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Regular Article |
a Institut National de la Santé et de la Recherche Médicale, 75014 Paris, France
ABSTRACT
The role of protein kinase C (PKC) in contraction of the human myometrium induced by endothelin-1 (ET-1) was investigated at the end of pregnancy. The expression and subcellular distribution of PKC isoforms were examined by Western blot analysis using isoform-specific antibodies. At least three conventional PKC isoforms (cPKC;
, ß1, and ß2), two novel PKC isoforms (
and 
), and an atypical PKC isoform (
) were detected in pregnant myometrium. Quantitative immunoblotting revealed that all these isoforms were mainly distributed in the particulate fraction. The lack of a calcium chelator to modify the particulate sequestration of cPKC suggests an interaction with an anchoring protein such as receptor-activated C kinase-1, which is evidenced in the particulate fraction of the pregnant myometrium. Of the six isoforms, only PKCß1, PKCß2, PKC, and PKC were translocated to the particulate fraction, and PKC to the cytoskeletal fraction, after stimulation with ET-1. Involvement of PKC in the ET-1-induced contractile response is supported by the inhibition caused by the PKC inhibitor calphostin C. However, we demonstrated that the selective cPKC isoform inhibitor, Gö 6976, as well as the substantial depletion of PKCß1 and PKC and the partial depletion of PKC and PKC by a long-term treatment with phorbol 12,13-dibutyrate did not prevent ET-1-induced contraction. Accordingly, our results suggest that PKC and PKC activation mediated ET-1-induced contraction, whereas cPKC isoforms were not implicated in the human pregnant myometrium.
female reproductive tract, kinases, other hormones, parturition, pregnancy, signal transducers, signal transduction, uterus
INTRODUCTION
During human pregnancy, development and contractile activity of uterine smooth muscle are regulated by numerous maternal and fetal signals. Steroid hormones, growth factors, catecholamines, eicosanoids, neuropeptides, and cytokines have all been implicated in the growth and differentiation of the myometrium as well as in its contractility [1]. However, the mechanisms involved in these phenomena remain unclear. The biochemical mechanisms that maintain uterine quiescence during pregnancy, but that also provoke uterine hypercontractility at the time of parturition, need to be identified and characterized. In this context, much can be learned from the study of intracellular signaling pathways related to the process of contraction.
The past 10 yr have seen the emergence of a new contractile agent, endothelin-1 (ET-1), which is a 21-residue peptide that was first isolated from the supernatant of porcine aortic endothelial cell culture [2]. The concentration of ET-1 in maternal blood is increased during pregnancy and reaches a peak at term [3], and its concentration in amniotic fluid is also high [4]. Human uterine muscle is sensitive to ET-1 [5], and ET-1-induced uterine contraction is markedly increased at the end of pregnancy [6]. In addition, rat uterine contractile responsiveness to ET-1 is elevated during labor and diminished postpartum. The increased responsiveness is accompanied by elevations in the myometrial receptor density of ET-1 [7]. Taken together, these data suggest a role for ET-1 in the initiation and maintenance of labor. Two types of endothelin-receptors, ETA and ETB, coexist in human myometrial tissues, but only the proportion of ETA receptor is increased in the pregnant myometrium [6–8]. The myometrial ETA receptor is linked to a membrane phosphoinositide-specific phospholipase C [8,9]. Phosphatidylinositol-4,5 biphosphate breakdown generates two intracellular second messengers: inositol 1,4,5-triphosphate, which releases Ca2+ from the sarcoplasmic store; and diacylglycerol (DAG), which acts in concert with free Ca2+ to stimulate protein kinase C (PKC). The Ca2+ mobilization and PKC activation appear to function synergistically in smooth muscle to promote contraction [10]. However, to our knowledge, the role of PKC in mediating ET-1-induced uterine contraction during pregnancy has not yet been elucidated.
