Navafenterol, a new drug for the treatment of chronic obstructive airway disease / 中国临床药理学与治疗学

Navafenterol is a new compound with both muscarinic receptor antagonist and β2 receptor agonist effects in a single molecule, who is being developed for the treatment of chronic obstructive airway disease such as chronic obstructive pulmonary disease and asthma. These pilot clinical studies found that it can significantly improve lung function and symptoms, and is safe and well tolerated. Common treatment emergent adverse events include headache, nasopharyngitis, and dizziness. It may become a next-generation bronchodilator for chronic obstructive airway disease. This review introduced the prospective of Navafenterol.

Evaluation of the gastrointestinal anti-motility effect of Anacardium occidentale stem bark extract:A mechanistic study of antidiarrheal activity / 药物分析学报

Diarrhea is a prevalent gastrointestinal problem associated with fatal implications.It is a huge public health concern that requires better alternatives to current drugs.This study investigated the mechanisms involved in the antidiarrheal activity ofAnacardium occidentale (Ao) stem bark extract,a plant commonly used in the management of diarrhea in Nigeria.Methanolic stem bark extract of the plant was parti-tioned into three fractions:hexane fraction,ethyl acetate fraction (AoEF) and methanol fraction.In vitro studies on the effect of these fractions on guinea pig ileum (GPI) strips,as well as the modulatory effect of AoEF on standard agonists-and antagonists-induced GPI contraction and relaxation,revealed AoEF as the most active fraction.In vivo studies to assess the effect of AoEF on the dopaminergic,muscarinic,and serotonergic pathways were carried out using gastric emptying (GE) and gastrointestinal transit (GT) as experimental end points.AoEF was subjected to GC-MS analysis,while the identified compounds were docked with the muscarinic acetylcholine receptor M3 (CHRM3) using AutodockVina.Results indicated that AoEF inhibited GE and GT via inhibition of CHRM3.In addition,GC-MS analysis revealed the presence of 24 compounds in AoEF,while docking indicated that octadecanoic acid 2-(2-hydroxylethoxy)ethyl ester exhibited the highest binding affinity to CHRM3.This study indicated that the antidiarrheal activity of Ao is through its antimotility effect via the inhibition of the muscarinic pathway.And since none of the identified compounds exhibited higher binding affinity to CHRM3 relative to loperamide,the antimotility activity of these phytoconstituents may be via synergism.

Pharmacological Modulation of Vagal Nerve Activity in Cardiovascular Diseases / 神经科学通报·英文版

Cardiovascular diseases are life-threatening illnesses with high morbidity and mortality. Suppressed vagal (parasympathetic) activity and increased sympathetic activity are involved in these diseases. Currently, pharmacological interventions primarily aim to inhibit over-excitation of sympathetic nerves, while vagal modulation has been largely neglected. Many studies have demonstrated that increased vagal activity reduces cardiovascular risk factors in both animal models and human patients. Therefore, the improvement of vagal activity may be an alternate approach for the treatment of cardiovascular diseases. However, drugs used for vagus nerve activation in cardiovascular diseases are limited in the clinic. In this review, we provide an overview of the potential drug targets for modulating vagal nerve activation, including muscarinic, and β-adrenergic receptors. In addition, vagomimetic drugs (such as choline, acetylcholine, and pyridostigmine) and the mechanism underlying their cardiovascular protective effects are also discussed.

Muscarinic receptor 3 agonist promotes epithelial-meschymal transition in human lung cancer A549 cells by activating PI3K/AKT signaling pathway / 中国病理生理杂志

