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Studies on the role of Ca²⁺ in the pancreatic acinar cell Ansah, Twum-Ampofo

Abstract

Calcium ions play an important role in the stimulation of enzyme secretion from the exocrine pancreas. It has been proposed that a secretagogue-induced rise in cytoplasmic free calcium triggers enzyme release from pancreatic acinar cells. The mechanism by which Ca²⁺ can regulate enzyme secretion was investigated using Isolated intact pancreatic acinar cells and plasma membrane-enriched preparations obtained from these cells. The viability of the cells isolated was greater than 95% as evaluated by the exclusion of trypan blue dye. Secretin, pancreozymin and carbachol produced a dose-dependent release of amylase in the acinar cells. The role of the calcium binding protein, calmodulin in enzyme secretion was studied indirectly by determining the effects of calmodulin antagonists on the secretory process. Trifluoperazine inhibited carbachol-stimulated amylase release (ED₅₀ of 10μM). Chlorpromazine at a concentration of 10μM inhibited both carbachol- and pancreozymin-stimulated amylase release to a significant degree without affecting secretin stimulated (non-calcium mediated) release. Propranolol did not show this selective inhibition. These studies indicate the possibility that phenothiazines inhibit calcium-mediated amylase release from pancreatic acinar cells by acting on a calmodulin-regulated step in the stimulus-secretion coupling process. A high ATP hydrolytic activity in the presence of either Mg or Ca was localized in acinar cell preparations enriched in plasma membranes. Kinetic analysis revealed that the enzyme had a significantly higher affinity (p < 0.05) for Ca²⁺ (K[sub d] of 1.73 μM) than Mg²⁺ (K[sub d] of 2.98 μM) but a similar maximal rate of activity. Further studies were carried out in order to determine if this ATPase activity represented one or more enzymes. A comparison of substrate requirements revealed very similar profiles for the Mg²⁺- and Ca²⁺-stimulated activities with ATP and GTP being the most effectivefollowed by ADP. The pH curves for both activities were identical. In addition, combinations of saturating concentrations of Mg²⁺ or Ca²⁺ produced the same degree of maximal activity. These data tend to indicate that the ATPase activity may be due to one enzyme with activation sites for both ions. Arrhenius plots though, revealed a difference in the transition temperatures. In addition, when SDS polyacrylamide gel electrophoresis was used to identify the phosphoprotein intermediates of the ATPase, one phosphoprotein intermediate (approx M.W. of 115,000) was observed in the presence of Ca²⁺ alone (endogenous Mg "r =1.5-2 μM) but upon the addition of both Ca²⁺ and Mg²⁺ , a second phosphoprotein intermediate was revealed (M.W. 130,000). Both of these phosphoproteins were hydroxylamine-sensitive, indicating that they were acyl phosphates. The presence of two acyl phosphoproteins would indicate possible separate intermediate reaction systems for the Mg²⁺ and Ca²⁺ stimulated ATPase activity. Calmodulin is known to regulate a number of enzyme systems including Ca²⁺ -transport ATPases. The effect of exogenous calmodulin on the Ca²⁺ -stimulated component of this ATPase activity was therefore investigated. In the presence of endogenous Mg²⁺ , significant stimulation by calmodulin of Ca²⁺ -ATPase activity was observed. This effect was dose-dependent with a for calmodulin of approximately 0.7 μM. Calmodulin increased the Ca²⁺ -sensitivity of this enzyme system; Mg²⁺ appeared to be required for this effect. This calmodulin stimulation was inhibited by trifluoperazine (ED₅₀ of 30μM), chlorpromazine (ED₅₀ of 55 (μM) and compound 48/80. Using an 125 ¹²⁵I-labeled calmodulin gel overlay technique, it was shown that calmodulin binds in a Ca²⁺ -dependent fashion to 133,000 and 230,000 dalton proteins present in the plasma membrane-enriched fraction. Further studies revealed that under conditions that favour Ca²⁺ -dependent kinase activity, calmodulin enhanced the phosphorylation of a 30,000 and a 19,000 dalton protein. The Ca²⁺ -ATPase was also stimulated by acidic phospholipids. Ca²⁺ -flux measurements in plasma membrane vesicles indicated the presence of two processes: there were significantly lower levels of 5 Ca²⁺ associated with vesicles 'loaded' in the presence of ATP than those 'loaded' in the absence of ATP indicating an energy-dependent efflux system. As well, a very rapid ATP-dependent Ca²⁺ -uptake system, that was Mg²⁺ -dependent was observed in sealed vesicles. In order to relate the (Mg²⁺ + Ca²⁺) ATPase activity and the Ca²⁺ fluxes observed to a possible functional role in the acinar cell, the orientation of the catalytic site of the enzyme was determined. The degree of ATPase activity observed when intact viable acinar cells were incubated with ATP and either Mg²⁺ or Ca²⁺ was similar to that observed in broken membranes. This provides evidence that this ATPase activity may be externally-oriented. The role of this system in acinar cell function remains to be elucidated.

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