Dopamine and D1-like receptors in the proximal tubules and other regions of the nephron play an important role in regulating renal sodium excretion. Preliminary studies have demonstrated that while dopamine concentration in the kidney and urine was similar in adult and old rats, dopamine and D1-like receptor agonist failed to inhibit Na,K-ATPase activity in the proximal tubules of old Fischer 344 rats. Therefore, it is hypothesized that defective dopamine receptors, G protein-coupling and/or cellular signaling mechanisms would lead to a reduced ability of endogenous and exogenous dopamine to promote sodium excretion in old rats. This hypothesis will be systematically tested both biochemically and functionally using Fischer 344 rat model (adult, middle aged and old). Since dopamine is known to cause natriuresis by inhibiting Na,K-ATPase and Na,H-exchanger activities on basolateral and brush border membranes, respectively, experiments are designed to compare the inhibitory effects of dopamine on both the enzymes in the proximal tubules of adult and old rat. Thereafter, D1-like receptor and the components of its cellular signaling cascade will be studied to investigate the mechanism(s) of reduced response of dopamine on Na,RATPase in old rats. The applicant will quantify D1-like receptor numbers and affinity in the basolateral and brush border membranes and measure D1A receptor mRNA in the proximal tubules in order to determine D1A receptor regulation in the old rats. The D1-like receptor-coupled G proteins (Gs and Gq/11) will be quantified by western blotting and their function measured using various agents such as GTP, NaF and a comparison will be made between adult and old rats. Phospholipase C, protein kinase C, adenylyl cyclase, protein kinase A and phospholipase A2 are the cellular signaling components that mediate D1-like receptor response on Na,K-ATPase and Na,H-exchanger activity in the proximal tubules, the functional status of these components will be studied to compare their role in D1-like receptor -mediated response in the adult and old rats. The role of endogenous kidney dopamine in promoting sodium excretion will be compared between adult and old rats placed on normal and high salt intake and during acute volume expansion. Natriuretic and diuretic effects of exogenously infused dopamine and D1-like receptor agonists will also be compared between old and adult rats. Identification of a defective dopamine response and the mechanism leading to this phenomenon in aging would require an alternate therapeutic approach in older patients requiring the use of dopamine for improvement of renal function.
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