Amplification of the rat M2 muscarinic receptor gene by the polymerase chain reaction: functional expression of the M2 muscarinic receptor

A selective amplification of the coding sequence of the rat M2 muscarinic receptor gene was achieved by the polymerase chain reaction. The error rate of this amplification system under conditions specified was 1 nucleotide substitution in 841 base pairs. In vitro expression of this gene in murine fibroblasts (B82) via the eukaryotic expression vector, pH beta APr-1-neo, resulted in high level expression of specific [3H] (-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of [3H] (-)MQNB binding with a Kd value of 265 pM and a Bmax value of 411 +/- 50 fmol/10(6) cells. Cardiac selective muscarinic antagonists such as himbacine and AF-DX 116 show high affinities for this binding site in the M2LKB2-2 cells. The rank order of potency of several antagonists in inhibiting [3H] (-)MQNB binding in these cells conformed to the characteristics of an M2 type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a Ki value of 2.0 microM. This receptor appeared to be inversely coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. Carbachol also had a slight stimulatory effect on the hydrolysis of inositol lipids. The polymerase chain reaction proves highly effective in cloning genes from genomic material, as demonstrated by the first in vitro functional expression of the rat M2 type muscarinic receptor.     

Light microscopic autoradiographic localization of [3H]pirenzepine and [3H](-)quinuclidinyl benzilate binding in human stellate ganglia

We have utilized the LKB Ultrofilm method of autoradiography to anatomically localize putative M1 and M2 muscarinic receptor subtypes in human stellate ganglia. Ten micron sections were labeled in vitro with either 1 nM of the classical antagonist [3H](-)quinuclidinyl benzilate ([3H](-)QNB) or 20 nM of the non-classical antagonist [3H]pirenzepine ([3H]PZ), using 1 microM atropine sulfate to define non-specific binding for both ligands. Our results indicate that [3H](-)QNB and [3H]PZ binding sites are distributed within the principal ganglion cells and nerve bundles.     

Necessity of intracellular cyclic AMP in inducing gastric acid secretion via muscarinic M3 and cholecystokinin2 receptors on parietal cells in isolated mouse stomach

The existence of a direct action of acetylcholine and gastrin on muscarinic M3 and cholecystokinin2 (CCK2) receptors on gastric parietal cells has not yet been convincingly established because these stimulated acid secretions are remarkably inhibited by histamine H2 receptor antagonists. In the present study, we investigated the necessity of intracellular cyclic AMP in inducing gastric acid secretion via muscarinic M3 and CCK2 receptors on parietal cells using an isolated mouse stomach preparation. Bethanechol (10-300 microM) produced a marked increase in acid output and this increase was completely blocked by famotidine (10 microM). In the presence of famotidine, bethanechol (1-30 microM) augmented the acid secretory response to dibutyryl AMP (200 microM) in a concentration-dependent manner. The augmentation was blocked by atropine (1 microM), 4-DAMP (0.1 microM), a muscarinic M3-selective antagonist, and by Ca2+ exclusion from the serosal nutrient solution. Pentagastrin (0.3-3 microM) also concentration-dependently stimulated gastric acid secretion, but the effect was completely inhibited by famotidine. In the presence of famotidine, pentagastrin (0.1-0.3 microM) elicited a definite potentiation of the acid secretory response to dibutyryl cyclic AMP (200 microM). This potentiation was inhibited by YM022 (1 microM), a CCK2 receptor antagonist, and by exclusion of Ca2+ from the serosal nutrient solution. The present results suggest that gastric acid secretion via the activation of muscarinic M3 and CCK2 receptors on the parietal cells is induced by activation of the cyclic AMP-dependent secretory pathway.     

Pharmacologic characterization of muscarinic receptors of insect brains.

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype-selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains. In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype-selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist, 11-[2-[dimethylamino)-methyl)1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepin-6 one (AF-DX 116), the M2-selective antagonist, and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) the M3-selective antagonist, was also the same as that of mammalian brains, i.e., 4-DAMP greater than PZ greater than AF-DX 116. The three insect brain receptors had 27-50-fold lower affinity for PZ (Ki 484-900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4-DAMP (Ki 18.9-56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.     

