Cholinergic Receptors in the Human vas Deferens

Abstract

This study represents the first investigation demonstrating the contractile response to exogenous acetylcholine (ACh) in the isolated human vas deferens. Pharmacological characterization of cholinergic receptors was achieved using selective antagonists to define receptor subtypes. In the HVD the effect of exogenous ACh is revealed as a dose-dependent sudden increase in the basal tension of the vasa. The ACh receptors of the HVD were competitively antagonized by atropine (ATR) with a high pA₂ value (8.78). The main finding of this study is the presence of cholinergic receptors of the pharmacologically defined M₂-ACh subtype in the isolated HVD, according to the pA₂ values obtained with pirenzepine (PRZ) 7.39, AF-DX 116 (AF) 5.92 and 4-DAMP 5.65, M₁-ACh, _M2-ACh and M₃-ACh selective antagonists, respectively. Prazosin (PZ), a selective α₁-adrenergic antagonist, displayed a similar competitive antagonism for the contractile response evoked both by ACh (pA₂ = 8.69) and NE (pA₂ = 8.58) in the HVD. The antagonism exerted by PZ on the ACh-induced contractile response of the HVD, suggests that ACh probably acts at a presynaptic level stimulating the release of NE from an adrenergic neuron. According to these findings, the receptor involved in this action, located in the proximity of the nerve terminals, seems to be of the M₂-ACh subtype.     

Expression of muscarinic acetylcholine receptors (M1-, M2-, M3- and M4-type) in the neuromuscular junction of the newborn and adult rat

Using intracellular recording and immunohistochemistry, we studied the presynaptic muscarinic autoreceptor subtypes controlling ACh release in the neuromuscular junctions of the newborn (3-6 days postnatal) and adult (30-40 days) rat. In the Levator auris longus muscles of both newborn and adult rats, acetylcholine release was modified by the M1-receptor selective antagonists pirenzepine (10 microM) and MT-7 (100 nM) and by the M2-receptor selective antagonists methoctramine (1 microM) and AF-DX 116 (10 microM). The M4-receptor selective antagonists tropicamide (1 microM) and MT-3 (100 nM) can also modify the neurotransmitter release in certain synapses of the newborn muscles. The neurotransmitter release was not altered by the M3-receptor selective antagonist 4-DAMP (1 microM) in the adult or newborn rats. However, we directly demonstrate by immunocytochemistry the presence of these receptors in the motor endplates and conclude that M1-, M2-, M3- and M4-type muscarinic receptors are present in all the neuromuscular junctions of the rat muscle both in newborn and adult animals. These receptors may be located in the perisynaptic glial cell as well as at the nerve terminals.     

Messenger RNA Levels and Binding Sites of Muscarinic Acetylcholine Receptors in Gastrointestinal Muscle Layers from Healthy Dairy Cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M₁to M₅ in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [³H]-QNB (1-quinuclidinyl-[phenyl-4-³H]-benzilate) binding by M antagonists [atropine (M_{1 ? 5}), pirenzepine (M₁), methoctramine (M₂), 4-DAMP (M₃), and tropicamide (M₄)] was used to identify receptors at the functional level. Maximal binding (B_max) was determined through saturation binding with atropine as a competitor. The mRNA levels of M₁, M₂, M₃, and M₅ represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M₄ was undetectable. The mRNA levels of M₂ and of M₃ in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [³H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [³H]-QNB binding. The [³H]-QNB binding was dose-dependent and saturable. B_max in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B_max and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.     

Functional characterization of a muscarinic receptor in the smooth muscle of the shark (Squalus acanthias) ventral aorta

A suite of muscarinic receptor blockers was used to characterize the receptor(s) mediating the contractile effect of acetylcholine (ACh) on isolated rings of ventral aorta from the dogfish shark, Squalus acanthias. The M₂/M₄-specific inhibitor N,N’-bis(6-{[(2-methoxyphenyl) methyl] amino} hexyl) -1,8- octane diamine tetrahydrochloride (methoctramine) did not reduce the efficacy of ACh, and the M₃-specific inhibitor 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) displaced the ACh concentration-response curve to the right at much lower concentrations than the M₁-specific inhibitor (5-11-dihydro-11- [4-methyl-l-piperazinyl)acetyl] -6H-pyrido[2,3-b] [1,4] benzodiazepin-6-one dihydrochloride) (pirenzepine). It appears, therefore, that an M₃-type muscarinic receptor is expressed in the aortic vascular smooth muscle of the dogfish shark.     

