Introduction

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Reversible phosphorylation of proteins on serine, threonine and/or tyrosine residues is a major mechanism implicated in the regulation of cellular signal transduction pathways. PP1 and PP2A are two of the four major serine/threonine phosphatases present in eukaryotic cells (Cohen et al., 1990). In the past few years, attention has focused on the physiological role of these enzymes taking advantage of the availability of potent, highly selective inhibitors (Ishihara et al., 1989a). Among these inhibitors, OA, a polyether toxin responsible for diarrhetic shellfish poisoning (Tachibana et al., 1981), has been widely used to characterize the role of PP1 and/or PP2A in the regulation of cellular processes (Cohen et al., 1990; Gong et al., 1992a).

Smooth muscle contraction is primarily regulated by the cytosolic calcium concentration ([Ca⁺⁺]_i). Phosphorylation of the LC₂₀ by the Ca⁺⁺/calmodulin-dependent enzyme MLCK is considered the essential step to initiate contraction (Adelstein and Klee, 1981; Somlyo and Somlyo, 1994). Dephosphorylation of LC₂₀ by MLCP causes relaxation (Somlyo and Somlyo, 1994). The mammalian smooth muscle MLCP holoenzyme has recently been purified and consists of the catalytic subunit of PP1 and two other regulatory subunits (Shirazi et al., 1994). The mechanisms that interfere with the activities of the phosphorylating (MLCK) and dephosphorylating (MLCP) enzymes are able to modulate the level of force and LC₂₀ phosphorylation achieved at a constant [Ca⁺⁺]_i (Gong et al., 1992b; Kubota et al., 1992). In good correlation with this statement, OA and other PP1 inhibitors (i.e, calyculin A, tautomycin) induce high-amplitude, well-sustained contractions which are accompanied by little or no increase in [Ca⁺⁺]_i (Ishihara et al., 1989b; Hartshorne et al., 1989; Hori et al., 1991) but are correlated with an increase in LC₂₀ phosphorylation in vascular and visceral smooth muscles (Bialojan et al., 1988; Obara et al., 1989; Gong et al., 1992a; Suzuki and Itoh, 1993). Nevertheless, in addition to LC₂₀, OA increases the phosphorylation state of many other cellular proteins (Walker and Watson, 1992). In smooth muscles, phosphorylation of proteins involved in some signaling cascades, i.e., PKA-, PKC- or PKG-dependent pathways, could additionally modulate the sensitivity of the contractile machinery (Somlyo and Somlyo, 1994). It thus appears that OA-induced contraction may result from various mechanisms.

In the estrogen-primed rat uterus, we showed previously that OA induces a contraction which is independent of neurotransmitter release and membrane receptor activation (Candenas et al., 1992; Arteche et al., 1995). The present work was designed to further characterize the signaling transduction pathways and the possible kinases involved in the contractile response induced by OA in this tissue.

	Methods

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Animals. Virgin female Wistar rats (200-250 g) were pretreated with 17-estradiol benzoate (20 µg·kg¹ i.p.) 24 h before the experiment. Rats were sacrificed, and myometrial tissue was removed. The estrus stage was confirmed by microscopic examination of a vaginal smear taken posthumously.

Tissue bath experiments. Longitudinal strips of uterine smooth muscle (8-10 mm in length and 1-2 mm in width) were prepared and mounted in isolated tissue baths containing 4 ml of a PSS1 of the following composition (mM): NaCl, 154; KCl, 5.6; CaCl₂, 0.54; MgCl₂, 0.95; NaHCO₃, 5.95; and glucose, 2.78, pH 7.4 with NaOH. The preparations were bubbled continuously with 95% O₂/5% CO₂ and warmed to 32°C. A low Ca⁺⁺ solution was used to avoid development of myometrial spontaneous activity, which has been shown to influence the amplitude of the subsequent contractile responses (Lalanne et al., 1984). Mechanical responses were recorded isometrically by means of force-displacement transducers (Grass FT-03) connected to a LETICA amplifier and a ABB GOERZ SE 130 multichannel recorder. The tissue was immersed in PSS1 and equilibrated for 45 min under a resting tension of 0.5 g. After the equilibration period, the preparation was challenged two or more times by administration of a maximally effective concentration of ACh (1 mM) until constant responses were obtained. The last response served as an internal standard for all subsequent contractions. Uterine strips were then allowed to equilibrate for a further 60-min period before OA or one of its derivatives was added. Only one concentration of OA or derivative was applied to each strip because we found in previous experiments that OA cannot be removed by washing (Candenas et al., 1992). We used OA at 20 µM because previous experiments revealed that this OA concentration induced a contraction of an amplitude similar to that of the maximal ACh-induced contraction (Arteche et al., 1995). OA derivatives were also used at this concentration for comparison. In some experiments, a single concentration of a protein kinase inhibitor (test tissues) or saline or drug vehicle (time-matched paired control tissues) was added to the bath 30 min before OA and maintained in contact with the preparation during the exposure to OA. Only one inhibitor was tested in each strip. The maximal contractile effect (E_max) induced by OA was expressed as a percentage of the maximal tension evoked by 1 mM ACh. Other parameters obtained for characterizing the biphasic response to OA were: time-to-peak tension, time for 50% relaxation and percent of relaxation at a given time. The maximal increase in tone induced by OA was considered as 100%.

