MOLECULAR DISSECTION OF DOPAMINE D5 RECEPTOR ACTIONS

除了作为一种重要的神经递质，多巴胺还有

In addition to its importance as a neurotransmitter, dopamine has profound effects on heart rate, blood pressure, renal blood flow, sodium absorption and water retention. In the clinical setting dopamine is used to treat patients suffering from shock or impaired renal function. At low doses the renal effects of dopamine predominate and are mediated by two receptor subtypes: DA1 and DA2. Little is known about renal DA2 receptor physiology. In contrast, DA1 dopamine receptors mediate natriuresis and diuresis in the tubules of the renal cortex and vasodilation of the mesenteric and renal vascular beds. DA1 receptors in proximal convoluted tubule (PCT) epithelial cells couple to the stimulation of cAMP and diacylglycerol (DAG) production. Increases in the concentration of these two second messengers activate protein kinases A and C, respectively. Two targets of these kinases are the luminal Na+/H+ exchanger (NHE) and the basolateral Na+-K+ATPase. Both the NHE and the ATPase are involved in sodium transport but it is the phosphorylation and subsequent inhibition of Na+/H+ exchanger activity that results in sodium excretion (natriuresis). Our results suggest that the dopamine D5 receptor that we recently cloned is identical to the renal PCT DA1 receptor subtype. We propose that dopamine D5 receptors in the renal PCT epithelia couple to second messenger systems that participate in the regulation of Na+/H+ exchanger activity. Therefore, any interference with the ability of D5/Da1 receptor's to regulate sodium transport may play an important role in the etiology of certain forms of essential hypertension. We propose to test several aspects of this hypothesis. Recently we demonstrated that activated dopamine D5 receptors stimulate cAMP production and the secretion of H+. This latter effect is sensitive to amiloride suggesting that a Na+/H+ exchanger (NHE) activity is involved. We propose to pursue the in vitro characterization of D5's coupling to adenylyl cyclase and phospholipase C. In addition, we have the opportunity to develop a very powerful in vitro system in which to dissect D5's regulation of NHE activity. Cell lines expressing each of the three recently cloned NHEs and the D5 receptor will be evaluated with respect to dopaminergic regulation of sodium transport. We also have evidence to suggest that recombinant vaccinia virus vectors can be used to generate polyclonal anti-receptor antiserum. We will use this technology to produce anti-D5 antiserum, a valuable reagent for the analysis of dopamine D5 receptor expression and post translational modification in normal and diseased renal cortex tissue. Abnormal renal responses to dopamine have also been reported in two well-documented rat models of inherited hypertension and we are now in a unique position to determine whether the genetic defect lies within the dopamine D5 receptor gene. finally we intend to generate transgenic mice that lack functional dopamine D5 receptors. The production of these "knockout" mice will provide a new and powerful mouse model system in which the role of dopamine D5 receptors in renal physiology in general, and sodium transport in particular, can be evaluated.