Cloning and functional analysis of a gene encoding a novel muscarinic acetylcholine receptor expressed in chick heart and brain

Previous studies have demonstrated that muscarinic acetylcholine receptors (mAChR) expressed in chick heart are pharmacologically, immunologically, and biochemically distinct from mAChR expressed in mammalian heart. A chicken genomic clone encoding a mAChR whose deduced amino acid sequence is most homologous to the mammalian m4 receptor has been isolated. Northern blot analysis demonstrated that this gene is expressed in both chick heart and brain. The receptor encoded by this gene was expressed in stably transfected Chinese hamster ovary (CHO) and Y1 adrenal carcinoma cells in order to examine its ligand binding and functional properties. The receptor expressed in CHO and Y1 cells exhibits high affinity binding for the muscarinic antagonists quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the cloned chick m4 receptor exhibits pharmacological properties similar to those previously reported for the chick cardiac receptor. This m4 receptor was able to mediate both agonist-dependent inhibition of forskolin-stimulated cAMP accumulation and agonist-dependent stimulation of phosphoinositide metabolism when expressed in CHO cells. In contrast, when expressed in Y1 cells, the chick m4 receptor mediated agonist-dependent inhibition of forskolin-stimulated cAMP accumulation, but not stimulation of phosphoinositide metabolism. Thus, as with the mammalian cardiac (m2) receptor, the functional specificity of the chick cardiac receptor appears to be dependent on the cell type in which it is expressed.     

A transfected m1 muscarinic acetylcholine receptor stimulates adenylate cyclase via phosphatidylinositol hydrolysis

The m1 muscarinic acetylcholine receptor gene was transfected into and stably expressed in A9 L cells. The muscarinic receptor agonist, carbachol, stimulated inositol phosphate generation, arachidonic acid release, and cAMP accumulation in these cells. Carbachol stimulated arachidonic acid and inositol phosphate release with similar potencies, while cAMP generation required a higher concentration. Studies were performed to determine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m1 muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messengers. Carbachol failed to stimulate adenylate cyclase activity in A9 L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated arachidonic acid release yet inhibited cAMP accumulation in response to carbachol. PMA also inhibited inositol phosphate release in response to carbachol, suggesting that activation of phospholipase C might be involved in cAMP accumulation. PMA did not inhibit prostaglandin E2-, cholera toxin-, or forskolin-stimulated cAMP accumulation. The phospholipase A2 inhibitor eicosatetraenoic acid and the cyclooxygenase inhibitors indomethacin and naproxen had no effect on carbachol-stimulated cAMP accumulation. Carbachol-stimulated cAMP accumulation was inhibited with TMB-8, an inhibitor of intracellular calcium release, and W7, a calmodulin antagonist. These observations suggest that carbachol-stimulated cAMP accumulation does not occur through direct m1 muscarinic receptor coupling or through the release of arachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. The generation of cytosolic calcium via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m1 muscarinic receptor stimulation of phospholipase C appears to generate the accumulation of cAMP.     

Muscarinic Receptor Stimulation Increases Inositol-Phospholipid Metabolism and Inhibits Cyclic AMP Accumulation in PC 12 Cells

Muscarinic receptor stimulation increased the accumulation of 3H-inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5'-N-ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol-phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 microM and 0.36 microM, respectively. PC12 cells contained a single class of N-[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 microM) and pirenzepine (Ki, 0.12 microM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an M1 receptor. Muscarine inhibited cAMP accumulation in cells made deficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of 3H-inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotremorine inhibited cAMP accumulation but it did not stimulate inositol-phospholipid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholine muscarinic m1 receptor regulation of cyclic AMP synthesis controls growth factor stimulation of Raf activity.

