Mechanisms for direct inhibition of canine gastric parietal cells by somatostatin

To examine the potential mechanisms by which somatostatin inhibits gastric acid secretion we studied its effects on isolated canine gastric parietal cells. Using 125I-[Leu8-D-Trp22-Tyr25]somatostatin-28 as ligand, we identified somatostatin-binding sites in parietal cell-enriched fractions of fundic mucosa. Two binding sites with respective dissociation constants of 3.2 X 10(-9) and 2.1 X 10(-7) M were identified. Somatostatin-14 and -28 were equally potent both in displacing bound ligand and in inhibiting parietal cell activity as measured by [14C]aminopyrine uptake. Pertussis toxin reversed the ability of somatostatin to inhibit the uptake of [14C]aminopyrine and production of cAMP by parietal cells stimulated with histamine and forskolin but not with dibutyryl cAMP or pentagastrin. Furthermore, somatostatin had no effect on parietal cell membrane inositol phospholipid turnover or changes in protein kinase C (Ca2+/phospholipid-dependent enzyme) activity induced by carbachol or pentagastrin. These data indicate that somatostatin directly inhibits parietal cell activity via mechanisms both dependent on and independent of the pertussis toxin-sensitive inhibitory guanine nucleotide-binding protein.     

Multiple effects of phorbol ester on secretory activity in rabbit gastric glands and parietal cells

Acid secretory activity and respiration in rabbit gastric glands are stimulated by cAMP-dependent and -independent agonists. Potentiation between agonists suggests interaction of the activation pathways. Regulation of secretory response by protein kinase C was investigated with 12-0-tetradecanoyl phorbol-13-acetate (TPA). TPA elevated basal respiration, pepsin release, and acid secretion but inhibited histamine and carbachol stimulation of acid secretion by gastric glands, as measured by [dimethylamino-14C]aminopyrine accumulation. The inhibition of histamine response was specific for protein kinase C activators, occurred after a 20-min lag, and was not reversed by removal of TPA after 3 min of preincubation. TPA pretreatment inhibited acid secretory responses to cholera toxin and forskolin but enhanced the response to cAMP analogues. Cholera toxin and pertussis toxin simulated ADP-ribosylation of 45 and 41 kDa proteins, respectively, in parietal cell membranes. Therefore, both stimulatory (Gs) and inhibitory (Gi) GTP binding proteins of adenylyl cyclase appear to be present in parietal cells. Pretreatment with pertussis toxin attenuated PGE2 but not TPA inhibition of histamine stimulation of aminopyrine accumulation. Thus, the inhibitory effect of TPA does not appear to be associated with an action on Gi. The results with histamine and carbachol suggest that protein kinase C may regulate both cAMP-dependent and -independent stimulation of parietal cell acid secretion.     

Involvement of a pertussis toxin-sensitive G protein in the action of gastrin on gastric parietal cells

The mechanism whereby gastrin triggers phosphoinositide breakdown was investigated in an enriched preparation of isolated rabbit parietal cells (approx. 75%). In a permeabilized preparation of myo-[3H]inositol-labelled cells, GTP[S], a non-hydrolysable GTP analogue, enhanced [3H]inositol trisphosphate ([3H]InsP3 accumulation in a dose-dependent manner; submaximal concentrations of GTP[S] (less than 10 microM), potentiated gastrin-induced [3H]InsP3 release; preincubation for 5 min with GDP[S], a non-hydrolysable GDP analogue, dose-dependently reduced [3H]InsP3 accumulation stimulated by gastrin even in presence of GTP[S]. Exposure of intact parietal cells for 3 h to pertussis toxin (PTx) (200 ng/ml) led to a 15-50% reduction in gastrin-induced [14C]aminopyrine [(14C]AP) uptake (an index of in vitro acid secretion) and [3H]inositol phosphate ([3H]InsP) accumulation. A decrease in the accumulation of the different [3H]inositol phosphate occurred in gastrin-stimulated parietal cells treated with PTx. A rightward shift of gastrin dose-response curves in the presence of PTx was observed for [14C]AP uptake (EC50 values: 0.125 +/- 0.045 nM without PTx and 1.05 +/- 0.63 nM with PTx), for [3H]InsP accumulation (EC50 values: 0.16 +/- 0.08 nM without PTx and 1.56 +/- 0.58 nM with PTx) and [125I]gastrin binding (IC50 values: 0.247 +/- 0.03 nM without PTx and 2.38 +/- 0.56 nM with PTx). In contrast, cholera toxin (CTx) treatment (100 ng/ml) for 3 h was without effect on gastrin-induced [3H]InsP accumulation. CTx induced a pronounced potentiation of gastrin-stimulated [14C]AP uptake; this effect can be mimicked by IBMX (a phosphodiesterase inhibitor) and by forskolin (an activator of adenylyl cyclase). We conclude that: (i) one or more than one G protein appeared to be involved in gastrin receptor coupling to phospholipase C (PL-C); (ii) these G proteins are not substrates for CTx; (iii) one of these appeared to be a PTx-sensitive 'Gi-like' protein which could be involved in hormone-induced acid secretion, (iiii) the potentiating effect of CTx observed on AP uptake stimulated by gastrin suggests the existence of a cooperative effect between cAMP pathway (CTx) and the gastrin-induced phosphoinositide breakdown in acid secretory activity of parietal 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.     

