Role of Rho and Rac in cholecystokinin induced pancreatic acinar morphological changes, actin redistribution and secretion

The purpose of this thesis is to evaluate the role that the small G proteins Rho and Rac play in CCK-induced pancreatic acinar secretion and in the pathophysiological changes induced by high concentrations of CCK that include morphological changes and inhibition of secretion. NIH3T3 cells transfected with CCKA receptors, were first used to show that CCK receptors can activate Rho using both a pulldown assay and stress fiber formation. This activation of Rho was shown to be mediated by Galpha12/13 rather than Galphaq. We next confirmed in isolated mouse pancreatic acini that CCK activated Rho and Rac in a time- and dose-dependent manner and that after activation, both of them translocated to the apical region of the cell. Primary cultured pancreatic acini were infected with adenovirus expressing either dominant negative or constitutively active mutants of Rho, Rac or C3 exotoxin which specifically inactivates Rho; cell morphology and actin redistribution were evaluated. Both dominant negative mutants of Rho and Rac and C3 decreased the acinar secretory response to CCK and simultaneous inhibition of both Rho and Rac resulted in a greater reduction across the dose-response curve. Dominant negative Rho inhibited both the early and late phases of secretion while dominant negative Rac decreased only the late phase; neither of them affected the intracellular calcium response to CCK. Both active Rho and Rac induced acinar morphological changes and actin redistribution, to some extent mimicking the effects of high concentrations of CCK. In addition, both active Rho and Rac increased the maximal release of amylase and partially ablated the inhibition of secretion induced by high concentrations of CCK. The effect on amylase release of dominant negative and constitutively active mutants of Rho and Rac could be mimicked by latrunculin, an actin depolymerizer, and jasplakinolide, an actin polymerizer, respectively. Moreover, jasplakinolide alone induced acinar morphological changes similar to high CCK and active Rho while latrunculin blocked the CCK induced acinar morphological changes. We conclude that CCK can activate Rho and Rac in pancreatic acini. Rho and Rac both play a positive role in regulated secretion probably through regulating the redistribution of actin cytoskeleton and are also involved in the acinar morphological changes induced by the high concentrations of CCK.