Acetylcholine muscarinic m2 receptors (m2R) couple to heterotrimeric Gi proteins and activate the Ras/Raf/mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase in Rat 1a cells. In contrast to the m2R, stimulation of the acetylcholine muscarinic m1 receptor (m1R) does not activate the Ras/Raf/mitogen-activated protein kinase regulatory pathway in Rat 1a cells but rather causes a pronounced inhibition of epidermal growth factor and platelet-derived growth factor receptor activation of Raf. In Rat 1a cells, m1R stimulation of phospholipase C beta and the marked rise in intracellular calcium stimulated cyclic AMP (cAMP) synthesis, resulting in the activation of protein kinase A. Stimulation of protein kinase A inhibited Raf activation in response to growth factors. Platelet-derived growth factor receptor stimulation of phosphatidylinositol 3-kinase activity was not affected by either m1R stimulation or protein kinase A activation in response to forskolin-stimulated cAMP synthesis. GTP loading of Ras in response to growth factors was unaffected by protein kinase A activation but was partially inhibited by carbachol stimulation of the m1R. Therefore, protein kinase A action at the Ras/Raf activation interface selectively inhibited only one branch of the signal transduction network initiated by tyrosine kinases. Specific adenylyl cyclases responding to different signals, including calcium, with enhanced cAMP synthesis will regulate Raf activation in response to Ras.GTP. Taken together, the data indicate that G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated mitogenic response pathways.     

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)     

Embryonic chick heart expresses multiple muscarinic acetylcholine receptor subtypes. Isolation and characterization of a gene encoding a novel m2 muscarinic acetylcholine receptor with high affinity for pirenzepine.

Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors which are highly conserved across mammalian species. Chick cardiac mAChR, however, have been shown to be pharmacologically, immunologically, and biochemically distinct from m2 mAChR expressed in mammalian heart. We previously reported the isolation and characterization of a novel chicken mAChR, cm4, which is expressed in chick heart and brain. We report here the isolation of an additional chicken mAChR gene whose deduced amino acid sequence is most homologous to the mammalian m2 receptor. Northern blot analysis demonstrated that this chicken m2 gene is also expressed in chick heart and brain. When stably transfected into Chinese hamster ovary (CHO) cells and Y1 adrenal carcinoma cells, the chicken m2 gene expresses a receptor protein which exhibits high affinity binding for the muscarinic antagonist quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the chick m2 receptor has pharmacological properties that are similar to the chick m4 receptor as well as those reported for endogenous mAChR in chick cardiac cells. Consistent with the properties of the chick m4, as well as mammalian m2 and m4 receptors, the chick m2 receptor was able to functionally couple to both the inhibition of adenylate cyclase and the stimulation of phosphoinositide metabolism when expressed in CHO cells, but only the inhibition of adenylate cyclase when expressed in Y1 cells. We conclude from this study that the embryonic chick heart expresses multiple subtypes of mAChR which are highly conserved with their mammalian counterparts. Furthermore, the high degree of conservation between the mammalian m2 and the chick m2 muscarinic receptors suggests that the pharmacological differences that exist between these receptors are due to a relatively small number of specific amino acid changes rather than larger changes in receptor sequence or structure.     

Neuropeptide Y secretion from bovine chromaffin cells inhibits cyclic AMP accumulation

Neuropeptide Y (NPY) is secreted from bovine chromaffin cells in response to nicotinic receptor stimulation and may exhibit autocrine, paracrine or endocrine effects. Stimulation of bovine chromaffin cells with nicotine followed by the addition of forskolin (FSK) to the media results in a decrease in cyclic AMP accumulation compared to that seen in the absence of nicotine. Pertussis toxin (PTX) treatment or the addition of BIBP 3226, a selective NPY Y1 receptor antagonist prevents the inhibitory effect of nicotine. Fractionation of media obtained from cells stimulated with nicotine reveals an NPY-like substance that inhibits FSK-stimulated cAMP accumulation. Thus, an NPY-like substance can be secreted from bovine chromaffin cells in quantities sufficient to inhibit FSK-stimulated cAMP accumulation. These results suggest that NPY can act in an autocrine fashion to regulate chromaffin cell function.     

