MOLECULAR DISSECTION OF DOPAMINE D5 RECEPTOR ACTIONS

除了作为神经递质的重要性外，多巴胺还具有 对心率、血压、肾血流量、钠 吸收和保水性。 在临床上， 以治疗患有休克或肾功能受损的患者。 在 低剂量时，多巴胺的肾脏效应占主导地位， 两种受体亚型：DA 1和DA 2。 关于肾脏DA 2的了解很少 受体生理学 相反，DA 1多巴胺受体介导 尿钠排泄和肾皮质小管中的利尿， 肠系膜和肾血管床的血管舒张。 DA 1受体 在近曲小管（PCT）上皮细胞中， 刺激cAMP和二酰基甘油（DAG）产生。 增加 这两种第二信使的浓度激活蛋白激酶 A和C，分别。 这些激酶的两个靶点是管腔 Na+/H+交换器（NHE）和基底外侧Na+-K+ ATP酶。 NHE 和ATP酶参与钠的转运， 磷酸化和随后的Na+/H+交换活性抑制 导致钠排泄（尿钠排泄）。 我们的结果表明 我们最近克隆的多巴胺D5受体 肾PCT DA 1受体亚型。 我们认为多巴胺D5受体 在肾脏PCT上皮细胞偶联到第二信使系统， 参与Na+/H+交换活性的调节。 因此，我们认为， 对D5/Da 1受体调节钠的能力的任何干扰 转运可能在某些形式的 原发性高血压 我们建议测试这方面的几个方面 假说. 最近我们证明了激活的多巴胺D5 受体刺激cAMP的产生和H+的分泌。 后一 作用对阿米洛利敏感，表明Na+/H+交换剂（NHE） 活动参与。 我们建议继续进行体外表征 D5与腺苷酸环化酶和磷脂酶C的偶联。 此外，本发明还提供了一种方法， 我们有机会开发一个非常强大的体外系统， 从而剖析D5对NHE活性的调节。 细胞系表达 最近克隆的三个NHE和D5受体中的每一个都将被 关于钠转运的多巴胺能调节进行评价。 我们也有证据表明重组牛痘病毒载体 可用于制备多克隆抗受体抗血清。 我们将使用 该技术制备的抗D5抗血清，是一种有价值的检测D5的试剂。 多巴胺D5受体表达和翻译后分析 在正常和患病肾皮质组织中的修饰。 异常肾 对多巴胺的反应也在两个有充分记录的大鼠中报道， 遗传性高血压的模型，我们现在处于一个独特的位置， 确定遗传缺陷是否位于多巴胺D5受体内 基因 最后，我们打算产生缺乏功能的转基因小鼠， 多巴胺D5受体 这些“基因敲除”小鼠的生产将 提供了一个新的和强大的小鼠模型系统，其中的作用， 多巴胺D5受体在肾脏生理学中的作用，以及钠 特别是交通运输，可以进行评估。

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.