Phorbol ester differentially regulates oxytocin receptor binding activity in hypothalamic cultured neurons and astrocytes

Peptides 22 (2001) 677– 683

Phorbol ester differentially regulates oxytocin receptor binding activity in hypothalamic cultured neurons and astrocytes Marie-The´re`se Strossera, Marie-Elisabeth Evrarda, Christophe Bretonb, Dominique Guenot-Di Scalaa,* a

Laboratoire de Neurophysiologie Cellulaire et Inte´gre´e, UMR 7519, CNRS ULP, 21 rue Rene´ Descartes, 67084 Strasbourg Cedex, France b Laboratoire d’Endocrinologie des Anne´lides, Universite´ des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France Received 1 August 2000; accepted 27 November 2000

Abstract Hypothalamic cultured neurons and astrocytes were used to investigate the cellular mechanisms underlying the oxytocin receptormediated downregulation through a possible involvement of protein kinase C (PKC). For this purpose, the effects of PKC activators, inhibitor and of OT on OT receptor binding activity were compared in both cultures. In neurons, phorbol-myristate-acetate (PMA), a potent PKC activator, increased the binding of an OT receptor antagonist whereas in astrocytes, a decrease was observed. Pre-treatment of the cells with bisindolylmaleimide (10⫺4 M), a PKC inhibitor, prevented the PMA-induced up- and downregulation. In contrast, receptor downregulation resulting from treatment of both cells with OT (10⫺9 M) was not affected by the PKC inhibitor. On the other hand, when PMA (10⫺7 M) was tested along with OT (10⫺9 M), a subsequent decrease in ligand binding was observed in astrocytes. In neurons, PMA attenuated the OT-induced downregulation. Structural analysis of neuron and astrocyte OT receptor mRNA by RT-PCR, subcloning and sequencing, demonstrated identical sequence to rat uterine receptor. In conclusion, these data suggest that activation of PKC has opposite effect on OT receptor binding activity in neurons and astrocytes but they do not support the involvement of PKC in the OT-induced downregulation. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Protein kinase C; Oxytocin receptor; Neurons; Astrocytes; Phorbol ester

1. Introduction Apart from its peripheral role, oxytocin (OT) exhibits also central neuromodulatory effects [30]. The use of brain cell cultures has allowed studies of the central OT receptors at a cellular level and the presence of specific OT receptors on cultured neurons and astrocytes has been conclusively demonstrated [9,10]. The astrocyte OT receptor has been more extensively studied and it could be demonstrated that the binding of OT on a G protein-coupled receptor [11] increases intracellular Ca2⫹ concentration mobilized from IP3-sensitive stores [12]. It is well established that hydrolysis of phosphoinositides produced IP3 and diacylglycerol (DAG) which are able to mobilize calcium from intracellular stores and activate protein kinase C (PKC) respectively * Corresponding author. Tel.: ⫹33-3-88-27-53-67; fax: ⫹33-3-88-2635-38. E-mail address: [email protected] (D. Guenot-Di Scala).

[2]. The PKC family consists of at least 12 isoforms which have closely related structures but differ in their individual properties [8,18]. So far, it is not clear whether all cell types in the brain express all members of the PKC isoforms [21,35] although analysis conducted on cell cultures demonstrated PKC localization in neurons and astrocytes but the isoform expression remains cell-type specific [23,29]. PKC activity in intact cells can be stimulated by tumourpromoting phorbol esters which bind to the diacylglycerol (DAG) binding site of PKC [6]. Due to the potency and stability of phorbol esters, the enzyme is permanently stimulated unless its down regulation makes it inactive [40]. A number of studies performed with phorbol esters have provided insight on PKC effects on cell functions including proliferation and differentiation [24,25,39], transmitter release [37] and control of receptor levels [32,33] in a great variety of cells. However, very few studies concerned the regulation of the OT receptor expression. At the periphery, there is clear evidence for the involvement of the phosphoinositide pathway in the OT receptor activation but only

