Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The involvement of Ca2+ and cyclic nucleotides in neurohormonal regulation of Na+-K+-ATPase (Na+-K+ pump) activity in guinea pig pancreatic acinar cells was investigated. Changes in Na+-K+ pump activity elicited by secretagogues were assessed by [3H]ouabain binding and by ouabain-sensitive 86Rb+ uptake. Carbachol (CCh) and cholecystokinin octapeptide (CCK-8) each stimulated both ouabain-sensitive 86Rb+ uptake and equilibrium binding of [3H]ouabain by approximately 60%. Secretin increased both indicators of Na+-K+ pump activity by approximately 40% as did forskolin, 8-bromo- and dibutyryl cAMP, theophylline, and isobutylmethylxanthine. Incubation of acinar cells in Ca2+-free HEPES-buffered Ringer (HR) with 0.5 mM EGTA reduced the stimulatory effects of CCh and CCK-8 by up to 90% but caused only a small reduction in the effects of secretin, forskolin, and cAMP analogues. In addition, CCh, CCK-8, secretin, and forskolin each stimulated ouabain-insensitive 86Rb+ uptake by acinar cells. The increase elicited by CCh and CCK-8 was greatly reduced in the absence of extracellular Ca2+, while that caused by the latter two agents was not substantially altered. The effects of secretagogues on free Ca2+ levels in pancreatic acinar cells also were investigated with quin-2, a fluorescent Ca2+ chelator. Basal intracellular Ca2+ concentration ([Ca2+]i) was 161 nM in resting cells and increased to 713 and 803 nM within 15 s after addition of 100 microM CCh or 10 nM CCK-8, respectively. Forskolin, secretin, and cAMP analogues had no effect on [Ca2+]i, nor did they either reduce or potentiate the rise in [Ca2+]i evoked by CCh.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Intracellular mediators of Na+-K+ pump activity in guinea pig pancreatic acinar cells. 241 68

The results of our investigations into the localization of Na+,K+-pump activity in pancreatic and parotid acinar cells and the effects of hormones and neurotransmitters on pump turnover can be integrated with data on other aspects of stimulus-response coupling to construct models of the neurohumoral control of protein, fluid, and electrolyte secretion (Fig. 23). In both tissues, Ca2+ and cyclic AMP serve as intracellular messengers. In pancreatic acinar cells, the Ca2+-dependent pathway activated by the occupation of CCK or cholinergic receptors provides the primary stimulus for digestive enzyme secretion. Cyclic AMP plays a comparatively minor role; VIP and secretin are much less effective stimulators of protein secretion. Conversely, cyclic AMP levels in parotid acinar cells, which are modulated primarily through occupation of beta-adrenergic receptors, are a major determinant of enzyme secretion. Activation of the Ca2+-dependent pathway by cholinergic or alpha-adrenergic agonists or substance P is less important. The presence of dual control processes in each gland suggests that the observed differences in effectiveness of cyclic AMP- versus Ca2+-dependent secretagogues may reflect not different mechanisms, but rather a shift in the relative emphasis placed on each pathway. This emphasis could conceivably result from subtle variations in the interaction between cellular protein kinases and phosphatases and their phosphoprotein substrates. Electrolyte secretion, on the other hand, appears to involve both discrete and common entities. In pancreatic acinar cells from rodent species, cholinergic or CCK receptor occupancy elicits a Ca2+-dependent increase in the open-state probability of nonselective cation channels in the basolateral plasma membrane. The resultant influx of Na+ and efflux of K+ is most probably the factor which activates Na+, K+-pumps. Based on electron probe studies of the effects of cholinergic agonists on acinar cell Na+ and K+ contents discussed earlier, a transient reduction in the intracellular K+/Na+ ratio of up to 4-fold may occur. A shift of this magnitude in the cytoplasmic microenvironment of the Na+, K+-pump clearly would have a stimulatory influence (see discussion by Jorgensen, 1980). In addition, Ca2+ itself may have direct effects on Na+,K+-pump activity. Calcium at levels much above 1 microM progressively inhibits Na+,K+-ATPase activity (Tobin et al., 1973; Yingst and Polasek, 1985). In unstimulated guinea pig pancreatic acinar cells, Ca2+i measured by quin-2 fluorescence was 161 +/- 13 nM (Hootman et al., 1985a) which increased to a maximal concentration of 803 +/- 122 nM following CCh stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Neuroendocrine control of secretion in pancreatic and parotid gland acini and the role of Na+,K+-ATPase activity. 287 3

