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Effect of Nitric Oxide on Arachidonic Acid Release from Human Amnion-like WISH Cells
Placenta (2002), 23, 575–583 doi:10.1053/plac.2002.0842, available online at http://www.idealibrary.com on
Effect of Nitric Oxide on Arachidonic Acid Release from Human Amnion-like WISH Cells C. Biondia,d, S. Fiorinia, I. Boarinia, L. Barbina, F. Cervellatib, M. E. Ferrettia and F. Vescec a Department of Biology, Section of General Physiology, and b Electron Microscopy Center, University of Ferrara, via L. Borsari, 46, 44100-I Ferrara, Italy and c Department of Biomedical Sciences and Advanced Therapy, Section of Obstetrics and Gynecology, University of Ferrara, corso Giovecca, 203, 44100-I Ferrara, Italy Paper accepted 16 May 2002
In order to clarify the possible interactions between nitric oxide (NO) and arachidonic acid (AA) pathways, human amnion-like WISH cells were perifused to measure the effects of the following substances on [3H]arachidonic acid release: (1) sodium nitroprusside (SNP), a nitric oxide donor; (2) 1,1,1-trifluoromethyl-6,9,12,15-heicosatetraen-2-one, a cytosolic phospholipase A2 (cPLA2) inhibitor; (3) -arginine, the substrate of nitric oxide synthase (NOS); (4) 3-(5 -Hydroxymethyl-2 -furyl)-1benzylindazole and 1H-[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one, activator and inhibitor of soluble guanylyl cyclase, respectively; (5) a membrane-permeable non-hydrolyzable analogue of guanosine-3 ,5 -cyclic monophosphate (cGMP). Furthermore, the effect of SNP on prostaglandin E2 (PGE2) release was tested. Exogenous and endogenous NO, as well as the guanylyl cyclase activator and cGMP analogue, significantly increased [3H]arachidonic acid release. Both soluble guanylyl cyclase and PLA2 inhibitors counteracted SNP response. Exogenous NO increased PGE2 release, although to a much lesser degree compared with arachidonic acid release. Our results indicate that NO stimulates AA release in WISH cells by activating PLA2 through a cyclic GMP-dependent mechanism. 2002 Published by Elsevier Science Ltd. All rights reserved. Placenta (2002), 23, 575–583
INTRODUCTION The mechanisms underlying uterine quiescence during pregnancy and the changes leading to onset of labour are not completely understood. It has long been known that prostaglandins (PGs) play a key role in the regulation of parturition by stimulating uterine contractility and causing cervical ripening during labour (Novy and Liggins, 1980). More recently NO, a free radical generated from -arginine by nitric oxide synthase, has been identified among the factors involved in the regulation of myometrial activity (Ledingham et al., 2000; Yallampalli et al., 1998). It has been shown in the rat and rabbit that NO maintains uterine relaxation during pregnancy, and that onset of labour is favoured by the decrease of myometrial and increase of cervical NOS activity (Ledingham et al., 2000; Buhimschi et al., 1996). In humans, there is controversy both about myometrial sensitivity to NO and about the presence and regulation of NOS activity in the myometrium (Buhimschi et al., 1995; Barber et al. 1999). Some authors have reported that enzyme expression is similar To whom correspondence should be addressed at: Dipartimento di Biologia, Sezione di Fisiologia Generale, Universita` di Ferrara, Via L. Borsari, 46, I44100 Ferrara, Italy. Tel.: 0532-291482; Fax: 0532207143; E-mail: [email protected] 0143–4004/02/$-see front matter
in labour and non-labour conditions (Dennes et al., 1999; Thomson et al., 1997) while others, in recent studies, have demonstrated that the expression of inducible NOS (iNOS) increased during the third trimester and declined towards term or with labour, and reported that the enzyme was undetectable in myometrial myocytes in non-pregnant subjects (Bansal et al., 1997; Norman et al., 1999). In spite of the known relaxing action on rat myometrium, a further controversial effect has been reported of NO on cyclooxygenase (COX), the rate-limiting enzyme of prostaglandin synthesis. Indeed, NO stimulates uterine cyclooxygenase activity and expression in the rat (Dong and Yallampally, 1996), thus apparently favouring PGE2-mediated uterine activity. Moreover, there are evidence for a coordinated expression of inducible nitric oxide synthase and cyclooxygenase-2 genes in mice uterine tissue (Swaisgood et al., 1997). On the other hand interleukin-1beta, which is known to stimulate PG release thus contributing to the onset of labour, is also known to increase iNOS expression and NO synthesis (Dong and Yallampally, 1996), the latter supposed to determine uterine relaxation. Interestingly, it has been reported that PGE2 and PGF2alpha in rat uterus inhibit the interleukin-1beta-stimulated NO production and the inducible nitric oxide synthase messenger ribonucleic acid, respectively (Dong and Yallampally, 1996; Dong et al., 1997). 2002 Published by Elsevier Science Ltd. All rights reserved.