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indirect studies suggest implication of PKC [15,28]. Concerning the central OT receptor, the transduction pathways involved are only speculative and based on observations of the peripheral OT receptor. Therefore the purpose of the current study was to investigate the potential regulatory influence of PKC, activated by a phorbol ester PMA or by agonist receptor occupation, on OT receptor binding activity in cultured neurons and astrocytes obtained from embryonic rat hypothalamus. 2. Materials and methods 2.1. Cell cultures Hypothalami from 16-day-old rat embryos were dissected out from the brain, placed in Earle’s balanced salt solution without Ca2⫹ and Mg2⫹ and their meningeal membranes carefully removed. Neuron-enriched cultures were obtained by triturating the tissues through a Pasteur pipette and the cell suspension, filtered through a 48 ␮m nylon mesh, was sedimented at 400 g for 10 min. The pellet was resuspended in Dulbecco’s modified Eagle’s medium (DMEM prepared in Volvic mineral water) containing 0.6% glucose and 20% foetal calf serum (FCS, DAP France). Cells were seeded in 12-well clusters precoated with poly-L-lysine at a density of 1/2 hypothalamus per well and medium was replaced by the chemically defined medium of Bottenstein and Sato [3] 24 hours after seeding. Astrocyte cultures were obtained by triturating the tissues through a syringe in DMEM supplemented with 15% FCS. Cell suspension was plated in 12-well clusters at a density of 1/3 hypothalamus per well. Both cell cultures were incubated at 37°C in a humidified incubator with 5% CO2-95% air until experiments were performed after 12 days in culture. 2.2. Treatment of the cells Before experiments were performed, the culture medium was removed and replaced by the defined medium of Bottenstein and Sato [3] then cells were treated with various agents for different periods of time. In all experiments the appropriate vehicle was added to the medium in control cells.

2.4. RT-PCR analysis Total RNA from parturient uterus, cultured neurons and astrocytes was extracted using Trizol (Gibco/BRL, France). 3 ␮g RNA were reverse transcribed into cDNA using random hexamers and Moloney Murine Leukemia Virus RT (Gibco/BRL) as previously described [4]. One sixth of the first strand synthesis reaction was amplified for 35 cycles using 1 U Taq polymerase (Eurobio) and 100 pmol of each forward and reverse primer. The cycling parameters were: 94°C for 90 sec, 70°C for 90 sec and 72°C for 120 sec. Negative control RT-PCR reactions were performed by omitting reverse transcriptase or RNA from the reaction mixture. The forward primer pair consisted of a 29-mer corresponding to the region encoding residues 298 –306 and the reverse primer (30-mer) was complementary to the region encoding residues 379 –388 of the rat OT receptor. The predicted size of the PCR products was 274 bp. The priming sites were separated on the OT receptor gene by a large intron (⬎12 kilobases), thus preventing amplification of any contaminating genomic DNA [31]. The PCR products were subcloned using TA cloning vector systems (Stratagene, France) and sequenced to verify the specificity of the amplification. 2.5. Data analysis Results are expressed as the percentage of specific binding of the radioiodinated ligand to control cells. Experimental data of saturation studies were analysed using the nonlinear regression program (Kell program-BiosoftCambridge). Statistical analysis were performed with the non parametric Wilcoxon test and differences of p ⬍ 0.05 were considered to be significant. 2.6. Chemicals The OT antagonist d(CH2)5[Tyr-(Me)2,Thr4,TyrNH92]OVT (OTA) was a gift of Dr. M. Manning (Medical College of Ohio, USA). OT was purchased from Bachem (Torance, CA, USA). Phorbol myristate acetate (PMA), bisindolylmaleimide (BIM), 1-oleoyl-2-acetylglycerol (OAG), cycloheximide and 4␣-phorbol 12–13 didecanoate (4␣-PDD) from Sigma (Saint-Louis, MO, USA). These reagents were dissolved in a stock solution and stored at ⫺20°C.

2.3. Binding assay 3. Results As described elsewhere [9], binding was performed on intact cells attached to the dish. The radioiodinated OT antagonist d(CH2)5[Tyr-(Me)2,Thr4,Tyr-NH92]OVT ([125I]OTA) used at a concentration of 0.03 nM in binding buffer (85 mM Tris-HCl pH 7.4, 5 mM Mg2⫹, 0.1% BSA) was allowed to incubate for 90 min at 37°C. The non specific binding was estimated by adding 10⫺6 M OT (Bachem, France) and never exceeded 20% of the total binding.

3.1. Effect of PMA on [125I]OTA binding in cultured neurons and astrocytes 3.1.1. Effect of PMA The potent PKC activator PMA induced an increase in ligand specific binding in neurons while the binding was decreased in astrocytes (Table 1).

