Résumé
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, Rhm
8
-PVA. Epifluorescence microscopy (3CCD camera) and fluorescence spectroscopy were used. Rhm
8
-PVA alone did not stimulate Ca
2+
signals and competitively blocked Ca
2+
signals (Kinact of 3.0 nM) evoked by arginine vasopressin (vasopressin). When rat hepatocytes were incubated with 10 nM of Rhm
8
-PVA for 30 min at 4C, the fluorescent antagonist bound to the surface of cells, presumably the plasma membrane. The V1a receptor specificity of Rhm
8
-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, Rhm
8
-PVA labeled the cell surface of rat hepatocytes. When cells labeled with Rhm
8
-PVA at 4C were warmed to 37C to initiate receptor-mediated internalization of the fluorescent complex, Rhm
8
-PVA remained at the cell surface. Incubation temperature at 4C or 37C had little effect on binding of Rhm
8
-PVA. We conclude that Rhm
8
-PVA is unable to evoke receptor-mediated endocytosis and can readily be used to visualize cell surface receptors in living cells.