapted tilapia, plasma PRL was strongly elevated in FW, whereas plasma GH was slightly elevated in SW. PRLR expression was reduced in the gill in SW, consistent with PRL's function in freshwater adaptation. GHR2 was elevated in the kidney in FW, and correlated negatively with plasma GH, whereas GHR1 was elevated in the gill in SW. Plasma IGF-I, but not GH, was reduced by 4 weeks of fasting. Transcript levels of GHR1 and GHR2 were elevated by fasting in the muscle. However

Proceedings of the International Symposium on Subtypes of Muscarinic Receptors (5th), Held in Newport Beach, California, October 22-24, 1992.

Science.gov (United States)

1993-02-22

AHMED , Chest 96 1285-1291 (1991). 29. P.J. BARNES, Thorax 44 161-167 (1989). 30. P.W. IND, C.M.S. DIXON, R.W. FULLER and P.J. BARNES, Am Rev Respir...mechanism by which muscarinic agonists, such as pilocarpiný_ lower the intraocular pressure of glaucoma patients. Pharmacological studies with subtype

Role of a Ubiquitously Expressed Receptor in the Vertebrate Olfactory System

Science.gov (United States)

DeMaria, Shannon; Berke, Allison P.; Van Name, Eric; Heravian, Anisa; Ferreira, Todd

2013-01-01

Odorant cues are recognized by receptors expressed on olfactory sensory neurons, the primary sensory neurons of the olfactory epithelium. Odorant receptors typically obey the “one receptor, one neuron” rule, in which the receptive field of the olfactory neuron is determined by the singular odorant receptor that it expresses. Odor-evoked receptor activity across the population of olfactory neurons is then interpreted by the brain to identify the molecular nature of the odorant stimulus. In the present study, we characterized the properties of a C family G-protein-coupled receptor that, unlike most other odorant receptors, is expressed in a large population of microvillous sensory neurons in the zebrafish olfactory epithelium and the mouse vomeronasal organ. We found that this receptor, OlfCc1 in zebrafish and its murine ortholog Vmn2r1, is a calcium-dependent, low-sensitivity receptor specific for the hydrophobic amino acids isoleucine, leucine, and valine. Loss-of-function experiments in zebrafish embryos demonstrate that OlfCc1 is required for olfactory responses to a diverse mixture of polar, nonpolar, acidic, and basic amino acids. OlfCc1 was also found to promote localization of other OlfC receptor family members to the plasma membrane in heterologous cells. Together, these results suggest that the broadly expressed OlfCc1 is required for amino acid detection by the olfactory system and suggest that it plays a role in the function and/or intracellular trafficking of other olfactory and vomeronasal receptors with which it is coexpressed. PMID:24048853

Muscarinic excitation of parvalbumin-positive interneurons contributes to the severity of pilocarpine-induced seizures

Science.gov (United States)

Yi, Feng; DeCan, Evan; Stoll, Kurt; Marceau, Eric; Deisseroth, Karl; Lawrence, J. Josh

2014-01-01

SUMMARY Objective A common rodent model in epilepsy research employs the muscarinic acetylcholine receptor (mAChR) agonist pilocarpine, yet the mechanisms underlying the induction of pilocarpine-induced seizures (PISs) remain unclear. Global M1 mAChR (M1R) knockout mice are resistant to PISs, implying that M1R activation disrupts excitation/inhibition balance. Parvalbumin-positive (PV) inhibitory neurons express M1 mAChRs, participate in cholinergically-induced oscillations, and can enter a state of depolarization block (DB) during epileptiform activity. Here, we test the hypothesis that pilocarpine activation of M1Rs expressed on PV cells contributes to PISs. Methods CA1 PV cells in PV-CRE mice were visualized with a floxed YFP or hM3Dq-mCherry adeno-associated virus, or by crossing PV-CRE mice with the RosaYFP reporter line. To eliminate M1Rs from PV cells, we generated PV-M1KO mice by crossing PV-CRE and floxed M1 mice. Action potential (AP) frequency was monitored during application of pilocarpine (200 µM). In behavioral experiments, locomotion and seizure symptoms were recorded in WT or PV-M1KO mice during PISs. Results Pilocarpine significantly increased AP frequency in CA1 PV cells into the gamma range. In the continued presence of pilocarpine, a subset (5/7) of PV cells progressed to DB, which was mimicked by hM3Dq activation of Gq-receptor signaling. Pilocarpine-induced depolarization, AP firing at gamma frequency, and progression to DB were prevented in CA1 PV cells of PV-M1KO mice. Finally, compared to WT mice, PV-M1KO mice were associated with reduced severity of PISs. Significance Pilocarpine can directly depolarize PV+ cells via M1R activation but a subset of these cells progress to DB. Our electrophysiological and behavioral results suggest that this mechanism is active during PISs, contributing to a collapse of PV-mediated GABAergic inhibition, dysregulation of excitation/inhibition balance, and increased susceptibility to PISs. PMID:25495999

Quantitative densitometry of neurotransmitter receptors

International Nuclear Information System (INIS)

Rainbow, T.C.; Bleisch, W.V.; Biegon, A.; McEwen, B.S.

1982-01-01

An autoradiographic procedure is described that allows the quantitative measurement of neurotransmitter receptors by optical density readings. Frozen brain sections are labeled in vitro with [ 3 H]ligands under conditions that maximize specific binding to neurotransmitter receptors. The labeled sections are then placed against the 3 H-sensitive LKB Ultrofilm to produce the autoradiograms. These autoradiograms resemble those produced by [ 14 C]deoxyglucose autoradiography and are suitable for quantitative analysis with a densitometer. Muscarinic cholinergic receptors in rat and zebra finch brain and 5-HT receptors in rat brain were visualized by this method. When the proper combination of ligand concentration and exposure time are used, the method provides quantitative information about the amount and affinity of neurotransmitter receptors in brain sections. This was established by comparisons of densitometric readings with parallel measurements made by scintillation counting of sections. (Auth.)

Basic and modern concepts on cholinergic receptor: A review

Directory of Open Access Journals (Sweden)

Prashant Tiwari

2013-10-01

Full Text Available Cholinergic system is an important system and a branch of the autonomic nervous system which plays an important role in memory, digestion, control of heart beat, blood pressure, movement and many other functions. This article serves as both structural and functional sources of information regarding cholinergic receptors and provides a detailed understanding of the determinants governing specificity of muscarinic and nicotinic receptor to researchers. The study helps to give overall information about the fundamentals of the cholinergic system, its receptors and ongoing research in this field.

Unique expression pattern of the three insulin receptor family members in the rat mammary gland

DEFF Research Database (Denmark)

Hvid, Henning; Klopfleisch, Robert; Vienberg, Sara Gry

2011-01-01

mammary gland. Using laser micro-dissection, quantitative RT-PCR and immunohistochemistry, we examined the expression of IR (insulin receptor), IGF-1R (IGF-1 receptor), IRR (insulin receptor-related receptor), ERα (estrogen receptor alpha), ERβ (estrogen receptor beta) and PR (progesteron receptor......) in young, virgin, female Sprague-Dawley rats and compared to expression in reference organs. The mammary gland displayed the highest expression of IRR and IGF-1R. In contrast, low expression of IR transcripts was observed in the mammary gland tissue with expression of the IR-A isoform being 5-fold higher...... than the expression of the IR-B. By immunohistochemistry, expression of IR and IGF-1R was detected in all mammary gland epithelial cells. Expression of ERα and PR was comparable between mammary gland and ovary, whereas expression of ERβ was lower in mammary gland than in the ovary. Finally, expression...