M.-T. Strosser et al. / Peptides 22 (2001) 677– 683 Table 1 Time- and dose-dependency of PMA on [125I]OTA specific binding in cultured neurons and astrocytes Time of treatment

NEURONS

ASTROCYTES

1/2 h 2h 18 h 24 h PMA concentrations 10⫺9 M 10⫺8 M 10⫺7 M 10⫺6 M

114 ⫾ 4a (12) 117 ⫾ 8a (6) 123 ⫾ 3.5a (17) 126 ⫾ 6a (9)

90 ⫾ 2a (12) 83 ⫾ 3a (17) 53 ⫾ 3a (22) 49 ⫾ 3a (10)

114 ⫾ 4 (4) 126 ⫾ 6a (4) 127 ⫾ 8a (9) 112 ⫾ 4a (4)

84 ⫾ 6 (5) 55 ⫾ 4a (5) 45 ⫾ 4a (7) 55 ⫾ 5a (5)

a

a

When cells were tested for time dependency, PMA was used at a concentration of 10⫺7 M. When different PMA concentrations were tested, cells were incubated for 18 hours with PMA. Data are Mean ⫾ S.E.M. for (n) experiments including 4 determinations in each experiment. a p ⬍ 0.05 compared to control.

In neurons, the PMA induced a significant increase of the ligand specific binding after 30 min incubation ( p ⬍ 0.05) and the effect was maximal with 10⫺7 M PMA after 18 hours of treatment (123 ⫾ 3.5% of the control value). The PMA-induced increase was not significantly time- and dosedependent (Table 1). In astrocytes, the PMA-induced decrease of the ligand specific binding was time- and dose-dependent and a significant decrease was observed after 30 min treatment ( p ⬍ 0.05) with a maximal effect at 18 hours with 10⫺7 M PMA (53 ⫾ 3% of the control value). A further slight decrease was observed after 24 hours. For the following studies, neurons and astrocytes were treated with 10⫺7 M PMA for 30 min and 18 hours. To examine whether the PMA-induced increase of ligand binding in neurons required protein synthesis, cells were treated with 10⫺7 M PMA and 2 ⫻ 10⫺6 M cycloheximide for 18 hours. Cycloheximide by itself slightly decreased the binding to 90 ⫾ 5% but a further decrease of the binding was observed when cells were treated with both drugs (from 123 ⫾ 3.5% with PMA to 72 ⫾ 3% with PMA ⫹ cycloheximide, p ⬍ 0.05). 3.1.2. Saturation experiments The Scatchard analysis of a saturation experiment for untreated and PMA-treated neurons and astrocytes as function of increasing ligand concentrations exhibited Hill coefficient close to 1 for both experimental conditions. This suggests that the ligand binds to a homogenous population of receptors (data not shown). PMA treatment resulted in a 29% increase of the number of binding sites in neurons and a 48% decrease in astrocytes without significantly altering their binding affinities (Table 2).

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Table 2 Scatchard analysis of saturation experiments NEURONS control with PMA ASTROCYTES control with PMA

Kd

B max ⫺11

3 ⫻ 10 M 5 ⫻ 10⫺11M 2.2 ⫻ 10⫺11M 2.4 ⫻ 10⫺11M

13.5 fmol/mg protein 17.5 fmol/mg protein 16 fmol/mg protein 7.8 fmol/mg protein

Saturations were performed in neurons and astrocytes treated for 18 h with PMA 10⫺7 M before binding experiments. K d is the dissociation constant at equilibrium and B max the maximal binding capacity.

3.1.3. Specificity The specificity of the PMA effect was evaluated by treating the cells with an inactive form of phorbol ester, 4␣-PDD. At a concentration of 10⫺7 M, 30 min or 18 hours treatment with this compound had no effect on ligand binding on both neurons and astrocytes (data not shown). The ability of the synthetic diacylglycerol (OAG) to affect ligand binding was compared to PMA on both cell types. OAG (10⫺6 M) was as potent as PMA to increase the specific ligand binding in neurons and to decrease it in astrocytes (Table 3). 3.1.4. Recovery Recovery of the up- and downregulatory PMA-induced effect in neurons and astrocytes was evaluated by treating the cells for 18 hours with 10⫺7 M PMA then allowing them to recover for 24 hours with the medium alone. In these conditions, the specific binding decreased in neurons from 123 ⫾ 3.5% with PMA to 107 ⫾ 5% ( p ⬍ 0.05) 24 hours after incubation with the medium alone. In astrocytes, the specific binding increased from 53 ⫾ 3% with PMA to 78 ⫾ 3% ( p ⬍ 0.05) with the medium alone.