Cellular mechanisms underlying the actions of antisecretory agents were studied with dispersed canine fundic cells; aminopyrine accumulation monitored parietal cell (PC) function. Canine PC have pharmacologically typical histamine (H) H2 and muscarinic (M) receptors. PC also have gastrin (G) receptors, which were selectively blocked by gastrin/CCK antagonists. Potentiating interactions occurred between secretagogues, one of the components of the interdependency between regulatory pathways. Prostaglandins (PG) E2 inhibited H-stimulated PC function. Treatment of PC with pertussis toxin (PT), which inactivates the inhibitory GTP-binding protein of adenylate cyclase (Gi), markedly reduced PG inhibition, indicating PG action via Gi. PC function can also be directly inhibited by H+/K+-ATPase inhibitors, such as omeprazole. When canine mucosal cells were studied, stimulatory G and inhibitory M receptors were present on fundic somatostatin (S) cells. Histamine was localized to canine fundic mast cells, which lacked G or M receptors, a conclusion that may not pertain to fundic histamine cells in other species. Nonparietal cell receptors may be important modulators of the regulation of acid secretion.
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PMID:Mechanisms of action of antisecretory drugs. Studies on isolated canine fundic mucosal cells. 288 44

The role of contractile proteins in secretory granule exocytosis was evaluated by determining whether myosin light chain phosphorylation was altered during stimulation of secretion in mouse pancreatic acini. Acinar myosin was purified by extraction into isosmotic sucrose solution containing 40 mM pyrophosphate followed by ammonium sulfate precipitation and Sepharose 4B-CL chromatography. Myosin was eluted as a single peak of K+-EDTA ATPase activity and was purified over 2,000-fold to a final ATPase specific activity of 0.96 mumol.min-1.mg protein-1. Three major myosin subunits of apparent Mr of 200,000, 20,000, and 17,000 were present in the purified myosin preparation. A fourth protein of Mr 21,000 was also present. Purification of myosin from 32P-labeled acini revealed the Mr 200,000, 21,000, and 20,000 proteins to be heavily labeled. The effect of cholecystokinin octapeptide (CCK-8) on myosin phosphorylation was studied after isolation of myosin from 32P-labeled acinar lysates by immunoprecipitation. Treatment of acini for 1-10 min with a concentration of CCK-8 that gives a maximal secretory response caused a 25-40% increase in light chain labeling. Treatment with a supramaximal CCK-8 concentration produced a 50-80% increase in light chain labeling. Phosphorylation of myosin heavy chain was not significantly affected by secretagogue treatment. These results indicate that stimulation of pancreatic acinar secretion is accompanied by an increase in myosin light chain phosphorylation.
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PMID:Evaluation of myosin light chain phosphorylation in isolated pancreatic acini. 333 33

ATP-dependent 45Ca2+ uptake was investigated in purified plasma membranes from rat pancreatic acinar cells. Plasma membranes were purified by four subsequent precipitations with MgCl2 and characterized by marker enzyme distribution. When compared to the total homogenate, typical marker enzymes for the plasma membrane, (Na+,K+)-ATPase, basal adenylate cyclase and CCK-OP-stimulated adenylate cyclase were enriched by 43-fold, 44-fold, and 45-fold, respectively. The marker for the rough endoplasmic reticulum was decreased by fourfold compared to the total homogenate. Comparing plasma membranes with rough endoplasmic reticulum, Ca2+ uptake was maximal with 10 and 2 mumol/liter free Ca2+, and half-maximal with 0.9 and 0.5 mumol/liter free Ca2+. It was maximal at 3 and 0.2 mmol/liter free Mg2+ concentration, at an ATP concentration of 5 and 1 mmol/liter, respectively, and at pH 7 for both preparations. When Mg2+ was replaced by Mn2+ or Zn2+ ATP-dependent Ca2+ uptake was 63 and 11%, respectively, in plasma membranes; in rough endoplasmic reticulum only Mn2+ could replace Mg2+ for Ca2+ uptake by 20%. Other divalent cations such as Ba2+ and Sr2+ could not replace Mg2+ in Ca2+ uptake. Ca2+ uptake into plasma membranes was not enhanced by oxalate in contrast to Ca2+ uptake in rough endoplasmic reticulum which was stimulated by 7.3-fold. Both plasma membranes and rough endoplasmic reticulum showed cation and anion dependencies of Ca2+ uptake. The sequence was K+ greater than Rb+ greater than Na+ greater than Li+ greater than choline+ in plasma membranes and Rb+ greater than or equal to K+ greater than or equal to Na+ greater than Li+ greater than choline+ for rough endoplasmic reticulum. The anion sequence was Cl greater than or equal to Br greater than or equal to 1 greater than SCN greater than NO3 greater than isethionate greater than cyclamate greater than gluconate greater than SO2(4) greater than or equal to glutarate and Cl- greater than Br greater than gluconate greater than SO2(4) greater than NO3 greater than 1 greater than cyclamate greater than or equal to SCN, respectively. Ca2+ uptake into plasma membranes appeared to be electrogenic since it was stimulated by an inside-negative K+ and SCN diffusion potential and inhibited by an inside-positive diffusion potential. Ca2+ uptake into rough endoplasmic reticulum was not affected by diffusion potentials. We assume that the Ca2+ transport mechanism in plasma membranes as characterized in this study represents the extrusion system for Ca2+ from the cell that might be involved in the regulation of the cytosolic Ca2+ level.
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PMID:Electrogenic calcium transport in plasma membrane of rat pancreatic acinar cells. 399 24