Messenger RNA levels and binding sites of muscarinic acetylcholine receptors in gastrointestinal muscle layers from healthy dairy cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M(1)to M(5) in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [(3)H]-QNB (1-quinuclidinyl-[phenyl-4-(3)H]-benzilate) binding by M antagonists [atropine (M(1 - 5)), pirenzepine (M(1)), methoctramine (M(2)), 4-DAMP (M(3)), and tropicamide (M(4))] was used to identify receptors at the functional level. Maximal binding (B(max)) was determined through saturation binding with atropine as a competitor. The mRNA levels of M(1), M(2), M(3), and M(5) represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M(4) was undetectable. The mRNA levels of M(2) and of M(3) in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [(3)H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [(3)H]-QNB binding. The [(3)H]-QNB binding was dose-dependent and saturable. B(max) in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B(max) and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.     

Muscarinic receptors involved in airway vascular leakage induced by experimental gastro-oesophageal reflux

Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Studies in humans or in animals have suggested that these responses involve cholinergic nerves. The purpose of this study was to investigate the role of the efferent vagal component on airway microvascular leakage induced by instillation of hydrochloric acid (HCl) into the oesophagus of guinea-pigs and the subtype of muscarinic receptors involved. Airway microvascular leakage induced by intra-oesophageal HCl instillation was abolished by bilateral vagotomy or by the nicotinic receptor antagonist, hexamethonium. HCl-induced leakage was inhibited by pretreatment with atropine, a non-specific muscarinic receptor antagonist, and also by pretreatment with either pirenzepine, a muscarinic M(1) receptor antagonist, or 4-DAMP, a muscarinic M(3) receptor antagonist. Pirenzepine was more potent than atropine and 4-DAMP. These antagonists were also studied on airway microvascular leakage or bronchoconstriction induced by intravenous administration of acetylcholine (ACh). Atropine, pirenzepine and 4-DAMP inhibited ACh-induced airway microvascular leakage with similar potencies. In sharp contrast, 4-DAMP and atropine were more potent inhibitors of ACh-induced bronchoconstriction than pirenzepine. Methoctramine, a muscarinic M(2) receptor antagonist, was ineffective in all experimental conditions. These results suggest that airway microvascular leakage caused by HCl intra-oesophageal instillation involves ACh release from vagus nerve terminals and that M(1) and M(3) receptors play a major role in cholinergic-mediated microvascular leakage, whereas M(3) receptors are mainly involved in ACh-induced bronchoconstriction.     

Age-Related Alterations In Pre-Synaptic and Receptor-Mediated Cholinergic Functions In Rat Brain

Fractional [³H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [³H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [³H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 M) significantly decreased stimulation-induced fractional [³H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [³H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [³H]ACh release from striatum of both age groups is modulated mainly by M₃ muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo-³H]inositol decreased with aging, carbachol-stimulated [³H]myo inositol-1-fosfat (IP₁) accumulation was found similar in striatal, cortical and hippocampal slices.     

