Role of Protein Kinase C in Endothelin-1-Induced Contraction of Human Myometrium¹

^a Institut National de la Santé et de la Recherche Médicale, Unité 361, Université René-Descartes, Pavillon Baudelocque, 75014 Paris, France

	ABSTRACT

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The role of protein kinase C (PKC) in the contraction of human myometrium induced by endothelin-1 was investigated. The PKC inhibitor, calphostin C, reduced the sustained phase of endothelin-1-induced contraction. The expression and subcellular distribution of PKC isoforms were determined in unstimulated myometrium by Western blotting using isoform-specific antisera. At least five PKC isoforms (PKC, PKCß₁, PKCß₂, PKC, and trace amounts of PKC) were detected. Quantitative immunoblotting revealed that all these isoforms were diversely distributed between the cytosolic and particulate fractions. After stimulation with phorbol 12,13-dibutyrate (PDB) and endothelin-1, differential redistribution occurred, suggesting a selective role of these isoforms in the physiological function of the myometrium. Biochemical assay confirmed that PDB as well as endothelin-1 evoked a decrease in cytosolic PKC activity. Taken together, these results suggest that PKC may play a role in endothelin-1-induced contraction of human uterine smooth muscle.

	INTRODUCTION

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Numerous studies have provided evidence for the involvement of endothelins in contraction of various smooth muscles, including vascular, airway, and uterine smooth muscles [1]. The mechanisms by which endothelin-1 causes contractions of myometrium have not been well elucidated. However, several lines of evidence have demonstrated that the endothelin ET_A receptor subtype predominates in human myometrium and is linked to a membranous phosphoinositide-specific phospholipase C (PLC) [2]. The products generated by phosphatidylinositol-4,5 bisphosphate breakdown consist of two intracellular second messengers: inositol 1,4,5-trisphosphate (IP₃), which releases Ca²⁺ from the sarcoplasmic store, and diacylglycerol (DAG), which, in concert with free Ca²⁺, stimulates protein kinase C (PKC).

It is widely accepted that activation of PKC, measured by its translocation from the cytosol to the membrane, plays an important role in agonist-induced contraction in smooth muscle by phosphorylating myosin light chains and modulating calcium channel activity and Ca²⁺ sensitivity of proteins such as calponin, caldesmon, and desmin, believed to be involved in the control of the contractile apparatus [3]. Rasmussen et al. [4] proposed a biphasic model for contraction in smooth muscle. It is speculated that the IP₃/Ca²⁺ system is responsible for the rapid and initial transient response, while the DAG/PKC system regulates the second and sustained phase, both cumulative phases constituting the final contractile response. Direct activators of PKC such as phorbol esters, which substitute for DAG, produce slowly developing, sustained contractions in various smooth muscles [5]. However, phorbol esters seem to have multiple effects in several smooth muscle cells, and especially in rat myometrium, where both stimulatory and inhibitory effects on uterine tension have been observed [6–9]. In addition, phorbol esters increased the L-type Ca²⁺ current in myometrial cells isolated from pregnant rats, suggesting the involvement of the PKC pathway in the control of uterine contractility [10]. This apparent variety of responses occurring after PKC activation may be partly explained by the existence of several isoforms and subgroups of PKCs.

Indeed, PKC is a family of at least 12 isoforms encoded by different genes. They are divided into three major groups based on their structure and mode of activation. These groups consist of the conventional PKC isoforms (, ß₁, ß₂, ), which require Ca²⁺, phospholipids, and DAG for full enzyme activity. The novel PKC isoforms (, , , , µ) are also activated by DAG and phospholipids, but not by Ca²⁺. The atypical PKC isoforms (, , ) are both DAG- and Ca²⁺-independent, but are phospholipid-dependent [11]. The distribution of PKC isoforms appears to be species-specific in smooth muscle [12]. While seven isoenzymes of PKC (, ß₁, ß₂, , , , ) are expressed in rat uterine smooth muscle [13, 14], no data concerning the identity, the subcellular distribution, or the individual function of PKC isoforms are available in the human myometrium.

