M2 Muscarinic Receptor Subtypes Research Articles

Carbachol effects on hippocampal neurons in vitro: dependence on the rate of rise of carbachol tissue concentration.

Nominally K-sensitive microelectrodes were used to measure carbachol (CCh) in order to study the dependence of muscarinic effects on CCh concentration and exposure time in guinea pig hippocampal slices. Interference presumably originating from tissue choline-compounds was neutralized by pre-equilibration of the slices with 500 microM choline and calibration of the CCh-sensitive microelectrodes in the presence of the same choline-concentration. Muscarinic depolarization and reduction of the afterhyperpolarization (AHP) following a train of action potentials by bath applied CCh were monitored in granule cells and CA3 pyramidal neurons by intracellular recording. A fast bath application mode of CCh was designed, by which CCh tissue concentration reached a peak after 2-3 min and was washed out with a half time of about 8 min. After application of 30 nmol CCh in this way, the AHP was reduced according to the variation of CCh concentration over time. Neurons depolarized with some delay after the reduction of the AHP and started to repolarize 1 min before the peak of tissue CCh concentration (0.6 microM) was reached. Pirenzepine (1-10 microM) blocked only the depolarization, while atropine (1-10 microM) blocked both the depolarization and the reduction of the AHP. When superfusing with CCh containing saline, 80% of the final concentration was reached in the bath after 12 min, but in the tissue only after 45 min. The slow increase of tissue CCh concentration was concurrent with the slow decrease of the AHP. No effect on the membrane potential was observed. Atropine, but not pirenzepine, blocked the reduction of the AHP. Superfusion with a high CCh concentration (100-300 microM) containing saline depolarized neurons and reduced the AHP. Then pirenzepine repolarized neurons, whereas atropine both repolarized the cells and restored the AHP. It is concluded tha the muscarinic depolarization depends not only on the CCh concentration, but also on the rate of rise of CCh, while the reduction of the AHP depends solely on the concentration. This result is discussed in terms of the possibility that the depolarization is mediated by a short term desensitizing M1 muscarinic receptor subtype and the reduction of the AHP is mediated by a M2 muscarinic receptor subtype.

Immunocytochemical and pharmacological evidence for an intrinsic cholinomimetic system modulating prolactin and growth hormone release in rat pituitary.

Pituitary cells were cultured as three-dimensional reaggregates in serum-free chemically defined medium supplemented with different concentrations of dexamethasone. Immunostaining of the cells using a polyclonal antiserum and three monoclonal antibodies raised against choline acetyl transferase (CAT), revealed the presence of CAT immunoreactivity in 4-10% of anterior pituitary cells depending on the antibody used. CAT immunoreactivity was also found in freshly dispersed anterior pituitary cells. CAT-immunoreactive cells could be enriched on BSA and Percoll gradients and codistributed with ACTH-immunoreactive cells in these gradients. Perifusion of the aggregates with the potent muscarinic receptor antagonist atropine (Atr) resulted in a dose-dependent (0.1-100 nM) increase in both basal PRL and GH secretion; the response was dependent on the dexamethasone concentration in the culture medium. A similar response to Atr was observed in organ-cultured pituitaries. The specificity of the Atr effect was supported by the findings that the potent and highly specific muscarinic receptor blocker dexetimide showed a similar action, whereas its inactive enantiomer levetimide and the nicotinic receptor blocker hexamethonium failed to do so. Two other muscarinic antagonists, benzatropine and pirenzepine, showed a dose-dependent hormone-releasing action similar to that of Atr, but were less potent than the latter. Pirenzepine was only effective at high molar concentrations, suggesting that an M2 muscarinic receptor subtype was mediating the present phenomenon. Atr also potentiated GH release stimulated by the beta-adrenergic agonist isoproterenol and PRL release stimulated by vasoactive intestinal peptide, but had no effect on GRF-stimulated GH release. The choline uptake blocker hemicholinium abolished the effect of Atr on GH and PRL release. These data suggest that certain pituitary cells can express CAT activity and that these cells exert a tonic inhibitory activity on GH and PRL release which is mediated by a cholinomimetic substance, possibly acetylcholine, through a muscarinic receptor.

Stimulation of acid secretion and phosphoinositol production by rat parietal cell muscarinic M2 receptors.

The muscarinic receptor system involved in hydrogen ion production by enriched rat gastric parietal cells was investigated. Muscarinic receptor density determined by [N-methyl-3H]scopolamine binding was 8,100/cell. The receptor appeared to be of the M2 muscarinic receptor subtype, since it had a low affinity (Kd, 189 nM) for the M1 receptor antagonist pirenzepine compared with atropine (Kd, 0.74 nM). Receptor activation by carbachol rapidly augmented levels of polyphosphoinositides, indicating an activation of a phospholipase C. The dose-response relations for the increase in inositol phosphates closely paralleled the binding of carbachol to muscarinic receptors with a Km of 17 microM. The inositol phosphate response was antagonized by pirenzepine with a Ki of 177 nM. The stimulation of inositol phosphate levels by carbachol correlated well with the stimulation of [14C]aminopyrine uptake, determined as an index of acid secretion. The muscarinic agonists oxotremorine, pilocarpine, and bethanechol elicited partial increases in inositol phosphates at maximal drug concentrations, and these partial increases correlated with their ability to stimulate [14C]aminopyrine uptake. These data indicate that inositol polyphosphates may be a second messenger of M2 receptors stimulating acid secretion.

