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C S Chew

Publications and source records attributed to C S Chew.

At least 37 records · Page 2Linked to original sources
PubMed1989

Carbachol-induced protein phosphorylation in parietal cells: regulation by [Ca2+]i.

1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), an intracellular calcium [( Ca2+]i) chelator, was used to investigate the role of [Ca2+]i in acid secretory activity and protein phosphorylation in parietal cells from rabbit. Chelation of extracellular calcium [( Ca2+]o) with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not prevent the initial carbachol-induced elevation of [Ca2+]i as measured with the fluorescent Ca2+ probe, fura-2, and only partially inhibited [14C]-aminopyrine (AP) accumulation, an indirect indicator of acid secretory activity. [Ca2+]i chelation with BAPTA/AM eliminated carbachol-stimulated increases in [Ca2+]i and AP accumulation but only transiently reduced histamine stimulation of AP accumulation. Carbachol increased phosphorylation of a 36-kDa, pI approximately 7 protein (pp36) and transient phosphorylation of a 28-kDa, pI approximately 5 protein (pp28), whereas histamine increased phosphorylation of 40-kDa, pI approximately 6.5 (pp40) and 27-kDa, pI approximately 6.2 (pp27) proteins. Phosphorylation of pp36 and pp28 were mimicked by the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore, ionomycin, respectively. Two other phosphoproteins with molecular weights of 66,000 and pIs of 5.7 and 5.9 were also phosphorylated in response to TPA and carbachol. Chelation of [Ca2+]i and [Ca2+]o blocked carbachol-induced phosphorylation of pp28 and pp36 and ionomycin phosphorylation of pp28 but not TPA-stimulated phosphorylation of pp36 or the two pp66s or histamine-stimulated phosphorylation of pp27 or pp40. Chelation of [Ca2+]i alone did not block increases in [Ca2+]i or phosphorylation of pp28 in response to ionomycin. Both pp28 and pp36 were localized in both microsomal and cytosolic fractions of cells, which suggests involvement in cytoskeleton-membrane interactions. These phosphoproteins could be common elements of Ca2+-dependent stimulus-secretion coupling as similar proteins were phosphorylated by carbachol and cholecystokinin (CCK) in chief cells. Based on data with TPA and ionomycin, both protein kinase C and an as yet unidentified Ca2+-dependent protein kinase(s) appear to be activated upon stimulation with cholinergic agonists and CCK.

Aminopyrine↗
PubMed1987

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.

Adenosine Diphosphate↗
PubMed1987

Histamine increases phosphorylation of 27- and 40-kDa parietal cell proteins.

Purified, hormonally responsive parietal cells from rabbit gastric mucosae were used as a model to study intracellular mechanisms controlling parietal cell HCl secretion. Using a high-resolution, two-dimensional electrophoretic technique, we demonstrate that histamine increases phosphorylation of two parietal cell proteins with approximate molecular weights of 27 and 40 kDa and respective pIs of 5.9 and 6.2. The increase in phosphorylation appears to be mediated via an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanism because cAMP analogues and forskolin stimulate phosphorylation of these proteins, whereas the cholinergic agonist, carbachol, which elevates parietal cell intracellular free calcium concentration but not cAMP content, and the calcium ionophore, ionomycin, do not. Both phosphoproteins are located in low-speed particulate fractions. The 40-kDa phosphoprotein was found in both enriched chief and parietal cells. This phosphoprotein may be cytoskeleton associated, since it is detected in a Triton-insoluble particulate fraction after prolonged exposure of parietal cells to Triton X-100. The 27-kDa phosphoprotein was detected in parietal but not in enriched chief cells and appeared to be localized in a low-speed fraction previously shown to contain increased H+-K+-ATPase activity after histamine stimulation. The location and rapid increase in phosphorylation of the 27-kDa phosphoprotein upon histamine stimulation make this protein an attractive candidate for future studies of intracellular regulation of parietal cell HCl secretion. The 40-kDa phosphoprotein may play a more general role in control of cytoskeletal activity, and perhaps, in morphological transformations associated with stimulus-secretion coupling.

Animals↗
PubMed1986

Release of intracellular Ca2+ and elevation of inositol trisphosphate by secretagogues in parietal and chief cells isolated from rabbit gastric mucosa.

