The purpose of the proposed studies is to further substantiate our working hypothesis that the diet-induced modifications in lipid composition and fluidity of plasma membranes of submandibular glands would result in a modification of the adenylate cyclase complex. The foregoing hypothesis will be tested by feeding rat essential fatty acid deficient-diets, trans fatty acids, saturated fats and cholesterol supplemented diets. All these dietary treatments are known to induce changes in membrane lipids and fluidity. Male, young rats will be fed different diets depending upon the type of study. At various time intervals, rats will be killed, the submandibular glands will be dissected out and plasma membranes will be prepared by using differential centrifugation and sucrose density gradient techniques. Basal, fluoride-, isoproterenol- and forskolin-stimulated adenylate cyclase activity will be determined. The concentration of beta-adrenergic receptor (B/max) and its dissociation constant (K/d) will also be measured. The levels of guanine-binding regulatory proteins and the characteristics of the catalytic subunit of adenylate cyclase will also be measured. In vitro studies with reconstituted membranes will be conducted to further assess the role of membrane lipids in adenylate cyclase activation. Beta-adrenergic stimulated secretion of peroxidase from the dispersed cells from the submandibular glands of rats fed various diets will be measured in vitro as a parameter of gland function. Changes in membrane fluidity will be determined by measuring the fluorescene polarization of diphenylhexatriene. Aliquots of plasma membranes from the submandibular glands will be extracted for lipids and the fatty acid composition of total lipids and phospholipids will be determined by gas chromatography. The degree of unsaturation of lipids, along with cholesterol to phospholipid ratios, will be used as additional criteria to assess membrane fluidity. Data will be stastically analyzed to evaluate the role of diet-induced changes in lipid composition and membrane fluidity in modifying the activities of adenylate cyclase and the secretory ability of the salivary glands.
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