Stephen J. Pandol

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

SummaryThe relative contributions of the Na+/Ca2+ exchange and the plasma membrane Ca2+ pump to active Ca2+ efflux from stimulated rat pancreatic acini were studied. Na+ gradients across the plasma membrane were manipulated by loading the cells with Na+ or suspending the cells in Na+-free media. The rates of Ca2+ efflux were estimated from measurements of [Ca2+]i using the Ca2+-sensitive fluorescent dye Fura 2 and45Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca2+ efflux is described by a single exponential function under control, Na+-loaded, and Na+-depleted conditions. Manipulation of Na+ gradients had no effect on the hormone-induced increase in [Ca2+]i. The results indicate that Ca2+ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca2+ pump. The effects of several cations, which were used to substitute for Na+, on cellular activity were also studied. Choline+ and tetramethylammonium+ (TMA+) released Ca2+ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na+, TMA+, choline+, or N-methyl-d-glucamine+ (NMG+) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG+, choline+, and TMA+ inhibited amylase release by 55, 40 and 14%, respectively. NMG+ also increased the Ca2+ permeability of the plasma membrane. Thus, to study Na+ dependency of cellular function, TMA+ is the preferred cation to substitute for Na+. The stimulatory effect of TMA+ can be blocked by atropine.

Calcium mobilizing hormones activate the plasma membrane Ca2+ pump of pancreatic acinar cells

Summary45Ca fluxes and free-cytosolic Ca2+ ([Ca2+]i) measurements were used to study the effect of Ca2+-mobilizing hormones on plasma membrane Ca2+ permeability and the plasma membrane Ca2+ pump of pancreatic acinar cells. We showed before (Pandol, S.J., et al., 1987.J. Biol. Chem.262:16963–16968) that hormone stimulation of pancreatic acinar cells activated a plasma membrane Ca2+ entry pathway, which remains activated for as long as the intracellular stores are not loaded with Ca2+. In the present study, we show that activation of this pathway increases the plasma membrane Ca2+ permeability by approximately sevenfold. Despite that, the cells reduce [Ca2+]i back to near resting levels. To compensate for the increased plasma membrane Ca2+ permeability, a plasma membrane Ca2+ efflux mechanism is also activated by the hormones. This mechanism is likely to be the plasma membrane Ca2+ pump. Activation of the plasma membrane Ca2+ pump by the hormones is time dependent and 1.5–2 min of cell stimulation are required for maximal Ca2+ pump activation. From the effect of protein kinase inhibitors on hormone-mediated activation of the pump and the effect of the phorbol ester 12-0-tetradecanoyl phorbol, 13-acetate (TPA) on plasma membrane Ca+ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca2+ pump.

Cholecystokinin-induced changes in polysome structure regulate protein synthesis in pancreas

Translational regulation of digestive enzyme synthesis during short-term stimulation by cholecystokinin-octapeptide (CCK-OP), was examined in minced rabbit pancreas by measuring protein synthesis and monitoring alterations in the size of polysomes attached to the rough endoplasmic reticulum (RER). The effect of CCK-OP on protein synthesis was determined by measuring [3H]leucine incorporation into trichloroacetic-acid-precipitable proteins. Concentrations of CCK-OP that caused maximal enzyme secretion (10 and 30 nM) decreased protein synthesis by approx. 50% compared to control. Protein synthesis returned to the control level 60 min after terminating the action of CCK-OP. Autoradiography of [35S]methionine-labeled proteins separated by one-dimensional SDS-polyacrylamide gel electrophoresis demonstrated that CCK-OP reversibly inhibited the synthesis of all of the major groups of digestive enzymes. Northern blot analysis revealed that CCK-OP did not alter the cellular content of amylase and elastase mRNA. Incubation with CCK-OP caused a decrease in the size distribution of RER-bound polysomes. Polysome profiles returned to the control pattern 60 min following termination of the stimulus. These results suggest that the inhibitory effects of CCK-OP on the synthesis of digestive enzymes is regulated at translation by decreasing the number of RER-bound ribosomes that are actively translating digestive enzyme mRNA.

Cellular Mechanisms Mediating Agonist-Stimulated Calcium Influx in the Pancreatic Acinar Cell

Figure 1 summarizes our current concept of a signaling mechanism to explain agonist-induced Ca2+ entry in the pancreatic acinar cell. We propose that cGMP can modulate Ca2+ entry under conditions of internal Ca2+ store depletion and that the NO signaling system may be involved in coupling Ca2+ depletion to cGMP formation. The finding that Ca2+ entry after Ca2+ store depletion can occur with no elevation in [Ca2+]i37 raises the possibility that alternative signaling pathways may converge to stimulate cGMP formation or that additional messengers may activate plasmalemmal Ca2+ entry mechanisms in parallel.

Calcium metabolism and bombesin-stimulated pancreatic enzyme secretion.

In dispersed guinea pig pancreatic acini, bombesin stimulates a rapid increase in the concentration of free cytosolic Ca2+ ([Ca2+]i) by releasing Ca2+ from an intracellular agonist-sensitive pool. This increase is transient, and it mediates the initial secretion of digestive enzymes from the acinar cell. During sustained bombesin stimulation, [Ca2+]i returns to the resting or near-resting level, but bombesin-stimulated enzyme secretion continues. Although [Ca2+]i is at the resting level during continued stimulation, 45Ca2+ flux measurements indicate that bombesin continues to release Ca2+ from an intracellular store that is probably the agonist-sensitive pool. Low concentrations (0.3 microM) of the non-fluorescent Ca2+ ionophore, 4-bromo-A23187 (Br-A23187), release Ca2+ from the agonist-sensitive pool without measurably increasing [Ca2+]i. This ionophore-induced Ca2+ release without production of an increase in [Ca2+]i does not by itself stimulate enzyme secretion, but it does augment the secretion caused by the phorbol ester, 4 beta-phorbol, 12-myristate, 13-acetate (PMA). These results suggest a role for intracellular Ca2+ release in bombesin-stimulated sustained secretion.