Visualization of Cell Surface Vasopressin V1a Receptors in Rat Hepatocytes with a Fluorescent Linear Antagonist

Abstract

To visualize cell surface V1a vasopressin receptors in rat hepatocytes in the absence of receptor-mediated endocytosis, we used a high-affinity fluorescent linear antagonist, Rhm8 -PVA. Epifluorescence microscopy (3CCD camera) and fluorescence spectroscopy were used. Rhm8 -PVA alone did not stimulate Ca2+ signals and competitively blocked Ca2+ signals (Kinact of 3.0 nM) evoked by arginine vasopressin (vasopressin). When rat hepatocytes were incubated with 10 nM of Rhm8 -PVA for 30 min at 4C, the fluorescent antagonist bound to the surface of cells, presumably the plasma membrane. The V1a receptor specificity of Rhm8 -PVA binding was confirmed by its displacement by the nonfluorescent antagonist V4253 and by the natural hormone vasopressin at 4C. Prior vasopressin-mediated endocytosis of V1a receptors at 37C abolished binding of the labeled antagonist, whereas in non-preincubated cells, Rhm8 -PVA labeled the cell surface of rat hepatocytes. When cells labeled with Rhm8 -PVA at 4C were warmed to 37C to initiate receptor-mediated internalization of the fluorescent complex, Rhm8 -PVA remained at the cell surface. Incubation temperature at 4C or 37C had little effect on binding of Rhm8 -PVA. We conclude that Rhm8 -PVA is unable to evoke receptor-mediated endocytosis and can readily be used to visualize cell surface receptors in living cells.