Parietal cell MAP kinases: multiple activation pathways.
Parietal cell MAP kinases: multiple activation pathways.
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壁细胞 MAP 激酶：多种激活途径。
DOI：
10.1152/ajpgi.1996.271.4.g640
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
C. Chew
中科院分区：
文献类型：
--
作者：
K. Nakamura;C. Zhou;J. Parente;C. Chew
表皮生长因子（EGF）是一种有效的有丝分裂原为许多细胞类型;然而，最为人所知的影响EGF对胃壁细胞盐酸分泌是抑制这种反应。使用原代培养的兔壁细胞，我们最近发现，EGF的效果是双相的急性抑制，然后持续增强酸分泌相关的反应。我们假设EGF可能激活壁细胞中的丝裂原活化蛋白（MAP）激酶信号通路，并且该通路可能在介导EGF对壁细胞酸分泌相关功能的持续和/或急性效应中起作用[C. S.乔，K。中村和A. C.嗜石油的Am. J. Physiol.267（Gastrointest. 30）：G818-G826，1994]。我们使用了几种方法来证明存在的MAP激酶（MAPK）亚型，细胞外信号调节激酶（ERK）1和2，在壁细胞和开始，以表征其在这种高度分化的细胞类型的激活机制。在急性分离的90-98%富集的壁细胞中，EGF双相激活ERK-1和ERK-2，峰值反应发生在约5分钟，随后持续较低水平的激活至少2小时。EGF的EC 50（1.2 +/- 0.4 nM）与先前测定的EGF对酸分泌反应的刺激作用的EC 50相似。与EGF相反，佛波酯蛋白激酶C激活剂12-O-十四烷酰基佛波醇13-乙酸酯（TPA）诱导ERK-1和ERK-2的持续激活至少2 h。卡巴胆碱也激活ERK-1和ERK-2;然而，这种反应较弱且为单相。无论是Ca 2+离子载体离子霉素也不腺苷酸环化酶激活剂毛喉素改变基础或刺激ERK活性。卡巴胆碱，而不是EGF或TPA，也激活了一个身份不明的70 kDa的蛋白激酶检测与凝胶内髓鞘碱性蛋白（MBP）激酶复性试验。壁细胞MAPK活化与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳凝胶上表观相对分子质量的变化无关，表明ERK亚型的基础磷酸化可能高于壁细胞中活跃增殖的细胞系。此外，与中性粒细胞中的观察结果相反，磷脂酰肌醇3-激酶（PtdIns 3-激酶）抑制剂渥曼青霉素（0.3-3 μ M）未能抑制响应EGF、卡巴胆碱或TPA的ERK激活。组合的数据表明：1）EGF、TPA和卡巴胆碱激活胃壁细胞中重叠的以及不同的细胞内信号传导途径，2）EGF激活ERK并通过具有相似亲和力的受体增强壁细胞酸分泌相关功能，以及3）与某些细胞类型相反，壁细胞ERK信号级联似乎不受PtdIns 3-激酶途径或升高的细胞内游离Ca 2+或腺苷3 ′，5 ′-环一磷酸浓度的直接调节。
Epidermal growth factor (EGF) is a potent mitogen for many cell types; however, the best known effect of EGF on gastric parietal cell HCl secretion is inhibition of this response. Using rabbit parietal cells in primary culture, we recently showed that the effect of EGF is biphasic with acute inhibition followed by sustained enhancement of acid secretory-related responses. We hypothesized that EGF might activate a mitogen-activated protein (MAP) kinase signaling pathway in parietal cells, and this pathway might play a role in mediating sustained and/or acute effects of EGF on parietal cell acid secretory-related functions [C. S. Chew, K. Nakamura, and A. C. Petropolous. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G818-G826, 1994]. We used several methodological approaches to demonstrate the presence of MAP kinase (MAPK) isoforms, extracellular signal-regulated kinases (ERKs) 1 and 2, in parietal cells and to begin to characterize their mechanisms of activation in this highly differentiated cell type. In acutely isolated, 90-98% enriched parietal cells, EGF biphasically activated ERK-1 and ERK-2, with peak response occurring at approximately 5 min followed by a sustained lower level of activation for at least 2 h. The EC50 for EGF (1.2 +/- 0.4 nM) was similar to the previously determined EC50 for the stimulatory effect of EGF on acid secretory responses. In contrast to EGF, the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induced a sustained activation of ERK-1 and ERK-2 for at least 2 h. Carbachol also activated ERK-1 and ERK-2; however, this response was weaker and monophasic. Neither the Ca2+ ionophore ionomycin nor the adenylyl cyclase activator forskolin altered basal or stimulated ERK activity. Carbachol, but not EGF or TPA, also activated an unidentified 70-kDa protein kinase as detected with in-gel myelin basic protein (MBP) kinase renaturation assays. Parietal cell MAPK activation was not correlated to a shift in apparent relative molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggesting that basal phosphorylation of ERK isoforms may be higher in parietal cells compared with actively proliferating cell lines. Also, in contrast to observations in neutrophils, the phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor, wortmannin (0.3-3 microM), failed to inhibit ERK activation in response to EGF, carbachol, or TPA. The combined data indicate that 1) EGF, TPA, and carbachol activate overlapping as well as distinct intracellular signaling pathways in gastric parietal cells, 2) EGF activates ERKs and enhances parietal cell acid secretory related functions via receptors with similar affinities, and 3) in contrast to some cell types, the parietal cell ERK-signaling cascade does not appear to be directly modulated by the PtdIns 3-kinase pathway or by elevated intracellular free Ca2+ or adenosine 3',5'-cyclic monophosphate concentrations.