The Na,K-pump (ie., Na,K-ATPase) is thought to be the major pharmacological receptor for cardiac glycosides such as digitalis and ouabain. It is a membrane-spanning protein complex that mediates the transmembrane exchange of Na+ and K+ at the expense of metabolic energy. Critical functions served by the pump include the generation and maintenance of the electrochemical gradients of Na+ and K+ across the plasma membrane, the absorption of Ca++, sugars and amino acids via cotransport systems, and the transport of salts and water across epithelia. The Na,K-pump is inhibited by cardiac glycosides, and within a given species, there is variation in ouabain affinity between tissues that may be as great as 500-fold. The basis for this variability is not well understood. There is also tissue variability in the expression of different isoforms of the component subunits of the Na,K-pump. This project will test the hypothesis that the basis of the variation in ouabain affinity is the cell-specific expression of different isoforms of the alpha and beta subunits. The subunit composition and the expressed ouabain affinity will be examined in a number of cultured cell lines by RNA hybridization analysis and enzymatic assays to correlate the presence of specific isoforms with a given binding affinity. DNA- mediated gene transfer will be used to introduce the various isoforms into recipient cell lines to observe directly their effects on ouabain affinity. Expression of the mRNAs that encode the native subunits will be monitored by hybridization analysis after transfer of a foreign isoform to determine its effects on the regulation of the native isoforms. From these studies, the molecular basis of the tissue variability in ouabain affinity will be elucidated.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HL039846-03
Application #
3471808
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1988-04-01
Project End
1993-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
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Kragenbrink, R; Higham, S C; Sansom, S C et al. (1996) Chronic stimulation of acetylcholine receptors: differential effects on Na,K-ATPase isoforms in a myogenic cell line. Synapse 23:219-23
Shanbaky, N M; Pressley, T A (1995) Transfection of Na,K-ATPase alpha-subunit: regulation of enzyme abundance. Biochem Cell Biol 73:261-8
Shanbaky, N M; Pressley, T A (1994) Mammalian alpha 1-subunit of Na(+)-K(+)-ATPase does not need its amino terminus to maintain cell viability. Am J Physiol 267:C590-7
Allen, J C; Pressley, T A; Odebunmi, T et al. (1994) Tissue specific membrane association of alpha 1T, a truncated form of the alpha 1 subunit of the Na pump. FEBS Lett 337:285-8
Scully, R R; Pressley, T A; O'Neil, R G (1993) A site-directed antibody recognizes a component of the ouabain-binding domain of the alpha 1 subunit of rat Na+,K(+)-ATPase. Biochem Cell Biol 71:538-43
Higham, S C; Melikian, J; Karin, N J et al. (1993) Na,K-ATPase expression in C2C12 cells during myogenesis: minimal contribution of alpha 2 isoform to Na,K transport. J Membr Biol 131:129-36
Pressley, T A (1992) Phylogenetic conservation of isoform-specific regions within alpha-subunit of Na(+)-K(+)-ATPase. Am J Physiol 262:C743-51
Allen, J C; Medford, R M; Zhao, X et al. (1991) Disproportionate alpha- and beta-mRNA sodium pump subunit content in canine vascular smooth muscle. Circ Res 69:39-44