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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Specialized Center (P50)
Project #
5P50HL050160-02
Application #
3759147
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Liedtke, C M; Cody, D; Cole, T S (2001) Differential regulation of Cl- transport proteins by PKC in Calu-3 cells. Am J Physiol Lung Cell Mol Physiol 280:L739-47
Liedtke, C M; Cole, T S (2000) PKC signaling in CF/T43 cell line: regulation of NKCC1 by PKC-delta isotype. Biochim Biophys Acta 1495:24-33
Steagall, W K; Drumm, M L (1999) Stimulation of cystic fibrosis transmembrane conductance regulator-dependent short-circuit currents across DeltaF508 murine intestines. Gastroenterology 116:1379-88
Nanduri, J; Mitra, S; Andrei, C et al. (1999) An unexpected link between the secretory path and the organization of the nucleus. J Biol Chem 274:33785-9
Kelley, T J; Cotton, C U; Drumm, M L (1998) Regulation of amiloride-sensitive sodium absorption in murine airway epithelium by C-type natriuretic peptide. Am J Physiol 274:L990-6
Kelley, T J; Drumm, M L (1998) Inducible nitric oxide synthase expression is reduced in cystic fibrosis murine and human airway epithelial cells. J Clin Invest 102:1200-7
Steagall, W K; Kelley, T J; Marsick, R J et al. (1998) Type II protein kinase A regulates CFTR in airway, pancreatic, and intestinal cells. Am J Physiol 274:C819-26
Liedtke, C M; Cole, T S (1998) Antisense oligonucleotide to PKC-epsilon alters cAMP-dependent stimulation of CFTR in Calu-3 cells. Am J Physiol 275:C1357-64
Liedtke, C M; Cole, T (1997) Antisense oligodeoxynucleotide to PKC-delta blocks alpha 1-adrenergic activation of Na-K-2Cl cotransport. Am J Physiol 273:C1632-40
Kelley, T J; Thomas, K; Milgram, L J et al. (1997) In vivo activation of the cystic fibrosis transmembrane conductance regulator mutant deltaF508 in murine nasal epithelium. Proc Natl Acad Sci U S A 94:2604-8

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