AIM: To investigate the possible signaling pathway that promotes epithelial-mesenchymal transi-tion(EMT)of the lung cancer A549 cells stimulated with muscarinic receptor 3(M3R)agonist carbachol.METHODS:The lung cancer cells A549 were treated with 400 μmol/L carbachol.The morphological changes of the cells were observed under inverted phase contrast microscope.The migration and invasion abilites were measured by Wound healing and Tran-swell assays.qPCR was used to detect the mRNA level of vimentin and E-cadherin.The protein levels of p-AKT,vimentin and E-cadherin were determined by Western blot.RESULTS:After treatment with carbachol,the A549 cells showed loss of the close connection and the cell morphology was transformed from irregular polygon to spindle -like cells.The results of Wound healing and Transwell assays showed that the migration and invasion abilites of the A 549 cells were enhanced.Car-bachol increased the vimentin expression and decreased the E-cadherin expression at mRNA and protein level(P<0.05). The phosphorylation of AKT in the A549 cells was up-regulated(P<0.05).These changes was inhibited by M3R antago-nist 4-DAMP.CONCLUSION:Carbachol promotes EMT in the human lung cancer A 549 cells by activating PI3K/AKT signaling pathway.

Study on the Molecular Docking of Penehyclidine Optical Isomers and Muscarinic Receptor Subtypes / 中国药房

OBJECTIVE:To study the affinity of penehyclidine optical isomers to muscarinic(M)receptor subtypes,and pro-vide reference for revealing the action targets and efficacy selectivity of penehyclidine. METHODS:Homology modeling,molecu-lar docking and other molecular simulation technologies were used to analyze and predict the binding energy of 4 optical isomers to M receptor subtypes and judge its affinity by comparing the binding energy of different optical isomers R1 (3R,2′R),R2 (3R, 2′S),S1(3S,2′R),S2(3S,2′S)with M receptor subtypes M1-M5. RESULTS:All the 4 optical isomers can dock into the ac-tive sites of M receptor subtypes,and different optical isomers showed great differences in the molecular docking with different M receptor subtypes. Penehyclidine isomers showed larger binding energy to M3,the binding energy of 4 optical isomers ranged in 5736.519-5907.143 kcal/mol. The binding energy of R1 to M1 was 1190.041 kcal/mol;while those of other optical isomers to each receptor subtype were lower or negative. CONCLUSIONS:R1 shows the affinity to M1 receptor. And all the 4 optical isomer show the affinity to M3.

Selective inhibition of ethanol on muscarinic receptor-or 5-HT receptor-mediated contraction in circular smooth muscle of rat stomach / 中国药理学通报

Aim To investigate the selective inhibition of ethanol on muscarinic receptor-or 5-HT receptor-me-diated contractile responses in the circular smooth mus-cle strips isolated from the different regions of rat stom-ach. Methods Circular muscle strips isolated from the rat gastric fundus, body, cardia and pylorus were prepared, and the contractile responses to carbachol ( CCh ) or 5-HT were recorded. Results Ethanol (0. 000 05~0. 000 5, V/V) did not affect the contrac-tile response to CCh in circular muscle strips from the rat gastric fundus and cardia, and that to 5-HT in the strips from rat gastric fundus and body ( P >0. 05 ) . However, ethanol(0. 000 1 and 0. 000 5) significantly inhibited the Emax value of the contraction by CCh from (12. 18 ± 0. 33) g of control level to (10. 88 ± 0. 41) g and -lgEC50 value from ( 6. 33 ± 0. 05 ) of control level to (6. 12 ± 0. 06)(P <0. 05) in the strips from rat gastric body. Ethanol(0. 000 1 and 0. 000 5) also significantly inhibited the Emax value of the contraction by CCh from (2. 87 ± 0. 15) g of control level to (2. 2 ± 0. 13) g and -lgEC50 value from (6. 49 ± 0. 10) of control level to (6. 05 ± 0. 09)(P<0. 01) in the strips from rat gastric pylorus. Moreover, ethanol ( 0. 000 1 and 0. 000 5) significantly inhibited the Emax value of the contraction by 5-HT from (2. 93 ± 0. 35) g of con-trol level to ( 2. 1 ± 0. 30 ) g ( P<0. 05 ) , but did not affect the -lgEC50 value in the strips from rat gastric cardia. Conclusions Ethanol inhibits the contractile responses to 5-HT only in the circular muscle strips of rat gastric cardia, and it inhibits the contractile respon-ses to CCh more strongly in the circular muscle strips of gastric pylorus than gastric body. In those gastric circular muscle strips, ethanol decreases both the ac-tivity and affinity of CCh to muscarinic receptors, but decreases only the activity of 5-HT to its receptors.