Antibody-induced cAMP accumulation in splenocytes from athymic nude mice

Products from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (IP3) can increase and/or potentiate cAMP accumulation in a variety of cells. Antibody to surface immunoglobulins activates IP3 hydrolysis in B-lymphocytes. In this study we have examined whether anti-Ig also stimulated and/or potentiated increases in the cAMP levels of splenocytes from athymic nude mice. Furthermore, since TPA potentiates anti-Ig-induced DNA synthesis and cAMP modulates DNA synthesis, the effects of TPA on any anti-Ig-induced changes in cAMP were also studied. Antibody (25 micrograms/ml) stimulated a rapid ris in cAMP which increased from 250 fmol/10(6) cells to 400 fmol/10(6) cells within 1 min and then subsided to 310 fmol/10(6) cells by 10 min. TPA (96 nM) suppressed the anti-Ig-induced cAMP accumulation at 1 min by 60%, but potentiated the forskolin (114 microM)-induced rise by 151%. Two other activators of protein kinase C, dioctanoylglycerol (5 microM), and anti-Ig (25 micrograms/ml), also potentiated the forskolin response by 198% and 52%, respectively. These results suggest that modulation of the adenylate cyclase system by anti-Ig may act in concert with cytokines and/or prostaglandins secreted by other lymphoid cells to define the state of proliferation or differentiation in B-cells.     

Inhibitory effect of NPY on isoprenaline-induced osteoclastogenesis in mouse bone marrow cells

The effect of neuropeptide Y (NPY), a co-transmitter with noradrenaline in peripheral sympathetic nerve fibers, on the osteoclastogenesis in mouse bone marrow cell cultures treated with isoprenaline, a beta-adrenergic receptor (beta-AR) agonist, was examined. The mouse bone marrow cells constitutively expressed mRNAs for the NPY-Y1 receptor and beta2-AR. NPY inhibited the formation of osteoclast-like cells induced by isoprenaline but not that by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) or soluble receptor activator of nuclear factor-kappaB ligand (RANKL); and it suppressed the production of RANKL and cyclic AMP (cAMP) increased by isoprenaline but not those increased by 1alpha,25(OH)2D3. NPY also inhibited osteoclastogenesis induced by forskolin, an activator of adenylate cyclase; however, it did not inhibit that induced by exogenously supplied dibutyryl cAMP, a cell-permeable cAMP analog that activates cAMP-dependent protein kinase. These results demonstrate that NPY inhibited the isoprenaline-induced osteoclastogenesis by blocking the agonist-elicited increases in the production of cAMP and RANKL in mouse bone marrow cells, suggesting an interaction between NPY and beta-AR agonist in bone resorption.     

Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA-). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA+ or PKA- cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA- cells. The m2 receptor was more sensitive to agonist-dependent internalization, and its rate of internalization was faster in PKA- cells than it was in PKA+ cells. Treatment of PKA+ cells with 8-(4-chlorophenylthio)-cAMP or forskolin did not result in internalization of either m1 or m2 receptors and did not alter the extent of agonist-dependent internalization of m2. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA+ and PKA- cells was accompanied by desensitization of functional responses. Exposure of PKA+ cells to 10(-7) M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 +/- 9% decrease in the number of m1 receptors on the cell surface. However, treatment of PKA- cells expressing the m1 receptor did not result in internalization, suggesting that PKA was required for some aspect of PMA-dependent internalization.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Carbachol-induced reverse transformation of Chinese hamster ovary cells transfected with and expressing the m5 muscarinic acetylcholine receptor

Reverse transformation was induced in Chinese hamster ovary (CHO) cells transfected with and stably expressing the m5 subtype of the muscarinic acetylcholine receptor when stimulated with the muscarinic agonist, carbachol. Atropine, a muscarinic antagonist, blocked the carbachol-stimulated reverse transformation. CHO cells not transfected with the muscarinic receptor did not change with added carbachol. PMA induced reverse transformation without increasing cAMP accumulation in CHO cells. Carbachol, prostaglandin E2, and cholecystokinin increased cAMP accumulation but only carbachol caused reverse transformation. Carbachol-stimulated cAMP accumulation occurred at a higher concentration (EC50 10 microM) than did carbachol-stimulated reverse transformation (EC50 63 nM). Muscarinic m5 acetylcholine receptor transfected into CHO cells can induce reverse transformation which may be independent of cAMP.     

