Isolation, sequence, and functional expression of the mouse M1 muscarinic acetylcholine receptor gene

A genomic clone encoding the gene for the mouse M1 muscarinic acetylcholine receptor has been isolated, placed under the control of the zinc-inducible mouse metallothionein promoter, and transfected into mouse Y1 adrenal cells. The receptor concentration was about 300 fmol/mg membrane protein in the absence of zinc and could be increased to 4000 fmol/mg membrane protein in the presence of increasing concentrations of zinc. The receptor expressed in zinc-induced cells exhibits the high affinity binding for quinuclidinyl benzilate, atropine, and pirenzepine expected of the M1 muscarinic receptor. The M1 receptor when expressed in Y1 or L cells is physiologically active, as measured by agonist-dependent stimulation of phosphatidylinositol metabolism, but does not inhibit forskolin stimulation of cAMP accumulation. In contrast, a cloned M2 muscarinic receptor when expressed in Y1 cells is able to inhibit forskolin stimulation of cAMP accumulation, but is unable to stimulate phosphatidylinositol metabolism. The stimulation of phosphatidylinositol metabolism mediated by the M1 receptor was not altered by prior treatment of Y1 cells with concentrations of islet-activating protein sufficient to eliminate M2 receptor-mediated inhibition of adenylate cyclase. The cloned M1 receptor gene thus exhibits both the pharmacological and physiological properties expected of the M1 muscarinic acetylcholine receptor. In addition, these results indicate that different subtypes of the muscarinic receptor are coupled to different physiological responses.     

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.     

The muscarinic receptor of chick embryo cells: correlation between ligand binding and calcium mobilization

In this report we characterize muscarinic cholinergic receptor on embryonic cells. We established dose-response curves by fluorometric measurement of Ca2+ mobilization in cell suspensions of whole chick embryos stage 23/24. Ca2+ mobilization was quantitated by standardization of chlorotetracycline (CTC) fluorescence changes after stimulation with muscarinic agonists. We determined ED50 values for the agonists acetylcholine and carbachol as 3.4 X 10(-6) and 2.7 X 10(-5) M, respectively. Pilocarpine and oxotremorine were found to act as reversible competitive antagonists with inhibition constants (Kl) of 5.0 X 10(-6) and 1.4 X 10(-6) M, respectively. Bethanechol, which induced only 23% of the maximal effect obtained by acetylcholine, was a partial agonist with an ED50 of 4.8 X 10(-4) M. Its antagonistic component is expressed by an inhibition constant of 1.9 X 10(-4) M. In parallel, binding studies were performed in a competition assay with [3H]-quinuclidinylbenzilate. For the agonists acetylcholine and carbachol, binding parameters were best fitted by a "two binding-sites model." Comparison with dose-response curves indicated that Ca2+ mobilization was triggered via the high-affinity binding site. The inhibition constants of antagonists derived from the shift of dose-response curves corresponded to the fitted KD values of the binding studies when a "one binding-site model" was applied. Combination of dose-response and binding data showed close proportionality between receptor occupancy and calcium mobilization. No spare receptors were present.     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.     

Effect of withdrawal from chronic ethanol ingestion on the cAMP response of cerebral cortical slices using the agonists histamine, serotonin, and other neurotransmitters.

The effect of ethanol withdrawal on the cAMP response of cerebral cortical brain slices was studied. The cAMP response was evoked in vitro by various neurotransmitters including norepinephrine (NE), histamine, serotonin, dopamine, acetylcholine, and gamma-aminobutyric acid (GABA). The cAMP response to NE and histamine was enhanced by ethanol withdrawal. Serotonin evoked a cAMP response in the brain slices from ethanol-withdrawal rats but not in pair-fed controls. The histamine and serotonin evoked responses were blocked by chlortripolon and methysergide, respectively. The responses to histamine and serotonin were also blocked by alpha- and beta-adrenergic antagonists, possibly because of the nonspecific membrane stablizing effect of these antagonists. GABA inhibited the NE stimulated cAMP response possibly through the hyperpolarizing action of GABA. The results support the hypothesis that ethanol withdrawal induces a nonspecific postjunctional supersensitivity. It is postulated that the supersensitivity involves a partial depolarization of the receptor membrane. Alternative hypotheses are reviewed.     

Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+-ATPase of Lumbricus terrestris body wall muscles

This work was aimed to identify the action of several ion channel and pump inhibitors as well as nicotinic, GABAergic, purinergic and serotoninergic drugs on the resting membrane potential (RMP) and assess the role of cholinergic and GABAergic sensitivity in earthworm muscle electrogenesis. The nicotinic agonists acetylcholine (ACh), carbacholine (CCh) and nicotine depolarize the RMP at concentrations of 5 μM and higher. The nicotinic antagonists (+)tubocurarine, α-bungarotoxin, muscarinic antagonists atropine and hexamethonium do not remove or prevent the CCh-induced depolarization. Verapamil, tetrodotoxin, removal of Cl(-) and Ca(2+) from the solution also cannot prevent the depolarization by CCh. In a Na(+)-free medium, however, CCh lost this depolarization ability and this indicates that the drug opens the sodium permeable pathway. Serotonin, glutamate, glycine, adenosine triphosphate (ATP) and cis-4-aminocrotonic acid (GABA(C) receptor antagonist) had no effect on the RMP. On the other hand, isoguvacin, γ-aminobutyric acid (GABA) and baclofen (GABA(B) receptor agonist) hyperpolarized the RMP. Ouabain, bicucullin (GABA(A) antagonist) and phaclofen (GABA(B) antagonist), as well as the removal of Cl(-), suppressed the effect of GABA and baclofen. CCh did not enhance the depolarization generated by ouabain but, on the other hand, hindered the hyperpolarizing activity of baclofen both in the absence and presence of atropine and (+)tubocurarine. The long-term application of CCh depolarizes the RMP primarily by inhibiting the Na(+)/K(+)-ATPase. The muscle membrane also contains A and B type GABA binding sites, the activation of which increases the RMP at the expense of increasing the action of ouabain- and Cl(-) -sensitive electrogenic pumps.     

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)     

Effects of Prolonged Depolarization on the Nicotinic Acetylcholine Receptors of PC12 Cells

To determine whether prolonged depolarization and/or changes in intracellular Ca2+ concentrations stimulate adaptive responses of neuronal nicotinic acetylcholine receptors, PC12 pheochromocytoma cells were grown in medium containing various concentrations of K+. Nicotinic receptor function was determined as carbachol-stimulated uptake of 86Rb+. Cells were exposed to 50 mM K+ for up to 4 days and then allowed to repolarize for 60 min. Under these conditions, no changes in basal or carbachol-stimulated uptake of 86Rb+ were observed. Furthermore, neither the time course of carbachol-stimulated uptake or the carbachol concentration dependence of 86Rb+ uptake was altered. Finally, concurrent depolarization did not affect the functional down-regulation produced by chronic exposure of the cells to carbachol. Thus, neuronal nicotinic acetylcholine receptors on PC12 cells do not appear to be regulated by depolarization or prolonged elevation of the intracellular Ca2+ level.     

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.     

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.     

G protein-independent activation of an inward Na(+) current by muscarinic receptors in mouse pancreatic beta-cells

Depolarization of pancreatic beta-cells is critical for stimulation of insulin secretion by acetylcholine but remains unexplained. Using voltage-clamped beta-cells, we identified a small inward current produced by acetylcholine, which was suppressed by atropine or external Na(+) omission, but was not mimicked by nicotine, and was insensitive to nicotinic antagonists, tetrodotoxin, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DiDS), thapsigargin pretreatment, and external Ca(2+) and K(+) removal. This suggests that muscarinic receptor stimulation activates voltage-insensitive Na(+) channels distinct from store-operated channels. No outward Na(+) current was produced by acetylcholine when the electrochemical Na(+) gradient was reversed, indicating that the channels are inward rectifiers. No outward K(+) current occurred either, and the reversal potential of the current activated by acetylcholine in the presence of Na(+) and K(+) was close to that expected for a Na(+)-selective membrane, suggesting that the channels opened by acetylcholine are specific for Na(+). Overnight pretreatment with pertussis toxin or the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) or guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S) instead of GTP to the pipette solution did not alter this current, excluding involvement of G proteins. Injection of a current of a similar amplitude to that induced by acetylcholine elicited electrical activity in beta-cells perifused with a subthreshold glucose concentration. These results demonstrate that muscarinic receptor activation in pancreatic beta-cells triggers, by a G protein-independent mechanism, a selective Na(+) current that explains the plasma membrane depolarization.     

