Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Endothelins are a family of 21 amino acid peptides that includes ET-1, ET-2 and ET-3. In addition to their vasoconstrictive activity, ETs induce various physiological effects that are mediated by distinct receptors with different specificities for the three peptides. The ET_A receptor is characterized by the rank order of potency ET-1 > ET-2  ET-3, the ET_B receptor displays equal affinities for the three ET isoforms, whereas ET_C receptor preferentially binds ET-3. Molecular characterization of ET receptors has revealed that they belong to the G-protein-coupled receptor superfamily. The mechanisms of ET action involve second messenger generation via different signal transduction processes. These include activation of the PLC degrading PtdInsP₂ with an increased production of InsP₃ and diacylglycerol, inhibition or activation of adenylyl cyclase and activation of phospholipase A₂. Recent observations also concern ET-1 interaction with the PLD pathway (Huggins et al., 1993; Masaki et al., 1994; Sokolovsky, 1994).

Evidence is accumulating that receptor-mediated hydrolysis of phospholipids by PLD is an important signaling pathway in many biological systems. PLD hydrolyzes PC with the production of PA. PA may serve as a potential lipid second messenger and/or constitute a source for diacylglycerol or lysophosphatidic acid (for reviews, see Cockcroft, 1992; Exton, 1994). The molecular details of the mechanisms involved in receptor signaling to PLD are incompletely defined. Besides heterotrimeric G proteins, a modulatory role for PKC and Ca⁺⁺ has been reported in the regulation of PLD activity (Cockcroft, 1992; Exton, 1994; Liscovitch et al., 1993). Activation of PLD has also been mediated by small G proteins (Bourgoin et al., 1995; Cockcroft et al., 1994; Malcolm et al., 1994), cytosolic factors (Bourgoin et al., 1995; Lambeth et al., 1995; Singer et al., 1996) and tyrosine kinases (Bourgoin and Grinstein, 1992).

The ability of ET-1 to contract isolated rat myometrium has recently been documented and binding sites for ET/Sarafotoxin have been identified in myometrial preparations from different species (Bousso-Mittler et al., 1989; Breuiller-Fouché et al., 1994). We have previously reported that in estrogen-treated rat myometrium ET-1 interacts with a specific ET_A receptor, which results in both activation of the PLC/PtdInsP₂ transducing system through a pertussis toxin-insensitive G protein, and inhibition of the adenylyl cyclase system through a pertussis toxin-sensitive G protein (Dokhac et al., 1994). Preliminary data (Dokhac et al., 1995) indicated that in the myometrium ET-1 also displays stimulatory effects on the PLD pathway. We now extend these observations by studying in more detail the receptor-mediated events at the level of PLD activation. The data further revealed that the two major signals which originated from the breakdown of PtdInsP₂ by PLC, namely increased [Ca⁺⁺]_i and PKC activation, are important regulators of PLD activity and that both signals largely contribute to ET-1-mediated PLD activation.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Chemicals. [³H]Myristic acid (30-40 Ci/mmol) was provided by N.E.N. (Les Ulis, France); endothelin-1, endothelin-3, and BQ123 were provided by Neosystem (Strasbourg, France); -estradiol 3-benzoate, bovine serum albumin, ionomycin, PA, PC, PS, PI, PE, PMA, PDBu, OAG, chelerythrine, calphostin C and H7 were from Sigma (St. Louis, MO); PBut was from Avanti Polar Lipids (Alabaster, AL), pertussis toxin from List Biological Laboratories (Campbell, CA) and Silica Gel 60 plates from Merck (Darmstad, Germany). Ro-31-8220 was generously provided by Dr. Bradshaw (Roche Ltd. Hertfordshire, United Kingdom). The ethanolic solution of [³H]myristic acid was evaporated under N₂ and the residue was dissolved in NH₄OH. All other chemicals were of the highest grade available.

Animals and tissue processing. Immature Wistar female rats (4-5 weeks of age) were treated with 30 µg of estradiol for 2 days and were sacrificed by decapitation the following day. Uteri were removed, and the myometrium was prepared free of endometrium as described previously (Amiot et al., 1993; Dokhac et al., 1994).