Nested Expression Domains for Odorant Receptors in Zebrafish Olfactory Epithelium

Science.gov (United States)

Weth, Franco; Nadler, Walter; Korsching, Sigrun

1996-11-01

The mapping of high-dimensional olfactory stimuli onto the two-dimensional surface of the nasal sensory epithelium constitutes the first step in the neuronal encoding of olfactory input. We have used zebrafish as a model system to analyze the spatial distribution of odorant receptor molecules in the olfactory epithelium by quantitative in situ hybridization. To this end, we have cloned 10 very divergent zebrafish odorant receptor molecules by PCR. Individual genes are expressed in sparse olfactory receptor neurons. Analysis of the position of labeled cells in a simplified coordinate system revealed three concentric, albeit overlapping, expression domains for the four odorant receptors analyzed in detail. Such regionalized expression should result in a corresponding segregation of functional response properties. This might represent the first step of spatial encoding of olfactory input or be essential for the development of the olfactory system.

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

Directory of Open Access Journals (Sweden)

Nan Hua

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

Expression of estrogen and progesterone receptors in astrocytomas: a literature review

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Cléciton Braga Tavares

Full Text Available Gliomas are the most common type of primary central nervous system neoplasm. Astrocytomas are the most prevalent type of glioma and these tumors may be influenced by sex steroid hormones. A literature review for the presence of estrogen and progesterone receptors in astrocytomas was conducted in the PubMed database using the following MeSH terms: “estrogen receptor beta” OR “estrogen receptor alpha” OR “estrogen receptor antagonists” OR “progesterone receptors” OR “astrocytoma” OR “glioma” OR “glioblastoma”. Among the 111 articles identified, 13 studies met our inclusion criteria. The majority of reports showed the presence of estrogen and progesterone receptors in astrocytomas. Overall, higher tumor grades were associated with decreased estrogen receptor expression and increased progesterone receptor expression.

Expression of estrogen and progesterone receptors in astrocytomas: a literature review

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Tavares, Cléciton Braga; Gomes-Braga, Francisca das Chagas Sheyla Almeida; Costa-Silva, Danylo Rafhael; Escórcio-Dourado, Carla Solange; Borges, Umbelina Soares; Conde, Airton Mendes; da Conceição Barros-Oliveira, Maria; Sousa, Emerson Brandão; da Rocha Barros, Lorena; Martins, Luana Mota; Facina, Gil; da-Silva, Benedito Borges

2016-01-01

Gliomas are the most common type of primary central nervous system neoplasm. Astrocytomas are the most prevalent type of glioma and these tumors may be influenced by sex steroid hormones. A literature review for the presence of estrogen and progesterone receptors in astrocytomas was conducted in the PubMed database using the following MeSH terms: “estrogen receptor beta” OR “estrogen receptor alpha” OR “estrogen receptor antagonists” OR “progesterone receptors” OR “astrocytoma” OR “glioma” OR “glioblastoma”. Among the 111 articles identified, 13 studies met our inclusion criteria. The majority of reports showed the presence of estrogen and progesterone receptors in astrocytomas. Overall, higher tumor grades were associated with decreased estrogen receptor expression and increased progesterone receptor expression. PMID:27626480

Decreased α1-adrenergic receptor-mediated inositide hydrolysis in neurons from hypertensive rat brain

International Nuclear Information System (INIS)

Feldstein, J.B.; Gonzales, R.A.; Baker, S.P.; Sumners, C.; Crews, F.T.; Raizada, M.K.

1986-01-01

The expression of α 1 -adrenergic receptors and norepinephrine (NE)-stimulated hydrolysis of inositol phospholipid has been studied in neuronal cultures from the brains of normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive (SH) rats. Binding of 125 I-1-[β-(4-hydroxyphenyl)-ethyl-aminomethyl] tetralone (HEAT) to neuronal membranes was 68-85% specific and was rapid. Competition-inhibition experiments with various agonists and antagonists suggested that 125 I-HEAT bound selectively to α 1 -adrenergic receptors. Specific binding of 125 I-HEAT to neuronal membranes from SH rat brain cultures was 30-45% higher compared with binding in WKY normotensive controls. This increase was attributed to an increase in the number of α 1 -adrenergic receptors on SH rat brain neurons. Incubation of neuronal cultures of rat brain from both strains with NE resulted in a concentration-dependent stimulation of release of inositol phosphates, although neurons from SH rat brains were 40% less responsive compared with WKY controls. The decrease in responsiveness of SH rat brain neurons to NE, even though the α 1 -adrenergic receptors are increased, does not appear to be due to a general defect in membrane receptors and postreceptor signal transduction mechanisms. This is because neither the number of muscarinic-cholinergic receptors nor the carbachol-stimulated release of inositol phosphates is different in neuronal cultures from the brains of SH rats compared with neuronal cultures from the brains of WKY rats. These observations suggest that the increased expression of α 1 -adrenergic receptors does not parallel the receptor-mediated inositol phosphate hydrolysis in neuronal cultures from SH rat brain

IL-21 Receptor Expression in Human Tendinopathy

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Abigail L. Campbell

2014-01-01

Full Text Available The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy.

Differential adipokine receptor expression on circulating leukocyte subsets in lean and obese children.

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Genoveva Keustermans

Full Text Available Childhood obesity prevalence has increased worldwide and is an important risk factor for type 2 diabetes (T2D and cardiovascular disease (CVD. The production of inflammatory adipokines by obese adipose tissue contributes to the development of T2D and CVD. While levels of circulating adipokines such as adiponectin and leptin have been established in obese children and adults, the expression of adiponectin and leptin receptors on circulating immune cells can modulate adipokine signalling, but has not been studied so far. Here, we aim to establish the expression of adiponectin and leptin receptors on circulating immune cells in obese children pre and post-lifestyle intervention compared to normal weight control children.13 obese children before and after a 1-year lifestyle intervention were compared with an age and sex-matched normal weight control group of 15 children. Next to routine clinical and biochemical parameters, circulating adipokines were measured, and flow cytometric analysis of adiponectin receptor 1 and 2 (AdipoR1, AdipoR2 and leptin receptor expression on peripheral blood mononuclear cell subsets was performed.Obese children exhibited typical clinical and biochemical characteristics compared to controls, including a higher BMI-SD, blood pressure and circulating leptin levels, combined with a lower insulin sensitivity index (QUICKI. The 1-year lifestyle intervention resulted in stabilization of their BMI-SD. Overall, circulating leukocyte subsets showed distinct adipokine receptor expression profiles. While monocytes expressed high levels of all adipokine receptors, NK and iNKT cells predominantly expressed AdipoR2, and B-lymphocytes and CD4+ and CD8+ T-lymphocyte subsets expressed AdipoR2 as well as leptin receptor. Strikingly though, leukocyte subset numbers and adipokine receptor expression profiles were largely similar in obese children and controls. Obese children showed higher naïve B-cell numbers, and pre-intervention also

Evaluation of stereoisomers of 4-fluoroalkyl analogues of 3-quinuclidinyl benzilate in in vivo competition studies for the M1, M2, and M3 muscarinic receptor subtypes in brain

International Nuclear Information System (INIS)

Kiesewetter, Dale O.; Eckelman, William C.; Jaetae, Lee; Paik, Chang H.; Park, Seok G.