The mechanism of the potentiating effect of vasoactive intestinal peptide (VIP) on cholecystokinin (CCK-8)-induced amylase release was studied in isolated and perifused pancreatic acini of the guinea pig. VIP (30 pM-10 nM) potentiated CCK-8 (100 pM)-induced amylase release. Unexpectedly, VIP inhibited CCK-8-induced intracellular Ca2+ oscillations. Forskolin (10 microM), an activator of adenylate cyclase, potentiated CCK-8 (100 pM)-induced amylase release with a time course similar to that observed with VIP. Caffeine (20 mM) inhibited both amylase release and Ca2+ oscillations in response to CCK-8, suggesting that inhibition of Ca2+ oscillations does not necessarily lead to a potentiation of amylase release. When intracellular Ca2+ concentration ([Ca2+]c) was raised by thapsigargin (10 microM), a selective inhibitor of Ca(2+)-ATPase in the endoplasmic reticulum (ER), VIP (10 nM) induced significantly greater amylase release than that induced by VIP alone. When [Ca2+]c was lowered by preincubation with BAPTA-AM (25 microM), a cell-permeant Ca2+ chelator, VIP-induced amylase release was completely abolished. These results suggest that VIP, in spite of its inhibitory action on Ca2+ oscillations, facilitates a Ca(2+)-dependent process distal to the increase in [Ca2+]c to potentiate CCK-8-induced amylase release.
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PMID:Potentiation of cholecystokinin-induced amylase release by peptide VIP in guinea pig pancreatic acini. 756 61

The roles of Ca2+ in agonist-induced pepsinogen secretion from guinea pig chief cells remain unclear. We used cholecystokinin octapeptide (CCK-8) or secretin alone or with thapsigargin (TG) to clarify these roles. TG releases Ca2+ from intracellular stores by inhibiting microsomal Ca(2+)-adenosinetriphosphatase (ATPase), thereby depleting intracellular Ca2+ (Cai2+) stores. In most cells TG also causes Ca2+ influx. In the present study, with an extracellular Ca2+ concentration ([Ca2+]o) of 1.5 mM, CCK-8 (0.1 microM) caused a rapid increase in pepsinogen secretion; however, the rate decreased with time. With [Ca2+]o = 0, the initial increase was similar but later secretion was abolished, suggesting that Ca2+ influx was important for sustained secretion. With [Ca2+]o = 1.5 mM, TG (0.1 microM) caused a 2.7-fold sustained increase in in Cai2+ concentration ([Ca2+]i) and a ninefold sustained increase in pepsinogen secretion. With [Ca2+]o = 0, TG caused a transient 66% increase in [Ca2+]i and a 50% increase in pepsinogen secretion. The time course of TG-induced pepsinogen secretion correlated with the time course of TG-induced increases in [Ca2+]i. These data demonstrated that Ca2+ influx itself was a potent stimulant of pepsinogen secretion. We further focused on the roles of increasing [Ca2+]i from Cai2+ stores. With or without extracellular Ca2+ (Cao2+) present, addition of CCK-8 (0.1 microM) 10 min after TG caused no further increase in [Ca2+]i, demonstrating depletion of the inositol 1,4,5-trisphosphate-sensitive pool. The Ca(2+)-mobilizing agent CCK-8 caused no pepsinogen secretion 10 min after TG preincubation, demonstrating that mobilization of Ca2+ from intracellular stores was important in the rapid initial phase stimulation of pepsinogen secretion caused by CCK-8. In contrast, preincubation with TG had no effect on pepsinogen secretion by secretin, an agent that increases adenosine 3',5'-cyclic monophosphate. A 6-min preincubation with TG potentiated the subsequent stimulation of pepsinogen secretion caused by secretin in the presence of Cao2+ where [Ca2+]i remained elevated. However, TG-induced potentiations of secretin-stimulated pepsinogen secretion was abolished once [Ca2+]i had returned to the basal level in the absence of Cao2+.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Thapsigargin defines roles of Ca2+ in initial, sustained, and potentiated stimulation of pepsinogen secretion. 817

