P2U(P2Y2)-PURINOCEPTOR AND WATER TRANSPORT IN RAT KIDNEY

P2U(P2Y2)-嘌呤受体和大鼠肾脏中的水转运

• 批准号: 6517989
• 负责人: BELLAMKONDA K KISHORE
• 金额: $ 22.31万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6517989
• 关键词: arginine vasopressin; body water dehydration; cellular polarity; eicosanoid metabolism; histology; hormone regulation /control mechanism; immunologic assay /test; laboratory rat; membrane permeability; nutrition related tag; osmotic pressure; protein localization; purinergic receptor; receptor expression; renal tubular transport; thirst; water

DESCRIPTION (Adapted from the Applicant's Abstract): Renal collecting duct water permeability is regulated by a complex interplay among different signaling pathways, which are linked to various membrane receptors. While arginine vasopressin (AVP), acting through its V2 receptor and the camp second messenger system, increases the water permeability of the collecting duct, a variety of autocrine and paracrine agents (e.g. prostaglandins, endothelin), acting through their respective membrane receptors and the phosphoinositide signaling pathway, down regulate the water permeability of the collecting duct. One such receptor is the P2Y2-purinoceptor (P2u-purinoceptor), which is activated by extracellular nucleotides (ATP or UTP). The long term goal of this application is to decipher the cellular and molecular mechanisms involved in the down regulation of AVP-induced water permeability in the IMCD by the activation of P2Y2-purinoceptor. The applicant has already (i) demonstrated that agonist activation of P2Y2-purinoceptor down regulates the AVP-induced osmotic water permeability (Pf) in microperfused rat inner medullary collecting duct (IMCD), (ii) developed a gene specific cDNA probe and peptide-derived polyclonal antibody to P2Y2-purinoceptor, and localized the receptor mRNA and protein in the rat IMCD, (iii) obtained preliminary evidence that agonist stimulation of P2Y2-purinoceptor in rat IMCD releases prostaglandin E2, and (iv) observed that P2Y2-purinoceptor mRNA and protein in the inner medullae of thirsted and hydrated rats are markedly altered, associated with an altered subcellular distribution of the receptor protein in the IMCD. Based on these observations of the applicant, the specific aims of the application are: (i) to investigate the role and contribution of apical and basolateral purinoceptor and the modulation of AVP-stimulated Pf of rat IMCD, (ii) to investigate the roles of cycloxygenase and cytosolic phospholipase A2 in the release of prostaglandins by the agonist stimulation of P2Y2-purinoceptor in rat IMCD, (iii) to investigate the role and contribution of P2Y2-purinoceptor in simple physiological rat models of thirsting and hydration. To achieve these, the applicant proposes to use rat models of thirsting and hydration and to perform (i) functional studies on in vitro microperfused IMCD, (ii) molecular studies to determine mRNA and protein expression levels, (iii) subcellular localization of the receptor protein by immunoperoxidase labeling on cryosections and immunogold labeling on ultrathin sections, (iv) studies on IMCD suspensions to detect the alterations in the arachidonic acid metabolism. Successful completion of these studies will lead to significant insights into the AVP-independent mechanisms of regulation of collecting duct water permeability.

描述（改编自申请人的摘要）：肾脏收集管道 水的渗透性受不同的相互作用的调节 信号通路，与各种膜受体有关。尽管 精氨酸加压素（AVP），通过其V2受体作用，第二个营地第二 Messenger系统，增加了收集管的水渗透性，A 多种自分泌和旁分泌剂（例如前列腺素，内皮素）， 通过各自的膜受体和磷酸肌醇作用 信号通路，向下调节收集管的水渗透性。 一个这样的受体是P2Y2-吡只受体（P2U-Purinoceptor），它是 被细胞外核苷酸（ATP或UTP）激活。这个长期目标 应用是破译参与的细胞和分子机制 AVP诱导的IMCD中AVP诱导的水的渗透性的下降调节 P2Y2-Purinoceptor的激活。申请人已经证明了 P2Y2-Purinoceptor的激动剂激活调节AVP诱导的 微孔大鼠内髓质收集中的渗透性水渗透率（PF） 导管（IMCD），（ii）开发了基因特异性cDNA探针和肽来源 抗P2Y2-纤维杆菌的多克隆抗体，并将受体mRNA和 大鼠IMCD中的蛋白质（iii）获得了激动剂的初步证据 大鼠IMCD中P2Y2-Purinoceptor的刺激释放前列腺素E2，并且 （iv）观察到p2y2- purinoceptor mRNA和蛋白 口渴和水合大鼠明显改变，与改变 IMCD中受体蛋白的亚细胞分布。基于这些 对申请人的观察，申请的具体目的是：（i） 研究顶端和基底外侧purinoceptor的作用和贡献 以及大鼠IMCD的AVP刺激的PF的调节（ii）研究 环加糖酶和胞质磷脂酶A2在释放中的作用 Prostaglandins通过大鼠IMCD中P2Y2-Purinoceptor的激动剂刺激， （iii）调查P2Y2-Purinoceptor在简单中的作用和贡献 渴求的生理大鼠模型。为了实现这些目标， 申请人建议使用渴求的大鼠模型，并执行 （i）关于体外微灌注IMCD的功能研究，（ii）分子研究 确定mRNA和蛋白质表达水平，（iii）亚细胞定位 免疫过氧化物酶在冷冻切片和 超薄切片上的免疫元标记，（iv）IMCD悬浮液的研究 检测蛛网膜酸代谢的改变。成功的 这些研究的完成将导致对 非AVP独立于收集管道渗透性调节的机制。