M1 Muscarinic Receptor Antagonist Research Articles

Sulfide analogues as potent and selective M2 muscarinic receptor antagonists

We have discovered highly potent, selective sulfide M2 receptor antagonists with low molecular weight and different structural features compared with our phase I clinical candidate Sch 211803. Analogue 30 showed superior M2 receptor selectivity profile over Sch 211803. More importantly, this study provided new leads for the discovery of M2 receptor antagonists as potential drug candidates.

Muscarinic receptor antagonist-induced lenticular opacity in rats.

Investigations on compound A, an M2-sparing M3 muscarinic receptor antagonist, showed that focal polar anterior subcapsular lenticular opacities, characterized by focal epithelial proliferation, developed in Sprague-Dawley rats. The incidence and bilateral localization of this change increased generally with dose and time, though plateauing after 8 months of treatment; however the severity progressed very slightly. Over a 1-year period, no anterior cortical lens fiber changes or other histological ocular changes developed. A decreased severity of the change and apoptosis suggested some regression after a 26-week recovery period. Two nonselective muscarinic receptor antagonists, atropine and tolterodine, induced similar lenticular changes in rats. A hypothesis in relation to an indirect effect of the drug, such as increased illumination of the lens due to mydriasis observed with all these compounds, was investigated and disproven. Because these opacities are induced by structurally unrelated muscarinic receptor antagonists (atropine and tolterodine), it is likely that these lenticular changes are the result of muscarinic receptor inhibition. However, hypotheses regarding a direct effect of the drug on muscarinic receptors in the lens epithelium, possibly mediated by drug and/or metabolite(s) in the aqueous humor and/or lens epithelium, remain to be investigated. This lenticular opacity is similar to that observed spontaneously in Sprague-Dawley rats, although the latter occur at a lower incidence. No such lenticular opacities have been reported in other animal species, including man, after treatment with muscarinic receptor antagonists.

Muscarinic modulation of hypoxia-induced release of catecholamines from the cat carotid body

Chemotransduction of arterial hypoxemia by the cat carotid body is generally thought to begin with a hypoxia-induced depolarization of the glomus cells (GCs) of the carotid body (CB). This depolarization activates voltage-gated calcium channels with the subsequent entry of calcium, movement of transmitter-containing vesicles to the synaptic-like juncture between the GC and apposed sensory afferent neuron. The vesicles exocytotically release their transmitters which then proceed to the receptors on both the postsynaptic neuron and on the GCs themselves (autoreceptors). Action potentials and their modulation in the sensory fibers are the result, along with the modulation of further neurotransmitter release from the GCs. The purpose of the present study was to: (1) determine the parameters of an incubated cat CB preparation capable of releasing measurable amounts of catecholamines (CAs) in response to hypoxia; (2) determine the impact of muscarinic activities on CA release during the hypoxic challenge; (3) determine if the muscarinic activity preferentially modified the release of one CA more than another; (4) determine if there were any differences in the pattern of hypoxia-induced release of dopamine (DA) vs. norepinephrine (NE). CBs were harvested from deeply anesthetized cats. Cleaned of fat and connective tissue, they were incubated in Krebs Ringer bicarbonate solution at 37 °C, and bubbled with a hyperoxic mixture of gases (95% O 2–5% CO 2) for 30 min. The first series of experiments to address the CB’s hypoxia-induced release of CAs explored the effects of incubating CBs for 2 h with hyperoxia vs. normoxia (21% O 2–6% CO 2) followed by a 30 min hypoxic challenge, with or without l-dihydroxyphenylalanine ( l-DOPA). In the second series of experiments the CBs, after the first 30 min of hyperoxia, were next challenged with hypoxia (4% O 2–5% CO 2) for intervals of 3–20 min with intervening recovery periods of hyperoxia to determine the effect of the duration of the hypoxic exposure on CA release. In the third series of experiments the CBs, after the first 30 min of hyperoxia, were challenged with hypoxia for intervals of 10–40 min in the presence or absence of an M1 or M2 muscarinic receptor antagonist. CAs released into the incubation medium were analyzed by means of high performance liquid chromatography–electrochemical detection using standard procedures. Incubated cat CBs challenged for 2 h with hyperoxia followed by 30 min of hypoxia, released much more measurable amounts of CAs in the presence of 40 μM l-DOPA than without it. Moving from hyperoxia to hypoxia produced a better yield than moving from normoxia to hypoxia, and at least 10–20 min exposures were needed for measurable amounts of CAs. The M1 muscarinic receptor antagonist, pirenzepine, reduced the hypoxia-induced release of CAs during each exposure. Further, the reduction appeared to be dose-related. The M2 muscarinic receptor antagonist, methoctramine, enhanced the hypoxia-induced release of CAs during each exposure. These data support a role for acetylcholine (ACh) in the hypoxia-induced release of CAs, and suggest a significant, if modest, muscarinic dimension to it. And although hypoxia induced a greater release of DA than of NE, the muscarinic modulation of the release (both decreasing it and increasing it) may have had a greater impact on NE release than on DA release. Finally, the patterns of hypoxia-induced release of DA and NE from incubated cat carotid bodies are significantly different.

