Calcium involvement in the muscarinic response of the gastric parietal cell

The influence of extracellular Ca2+ on the mediation of carbachol stimulation in isolated rabbit gastric parietal cells was studied. Removing Ca2+ from extracellular medium caused a 42% decrease of the aminopyrine accumulation due to carbachol with the same EC50 value (approximately 5 microM). A short time depletion in extracellular calcium suppressed the carbachol-dependent Ca2+ influx without affecting Ca2+ release from internal stores (fura-2 measurements). Similarly, the production of inositol phosphates under cholinergic stimulation was reduced by 29%. A rapid increase in Ins(1,4,5)P3 was obtained 5 s after carbachol stimulation, and this increase was not changed in Ca2(+)-depleted medium. In contrast, a 20 min incubation with carbachol caused a 50% reduction in both basal and carbachol-stimulated inositol phosphate accumulations. In conclusion, phospholipase C activation, intracellular Ca2+ release and aminopyrine accumulation were sequentially observed following carbachol stimulation of the isolated gastric parietal cell and extracellular calcium contributed to sustain this acid secretory response.     

Control by calcium of protein discharge and membrane permeability to potassium in the rat lacrimal gland

Discharge of protein from slices of rat exorbital lacrimal gland was stimulated by 10^?5 M carbachol. This response was blocked by 10^?4 M atropine or by the omission of extracellular calcium. In the latter case, secretion could be restored by the reintroduction of calcium to the medium. Carbachol (10^?5 M) also stimulated the release of ⁸⁶Rb (a marker for potassium) from the slices. This effect was completely blocked by 10^?4 M atropine. The initial transient release of ⁸⁶Rb was only partially inhibited by Ca removal, but the later sustained phase of release was completely blocked. As with protein secretion, this effect of Ca removal could be reversed by re-introduction of Ca to the medium. It is concluded that activation of cholinergic receptors in the lacrimal gland stimulates protein discharge and increases potassium permeability by mechanisms utilizing extracellular calcium ions.     

M2 muscarinoceptor-associated ionophore at the cat adrenal medulla

Atropine and pirenzepine displaced 3H-quinuclydinyl-benzylate binding and inhibited methacholine-evoked catecholamine release with a similar order of potencies, atropine being 200 fold more potent than pirenzepine. In contrast to high-K, methacholine-evoked 45Ca uptake or catecholamine release were not blocked by (+)PN200-110. Bay-K-8644 did not modify the secretory response to methacholine either in the presence of Ca or Sr but potentiated K-evoked secretion. In depolarized glands, methacholine still evoked its usual secretory response. The results suggest that muscarinic stimulation of cat adrenal chromaffin cells stimulates Ca entry though an ionophore other than voltage-dependent Ca channels; such ionophore seems to be chemically operated through a M2 muscarinoceptor.     

Involvement of Ca2+ entry and inositol trisphosphate-induced internal Ca2+ mobilization in muscarinic receptor-mediated catecholamine release in dog adrenal chromaffin cells.

Catecholamine (CA) release from adrenal medulla evoked by muscarinic receptor stimulation has been studied using isolated perfused adrenal gland and cultured chromaffin cells from dogs. Muscarine and oxotremorine (1-100 microM), and bethanechol (0.1-1 mM) dose-dependently stimulated CA release. Muscarine-evoked CA release was antagonized with M1-antagonist, pirenzepine and, to a lesser extent, with atropine; and was reduced either by removal of extracellular Ca2+ or treatment with Ca2+ channel blockers. Muscarine caused an increase of 45Ca uptake and 22Na uptake. Tetrodotoxin (TTX) did not affect muscarine-evoked increase of 22Na uptake and CA release. Under the absence of extracellular Ca2+, muscarine stimulated a 45Ca efflux. Muscarine-induced CA release was attenuated by treating the cells with 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate-HCl (TMB-8) which blocks Ca2+ release from the intracellular store. A phospholipase C inhibitor, neomycin, markedly reduced muscarine-induced CA release but not nicotine- and high K(+)-evoked release. Cinnarizine, a Ca2+ channel blocker, attenuated muscarine-evoked but not caffeine-induced CA release and 45Ca efflux in the absence of extracellular Ca2+. Muscarine caused an increase in intracellular free Ca2+ concentration ([Ca2+]i) in the presence of extracellular Ca2+. It caused a similar increase, but to a lesser extent, in the absence of extracellular Ca2+. The increase of [Ca2+]i induced by muscarine without extracellular Ca2+ was reduced by neomycin and cinnarizine. Polymixin B and retinal, which reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CA release, had little effect on muscarine-induced CA release. Muscarine increased cellular Ins(1,4,5)P3 production, and atropine inhibited this increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium Dependence of Muscarinic Receptor-Mediated Catecholamine Secretion from the Perfused Rat Adrenal Medulla

