Muscarinic receptor enhancement of nicotine-induced catecholamine secretion may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells

Bovine adrenal chromaffin cells possess both nicotinic and muscarinic cholinergic receptors, but only nicotinic receptors have heretofore appeared to mediate Ca2+-dependent exocytosis. We have now found that muscarinic receptor stimulation in bovine adrenal chromaffin cells leads to enhanced inositol phospholipid metabolism as evidenced by the rapid (less than 1 min) formation of inositol trisphosphate (IP3) and inositol bisphosphate (IP2). Muscarinic receptor-mediated accumulation of IP3 and IP2 continues beyond 1 min in the presence of LiCl and is accompanied by large increases in inositol monophosphate. Muscarinic receptor stimulation was also found to enhance nicotine-induced catecholamine secretion by 1.7-fold if muscarine was added 30 s before nicotine addition. Moreover, since the muscarinic antagonist atropine reduces acetylcholine-induced secretion, we conclude that muscarinic receptor stimulation somehow primes these cells for nicotinic receptor-mediated secretion, perhaps by causing small nonstimulatory increases in cytosolic free Ca2+ mediated by IP3. Furthermore, we show that small depolarizations of these cells with 10 mM K+, which themselves do not affect basal secretion, also enhance nicotine-induced secretion. Thus, small increases in cytosolic free Ca2+ produced either by physiologic muscarinic receptor stimulation or by small experimental depolarizations with K+ may prime the chromaffin cells for nicotinic receptor-mediated secretion.     

Properties of receptor-controlled inositol trisphosphate formation in parotid acinar cells.

Activation of muscarinic receptors in rat parotid cells results in breakdown of polyphosphoinositides liberating inositol phosphates, including inositol trisphosphate. Formation of inositol trisphosphate appears independent of agonist-induced Ca2+ mobilization, since neither formation nor degradation of inositol trisphosphate are appreciably altered in low-calcium media, and elevation of cytosolic Ca2+ with a calcium ionophore does not cause an increase in cellular inositol trisphosphate. Further, activation of substance P receptors and alpha 1-adrenoreceptors, but not beta-adrenoreceptors, increases inositol trisphosphate formation. The dose-response curve for methacholine activation of inositol trisphosphate formation more closely approximates the curve for receptor occupancy than for Ca2+-activated K+ release. These results are all consistent with the suggestion that inositol trisphosphate could function as a second messenger linking receptor occupation to cellular Ca2+ mobilization.     

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophosphate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mM Ca2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+-dependent [3H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3-10 microM) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catecholamine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and myo-inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.     

Antigen-stimulated metabolism of inositol phospholipids in the cloned murine mast-cell line MC9.

Cells of the murine mast-cell clone MC9 grown in suspension culture were sensitized with an anti-DNP (dinitrophenol) IgE and subsequently prelabelled by incubating with [32P]Pi. Stimulation of these cells with DNP-BSA (bovine serum albumin) caused marked decreases in [32P]polyphosphoinositides (but not [32P]phosphatidylinositol) with concomitant appearance of [32P]phosphatidic acid. Whereas phosphatidylinositol monophosphate levels returned to baseline values after prolonged stimulation, phosphatidylinositol bisphosphate levels remained depressed. Stimulation of sensitized MC9 cells with DNP-BSA increased rates of incorporation of [32P]Pi into other phospholipids in the order: phosphatidylcholine greater than phosphatidylinositol greater than phosphatidylethanolamine. In sensitized cells prelabelled with [3H]inositol, release of inositol monophosphate, inositol bisphosphate and inositol trisphosphate, was observed after stimulation with DNP-BSA. When Li+ was added to inhibit the phosphatase activity that hydrolysed the phosphomonoester bonds in the sugar phosphates, greater increases were observed in all three inositol phosphates, particularly in inositol trisphosphate. The IgE-stimulated release of inositol trisphosphate was independent of the presence of extracellular Ca2+. In addition, the Ca2+ ionophore A23187 caused neither the decrease in [32P]polyphosphoinositides nor the stimulation of the release of inositol phosphates. These results demonstrate that stimulation of the MC9 cell via its receptor for IgE causes increased phospholipid turnover, with effects on polyphosphoinositides predominating. These data support the hypothesis that hapten cross-bridging of IgE receptors stimulates phospholipase C activity, which may be an early event in stimulus-secretion coupling of mast cells. The results with the Ca2+ ionophore A23187 indicate that an increase in intracellular Ca2+ alone is not sufficient for activation of this enzyme.     