Measurement of [Ca⁺⁺]_i. Uteri were removed and placed in a PSS2 of the following composition (mM): NaCl, 130; KCl, 5.6; CaCl₂, 1.9; MgCl₂, 0.9; glucose, 11; N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 10, pH 7.4 with NaOH. The uterine horn was open longitudinally and under binocular control the endometrium and the circular muscle layer were removed. The myometrium was cut in several pieces (1 × 1 mm), incubated for 10 min in low Ca⁺⁺ (200 µM) PSS2 and then incubated in Ca⁺⁺- and Mg⁺⁺-free PBS (Biochrom KG, Berlin, Germany) containing 0.09% collagenase, 0.045% pronase and 2% bovine serum albumin at 37°C for two successive periods of 20 min. After these periods, the solution was removed and the myometrial pieces were incubated again in a fresh enzyme-free solution and triturated with a fire-polished Pasteur pipette to release cells. Cells were stored on glass coverslips at 4°C in PSS2 containing 0.8 mM Ca⁺⁺ and used on the same day. To assess the dynamic changes in [Ca⁺⁺]_i of individual myometrial cells, we used the Ca⁺⁺-sensitive fluorophore indo 1. Cells were loaded with 1 µM indo 1 acetoxymethyl ester (AM), the cell-permeant acetoxymethyl derivative of indo 1, for 30 min at room temperature (20 ± 0.5°C). Coverslips containing indo 1-loaded cells were then washed for 25 min with fresh PSS2 to remove extracellular indo 1-AM. [Ca⁺⁺]_i was estimated from the indo 1 fluorescence ratio method using single-wavelength excitation (360 ± 10 nm) and dual emission (405 ± 10 and 480 ± 10 nm) (Guibert et al., 1996). A microspectrofluorimeter was constructed from a Nikon (Diaphot 300) inverted microscope fitted with epifluorescence (×40 oil immersion objective). The intensities of transmitted light were recorded by two photometers (P100, Nikon), and single photon currents were converted to voltage signals. Signals at each wavelength were digitized and stored on a PC using a PC-Lab Card 812 interface. Sampling was at 17 Hz. The ratio (R = F405/F480) was calculated on-line and displayed with the two voltage signals on a monitor. [Ca⁺⁺]_i was estimated from the ratio of the fluorescence (Grynkiewicz et al., 1985), with use of a specific calibration for indo 1 determined within myometrial cells. Isolated cells were superfused with OA (20 µM) for 30 or 60 min. ACh was applied to the recorded cell by pressure ejection from a glass pipette for the period indicated on the records. No change in [Ca⁺⁺]_i was observed during control ejection of PSS2. Each record of [Ca⁺⁺]_i response to OA or ACh was obtained from a different cell. Each type of experiment was repeated for the number of cells indicated in the text.

