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首页> 外文期刊>Journal of Cellular Physiology >Effects of mutations in cAMP-dependent protein kinase on chloride efflux in Caco-2 human colonic carcinoma cells.
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Effects of mutations in cAMP-dependent protein kinase on chloride efflux in Caco-2 human colonic carcinoma cells.

机译：cAMP-dependent突变蛋白的影响激酶在人类结肠Caco-2氯流出癌的细胞。

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摘要

In order to evaluate the importance of cAMP and cAMP-dependent protein kinase (cAMPdPK) in the regulation of chloride efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, Caco-2, human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of regulatory subunit of cAMPdPK under control of the mouse metallothionein 1 promoter. Four stable transformants were isolated that expressed the mutant subunit in a Zn(2+)-inducible manner and exhibited Zn(2+)-inducible inhibition of cAMPdPK activity. The parental and transformed Caco-2 cells were examined for their abilities to regulate chloride efflux in response to various secretagogues using a radioactive iodide-efflux assay. In the transformants, induction of the protein kinase mutation with ZnSO4 markedly decreased chloride efflux in response to forskolin, the 8-(4-chlorophenylthio) analog of cAMP, vasoactive intestinal polypeptide, prostaglandin E2 and isoproterenol, whereas Zn(2+)-treated parental cells remained responsive to these secretagogues. Treatment with carbachol, calcium ionophores or phorbol ester did not acutely affect chloride efflux. Together, these studies indicate that cAMP and cAMPdPK are essential components of secretagogue-regulated chloride channel activity in the Caco-2 cell line. In whole cell patch clamp recordings, induction of the cAMPdPK mutation inhibited anionic conductances indicative of the CFTR chloride channel, whereas purified catalytic subunit of cAMPdPK, added intracellularly, reversed the inhibition. These latter results demonstrate that the CFTR chloride channels in the protein kinase-defective transformants are normal and that the protein kinase mutation specifically affects their regulation, presumably by direct phosphorylation.
机译：为了评估阵营和的重要性cAMP-dependent蛋白激酶(cAMPdPK)通过囊性调节氯流出囊性纤维化跨膜电导调节(雌性生殖道)氯通道、Caco-2人类结肠癌细胞转染的表达载体编码的一种变异形式调节亚基的cAMPdPK控制的鼠标金属硫蛋白1启动子。转化株是孤立的,表达了突变体亚基锌(2 +)诱导的方式表现出锌(2 +)诱导抑制cAMPdPK活动。细胞被检查的能力调节氯流出来响应不同使用放射性iodide-efflux促分泌素化验。蛋白激酶与ZnSO4明显突变减少氯射流在回应forskolin, 8 - (4-chlorophenylthio)模拟营地,——血管活性肠多肽,前列腺素E2和异丙肾上腺素,而锌(2 +)对待父母的细胞反应这些促分泌素。没有钙离子载体或佛波醇酯严重影响氯流出。研究表明,营地和cAMPdPKsecretagogue-regulated必不可少的组成部分氯通道Caco-2细胞的活动线。cAMPdPK突变抑制的感应阴离子电导雌性生殖道的象征氯通道,而净化催化cAMPdPK亚基,添加细胞,扭转了抑制。证明的雌性生殖道氯通道蛋白质kinase-defective转化株正常,蛋白激酶突变具体影响他们的监管,大概通过直接磷酸化。

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