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: 6635405
• 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/6635405
• 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(摘自申请者摘要)：肾脏集合管 水的渗透性由不同物种之间复杂的相互作用来调节 信号通路，连接到不同的膜受体。而当 精氨酸加压素(AVP)通过其V2受体和cAMP第二受体发挥作用 信使系统，增加了收集管道的透水性， 多种自分泌和旁分泌药物(如前列腺素、内皮素)， 通过它们各自的膜受体和肌醇磷脂发挥作用 信号通路，下调集合管的透水性。 其中一个这样的受体是P2Y2-嘌呤受体(P2u-Purinoeptor)，它是 被细胞外核苷酸(ATP或UTP)激活。这样做的长期目标是 应用是破译细胞和分子机制涉及的 血管紧张素转换酶对血管紧张素转换酶诱导的IMCD通透性的下调作用 P2Y2-嘌呤受体的激活。申请人已(I)证明 P2Y2-嘌呤受体激动剂的激活下调AVP诱导的 微灌流大鼠髓内集束的渗透水通透性 Date(IMCD)，(II)开发了一种基因特异的cDNA探针和多肽 抗P2Y2-嘌呤受体的多克隆抗体，并定位该受体的mRNA和 大鼠IMCD中的蛋白质，(III)获得了初步证据，即激动剂 刺激大鼠IMCD中的P2Y2-嘌呤受体释放前列腺素E_2和 (4)观察到大鼠内髓中的P2Y2-嘌呤受体基因和蛋白。 口渴和补充水分的大鼠明显改变，与改变的 IMCD中受体蛋白的亚细胞分布。基于这些 申请人的意见，申请的具体目的是：(I) 探讨心尖和基底外侧嘌呤感受器的作用和贡献 以及AVP刺激的大鼠IMCD对PF的调节作用，(Ii)研究AVP对大鼠IMCD 环氧合酶和胞浆磷脂酶A2在细胞释放中的作用 前列腺素通过激动剂刺激大鼠IMCD中的P2Y2-嘌呤受体， (3)研究P2Y2-嘌呤受体在SIMP中的作用和贡献 口渴和补水的生理性大鼠模型。为了实现这些目标， 申请人提议使用大鼠口渴和水分的模型，并进行 (I)体外微灌流IMCD的功能研究；(Ii)分子研究 确定信使核糖核酸和蛋白质的表达水平；(三)亚细胞定位 冰冻切片上免疫过氧化物酶标记的受体蛋白 超薄切片免疫金标记法，(Iv)IMCD悬浮液研究 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 不依赖于AVP的集合管透水率调节机制。