The fluorescent intracellular Ca2+ indicator, fura2/AM, was used to determine the effects of carbachol, cholecystokinin octapeptide (CCK-8), gastrin and histamine on intracellular Ca2+ ([Ca2+]i) in parietal cells from rabbit gastric mucosa enriched to more than 95% purity by a new Nycodenz gradient/centrifugal elutriation technique. Changes in [Ca2+]i in response to the same agonists were also measured in enriched chief cells. Carbachol, histamine, gastrin and CCK-8 increased parietal cell [Ca2+]i with the response to carbachol greater than CCK -8 = histamine = gastrin. Prestimulation with msximal doses of carbachol blocked histamine-induced increases in [Ca2+]i. In chief cells, carbachol increased [Ca2+]i but to a lesser degree than CCK-8, while histamine had no significant effect on [Ca2+]i. Neither removal of extracellular Ca2+ coupled with acute addition of 1 mM EGTA nor addition of the Ca2+-channel blocker nicardipine prevented agonist-induced changes in [Ca2+]i in either cell type. In the presence and absence of 10 mM LiCl2, carbachol and CCK-8 were found to increase inositol trisphosphate (IP3) content in both parietal and chief cells while histamine had no significant effect on this phosphoinositide hydrolysis product. From these results and previous observations with gastric glands (Chew, C.S. (1986) Am. J. Physiol. 13, G814-G823) we conclude that: carbachol, CCK-8, gastrin and histamine increase parietal cell [Ca2+]i initially by release of Ca2+ from the same intracellular store(s); the release of [Ca2+]i in response to carbachol and CCK-8 in both chief and parietal cells appear to be mediated by IP3; however, other mechanisms may be involved in histamine-induced release of parietal cell Ca2+.

Animals↗
PubMed1986

Cholecystokinin, carbachol, gastrin, histamine, and forskolin increase [Ca2+]i in gastric glands.

The Ca2+-selective fluorescent probe quin 2 was used to measure changes in the concentration of free cytosolic [Ca2+] in isolated rabbit gastric glands. Both carbachol and cholecystokinin octapeptide (CCK-8) were found to increase transiently intracellular Ca2+ concentration, [( Ca2+]i) with maximal increases from approximately 0.15 to 0.5 microM occurring within 4-6 s following secretagogue addition. Increases in [Ca2+]i were dose dependent and inhibited by appropriate antagonists. Prestimulation with either carbachol or CCK-8 effectively prevented increases in [Ca2+]i in response to the other agonist. Acute removal of extracellular Ca2+ slightly reduced the increase in [Ca2+]i that occurred following secretagogue addition but had no effect on pepsinogen secretion. Severe Ca2+ depletion resulted in potent inhibition of the quin 2 signal and suppressed basal pepsinogen release and reduced, but did not totally block, pepsinogen release in response to carbachol and CCK-8. Gastrin stimulation also elevated [Ca2+]i in glands, but this agonist was only 40-50% as effective as CCK-8. The cAMP-dependent agonists histamine and forskolin increased [Ca2+]i to approximately the same degree as gastrin. There was a definite lag in the rise in [Ca2+]i following simulation with histamine and forskolin compared with carbachol and CCK-8, which suggests that additional biochemical events occur between agonist-receptor binding and the rise in [Ca2+]i observed with the cAMP-dependent agonists. Both cAMP-dependent and -independent agonists induced an increase in autofluorescence that was slower than the rise in [Ca2+]i but equally affected by extracellular Ca2+ depletion.

Aminoquinolines↗
PubMed1985

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.

Aminopyrine↗
PubMed1985

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.

Adenylyl Cyclase Inhibitors↗
PubMed1983

Forskolin stimulation of acid and pepsinogen secretion in isolated gastric glands.

Forskolin, a specific diterpene activator of adenylate cyclase in intact cells and cellular homogenates, was used to examine the relationship among adenosine 3',5'-cyclic monophosphate (cAMP) metabolism, gastric acid, and pepsinogen secretion in isolated gastric glands. This agent was found to stimulate [14C]aminopyrine (AP) accumulation and respiration, both measurements of which are indexes of parietal cell acid secretory responsiveness and pepsinogen secretion, which is a measure of chief cell activity. Forskolin also increased cAMP content and activated cAMP-dependent protein kinase in the glands. The histamine H2-receptor antagonist, cimetidine, inhibited forskolin-stimulated increases in AP accumulation and respiration when submaximal concentrations of forskolin were used but had not effect on the other response parameters. Forskolin also potentiated the action of the muscarinic agonist, carbachol, in both the presence and absence of cimetidine. Since there was a close kinetic and temporal correlation between the secretory response parameters and cAMP-dependent protein kinase activation, it appears that cAMP plays an important role in the mediation of gastric acid and pepsinogen secretion. The inhibitory action of cimetidine on forskolin-stimulated AP accumulation and respiration suggest that forskolin potentiates the action of endogenous histamine present in the glands. Forskolin potentiation of carbachol in the presence of maximum inhibitory concentrations of cimetidine indicates that previously observed potentiating interactions between carbachol and histamine, secretagogues which appear to act via cAMP-independent and cAMP-dependent mechanisms, respectively, involve intracellular events that occur subsequent to the binding of these agents to their respective receptors and subsequent to an increase in intracellular cAMP content.