The PKCs are a multigene family of serine-threonine protein kinases involved in the regulation of various cellular processes, such as proliferation, differentiation, tumorigenesis, and contractility. The translocation and binding of PKC isoforms to different intracellular structures suggest distinct physiologic roles for individual isoforms [11]. A total of 12 isoforms have been cloned to date, and they are classified into three groups on the basis of their cofactor requirements. Conventional PKC isoforms (, ß1, ß2, and 
) are activated by DAG or phorbol esters in a Ca2+-dependent manner. Novel PKC isoforms (, , 
, 
, and µ) are also activated by DAG or phorbol esters, but in a Ca2+-independent manner. Atypical PKC isoforms (, 
, and 
) are neither activated by Ca2+ nor by DAG or phorbol esters but are regulated by other phospholipidic mediators [12]. Structurally, PKC is divided into regulatory and catalytic domains. The primary amino acid sequence of PKC includes four conserved functional domains (C1–C4) that are separated from each other by variable regions (V1–V5). Deletions in the C2 region appear to influence the affinity of conventional PKC for Ca2+, whereas deletions within the C1 region result in loss of phorbol ester and DAG binding. Therefore, the C1 domain is an important region in mediating interactions with lipids and a possible recognition motif for protein-protein interaction, whereas the C2 domain is important for interactions with Ca2+. Translocation of PKC to the particulate fraction was thought to reflect direct binding of the enzyme to lipids at the plasma membrane. However, other data have demonstrated that translocated PKC interacts with protein called receptor-activated C kinase (RACK) at the site of translocation. A 36-kDa protein cloned from rat brain, RACK1 is the best-characterized member of the RACK family, and it is thought to preferentially interact with the conventional PKC isoforms [13, 14]. Such an interaction with an anchoring protein is alleged to maintain an active pool of enzyme in the nearby area of its substrates, which can then be easily and rapidly phosphorylated.
We previously reported the existence of multiple PKC isoforms in the myometrium from nonpregnant women, and that PKC is a component of the signal transduction pathway involved in ET-1-induced contractions in vitro [15]. It seems to be less obvious whether PKC plays a role in the kinase cascade involved in the myometrial contractile process during human pregnancy. To address this issue, we examined the expression and cellular distribution of PKC isoforms in the myometrium from pregnant women. Because binding to RACK is required for the enzyme to perform its cellular function, we examined whether RACK1 protein might be expressed in the myometrium. Measuring its translocation from the cytosol to the particulate fraction assessed the activation of PKC isoforms by ET-1. In addition, we studied the role of PKC isoforms in the contractile process, using a generic inhibitor of PKC (calphostin C) and a selective inhibitor of conventional PKC (Gö 6976) to elucidate the contribution of each PKC isoform in human myometrial contractility. Lastly, we analyzed the impact of the downregulation of PKC by phorbol 12,13-dibutyrate (PDBu) to identify the role of phorbol ester-sensitive PKC isoforms on ET-1-induced contractions in myometrial strips isolated from pregnant women.
MATERIALS AND METHODS
Subjects and Biological Samples
Biopsies of myometrium were obtained from 20 pregnant women (26–43 yr old) who presented normal, uncomplicated pregnancies but were delivered by elective caesarean section performed before the onset of labor, between the 37th and the 40th wk of pregnancy, because of diagnosed cephalopelvic disproportion. Myometrial strips were excised from the uterine body at the antiplacental site from the longitudinal layer and immediately placed on ice. Written informed consent was obtained from all donors. This study was approved by the Consultative Committee for the Protection of Persons in Biomedical Research (Paris-Cochin, France).