PLD assay. The activity of PLD was determined in [³H]myristic acid-labeled myometrium by measuring the formation of [³H]PA and [³H]PBut in the absence and presence of 0.3% butanol, respectively. [³H]PBut, which is formed exclusively through the PLD-catalyzed transphosphatidylation reaction, is considered to be a specific marker of PLD activity (Billah and Anthes, 1990; Liscovitch et al., 1993). Myometrial strips (about 25 mg) were equilibrated for 20 min in 5 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) containing (in mM): NaCl, 117; KCl, 4.7; MgSO₄, 1.1; KHPO₄, 1.2; NaHCO₃, 2.4; CaCl_2, 0.8 and glucose, 1 (gas phase 95% O₂-5% CO₂) under constant agitation. The tissues were then incubated with 8 µCi/ml of [³H]myristic acid in 600 µl of fresh buffer for 5 h by which time the incorporation of [³H]myristic acid into phospholipids had reached a plateau (data not shown). Tissues were then washed once with 5 ml of nonradioactive buffer containing 1 mg/ml of serum albumin, then twice with buffer without serum albumin. After another 20-min incubation in 5 ml of buffer, myometrial strips were transferred to 1 ml of fresh buffer and incubated with 0.3% butanol for 10 min before exposure to the indicated agents. The reactions were stopped at the time indicated in the legends to figures by the rapid immersion of myometrial strips in liquid nitrogen, and lipids were extracted by a modification of the method of Bligh and Dyer (1959). The tissues were homogenized in 1.8 ml of chloroform/methanol/HCl (50:100:1, v/v/v) and left at room temperature for 30 min. One-half milliliter of H₂0 was then added and, after a brief homogenization, the monophase was split by the addition of 0.6 ml of 2 M KCl and 0.6 ml of chloroform. After a vigorous mixing, phases were separated by a 10-min centrifugation at 1000 × g and the aqueous phase was removed. The chloroform extract was dried with a speed vacuum concentrator and was then resuspended in 50 µl of methanol/chloroform (95:5, v/v). Relevant standards were added to each sample before thin-layer chromatography on heat-activated precoated 20 × 20 cm silica gel plates. To determine the incorporation of radiolabeled myristic acid into various phospholipids, plates were developed by three consecutive solvent systems with intermittent drying (Irvine et al., 1984): the first run with petrol ether/acetone (90:30; v/v), with use of the full length of the plate (16 cm); the second run in CHCl₃/methanol/acetic acid/H₂O (75:60:9:0.9; v/v/v/v/) up to 11.5 cm; and the third run in ethyl acetate/acetone/H₂O/acetic acid (40:40:2:1; v/v/v/v/), up to 13 cm. PC, (R_f: 0.01), PI (R_f, 0.32), PS (R_f, 0.42), PE (R_f, 0.78) were well separated from both neutral lipids and free myristic acid which comigrated at the solvent front. To analyze the production of [³H]PBut and [³H]PA, plates were developed in the organic phase of a mixture of ethyl acetate/2,2,4-trimethylpentane/acetic acid/water (13:2:3:10; v/v/v/v). In this solvent system (Van Blitterswijk et al., 1991), PBut (R_f, 0.5) and PA (R_f, 0.26) were separated from each other and from both PC (R_f, 0.01) and neutral lipids, the latter compounds migrating at the solvent front. PC was separated from PE but comigrated with two other phospholipids, PS and PI. After the appropriate runs, the plates were stained with iodine vapor and were analyzed, after iodine sublimation, by a computerized radioactivity scanner. The production of [³H]PA and [³H]PBut was expressed as the percentage of the radioactivity in PC obtained from the same sample.

Data analysis. The results are expressed as the mean ± S.E. and were analyzed statistically by the Student's t test. P < .05 was considered significant.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Distribution of [³H]myristic acid in total lipid extracts of myometrium. Analysis of ³H-labeled lipid extracts obtained from myometrial strips incubated with [³H]myristic acid revealed (table 1) that 80% of [³H]myristic acid was associated with neutral lipids versus 20% of the label incorporated into phospholipids. The major labeled phospholipid was PC, which accounted for 97% of the total labeled phospholipids. PE (contaminated with PA) was next but represented at most 2% of the label, whereas less than 0.5% of the label was associated with PS plus PI. The results are consistent with observations made for other tissues (Cockroft, 1992; Exton, 1994; Liscovitch et al., 1993) and clearly indicate that in the myometrium [³H]myristic acid preferentially labeled the lipid domain of PC and that this fatty acid was not readily incorporated into phosphoinositides. In subsequent experiments, with thin-layer chromatography with a developing system (Van Blitterswijk et al., 1991) that allowed an adequate separation of PA from PC, it was found that the label associated with PA represented 0.56 ± 0.07% of label in PC (see legend to fig. 1).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Time course of ET-1-induced [3H]PA and [3H]PBut accumulation. [3H]Myristic acid-labeled myometrial strips were stimulated with 0.2 µM ET-1 in the absence (A) or presence (B) of 0.3% butanol, added 10 min before ET-1. Incubations were stopped at the indicated times. [3H]PBut () and [3H]PA () production was estimated as described under "Materials and Methods" and expressed as % of label in PC. Values were obtained after substraction of basal values: 0.65% and 0.57% of label in PC for [3H]PA in (A) and (B), respectively, and 0.2% of label in PC for [3H]PBut. Data represent the mean of six independent experiments, each done in duplicate, and the standard error did not exceed 10% of mean.