Measurement of arachidonic acid. The whole uterus from a estrogen-primed rat was used in these experiments. After removal of adhering fat and mesenteric attachments, each uterine horn was opened longitudinally and mounted in an isolated tissue bath containing 10 ml of PSS1 continuously gassed with 95% O₂/5% CO₂ and maintained at 32°C. Uteri were then equilibrated in PSS1 containing 0.25% fatty acid-free bovine serum albumin for 60 min, with washing every 15 min. After the equilibration time, one uterine horn was incubated with saline (control tissue) and the other with okadaic acid (test tissue) for different times. The reaction was stopped by adding 10 ml of methanol chilled in dry ice and uteri were rapidly transferred to tubes placed in dry ice and containing 0.25% of the antioxidant butylated hydroxytoluene in 2 ml of chloroform/methanol (2:1, v/v). The mixture also contained 7.5 µg of arachidate used as an internal standard and 50 µg of linoleic and linolenic acid to aid in arachidonic acid recovery. Samples were then shaken vigorously and vortexed twice for about 2 min. After addition of 0.2 ml of water to aid in separation of the aqueous and organic phases, the samples were centrifuged (800 × g) for 10 min at 4°C. The lower organic phase was then transferred to a clean silanized tube, and the remaining aqueous phase was re-extracted with chloroform and the extract combined with the previous organic phase. The organic phase was then dried under a N₂ stream and derivatized to the pentafluorobenzyl ester in 100 µl of acetonitrile with 50 µl of 10% isopropylethylamine in acetonitrile and 50 µl of 33% pentafluorobenzylbromide in acetonitrile, as described previously (Gong et al., 1995). The samples were reacted for 10 min at room temperature, dried under N₂ and reconstituted in 20 µl hexane. The samples were analyzed by gas-liquid chromatography by a gas chromatograph (KNK 3000-HRGC, Konik Instruments, Miami, FL) equipped with flame ionization detectors. A 2-µl aliquot of the sample was injected on a 25 m × 0.22 mm column (0.25 µm BP-5, Scientific Glass Engineering, Austin, TX). The carrier gas was hydrogen, at 3.5 ml/min flow rate. The injection port temperature was 100°C and the detector temperature was 275°C. The oven temperature was programmed to run for 2 min isothermically, followed by a temperature increase of 10°C/min to 200°C and then of 8°C/min to 280°C maintained for 10 min. Retention times (tr) were 15.7 and 20.0 min for arachidate and arachidonate, respectively. The amount of arachidonic acid in terms of free acid was calculated according to the equation: where factor was calculated from a calibration curve according to the equation:

Isolation of okadaic acid and 7-hydroxy-4-methyl-2-methylen-hept-4(E)-enyl okadaate. OA and its ester derivative (fig. 1) were obtained from unialgal cultures of two different strains of the marine dinoflagellate Prorocentrum lima coded PL-4 and PL-2, respectively, as previously described (Norte et al., 1991, 1994).

View larger version (K): [in this window] [in a new window]   Fig. 1.   Chemical structure of OA and its derivatives.

Preparation of okadanol and methyl okadaate. Methyl okadaate and okadanol (fig. 1) were prepared from OA by the following procedure: OA (5 mg) was dissolved in 0.5 ml of ethyl ether. One milliliter of diazomethane (CH₂N₂), prepared from Diazald, was added to the ether solution to obtain the methyl ester of okadaic acid. To prepare okadanol, the previous reaction mixture was stirred at 0°C for 4 h, and the solvents were then completely removed under vacuum. The residue was dissolved in 1 ml CH₂Cl₂ and cooled at 70°C. One-half milliliter of a solution of DIBAL-H (1 M) in CH₂Cl₂ was cautiously added and then, for 6 h while, the temperature was slowly raised to 0°C. After addition of 2 ml water, the product was extracted three times with 3 ml CH₂Cl₂. The organic extracts were washed with 2 ml of 2% HCl, dried over anhydrous Na₂SO₄, filtered and the solvent removed under vacuum. Final purification was achieved by using a mixture of methanol/water (85:15, v/v) as eluent on a µBondapak C-18 column HPLC reverse-phase chromatography.

Materials. Collagenase (type CLS1) was obtained from Worthington (Freehold, NJ). Indo 1-AM was from Calbiochem (France Biochem, Meudon, France). Pronase (type E), bovine serum albumin, fatty acid-free bovine serum albumin, acetylcholine hydrochloride, W-7, KN-62, neomycin sulfate, mepacrine, staurosporine, calphostin C, H-7, ML-9, linoleic acid, linolenic acid, methyl arachidate, arachidonic acid, were from Sigma (St. Louis, MO).

Expression and statistical analysis of results. All values in the text and tables are expressed as mean ± S.E.M. for n number of experiments. Statistical significance of differences between two means was assessed by Student's t test. Multiple means were compared by one-way analysis of variance (ANOVA). P values of less than .05 were considered to represent significant differences.