Table 3 Effects of PMA 10⫺7 M, OAG 10⫺4 M and BIM 10⫺6 M on [125I]OTA specific binding in cultured neurons and astrocytes NEURONS

1/2 h

18 h

PMA OAG BIM PMA ⫹ BIM ASTROCYTES PMA OAG BIM PMA ⫹ BIM

114 ⫾ 4 (12) 111 ⫾ 4a (5) 95.2 ⫾ 6.8 (5) 86.7 ⫾ 6.4b (8)

123 ⫾ 3.5a (17) 123 ⫾ 8a (6) 99.8 ⫾ 7 (5) 108 ⫾ 4.6b (5)

89.9 ⫾ 1.9a (12) 75.3 ⫾ 4.4a (3) 98.2 ⫾ 2 (5) 99.7 ⫾ 6 (6)

53.7 ⫾ 3a (22) 58.7 ⫾ 6.7a (4) 99.5 ⫾ 5.4 (5) 89.7 ⫾ 6.5b (7)

a

BIM was added 30 min prior to the other drugs. Data are Mean ⫾ S.E.M. for (n) experiments including 4 determinations in each experiment. a p ⬍ 0.05 compared to control cells. b p ⬍ 0.05 compared to PMA-treated cells.

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3.2. Effects of PKC inhibitor on PMA-treated cells The effect of a 30 min pre-treatment with bisindolylmaleimide (BIM, 10⫺6 M) on PMA-mediated change in specific binding was examined. BIM alone had no effect on both cell types (Table 3). In neurons, BIM blocked the PMA-induced upregulation after 30 min- and 18 hour-treatments (Table 3). In astrocytes, the PMA-induced downregulation was blocked by BIM after 30 min but it was significant only after a 18 hour-treatment (Table 3). 3.3. Involvement of PKC in OT-induced downregulation 3.3.1. Effect of a PKC inhibitor OT (10⫺9 M) tested for 30 min or 18 hours produced a significant decrease of the binding on both neurons and astrocytes. This downregulatory effect of OT could never be blocked by BIM when added for 30 min or 18 hours (Table 4).

3.4. OT receptor gene expression in cultured neurons and astrocytes Structural analysis of neuron and astrocyte OT receptor mRNA was undertaken using RT-PCR analysis from three independent samples. Uterine OT receptor mRNA was used as a positive control. The sizes of the PCR amplification products corresponded to the sizes predicted from the genomic sequences. The size of the PCR bands generated were identical to the ones generated when uterine cDNA was used as a template, suggesting that, at least, within the boundaries of the primer pair used, the transcripts detected corresponded to the ones found in uterus (data not shown). The nature of the PCR products was further assessed after subcloning and sequencing of the specific bands. cDNA sequences obtained for rat uterus, neurons and astrocytes were the same and were identified as the sequence of rat OT receptor.

4. Discussion 3.3.2. Effect of PMA on OT-induced downregulation When neurons were simultaneously treated with OT (10⫺9 M) and PMA (10⫺7 M), the OT-induced downregulation was impaired by simultaneous addition of PMA (Table 4) and this effect was significant at 30 min and 18 hours when compared to OT or PMA alone (also see Table 3). The PKC inhibitor, BIM, was unable to block the effect of PMA on OT-induced downregulation (Table 4). In astrocytes, the OT-induced downregulation was further decreased by addition of PMA (Table 4). This effect was significant at both times tested when compared to PMA or OT alone (see also Table 3). The PKC inhibitor, BIM, was unable to reverse the effect of PMA-enhanced downregulation (Table 4).

