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: 6447274
• 负责人: BELLAMKONDA K KISHORE
• 金额: $ 21.34万
• 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/6447274
• 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（改编自申请人摘要）：肾集合管 水的渗透性是由不同物质之间复杂的相互作用来调节的。 信号通路，其与各种膜受体相连。而 精氨酸加压素（AVP），通过其V2受体和cAMP第二 信使系统，增加了收集管道的透水性， 各种自分泌和旁分泌剂（如肾上腺素、内皮素）， 通过它们各自的膜受体和磷酸肌醇 信号通路，下调集合管的水渗透性。 一种这样的受体是P2 Y2-嘌呤受体（P2 u-嘌呤受体），其是 由细胞外核苷酸（ATP或UTP）激活。长期目标是 应用程序是破译参与的细胞和分子机制 AVP诱导的IMCD水渗透性的下调， P2 Y2-嘌呤受体的激活。申请人已证明（i） P2 Y2-嘌呤受体的激动剂激活下调AVP诱导的 微灌流大鼠内髓集合体的渗透水通透性 导管（IMCD），（ii）开发了基因特异性cDNA探针和肽衍生的 P2 Y2-嘌呤受体的多克隆抗体，并定位受体mRNA， 蛋白质在大鼠IMCD，（iii）获得的初步证据表明，激动剂 刺激大鼠IMCD中P2 Y2-嘌呤受体释放前列腺素E2， (iv)观察到P2 Y2-嘌呤受体mRNA和蛋白质在延髓内， 口渴和水合的大鼠明显改变，与改变的 IMCD中受体蛋白的亚细胞分布。基于这些 （一）申请人的姓名或者名称; 研究顶侧和基底侧嘌呤受体的作用和贡献 和AVP刺激的大鼠IMCD Pf的调制，（ii）研究 环氧合酶和胞浆磷脂酶A2在释放 通过P2 Y2-嘌呤受体激动剂刺激大鼠IMCD， (iii)探讨P2 Y2-嘌呤受体在单纯性肝癌中的作用和贡献， 生理大鼠模型的口渴和水合作用。为了实现这些目标， 申请人提出使用大鼠口渴和水合模型， (i)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 通过免疫过氧化物酶标记冷冻切片上的受体蛋白， 免疫金标记的切片，（iv）研究IMCD悬浮液， 检测花生四烯酸代谢的变化。成功 这些研究的完成将导致对 集合管水渗透性调节的AVP独立机制。