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This suggests that increased PG production at term may down-regulate uterine NO production, thus facilitating labour. In human pregnancy NO is also produced by foetal membranes and placental tissue, although its role has not been established. The expression of both constitutive (cNOS) and inducible NO synthase has been demonstrated in amnion, chorion-decidua and placenta at term (Thomson et al., 1997; Marinoni et al., 1997). Furthermore the expression of cNOS and iNOS mRNA has been reported in human amnion (Dennes et al., 1997) and placenta (Conrad et al., 1993). However, according to some authors, it appears that there is no difference in NOS activity before, during, and after labour (Thomson et al., 1997), even if a decrease of the enzyme has been reported by others (Marinoni et al., 2000). Since it is known that NO modulates PG release in several tissues, we decided to investigate the effect of nitric oxide on the arachidonic acid (AA) pathway in human amnion-like WISH cells. Knowing that amniotic PG release triggers the onset of labour, our work hypothesis was that the possible influences of NO on AA metabolism were in same way related to its relaxing effect on uterine muscle during pregnancy.
MATERIALS AND METHODS Chemicals [5,6,8,9,11,12,14,15-3H]arachidonic acid (205 Ci/mmol) and [5,6(n)-3H]PGE2 (181 Ci/mmol), were purchased from Amersham Italia Srl, Milan, Italy. PGE2 antiserum, oxytocin, sodium nitroprusside, -arginine, -nitroarginine methyl esther, 3-(5 -Hydroxymethyl-2 -furyl)-1-benzylindazole, 1H[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one, 1,1,1-trifluoromethyl6,9,12,15-heicosatetraen-2-one and N2,2 -O-dibutyrylguanosine3 ,5 -cyclic monophosphate were purchased from SigmaAldrich. All tissue culture media and sera were purchased from GIBCO/BRL (Paisley, Scotland). All other chemicals were the highest reagent grades commercially available.
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confluent (2105 cells/well). Each experiment was performed in parallel on four wells. Radiolabelling of the cells with [3H]arachidonic acid was achieved by including 0.5 Ci/well in the serum-free medium 18 h before assay. After incubation, the cells were washed three times with pseudoamniotic fluid, containing: 118.5 m NaCl, 4.8 m KCl, 2.5 m CaCl2, 1.15 m KH2PO4, 1.15 m MgSO4 and 25.0 m NaHCO3, supplemented with 2.0 m glucose, 6.0 m urea and 0.2 per cent BSA, pH 7.0, previously gassed with a 95 per cent O2 : 5 per cent CO2 mixture. Cells were supplied with pseudoamniotic fluid at a constant flow rate of 0.5 ml/min by a fourchannel peristaltic pump (Gilson) and perifused with the same fluid for 1 h before treatment to obtain a stable [3H]arachidonic acid basal release. The test substances were dissolved in gassed pseudoamniotic fluid and infused into the wells. The inhibitors were administered five fractions (15 min) before SNP treatment. Fractions of perifusate were set apart every 3 min, and radioactivity of the superfusate solution was determined by a Beckman LS 6500 scintillation spectrometer. The percent stimulation was determined by evaluating the dpm values of [3H]arachidonic acid released following the cell treatments vs its spontaneous release in the ‘corresponding’ fractions, calculated through linear regression of the baseline values before and after stimulation. Basal values ranged from 1800 to 2500 dpm/3 min fraction among the different cell cultures. Preliminary trials showed that spontaneous release remained unchanged throughout the entire experiment, which lasted up to 2.5 h.
