Lanthanum as a tool to study the role of phosphatidylinositol in the calcium transport in rat parotid glands upon cholinergic stimulation.

We describe the effects of lanthanum on protein secretion, potassium efflux, calcium uptake and phosphatidylinositol turnover stimulated by cholinergic agonists in rat parotid glands. Carbachol increases in vitro calcium uptake, protein secretion and K+ efflux through muscarinic receptor; however it fails to stimulate protein discharge or K+ release in a incubation medium free of calcium. Lanthanum inhibits calcium uptake, protein secretion and K+ efflux induced by carbachol without impairing protein discharge stimulated by norepinephrine through the beta-adrenergic receptor. Norepinephrine, in the presence of calcium in the incubation medium, stimulates the K+ efflux through the alpha-adrenergic receptor: this effect is suppressed by lanthanum. These results emphasize the role of increased influx of calcium in the cellular phenomena controlled by muscarinic or alpha-adrenergic receptors. Carbachol increases phosphatidylinositol turnover in the absence of calcium in extracellular medium; indeed it is shown that carbachol increases the rate of phosphatidylinositol breakdown and that lanthanum impairs this cholinergic effects. From these data it is suggested that the interaction between cholinergic agonist and muscarinic receptor could induce a stimulation of 'phosphatidylinositol turnover' which could control the calcium influx according to the gradient through the plasmalemma membrane.     

Stimulation of phosphatidylinositol turnover in various tissues by cholinergic and adrenergic agonists, by histamine and by caerulein

Studies are reported of the biochemical and pharmacological characteristics of the stimulation of phosphatidylinositol metabolism that is produced in appropriate target tissues by stimulation of various receptors that use Ca(2+) as their second messenger. (1) Muscarinic cholinergic and alpha-adrenergic phosphatidylinositol responses were observed in rat lacrimal gland, and a response to caerulein was detected in the longitudinal smooth muscle of guinea-pig ileum. (2) The muscarinic cholinergic phosphatidylinositol response of rat lacrimal gland, like that of several other tissues, is not dependent on the availability of extracellular Ca(2+). (3) Three phosphatidylinositol responses, namely to histamine in guinea-pig ileum smooth muscle, to alpha-adrenergic stimulation in rat vas deferens and to muscarinic cholinergic stimulation in rat lacrimal gland, were all found to involve phosphatidylinositol breakdown. (4) The stereospecificity of the muscarinic receptor responsible for the phosphatidylinositol response of guinea-pig pancreas was tested by using the two stereoisomeric forms of acetyl-beta-methylcholine; the S-isomer was very much more active than the R-isomer in provoking both phosphatidylinositol breakdown and its labelling with (32)P, as it is in provoking other physiological responses such as contractility or secretion. (5) Pilocarpine, a muscarinic partial agonist, provoked a significantly smaller phosphatidylinositol breakdown in rat parotid fragments than did carbamoylcholine, a potent muscarinic agonist. (6) All of these results are consistent with, but do not prove, a previously offered hypothesis that suggests that phosphatidylinositol breakdown is a reaction essential to stimulus-response coupling at a variety of cell-surface receptors that mobilize Ca(2+) from and through the plasma membranes of target tissues.     

Pertussis toxin does not inhibit muscarinic-receptor-mediated phosphoinositide hydrolysis or calcium mobilization.

Pertussis toxin was used to examine the role of the inhibitory guanine nucleotide regulatory protein, Ni, in muscarinic-receptor-mediated stimulation of phosphoinositide turnover and calcium mobilization. In cultured chick heart cells, pertussis-toxin treatment inhibited muscarinic-receptor-mediated attenuation of isoprenaline-stimulated cyclic AMP accumulation. This finding is consistent with the proposal that pertussis toxin blocks the capacity of Ni to couple muscarinic receptors to adenylate cyclase. In contrast, treatment of chick heart cells or 1321N1 human astrocytoma cells with pertussis toxin did not block muscarinic-receptor-mediated stimulation of phosphoinositide hydrolysis, as measured by [3H]inositol phosphate accumulation in the presence of Li+. Pertussis-toxin treatment also had little effect on basal and muscarinic-receptor-stimulated phosphatidylinositol synthesis, as measured by the incorporation of [3H]inositol into phosphatidylinositol. Activation of muscarinic receptors also enhances the rate of unidirectional 45Ca2+ efflux in 1321N1 cells; this response, like phosphoinositide hydrolysis, was not prevented by pertussis-toxin treatment. Our data suggest that muscarinic receptors are not coupled to phosphoinositide hydrolysis or calcium mobilization through Ni.     