Phorbol esters alter muscarinic receptor binding and inhibit polyphosphoinositide breakdown in human neuroblastoma (SH-SY5Y) cells

Many recent reports have indicated that the effect of the phorbol ester tumor promoters is mediated through the Ca2+/phospholipid dependent protein kinase C. We have investigated the effect of two biologically active phorbol esters, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) and 4 beta-phorbol 12 beta,13 alpha-didecanoate (beta PDD) on muscarinic agonist binding and receptor-stimulated phosphoinositide breakdown in cultured human neuroblastoma (SH-SY5Y) cells. Preincubation of these cells with phorbol esters significantly reduced the carbachol-stimulated breakdown of inositol phospholipids and caused a decrease of agonist affinity for [3H](-)methyl quinuclidinyl benzilate ([3H](-)MQNB) binding without affecting the affinity of antagonist to the muscarinic receptor. The nontumor promoting 4 alpha-phorbol 12 beta,12 alpha-didecanoate (alpha PDD) was ineffective in our studies. These results suggest that the activation of protein kinase C may play an important role in regulating the muscarinic receptor system.     

Cloning of the rat M3, M4 and M5 muscarinic acetylcholine receptor genes by the polymerase chain reaction (PCR) and the pharmacological characterization of the expressed genes.

The coding sequence of the rat m3, m4 and m5 subtypes of muscarinic acetylcholine receptor (mAChR) genes was amplified by the polymerase chain reaction (PCR), cloned, and expressed in the murine fibroblast (B82) cell line. Sequencing of the cloned genes revealed some nucleotide differences when compared with the DNA sequence published in the literature. When the different sequence appeared in only one clone obtained by PCR, it was considered an error of the polymerase. The overall error frequency in the 25 cycles of PCR with either Taq polymerase or Replinase was 1 nucleotide in 1,692 base pairs. In order to evaluate the different nucleotide sequence from a PCR product as an error or as an allelic variant, at least three different clones were sequenced. The cloned genes were each stably expressed in a B82 cell line and pharmacologically evaluated. The affinity of the different antagonists to the muscarinic receptor subtypes was determined by [3H](-)MQNB/ligand inhibition experiments. In the m3, m4 and m5 transfected cells, carbachol appeared to stimulate [3H]inositol monophosphate (IP1) accumulation. Carbachol, at 3 microM, appeared to suppress the forskolin-stimulated cAMP formation in the m4 transfected cells. These findings suggest these mAChRs amplified by PCR, cloned, and expressed in the B82 cell lines exhibit the pharmacological characteristics of the muscarinic receptor subtypes.     

The effects of ovarian hormones on beta-adrenergic and muscarinic receptors in rat heart

The in vitro and in vivo effects of estrogen and progesterone on muscarinic and beta-adrenergic receptors of cardiac tissue were studied in ovariectomized (OVX) rats. The binding assay for muscarinic receptors was performed under a nonequilibrium condition; whereas the binding assay for beta-adrenergic receptors, under an equilibrium condition. Estrogenic compounds and progesterone were found to have no effect on the binding of the radioligand, [3H]-dihydroalprenolol, to beta-adrenergic receptors in vitro. However, progestins but not estrogenic compounds inhibited the binding of the radioligand, [3H]-quinuclidinyl benzilate, to muscarinic receptors in vitro, with progesterone as the most potent inhibitor (IC50 = 37 microM, apparent Ki = 13 microM). Progesterone was found to decrease the apparent affinity of muscarinic receptors for [3H](-)QNB in vitro. Daily treatment of OVX rats with estradiol benzoate (4 micrograms) or progesterone (2.5 mg) for 4 days had no effect on the muscarinic or beta-adrenergic receptors with respect to the binding affinity and receptor density. However, administrations of these hormones together for 4 days caused an increase in the receptor density of muscarinic receptors without a significant effect on their apparent binding affinity; also these hormones induced a decrease in the binding affinity and an increase in the receptor density of beta-adrenergic receptors. The results of this study demonstrate that progestins are capable of interacting with the cardiac muscarinic receptors in vitro, and indicate that estrogen and progesterone have a synergistic effect to increase the receptor densities of muscarinic and beta-adrenergic receptors as well as to cause a decrease in the binding affinity of beta-adrenergic receptors in vivo.     