PGE2 determination Experiments were performed on cells grown to confluence in 24-well plates in F12/DMEM+10 per cent FBS. For PGE2 determination, the medium was removed and replaced with fresh serum-free F12/DMEM, containing the test substances. After 30 min at 37C, the supernatants were collected and stored at 80C until PGE2 radioimmunoassay was performed.
Cell culture PGE2 radioimmunoassay Amnion-like WISH cells were obtained from the American Type Culture Collection (ATCC CCL-25) and maintained in the laboratory. Cells were grown at 37C in an atmosphere of 5 per cent CO2/95 per cent air, in a mixture of Ham F12 and Dulbecco Modified Eagle Medium (F12/DMEM) (1 : 1 vol/vol) supplemented with 10 per cent foetal bovine serum (10 per cent FBS), 30 g/ml gentamicin and 0.25 g/ml amphotericin B.
Incorporation of [3H]arachidonic acid The cells were seeded in 24-well plates in F12/DMEM containing 10 per cent foetal bovine serum until 80–90 per cent
The amount of PGE2 was assayed in the collected media by a specific RIA procedure according to the manufacturer’s protocol (Sigma-Aldrich). A specific antiserum for both PGE1 and PGE2 (cross reactions 100 per cent and 165 per cent respectively) was used. Label [3H]PGE2 (3nCi) was added to each tube. The level of PGE2 in each serially diluted sample was determined by comparison with a displacement standard curve (15–500 pg/0.1 ml). Incubation was performed for 90 min at 4C. Bound and free radioligands were separated by dextrancoated charcoal and centrifugation of tubes for 10 min at 2000g. Assay sensitivity was 15 pg/tube, and the intra-assay or inter-assay coefficients of variations were <10 per cent. Data were expressed as nanograms of PGE2 produced per 106 cells.
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Figure 1. Effect of two different oxytocin concentrations on [3H]AA release by perifused WISH cells. Duration of treatments is indicated by shaded areas. [3H]AA release, measured as dpm/fraction (one fraction=3 min), is expressed as percent with respect to basal release. Each point represents the mean of four independent experiments performed with different cell cultures, each run in parallel on four wells. *P<0.05 compared to normalized basal value.
Specimen preparation for scanning electron microscopy Samples for imaging were fixed with 2.5 per cent glutaraldehyde (EMS) buffered in 0.1 phosphate buffer, pH 7.2– 7.4. The samples were fixed for 2 h or longer at 4C. After fixation, the samples were rinsed three times with 0.1 phosphate buffer and post-fixed in 2 per cent (w/v) osmium tetroxide/0.1 phosphate buffer for 1 h at room temperature. After dehydratation in a graded ethanol series and critical point drying (Balzer CPD), the specimens were sputter coated with gold in EDWARDS sputter coating S 150 and examined in a Cambrige S 360 operating at 20 kV. Calculations and statistics Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Bonferroni’s test for AA release data and Dunnet’s test for dose-response curve of PGE2. Differences were considered statistically significant at P<0.05. RESULTS Release of [3H]arachidonic acid Time courses of radioactivity incorporated into WISH cell culture revealed that incorporation was maximal at 18 h (data
not shown). In a first series of experiments we measured AA output from WISH cells in the absence and presence of oxytocin, a hormone able to increase PGE2 output from these cells. We tested 10 7 and 10 6 oxytocin because these concentrations were the most efficacious on PGE2 release (Pavan et al., 2000). As shown in Figure 1, 10 7 oxytocin significantly increased AA release (190 per cent, peak value vs basal level); this stimulatory effect was less evident, but highly significant, at 10 6 oxytocin (160 per cent, peak value vs basal level). We thus concluded that our cell model is suitable for study of AA release. To evaluate the exogenous NO effect on AA release we used SNP, a classic NO donor. Since SNP was used at high concentrations to ensure optimal NO release, before the perifusion experiments we evaluated the possibile toxic effects of this compound, observing the cells by scanning electron microscopy before and after the treatments. As shown in Figure 2, the cells produced an epithelial-like monostratal organization. The monolayer culture showed homogeneously arranged cells, connected with close filopodial extension. When we treated the cells for 30 min with 10 2 SNP, no significant alteration in cellular organization was observed in the presence of either pseudoamniotic fluid or serum-free medium, which are the experimental conditions for measuring AA and PGE2 release, respectively. Indeed, in both conditions the rate of cell death was about 20–25 per cent, in both control conditions and in treated cells.