Parietal cell protein kinases. Selective activation of type I cAMP-dependent protein kinase by histamine

cAMP-dependent protein kinases have been characterized in parietal cells isolated from rabbit gastric mucosa. Both Type I and Type II cAMP-dependent protein kinase isozymes are present in these cells. Type II isozymes were detected in 900, 14,000, and 100,000 X g particulate fractions as well as 100,000 X g cytosolic fractions; Type I isozymes were found predominately in the cytosolic fraction. When parietal cells were stimulated with histamine, an agent that elevates intracellular cAMP content and initiates parietal cell HCl secretion, cAMP-dependent protein kinase activity was increased in homogenates of these cells as measured by an increase in the cAMP-dependent protein kinase activity ratio. Histamine activation of cAMP-dependent protein kinase was correlated with parietal cell acid secretory responses which were measured indirectly as increased cellular uptake of the weak base, [14C]aminopyrine. These results suggest that cAMP-dependent protein kinase(s) is involved in the control of parietal cell HCl secretion. The parietal cell response to histamine may be compartmentalized because histamine appears to activate only a cytosolic Type I cAMP-dependent protein kinase isozyme, as determined by three different techniques including 1) ion exchange chromatography; 2) Sephadex G-25 to remove cAMP and allow rapid reassociation of the Type II but not the Type I isozyme; and 3) 8-azido-[32P]cAMP photoaffinity labeling. Forskolin, an agent that directly stimulates adenylate cyclases, was found to activate both the Type I and Type II isozymes. Several cAMP-dependent protein kinases were also detected in parietal cell homogenates, including a Ca2+-phospholipid-sensitive or C kinase and two casein kinases which were tentatively identified as casein kinase I and II. At least two additional protein kinases with a preference for serine or lysine-rich histones, respectively, were also detected. The function of these enzymes in parietal cells remains to be shown.     

Aminopyrine uptake by guinea pig gastric mucosal cells. Mediation by cyclic AMP and interactions among secretagogues

The role of cyclic nucleotides in regulating acid secretion by dispersed mucosal cells from guinea-pig stomach was examined by measuring first the ability of histamine and carbachol to stimulate [dimethylamine-14C]aminopyrine uptake and cyclic nucleotide metabolism and secondly, the effect of exogenous cyclic nucleotides on basal and stimulated [14C]aminopyrine uptake. The [14C]aminopyrine was found in an acidic, osmotically sensitive compartment, probably associated with the initial steps in acid secretion by these cells. Although histamine increased [14C]aminopyrine uptake and cyclic AMP synthesis as expected, histamine was approx. 10-fold more potent in inducing [14C]aminopyrine uptake. This dissociation of [14C]aminopyrine uptake and cyclic AMP metabolism process was further manifested by the observation that prostaglandin E1 failed to increase [14C]aminopyrine uptake, although it did cause a rise in cellular cyclic AMP. Furthermore, prostaglandin E1 did not alter the [14C]-aminopyrine uptake caused by histamine. Carbachol was found to increase the [14C]aminopyrine uptake and also to potentiate the ability of histamine to increase [14C]aminopyrine uptake. Carbachol, however, affected neither the histamine-induced increase in cyclic AMP nor the binding of [3H]histamine to the cells. Cimetidine, a histamine H2 receptor antagonist, blocked the [14C]aminopyrine uptake induced either by histamine alone or by the potentiating combination of histamine plus carbachol. These results suggest that cyclic AMP is mediating the action of histamine on [14C]aminopyrine uptake but changes in cyclic AMP per se are not necessarily the cause for the potentiated increase in [14C]aminopyrine uptake. Furthermore, the potentiated response observed with histamine plus carbachol on [14C]aminopyrine uptake occurs at a biochemical step distal to and not obviously related to cyclic AMP generation.     