Animals↗
PubMed1983

Inhibitory action of somatostatin on isolated gastric glands and parietal cells.

The action of somatostatin in vitro was characterized using glands and parietal cells isolated from rabbit gastric mucosa. In the presence of the reducing agent dithiothreitol, somatostatin was found to inhibit gastrin- and histamine-stimulated acid formation in glands as measured by [14C]aminopyrine (AP) accumulation and oxygen consumption, both measurements that appear to be reliable indexes of parietal cell acid formation. In glands the inhibition of the secretory response to gastrin was more potent (60-80%) than that to histamine (15-25%). The kinetics of somatostatin inhibition of responses to both agents were noncompetitive. The apparent IC50 for the partial somatostatin inhibition of histamine-stimulated AP accumulation was similar to that for gastrin (approx 3 X 10(-9) M) when maximum concentrations of histamine (10(-4) M) or gastrin (10(-7) M) were used. The inhibitory action of somatostatin appeared to be specific, inasmuch as this peptide had no significant effect on basal secretion or secretion stimulated by carbachol, dibutyryl cAMP, cholera toxin, or elevated extracellular K+. In purified parietal cell preparations, somatostatin inhibited histamine- but not gastrin-stimulated AP accumulation. Moreover, the inhibition of histamine-stimulated AP accumulation in parietal cells was more pronounced than in glands. These results suggest that somatostatin acts directly on parietal cells to inhibit histamine activation of H+ secretion. Somatostatin also acts indirectly to inhibit gastrin, perhaps by blocking the release of histamine from paracrine- or endocrinelike cells present in the glands.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Gastrin stimulation of isolated gastric glands.

The ability of gastrin to stimulate acid formation was studied in gastric glands and isolated parietal cells obtained from rabbit gastric mucosa. Accumulation of the weak base aminopyrine and increases in oxygen consumption were used as measures of acid secretory activity. The responses to gastrin were found to be very small (10-15% increase). However, inclusion of dithiothreitol (0.5 mM) in the incubation medium enhanced the responses in both glands and isolated cells to easily detectable levels. For the gastric gland preparation, gastrin stimulation was maximal at 1 X 10(-7) M, with an apparent ED50 of 5 nM. The response reached a maximum at about 30 min and was stable for at least an hour. The gastrin response was enhanced by the phosphodiesterase inhibitor isobutylmethylxanthine and partially inhibited by cimetidine, a histamine H2-receptor antagonist. Combinations of gastrin and histamine showed an additive response over a wide range of histamine concentrations. However, time-course studies revealed a transient potentiation of gastrin response by histamine, which reached a peak at 15 min and was reduced to an additive response by 30 min. Studies using isolated cell populations enriched in parietal cells (approximately 70%) revealed a gastrin stimulation that was not inhibited by cimetidine. The transient potentiation of the gastrin response by histamine was also found in the isolated cell preparation. Gastrin had no effect on cellular cAMP levels or adenylyl cyclase activity. The results are interpreted to indicate that gastrin stimulates acid secretion through three separate actions: 1) a direct stimulation of parietal cell activity, 2) a potentiating interaction with histamine, and 3) for more intact preparations, a release of histamine, which in turn acts as a paracrine stimulus. Quantitatively, the most important action appears to be the release of histamine. None of the actions of gastrin appear to involve a change in cAMP metabolism.

Adenylyl Cyclases↗
PubMed1982

Pepsinogen release from isolated gastric glands.