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Figure 2. Scanning electron micrography of WISH cells in a FBS free medium treated in absence (A) and in presence (B) of 10 2 SNP and in pseudoamniotic fluid without (C) and with (D) 10 2 SNP.
The effect of exogenous and endogenous NO on [3H]AA release from perifiused WISH cells is reported. As shown in Figure 3A, SNP at concentrations below 10 3 was ineffective while at higher doses the NO donor dose-dependently and greatly increased AA release, reaching the maximum (450 per cent, peak value vs basal level) at 10 2 . This effect was counteracted by 1,1,1-trifluoromethyl-6,9,12,15heicosatetraen-2-one (AACOCF3), a specific cytosolic PLA2 inhibitor (Street et al., 1993). The drug at 10 5 , per se ineffective on basal release, completely blocked the effect of a second pulse of SNP (10 2 ) (Figure 3B). The effect of 10 4 and 10 3 -arginine on AA release is shown in Figure 3C. These concentrations caused a significant increase in AA output (150 per cent and 210 per cent, peak value vs basal level, respectively). -nitroarginine methyl esther (L-NAME), a competitive inhibitor of NOS, instead of counteracting -arginine response, paradoxically potentiated it (data not shown). In order to examine the mechanisms of NO involved in AA mobilization, WISH cells were perifused with drug able to
affect the cGMP pathway, since the soluble isoform of guanylyl cyclase is a known target of NO action. In Figure 4A it is shown that 3-(5 -Hydroxymethyl-2 -furyl)-1-benzyl indazole (YC-1), an activator of soluble guanylyl cyclase at 10 6 and 10 5 significantly increased AA release (170 per cent and 340 per cent, peak value vs basal level, respectively). In addition the treatment with N2,2 -O-dibutyrylguanosine-3 ,5 cyclic monophosphate (db-cGMP), a membrane-permeable, non-hydrolyzable analogue of cGMP, at 10 6 and 10 5 significantly enhanced AA output by 150 per cent and 185 per cent (peak value vs basal level), respectively (Figure 4B). To provide further evidence of the involvement of guanylyl cyclase in this response, WISH cells were perifused with the optimal dose of 10 5 1H-[1,2,4]oxadiazolo[4,3-] quinoxalin-1-one (ODQ), an inhibitor of the soluble form of the cyclic GMP producing enzyme. As shown in Figure 4C, the drug was able to completely counteract the effect of SNP when the NO donor was applied at 10 3 . A further putative inhibitor of guanylyl cyclase, methylene blue, was tested. However, instead of the espected inhibition, a
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Figure 3. Effect of exogenous and endogenous NO on [3H]AA release by WISH cells. A: dose response effect of SNP. B: effect of the AACOCF3 (in frame) on SNP-induced [3H]AA output. C: effect of two different -arginine concentrations on [3H]AA release. Shaded areas represent administration of the substances different from AACOCF3. [3H]AA release, measured as dpm/fraction, is expressed as percent with respect to basal release. Each point represents the mean of four independent experiments performed with different cell cultures, each run in parallel on four wells. *P<0.05 compared to normalized basal value.
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Figure 4. Influence of cGMP pathway modulators on [3H]AA release from WISH cells. A: effect of guanylyl cyclase activator YC-1. B: effect of two different db-cGMP concentrations. C: effect of guanylyl cyclase inhibitor ODQ (in frame) on SNP-induced [3H]AA release. Shaded areas represent administration of the substances different from ODQ. [3H]AA release, measured as dpm/fraction, is expressed as percent with respect to basal release. Each point represents the mean of four independent experiments performed with different cell cultures, each run in parallel on four wells. *P<0.05 compared to normalized basal value. P<0.05 vs value obtained with 510 3 SNP.