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.     

Inositol trisphosphate mediates thyrotropin-releasing hormone mobilization of nonmitochondrial calcium in rat mammotropic pituitary cells

Thyrotropin-releasing hormone (TRH) stimulation of prolactin secretion from GH3 cells, cloned rat pituitary tumor cells, is associated with 1) hydrolysis of phosphatidylinositol 4,5-bisphosphate to yield inositol trisphosphate (InsP3) and 2) elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i), caused in part by mobilization of cellular calcium. We demonstrate, in intact cells, that TRH mobilizes calcium and, in permeabilized cells, that InsP3 releases calcium from a nonmitochondrial pool(s). In intact cells, TRH caused a loss of 16 +/- 2.7% of cell-associated 45Ca which was not inhibited by depleting the mitochondrial calcium pool with uncoupling agents. Similarly, TRH caused an elevation of [Ca2+]i from 127 +/- 6.3 nM to 375 +/- 54 nM, as monitored with Quin 2, which was not inhibited by depleting mitochondrial calcium. Saponin-permeabilized cells accumulated Ca2+ in an ATP-dependent manner into a nonmitochondrial pool, which exhibited a high affinity for Ca2+ and a small capacity, and into a mitochondrial pool which had a lower affinity for Ca2+ but was not saturated under the conditions tested. Permeabilized cells buffered free Ca2+ to 129 +/- 9.2 nM when incubated in a cytosol-like solution initially containing 200 to 1000 nM free Ca2+. InsP3, but not other inositol sugars, released calcium from the nonmitochondrial pool(s); half-maximal effect occurred at approximately 1 microM InsP3. Ca2+ release was followed by reuptake into a nonmitochondrial pool(s). These data suggest that InsP3 serves as an intracellular mediator (or second messenger) of TRH action to mobilize calcium from a nonmitochondrial pool(s) leading to an elevation of [Ca2+]i and then to prolactin secretion.     

Chick embryo muscarinic and purinergic receptors activate cytosolic Ca2+ via phosphatidylinositol metabolism

In dissociated cells from chick embryos or from chick limb buds, acetylcholine (ACh) induced an increase in cellular levels of inositol 1,4,5-trisphosphate (Ins-P3) and of inositol 1,3,4,5-tetrakisphosphate (Ins-P4). The concentration of Ins-P3 was enhanced transiently, whereas the level of Ins-P4 remained elevated for at least 20 min after addition of ACh. In most cases the increase in Ins-P4 levels was more pronounced than that of Ins-P3 levels. The inhibition of the ACh-induced inositol-phosphate response by atropine (half-maximal inhibition at 10 nM) indicates the involvement of muscarinic receptors, which in chick embryo cells induce a transient rise and a following persistent elevation of cytosolic Ca2+ activity (G. Oettling et al. (1989) J. Dev. Physiol. 12, 85-94). Adenosine 5'-triphosphate (ATP) elicited a similar transient rise in cytosolic Ca2+ activity, however, without a subsequent plateau. ATP also caused an increase in inositol-oligophosphate levels. Thus, both muscarinic and purinergic receptors in chick embryo cells are coupled to phospholipase C. The enzymatically formed Ins-P3 mediates the release of Ca2+ from internal stores. The Ca2+ signal could be involved in embryonic cell migration during morphogenesis.     

Inositol Trisphosphate Mobilizes Intracellular Calcium in Permeabilized Adrenal Chromaffin Cells

Using permeabilized chromaffin cells and the fluorescent probe Quin 2 (an indicator of free Ca2+), we found that inositol trisphosphate (IP3) specifically triggered an immediate and dose-dependent release of Ca2+ from intracellular stores. Desensitization of the response was observed at nonsaturating concentrations of inositol trisphosphate and resequestration of Ca2+ was not observed. While representing only a small fraction of the total cellular Ca2+, the amount released by IP3 could significantly raise cytosolic Ca2+ and may account for muscarinic effects on Ca2+ metabolism in chromaffin cells.     

