Several of the dopamine receptor genes cloned from brain are expressed in the kidney. This proposal will test the hypothesis that the dopamine receptors associated with the stimulation of adenylyl cyclase (D1A and D1B, also known as D5) are differentially expressed in specific nephron segments and this differential expression regulates net sodium transport. These receptors mediate decreases in sodium transport by a cAMP dependent and a cAMP independent mechanism. Since the D1B receptor is more potent and efficacious than the D1A receptor in the stimulation of adenylyl cyclase in transfected cells, the net effect of these receptors on sodium transport may be due to inherent differences in signal transduction. One cAMP independent pathway may be via a dopamine receptor, which thus far has not been cloned. This receptor, unlike the D1A and D1B receptor, is linked to the stimulation of phosphoinositol specific phospholipase C (PI-PLC). This receptor shares some properties in common with the cloned D1A and D1B receptor genes, since all are blocked by """"""""D1"""""""" antagonists like SCH 23390 and stimulated by """"""""D1"""""""" agonists like fenoldopam and SKF 38393. In renal tubular membranes, D1 agonists stimulate the PLC isoform, PLC-beta, and inhibit the PLC isoform, PLC-gamma. The stimulation of PLC-beta leads to the stimulation of the protein kinase C (PKC) isoform, PKC-gamma. We hypothesize that PKC-gamma in renal tubular cells increases cAMP concentrations; cAMP in turn inhibits Na+/K+ ATPase activity. PKC-gamma may also inhibit Na+?K+ ATPase activity independently of cAMP. Both of these effects on Na+/K+ ATPase, in conjunction with an inhibitory action on Na+/H+ exchange activity, result in a decrease in sodium transport. In order to test the above hypotheses, the following specific aims will be pursued: 1. To determine by quantitative reverse transcriptase/polymerase chain reaction (RT/PCR), D1A and D1B receptor gene expression in microdissected nephron segments (specifically, the proximal convoluted tubule, thick ascending limb of Henle, and cortical collecting duct). The quantity of the receptor protein will be measured in proximal tubule and thick ascending limb of Henle cells by Western blotting and ELISA using antibodies directed against D1A and D1B receptors. 2. To determine the role of cAMP- dependent mechanisms mediated by D1A nd D1B receptors on inhibition of Na+/H+ exchange and Na+/K+ ATPase activity in murine LM-TK cell line stably transfected with either the D1A and/or the D1B receptor cDNA as well as specific nephron segments. 3. To determine the role of the as yet uncloned D1 receptor linked to PI-PLC on the inhibition of Na+/K+ ATPase activity in renal tubules. 4. To determine the mechanism by which the inhibitory action of dopamine on sodium transport in renal tubules changes with maturation. The knowledge of these dopamine-regulated processes is necessary to understand the important paracrine function of dopamine in regulating renal sodium handling. Indeed, intrarenal dopamine may be responsible for more than 50% of the natriuresis associated with saline loading (3-8% of body weight).
Showing the most recent 10 out of 152 publications