The in vitro release of pepsinogen was studied using a preparation of isolated gastric glands from rabbits. The pepsinogen content of the glands was estimated to be about 700 U/mg dry wt. Spontaneous release of pepsinogen was found to be less than 1% of the total per hour and relatively constant for at least 2 h. Pepsinogen release was stimulated in a dose-dependent manner by both carbachol and isoproterenol. The cholinergic and beta-adrenergic stimulation was selectively inhibited by atropine and propranolol, respectively. Removal of external calcium inhibited the responses to both isoproterenol (partially) and carbachol (completely). Several agents, including histamine, prostaglandin (E2), and synthetic secretin, were found not to stimulate pepsinogen release. However, a crude secretin preparation (Boots) was found to produce significant stimulation. Dibutyryl cAMP increased pepsinogen release in a dose-dependent manner. Isoproterenol was found to increase the cAMP content of gastric glands and to stimulate adenylyl cyclase activity in homogenates. The beta-adrenergic stimulation of adenylyl cyclase was found to be selective for a population of gastric cells that was relatively depleted of parietal cells and distinct from the histamine-stimulated adenylyl cyclase activity. The results indicate that pepsinogen secretion by the gastric chief cell is regulated, in part, by separate cholinergic and beta-adrenergic mechanisms and that both calcium and cAMP play a role in this regulation.

Animals↗
PubMed1981

Mechanism of action of SCN in isolated gastric glands.

Isolated gastric glands were used to study the mechanism of acid secretory inhibition by thiocyanate (SCN). It was found that SCN does not act as a competitive antagonist of histamine nor does SCN prevent the increase in cellular cAMP associated with histamine stimulation. SCN modifies but does not prevent the expansion of parietal cell canaliculi, indicating that this characteristic morphological transition does not require the actual formation of hydrochloric acid. Low doses (less than 5 mM) of SCN were found to inhibit aminopyrine accumulation, an index of acid formation, but do not inhibit either resting or stimulated respiration. Higher doses (greater than 10 mM) of SCN produce significant inhibition of stimulated but not resting respiration. These results indicate that SCN has two actions, i.e., inhibition of acid formation that requires low doses and inhibition of oxidative metabolism that requires higher doses. Incubation of glands in high-K+ (108 mM) medium leads to formation of an acid gradient in the absence of other secretagogues. The gradient was found to be transient, and its formation does not require oxidative metabolism, indicating that continued proton pumping is not required. Low doses of SCN were found to be more effective in dissipating the high-K+-induced gradient than a similar gradient induced by histamine stimulation. These results support the hypothesis that SCN inhibits acid secretion by increasing the rate of proton-gradient dissipation rather than interfering with a proton-pump mechanism.

Aminopyrine↗
PubMed1980

Histamine responsiveness of isolated gastric glands.

Gastric glands isolated from rabbit were employed to perform a pharmacological characterization of the histamine receptor associated with physiological and biochemical responses in gastric cells. Five separate response parameters were characterized using histamine analogues and histamine antagonists. The following parameters were studied: respiration, accumulation of the weak base aminopyrine, adenylate cyclase activity, cAMP accumulation, and the uptake of histamine. All parameters were examined for agonist and antagonist potency using dose-response curves, ED50 and pA2 values. Comparison of the ED50-agonist and pA2-cimetidine values showed a remarkable similarity for respiration, aminopyrine accumulation, adenylate cyclase activity, and cAMP accumulation. The agonist potency sequence and pA2 values for H2- vs. H1-receptor antagonists characterized the histamine receptor associated with these four parameters as being of the H2 type. Moreover, the similarity of pharmacological characteristics provides evidence for a similar, if not common, receptor for these responses. The histamine uptake system shows a generally lower affinity for most agonists. Although the general agonist potency sequence is similar to the other parameters, notable exceptions were found for antagonists and the typical H2-agonist, dimaprit. Thus, the uptake system does not appear to be related directly to the activation of secretion and the carrier binding site cannot be simply defined by H1 or H2 properties.

Adenylyl Cyclases↗
PubMed1978

Ultrastructural changes and cyclic AMP in frog oxyntic cells.

In vitro frog gastric mucosa was employed as a model for a combined physiological, biochemical, and ultrastructural study of the morphological changes which accompany the onset of acid secretion by the oxyntic cell. The histamine H2-receptor antagonist metiamide was used to provide a reproducible control state. Stimulation of acid production by theophylline resulted in a 10-fold increase in plasma membrane surface area and a distinct change in the conformation of mitochondrial cristae. Studies using the acid secretion inhibitors, thiocyanate and anoxia, demonstrated that neither acid production per se nor oxidative metabolism is essential for the theophylline-dependent changes in surface area. Increases in tissue cyclic AMP levels were observed under the conditions producing morphological changes. It is postulated that surface area changes induced by theophylline are controlled by cellular cyclic AMP levels.

Animals↗