Selective inactivation of muscarinic receptor subtypes

Muscarinic receptors exist in multiple subtypes, denoted as M₁, M₂ M₃ and M₄, encoded by four distinct but related genes. A fifth gene product, m5, has also been predicted although this sequence awaits a pharmacological equivalent. Many tissues express more than one muscarinic receptor subtype, which may couple to different intracellular effectors and thus have different physiological roles. One way to characterize the role of each receptor is to selectively inactivate one receptor population, thus pharmacologically ‘isolating’ the muscarinic receptor subtype of interest. Selective receptor inactivation can be achieved using either a selective, irreversible antagonist, or protection using a selective, reversible antagonist against a non-selective irreversible antagonist. Therefore, combination of these two approaches may provide optimal selective inactivation. Several muscarinic alkylating agents have been identified, including phenoxybenzamine, EEDQ (N-Ethoxycarbonyl-1-ethoxy-1,2-dihydroquinoline) and propylbenzilylcholine mustard. These irreversible antagonists do not, in general, discriminate between muscarinic receptor subtypes and are frequently used to estimate the affinity and relative efficacy of muscarinic agonists. Consequently, use of these irreversible antagonists provides estimations of the ‘receptor reserve’ associated with a response mediated by muscarinic receptor activation. In contrast, 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl)piperidine) selectively inactivates M₃ receptors, but will not discriminate between M₁ M ₂ or M₄ receptors. In the absence of highly selective alkylating agents, receptor protection by reversible antagonists may be used. Thus, reversible antagonists, such as pirenzepine, methoctramine or para-fluorohexahydrosiladifenidol, at appropriate fractional receptor occupancies, may protect M₁ M₂ or M₃ receptors against alkylation by phenoxybenzamine. Selective alkylation of M₃ receptors by 4-DAMP mustard is enhanced with concurrent M₂ protection. This approach has been applied to defining the role of these muscarinic receptor subtypes in the control of ileal smooth muscle tone. These data suggest that, in ileum, M₂ receptors may act to inhibit β-adrenoceptor activation, thereby offsetting relaxation, while M₃ receptors directly mediate contraction.     

Muscarinic M1 receptors stimulate phosphoinositide hydrolysis in bovine cerebral arteries

The muscarinic agonist oxotremorine-M produced a concentration-dependent increase in phosphoinositide hydrolysis in bovine pial arteries. The maximal effect was 5.9 +/- 0.89 fold over basal levels, and the EC50 for oxotremorine-M was 8.9 x 10(-6) M. The phosphoinositide response in arteries with the luminal endothelium removed was similar to the response in intact arteries. The specific muscarinic antagonists pirenzepine, 4-DAMP and methoctramine produced parallel shifts of the concentration-response curve to oxotremorine-M, with the following order of potency (pKB): 4-DAMP (8.59 +/- 0.10) greater than pirenzepine (8.12 +/- 0.11) greater than methoctramine (6.77 +/- 0.20). These results indicate that muscarinic stimulation activates phosphoinositide hydrolysis in cerebral arteries, and that the muscarinic receptors mediating this increase are similar to the M1 subtype.     

Pharmacological Profiles of the Subtypes of Muscarinic Cholinoceptors That Mediate Aggregation of Pigment in the Melanophores of Two Species of Catfish

Using selective antagonists, including pirenzepine, adiphenine, AF-DX116, gallamine, and 4-DAMP we attempted to characterize the muscarinic cholinoceptors on the melanophores of the translucent glass catfish Kryptopterus bicirrhis and the mailed catfish Corydoras paleatus. The M₃ receptor-selective antagonist, 4-DAMP, potently inhibited the acetylcholine-induced aggregation of pigment in both species. It appeared, therefore, that the receptors that mediated the cholinergically evoked aggregation of melanosomes in these species were of the M₃ muscarinic subtype.     

Roles of muscarinic receptor subtypes in small intestinal motor dysfunction in acute radiation enteritis

Administration of abdominal radiotherapy results in small intestinal motor dysfunction. We have developed a rat radiation enteritis model that, after exposure in vivo, shows high-amplitude, long-duration (HALD) pressure waves in ex vivo ileal segments. These resemble in vivo dysmotility where giant contractions migrate both antegradely and retrogradely. Mediation of these motor patterns is unclear, although enteric neural components are implicated. After the induction of acute radiation enteritis in vivo, ileal segments were isolated and arterially perfused. TTX, hexamethonium, atropine, or the selective muscarinic antagonists pirenzepine (M(1)), methoctramine (M(2)), and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; M(3)) were added to the perfusate. The baseline mean rate per minute per channel of HALD pressure waves was 0.35 +/- 0.047. This was significantly reduced by TTX (83.3%, P < 0.01), hexamethonium (90.3%, P < 0.03), and atropine (98.4%, P < 0.01). The HALD pressure wave mean rate per minute per channel was significantly reduced by pirenzepine (81.1%, P < 0.03), methoctramine (96.8%, P < 0.001), and 4-DAMP (93.1%, P < 0.03) compared with predrug baseline data. As an indicator of normal motility patterns, the frequency of low-amplitude, short-duration pressure waves was also assessed. The mean rate per minute per channel of 5.15 +/- 0.98 was significantly increased by TTX (19%, P < 0.05) but significantly reduced by pirenzepine (35.1%, P < 0.02) and methoctramine (75%, P < 0.0003). However, the rate of small-amplitude pressure waves was not affected by hexamethonium, atropine, or the M(3) antagonist 4-DAMP. The data indicate a role for neuronal mechanisms and the specific involvement of cholinergic receptors in generating dysmotility in acute radiation enteritis. The effect of selective M(3) receptor antagonism suggests that M(3) receptors may provide specific therapeutic targets in acute radiation enteritis.     

Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulin-secreting β cell line

Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal β cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1–m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 μM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 μM of methoctramine (M2 antagonist) increased ACh (100 μM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release.     

Effects of cholinergic agents on the vasopressin-mediated water transport in the isolated toad bladder

The aim of this work was to study the effect of some pharmacological cholinergic agents on the events that follow the interaction of arginine vasopressin with toad bladder membrane receptors related to synthesis of 3′5′_cAMP. The water flow through the membrane was measured gravimetrically in sac preparations of the membrane. In the absence of arginine vasopressin (AVP), carbachol induced a significant increase in the water flow (37%) related to the basal (Ringer's solution). On the other hand, when carbachol and AVP were associated, a significant decrease of AVP hydrosmotic activity occurred (23%). The inhibitory effect of carbachol on the AVP action was almost completely abolished by the cholinergic antagonists atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and the calcium antagonist lanthanum. Similarly, when carbachol and 3′5′ cyclic adenosine monophosphate (3′5′_cAMP) were associated, a decrease of nucleotide hydrosmotic activity was observed (12.80%). This effect was partially restored by the addition of pirenzepine or 4-DAMP in the bath solution. These results suggest a role for muscarinic receptors of sub-type M₁ and M₃, which are involved in the intracellular calcium release. The increase of calcium concentration in the intracellular medium acts as a negative modulator in the hydrosmotic action of antidiuretic hormone.     

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

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M₂ receptors, and strychnine did so also at the M₄ receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M₂) and scopolamine (M₂), between strychnine and butylscopolamine (M₄), L-hyoscyamine (M₂ and M₄) and scopolamine (M₄), and between brucine and scopolamine (M₂). Positive effects of alcuronium, strychnine and brucine on the affinity of the M₂ receptors for 4-DAMP have been confirmed by direct measurements of the binding of [³H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M₂ receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.     

M3 muscarinic receptor activation of a delayed rectifier potassium current in canine atrial myocytes.

Growing body of evidence indicates that the functional responses of cells to muscarinic acetylcholine receptors (mAChRs) are mediated by multiple receptor subtypes. It is commonly thought that the M2 receptor is the only functional mAChR subtype in the heart and little data regarding the potential roles of other subtypes in cardiac tissues has been reported. In the present study, we provide functional evidence for the presence and physiological function of an M3 receptor in canine atrial myocytes. Using whole-cell patch-clamp techniques, we consistently found that pilocarpine, an mAChR agonist, induced a K+ current similar to but distinct from the classical delayed rectifier K+ current. Same observations were obtained when choline or tetramethylammonium (TMA) was applied to the bath. The currents were abolished by 1 microM atropine. Antagonists selective to M1 (pirenzepine, 100 nM), M2 (methoctramine 100 nM), or M4 (tropicamide 200 nM) receptors failed to alter the currents. Conversely, three different M3-selective inhibitors, p-F-HHSiD (20-200 nM), 4-DAMP methiodide (2-10 nM) and 4-DAMP mustard (4-20 nM), all produced concentration-dependent suppression of the currents. A cDNA fragment representing the M3 receptor was isolated from dog atrial RNA and the mRNA level of this construct was 0.7 +/- 0.1 pg/microg total RNA, as quantified by the competitive RT-PCR methods. Our data strongly suggested that an M3 receptor exists and is coupled to a K+ channel in the heart.     

