STIMULATION OF INTESTINAL CL- TRANSPORT BY HEAT-STABLE ENTEROTOXIN - ACTIVATION OF CAMP-DEPENDENT PROTEIN-KINASE BY CGMP
STIMULATION OF INTESTINAL CL- TRANSPORT BY HEAT-STABLE ENTEROTOXIN - ACTIVATION OF CAMP-DEPENDENT PROTEIN-KINASE BY CGMP
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DOI：
10.1152/ajpcell.1992.263.3.c607
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发表时间：
1992-09-01
影响因子：
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通讯作者：
CORBIN, JD
中科院分区：
文献类型：
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作者：
FORTE, LR;THORNE, PK;CORBIN, JD
热稳定的肠毒素激活鸟苷酸环化酶，而热不稳定的肠毒素则刺激腺苷酸环化酶。这两类毒素至少部分是通过刺激肠道中的 Cl- 分泌而引起分泌性腹泻。使用培养的 T84 肠细胞作为肠隐窝细胞模型，研究了 3',5'-环单磷酸鸟苷 (cGMP) 调节 Cl- 分泌的机制。大肠杆菌热稳定肠毒素 (ST) 显着刺激 T84 细胞中 cGMP 的产生。大肠杆菌 ST、霍乱毒素或 8-BrcAMP 刺激培养在渗透性滤器上的 T84 细胞单层的 Cl- 分泌，但 8-BrcGMP 无效。众所周知，cGMP 类似物是 cGMP 依赖性蛋白激酶（cG 激酶）的有效和特异性激活剂，对塑料培养皿中培养的 T84 细胞对 Cl-36- 的吸收也几乎没有影响。大肠杆菌 ST、毛喉素、霍乱毒素或膜渗透 cAMP 类似物显着增加 T84 细胞对 Cl-36- 的摄取。一般蛋白激酶抑制剂星形孢菌素可抑制大肠杆菌 ST、血管活性肠肽 (VIP) 或 8-BrcAMP 引起的 Cl-通透性刺激。 DEAE-Sephacel 色谱法显示 T84 细胞中主要存在 cAMP 依赖性蛋白激酶（cA 激酶）的 II 型亚型，而胞质 cG 激酶活性很少或没有发现。如果在低温（冰上）下测定胞质酶，则用大肠杆菌 ST 或 VIP 处理 T84 细胞会导致 cA 激酶活性比 (-cAMP/+cAMP) 增加。总而言之，这些数据支持这样的假设：大肠杆菌ST对Cl-通道的刺激不是通过cG激酶激活而发生的，而是可能通过cGMP与T84细胞中的cA激酶的结合和激活而发生。因此，大肠杆菌 ST 引起肠内盐和液体分泌的一种可能机制可能是通过隐窝细胞中 cA 激酶的 cGMP 交叉激活而发生的。这种机制是否存在于肠道中是我们用模型上皮进行的实验提出的一个关键问题，并且作为肠道中 cGMP 的假定信号通路仍有待探索。
Heat-stable enterotoxins activate guanylate cyclase, whereas heat-labile enterotoxins stimulate adenylate cyclase. Both classes of toxins cause secretory diarrhea at least in part by stimulating Cl- secretion in the intestine. The mechanism for regulation of Cl- secretion by guanosine 3',5'-cyclic monophosphate (cGMP) was investigated using cultured T84 intestinal cells as a model for intestinal crypt cells. Escherichia coli heat-stable enterotoxin (ST) markedly stimulated cGMP production in T84 cells. Cl- secretion across T84 cell monolayers cultured on permeable filters was stimulated by E. coli ST, cholera toxin, or 8-BrcAMP, but 8-BrcGMP was ineffective. cGMP analogues that are known to be potent and specific activators of cGMP-dependent protein kinase (cG-kinase) also had little effect on Cl-36- uptake by T84 cells cultured in plastic dishes. E. coli ST, forskolin, cholera toxin, or membrane-permeant cAMP analogues markedly increased Cl-36- uptake into T84 cells. The general protein kinase inhibitor, staurosporine, inhibited the stimulation of Cl- permeability elicited by E. coli ST, vasoactive intestinal peptide (VIP), or 8-BrcAMP. DEAE-Sephacel chromatography revealed a predominant type II isoform of cAMP-dependent protein kinase (cA-kinase) in T84 cells, whereas little or no cytosolic cG-kinase activity was found. Treatment of T84 cells with E. coli ST or VIP resulted in an increase in the cA-kinase activity ratio (-cAMP/+cAMP) if the cytosolic enzyme was assayed at reduced temperature (on ice). Taken together, these data supported the hypothesis that the stimulation of Cl- channels by E. coli ST does not occur by cG-kinase activation but may occur by cGMP binding to and activation of cA-kinase in T84 cells. Thus one possible mechanism for the action of E. coli ST to elicit salt and fluid secretion in the intestine could occur by cGMP cross-activation of cA-kinase in the crypt cells. Whether such a mechanism exists in the intestine is a key question raised by our experiments with a model epithelium and remains to be explored as a putative signaling pathway for cGMP in the intestine.