It had previously been thought that muscarinic cholinergic receptors utilize an influx of extracellular calcium for activation of adrenomedullary catecholamine secretion. However, it has recently been demonstrated that muscarinic receptors on isolated adrenal chromaffin cells can elevate cytosolic free calcium levels in a manner independent of extracellular calcium, presumably by mobilizing intracellular calcium stores. We now demonstrate that muscarinic receptor-mediated catecholamine secretion from perfused rat adrenal glands can occur under conditions of extracellular calcium deprivation that are sufficient to block both nicotine- and electrically stimulated release. Three independent conditions of extracellular calcium deprivation were used: nominally calcium-free perfusion solution (no calcium added), EGTA-containing calcium-free perfusion solution, and perfusion solution containing the calcium channel blocker verapamil. Secretion was evoked from the perfused glands by either transmural electrical stimulation or injection of nicotine or muscarine into the perfusion stream. Each condition of calcium deprivation was able to block nicotine- and electrically stimulated catecholamine release in an interval that left muscarine-evoked release largely unaffected. The above results demonstrate that muscarine-evoked catecholamine secretion from perfused rat adrenal glands can occur in the absence of extracellular calcium, presumably by mobilization of intracellular calcium. The latter may be due to muscarinic receptor-mediated generation of inositol trisphosphate.     

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.     

Carbachol induces secretion in a mast cell line (RBL-2H3) transfected with the ml muscarinic receptor gene.

The possibility that the ml muscarinic receptor subtype can induce release of intracellular granules and transmitters was studied by transfecting a cultured mast cell line. RBL-2H3 cells, with the ml receptor gene. Comparisons were made between carbachol- and antigen-induced activation of various secretory responses. Like antigen, carbachol stimulated inositol phospholipid hydrolysis and release of arachidonic acid with concomitant dose-dependent secretion of granular contents. Carbachol also stimulated a biphasic increase in intracellular calcium, as measured by single cell fura-2 measurements. Although the kinetics of the carbachol-induced rise in intracellular calcium differed from that induced by antigen, they both utilized the same intracellular pool of calcium, and the second phase of the rise in intracellular calcium was dependent on extracellular calcium in both cases. Thus, the ml muscarinic receptor activates release of granules by a mechanism ostensibly similar to that of antigen.     

Muscarinic Agonists Cause Calcium Influx and Calcium Mobilization in Forebrain Neurons In Vitro

We have examined the effects of the muscarinic agonist carbachol on the intracellular free Ca2+ concentration ([Ca2+]i) in primary cultures of neurons from rat forebrain using the Ca2+-sensitive fluorescent dye fura-2. Addition of carbachol increased the [Ca2+]i in approximately 60% of cells studied. Oxotremorine-M, but not pilocarpine, mimicked the effects of carbachol. The response was reduced by 60% on removal of extracellular Ca2+, a finding suggesting that muscarinic receptor activation causes Ca2+ influx in addition to intracellular Ca2+ mobilization. Tetrodotoxin and nitrendipine also significantly reduced the response to carbachol. These studies suggest that the changes in [Ca2+]i produced by activation of muscarinic receptors result in part from mobilization of intracellular Ca2+ and that influx through voltage-sensitive Ca2+ channels also provides a significant contribution to the net [Ca2+]i change observed.     

