Cell. Signal. Vol. 10, No. 3, pp. 217–223, 1998 ISSN 0898-6568/98 $19.00Copyright 1998 Elsevier Science Inc. PII S0898-6568(97)00124-1

Homologous andHeterologous Acute Desensitization

of Vasopressin V1a Receptor in Xenopus OocytesNicolas Ancellin† and Alain Morel‡*

†Department de Biologie Cellulaire et Moleculaire,Service de Biologie Cellulaire, CEA Saclay, 91191 Gif-sur-Yvette

Cedex, France and ‡Universite d’Angers, 2 rue Lavoisier, 49000 Angers, France

ABSTRACT. The mechanism of short-term desensitisation of the V1a vasopressin receptor, a phospholipase-Cb linked receptor, was investigated in albino Xenopus oocytes. V1a receptors showed rapid agonist-dependentmobilisation of intracellular calcium, as detected by aequorin photon emission. Agonist-induced homologousshort-term desensitisation was evidenced within minutes after stimulation. Injection of the second messengerscalcium or inositol triphosphate inside the cell did not desensitise the receptors. In contrast, protein kinase C(PKC) activators 1-oleoyl-2-acetyl-sn-glycerol (OAG) (50 mM) and 1,2-dioctanoyl-glycerol (DIC8) (10 mM),as well as phorbol -12-myristate-13-acetate (1 mM) and phorbol -12,13-dibutyrate (1 mM) blunted the calciumresponsiveness of the V1a receptors. The specific PKC inhibitor bisindolylmaleimide (GF109203X) (1 mM) pre-vented the effect of DIC8 and OAG on V1a receptor desensitisation. Heterologous desensitisation induced byagonist occurred in oocytes that co-expressed the V1a receptor and the PKC-activating M5 muscarinic receptor.It was concluded that PKC activation has a role in short-term desensitisation of the V1a receptor. cell signal10;3:217–223, 1998. 1998 Elsevier Science Inc.

KEY WORDS. V1a vasopressin receptor, M5 muscarinic receptor, Desensitisation, DAG analogs, Phorbolesters, PKC, Aequorin

INTRODUCTION of GRKs in their desensitisation processes. The role of pro-tein kinase activation is still under debate, depending on re-G protein–coupled receptor (GPCR) activity is preciselyceptors. In addition, how different PLCb receptors sharingregulated in order to tune the cellular response to continueddiacyglycerol (DAG) and inositol triphosphate (IP3) as sec-or repeated hormonal exposure. When such a receptor hasond messengers were desensitised through similar or differ-been activated by its own ligand, its further responsivenessent mechanisms inside the same cell remains unknown.is attenuated, indicating a homologous desensitisation.

The aim of this study was, in this respect, to investigateConversely, heterologous desensitisation takes place betweenthe homologous and heterologous desensitisation pathwaysdifferent receptors [1]. Both acute desensitisation—withinof the V1a vasopressin receptor expressed in the Xenopusminutes of agonist exposure—and chronic desensitisation—oocyte, a powerful bio-sensor in receptor function research.agonist exposure for hours—have been reported for almost allThe patterns of rapid desensitisation of rat vasopressin V1athe studied G protein–coupled receptors.and muscarinic M5 receptors were defined, and the roles ofThe mechanisms of acute desensitisation have been ex-PKC activation and intracellular calcium in this mecha-tensively explored for adenylate cyclase–activating recep-nism were studied.tors, such as the b2 adrenergic receptor [2]. It is widely ac-

cepted that its desensitisation undergoes post-translationalmodifications such as phosphorylation. G protein–coupled METHODSreceptor kinase (GRK), second messenger–activated pro- In Vitro Transcriptiontein kinase A (PKA), and protein kinase C (PKC) take part

Synthetic messenger cap RNA (cRNA) corresponding toin these mechanisms of acute desensitisation [3, 4]. How-rat vasopressin V1a receptor [5] and rat muscarinic M5 re-ever, most phospholipase-Cb (PLCb)–linked receptors didceptor (kindly provided by Dr. TI Bonner, Laboratory ofnot seem to activate PKA, and little is known about the roleCell Biology, NIMH, Washington DC, USA) was producedby in vitro transcription with the use of SP6 RNA polymer-

