DIGLYCERIDES AS REGULATORS OF ION TRANSPORT

Cf的基本缺陷表现为氯离子渗透性异常。 呼吸道上皮细胞。这现在归因于营地的减少或缺席。 心尖氯通道的调节。缓解疾病的治疗方法 这种异常是为了改变心尖离子通道的活动 实现更多正常的氯离子分泌。然而，基底外侧的激活 NaC_1(K)共转运体，为分泌提供氯是必不可少的 可能是一种重要的辅助治疗干预措施。我们的研究 结果表明，与心尖氯通道相比，共转运是受调控的 通过涉及百日咳毒素敏感的α-肾上腺素能机制 激活PtdIns-4，5-P2敏感的磷脂酶C以产生 细胞内第二信使钙和二甘油酯。这一发现加上 观察到蛋白激酶C(PKC)激活剂刺激 辅转运体和蛋白激酶C抑制剂阻断激素α-肾上腺素能 刺激是我们假设PKC是一个关键的 通过特异性磷酸化调节共转运蛋白的活性 传送器。我们建议，在这个项目中，更详细地研究 PKC的作用：1)鉴定PKC亚型及其活性 未刺激和刺激的呼吸道上皮细胞(AEC)，2)测定 蛋白激酶C亚型的脂质辅因子特异性，3)调查 内源性脂类辅助因子激活共转运蛋白，以及4) 重组共转运蛋白磷酸化位点的测定 由共转运蛋白基因产生。从这项研究中获得的信息 会指出可能的治疗方法来操纵供应 通过改变PKC和共转运来调节细胞内氯的分泌 活动。此外，对PKC和PKC亚型的专门知识 他们在AEC中的激活剂在项目#4中可能有用，以协助PKC 启动突变形式的CFTR的PKA激活。

The basic defect in CF manifests itself as abnormal Cl permeability in airway epithelium. This is now attributed to diminished or absent cAMP regulation of apical Cl channels. Therapeutic approaches to alleviate this abnormality are geared to altering apical ion channel activities to achieve more normal Cl secretion. However, activation of basolateral NaC1(K) cotransporter which is essential for supplying Cl for secretion may be an important adjunctive therapeutic intervention. Our studies show that, in contrast to apical Cl channels, cotransport is regulated by an alpha-adrenergic mechanism involving pertussis toxin-sensitive activation of PtdIns-4,5-P2-sensitive phospholipase C to generate the intracellular second messengers Ca and diglycerides. This finding plus the observation that a protein kinase C (PKC) activator stimulates the cotransporter and a PKC inhibitor blocks hormone alpha-adrenergic stimulation serves as the basis for our hypothesis that PKC is a critical regulator of cotransport activity through specific phosphorylation of the transporter. We propose, in this project, to examine, in greater detail the role of PKC by 1) identifying PKC isotypes and their activities in unstimulated and stimulated airway epithelial cells (AEC), 2) determining the lipid cofactor specificity of PKC isotypes, 3) investigating cotransporter activation by endogenous lipid cofactors, and 4) determining sites of phosphorylation of a recombinant cotransporter generated from cotransporter cDNA. Information gained from this research will point to possible therapeutic approaches to manipulate the supply of intracellular Cl for secretion by altering PKC and cotransport activities. In addition, specific knowledge of the isotypes of PKC and their activators in AEC may be useful in project #4 to assist in PKC priming of PKA activation of mutant forms of CFTR.