Intracellular calcium mobilization on stimulation of the muscarinic cholinergic receptor in chick limb bud cells

Summary Cell suspensions of chick limb buds (stage 23/24) were loaded with the fluorescent Ca²⁺ chelator chlorotetracycline. Fluorescence was monitored in a spectrofluorometer. Stimulation with acetylcholine induced a fluorescence decrease, indicating intracellular Ca²⁺ mobilization. The fluorescence decrease triggered by acetylcholine was inhibited by muscarinic but not by nicotinic antagonists, indicating that a muscarinic acetylcholine receptor is involved. The muscarinic receptor in the chick limb bud has a characteristic pharmacological profile: acetylcholine, carbachol and acetyl--methylcholine functioned as full agonists triggering maximal fluorescence decrease. Bethanechol was less effective, producing only one-third of the maximum response. Pilocarpine and oxotremorine, two classical agonists in other systems, were ineffective and functioned as antagonists. In the chick limb bud, cholinesterase, choline acetyltransferase and the presence of a muscarinic receptor have been demonstrated in previous studies. The present experiments show that stimulation of the embryonic muscarinic receptor leads to intracellular Ca²⁺ mobilization.     

Acetylcholine and cholecystokinin receptors functionally couple by different G-proteins to phospholipase C in pancreatic acinar cells

We have studied the involvement of GTP-binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin-octapeptide (CCK-OP) and GTPγS but not carbachol (CCh)-induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator of adenylyl cyclase, nor the cAMP-analogue, 8-bromo cAMP, mimicked the inhibitory effect of cholera toxin on agonist-induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated G_s with phospholipase C or to an elevation of cAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP-ribosylated a 40 kDa protein, which was inhibited by CCK-OP but not by CCh. We conclude from these data that both CCK- and muscarinic acetylcholine receptors functionally couple to phospholipase C by two different GTP-binding proteins.     

A role for acetylcholine receptors in the fusion of chick myoblasts

The role of acetylcholine receptors in the control of chick myoblast fusion in culture has been explored. Spontaneous fusion of myoblasts was inhibited by the nicotinic acetylcholine receptor antagonists alpha-bungarotoxin, Naja naja toxin and monoclonal antibody mcAb 5.5. The muscarinic antagonists QNB and n-methyl scopolamine were without effect. Atropine had no effect below 1 microM, where it blocks muscarinic receptors; at higher concentrations, when it blocks nicotinic receptors also, atropine inhibited myoblast fusion. The inhibitions imposed by acetylcholine receptor antagonists lasted for approximately 12 h; fusion stimulated by other endogenous substances then took over. The inhibition was limited to myoblast fusion. The increases in cell number, DNA content, the level of creatine phosphokinase activity (both total and muscle-specific isozyme) and the appearance of heavy chain myosin, which accompany muscle differentiation, followed a normal time course. Pre-fusion myoblasts, fusing myoblasts, and young myotubes specifically bound labeled alpha-bungarotoxin, indicating the presence of acetylcholine receptors. The nicotinic acetylcholine receptor agonist, carbachol, induced uptake of [14C]Guanidinium through the acetylcholine receptor. Myoblasts, aligned myoblasts and young myotubes expressed the synthetic enzyme Choline acetyltransferase and stained positively with antibodies against acetylcholine. The appearance of ChAT activity in myogenic cultures was prevented by treatment with BUDR; nonmyogenic cells in the cultures expressed ChAT at a level which was too low to account for the activity in myogenic cultures. We conclude that activation of the nicotinic acetylcholine receptor is part of the mechanism controlling spontaneous myoblast fusion and that myoblasts synthesize an endogenous, fusion-inducing agent that activates the nicotinic ACh receptor.     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.     

Regulation of the solubilized bovine cerebral cortex muscarinic receptor by GTP and Na+

The muscarinic acetylcholine receptor was solubilized, in a sensitive form for GTP and Na+, from bovine cerebral cortex using a zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The solubilized muscarinic receptor displayed characteristics as follows: (1) high affinity to nanomolar concentration of Z-[3H]quinuclidinyl benzilate; (2) muscarinic agonists and antagonists had similar inhibitory potencies as on the membrane-bound receptor; (3) without Na+, GTP did not significantly alter the binding affinity of muscarinic agonists and antagonists; (4) GTP in the presence of Na+, selectively decreased the affinity of muscarinic agonists, carbamylcholine and oxotremoline, but not the antagonist binding affinity; (5) Na+ in the absence or presence of GTP, reduced both muscarinic agonist and antagonist affinities.     

