The long term objective of this proposal is to determine the cellular signal transduction mechanism(s) by which vasopressin, a hormone essential to the regulation of body fluid homeostasis, exerts control over the terminal nephron. ln addition to the considerable evidence supporting the role for cyclic AMP as a second messenger, cytosolic free calcium has also been proposed to regulate the hormonal responses, either independently or synergistically with cAMP. The hypothesis to be tested by the proposed studies is that vasopressin affects cellular function in the collecting tubule through two distinct receptor-effector systems localized on the same cell, and that the regulation of vasopressin signal occurs by the activation and perhaps interaction of the two systems. Understanding the molecular mechanism by which vasopressin regulates nephron function is impaired by the limited amount, heterogeneity, and relatively inaccessibility of the cells which it regulates. The use of monoclonal antibodies as immunoaffinity reagents for the isolation of large quantities of homogenous cell populations has recently made it possible to isolate segment- specific renal cells. This approach will be used to isolate collecting tubule-specific cells to determine: 1) what is the cellular mechanism of vasopressin action 2) are both vasopressin receptors (Vl and V2) present on the same cell 3) is this dual signal transduction system acting independently or synergistically, and 4) is the sodium uptake and oxygen consumption in these cells regulated by this dual signal-transduction system. These studies will provide information on the molecular mechanism by which vasopressin regulates water and solute transport in the distal segment. In addition, it will further our understanding of a dual signal-transduction systems in general.