Attenuation of inositol trisphosphate generation and cytosolic Ca2+ elevation in dispersed rat parotid acini stimulated simultaneously at muscarinic and alpha 1-adrenergic receptors

We have examined intracellular signalling events, peak cytosolic [Ca2+] and inositol trisphosphate levels, in rat parotid acini simultaneously stimulated with two Ca2+ mobilizing agonists, carbachol (muscarinic-cholinergic) and epinephrine (alpha 1-adrenergic). When the agonists were added together, either at sub-maximal (200 nM each, i.e. 400 nM total agonist concentration) or maximal (10 uM each, i.e. 20 uM total) stimulatory concentrations, the resulting elevations in both cytosolic [Ca2+] and inositol trisphosphate levels were not greater than those achieved when each agonist was added individually. However, with 400 nM carbachol these responses were significantly greater than those seen with either 200 nM carbachol or 200 nM carbachol + 200 nM epinephrine. The data indicate that when muscarinic and alpha 1-adrenergic receptors of rat parotid acini are simultaneously stimulated a novel regulatory mechanism is induced, which attenuates inositol trisphosphate generation and, consequently, intracellular Ca2+ release.     

Receptor-mediated metabolism of the phosphoinositides and phosphatidic acid in rat lacrimal acinar cells.

The metabolism of the inositol lipids and phosphatidic acid in rat lacrimal acinar cells was investigated. The muscarinic cholinergic agonist methacholine caused a rapid loss of 15% of [32P]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and a rapid increase in [32P]phosphatidic acid (PtdA). Chemical measurements indicated that the changes in 32P labelling of these lipids closely resembled changes in their total cellular content. Chelation of extracellular Ca2+ with excess EGTA caused a significant decrease in the PtdA labelling and an apparent loss of PtdIns(4,5)P2 breakdown. The calcium ionophores A23187 and ionomycin provoked a substantial breakdown of [32P]PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P); however, a decrease in [32P]PtdA was also observed. Increases in inositol phosphate, inositol bisphosphate and inositol trisphosphate were observed in methacholine-stimulated cells, and this increase was greatly amplified in the presence of 10 mM-LiCl; alpha-adrenergic stimulation also caused a substantial increase in inositol phosphates. A23187 provoked a much smaller increase in the formation of inositol phosphates than did either methacholine or adrenaline. Experiments with excess extracellular EGTA and with a protocol that eliminates intracellular Ca2+ release indicated that the labelling of inositol phosphates was partially dependent on the presence of extracellular Ca2+ and independent of intracellular Ca2+ mobilization. Thus, in the rat lacrimal gland, there appears to be a rapid phospholipase C-mediated breakdown of PtdIns(4,5)P2 and a synthesis of PtdA, in response to activation of receptors that bring about an increase in intracellular Ca2+. The results are consistent with a role for these lipids early in the stimulus-response pathway of the lacrimal acinar cell.     

Relationship between inositol polyphosphate production and the increase of cytosolic free Ca2+ induced by vasopressin in isolated hepatocytes

Addition of vasopressin to rat hepatocytes prelabeled with myo-[2-3H]inositol resulted in a very rapid decrease [3H]phosphatidylinositol 4,5-bisphosphate (Ptd-Ins-4,5-P2) which was paralleled by increases of up to 3-fold in the levels of [3H]inositol trisphosphate (Ins-P3) and [3H]inositol bisphosphate (Ins-P2). Increases of [3H]inositol phosphate (Ins-P) were not detected until about 5 min after hormone addition. These data indicate that the major pathway for hormone-induced lipid breakdown in liver is through a phosphodiesterase for PtdIns-4,5-P2 and that decreases of phosphatidylinositol are a secondary result of increased PtdIns-4,5-P2 resynthesis. Using the fluorescent Ca2+ indicator Quin 2, cytosolic free Ca2+ increased from 160 nM to about 400 nM after vasopressin addition to hepatocytes and preceded the conversion of phosphorylase b to a. Half-maximal and maximal increases of cytosolic free Ca2+ and phosphorylase a activity were observed at 0.2 and 1 nM vasopressin, respectively. The dose-response curve for the initial rate of cytosolic free Ca2+ increase was very similar to those obtained for the initial rates of Ins-P3 production and PtdIns-4,5-P2 breakdown. Pretreatment of hepatocytes with Li+ caused a 3--4-fold potentiation of vasopressin-induced elevations of Ins-P, Ins-P2, and Ins-P3, with half-maximal effects at 0.5, 1, and 5 mM, respectively. The calculated maximal concentrations of Ins-P3 in cells treated with 20 nM vasopressin were 10 and 30 microM, respectively, without and with Li+. Lithium did not affect the initial rate of inositol polyphosphate production or Ca2+ mobilization. The increase of Ins-P3 which correlated with peak cytosolic free Ca2+ elevation was about 0.6 microM. In a saponin-permeabilized hepatocyte preparation, Ins-P3 (1 microM) caused Ca2+ release from a vesicular, ATP-dependent Ca2+ pool. The data presented here suggest that Ins-P3 may be a second messenger for the mobilization of intracellular Ca2+ by hormones in liver.     

