M1-selective Antagonist Research Articles

Muscarinic agonist-mediated heterologous desensitization in isolated ileum requires activation of both muscarinic M2 and M3 receptors.

We investigated the subtypes of the muscarinic receptor mediating short-term heterologous desensitization in the isolated ileum. Treatment of the ileum from C57BL/6 mice with acetylcholine (30 microM) for 20 min caused a subsequent decrease in contractile sensitivity to both prostaglandin F2alpha (PGF2alpha) and the muscarinic agonist, oxotremorine-M. This subsensitivity was characterized by 7- and 3-fold increases in the EC50 values of the agonists, respectively, with no significant effect on the maximal response. The subsensitivity to PGF2alpha was prevented in both M2 and M3 muscarinic receptor knockout mice. Similarly, the subsensitivity to oxotremorine-M was prevented in M2 knockout mice. Acetylcholine-mediated desensitization of histamine-induced contractions in the guinea pig ileum was inhibited by both M2- and M3-selective muscarinic antagonists with high potency, although careful analysis of the data suggested behavior more consistent with an M2 antagonistic profile. Modeling studies showed that the competitive antagonism of response contingent upon activation of two receptor subtypes should exhibit a pharmacological profile similar to that of the least sensitive signaling pathway. Our results demonstrate that muscarinic agonist-mediated short-term heterologous desensitization of intestinal smooth muscle is contingent upon activation of both M2 and M3 muscarinic receptors and that activation of either receptor by itself is insufficient to cause desensitization.

Aging-related alterations in the contractile responses to acetylcholine, muscarinic cholinoceptors and cholinesterase activities in jejunum and colon of the male Fischer 344 rats

In an attempt to examine whether the muscarinic receptor-activated intestinal function is altered by aging, we studied the changes in (1) contractile responses to acetylcholine (Ach), (2) muscarinic cholinoceptors and (3) cholinesterase (ChE) activities, in jejunum and colon of the young (2–3 months) and aged (24–28 months) Fischer 344 rats. In the physiological contraction experiments of jejunum and colon, Ach concentration-dependently increased the force of contraction, and the contractile responses to Ach were not affected by aging. In addition, the true- and pseudo-ChE activities were not significantly changed by aging. The Ach-induced contraction was competitively inhibited by muscarinic M3-selective antagonist hexahydro-sila-difenidolhydrochloride p-fluoroanalog ( p-F-HHSiD), suggesting that the contractile responses in the rat jejunum and colon were mediated through M3-cholinoceptor. Age-related changes in muscarinic cholinoceptors of jejunum and colon were determined with the use of specific muscarinic radioligand [ 3H]-quinuclidinylbenzilate (QNB). The [ 3H]QNB saturation binding experiments revealed that the maximal binding ( B max) was increased only in aged jejunum without changes in K D values. These results suggest that aging may not attenuate the Ach-induced intestinal contraction via muscarinic M3 receptor, although the expression of muscarinic cholinoceptor is differentially modulated in jejunum and colon.

Antinociception of intrathecal cholinesterase inhibitors and cholinergic receptors in rats.

Intrathecal cholinesterase inhibitors have been shown to have an antinociceptive effect which is mediated through the spinal cholinergic receptors, mainly muscarinic receptor. Spinal nicotinic receptor also has been involved in the control of nociception. Authors characterized the respective role of muscarinic or nicotinic receptor for the antinociception of cholinesterase inhibitors and further determined the antinociceptive potency of them. Rats were prepared with intrathecal catheters. Formalin-induced flinching response was regarded as a nociceptive behavior. Intrathecal neostigmine, physostigmine and edrophonium produced a dose-dependent suppression of flinching in both phases. Atropine and the M1 selective antagonist attenuated the effect of them, while the M2 selective antagonist did not affect. M3, M4 selective, and nicotinic receptor antagonists reversed the antinociception induced by edrophonium, but by neither neostigmine nor physostigmine. The ordering of potency was neostigmine > physostigmine > > edrophonium. These data indicate that the nicotinic receptor may be involved, at least in part, in the antinociceptive action of cholinesterase inhibitor at the spinal level, and M1 receptor subtype may be a common pharmacologic site of action. Moreover, neostigmine is more potent than physostigmine and edrophonium.

