PubMed Health⌕ Search

Biomedical subjects

F J Ehlert

Publications and source records attributed to F J Ehlert.

At least 37 records · Page 2Linked to original sources
PubMed1996

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.

Adrenergic beta-Agonists↗
PubMed1996

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.

1-Methyl-3-isobutylxanthine↗
PubMed1996

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.

Alkylation↗
PubMed1996

The quaternary transformation products of N-(3-chloropropyl)-4-piperidinyl diphenylacetate and N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) have differential affinity for subtypes of the muscarinic receptor.

A 3-chloropropylamine derivative (N-(3-chloropropyl)-4-piperidinyl diphenylactate) of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate was synthesized and its conversion to a stable azetidinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4, N-(3-chloropropyl)-4-piperidinyl diphenylactate formed a stable azetidinium ion with a half-time of approximately 3.6 hr. The selectivity of the azetidinium ion for native M1, M2 and M3 subtypes of the muscarinic receptors was investigated in competitive binding experiments on the hippocampus, heart and submaxillary gland of rats, respectively, using N-[3H]methylscopolamine as the radioligand. The azetidinium ion exhibited equivalent high affinities for the M1 and M3 mucarinic receptor subtypes (KD = approximately 5 nM), but 10-fold lower affinity for the M2 muscarinic receptor subtype (KD = 44 nM). Similar competitive binding experiments were carried out on Chinese hamster ovary cells transfected with the M1 through M5 subtypes of the muscarinic receptor. In these experiments, the azetidinium ion exhibited similar high affinities for the M1, M3, M4 and M5 muscarinic receptor subtypes (KD = approximately 2.4 nM), but approximately 14-fold lower affinity for the M2 muscarinic receptor subtype (KD = 34 nM). In contrast to the azetidinium ion, the parent N-(3-chloropropyl)-4-piperidinyl diphenylactate compound was 130-fold less potent. An analogous series of experiments were carried out with the aziridinium ion derived from the muscarinic receptor alkylating agent, N-(2-chloroethyl)-4-piperidinyl diphenylactate. For these binding experiments, the incubations were carried out at 0 degrees C to prevent the aziridinium ion from alkylating muscarinic receptors. The aziridinium ion was found to have equivalent high affinities for the M1, M3, M4 and M5 subtypes of the muscarinic receptor (KD = approximately 6.6 nM), but about 11-fold lower affinity for the M2 muscarinic receptor subtype (KD = 72 nM). Our results suggest that 3-haloalkylamine derivatives of 4-piperidinyl diphenylactate may be candidate prodrugs that may penetrate into brain and form azetidinium ions that have a long-lasting central anticholinergic effect.

Animals↗
PubMed1996

The interaction of the enantiomers of aceclidine with subtypes of the muscarinic receptor.

The pharmacological activity of the enantiomers of aceclidine was investigated in Chinese hamster ovary cells transfected with the M1 through M5 subtypes of the muscarinic receptor and also in the rat heart and parotid gland that express primarily M2 and M3 receptors, respectively. When measured by stimulation of phosphoinositide hydrolysis in Chinese hamster ovary cells transfected with the M1, M3 and M5 muscarinic subtypes, the potency of S-(+)-aceclidine was approximately 2- to 4-fold greater than that of R-(-)-aceclidine, whereas the maximal response of the R-(-)-isomer was only 44 to 64% that of the S-(+)-isomer. When measured by inhibition of forskolin-stimulated cyclic AMP accumulation in Chinese hamster ovary cells transfected with the M2 and M4 muscarinic subtypes, the potency of S-(+)-aceclidine was approximately 3.5-fold greater than that of R-(-)-aceclidine. In cells transfected with the M2 muscarinic receptor, the maximal responses of the enantiomers were the same, whereas the maximal response of R-(-)-aceclidine was 86% that of S-(+)-aceclidine in cells transfected with the M4 muscarinic subtype. The activities of the enantiomers of aceclidine at native M2 and M3 muscarinic receptors coupled to inhibition of adenylyl cyclase activity in the heart and stimulation of phosphoinositide hydrolysis in the parotid gland, respectively, were similar to those observed in Chinese hamster ovary cells transfected with the corresponding receptor subtypes. We devised a simple quantitative method for using our data in Chinese hamster ovary cells to predict the relative potencies of agonists in a more sensitive assay in which the agonists produce a full maximum response. By using this method, we were able to predict the relative potencies of the enantiomers for eliciting contractions in the guinea pig ileum, an M3 muscarinic response, from their activity in Chinese hamster ovary cells transfected with the M3 muscarinic subtype. Our method of analysis should have application in a variety of studies in which transfected cells are used to determine the pharmacological activity of agonists.