Muscarinic release of inositol trisphosphate without mobilization of calcium in bovine adrenal chromaffin cells

Chromaffin cells of bovine adrenal medulla release catecholamines in response to activation of nicotinic ACh receptors which open voltage-sensitive calcium channels. Catecholamine secretion by exocytosis requires an increase in cytosolic free calcium. The cells also possess muscarinic ACh receptors but muscarinic agents do not provoke catecholamine release. Quin-2 studies show that they do not increase cytosolic free Ca2+ concentration, but unlike the nicotinic agents, they cause phosphoinositide hydrolysis. Muscarinic stimulation leads to rapid loss of labelled phosphatidylinositol 4-phosphate and of phosphatidylinositol 4,5-bisphosphate. At the same time there is release of inositol trisphosphate, inositol bisphosphate and inositol phosphate. In a number of other cells inositol trisphosphate may act as a second messenger releasing Ca2+ from storage sites in the endoplasmic reticulum but this is not its function in bovine chromaffin cells.     

Modulation of Second Messengers in the Nervous System of Larval Manduca sexta by Muscarinic Receptors

Abstract: Measurements were made of the effects of muscarinic agents on endogenous levels of cyclic AMP and cyclic GMP, and the turnover of radiolabeled inositol phosphates in the abdominal nervous system of larval Manduca sexta . Cyclic AMP levels were increased by treatment with 3-isobutyl-1-methylxanthine or tetrodotoxin, but the muscarinic agonist oxotremorine-M and the muscarinic antagonist scopolamine had no consistent effects. In contrast, cyclic GMP levels were significantly increased by oxotremorine-M and by oxotremorine-M in the presence of 3-isobutyl-1-methylxanthine and tetrodotoxin but not in the presence of scopolamine. Using lithium to inhibit the recycling of inositol phospholipid metabolites in isolated nerve cords, we detected a small but consistent increase in inositol phosphate production by oxotremorine-M. The primary inositol metabolite generated during a 5-min exposure to oxotremorine-M co-eluted from ion-exchange columns with inositol-1-monophosphate, although other more polar metabolites were also detected. This agonist-evoked increase in inositol phosphate production was unaffected by tetrodotoxin but inhibited by scopolamine, suggesting that it is directly mediated by muscarinic receptors. Further evidence for coupling between muscarinic receptors and inositol metabolism was obtained using a cell-free preparation of nerve cord membranes labeled with [³H]inositol. Incubation with oxotremorine-M evoked a significant increase in labeled inositol bisphosphate, consistent with muscarinic receptors coupling to phosphatidylinositol metabolism. The accumulation of inositol bisphosphate in cell-free preparations suggests that the normal breakdown to inositol monophosphate requires cytosolic components. Together, these results indicate that muscarinic acetylcholine receptors in Manduca couple predominantly to the inositol phospholipid signaling system, although some receptors may modulate cyclic GMP.     

Characterization of Muscarinic Receptors: Type M2 (Subtype B) on Neuro-2A Neuroblastoma Cells