1995-01-01

To develop a subtype selective muscarinic acetylcholine receptor (mAChR) antagonist for PET, fluorine-19 labeled alkyl analogues of quinuclidinyl benzilate (QNB) were synthesized by stereoselective reactions. To investigate these analogues for tissue subtype specificity, in vivo competitive binding studies were performed in rat brain using (R)-3-quinuclidinyl (R)-4-[ 125 I]Iodobenzilate (IQNB). Five, fifty, or five-hundred nmol of the non-radioactive ligands were coinjected intravenously with 8 pmol of the radioligand. Cold (R,R)-IQNB blocked (R,R)-[ 125 I]IQNB in a dose-dependent manner, without showing regional specificity. For the (R,S)-fluoromethyl, -fluoroethyl, and -fluoropropyl derivatives, a higher percent blockade was seen at 5 and 50 nmol levels in M2 predominant tissues (medulla, pons, and cerebellum) than in M1 predominant tissues (cortex, striatum and hippocampus). The blockade pattern of the radioligand also correlated qualitatively with the percentage of M2 receptors in the region. The S-quinuclidinyl analogues showed M2 selectivity but less efficient blockade of the radioligand, indicating lower affinities. Radioligand bound to the medulla was inversely correlated to the M2 relative binding affinity of the fluoroalkyl analogues. These results indicate that the nonradioactive ligand blocks the radioligand based on the affinity of the nonradioactive ligand for a particular receptor subtype compared to the affinity of the radioligand for the same receptor subtype. Of the seven compounds evaluated, (R,S)-fluoromethyl-QNB appears to show the most selectivity for the M2 subtypes in competition studies in vivo

Cloning and expression of a widely expressed receptor tyrosine phosphatase

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Sap, J; D'Eustachio, P; Givol, D

1990-01-01

We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

Effects of acute administration of nicotinic and muscarinic cholinergic agonists and antagonists on performance in different cost-benefit decision making tasks in rats.

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Mendez, Ian A; Gilbert, Ryan J; Bizon, Jennifer L; Setlow, Barry

2012-12-01

Alterations in cost-benefit decision making accompany numerous neuropsychiatric conditions, including schizophrenia, attention deficit hyperactivity disorder, and addiction. Central cholinergic systems have been linked to the etiology and/or treatment of many of these conditions, but little is known about the role of cholinergic signaling in cost-benefit decision making. The goal of these experiments was to determine how cholinergic signaling is involved in cost-benefit decision making, using a behavioral pharmacological approach. Male Long-Evans rats were trained in either "probability discounting" or "delay discounting" tasks, in which rats made discrete-trial choices between a small food reward and a large food reward associated with either varying probabilities of omission or varying delays to delivery, respectively. The effects of acute administration of different doses of nicotinic and muscarinic acetylcholine receptor agonists and antagonists were assessed in each task. In the probability discounting task, acute nicotine administration (1.0 mg/kg) significantly increased choice of the large risky reward, and control experiments suggested that this was due to robust nicotine-induced impairments in behavioral flexibility. In the delay discounting task, the muscarinic antagonists scopolamine (0.03, 0.1, and 0.3 mg/kg) and atropine (0.3 mg/kg) both significantly increased choice of the small immediate reward. Neither mecamylamine nor oxotremorine produced reliable effects on either of the decision making tasks. These data suggest that cholinergic receptors play multiple roles in decision making contexts which include consideration of reward delay or probability. These roles should be considered when targeting these receptors for therapeutic purposes.

Notch receptor expression in neurogenic regions of the adult zebrafish brain.

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Vanessa de Oliveira-Carlos

Full Text Available The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90 [Formula: see text] of proliferating radial glia express notch1a, notch1b and notch3. In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA [Formula: see text] cells. In this region notch3 expression is mostly in Bergmann glia and at low levels in few PCNA [Formula: see text] cells. Additionally, we found that in the proliferation zone of the ventral telencephalon, Notch receptors display an apical high to basal low gradient of expression. Notch receptors are also expressed in subpopulations of oligodendrocytes, neurons and endothelial cells. We suggest that the partial regional heterogeneity observed for Notch expression in progenitor cells might be related to the cellular diversity present in each of these neurogenic niches.

Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken.

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Cooley, James R; Yatskievych, Tatiana A; Antin, Parker B

2014-03-01

Transforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published. Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis. Copyright © 2013 Wiley Periodicals, Inc.

Expression of steroid receptors in ameloblasts during amelogenesis in rat incisors

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Sophia Houari

2016-11-01

Full Text Available Endocrine disrupting chemicals (EDCs play a part in the modern burst of diseases and interfere with the steroid hormone axis. Bisphenol A (BPA, one of the most active and widely used EDCs, affects ameloblast functions, leading to an enamel hypomineralization pattern similar to that of Molar Incisor Hypomineralization (MIH. In order to explore the molecular pathways stimulated by BPA during amelogenesis, we thoroughly investigated the receptors known to directly or indirectly mediate the effects of BPA. The expression patterns of high affinity BPA receptors (ERRγ, GPR30, of ketosteroid receptors (ERs, AR, PGR, GR, MR, of the retinoid receptor RXRα and PPARγ were established using RT-qPCR analysis of RNAs extracted from microdissected enamel organ of adult rats. Their expression was dependent on the stage of ameloblast differentiation, except that of ERβ and PPARγ which remained undetectable. An additional large scale microarray analysis revealed three main groups of receptors according to their level of expression in maturation stage ameloblasts. The expression level of RXRα was the highest, similar to the vitamin D receptor (VDR, whereas the others were 13 to 612 fold lower, with AR and GR being intermediate. Immunofluorescent analysis of VDR, ERα and AR confirmed their presence mainly in maturation- stage ameloblasts. These data provide further evidence that ameloblasts express a specific combination of hormonal receptors depending on their developmental stage. This study represents the first step towards understanding dental endocrinology as well as some of the effects of EDCs on the pathophysiology of amelogenesis.

Expression of Steroid Receptors in Ameloblasts during Amelogenesis in Rat Incisors.

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Houari, Sophia; Loiodice, Sophia; Jedeon, Katia; Berdal, Ariane; Babajko, Sylvie

2016-01-01

Endocrine disrupting chemicals (EDCs) play a part in the modern burst of diseases and interfere with the steroid hormone axis. Bisphenol A (BPA), one of the most active and widely used EDCs, affects ameloblast functions, leading to an enamel hypomineralization pattern similar to that of Molar Incisor Hypomineralization (MIH). In order to explore the molecular pathways stimulated by BPA during amelogenesis, we thoroughly investigated the receptors known to directly or indirectly mediate the effects of BPA. The expression patterns of high affinity BPA receptors (ERRγ, GPR30), of ketosteroid receptors (ERs, AR, PGR, GR, MR), of the retinoid receptor RXRα, and PPARγ were established using RT-qPCR analysis of RNAs extracted from microdissected enamel organ of adult rats. Their expression was dependent on the stage of ameloblast differentiation, except that of ERβ and PPARγ which remained undetectable. An additional large scale microarray analysis revealed three main groups of receptors according to their level of expression in maturation-stage ameloblasts. The expression level of RXRα was the highest, similar to the vitamin D receptor (VDR), whereas the others were 13 to 612-fold lower, with AR and GR being intermediate. Immunofluorescent analysis of VDR, ERα and AR confirmed their presence mainly in maturation- stage ameloblasts. These data provide further evidence that ameloblasts express a specific combination of hormonal receptors depending on their developmental stage. This study represents the first step toward understanding dental endocrinology as well as some of the effects of EDCs on the pathophysiology of amelogenesis.

Chemokine receptor expression by inflammatory T cells in EAE

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Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

2014-01-01

Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20...... receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed...... whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays...

Serotonin 5-HT4 receptors and forebrain cholinergic system: receptor expression in identified cell populations.

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Peñas-Cazorla, Raúl; Vilaró, M Teresa

2015-11-01

Activation of serotonin 5-HT4 receptors has pro-cognitive effects on memory performance. The proposed underlying neurochemical mechanism is the enhancement of acetylcholine release in frontal cortex and hippocampus elicited by 5-HT4 agonists. Although 5-HT4 receptors are present in brain areas related to cognition, e.g., hippocampus and cortex, the cellular localization of the receptors that might modulate acetylcholine release is unknown at present. We have analyzed, using dual label in situ hybridization, the cellular localization of 5-HT4 receptor mRNA in identified neuronal populations of the rat basal forebrain, which is the source of the cholinergic innervation to cortex and hippocampus. 5-HT4 receptor mRNA was visualized with isotopically labeled oligonucleotide probes, whereas cholinergic, glutamatergic, GABAergic and parvalbumin-synthesizing neurons were identified with digoxigenin-labeled oligonucleotide probes. 5-HT4 receptor mRNA was not detected in the basal forebrain cholinergic cell population. In contrast, basal forebrain GABAergic, parvalbumin synthesizing, and glutamatergic cells contained 5-HT4 receptor mRNA. Hippocampal and cortical glutamatergic neurons also express this receptor. These results indicate that 5-HT4 receptors are not synthesized by cholinergic cells, and thus would be absent from cholinergic terminals. In contrast, several non-cholinergic cell populations within the basal forebrain and its target hippocampal and cortical areas express these receptors and are thus likely to mediate the enhancement of acetylcholine release elicited by 5-HT4 agonists.