19F Solid-State NMR Spectroscopic Investigation of Crystalline and Amorphous Forms of a Selective Muscarinic M3 Receptor Antagonist, in Both Bulk and Pharmaceutical Dosage Form Samples

ABSTRACTThe purpose of the following investigation was to display the utility of 19F solid-state nuclear magnetic resonance (NMR) in both distinguishing between solid forms of a selective muscarinic M3 receptor antagonist and characterizing the active pharmaceutical ingredient in low-dose tablets. Ambient- and elevated-temperature solid-state 19F fast (15 kHz) magic-angle spinning (MAS) NMR experiments were employed to obtain desired spectral resolution in this system. Ambient sample temperature combined with rotor frequencies of 15 kHz provided adequate 19F peak resolution to successfully distinguish crystalline and amorphous forms in this system. Additionally, elevated-temperature 19F MAS NMR further characterized solid forms through 19F resonance narrowing brought about by the phenomenon of solvent escape. Similar solvent dynamics at elevated temperatures were utilized in combination with ambient-temperature 19F MAS NMR analysis to provide excipient-free spectra to unambiguously identify the active pharmaceutical ingredient (API) conversion from crystalline Form I to the amorphous form in low-dose tablets. It is shown that 19F solid-state NMR is exceptionally powerful in distinguishing amorphous and crystalline forms in both bulk and formulation samples.

Forebrain muscarinic control of micturition reflex in rats

Functional contribution of the cholinergic pathway between the frontal cortex and basal nucleus of Meynert to micturition reflex was investigated. Male Wistar rats were subjected to bilateral lesion of the basal forebrain by ibotenic acid (IA) injection (7.5 μg/rat on each side) (BF rats). Phosphate buffered saline (PBS) was injected into control rats (sham operated rats; SO rats). Cystometrograms were obtained from conscious BF and SO rats 7–10 days after IA/PBS injection. Bladder capacity (BC) of BF rats was significantly smaller than that of SO rats (approximately 43.7%) and was accompanied by decrease in choline-acetyltransferase activity in the frontal cortices. Oxotremorine M, a muscarinic receptor agonist, increased BC in BF rats, while pirenzepine, an M1 muscarinic receptor antagonist, counteracted the effect of the oxotremorine M-induced increase in BC. Injection of oxotremorine M into the dorsal pontine tegmentum (DPT) reduced BC in BF and SO rats, while injection of pirenzepine had no effect on cystometrograms. These findings indicate that the M1 muscarinic receptor plays a part in the forebrain inhibitory mechanisms involved in the micturition reflex and that muscarinic receptor in the DPT contributes to excitatory control of micturition reflex.