Table 4 Effect of OT alone or combined to a PKC inhibitor (BIM) or to PMA on [125I]OTA specific binding in cultured neurons and astrocytes NEURONS

1/2 h

18 h

OT OT ⫹ BIM OT ⫹ PMA OT ⫹ PMA ⫹ BIM ASTROCYTES OT OT ⫹ BIM OT ⫹ PMA OT ⫹ PMA ⫹ BIM

73.6 ⫾ 3.8 (8) 80 ⫾ 7.8a (5) 87 ⫾ 4b (6) 86 ⫾ 4b (5) a

82.9 ⫾ 2.8a (12) 79.3 ⫾ 1.4b (5) 68.5 ⫾ 4b (9) 67 ⫾ 4 (5)

43 ⫾ 5a (6) 50 ⫾ 7.6a (5) 73 ⫾ 7b (7) 69 ⫾ 6b (5) 34.5 ⫾ 2.5a (9) 34.4 ⫾ 1.6b (5) 14 ⫾ 2.5b (6) 22 ⫾ 3 (5)

BIM (10⫺6 M) was added 30 min prior to OT 10⫺9 M and PMA 10⫺7 M. Data are means ⫾ S.E.M. for (n) experiments including 4 determinations in each experiment. a p ⬍ 0.05 compared to control cells. b p ⬍ 0.05 compared to OT or PMA alone (see Table 3).

The results presented here provide evidence that PKC activation by PMA produced opposite effect on OT receptor expressed by hypothalamic neurons and astrocytes in culture. The PKC activation induced an upregulation of the OT receptor in neurons but a downregulation in astrocytes. Both effects could be blocked by a PKC inhibitor. Experiments were also performed with staurosporine, another PKC inhibitor, although non specific, which at short term, also blocked PMA effects on both cell types (data not shown). On the other hand, the OT-induced downregulation in neurons and astrocytes was never affected by pretreatment with a PKC inhibitor. This would suggest that PKC is not directly involved in the OT-induced receptor activation but could modulate OT receptor by heterologous regulation. The further increase of the OT-induced downregulation by PMA in astrocytes and the reduced decrease of the OT-induced downregulation in neurons corroborates this hypothesis. The specificity of the PMA effect on both cell types is evidenced by 1) the inability of an inactive form of phorbol ester (PDD) to alter the ligand binding; 2) the ability of a synthetic analogue of diacylglycerol, OAG, to substitute for PMA to produce similar effects; 3) the ability of a PKC inhibitor to prevent PMA-induced downregulation in astrocytes and upregulation in neurons. Therefore, PMA is not acting on OT receptor regulation through non-specific chemical action. In neurons, cycloheximide a protein synthesis inhibitor, decreased the specific binding by itself (to 90%), however an additive negative effect of PMA and cycloheximide was observed (from 123% with PMA alone to 72% with cycloheximide). This suggests that in neurons, the PMA-induced upregulation implies new receptor synthesis but also that synthesis of cellular elements involved in receptor trafficking and/or structure maintenance might be inhibited as well.

M.-T. Strosser et al. / Peptides 22 (2001) 677– 683

On the other hand, we have to assume that PKC activation is directly implicated in this process as it was blocked by BIM, a PKC inhibitor. It could be postulated that PKC phosphorylates a membrane protein, thus unmasking OT receptor or that PKC directly phosphorylates OT receptor inducing a conformational change that permits ligand binding. PKC could also phosphorylate a cellular component involved in the translocation of the OT receptor to membrane surface. This has already suggested for angiotensin II binding sites expressed in neuronal cultures [19]. In astrocytes, the downregulatory effect of PMA also directly involves PKC since it can also be blocked by BIM, but in this case, phosphorylation of the OT receptor may induce uncoupling of the receptor from its G proteins, internalisation and subsequent degradation of the receptor. As reported in the literature, differential sensitivity to PMA according to the cell type is described. Indeed, PMA has no effect on angiotensin II receptor in astrocytes but increases the receptor number in neurons [34] whereas insulin receptors are increased by PMA in astrocytes but not in neurons [22]. These data may suggest expression of different receptor subtypes in neurons and astrocytes as shown for insulin and angiotensin receptors. However, the structural analysis of neuron and astrocyte OT receptor mRNA by RT-PCR suggests that they derive from the same gene as the uterine OT receptor. However, the presence of an OT receptor subtype that is not recognized by our probe cannot be ruled out though pharmacological characterisation of OT receptors in neurons and astrocytes do not support the idea of two receptor subtypes. In addition Scatchard plot analysis of binding data did not provide any evidence for the existence of more than one class of binding sites in our cultures. On the other hand, it cannot be excluded that the PKC cellspecific effect involves changes in the 5⬘-untranslated region and several transcriptional initiation sites, thus placing OT receptor gene expression under the control of a different promoter. Cell-specific effects do not necessarily imply the existence of receptor subtypes, as in the rat brain, steroids modulate the OT receptor only in some brain structures [36] but a single type of OT receptor was identified, which is structurally related to the uterine-type [5]. On the other hand, it could be postulated that the PKC isoforms activated by PMA may be different in astrocytes and in neurons since distribution of the PKC isoforms differs both in vivo [21,35] and in vitro in these two cell types [23,29]. Surprisingly, effect of PMA in neurons and astrocytes was not abolished or reduced after 18 h treatment with PMA, time which should deplete the cells of PKC activity. However, the precise schedule of PKC inactivation is not precisely known and may relay on the cell types studied. On the other hand, some PKC isoforms, members of the atypical PKC family are known not to be activated by phorbol esters and therefore may not be downregulated by PMA. The possibility exists that in our cultured cells, the prominent PKC isoforms are resistant to PMA-induced inactivation [see 38]. Similarly, in myometrial tissue, PKC which