Expression and purification of functional human mu opioid receptor from E.coli.

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Yanbin Ma

Full Text Available N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3-0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a K(D of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.

M3 receptor is involved in the effect of penehyclidine hydrochloride reduced endothelial injury in LPS-stimulated human pulmonary microvascular endothelial cell.

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Yuan, Qinghong; Xiao, Fei; Liu, Qiangsheng; Zheng, Fei; Shen, Shiwen; He, Qianwen; Chen, Kai; Wang, Yanlin; Zhang, Zongze; Zhan, Jia

2018-02-01

LPS has been recently shown to induce muscarinic acetylcholine 3 receptor (M 3 receptor) expression and penehyclidine hydrochloride (PHC) is an anticholinergic drug which could block the expression of M 3 receptor. PHC has been demonstrated to perform protective effect on cell injury. This study is to investigate whether the effect of PHC on microvascular endothelial injury is related to its inhibition of M 3 receptor or not. HPMVECs were treated with specific M 3 receptor shRNA or PBS, and randomly divided into LPS group (A group), LPS+PHC group (B group), LPS + M 3 shRNA group (C group) and LPS + PHC + M 3 shRNA group (D group). Cells were collected at 60 min after LPS treatment to measure levels of LDH, endothelial permeability, TNF-α and IL-6 levels, NF-κB p65 activation, I-κB protein expression, p38MAPK, and ERK1/2 activations as well as M 3 mRNA expression. PHC could decrease LDH levels, cell permeability, TNF-α and IL-6 levels, p38 MAPK, ERK1/2, NF-κB p65 activations and M 3 mRNA expressions compared with LPS group. When M 3 receptor was silence, the changes of these indices were much more obvious. These findings suggest that M 3 receptor plays an important role in LPS-induced pulmonary microvascular endothelial injury, which is regulated through NF-κB p65 and MAPK activation. And knockout of M 3 receptor could attenuate LPS-induced pulmonary microvascular endothelial injury. Regulative effects of PHC on pulmonary microvascular permeability and NF-κB p65 as well as MAPK activations are including but not limited to inhibition of M 3 receptor. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparison of the pharmacokinetics of different analogs of 11C-labeled TZTP for imaging muscarinic M2 receptors with PET

International Nuclear Information System (INIS)

Reid, Alicia E.; Ding Yushin; Eckelman, William C.; Logan, Jean; Alexoff, David; Shea, Colleen; Xu Youwen; Fowler, Joanna S.

2008-01-01

Introduction: The only radiotracer available for the selective imaging of muscarinic M2 receptors in vivo is 3-(3-{3-[ 18 F]fluoropropyl)thio}-1,2,5-thiadiazol-4-yl) -1,2,5,6-tetrahydro-1-methylpyridine) ([ 18 F]FP-TZTP). We have prepared and labeled 3-(3-(3-fluoropropylthio)-1,2,5-thiadiazol-4-yl) -1,2,5,6-tetrahydro-1-methylpyridne (FP-TZTP, 3) and two other TZTP derivatives with 11 C at the methylpyridine moiety to explore the potential of using 11 C-labeled FP-TZTP for positron emission tomography imaging of M2 receptors and to compare the effect of small structural changes on tracer pharmacokinetics (PK) in brain and peripheral organs. Methods: 11 C-radiolabeled FP-TZTP, 3-(3-propylthio)-TZTP (6) and 3,3,3-(3-(3-trifluoropropyl)-TZTP (10) were prepared, and log D, plasma protein binding (PPB), affinity constants, time-activity curves (TACs), area under the curve (AUC) for arterial plasma, distribution volumes (DV) and pharmacological blockade in baboons were compared. Results: Values for log D, PPB and affinity constants were similar for 3, 6 and 10. The fraction of parent radiotracer in the plasma was higher and the AUC lower for 10 than for 3 and 6. TACs for brain regions were similar for 3 and 6, which showed PK similar to the 18 F tracer, while 10 showed slower uptake and little clearance over 90 min. DVs for 3 and 6 were similar to the 18 F tracer but higher for 10. Uptake of the three tracers was significantly reduced by coinjection of unlabeled 3 and 6. Conclusion: Small structural variations on the TZTP structure greatly altered the PK in brain and behavior in blood with little change in the log D, PPB or affinity. The study suggests that 11 C-radiolabeled 3 will be a suitable alternative to [ 18 F]FP-TZTP for translational studies in humans

Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue

International Nuclear Information System (INIS)

Smith, Robert A; Lea, Rod A; Curran, Joanne E; Weinstein, Stephen R; Griffiths, Lyn R

2003-01-01

Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue

Advantage of the Highly Restricted Odorant Receptor Expression Pattern in Chemosensory Neurons of Drosophila.

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Tharadra, Sana Khalid; Medina, Adriana; Ray, Anandasankar

2013-01-01

A fundamental molecular feature of olfactory systems is that individual neurons express only one receptor from a large odorant receptor gene family. While numerous theories have been proposed, the functional significance and evolutionary advantage of generating a sophisticated one-receptor-per neuron expression pattern is not well understood. Using the genetically tractable Drosophila melanogaster as a model, we demonstrate that the breakdown of this highly restricted expression pattern of an odorant receptor in neurons leads to a deficit in the ability to exploit new food sources. We show that animals with ectopic co-expression of odorant receptors also have a competitive disadvantage in a complex environment with limiting food sources. At the level of the olfactory system, we find changes in both the behavioral and electrophysiological responses to odorants that are detected by endogenous receptors when an olfactory receptor is broadly misexpressed in chemosensory neurons. Taken together these results indicate that restrictive expression patterns and segregation of odorant receptors to individual neuron classes are important for sensitive odor-detection and appropriate olfactory behaviors.

Nuclear hormone receptor expression in mouse kidney and renal cell lines.

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Daisuke Ogawa

Full Text Available Nuclear hormone receptors (NHRs are transcription factors that regulate carbohydrate and lipid metabolism, immune responses, and inflammation. Although several NHRs, including peroxisome proliferator-activated receptor-γ (PPARγ and PPARα, demonstrate a renoprotective effect in the context of diabetic nephropathy (DN, the expression and role of other NHRs in the kidney are still unrecognized. To investigate potential roles of NHRs in the biology of the kidney, we used quantitative real-time polymerase chain reaction to profile the expression of all 49 members of the mouse NHR superfamily in mouse kidney tissue (C57BL/6 and db/m, and cell lines of mesangial (MES13, podocyte (MPC, proximal tubular epithelial (mProx24 and collecting duct (mIMCD3 origins in both normal and high-glucose conditions. In C57BL/6 mouse kidney cells, hepatocyte nuclear factor 4α, chicken ovalbumin upstream promoter transcription factor II (COUP-TFII and COUP-TFIII were highly expressed. During hyperglycemia, the expression of the NHR 4A subgroup including neuron-derived clone 77 (Nur77, nuclear receptor-related factor 1, and neuron-derived orphan receptor 1 significantly increased in diabetic C57BL/6 and db/db mice. In renal cell lines, PPARδ was highly expressed in mesangial and proximal tubular epithelial cells, while COUP-TFs were highly expressed in podocytes, proximal tubular epithelial cells, and collecting duct cells. High-glucose conditions increased the expression of Nur77 in mesangial and collecting duct cells, and liver x receptor α in podocytes. These data demonstrate NHR expression in mouse kidney cells and cultured renal cell lines and suggest potential therapeutic targets in the kidney for the treatment of DN.