PKC Redistribution
A 150- to 200-mg sample of human myometrium was placed in 1.5–2 ml of Krebs-Ringer buffer containing 12 mM glucose and preincubated at 37°C for 15 min under an atmosphere of 95% O2/5% CO2. The ET-1 (Neosystem Laboratoire, Strasbourg, France) was diluted in H2O, and calphostin C (Calbiochem, Meudon, France), Gö 6976 (Calbiochem), 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM; Sigma-Aldrich, St. Louis, MO), and PDBu (Sigma Aldrich) were diluted in dimethyl sulfoxide (DMSO). Aliquots of 1 µM ET-1, 300 µM BAPTA-AM, 1 µM PDBu, or vehicle (DMSO) were added, and incubation was continued for 0.5 to 10 min and 4 h. Reactions were stopped by cooling the mixture to 4°C. Samples were homogenized (200 mg/ml) using an Ultra-Turrax apparatus in ice-cold homogenization buffer: 20 mM Tris-HCl (pH 7.5) containing 250 mM sucrose, 1 mM EGTA, 2 mM EDTA, 50 mM ß-mercaptoethanol, and 2 mM PMSF plus 5% glycerol and 20 µg/ml of leupeptin. Homogenates were centrifuged at 2000 x g for 15 min at 4°C to remove cell debris. Aliquots of homogenate were immediately stored at -20°C until use, and preparations were further ultracentrifuged for 60 min at 100 000 x g. The resulting supernatants (cytosolic fraction) were collected and stored at 4°C. The pellets were resuspended in the homogenization medium containing 1% Nonidet P-40, gently mixed for 45 min on ice, and ultracentrifuged at 100 000 x g for 30 min. The resulting supernatant was the solubilized particulate PKC fraction. The pellets were then resuspended in the homogenization buffer without Nonidet P-40 and gently mixed; this was the PKC cytoskeletal fraction. Protein concentrations were measured according to the method described by Bradford [16].
Western Blot Analysis
Equal amounts of protein (40 µg/lane) from cytosolic, particulate, and cytoskeletal fractions or crude homogenates were separated by SDS-PAGE on 8% gels according to the method described by Laemmli [17], and the separated proteins were transferred to a nitrocellulose membrane overnight as described elsewhere [18]. Nonspecific binding sites were blocked by incubating the membrane with 5% fat-free dried milk in TBST (10 mM Tris-HCl [pH 7.5], 0.15 M NaCl, and 0.1% Tween 20). Polyclonal antibodies against PKC and RACK1 were added at the appropriate concentration (PKC, 1:20 000; PKCß1, 1:50; PKCß2, 1:100; PKC, 1:15 000; PKC, 1:2000; PKC, 1:5000; PKC, 1:500; and RACK1, 1:400) and incubated for different times at room temperature. Antibodies against PKC, PKC, and PKC were from Sigma-Aldrich and were raised in rabbits against peptides corresponding to amino acid sequences 659–672, 726–737, and 577–592, respectively. Antibodies against PKCß1, PKCß2, and PKC were from Santa Cruz Biotechnology, Inc. (Le Perray en Yvelines, France) and were raised in rabbits against peptides corresponding to amino acid sequences 656–671, 657–673, and 656–673, respectively. Antibody against PKC was from Gibco BRL (Cergy Pontoise, France) and was raised in rabbits against peptides corresponding to the amino acid sequence 306–318. Antibody against RACK1 was from Transduction Laboratories (Lexington, KY) and was raised in mice against peptides corresponding to the amino acid sequence 113–317. Membranes were washed with TBST and incubated with the secondary antibody, a donkey antirabbit immunoglobulin (Ig) G (dilution 1:5000) or a donkey antimouse IgG (dilution 1:5000) conjugated to horseradish peroxidase (Amersham International, Chalfont, Buckinghamshire, UK). The blots were developed with enhanced chemiluminescence reagents and visualized on Kodak x-ray films (Rochester, NY); the immunoreactive bands were quantified by densitometric scanning (Studio Scan IISI, Agfa, Morstel, Belgium) processed by the National Institutes of Health (NIH) Image 1.6 software package (NIH, Bethesda, MD). Results are expressed as the mean ± SEM. Molecular weight markers (Bio-Rad Laboratories, Richmond, CA) were run in parallel, and rat brain extract was used as a control. Specific blocking in the presence of the respective antigen peptides against which the antibodies had been raised showed the specificity of each immunoreactive band of PKC isoforms.
