A variety of functions of the glomerular mesangial cells are regulated by humoral signals, including contraction and eicosanoid production. These functions are altered in glomeruli removed from rats with experimental diabetes, and these functional abnormalities may contribute to glomerular hypertension and development of diabetic glomerulosclerosis. Modulation of cell function in response to extracellular signals frequently involves regulatory protein phosphorylation as an intermediate reaction. A likely site of regulation of mesangial prostanoid production is at the level of phospholipase A2 activity, through the action of lipocortin. This protein is a potent inhibitor of phospholipase A2, and its inhibitory activity is regulated by tyrosine and serine/threonine-specific phosphorylation. The proposed studies will characterize the pathways of regulatory protein phosphorylation and the endogenous phosphoprotein substrates in the cultured glomerular mesangial cell, in response to insulin and growth substrates in the cultured glomerular mesangial cell, in response to insulin and growth factors, vasoconstrictor peptides, Atrial Naturetic hormone and a variety of pharmacologic pertubants (cyclic nucleotides, phorbol esters, divalent cation ionophores). Lipocortin phosphorylation will be specifically determined by immunoprecipitation, and compared by peptide mapping to the phosphorylation of the partially purified protein by known protein kinases in vitro. Correlative measurements of lipocortin phosphorylation with concomitant phospholipase A2 inhibitory activity and overall eicosanoid production will be carried out. Subsequent to elucidation of regulation in the normal mesangial cell, the integrity of these systems will be defined in mesangial cells cultured from animals (rats) with experimental diabetes. These studies will elucidate the role of lipocortin in mesangial eicosanoid production, and the regulation of this function by humoral signals in the normal and diabetic animal.
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