Time-dependent accumulation of [³H]PA and [³H]PBut induced by ET-1. Figure 1A shows that addition of 0.2 µM ET-1 to [³H]myristic acid-labeled myometrial strips rapidly increased [³H]PA formation. A significant increase was detected within 1 min and reached a maximum at 5 min with a formation of [³H]PA amounting to 0.8% label in PC. The [³H]PA level triggered by ET-1 remained elevated for at least 10 min and then declined progressively with time. With this procedure, the source of PA is ambiguous because it could have been generated by the combined actions of PLC and diacylglycerol kinase rather than PLD (Billah and Anthes, 1990). We therefore took advantage of the unique PLD-catalyzed transphosphatidylation reaction (Billah and Anthes, 1990; Liscovitch et al., 1993), which results in the formation of [³H]PBut in the presence of butanol. Thus, 0.3% butanol was added 10 min before exposure of myometrium to 0.2 µM ET-1. At this concentration, butanol had no effect on inositol phosphate accumulation induced by ET-1 (data not shown). In the absence of ET-1, the percentage of conversion to [³H]PBut was somewhat small (0.15-0.3% of the radioactivity in PC). Addition of ET-1 markedly enhanced [³H]PBut formation with a time course that paralleled that of [³H]PA synthesis. As illustrated in figure 1B, the formation of [³H]PBut triggered by ET-1 increased at the expense of [³H]PA. Thus, at 10 min stimulation, [³H]PBut and [³H]PA formation caused by ET-1 amounted to 1.0 ± 0.1% and 0.3 ± 0.05% of label in PC, respectively. These findings are consistent with the notion that [³H]PBut synthesis stimulated by ET-1 occurred by a PLD-catalyzed transphosphatidylation reaction. Considering that PC appears to be the preferred substrate for PLD (Billah and Anthes, 1990; Exton, 1994), it is reasonable to assume that in rat myometrium PC was the major source for [³H]PBut generated through PLD activated by ET-1.

Dose-dependent effects of ET-1 and ET-3 on [³H]PBut accumulation. Effect of BQ123. ET-1-evoked PLD activation, as determined by 10 min [³H]PBut accumulation, was dose dependent with EC₅₀ = 50 ± 5.2 nM and a maximal response at 0.2 µM ET-1 (fig. 2). ET-3 also stimulated [³H]PBut accumulation, but with a rightward shift of its concentration-response curve and a stimulatory effect at 10 µM that did not exceed 50% of the response triggered by ET-1. The rank order of potency ET-1  ET-3 was consistent with an ET_A receptor (Huggins et al., 1993; Masaki et al., 1994) coupled to PLD activation. This interpretation was confirmed by the ability of BQ123 (Eguchi et al., 1992), a selective ET_A receptor antagonist, to suppress ET-1-mediated [³H]PBut accumulation.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Dose-dependent effects of ET-1 and ET-3 on [3H]PBut accumulation. Inhibitory effect of BQ123. [3H]Myristic acid-labeled myometrial strips were exposed to the indicated concentrations of ET-1 () and ET-3 () for 10 min in the presence of 0.3% butanol added 10 min before the agonist. BQ123 (1 µM), when tested, was added 5 min before the addition of 0.2 µM ET-1. [3H]PBut production was estimated as described under "Materials and Methods" and expressed as % label in PC. Values represent the mean ± S.E. of three independent experiments, each done in duplicate.

