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.