Deficits in avoidance responding after paradoxical sleep deprivation are not associated with altered [ 3H]pirenzepine binding to M1 muscarinic receptors in rat brain

Previous work had indicated that animals that were sleep-deprived and then trained on a passive avoidance task show poor retention of the task 24 h later after being allowed to sleep freely again. Cholinergic involvement is suggested by the fact that this effect is prevented by treatment with the muscarinic agonist pilocarpine during sleep deprivation. The observation that similar deficits are observed in non-deprived rats after treatment with M1-selective antagonist compounds such as dicyclomine or pirenzepine cause similar impairments, and gave rise to the hypothesis that sleep deprivation might induce significant reductions in M1 binding in brain areas involved in learning and memory processes. Rats were deprived of sleep for 96 h and then either immediately killed, or allowed to recover sleep for 24 h before being killed. [ 3H]pirenzepine binding to M1 sites was examined by quantitative autoradiography in 39 different brain areas in cage controls, sleep-deprived and sleep-recovered animals ( N=8 per group). No significant differences among groups were found in any brain region. A separate group of animals was subjected to the sleep deprivation procedure and then trained in a simple avoidance task. Animals were then allowed to sleep and retested 24 h later. This group showed a significant impairment in the avoidance task compared to cage controls, in agreement with previous observations. These data suggest that proactive learning/memory deficits induced by sleep deprivation cannot be attributed to altered M1 binding either immediately after deprivation (when avoidance training occurs) or after sleep has recovered (when acquisition/retention are tested). The possibility remains that alterations in M1 function occur at post-membrane second messenger systems.

A mechanism for rapacuronium-induced bronchospasm: M2 muscarinic receptor antagonism.

A safe and effective ultra-short-acting nondepolarizing neuromuscular blocking agent is required to block nicotinic receptors to facilitate intubation. Rapacuronium, which sought to fulfill these criteria, was withdrawn from clinical use due to a high incidence of bronchospasm resulting in death. Understanding the mechanism by which rapacuronium induces fatal bronchospasm is imperative so that newly synthesized neuromuscular blocking agents that share this mechanism will not be introduced clinically. Selective inhibition of M2 muscarinic receptors by muscle relaxants during periods of parasympathetic nerve stimulation (e.g., intubation) can result in the massive release of acetylcholine to act on unopposed M3 muscarinic receptors in airway smooth muscle, thereby facilitating bronchoconstriction. Competitive radioligand binding determined the binding affinities of rapacuronium, vecuronium, cisatracurium, methoctramine (selective M2 antagonist), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; selective M3 antagonist) for M2 and M3 muscarinic receptors. Rapacuronium competitively displaced 3H-QNB from the M2 muscarinic receptors but not from the M3 muscarinic receptors within clinically relevant concentrations. Fifty percent inhibitory concentrations (mean +/- SE) for rapacuronium were as follows: M2 muscarinic receptor, 5.10 +/- 1.5 microm (n = 6); M3 muscarinic receptor, 77.9 +/- 11 microm (n = 8). Cisatracurium and vecuronium competitively displaced 3H-QNB from both M2 and M3 muscarinic receptors but had affinities at greater than clinically achieved concentrations for these relaxants. Rapacuronium in clinically significant doses has a higher affinity for M2 muscarinic receptors as compared with M3 muscarinic receptors. A potential mechanism by which rapacuronium may potentiate bronchoconstriction is by blockade of M2 muscarinic receptors on prejunctional parasympathetic nerves, leading to increased release of acetylcholine and thereby resulting in M3 muscarinic receptor-mediated airway smooth muscle constriction.

THIS MONTH IN ANESTHESIOLOGY

THIS MONTH IN ANESTHESIOLOGY

Intracellular calcium signaling through the cADPR pathway is agonist specific in porcine airway smooth muscle.

Cyclic ADP-ribose (cADPR) induces intracellular Ca2+ ([Ca2+]i) release in airway smooth muscle, and the cADPR antagonist, 8-amino-cADPR, abolishes [Ca2+]i oscillations elicited by acetylcholine (ACh), suggesting that cADPR is involved during muscarinic receptor activation. Whether the cADPR signaling pathway is common to agonists acting through different G protein-coupled receptors is not known. Using digital video imaging of Fura2-AM loaded porcine airway smooth muscle cells, we examined the effects of the membrane-permeant cADPR antagonist, 8-bromo-cADPR (8Br-cADPR), on the [Ca2+]i responses to ACh, histamine and endothelin-1 (ET-1). In cells preincubated with 100 microM 8Br-cADPR, the [Ca2+]i responses to ACh and ET-1 were significantly attenuated, whereas responses to histamine were not, suggesting agonist specificity of cADPR signaling. The effects of 8Br-cADPR were concentration dependent. We further examined whether muscarinic receptor subtypes specifically couple to this pathway, because in porcine airway smooth muscle cells, ACh activates both M2 and M3 muscarinic receptors coupled to Gai and Gaq, respectively. Methoctramine, an M2-selective antagonist, attenuated the [Ca2+]i responses to Ach, and there was no further attenuation by 8Br-cADPR. In airway smooth muscle, the CD38/cADPR signaling pathway is involved in [Ca2+]i responses to contractile agonists in an agonist-specific manner.