*Author to whom correspondence should be addressed. E-mail: amorel2@ase (New England Biolabs, USA) in the presence of CAPcea.fr

Received 10 June 1997; and accepted 14 July 1997. analogue (New England Biolabs, USA), as previously de-

218 N. Ancellin and A. Morel

scribed [6]. Synthetic cRNA was checked and quantified by measured at 12-s intervals was determined for each singleoocyte. Cells with high background were rejected as un-gel electrophoresis and was then stored in aliquots at

2708C until use. Each cRNA aliquot was thawed just prior healthy or leaky. Results were expressed as arbitrary units(a.u.) of light emission corresponding to the percentage ofto injection and used once only.the maximal response obtained on the day of the assays.Each experiment was repeated at least three times, over

Oocyte Expression months, with different frogs.Female albino oocyte-positive Xenopus laevis, purchasedfrom Xenopus One (Ann Arbor, MI, USA), were fed and

Desensitisation Assaymaintained as previously described [7]. Xenopus were cold-anaesthetised and Stage V and VI albino oocytes were ob- Receptors expressing oocytes were incubated for 2 min in OR2

buffer containing 0.1% BSA and (Arg8)-vasopressin (AVP)tained after ovary section by laparotomy [8]. They were fur-ther defolliculated by exposure to OR2 buffer (82.5 mM and were then washed five times with 100 volumes of medium.

Fifteen minutes after the first exposure, a second challenge wasNaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mMNa2HPO4, 5 mM Hepes, pH 7.8) [9] containing 2 mg/mL made with 1027 M AVP, and light emission was monitored.collagenase, for 2 h at room temperature, with gentle con- Results were expressed as a percentage of the signal obtainedtinuous rocking. After extensive wash with OR2 solution in absence of the first agonist administration.and manual selection, healthy oocytes were transferred tofresh OR2 medium. Each oocyte was injected on the same

Chemicalsday with 40 nL of cRNA sample dissolved in distilled sterilewater at 1 mg/mL. Injection was performed with a glass mi- 1,2-Dioctanoyl-glycerol (DIC8), 1-oleoyl-2-acetyl-sn-glyc-cropipette connected to a pneumatic injector (Inject- erol (OAG), phorbol-12-myristate-13-acetate (PMA), phor-Matic, Geneva, Switzerland). Injected oocytes were main- bol-12,13-dibutyrate (PDBu), 4a-phorbol-12,13-didecanoatetained in the OR2 medium at 188C until use. (4aPDD), bisindolylmaleimide (GF109203X), and IP3 were

from France Biochem. Collagenase type IA, carbachol AVP,and BSA were from Sigma (France). b-Mercapto-b, b-cyclo-

Intracellular Calcium pentamethylene-propionyl-Tyr ( Me )-Phe-Gin-Asn-Cys-Pro-Detection by Photoprotein Aequorin Arg-Gly-NH2 (mAVP) and (deamino-Cys1, d-Arg8-vasopres-Cytoplasmic calcium mobilisation was detected as light sin (dDAVP) were from Bachem (France).emission generated by the photoprotein aequorin (obtainedfrom Dr. John R. Blinks, Friday Harbour Laboratory, USA),

RESULTSessentially as previously described [5, 10]. Briefly, healthyPLCb-Coupled Receptor Assay inoocytes were injected with 20 nL of 1 mg/mL aequorin inXenopus Oocytes: Characterisation and Validationcalcium-free water and stored in a glass scintillation vial

containing 500 mL OR2 with 0.1% bovine serum albumin The presence of functional receptor at the surface of the oo-cyte 2 days after injection of its synthetic messenger cRNA(BSA). Expression of the receptor was detected by light

emission after exposure to drugs in a scintillation counter was tested by aequorin assay. Oocytes that expressed theV1a receptor are characterised by a large and transient light(Intertechnique SL 3000) with the coincidence gate

switched off. Prior to each experiment, basal light emission emission within seconds after exposure to 1027 M AVP

