45Ca 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 Ca2+ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca2+ pump.

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The biochemical and Ca2+ transport pathways involved in generating the hormone-evoked Ca2+ signal are reported to be influenced by pH. The present study was designed to determine the effect of extracellular pH (pHo) and intracellular pH (pHi) on hormone-stimulated Ca2+ transport. We used rat pancreatic acini and measured free cytosolic Ca2+ concentration ([Ca2+]i) with fura-2, pHi with 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and Ca2+ fluxes with 45Ca2+. In the presence of external Ca2+, increasing pHo increased steady-state [Ca2+]i during sustained agonist stimulation; in the absence of external Ca2+, this increase in [Ca2+]i did not occur. The addition of an antagonist or blocking plasma membrane Ca2+ influx with La3+ in stimulated cells suspended at pHo 8.2 resulted in a reduction in [Ca2+]i. Increasing pHo increased the rate and extent of 45Ca2+ uptake into stimulated cells and the rate and extent of Ca2+ reloading of intracellular stores. The increased Ca2+ content of the intracellular stores with increased pHo indicated that at physiological pHo and pHi the agonist-mobilizable internal stores are not saturated with Ca2+. Changes in pHo affected pHi. However, changes in pHi at constant pHo had no effect on hormone-evoked [Ca2+]i increase, reduction in [Ca2+]i after hormone stimulation, or reloading of intracellular stores. We conclude that the hormone-activated plasma membrane Ca2+ entry pathway responsible for Ca2+ reloading is directly modulated by external H+.

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The 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 and 45Ca 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 across the plasma membrane had minimal effects on resting [Ca2+]i, the rate constant of Ca2+ efflux, and [Ca2+]i levels attained by the cells after 5 min of stimulation. Changing 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.