Desensitization of the [³H]PBut response to ET-1. As shown above (fig. 1), after a linear increase, the formation of [³H]PBut caused by ET-1 rapidly ceased. Considering that [³H]PBut is metabolically stable, the plateau phase of its formation in response to ET-1 tended to indicate that PLD was transiently activated. To address the possibility of a desensitization phenomenon, myometrial strips were exposed to 0.2 µM ET-1 for various times in the absence of butanol so that no [³H]PBut was formed. The alcohol was then added during the last 10 min of ET-1 treatment to determine any subsequent ET-1-mediated PLD activation. It was previously verified that during the 5- to 30-min pretreatment, ET-1 was not degraded in the medium (Dokhac et al., 1994). Data in figure 3 clearly show that, compared with the nontreated preparation, pretreatment with ET-1 caused a progressive reduction in the stimulatory effect of the peptide in terms of [³H]PBut accumulation. After a 5-min pretreatment with ET-1, the formation of [³H]PBut was reduced by almost 80%. Under these conditions of ET-1-induced self-desensitization, there was no loss of PLD activation triggered by AlF₄ and by bombesin, two activators of PLD (see fig. 4). Thus, during the first 5 min, desensitization of ET-1-evoked [³H]PBut accumulation was an homologous process.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   ET-1 induced a homologous desensitization of the [3H]PBut responses. [3H] myristic acid-labeled myometrial strips were pretreated in the absence of butanol, with 0.2 µM ET-1. At the indicated times, the formation of [3H]PBut was initiated by adding 0.3% butanol and the incubations were continued for a further 10 min (). For comparison with AlF4 () and bombesin (BN, ), tissues were pretreated with 0.2 µM ET-1 for 5 min and then challenged with NaF (20 mM) + AlCl3 (10 µM) and bombesin (100 nM) in the presence of butanol. [3H]PBut production was estimated as described under "Materials and Methods." Results are expressed as a percentage of the control [3H]PBut responses to the indicated agonists (1.15, 1.26 and 0.6% of label in PC, for ET-1, AlF4 and bombesin, respectively). Values represent the mean ± S.E. of three independent experiments, each done in duplicate. * P < .01 vs. ET-1 alone. NS, no significant difference with ET-1 alone. Values for AlF4 and BN were not significantly different from their respective controls that were not pretreated with ET-1.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Effect of bombesin and ionomycin on [3H]But accumulation. [3H]Myristic acid-prelabeled myometrial strips were treated for 10 min with 0.3% butanol. Bombesin (BN, ) at 100 nM and ionomycin (Iono, ) at 10 µM were then added either alone or combined with 0.2 µM ET-1 () and incubations were continued for a further 10 min. [3H]PBut was estimated as described under "Materials and Methods" and expressed as % of label in PC. Values represent the mean ± S.E. of three independent experiments, each done in duplicate. (a), no significant difference with ET-1 stimulation.

Stimulatory effect of fluoroaluminates on [³H]PBut accumulation. Insensitivity to pertussis toxin of ET-1-induced production of [³H]PBut. ET receptors have been characterized as members of the G protein-coupled receptor superfamily (Huggins et al., 1993; Masaki et al., 1994; Sokolovsky, 1994). On the other hand, several studies implicated a heterotrimeric G protein in the activation of PLD (Cockroft, 1992; Exton, 1994; Liscovitch et al., 1993). We previously demonstrated that treatment of myometrium with AlF₄ (20 mM NaF + 10 µM AlCl₃) for 20 min caused the activation of G proteins which are coupled to the PLC and adenylyl cyclase pathways (Amiot et al., 1993; Dokhac et al., 1994). Data in table 2 show that, under these conditions of fluoroaluminate treatment, the production of [³H]PBut was enhanced. These results indicate that a heterotrimeric G protein contributed to the activation of PLD in myometrium. Table 2 further shows that pretreatment of myometrium with 300 ng/ml pertussis toxin for 6 h, conditions under which the pertussis toxin-sensitive G proteins in the cells are completely ADP-ribosylated (Tanfin and Harbon, 1987), did not affect the production of [³H]PBut caused by ET-1.

View this table: [in this window] [in a new window]   TABLE 2 Fluoroaluminates induced accumulation of [3H]PBut; ET-1 stimulatory effect on PLD insensitive to pertussis toxin [3H]Myristic acid-labeled myometrial strips were incubated either with 0.2 µM ET-1 for 10 min or with AlF4 (20 mM NaF + 10 µM AlCl3) for 20 min in the presence of 0.3% butanol added 10 min before the agonist. When indicated, tissues were incubated for 6 h with pertussis toxin at 300 ng/ml; [3H]myristic acid was present during the last 5 h of treatment. [3H]PBut production was evaluated and expressed as described under "Materials and Methods." Values represent the mean ± S.E. of three to five independent experiments, each done in duplicate.