Abstract

The binding of the nonselective muscarinic antagonist, [³H]N-methylscopolamine (NMS) to a mouse neuroblastoma cell line (Neuro-2A) and its coupling to the inhibition of adenylate cyclase were characterized. Specific [³H]NMS binding to membrane preparations was rapid, saturable, and of high affinity. Saturation experiments revealed a single class of binding sites for the radioligand. Competition experiments with the muscarinic drugs pirenzepine, AF DX 116, dicyclomine and atropine revealed that the muscarinic receptors present on these cells are predominantly of a single class, subtype B (M2). In addition, agonist binding demonstrated existence of a GTP-sensitive high affinity binding state of the receptors. Coupling of these muscarinic receptors to the adenylate cyclase system was investigated using the muscarinic agonist carbachol which was able to inhibit the prostaglandin (PGE₁)-stimulated activation of adenylate cyclase. The agonist carbachol did not stimulate the formation of IP₃ above basal levels, which indicated that the receptors are not coupled to phosphatidylinositol metabolism. In conclusion, we show that possessing predominantly one subtype of muscarinic receptor, the Neuro-2A cells provide a useful model for the investigation of the heterogeneity of muscarinic receptors and the relationship of subtype to the coupling of different effectors.     

Enhancement of muscarinic receptor-coupled phosphatidyl inositol hydrolysis in diabetic bladder

We previously have shown an increase in muscarinic receptor density in streptozotocin (STZ)-induced diabetic and sucrosefed diuretic rat detrusor that correlates with an increase in the contractile response to muscarinic agonist (J Pharmacol Exp Ther 248: 81, 1989; Diabetes 40: 265, 1991). To investigate the signal transduction pathway involved in this altered functional response, we examined muscarinic receptor-coupled phosphatidylinositol metabolism in STZ-diabetic, sucrose-fed diuretic and age-matched control rat bladders. [³H]myo-inositol uptake was similar in all groups, but incorporation of myo-inositol into phosphatidylinositol (PI) was significantly increased in the diabetic bladder compared to the sucrose-fed and control rat bladders. Carbachol-induced increase in inositol phosphate (IPs) production was higher in the diabetic bladder than in bladders from control and sucrose-fed animals although the EC₅₀ values were similar for all groups. Enhanced inositol phosphate production after muscarinic agonist stimulation may be due not only to the upregulation of muscarinic receptors but also to the increased incorporation of myo-inositol into PI in the STZ-induced diabetic bladder.     

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.     

Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands.

The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.     

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.     

Muscarinic regulation of phosphatidylinositol turnover and cyclic nucleotide metabolism in the heart

Stimulation of cardiac muscarinic receptors leads to increases in the synthesis and hydrolysis of the membrane phospholipid phosphatidylinositol (PI). Carbachol stimulates PI hydrolysis in right and left murine atria as well as in murine ventricule and dissociated embryonic chick heart cells. Muscarinic stimulation of PI hydrolysis is markedly attenuated in calcium-free medium, is not antagonized by isoproterenol, occurs after a latency of several minutes, and is half-maximally activated by approximately 10 microM carbachol. In contrast, muscarinic inhibition of cyclic AMP accumulation in the same preparations is calcium independent, is opposed by the effect of isoproterenol, is maximal in minutes, and is half-maximally activated by 0.1 microM carbachol. These differences demonstrate that the two muscarinic receptor-mediated events are probably unrelated and independent responses. The concentration of carbachol that causes half-maximal activation of PI hydrolysis is almost identical to that causing half muscarinic receptor occupancy as assessed by 3H-labeled (-)-quinuclidinyl benzilate binding. Thus activation of the PI response by carbachol appears to be closely linked to receptor occupancy, whereas cyclase inhibition may occur when only a small percentage of receptors are occupied. The possible role of the PI response in generating intracellular signals such as arachidonic acid release, cyclic GMP synthesis, or C-kinase activation is discussed.     

Enhancement of the muscarinic synaptosomal phospholipid labeling effect by the ionophore A23187