EFFECTS OF HEXASULFOBUTYLATED C60 ON THE GASTRIC CIRCULAR MUSCLE OF GUINEA PIG

The effects of hexasulfobutylated (FC4S), the fullerene derivative on the contraction of smooth muscle were tested pharmacologically on the circular muscle of stomach of guinea pigs. The effects of monomalonic acid C60 (MMA C60) on the same preparations were compared. The effects of those compounds on the taenia coli, portal vein and vas deferens of guinea pigs were also tested. The FC4S did, while MMA C60 did not elicit contracture of the circular muscle of stomach and taenia coli. Both FC4S and MMA C60 did not elicit contraction on the portal vein and on the vas deferens of the guinea pig. Prazosin (0.5 μM) or propranolol (0.5 μM) did not alter the FC4S elicit contracture of the circular muscle of stomach. However, atropine (0.01, 0.1 and 1 μM), tetrodotoxin (0.1 μM) or low calcium medium decreased reversibly the FC4S elicited contracture of the circular muscle of stomach. The effect of FC4S on the contracture of the gastric muscles was also tested using various muscarinic receptor subtype antagonists. 4-DAMP (1 μM), muscarinic M3 receptor antagonist, and tropicamide (1 μM), muscarinic M4 receptor antagonist, did not alter the contracture elicited by FC4S. Pirenzepine (0.1 μM), muscarinic M1 receptor antagonist, and methoctramine (0.25 μM), muscarinic M2 receptor antagonist, significantly decreased the FC4S elicited contracture of the circular muscle. Atropine (1 μM) or tetrodotoxin (0.1 μM) completely blocked the FC4S elicited contracture of the circular muscle of stomach. It is concluded that FC4S elicited contracture of the circular muscle of stomach. The effect may be due to FC4S acts on the cholinergic cells existed in the gastric muscle and indirectly activating the tetrodotoxin dependent releasing of the transmitters from the cells, then, activating the muscarinice M1, M2 receptors in the muscle eliciting the contractures.

At physiological concentrations, deoxycholic acid conjugates are M3 muscarinic receptor antagonists

At physiological concentrations, deoxycholic acid conjugates are M3 muscarinic receptor antagonists

At physiological concentrations, deoxycholic acid conjugates are M3 muscarinic receptor antagonists

At physiological concentrations, deoxycholic acid conjugates are M3 muscarinic receptor antagonists

Muscarinic receptors of the M2 subtype in human and bovine trabecular meshwork.

The trabecular meshwork is a tissue actively involved in the regulation of intraocular pressure via contractile mechanisms. The present study was performed to investigate the effects of muscarinic m2-receptor antagonists on trabecular meshwork contractility and to identify the m2 muscarinic receptor in human and bovine trabecular meshwork cells. Isometric tension measurements of bovine trabecular meshwork strips were performed using a custom-made force length transducer. Western blot and immunoprecipitation analysis was used to detect the m2-receptor proteins in membrane preparations of human and bovine trabecular meshwork cells. Immunoblotting results showed the expression of an m2-receptor protein band at 56 kDa in both human and bovine trabecular meshwork cells. Two different m2-receptor antagonists were tested on trabecular meshwork contractility. After carbachol-induced contraction (10(-6) M set to 100% contractile force), specific m2-receptor antagonists were applied. 3 alpha-Chloroimperaline (10(-6) M) had no effect on the maximal carbachol-induced contraction in trabecular meshwork strips. Methoctramine induced a significant relaxation at concentrations of 10(-7), 10(-6) and 5 x 10(-6) M even in the presence of m1- and m3-receptor antagonists. These data indicate that in addition to the m3-receptor subtype present in the trabecular meshwork this tissue also features the m2 receptor. This receptor is partly involved in the regulation of trabecular meshwork contractility, suggesting that outflow facility might be influenced through this receptor.

