C-KINASE AND CA+2 REGULATION OF INTESTINAL ION TRANSPORT

细胞内钙(CaI)升高和蛋白激酶C的激活 (PKC)似乎在调节肠道钠吸收方面起着重要作用。 我们之前的研究表明，神经体液刺激 CAI的增加至少可以通过三种不同的机制发生：(1) 刺激磷脂酰肌醇(PI)代谢，(2)血浆升高 膜钙通透性，以及(3)刺激内源性钙释放。 环核苷酸。此外，他们还证明了药理作用 佛波醇酯对PKC的激活可显著抑制 通过阻断刷状缘膜(BBM)的Na/H交换来吸收Na。 我们现在想更详细地处理其中许多问题。我们 希望定义生理激活后的细胞事件 鸡离体肠上皮细胞的PI代谢。我们将努力澄清 PKC和升高的CaI在Na/H调节中的相对作用 交换。这些研究将涉及CAI和CAI的相关测量 用荧光指示剂研究完整细胞的pH，钠的转运 BBM囊泡、PKC易位测定及生化研究 与生理相关的依赖于PKC和CaI的磷酸蛋白。至 为了进一步解决这些问题，还将单独进行研究 肠上皮细胞和自发分化的Caco-2结肠细胞 CAI中的变化被缓冲，或者PKC已经被下调。下一首, 我们将确定为什么荷尔蒙刺激的PKC易位 胞浆到膜部分是瞬变的，这是否可能 包括随后形成可溶的但可激活的PKC 我们最近鉴定的蛋白水解性片段。循环的作用机制 核苷酸刺激的细胞内钙离子浓度升高和质膜钙离子增多 受刺激的PI水解后的渗透性将得到更多的研究 广泛存在于微粒体制剂和基侧膜中 水泡。最后，我们将调查两个以前没有勘探过的区域 肠细胞离子转运，即肠上皮细胞的分布和相关作用 绒毛-隐窝轴上的PKC同工酶及其鉴定 BBM钠氢交换器。 这些研究将有助于确定 肠肽和神经递质调节肠道盐分和 水路运输。这些信息可能会扩大应用范围，以了解 腹泻病的病理生理基础及其制剂 治疗他们的策略。

Increases in cytosolic calcium (Cai) and activation of protein kinase C (PKC) appear to have a major role in regulating intestinal Na absorption. Our previous investigations have shown that neurohumoral-stimulated increases in Cai can occur by at least 3 different mechanisms: (1) stimulation of phosphatidylinositol (PI) metabolism, (2) increased plasma membrane Ca permeability, and (3) stimulation of endogenous Ca release by cyclic nucleotides. Furthermore, they demonstrated that pharmacological activation of PKC with phorbol esters causes a significant inhibition of Na absorption by blocking brush-border membrane (BBM) Na/H exchange. We would now like to address many of these issues in greater detail. We wish to define the cellular events following physiological activation of PI metabolism in isolated chicken enterocytes. We will try to elucidate the relative roles PKC and increased Cai in the regulation of Na/H exchange. These studies will involve correlative measurements of Cai and pH in intact cells using fluorescent indicators, Na transport studies in BBM vesicles, PKC translocation determinations and biochemical studies of physiologically-relevant PKC- and Cai-dependent phosphoproteins. To further address these issues, studies will also be performed in isolated enterocytes and in spontaneously-differentiating Caco-2 colon cells where changes in Cai are buffered or where PKC has been downregulated. Next, we will determine why hormonally-stimulated PKC translocation from cytosol to the membrane fraction is transient and whether this may involve the subsequent formation of a soluble, but activatable PKC proteolytic fragment we recently identified. The mechanisms of cyclic nucleotide-stimulated increases in Cai and increased plasma membrane Ca permeability following stimulated PI hydrolysis will be studied more extensively in microsomal preparations and in basolateral membrane vesicles. Finally, we will investigate 2 previously unexplored areas of enterocyte ion transport, i.e., the distribution and relevant roles of PKC isoenzymes along the villus-crypt axis and the identification of the BBM Na/H exchanger. These studies will help define the physiological mechanisms by which intestinal peptides and neurotransmitters regulate intestinal salt and water transport. This information may widen application to understanding the pathophysiological basis of diarrheal diseases and to the formulation of strategies to treat them.