In Vitro Contractile Studies
Myometrial segments (8–12 x 3 x 2 mm) were suspended in parallel for isometric tension recordings using Bioscience UF1 tension transducers (Phymep, Paris, France) in 6-ml organ baths containing aerated (95% O2/5% CO2) Krebs solution (in mmol/L: glucose, 11.1; KCl, 6.2; NaCl, 144; CaCl2, 2.5; MgCl2, 0.5; NaH2PO4, 1; and NaHCO3, 30) maintained at 33°C. A resting tension of 600 mg was applied to each segment, and a spontaneous tone was allowed to develop. As described elsewhere [5], the myometrial strips were allowed to equilibrate for 2 h until spontaneous contractions became regular in frequency and intensity. Muscle strips, which did not develop spontaneous contractions at this stage, were discarded. After equilibration, agonists were cumulatively added at 10-min intervals or applied as a single dose. Calphostin C and Gö 6976 were added to the bathing medium 30 min before adding ET-1. We verified that pretreatment with calphostin C (1 nM to 1 µM) and Gö 6976 (1 nM to 1 µM) did not alter the spontaneous contractions. The PDBu was added to the bath 4 h before adding the ET-1. Measurements were processed by the Maclab/8e software package (ADInstruments LtD, Hastings, UK). Area under the tension curve was measured for a given time (contraction area). Results are expressed as g tension/5 or 20 min.
RESULTS
Expression and Intracellular Distribution of PKC Isoforms in Myometrium from Pregnant Women
Figure 1A shows a representative Western blot analysis from a series of 13 experiments with antibodies specific for individual PKC isoforms. At least six PKC isoforms were found in the myometrium from pregnant women: three conventional PKC isoforms (PKC, PKCß1, and PKCß2), two novel PKC isoforms (PKC and PKC), and one atypical PKC isoform (PKC). However, PKC was not identified, and the presence of other isoforms was not investigated. The amounts of these isoforms were not quantified, because the antisera had different affinities for their respective antigen and, thus, for the corresponding PKC isoforms. However, the intensity of the immunoreactive bands in the cytosolic (C) and particulate (P) fractions for a given isoform was determined by scanning densitometric analysis and is shown in Figure 1B. Under basal conditions (unstimulated strips), all PKC isoforms were found mainly in the particulate fraction. Specifically, PKC, PKCß1, PKCß2, PKC, PKC, and PKC in the particulate fraction represented, respectively, 63% ± 9%, 71% ± 9%, 77% ± 7%, 81% ± 5%, 100% ± 1%, and 64% ± 11% of the total amount of each isoform (C + P = 100%; n = 13).
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Redistribution of PKC Isoforms in Response to BAPTA-AM Treatment
Myometrial strips were incubated with the chelator of Ca2+, BAPTA-AM, to analyze the type of interaction of PKC with plasma membrane. The calcium chelator allows easier dissociation of PKC from the membrane, even if PKC was linked to DAG in the membrane [19]. The relative level of particulate PKC was reduced (53% ± 13% of the control level, n = 2) after BAPTA-AM treatment (Fig. 2). The intensity of the immunoreactive band revealed dissociation of PKC from the membrane. In contrast, the relative level of particulate PKC, PKCß1, PKC, and PKC were unchanged by BAPTA-AM treatment. These isoforms were not dissociated from the plasma membrane.
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Expression of RACK1
Figure 3 shows a representative Western blot from a series of five experiments. Under basal conditions, RACK1 was present. The specific antibody to RACK1 recognized an immunoreactive species at 36 kDa. More RACK1 protein was in the particulate fraction (75% ± 5%) than in the cytosolic fraction (25% ± 4%).
