The objective of this proposal is to determine the regulatory role of cAMP phosphodiesterase isozymes (PDEs) in renal cells, namely in cells of inner medullary collecting ducts (IMCD). We will test the hypothesis that cAMP- mediated vasopressin (AVP) effects are modulated by specific PDE isozymes via action of other 2nd messengers (Ca2+, cGMP), and extrarenal hormones, namely glucocorticoids (GCD). In hereditary strain of mice with nephrogenic diabetes insipidus (NDI mice), excessive and/or anomalous cAMP PDE(s) account for complete resistance to AVP, a paradigm of essential role of PDEs in cAMP-mediated action of AVP. Specific objectives include: 1) To identify PDEs which control AVP-dependent cAMP pool(s) in IMCD cells. To define the mechanism by which cAMP generated in response to AVP by acting on PDEs autoregulates the AVP response. To clarify the mechanism by which other hormones (e.g. bradykinin) modulate action of AVP by changing activities of PDEs via other 2nd messengers (cGMP, Ca2+i). 2) To elucidate how glucocorticoids (GCD) regulate the response of IMCD cells to AVP at the step of the PDEs. We will determine which one of PDE isozymes is down-regulated by GCD, and we will delineate the molecular- biochemical mechanism of GCD effect upon PDE in IMCD cells. 3) To determine the mechanism by which PDE isozyme(s) of type-IV, present in IMCD cells of NDI mice, prevents cAMP accumulation and the end-response to AVP. a) We will explore whether higher PDE-IV activity is due to increased quantum of enzyme protein, mRNA, transcription or translation rates in PDE- IV synthesis, or to post-translational modification. Also, whether a mutant PDE-IV with high Vmax or resistance to proteolysis may account for high PDE-IV activity. b) We will test whether stable transfection of cells in vitro by PDE-IV gene will reproduce the resistance to AVP as in NDI mice. In studies on PDE-IV - transfected cells, we will determine, in general, the relationship between cAMP response of IMCD cells to AVP and endogenous activity of PDE- IV isozymes. These studies will be conducted on IMCD cells freshly prepared from rat, rabbit or mouse kidneys, rat IMCD cells grown in primary or continuous in vitro culture, and on established epithelial cell lines.
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