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The Journal of Membrane Biology

, Volume 93, Issue 2, pp 133–139 | Cite as

Protein kinase C activates the renal apical membrane Na+/H+ exchanger

  • E. J. Weinman
  • S. Shenolikar
Articles

Summary

Studies were performed on purified brush-border membranes from the kidney of the rabbit to examine the relation between protein kinase C and the Na+/H+ exchanger in these membranes. The brush-border membranes were transiently opened by exposure to hypotonic media and the membrane proteins phosphorylated by exposure to ATP and phorbol esters or partially purified protein kinase C. The membranes were resealed and the intravesicular space acidified by incubation in a sodium-free isotonic solution (pH 5.5). The rate of uptake of 1mm22Na+ (pH 7.5), with and without amiloride (1mm), was assayed and the proton gradient-stimulated, amiloride-inhibitable component of22Na+ taken as a measure of the activity of the Na+/H+ exchanger. 12-0-tetradecanoyl phorbol-13-acetate (TPA) increased the amiloride-sensitive component of22Na+ uptake TPA did not affect the amiloride-insensitive component of22Na+ uptake or the equilibrium concentration of sodium. TPA also did not affect the rate of dissipation of the proton gradient in the absence of sodium or the rate of sodium-dependent or-independent uptake ofd-glucose. Other “active” phorbol esters stimulated the rate of Na+/H+ exchange, but phorbol esters of the 4 α configuration did not. Incubation of the opened membranes in partially purified protein kinase C increased the rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake. The stimulatory effect of TPA and protein kinase C was not additive. In the absence of ATP, neither TPA nor protein kinase C affected Na+/H+ exchange transport. To determine the membrane-bound protein substrates, parallel experiments were conducted with γ-[32P] ATP in the phosphorylating solutions. The reaction was stopped by SDS and the phosphoproteins resolved by PAGE and autoradiography. TPA stimulation of protein kinase C resulted in phosphorylation of approximately 13 membrane-bound proteins ranging in apparent molecule from 15,000 to 140,000 daltons. These studies indicate that activation of endogenous renal brush-border protein kinase C by phorbol esters or exposure of these membranes to exogenous protein kinase C increases the rate of proton gradient-stimulated, amiloride-inhibitable sodium transport. Protein kinase C activation also results in phosphorylation of a finite number of membrane-bound proteins.

Key Words

protein phosphorylation acid/base regulation protein kinase C cAMP-dependent protein kinase sodium transport renal proximal tubule 

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Copyright information

© Springer-Verlag New York Inc 1986

Authors and Affiliations

  • E. J. Weinman
    • 1
  • S. Shenolikar
    • 1
  1. 1.Department of Internal MedicineThe University of Texas Medical SchoolHouston

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