The action of the posterior pituitary hormone vasopressin to increase water permeability of the apical surface of renal collecting tubular cells is critical in the maintenance of normal body water balance. Clarification of the mechanism of the hydro-osmotic effect of vasopressin is the long-range goal of the applicant. As a step toward this goal, the present studies will examine the role of the calcium ion and calcium binding proteins in modulating the water permeability effect of vasopressin. Rabbit collecting tubules, perfused in vitro, will be used to determine the effect of altering collecting tubular cell calcium and calmodulin activities on water permeability response to vasopressin. The effect of vasopressin on net water flux and hydraulic conductivity in the presence and absence of calcium ion, calcium ionophores and inhibitors of cellular calcium uptake and calmodulin will be measured. To characterize any observed effects, both diffusional and osmotic water permeabilities as well as electrical potential difference across the tubule will be measured. Two additional types of studies will be performed to elucidate a potential role for cyclic necleotides in any observed effect: (1) perfused collecting tubule studies utilizing pharmacologic probes which manipulate renal collecting tubular cell content of cyclic nucleotides (for example, forskolin, diterpene compound which directly activates the catalytic unit of adenylate cyclase) and (2) collecting tubular biochemical assays for adenylate cyclase and phosphodiesterase. The proposed studies combine physiologic, pharmacologic, and biochemical approaches to clarify the role of the calcium ion and calcium-activated proteins in mediating the effect of vasopressin to increase collecting tubular cell apical water permeability. These studies should help clarify the mechanism of action of vasopressin in the mammalian nephron. These studies have health-related implications in view of the importance of the wather retaining effect of vasopressin in maintenance of normal tonicity and volume of body fluids.
