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Journal of Histochemistry and Cytochemistry, Vol. 47, 401-410, March 1999, Copyright © 1999, The Histochemical Society, Inc.

TECHNICAL NOTE

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

Dien Trana, Thierry Durrouxb, Nicole Stellya, René Seyerb, Thierry Tordjmanna, Laurent Combettesa, and Michel Clareta
a INSERM U.442, IFR-FR 46, Paris Sud, Orsay, France
b INSERM U.469 and CNRS UPR.9023, CCIPE, Montpellier, France

Correspondence to: Michel Claret, Signalisation Cellulaire et Calcium INSERM U.442, IFR-FR 46, Université Paris Sud, bât. 443, 91405 Orsay Cedex, France. E-mail: u442.inserm@ibaic.u-psud.fr

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. (J Histochem Cytochem 47:401–409, 1999)

Key Words: localization, V1a vasopressin receptor, fluorescent antagonist, rat hepatocytes


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