Expression of Estrogen Receptor Alpha in Malignant Melanoma

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Parvin Rajabi

2017-01-01

Full Text Available Background: Features of malignant melanoma (MM vary in the different geographic regions of the world. This may be attributable to environmental, ethnic, and genetic factors. The aim of this study was to determine the expression of estrogen receptor alpha (ER-α in MM in Isfahan, Iran. Materials and Methods: This study was planned as a descriptive, analytical, cross-sectional investigation. During this study, paraffin-embedded tissue blocks of patients with a histopathologic diagnosis of MM was studied for ER-α using immunohistochemistry (IHC. Results: In this study, 38 patients (female/male; 20/18 with a definite diagnosis of malignant cutaneous melanoma and mean age of 52.4 ± 11.2 years were investigated. Using envision IHC staining, there were not any cases with ER-α expression. Conclusion: In confirmation to the most previous studies, expression of ER-α was negative in MM. It is recommended to investigate the expression of estrogen receptor beta and other markers in MM.

Pharmacological receptors of nematoda as target points for action of antiparasitic drugs

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Trailović Saša M.

2010-01-01

Full Text Available Cholinergic receptors of parasitic nematodes are one of the most important possible sites of action of antiparasitic drugs. This paper presents some of our own results of electrophysiological and pharamcological examinations of nicotinic and muscarinic receptors of nematodes, as well as data from literature on a new class of anthelmintics that act precisely on cholinergic receptors. The nicotinic acetylcholine receptor (nAChR is located on somatic muscle cells of nematodes and it is responsible for the coordination of parasite movement. Cholinomimetic anthelmintics act on this receptor, as well as acetylcholine, an endogenic neurotransmitter, but they are not sensitive to enzyme acetylcholineesterase which dissolves acetylcholine. As opposed to the nicotinic receptor of vertebra, whose structure has been examined thoroughly, the stoichiometry of the nicotinic receptor of nematodes is not completely known. However, on the grounds of knowledge acquired so far, a model has been constructed recently of the potential composition of a type of nematodes nicotinic receptor, as the site of action of anthelmintics. Based on earlier investigations, it is supposed that a conventional muscarinic receptor exists in nematodes as well, so that it can also be a new pharamocological target for the development of antinematode drugs. The latest class of synthesized anthelmintics, named aminoacetonitriles (AAD, act via the nicotinic receptor. Monepantel is the first drug from the AAD group as a most significant candidate for registration in veterinary medicine. Even though several groups of cholinomimetic anthelmintics (imiodazothiazoles, tetrahydropyrimidines, organophosphat anthelmintics have been in use in veterinary practice for many years now, it is evident that cholinergic receptors of nematodes still present an attractive place in the examinations and development of new antinematode drugs. .

Expression of nicotinic acetylcholine receptors on human B-lymphoma cells

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Skok M. V.

2009-12-01

Full Text Available Aim. To find a correlation between the level of nicotinic acetylcholine receptor (nAChR expression and B lymphocyte differentiation or activation state. Methods. Expression of nAChRs in the REH, Ramos and Daudi cell lines was studied by flow cytometry using nAChR subunit-specific antibodies; cell proliferation was studied by MTT test. Results. It is shown that the level of 42/4 and 7 nAChRs expression increased along with B lymphocyte differentiation (Ramos > REH and activation (Daudi > > Ramos and depended on the antigen-specific receptor expression. The nAChR stimulation/blockade did not influence the intensity of cell proliferation.

Comparative functional expression of nAChR subtypes in rodent DRG neurons.

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Smith, Nathan J; Hone, Arik J; Memon, Tosifa; Bossi, Simon; Smith, Thomas E; McIntosh, J Michael; Olivera, Baldomero M; Teichert, Russell W

2013-01-01

We investigated the functional expression of nicotinic acetylcholine receptors (nAChRs) in heterogeneous populations of dissociated rat and mouse lumbar dorsal root ganglion (DRG) neurons by calcium imaging. By this experimental approach, it is possible to investigate the functional expression of multiple receptor and ion-channel subtypes across more than 100 neuronal and glial cells simultaneously. Based on nAChR expression, DRG neurons could be divided into four subclasses: (1) neurons that express predominantly α3β4 and α6β4 nAChRs; (2) neurons that express predominantly α7 nAChRs; (3) neurons that express a combination of α3β4/α6β4 and α7 nAChRs; and (4) neurons that do not express nAChRs. In this comparative study, the same four neuronal subclasses were observed in mouse and rat DRG. However, the expression frequency differed between species: substantially more rat DRG neurons were in the first three subclasses than mouse DRG neurons, at all developmental time points tested in our study. Approximately 70-80% of rat DRG neurons expressed functional nAChRs, in contrast to only ~15-30% of mouse DRG neurons. Our study also demonstrated functional coupling between nAChRs, voltage-gated calcium channels, and mitochondrial Ca(2) (+) transport in discrete subsets of DRG neurons. In contrast to the expression of nAChRs in DRG neurons, we demonstrated that a subset of non-neuronal DRG cells expressed muscarinic acetylcholine receptors and not nAChRs. The general approach to comparative cellular neurobiology outlined in this paper has the potential to better integrate molecular and systems neuroscience by uncovering the spectrum of neuronal subclasses present in a given cell population and the functionally integrated signaling components expressed in each subclass.

Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

Directory of Open Access Journals (Sweden)

Lili Jiang

Full Text Available Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2 receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

Evaluation and metabolite studies of {sup 125}I- and {sup 123}I-labelled E-(R,R)-IQNP: potential radioligands for visualization of M{sub 1} muscarinic acetylcholine receptors in brain

Energy Technology Data Exchange (ETDEWEB)

Bergstroem, Kim A.; Halldin, Christer; Hiltunen, Jukka; Swahn, Carl-Gunnar; Ito, Hiroshi; Ginovart, Nathalie; Hall, Haakan; McPherson, Daniel W.; Knapp, F. F. (Russ); Larsson, Stig; Schnell, Per-Olof; Farde, Lars

1998-04-01

A new ligand for the M{sub 1} muscarinic receptor subtype, E-(R,R)-1-azabicyclo[2.2.2]oct-3-yl {alpha}-hydroxy-{alpha}-(1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (E-IQNP), was labelled with {sup 125}I and {sup 123}I for autoradiographic studies on human whole-brain cryosections and SPET studies, respectively, in Cynomolgus monkey. Autoradiography demonstrated E-[{sup 125}I]IQNP binding in M{sub 1} receptor-rich regions such as the neocortex and the striatum. The binding was displaceable by the selective M{sub 1} antagonist biperiden. In vivo single photon emission tomography (SPET) studies with E-[{sup 123}I]IQNP demonstrated a high accumulation of radioactivity in the monkey neocortex. Rapid hydrolysis of the quinuclidinyl ester to the free acid was found to be a major biotransformation route for E-[{sup 123}I]IQNP. The free acid of E-[{sup 123}I]IQNP does not pass the blood-brain barrier, but the plasma concentration was high as compared to the total radioactivity in brain. It is thus necessary to correct for the high concentration of radioactive metabolites in parenchymal blood (CBV) to obtain accurate values for E-[{sup 123}I]IQNP binding in brain.

Labeled receptor ligands for spect

International Nuclear Information System (INIS)

Kung, H.F.

