Dopamine promotes an increase in renal sodium excretion by activating dopamine D1-like receptors in proximal tubules (PTs) and causing inhibition of Na,H-exchanger and Na,K-ATPase. We have shown that the ability of dopamine to inhibit these sodium transporters is reduced in old rats and the natriuretic response to dopamine is also diminished in older animals. This is due to a defective D1-like receptor-coupled signal transduction pathway, caused by hyper-serine-phosphorylation of D1A receptor in old rats, and an increase in protein kinase C (PKC) activity in the PTs. G-protein coupled receptor kinases (GRKs) are known to phosphorylate and desensitize dopamine D1 receptors. In preliminary studies, we found an increase in oxidative stress in old rats and antioxidant supplementation lowered oxidative stress, decreased basal PKC activity, and restored natriuretic response to D1 receptor activation. This application will test the hypothesis that increase in oxidative stress causes increase in PKC activity, which via activation of GRKs, produces an increase in the basal serine-phosphorylation of D1A receptors, causing it's uncoupling from G-proteins. Experiments are designed to determine the mechanism of oxidative stress-induced increase in basal PKC activity, role of specific PKC isoforms (beta &delta) and GRK isoform (GRK-2) in hyper-serine-phosphorylation of D1A receptor and G-protein uncoupling in proximal tubular cell cultures exposed to oxidants and in old rats. In order to examine the role of oxidative stress in D1A receptor G-protein uncoupling in old rats, animals will be given antioxidants supplementation followed by measurements of oxidant levels, PKC and GRK activities and D1A receptor signaling and natriuretic response to D1-like agonist, fenoldopam. The results will allow us to identify the molecular basis of renal D1 receptor dysfunction in old rats. Our findings will have a far reaching significance as it relates to the use of antioxidants in restoring defective G-protein coupled receptor function and drug responsiveness associated with increased oxidative stress in aging.
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