Adenylyl Cyclases↗
PubMed1995

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.

Animals↗
PubMed1994

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.

Aging↗
PubMed1994

Pertussis toxin blocks M2 muscarinic receptor-mediated effects on contraction and cyclic AMP in the guinea pig ileum, but not M3-mediated contractions and phosphoinositide hydrolysis.

The effects of pertussis toxin on muscarinic receptor-induced contractions of the isolated guinea pig ileum were investigated. In control tissues, isoproterenol (1 microM) only slightly inhibited contractions in response to oxotremorine-M; however, in pertussis toxin-treated tissues, isoproterenol exhibited an enhanced inhibition of the concentration-effect curve. Under basal conditions, pertussis toxin had no dampening effect on oxotremorine-M-induced contractions. When ilea were precontracted with histamine (1 microM) and then relaxed back to base line using forskolin (10 microM) before contractile responses to oxotremorine-M were measured, pertussis toxin shifted the concentration-effect curve to oxotremorine-M by a 6.1-fold increase in the EC50 value. Ilea were then incubated with [N-(2-chloroethyl)-4-piperidinyl diphenylacetate] (40 nM; 1 hr) in the presence of [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (1 microM) to selectively inactivate the M3 muscarinic receptors. Under these conditions, pertussis toxin blocked the concentration-effect curve to oxotremorine-M by a 30-fold increase in the EC50 value. Prior treatment of guinea pigs in vivo with pertussis toxin diminished the labeling of a 41-kDa protein in membranes suspensions of the longitudinal muscle incubated with [32P]nicotinamide adenine dinucleotide] and pertussis toxin. This ADP-ribosylation caused a functional uncoupling of the G protein from its receptor as indicated by radioligand-binding studies and second messenger assays. Our results verify that the M2 muscarinic acetylcholine receptor, like the M3 receptor, can elicit contractions of the guinea pig ileum and that the mechanism of this action involves inhibition of adenylate cyclase.

Adenosine Diphosphate Ribose↗
PubMed1993

Kinetics of activation and in vivo muscarinic receptor binding of N-(2-bromoethyl)-4-piperidinyl diphenylacetate: an analog of 4-DAMP mustard.

4-DAMP mustard (N-2-chloroethyl)-4-piperidinyl diphenylacetate) has been shown to selectively and irreversibly inhibit muscarinic receptors. In an attempt to increase the rate of formation and peak concentration of the reactive intermediate, an analog [N-(2-bromoethyl)-4-piperidinyl diphenylacetate (4-DAMP bromo mustard)] was synthesized and the molecular formula confirmed by mass analysis. The 4-DAMP bromo mustard was shown to cyclize in phosphate buffer (pH 7.4) to the corresponding aziridinium ion with a first-order rate constant (k1) of 0.071 min-1 at 0 degrees C. At 25 degrees C and 37 degrees C, the formation of the aziridinium ion was nearly instantaneous (100% cyclized within 15 sec) at neutral pH. The rate constants (k2) for the hydrolysis of the aziridinium ion at 25 degrees C and 37 degrees C (pH 7.4) were 0.0027 and 0.010 min-1, respectively, in excellent agreement with the published rate constants for the hydrolysis of the aziridinium ion formed from 4-DAMP mustard. In vivo treatment with 4-DAMP bromo mustard in rats resulted in irreversible inhibition of muscarinic receptor binding in peripheral, but not central nervous system, tissues, suggesting that the quickly formed aziridinium ion does not penetrate the blood-brain barrier.

Animals↗
PubMed1993

Functional role for the M2 muscarinic receptor in smooth muscle of guinea pig ileum.

