Duan R D, Williams J A
Department of Physiology, University of Michigan, Ann Arbor 48109-0622.
Am J Physiol. 1994 Sep;267(3 Pt 1):G401-8. doi: 10.1152/ajpgi.1994.267.3.G401.
The existence and activation of mitogen-activated protein (MAP) kinase in isolated pancreatic acini have been demonstrated. Immunoblotting and immunoprecipitation revealed two forms of MAP kinase in pancreatic acini, with relative molecular masses of approximately 42 and 44 kDa. Both forms of MAP kinase were activated by cholecystokinin (CCK). The threshold concentration of CCK was approximately 3 pM, and the maximal effect occurred at 1 nM, which enhanced MAP kinase activity by 2.5-fold, as determined in polyacrylamide gel copolymerized with substrate myelin basic protein. Activation of MAP kinase by CCK was rapid, reaching a maximum within 5-10 min that subsequently declined. Bombesin and carbachol but not secretin or vasoactive intestinal peptide also activated MAP kinase. CCK-induced activation of MAP kinase may be mediated by protein kinase C, since 12-O-tetradecanoylphorbol 13-acetate (TPA) mimicked the effect of CCK and staurosporine concentration dependently inhibited the action of CCK. Treatment of acini with thapsigargin, ionomycin, or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not influence MAP kinase, indicating that mobilization of intracellular calcium by CCK is not important in activation of acinar MAP kinase. CCK and TPA increased tyrosine phosphorylation of both 42- and 44-kDa forms. Genistein and tyrphostin 23, the inhibitors of tyrosine kinase, suppressed the activation of MAP kinase by CCK. In conclusion, MAP kinase in pancreatic acini is activated by agonists related to hydrolysis of phosphoinositide, via a mechanism involving protein kinase C and tyrosine kinase.
已证实丝裂原活化蛋白（MAP）激酶在分离的胰腺腺泡中存在并被激活。免疫印迹和免疫沉淀显示胰腺腺泡中有两种形式的MAP激酶，相对分子质量约为42 kDa和44 kDa。两种形式的MAP激酶均被胆囊收缩素（CCK）激活。CCK的阈值浓度约为3 pM，最大效应出现在1 nM，在与底物髓鞘碱性蛋白共聚的聚丙烯酰胺凝胶中测定，其使MAP激酶活性增强2.5倍。CCK对MAP激酶的激活迅速，在5 - 10分钟内达到最大值，随后下降。蛙皮素和卡巴胆碱可激活MAP激酶，而促胰液素或血管活性肠肽则不能。CCK诱导的MAP激酶激活可能由蛋白激酶C介导，因为12 - O - 十四烷酰佛波醇13 - 乙酸酯（TPA）模拟了CCK的作用，而星形孢菌素浓度依赖性地抑制了CCK的作用。用毒胡萝卜素、离子霉素或乙二醇双（β - 氨基乙基醚）- N，N，N'，N' -四乙酸处理腺泡不影响MAP激酶，表明CCK动员细胞内钙在腺泡MAP激酶激活中并不重要。CCK和TPA增加了42 kDa和44 kDa两种形式的酪氨酸磷酸化。酪氨酸激酶抑制剂染料木黄酮和 tyrphostin 23抑制了CCK对MAP激酶的激活。总之，胰腺腺泡中的MAP激酶通过涉及蛋白激酶C和酪氨酸激酶的机制，被与磷酸肌醇水解相关的激动剂激活。