Secretin induces rapid increases in inositol trisphosphate, cytosolic Ca2+ and diacylglycerol as well as cyclic AMP in rat pancreatic acini.

Previous studies have shown that the dose-response relationship for secretin-stimulated cyclic AMP accumulation is different from that for secretin-stimulated enzyme secretion in the rat exocrine pancreas. Here we show that secretin concentrations of 10(-10) M and higher stimulated a rise in cyclic AMP levels, with maximum effect on cyclic AMP accumulation being achieved already with 10(-8) M-secretin. However, at this concentration of secretin, enzyme secretion rates were approximately half-maximal. Unexpectedly, at concentrations of secretin greater than 10(-8) M there was evidence suggestive of phosphatidylinositol bisphosphate hydrolysis with rapid increases in inositol trisphosphate, cytosolic free calcium and diacylglycerol content of rat pancreatic acini. Furthermore, there was a dose-response relationship among secretin concentration (in the range 10(-8) M-2 X 10(-6) M), increases in inositol trisphosphate and increases in cytosolic free calcium ([Ca2+]i). Contrary to what has been previously believed, these results clearly indicate that in rat pancreatic acini secretin not only stimulates cyclic AMP accumulation but also raises inositol trisphosphate, [Ca2+]i and diacylglycerol. Thus, two second messenger systems may play a role in the regulation of secretin-induced amylase release.     

Calcium mediated neurohumoral inhibition of chicken enterocyte Na influx: role of phosphatidylinositol metabolites.

Carbachol (CCH), serotonin (5HT), divalent ionophore A23187, cAMP, and certain neuropeptides, i.e. substance P (SP), inhibit the initial rate of uptake (influx) of 22Na into isolated chicken villus enterocytes. All these agents also increase cytosolic Ca. However, the increases stimulated by CCH, 5HT, and cAMP are not blocked by chelation of extracellular Ca, whereas those of A23187 and SP are. Only CCH and 5HT stimulate hydrolysis of membrane phosphoinositides to form inositol phosphates. CCH and 5HT also stimulate incorporation of [32P]-PO4 into membrane polyphosphoinositides. These studies suggest that at least three mechanisms exist to increase cytosolic Ca in chicken enterocytes and thereby inhibit Na influx. Certain neurohumoral agents such as SP open a plasma membrane permeability for Ca, permitting extracellular Ca to enter the cell down its electrochemical gradient. These agents do not stimulate phosphatidylinositol breakdown. CCH and 5HT stimulate phosphatidylinositol breakdown and via the formation of inositol trisphosphate release Ca from intracellular stores. A third mechanism exists for cAMP which mobilizes Ca from intracellular stores, but does not involve the metabolism of membrane phosphatidylinositols.     

Synthetic diacylglycerols trigger an increase of intracellular free calcium in promyelocytic HL60 cells

Phosphoinositide turnover is known to play an important role in intracellular free calcium homeostasis through the inositol trisphophate-mediated release of calcium from intracellular stores. We find that the other product of phosphoinositide turnover, 1,2-diacylglycerol, elicits an increase in intracellular free calcium in HL60 cells which is due, at least in part, to release of calcium from intracellular stores. This effect is specific for calcium, since intracellular sodium and potassium levels and cellular volume were unaffected. Concomitant with the intracellular calcium increase, we find an increase in cellular inositol trisphosphate levels, suggesting that the effect of diacylglycerol on calcium may be mediated by inositol trisphosphate. Diacylglycerols also stimulate calcium efflux. This stimulation is not simply due to the increase in intracellular calcium. These effects appear not to be mediated through stimulation of a phorbol ester-activatable protein kinase C (Ca2+/phospholipid-dependent enzyme) since phorbol esters do not elicit an increase in cytoplasmic free calcium or an increase in calcium efflux.     