Effect of bombesin on [³H]PBut accumulation. It has been observed for various cellular systems that agonists which elicit PtdInsP₂ hydrolysis also stimulate PLD activity (Cockcroft, 1992; Exton, 1994; Liscovitch et al., 1993). Figure 4 shows that, in addition to ET-1, bombesin, another Ca⁺⁺-mobilizing agonist, when tested at 100 nM, a concentration which has been reported to maximally stimulate inositol phosphate generation (Amiot et al., 1993), also stimulated [³H]PBut accumulation. Simultaneous addition of bombesin to ET-1 did not result in any further enhancement of [³H]PBut accumulation induced by ET-1 alone, which suggested that both agonists share a common pathway in the activation of PLD. These observations raise the possibility that in myometrium activation of PLC and PLD may be interrelated responses to the stimulatory agonists.

Role of PKC on [³H]PBut accumulation. The activation of PLD by tumor-promoting phorbol esters has been reported in a wide variety of cells (Cockcroft, 1992; Exton, 1994; Liscovitch et al., 1993). Figure 5A shows that a 10-min exposure of myometrium to various concentrations of PDBu resulted in a dose-dependent accumulation of [³H]PBut. Half and maximal responses were obtained at 0.5 µM and 2 µM, respectively, in close agreement with the responses obtained for PDBu activation of PKC in several tissues. The response to PDBu was slower in onset than ET-1, becoming apparent by 1 to 2 min, then continuing to accumulate for up to 30 min after addition of PDBu (fig. 5B). After a 10-min stimulation, PDBu was found to be more effective than ET-1 at eliciting [³H]PBut formation. Data in figure 5 (A and B) show that two other activators of PKC, the phorbol ester PMA and the permeable analog of diacylglycerol, OAG, both at 1 µM, also increased [³H]PBut accumulation. In contrast, the inactive 4-phorbol (1 µM) was without effect (not shown). To further determine the role of PKC in the generation of [³H]PBut induced by PDBu, we tested the effects of three PKC inhibitors. It can be seen (fig. 6, upper panel) that calphostin C and chelerythrine (Herbert el al., 1990), at 1 and 10 µM, respectively, inhibited by 40% [³H]PBut accumulation induced by PDBu. Higher concentrations of chelerythrine were not used because of the possibility of chelerythrine reducing cell viability. However, the most potent PKC inhibitor, Ro-31-8220 (Davis et al., 1989), greatly attenuated the generation of [³H]PBut promoted by PDBu. More than 80% inhibition was achieved at 5 µM Ro-31-8220, which suggested that almost the entire stimulatory effect of the phorbol ester on the accumulation of [³H]PBut was mediated by activation of PKC. In additional experiments, a prolonged (5-h) exposure of the tissue to 1 µM PMA led to decrease (down-regulation) in the amounts of PKC determined by immunoreactivity (data not shown). This procedure similarly abolished about 80% of the PDBu-promoted [³H]PBut response (fig. 6, upper panel).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effects of various activators of PKC on [3H]PBut accumulation. [3H]Myristic acid-prelabeled myometrial strips were incubated for 10 min in the presence of 0.3% butanol and then (A) stimulated for an additional 10 min with the indicated concentrations of PDBu, OAG and PMA and (B) stimulated with 1 µM PDBu and 1 µM PMA for the stated times. [3H]PBut accumulation was estimated as described under "Materials and Methods" and expressed as % of label in PC. Values, obtained after substraction of basal values (0.22 ± 0.03% of label in PC) represent the mean ± S.E. of three independent experiments, each done in duplicate.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Effects of PKC inhibitors and prolonged PMA pretreatment on [3H]PBut accumulation induced by PDBu, ET-1 and ionomycin. To test the effects of PKC inhibitors (left part), [3H]myristic acid-labeled myometrial strips were pretreated for 10 min with 10 µM chelerythrine (), 1 µM calphostin C () and the indicated concentrations of Ro-31-8220 (). For down-regulation of PKC, (right part) tissues were exposed to 1 µM PMA for 5 h during the [3H]myristic acid labeling period, then washed with buffer (). The [3H]PBut responses to a 10 min stimulation with ET-1 (0.2 µM), PDBu (1 µM) and ionomycin (10 µM) were estimated in the presence of 0.3% butanol as described under "Materials and Methods." Data were expressed as % of label in PC. Values represent the mean ± S.E. of three separate experiments, each done in duplicate. *P < .01 as compared with their respective controls. NS, no significant difference with their respective controls.

