Pollo D A, Baldassare J J, Honda T, Henderson P A, Talkad V D, Gardner J D
Department of Internal Medicine, Saint Louis University Health Sciences Center, MO 63104.
Biochim Biophys Acta. 1994 Oct 20;1224(1):127-38. doi: 10.1016/0167-4889(94)90120-1.
We used rat pancreatic acini and measured the effects of various agents on digestive enzyme secretion, diacylglycerol (DAG) and the cellular distribution of protein kinase C (PKC) enzyme activity as well as isoforms of PKC determined by quantitative immunoblot analysis. TPA, but not CCK-8, caused translocation of PKC enzyme activity from the cytosol fraction to the membrane fraction. Immunoblot analysis detected PKC-alpha, PKC-delta, PKC-epsilon and PKC-zeta. PKC-beta, PKC-gamma and PKC-eta were not detected. TPA caused translocation of all isoforms from cytosol to membrane, whereas CCK-8 caused translocation of PKC-delta and PKC-epsilon, carbachol caused translocation of PKC-epsilon, and bombesin and secretin caused no detectable translocation of any isoform. Specific receptor antagonists could prevent, as well as reverse completely, the translocation of PKC isoforms caused by CCK-8 or carbachol. Agonists added in sequence with an interposed addition of a specific receptor antagonist caused cycling of PKC-epsilon between cytosol and membrane fractions. Each receptor-mediated agonist that caused translocation of PKC also increased DAG, and with CCK-8 and carbachol cycling of PKC-epsilon between cytosol and membrane was accompanied by corresponding cyclic changes in cellular DAG. CCK-JMV-180, bombesin and secretin increased DAG but did not cause translocation of any PKC isoform. Translocation of a PKC isoform could be accounted for by whether the increased DAG originated from PIP2 (accompanied by translocation) or from phosphatidylcholine (no accompanying translocation). Thus it appeared that DAG, in pancreatic acini, is functionally compartmentalized depending on the source of the lipid. Studies using CCK-8 and CCK-JMV-180 indicated that occupation of the low affinity state of the CCK receptor by either peptide increased DAG from phosphatidylcholine, whereas occupation of the very low affinity state by CCK-8 increased DAG from PIP2 and caused translocation of PKC-delta and PKC-epsilon. TPA stimulated amylase secretion, indicating that activation of PKC can stimulate enzyme secretion; however, with the various receptor-mediated secretagogues there was no consistent, unequivocal correlation between translocation of an isoform of PKC and accompanying changes in enzyme secretion.
我们使用大鼠胰腺腺泡，测量了各种试剂对消化酶分泌、二酰基甘油（DAG）以及蛋白激酶C（PKC）酶活性的细胞分布的影响，以及通过定量免疫印迹分析确定的PKC亚型。佛波酯（TPA）可导致PKC酶活性从胞质组分转位至膜组分，而八肽胆囊收缩素（CCK-8）则不能。免疫印迹分析检测到了PKC-α、PKC-δ、PKC-ε和PKC-ζ，未检测到PKC-β、PKC-γ和PKC-η。TPA可导致所有亚型从胞质转位至膜，而CCK-8可导致PKC-δ和PKC-ε转位，卡巴胆碱可导致PKC-ε转位，蛙皮素和促胰液素未导致任何亚型的可检测到的转位。特异性受体拮抗剂可预防并完全逆转由CCK-8或卡巴胆碱引起的PKC亚型转位。依次添加激动剂并插入特异性受体拮抗剂可导致PKC-ε在胞质和膜组分之间循环。每种引起PKC转位的受体介导的激动剂也会增加DAG，并且CCK-8和卡巴胆碱引起的PKC-ε在胞质和膜之间的循环伴随着细胞DAG的相应周期性变化。CCK-JMV-180、蛙皮素和促胰液素增加了DAG，但未导致任何PKC亚型转位。PKC亚型的转位可由增加的DAG是源自磷脂酰肌醇-4,5-二磷酸（PIP2）（伴随转位）还是源自磷脂酰胆碱（无伴随转位）来解释。因此，在胰腺腺泡中，DAG在功能上似乎根据脂质来源进行了区室化。使用CCK-8和CCK-JMV-180的研究表明，任一肽占据CCK受体的低亲和力状态都会增加源自磷脂酰胆碱的DAG，而CCK-8占据极低亲和力状态会增加源自PIP2的DAG并导致PKC-δ和PKC-ε转位。TPA刺激淀粉酶分泌，表明PKC的激活可刺激酶分泌；然而，对于各种受体介导的促分泌剂，PKC亚型的转位与伴随的酶分泌变化之间没有一致、明确的相关性。