Prostaglandin E receptor enhancement of catecholamine release may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells

In the presence of ouabain, prostaglandin (PG) E2 stimulated a gradual secretion of catecholamines from cultured bovine adrenal chromaffin cells. PGE2 or ouabain alone evoked a marginal secretory response. The synergism of ouabain was also observed with muscarine. PGE2, like muscarine, induced a concentration-dependent formation of inositol phosphates: rapid rises in inositol trisphosphate and inositol bisphosphate followed by a slower accumulation of inositol monophosphate. This effect on phosphoinositide metabolism was accompanied by an increase in cytosolic free Ca2+. The potency of PGs (PGE2 greater than PGF2 alpha greater than PGD2) to stimulate catecholamine release was well correlated with that to affect phosphoinositide metabolism and that to increase the level of intracellular Ca2+. PGE2 did not stimulate cAMP generation significantly in bovine chromaffin cells. The effect of PGE2 on catecholamine release was mimicked by 12-O-tetradecanoylphorbol 13-acetate and A23187, but not by the cAMP analogue dibutyryl cAMP nor by forskolin. These results indicate that PGE2 may enhance catecholamine release from chromaffin cells by activating protein kinase C in concert with the increment of intracellular Ca2+.     

Cyclic ADP-ribose regulation of ryanodine receptors involved in agonist evoked cytosolic Ca2+ oscillations in pancreatic acinar cells.

We have investigated the role of the ryanodine-sensitive intracellular Ca2+ release channel (ryanodine receptor) in the cytosolic Ca2+ oscillations evoked in pancreatic acinar cells by acetylcholine (ACh) or cholecystokinin (CCK). Ryanodine abolished or markedly inhibited the agonist evoked Ca2+ spiking, but enhanced the frequency of spikes evoked by direct internal inositol trisphosphate (InsP3) application. We have also investigated the possibility that cyclic ADP-ribose (cADP-ribose), the putative second messenger controlling the ryanodine receptor, plays a role in Ca2+ oscillations. We found that cADP-ribose could itself induce repetitive Ca2+ spikes localized in the secretory pole and that these spikes were blocked by ryanodine, but also by the InsP3 receptor antagonist heparin. Our results indicate that both the ryanodine and the InsP3 receptors are involved in Ca2+ spike generation.     

Calcium mobilization and catecholamine secretion in adrenal chromaffin cells. A Quin-2 fluorescence study

To better understand the relation between cell calcium and exocytotic secretion, a quantitative dependence of adrenal catecholamine secretion on cytosolic free calcium has been determined for isolated, intact, bovine chromaffin cells, using the fluorescent probe Quin-2. The cells required a threshold of 250-300 nM cytosolic calcium to be reached before detectable secretion occurred and half-maximal secretion occurred near 2 microM cytosolic calcium. Nicotinic receptors mediated an increase of cytosolic calcium from resting levels near 100 nM to levels in the 1-10 microM range within seconds followed by a decay back to resting levels over several minutes. Muscarinic receptors mediated a smaller rise in cytosolic free calcium from 100 to about 200 nM, within seconds. The nicotinic response required extracellular calcium, while the muscarinic response was largely independent of extracellular calcium, suggesting the latter mobilizes intracellular calcium. The acetylcholine-evoked rise in cytosolic calcium decayed by at least two kinetically distinct processes with half-time constants: t1 = 0.6 min and t2 = 3.2 min. Extracellular Na+ deprivation caused a more prolonged elevation of the acetylcholine-evoked calcium transient, suggesting a possible role of Na+/Ca2+ exchange and/or other Na+ -dependent processes in lowering cytosolic calcium following stimulation. The possible perturbing effects of Quin-2 on resting and stimulated cytosolic calcium levels and on secretion were examined and a novel use of Quin-2 to measure membrane calcium flux was demonstrated.     

Inositol 1,4,5-trisphosphate releases Ca2+ from intracellular store sites in skinned single cells of porcine coronary artery

Effects of inositol 1,4,5- trisphosphate , extracted from human erythrocyte ghosts, on Ca2+ release from intracellular store sites were studied in saponin-treated single muscle cells of the porcine coronary artery. Application of micromolar concentrations of inositol 1,4,5- trisphosphate released Ca2+ from the intracellular non-mitochondrial store sites, within 1 min. However, when the concentrations of free Ca2+ were over 1.5 X 10(-6) M, the release of Ca2+ by this agent was inhibited. The Ca2+ releasing mechanism differed from that seen with A23187, therefore this release of Ca2+ from store sites was not due to Ca2+ ionophore actions. This agent may play the role of messenger in increasing the cytosolic Ca2+, provoking pharmaco-mechanical coupling, and thus producing the contraction.     

