The long term goal of our investigations is elucidation of the cellular mechanism by which vasopressin (VP) regulates H2O permeability in in collecting tubules (CT) and the cellular pathogenesis of nephrogenic diabetes insipidus (NDI). In particular, we will clarify the role of cAMP phosphodiesterase (PDE) and cAMP-protein kinase (cA-PK) isozyme systems as an essential link in the pathway which connects V2-receptor signal at the basolateral membrane with H2O permeability end-response at luminal membrane (LM). Further, we will identify the defect(s) of PDE and cA-PK system in mice with hereditary sever NDI. Specific objectives include: 1. The determination of basic properties and modulatory role of cyclic 3', 5'-nucleotide phosphodiesterases (PDE) isozymes in cortical, medullary and papillary subsegments of CT. The studies will focus on the a) acute modulatory effects of atrial natriuretic peptide (ANP) via cGMP b) long-term effects of glucocorticoids and c) pharmacologic effects of novel """"""""second generation"""""""" synthetic PDE inhibitors. 2. To elucidate segmental localization, isozyme composition, and dynamics of enzymes and modulators cAMP-protein phosphorylation system. Further, we will identify, using in situ phosphorylation system, endogenous protein substrate(s) with special attention to i) cytoskeletal and contractile protein, ii) the feedback effect on PDE and V2-receptor/adenylate cyclase complex, and iii) glycolytic and glycogenolytic enzymes. 3. To clarify role of PDE isozymes in refractoriness of CT of NDI mice to VP. Studies will design aimed pharmacologic correction by new synthetic compounds and by glucocorticoids. We will explore possible anomalies in cA-PK and their relation to PDE in CT of NDI mice. Studies will be conducted on microdissected subsegments of collecting tubules from mouse and rat kidney, and on cells from these segments grown in primary culture. In short, the outlined studies will decipher the role of PDE and cA-PK isozymes in intracellular transmission of the VP-generated cAMP signal in CT, and discover anomalies at these steps as a cause of resistance to antidiuretic action of VP in NDI mice.
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