Role of Ca⁺⁺ in the generation of [³H]PBut. To determine whether activation of PLD in the myometrium could arise simply by increasing [Ca⁺⁺]_i, the ability of the calcium ionophore ionomycin to stimulate [³H]PBut formation was examined. Figure 4 shows the increased production of [³H]PBut promoted by a 10-min incubation with 10 µM ionomycin. The amount of accumulated [³H]PBut was similar to that induced by a maximally effective concentration of ET-1. Simultaneous addition of ET-1 to ionomycin did not result in any further enhancement of [³H]PBut accumulation triggered by each agent alone. Hence, both ET-1 and ionomycin appear to share a common pathway in the activation of PLD. As shown in figure 6 (lower panel), treatment of myometrial preparations with chelerythine (10 µM) and with the most potent PKC inhibitor, Ro-31-8220, at its maximal effective concentration, caused 30% and 70% reduction, respectively, of the ionomycin response. Also, the ability of ionomycin to stimulate the generation of [³H]PBut was similarly attenuated (45%) in tissue preparations depleted of PKC. The data demonstrate that a major part of the effect of Ca⁺⁺ entry on PLD activity was mediated by PKC activation.

View larger version (36K): [in this window] [in a new window]   Fig. 7.   Effect of Ca++ on [3H]PBut accumulation induced by ET-1, PDBu, PMA and ionomycin. After the [3H]myristic acid prelabeling step, myometrial strips were transferred to 1 ml of buffer with the indicated concentrations of Ca++ or to a Ca++-depleted medium containing 1 mM EGTA and were allowed to equilibrate for 10 min before a further incubation in the presence of 0.3% butanol. When used, nifedipine at 250 nM () was added in a 800 µM Ca++-containing medium during the 10 min of butanol treatment. Tissues were then incubated for 10 min with 0.2 µM ET-1, 10 µM ionomycin (Iono), 1 µM PMA and 1 µM PDBu. [3H]PBut accumulation was estimated as described under "Materials and Methods" and was expressed, after substraction of basal values (0.2 ± 0.02% of label in PC) as % of label in PC. Values represent the mean ± S.E. of three separate experiments, each done in duplicate. * P < .01 vs. control values in 800 µM Ca++ medium. NS, no significant difference with control values in 800 µM Ca++ medium.

View larger version (30K): [in this window] [in a new window]   Fig. 8.   Combined effects of PKC and Ca++ on [3H]PBut accumulation induced by ET-1. After the [3H]myristic acid prelabeling step, myometrial strips were transferred to a fresh medium containing 800 µM or 1 µM Ca++ and were allowed to equilibrate for 10 min. 0.3% butanol was then added and incubations were continued for an additional 10 min in the presence and absence of 10 µM chelerythrine before exposure of the tissues to 0.2 µM ET-1. [3H]PBut accumulation was estimated 10 min later, as described under "Materials and Methods" and expressed as % of labeled PC. Values represent the mean ± S.E. of three independent experiments, each done in duplicate. * P < .01 versus control.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The results presented in this study provide evidence for the expression in rat myometrium of a PLD pathway that can be stimulated through receptor as well as heterotrimeric G protein activation. The data demonstrate that the potent contractile agonist, ET-1, was able to enhance the accumulation of PBut, the unambiguous transphosphatidylation product of PLD, via the ET_A receptor subtype involving a pertussis toxin-insensitive G protein. Moreover, we have demonstrated that in the myometrium, PLD was activated by pathways involving PKC and Ca⁺⁺ and that the two signals make a major, although not exclusive, contribution to ET-1-mediated PLD activation.

Numerous studies have demonstrated that PA can be generated through the action of PLD that preferentially hydrolyzes PC (Cockcroft, 1992; Exton, 1994; Liscovitch et al., 1993). In rat myometrium labeled with [³H]myristic acid, with PC constituting the major (97%) labeled phospholipid, there was a rapid production of [³H]PA in response to ET-1. The observations that, in the presence of butanol, the production of [³H]PA was diverted largely to the accumulation of [³H]PBut, a specific product of the transphosphatidyltransferase activity of PLD, support the contention that the PLD/PC pathway is the predominant route used by ET-1 to accumulate PA in the myometrium. In view of the relatively higher metabolic stability of PBut, compared with PA, the production of [³H]PBut appeared a suitable indicator for PLD activity in the myometrium.