In order to study the role of PKC activation in human uterine smooth muscle, we explored 1) the possible antagonistic effect of a selective inhibitor of PKC, calphostin C, on endothelin-1-induced contractions on isolated human myometrial strips; 2) the expression and the subcellular distribution of PKC isoforms and their differential activation induced by endothelin-1 and phorbol 12,13-dibutyrate (PDB); and 3) the translocation of PKC activity in response to PDB and endothelin-1.

	MATERIALS AND METHODS

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Subjects and Sample Preparation

Human myometrium was obtained from premenopausal women (34–46 yr old) undergoing total hysterectomy for benign gynecological indications such as leiomyomas. All subjects had regular menstrual cycles and for at least 3 mo before the study had taken no hormonal medication. The uteri were examined by a pathologist to exclude adenomyosis or malignant change. Tissue samples were excised with a scalpel in the uterine corpus from normal muscle in areas free of macroscopically visible anomalies. This study was approved by the Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale (Paris-Cochin, France). The stage of the menstrual cycle was estimated histologically by dating the endometrium according to the criteria of Noyes et al. [15]. Biopsy specimens (7 from the proliferative and 5 from the secretory phase of the menstrual cycle) were taken in the external muscle layer to avoid contamination by endometrium. The operations were performed with the patients under thiopenthal sodium-succinylcholine anesthesia.

In Vitro Contractile Studies

Isometric contraction experiments were carried out with myometrial strips (5–7 x 2 x 1 mm) suspended in a 10-ml organ bath containing physiological salt solution (composition: glucose, 2.8 mM; KCl, 6.2 mM; NaCl, 144 mM; CaCl₂, 2 mM; MgCl₂, 0.5 mM; NaH₂PO₄, 1 mM; NaHCO₃, 30 mM) at 32°C bubbled with 95% O₂:5% CO₂ as previously described [16].

Contractile activity was measured isometrically using Bioscience UF1 tension transducers (Phymep, Paris, France) and was recorded on an IBM-PC and Gould (Paris, France) recorder. The preparations were allowed to equilibrate for 2–3 h under a resting tension of 0.5 g until spontaneous contractions became regular in frequency and intensity.

Muscle strips that did not develop spontaneous contractions at this stage were considered not to be viable and were discarded. After equilibration, agonists were cumulatively added or applied as a single dose. Calphostin C was added to the bathing medium 30 min before the application of endothelin-1 (Neosystem Laboratoire, Strasbourg, France). Contractions were expressed as g/10 min or percentage of the maximal tension (E_max) elicited by the agonist.

PKC Redistribution

A total of 150 mg to 200 mg of muscle samples was isolated from human myometrium, added to 1.5–2 ml Krebs-Ringer buffer containing 12 mM glucose, and preincubated at 37°C for 15 min under an atmosphere of 95% O₂:5% CO₂. After addition of 1 µM PDB, 1 µM endothelin-1, or vehicle, the reactions were carried out for 0.5–20 min and stopped by holding at 4°C. For enzyme extraction, the strips were homogenized in 5 volumes of a 20 mM Tris-HCl buffer, pH 7.5, containing 250 mM sucrose, 1 mM EGTA, 2 mM EDTA, 50 mM mercaptoethanol, and 2 mM PMSF in the presence of 5% glycerol and 20 µg/ml of leupeptin using an Ultra-Turrax (Janke & Kinkel KG, Staufen Germany) apparatus. The homogenate was centrifuged at 1000 x g for 15 min to remove debris and nuclei, and the supernatant was then ultracentrifuged for 60 min at 100 000 x g. The resulting supernatant (cytosolic fraction) was removed and held at 4°C. The pellet was resuspended in the homogenization medium containing 1% Nonidet P-40, gently mixed for 45 min on ice, and centrifuged at 100 000 x g for 30 min. The resulting supernatant constituted the solubilized particulate PKC fraction.