ChemInform Abstract: Asymmetric Synthesis: Application to the Synthesis of a Platelet Aggregation Inhibitor and an M3 Muscarinic Receptor Antagonist

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Prevention of Experimental Autoimmune Cardiomyopathy in Rabbits by Receptor Blockers

We investigated the effects of β1-adrenoceptor blockade and M2-muscarinic receptor antagonist in rabbits which have developed dilated cardiomyopathy-like changes after immunization with the peptides from the second extracellular loop of human pi-adrenoceptor (βl-peptide) and M2-muscarinic receptor (M2-peptide). Ten rabbits, which were immunized with (β1-peptide once a month for one year, were treated with bisoprolol and 10 rabbits, which were immunized with M2-peptide, were treated with otenzepad. Although both groups treated with receptor blockade or antagonist showed an increased titer of anti-β-adrenoceptor or anti-M2-muscarinic receptor antibodies, myocardial damages were markedly less than those in pi-peptide-or M2-peptide-immunized rabbits. This study indicates that anti-pi-adrenoceptor and anti-M2-muscarinic receptor antibodies are of pathogenic importance in the development of human dilated cardiomyopathy, and that β-adrenoceptor blockade, bisoprolol, and M2-muscarinic receptor antagonist, otenzepad, might be clinically useful for treatment of dilated cardiomyopathy.

Effects of YM905, a Novel Muscarinic M3-Receptor Antagonist, on Experimental Models of Bowel Dysfunction In Vivo

We investigated the effects of YM905 [(+)-(1S,3'R)-quinuclidin-3'-yl 1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-carboxylate monosuccinate], a new orally active muscarinic M3-receptor antagonist, on bowel dysfunction in vivo using experimental models that reproduce the symptoms present in irritable bowel syndrome (IBS). YM905 potently inhibited restraint stress-induced fecal pellet output in fed rats (ED50: 4.0 mg/kg) and diarrhea in fasted rats (ED50: 1.7 mg/kg), with similar potencies to the inhibition of bethanechol-, neostigmine- and nicotine-induced fecal pellet output in rats (ED50: 3.3, 7.9 and 4.5 mg/kg, respectively). YM905 also inhibited 5-hydroxytryptamine (5-HT)-, prostaglandin E2- and castor oil-induced secretory diarrhea in mice (ED50: 5.5, 14 and 6.3 mg/kg, respectively), but showed no significant effect on cholera toxin-induced intestinal secretion in mice. In addition, YM905 (3, 10 mg/kg) reversed morphine-decreased postprandial defecation in ferrets, a model of spastic constipation, whereas remosetron, a 5-HT3-receptor antagonist, was not effective. The mode of YM905 action was similar to that of darifenacin, a selective M3-receptor antagonist, with equivalent potencies. By contrast, propantheline, an antimuscarinic drug that has been used for IBS, was much less potent. These results show that YM905 ameliorates a wide spectrum of bowel dysfunctions through the blockade of M3 receptors, suggesting its therapeutic potential for treating IBS.

Presynaptic dopamine D2 and muscarine M3 receptors inhibit excitatory and inhibitory transmission to rat subthalamic neurones in vitro

Whole-cell patch-clamp recordings were made from subthalamic nucleus (STN) neurones in brain slices from rats. Stimulation with bipolar electrodes evoked synaptic currents mediated by glutamate (EPSCs) and GABAA (IPSCs) receptors. Dopamine reversibly reduced the amplitude of GABAA IPSCs by up to 48 % with an IC50 value of 3.4 +/- 0.8 microM. The dopamine D2 receptor agonist quinpirole, but not the D1 receptor agonist SKF 82958, also inhibited GABAA IPSCs. This effect was completely reversed by the D2 receptor antagonist sulpiride but not by SCH 23390, a D1 antagonist. Muscarine reversibly reduced the amplitude of GABAA IPSCs by up to 70 % with an IC50 value of 0.6 +/- 0.1 microM. Inhibition of IPSCs by muscarine was completely blocked by scopolamine (10 microM), a muscarinic receptor antagonist. The M3 muscarinic receptor antagonist 4-DAMP effectively reversed muscarine-induced inhibition of IPSCs with an IC50 of 0.11 +/- 0.03 microM. Although the M1 receptor antagonist pirenzepine also reversed the inhibition of IPSCs by muscarine, this effect was only observed at relatively high concentrations (IC50 = 21.7 +/- 9.4 microM). Dopamine and muscarine both increased the paired-pulse ratio of GABAA IPSCs. Neither agent produced sustained changes in postsynaptic holding current. Glutamate EPSCs were also inhibited reversibly by dopamine (by up to 29%; IC50 = 16 +/- 3 microM) and muscarine (by up to 41%; IC50 = 1.0 +/- 0.4 microM). However, both agents were more potent and efficacious for reducing GABA IPSCs compared with glutamate EPSCs. These results suggest that the most significant effect of dopamine and muscarine in the STN is to reduce inhibitory synaptic input by acting at presynaptic dopamine D2 and muscarinic M3 receptors, respectively.