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activation blocks the OT-induced contractions, did not downregulate after a long term treatment with phorbol ester [28] and the author suggested that such sustained PKC activation could occur during gestation and facilitate the maintenance of the myometrial quiescence that is observed along gestation. Previous studies in our laboratory have shown that OT causes desensitization [12] and downregulation of OT receptor expressed in cultured astrocytes [13] and here we present also direct evidence for an OT-induced downregulation in neurons. Although bisindolylmaleimide abolished the up- and downregulation induced by PMA, it did not affect the rate of OT-induced downregulation suggesting that PKC activation may not be the principal mechanism involved in OT receptor regulation. Thus, it suggests that, although the agonist-downregulation appears independent of PKC activity, this kinase may be involved in the heterologous regulation of the OT receptor possibly through receptors that are coupled to phosphoinositide turnover. From the literature, this dual mode of receptor regulation, one PKC dependent and the other PKC independent, has been reported for many receptors as opioid [27], histamine H1 [41], or muscarinic [20] receptors. Other studies also demonstrated that PKC activated by phorbol ester enhanced agonist-induced downregulation of the ␦-opioid receptors [16]. The OT receptor is a member of the G protein family demonstrated to be coupled to PLC activation with increase of intracellular calcium concentration and DAG accumulation in many peripheral targets [for references see 1]. However, little information concerns OT-mediated PKC activation. A report from Phillippe [28] indicated that PKC stimulation with phorbol ester reduces the OT-induced rat myometrium contractions but a PKC inhibitor was unable to block this contractile activity. Recently, it was demonstrated that in a human breast cell line, PKC inhibitors blocked the dexamethasone-induced up regulation of OT receptor [7] and that oxytocin stimulated ERK-2 phosphorylation and prostaglandin E2 synthesis via protein kinase C activity [17]. In the central nervous system, no information is available concerning OT receptor-associated transduction pathways. We demonstrated that cultured astrocytes increased their intracellular calcium concentration after OT application [12] and that repetitive OT applications desensitized the receptor. The absence of a direct implication of PKC on oxytocin-mediated downregulation may reflect differences in the PKC isoforms activated by PMA on one hand and by OT on the other hand. Indeed, PKC consists of multiple isoforms closely related with different pattern of cell and tissue expression [8,18]. Thus, PKC activity within a cell can involve the combined activities of more than one isoform. OT receptor activation may lead to the activation of a subset of PKC isoforms which will induce attenuation of cellular activity but no homologous downregulation whereas PMA will activate other PKC isoforms initiating the heterologous downregulation.

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Although it is well known that PKC is an important effector in the mechanism of action of calcium-mobilising hormones, it cannot be ruled out that other types of kinases may be involved in receptor regulation. Indeed, Nohara et al., [26] demonstrated that OT caused rapid tyrosine phosphorylation of mitogen-activated protein kinase in both human and rat uterine myometrial cultured cells. Other G protein-coupled receptor kinases may be implicated, as shown for ␦-opioid [33], or substance P [14] receptors phosphorylated by a ␤-adrenergic kinase. We have shown here that phorbol ester-induced heterologous regulation is functionally distinct from OT-induced homologous downregulation and that PKC activation is not a prominent component of the latter.

Acknowledgments We are grateful to Ch. Reiss and B. Waltisperger for their excellent technical assistance. This work was supported by a grant from the CNRS and University Louis Pasteur (UMR 7519), Strasbourg France.

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