Western Blotting Analysis

Samples of cytosolic and particulate fraction were separated by SDS-PAGE on 8% gels according to the method of Laemmli [17]. The separated proteins were electrophoretically transferred to a Hybond-C membrane (Amersham International, Buckinghamshire, UK) overnight. 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, 0.05% Tween 20). Anti-PKC antibodies were added at the appropriate concentration (1:100 to 1:500), and incubation was performed for various times at room temperature. Antibodies against PKC, PKC, PKC, and PKC were from Gibco BRL (Cergy Pontoise, France). They were raised in the rabbit against peptides 313–326 from PKC, 662–673 from PKC, 726–737 from PKC, and 577–592 from PKC. Antibodies against PKCß₁ and PKCß₂ were from Santa Cruz Biotechnology Inc. (Le Perray en Yvelines, France). They were raised in rabbits against peptides corresponding to amino acid sequences 656–671 and 657–673, respectively. The membrane was then washed with TBST and incubated with the second antibody, a donkey anti-rabbit IgG conjugated to horseradish peroxidase (Amersham International) at dilutions of 1:20 000 to 1:5000. The blots were developed using enhanced chemiluminescence (ECL; Amersham International, Buckinghamshire, UK) reagents visualized on Kodak x-ray films, and the immunoreactive bands were quantitated by scanning laser densitometry (Studio Scann IISI; Agfa, Mortsel, Belgium). Molecular weight markers were run in parallel, and all the PKC isoforms examined were compared to rat brain extract used as control. The specificity of each immunoreactive band was shown by specific blocking in the presence of the respective peptide antigen against which the antibodies had been raised.

PKC Activity Assay

PKC activity was determined by measuring the transfer of ³²P from [-³²P]ATP (Amersham International) to histone III S. Aliquots of each sample (50 µl) were incubated in a reaction mixture (final volume 250 µl) containing 20 mM Tris-HCl, pH 7.5, 4 mM MgCl₂, 100 µM ATP, 2 µCi/ml [-³²P]ATP, 0.6 mM Ca²⁺, 160 µg/ml histone III S, 20 µg/ml phosphatidylserine, and 3 µg/ml 1,2-diolein. The reaction was initiated by addition of [-³²P]ATP. Each fraction was assayed in the absence of phospholipids and Ca²⁺ and in the presence of 1 mM EGTA. The incubation was carried out for 5 min at 30°C and was stopped by addition of 1 ml of 20% ice-cold (w:v) trichloroacetic acid in 10 mM potassium monophosphate followed by 500 µg of BSA added as carrier. After centrifugation (2500 x g, 15 min), the pellet was solubilized with 1 N NaOH and precipitated again with trichloroacetic acid. This procedure was repeated twice. Finally, the pellet was dissolved in 1 N NaOH, and its radioactivity was then counted by liquid scintillation. PKC activity was expressed as the difference between the activity assayed in the presence and in the absence of Ca²⁺, phosphatidylserine, and diolein. Values were expressed as cpm/mg protein. Protein determinations were performed by the method of Bradford [18].

Data Analysis

Statistical comparisons between multiple means were performed using one-way ANOVA followed by Scheffe's test for differences among means or Student's t-test as appropriate. Significance was set at p < 0.05. Values are expressed as means ± SE.

	RESULTS

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Effects of a PKC Inhibitor on Endothelin-1-Induced Uterine Contractions

We previously demonstrated that endothelin-1 produces a concentration-dependent contraction of myometrial strips, with a mean EC₅₀ value of 5.8 nM. Maximal stimulatory effect (2.27 g/10 min) was achieved at a concentration of 30 nM endothelin-1 [16]. Figure 1A shows a typical example of the effect of endothelin-1 on human myometrium contractility. When a maximal dose of endothelin-1 (30 nM) was added to the organ bath, the immediate response was an elimination of rhythmic activity and a rapid increase in tension that quickly became tetanic. There was no difference in the contractile responses induced by endothelin-1 in the proliferative phase and the responses in the secretory phase of the cycle.