	Results

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Effects of okadaic acid and its derivatives on the mechanical activity in the rat uterus. OA (20 µM) caused a slowly developing contraction (40-45 min) which after plateauing, was followed by a gradual decay in tension. The characteristics of this response are shown in table 1. The structurally related compound okadanol (20 µM) failed to induce any significant contraction (fig. 2). Conversely, the synthetic compound methyl okadaate (20 µM) and the naturally occurring ester 7-hydroxy-4-methyl-2-methylen-hept-4(E)-enyl okadaate (20 µM) induced a contraction similar both in amplitude and kinetics to that evoked by the free acid (fig. 2).

View larger version (K): [in this window] [in a new window]   Fig. 2.   Time course of the effect of OA (20 µM, ), 7-hydroxy-4-methyl-2-methylen-hept-4(E)-enyl okadaate (20 µM, ), methyl okadaate (20 µM, ) and okadanol (20 µM, ) on the mechanical activity in the estrogen-primed rat uterus. Data are means ± S.E.M. of four to eight experiments, expressed as a percentage of the maximal response to ACh (1 mM).

Effect of protein kinase inhibitors on the contractile response to okadaic acid. The data summarized in table 1 show that the OA (20 µM)-induced contraction was unaffected in the presence of one of the following compounds: the PKC inhibitor calphostin C (3 µM), the PKC and PKA inhibitor H-7 (30 µM), the Ca⁺⁺/calmodulin-dependent protein kinase II inhibitor KN-62 (10 µM), the phospholipase C inhibitor neomycin (5 mM) and the phospholipase A₂ inhibitor mepacrine (30 µM). The calmodulin inhibitor W-7 (100 µM) did not modify the amplitude of the OA-induced contraction but significantly decreased the time needed to reach 50% relaxation (fig. 3B). The MLCK inhibitor ML-9 (1 mM) significantly reduced the peak amplitude of the contraction to OA (fig. 3C). The nonselective protein kinase inhibitor staurosporine (0.03-0.1 µM) did not modify the contractile component of the OA-induced mechanical response, but it inhibited the subsequent decay in tension (fig. 3D).

View larger version (K): [in this window] [in a new window]   Fig. 3.   Typical tracings showing the response to OA (20 µM) before (control, A) and after pretreatment of the rat uterine strip for 30 min with (B) W-7 (100 µM); (C) ML-9 (1 mM);and (D) staurosporine (Staur., 0.03 µM). A first response to ACh (1 mM) was used as internal control. The records were obtained in different strips from the same uterus and are representative of typical results in four to five experiments.

Effect of okadaic acid on [Ca⁺⁺]_i. In a large series of myometrial cells (n = 30), incubation with OA (20 µM) for 30 min did not induce any significant change in the average basal [Ca⁺⁺]_i value, 114 ± 4 nM in cells maintained in PSS2 (n = 30) and 132 ± 4 nM in cells treated with OA for 30 min (P > .05). A similar [Ca⁺⁺]_i value was recorded in cells pretreated with OA (20 µM) for 60 min (n = 6). In another set of experiments, short (30 s) microejection of ACh (10 µM) near myometrial cells caused a biphasic [Ca⁺⁺]_i response consisting in a rapid transient peak followed by a lower sustained plateau phase which persisted throughout the ACh stimulation (fig. 4A). The initial transient peak increased [Ca⁺⁺]_i from a resting value of 113 ± 4 nM to 310 ± 15 nM (n = 14). The [Ca⁺⁺]_i level during the plateau phase averaged 175 ± 5 nM (measured at the end of the stimulation) (n = 14). The delay between the beginning of ACh ejection and the transient peak in [Ca⁺⁺]_i was 4 s (n = 14). [Ca⁺⁺]_i rapidly returned to basal levels after the cessation of ACh microejection. Pretreatment of the cells with OA for 30 (fig. 4B) or 60 min (n = 4, not shown) did not alter the ACh-induced [Ca⁺⁺]_i response.

View larger version (K): [in this window] [in a new window]   Fig. 4.   Effect of ACh on [Ca++]i in freshly dispersed myometrial cells. (A) control cells; (B) cells preincubated with OA (20 µM, 30 min). Ejection of ACh (10 µM) near the cell induced a transient peak increase in [Ca++]i followed by a sustained phase during which [Ca++]i remained slightly over base-line levels. Records are representative of typical results in 14 (a) or 10 (b) different cells.