Functional and direct binding studies using subtype selective muscarinic receptor antagonists.

1. Muscarinic receptor antagonists were examined in direct binding studies on guinea-pig cardiac and cortical muscarinic receptors. Pirenzepine, dicyclomine and hexahydroadiphenine were shown to be selective ligands for the putative M1-muscarinic receptor. 2. Functional affinity estimates of the muscarinic ligands studied was determined from their ability to inhibit carbachol-stimulated inositol phosphate (IP) accumulation in guinea-pig cortical slices. 3. The affinity estimates for the inhibition of muscarinic agonist-stimulated IP accumulation were better correlated with affinity estimates obtained from binding studies on the M1 than the M2 muscarinic receptor. 4. These data provide additional evidence, both from direct binding and functional studies, for the presence of M1 and M2 muscarinic receptor subtypes.

Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands 3H-N-methylscopolamine (3H-NMS), 3H-quinuclidinyl benzilate (3H-QNB), and 3H-pirenzepine. 3H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, 3H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by 3H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for 3H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure. In conclusion, both M1 and M2 muscarinic receptor subtypes and an adenylate cyclase system linked to at least some of these receptors are present as intrinsic components of myelin. The possibility that some of these muscarinic receptors may be involved in regulation of phosphinositide metabolism and the protein kinase activities of myelin is considered.

The M1-muscarinic acetylcholine receptor subtype is linked to the inositide cycle in rat brain synaptosomes

The M1-muscarinic acetylcholine receptor subtype is linked to the inositide cycle in rat brain synaptosomes

Binding and functional profiles of the selective M1 muscarinic receptor antagonists trihexyphenidyl and dicyclomine

The selectivity profiles of the muscarinic receptor antagonists dicyclomine and trihexyphenidyl have been examined in binding and functional studies and compared with those of pirenzepine and atropine. Dicyclomine, trihexyphenidyl and pirenzepine demonstrated the highest affinity for the M1 muscarinic receptor subtype as revealed in competition experiments against [3H]-pirenzepine labelling of cortical membranes. Their affinity values lay in a narrow range (3.7-14 nM) approaching that of atropine (1.6 nM). Competition experiments against [3H]-N-methylscopolamine in cardiac and glandular (salivary) membranes revealed differences between the drugs examined. Dicyclomine, trihexyphenidyl and pirenzepine displayed low affinity for the cardiac and intermediate affinity for the glandular receptors. Thus, the drugs appeared to discriminate between the M1 (cortical) and the peripheral muscarinic subtypes (cardiac and glandular). However, atropine displayed similar affinities for either subtype with IC50s varying only slightly (1.6-4.6 nM). The rank order of selectivity was: pirenzepine greater than dicyclomine greater than trihexyphenidyl greater than atropine. Mirroring the binding data, pirenzepine, dicyclomine and trihexyphenidyl showed a tenfold greater ability at inhibiting M1-receptor mediated ganglionic responses (McN A-343 pressor effect in pithed rats and nictitating membrane contraction in cats) than at inhibiting peripheral muscarinic responses in the heart and cardiovascular smooth muscle (vagal bradycardia in rats and cats and vagally-induced vasodilatation in cats). The muscarinic antagonists so far examined can be categorized into two groups. Trihexyphenidyl, dicyclomine and pirenzepine, included in one group, are characterized by a higher affinity for the neuronal (M1) muscarinic receptor, hence they antagonize functional responses mediated by the M1 subtype. Atropine, a member of the other group, shows essentially no selectivity. 6 Differentiation of M1 and peripheral muscarinic receptor subtypes appears to be a property not confined to tricyclics such as pirenzepine but shared by diverse chemical structures. Both trihexyphenidyl and dicyclomine appear to be useful pharmacological tools in the classification of muscarinic receptor subtypes.

The relative selectivity of anticholinergic drugs for the M1 and M2 muscarinic receptor subtypes.

Anticholinergic drugs are used to treat a number of neurologic disorders, including parkinsonism, vestibular disturbances, and dystonia. Traditionally, these drugs have been thought to act in similar fashion, as competitive antagonists at a single class of muscarinic receptors, and not to differ significantly in their therapeutic efficacy. Recently, however, pharmacologic studies have shown that the novel antagonist pirenzepine is capable of recognizing heterogeneity among muscarinic receptors; high-affinity pirenzepine sites have been classified as M1 sites and low-affinity sites as M2. This study examined whether the anticholinergics currently available for treatment of neurologic symptoms have selectivity for these subtypes and whether they differ in their degree of selectivity; the study showed that these drugs do demonstrate selectivity. All had greater affinity for the M1 site, indicated by higher affinity for rat forebrain membranes, where M1 predominates, than hindbrain preparations, where M2 predominates. The degree of selectivity varied greatly; some compounds, such as ethopropazine, had little M1 selectivity, whereas others, such as scopolamine, trihexyphenidyl, and biperiden, were quite selective, like pirenzepine. It is unknown whether these differences in selectivity have any immediate therapeutic implications. However, these results support the emerging concept of muscarinic receptor subtypes and the prospect of developing more selective agents, with enhanced therapeutic efficacy.

Chemical Nature of Synaptic Transmission in Vertebrates

Chemical Nature of Synaptic Transmission in Vertebrates