Modulating effect of thiol-disulfide status on [14C]aminopyrine accumulation in the isolated parietal cell

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.     

Pancreastatin: a novel peptide inhibitor of parietal cell signal transduction

The direct inhibition of secretion by pancreastatin was investigated in rabbit isolated parietal cells. Pancreastatin exerted no influence on basal aminopyrine uptake. Pancreastatin inhibited histamine stimulated aminopyrine uptake through a decrease in intracellular cAMP. Pancreastatin inhibition of histamine stimulated uptake was blocked in the presence of pertussis toxin. Pancreastatin also inhibited the carbachol stimulated increase in aminopyrine accumulation. However, the effects of pancreastatin on carbachol stimulation were not reversed by pertussis toxin. Pancreastatin did not alter the carbachol induced increase in cytosolic free calcium. Thus, pancreastatin appears to inhibit parietal cell signal transduction at multiple points along the second messenger pathways.     

A proposed mechanism for the potentiation of cAMP-mediated acid secretion by carbachol

Acid secretion in isolated rabbit gastric glands was monitored by the accumulation of [(14)C]aminopyrine. Stimulation of the glands with carbachol synergistically augmented the response to dibutyryl cAMP. The augmentation persisted even after carbachol was washed out and was resistant to chelated extracellular Ca(2+) and to inhibitors of either protein kinase C or calmodulin kinase II. Cytochalasin D at 10 microM preferentially blocked the secretory effect of carbachol and its synergism with cAMP, whereas it had no effect on histamine- or cAMP-stimulated acid secretion within 15 min. Cytochalasin D inhibited the carbachol-stimulated intracellular Ca(2+) concentration ([Ca(2+)](i)) increase due to release from the Ca(2+) store. Treatment of the glands with cytochalasin D redistributed type 3 inositol 1,4,5-trisphosphate receptor (the major subtype in the parietal cell) from the fraction containing membranes of large size to the microsomal fraction, suggesting a dissociation of the store from the plasma membrane. These findings suggest that intracellular Ca(2+) release by cholinergic stimulation is critical for determining synergism with cAMP in parietal cell activation and that functional coupling between the Ca(2+) store and the receptor is maintained by actin microfilaments.     

Cellular origin and release of intrinsic factor from isolated rat gastric mucosal cells

The cellular content and secretion of intrinsic factor was measured by [57Co]cyanocobalamin binding using isolated rat gastric mucosal cells. The intrinsic factor/R-protein ratio was above 9:1 as evaluated by specific anti-intrinsic factor antibodies. In unfractionized cells with 23 +/- 1.3% parietal cells the intrinsic factor content of 148 +/- 47 fmol/10(6) cells remained almost unchanged over 3 h, whereas basal secretion rose up to 57 +/- 10. In fractionized cells (Percoll) with 3-85% parietal cells most intrinsic factor was found in the parietal cell-depleted fraction (content: 441 +/- 30, secretion/3 h: 139 +/- 16, mean formation/h: 50 +/- 12 fmol/10(6) cells). The intrinsic factor content of the different cell fractions correlated with that of pepsin. [14C]Aminopyrine uptake, an indirect measure of parietal cell H+ production, was inversely related. Carbachol (1 X 10(-6)-10(-3) mol/l) stimulated intrinsic factor secretion, 1 X 10(-3) mol/l being maximally effective (90 +/- 8% above basal). This response was inhibited by atropine and pirenzepine, but not by prostaglandin E2 (PGE2) and somatostatin. Dibutyryl cyclic adenosine monophosphate (dibutyryl cAMP, 43 +/- 7%) and hexoprenaline (24 +/- 5%) enhanced intrinsic factor secretion less effectively and pentagastrin like histamine lacked any stimulatory effect. We conclude that in the rat intrinsic factor is produced and released from chief cells mainly under cholinergic control.     

