DIGLYCERIDES AS REGULATORS OF ION TRANSPORT

CF的基本缺陷表现为Cl渗透性异常， 气道上皮 这是由于cAMP的减少或缺失 顶端Cl通道的调节。 缓解的治疗方法 这种异常改变了顶端离子通道的活性， 实现更正常的Cl分泌。 然而，基底外侧的激活 NaC 1（K）协同转运蛋白，为分泌提供Cl所必需 可能是一种重要的连续性治疗干预。 我们的研究 显示，与顶端Cl通道相反，共转运受到调节， 通过涉及百日咳毒素敏感的α-肾上腺素能机制， 活化PtdIns-4，5-P2-敏感性磷脂酶C以产生 细胞内第二信使Ca和甘油二酯。 这一发现加上 蛋白激酶C（PKC）激活剂刺激 协同转运蛋白和PKC抑制剂阻断激素α-肾上腺素能 刺激作为我们假设PKC是一个关键的基础， 通过特异性磷酸化的共转运活性的调节剂 传送器 我们建议，在这个项目中，更详细地审查， PKC的作用：1）鉴定PKC同种型及其活性， 未刺激和刺激的气道上皮细胞（AEC），2）确定 PKC亚型的脂质辅因子特异性，3）研究 通过内源性脂质辅因子激活协同转运蛋白，和4） 确定重组协同转运蛋白的磷酸化位点 由协同转运蛋白cDNA产生。 从这项研究中获得的信息 将指出可能的治疗方法来操纵供应 通过改变PKC和共转运来分泌细胞内Cl 活动 此外，PKC亚型的具体知识， 它们在AEC中的激活剂可能在项目#4中有助于PKC 引发CFTR突变形式的PKA活化。

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.