Postsynaptic muscarinic M1 receptors activate prefrontal cortical EEG of C57BL/6J mouse.

Recent pharmacological studies exploring the functional roles of muscarinic cholinergic receptor (mAChR) subtypes in prefrontal cortex of C57BL/6J (B6) mouse have provided evidence for a presynaptic M2 autoreceptor. The B6 mouse was chosen for these studies because it is a genetically well-characterized model that also provides the genomic background for many genetically modified mice. In addition to increasing ACh release, one functional consequence of pharmacologically blocking the cortical M2 autoreceptor is activation of the contralateral prefrontal cortical EEG. To date, the mechanisms through which M2 autoreceptor antagonism causes cortical EEG activation have not been investigated. The present study tested the hypothesis that, in the B6 mouse, prefrontal cortical ACh activates the contralateral prefrontal EEG via postsynaptic M1 receptors. This hypothesis was tested in 15 mice using in vivo microdialysis delivery of muscarinic antagonists with simultaneous quantification of ACh release, number of 7- to 14-Hz EEG spindles, and fast Fourier transformation analysis of prefrontal EEG. Dialysis delivery of the nonsubtype selective muscarinic antagonist scopolamine (10 nM) significantly (P = 0.01) increased ACh release. Quantitative EEG analysis showed that scopolamine did not alter contralateral prefrontal cortical EEG. To differentiate mAChR subtypes mediating pre- versus postsynaptic responses, additional experiments used muscarinic antagonists with different affinities for the five mAChR subtypes. Microdialysis delivery of 3 nM AF-DX 116, a muscarinic antagonist with relatively high affinity for the M2 and M4 subtypes, significantly (P < 0.01) increased prefrontal cortical ACh release and activated EEG in the contralateral prefrontal cortex. EEG activation was characterized by a significant decrease in number of 7- to 14-Hz EEG spindles (P < 0.0001) and power (Vrms) of EEG slow waves (P < 0.05). Microdialysis delivery of 3 nM AF-DX 116 plus 3 nM pirenzepine, a relatively selective M1 and M4 muscarinic antagonist, also significantly (P < 0.01) increased ACh release but did not decrease the number of EEG spindles and did not change EEG slow waves. The differential EEG and ACh responses to dialysis delivery of the muscarinic antagonists support the conclusion that, in B6 mouse, postsynaptic muscarinic receptors of the M1 subtype are a primary site by which ACh activates the EEG.

Theta-Frequency Synaptic Potentiation in CA1In VitroDistinguishes Cognitively Impaired from Unimpaired Aged Fischer 344 Rats

Hippocampal-dependent learning and memory deficits have been well documented in aging rodents. The results of several recent studies have suggested that these deficits arise from weakened synaptic plasticity within the hippocampus. In the present study, we examined the relationship between hippocampal long-term potentiation (LTP) in vitro and spatial learning in aged (24-26 months) Fischer 344 rats. We found that LTP induced in the CA1 region using theta-frequency stimulation (5 Hz) is selectively impaired in slices from a subpopulation of aged rats that had shown poor spatial learning in the Morris water maze. LTP at 5 Hz in aged rats that did not show learning deficits was similar to that seen in young (4-6 months) controls. We also found that 5 Hz LTP amplitude strongly correlated with individual learning performance among aged rats. The difference in 5 Hz LTP magnitude among aged rats was not attributable to an altered response to 5 Hz stimulation or to differences in the NMDA receptor-mediated field EPSP. In addition, no performance-related differences in LTP were seen when LTP was induced with 30 or 70 Hz stimulation protocols. Finally, both 5 Hz LTP and spatial learning in learning-impaired rats were enhanced with the selective muscarinic M2 antagonist BIBN-99 (5,11-dihydro-8-chloro-11-[[4-[3-[(2,2-dimethyl-1-oxopentyl)ethylamino]propyl]-1-piperidinyl]acetyl]-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one). These findings reinforce the idea that distinct types of hippocampal LTP offer mechanistic insight into age-associated cognitive decline.