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Figure 5. Concentration-dependent enhancement of PGE2 release by WISH cells treated with SNP. Values are mean of four independent determinations. *P<0.05 compared to basal value.
large increase of the basal AA release was observed (data not shown).
PGE2 output by WISH cells In order to evaluate the fate of AA released by NO stimulus, we tested the effect of SNP concentrations ranging from 10 4 to 10 2 on PGE2 synthesis. The donor increased PGE2 production, dose-dependently, even if only slightly, with a maximal effect (140 per cent with respect to basal value) at 510 3 , while for higher concentrations no further increase was observed (Figure 5).
DISCUSSION The contractility of the uterus during pregnancy and labour is modulated by a number of agents, among which prostaglandins and nitric oxide. It is generally accepted that PGs, produced by both uterus and foetal membranes during pregnancy and in higher amounts during labour, play a significant role in parturition (Skinner and Challis, 1984). NO, on the other hand, has been reported to induce uterine relaxation during pregnancy in both animals and humans. This response is achieved through the production of cGMP generated from the
interaction of NO with the haem moiety of soluble guanylyl cyclase (Ledingham et al., 2000). The radical has moreover been shown to act also on COX, and a possible interaction between the two pathways seems to be involved in the regulation of uterine activity during pregnancy and labour in the rat. Indeed, in this animal model it has been clearly demonstrated that NO stimulates COX whereas, by means of a sort of negative feedback, the enzyme products PGE2 and PGF2alpha inhibit both NO production and NOS expression (Dong and Yallampally, 1996; Dong et al., 1997). Inhibition of NOS by COX products has in fact been observed also in other systems (Tetsuka et al., 1994), and a biphasic effect of PGE2 on NO production has been shown in macrophage cell line J774 (Milano et al., 1995). The action of NO on COX can be mediated in two ways: either by a cGMP-dependent or -independent mechanism. In the first case, the nucleotide influences the transcriptional processes of the COX gene (Tetsuka et al., 1996), whereas in the second, NO can act at the haem level or as peroxynitrite both on the haem and on the cysteine or tyrosine residues of the enzyme. The consequence of these interactions can be either stimulatory or inhibitory, depending on the experimental model (Goodman et al., 1999). We studied the action of NO on the AA pathway in human amnion-like WISH cells, considered a good model for studying gestational prostanoid metabolism. A criticism recently raised against the utilization of this cellular model is that WISH cells
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show the HeLa genotype due to contamination in culture (Kniss et al., 2002). However, in our experience the modulation of AA metabolism in these cells is comparable to that observed in amnion tissue (Buzzi et al., 1999; Pavan et al., 2000; Biondi et al. 2001). The data here reported demonstrate that exogenous NO induces a large increase of AA release. Since treatment with a selective PLA2 inhibitor completely counteracts SNP-induced AA output, it must be concluded that the enzyme mediates the stimulatory NO actions. In order to verify if this NO action is mediated by soluble guanylyl cyclase, we tested the direct addition of cGMP as well as that of the enzyme activator or inhibitor. Addition of db-cGMP or the activator YC-1, both of which are supposed to mimic NO action, significantly enhances AA output. On the contrary, in other systems, both stimulation and inhibition of phospholipase A2 (PLA2) by exogenous NO have been reported as independent of cyclic GMP-mediated signal transduction (Rupprecht et al., 1999, Thang et al., 2000). In the case of guanylyl cyclase inhibitors, we found that ODQ was able to inhibit SNP-induced AA release, whereas surprisingly methylene blue, that is considered a classic inhibitor of the enzyme, greatly enhanced its basal output. In this context, it has been shown that, in guinea pig airway smooth muscle, methylene blue either fails to modify or only partially inhibits both cyclic GMP accumulation and the relaxant responses induced by SNP (Hwang et al., 1998). On the basis of the above results, we can affirm that methylene blue does not exhibit the behavior of a general soluble guanylyl cyclase inhibitor. As for endogenous NO production, we found that the stimulatory action of -arginine on AA release instead of being