FIGURE 1. Light emission induced by AVP in Xenopus oocyte expressing AVP V1a receptors. (A) Aequorin-loaded albino oocytesafter 2-day translation of 40 ng synthetic cap messenger RNA coding for AVP V1a receptor (h) and oocytes injected with vehiclesas control (s) were exposed to 1027 M AVP in OR2 BSA 0.1% solution. Light emission was recorded every 12 s at room temperature.Data were means 6 SEM from five oocytes expressing V1a receptor and five control oocytes. (B) Concentration-dependent effect ofAVP on the light emission peak in V1a expressing oocyte (j). Results are mean 6 SEM for at least seven oocytes for each AVP concen-tration.

V1a Receptor Desensitisation 219

(Fig. 1A). This effect can be completely blocked by mAVP[11], a specific V1a antagonist. Addition of dDAVP [12], aselective V2 agonist, at a dose as high as 1026 M did not in-duce intracellular calcium mobilisation. Oocytes injectedwith cRNA vehicle and loaded with aequorin did not pro-duce any photons in response to the same AVP concentra-tion. These results indicate that intracellular calcium mobi-lisation by AVP is tightly dependent on the injected V1acRNA. The concentration-response curve of aequorin stim-ulation by AVP is shown in Fig. 1B. The mean EC50 was 5 310210 M, a concentration close to the Kd of the V1a recep-tor. Taken together, these results show that the rat V1a re-ceptor expressed in oocytes retains the pharmacologicalproperties previously established in other tissues.

Acute Desensitisation of the AVP V1a Receptor

Desentisation of V1a-mediated Ca21 mobilisation in oocyteswas examined by sequenced hormonal stimulation. Whenfirst exposed to 1027 M AVP, oocytes injected with thecRNA produced a large calcium response (Fig. 2A). Twominutes later, cells were extensively washed with OR2 solu-tion to remove AVP from the medium. Fifteen minuteslater, they were submitted to a second exposure to 1027 MAVP. This second stimulation did not elicit any light emis-sion. When oocytes were first challenged by 10210 M AVP for2 min (Fig. 2B), the subsequent exposure to 1027 M AVP in-duced a second signal representing 20 6 6% of the light emis-sion obtained by 1027 M AVP on control oocytes (Fig. 2C).A second exposure of 1027 M AVP 24 h later resulted in aresponse comparable to the first one, indicating that the at-tenuation or the absence of vasopressin response to repeated FIGURE 2. V1a homologous desensitisation by AVP in Xeno-AVP stimulations was not the consequence of a limitation pus oocytes. V1a-expressing oocytes were tested by aequorin

assay 48 h after cRNA injection. Individual cells were first chal-in functional aequorin inside the cell (data not shown).lenged by (A) 1027 M AVP, (B) 10210 M AVP, or (C) vehicleOR2; light emission was recorded immediately. After 2 min, oo-cytes were extensively washed with OR2 0.1% BSA. FifteenEffect of Intracellular Calcium and IP3 minutes after the first AVP challenge, a second exposure to 1027

on Acute Attenuation of the V1a Receptor M AVP concentration was performed and light emission was re-corded. Data, expressed as a percentage of the peak light emis-One of the major events in the signalling pathway of thesion, are means 6 SEM from five oocytes.V1a receptor is an elevation of intracellular calcium con-

centration. The hypothesis that calcium is responsible forEffect of Diacylglycerol Analoguesthe desensitisation process was tested by injecting 30 nL ofand Phorbol Esters on V1a Responsiveness10 mM calcium inside the aequorin-loaded cell. The light