[3H]N-Methylscopolamine Binding Studies Reveal M2 and M3 Muscarinic Receptor Subtypes on Cerebellar Granule Cells in Primary Culture

Saturation experiments with the muscarinic antagonist [3H]N-methylscopolamine ([3H]NMS) indicated that cerebellar granule cells in primary culture possess a high density of muscarinic acetylcholine receptors (mAChRs): Bmax = 1.85 +/- 0.01 pmol/mg of protein at 10 days in culture; KD = 0.128 +/- 0.01 nM. The selective M1 antagonist pirenzepine displaced [3H]NMS binding with a low affinity (Ki = 273 +/- 13 nM), whereas the M2/M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide competed with [3H]NMS with Ki values in the nanomolar range, a result suggesting that some of the mAChRs on cerebellar granule cells belong to the M3 subtype. Methoctramine, which discriminates between M2 and M3 subtypes with high and low affinity, respectively, displayed a high and low affinity for [3H]NMS binding sites (Ki(H) = 31 +/- 5 nM; Ki(L) = 2,620 +/- 320 nM). These results provide the first demonstration that both M2 and M3 mAChR subtypes may be present on cultured cerebellar cells. In addition, complete death of neurons induced by N-methyl-D-aspartate (100 microM for 1 h) reduced by 85% the specific binding of [3H]NMS, a result indicating that most mAChRs were associated with neuronal components. Finally, the evolution of the density of mAChRs, labeled by [3H]NMS, correlated with the neuronal maturation during the in vitro development of these cells.     

Histamine Stimulation of Cyclic AMP Accumulation in Astrocyte-Enriched and Neuronal Primary Cultures from Rat Brain

Histamine stimulates cyclic AMP accumulation in astrocyte-enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax = 304 +/- 44% over basal, EC50 = 43 +/- 5 microM) was much higher than in neuronal cultures (Emax = 24 +/- 2%, EC50 = 14 +/- 7 microM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki = 1.1 +/- 0.2 microM) and ranitidine (Ki = 46 +/- 10 nM) but was insensitive to the H1 antagonist mepyramine (1 microM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2-thiazolylethylamine (0.01-1 mM) did not potentiate the response to impromidine (10 microM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine-induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors.     

Subtype of muscarinic receptor coupled to the attenuation of hormone-stimulated cAMP accumulation in NG108-15 neuroblastoma x glioma hybrid cells.

The subtype of muscarinic receptor which mediates cAMP attenuation is not established. Therefore, several selective muscarinic antagonists were used to characterize the subtype of muscarinic receptor coupled to the inhibition of hormone-stimulated cAMP accumulation using NG108-15 neuroblastoma x glioma hybrid cells. These cells were prelabeled with [2-3H]-adenine, washed, and resuspended in a culture medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). The labeled cells were preincubated with the different antagonists 12-15 min. before they were challenged with agonists. The formation of [3H]-cAMP was activated by PGE1 (1 microM) or forskolin (1 microM). In all cases, [3H]-cAMP formed was separated and measured. Carbachol (100 microM) and McN-A343 (10 mM) were used as standard muscarinic agonists. These studies gave the following results: a) McN-A343 (10 mM), an M1 receptor agonist, was only a partial agonist causing 40% inhibition of cAMP accumulation indicating that this effect was not mediated by an M1 receptor; b) The M1-selective antagonist, pirenzepine, exhibited low affinity (pA2 6.2) further suggesting that an M1 receptor was not coupled to the attenuation of cAMP accumulation; c) Two selective M2 antagonists (AF-DX 116 and methoctramine) and M3 antagonist (HHSiD) were used to further characterize these muscarinic receptors. The order of all antagonists based on their affinities (pA2 values) could be arranged in the following order: atropine (9.0) > methoctramine (7.6) > HHSiD (6.9) > AF-DX 116 (6.6) > pirenzepine (6.2). HHSiD exhibits the same degree of affinity to M2 receptors of other tissues as it does to those of NG cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the subtype of muscarinic receptor coupled to the stimulation of phosphoinositide hydrolysis in 132-1N1 human astrocytoma cells.

