TECHNICAL NOTE |
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
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Summary |
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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 47:401409, 1999)
Key Words: localization, V1a vasopressin receptor, fluorescent antagonist, rat hepatocytes
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Introduction |
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Vasopressin V1a receptors are found in a variety of tissues, including liver, blood vessels, kidney, choroid plexus, and central nervous system (for reviews see 11/G
q proteins (
V1a vasopressin receptors have been visualized in several tissues by different methods (for reviews see
To visualize cell surface V1a vasopressin receptors in rat hepatocytes in the absence of receptor-mediated endocytosis (
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Materials and Methods |
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Preparation of Rat Hepatocytes
Single cells (70%) and multicellular systems (30%), called multiplets (doublets and triplets), were prepared from fed female Wistar rats by limited collagenase (Boehringer Mann-heim; Meylan, France) digestion of rat liver, as previously described (
Synthesis of the Fluorescent Vasopressin Antagonist
A fluorescent vasopressin antagonist, 4-HO-Ph(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5-carboxytetramethylrhodamyl)-NH2 (Rhm8-PVA), which binds to vasopressin V1a receptors, has been recently synthesized. The synthetic strategy involved Rhm8-PVA by coupling 5-carboxy tetramethyl rhodamine to a Lys8 linear vasopressin antagonist (
Cell Loading with Quin2 for Measuring [Ca2+]i in Suspensions of Rat Hepatocytes
Suspensions of rat hepatocytes were loaded with quin2 by incubation with the permeant acetoxymethyl ester form of the dye (Molecular Probes; Leiden, The Netherlands) and [Ca2+]i was measured as previously described (
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Visualization of Cell Surface V1a Receptors with Rhm8-PVA in Rat Hepatocytes
Single cells and multiplets were suspended in Williams' medium E (5 x 105 cells/2 ml), supplemented with 10% fetal calf serum, glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco BRL; Gaithersburg, MD) and plated on collagen-coated glass coverslips for 2 hr at 37C under an atmosphere containing 5% CO2. Cells were rinsed twice with MEM and preincubated with vasopressin or with the nonfluorescent vasopressin antagonist V4253 ([ß-Mercapto-ß-ß-cyclopenta-methylenepropionyl1,O-Et-Tyr2,Val4,Arg8]-vasopressin) (Sigma) at concentrations, temperatures, and time periods of incubation indicated in the figure legends. At the end of these preincubation periods, cells were rinsed twice with MEM, then further incubated for 30 min at the same temperature with 10 nM Rhm8-PVA in MEM supplemented with 1 mg/ml bovine serum albumin (Miles Laboratories; Kankakee, IL). Cells were washed twice at 4C with MEM, fixed by incubation for 15 min in 4% formaldehyde (FA), and mounted in buffered glycerin (Sanofi Diagnostics Pasteur; Marnes-la-Coquette, France). In some experiments, fixed cells were further incubated with FITCphalloidin (Sigma) (1:100 for 15 min at 22C) to label actin.
All images were observed with an epifluorescent photomicroscope Axioskop (Carl Zeiss; Le Pecq, France) equipped with a high-resolution (752 x 582 pixels) and sensitive 3CCD cooled camera LH750RC3 (Lhesa; Cergy Pontoise, France). The excitation wavelengths ex were 546 nm for Rhm8-PVA and 490 nm for FITCphalloidin. Throughout the study, and for longitudinal studies over longer time periods, the 3CCD camera had its mode "auto mode" turned off and manual controls were used to adjust the image intensity and the acquisition time to 4 sec. These controls were kept at an identical level during the studies. Standardized controls of fluorescence were used every day, with proper calibrations of the microscope and 3CCD camera to yield comparable conditions for image collections and analyses. Red and green fluorescence controls were obtained using Inspeck Red (580/605) and Green (505/515) Microscope image intensity calibration standards (Molecular Probes). Digitalized images were acquired with Photomat software and were stored on an external Magneto Optical Disk Device (Sony; Tokyo, Japan).
All results are expressed as means ± SEM.
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Results and Discussion |
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Synthesis and Characterization of Rhm8-PVA
The synthesis and characterization of the fluorescent vasopressin antagonist Rhm8-PVA are described elsewhere (
Rhm8-PVA Does Not Stimulate Ca2+ Signals and Competitively Blocks Vasopressin-mediated Ca2+ Signals
The ability of Rhm8-PVA to activate hepatocyte V1a receptors was assessed by fluorescence spectroscopy to evaluate its ability to evoke Ca2+ signals in cell suspensions. The presence of a rhodamine-labeled antagonist did not affect [Ca2+]i signals, as measured using the fluorescent indicator quin2. Addition of 20 nM Rhm8-PVA abolished Ca2+ signals elicited by 1 nM vasopressin within 1 min (Figure 1). Rhm8-PVA itself, at concentrations from 0.1 nM to 100 nM, was unable to evoke Ca2+ signals (see the control curve in Figure 2). These concentrations are above its Ki for binding to V1a vasopressin receptors and its Kinact for inhibiting vasopressin-mediated synthesis of InsP3 in the CHO cell line. The fluorescent probe did not prevent the [Ca2+]i rises generated by the 1-adrenergic agonist norepinephrine (not shown). Because vasopressin and norepinephrine use different hormone receptors (
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Competition inhibition assays were used to establish the inactivation constant of Rhm8-PVA in suspensions of rat hepatocytes. The inhibition by 20 nM Rhm8-PVA of increases in [Ca2+]i caused by various concentrations of vasopressin (not shown), and the effects of various concentrations of Rhm8-PVA on Ca2+ signals elicited by a concentration of vasopressin near the Kinact (0.4 nM), were determined (Figure 2). The control curve shows that Rhm8-PVA at concentrations of 0.1100 nM did not alter basal [Ca2+]i. The concentration of Rhm8-PVA that resulted in half-maximal inhibition of Ca2+ signals induced by 0.4 nM vasopressin was 7 nM. The Kinact for Rhm8-PVA inactivation of V1a receptors in rat hepatocytes was calculated to be 3.0 nM. These results show that, as in CHO cells expressing V1a vasopressin receptors (