View larger version (17K): [in this window] [in a new window]   FIG. 1. A) Original tracings of the effect of endothelin-1 on the contractile activity of human myometrial strips in vitro. Endothelin-1 (30 nM) was applied during the periods indicated by the labeled bracket below the tracing. Similar records were obtained in 8 other experiments. B) Mean contractile response to endothelin-1 (30 nM) at a single dose in the absence (circles) and presence (triangles) of calphostin C (1 µM) in human myometrial strips. Endothelin-1 was applied at Time 0, and the time (min) after addition of endothelin-1 is represented on the abscissa. When present, calphostin C was added 30 min before addition of endothelin-1. Mean responses for 6 myometrial strips isolated from 3 different patients are expressed as a percentage of the response to 30 nM endothelin-1 (2.28 ± 0.18 g/10 min). The SE of the mean values is shown as vertical bars, and significant differences between control and calphostin C-treated strips are indicated. * p < 0.01.

To assess the role of PKC in endothelin-1-mediated contraction, myometrial tissue was incubated with the potent and specific PKC inhibitor calphostin C for 30 min prior to elicitation of an endothelin-1 maximal contraction. After addition of a single dose of 30 nM endothelin-1, the tension rapidly reached a peak at 1–2 min (the first component) and then, at 15 min, slowly and gradually declined to a steady-state level (the second component). The extent of tension development at 20 min after application of endothelin-1 was about 90% of the initial response. When 1 µM calphostin C was applied, the tension development induced by endothelin-1 was not significantly modified during the first 3 min. However, after this time, the second component of the tension was significantly inhibited to a lower steady-state level. Thus, 20 min after application of calphostin C, the tension induced by endothelin-1 was reduced to 28% of the initial response (Fig. 1B). We verified that pretreatment with this inhibitor (0.1–10 µM) did not alter spontaneous contractions.

Expression and Intracellular Distribution of PKC Isoforms in Human Myometrium

Western blot analysis with antibodies specific for individual isoforms revealed that human myometrium expresses at least five PKC isoforms (results not shown): three Ca²⁺-dependent PKC isoforms (PKC, PKCß₁ and PKCß₂), as well as two Ca²⁺-independent PKC isoforms (PKC and PKC). PKC and PKC were not detectable in our conditions. The presence of other isoforms has not been investigated. All these isoforms were observed in both the cytosolic and particulate fractions of untreated tissue. PKC gave a single specific band at approximately 80 kDa. Several bands were recognized by the PKCß₁ antibody: a major band with apparent molecular mass of 80 kDa and a minor band with apparent molecular mass of 76 kDa. This doublet could represent different phosphorylation states of the enzyme [19]. Several forms of PKCß₂ were also detected by the PKCß₂ antibody and were specifically blocked by the peptide antigen. In contrast, PKC immunoreactivity was poorly represented and appeared as a single band of higher molecular mass of approximately 90 kDa. As previously observed in many tissue and cell types, PKC was detected as two or three immunoreactive bands [20].

The amount of immunoreactivity in the cytosolic fraction relative to the particulate fraction was determined by densitometric analysis of the immunobands and is shown in Table 1. Under basal conditions (unstimulated tissue), PKC and PKC were distributed almost equally between the two fractions, while PKCß₁, PKCß₂, and PKC were predominantly localized in the particulate fraction. Relative expression of the various PKC isoforms was not valid, since the respective antibodies had different affinities for the corresponding PKC isoforms.

Redistribution of PKC Isoforms in Response to PDB and Endothelin-1 Treatment

Myometrial explants were incubated for various times in the presence of 1 µM PDB or 1 µM endothelin-1 and then fractionated into cytosolic and particulate fractions as described in Materials and Methods.

Figure 2 shows a representative Western blot from a series of three experiments. Densitometric scanning of the immunoblots revealed that PDB provoked a marked time-dependent decrease in PKC, PKCß₁, and PKCß₂ levels in the cytosolic fraction. A maximal decrease occurred within 5–10 min (69 ± 5%, 72 ± 8%, and 50 ± 3%, respectively) and was sustained for up to 20 min (Fig. 3A). As shown in Figure 3B, a concomitant increase in the particulate fraction was observed for PKC and PKCß₁ (70 ± 10% and 95 ± 12%, respectively); this was followed by a slight decline at 20 min, reflecting rapid proteolytic degradation and/or rapid down-regulation of the enzyme. In contrast, no increase in PKCß₂ could be detected in this fraction. Although a marked elevation of PKC immunoreactivity was detected in the particulate fraction (46 ± 10%), only a slight decrease was apparent in the cytosolic compartment (20 ± 7%). Since PKC is poorly expressed in this tissue, its translocation was not studied.