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counteracted by the competitive inhibitor of NOS, L-NAME, was enhanced. However, it has recently been reported that different -arginine analogs can be also a source for nonenzymatically produced NO both in vitro and ex vivo (Moroz et al., 1998). Our data also show that optimal oxytocin concentrations in increasing PGE2 output from WISH cells (Pavan et al., 2000) are able to increase AA release by the same magnitude, while the stimulatory effect of exogenous NO is strong on AA but weak on PGE2 output. As for foetal membranes, it has been reported that NO exerts a strong stimulatory action on PGE2 release. However, since these results were obtained from a complex of amnion and chorion-decidua (Ticconi et al., 1996), it is not possible to establish the contribution of amnion cells to prostaglandin release. With respect to WISH cells, it can therefore be supposed that the AA released by oxytocin is completely converted to PGE2 production. On the contrary the slight increase of PGE2 produced by NO, in spite of the wide availability of the substrate, could represent the expression of different actions of the radical on enzymes downstream to PLA2, other than COX. At this regard, it has recently been demonstrated that NO stimulates the constitutive form of COX (COX-1), but inhibits the inducible form of the enzyme (COX-2) in macrophages and in COX-1- or COX-2-deficient cells (Clancy et al., 2000). Therefore no conclusions can be drawn until NO influences on all the enzymes of the AA cascade will be investigated. In summary, our finding of a strong stimulation of arachidonic acid output coupled with a weak release of PGE2 may represent one aspect of NO protective function, which is open to investigation and debate.
ACKNOWLEDGEMENTS This work was supported by Grants from Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (PRIN 2000) and from University of Ferrara (ex 60 per cent).
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583 Skinner KA, Challis JA (1984) Changes in the synthesis and metabolism of prostaglandins by human fetal membranes and decidua at labor. Am J Obstet Gynecol, 151, 519–523. Street IP, Lin HK, Laliberte F, Ghomashchi F, Wang Z, Perrier H, Tremblay NM, Huang Z, Weech PK, Gelb MH (1993) Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2. Biochemistry, 32, 5935–5940. Swaisgood CM, Zu HX, Perkins DJ, Wu S, Garver CL, Zimmerman PD, Iams JD, Kniss DA (1997) Coordinate expression of inducible nitric oxide synthase and cyclooxygenase-2 genes in uterine tissues of endotoxin-treated pregnant mice. Am J Obstet Gynecol, 177, 1253–1262. Tetsuka T, Daphna-Iken D, Miller BW, Guan Z, Baier LD, Morrison AR (1996) Nitric oxide amplifies interleukin 1-induced cyclooxygenase 2 expression in rat mesangial cells. J Clin Invest, 97, 2051–2056. Tetsuka T, Daphna-Iken D, Srivastava SK, Baier LD, DuMaine J, Morrison AR (1994) Cross-talk between cyclooxygenase and nitric oxide pathways: prostaglandin E2 negatively modulates induction of nitric oxide synthase by interleukin 1. Proc Natl Acad Sci USA, 91, 12168–12172. Thang SH, Yasuda Y, Umezawa M, Murayama T, Nomura Y (2000) Inhibition of phospholipase A(2) activity by S-nitroso-cysteine in a cyclic GMP-indipendent manner in PC12 cells. Eur J Pharmacol, 395, 183–191. Thomson AJ, Telfer JF, Kohnen G, Young A, Cameron IT, Greer IA, Normann JE (1997) Nitric oxide synthase activity and localization do not change in uterus and placenta during human parturition. Hum Reprod, 12, 2546–2552. Ticconi C, Zicari A, Losardo A, Pontieri G, Pasetto N, Piccione E (1996) Nitric oxide in human fetal membranes at term gestation: effect on prostaglandin E2 release. Eur J Obstet Gynecol Reprod Biol, 69, 135–139. Yallampalli C, Dong IL, Gangula PR, Fang L (1998) Role and regulation of nitric oxide in the uterus during pregnancy and parturition. Soc Gynecol Invest, 5, 58–67.
