Cyclic-3',5'-AMP (cAMP) plays a major role in regulating sodium transport across frog skin and other tight epithelia. This second messenger is thought to stimulate transepithelial Na+ movement by activating a cAMP-dependent kinase, which phosphorylates sites gating the apical Na+ channels. An increasing number of observations from this laboratory have suggested that the ubiquitous enzyme protein kinase C (PKC) may also be an important regulator of sodium transport across frog skin. The hypothesis has been developed that: (i) PKC phosphorylates sites identical with, or near, the regulatory sites phosphorylated by cAMP kinase, and (ii) that the natriferic action of insulin at the apical membrane is mediated by activation of PKC. The present proposal is focussed on this hypothesis, and aims at: (i) more rigorously examining whether PKC activation increases apical Na+ permeability (papNa), (ii) determining whether the apical natriferic action of insulin is mediated by PKC, (iii) examining whether translocation of PKC from cytosol to apical membrane is necessary for PKC activation to increase papNa, and (iv) determining whether the intracellular alkalinization (produced by both PKC activation and insulin in other cells) is important in the expression of this natriferic effect. Apical Na+ permeability, transepithelial Na+ transport and intracellular pH will be monitored with intracellular micropipets and pH-selective microelectrodes and transepithelial voltage clamping of intact isolated-epithelial and whole-thickness preparations of frog skin. Parallel studies of PKC activity and translocation will be conducted, using both enzymatic assays and monoclonal and polyclonal antibodies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK040145-04
Application #
3240247
Study Section
Physiology Study Section (PHY)
Project Start
1988-04-01
Project End
1993-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Garty, H; Peterson-Yantorno, K; Asher, C et al. (1994) Effects of corticoid agonists and antagonists on apical Na+ permeability of toad urinary bladder. Am J Physiol 266:F108-16
Civan, M M; Robbins, J; Broad, S et al. (1993) Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS, of protein kinase C. J Membr Biol 133:51-9
Civan, M M; Sinnett-Smith, J; Bouzyk, M et al. (1993) Bombesin treatment enhances vasopressin receptors in Swiss 3T3 cells. Am J Physiol 265:C1658-62
Wu, M M; Civan, M M (1991) Voltage dependence of current through the Na,K-exchange pump of Rana oocytes. J Membr Biol 121:23-36
Civan, M M; Oler, A; Peterson-Yantorno, K et al. (1991) Ca(2+)-independent form of protein kinase C may regulate Na+ transport across frog skin. J Membr Biol 121:37-50
Tang, C S; Peterson-Yantorno, K; Civan, M M (1989) Coupling of volume and Na+ transport in frog skin epithelium. Biol Cell 66:183-90
Civan, M M; Peterson-Yantorno, K; George, K et al. (1989) Interactions of TPA and insulin on Na+ transport across frog skin. Am J Physiol 256:C569-78