Stimulation of muscarinic receptors increases phosphoinositide (PI) hydrolysis in 132-1N1 human astrocytoma cells. To evaluate the subtype of receptors which mediate PI hydrolysis in 132-1N1 cells, the effects of: a) the nonselective M1 agonist, carbachol; b) the selective M1 agonist, 4-hydroxy-2-butynyl-trimethylammonium chloride-m-chlorocarbinilate (McN-343); c) the nonselective antagonists, atropine and scopolamine; d) the relatively selective M1 antagonist, pirenzepine; e) the relatively selective M2 antagonists, AF-DX 116 (11-2-diethylaminomethyl-1-piperidinylacetyl-5, 11-dihydro-6H-pyrido-2,3-b-1,4-benzodiazepine-6-one) and methoctramine and f) the relatively selective M3 antagonist, hexahydrosila-difenidol (HHSiD) on PI hydrolysis in 132-1N1 cells were studied. The cell pools of inositol-phospholipids were prelabelled by incubating 132-1N1 cells in a low inositol containing medium (CMRL-1066) supplemented with [3H]inositol (2 microCi/ml) for 20-24 hours at 37 degrees C. The cells were washed and resuspended in a physiological salt solution, and PI hydrolysis was measured by accumulation of [3H]inositol-1-phosphate (IP) in the presence of 10 mM LiCl. Carbachol produced time and concentration dependent PI hydrolysis (EC50, 37 microM). McN-A343 did not cause significant hydrolysis of PI in 132-1N1 cells indicating that the receptor was not of M1 type. All the above muscarinic antagonists caused a concentration dependent decrease in the level of IP in response to carbachol (100 microM). The rank order of their affinities (pA2 values) was: atropine (8.8) > HHSiD (7.6) > pirenzepine (6.8) > methoctramine (6.0) > AF-DX 116 (5.8). This rank order supports the concept that M3 (other names, M2 beta, glandular M2) receptors are linked to PI hydrolysis in 132-1N1 cells. HHSiD, which is selective for M3 receptors of the smooth muscle has higher affinity for muscarinic receptors in 132-1N1 cells than AF-DX 116 which is selective for M2 receptors in cardiac tissue. If the receptor in 132-1N1 cells had been M2, part of the rank order for affinities would have been methoctramine > AF-DX 116 > HHSiD > pirenzepine. From all of these observations, the muscarinic receptor for PI hydrolysis in 132-1N1 cells is tentatively characterized as of M3 type.     

Molecular probes for muscarinic receptors: derivatives of the M1-antagonist telenzepine.

Functionalized congeners of the M1-selective muscarinic antagonist telenzepine (4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]-10H- thieno[3,4-b][1,5]benzodiazepin-10-one) were developed and found to bind to the receptor with affinities (Ki values) in approximately the nanomolar range. The derivatives contain a 10-aminodecyl group, which provides a nucleophilic functionality for further derivatization. The attachment of a spacer chain to the distal piperazinyl nitrogen was based on previous findings of enhanced affinity at muscarinic receptors in an analogous series of alkylamino derivatives of pirenzepine [J. Med. Chem. (1991) 34, 2133-2145]. The telenzepine derivatives contain prosthetic groups for radioiodination, protein cross-linking, photoaffinity labeling, and fluorescent labeling and biotin for avidin complexation. The affinity for muscarinic receptors in rat forebrain (mainly m1 subtype) was determined in competitive binding assays vs [3H]-N-methylscopolamine. A (p-aminophenyl)-acetyl derivative for photoaffinity labeling had a Ki value of 0.29 nM at forebrain muscarinic receptors (16-fold higher affinity than telenzepine). A biotin conjugate displayed a Ki value of 0.60 nM at m2-receptors and a 5-fold selectivity versus forebrain. The high affinity of these derivatives makes them suitable for the characterization of muscarinic receptors in pharmacological and spectroscopic studies, for peptide mapping, and for histochemical studies.     

Antagonism by 8-hydroxy-2(di-n-propylamino)tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells.

IMR-32 and SK-N-MC cells were found to contain [3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC50 value of about 50 microM in both cases. Pirenzepine inhibited the carbachol (100 microM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT (10-100 microM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.     

Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors

Incubation of 1321N1 human astrocytoma cells with carbachol resulted in a rapid loss of binding of [3H]N-methylscopolamine ([3H]NMS) to muscarinic cholinergic receptors measured at 4 degrees C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate [( 3H]QNB) at 37 degrees C occurred at a slower rate. Upon removal of agonist from the medium, the lost [3H]NMS binding sites measured on intact cells recovered with a t1/2 of approximately 20 min, but only to the level to which [3H]QNB binding sites had been lost; no recovery of "lost" [3H]QNB binding sites occurred over the same period. Based on these data and the arguments of Galper et al. (Galper, J. B., Dziekan, L. C., O'Hara, D. S., and Smith, T. W. (1982) J. Biol. Chem. 257, 10344-10356) regarding the relative hydrophilicity of [3H]NMS versus [3H]QNB, it is proposed that carbachol induces a rapid sequestration of muscarinic receptors that is followed by a loss of these receptors from the cell. These carbachol-induced changes are accompanied by a change in the membrane form of the muscarinic receptor. Although essentially all of the muscarinic receptors from control cells co-purified with the plasma membrane fraction on sucrose density gradients, 20-35% of the muscarinic receptors from cells treated for 30 min with 100 microM carbachol migrated to a much lower sucrose density. This conversion of muscarinic receptors to a "light vesicle" form occurred with a t1/2 approximately 10 min, and reversed with a t1/2 approximately 20 min. In contrast to previous results in this cell line regarding beta-adrenergic receptors (Harden, T. K., Cotton, C. U., Waldo, G. L., Lutton, J. K., and Perkins, J. P. (1980) Science 210, 441-443), agonist binding to muscarinic receptors in the light vesicle fraction obtained from carbachol-treated cells was still regulated by GTP. One interpretation of these data is that agonists induce an internalization of muscarinic receptors with the retention of their functional interaction with a guanine nucleotide regulatory protein.     

M1 and M3 muscarinic antagonists inhibit human nasal glandular secretion in vitro.

Mucus glycoproteins (MGP) are high-molecular-weight glycoconjugates that are released from submucosal glands and epithelial goblet cells in the respiratory tract. Muscarinic receptors have an important role in the regulation of human nasal glandular secretion and mucus production, but it is not known which of the five muscarinic receptor subtypes are involved. The effect of nonselective and M1-, M2-, and M3-selective muscarinic antagonists on methacholine (MCh)-induced MGP secretion from human nasal mucosal explants was tested in vitro. MGP was assayed by enzyme-linked immunosorbent assay using a specific anti-MGP monoclonal antibody (7F10). MCh (100 microM) induced MGP secretion up to 127% compared with controls. MCh-induced MGP release was significantly inhibited by atropine (100 microM), the M, receptor antagonist pirenzepine (10-100 microM), and the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 1-100 microM). 4-DAMP significantly inhibited MCh-induced MGP release at a lower concentration (1 microM) than pirenzepine (10 microM). The M2 receptor antagonists AF-DX 116 and gallamine (both at 100 microM) had no effect. No antagonist alone had a significant effect on MGP release. These results indicate that the M1 and M3 muscarinic receptor subtypes regulate MGP secretion from human nasal mucosa and suggest that the M3 receptor has the predominant effect.     

D1 Dopamine Receptors of NS20Y Neuroblastoma Cells Are Functionally Similar to Rat Striatal D1 Receptors

Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.     

Heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex demonstrated by the selective antagonist AF-DX 116

Recent studies have demonstrated that the majority of muscarinic receptors in rabbit peripheral lung homogenates bind pirenzepine with high affinity (putative M1 subtype). In experiments of AF-DX 116 inhibiting [3H](-)quinuclidinyl benzilate or [3H]pirenzepine, we found similar inhibitory constants for AF-DX 116 binding in rat heart and rabbit peripheral lung that were 4-fold smaller (i.e. of higher affinity) than the inhibitory constant for rat cerebral cortex. This result demonstrates heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex.     

Stereoselective binding and activity of oxotremorine analogs at muscarinic receptors in rat brain.