emission obtained by this procedure was similar to that ob- DAG, which is also produced from phosphatidyl inositol bi-tained by 1027 M AVP exposure. Oocytes stimulated 10 phosphate hydrolysis by PLCb, could be another signallingmin later by 1027 M AVP were still able to mobilise intracy- candidate participating in acute attenuation of the V1a re-toplasmic calcium (Fig. 3A). There was no significant dif- ceptor activity. This possibility was investigated by usingference in the response to AVP between oocytes injected OAG and DIC8 [13, 14], two cell-permeable DAG ana-with calcium, and those that were not. Another potential logues. As shown in Fig. 4, incubation of the oocytes withcandidate for desensitisation is the IP3 produced by activa- either OAG (50 mM) or DIC8 (10 mM) for 10 min attenu-tion of the PLCb, either by desenstitisation of the IP3 recep- ated the subsequent response of the V1a receptors to 1027 Mtor inside the cell or be calcium store depletion or by any AVP by respectively 86 6 6% and 82 6 7%. To test the possi-other mechanisms. When 20 nL of 1023 M IP3 was injected bility of an attenuation by OAG of the IP3 receptor func-per oocyte, a large calcium mobilisation was detected (Fig. tionality, oocytes incubated with 50 mM OAG for 10 min3B). A 1027 M AVP stimulation 10 min later induced a sec- were injected with IP3, and intracellular luminescence wasond intracellular calcium peak. A subsequent equivalent IP3 recorded. No differences between oocytes incubated with

OAG and control oocytes was evidenced (data not shown).injection also induced a new calcium signal.

220 N. Ancellin and A. Morel

FIGURE 3. Effect of CaCl2 or IP3 on the desensitisation of the V1a receptor. (A) V1a-expressing oocytes injected with 30 nL of 10mM CaCl2 were stimulated 10 min later with 1027 M AVP. Controls from the same batch of oocytes were not injected with CaCl2.Responses are the means 6 SEM from 12 oocytes and expressed as the percentage of the peak light emission obtained in control oocytes.(B) Light emission was followed before and after 20 nL injection of 1 mM IP3 into V1a expressing oocytes loaded with aequorin. Thesame cells were then exposed to 1027 M AVP and again injected with IP3. Representative of the results from 10 experiments.

PKC is the principal target of DAG inside the cell. Its with 1 mM GF109203X (bisindolylmaleimide), a highly se-lective cell-permeable PKC inhibitor (Fig. 6) [17].involvement in acute homologous attenuation of the V1a was

assessed with the potent PKC activators PMA and PDBu [15].Under our conditions (1 mM, 10 min), both phorbol esters

Acute Heterologousblunted the response of the V1a receptor (91 6 5% for PMADesensitisation Between V1a and M5 Receptorsand 94 6 6% for PDBu), whereas incubation with 4aPDD

[16], an inactive phorbol ester, had no effect of AVP stimu- Muscarinic M5 receptor was expressed in oocytes to stimu-lation (Fig. 5). late the signalling pathways shared with the V1a receptor.

V1a acute desentitisation by the DAG agonists OAG and Oocytes injected with the cRNA coding for the M5 recep-tor and challenged with carbachol 1024 M showed mobilisa-DIC8 was prevented by pre-incubation of oocytes for 30 min

FIGURE 4. Effect of diacylglyc-erol analogues on the V1a re-ceptor signalling pathway. Oo-cytes expressing the V1a receptorwere incubated, either with 50mM OAG (h) or 10 mM DIC8(d) OR2 0.1% BSA for 10 minprior to functional assay with1027 M AVP. Control (D) cor-responded to oocytes incubatedwith the drug vehicle (DMSO)diluted to the same final concen-tration (,0.2%). For each setof conditions, data are themeans 6 SEM from 12 oocytesand expressed as a percentage ofthe peak light emission.