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Redistribution of PKC Isoforms in Response to ET-1 Treatment
The effect of 1 µM ET-1 on the relative levels of particulate-associated PKC isoforms is shown in Figure 4. The intensity of the immunoreactive bands 0.5 min after stimulation revealed that treatment with 1 µM ET-1 caused the transient translocations to the particulate fraction of PKCß1 (117% ± 4% of the control level, n = 3), PKCß2 (122% ± 7% of the control level, n = 3), PKC (223% ± 7% of the control level, n = 3), and PKC (152% ± 1% of the control level, n = 3). This was followed by a decline at 10 min, probably reflecting rapid proteolytic degradation (Fig. 4B). By contrast, ET-1 had no detectable effect on the increase in immunoreactive PKC and PKC in the particulate fraction, whatever the incubation treatment.
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We also studied the redistribution of each PKC isoform in the cytoskeletal fraction. Among the PKC isoforms that were not translocated in the particulate fraction (PKC and PKC), only PKC was redistributed to the cytoskeletal fraction (217% ± 4% of the control level, n = 3) 0.5 and 10 min after stimulation (Fig. 4A). In addition, PKC was not translocated to the cytoskeleton (data not shown).
Effect of Inhibition of PKC Isoforms on ET-1-Induced Uterine Contraction
Endothelin-1 (1 pM to 1 µM) caused a concentration-dependent contraction of myometrial strips (n = 3) (Fig. 5A). Concentrations of ET-1 as low as 10 nM increased the frequency and tonus of contractions. Administration of 30 nM ET-1 produced an additional effect, namely an increase in uterine tone culminating in marked tetanic contraction. To assess the role of PKC in ET-1-induced contraction, myometrial strips were incubated with the potent generic PKC inhibitor calphostin C for 30 min before adding ET-1 to assess the role of PKC in ET-1-mediated contraction. When 1 µM calphostin C was applied (Fig. 5B), tension development induced by the addition of a single dose of 30 nM ET-1 was markedly reduced. Other concentrations of calphostin C (1–100 nM) had no effect on ET-1-induced contraction (data not shown).
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We also evaluated the role of conventional PKC isoforms in ET-1-mediated contraction by incubating myometrial strips with the selective potent conventional PKC inhibitor Gö 6976 for 30 min before adding ET-1. Figure 5C shows that a concentration of Gö 6976 as high as 1 µM did not affect the response to ET-1. The same results were observed regardless of the concentration of Gö 6976 used (1–100 nM; data not shown).
In addition, we incubated myometrial strips for a prolonged treatment (4 h) with the phorbol ester PDBu. Myometrial strips responded to 1 µM PDBu by developing a gradual increase in contractile force, which reached a plateau after 4 h (Fig. 5D). Adding ET-1 to myometrium treated in this way always produced a tetanic contraction. We have verified that a prolonged exposure of myometrial tissue to PDBu for 4 h failed to have an effect on 80 mM KCl-induced contractions compared with the effect observed in control strips (data not shown). Because of the reduced contractile response of the control strips, we never performed contractile studies to 4 h. This duration of treatment with PDBu can deplete conventional and novel PKC isoforms. The intensity of the immunoreactive bands from particulate fraction obtained by Western blot analysis (Fig. 5D) revealed that the relative levels of PKCß and PKC were strongly reduced (42% ± 5% and 29% ± 7%, respectively, of the untreated tissue; n = 3), but those of PKC and PKC were only slightly reduced (85% ± 3% and 82% ± 5%, respectively, of the untreated tissue; n = 3). However, PKC was insensitive to PDBu (99% ± 2% of the untreated tissue, n = 3).
DISCUSSION
The present study provides evidence for the coupling of ET-1 to PKC activation in human myometrium as a possible pathway leading to contraction at the end of pregnancy. Furthermore, our results suggest that PKC and PKC are necessary for ET-1-induced myometrial contractions, and that conventional PKC isoforms (, ß1, and ß2) are not implicated in this process.
