Willems P H, Van Emst-de Vries S E, De Pont J J
Department of Biochemistry, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Pflugers Arch. 1995 Sep;430(5):626-35. doi: 10.1007/BF00386156.
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.
为了确定磷酸化蛋白在受体刺激的酶分泌中的调节作用，在不存在和存在星形孢菌素和/或十四烷酰佛波醇-13-乙酸酯（TPA）或福斯可林的情况下，用胆囊收缩素（CCK8）的羧基末端八肽刺激分散的兔胰腺腺泡细胞。CCK8对淀粉酶分泌的刺激作用的剂量/反应曲线是双相的，平均半数最大浓度（EC50）为21 pM。星形孢菌素（1 microM）不影响CCK8浓度低于0.1 nM时引起的分泌，但降低了对CCK8浓度高于0.1 nM时的反应。结果，CCK8的平均EC50降至8 pM，其效力降至70%。佛波酯TPA（0.1 microM）减弱了CCK8浓度低于0.1 nM时引起的分泌，并增强了对CCK8浓度高于0.1 nM时的反应。结果，CCK8的平均EC50增加到0.14 nM，其效力增加到300%。星形孢菌素消除了TPA的抑制和增强作用，从而将抑制作用转变为强烈的增强作用。结果，CCK8的平均EC50降至3 pM，而其效力增加到190%。福斯可林（30 microM）增强了对较低和较高CCK8浓度的反应。结果，CCK8的平均EC50增加到28 pM，其效力增加到300%。星形孢菌素在CCK8浓度低于0.1 nM时增强了福斯可林的增强作用，但在CCK8浓度高于0.1 nM时消除了增强作用。结果，CCK8的平均EC50降至1.4 pM，而其效力增加到260%。所呈现的数据表明，当激酶被激活时，分散的胰腺腺泡细胞对CCK8刺激酶分泌过程的明显敏感性降低，而当激酶失活时则增加。此外，它们表明CCK8的效力通过对星形孢菌素敏感和不敏感的激酶的作用而增加。