Effect of Nitric Oxide on Arachidonic Acid Release from Human Amnion-like WISH Cells C. Biondia,d, S. Fiorinia, I. Boarinia, L. Barbina, F. Cervellatib, M. E. Ferrettia and F. Vescec a Department of Biology, Section of General Physiology, and b Electron Microscopy Center, University of Ferrara, via L. Borsari, 46, 44100-I Ferrara, Italy and c Department of Biomedical Sciences and Advanced Therapy, Section of Obstetrics and Gynecology, University of Ferrara, corso Giovecca, 203, 44100-I Ferrara, Italy Paper accepted 16 May 2002
In order to clarify the possible interactions between nitric oxide (NO) and arachidonic acid (AA) pathways, human amnion-like WISH cells were perifused to measure the effects of the following substances on [3H]arachidonic acid release: (1) sodium nitroprusside (SNP), a nitric oxide donor; (2) 1,1,1-trifluoromethyl-6,9,12,15-heicosatetraen-2-one, a cytosolic phospholipase A2 (cPLA2) inhibitor; (3) -arginine, the substrate of nitric oxide synthase (NOS); (4) 3-(5 -Hydroxymethyl-2 -furyl)-1benzylindazole and 1H-[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one, activator and inhibitor of soluble guanylyl cyclase, respectively; (5) a membrane-permeable non-hydrolyzable analogue of guanosine-3 ,5 -cyclic monophosphate (cGMP). Furthermore, the effect of SNP on prostaglandin E2 (PGE2) release was tested. Exogenous and endogenous NO, as well as the guanylyl cyclase activator and cGMP analogue, significantly increased [3H]arachidonic acid release. Both soluble guanylyl cyclase and PLA2 inhibitors counteracted SNP response. Exogenous NO increased PGE2 release, although to a much lesser degree compared with arachidonic acid release. Our results indicate that NO stimulates AA release in WISH cells by activating PLA2 through a cyclic GMP-dependent mechanism. 2002 Published by Elsevier Science Ltd. All rights reserved. Placenta (2002), 23, 575–583
INTRODUCTION The mechanisms underlying uterine quiescence during pregnancy and the changes leading to onset of labour are not completely understood. It has long been known that prostaglandins (PGs) play a key role in the regulation of parturition by stimulating uterine contractility and causing cervical ripening during labour (Novy and Liggins, 1980). More recently NO, a free radical generated from -arginine by nitric oxide synthase, has been identified among the factors involved in the regulation of myometrial activity (Ledingham et al., 2000; Yallampalli et al., 1998). It has been shown in the rat and rabbit that NO maintains uterine relaxation during pregnancy, and that onset of labour is favoured by the decrease of myometrial and increase of cervical NOS activity (Ledingham et al., 2000; Buhimschi et al., 1996). In humans, there is controversy both about myometrial sensitivity to NO and about the presence and regulation of NOS activity in the myometrium (Buhimschi et al., 1995; Barber et al. 1999). Some authors have reported that enzyme expression is similar To whom correspondence should be addressed at: Dipartimento di Biologia, Sezione di Fisiologia Generale, Universita` di Ferrara, Via L. Borsari, 46, I44100 Ferrara, Italy. Tel.: 0532-291482; Fax: 0532207143; E-mail: [email protected] 0143–4004/02/$-see front matter
in labour and non-labour conditions (Dennes et al., 1999; Thomson et al., 1997) while others, in recent studies, have demonstrated that the expression of inducible NOS (iNOS) increased during the third trimester and declined towards term or with labour, and reported that the enzyme was undetectable in myometrial myocytes in non-pregnant subjects (Bansal et al., 1997; Norman et al., 1999). In spite of the known relaxing action on rat myometrium, a further controversial effect has been reported of NO on cyclooxygenase (COX), the rate-limiting enzyme of prostaglandin synthesis. Indeed, NO stimulates uterine cyclooxygenase activity and expression in the rat (Dong and Yallampally, 1996), thus apparently favouring PGE2-mediated uterine activity. Moreover, there are evidence for a coordinated expression of inducible nitric oxide synthase and cyclooxygenase-2 genes in mice uterine tissue (Swaisgood et al., 1997). On the other hand interleukin-1beta, which is known to stimulate PG release thus contributing to the onset of labour, is also known to increase iNOS expression and NO synthesis (Dong and Yallampally, 1996), the latter supposed to determine uterine relaxation. Interestingly, it has been reported that PGE2 and PGF2alpha in rat uterus inhibit the interleukin-1beta-stimulated NO production and the inducible nitric oxide synthase messenger ribonucleic acid, respectively (Dong and Yallampally, 1996; Dong et al., 1997). 2002 Published by Elsevier Science Ltd. All rights reserved.