Epidermal growth factor (EGF) inhibits both intrinsic factor secretion and acid secretion in histamine-stimulated isolated gastric glands

Epidermal growth factor (EGF) is a polypeptide present in mammalian salivary glands which has been shown to have mitogenic and gastric acid inhibitory properties in vivo. The mechanisms of action of EGF at the level of the parietal cell are not clear. In the present study, we have examined the effects of EGF on both acid and macromolecular (intrinsic factor, IF) secretion stimulated by the cyclic AMP-mediated agonist histamine using the rabbit isolated gastric gland model. Acid secretion was assessed by the accumulation of [14C]aminopyrine (AP) in glands and IF in the supernatants by the binding of [57Co]cyanocobalamin. Histamine (10(-6) to 5 x 10(-5) M) resulted in a 4-6 fold increase in [14C]AP and IF (P less than 0.05). EGF alone (10(-8) M, 10(-7) M) had no significant effect on basal [14C]AP accumulation or IF secretion (P less than 0.05). EGF (10(-7) M) significantly inhibited the histamine dose-response curve for [14C]AP and IF, but a relatively greater inhibition was observed at higher histamine concentration. These data demonstrate that EGF inhibits both acid and IF secretion in vitro at concentrations consistent with those observed in vivo. The observations further support the hypothesis that EGF may play a role in the regulation of parietal cell secretion.     

Inhibition of gastric acid secretion by human calcitonin gene-related peptide with picomolar potency in guinea-pig parietal cell preparations

The effect of CGRP on [14C]-aminopyrine accumulation in isolated parietal cell preparations from guinea-pig fundic mucosa was studied. Parietal cells consisted of 60% of the preparations. [14C]-Aminopyrine accumulation was used as an index of physiological response of parietal cells to secretagogues. CGRP dose-dependently (10(-12)-10(-9) M) inhibited parietal cell aminopyrine accumulation stimulated by histamine (10(-4) M), carbachol (10(-4) M), and pentagastrin (5 X 10(-6) M). The concentration of CGRP exerting half-maximal inhibition of [14C]-aminopyrine accumulation was 8.7 X 10(-11) M for histamine, 9.1 X 10(-11) M for carbachol, and 4.7 X 10(-11) M for pentagastrin. The inhibitory effect was much more potent than cimetidine, pirenzepine or benzotript. CGRP but not cimetidine inhibited DBcAMP stimulated aminopyrine accumulation (IC50 = 7.5 X 10(-11) M). These results suggest that CGRP may exert its inhibitory action on gastric acid secretion by a direct action on the parietal cell or the somatostatin-producing D cell.     

Stimulation of inositol phosphate and diacylglycerol production by RHC 80267, a diacylglycerol-lipase inhibitor, in rat gastric parietal cells: effects on hydrogen ion secretion

RHC 80267, on inhibitor of diacylglycerol lipase, was used to investigate the role of diacylglycerol in acid secretion by isolated rat gastric parietal cells. Unexpectedly, RHC 80267 stimulated the production of inositol phosphates in [3H]inositol-prelabeled cells and increased levels of 32P-labeled phosphatidic acid to the same degree as did carbachol. RHC 80267 increased diacylglycerol to a greater extent than did carbachol, and additionally decreased levels of [3H]arachidonic acid. This suggests that RHC 80267 stimulated phospholipase C and inhibited diacylglycerol lipase in parietal cells. RHC inhibited [14C]aminopyrine uptake, a measure of acid secretion, stimulated by carbachol or by simultaneous addition of carbachol and dibutyryl-cAMP. These data support the model that the diacylglycerol/protein kinase C branch of the phosphoinositide system is inhibitory to acid secretion.     

Targeted disruption of the Lasp-1 gene is linked to increases in histamine-stimulated gastric HCl secretion

Lasp-1 (LIM and SH3 domain protein 1) is a multidomain actin-binding protein that is differentially expressed within epithelial tissues and brain. In the gastric mucosa, Lasp-1 is highly expressed in the HCl-secreting parietal cell, where it is prominently localized within the F-actin-rich subcellular regions. Histamine-induced elevation of parietal cell [cAMP]i increases Lasp-1 phosphorylation, which is correlated with activation of HCl secretion. To determine whether Lasp-1 is involved in the regulation of HCl secretion in vivo, we generated a murine model with a targeted disruption of the Lasp-1 gene. Lasp-1-null mice had slightly lower body weights but developed normally and had no overt phenotypic abnormalities. Basal HCl secretion was unaffected by loss of Lasp-1, but histamine stimulation induced a more robust acid secretory response in Lasp-1-null mice compared with wild-type littermates. A similar effect of histamine was observed in isolated gastric glands on the basis of measurements of accumulation of the weak base [14C]aminopyrine. In addition, inhibition of the acid secretory response to histamine by H2 receptor blockade with ranitidine proceeded more slowly in glands from Lasp-1-null mice. These findings support the conclusion that Lasp-1 is involved in the regulation of parietal HCl secretion. We speculate that cAMP-dependent phosphorylation of Lasp-1 alters interactions with F-actin and/or endocytic proteins that interact with Lasp-1, thereby regulating the trafficking/activation of the H+, K+-ATPase (proton pump).     

