The M2-muscarinic receptor inhibits the development of streptozotocin-induced neuropathy in mouse urinary bladder.

We investigate the role of M(2)-muscarinic receptors in maintaining neurogenic bladder contraction during hyperglycemia. Mice were injected with a single dose of streptozotocin (125 mg/kg), and neurogenic contraction of urinary bladder from wild type and M(2)-muscarinic receptor knockout (M(2) KO) mice was measured at 8 to 24 weeks after treatment. In wild-type bladder lacking urothelium, the summation of the cholinergic (64%) and purinergic (56%) components of the electrical-field-stimulated response exceeded 100%, indicating a reserve capacity. Although the cholinergic component was slightly less in the M(2) KO mouse, the total electrical-field-stimulated contraction was the same as wild type. The cholinergic and purinergic components of contraction in wild-type bladder were minimally affected by streptozotocin treatment. In M(2) KO bladder, streptozotocin treatment reduced both the cholinergic (after 8-9 and 20-24 weeks) and purinergic (after 20-24 weeks only) components. The loss of function was approximately 50 to 70%. Similar results were observed in bladder with intact urothelium. M(2) KO bladder was more sensitive to the relaxant effect of isoproterenol compared with wild type, and this difference significantly increased at the early and late time points after streptozotocin treatment. In the presence of urothelium, however, this difference in isoproterenol sensitivity was smaller with streptozotocin treatment, but this trend reversed over time. Our results show that M(2) receptors oppose urinary bladder distension in wild-type bladder and inhibit streptozotocin-induced neuropathy.

Tertiary 2-haloethylamine derivatives of the muscarinic agent McN-A-343, [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride.

4-[(2-Chloroethyl)methylamino]-2-butynyl N-(3-chlorophenyl)carbamate (2) and 4-[(2-bromoethyl)methylamino]-2-butynyl N-(3-chlorophenyl)carbamate (3) were synthesized. Compounds 2 and 3 cyclized at neutral pH to an aziridinium ion (4). The rate constants for the cyclization of 2 and 3 at 37 degrees C were about 0.01 and 0.4 min-1, respectively, as measured by titrimetric analysis and by 1H NMR spectroscopy. The aziridinium ion had 1/4 the potency of McN-A-343 (1) as a ganglionic muscarinic stimulant in the anesthetized, pentolinium-treated rat but showed no muscarinic effects on the isolated guinea pig ileum. It caused alkylation of muscarinic receptors in homogenates of the rat cerebral cortex. An irreversible blockade of central muscarinic receptors was also observed after intravenous administration of 3 to mice. Because of its selectivity, irreversible actions, and ability to pass into the central nervous system, 3 should become a valuable tool in studies of muscarinic receptors.

Functional role of muscarinic M(2) receptors in alpha,beta-methylene ATP induced, neurogenic contractions in guinea-pig ileum.

1. The muscarinic acetylcholine receptors mediating the contractile response elicited to endogenous acetylcholine released by the selective P2X receptor agonist alpha,beta-methylene ATP (mATP) were investigated in guinea-pig ileum. 2. mATP (0.1 - 30 microM) elicited a concentration-dependent neurogenic contractile response inhibited by tetrodotoxin (TTX) and antagonized by the non-selective muscarinic receptor antagonist N-methylscopolamine (NMS). 3. The contractile response to mATP was pertussis toxin-insensitive, irreversibly antagonized by N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), and unaffected by the muscarinic M(2)/M(4) receptor selective antagonist AF-DX 116 (1 microM). 4. When measured in the presence of histamine and isoproterenol after treatment with 4-DAMP mustard, mATP elicited a pertussis toxin-sensitive contractile response potently antagonized by AF-DX 116. 5. Collectively, our data suggest that endogenous acetylcholine released by mATP can elicit a direct contractile response through the muscarinic M(3) receptor and an indirect contractile response through the muscarinic M(2) receptor by antagonizing the relaxant effects of isoproterenol on histamine induced contraction.

Involvement of the M2 muscarinic receptor in contractions of the guinea pig trachea, guinea pig esophagus, and rat fundus.