Calcium promotes the accumulation of polyphosphoinositides in intact and permeabilized bovine adrenal chromaffin cells

1. Because cellular pools of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate turn over rapidly during phospholipase C stimulation, the continuing production of inositol phosphates requires continuing synthesis from phosphatidylinositol of the polyphosphoinositides. In the present study in adrenal chromaffin cells, we examined the effects of nicotinic stimulation and depolarization in intact cells and micromolar Ca2+ in permeabilized cells on the levels of labeled polyphosphoinositides. We compared the effects to muscarinic stimulation in intact cells and GTP gamma S in permeabilized cells. 2. Nicotinic stimulation, elevated K+, and muscarinic stimulation cause similar production of inositol phosphates (D. A. Eberhard and R. W. Holz, J. Neurochem. 49:1634-1643, 1987). Nicotinic stimulation and elevated K+ but not muscarinic stimulation increased the levels of [3H]inositol-labeled phosphatidylinositol phosphate by 30-60% and [3H]phosphatidylinositol bisphosphate by 25-30%. The increase required Ca2+ in the medium, was maximal by 1-2 min, and was not preceded by an initial decrease in phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. 3. In digitonin-permeabilized cells, Ca2+ caused as much as a twofold increase in [3H]phosphatidylinositol phosphate and [3H]phosphatidylinositol bisphosphate. Similarly, Ca2+ enhanced the production of [32P]phosphatidylinositol phosphate and [32P]phosphatidylinositol bisphosphate in the presence of [gamma-32P]ATP. In contrast, GTP gamma S in permeabilized cells decreased polyphosphoinositides in the presence or absence of Ca2+. 4. The ability of Ca2+ to increase the levels of the polyphosphoinositides decayed with time after permeabilization. The effect of Ca2+ was increased when phosphoesterase and phospholipase C activities were inhibited by neomycin. 5. These observations suggest that Ca2+ specifically enhances polyphosphoinositide synthesis at the same time that it activates phospholipase C.     

Activation of muscarinic receptors in PC12 cells. Correlation between cytosolic Ca2+ rise and phosphoinositide hydrolysis.

The intracellular signals generated by carbachol activation of the muscarinic receptor [release of inositol phosphates as a consequence of phosphoinositide hydrolysis and rise of the cytosolic Ca2+ concentration ([Ca2+]i, measured by quin2)] were studied in intact PC12 pheochromocytoma cells that had been differentiated by treatment with nerve growth factor. When measured in parallel samples of the same cell preparation 30 s after receptor activation, the release of inositol trisphosphate and of its possible metabolites, inositol bis- and mono-phosphate, and the [Ca2+]i rise were found to occur with almost superimposable carbachol concentration curves. At the same time carbachol caused a decrease in the radioactivity of preloaded phosphatidylinositol 4,5-bisphosphate, the precursor of inositol trisphosphate. Neither the inositol phosphate nor the [Ca2+]i signal was modified by preincubation of the cells with either purified Bordetella pertussis toxin or forskolin, the direct activator of adenylate cyclase. Both signals were partially inhibited by dibutyryl cyclic AMP, especially when the nucleotide analogue was applied in combination with the phosphodiesterase inhibitors RO 201724 and theophylline. The latter drug alone profoundly inhibited the carbachol-induced [Ca2+]i rise, with only minimal effect on phosphoinositide hydrolysis. Because of the diverging results obtained with forskolin on the one hand, dibutyryl cyclic AMP and phosphodiesterase inhibitors on the other, the effects of the latter drugs are considered to be pharmacological, independent of the intracellular cyclic AMP concentration. Two further drugs tested, mepacrine and MY5445, inhibited phosphoinositide hydrolysis at the same time as the 45Ca2+ influx stimulated by carbachol. Taken together, our results concur with previous evidence obtained with permeabilized cells and cell fractions to indicate phosphatidylinositol 4,5-bisphosphate hydrolysis and [Ca2+]i rise as two successive events in the intracellular transduction cascade initiated by receptor activation. The strict correlation between the carbachol concentration curves for inositol trisphosphate generation and [Ca2+]i rise, and the inhibition by theophylline of the Ca2$ signal without major effects on inositol phosphate generation, satisfy important requirements of the abovementioned interpretation.