V1a Receptor Desensitisation 221

FIGURE 5. Effect of phorbol esters on the V1a receptor signal- FIGURE 6. Effects of GF109203X on desensitisation inducedling pathway. Oocytes expressing the V1a receptor were incu- by DAG analogues. Oocytes micro-injected with V1a cRNA werebated, either with 1 mM PMA, 1 mM PDBu, or 1 mM aPDD incubated for 10 min with the studied compounds. Where indi-in OR2 0.1%BSA for 10 min prior to functional assay with 1027 cated, oocytes were pretreated for 30 min with 1 mM GF109203XM AVP. Controls were oocytes incubated with the drug vehicle in OR2 0.1% BSA prior to drug exposure, and cells were subse-(DMSO) diluted to the same final concentration (,0.2%). For quently tested for 1027 M AVP response. Results are the means 6each set of conditions, data are the means 6 SEM from 10 oo- SEM for at least 12 oocytes for each experiment.cytes and expressed as a percentage of the peak light emission.

tion of intracellular calcium similar to that of the V1a re- sengers and non-permeable drugs, offers a unique opportu-ceptor, as detected by aequorin (Fig. 7). In addition, the M5 nity to address several questions relative to acute homolo-receptor underwent a homologous desensitisation process gous and heterologous receptor regulation.similar to that presented in Fig. 1 for the V1a receptor (data Rat V1a cRNA or rat M5 cRNA was injected into oo-not shown). Acute desensitisation of the M5 receptor was cytes and, after its translation, the aequorin assay [5, 10] wasalso evidenced either by 10 mM DIC8 (88 6 7% loss of cal- used to characterise the functional properties of the recep-cium signal compared with the vehicle-treated oocytes) or tors. We first reported that the V1a receptor expressed in1 mM PMA (92 6 5% of desensitization) for 10 min. As ex- Xenopus oocytes could undergo a marked acute homologouspected, the effect of DIC8 was prevented by pre-incubation desensitisation after a 2-min exposure to its agonist AVP.with 1 mM GF109203X for 30 min. Heterologous attenuation of the V1a receptor by another

The V1a and M5 cRNA were further co-injected into activated PLCb-linked receptor, the muscarinic M5 chal-Xenopus oocytes to obtain V1a-M5–expressing cells. As lenged by 1024 M carbachol for 2 min also was evidenced.shown in Fig. 7, AVP was unable to raise the cytoplasmic Taken together, these results indicate that, in Xenopus oo-calcium concentration when the V1a-M5–expressing oo- cyte, two different PLCb receptors linked to similar signal-cytes were first challenged by carbachol. Moreover, carba- ling pathways may be acutely desensitised in homologous orchol did not induce a calcium response after exposure to heterologous ways. Internalisation of the receptor may beAVP. This experiment indicated that heterologous desen- partly responsible for the desensitisation process [1]. Thissitisation between V1a and M5 occurred in both ways in does not seem to apply to our experiments. It was previouslythis co-expressing system. reported [19] that, in V1a-expressing oocytes, only 50% of

the AVP-binding sites were internalised after a 10-min ex-posure to 1027 M AVP. In addition, the present study shows

DISCUSSION that a 2-min exposure to 10210 M AVP, which would havea small, if any, effect on internalisation, induces an acuteThe capacity of Xenopus oocytes to translate foreign mes-desensitisation (80 6 6% loss of calcium signal with regardsenger RNA has been extensively used in recent years to ex-to the control oocytes). The heterologous desensitisationpress functional proteins. In particular, they have been usedbetween the M5 and the V1a receptor induced by DAG an-for numerous G protein–coupled receptors linked to PLCb,alogues also suggests that internalisation of the ligand re-including biogenic amine and peptide receptors [7, 18]. In

addition, the possibility of injecting proteins, second mes- ceptor complex for vasopressin is not involved in acute de-