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.     

Isolation and functional characterization of the chick M5 muscarinic acetylcholine receptor gene

The chick is a widely used system for study of the actions of muscarinic acetylcholine receptors in the cardiovascular, visual, and nervous systems. We report the isolation and functional analysis of the gene encoding the chick M5 muscarinic receptor. RT-PCR analysis indicates that the M5 receptor is expressed at low levels in embryonic chick brain and heart. When expressed in stably transfected Chinese hamster ovary cells, the M5 receptor exhibits high-affinity binding to muscarinic antagonists and mediates robust activation of phospholipase C activity.     

The relationship of calcium to receptor-controlled stimulation of phosphatidylinositol turnover. Effects of acetylcholine, adrenaline, calcium ions, cinchocaine and a bivalent cation ionophore on rat parotid-gland fragments.

The possibility that Ca2+ ions are involved in the control of the increased phosphatidylinositol turnover which is provoked by alpha-adrenergic or muscarinic cholinergic stimulation of rat parotid-gland fragments has been investigated. Both types of stimulation provoked phosphatidylinositol breakdown, which was detected either chemically or radiochemically, and provoked a compensatory synthesis of the lipid, detected as an increased rate of incorporation of 32Pi into phosphatidylinositol. Acetylcholine had little effect on the incorporation of labelled glycerol, whereas adrenaline stimulated it significantly, but to a much lower extent than 32P incorporation: this suggests that the response to acetylcholine was entirely accounted for by renewal of the phosphorylinositol head-group of the lipid, but that some synthesis de novo was involved in the response to adrenaline. The responses to both types of stimulation, whether measured as phosphatidylinositol breakdown or as phosphatidylinositol labelling, occurred equally well in incubation media containing 2.5 mm-Ca2+ or 0.2 mm-EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid]. Incubation with a bivalent cation ionophore (A23187) led to a small and more variable increase in phosphatidylinositol labelling with 32Pi, which occurred whether or not Ca2+ was available in the extracellular medium: this was not accompanied by significant phosphatidylinositol breakdown. Cinchocaine, a local anaesthetic, produced parallel increases in the incorporation of Pi and glycerol into phosphatidylinositol. This is compatible with its known ability to inhibit phosphatidate phosphohydrolase (EC 3.1.3.4) and increase phosphatidylinositol synthesis de novo in other cells. These results indicate that the phosphatidylinositol turnover evoked by alpha-adrenergic or muscarinic cholinergic stimuli in rat parotid gland probably does not depend on an influx of Ca2+ into the cells in response to stimulation. This is in marked contrast with the K+ efflux from this tissue, which is controlled by the same receptors, but is strictly dependent on the presence of extracellular Ca2+. The Ca2+-independence of stimulated phosphatidylinositol metabolism may mean that it is controlled through a mode of receptor function different from that which controls other cell responses. Alternatively, it can be interpreted as indicating that stimulated phosphatidylinositol breakdown is intimately involved in the mechanisms of action of alpha-adrenergic and muscarinic cholinergic receptor systems.     

Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene.

To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.     

Interaction between phosphoinositide turnover system and cyclic AMP pathway for the secretion of pancreastatin and somatostatin from QGP-1N cells.