1989-01-01

Receptor specific imaging agents for single photon emission computed tomography (SPECT) can potentially be useful in the understanding of basic biochemistry and pharmacology of receptors. SPECT images may also provide tools for evaluation of density and binding kinetics of a specific receptor, information important for diagnosis and patient management. Basic requirements for receptor imaging agents are: (a) they are labeled with short-lived isotopes, (b) they show high selectivity and specific uptake, (c) they exhibit high target/background ratio, and (d) they can be modeled to obtain quantitative information. Several good examples of CNS receptor specific ligands labeled with I-123 have been developed, including iodoQNB, iodoestrogen iodobenzadiazepine, iodobenazepine, iodobenzamides for muscarinic, estrogen benzadiazepine, D-1 and D-2 dopamine receptors. With the advent of newer and faster SPECT imaging devices, it may be feasible to quantitate the receptor density by in vivo imaging techniques. These new brain imaging agents can provide unique diagnostic information, which may not be available through other imaging modalities, such as CT and MRI

Abnormal Sensory Protein Expression and Urothelial Dysfunction in Ketamine-Related Cystitis in Humans

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Yao Chou Tsai

2016-09-01

Full Text Available Purpose The aim of this study was to analyze patterns of sensory protein expression and urothelial dysfunction in ketamine-related cystitis (KC in humans. Methods Biopsies of bladder mucosa were performed in 29 KC patients during cystoscopy. Then specimens were analyzed for tryptase, zonula occludens-1 (ZO-1, E-cadherin, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL with immunofluorescence staining and quantification. In addition, 10 healthy control bladder specimens were analyzed and compared with the KC specimens. Another 16 whole bladder specimens obtained from partial cystectomy were also analyzed for the muscarinic receptors M2 and M3, endothelial nitric oxide synthase (eNOS, inducible nitric oxide synthase (iNOS, β-3 adrenergic receptors (β3-ARs, and the P2X3 receptor by western blotting. In addition, 3 normal control bladder specimens were analyzed and compared with the KC specimens. Results The KC bladder mucosa revealed significantly less expression of ZO-1 and E-cadherin, and greater expression of TUNEL and tryptase activity than the control samples. The expression of M3 and β3-AR in the KC specimens was significantly greater than in the controls. The expression of iNOS, eNOS, M2, and P2X3 was not significantly different between the KC and control specimens. Conclusions The bladder tissue of KC patients revealed significant urothelial dysfunction, which was associated with mast-cell mediated inflammation, increased urothelial cell apoptosis, and increased expression of the M3 and β3-AR.

Testosterone-Dependent Interaction between Androgen Receptor and Aryl Hydrocarbon Receptor Induces Liver Receptor Homolog 1 Expression in Rat Granulosa Cells

Science.gov (United States)

Wu, Yanguang; Baumgarten, Sarah C.; Zhou, Ping

2013-01-01

Androgens play a major role in the regulation of normal ovarian function; however, they are also involved in the development of ovarian pathologies. These contrasting effects may involve a differential response of granulosa cells to the androgens testosterone (T) and dihydrotestosterone (DHT). To determine the molecular pathways that mediate the distinct effects of T and DHT, we studied the expression of the liver receptor homolog 1 (LRH-1) gene, which is differentially regulated by these steroids. We found that although both T and DHT stimulate androgen receptor (AR) binding to the LRH-1 promoter, DHT prevents T-mediated stimulation of LRH-1 expression. T stimulated the expression of aryl hydrocarbon receptor (AHR) and its interaction with the AR. T also promoted the recruitment of the AR/AHR complex to the LRH-1 promoter. These effects were not mimicked by DHT. We also observed that the activation of extracellular regulated kinases by T is required for AR and AHR interaction. In summary, T, but not DHT, stimulates AHR expression and the interaction between AHR and AR, leading to the stimulation of LRH-1 expression. These findings could explain the distinct response of granulosa cells to T and DHT and provide a molecular mechanism by which DHT negatively affects ovarian function. PMID:23689136

Expansion of microsatellite in the thyroid hormone receptor-alpha1 gene linked to increased receptor expression and less aggressive thyroid cancer

DEFF Research Database (Denmark)

Onda, Masamitsu; Li, Daisy; Suzuki, Shinichi

2002-01-01

PURPOSE: The purpose of this study was to determine whether the length of the THRA1 microsatellite, which resides in a noncoding portion of the thyroid hormone receptor-alpha1 gene, affects receptor expression and is linked to clinicopathological parameters in thyroid cancer. EXPERIMENTAL DESIGN......: In 30 cases of surgically resected sporadic thyroid cancer, the length of the THRA1 microsatellite was determined by DNA sequence analysis, and expression of thyroid hormone receptor-alpha1 was assessed immunohistochemically in thin sections cut from tumor blocks. The length of THRA1 and expression...... of thyroid hormone receptor-alpha1 were also assessed in seven cancer cell lines. Regression analysis was used to gauge the correlation between the size of THRA1 and receptor expression. Multivariate analysis was used to test for links to the clinical parameters of gender, age, histology, stage, nodal...

Steroid receptor expression in the fish inner earvaries with sex, social status, and reproductive state

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Fernald Russell D

2010-04-01

Full Text Available Abstract Background Gonadal and stress-related steroid hormones are known to influence auditory function across vertebrates but the cellular and molecular mechanisms responsible for steroid-mediated auditory plasticity at the level of the inner ear remain unknown. The presence of steroid receptors in the ear suggests a direct pathway for hormones to act on the peripheral auditory system, but little is known about which receptors are expressed in the ear or whether their expression levels change with internal physiological state or external social cues. We used qRT-PCR to measure mRNA expression levels of multiple steroid receptor subtypes (estrogen receptors: ERα, ERβa, ERβb; androgen receptors: ARα, ARβ; corticosteroid receptors: GR2, GR1a/b, MR and aromatase in the main hearing organ of the inner ear (saccule in the highly social African cichlid fish Astatotilapia burtoni, and tested whether these receptor levels were correlated with circulating steroid concentrations. Results We show that multiple steroid receptor subtypes are expressed within the main hearing organ of a single vertebrate species, and that expression levels differ between the sexes. We also show that steroid receptor subtype-specific changes in mRNA expression are associated with reproductive phase in females and social status in males. Sex-steroid receptor mRNA levels were negatively correlated with circulating estradiol and androgens in both males and females, suggesting possible ligand down-regulation of receptors in the inner ear. In contrast, saccular changes in corticosteroid receptor mRNA levels were not related to serum cortisol levels. Circulating steroid levels and receptor subtype mRNA levels were not as tightly correlated in males as compared to females, suggesting different regulatory mechanisms between sexes. Conclusions This is the most comprehensive study of sex-, social-, and reproductive-related steroid receptor mRNA expression in the peripheral

Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

International Nuclear Information System (INIS)

Teodorov, E.; Ferrari, M.F.R.; Fior-Chadi, D.R.; Camarini, R.; Felício, L.F.

2012-01-01

The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

Energy Technology Data Exchange (ETDEWEB)

Teodorov, E. [Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Paulo, SP (Brazil); Ferrari, M.F.R. [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Fior-Chadi, D.R. [Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Camarini, R. [Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil); Felício, L.F. [Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP (Brazil)

2012-06-01

The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

Directory of Open Access Journals (Sweden)

E. Teodorov

2012-10-01

Full Text Available The periaqueductal gray (PAG has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc or 0.9% saline (up to 1 mL/kg and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05 because a lower percentage of kappa group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR. A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05 and lactating female rats (P < 0.01, with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in

Effects of acute administration of nicotinic and muscarinic cholinergic agonists and antagonists on performance in different cost–benefit decision making tasks in rats

Science.gov (United States)

Mendez, Ian A.; Gilbert, Ryan J.; Bizon, Jennifer L.