Using Western blot analysis with isoform-specific antibodies to PKC, we identified at least six PKC isoforms in myometrium from pregnant women: three conventional PKC isoforms (PKC, PKCß1, and PKCß2), two novel PKC isoforms (PKC and PKC), and an atypical PKC isoform (PKC). In myometrium from pregnant women, all the identified PKC isoforms were mainly located in the particulate fraction. Our results are compatible with the predominant expression of PKC isoforms as found in the membrane fraction of rat uterine tissue [20]. This result suggested that these isoforms, without any stimuli, were in an activated form. The membrane-associated activated enzyme is closed to its substrates and should easily and rapidly phosphorylate them. As reported by Ruzycky and Kulick [21], this large increase in membrane-associated PKC levels might be indicative of the estrogen-dominating environment. This increased distribution of PKC isoforms in the particulate fraction of human, term myometrium is consistent with our previous results showing an increase in the myometrial estradiol-17ß:progesterone ratio at the time of parturition [22].
Sequestering of PKC isoforms in the particulate fraction of the unstimulated myometrium could result from a direct link between PKC and phospholipid-containing membranes or from an indirect association between PKC and plasma membranes via an anchoring protein. Binding of PKC to an anchoring protein is often required for PKC-mediated signal transduction. Mochly-Rosen et al. [14] have cloned the first cDNA-encoding receptor for C kinase (i.e., RACK). The RACKs are proteins in the particulate fraction that bind PKC. The interaction site becomes exposed only when the enzyme has been activated by cofactors such as DAG, phosphatidylserine, and Ca2+. The interaction between PKC and RACK involves the C2 region of PKC [23], which is also known for its high affinity for Ca2+. Treatment with BAPTA-AM was used to analyze the type of interaction between PKC and plasma membranes; BAPTA-AM caused the dissociation of the PKC from the membrane. Results of previous studies have shown that the interaction of PKC with phospholipidic vesicles is rapidly disrupted by Ca2+ chelator [19], and dissociation of PKC from the membrane revealed an interaction between this enzyme and phospholipid elements such as DAG. We also found that the Ca2+ chelator does not alter the interaction between PKC, PKCß1, PKC, and PKC and the membrane, probably reflecting an indirect association between PKC and the plasma membranes via an anchoring protein. We have demonstrated, to our knowledge for the first time, that the anchoring protein RACK1 can be recognized in the particulate fraction of the myometrium from pregnant women. This suggests, but does not demonstrate, that RACK1 may interact with Ca2+-dependent PKC isoforms (PKC and PKCß1) to mediate contractile or growth functions of the myometrium throughout pregnancy. Such an interaction is supposed to maintain an active pool of PKC in the vicinity of its substrates; the coordinated regulation of the PKC/RACK system by ET-1 could be a molecular mechanism involved in ET-1-contraction.
We also analyzed activation of the PKC isoforms identified in myometrium from pregnant women after treatment with ET-1 before identifying the role of each in ET-1-induced contraction. For this purpose, we measured the increases in PKC isoforms in the particulate and cytoskeletal fractions, and we found that ET-1 caused a selective and transient activation of PKCß1, PKCß2, PKC, PKC, and PKC. Previous authors have reported that PKCß is involved in the regulation of smooth muscle contractile activity [24], and PKC is involved in the contractile potentiation of ET-1 in the porcine coronary artery [25]. In addition, PKC, which is a Ca2+-independent isoform, has been described as being a possible partner in the sustained phase of the contraction of bovine carotid arterial smooth muscle [26]. The translocation of PKC to the cytoskeletal fraction after ET-1 treatment can reveal any interaction between this PKC isoform and the actin-rich microfilaments of the cytoskeleton. This novel PKC isoform seems to act as a regulator of smooth muscle contractility [27], and it stimulates cell growth in vascular smooth muscle [28]. The inability to detect an ET-1-induced increase in the particulate fraction of PKC may result from a redistribution of this isoform to other subcellular sites, such as the nucleus. In various systems, PKC translocation to the nucleus appears to be an essential step in eliciting a mitogenic action [29]. The role of this ubiquitous isoform in myometrium from pregnant women may correlate with its considerable enlargement, which is thought to result from smooth muscle hypertrophy.
