UBC Theses and Dissertations
Calcium transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes Mahey, Rajesh
The aim of the present investigation was to determine whether a plasma membrane high affinity Ca²+-ATPase plays an integral role in the maintenance of cytoplasmic free Ca²+ in pancreatic acinar cells. To achieve this, the Ca²+-transport and Ca²+-ATPase activities were characterized and their properties compared. Plasma membranes from guinea-pig pancreatic acini were shown to contain an ATP-dependent high affinity Ca²+-pump and a high affinity Ca²+-dependent ATPase activity. In addition, a low affinity ATPase activity was also observed. The high affinity Ca²+-ATPase activity as well as the Ca²+-transport were found to be dependent on Mg²+, whereas the low affinity ATPase activity appeared to be inhibited by Mg²+. The high affinity ATPase activity was 7-fold greater in magnitude than the Ca²+-transport. Whereas the Ca²+-transport was very specific for ATP as a substrate, the high affinity Ca²+-ATPase showed little specificity for various nucleotide triphosphates. These data would suggest that the Ca²+-transport and the high affinity Ca²+-dependent ATPase in guinea-pig pancreatic acinar plasma membranes may be two distinct activities To further investigate whether the two activities were related, we investigated how the Ca²+-transport and Ca²+-ATPase activities were regulated by intracellular mediators. Regulation of the two activities by calmodulin, cyclic AMP-dependent protein kinase, Protein kinase C and inositol phosphates was investigated. Calmodulin failed to stimulate either activity. In addition, calmodulin antagonists, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca²+-transport. These data suggested the presence of endogenous calmodulin. Both antagonists failed to influence the Ca²+-dependent ATPase activity. Experiments using boiled extracts from guinea-pig pancreatic acinar plasma membranes and erythrocyte plasma membranes Ca²+-ATPase confirmed the presence of endogenous calmodulin. The catalytic subunit of cyclic AMP-dependent protein kinase stimulated Ca²+ transport, suggesting that cyclic AMP may have a role in the regulation of Ca²+-pump-mediated Ca²+ efflux from pancreatic acini. Ca²+-dependent ATPase activity, on the other hand, was not affected by the catalytic subunit. HA 1004, a specific inhibitor of cAMP-dependent protein kinase, failed to inhibit the Ca²+-transport and Ca²+-dependent ATPase activities. Since, this inhibitor was also ineffective at inhibiting the catalytic-subunit-stimulated Ca²+ transport, it may be concluded that HA 1004 is ineffective in blocking the actions of cAMP-dependent protein kinase in pancreatic acinar plasma membranes. In our studies, purified protein kinase C, the phorbol ester TPA and the diacylglycerol derivative, SA-DG, failed to stimulate the Ca²+-uptake activity. However, these agents produced stimulation of the Ca²+-dependent ATPase activity in the presence of phosphatidylserine. CGP 41 251, a potent and selective inhibitor of protein kinase C, did not inhibit the Ca²+-transport or Ca²+-dependent ATPase activities. These observations suggest that protein kinase C may not be involved in the regulation of the plasma membrane Ca²+-pump in guinea-pig pancreatic acinar cells. These results also point to another difference between Ca²+-transport and the Ca²+-ATPase activities in guinea-pig pancreatic acinar plasma membranes. Neither inositol trisphosphate nor inositol tetrakisphosphate produced a statistically significant effect on Ca²+-uptake, suggesting that IP₃- and/or IP₄-mediated Ca²+ releasing pathways may not operate in the isolated guinea-pig pancreatic acinar plasma membrane vesicles. In summary, the results presented here provide evidence to suggest that the high affinity Ca²+-ATPase is not the biochemical expression of plasma membrane Ca²+-transport in panreatic acini. Our results imply a role for calmodulin and cAMP-dependent protein kinase, but not protein kinase C, in the regulation of Ca²+ efflux from pancreatic acinar cells.
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