Li Chenwei, Chen Xuequn, Williams John A
Dept. of Molecular and Integrative Physiology, Univ. of Michigan, 7744 Medical Science II, Ann Arbor, MI 48109-0622, USA.
Am J Physiol Gastrointest Liver Physiol. 2004 Oct;287(4):G764-71. doi: 10.1152/ajpgi.00111.2004. Epub 2004 Jun 24.
PKC is known to be activated by pancreatic secretagogues such as CCK and carbachol and to participate along with calcium in amylase release. Four PKC isoforms, alpha, delta, epsilon, and zeta, have been identified in acinar cells, but which isoforms participate in amylase release are unknown. To identify the responsible isoforms, we used translocation assays, chemical inhibitors, and overexpression of individual isoforms and their dominant-negative variants by means of adenoviral vectors. CCK stimulation caused translocation of PKC-alpha, -delta, and -epsilon, but not -zeta from soluble to membrane fraction. CCK-induced amylase release was inhibited approximately 30% by GF109203X, a broad spectrum PKC inhibitor, and by rottlerin, a PKC-delta inhibitor, but not by Gö6976, a PKC-alpha inhibitor, at concentrations from 1 to 5 microM. Neither overexpression of wild-type or dominant-negative PKC-alpha affected CCK-induced amylase release. Overexpression of PKC-delta and -epsilon enhanced amylase release, whereas only dominant-negative PKC-delta inhibited amylase release by 25%. PKC-delta overexpression increased amylase release at all concentrations of CCK, but dominant-negative PKC-delta only inhibited the maximal concentration; both similarly affected carbachol and JMV-180-induced amylase release. Overexpression of both PKC-delta and its dominant-negative variant affected the late but not the early phase of amylase release. GF109203X totally blocked the enhancement of amylase release by PKC-delta but had no further effect in the presence of dominant-negative PKC-delta. These results indicate that PKC-delta is the PKC isoform involved with amylase secretion.
已知蛋白激酶C（PKC）可被诸如胆囊收缩素（CCK）和卡巴胆碱等胰腺促分泌素激活，并与钙共同参与淀粉酶的释放。在腺泡细胞中已鉴定出四种PKC亚型，即α、δ、ε和ζ，但尚不清楚哪些亚型参与淀粉酶的释放。为了确定相关亚型，我们采用了转位分析、化学抑制剂以及通过腺病毒载体对单个亚型及其显性负变体进行过表达等方法。CCK刺激导致PKC-α、-δ和-ε从可溶性部分转位至膜部分，而PKC-ζ则未发生转位。在1至5微摩尔浓度下，广谱PKC抑制剂GF109203X和PKC-δ抑制剂rottlerin可使CCK诱导的淀粉酶释放受到约30%的抑制，而PKC-α抑制剂Gö6976则无此作用。野生型或显性负性PKC-α的过表达均不影响CCK诱导的淀粉酶释放。PKC-δ和-ε的过表达增强了淀粉酶的释放，而只有显性负性PKC-δ可使淀粉酶释放受到25%的抑制。PKC-δ的过表达在所有CCK浓度下均增加了淀粉酶的释放，但显性负性PKC-δ仅抑制最大浓度时的释放；二者对卡巴胆碱和JMV-180诱导的淀粉酶释放的影响相似。PKC-δ及其显性负变体的过表达均影响淀粉酶释放的后期阶段，但不影响早期阶段。GF109203X完全阻断了PKC-δ对淀粉酶释放的增强作用，但在存在显性负性PKC-δ时没有进一步影响。这些结果表明，PKC-δ是参与淀粉酶分泌的PKC亚型。
