Ca2+-independent protein kinase C isoforms may modulate parietal cell HCl secretion
Ca2+-independent protein kinase C isoforms may modulate parietal cell HCl secretion
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DOI：
10.1152/ajpgi.1997.272.2.g246
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发表时间：
1997-02-01
影响因子：
4.5
通讯作者：
Parente, JA
中科院分区：
文献类型：
--
作者：
Chew, CS;Zhou, CJ;Parente, JA
虽然组胺激活3‘，5’-环磷酸腺苷和胆碱能激动剂激活钙依赖的信号通路是增加壁细胞HCl分泌的公认机制，但蛋白激酶C(PKC)在这一过程中的作用仍存在争议。在这项研究中，兔胃腺的酸分泌反应[测量为氨基比林(AP)的积聚]被发现对PKC抑制剂Calphostin C、Chelerythine Cli、Stauroporine和双吲哚马来酰亚胺类抑制剂Ro31-8220、GO 6976和双吲哚马来酰亚胺I盐酸盐具有相对抗性。对高度浓缩的壁细胞(98%纯度)进行的蛋白激酶C同工酶图谱分析表明，该细胞类型表达丰富的新的PKC-epsilon和PKC-Mu亚型，以及大量的非典型亚型PKC-iota、PKC-lambda和PKC-Zeta。相反，经典异构体PKC-a和PKC-beta(1)/beta(2)的表达似乎很低或检测不到。相对较高浓度的Ro 31-8220可增强卡巴胆碱和组胺刺激的AP积聚(IC50857+/-100和910+/-98 nM)。Ro 31-8220抑制壁细胞磷酸蛋白pp66的原位磷酸化也有类似的剂量依赖关系(IC50 750+/-120 nM)。相似浓度的Ro31-8220也抑制细胞骨架、肌动蛋白膜交联型磷蛋白Ezrin的磷酸化，但不抑制其他磷蛋白的磷酸化。卡巴胆碱和12-O-十四酰佛波醇13-乙酸酯(TPA)可促进Ezrin的磷酸化。由于卡巴胆碱和TPA以不依赖于钙离子的方式刺激pp66的磷酸化，我们的结果表明，一个或多个新的PKC亚型可能参与了对HCl分泌的负调节。在相关实验中，共聚焦显微镜将PKC-epsilon而不是PKC-u免疫定位于一个壁细胞室，该细胞室与含有丝状肌动蛋白的细胞室有惊人的相似之处。此外，pp66富含Triton X-100不溶的壁细胞部分，这表明这个未知蛋白可能存在细胞骨架定位。鉴于它们的位置和对RO31-8220的敏感性，pp66和Ezrin可能以依赖PKC的方式相互作用，调节与激动剂依赖的壁细胞HCl分泌激活相一致的众所周知的形态变化。
Although activation of adenosine 3',5'-cyclic monophosphate by histamine and of Ca2+-dependent signaling pathways by cholinergic agonists is a generally recognized mechanism for increasing parietal cell HCl secretion, the role of protein kinase C (PKC) in this process is controversial. In this study, acid-secretory responses of gastric glands from rabbits [measured as accumulation of aminopyrine (AP)] were found to be relatively resistant to the PKC inhibitors calphostin C, chelerythrine chloride, staurosporine, and the bisindolylmaleimide-like inhibitors Ro 31-8220, Go 6976, and bisindolylmaleimide I hydrochloride. Western analyses of the PKC isozyme profile in highly enriched parietal cells (98% purity) indicated that this cell type expresses abundant levels of the novel isoforms PKC-epsilon and PKC-mu and abundant levels of the atypical isoforms PKC-iota, PKC-lambda, and PKC-zeta. In contrast, there appeared to be low to undetectable expression of the classical isoforms PKC-a and PKC-beta(1)/beta(2), respectively. Relatively high concentrations of Ro 31-8220 potentiated both carbachol- and histamine-stimulated AP accumulation (IC50 857 +/- 100 and 910 +/- 98 nM, respectively). There was a similar dose dependence for Ro 31-8220 inhibition of in situ phosphorylation of a parietal cell phosphoprotein, pp66 (IC50 750 +/- 120 nM). Similar concentrations of Ro 31-8220 also inhibited phosphorylation of the cytoskeletal, actin membrane cross-linking phosphoprotein ezrin, but not other phosphoproteins. Ezrin phosphorylation was increased by carbachol and 12-O-tetradecanoylphorbol 13-acetate (TPA). Because carbachol and TPA stimulate pp66 phosphorylation in a Ca2+-independent manner, our results suggest that one or more novel PKC isoforms may be involved in negative regulation of HCl secretion. In related experiments, PKC-epsilon, but not PKC-mu, was immunolocalized by confocal microscopy to a parietal cell compartment that bore a striking resemblance to that containing filamentous actin. Moreover, pp66 was enriched in a Triton X-100-insoluble parietal cell fraction, suggesting a potential cytoskeletal localization for this unknown protein. Given their location and sensitivity to Ro 31-8220, it is possible that pp66 and ezrin interact in a PKC-dependent manner to regulate the well-known morphological changes that occur in concert with agonist-dependent activation of parietal cell HCl secretion.