The components and mechanisms involved in receptor signaling to PLD are not yet totally defined and may even be distinct for different receptors and cellular systems. Besides the receptors themselves, Ca⁺⁺ and/or PKC have been shown to modulate cellular PLD activity. As far as the many cases of stimulation of the PLC/PtdInsP₂ pathway by receptors in which PLD activation has also been reported to occur, it has been proposed that receptor-mediated activation of PLD may be secondary to prior activation of PLC degrading PtdInsP₂. In the myometrium, in addition to its stimulatory effect on PLD activity, ET-1 has enhanced the PLC/PtdInsP₂ pathway (Dokhac et al., 1994). Examination of the rank order of potencies of ET-1 and ET-3, both in inducing the accumulation of inositol phosphates (Dokhac et al., 1994) and the production of PBut, indicated that both signaling pathways responded in complete agreement with the ET peptide selectivity for ET_A receptors. Thus, ET-1 stimulated inositol phosphate accumulation and PBut formation with very similar EC₅₀ values (70 ± 5.7 and 50 ± 5.2 nM, respectively). ET-3 similarly increased the production of both inositol phosphates and PBut but was less potent. Also, BQ123, a selective antagonist of the ET_A receptor subtype, significantly abolished both ET-1-mediated effects. The findings clearly illustrate that both the PLC and the PLD signaling pathways were triggered by the same ET_A subclass receptors. Furthermore, the stimulatory effects of ET-1, both on the accumulation of inositol phosphates (Dokhac et al., 1994) and on the production of PBut, were insensitive to pertussis toxin and were susceptible to self-induced desensitization involving a receptor-mediated homologous process. Such a close relationship between the two phospholipid signaling pathways stimulated by ET-1, added to the ability of another Ca⁺⁺-mobilizing agonist, bombesin (Amiot et al., 1993), to stimulate PLD, suggest that ET-1 may activate PLD via the intracellular signals generated by its stimulatory effect on the PLC/PtdInsP₂ pathway.

It is well established that activation of PKC by phorbol esters can stimulate PLD activity (for reviews, see Cockcroft, 1992; Exton, 1994; Liscovitch et al., 1993). The present results in myometrium show that two phorbol esters, PMA and PDBu, enhanced the production of PBut, whereas the inactive 4-phorbol ester was without effect. PDBu-mediated PBut production was markedly attenuated (80%) by inhibitors of PKC (Davis et al., 1989; Herbert et al., 1990,), particularly Ro-31-8220, as well as after a prolonged treatment of the myometrium with PMA, conditions that led to PKC depletion, which indicated that PDBu exerted its effect on PLD via PKC. The demonstration that PKC serves as an up-stream regulator of PLD in the ET-1 signal transduction cascade was provided by the findings that different PKC inhibitors (chelerythrine, Ro-31-8220), as well as PKC down-regulation, attenuated the production of PBut caused by ET-1. However, ET-1-stimulated PLD activity was inhibited by no more than 40% under conditions that abrogated the PDBu response. It clearly appeared that the PKC-triggered process accounted only for a part of the ET-1 effect on PLD, which suggested an additional PKC-independent mechanism(s).

In addition to PKC, Ca⁺⁺ has also been reported to modulate PLD activity in different intact cell preparations (for reviews, see Billah and Anthes, 1990; Exton, 1994; Liscovitch et al., 1993). Our present findings illustrate that the Ca⁺⁺ ionophore, ionomycin, enhanced the accumulation of PBut in the myometrium, to an extent similar to that displayed by ET-1. The stimulation of PLD activity produced by ionomycin was highly dependent on extracellular Ca⁺⁺ and was almost completely inhibited in the presence of the EGTA used to buffer extracellular Ca⁺⁺ to 1 µM. Thus, increases in [Ca⁺⁺]_i are able to activate PLD. Similarly to previous reports (Cook et al., 1991; Llahi and Fain, 1992), an important attenuation of ionomycin effect was noted with Ro-31-8220 and in PKC-depleted myometrial preparations, which suggested that at least part of ionomycin-stimulated PLD activity was perhaps caused by Ca⁺⁺, acting as a cofactor in activation of PKC. In contrast, the PDBu stimulatory effect on PLD was not modified in the Ca⁺⁺-poor medium, consistent with PDBu exerting its effect solely by activation of PKC. The dependence on the rise of Ca⁺⁺ for the activation of PLD mediated by receptor agonists has been reported previously (Billah and Anthes, 1990; Exton, 1994; Liscovitch et al., 1993). The observation that, in the Ca⁺⁺-poor medium, ET-1-stimulated PLD activity was attenuated by about 60%, suggested that a sustained rise in [Ca⁺⁺]_i was partly involved in the peptide-mediated activation of PLD. The findings that thapsigargin failed to alter the accumulation of [³H]PBut in response to ET-1 precluded the contribution of the intracellular InsP₃-sensitive Ca⁺⁺ pool (Arnaudeau et al., 1994; Molnar and Hertelendy, 1995) in the activation of PLD caused by the peptide. Similar findings were reported for the activation of PLD by bombesin (Cook et al., 1991) and by bradykinin (Pyne and Pyne, 1995). Of interest, the inhibition noted with nifedipine supports the notion that, similar to other Ca⁺⁺-mobilizing agonists (Dokhac et al., 1996), ET-1 is capable of activating an ion channel which elicits specific Ca⁺⁺ influx in the myometrium and thus contributes to the regulation of PLD activity.