Model of rapid gastrointestinal transit in dogs: effects of muscarinic antagonists and a nitric oxide synthase inhibitor.

Our aims were to establish a canine model of rapid gastrointestinal transit, and to test the effects of muscarinic receptor antagonists (atropine, pirenzepine, AF-DX116, and darifenacin), and an NOS inhibitor, L-nitro-N-arginine (L-NNA) in this model. For gastric emptying and small bowel transit, 99mTc-labelled DTPA were added to a meal of skimmed milk (236 mL) that contained 2.4 g of magnesium hydroxide. Regional colonic transit was measured by111In-labelled beads placed in a capsule that released isotope in the proximal colon. Scintiscans were taken at regular intervals and indices of transit were calculated. Drugs were administrated intravenously. Gastric emptying, small bowel and colonic transit were rapid. Atropine and darifenacin (a selective M3 antagonist) delayed gastric emptying and colonic transit, the selective M1 and M2 muscarinic antagonists did not. The muscarinic blockers did not slow small bowel transit. L-NNA delayed small bowel and colonic transit but did not slow gastric emptying. A model suitable for the preclinical study of antidiarrhoeals was established. M3 receptors are important in the control of gastric emptying and colonic transit, and NOS inhibition slowed small bowel and colonic transit.

Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction.

M(3) muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M(2) receptors participate in contraction because M(3)-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M(2)-selective antagonist methoctramine in the denervated bladder is consistent with M(3) receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M(2) receptor and one by the M(3) receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M(2) and M(3) receptors can induce contraction. In the denervated bladder, the M(2) and the M(3) receptors interact in a facilitatory manner to mediate contraction.

The Role of M2 Muscarinic Receptor Subtypes Mediating Contraction of the Circular and Longitudinal Smooth Muscle of the Pig Proximal Urethra

We investigated the characterization of muscarinic receptor subtypes in the female pig urethra. The affinities of muscarinic antagonists against carbachol responses were calculated in normal and cyclic adenosine monophosphate (cAMP) elevated tissues (contraction with KCl and relaxation with isoprenaline) using longitudinal and transverse strips of urethra. In displacement experiments with 1-quinuclidinyl [phenyl-4-3H] benzilate inhibitory constant (pKi) values of 4-diphenylacetoxyl-N-methyl-piperidine methiodide (DAMP) M3 selective antagonist) and methoctramine (M2 antagonist) indicated the presence of the M2 receptor. In functional studies contraction responses to carbachol were greater in longitudinal than in circular muscle. After cAMP elevation the contraction response increased in circular muscles to the level close to that of longitudinal muscles but did not change significantly in cAMP elevated longitudinal muscle. On normal circular tissues 4-DAMP had high mean affinity (mean apparent pKB value 9.3) but with a Schild slope of less than unity (0.76). Methoctramine competitively antagonized carbachol responses (mean affinity [pA2] value 6.9). On normal longitudinal tissues 4-DAMP and methoctramine competitively antagonized carbachol responses (mean pA2 9.0 and 6.2, respectively). After cAMP elevation in circular tissues mean pA2 values for 4-DAMP (8.7) were significantly lower (p = 0.0015), and those for methoctramine (7.3) were significantly higher (p = 0.0193) than in normal tissues. In longitudinal tissues the mean pA2 value for methoctramine (6.9) was significantly greater than in normal tissues (p <0.0001) but the value for 4-DAMP (8.8) did not alter. Pig urethra appears to have predominantly M2 muscarinic receptors. Contraction of the normal urethra appears to be predominantly mediated by M2 and M3 receptors in circular muscle but by M3 receptors in longitudinal muscle. After cAMP elevation a contribution to contraction of M2 receptors appeared to be demonstrated in the 2 tissues but the involvement of M2 receptors appeared greater in circular muscle.

A novel short convergent entry into himbacine derivatives.

[reaction: see text]. The IMDA reaction of 9 leads with good stereoselectivity to exo-adduct 10b. The functionalized ABC-ring core in 10 is well suited for the convergent synthesis of analogues of himbacine, a naturally occurring M2 selective muscarine receptor antagonist, as illustrated with the further synthesis of the dehydro-derivative 5.