Additive interaction of intrathecal ginsenosides and neostigmine in the rat formalin test / 대한마취과학회지

BACKGROUND: The authors evaluated the effect of intrathecal mixture of ginsenosides with neostigmine on formalin-induced nociception and made further clear the role of the spinal muscarinic (M) receptors on the activity of ginsenosides. METHODS: A catheter was located in the intrathecal space of male Sprague-Dawley rats. Pain was evoked by injection of formalin solution (5%, 50 microl) to the hindpaw. Isobolographic analysis was done to characterize drug interaction between ginsenosides and neostigmine. The antagonism of ginsenosides-mediated antinociception was determined with M1 receptor antagonist (pirenzepine), M2 receptor antagonist (methoctramine), M3 receptor antagonist (4-DAMP), M4 receptor antagonist (tropicamide). The expression of muscarinic receptor subtypes was examined with RT-PCR. RESULTS: Intrathecal ginsenosides and neostigmine produced an antinociceptive effect during phase 1 and phase 2 in the formalin test. Isobolographic analysis revealed an additive interaction between ginsenosides and neostigmine in both phases. Intrathecal pirenzepine, methoctramine, 4-DAMP, and tropicamide reversed the antinociception of ginsenosides in both phases. M1-M4 receptors mRNA detected in spinal cord of naive rats and the injection of formalin decreased the expression of M1 receptor mRNA, but it had no effect on the expression of other three muscarinic receptors mRNA. Intrathecal ginsenosides little affected the expression of all of muscarinic receptors mRNA in formalin-injected rats. CONCLUSIONS: Intrathecal ginsenosides additively interacted with neostigmine in the formalin test. Furthermore, M1-M4 receptors exist in the spinal cord, all of which contribute to the antinocieption of intrathecal ginsenosides.

Changes in the Muscarinic Receptors on the Colonic Smooth Muscles of Rats with Spinal Cord Injury

OBJECTIVE: To investigate changes in (1) the colonic response to acetylcholine (Ach), (2) the muscarinic (M) receptors in the colon, and (3) the levels of colonic contraction-related proteins after a spinal cord injury (SCI). METHOD: We divided 16 Sprague-Dawley rats into 2 groups: the control group and the SCI group. A spinal cord transection was performed surgically at the T10 vertebral level. After 1 week, the entire colon was divided into 2 segments, the proximal and distal colon. Each segment was mounted in a longitudinal or circular muscle direction in a 10-ml organ bath. We determined the intergroup differences as percentage changes in contractility after Ach treatment alone, Ach treatment with M2 receptor antagonist (AQ-RA741) pretreatment, and Ach treatment with M3 receptor antagonist (4-DAMP) pretreatment. Western blot analyses were performed to determine the expression level of RhoA, and heat shock protein 27 (HSP27). RESULTS: Compared to the control rats, the SCI rats showed an increased response to Ach along both the directions in the proximal colon (p<0.05). Compared to the control group, in the SCI group, the Ach response was significantly different in the proximal segment under AQ-RA741 pretreatment (p<0.05) and in the distal segment under 4-DAMP pretreatment (p<0.05). Findings of the western blot analyses showed a significant decrease in the level of protein gene product 9.5 in the proximal and distal colon and a significant increase in the level of RhoA and HSP27 in the proximal colon of the SCI rats. CONCLUSION: Our results suggest that changes in colonic contractility after SCI are partly attributable to changes in the M receptor subtypes.

Efectos de la administración crónica de alcohol sobre la conducta motora y su relación con el sistema colinérgico muscarinico en ratas sprague dawley bajo estrés discontinuos / Effects of chronic administration of alcohol on motor behavior and its relation to the muscarinic cholinergic system in Sprague Dawley rats under stress discontinuous