Cholinergic Dilatation and Constriction of Feline Cerebral Blood Vessels Are Mediated by Stimulation of Phosphoinositide Metabolism via Two Different Muscarinic Receptor Subtypes

Abstract: The muscarinic receptors involved in phosphoinositide (PI) hydrolysis have been pharmacologically characterized in cat cerebral blood vessels. Carbachol elicited a concentration-dependent increase in inositol phosphate accumulation [inositol monophosphate, bisphosphate, trisphosphate (IP₃) and tetrakisphosphate] in both major cerebral arteries and small pial vessels, which reached 140–280% of baseline at 10^?3M carbachol (referred to as maximal effect). However, the inositol phosphate accumulation response was found to be biphasic with a submaximal effect (30–50% of the maximal stimulation) obtained at low carbachol concentrations (<10^?5M). Endothelial denudation induced a virtual disappearance of the submaximal PI response without affecting that elicited by high concentrations of carbachol. The pharmacology of the two carbachol-induced PI responses was investigated by comparing the potency of selected muscarinic antagonists to block the IP₃ accumulation induced by 10^?7M (endothelium-dependent submaximal effect) and 10^?4M (endothelium-independent near-maximal effect) carbachol. In both major arteries and pial vessels, the activation of IP₃ production by 10^?4M carbachol was similarly inhibited by muscarinic antagonists with the following averaged rank order of potency (in -log IC₅₀): 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 8.65) > pirenzepine (8.28) > 6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H-dibenzo[b,e][1,4]diazepine-11-one (UH-AH 371; 7.87) > 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,-11-dihydro-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116; 6.62), a pharmacological profile compatible with an M₁ receptor subtype. In contrast, the submaximal stimulation of the PI metabolism elicited by 10^?7M carbachol in major arteries was blocked by the same antagonists with the following order of potency (in -log IC₅₀): 4-DAMP (8.38) > pirenzepine (7.25) > UH-AH 371 (6.25) > AF-DX 116 (5.72), which was reminiscent of an M₃ pharmacological profile. These findings indicate that stimulation of cerebrovascular muscarinic receptors is accompanied by PI hydrolysis via two distinct receptors, most probably the M₁ and M₃ subtypes that have been associated with constriction and dilatation, respectively, of cat cerebral arteries. Furthermore, these results provide strong evidence for an endothelial localization of the M₃ dilatatory receptors within the vessel wall.     

Cyclic GMP regulates M3AChR activity at plasma membranes from Airway smooth muscle

Abstract

Muscarinic acetylcholine receptors MAChRs from Bovine Tracheal Smooth Muscle (BTSM) plasma membranes are responsible for the cGMP rise and signal-amplitude peaks associated with smooth muscle contraction present in bronchial asthma. These MAChRs bind [³H]QNB and exhibit the classic G Protein Coupled-Receptor (GPCR) behavior towards muscarinic agonist and antagonists that is sensitive to sensitive to GTP analogs. Interestingly, the [³H]QNB binding activity was stimulated by cGMP and ATP, and was enhanced by IBMX and Zaprinast, inhibitors of cGMP-PDE. Cyclic GMP plus ATP affected the agonist-antagonist muscarinic binding activities. Thus, the high affinity agonist (Carbamylcholine) binding sites disappeared, whereas, 4-DAMP, a M₃ selective antagonist displayed an additional high affinity-binding site. In contrast, non-selective (atropine) and M₂-selective (methoctramine and gallamine) antagonists revealed one low binding site. Moreover, the 4-DAMP-mustard alkylation of the MAChRs blocked the cGMP effect indicating that the M₃AChR is the main receptor target of cGMP. Interestingly, these cGMP effects were potentiated by an activator (Sp-8-pCPT-cGMPS), and diminished by an inhibitor (Rp-8-pCPT-CGMPS), of cGMP-dependent protein kinase (PKG-II), which was detected by Western blotting using specific PKG II antibodies. Finally, plasma membrane M₃AChRs were phosphorylated in a cGMP-dependent manner and this novel post-translational reversible modification at M₃AChRs may act as a feedback mechanism to terminate the cGMP dependent muscarinic signal transduction cascades at the sarcolema of BTSM.     

The significance of extracellular Ca<Superscript>2+</Superscript> in contractile responses of chick amnion

Neurotransmitter receptors are formed during chick embryo development in the amnion, an avascular extraembryonic membrane devoid of innervation. Carbachol induces phasic and tonic contractions mediated by M₃ cholinoceptors in an amniotic membrane strip isolated from 11–14-day-old chick embryo. The carbachol effect on the amnion contractile activity was studied in normal physiological salt solution, during depolarization by K⁺, exposure to nifedipine, and in calcium-free medium. Voltage-dependent and receptor-operated Ca²⁺ channels as well as calcium from intracellular stores are involved in the contractile response to carbachol. Phasic contractions of the amnion are mainly induced by calcium ions entering through voltage-dependent calcium channels, while tonic contractions are also maintained by receptor-operated channels. Ca²⁺-activated potassium channels can serve as a negative feedback factor in regulation of the amnion contractile responses.     

