ATP-dependent Ca2+ uptake into isolated pancreatic acinar cells with permeabilized plasma membranes, as well as into isolated endoplasmic reticulum prepared from these cells, was measured using a Ca2+ -specific electrode and 45Ca2+. Endoplasmic reticulum was purified on an isopycnic Percoll gradient and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the rough endoplasmic reticulum RNA was enriched threefold and the typical marker for the plasma membrane Na+,K+(Mg2+)ATPase was decreased 20-fold. When different fractions of the Percoll gradient were compared, 45Ca2+ uptake correlated with the RNA content and not with the Na+,K+(Mg2+)ATPase activity. The characteristics of nonmitochondrial Ca2+ uptake into leaky isolated cells and 45Ca2+ uptake into isolated endoplasmic reticulum were very similar: Calcium uptake was maximal at 0.3 and 0.2 mmol/liter free Mg2+, at 1 and 1 mmol/liter ATP, at pH 6.0 and 6.5, and free Ca2+ concentration of 2 and 2 mumol/liter, respectively. Calcium uptake decreased at higher free Ca2+ concentration. 45Ca2+ uptake was dependent on monovalent cations (Rb+ greater than K+ greater than Na+ greater than Li+ greater than choline+) and different anions (Cl- greater than Br- greater than SO4(2-) greater than NO3- greater than I- greater than cyclamate- greater than SCN-) in both preparations. Twenty mmol/liter oxalate enhanced 45Ca2+ uptake in permeabilized cells 10-fold and in vesicles of endoplasmic reticulum, fivefold. Calcium oxalate precipitates in the endoplasmic reticulum of both preparations could be demonstrated by electron microscopy. The nonmitochondrial Ca2+ pool in permeabilized cells characterized in this study has been previously shown to regulate the cytosolic free Ca2+ concentration to 0.4 mumol/liter. Our results provide firm evidence that the endoplasmic reticulum plays an important role in the regulation of the cytosolic free Ca2+ concentration in pancreatic acinar cells.
Many types of secretory granule have been observed to swell as a result of cell stimulation implying a degree of osmotic control, although the regulation of granule fusion with the apical plasma membrane is not clearly understood. In the present study we have investigated the ionic and osmotic dependency of basal and stimulated 3H-protein release from rat pancreatic acini, permeabilised by either digitonin or high voltage electric discharge. Acini were stimulated with either cholecystokinin-pancreozymin octapeptide (CCK-8), carbachol (CCh), or with phorbol ester (TPA) plus cAMP. Stimulated secretion was significantly reduced when 130 mmol/l Cl- in the buffer was replaced by I-, NO3-, SCN- or cyclamate-. Secretion in Cl- buffers was inhibited by the anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), by 40% of the control response. Neither Na+ nor N-methyl-D-glucamine+ could replace K+ in the buffer. Ba2+ and quinine, which block K+ conductance pathways, inhibited stimulated secretion by 50%. Finally, stimulated secretion from leaky cells was nearly abolished by doubling buffer osmolarity. The data suggest that when the cell is stimulated, a Cl- and a K+ permeability appear in the zymogen granule membrane and the ions enter down their electrochemical gradients. The increased intragranular osmolarity results in granular swelling which is intimately associated with secretion.
We have studied the involvement of GTP-binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin-octapeptide (CCK-OP) and GTPyS but not carbachol (CCh)-induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator, of adenylyl cyclase, nor the CAMP-analogue, 8-bromo CAMP, mimicked the inhibitory effect of cholera toxin on agonist-induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated G, with phospholipase C or to an elevation of CAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP-ribosylated a 40 kDa protein, which was inhibited by CCK-OP but not by CCh. We conclude from these data that both CCK-and muscarinic acetylcholine receptors functionally couple to phospholipase C by two different GTP-binding proteins.
Enzyme secretion from the exocrine pancreas is stimulated by receptor-activated breakdown of phosphatidylinositol 4,5-bisphosphate and consequent rise of both inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, which leads to Ca2+ release and to activation of protein kinase C, respectively. Another way involves receptor-mediated stimulation of adenylate cyclase and consequent rise of cAMP and activation of protein kinase A. In the present work we have studied direct stimulation, inhibition, and mutual interaction of these pathways on enzyme secretion from isolated rat pancreatic acini that had been permeabilized by treatment with saponin or digitonin. The data were compared with those obtained in isolated intact acini. The data show that with increasing free Ca2+ concentrations greater than 10(-6) M protein release increases in "leaky" but not in "intact" cells and is maximal at approximately 10(-3) M, increasing about twofold compared with that in the absence of Ca2+. In the presence of the acetylcholine analogue carbachol, this effect of Ca2+ is enhanced by about threefold in leaky cells and is also present in intact cells to a similar extent. cAMP and its analogues, dibutyryl cAMP (dbcAMP) and 8-bromo-cAMP stimulate protein release by about twofold in the presence of Ca2+ in leaky cells. In intact acini cAMP has no effect, and cAMP analogues stimulate enzyme secretion by about twofold in some but not all experiments. Similarly, forskolin, an activator of adenylate cyclases and inhibitors of cyclic nucleotide-dependent phosphodiesterases, such as 3-isobutyl-1-methylxanthine (IBMX) and R0 201724, stimulate protein release in permeabilized acini. The Ca2+-binding protein calmodulin has no effect on enzyme secretion, whereas the calmodulin antagonist trifluoperazine dihydrochloride stimulates protein release in leaky but not in intact acini. The activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates protein release in a Ca2+-dependent manner and enhances cAMP-induced secretion. The effects of carbachol, TPA, cAMP, and a combination of both TPA and cAMP are inhibited by the polyamine spermine in permeabilized cells. Spermine has no effect on carbachol-induced enzyme secretion in intact cells. The data suggest that enzyme secretion from pancreatic acinar cells is mediated by cAMP protein kinase A and by Ca2+ phospholipid protein kinase C in a Ca2+-dependent way and that interaction occurs between both pathways.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.