Effects of muscarinic receptor agonists and antagonists on response of non-extensor rats to maximal electroshock

Rats which do not respond consistently to maximal electroshock by exhibiting the classical hindlimb extensor response, are designated as 'flexors', and can serve as a useful experimental model for investigating seizure mechanisms. 20-25% Charles Foster rats exhibit the flexor status and were used in this study. The flexor rats were converted to extensors by acetylcholine (icv), physostigmine (ip) and the selective muscarinic M1 receptor agonists, arecholine (ip) and McN-A-343 (icv). This conversion of flexors to extensors was significantly attenuated by M1 receptor antagonists scopolamine (ip) and pirenzepine (icv). The M2 receptor agonist, carbachol (icv), had no effect in lower doses but induced conversion of flexor rats to the extensor status only in very high doses which may be due to loss of receptor specificity on dose increment. The M2 receptor antagonists, gallamine (icv) and AF-DX 116 (ip), also induced significant conversion of flexors to extensors, which was dependent upon the availability of neuronal acetylcholine since the effects were attenuated following pretreatment with hemicholinium, an inhibitor of acetylcholine synthesis. The results suggest that the central cholinergic system has a facilitatory pro-convulsant effect, mediated through the muscarinic M1 receptors, an action modulated by the M2 receptors.     

Characterization of the m4 muscarinic receptor Ca2+ response in a subclone of PC-12 cells by single cell flow cytometry. Inhibition of the response by bradykinin

We have studied Ca2+ mobilization mediated by the constitutively expressed muscarinic receptor on a subclone of PC-12 cells. The subclone, ACH2, was isolated with a flow cytometer by selection of single cells that exhibited a strong intracellular Ca2+ response to acetylcholine (ACh). Cell to cell heterogeneity of resting Ca2+ levels was markedly reduced in the subclone and homogeneity of the population response was also dramatically improved. ACH2 cells were highly sensitive to ACh and the Ca2+ response in all cells was blocked by muscarinic antagonists. Membranes from ACH2 exhibited muscarinic binding affinities which were not typical of M1, M2, or M3 receptors but were consistent with the profile of the putative m4 receptor. The same percentage of cells responded to ACh whether or not extracellular Ca2+ was reduced with EGTA, but the response was eliminated in all cells by preincubation with pertussis toxin. Thus, the constitutive m4 receptor on ACH2 cells is efficiently coupled to intracellular Ca2+ release by a pertussis toxin-sensitive mechanism. Stimulation of the ACH2 cells by bradykinin (BK) evoked a Ca2+ response in 90% of the cells. Prestimulation with BK diminished the magnitude of the muscarinic Ca2+ response but did not reduce the number of cells which responded to ACh. Inhibition was partially attributed to inhibition of a Ca2+ influx pathway in resting cells. Thus, the signaling mechanism coupled to the m4 muscarinic receptor can be inhibited by signals initiated by the BK receptor.     

The role of cyclic GMP in cells with the properties of smooth muscle cultured from the rat myometrium

Cells growing in culture with previously described properties of rat uterine smooth muscle accumulated 45Ca2+ from the medium. Ca2+ uptake by these cells was stimulated by the addition to the medium of 8-bromo-cGMP but not by 8-bromo-cAMP. Ca2+ uptake was also stimulated by carbachol and by the nitro-vasodilator nitroprusside. Although cholinergic agonists have been shown previously to stimulate contraction but not cGMP synthesis in the rat myometrium, both carbachol and nitroprusside stimulated cGMP production by the cultured cells. These results suggested the cells had cholinergic receptor-mediated functions that reflected some neurotransmitter-sensitive properties of uterine smooth muscle in situ. When determined by a specific radioligand binding assay, subcellular fractions of the cultured cells bound muscarinic cholinergic agonists and antagonists with affinities expected of the muscarinic receptor. The cells were also sensitive to the beta-adrenergic catecholamine agonist isoproterenol, which stimulated cAMP production but not Ca2+ uptake. Carbachol failed to inhibit isoproterenol-dependent cAMP production, which is an important property of the cholinergic receptor in uterine smooth muscle in situ. These results suggest some but not all acetylcholine-sensitive properties of uterine smooth muscle may be retained in cell culture.     

Direct measurement of the action of acetylcholine on isolated outer hair cells of the guinea pig cochlea.

Acetylcholine has long been thought to be the neurotransmitter of the cochlear efferent system in mammals although the evidence is largely indirect. By using whole-cell recordings from isolated outer hair cells, we show that acetylcholine activates a large rapidly desensitizing outward potassium current. This corresponds to hyperpolarization of the membrane potential from rest. The half maximal dose for acetylcholine was 13.5 microM with a cooperativity of 2. The response was not due to a conventional muscarinic action of acetylcholine for it was not blocked by 0.1 microM atropine and muscarinic antagonists but it could be blocked by 0.1 microM curare, suggesting that it shared many properties of a nicotinic receptor. It was, however, inhibited by 10 microM strychnine. The potassium current activated by acetylcholine required external calcium and was characterized by a significant delay at room temperature. This points to the involvement of a second messenger system, possibly calcium itself.