Addition of either acetylcholine (ACh) or the ionophore A23187 to synaptopsomes resulted in a selective stimulation of 32Pi incorporation into phosphatidate (PhA) and phosphatidylinositol (PhI), while the labeling of phosphatidylinositol phosphate (PhIP) and phosphatidylinositol diphosphate (PHIP2) was reduced. The inclusion of both ACh and A23187 resulted in a synergistic increase in PhA and PhI labeling, and a synergistic decrease in the labeling of the polyphosphoinositides. Added calcium was not required, although inclusion of EGTA prevented the alterations in lipid labeling. The enhanced labeling of PhA and PhI by ACh or A23187 was not the result of either an increase in the radioactivity of the precursor [32P]ATP pool, or increased de novo synthesis of these lipids as judged from the incorporation of [3H]glycerol, [3H]glucose or [3H]myo-inositol. The synergistic alterations in PhA, PhI, and polyphosphoinositide labeling were observed with ionophore only in the presence of selected muscarinic agonists, and with the inclusion of atropine or scopolamine the labeling reverted to a value which approximated that seen with the ionophore alone. Synergistic effects on phospholipid labeling with muscarinic agonists were also obtained with the calcium ionophore, ionomycin, but not with X537A, monensin, or valinomycin. Neither the apparent number of muscarinic receptors present, nor their affinity for the ligand were altered by the presence of A23187. In prelabeling experiments, A23187 accelerated the loss of [32P]label from PhIP and PhIP2, and the rate of loss was further augmented by the addition of ACh. Neither agent produced comparable effects on the breakdown of prelabeled PhA or PhI. It is suggested that phosphodiesteratic cleavage of the polyphosphoinositides might account for both the decrease in labeled PhIP and PhIP2 and increased labeling of PhA and PhI via the availability of resultant diglyceride. In any event, the results demonstrate that the turnover of polyphosphoinositides, in addition to that of PhA and PhI, is linked to the activation of muscarinic receptors.     

Breakdown of phosphatidylinositol provoked by muscarinic cholinergic stimulation of rat parotid-gland fragments

When rat parotid fragments that had been labelled with (32)P in vivo were exposed to high concentrations of acetylcholine, radioactivity was lost from phosphatidylinositol but not from other phospholipids. Simultaneously the concentration of phosphatidylinositol in the tissue decreased. If previously unlabelled tissue was incubated with (32)P(i) an increase in incorporation of radioactivity into phosphatidylinositol was observed during this decrease in concentration. The effects of acetylcholine were blocked by atropine, but not by tubocurarine. The response to acetylcholine was rapid, with up to one-third of the tissue's phosphatidylinositol disappearing within 5min. Similar effects were evoked by stimulation with methacholine and by high concentrations of tetramethylammonium ion; these responses were also atropine-sensitive and tubocurarine-insensitive. It is concluded that the event in inositol lipid metabolism that is affected by acetylcholine stimulation is removal of the phosphorylinositol group from the molecule; this is mediated through muscarinic cholinergic receptors. This is followed by a compensatory increase in the rate of synthesis of phosphatidylinositol, which has been described in detail in the past. These observations are compared with those of previous workers and are discussed in relation to the existing hypotheses relating to the significance of stimulus-provoked phosphatidylinositol turnover.     

Calcium and the Muscarinic Synaptosomal Phospholipid Labeling Effect

Abstract: The role of calcium in the muscarinic phospholipid labeling effect in synaptosomes has been investigated. In the absence of added calcium, acetylcholine doubled phosphatidate labeling and increased phosphatidy linositol labeling 40% in synaptosomes when incubated in a medium that contained [³²P]orthophosphate. Inclusion of calcium or the omission of magnesium resulted in a marked elevation of acetylcholine-stimulated phosphatidylinositol labeling (70–80%) while phosphatidate stimulation was unaltered. Calciumchelating agents, EGTA and EDTA, reduced the stimulated labeling of both phosphatidate and phosphatidylinositol, but this inhibition could be reversed by calcium addition. The calcium ionophore A23187, which promotes entry of calcium into cells, selectively increased labeling of both phosphatidate and phosphatidyl-inositol. This effect, unlike acetylcholine-stimulated labeling, was not blocked by the addition of atropine. The calcium dependency of the acetylcholine stimulation, on the one hand, and the insensitivity of the ionophore to a muscarinic antagonist, on the other, argue strongly that the acetylcholine-receptor interaction regulates calcium mobilization and that the latter is linked to the stimulated labeling of phosphatidate and phosphatidylinositol.     