A functional role for the M2 muscarinic receptor in smooth muscle contraction was investigated in isolated guinea pig ileum. Contractile responses to the muscarinic agonist oxotremorine-M (oxo-M) were measured in isolated ilea that had been pretreated with histamine (0.32 microM) and isoproterenol (0.64 microM) to achieve conditions of elevated cAMP. The resulting concentration-effect curve was biphasic, consisting of high (0-50 nM) and low (> 50 nM) potency components. The reversible M2-selective antagonist AF-DX 116 ([[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one) (1 and 10 microM) shifted this curve in a manner that was inconsistent with competitive antagonism at a single receptor site; the high affinity component was significantly blocked, whereas there was little effect on the low affinity portion of the curve. To inactivate the M3 muscarinic receptors selectively, ilea were incubated with the irreversible M1/M3-selective muscarinic antagonist 4-DAMP mustard [N-(2-chloroethyl)-4-piperidinyldiphenylacetate] (40 nM) for 1 hr in the presence of AF-DX 116 (1 microM) and were then washed extensively. Under these conditions, the contractile responses to oxo-M, in the presence of histamine and isoproterenol or forskolin, were antagonized by AF-DX 116 (1 microM) in a manner consistent with that mediated by an M2 receptor. AF-DX 116 caused 6.6- and 11-fold increases in the EC50 value for oxo-M for ilea pretreated with isoproterenol and forskolin, respectively, and a significant increase in the Hill coefficient in both cases. Under basal conditions, AF-DX 116 caused only a 1.34-fold increase in the EC50 value and no change in the Hill coefficient. In addition, under basal conditions 4-DAMP mustard treatment shifted the oxo-M contractile response curve to the right approximately 20-fold. However, when histamine was present in combination with isoproterenol or forskolin 4-DAMP mustard treatment shifted the concentration-effect curves for oxo-M to the right only about 3.5-fold. Oxo-M produced an M3-mediated stimulation of phosphoinositide hydrolysis in the longitudinal muscle of rat ileum with an EC50 value of 30 microM. 4-DAMP mustard (10 nM; 1 hr) prevented this response, resulting in a 6.6-fold increase in the EC50 value with a 65% reduction of the maximal response. In contrast, this treatment blocked M2-mediated inhibition of isoproterenol-stimulated adenylate cyclase with only a 2-fold increase in EC50, without affecting maximum inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Heparin, dextran and trypan blue allosterically modulate M2 muscarinic receptor binding properties and interfere with receptor-mediated inhibition of adenylate cyclase.

The influences of heparin, dextran and trypan blue on muscarinic receptor binding properties and inhibition of adenylate cyclase were investigated in homogenates of the rat heart. These compounds caused a concentration-dependent enhancement in the specific binding of the muscarinic antagonist [3H]N-methylscopolamine ([3H]NMS) when measured at a radioligand concentration of approximately 0.5 nM in magnesium-containing, low ionic strength buffer. The maximal enhancements of [3H]NMS binding were 2.89-, 1.68- and 1.43-fold increases for heparin, dextran and trypan blue, respectively; the EC50 values for this effect were 0.12, 0.033 and 4.6 microM, respectively. The effects of heparin, dextran and trypan blue on [3H]NMS binding were attributed mainly to an increase in the overall affinity of muscarinic receptors for [3H]NMS, and were greatly attenuated by 100 mM NaCl. These effects were qualitatively similar to those produced by GTP. Heparin, dextran and trypan blue also affected the binding of the muscarinic agonist oxotremorine-M in a manner similar to that of GTP; that is, in the presence of these compounds, agonist affinity was decreased. Our experiments also showed that heparin and dextran attenuate the inhibition of adenylate cyclase activity caused by oxotremorine-M in myocardial homogenates without influencing basal adenylate cyclase activity. We conclude that heparin and dextran interfere with the muscarinic receptor-G protein coupling in the rat heart.

Adenylyl Cyclase Inhibitors↗
PubMed1992

Specific inhibition of isoproterenol-stimulated cyclic AMP accumulation by M2 muscarinic receptors in rat intestinal smooth muscle.

The ability of oxotremorine-M to inhibit cyclic AMP accumulation in the presence of a variety of adenylate cyclase activators was studied in slices from the longitudinal muscle of the rat ileum. Oxotremorine-M was found to inhibit forskolin- and isoproterenol-stimulated cyclic AMP accumulation maximally by 17 and 32%, respectively, but not the stimulation due to other activators of adenylate cyclase. Inhibition of cyclic AMP accumulation by oxotremorine-M was unaffected by tetrodotoxin and was completely reversed by atropine. AF-DX 116 (11[[2-[(diethylamino)methyl]-1- piperidynyl]acetyl]-5,11-dihydro-6H-pyrido[2,3- b][1,4]benzodiazepine-6-one) an M2-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant (KB) of 0.20 microM, consistent with the dissociation constants for binding at the M2 muscarinic receptor site (KD = 0.092 microM) and inhibition of adenylate cyclase activity (KB = 0.13 microM). Hexahydrosiladifenidol, an M3-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant of 0.67 microM, again consistent with the dissociation constant for binding at the M2 site (KD = 0.83 microM). The agreement between the estimates of the dissociation constants of muscarinic antagonists for binding and for inhibition of cyclic AMP accumulation suggest that oxotremorine-M inhibition of isoproterenol-stimulated cyclic AMP accumulation in slices of rat intestinal smooth muscle is mediated by the M2 receptor.

Adenylyl Cyclases↗
PubMed1992

Conversion of N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) to an aziridinium ion and its interaction with muscarinic receptors in various tissues.

