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Cholecystokinin-stimulated enzyme secretion from dispersed rabbit pancreatic acinar cells: phosphorylation-dependent
P H Willems1, S E Van Emst-de Vries, J J De Pont
1Department of Biochemistry, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Abstract:
In order to establish a regulatory role for phosphoproteins in receptor-stimulated enzyme secretion, dispersed rabbit pancreatic acinar cells were stimulated with the COOH-terminal octapeptide of cholecystokinin (CCK8) in the absence and presence of staurosporine and/or 12-O-tetradecanoylphorbol 13-acetate (TPA) or forskolin. The dose/response curve for the stimulatory effect of CCK8 on amylase secretion was biphasic, with a mean half-maximal concentration (EC50) of 21 pM. Staurosporine (1 microM) did not affect secretion elicited by CCK8 concentrations below 0.1 nM, but reduced the response to CCK8 concentrations above 0.1 nM. As a result, the mean EC50 for CCK8 decreased to 8 pM and its efficacy to 70%. The phorbol ester TPA (0.1 microM) attenuated secretion evoked by CCK8 concentrations below 0.1 nM and potentiated the response to CCK8 concentrations above 0.1 nM. As a result, the mean EC50 for CCK8 increased to 0.14 nM and its efficacy to 300%. Staurosporine abolished both the inhibitory and the potentiating effect of TPA, thereby turning the inhibitory effect into a strong potentiating effect. As a result, the mean EC50 for CCK8 decreased to 3 pM, whereas its efficacy increased to 190%. Forskolin (30 microM) potentiated the response to both the lower and the higher CCK8 concentrations. As a result, the mean EC50 for CCK8 increased to 28 pM and its efficacy to 300%. Staurosporine enhanced the potentiating effect of forskolin at CCK8 concentrations below 0.1 nM, but abolished potentiation at CCK8 concentrations above 0.1 nM. As a result, the mean EC50 for CCK8 decreased to 1.4 pM, whereas its efficacy increased to 260%. The data presented demonstrate that the apparent sensitivity of dispersed pancreatic acinar cells to stimulation of the process of enzyme secretion by CCK8 decreases when kinases are activated and increases when kinases are inactivated. Moreover, they show that the efficacy of CCK8 increases by the action of kinases, both sensitive and insensitive to staurosporine.