The involvement of the M2 muscarinic receptor in contractile responses of the guinea pig trachea, guinea pig esophagus, and rat fundus was investigated. In the standard assay, oxotremorine-M elicited contractions of the trachea with an EC50 value of approximately 73 nanoM.--2- -(Diethylamino)methyl- -1-piperidinyl-acetyl--5,11- dihydro-6H-pyrido-2,3-b--1,4- benzodiazepine-6-one (AF-DX 116) at 1 and 10 microM antagonized these contractions by 2.1- and 9.0-fold increases in the EC50 value for oxotremorine-M. These effects are consistent with antagonism of an M3-mediated contractile response. In subsequent experiments, the M3 receptors were first inactivated selectively by incubation with N-(2-chloroethyl)-4- piperidinyl diphenylacetate (4-DAMP mustard) (40 nanoM) for 1 hr in the presence of AF-DX 116 (1 microM) followed by extensive washing. In 4-DAMP mustard treated trachea, oxotremorine-M elicited contractions with an EC50 value of 0.31 microM in the presence of histamine (10 microM) and forskolin (4 microM). Under these conditions, AF-DX 116 at 1 and 10 microM antagonized contractions to oxotremorine-M by 8- and 59-fold increases in the EC50, respectively, while para- fluorohexahydrosiladiphenidol(p-F-HHSiD) (0.1 microM) had no effect. These effects are consistent with a contraction being mediated by an M2 receptor. In the guinea pig esophagus and rat fundus, AF-DX 116 and p-F-HHSiD blocked contractions measured under similar conditions with magnitudes intermediate between what would be expected from an M2 and an M3 receptor, suggesting that perhaps both subtypes contribute to the overall contractile response under these conditions. In addition, contractions of the guinea pig trachea measured in the presence of histamine and forskolin were pertussis toxin sensitive. These results that, in the trachea, M2 receptors can dominate the contractile response after a majority of the M3 receptors have been inactivated, whereas in the guinea pig esophagus and rat fundus, M2 receptors may contribute to, but do not play a dominant role in the overall response.

Stimulation of cyclic AMP accumulation and phosphoinositide hydrolysis by M3 muscarinic receptors in the rat peripheral lung.

The effects of oxotremorine-M (oxo-M), a muscarinic agonist, on cyclic AMP (cAMP) accumulation in slices of the rat peripheral lung were investigated. Oxo-M stimulated cAMP accumulation in a concentration-dependent manner with an EC50 value of 4.2 microM and a maximal effect of 2.4 +/- 0.39-fold over basal. In the presence of forskolin (25 microM), the maximal effect of oxo-M was increased to 14.1 +/- 4.0-fold over basal. Forskolin alone caused a 5.9 +/- 2.2-fold increase in cAMP relative to basal; therefore, the combination of both drugs was more than additive. The effects of oxo-M on cAMP accumulation were unaffected by tetrodotoxin, indicating that the action of oxo-M was not mediated by neuronal release of neurotransmitters. Oxo-M had a small inhibitory effect on cAMP in a homogenate preparation, indicating that the stimulatory response to oxo-M in slices of the lung is not due to direct stimulation of adenylyl cyclase. Characterization of the oxo-M potentiation of forskolin-stimulated cAMP accumulation using different muscarinic antagonists yielded calculated pKB values that agreed with binding affinities for the M3 subtype. Oxo-M elicited phosphoinositide hydrolysis in the lung, and the nature of the antagonism of this response was also consistent with that expected for an M3-mediated response. cAMP accumulation in the presence of oxo-M (100 microM), forskolin (12 microM), or both drugs combined was inhibited by indomethacin (1 microM). These results demonstrate that the M3 receptor stimulates cAMP accumulation and phosphoinositide hydrolysis in the rat peripheral lung, and the mechanism for cAMP stimulation may involve arachidonic acid metabolites.

Relation between behaviorally augmented tolerance and upregulation of muscarinic receptors in the CNS: effects of chronic administration of chronic administration of scopolamine.