Intrinsic factor secretion from isolated human gastric mucosal cells

Human gastric mucosal cells were isolated from the resected fundic mucosa of peptic ulcer patients. The intracellular content and secretion of intrinsic factor were estimated by binding to cyano[⁵⁷Co]cobalamin. The content was maximal in the enriched parietal cell fraction which also displayed the highest H⁺ production as measured by amino[¹⁴C]pyrine uptake. Secretagogues evoked full response after 15 min of incubation: pentagastrin (181% of basal secretion), carbachol (208%), histamine (250%) and dibutyryl cyclic adenosine monophosphate (304%). The phosphodiesterase inhibitor isobutylmethylxanthine was slightly more effective even than dibutyryl cAMP. The response to histamine was abolished by ranitidine, indicating activation of adenylate cyclase via histamine H₂ receptors, but remained unaffected by atropine, which in turn blocked the carbachol effect, whereas ranitidine was ineffective. The mean formation rate was 8.4 fmol intrinsic factor/10⁶ cells per h under basal conditions and 14.3 fmol in response to histamine.     

ATP dependence of H+ secretion

Cells in isolated rabbit gastric gland were made permeable to ATP by high voltage discharge across a gland suspension. In both normal (5.4 mM K+) and high K+ (108 mM) medium, this electrical shock resulted in a marked reduction in the ability of the parietal cell to produce and accumulate acid. Acid production was monitored both microscopically by acridine orange accumulation in the secretory canaliculus and by accumulation of the weak base [14C]aminopyrine. In 108 mM K+ solutions but not in 5.4 mM K+ solutions 5, mM ATP was able to restore the accumulation of these probes to control (unshocked) levels. When shocked glands had been previously stimulated by secretagogues, the aminopyrine accumulation ratio was only partly restored by ATP. Inhibition of mitochondrial respiration by cyanide, azide, or Amytal abolished acid secretion; the subsequent addition of ATP to shocked glands increased the aminopyrine accumulation ratio to 47 and resulted in an acridine orange fluorescence indistinguishable from that of histamine-stimulated, unshocked glands. We conclude that ATP can act as a substrate for H+ secretion in the parietal cell, and that perhaps no additional energy source is necessary.     

Gastrin and CCK-8 induce inositol 1,4,5-trisphosphate formation in rabbit gastric parietal cells

The role of phosphoinositide turnover in the mediation of acid secretion was examined in an enriched preparation of isolated rabbit parietal cells (75%). Both gastrin and CCK-8 (octapeptide of cholecystokinin) stimulated [14C]aminopyrine (AP) uptake by cells (EC50 0.07 +/- 0.03 nM (gastrin) and 0.093 +/- 0.065 nM (CCK-8] and increased [3H]inositol phosphates cellular contents (EC50 0.142 +/- 0.016 nM (gastrin) and 0.116 +/- 0.027 nM (CCK-8] in a parallel fashion. In addition, the EC50 values for both phenomenon were quite similar to the Kd values obtained from binding experiments. HPLC analysis of the different [3H]inositol phosphates produced under gastrin or CCK-8 stimulation showed a 2-fold increase in [3H]Ins(1,4,5)P3 levels within 5 s with a concomitant increase in [3H]Ins(1,4)P2 content within 15 s. A low but significant rise in [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,3,4)P3 cellular contents was also observed. No difference between gastrin- and CCK-8-induced inositol phosphates production could be shown. We can conclude that gastrin and CCK-8 display an identical profile of action, suggesting that they stimulate the acid secretory function of parietal cells through the same receptor site coupled to the Ins(1,4,5)P3 production.     

Thapsigargin potentiates histamine-stimulated HCl secretion in gastric parietal cells but does not mimic cholinergic responses.

