Warhurst G, Turnberg L A, Higgs N B, Tonge A, Grundy J, Fogg K E
Epithelial Membrane Research Centre, University of Manchester, Hope Hospital, Salford, United Kingdom.
J Clin Invest. 1993 Aug;92(2):603-11. doi: 10.1172/JCI116627.
Using the functionally differentiated colonic cell line, HT29-19A, we have examined sites at which inhibitory G-proteins mediate the antisecretory actions of somatostatin (SST) and the alpha 2-adrenergic agonist, clonidine (CLON) at the epithelial level. Both agents caused a dose-dependent inhibition (EC50:SST 35 nM; CLON 225 nM) of Cl- secretion (assessed by changes in short circuit current) activated by cAMP-mediated agonists, PGE2 and cholera toxin. Inhibition was accompanied by a reduction in intracellular cAMP accumulation and could be blocked by pretreatment with pertussis toxin at a concentration (200 ng/ml) which activated ADP-ribosylation of a 41-kD inhibitory G protein in HT29-19A membranes. Secretion stimulated by the permeant cAMP analogue, dibutyryl cAMP, was also inhibited by SST and CLON (30-50%; P < 0.005), indicating additional inhibitory sites located distal to cAMP production. Both agents were effective inhibitors of secretion mediated through the Ca2+ signaling pathway. SST (1 microM) and CLON (10 microM) reduced the Isc response to the muscarinic agonist, carbachol, by 60-70%; inhibition was reversed in pertussis toxin-treated cells. These effects did not, however, involve inhibition of the carbachol-induced increase in cellular inositol 1,4,5-trisphosphate levels or the rise in cytosolic calcium, [Ca]i. Inhibition by SST of secretion induced by phorbol 12,13 dibutyrate but not by the calcium agonist, thapsigargin, suggests that SST may act at a distal inhibitory site in the Ca(2+)-dependent secretory process activated by protein kinase C. We conclude that SST and alpha 2-adrenergic agonists can act directly on intestinal epithelial cells to exert a comprehensive inhibition of Cl- secretion mediated through both cAMP and Ca2+/protein kinase C signaling pathways. Inhibition is mediated via pertussis toxin-sensitive G-proteins at sites located both proximal and distal to the production of second messengers.
利用功能分化的结肠细胞系HT29-19A，我们研究了抑制性G蛋白在上皮水平介导生长抑素（SST）和α2肾上腺素能激动剂可乐定（CLON）抗分泌作用的位点。两种药物均对由cAMP介导的激动剂、前列腺素E2（PGE2）和霍乱毒素激活的Cl-分泌（通过短路电流变化评估）产生剂量依赖性抑制（半数有效浓度：SST为35 nM；CLON为225 nM）。抑制作用伴随着细胞内cAMP积累的减少，并且可以被百日咳毒素（浓度为200 ng/ml）预处理阻断，该浓度可激活HT29-19A细胞膜中41-kD抑制性G蛋白的ADP核糖基化。渗透性cAMP类似物二丁酰cAMP刺激的分泌也受到SST和CLON的抑制（30%-50%；P<0.005），表明在cAMP产生的远端存在其他抑制位点。两种药物都是通过Ca2+信号通路介导的分泌的有效抑制剂。SST（1 μM）和CLON（10 μM）使对毒蕈碱激动剂卡巴胆碱的短路电流反应降低60%-70%；在百日咳毒素处理的细胞中抑制作用被逆转。然而，这些作用并不涉及抑制卡巴胆碱诱导的细胞内肌醇1,4,5-三磷酸水平的升高或胞质钙[Ca]i的升高。SST抑制佛波醇12,13-二丁酸酯诱导的分泌，但不抑制钙激动剂毒胡萝卜素诱导的分泌，这表明SST可能作用于蛋白激酶C激活的Ca(2+)依赖性分泌过程中的远端抑制位点。我们得出结论，SST和α2肾上腺素能激动剂可直接作用于肠上皮细胞，对通过cAMP和Ca2+/蛋白激酶C信号通路介导的Cl-分泌产生全面抑制。抑制作用是通过百日咳毒素敏感的G蛋白在第二信使产生的近端和远端位点介导的。