Synthesis of new cardioselective M2 muscarinic receptor antagonists.

A series of 5H-dibenz[b,f]azepine derivatives was prepared and evaluated for binding affinities to muscarinic receptors in vitro. Among them, compound 8 showed a high affinity for human recombinant M2 receptors (Ki=2.6 nm), a low affinity for M4 receptors (39-fold less than for M2 receptors) and a very low affinity for M1 and M3 receptors (119- and 112-fold less than for M2 receptors, respectively). The high M2 selectivity of 8 may be attributed to the olefinic bond of the azepine ring. Functional experiments showed 8 to be a competitive antagonist with high affinity to the cardiac (pA2=7.1) and low affinity to the intestinal muscarinic receptors (IC50=0.54 microM). In vivo experiments confirmed the in vitro M, selectivity of 8. Acetylcholine-induced bradycardia was dose-dependently antagonized in rats after both intravenous and intraduodenal administration of 8. In rats, cholinergic functions mediated by M1 or M3 receptors (salivary secretion, pupil diameter, gastric emptying, intestinal transit time) were not affected by the oral administration of 8 even at doses as high as 30 times the antibradycardic effective dose. Furthermore, 8 had no analgesic activity in mice, indicating poor central nervous system penetration. In dogs, nocturnal bradycardia was dose-dependently inhibited by the oral route with a duration of action of about 24 h. Compound 8 appears to be a promising cardioselective antimuscarinic agent for the treatment of dysfunctions of the cardiac conduction system such as sinus or nodal bradycardia ("sick-sinus syndrome") and atrioventricular block.

Spatial working memory is independent of hippocampal CA1 long-term potentiation in rats.

This study investigated the relationship between spatial working memory and hippocampal long-term potentiation (LTP) using the allocentric place discrimination task (APDT) in rats, in which the selection accuracy is a good index for spatial working memory. Either the selective M1 muscarinic receptor antagonist pirenzepine (50 microg) or the choline uptake inhibitor hemicholinium-3 (5 microg) impaired APDT selection accuracy, but neither affected the induction of LTP in the hippocampal CA1 region in anesthetized rats. In contrast, the selective N-methyl-D-aspartate receptor antagonist D-amino-5-phosphonopentanoate (200 nmol) did not impair APDT selection accuracy but completely blocked hippocampal CA1 LTP. These results suggest that spatial working memory is independent of hippocampal CA1 LTP and that the central cholinergic system is involved in spatial working memory, but not through the modulation of hippocampal CAI LTP.

J-104129, a novel muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors

A new class of 4-acetamidopiperidine derivatives has been synthesized and investigated for human muscarinic receptor subtype selectivity. Introduction of a hydrocarbon chain of appropriate length into the piperidine nitrogen of the racemic N-(piperidin-4-yl)-2-cyclobutyl-2-hydroxy-2-phenylacetamide platform conferred up to 70-fold selectivity for human muscarinic M3 receptors over M2 receptors. Subsequent synthetic derivatizations resulted in highly potent M3 receptor antagonists with selectivity greater than two orders of magnitude for M3 over M2 receptors, from which the analogue 4r was selected. Preparation of both enantiomers of 4r led to the identification of (2R)-N-[1-(4-methyl-3-pentenyl)piperidin-4-yl]-2-cyclopentyl-2-hydroxy-2-phenylacetamide (J-104129, (R)-4r), which exhibited 120-fold selectivity for M3 receptors (Ki=4.2 nM) over M2 receptors (Ki=490 nM). In isolated rat trachea, (R)-4r potently and specifically antagonized acetylcholine (ACh)-induced responses with a KB value of 3.3 nM. The highly subtype-selective profile was also seen in isolated rat tissue assays (50-fold) and in anesthetized rats (>250-fold). Oral administration of J-104129 ((R)-4r) antagonized ACh-induced bronchoconstriction with an ED50 value of 0.58 mg/kg in rats. Thus, J-104129 ((R)-4r) may effectively facilitate bronchodilation in the treatment of obstructive airway disease.