The present experiment was planned to provide information about relations between behaviorally augmented tolerance and accompanying upregulation of muscarinic receptors (mAChR) (physiological tolerance) in the CNS during chronic administration of the cholinergic antagonist scopolamine. Analyses of the data on mAChR binding established significant upregulation (Bmax) had occurred in the cortex, hippocampus, and striatum of animals treated with scopolamine, but not of those in the saline or methylscopolamine groups. There were no treatment effects in affinity (KD). The effect of scopolamine administered before a behavioral test session was to cause an acute decrease in FR5 responding to water reinforcement, and hence in resulting water consumption. Animals immediately compensated for this decrement by higher response rates during a free drinking (FDR) period which followed. When scopolamine was injected between the FR5 and FDR periods, FR5 responding increased to compensate for the drug's effect on the FDR. There was evidence that physiological tolerance also occurred as indicated by a more slowly developing trend toward recovery of levels of behavioral responding related to mAChR upregulation, although full recovery to pretreatment baselines did not occur within the 25 days of chronic treatments. The results as a whole are consistent with a multifactorial model of tolerance development, to which both behavioral and neurochemical processes contribute.

Human urotensin II mediates vasoconstriction via an increase in inositol phosphates.

The cyclic peptide urotensin II has recently been cloned from human and reported to potently constrict primate blood vessels. To elucidate the cellular signalling mechanisms of this peptide, we investigated a possible relationship of vasomotor effects of human urotensin II and phosphoinositide turnover in isolated rabbit thoracic aorta. Human urotensin II produced a slowly developing increase in isometric contractile force (pEC(50)=9.0) that was endothelium-independent. The contractile effect of urotensin II was significantly inhibited by the phospholipase C inhibitor, 2-nitro-4-carboxyphenyl-N,N,-diphenylcarbamate (NCDC), but not by the cyclooxygenase inhibitor, indomethacin. In slices of rabbit thoracic aorta, human urotensin II increased phosphoinositide hydrolysis, and this effect was also inhibited by NCDC. The potency of urotensin II (pEC(50)=8.6) was similar to that found in the contractile studies. Thus, vasoconstrictor effects of human urotensin II appear to be mediated by a phospholipase C-dependent increase in inositol phosphates, suggesting that the peptide acts via a G(q) protein-coupled receptor.

Modulation of benzodiazepine receptor binding: insight into pharmacological efficacy.

The effects of GABA on the binding of analogues of benzodiazepines, triazolopyridazines, beta-carbolines and imidazodiazepines were examined in ligand/[3H] flunitrazepam competition experiments. GABA increased the potency of anxiolytics, like flunitrazepam, whereas the potency of benzodiazepine antagonists, like Ro15-1788, was largely insensitive to the influence of GABA. Several other agents including pyrazolopyridines, barbiturates and etomidate caused a chloride dependent enhancement of [3H] flunitrazepam binding but not an enhancement of [3H] propyl-beta-carboline-3-carboxylate binding.

Relative efficacies of cannabinoid CB1 receptor agonists in the mouse brain.

We measured (-)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohe xyl]-phenol (CP 55,940)-, (-)11-OH-delta8-tetrahydrocannabinol-dimethylheptyl (HU-210)-, anandamide- and delta9-tetrahydrocannabinol-stimulated G protein activation in mouse brain using the [35S]GTPgammaS functional assay. The Ki values for these drugs were determined by agonist competition binding with the cannabinoid CB1 receptor antagonist [3H]N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamidehydrochloride ([3H]SR141716A). This information was used to calculate the efficacy for drug stimulation of G protein activity. The rank order of efficacy was CP 55,940 > HU-210 > anandamide > delta9-tetrahydrocannabinol with the latter two drugs being partial agonists. Since efficacy values relate receptor occupancy to functional responses, we believe efficacy values are a better measure of drug-mediated functional responses compared with measurements of drug potency.

The interaction of 4-DAMP mustard with subtypes of the muscarinic receptor.