THE ROLE OF M2 MUSCARINIC RECEPTOR SUBTYPES IN MEDIATING CONTRACTION OF THE PIG BLADDER BASE AFTER CYCLIC ADENOSINE MONOPHOSPHATE ELEVATION AND/OR SELECTIVE M3 INACTIVATION

In the bladder body M2 muscarinic receptors predominate but a smaller population of M3 receptors mediates direct detrusor contraction. M2 receptors have an indirect role by inhibiting cyclic adenosine monophosphate mediated relaxation in the bladder body. We investigated whether a similar mechanism also exists in the bladder base. Experiments were performed on pig detrusor muscle. In receptor binding studies using l-quinuclidinyl [phenyl-4-(3)H] benzilate ([(3)H]QNB) (NEN Life Science Products, Inc., Boston, Massachusetts) displacement experiments were performed with subtype selective antagonists to determine the M2-to-M3 receptor ratio. In functional studies the affinity of these antagonists against carbachol induced contractions was calculated in normal tissues and in tissues after cyclic adenosine monophosphate elevation by pre-contraction with KCl and relaxation with isoprenaline, and/or selective M3 inactivation by incubation with 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) mustard (Sigma Chemical Co., St. Louis, Missouri) in the presence of methoctramine (Sigma Chemical Co.) to protect M2 receptors. In saturation binding studies receptor density was 130.5 of fmol./mg. protein and the dissociation constant for [(3)H]QNB was 0.27 nM. Displacement of [(3)H]QNB by the M3 selective antagonist 4-DAMP and the M2 antagonist methoctramine indicated an M2-to-M3 ratio of about 3:1. In functional studies 4-DAMP and methoctramine caused competitive antagonism of responses with affinity values of 9.5 and 6.3 in normal tissues, and 9.3 and 6.1, respectively, in cyclic adenosine monophosphate elevated tissues, suggesting the involvement of M3 receptors only. In tissues in which M3 receptors were inactivated and cyclic adenosine monophosphate levels were elevated the affinity of 4-DAMP was significantly reduced to 8.5 but that of methoctramine was significantly increased to 6.5, suggesting the involvement of M2 receptors. The M3 subtype appears to mediate contraction of the normal and cyclic adenosine monophosphate elevated tissues of the bladder base. Involvement of M2 receptors was only noted after selective M3 inactivation and cyclic adenosine monophosphate elevation.

A new enzymic method for the isolation and culture of human bladder body smooth muscle cells.

Cultured cells of the human urinary bladder smooth muscle are useful for investigating bladder function, but methods for culturing them are not well developed. We have now established a novel enzymic technique. The smooth muscle layer was separated out and incubated with 0.2% trypsin for 30 min at 37 degrees C. The samples were then minced and incubated with 0.1% collagenase for 30 min and centrifuged at 900 g. The pellets were resuspended in RPMI-1640 medium containing 10% fetal calf serum (FCS) and centrifuged at 250 g. The smooth muscle cells from the supernatant were cultured in RPMI-1640 containing 10% FCS. The cells grew to confluence after 7-10 days, forming the "hills and valleys" growth pattern characteristic of smooth muscle cells. Immunostaining with anti-alpha-actin, anti-myosin, and anti-caldesmon antibodies demonstrated that 99% of the cells were smooth muscle cells. To investigate the pharmacological properties of the cultured cells, we determined the inhibitory effect of muscarinic receptor antagonists on the binding of [3H]N-methylscopolamine to membranes from cultured cells. The pKi values obtained for six antagonists agreed with the corresponding values for transfected cells expressing the human muscarinic M2 subtype. Furthermore, carbachol produced an increase in the concentration of cytoplasmic free Ca2+ an action that was blocked by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 selective antagonist. This result suggests that these cells express functional M3 muscarinic receptors, in addition to M2 receptors. The subcultured cells therefore appear to be unaffected by our new isolation method.

Which muscarinic receptor is important in the bladder?

