Visualization of cell surface vasopressin V1a receptors in rat hepatocytes with a fluorescent linear antagonist
| Autor: | René Seyer, Thierry Durroux, Laurent Combettes, Dien Tran, Thierry Tordjmann, Nicole Stelly, Michel Claret |
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| Rok vydání: | 1999 |
| Předmět: |
0301 basic medicine
Vasopressin Receptors Vasopressin Histology Arginine Vasopressins Receptors Cell Surface Biology Endocytosis Ligands Cell membrane 03 medical and health sciences medicine Fluorescence microscope Animals Rats Wistar Receptor Vasopressin receptor Fluorescent Dyes integumentary system 030102 biochemistry & molecular biology Dose-Response Relationship Drug Cell Membrane Antagonist Molecular biology Rats 030104 developmental biology medicine.anatomical_structure Liver Calcium Female Anatomy Antidiuretic Hormone Receptor Antagonists |
| Zdroj: | ResearcherID |
| ISSN: | 0022-1554 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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