The basic underlying hypothesis of this proposal is that (Na+)i plays a role in regulating the amount of contractile proteins and the number of Na+ pump sites in cultured vascular smooth muscle cells. We will grow both arterial and venous smooth muscle cells in culture and quantitate contractile protein content (actin and myosin) by gel electrophoresis, Na+ pump site number by H3-ouabain binding and Na+ pump turnover rate by ouabain sensitive Rb86 uptake. After control characteristics have been established for these protein complexes, the cells will be grown under 4 specific conditions which will alter (Na+)i by significantly varied mechanisms: 1) O K+ or ouabain, which will inhibit the Na+ pump and increase (Na+)i; 2) monensin, aldosterone, angiotensin II and veratridine, which will increase (Na+)i and stimulate the pump; 3) amiloride which will decrease (Na+)i and inhibit the pump; and 4) cyclical stretch. The contractile protein content and Na+ pump site number will then be assessed after the appropriate growth period. In addition to these specific parameters, important additional parameters will be assessed to characterize the affect of these altered conditions. These will include determining the cell content of cAMP, DNA, Na+, K+, Ca++, the measurement of cell volume, intermediate filament content and H3-thymidine incorporation into DNA and morphological characterization (scanning and TEM). If an increase in contractile protein content or Na+ pump site number occurs, the role of Ca++ and amino acids in this process will be assessed by determining the amont of Na+i-Ca++o exchange and H3-alpha aminoisobutyric acid uptake. In addition, the effect of protein synthesis inhibitors (anisomycin, cyclohexamide) will be determined. Assessment of the amount of contractile protein and the number of Na+ pump sites under these systematically controlled conditions will lead to a better understanding concerning the role of the (Na+)i in regulating the amount of these proteins. Because of the similarity between these growth conditions and certain models of hypertension which involve increased (Na+)i, increased strain, and Na+ pump alterations, this work may lead to a hypothesis regarding the etiology of this disease.
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