El alcohol y el estrés son problemas de salud pública que afectan el Receptor Colinérgico Muscarínico (RCM). En el presente trabajo se estudia el efecto de ambos fenómenos sobre la funcionalidad y densidad del RCM. Métodos: 43 ratas Sprague Dawley se dividieron en 4 grupos: Control (n=12), Estrés (n=11), Alcohol (n=10) y Alcohol-Estrés (n=10). A los grupos alcohol se le administró diariamente etanol al 10 % ad libitum y los grupos Estrés se sometieron a nado forzado a 5°C por 5 min tres veces/semana. Resultados: las ratas tratadas con alcohol presentaron adicción e hipermotilidad, siendo el efecto mayor en el grupo alcohol-estrés. Escopolamina incremento la motilidad en todos los grupos. No hubo diferencias significativas entre los grupos en el desempeño en el Rotarod. La densidad de los RCM estuvo disminuida significativamente en Hipocampo en el grupo alcohol. Conclusión: el alcohol induce trastornos del RCM relacionados a hipermotilidad

Alcohol consumption and stress are health problems, which affects the Cholinergic Muscarinic Receptor (CMR) system. Here we studied the effect of both phenomena on CMR functionality and densities. Methods: 43 Sprague Dawley rats were divided in 4 groups: Control (n=11), Stress (n=10), Alcohol (n=10) and Alcohol-Stress (n=9). Alcohol groups received 10% ethanol ad libitum in substitution of water every day, stress groups were submitted 3 days at week to 5 min force swimming at 5°C. Results: rats that had alcohol displayed addiction and hypermotility, the effect was higher at alcohol-stress group. Scopolamine significantly increased motility in all groups. No differences were observed at Rotarod performance. CMR density was decreased in hippocampus of rats belonging to alcohol group. Conclusion: alcohol induces motor disturbances related to CMR system

The altered expression of M_2 muscarinic receptor in the neurons of the pedunculopontine nucleus in 6-hydrodopamine unilaterally lesioned rats and its significance / 西安交通大学学报(医学版)

Objective To investigate the altered expression of muscarinic receptor 2 (M_2 receptor) in the pedunculopontine nucleus (PPN) of Parkinson's disease (PD) model rat by immunocytochemical technique and explore the role of M_2 receptor in etiopathogenesis and pathophysiological changes of PD. Methods Sixteen healthy SD rats were divided randomly into two groups. Rat monoclonal antibody against the M_2 receptor was used. Then we used positive cell counting and optical density as indicators to analyze the altered expression of M_2 receptor in PPN of PD model rats. Results The counting of M_2 receptor positive cells in the PPN was not obviously changed in normal rats and the unlesioned side of PD rats (P>0.05), whereas a significant decrease was observed when compared to that in normal rats and the lesioned side of PD rats, respectively (P<0.05). However, the positive intensity in the three groups did not differ significantly. Conclusion The results indicate that there was a degenerative death or receptor loss of M_2 receptor positive cells in the lesioned PPN of PD rats. The expression intensity of M_2 receptor positive cells without degenrative death or receptor loss was not affected. It was also found that the factor affecting the change of M_2 receptor positive cells in the PPN involved only one side.

The Effect of Muscarinic Receptor Subtype Antagonists on Detrusor Overactivity Induced by Bladder Outlet Obstruction in Rats / 대한비뇨기과학회지

PURPOSE: We studied the role of the muscarinic receptor subtype on the urothelium and detrusor activity in rats with detrusor overactivity induced by bladder outlet obstruction(BOO). MATERIALS AND METHODS: Forty Sprague-Dawley rats were used for this study. They were divided into 15 controls and 25 experimental rats. Partial BOO was induced for 3 weeks and a sham operation was performed on the control group animals. A cystometrogram(CMG) was performed in 20 BOO and 10 control rats. During the CMG, M2 selective muscarinic receptor antagonist(methoctramine) and nonselective muscarinic receptor antagonist(tolterodine) drugs were administrated intravesically. The contraction intervals and pressure were evaluated. Bladder muscle strips were prepared from five BOO and five control rats. The contractile responses were evaluated at 2-, 4-, 8-, 16-, and 32Hz field stimulation in the control and BOO groups and after administration of the methoctramine and tolterodine. RESULTS: The results of the CMG showed that the rats in the BOO group had a decreased contraction interval and increased contraction pressure compared to the control group(p<0.05). The contraction intervals were increased after intravesical administration of methoctramine and tolterodine compared to the BOO group(p<0.05). However, the contraction pressures were similar among the methoctramine, tolterodine and BOO groups. For the muscle strip study, the BOO group demonstrated increased contractile responses compared to the control group(p<0.05). However, the contractile responses were decreased after administration of methoctramine(only in 32Hz) and tolterodine(p<0.05). CONCLUSIONS: The results of this study showed that detrusor overactivity induced by BOO in rats is primarily mediated by M3 muscarinic receptors in the detrusor muscle, and by M2 and M3 muscarinic receptors in the urothelium.

