Evidence for the presence of cGMP-dependent protein kinase-II in human distal colon and in T84, the colonic cell line.
Evidence for the presence of cGMP-dependent protein kinase-II in human distal colon and in T84, the colonic cell line.
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DOI：
10.1016/s0167-4889(00)00075-6
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发表时间：
2000-10
期刊：
影响因子：
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通讯作者：
N. Selvaraj;R. Prasad;J. Goldstein;M. Rao
中科院分区：
文献类型：
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作者：
N. Selvaraj;R. Prasad;J. Goldstein;M. Rao
热稳定肠毒素（STa）通过激活鸟苷酸环化酶C（GCC）来刺激肠道Cl−分泌，从而增加细胞内的环GMP（cGMP）。在结肠中，cGMP的作用可能涉及蛋白激酶（PK）G-II或PKA途径，这取决于片段和物种。在人类结肠中，PKG和PKA途径都有牵连，因此，本研究在人类远端结肠细胞和结肠细胞系T84的原代培养物中研究了cGMP介导的Cl−转运机制。通过RT-PCR检测，两种细胞制备物均表达CFTR、Na+-K+-2Cl-β协同转运蛋白（NKCC 1）、GCC和PKG-II的mRNA。使用卤化物敏感探针测量了STa和PKG特异性cGMP类似物8Br-cGMP和8 pCPT-cGMP对Cl−转运的影响。在原代人类结肠细胞和T84细胞中，STa、cGMP类似物和cAMP依赖性促分泌素前列腺素E1（PGE 1）增强了Cl−转运。8Br-cGMP和8 pCPT-cGMP的作用表明PKG参与，并在T84细胞中进一步探索。8 pCPT-cGMP的作用呈剂量依赖性，对PKG抑制剂H8（70 μM）敏感，但H8对PGE 1诱导的Cl−分泌无影响。相比之下，PKA抑制剂H7（50 μM）阻断PGE 1介导的Cl −转运，但不阻断8 pCPT-cGMP诱导的Cl−转运。8 pCPT-cGMP在体外增强T84膜对PKG特异性底物2A 3的磷酸化。这种磷酸化被H8抑制。这些结果强烈表明，cGMP通过PKG-II途径激活了原代细胞和人结肠T84细胞系中的Cl−转运。
Heat-stable enterotoxin (STa) stimulates intestinal Cl−secretion by activating guanylate cyclase C (GCC) to increase intracellular cyclic GMP (cGMP). In the colon, cGMP action could involve protein kinase (PK) G-II or PKA pathways, depending on the segment and species. In the human colon, both PKG and PKA pathways have been implicated, and, therefore, the present study examined the mechanism of cGMP-mediated Cl−transport in primary cultures of human distal colonocytes and in T84, the colonic cell line. Both cell preparations express mRNA for CFTR, Na+-K+-2Cl−cotransporter (NKCC1), GCC and PKG-II as detected by RT-PCR. The effects of STa and the PKG-specific cGMP analogues, 8Br-cGMP and 8pCPT-cGMP, on Cl−transport were measured using a halide-sensitive probe. In primary human colonocytes and T84 cells, STa, the cGMP analogues and the cAMP-dependent secretagogue, prostaglandin E1(PGE1), enhanced Cl−transport. The effects of 8Br-cGMP and 8pCPT-cGMP suggested the involvement of PKG, and this was explored further in T84 cells. The effects of 8pCPT-cGMP were dose-dependent and sensitive to the PKG inhibitor, H8 (70 μM), but H8 had no effect on PGE1-induced Cl−secretion. In contrast, a PKA inhibitor, H7 (50 μM), blocked PGE1-mediated but not 8pCPT-cGMP-induced Cl−transport. 8pCPT-cGMP enhanced phosphorylation of the PKG-specific substrate, 2A3, by T84 membranes in vitro. This phosphorylation was inhibited by H8. These results strongly suggest that cGMP activates Cl−transport through a PKG-II pathway in primary cells and in the T84 cell line of the human colon.