Visualization of Rhm8-PVA Binding to Cell Surface of Rat Hepatocytes
The ability of Rhm8-PVA to bind and to be internalized was evaluated by epifluorescence microscopy with a high resolution and sensitive 3CCD camera to improve the resolution of the image and highlight the binding of the fluorescent vasopressin analogue Rhm8-PVA to V1a receptors (see Materials and Methods). The experiments were carried out with single cells and also with doublets and triplets of rat hepatocytes that retain certain aspects of structural and functional epithelial polarity, particularly the expression of basal, lateral and canalicular domains found in the intact liver (
The 3CCD camera revealed the fluorescent signal at the periphery of cells. In cells incubated with 10 nM Rhm8-PVA for 30 min at 4C, the fluorescent antagonist bound to the surface of cells, presumably the plasma membrane of rat hepatocytes (compare the peripheral labeling of the single cell and the triplet observed in Figure 3A to that seen in Figure 3B). Significant fluorescent signal was also observed intracellularly, close to nuclei. Because this labeling was present in cells incubated without Rhm8-PVA (Figure 3B), it may have been due to the high autofluorescence signal consistently found in rat hepatocytes (
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The cell doublet system has been widely used to investigate a variety of cytoskeletal-dependent functions because the networks of actin and tubulin remain polarized in these preparations (
Rat hepatocyte doublets were incubated (as described for Figure 3) with 10 nM Rhm8-PVA for 30 min at 4C to label V1a receptors, washed, fixed, and then incubated with FITCphalloidin (see Materials and Methods) for labeling actin. Both Rhm8-PVA (Figure 4A) and FITCphalloidin (Figure 4B) labeled a very thin layer at the periphery of the cells, corresponding to the basolateral domain of the plasma membrane. The fluorescent V1a receptor antagonist was not observed in the apical area, a domain strongly labeled by FITCphalloidin in the hepatocyte doublet (
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Specificity of Rhm8-PVA Binding to V1a Receptors
The V1a receptor specificity of Rhm8-PVA binding was assessed by investigating its displacement by the nonfluorescent antagonist V4253 and by the natural hormone vasopressin. The experiment was performed at 4C throughout to prevent any receptor-mediated endocytosis (
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Vasopressin Induces Internalization of V1a Receptors
We compared the internalization of Rhm8-PVA and vasopressin in rat hepatocytes. We investigated whether Rhm8-PVA binding to the cell surface of rat hepatocytes was abolished by prior endocytosis of the V1a receptors. V1a receptor-mediated endocytosis was initiated by preincubation for 560 min with 100 nM vasopressin at 37C, and then cells were cooled at 4C to examine binding of Rhm8-PVA (10 nM for 30 min). For all periods of preincubation, prior vasopressin-mediated endocytosis of V1a receptors abolished antagonist binding to the cell surface (Figure 6BD) whereas in non-preincubated cells, Rhm8-PVA bound to the cell surface of rat hepatocytes (Figure 6A). The V1a receptor of rat hepatocytes is therefore specifically internalized by the natural agonist vasopressin. In addition, the development of vasopressin-mediated receptor endocytosis detected in fluorescence microscopy is as rapid (less than 5 min) as that previously observed using tritiated vasopressin in suspensions of rat hepatocytes (
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Rhm8-PVA Does Not Induce Internalization of V1a Receptors
Because Rhm8-PVA is a specific antagonist of V1a receptors in rat hepatocytes, we investigated whether incubation of cells at 37C can elicit endocytosis of the Rhm8-PVA/V1a receptor complex. Rat hepatocytes were equilibrated with 10 nM Rhm8-PVA for 30 min at 4C to label V1a receptors (Figure 7A) and then warmed to 37C to initiate receptor-mediated internalization of the fluorescent complex for 1, 3, 5, 15, and 30 min (Figure 7BD). The Rhm8-PVA label remained at the cell surface even after 30 min. Therefore, unlike vasopressin, Rhm8-PVA is unable to evoke receptor-mediated endocytosis of V1a receptors in rat hepatocytes.
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Effect of Temperature on Rhm8-PVA Binding
The incubation temperatures at 4C and 37C had little effect on binding and noninternalization of 10 nM Rhm8-PVA by rat hepatocytes. At the two incubation temperatures, Rhm8-PVA remained bound to the cell surface for 15 and 30 min (not shown), whereas vasopressin-mediated endocytosis of V1a receptors is complete by that time (see Figure 7).
Conclusion
In summary, the vasopressin fluorescent linear antagonist Rhm8-PVA binds V1a receptors with a high affinity and does not elicit cellular responses in single cells and multiplets of rat hepatocytes. The inability of Rhm8-PVA bound to the cell surface to be internalized indicates that occupancy of the hepatocyte vasopressin V1a receptor in the absence of receptor activation is insufficient to elicit receptor-mediated endocytosis, as previously observed with another fluorescent antagonist in the A10 rat smooth muscle cell line expressing V1a vasopressin receptors (
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Acknowledgments |
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Supported by the Association pour la Recherche sur le Cancer, la Fondation pour la Recherche Médicale, and the Association Française Contre les Myopathies.
We thank Raymonde Leuillet and Denise Reuter for excellent technical assistance and A. Edelman for help in editing the manuscript.
Received for publication July 31, 1998; accepted October 27, 1998.
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