BAY-K-8644-stimulated amylase secretion from pancreatic acinar cells

Pancreatic acinar cells do not contain depolarization-sensitive calcium channels. Nonetheless, in the current study, the calcium channel activator, BAY-K-8644, was found to stimulate a time- and concentration-dependent increase in the spontaneous release of amylase. Secretion was dependent on the presence of extracellular calcium in the incubation medium. Racemic BAY-K-8644 and (or) its S(-)optical isomer did not enhance the secretory response to either carbachol or cholecystokinin octapeptide; however, when co-applied with either phorbol ester, vasoactive intestinal peptide, or forskolin, they potentiated amylase secretion. Nifedipine and the R(+)isomer of BAY-K-8644, which are both calcium channel antagonists, did not alter basal or forskolin-stimulated amylase secretion, and [3H]nitrendipine did not bind to acinar cell membranes. Neither atropine nor dibutyryl cGMP, inhibitors of cholinergic and cholecystokininergic receptors, respectively, affected BAY-K-8644-induced amylase secretion. While BAY-K-8644 stimulated concentration-dependent cGMP synthesis in acinar cells, it had no effect on basal or forskolin-stimulated cAMP formation. The data suggest that BAY-K-8644 may bind to acinar cell sites that are not functional calcium channel proteins but are coupled nevertheless to the secretory response, and that calcium channel antagonists do not bind to these sites. The mechanism of the secretagogue action of BAY-K-8644 remains to be elucidated.     

Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium

The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+- sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation.     

Muscarinic Receptors in Chromaffin Cell Cultures Mediate Enhanced Phospholipid Labeling but Not Catecholamine Secretion

Abstract: The addition of either carbachol or muscarinic agonists to cultured bovine adrenal chromaffin cells results in a selective stimulation of phosphatidate (PhA) and phosphatidylinositol (PhI) labeling from ³²Pⁱ and [³H]glycerol that can be inhibited by the inclusion of atropine, but not d -tubocurarine. In contrast, increased catecholamine secretion is observed on the addition of carbachol or nicotinic agonists and is inhibited by d -tubocurarine but not by atropine. Added calcium is essential for catecholamine secretion but not for stimulated phospholipid labeling. Chelation of endogenous Ca²⁺ with EGTA does, however, inhibit the stimulated phospholipid labeling. These results suggest that stimulated phospholipid labeling in the bovine chromaffin cell and catecholamine secretion are separate and distinct processes.     

Protein kinase C mediated regulation of calcium channels in PC-12 pheochromocytoma cells

Depolarization of PC-12 pheochromocytoma cells with K+ produces an immediate increase in catecholamine release. The stimulation of release is blocked by Co2+, removal of extracellular Ca2+ or by dihydropyridine drugs such as nitrendipine. Release is enhanced by other dihydropyridines such as BAY K8644. Release is accompanied by a voltage dependent uptake of 45Ca2+ which is also blocked by Co2+ or nitrendipine and enhanced by BAY K8644. The phorbol ester phorbol 12-myristate-13-acetate (TPA) in the range 10(-9)-10(-6) M produced little effect by itself but augmented the K+ evoked release of catecholamine. An analog of TPA which does not activate protein kinase C was ineffective. In contrast, TPA in the same concentration range blocked influx of 45Ca2+ induced by 70 mM K+ or 70 mM K+/BAY K8644. 45Ca2+ influx produced by A23187 was not blocked by TPA. The results suggest a system by which protein kinase C may regulate the output of transmitters from secretory cells.     

