Age-Related Changes in Renal Dopamine Receptor Function

描述(申请人提供)：多巴胺通过激活近端小管(PTS)上的多巴胺D1样受体，抑制Na，H-交换器和Na，K-ATPase，促进肾脏钠的排泄增加。我们已经证明，在老年大鼠中，多巴胺抑制这些钠转运体的能力降低，在老年动物中，对多巴胺的利钠反应也减少。这是由于老年大鼠D1a受体的高丝氨酸磷酸化导致的D1样受体偶联信号转导通路的缺陷，以及PTS中蛋白激酶C(PKC)活性的增加。G蛋白偶联受体激酶(GRKs)可磷酸化多巴胺D1受体并使其脱敏。在初步研究中，我们发现老年大鼠氧化应激增加，补充抗氧化剂降低了氧化应激，降低了基础PKC活性，并恢复了对D1受体激活的利钠反应。这一应用将检验这样一种假设，即氧化应激增加会导致PKC活性增加，而PKC活性通过激活GRKs，产生D1a受体基础丝氨酸磷酸化的增加，导致其与G蛋白解偶联。本实验旨在探讨氧化应激诱导肾小管上皮细胞基础PKC活性升高的机制，以及特定的PKC亚型(β和Delta)和GRK亚型(GRK-2)在氧化应激致肾小管上皮细胞D1a受体高丝氨酸磷酸化和G蛋白解偶联中的作用。为了研究氧化应激在老年大鼠D1a受体G蛋白解偶联中的作用，动物补充抗氧化剂，然后测量氧化水平、PKC和GRK活性、D1a受体信号转导和对D1样激动剂非诺多巴的利钠反应。这一结果将使我们能够确定老年大鼠肾脏D1受体功能障碍的分子基础。我们的发现将具有深远的意义，因为它涉及使用抗氧化剂来恢复有缺陷的G蛋白偶联受体功能，以及与衰老过程中氧化应激增加相关的药物反应性。

DESCRIPTION (provided by applicant): 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.