View larger version (44K): [in this window] [in a new window]   FIG. 2. A) Western blot analysis of PKC isoforms in the cytosolic (C) and particulate (P) fractions of human myometrium after acute treatment with 1 µM PDB for the indicated periods of time. Proteins from the cytosolic (40–80 µg of protein) and particulate (20–80 µg of protein) fractions were separated on 8% SDS-polyacrylamide gel and subsequently transferred to a nitrocellulose membrane as described in Materials and Methods. PKC isoforms were detected with specific antibodies, and the immunoreactive bands were visualized by the ECL reagent. Blots are representative of two or three experiments. The arrows on the left indicate the migration position of the molecular size standard 80 kDa. Rat brain cytosolic extract (b) was used as a positive control. This is the typical result of at least 3 independent experiments. B) Western blot analysis of PKC isoforms in the cytosolic and particulate fractions of human myometrium after treatment with 1 µM endothelin-1 for the indicated periods of time. Proteins from the cytosolic (80 µg of protein) and particulate (30–60 µg of protein) fractions were separated on 8% SDS-polyacrylamide gel and subsequently transferred to nitrocellulose membrane as described in Materials and Methods. PKC isoforms were detected with specific antibodies, and the immunoreactive bands were visualized by the ECL reagent. Blots are representative of two or three experiments. The arrows on the left indicate the migration position of the molecular standard (Mr 80 000). Rat brain cytosolic extract (b) was used as a positive control. This is the typical result of at least 3 independent experiments.

View larger version (16K): [in this window] [in a new window]   FIG. 3. Subcellular distribution of PKC isoforms after treatment with 1 µM PDB. A) Time-dependent decrease in PKC, PKCß1, PKCß2, and PKC after exposure of human myometrium to 1 µM PDB. Immunoreactive bands were quantified densitometrically, and values (means ± SE, n = 3) are expressed as percentage of control values. B) Time-dependent increase in PKC, PKCß1, PKCß2, and PKC in the particulate fraction after exposure of human myometrium to 1 µM PDB.

Treatment with 1 µM endothelin-1 induced a modest time-dependent translocation of PKC isoforms. Endothelin-1 caused a decrease in PKCß₁, PKCß₂, and PKC in the cytosolic fraction (30 ± 3%, 20 ± 5%, and 20 ± 4%, respectively). The kinetics of their disappearance presented some differences. PKCß₁ and PKC decreased from the soluble fraction within 2 min, whereas a more prolonged time period of incubation (20 min) was necessary to show maximal loss of PKCß₂ (Fig. 4A). This effect was accompanied by an increase in the particulate-associated forms (40 ± 15% in the case of PKCß₁, 70 ± 8% in the case of PKCß₂, and 65 ± 5% in the case of PKC). In contrast, the increase in PKC detected in the particulate fraction (70 ± 10%) was not associated with a detectable loss in the soluble fraction (Fig. 4B).

View larger version (16K): [in this window] [in a new window]   FIG. 4. Subcellular distribution of PKC isoforms after treatment with 1 µM endothelin-1. A) Time-dependent decrease in PKC, PKCß1, PKCß2, and PKC from the cytosolic fraction after exposure of human myometrium to 1 µM endothelin-1. Immunoreactive bands were quantified densitometrically, and values (means ± SE, n = 3) are expressed as percentage of control values. B) Time-dependent increase in PKC, PKCß1, PKCß2, and PKC in the particulate fraction after exposure of human myometrium to 1 µM endothelin-1.

Effects of Phorbol Esters and Endothelin-1 on the Redistribution of PKC Activity

PKC activity was measured in both the cytosolic and particulate fractions prepared from human myometrium. Under basal conditions (unstimulated state), PKC activity was predominantly cytosolic (95 ± 4% of total activity, n = 6).