2012-01-01

Rationale Alterations in cost–benefit decision making accompany numerous neuropsychiatric conditions, including schizophrenia, attention deficit hyperactivity disorder, and addiction. Central cholinergic systems have been linked to the etiology and/or treatment of many of these conditions, but little is known about the role of cholinergic signaling in cost–benefit decision making. Objectives The goal of these experiments was to determine how cholinergic signaling is involved in cost–benefit decision making, using a behavioral pharmacological approach. Methods Male Long-Evans rats were trained in either “probability discounting” or “delay discounting” tasks, in which rats made discrete-trial choices between a small food reward and a large food reward associated with either varying probabilities of omission or varying delays to delivery, respectively. The effects of acute administration of different doses of nicotinic and muscarinic acetylcholine receptor agonists and antagonists were assessed in each task. Results In the probability discounting task, acute nicotine administration (1.0 mg/kg) significantly increased choice of the large risky reward, and control experiments suggested that this was due to robust nicotine-induced impairments in behavioral flexibility. In the delay discounting task, the muscarinic antagonists scopolamine (0.03, 0.1, and 0.3 mg/kg) and atropine (0.3 mg/kg) both significantly increased choice of the small immediate reward. Neither mecamylamine nor oxotremorine produced reliable effects on either of the decision making tasks. Conclusions These data suggest that cholinergic receptors play multiple roles in decision making contexts which include consideration of reward delay or probability. These roles should be considered when targeting these receptors for therapeutic purposes. PMID:22760484

Glucose transporters are expressed in taste receptor cells.

Science.gov (United States)

Merigo, Flavia; Benati, Donatella; Cristofoletti, Mirko; Osculati, Francesco; Sbarbati, Andrea

2011-08-01

In the intestine, changes of sugar concentration generated in the lumen during digestion induce adaptive responses of glucose transporters in the epithelium. A close matching between the intestinal expression of glucose transporters and the composition and amount of the diet has been provided by several experiments. Functional evidence has demonstrated that the regulation of glucose transporters into enterocytes is induced by the sensing of sugar of the enteroendocrine cells through activation of sweet taste receptors (T1R2 and T1R3) and their associated elements of G-protein-linked signaling pathways (e.g. α-gustducin, phospholipase C β type 2 and transient receptor potential channel M5), which are signaling molecules also involved in the perception of sweet substances in the taste receptor cells (TRCs) of the tongue. Considering this phenotypical similarity between the intestinal cells and TRCs, we evaluated whether the TRCs themselves possess proteins of the glucose transport mechanism. Therefore, we investigated the expression of the typical intestinal glucose transporters (i.e. GLUT2, GLUT5 and SGLT1) in rat circumvallate papillae, using immunohistochemistry, double-labeling immunofluorescence, immunoelectron microscopy and reverse transcriptase-polymerase chain reaction analysis. The results showed that GLUT2, GLUT5 and SGLT1 are expressed in TRCs; their immunoreactivity was also observed in cells that displayed staining for α-gustducin and T1R3 receptor. The immunoelectron microscopic results confirmed that GLUT2, GLUT5 and SGLT1 were predominantly expressed in cells with ultrastructural characteristics of chemoreceptor cells. The presence of glucose transporters in TRCs adds a further link between chemosensory information and cellular responses to sweet stimuli that may have important roles in glucose homeostasis, contributing to a better understanding of the pathways implicated in glucose metabolism. © 2011 The Authors. Journal of Anatomy © 2011

Development of gamma emitting receptor-binding radiotracers for imaging the brain and pancreas. Progress report, February 1983-September 1984

International Nuclear Information System (INIS)

Reba, R.C.

1984-01-01

The possibility of measuring the change in receptor concentration as a function of disease by external imaging was investigated. The structure-binding-relationship which provides optimal localization of radiolabelled antagonist of the muscarinic acetylcholine receptors in the brain was studied. These relationships were also studied with respect to localization in the pancreas

Cloning and expression of a rat brain α2B-adrenergic receptor

International Nuclear Information System (INIS)

Flordellis, C.S.; Handy, D.E.; Bresnahan, M.R.; Zannis, V.I.; Gavras, H.

1991-01-01

The authors isolated a cDNA clone (RBα 2B ) and its homologous gene (GRα 2B ) encoding an α 2B -adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (α 2 -C4) and divergent from the rat kidney nonglycosylated α 2B subtype (RNGα 2 ). Transient expression of RBα 2B in COS-7 cells resulted in high-affinity saturable binding for [ 3 H]rauwolscine and a high receptor number in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine > yohimbine > prazosin > oxymetazoline, with a prazosin-to-oxymetazoline K i ratio of 0.34. This profile is characteristic of the α 2B -adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major and two minor mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GRα 2B may be transcriptionally active. These findings show that rat brain expresses an α 2B -adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated α 2B subtype. Thus the rat expresses at least two divergent α 2B -adrenergic receptors

Expression of androgen and estrogen receptors in the testicular ...

African Journals Online (AJOL)

enoh

2012-04-10

Apr 10, 2012 ... 66: 1161-1168. Oliveira CA, Mahecha GA, Carnes K, Prins GS, Saunders PT, Franca. LR, Hess RA (2004). Differential hormonal regulation of estrogen receptors ERα and ER and androgen receptor expression in rat efferent ductules. Reproduction, 128(1): 73-86. O'Shaughnessy PJ, Johnston H, Willerton L ...

Spinal cord regeneration by modulating bone marrow with neurotransmitters and Citicholine: Analysis at micromolecular level.

Science.gov (United States)

Paulose, Cheramadathukudiyil Skaria; John, Ponnezhathu Sebastian; Chinthu, Romeo; Akhilraj, Puthenveetil Raju; Anju, Thoppil Raveendran

2017-04-01

Spinal cord injury results in disruption of brain-spinal cord fibre connectivity, leading to progressive tissue damage at the site of injury and resultant paralysis of varying degrees. The current study investigated the role of autologous bone marrow modulated with neurotransmitters and neurotransmitter stimulating agent, Citicholine, in spinal cord of spinal cord injured rats. Radioreceptor assay using [3H] ligand was carried out to quantify muscarinic receptor. Gene expression studies were done using Real Time PCR analysis. Scatchard analysis of muscarinic M1 receptor showed significantly decreased B max (p neurotransmitters combination along with bone marrow or Citicholine with bone marrow can reverse the muscarinic receptor alterations in the spinal cord of spinal cord injured rats, which is a promising step towards a better therapeutic intervention for spinal cord injury because of the positive role of cholinergic system in regulation of both locomotor activity and synaptic plasticity. Copyright © 2017 Chang Gung University. Published by Elsevier B.V. All rights reserved.

Postnatal Expression of V2 Vasopressin Receptor Splice Variants in the Rat Cerebellum

Science.gov (United States)

Vargas, Karina J.; Sarmiento, José M.; Ehrenfeld, Pamela; Añazco, Carolina C.; Villanueva, Carolina I.; Carmona, Pamela L.; Brenet, Marianne; Navarro, Javier; Müller-Esterl, Werner; Figueroa, Carlos D.; González, Carlos B.

2010-01-01

The V2 vasopressin receptor gene contains an alternative splice site in exon-3, which leads to the generation of two splice variants (V2a and V2b) first identified in the kidney. The open reading frame of the alternatively spliced V2b transcripten codes a truncated receptor, showing the same amino acid sequence as the canonical V2a receptor up to the 6th transmembrane segment, but displaying a distinct sequence to the corresponding 7th transmembrane segment and C-terminal domain relative to the V2a receptor. Here, we demonstrate the postnatal expression of V2a and V2b variants in the rat cerebellum. Most importantly, we showed by in situ hybridization and immunocytochemistry that both V2 splice variants were preferentially expressed in Purkinje cells, from early to late postnatal development. In addition, both variants were transiently expressed in the neuroblastic external granule cells and Bergmann fibers. These results indicate that the cellular distributions of both splice variants are developmentally regulated, and suggest that the transient expression of the V2 receptor is involved in the mechanisms of cerebellar cytodifferentiation by AVP. Finally, transfected CHO-K1 .expressing similar amounts of both V2 splice variants, as that found in the cerebellum, showed a significant reduction in the surface expression of V2a receptors, suggesting that the differential expression of the V2 splice variants regulate the vasopressin signaling in the cerebellum. PMID:19281786

Insulin decreases atherosclerosis by inducing endothelin receptor B expression

DEFF Research Database (Denmark)

Park, Kyoungmin; Mima, Akira; Li, Qian

2016-01-01

Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1...... induction of NO action, which increases endothelin receptor B (EDNRB) expression and intracellular [Ca(2+)]. Using the mice with knockin mutation of eNOS, which had Ser1176 mutated to alanine (AKI), deleting the only known mechanism for insulin to activate eNOS/NO pathway, we observed that IRS1...... overexpression in the endothelia of Aki/ApoE(-/-) mice significantly decreased atherosclerosis. Interestingly, endothelial EDNRB expression was selectively reduced in intima of arteries from diabetic patients and rodents. However, endothelial EDNRB expression was upregulated by insulin via P13K/Akt pathway...

