The objective of this proposal is to examine the biochemical mechanisms of renal resistance to vasopressin (AVP) in two settings associated with nephrogenic diabetes insipidus (NDI): administration of pharmacologic agents (lithium and demeclocycline) that are known to cause a renal concentrating defect and congenital NDI. The system in which studies will be performed is a primary culture of inner medullary collecting tubule cells that responds specifically to AVP by forming the intermediate effector of the hormone's action, cAMP, in a dose dependent manner. Vasopressin-stimulated cAMP formation will be determined in the presence and absence of phosphodiesterase inhibition. Prostaglandin E2 production in these settings will be measured and the effect of prostaglandin synthesis inhibitors on cAMP production will be assessed. The site of the receptor-adenylate cyclase complex at which any changes occur will be defined. A binding assay will examine receptor function and forskolin will be employed to directly stimulate the catalytic unit. Studies directed at assessing whether a guanine nucleotide binding regulatory subunit defect is operant will employ agents (e.g. cholera toxin, pertussis toxin) known to stimulate at this site. In conjunction with these latter studies an adenylate cyclase complementation assay employing the cyc- mutant of murine S49 lymphoma cells will be performed. These studies will significantly enhance our understanding of the biochemical mechanisms that underly commonly encountered AVP resistant states characterized clinically by polyuria, polydipsia and impaired urinary concentration.