Local anesthetics inhibit muscarinic receptor-mediated activation of extracellular signal-regulated kinases in rat pheochromocytoma PC12 cells.

Because protein phosphorylation is a key mechanism for controlling cellular functions and extracellular signal-regulated kinase (ERK) plays a role in cellular signal transduction, the authors wanted to determine whether local anesthetics interfere with biochemical signaling molecules. Protein tyrosine phosphorylation and ERK activation induced by carbachol, an agonist for muscarinic acetylcholine receptors, were examined in rat pheochromocytoma PC12 cells, a model for investigating signal transduction. Carbachol-induced tyrosine-phosphorylated proteins of 44 and 42 kd were determined by Western blot analysis and identified as activated ERK1 and ERK2 using anti-ERK antibody. The ERK activation was blocked by preincubation with atropine or an M3 muscarinic acetylcholine receptor antagonist 4-diphenyacetooxy-1, 1-dimethylpiperidinium, indicating that is was mediated by M3 muscarinic acetylcholine receptor activation. Then, in the presence of local anesthetic, the carbachol-induced tyrosine phosphorylation and ERK activation were evaluated. The effects of three Na+ current-modifying reagents on carbachol-induced ERK activation were also evaluated. Procaine (10(-4) to 10(-3) M) inhibited carbachol-induced tyrosine phosphorylation and ERK activation in a concentration-dependent manner. Although tetracaine, lidocaine, and bupivacaine similarly suppressed carbachol-induced tyrosine phosphorylation and ERK activation, neither tetrodotoxin, veratridine, nor ouabain affected the carbachol-induced ERKs activation. Both ERKs were also activated by 4beta-phorbol 12-myristate 13-acetate, an activator of protein kinase C, and fluoroaluminate (AlF4-), respectively, but procaine did not affect ERK activation induced by these two substances. The inhibition of carbachol-induced ERK activation by procaine was not modified by a phosphatase inhibitor, calyculin A. The current results indicate that local anesthetics inhibit the activity of the signal-transducing molecule(s) leading to M3 muscarinic acetylcholine receptor-mediated ERK activation in PC12 cells. Such action is unlikely to be a result of the drug's action on Na+ channels or on the electrochemical gradients of the neuronal cell membrane.

Muscarinic receptor modulation of GABA-mediated giant depolarizing potentials in the neonatal rat hippocampus.

1. The whole-cell patch clamp technique was used to study the role of muscarinic receptors in regulating the frequency of giant depolarizing potentials (GDPs) in CA3 hippocampal neurones in slices from postnatal (P) P1-P8 rats. 2. Atropine (1 microM) reduced the frequency of GDPs by 64.2 +/- 2.9 %. The acetylcholinesterase inhibitor edrophonium (20 microM) increased the frequency of GDPs in a developmentally regulated way. This effect was antagonized by the M1 muscarinic receptor antagonist pirenzepine. 3. In the presence of edrophonium, tetanic stimulation of cholinergic fibres induced either an enhancement of GDP frequency (179 +/- 79 %) or a membrane depolarization (27 +/- 16 mV) associated with an increase in synaptic noise. These effects were prevented by atropine. 4. Application of carbachol (3 microM) produced an increase in GDP frequency that at P5-P6 was associated with a membrane depolarization and an increase in synaptic noise. These effects were prevented by atropine, pirenzepine (3 microM) and bicuculline (10 microM). 5. In the presence of pirenzepine, carbachol reduced GDP frequency by 50 +/- 4 %. Conversely, in the presence of methoctramine (3 microM), carbachol enhanced GDP frequency by 117 +/- 4 %. 6. It is concluded that endogenous acetylcholine, through the activation of M1 receptors, enhances the release of gamma-aminobutyric acid (GABA), in a developmentally regulated way. On the other hand, carbachol exerts both an up- and downregulation of GABA release through the activation of M1 and M2 receptors, respectively.