Antimuscarinic agents are the most widely used therapy for urge incontinence, but have side effects such as constipation, tachycardia and dry mouth, resulting from a lack of selectivity for the bladder. M2 receptors are the predominant cholinoceptors present in urinary bladder, but mainly the minor population of M3 receptors mediate its contraction. M2 receptors modulate detrusor contraction by several mechanisms, and may contribute more to contraction of the bladder in pathological states such as bladder denervation or spinal cord injury. Prejunctional inhibitory M2 or M4 receptors and prejunctional facilitatory muscarinic Ml receptors in the bladder have all been reported. In clinical studies, tolterodine, a non-selective muscarinic antagonist, has been reported to be as effective as oxybutynin but inducing less dry mouth. Thus, although it is not certain which antimuscarinic drugs have the better efficacy and tolerability, the non-selective antimuscarinic drugs seem to be better than M3-selective antagonists in their clinical efficacies. However, controlled release, or intravesical, intravaginal, or rectal administrations of oxybutynin have been reported to cause fewer side effects. Darifenacin, a new M3 selective antagonist, has been reported to have selectivity for the bladder over the salivary gland in vivo. To verify which antimuscarinic drugs selective for the muscarinic subtypes have the best efficacy and tolerability, comparative clinical trials between M3 selective antagonists and non-selective compounds, such as olterodine, are required in the future.

The minor population of M3-receptors mediate contraction of human detrusor muscle in vitro.

1 The objective was to determine the role of muscarinic receptor subtypes in mediating contraction of the human detrusor smooth muscle in vitro. 2 Contractile responses of human detrusor muscle strips to carbachol were obtained in the absence and presence of a range of muscarinic antagonists (pirenzepine, methoctramine, 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP), tropicamide, oxybutynin and tolterodine). Affinity estimates (pKB values) were calculated for the antagonists and correlated with values at the cloned muscarinic receptor subtypes quoted in the literature. 3 Pirenzepine, methoctramine and tropicamide drugs that have high affinities at M1, M2 and M4-receptors, respectively, all had low affinities on the human detrusor (pKB values of 6.8, 6.9 and 6.5, respectively), whilst the M3-selective antagonist 4-DAMP had a high affinity (9.5). Schild plots for all four antagonists had slopes of unity indicating an action at a single receptor. Oxybutynin and tolterodine also acted as competitive antagonists with affinity estimates of 7.6 and 8.1, respectively. 4 When the antagonist affinities obtained on the bladder were plotted against the values published for these antagonists at the cloned muscarinic receptor subtypes, the best correlations were obtained for the m3- and m5-muscarinic receptor subtypes. 5 These data suggest that direct contractile responses of the human detrusor muscle to muscarinic receptor stimulation in vitro are mediated solely via the M3-muscarinic receptor subtype with no contribution from the major M2-receptor population.

Inhibition of field stimulation-induced contractions of rabbit vas deferens by muscarinic receptor agonists: selectivity of McN-A-343 for M1 receptors.

Inhibition of the field stimulation-induced twitch responses of the rabbit vas deferens by the muscarinic receptor agonist, McN-A-343, has been attributed to presynaptic muscarinic receptors of the M1 subtype located on noradrenergic nerve terminals. Stimulation of these receptors causes inhibition of transmitter release and inhibition of the contractile response. However, the selectivity of McN-A-343 for M1 receptors has been questioned and this throws doubt on whether the prejunctional receptors of the rabbit vas deferens are of the M1 subtype. In this study we have undertaken a comprehensive re-evaluation of the inhibition of prostatic and epididymal portions of the rabbit isolated field-stimulated vas deferens by several agonists, including McN-A-343, and quantified the antagonism by M1-selective antagonists, pirenzepine and telenzepine. Prostatic and epididymal portions of vasa deferentia from New Zealand White rabbits were immersed in a low Ca2+ Krebs solution at 32+/-0.5 degrees C gassed with 5% CO2 in oxygen. Yohimbine (1.0mM) was present throughout to block prejunctional alpha2-adrenoceptors. Field stimulation was applied by repeated application of single pulses (30 V, 0.05 Hz, 0.5 ms) and isometric contractions recorded. Carbachol and oxotremorine initially potentiated the epididymal contractions but at higher concentrations there was inhibition. In the prostatic portion, oxotremorine only inhibited. McN-A-343 produced inhibitory responses only in both epididymal and prostatic portions. Pirenzepine shifted the concentration-response curves forthe inhibitory responses to oxotremorine to the right. However, the potentiation of the twitches also became more apparent with the lower concentrations of oxotremorine. Schild plots for the antagonism by pirenzepine yielded pA2 values of 7.96+/-0.004 and 7.7+/-0.02 for the epididymal and prostatic portions, respectively. The concentration-response curves for the inhibition of twitches by McN-A-343 were displaced to the right in a parallel manner by pirenzepine in both prostatic and epididymal portions with no potentiation of the twitches. The Schild plot for this antagonism generated pA2 values of 7.68+/-0.01 and 8.07+/-0.01, respectively. Telenzepine caused parallel shifts of the McN-A-343 concentration-response curves to the right in prostatic portions, the pA2 value being 8.70+/-0.13. Telenzepine (10(-7) M) abolished the inhibitory effect of carbachol to reveal only concentration-dependent potentiation of the contractions. The Schild plot for antagonism of this contractile effect yielded a pA2 value (7.07+/-0.09) that was significantly less by almost two orders of magnitude (1.70) than the value for the antagonism by telenzepine of the McN-A-343-induced inhibitory response. The pA2 values of pirenzepine and telenzepine against the inhibitory responses of the rabbit vas deferens are consistent with the involvement of M1 receptors. This leads to the conclusion that McN-A-343 causes inhibition through this receptor type. The doubts concerning the selectivity of McN-A-343 for M1 receptors are therefore unfounded. The fact that McN-A-343 does not display a selective binding profile suggests that its selectivity does not arise from affinity differences but probably resides in its intrinsic efficacy.