ATP is an extracellular regulator in numerous physiological and pathologic processes. Recently, 7 different subtypes of purinoceptors were identified on either the basolateral or the luminal membrane of pancreatic duct cells. However, the in vivo regulatory role of ATP in pancreatic function has not been established. We investigated the possible regulatory role of endogenous ATP in pancreatic function by measuring ATP concentrations and ATPase activity in pancreatic juice obtained from anesthetized rats and guinea pigs and from human patients undergoing endoscopy. Juice was collected from the main pancreatic duct in rats and guinea pigs under basal conditions or during stimulation with CCK, bombesin, or secretin. In guinea pigs, CCK, bombesin, and secretin did not affect ATP output, although they did stimulate fluid secretion. ATPase activity in the juice was evaluated by measuring the rate of hydrolysis of added ATP. Consistent with the low ATP concentrations in rat pancreatic juice, we found high levels of ATPase activity in this species. This was confirmed by HPLC, which also showed the metabolites of ATP hydrolysis. Ecto-ATPase activity was demonstrated by enzyme histochemistry in both the pancreatic acini and ducts in rats, but it was not detectable in guinea pigs and humans. These differences in ATP levels and ATPase expression may indicate significant species differences in the purinergic regulation of pancreatic secretion.
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PMID:ATP and ATPase secretion by exocrine pancreas in rat, guinea pig, and human. 1521 Nov 12

Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca(2+) responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-gamma, regulates Ca(2+) responses and the Ca(2+)-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3-kinase involvement in Ca(2+) responses induced in these cells by CCK and carbachol. The PI3-kinase inhibitors inhibited both Ca(2+) influx and mobilization from intracellular stores induced by stimulation of acini with physiological and pathological concentrations of CCK, as well as with carbachol. PI3-kinase inhibition facilitated the decay of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) oscillations observed in individual acinar cells. The PI3-kinase inhibitors decreased neither CCK-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] production nor Ins(1,4,5)P(3)-induced Ca(2+) mobilization, suggesting that the effect of PI3-kinase inhibition is not through Ins(1,4,5)P(3) or Ins(1,4,5)P(3) receptors. PI3-kinase inhibition did not affect Ca(2+) mobilization induced by thapsigargin, a specific inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacological and genetic PI3-kinase inhibition on [Ca(2+)](i) signals, suggesting SERCA as a downstream target of PI3-kinase. Both pharmacological PI3-kinase inhibition and genetic deletion of PI3-kinase-gamma increased the amount of Ca(2+) in intracellular stores during CCK stimulation. Finally, addition of the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate to permeabilized acini significantly attenuated Ca(2+) reloading into the endoplasmic reticulum. The results indicate that PI3-kinase regulates Ca(2+) signaling in pancreatic acinar cells through its inhibitory effect on SERCA.
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PMID:Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase. 1527 49

The existence of functionally distinct intracellular Ca(2+) stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca(2+) stores in the gallbladder by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca(2+)](i) that was insensitive to 2-APB but was abolished by pretreatment with 10 muM ryanodine. These data support the idea that both inositol trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca(2+)-free solution, the [Ca(2+)](i) transients decreased as the interval between Ca(2+) removal and caffeine application was increased, indicating a possible leakage of Ca(2+) in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca(2+)-ATPase activation, similar to IP(3)-sensitive stores. The moderate Ca(2+) elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 muM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca(2+) pools do not exist, but IP(3)R and RyR must be spatially separated because Ca(2+) release via these pathways leads to opposite responses.
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PMID:Characterization of intracellular Ca(2+) stores in gallbladder smooth muscle. 1549 78


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