Muscarinic M5 receptor subtype and its biologic characterizations / 中国药理学通报

The fifth muscarinic receptor (M5), the last one of the muscarinic receptor family to be cloned, has the same basic formation characterization as G-protein coupled receptor family. M5 transduces signals by coupling with G-proteins, which then modulate the activities of a number of effector enzymes and ion channels. As M5 also plays a variety of prominent physiological roles by regulating central transmitters NO and DA, it has been considered as a novel drug therapy target for drug addiction, dysfunction of dopamine-ergic nervous system, Alzheimers disease and cerebral ischemia.

The Effect of Enflurane on the Tracheal Smooth Muscle Contracted with Electrical Field Stimulation in the Guinea Pig / 대한마취과학회지

BACKGORUND: inhalation anesthetics have been known as bronchodilators, and there are reports that enflurane has some relaxing effects on tracheal smooth muscles. However, there are not so many reports on the ACh release in the postganglion nerve endings. We tried to evaluate the effect of enflurane on the contraction of the tracheal smooth muscle in the postganglion nerve ending in guinea pigs. METHODS:isolated tracheal preparations of guinea pigs were used and contractions were induced by electrical field stimulation (3 Hz & 30 Hz). in the pilocarpine- enflurane group, pilocarpine (10(-5) M) was administrated and enflurane (1 MAC and 2 MAC) was administered. in the gallamine-enflurane group, gallamine (10(-6) M) was administrated and enflurane (1 MAC and 2 MAC) was administered. in the enflurane 1 MAC group and 2 MAC group, contractions were induced by electrical field stimulation before and after administration of enflurane. The percentile contraction to the contraction induced by acetylcholine (10(-4) M) were evaluated. RESULTS: The potentiation of the contraction which was induced by electrical field stimulation was observed by enflurane administration and with prior administration of pilocarpine (10(-6) M), with prior administration of gallamine (10(-5) M). There was no potentiation of contractions, but potentiation of the contraction was observed with enflurazne (2 MAC, 30 Hz). CONCLUSiONS:Enflurane potentiates the contraction induced by electrical field stimulation in guinea pig tracheal smooth muscle. These findings seem to be related with prejunctional M2 receptor in the postganglionic nerve endings.

Protective effects of muscarinic receptor on apoptosis of PC12 cells induced by hydrogen peroxide / 中国药理学通报

Aim To find out the relationship between muscarinic receptor and reactive oxygen species (ROS) and the probable differences between the four muscarinic receptor subtypes. Methods We transfected the plasmid encoding muscarinic receptor (including subtypes: M_1, M_2, M_3 and M_4) into PC12 cells. Then PC12 cells were exposed to hydrogen peroxide (H_2O_2), carbachol and other inhibitors such as atropine, LY294002 and PD98059. Results The results showed that activation of muscarinic receptor by carbachol protected PC12-M_1, PC12-M_2,PC12-M_3 and PC12-M_4 cells from apoptosis induced by H_2O_2. There was no statistical difference in the protective effect between these four muscarinic receptor subtypes. By using the inhibitors, we found that atropine and LY294002 blocked the protective effect of activation of muscarinic receptor on apoptosis induced by H_2O_2. Conclusion Activation of muscarinic receptor retarded the apoptosis induced by H_2O_2. There was no difference between the four muscarinic receptor subtypes. The protective effect was mainly mediated by the activation of muscarinic receptor and phosphatidylinositol-3 kinase (PI3K).