Differential effects of extracellular calcium removal and nonspecific effects of Ca2+ antagonists on acid secretory activity in isolated gastric glands

The role of extracellular calcium in the action of the secretagogues, carbachol, histamine and forskolin, on parietal cell HCl secretion was investigated using glands isolated from rabbit gastric mucosa. Omission of calcium from the cellular incubation medium and chelation of a major portion of contaminating calcium with EGTA resulted in a disappearance of the initial transient response to carbachol (as measured by uptake of the weak base, amino[14C]pyrine), but the sustained response to carbachol persisted. Neither histamine nor forskolin-stimulated increase in amino[14C]pyrine uptake were affected by omission of extracellular calcium. Furthermore, the potentiating interactions between histamine and carbachol and between forskolin and carbachol appeared to occur independent of extracellular calcium. Attempts to assess the contribution of intracellular calcium to secretory activity using the Ca2+ antagonists, verapamil, nifedipine, nicardipine and lanthanum, and the putative intracellular Ca2+ antogonist, TMB-8 (3,4,5-trimethyloxybenzoic acid 8-(diethyl-amino)-octyl ester) were unsuccessful. Nifedipine had no effect on secretagogue stimulated amino[14C]pyrine accumulation even at concentration well above the pA2 reported for excitable tissues. Verapamil, nicardipine, lanthanum and TMB-8 all appeared to have nonspecific inhibitory effects on amino [14C]pyrine uptake. From these results we conclude that: (1) parietal cell HCl secretion can occur independent of extracellular Ca2+; (2) influx of extracellular Ca2+ enhances the response to carbachol but has little influence on the secretory response initiated by cAMP-dependent secretagogues; and (3) parietal cell Ca2+ channels have a different molecular configuration than Ca2+ channels in excitable cells.     

Forskolin potentiates calcium-dependent amylase secretion from rat pancreatic acinar cells

The cellular and molecular effects of forskolin, a direct, nonhormonal activator of adenylate cyclase, were assessed on the enzyme secretory process in dispersed rat pancreatic acinar cells. Forskolin stimulated adenylate cyclase activity in the absence of guanyl nucleotide. It promoted a rapid and marked increase in cellular accumulation of cyclic AMP alone or in combination with vasoactive intestinal peptide (VIP) but was itself a weak pancreatic agonist and did not increase the secretory response to VIP or other cyclic AMP dependent agonists. Somatostatin was a partial antagonist of forskolin stimulated cyclic AMP synthesis and forskolin plus cholecystokinin-octapeptide (CCK-OP) induced amylase release. Forskolin potentiated amylase secretion in response to calcium-dependent agonists such as CCK-OP, carbachol and A-23187, but did not affect the ability of CCK-OP and (or) carbachol to mobilize 45Ca from isotope preloaded cells; forskolin alone did not stimulate 45Ca release. In calcium-poor media, the secretory response to forskolin and CCK-OP was reduced in a both absolute and relative manner. The data suggests that calcium plays the primary role as intracellular mediator of enzyme secretion and that the role of cyclic AMP may be to modulate the efficiency of calcium utilization.     

Does cyclic AMP mobilize Ca2+ for amylase secretion from rat parotid cells?

Both dibutyryl cAMP and carbachol stimulated amylase released from rat parotid cells incubated in Ca2+-free medium containing 1 mM EGTA. Cells preincubated with 10 microM carbachol in Ca2+-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 microM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of 45Ca from cells preequilibrated with 45Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular 45Ca level. Inositol trisphosphate, but not cAMP, evoked 45Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.     

Acetylcholine release from the carotid body by hypoxia: evidence for the involvement of autoinhibitory receptors.