It is found that secretion of pancreastatin and somatostatin from QGP-1N cells is regulated through muscarinic receptor-mediated activation of phosphatidylinositide hydrolysis system. In this report, whether the cAMP pathway interacts with the phosphoinositide turnover system for the secretion of pancreastatin and somatostatin from QGP-1N cells through muscarinic receptors was studied. Stimulation of QGP-1N cells with carbachol increased intracellular cAMP levels. The carbachol-induced increase in cAMP levels was inhibited by atropine. Calcium ionophore (A23187) and phorbol 12-myristate 13-acetate increased cAMP synthesis. Dibutyryl cAMP, forskolin and theophylline stimulated secretion of pancreastatin and somatostatin. When either dibutyryl cAMP, forskolin or theophylline was added in culture medium with A23187, phorbol ester or carbachol, a synergistic effect was found on pancreastatin and somatostatin secretion. These results suggest that interaction between the phosphoinositide turnover system and the cAMP pathway occurs in QGP-1N cells through muscarinic receptor stimulation for the secretion of pancreastatin and somatostatin.     

Acetylcholine inhibits cell cycle progression in rat Schwann cells by activation of the M2 receptor subtype

Cultures of Schwann cells from neonatal rat sciatic nerves were treated with acetylcholine agonists and the effects on cell proliferation evaluated. (3)[H]-thymidine incorporation shows that acetylcholine (ACh) receptor agonists inhibit cell proliferation, and FACS analysis demonstrates cell-cycle arrest and accumulation of cells in the G1 phase. The use of arecaidine, a selective agonist of muscarinic M2 receptors reveals that this effect depends mainly on M2 receptor activation. The arecaidine dependent-block in G1 is reversible because removal of arecaidine from the culture medium induces progression to the S phase. The block of the G1-S transition is also characterized by modulation of the expression of several cell-cycle markers. Moreover, treatment with ACh receptor agonist causes both a decrease in the PCNA protein levels in Schwann cell nuclei and an increase in p27 and p53 proteins. Finally, immuno-electron microscopy demonstrates that M2 receptors are expressed by Schwann cells in vivo. These results indicate that ACh, by modulating Schwann cell proliferation through M2 receptor activation, might contribute to their progression to a more differentiated phenotype.     

Inhibition of hormone-stimulated adenylate cyclase activity after altering turkey erythrocyte phospholipid composition with a nonspecific lipid transfer protein. Phosphatidylinositol uncouples catecholamine binding from adenylate cyclase activation

The nonspecific lipid transfer protein from beef liver was used to modify the phospholipid composition of intact turkey erythrocytes in order to study the dependence of isoproterenol-stimulated adenylate cyclase activity on membrane phospholipid composition. Incorporation of phosphatidylinositol into turkey erythrocytes inhibited isoproterenol-stimulated cyclic AMP accumulation in a linear, concentration-dependent manner. Inhibition was relatively specific for phosphatidylinositol; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid were from 3 to 7 times less effective as inhibitors of hormone-stimulated cyclase activity. Inhibition by phosphatidylinositol was not reversible when up to 90% of the incorporated phosphatidylinositol was removed, either by incubation with phosphatidylinositol-specific phospholipase C or a second incubation with transfer protein; possibly adenylate cyclase activity depends on a small pool of phosphatidylinositol that is inaccessible to either phospholipase C hydrolysis or removal by lipid transfer protein. Phosphatidylinositol incorporation inhibits adenylate cyclase activity by uncoupling beta-adrenergic receptors from the remainder of the cyclase complex. Phosphatidylinositol incorporation had no effect on stimulation of cAMP accumulation by either cholera toxin or forskolin, indicating that inhibition occurs only at the level of receptor. Phosphodiesterase activity was not altered in phosphatidylinositol-modified cells. Inhibition of cAMP accumulation was not the result of changes in either membrane fluidity or in cAMP transport out of modified turkey erythrocytes. Phosphatidylinositol inhibition of isoproterenol-stimulated cyclase activity may serve as a useful model system for hormone-induced desensitization.