In vitro macro- and microautoradiographic localization of V1 and V2 receptors in the rat kidney using OPC-21268 and OPC-31260.

Abstract

To elucidate the precise localization of vasopressin (VP) V1 and V2 receptors in the kidney, we utilized in vitro macroautoradiography (macro-ARG) and microautoradiography (micro-ARG) of these receptors in Wistar rat kidneys. This was done by using OPC-21268 and OPC-31260, two newly developed selective V1 (OPC-21268) and V2 (OPC-31260) receptor antagonists. For macro-ARG, 10-microm kidney sections were incubated with Tris-HCl buffer containing [3H]-VP with or without unlabeled ligand (VP, OPC-21268, or OPC-31260) at 20 degrees C for 40 min. These sections were then loaded into X-ray cassettes with Hyperfilm-[3H] and exposed in the dark for 2 months. The autoradiograms were quantitatively analyzed by using the research analysis system RAS 1,000; the V1 and V2 receptors were quantitated by subtracting the nonspecific binding (incubated with OPC-21268 and OPC-31260, respectively) from the total binding. To assess a more precise localization of the V1 and V2 receptors, we also investigated the micro-ARG of the renal V1 and V2 receptors by dipping the kidney section slides used for macro-ARG into a photographic emulsion and observing the receptors under light microscopy. [3H]-VP binding to the rat kidney was completely displaced by unlabeled excess VP, but not by unlabeled angiotensin II, indicating that [3H]-VP binding was specific for VP receptors. Computerized quantification showed that V2 receptors, visualized by OPC-31260, were the predominant type of VP receptor in the kidney. Conversely, V1 receptors, visualized by OPC-21268, were fewer in number. V1 receptors were partly localized to the glomerulus, cortical vessels, interstitial cells, and the medullary vessels. The V2 receptors localized to the collecting ducts and medullary tubules. Our findings indicated that renal V1 and V2 receptors can be detected by in vitro macro- and micro-ARG by using OPC-21268 and OPC-31260.