Rapid accumulation of inositol phosphates in isolated rat superior cervical sympathetic ganglia exposed to V1-vasopressin and muscarinic cholinergic stimuli.

An accumulation of 3H-labelled inositol phosphates is observed when prelabelled rat superior cervical sympathetic ganglia are exposed to [8-arginine]vasopressin or to muscarinic cholinergic stimuli. The response to vasopressin is much greater than the response to cholinergic stimuli. The response to vasopressin is blocked by a V1-vasopressin antagonist, and oxytocin is a much less potent agonist than vasopressin. Vasopressin causes no increase in the cyclic AMP content of ganglia. These ganglia therefore appear to have functional V1-vasopressin receptors that are capable of activating inositol lipid breakdown, but no V2-receptors coupled to adenylate cyclase. The first [3H]inositol-labelled products to accumulate in stimulated ganglia are inositol trisphosphate and inositol bisphosphate, suggesting that the initiating reaction in stimulated inositol lipid metabolism is a phosphodiesterase-catalysed hydrolysis of phosphatidylinositol 4,5-bisphosphate (and possibly also phosphatidylinositol 4-phosphate). This response to exogenous vasopressin occurs in ganglia incubated in media of reduced Ca2+ concentration. The physiological functions of the V1-vasopressin receptors of these ganglia remain unknown.     

Comparison of muscarinic and alpha-adrenergic receptors in rat atria based on phosphoinositide turnover

The effects of muscarinic and alpha-adrenergic receptor stimulation on phosphoinositide turnover in rat atria have been compared. Despite the similar densities of muscarinic receptors in rat left and right atria, 0.1 mM carbachol increased [32P]phosphate incorporation into phosphatidylinositol (PI) by 35% (p less than 0.05) in left atria but had no effect in right atria. By contrast to the small muscarinic receptor effect, stimulation of alpha 1-adrenergic receptors by 0.1 mM methoxamine produced a more than two fold increase in [32P]phosphate incorporation into PI in both left and right atria, despite the reported smaller density of alpha-adrenergic receptors in rat atria compared to muscarinic receptors. Enhanced phosphate labelling by methoxamine did not occur in phospholipids other than PI, and was blocked by the alpha-adrenergic antagonist, phentolamine (20 microM). The results indicate that the majority of the muscarinic receptors in rat atria are not coupled to phosphoinositide turnover. If indeed the observed enhancement in [32P]-phosphate labelling by carbachol reflects phosphoinositide turnover, and assuming equal coupling efficiencies of muscarinic and adrenergic receptors, it is calculated that not more than 2% of the muscarinic receptors in rat left atria are coupled to this response.     

Activation of a common effector system by different brain neurotransmitter receptors in Xenopus oocytes

Xenopus oocytes possess 'native' muscarinic receptors, which give rise to oscillatory chloride currents; similar responses are elicited by activation of foreign receptors to serotonin, glutamate and noradrenaline, expressed in oocytes after injection of messenger RNA from rat brain. When low concentrations of two agonists are applied together, the combined response is greater than would be expected from the sum of the responses to each agonist applied alone. Potentiation of acetylcholine by serotonin is blocked by the serotonin antagonist methysergide; conversely, the potentiation of serotonin by acetylcholine is blocked by the muscarinic antagonist atropine. This indicates that each agonist acts on a distinct receptor. The interactions between serotonin, acetylcholine and other agonists provide further evidence that the different receptors may all 'link in' to a common receptor-channel coupling system, in which phosphoinositide metabolism and calcium liberation lead to the opening of chloride channels in the oocyte membrane.     

