DIGLYCERIDES AS REGULATORS OF ION TRANSPORT

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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.