Affinities of muscarinic drugs for [3H]N-methylscopolamine (NMS) and [3H]oxotremorine (OXO) binding to a mixture of M1−M4 muscarinic receptors: Use of NMS/OXO-M ratios to group compounds into potential agonist,partial agonist,and antagonist classes

The relative affinities of various muscarinic drugs in the antagonist ([³H]N-methyl scopolamine ([³H]NMS)) and agonist ([³H]Oxotremorine-m ([³H]OXO-M)) binding assays using a mixture of tissues containing M₁–M₄ receptor subtypes have been determined. [³H]NMS bound with high affinity (K_d=25±5.9 pM; n=3) and to a high density (B_max=11.8±0.025 nmol/g wet weight) of muscarinic receptors. [³H]OXO-M appeared to bind to two binding sites with differing affinities (K_d1=2.5±0.1 nM; K_d2=9.0±4.9 M; n=4) and to a different population of binding sites (B_max1=5.0±0.26 nmol/g wet weight; B_max2=130±60 nmol/g wet weight). Well known antagonists exhibited high affinity for [³H]NMS binding but a lower affinity for [³H]OXO-M binding. The opposite was true for acetylcholine and other known agonists. However, pilocarpine and McN-A-343 had similar affinities for sites labeled by both radioligands. Using the ratios of antagonist-to-agonist binding affinities, it was possible to group compounds into apparently distinct full agonist (ratios of 180–665; e.g. carbachol, muscarine, OXO-M, OXO-S and arecoline), partial agonist (ratios of 14–132; e.g. McN-A-343, pilocarpine, aceclidine, bethanechol, OXA-22 and acetylcholine) and antagonist (ratios of 0.22–1.9; e.g. atropine, NMS, pirenzepine, methoctramine, 4-DAMP and p-fluorohexahydrosialo-difenidol) classes. These data suggest that the NMS/OXO-M affinity ratios using a mixture of M₁–M₄ muscarinic receptors may be a useful way to screen and group a large number of compounds into apparent agonist, partial agonist, and antagonist classes of cholinergic agents.     

Change in muscarinic modulation of transmitter release in the rat urinary bladder after spinal cord injury

Muscarinic facilitation of 14C-ACh release from post-ganglionic parasympathetic nerve terminals was studied in bladder strips prepared from spinal intact (SI) and spinal cord transected (SCT) rats. The spinal cord was transected at the lower thoracic spinal segments 3 weeks prior to the experiments. Using non-facilitatory stimulation (2 Hz) the release of ACh in spinal intact rats did not change in the presence of a non-specific muscarinic antagonist, atropine (100 nM), an M(1) specific antagonist (pirenzepine, 50 nM) or an M(1)-M(3) specific antagonist (4-DAMP, 5 nM). However, during a facilitatory stimulation paradigm (10 Hz or 40 Hz, 100 shocks) atropine and pirenzepine, but not 4-DAMP inhibited the release of ACh in bladders from spinal intact rats, indicating an M(1) receptor-mediated facilitation. In spinal cord transected rats, 2 Hz stimulation-induced release was significantly inhibited by atropine or 4-DAMP but not by pirenzepine indicating that a pre-junctional facilitatory mechanism mediated via M(3) muscarinic receptors could be induced by a non-facilitatory stimulation paradigm after spinal injury. In bladders of spinal cord transected rats, 10 Hz stimulation-evoked release of ACh was also inhibited by atropine and 4-DAMP (5 nM) but not by pirenzepine (50 nM). These results indicate that pre-junctional muscarinic receptors at cholinergic nerve endings in the bladder change after chronic spinal cord injury. It appears that low affinity M(1) muscarinic receptors are replaced by high affinity M(3) receptors. This change in modulation of ACh release may partly explain the bladder hyperactivity after chronic spinal cord injury.