The gene expression profiles through activating endothelial target for acetylcholine / 中国药理学通报

Aim To investigate the effects of gene expression through activating endothelial target for acetylcholine (ETA).Methods Comparative studies were carried out to explore the distinct gene expression of carbachol and pilocarpine.Results Human coronary aortic endothelial cells were incubated with carbachol which activates ETA and muscarinic receptors and with pilocarpine which activates muscarinic receptors distinctly for 10 hours at the concentration of 100 ?mol?L -1.The endothelial gene expression was detected by BiostarH-14112S cDNA expression profiling microarrays containing 14 000 human unigenes. There were 801 differential genes totally (491 differentially expressed both by carbachol and by pilocarpine, 310 differentially expressed by carbachol exclusively). We found a significant increase in expression of 119 and a significant decrease in expression of 191 genes after treatment by carbachol preferentially. And these genes were not affected by pilocarpine. Further globally functional catagorization indicates that there are seven types of distinct expressed genes induced by carbachol selectively including membrane receptors and G proteins, ion channels, G protein coupled receptors, transcriptional factors, apoptosis-related genes, cell adhesive factors, thrombosis and atherosclerosis-related genes.Conclusion The differentially expressed genes evoked by carbachol exclusively are closely associated with ETA and its effectors.

Relationship between muscarinic receptor subtype density change and unstable bladder in rats / 第三军医大学学报

Objective To study the relationship between muscarinic receptor(MR) subtype density changes and the producing of unstable bladder Methods The rat model of unstable bladder was established and the MR subtype density of control and unstable bladders were defermined by RT-PCR Results There were only m 2RNA and m 3RNA in the detrusor of control and unstable bladders, but no m 1, m 2 and m 3 mRNA, the amount of m 2RNA(1 67?0 42)was larger obviously than m 3RNA(0 64?0 19),the ratio was 2 59∶1 in control group, and the density of both m 2 and m 3 in unstable bladder increased to (2 03?0 65) and(1 53?0 46)respectively, and the increase of m 3 was more significant ( P

Relationship between IP_3 and detrusor cell contraction mediated by M_3R / 中华泌尿外科杂志

Objective To study the relationship between IP 3 and detrusor cell contraction mediated by M 3R. Methods [3H]-IP contents of human cultured detrusor cells were detected after stimulated by carbachol,atropine,methoctramine and 4-DAMP respectively. Results [3H]-IP contents increased with carbachol concentration.On the different concentrations (10 -9、10 -8、10 -7、10 -6、10 -5、10 -4 mmol/L )of 4-DAMP,[3H]-IP contents were 3 926.57?273.29、2 780.52?211.09、2436.84?153.62、1 973.22?164.71、1 372.38?141.35 and 1 107.98?920.45 cpm respectively.On the some concertrations of Atropine,[3H]-IP contents were 3 602.69?280.17,2 891.31?207.45,1 983.97?145.74,1 269.57? 105.31,1 106.37?75.23,927.50?77.36/min,respectively.On the same concentrations of methoctramine, the [3H]-IP contents was 4 462.74?360.69、3 938.61?327.13、3 315.45?270.36、3 063.19? 246.79、2927.37?226.45 and 2 836.55?241.63 cpm.This donoted that 4-DAMP and atropine signficantly inhibited the effect of carbachol on PI decomposition(P0.05). Conclusions M 3R is much related to IP 3.

Expression of ? and ? opiate receptor in spinal cord andbrainstem during morphine withdrawal in rats / 中国临床药理学与治疗学