Blood Pressure and Heart Rate Are Increased by AF-DX 116, a Selective M2 Antagonist, in Autonomic Imbalanced and Hypotensive Rats Caused by Repeated Cold Stress

Rats exposed to SART (specific alternation of rhythm in temperature) stress, which are ideal animal models for vagotonia-type dysautonomia, show various changes in cardiac and circulatory systems. In this study, attention was directed to cholinergic function in the SART-stressed rat heart and the effects of AF-DX 116, a specific muscarinic M2 antagonist, on blood pressure and heart rate. The results were compared with those obtained for atropine and pirenzepine. In SART-stressed rats, systolic and diastolic blood pressures (SBP and DBP) were lower than in unstressed rats. Oral AF-DX 116 resulted in greater elevation of DBP than SBP in unstressed rats. In stressed rats, greater and more prolonged elevation of SBP than in unstressed rats was noted, particularly at higher doses. A dose-dependent SBP change in stressed rats, caused by intravenous AF-DX 116, was shifted upward in parallel with that in unstressed groups, unlike with oral administration. The positive chronotropic effect of this drug was smaller in stressed rats than in unstressed rats, in contrast to the pressor effect. SART-stressed rats may thus have an enhanced sympathetic tone in the heart, as well as changes in muscarinic M2 receptors at sympathetic nerve endings and at the heart muscle. The effects of AF-DX 116 on blood pressure and heart rate thus may arise from peripheral action and AF-DX 116 may be useful for treating hypotension related to autonomic imbalance of the vagotonia type.

Human esophageal smooth muscle cells express muscarinic receptor subtypes M(1) through M(5).

Receptor characterization in human esophageal smooth muscle is limited by tissue availability. We used human esophageal smooth muscle cells in culture to examine the expression and function of muscarinic receptors. Primary cultures were established using cells isolated by enzymatic digestion of longitudinal muscle (LM) and circular muscle (CM) obtained from patients undergoing esophagectomy for cancer. Cultured cells grew to confluence after 10-14 days in medium containing 10% fetal bovine serum and stained positively for anti-smooth muscle specific alpha-actin. mRNA encoding muscarinic receptor subtypes M(1)-M(5) was identified by RT-PCR. The expression of corresponding protein for all five subtypes was confirmed by immunoblotting and immunocytochemistry. Functional responses were assessed by measuring free intracellular Ca(2+) concentration ([Ca(2+)](i)) using fura 2 fluorescence. Basal [Ca(2+)](i), which was 135 +/- 22 nM, increased transiently to 543 +/- 29 nM in response to 10 microM ACh in CM cells (n = 8). This response was decreased <95% by 0.01 microM 4-diphenylacetoxy-N-methylpiperidine, a M(1)/M(3)-selective antagonist, whereas 0.1 microM methoctramine, a M(2)/M(4)-selective antagonist, and 0.1 microM pirenzepine, a M(1)-selective antagonist, had more modest effects. LM and CM cells showed similar results. We conclude that human smooth muscle cells in primary culture express five muscarinic receptor subtypes and respond to ACh with a rise in [Ca(2+)](i) mediated primarily by the M(3) receptor and involving release of Ca(2+) from intracellular stores. This culture model provides a useful tool for further study of esophageal physiology.