The role of calcium in control of HCl secretion by the gastric parietal cell was examined using a recently available intracellular calcium-releasing agent, thapsigargin, which has been shown, in some cell types, to induce sustained elevation of intracellular calcium ([Ca2+]i), an action that appears to be independent of inositol lipid breakdown and protein kinase C activation and to be mediated, at least partially, by selective inhibition of endoplasmic reticulum Ca2(+)-ATPase. Using the calcium-sensitive fluorescent probe, fura-2, in combination with digitized video image analysis of single cells as well as standard fluorimetric techniques, we found that thapsigargin induced sustained elevation of [Ca2+]i in single parietal cells and in parietal cells populations. Chelation of medium calcium led to a transient rise and fall in [Ca2+]i, indicating that the sustained elevation in [Ca2+]i in response to thapsigargin was due to both intracellular calcium release and influx. Although thapsigargin appeared to affect the same calcium pool(s) regulated by the cholinergic agonist, carbachol, and the pattern of thapsigargin-induced increases in [Ca2+]i were similar to the plateau phase of the cholinergic response, thapsigargin did not induce acid secretory responses of the same magnitude as those initiated by carbachol (28 vs 600% of basal). The protein kinase C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) potentiated the secretory response to thapsigargin but this combined response also did not attain the same magnitude as the maximal cholinergic response. In the presence but not the absence of medium calcium, thapsigargin potentiated acid secretory responses to histamine, which elevate both cyclic AMP (cAMP) and [Ca2+]i in parietal cells, as well as forskolin and cAMP analogues but had no effect on submaximal and an inhibitory effect on maximal cholinergic stimulation. Furthermore, thapsigargin did not fully mimic potentiating interactions between histamine and carbachol, either in magnitude or in the pattern of temporal response. Assuming that the action of thapsigargin is specific for intracellular calcium release mechanisms, these data suggest that 1) sustained influx of calcium is necessary but not sufficient for cholinergic activation of parietal cell HCl secretion and for potentiating interactions between cAMP-dependent agonists and carbachol; 2) mechanisms in addition to elevated [Ca2+]i and protein kinase C activation may be involved in cholinergic regulation; and 3) increases in [Ca2+]i in response to histamine are not directly involved in the mechanism of histamine-stimulated secretion.     

Gastrin induces intracellular Ca2+ release and acid secretion regulation by the microtubular-microfilamentous system

In the absence of extracellular Ca2+ gastrin induced accumulation of amino [14C]pyrine as an index of acid secretion and evoked a cytosolic Ca2+ rise (measured by quin2/AM), suggesting that an intracellular Ca2+ release after the activation of the gastrinergic pathway may trigger acid secretion. In isolated parietal cells pretreatment with colchicine or cytochalasin B abolished the amino [14C]pyrine accumulation and the rise in cytosolic Ca2+ concentration evoked by gastrin. The results suggest that the microtubular-microfilamentous system regulates gastrin-induced intracellular Ca2+ release and acid secretion.     

Metabolism and gastric acid secretion. Substrate-dependency of aminopyrine accumulation in isolated rat parietal cells.

The substrate-dependency of gastric acid secretion was investigated in isolated rat parietal cells by using the accumulation of the weak base aminopyrine as an index of acid secretion. Exogenous substrates enhanced accumulation of aminopyrine in rat parietal cells stimulated by secretagogues, and this effect was probably directly related to the provision of energy for acid secretion. At physiological concentrations, certain of the substrates (glucose, oleate, lactate, D-3-hydroxybutyrate, L-isoleucine, L-valine and acetoacetate) could support acid secretion, with glucose being the most effective. L-Leucine and acetate were only effective stimulators of parietal-cell aminopyrine accumulation at high concentrations (5mM). L-Glutamine was unable to stimulate aminopyrine accumulation even at high concentrations, and glutaminase activity in parietal cells was estimated to be low by comparison with small-intestinal epithelial cells. Variation in the concentrations of D-3-hydroxybutyrate and L-isoleucine, but not of glucose, within the physiological range affected their ability to support aminopyrine accumulation. The presence of 5 mM-L-isoleucine, 5 mM-lactate and combinations of certain substrates at physiological concentrations produced aminopyrine accumulation in stimulated parietal cells that was greater than that obtained in cells incubated with 5 mM-glucose alone. In conclusion, fulfillment of the metabolic requirements of the acid-secreting parietal cell under physiological circumstances requires a combination of substrates, and integration of the results with previous data [Anderson & Hanson (1983) Biochem. J. 210, 451-455; 212, 875-879] suggests that after overnight starvation in vivo metabolism of glucose, D-3-hydroxybutyrate and L-isoleucine may be of particular importance.