Cholinergic modulation of non-adrenergic, non-cholinergic relaxation in isolated, small coronary arteries from lambs.

The presence of cholinergic innervation of small coronary arteries in the lamb was investigated by measuring choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities and by performing in vitro experiments in a microvascular myograph to establish whether or not there is a cholinergic component in the response to electrical field stimulation (EFS). ChAT-specific activity was present in proximal coronary segments, but was significantly higher in small coronary arteries. AChE-positive ganglia and fibres were distributed within the adventitia and outer third of the media in proximal coronary segments, and dense perivascular nerve plexuses were observed in small coronary arteries. Acetylcholine induced contractions in all preparations examined and relaxations in 20% of the segments contracted with the thromboxane analogue U46619. EFS did not induce neurogenic contractions in lamb small coronary arteries. In the presence of the alpha-adrenoceptor antagonist, phentolamine, EFS caused frequency-dependent reproducible relaxations that were enhanced by the blocker of cholinergic transmission, botulinum neurotoxin. An inhibitor of AChE, physostigmine, had no significant effect on the relaxations caused by EFS, while both the muscarinic receptor antagonist, atropine, and the muscarinic M2-receptor antagonist, AFDX 116, enhanced these responses. Blockade of sympathetic neurotransmission with guanethidine or incubation with the P2-receptor antagonist, suramin, abolished the relaxations induced by EFS, whereas propranolol was without effect. Low-frequency EFS caused less relaxation in preparations activated by acetylcholine than in those contracted with U46619, while sensitivity and maximal relaxation induced by adenosine 5'-triphosphate (ATP) were not different in U46619- and acetylcholine-contracted arteries. The presence of the enzymes necessary for both biosynthesis and degradation of acetylcholine and the finding that blockers of cholinergic neurotransmission enhance EFS-induced relaxations suggest that small coronary arteries are cholinergically innervated.

Inhibition of GTPase activity of Gi proteins and decreased agonist affinity at M2 muscarinic acetylcholine receptors by spermine and methoctramine.

1. The effects of spermine and methoctramine, a selective M2 muscarinic receptor antagonist, were studied on the high-affinity GTPase activity of G proteins, and on ligand binding to M2 muscarinic receptors in pig heart sarcolemma. 2. The spontaneous GTP hydrolysis by pig heart sarcolemma and its stimulation by mastoparan or carbachol were prevented by pertussis toxin and inhibited by methoctramine (IC50s: 21, 13 and 0.005 microM, respectively), and spermine (IC50s: 967, 278 and 11 microM). Spermine and methoctramine also inhibited spontaneous GTP hydrolysis by rat peritoneal mast cell membranes which do not respond to carbachol. 3. The neutral muscarinic antagonists, AF-DX 116 and atropine, did not modify the inhibitory effect of high concentrations of methoctramine, indicating that this effect was not related to the antagonist binding site of muscarinic receptors. We suggest that methoctramine behaves as a receptor antagonist at nanomolar concentrations and interacts with G proteins at micromolar concentrations. 4. Spermine did not modify the binding of the tritiated muscarinic antagonist [3H]-NMS, but decreased the binding of the agonist [3H]-Oxo-M. Spermine elicited a rightward shift of the carbachol/[3H]-NMS binding isotherm with a decrease in the proportion of sites with high-affinity for carbachol, suggesting that polyamines uncouple Gi proteins from receptors. 5. The inhibition of GTPase activity by polyamines, preventing the re-association of alpha and betagamma subunits of Gi proteins, might sustain the regulatory effect of Gi subunits on downstream effectors. The level of intracellular polyamines might be important for the control of the transduction of extracellular signals through Gi protein-coupled receptors.