The compound 4-DAMP mustard (N-2-chloroethyl-4-piperidinyl diphenylacetate) is a 2-chloroethylamine derivative of the selective muscarinic antagonist 4-DAMP (N,N-dimethyl-4-piperidinyl diphenylacetate). At neutral pH, 4-DAMP mustard cyclizes spontaneously into an oziridinium ion that binds covalently with muscarinic receptors. Analysis of the kinetics of receptor alkylation showed that the interaction of 4-DAMP mustard with M2 and M3 receptors was consistent with a model in which the aziridinium ion rapidly forms a reversible complex with the receptor which converts to a covalent complex at a relatively slower rate. The rate constant (k2) for alkylation of M2 and M3 receptors was approximately the same (k2 = 0.1 min-1); however, the affinity of the aziridinium ion for the M3 receptor (KD = 7.2 nM) was approximately 6.3-fold greater than that for the M2 receptor (KD = 43 nM). The results of competitive binding experiments on Chinese hamster ovary cells transfected with the M1 - M5 subtypes of the muscarinic receptor showed that the affinity of the aziridinium ion for the M1, M3, M4 and M5 subtypes was approximately the same and about 11-fold greater than that for the M2 receptor. 4-DAMP mustard is a useful tool for selectively inactivating all non-M2 muscarinic receptors, particularly when it is used in the presence of a reversible M2 selective antagonist to protect the M2 receptor from alkylation. The results of studies on isolated smooth muscle preparations that have had their M3 receptors alkylated with 4-DAMP mustard are consistent with the postulate that the M2 receptor can elicit contraction by inhibiting the relaxant effect of isoproterenol and forskolin on histamine induced contractions.

Functional role of M2 muscarinic receptors in the guinea pig ileum.

Muscarinic agonists elicit contraction in the standard guinea pig ileum bioassay through activation of M3 muscarinic receptors that are also linked to phosphoinositide hydrolysis. Surprisingly, the most abundant muscarinic receptor in the ileum is the M2 which causes a specific inhibition of cyclic AMP accumulation elicited by the beta-adrenergic receptor. After most of the M3 receptors are inactivated, the ileum still retains high sensitivity to muscarinic agonists provided that the contractile responses are measured in the presence of histamine and forskolin, which together, have no effect on contraction. Under these conditions, the potencies of antagonists for blocking the contractile response are consistent with those expected for an M2 response. Moreover, the muscarinic contractile response measured in the presence of histamine and forskolin after inactivation of M3 receptors is pertussis toxin sensitive. In contrast, muscarinic contractions in the standard bioassay are pertussis toxin insensitive. These results demonstrate that the M2 muscarinic receptor can cause an indirect contraction of the guinea pig ileum by preventing the relaxing effect of agents that increase cAMP.

A comparison of the effects of cinnarizine and related compounds on [3H]nitrendipine binding in the brain, heart and ileum.

The influence of cinnarizine, flunarizine and lidoflazine on [3H]nitrendipine binding in the cerebral cortex, heart and longitudinal muscle of the ileum was investigated. When assays were run in Ca++ free Tris/HCl buffer on extensively washed tissue homogenates, cinnarizine, flunarizine and lidoflazine were approximately equipotent inhibitors of [3H] nitrendipine binding in the heart and cerebral cortex, with Ki values of approximately 10(-6) M. In contrast, the compounds were 4 to 100 times more potent in the ileum with the rank order of potency being: lidoflazine greater than flunarizine greater than cinnarizine. The same rank order of potency was observed in the ileum when assays were run in the presence of 1 mM Ca++, although all three drugs were somewhat less potent. Similarly, Ca++ inhibited the binding of the cinnarizine-like drugs in the cerebral cortex and heart as well, with all drugs being less potent than that observed in the ileum under similar assay conditions.

The use of irreversible ligands to inactivate receptor subtypes: 4-DAMP mustard and muscarinic receptors in smooth muscle.

Irreversible ligands are useful tools for investigating the function of receptor subtypes in various physiological processes. The mechanism for alkylation involves the formation of a reversible receptor complex followed by a covalent reaction. The extent of receptor alkylation is determined by the dissociation constant of the reversible complex and the rate constant for conversion to the covalent complex. Selectivity can be achieved if the irreversible ligand exhibits a difference in its dissociation constants for receptor subtypes. Selective alkylation can also be achieved using a selective competitive inhibitor to protect the desired receptor subtype. By using the non-M2-selective irreversible antagonist, 4-DAMP mustard, in combination with the competitive M2-selective antagonist, AF-DX 116, it has been possible to achieve a highly selective inactivation of all non-M2 subtypes of the muscarinic receptors in smooth muscle and has enabled the discovery of the functional role of M2 receptors in smooth muscle.

Inhibition of muscarinic stimulated phosphoinositide hydrolysis in the rat parotid gland by cAMP.