Aim To observe the gene expression of ? and ? opiate receptor in spinal cord and brainstem,and the effects of muscarinic receptor antagonist, NMDA receptor antagonists and inhibitor of nitric oxide synthase on the expression of these genes during morphine withdrawal in rats. Methods The mRNA levels of ? and ? opiate receptor mRNA were assayed by reverse transcription polymerase chain reaction (RT_PCR) with the beta_actin mRNA as an internal control. Results The ? opiate receptor mRNA levels were increased significantly in spinal cord and brainstem during morphine dependence, and decreased after injection of naloxone during morphine withdrawal in rats. The ? opiate receptor mRNA levels in spinal cord and brainstem were changed conversely compared with the ? opiate receptor mRNA levels during morphine dependence and withdrawal. The ? and ? opiate receptor mRNA levels in spinal cord and brainstem were decreased by administration of either Rp_cAMPs or calyculin A while these levels were not changed by Sp_cAMPs at half hour before injection of naloxone in morphine dependent rats. Administration of l_N_nitric arginine methylester(10 mg?kg-1) resulted in a decrease of ? opiate receptor and ? opiate receptor levels in spinal cord , and ? opiate receptor levels in spinal cord and ? opiate receptor levels in brainstem were dedcreased by pretreatment with methyl_scopolamine (0.5 mg?kg-1) during morphine withdrawal. However, the ? and ? opiate receptor levels in both spinal cord and brainstem were not different from those of morphine withdrawal rats pretreated with either MK801 (0.125 mg?kg-1) or pirezenpine(10 mg?kg-1). In adddition, ?_actin mRNA levels were not different in each group.Conclusion The expression of ? opiate receptor and ? opiate receptor mRNA plays an important role in mediating the process of morphine dependence and withdrawal, and the expression of ? opiate receptor and ? opiate receptor mRNA in spinal cord and brainstem could be inhibited by block of muscarinic receptor or inhibition of nitric oxide production.

Muscarinic receptor subtype controlling the carbachol-induced muscle contraction in guinea pig gastric antrum

Stimulation of muscarinic receptors by carbachol (CCh) in the circular smooth muscle of the guinea pig gastric antrum causes muscle contraction. In the present study, muscarinic receptor subtype controlling the muscle contraction in response to CCh was studied using putative muscarinic receptor antagonists. Isometric force of the isolated circular muscle strips was measured in an organ bath. CCh contracted the muscle in a dose-dependent way, and each of the three muscarinic receptor antagonists, 4-diphenylacetoxy-N-methylpeperdine methiodide (4-DAMP), methoctramine and pirenzepine shifted the concentration-response curves to the right without significantly reducing the maximum force. The affinities of the muscarinic antagonists (pA2 values) obtained from Schild plot analysis were 10.15, 7.05 and 6.84 for 4-DAMP, methoctramine and pirenzepine, respectively. These results suggest that the M3-subtype mainly mediate the muscle contraction in response to CCh in guinea pig gastric antrum.

Effect of K+-channel blockers on the muscarinic- and A|1-adenosine-receptor coupled regulation of electrically evoked acetylcholine release in the rat hippocampus

It was attempted to clarify the participation of K+ channels in the post-receptor mechanisms of the muscarinic and A1-adenosine receptor-mediated control of acetylcholine (ACh) release in the present study. Slices from the rat hippocampus were equilibrated with (3H)choline and the release of the labelled products was evoked by electrical stimulation (3 Hz, 5 V/cm, 2 ms, rectangular pulses), and the influence of various agents on the evoked tritium-outflow was investigated. Oxotremorine (Oxo, 0.1~10 micrometer), a muscarinic agonist, and N6-cyclopentyladenosine (CPA, 1~30 micrometer), a specific A1-adenosine agonist, decreased the ACh release in a dose-dependent manner, without affecting the basal rate of release. 4-aminopyridine (4AP), a specific A-type K+-channel blocker (1~100 micrometer), increased the evoked ACh release in a dose-related fashion, and the basal rate of release is increased by 3 and 100 micrometer. Tetraethylammonium (TEA), a non-specific K+-channel blocker (0.1~10 mM), increased the evoked ACh release in a dose-dependent manner without affecting the basal release. The effects of Oxo and CPA were not affected by 3 micrometer 4AP co-treatment, but 10 mM TEA significantly inhibited the effects of Oxo and CPA. 4AP (10 micrometer- and TEA (10 mM)-induced increments of evoked ACh release were completely abolished in Ca2+-free medium, but these were recovered in low Ca2+ medium. And the effects of K+-channel blockers in low Ca2+ medium were inhibited by Mg2+ (4 mM) and abolished by 0.3 micrometer tetrodotoxin (TTX). These results suggest that the changes in TEA-sensitive potassium channel permeability and the consequent limitation of Ca2+ influx are partly involved in the presynaptic modulation of the evoked ACh-release by muscarinic and A1-adenosine receptors of the rat hippocampus.