Rogers K V, Goldman P S, Frizzell R A, McKnight G S
Department of Pharmacology, University of Washington, Seattle 98195.
Proc Natl Acad Sci U S A. 1990 Nov;87(22):8975-9. doi: 10.1073/pnas.87.22.8975.
Cl- channels in the apical membranes of salt-secreting epithelia are activated by both cAMP and Ca2+ second-messenger systems, and dysfunctions in their hormonal regulation have been demonstrated in patients with cystic fibrosis. We have transfected the epithelial cell line T84 with an expression vector containing a mutant form of the regulatory subunit of the cAMP-dependent protein kinase. Stable transformants that express this construct have reduced basal cAMP-dependent protein kinase activity and do not increase kinase activity beyond the basal level of control cells in response to cAMP. Forskolin, vasoactive intestinal peptide, and prostaglandin E2 each stimulate intracellular cAMP accumulation in both mutant and control clones; however, the activation of Cl- channels in response to elevated cAMP is blocked in mutant clones, indicating direct involvement of the cAMP-dependent protein kinase. In contrast, Ca2+ ionophores retain their ability to activate the Cl- channel in T84 cells expressing the mutant regulatory subunit, suggesting that activation of the channel by means of Ca2+ does not require the participation of cAMP-dependent protein kinase activity. These clones will be useful for further studies of the interactions between the cAMP- and Ca2(+)-dependent regulatory pathways in salt-secreting epithelial cells. They can also be used to identify the mediators of Ca2(+)-dependent Cl- channel activation in isolation from interactions with the cAMP second-messenger pathway.
分泌盐分的上皮细胞顶端膜中的氯离子通道可被环磷酸腺苷（cAMP）和钙离子（Ca2+）这两种第二信使系统激活，囊性纤维化患者体内已证实其激素调节存在功能障碍。我们用含有环磷酸腺苷依赖性蛋白激酶调节亚基突变形式的表达载体转染了上皮细胞系T84。表达此构建体的稳定转化体的基础环磷酸腺苷依赖性蛋白激酶活性降低，且在响应环磷酸腺苷时，激酶活性不会超过对照细胞的基础水平。福斯可林、血管活性肠肽和前列腺素E2均可刺激突变体和对照克隆细胞内的环磷酸腺苷积累；然而，在突变体克隆中，响应环磷酸腺苷升高而激活的氯离子通道被阻断，这表明环磷酸腺苷依赖性蛋白激酶直接参与其中。相比之下，钙离子载体在表达突变调节亚基的T84细胞中仍保留激活氯离子通道的能力，这表明通过钙离子激活通道不需要环磷酸腺苷依赖性蛋白激酶活性的参与。这些克隆将有助于进一步研究分泌盐分的上皮细胞中环磷酸腺苷和钙离子依赖性调节途径之间的相互作用。它们还可用于在与环磷酸腺苷第二信使途径相互作用分离的情况下，鉴定钙离子依赖性氯离子通道激活的介质。