222 N. Ancellin and A. Morel

To test this hypothesis, calcium was injected into the cellsto a final concentration of 1026 M. In these conditions,there was no detectable alteration in the response of the oo-cytes to a further challenge with AVP, which strongly sug-gests that calcium alone, at a level similar to that obtainedby hormone stimulation, does not take part in the processof acute desensitisation. Activation of PKC is mainly medi-ated by the DAG produced by the hydrolysis of phosphati-dyl inositol biphosphate. The importance of PKC on desen-sitisation of the V1a receptor has been studied by others[19–21]. Their results, however, are conflicting. In the pres-ent study, we report that OAG and DIC8, two specific PKCactivators, induced AVP V1a desensitisation, as did thephorbol ester PMA. This observation contrasts with the re-sults of Nathanson et al. [19], who showed that AVP V1areceptor desensitisation in Xenopus oocytes was insensitiveto PDBu. These differences in the results are probably re-lated to the kinetics of the PDBu effect. Indeed, we foundthat a 3-min incubation with PDBu—the time used by Na-thanson et al.—results in a 14 6 7% attenuation of the re-ceptor activity; a 6-min incubation, 82 6 9%; and a 10-minincubation, 94 6 6% (data not shown). The discrepancybetween Nathanson’s results and ours may just be the con-sequence of a longer incubation time with PDBu. The ab-sence of desensitisation with inactive phorbol ester 4aPDDand the fact that GF109203X, a PKC inhibitor, preventedthe action of OAG and DIC8 also argue for an involvementof PKC in desentitisation control of the V1a receptor. AnFIGURE 7. Heterologous desensitisation in oocytes co-express-increase in intracellular calcium after IP3 injection in oo-ing the vasopressin V1a and muscarinic M5 receptors. Oocytes

were micro-injected 2 days before experiments with V1a and M5 cytes treated with OAG also indicates that the PKC targetscRNA. Oocytes were first stimulated by 1024 M carbachol were upstream of the IP3 receptor system.(Cch) or 1027 M AVP, and the response was recorded as pre- Cross desensitisation by receptors having similar signal-viously described. After 15 min, when light emission returned to

ling pathways has been detected by co-expressing V1a andbaseline, the same cell was exposed to 1027 M AVP or 1024 MM5 receptors in oocytes. Addition of vasopressin or carba-Cch, respectively. Each point represents the mean percentage of

the peak light signal 6 SEM from 10 oocytes. chol on V1a-M5–expressing oocytes showed that heterolo-gous desensitisation occurred between the two receptors.This indicates that the desensitisation of a receptor may oc-

sensitisation. Response attenuation of the receptor thuscur in the presence or in the absence of its own ligand.seems to be mainly the consequence of a rapid uncouplingCommon desensitisation pathways for these receptors pro-of the receptor from the downstream signalling pathwayducing similar second messengers was evidenced by theirrather than internalisation.comparable pharmacological properties. The effects ofThe mechanisms of this acute desensitisation were fur-PMA and DIC8 on desensitisation of M5 receptors were in-ther investigated. Our data indicated that the second mes-deed similar to those obtained with V1a (respectively 92 6senger IP3 alone, directly injected into oocytes (Fig. 3), was5% and 88 6 7% of desensitisation) and, as expected,not able to attenuate the response evoked by subsequent ex-DIC8-induced desensitisation was inhibited by GF109203Xposure to AVP. Moreover, because serial IP3 injections(data not shown), implicating PKC in M5 desensitisation.could repeatedly induce a calcium response, alteration of

In conclusion, acute homologous desensitisation of vaso-the calcium mobilisation by this PLCb-linked receptorpressin V1a receptor is independent of the rise in intracellu-seemed to be independent of intracellular IP3 receptor de-lar calcium or inositol phosphates. In contrast, stimulationsensitisation itself, at least in this expression system. Fromof PKC activity induced a marked desensitisation of thethese results, we concluded that the observed short-term de-V1a receptor. This desensitisation due to PKC activationsensitisation of the V1a receptor was not the consequencewas independent of occupancy of the receptor by vasopres-of a depletion of intracellular calcium stores.sin. Acute desensitisation may also be induced by anotherCalcium released by the IP3-sensitive intracellular storePLCb-coupled receptor expressed in the same cell, indicat-may activate some protein kinases or calcium-dependent

protein potentially participating in acute desensitisation. ing heterologous desensitisation.

V1a Receptor Desensitisation 223

10. Sandberg K., Markwicki A. J., Trinh D. P. and Catt K. J.(1988) FEBS Lett. 241, 177–180.We wish to thank B. Corman for the critical reading of the manuscript.

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12. Kruszynski M., Lammek B., Manning M. M., Seto J., HaldarJ. and Sawyer W. H. (1980) J. Med. Chem. 23, 364–368.

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