Effect of okadaic acid on the intracellular levels of free arachidonic acid. The intracellular basal level of arachidonic acid in uteri from estrogen-primed rats was 0.42 ± 0.05 ng/mg tissue wet weight (n = 10). OA (20 µM; 5, 15, 45 and 90 min contact) slightly increased the levels of arachidonic acid, but the increase did not reach statistical significance (P > .05, one-way ANOVA). In similar experimental conditions, a high K⁺ (60 mM) solution (5 min contact) increases intracellular free arachidonic acid levels by 436 ± 62% (n = 8), respective to base-line values.

	Discussion

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The present study shows that OA (20 µM) and some of its derivatives induce a transient contraction in the estrogen-primed rat uterus. The mechanism of action of OA is generally ascribed to its binding to the catalytic subunit of PP1 and/or PP2A (Ishihara et al., 1989a; Takai et al., 1992), leading to phosphatase inhibition and then to an increase in the phosphorylation state of many cellular proteins including LC₂₀. Nevertheless, it has also been reported that smooth muscle contraction caused by OA is due to a direct effect on the ATP-dependent interaction between actin and myosin. This latter effect is independent of the inhibitory one on protein phosphatases (Hayakawa et al., 1991). Studies on the structure-activity relationship of OA and its derivatives have shown that the inhibitory activity on protein phosphatases depends on the presence of the free acid group in OA (Nishiwaki et al., 1990; Takai et al., 1992). In the present study, okadanol, the chemical structure of which only differs from that of OA by the loss of the carboxyl group, is ineffective as a phosphatase inhibitor (Nishiwaki et al., 1990) and failed to contract uterine smooth muscle. This suggests that the contractile effect caused by OA is specifically mediated by inhibition of PP1 and/or PP2A. The synthetic ester methyl okadaate, previously shown to be ineffective as an inhibitor of purified protein phosphatases (Nishiwaki et al., 1990; Takai et al., 1992), and the naturally occurring ester 7-hydroxy-4-methyl-2-methylen-hept-4(E)-enyl okadaate were as active as the free acid. Esterification is a common strategy to obtain cell-permeant compounds which then are deesterified intracellularly by specific esterases (Grynkiewicz et al., 1985). In addition to the naturally occurring ester used in the present study (Norte et al., 1994), other esters with similar properties have also been isolated from diarrhetic shellfish poisoning toxin-producing Prorocentrum species (Hu et al., 1995; Needham et al., 1995). The finding that the ester derivatives could be active on intact cells is important from a toxicological point of view and suggest that the presence in shellfish of levels of OA or its derivatives toxic for human health must be studied by use of a mouse bioassay. Alternative immunological or analytical methods would only detect OA.

OA (20 µM) failed to alter both basal and ACh-induced [Ca⁺⁺]_i values. Because transient and sustained phases of the ACh response in rat uterus have been ascribed previously to a Ca release from intracellular stores and a calcium influx through the plasmalemma, respectively (Arnaudeau et al., 1994), these data indicate that OA has no effect on Ca⁺⁺ movements in myometrial cells. Consequently, OA-induced contraction in the estrogen-primed rat uterus does not depend on a previous increase in [Ca⁺⁺]_i and is probably caused by a direct interaction with the contractile machinery. In this connection, simultaneous measurements of tension and [Ca⁺⁺]_i in different smooth muscles have revealed that the contraction induced by OA or other phosphatase inhibitors, i.e., calyculin A and tautomycin, is accompanied by little or no change in [Ca⁺⁺]_i (Hartshorne et al., 1989; Ishihara et al., 1989b; Hori et al., 1991).

The present work also suggests that calmodulin or a calmodulin-regulated protein could be involved in sustaining the response to OA. This suggestion is drawn from the following observations: 1) the contraction to OA was significantly more sustained in Ca⁺⁺-containing solution (this study) than in Ca⁺⁺-free solution (Arteche et al., 1995) and 2) inhibition of calmodulin by W-7 caused a significant increase in the rate of tension decay of the response to OA without altering the amplitude of this response. Moreover, the contraction induced by OA was not accompanied by any change in [Ca⁺⁺]_i; and ML-9, a selective inhibitor of MLCK, significantly reduced the amplitude of this contraction. Collectively, these findings suggest that a constitutively active MLCK may be involved in the contraction to OA independently of Ca⁺⁺ and calmodulin, as previously suggested in other smooth muscles (Hartshorne et al., 1989; Suzuki and Itoh, 1993).