Furthermore, when the PKC inhibitor, chelerythrine, was added to a poor Ca⁺⁺ medium, an additive attenuation of ET-1-mediated PBut accumulation was observed, consistent with the interpretation that both Ca⁺⁺- and PKC-mediated mechanisms contributed to the peptide stimulation. Nevertheless, inhibitions obtained by the combined effects of decreased Ca⁺⁺ and PKC inhibition were not completely additive, which supported the interpretation that some of the effects of increased [Ca⁺⁺]_i can be attributed to activation of PKC. Our data are similar to those previously reported for bombesin (Cook et al., 1991)- and norepinephrine (Llahi and Fain, 1992)-mediated PLD activation. They are in variance with recent reports demonstrating that prostaglandin F₂ stimulated PLD in osteoblast-like cells via a Ca⁺⁺-calmodulin process which was totally independent of PKC activation (Imamura et al., 1995). Despite many studies, the exact mechanism for the implication of PKC in PLD activation has not yet been defined. It may be possible that PKC interacts directly with PLD in membranes or that PKC interacts with other membrane-associated proteins that in turn activate PLD (Conricode et al., 1992; Singer et al., 1996)

Our observations that AlF₄ enhanced the production of PBut support the contention that a heterotrimeric G protein is contributing to the modulation of PLD activity in the myometrium. The ET-1 stimulatory effect on PLD is shown to be insensitive to pertussis toxin. Because pertussis toxin does not prevent PtdInsP₂ degradation caused by ET-1 (Dokhac et al., 1994), it remains unclear as to whether the pertussis toxin insensitivity at the level of PLD activation may reflect a potential link between the PLC and PLD pathways, via Ca⁺⁺ and PKC, or may be suggestive of a pertussis toxin-insensitive G protein that directly couples to PLD. Although the activation of PLD by ET-1 in the myometrium appears to be determined by the two major signals derived from PtdInsP₂ breakdown, increased [Ca⁺⁺]_i and PKC activation, it is worth noting that with simultaneous inhibition of both PKC activity and Ca⁺⁺ entry into the cell, ET-1 still retained the ability to trigger a small (30%) but consistent production of PBut. The possibility that receptor-mediated up-regulation of PLD activity in the myometrium could be modulated by other components such as small G proteins (Cockcroft et al., 1994; Malcolm et al., 1994), tyrosine phosphorylation (Bourgoin and Grinstein, 1992) and an yet undefined cytosolic factor (Bourgoin et al., 1995; Lambeth et al., 1995; Singer et al., 1996) should be worth considering.

A large body of evidence emphasizes the important role of ET-1 in the regulation of different functions of smooth muscle cells (Huggins et al., 1993). We previously reported that in the myometrium, activation of ET_A receptors are coupled to both the stimulation of the Ca⁺⁺/PtdInsP₂ pathway and the inhibitory arm of the adenylyl cyclase. The resulting increase in Ca⁺⁺ and the decline in cAMP provide major determinants of ET-1-induced uterine contractions (Dokhac et al., 1994). The present findings demonstrate that in the myometrium, activated ETA receptors are associated with an additional signaling system, namely the PLD pathway which degrades PC and leads to an increased production of PA. The functional significance of PA in the myometrium remains to be delineated. PA has been proposed to mediate a variety of cellular processes, including promotion of entry and mobilization of Ca⁺⁺, as well as cell proliferation (Cockcroft, 1992; Exton, 1994; Liscovitch et al., 1993). Such potentially regulatory processes are particularly interesting in view of the key role of both motility and cell proliferation in the physiology of the myometrium.