In vitro Macro-and Microautoradiographic Localization of V₁ and V₂ Receptors in the Rat Kidney Using OPC-21268 and OPC-31260

To elucidate the precise localization of vasopressin(VP) Vj 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 Vi (OPC-21268) and V₂ (OPC-31260)receptor antagonists. For macro-ARG, 10-μm kidney sections were incubated with Tris-HCl buffer containing [³H]-VP with or without unlabeled ligand (VP, OPC-21268, orOPC-31260) at 20°C for 40 min. These sections were then loaded intoX-ray cassettes with Hyperfilm-[³H] and exposed inthe dark for 2 months. The autoradiograms were quantitativelyanalyzed by using the research analysis system RAS 1,000; the V₁ and V₂ receptors were quantitated by inftracting the nonspecific binding (incubatedwith OPC-21268 and OPC-31260, respectively) from the total binding. To assess a more precise localization of the V₁and V₂ receptors, we also investigated the micro-ARG of the renal V₁ and V₂ receptorsby 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, V₁receptors, visualized by OPC-21268, werefewer in number. V) receptors were partly localized to the glomerulus, cortical vessels, interstitial cells, and the medullary vessels. The V₂receptors 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.