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

胞浆钙（Cai）增加和蛋白激酶C活化 (PKC)似乎在调节肠钠吸收方面具有主要作用。 我们以前的研究表明，神经体液刺激 Cai的增加可以通过至少3种不同的机制发生：（1） 刺激磷脂酰肌醇（PI）代谢，（2）增加血浆 膜钙渗透性，和（3）刺激内源性钙释放 环核苷酸 此外，他们还证明， 用佛波醇酯激活PKC可显著抑制 通过阻断刷状缘膜（BBM）Na/H交换的Na吸收。 我们现在要更详细地讨论其中许多问题。 我们 希望定义生理激活后的细胞事件， 鸡离体肠上皮细胞PI代谢的研究。 我们会尽力解释 PKC和CaI增加在Na/H调节中的相对作用 交易所 这些研究将涉及相关的测量蔡和 使用荧光指示剂的完整细胞中的pH， BBM囊泡、PKC转位测定和BBM的生化研究 生理相关的PKC和CaI依赖性磷蛋白。 到 为了进一步解决这些问题，还将在隔离的 肠细胞和自发分化的Caco-2结肠细胞中， Cai的变化被缓冲或PKC被下调。 下一个是 我们将确定为什么从细胞内刺激的PKC易位， 细胞质到膜部分是短暂的，这是否可能 涉及随后形成可溶性但可活化的PKC 我们最近发现的蛋白水解片段 循环经济的机制 核苷酸刺激的Cai增加和质膜Ca 将进一步研究刺激PI水解后的渗透性 广泛存在于微粒体制剂和基底外侧膜中 囊泡 最后，我们将研究2个以前未探索的领域， 肠上皮细胞离子转运，即，的分布和相关作用 绒毛-腺窝轴蛋白激酶C同工酶的沿着分布及蛋白激酶C同工酶的鉴定 BBM Na/H交换器。 这些研究将有助于确定生理机制， 肠肽和神经递质调节肠盐， 水上运输。 这一信息可以扩大应用范围， 牙周病的病理生理学基础和制剂 治疗它们的策略。

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.