The activities of the enantiomers of BM-5 were examined to measure muscarinic cholinergic selectivity in the central nervous system. Autoradiographic studies assessed the ability of each enantiomer to inhibit the binding of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) to muscarinic receptors in the rat brain. (+)-(R)-BM-5 inhibited [3H]-(R)-QNB binding to rat brain sections at concentrations below 1.0 microM, while 100-fold higher concentrations of (-)-(S)-BM-5 were required for comparable levels of inhibition. Analysis of the autoradiograms indicated that both stereoisomers had a similar distribution of high affinity binding sites. Each enantiomer displayed higher affinity for muscarinic receptors in the superior colliculi and lower affinity for receptors in the cerebral cortex and hippocampus. (+)-(R)-BM-5 and oxotremorine inhibited adenylyl cyclase activity in the cerebral cortex with efficacies comparable to that for acetylcholine. (+)-(R)-BM-5 was 26-fold more potent than (-)-(S)-BM-5 in inhibiting adenylyl cyclase. Oxotremorine-M and carbamylcholine stimulated phosphoinositide turnover in the cerebral cortex. Oxotremorine had lower activity and (+)-(R)-BM-5 was essentially inactive at comparable concentrations. The difference in activity of the two enantiomers indicates a remarkable stereochemical selectivity for muscarinic receptors. The stereoselectivity index is comparable for both the autoradiographic assays (48) and measures of adenylyl cyclase activity (26) in the cerebral cortex.     

Deoxamuscaroneoxime derivatives as useful muscarinic agonists to explore the muscarinic subsite: demox, a modulator of orthosteric and allosteric sites at cardiac muscarinic M2 receptors

A series of muscarinic agonists, straight chained, branched, cyclic alkyl and aromatic derivatives of the oxime 1 (demox) was designed with the aim of investigating their activity on muscarinic receptor subtypes. Effects on M1 receptor were assessed functionally by a microphysiometer apparatus, while M2, M3, and M4 receptor potency and affinity were studied on isolated preparations of guinea pig heart, ileum, and lung, respectively. The results suggest that the substitution of a hydrogen with a long side-chain or bulky group generally induces a decrease in potency at M1 and M3 subtypes, while a general increase in this parameter is obtained at M2 subtype. Among the agonists 2-18, compound 4 behaves as a full agonist with a preference for M3 subtype. Moreover, compound 12 is inactive at M1 and M4 receptors while it displays a full agonist activity at M2 and M3 subtypes. Since demox displays a variable response on cardiac M2 receptors regulating heart force, an in-depth inquiry of the functional behaviour of this compound was carried out at M2 receptors. In presence of 10(-11) and 10(-10) M demox, the binding of [3H]-NMS was increased by approximately 30% as a consequence of an increase of the association of [3H]-NMS to membranes; this effect was not observed in presence of a higher concentration of [3H]-NMS. Higher concentrations of demox decreased the binding of [3H]-NMS to heart atrial membranes but significantly retarded the dissociation of this radioligand. Our results suggest that demox may interact with orthosteric and allosteric sites of atrial M2 muscarinic receptor.     

Proliferative effect of acetylcholine on rat trachea epithelial cells is mediated by nicotinic receptors and muscarinic receptors of the M1-subtype

Acetylcholine (ACh), synthesized in mammalian non-neuronal cells such as epithelial cells of the airways, digestive tract and skin, is involved in the regulation of basic cell functions (so-called non-neuronal cholinergic system). In the present experiments rat trachea epithelial cells have been cultured to study the proliferative effect of applied ACh by [3H]thymidine incorporation. ACh (exposure time 24 h) caused a concentration-dependent increase in cell proliferation with a doubling of the [3H]thymidine incorporation at a concentration of 0.1 microM. This effect was partly reduced by 30 microM tubocurarine and completely abolished by the additional application of 1 microM atropine. The stimulatory effect of acetylcholine, remaining in the presence of tubocurarine, was prevented by 1 microM pirenzepine (preferentially acting at M1-receptors), but neither by 1 microM AFDX 116 (preferentially acting at M2-receptors) nor by 1 microM hexahydrosiladifenidol (preferentially acting at M3-receptors). The combination of tubocurarine and pirenzepine halved the basal [3H]thymidine incorporation. In conclusion, ACh produces a proliferative effect in rat trachea epithelial cells, the effect being mediated by both nicotinic receptors and muscarinic receptors of the M1-subtype.