Figure 5 shows the percentage of decrease in cytosolic PKC activity induced by 15-min incubation treatment in the presence of two different phorbol esters, PDB and 4-phorbol 12,13-didecanoate (PDD), and in response to increasing doses of endothelin-1. One micromolar PDB induced a significant decrease in cytosolic PKC activity (64 ± 7%), whereas PDD, an inactive phorbol ester, had no effect (113 ± 3% of the control level). A modest but significant decrease in cytosolic PKC activity was also observed in the presence of 1 µM endothelin-1 (23 ± 6%). No concomitant increase in membrane-bound PKC activity was observed in these conditions.

View larger version (55K): [in this window] [in a new window]   FIG. 5. Effects of two phorbol esters and increasing concentrations of endothelin-1 on cytosolic levels of PKC activity in human myometrium. PKC activity was determined as described in Materials and Methods. Data are means ± SEM of 4 independent series of experiments. * p < 0.05; ** p < 0.01: significant difference between the values obtained in the absence (control) and presence of the indicated treatment.

	DISCUSSION

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The present results provide evidence for the coupling of endothelin-1 to PKC activation in human myometrium as a possible pathway leading to contraction. Endothelin-1 is the most potent uterotonic agent known in various species. We recently demonstrated that responses to endothelin-1 are long-lasting and are mediated exclusively via ET_A receptors in human myometrial tissue [16]. As smooth muscle contractions mediated by activation of PKC are known to be sustained [5], these results prompted us to consider a role for PKC in the contractile response induced by endothelin-1 in human myometrium.

Involvement of PKC in the endothelin-1-induced contractile response in human myometrium is supported by the substantial inhibition caused by a specific PKC inhibitor, calphostin C. This compound specifically binds to the DAG/phorbol ester site present on the regulatory moiety of the molecule and is a highly specific inhibitor of PKC [21]. Attempts were made to correlate the attenuation by calphostin C of the endothelin-1-induced contractile response and changes in PKC isoform activation in human myometrial tissue. Using isoform-specific antibodies, we demonstrated for the first time the presence of at least five PKC isoforms in human myometrium: PKC, PKCß₁, PKCß₂, PKC, and trace amounts of PKC. In addition, PKC, PKCß₁, PKC, PKC isoforms, and the predominant expression of PKCß₂ and PKC, have recently been reported in rat myometrium [13]. The failure to detect PKC and PKC in human myometrium suggests a species-specific pattern of PKC expression. The existence of multiple PKC isoforms in the human myometrium may reflect the various metabolic functions mediated by this kinase. Their distinct cofactor requirement suggests specific roles for individual PKC isoforms. Some of these isoforms are ubiquitous (PKC, PKC), indicating a general cellular role, while others are restricted in their location and mediate more specialized cellular functions.

It is well established that activation of PKC is associated with its translocation into various subcellular sites presumably containing appropriate substrates [22]. Two approaches were used to assess the activation of PKC in response to PDB and endothelin-1: the redistribution of PKC isoforms examined by immunoblot analysis and the measurement of PKC activity in both the cytosolic and particulate fractions.

Densitometric scanning of the immunoblots revealed that treatment with PDB and endothelin-1 produced a differential redistribution of the four predominantly expressed PKC isoforms. As classically reported in several systems, incubation with PDB induced a more rapid and extensive translocation than was observed with the physiological agonist endothelin-1 [23]. Interestingly, whereas endothelin-1 causes redistribution of PKCß₂, PDB treatment leads to the disappearance of that isoform in the cytosol without a concomitant increase in the particulate fraction. The translocation of PKCß₂ toward another subcellular compartment was suggested by Goodnight et al. [24]. By in situ immunocytochemical analyses, these authors recently demonstrated a specific association of PKCß₂ with actin-rich microfilaments of the cytoskeleton. Redistribution of PKC was observed after exposure to PDB, although this atypical isoform, which contains only a single cysteine-rich motif, is known to be phorbol ester-insensitive [25]. Similar translocation was observed in a variety of cellular types, and PKC is thought to have a low-affinity binding site for this compound. Thus, the use of a high dose of PDB would trigger its redistribution [26]. Furthermore, it remains to be determined whether apparent translocation of PKC resulted from cross-reaction between conventional PKCs and anti-PKC antibody [27]. The translocation was not a stoichiometric phenomenon. For instance, a significant increase in the particulate fraction was not always associated with an apparent decrease in the cytosolic fraction, suggesting that only a small percentage of PKC isoforms may be translocated to the particulate fraction [28]. Both PDB and endothelin-1 were able to induce PKC redistribution and thereby its activation. Furthermore, the differential translocation of PKC isoforms in response to two different stimuli suggests that they may phosphorylate specific substrates located at different sites, leading to selective regulation of uterine function. The specific role for each PKC isoform in regulating uterine contractility has not yet been elucidated. Overexpression of specific PKC isoforms has been observed in estrogen-dominated rat myometrial tissue, suggesting that individual PKC isoforms may selectively elicit specific cellular responses when the steroidal environment mimics parturition [13].

