Age-Related Changes in Renal Dopamine Receptor Function

性状（由申请方提供）：多巴胺通过激活近端小管（PT）中的多巴胺D1样受体并抑制Na，H-交换器和Na，K-ATP酶，促进肾钠排泄增加。我们已经证明，多巴胺抑制这些钠转运蛋白的能力在老年大鼠中降低，对多巴胺的利钠反应在老年动物中也减少。这是由于老年大鼠D1 A受体的过度丝氨酸磷酸化引起的D1样受体偶联信号转导通路缺陷以及PT中蛋白激酶C（PKC）活性增加所致。已知G蛋白偶联受体激酶（GRKs）使多巴胺D1受体磷酸化和脱敏。在初步研究中，我们发现老年大鼠氧化应激增加，补充抗氧化剂降低氧化应激，降低基础PKC活性，并恢复对D1受体激活的利钠反应。本申请将测试氧化应激增加导致PKC活性增加的假设，PKC活性通过GRKs的激活，产生D1 A受体的基础丝氨酸磷酸化增加，导致其与G蛋白解偶联。实验旨在确定氧化应激诱导的基础PKC活性增加的机制，特异性PKC亚型（β和δ）和GRK亚型（GRK-2）在暴露于氧化剂的近端肾小管细胞培养物和老年大鼠中D1 A受体的过度丝氨酸磷酸化和G蛋白解偶联中的作用。为了检查氧化应激在老年大鼠中D1 A受体G蛋白解偶联中的作用，将给予动物抗氧化剂补充，然后测量氧化剂水平、PKC和GRK活性以及D1 A受体信号传导和对D1样激动剂非诺多泮的利钠反应。这些结果将使我们能够确定老年大鼠肾D1受体功能障碍的分子基础。我们的研究结果将具有深远的意义，因为它涉及到使用抗氧化剂来恢复有缺陷的G蛋白偶联受体功能和药物反应性，这些功能和药物反应性与衰老中氧化应激增加有关。

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.