Polarized expression of the GFP-tagged rat V(1a) vasopressin receptor.

Science.gov (United States)

Campos, D M; Reyes, C E; Sarmiento, J; Navarro, J; González, C B

2001-11-30

We investigated the targeting of the V(1a) receptor fused with the green fluorescence protein (V(1a)R-GFP) in polarized MDCK cells. Cells expressing V(1a)R-GFP displayed binding to vasopressin (AVP) and AVP-induced calcium responses, similar to cells expressing the wild-type V1a receptor. Interestingly, as with the wild-type V(1a)R, V(1a)R-GFP is preferentially distributed in the basolateral side of MDCK cells as monitored by confocal microscopy. Furthermore, AVP induced internalization of GFP-tagged receptors. Therefore, the GFP-tagged V(1a) receptor retains all the sorting signals of the wild-type receptor and offers an excellent system to elucidate the mechanisms of cell trafficking of V(1a) receptors.

Upregulation of neurokinin-1 receptor expression in the lungs of patients with sarcoidosis.

LENUS (Irish Health Repository)

O'Connor, Terence M

2012-02-03

Substance P (SP) is a proinflammatory neuropeptide that is secreted by sensory nerves and inflammatory cells. Increased levels of SP are found in sarcoid bronchoalveolar lavage fluid. SP acts by binding to the neurokinin-1 receptor and increases secretion of tumor necrosis factor-alpha in many cell types. We sought to determine neurokinin-1 receptor expression in patients with sarcoidosis compared with normal controls. Neurokinin-1 receptor messenger RNA and protein expression were below the limits of detection by reverse transcriptase-polymerase chain reaction and immunohistochemistry in peripheral blood mononuclear cells of healthy volunteers (n = 9) or patients with stage 1 or 2 pulmonary sarcoidosis (n = 10), but were detected in 1\\/9 bronchoalveolar lavage cells of controls compared with 8\\/10 patients with sarcoidosis (p = 0.012) and 2\\/9 biopsies of controls compared with 9\\/10 patients with sarcoidosis (p = 0.013). Immunohistochemistry localized upregulated neurokinin-1 receptor expression to bronchial and alveolar epithelial cells, macrophages, lymphocytes, and sarcoid granulomas. The patient in whom neurokinin-1 receptor was not detected was taking corticosteroids. Incubation of the type II alveolar and bronchial epithelial cell lines A549 and SK-LU 1 with dexamethasone downregulated neurokinin-1 receptor expression. Upregulated neurokinin-1 receptor expression in patients with sarcoidosis may potentiate substance P-induced proinflammatory cytokine production in patients with sarcoidosis.

Comparison of the pharmacokinetics of different analogs of {sup 11}C-labeled TZTP for imaging muscarinic M2 receptors with PET

Energy Technology Data Exchange (ETDEWEB)

Reid, Alicia E. [Medical Department, Brookhaven National Laboratory, Upton, NY 11973 (United States)], E-mail: areid@bnl.gov; Ding Yushin [Radiology Department, Yale University School of Medicine, New Haven, CT 06510 (United States); Eckelman, William C. [Molecular Tracer LLC, Bethesda, MD 20892 (United States); Logan, Jean; Alexoff, David; Shea, Colleen; Xu Youwen; Fowler, Joanna S. [Medical Department, Brookhaven National Laboratory, Upton, NY 11973 (United States)

2008-04-15

Introduction: The only radiotracer available for the selective imaging of muscarinic M2 receptors in vivo is 3-(3-{l_brace}3-[{sup 18}F]fluoropropyl)thio{r_brace}-1,2,5-thiadiazol-4-yl) -1,2,5,6-tetrahydro-1-methylpyridine) ([{sup 18}F]FP-TZTP). We have prepared and labeled 3-(3-(3-fluoropropylthio)-1,2,5-thiadiazol-4-yl) -1,2,5,6-tetrahydro-1-methylpyridne (FP-TZTP, 3) and two other TZTP derivatives with {sup 11}C at the methylpyridine moiety to explore the potential of using {sup 11}C-labeled FP-TZTP for positron emission tomography imaging of M2 receptors and to compare the effect of small structural changes on tracer pharmacokinetics (PK) in brain and peripheral organs. Methods: {sup 11}C-radiolabeled FP-TZTP, 3-(3-propylthio)-TZTP (6) and 3,3,3-(3-(3-trifluoropropyl)-TZTP (10) were prepared, and log D, plasma protein binding (PPB), affinity constants, time-activity curves (TACs), area under the curve (AUC) for arterial plasma, distribution volumes (DV) and pharmacological blockade in baboons were compared. Results: Values for log D, PPB and affinity constants were similar for 3, 6 and 10. The fraction of parent radiotracer in the plasma was higher and the AUC lower for 10 than for 3 and 6. TACs for brain regions were similar for 3 and 6, which showed PK similar to the {sup 18}F tracer, while 10 showed slower uptake and little clearance over 90 min. DVs for 3 and 6 were similar to the {sup 18}F tracer but higher for 10. Uptake of the three tracers was significantly reduced by coinjection of unlabeled 3 and 6. Conclusion: Small structural variations on the TZTP structure greatly altered the PK in brain and behavior in blood with little change in the log D, PPB or affinity. The study suggests that {sup 11}C-radiolabeled 3 will be a suitable alternative to [{sup 18}F]FP-TZTP for translational studies in humans.

Pharmacological identification of cholinergic receptor subtypes on Drosophila melanogaster larval heart.

Science.gov (United States)

Malloy, Cole A; Ritter, Kyle; Robinson, Jonathan; English, Connor; Cooper, Robin L

2016-01-01

The Drosophila melanogaster heart is a popular model in which to study cardiac physiology and development. Progress has been made in understanding the role of endogenous compounds in regulating cardiac function in this model. It is well characterized that common neurotransmitters act on many peripheral and non-neuronal tissues as they flow through the hemolymph of insects. Many of these neuromodulators, including acetylcholine (ACh), have been shown to act directly on the D. melanogaster larval heart. ACh is a primary neurotransmitter in the central nervous system (CNS) of vertebrates and at the neuromuscular junctions on skeletal and cardiac tissue. In insects, ACh is the primary excitatory neurotransmitter of sensory neurons and is also prominent in the CNS. A full understanding regarding the regulation of the Drosophila cardiac physiology by the cholinergic system remains poorly understood. Here we use semi-intact D. melanogaster larvae to study the pharmacological profile of cholinergic receptor subtypes, nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs), in modulating heart rate (HR). Cholinergic receptor agonists, nicotine and muscarine both increase HR, while nAChR agonist clothianidin exhibits no significant effect when exposed to an open preparation at concentrations as low as 100 nM. In addition, both nAChR and mAChR antagonists increase HR as well but also display capabilities of blocking agonist actions. These results provide evidence that both of these receptor subtypes display functional significance in regulating the larval heart's pacemaker activity.

Rapamycin down-regulates LDL-receptor expression independently of SREBP-2