The ability of agents that increase or mimic cAMP to affect muscarinic receptor mediated phosphoinositide hydrolysis was investigated in the rat parotid gland. Forskolin (10 microM) and isoproterenol (10 microM) elevated cAMP in the parotid gland by 2-fold and 7-fold, respectively, and these agents also inhibited oxotremorine-M (3 microM) mediated phosphoinositide hydrolysis by 14% and 26%, respectively. Forskolin (1, 4.3, 18, and 75 microM) increased cAMP accumulation and inhibited PIP2 hydrolysis in a concentration-dependent manner. Forskolin (75 micrometers) shifted the concentration-response curve for the full agonist oxotremorine-M rightward by 4.2-fold. Pre-treatment with the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) reduced the maximum effect of oxotremorine-M by 31%. The inhibitory effect of isoproterenol and forskolin on muscarinic receptor-mediated phosphoinositide hydrolysis was unaffected by the removal of extracellular Ca2+. Moreover, isoproterenol and forskolin dampened sodium fluoride and oxotremorine-M mediated phosphoinositide hydrolysis to the same extent suggesting that the inhibitory effect of cAMP is downstream from the muscarinic receptor.

An alkylating derivative of oxotremorine interacts irreversibly with the muscarinic receptor.

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

Muscarinic receptors and novel strategies for the treatment of age-related brain disorders.

The muscarinic class of acetylcholine receptors is widely distributed throughout the body and mediates numerous vital functions in both the brain and autonomic nervous system. Within the brain, muscarinic receptors play an important role in learning, memory and the control of posture. There is a decrease in the synthesizing enzyme for acetylcholine in Alzheimer's disease, and damage to the ascending cholinergic system is thought to be an important determinant of the loss of memory and other functional deficits of this disease. Five subtypes of the muscarinic receptor (m1-m5) have been identified, and these receptors have a differential distribution throughout the body. The differential distribution of subtypes of the muscarinic receptor in the body suggests that centrally acting m1 and m4 muscarinic agonists might be efficacious in the treatment of age-related memory disorders, without causing peripheral side effects. In addition to the primary ligand binding site, muscarinic receptors also possess a secondary allosteric site that appears to be the target for some novel cardioselective muscarinic antagonists including the neuromuscular blocking agent gallamine. The existence of a secondary allosteric site on the muscarinic receptor suggests that it might be possible to develop novel allosteric muscarinic agonists that potentiate the effects of endogenous acetylcholine much in the same way that benzodiazepines potentiate GABA. Although no such allosteric muscarinic agonists have been identified to date, they could be very efficacious in the treatment of Alzheimer's disease.

The binding of [3H]nitrendipine to receptors for calcium channel antagonists in the heart, cerebral cortex, and ileum of rats.

The binding properties of the calcium channel antagonist, [3H]nitrendipine, were investigated in homogenates of the rat cerebral cortex, heart and ileum. The specific component of [3H]nitrendipine binding was consistent with mass-action behavior and was characterized by a high affinity dissociation constant in the range of 0.1-0.3 nM. A variety of other calcium channel antagonists inhibited the binding of [3H]nitrendipine with Ki's that agree generally with the ability of these drugs to block contractions of cardiac and smooth muscle. The inhibition of [3H]nitrendipine binding by other dihydropyridines was consistent with competitive antagonism whereas the inhibition caused by verapamil and D600 resembled negative heterotropic cooperativity. Consistent with this latter postulate was the observation that the kinetics of [3H]nitrendipine binding are altered by verapamil, with both the association rate and the dissociation rate being increased. La+3 and several divalent cations caused an inhibition of [3H]nitrendipine with the rank order of potency being Cd+2 greater than La+3 greater than Ni+2 greater than Co+2 = Mn+2 greater than Mg+2 = Ba+2 greater than Ca+2.

Subtypes of the muscarinic receptor in smooth muscle.