The purpose of the present study was to investigate whether hypoxia influences acetylcholine (ACh) release from the rabbit carotid body and, if so, to determine the mechanism(s) associated with this response. ACh is expressed in the rabbit carotid body (5.6 +/- 1.3 pmol/carotid body) as evidenced by electrochemical analysis. Immunocytochemical analysis of the primary cultures of the carotid body with antibody specific to ACh further showed that ACh-like immunoreactivity is localized to many glomus cells. The effect of hypoxia on ACh release was examined in ex vivo carotid bodies harvested from anesthetized rabbits. The basal release of ACh during normoxia ( approximately 150 Torr) averaged 5.9 +/- 0.5 fmol.min-1.carotid body-1. Lowering the Po2 to 90 and 20 Torr progressively decreased ACh release by approximately 15 and approximately 68%, respectively. ACh release returned to the basal value on reoxygenation. Simultaneous monitoring of dopamine showed a sixfold increase in dopamine release during hypoxia. Hypercapnia (21% O2 + 10% CO2) as well as high K+ (100 mM) facilitated ACh release from the carotid body, suggesting that hypoxia-induced inhibition of ACh release is not due to deterioration of the carotid body. Hypoxia had no significant effect on acetylcholinesterase activity in the medium, implying that increased hydrolysis of ACh does not account for hypoxia-induced inhibition of ACh release. In the presence of either atropine (10 microM) or domperidone (10 microM), hypoxia stimulated ACh release. These results demonstrate that glomus cells of the rabbit carotid body express ACh and that hypoxia overall inhibits ACh release via activation of muscarinic and dopaminergic autoinhibitory receptors in the carotid body.     

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.     

Agonist activation of cytosolic Ca2+ in subfornical organ cells projecting to the supraoptic nucleus

The subfornical organ (SFO) is sensitive to both ANG II and ACh, and local application of these agents produces dipsogenic responses and vasopressin release. The present study examined the effects of cholinergic drugs, ANG II, and increased extracellular osmolarity on dissociated, cultured cells of the SFO that were retrogradely labeled from the supraoptic nucleus. The effects were measured as changes in cytosolic calcium in fura 2-loaded cells by using a calcium imaging system. Both ACh and carbachol increased intracellular ionic calcium concentration ([Ca2+]i). However, in contrast to the effects of muscarinic receptor agonists on SFO neurons, manipulation of the extracellular osmolality produced no effects, and application of ANG II produced only moderate effects on [Ca2+]i in a few retrogradely labeled cells. The cholinergic effects on [Ca2+]i could be blocked with the muscarinic receptor antagonist atropine and with the more selective muscarinic receptor antagonists pirenzepine and 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP). In addition, the calcium in the extracellular fluid was required for the cholinergic-induced increase in [Ca2+]i. These findings indicate that ACh acts to induce a functional cellular response in SFO neurons through action on a muscarinic receptor, probably of the M1 subtype and that the increase of [Ca2+]i, at least initially, requires the entry of extracellular Ca2+. Also, consistent with a functional role of M1 receptors in the SFO are the results of immunohistochemical preparations demonstrating M1 muscarinic receptor-like protein present within this forebrain circumventricular organ.     

Stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system. Determination of GTP/peptide bond ratios during codon-specific elongation and misreading

Pancreatic secretory factor (PSF), a 17.5-kDa protein purified from the venom of Gila monster (Heloderma suspectum), stimulated amylase secretion from dispersed rat pancreatic acini more efficiently than CCK-8, bombesin, carbachol and secretin, and without increasing 45Ca2+ efflux and cyclic AMP levels. The secretory action was dependent on the presence of extracellular calcium and was additive to the secretion induced by agents acting via cyclic AMP or via Ca2+ efflux.     

Role of calcium in the preparation and secretory activity of cells isolated from the gastric mucosa of rabbits

The role of calcium in the preparation and the acid secretory activity of parietal cells was studied using cells isolated from rabbit gastric mucosa. The preparation of isolated cells was performed by enzymatic dissociation (collagenase) in the presence of EDTA; without EDTA, only isolated gastric glands were obtained. The acid secretory activity of parietal cells was determined by the 14C-aminopyrine accumulation method; the stimulation induced by histamine or isobutylmethylxanthine (IBMX) was not significantly affected by a reduction of extracellular Ca2+ level (20% diminution in a Ca2+-free medium). The carbachol induced stimulation was highly dependent upon the concentration of extracellular Ca2+: incubation of parietal cells in a Ca2+-free medium reduced the response to 100 microM carbachol by about 60%.