Enzymatic data show that PDB induces a substantial decrease in cytosolic PKC activity. In contrast, the inactive phorbol ester PDD had no significant effect. As expected, we observed a significant decrease in cytosolic PKC activity upon endothelin-1 incubation over a concentration range similar to that found in our study to stimulate inositol phosphate accumulation in human myometrium [2]. As observed by immunoblot analysis, the magnitude of this decrease was weaker than that induced by the phorbol ester. The increase in PKC immunoreactivity in the particulate fraction was not associated with a concomitant stimulation of Ca²⁺-phospholipid-dependent PKC activity. This inability to detect membrane-associated PKC activity, already reported in some systems [29, 30], may be explained by the instability of the DAG-PKC complex or by rapid dephosphorylation of the activated enzyme leading to its inactivation [31]. The presence of an endogenous inhibitor can be excluded, since no increase occurred after partial purification of the enzyme by chromatography DEAE-cellulose (unpublished results).

The disappearance of cytosolic PKC activity after endothelin-1 and PDB treatment provides a second line of evidence for translocation of the investigated PKC isoforms by these agonists. As previously reported in vascular smooth muscle [32], we observed that the activation of PKC by endothelin-1 occurred during the contraction of human uterine smooth muscle elicited by this physiologic agonist. However, the role of Ca²⁺ in the activation process of PKC remains to be determined. Besides human myometrial contractile effects, endothelin-1 exhibits growth factor-like actions in human uterine smooth muscle [33]. A better understanding of the signaling pathways will clarify the participation of endothelins in both normal and pathological uterine functions such as dysmenorrhea, leiomyoma, and premature labor.

In conclusion, these findings support, but do not confirm, the hypothesis that PKC is a component of the signal transduction mechanism involved in endothelin-1-induced contractile activity in human myometrium. This ubiquitous enzyme, located at the crossroads of many intracellular signaling pathways, may exert a pivotal role in the control of uterine functions. These various signaling pathways (phospholipases C, D, A₂, etc.) converge in a multiplicity of PKC isoforms to provide the cellular specificity of endothelin-1 responses such as contractility, modulation of cell proliferation, and differentiation. However, further investigations are necessary to identify the specific functional roles of individual PKC isoforms.

	ACKNOWLEDGMENTS

 
We are grateful to the Department of Obstetrics and Gynecology of Cochin-Port-Royal for assistance in obtaining uterine tissues and to Dr. M.C. Vacher-Lavenu of the Pathological Anatomy Department for histological examinations. The contribution of the medical student Milad Pooran (School of Medicine, University of Maryland, Baltimore, USA) is acknowledged. We thank J. Bram for reviewing the English text and M. Verger for secretarial assistance.

	FOOTNOTES

 
¹ Supported by Institut National de la Santé et de la Recherche Médicale and by University René-Descartes.

² Correspondence: M. Breuiller-Fouché, INSERM U. 361, Pavillon Baudelocque, 123, Bd de Port-Royal, 75014 Paris, France. FAX: 00.1.43.26.44.08; u361{at}cochin.inserm.fr

Accepted: February 27, 1998.

Received: April 25, 1997.

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