Muscarinic receptors are expressed in smooth muscle throughout the body. In most instances, the muscarinic receptor population in smooth muscle is composed of mainly the M2 and M3 subtypes in an 80% to 20% mixture. The M3 subtype mediates phosphoinositide hydrolysis and calcium mobilization, whereas the M2 subtype mediates an inhibition of cAMP accumulation. In addition, a variety of ionic conductances are elicited by muscarinic receptors. Muscarinic agonists stimulate a nonselective cation conductance that is pertussis toxin-sensitive and dependent on calcium. The pertussis toxin-sensitivity of this response suggests that it is mediated by M2 receptors. Following agonist induced depolarization of smooth muscle, voltage dependent calcium channels are activated to enable an influx of calcium. In some instances, muscarinic agonists enhance this conductance through a mechanism involving protein kinase C, whereas in other instances, muscarinic agonists suppress this calcium conductance. Smooth muscle often contains calcium activated potassium channels that tend to repolarize the membrane following calcium influx. Activation of muscarinic receptors suppresses this potassium conductance in some smooth muscles. Under standard conditions, muscarinic agonists elicit pertussis toxin-insensitive contractions through activation of the M3 receptor. When most of the M3 receptors are inactivated, it is possible to measure a pertussis toxin-sensitive contractile response to muscarinic agonists that is most likely mediated through M2 receptors. M2 receptors also cause an indirect contraction by inhibiting the relaxant effects of agents that increase cAMP (e.g., forskolin and isoproterenol).

Contractile role of M2 and M3 muscarinic receptors in gastrointestinal smooth muscle.

Muscarinic agonists elicit contraction through M3 receptors in most isolated preparations of gastrointestinal smooth muscle, and not surprisingly, several investigators have identified M3 receptors in smooth muscle using biochemical, immunological and molecular biological methods. However, these studies have also shown that the M2 receptor outnumbers the M3 by a factor of about four in most instances. In smooth muscle, M3 receptors mediate phosphoinositide hydrolysis and Ca2+ mobilization, whereas M2 receptors mediate an inhibition of cAMP accumulation. The inhibitory effect of the M2 receptor on cAMP levels suggests an indirect role for this receptor; namely, an inhibition of the relaxant action of cAMP-stimulating agents. Such a function has been rigorously demonstrated in an experimental paradigm where gastrointestinal smooth muscle is first incubated with 4-DAMP mustard to inactivate M3 receptors during a Treatment Phase, and subsequently, the contractile activity of muscarinic agonists is characterized during a Test Phase in the presence of histamine and a relaxant agent. When present together, histamine and the relaxant agent (e.g., isoproterenol or forskolin) have no net contractile effect because their actions oppose one another. However, under these conditions, muscarinic agonists elicit a highly potent contractile response through the M2 receptor, presumably by inhibiting the relaxant action of isoproterenol or forskolin on histamine-induced contractions. This contractile response is pertussis toxin-sensitive, unlike the standard contractile response to muscarinic agonists, which is pertussis toxin-insensitive. When measured under standard conditions (i.e., in the absence of histamine and without 4-DAMP mustard-treatment), the contractile response to muscarinic agonists is moderately sensitive to pertussis toxin if isoproterenol or forskolin is present. Also, pertussis toxin-treatment enhances the relaxant action of isoproterenol in the field-stimulated guinea pig ileum. These results demonstrate that endogenous acetylcholine can activate M2 receptors to inhibit the relaxant effects of beta-adrenoceptor activation on M3 receptor-mediated contractions. An operational model for the interaction between M2 and M3 receptors shows that competitive antagonism of the interactive response resembles an M3 profile under most conditions, making it difficult to detect the contribution of the M2 receptor.

A simple and rapid radio-receptor assay for the estimation of acetylcholine.

The high potency with which acetylcholine (ACh) inhibits the binding of the specific muscarinic agonist, [3H]cis methyldioxolane ([3H]CD), has provided the basis for the development of a radioreceptor assay for estimation of ACh. A synaptosomal preparation of the rat cerebral cortex was used as a source of muscarinic receptors. When binding assays were run at 0 degree C, the IC50 value of ACh was approximately 5 x 10(-9)M, which corresponds to 2.5 - 10 pmoles of ACh, depending upon the assay volume. The ACh content of the rat cerebral cortex and corpus striatum was measured following fast microwave irradiation. By measuring the displacement of [3H]CD binding caused by aliquots of the supernatant from tissue homogenates and comparing the displacement values with an ACh standard curve, the ACh content of the cerebral cortex and corpus striatum was calculated to be 19 and 55 nmoles/g wet tissue weight, respectively.