The objective of this proposal is to examine the biochemical mechanisms of renal resistance to vasopressin (AVP) in three settings associated with nephrogenic diabetes insipidus (NDI): hypercalcemia, pharmacologic agents (lithium and demeclocycline) and congenital NDI. The primary system in which studies will be performed is a primary culture of inner medullary collecting tubule cells that respond specifically to AVP by forming the intermediate effector of the hormone's action, cAMP in a dose dependent manner. The measurement of cAMP formation, in the presence and absence of phosphodiesterase inhibition, prostaglandin E2 production and adenylate cyclase activity will be employed. The effects of changes in extracellular calcium (Ca), agents that impair cellular Ca uptake, agents that enhance entry of Ca into cells, calmodulin inhibitors, and prostaglandin synthesis inhibitors on cAMP production will be assessed. Definition of the site of the cyclase at which any changes occur will be performed. A binding assay will examine receptor function, guanine nucleotide regulatory unit stimulation will examine the regulatory subunit and forskolin will be employed to look at the catalytic unit. The latter will be complemented by studies on cells that are deficient in the regulatory subunit, the cyc mutant of murine S49 lymphoma cells. Similar examination of the cyclase system and prostaglandin dependent mechanisms will be undertaken with lithium and demeclocycline. The culture technique will be applied to mice in order to define the defect in mice with severe congenital NDI. Studies directed to assess whether a guanine nucleotide regulatory subunit defect is operant will employ a complementation adenylate cyclase assay employing the murine S49 lymphoma cells. These studies should significantly enhance our understanding of the biochemical mechanisms that underline commonly encountered AVP resistance states characterized clinically by polyuria, polydipsia and impaired urinary concentration.
| Anger, M S; Shanley, P; Mansour, J et al. (1990) Effects of lithium on cAMP generation in cultured rat inner medullary collecting tubule cells. Kidney Int 37:1211-8 |
| Teitelbaum, I; Strasheim, A; Berl, T (1989) Adrenergic control of cAMP generation in rat inner medullary collecting tubule cells. Kidney Int 35:647-53 |