OA enhances phosphorylation of many cellular proteins and this results in the apparent activation of protein kinases (Cohen et al., 1990; Walker and Watson, 1992). Among them, PKC, PKA and PKG are likely candidates (Ashizawa et al., 1989; Walker and Watson, 1992). These kinases could then modify the sensitivity of the contractile proteins (Somlyo and Somlyo, 1994; Savineau and Marthan, 1994) thus influencing the contractile response to OA. We previously reported that the uterine OA-induced contraction was not altered in the presence of various cAMP- and/or cGMP-elevating agents (Candenas et al., 1992). The present study shows that OA-induced contraction was also unaffected in the presence of the PKC inhibitor calphostin C, the PKC and PKA inhibitor H-7 and the Ca⁺⁺/calmodulin kinase II inhibitor KN-62. The phospholipase C inhibitor neomycin and the phospholipase A₂ inhibitor mepacrine also failed to modify the OA-induced contraction. These results suggest that these kinases or phospholipases do not play a major role in sustaining the contractile response to OA in the rat myometrium.

Smooth muscle MLCP consists of the catalytic subunit of PP1 and two other regulatory subunits (Shirazi et al., 1994). OA, which is an inhibitor of PP1, induces LC₂₀ phosphorylation and inhibited dephosphorylation of phosphorylated LC₂₀ in virtually all smooth muscles that have been studied (Bialojan et al., 1988; Gong et al., 1992a; Erdödi et al., 1988). In rat uterus, ML-9 decreased the OA-induced contraction, which suggests that OA may also induce LC₂₀ phosphorylation in this tissue. However, the OA (20 µM)-induced contraction was not sustained. It has been shown that: 1) calyculin A also induces LC₂₀ phosphorylation and transient contraction in the skinned rabbit mesenteric artery (Suzuki and Itoh, 1993); 2) relaxation by OA of tracheal smooth muscle precontracted with carbachol is not accompanied by LC₂₀ dephosphorylation (Tansey et al., 1990); 3) isoproterenol and sodium nitroprusside relax precontracted porcine uterine muscle without dephosphorylation of LC₂₀ (Barány and Barány, 1993). Collectively, these data suggest that LC₂₀ phosphorylation, although required to initiate contraction, may not be sufficient to maintain contraction. In our study, staurosporine was able to inhibit the relaxant component of the OA-induced mechanical response without affecting the amplitude of the initial, contractile component. This favors the suggestion that a protein kinase sensitive to staurosporine could mediate the inhibitory phase of the response to OA. Alternatively, the effect of staurosporine could be caused by inhibition of a kinase which negatively regulates Ca⁺⁺/calmodulin or a Ca⁺⁺/calmodulin-regulated protein. In any case, the inhibition of tension decay was unique to staurosporine and, therefore, not mediated by an effect on MLCK, PKC, PKA or Ca⁺⁺/calmodulin kinase II, kinases which have been reported to be inhibited by staurosporine (Tamaoki et al., 1986; Nakano et al., 1987; Yanagihara et al., 1991).

Finally, it has recently been suggested that arachidonic acid could act as a messenger regulating myosin phosphatase and coupling stimulation of G-protein-associated membrane receptors and Ca⁺⁺-sensitizing effects caused by agonists (Gong et al., 1992b, 1995). OA stimulates the release of arachidonic acid and its metabolites in several cell types, i.e., rat cardiomyocytes, aortic smooth muscle and liver cells (Levine, 1991; Braconi et al., 1992). An increase in arachidonic acid levels caused by OA could then affect or participate in the observed contraction. In our hands, OA induced a slight but not significant increase in the intracellular levels of arachidonic acid in the rat uterus. This finding does not support a role for arachidonic acid in influencing the OA-induced mechanical response in myometrium.

In conclusion, the present data show that, in the rat uterus, OA induces a contractile response which is specifically mediated by PP1 and/or PP2A inhibition and derived from a direct interaction with the contractile machinery. The mechanical activity of the uterus increasing throughout the pregnancy and the parturition, the relative role of cellular mechanisms identified using OA as a tool remains to be assessed in these conditions.