Finally, we investigated the possibility that the various PKC isoforms play specific roles in uterine contractility. Involvement of PKC in the ET-1-induced contractile response was supported by the substantial inhibition caused by a generic PKC inhibitor, calphostin C. This compound binds specifically to the DAG/phorbol ester site on the regulatory domain of the molecule [30]. Calphostin C (1 µM) strongly inhibited the maximum ET-1-induced contraction. These results suggest that PKC is necessary for ET-1-induced contraction in human pregnant myometrium, but we did not exclude that other transduction signals, such as mitogen-activated protein (MAP) kinase and tyrosine kinase, could be implicated. Kimura et al. [31] demonstrated that the MAP kinase cascade was involved in ET-1-induced rat puerperal uterine contraction, and Ascher-Landsberg et al. [32] showed that tyrosine kinase mediated the activation of cytosolic calcium oscillations and phasic myometrial contractions in rats.
We used a specific, conventional PKC isoform, Gö 6976, that clearly discriminates in vitro between Ca2+-dependent subtypes of PKC and PKCß and the Ca2+-independent PKC isoforms , , and . The mechanism of inhibition of Gö 6976 has been shown to implicate the ATP-binding site on the kinase, and even micromolar concentrations of Gö 6976 have no effect on the kinase activity of the Ca2+-independent subtypes of PKC [33]. Our results show that different concentrations (1 nM to 1 µM) of Gö 6976 failed to modify ET-1-induced contraction. We conclude that the conventional isoforms PKC, PKCß1, and PKCß2, as evidenced in myometrium from pregnant women, are not implicated in the ET-1-induced contraction. So, that PKC, PKCß1, and PKCß2 might be linked to the membrane via an anchoring protein, but are not implicated in the ET-1-contractile effect, suggests that they must be involved in some other myometrial function, such as smooth muscle hypertrophy.
After PKC downregulation induced by phorbol ester long-term treatment, PKC is rapidly degraded by membrane-bound proteases, and chronic activation over more than 1 h leads to depletion of PKC in the cell and attenuation of the signal. Biologically active phorbol esters such as PDBu replace DAG and, thus, serve as a useful tool to investigate the effect of PKC [34]. Therefore, PKC downregulation was also used to identify the role of phorbol ester-sensitive PKC isoforms in myometrium contractility. Activation of PKC by PDBu produced a slowly developing, contractile tension in myometrial strips, and the uterotonic action of ET-1 was preserved after 4 h of treatment. This observation was consistent with the results of Katoch [35] in vascular smooth muscle, in which PKC downregulation suppressed the potentiated ET-1 contractile response. The differential susceptibility of the PKC isoforms to phorbol ester-induced downregulation has been demonstrated in myometrial tissues. Western blot analysis revealed a discrete depletion of PKC and PKC and a consistent depletion of PKCß and PKC. The persistence of PKC is consistent with its known properties as a phorbol ester-insensitive isoform.
In conclusion, results of our functional experiments strongly support the idea that PKC may play a crucial role in the contractile activity induced by ET-1 in human myometrium at the end of pregnancy. Our results are consistent with the idea that PKC and PKC, but not conventional PKC isoforms (, ß1, and ß2), regulate contractility of the myometrium. Further studies are necessary to examine whether, in addition to its contractile action, ET-1 has properties as a growth factor and participates in the process of hypertrophy, which occurs in the uterus throughout pregnancy.
ACKNOWLEDGMENTS
We are very grateful to T. Fournier for excellent technical assistance (INSERM U.361), G. Delrue (SC6 INSERM) for photographic expertise, D. Joubert for helpful discussions (INSERM U.469), and G. Watts and O. Parkes for reviewing the English text.
FOOTNOTES
1 Correspondence: M. Breuiller-Fouché, INSERM U.361, Pavillon Baudelocque, 123, Bd de Port-Royal, 75014 Paris, France. FAX: 33 1 43 26 44 08; breuiller-fouche{at}cochin.inserm.fr 
Accepted: July 7, 2000.
Received: May 1, 2000.
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