Muscarinic receptors on bovine chromaffin cells mediate a rise in cytosolic calcium that is independent of extracellular calcium

Although the mechanism by which nicotinic receptors on adrenal chromaffin cells regulate catecholamine secretion is reasonably well understood, that of the muscarinic receptors remains obscure. The effects of both acetylcholine and specific muscarinic agonists on cytosolic free calcium in isolated bovine adrenal chromaffin cells have been measured using the fluorescent probe Quin-2. Acetylcholine (0.1 mM) evokes a large increase in cytosolic free calcium from resting levels near 100 nM into the microM range, most of which is blocked by hexamethonium (0.5 mM) or removal of extracellular calcium. A small component of the acetylcholine-evoked rise in cytosolic free calcium (approximately 50-100 nM) is independent of extracellular calcium and is unaffected by 0.5 mM hexamethonium, but is totally blocked by 0.5 microM atropine. The muscarinic nature of this component is further confirmed by the fact that the muscarinic agonists, muscarine (0.1 mM) and methacholine (0.3 mM), stimulate a 50-100 nM rise in chromaffin cell cytosolic calcium which is blocked by 0.5 microM atropine and is largely independent of extracellular calcium. These results suggest that muscarinic receptors regulate cytosolic calcium in chromaffin cells by a new mechanism different from that of nicotinic receptors, a mechanism utilizing an intracellular calcium source. The small size of the muscarinic-induced rise in cytosolic calcium in the bovine chromaffin cell would explain why no secretion is evoked by muscarinic agonists in this species.     

Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, and regulation of M current

We have further tested the hypothesis that receptor-mediated modulation of KCNQ channels involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC). We used four parallel assays to characterize the agonist-induced PLC response of cells (tsA or CHO cells) expressing M1 muscarinic receptors: translocation of two fluorescent probes for membrane lipids, release of calcium from intracellular stores, and chemical measurement of acidic lipids. Occupation of M1 receptors activates PLC and consumes cellular PIP2 in less than a minute and also partially depletes mono- and unphosphorylated phosphoinositides. KCNQ current is simultaneously suppressed. Two inhibitors of PLC, U73122 and edelfosine (ET-18-OCH3), can block the muscarinic actions completely, including suppression of KCNQ current. However, U73122 also had many side effects that were attributable to alkylation of various proteins. These were mimicked or occluded by prior reaction with the alkylating agent N-ethylmaleimide and included block of pertussis toxin-sensitive G proteins and effects that resembled a weak activation of PLC or an inhibition of lipid kinases. By our functional criteria, the putative PLC activator m-3M3FBS did stimulate PLC, but with a delay and an irregular time course. It also suppressed KCNQ current. The M1 receptor-mediated activation of PLC and suppression of KCNQ current were stopped by lowering intracellular calcium well below resting levels and were slowed by not allowing intracellular calcium to rise in response to PLC activation. Thus calcium release induced by PLC activation feeds back immediately on PLC, accelerating it during muscarinic stimulation in strong positive feedback. These experiments clarify important properties of receptor-coupled PLC responses and their inhibition in the context of the living cell. In each test, the suppression of KCNQ current closely paralleled the expected fall of PIP2. The results are described by a kinetic model.     

Agonist-Induced Endocytosis of Muscarinic Cholinergic Receptors: Relationship to Stimulated Phosphoinositide Turnover

Abstract: The ability of muscarinic cholinergic receptors to activate phosphoinositide turnover following agonist-induced internalization has been investigated. Incubation of SH-SY5Y neuroblastoma cells with oxotremorine-M resulted in a time-dependent endocytosis of both muscarinic receptors and α subunits of G_q and G₁₁, but not of isoforms of phosphoinositide-specific phospholipase C, into a subfraction of smooth endoplasmic reticulum (V₁). Agonist-induced increases in diacylglycerol mass and in ³²P-phosphatidate labeling, much of which was of the tetraenoic species, were also observed in the V₁ fraction, but these increases persisted when the agonist-induced translocation of receptors into the V₁ fraction was blocked. All enzymes of the phosphoinositide cycle were detectable in the V₁ fraction. However, with the exception of phosphatidylinositol 4-kinase, none was enriched when compared with cell lysates. Both ³²P-labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinositol 4,5-bisphosphate, whereas the synthesis of phosphatidylinositol 4-phosphate is robust. These results indicate that endocytosed receptors do not appear to retain their ability to activate phosphoinositide turnover. The availability of the substrate for phospholipase C, phosphatidylinositol 4,5-bisphosphate, may be one factor that limits the activity of muscarinic receptors in this subcellular compartment.