A 2-chloroethylamine derivative [N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard)] of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) was synthesized, and its conversion to an aziridinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4 and 37 degrees, 4-DAMP mustard released an equivalent amount of chloride. The release of chloride was consistent with a first-order process having a half-time of 5.7 min. The aziridinium ion reached a peak concentration at 32 min, corresponding to 75% of the initial concentration of 4-DAMP mustard. When homogenates of rat brain, heart, and submaxillary gland were incubated with 4-DAMP mustard (9 nM) for 1 hr, washed extensively, and then assayed for muscarinic receptor binding properties, a 56% decrease in the binding capacity of N-[3H]methylscopolamine in the heart and brain and a 71% decrease in the gland were observed, without a significant change in the dissociation constants. The affinity of 4-DAMP mustard and its transformation products for muscarinic receptors was determined in competitive binding experiments with N-[3H] methylscopolamine, and the results show that the aziridinium ion of 4-DAMP mustard was the most potent form, compared with the parent 2-chloroethylamine (4-DAMP mustard) and the alcoholic hydrolysis product. The rates of receptor alkylation by 4-DAMP mustard were measured in the rat heart and gland. Virtually no alkylation (less than 1%) occurred in the heart at a 4-DAMP mustard concentration of 1.6 nM, after 30 min, whereas almost 50% alkylation was observed in the gland under the same conditions. Almost complete alkylation of receptors in the gland could be achieved at a 4-DAMP mustard concentration of 200 nM, after 1 hr. Treatment of the isolated rat ileum with 4-DAMP mustard caused an irreversible blockade of contractions elicited by the muscarinic agonist oxotremorine-M, and this blockade persisted after extensive washing. The results presented here show that 4-DAMP mustard forms an aziridinium ion that binds irreversibly to muscarinic receptors and exhibits selectivity for M3, compared with M2 muscarinic receptors.

Alkylation↗
PubMed1991

Heterogeneity of muscarinic receptor subtypes in cerebral blood vessels.

The identity and distribution of muscarinic cholinergic receptor subtypes and associated signal transduction mechanisms was characterized for the cerebral circulation using correlated functional and biochemical investigations. Subtypes were distinguished by the relative affinities of a panel of muscarinic antagonists, pirenzepine, AF-DX 116 [11-2-[[2-[diethylaminomethyl]- 1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one], hexahydrosiladifenidol, methoctramine, 4-diphenylacetoxy-N-methylpiperidine methobromide, dicyclomine, para-fluoro-hexahydrosiladifenidol and atropine. Muscarinic receptors characterized by inhibition of [3H]quinuclidinylbenzilate binding in membranes of bovine pial arteries were of the M2 subtype. In contrast pharmacological analysis of [3H]-quinuclidinylbenzilate binding in bovine intracerebral microvessels suggests the presence of an M4 subtype. Receptors mediating endothelium-dependent vasodilation in rabbit pial arteries were of the M3 subtype, whereas muscarinic receptors stimulating endothelium-independent phosphoinositide hydrolysis in bovine pial arteries were of the M1 subtype. These findings suggest that characteristics of muscarinic receptors in cerebral blood vessels vary depending on the type of vessel, cellular location and function mediated.

Animals↗
PubMed1991

N-[3H]methylscopolamine labeling of non-M1, non-M2 muscarinic receptor binding sites in rat brain.

Radioligand binding and quantitative autoradiographic techniques were used to characterize the pharmacological profile and anatomical distribution of N-[3H]methylscopolamine [( 3H] NMS)-labeled sites under assay conditions in which radioligand binding to M1 and M2 muscarinic receptors was blocked by addition of pirenzepine and AF-DX 116 (11[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b] [1,4]benzodiazepine-6-one) to the incubation buffer. Nonlinear regression analysis of saturation data demonstrated that a large proportion of atropine-displaceable [3H]NMS binding persisted in the presence of saturating concentrations of M1 and M2 blockers. The residual [3H]NMS [( 3H]NMSb) sites were widely distributed throughout rat brain and represented the predominant muscarinic receptor population. The autoradiographic distribution of [3H]NMSb sites did not correspond to that of [3H]pirenzepine or [3H]AF-DX 116, indicating that [3H]NMSb labeled non-M1, non-M2 muscarinic sites. Moreover, the pharmacological profile of [3H]NMSb differed from that of [3H]pirenzepine at M1 sites, and was inconsistent with that of M2 receptor binding sites. Although we were unable to pharmacologically distinguish subpopulations of non-M1, non-M2 binding sites, the anatomical distribution of [3H]NMSb sites corresponded to that of the combined mRNA distributions for m3 and m4.

Animals↗