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This suggests that increased PG production at term may down-regulate uterine NO production, thus facilitating labour. In human pregnancy NO is also produced by foetal membranes and placental tissue, although its role has not been established. The expression of both constitutive (cNOS) and inducible NO synthase has been demonstrated in amnion, chorion-decidua and placenta at term (Thomson et al., 1997; Marinoni et al., 1997). Furthermore the expression of cNOS and iNOS mRNA has been reported in human amnion (Dennes et al., 1997) and placenta (Conrad et al., 1993). However, according to some authors, it appears that there is no difference in NOS activity before, during, and after labour (Thomson et al., 1997), even if a decrease of the enzyme has been reported by others (Marinoni et al., 2000). Since it is known that NO modulates PG release in several tissues, we decided to investigate the effect of nitric oxide on the arachidonic acid (AA) pathway in human amnion-like WISH cells. Knowing that amniotic PG release triggers the onset of labour, our work hypothesis was that the possible influences of NO on AA metabolism were in same way related to its relaxing effect on uterine muscle during pregnancy.
MATERIALS AND METHODS Chemicals [5,6,8,9,11,12,14,15-3H]arachidonic acid (205 Ci/mmol) and [5,6(n)-3H]PGE2 (181 Ci/mmol), were purchased from Amersham Italia Srl, Milan, Italy. PGE2 antiserum, oxytocin, sodium nitroprusside, -arginine, -nitroarginine methyl esther, 3-(5 -Hydroxymethyl-2 -furyl)-1-benzylindazole, 1H[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one, 1,1,1-trifluoromethyl6,9,12,15-heicosatetraen-2-one and N2,2 -O-dibutyrylguanosine3 ,5 -cyclic monophosphate were purchased from SigmaAldrich. All tissue culture media and sera were purchased from GIBCO/BRL (Paisley, Scotland). All other chemicals were the highest reagent grades commercially available.
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confluent (2105 cells/well). Each experiment was performed in parallel on four wells. Radiolabelling of the cells with [3H]arachidonic acid was achieved by including 0.5 Ci/well in the serum-free medium 18 h before assay. After incubation, the cells were washed three times with pseudoamniotic fluid, containing: 118.5 m NaCl, 4.8 m KCl, 2.5 m CaCl2, 1.15 m KH2PO4, 1.15 m MgSO4 and 25.0 m NaHCO3, supplemented with 2.0 m glucose, 6.0 m urea and 0.2 per cent BSA, pH 7.0, previously gassed with a 95 per cent O2 : 5 per cent CO2 mixture. Cells were supplied with pseudoamniotic fluid at a constant flow rate of 0.5 ml/min by a fourchannel peristaltic pump (Gilson) and perifused with the same fluid for 1 h before treatment to obtain a stable [3H]arachidonic acid basal release. The test substances were dissolved in gassed pseudoamniotic fluid and infused into the wells. The inhibitors were administered five fractions (15 min) before SNP treatment. Fractions of perifusate were set apart every 3 min, and radioactivity of the superfusate solution was determined by a Beckman LS 6500 scintillation spectrometer. The percent stimulation was determined by evaluating the dpm values of [3H]arachidonic acid released following the cell treatments vs its spontaneous release in the ‘corresponding’ fractions, calculated through linear regression of the baseline values before and after stimulation. Basal values ranged from 1800 to 2500 dpm/3 min fraction among the different cell cultures. Preliminary trials showed that spontaneous release remained unchanged throughout the entire experiment, which lasted up to 2.5 h.
PGE2 determination Experiments were performed on cells grown to confluence in 24-well plates in F12/DMEM+10 per cent FBS. For PGE2 determination, the medium was removed and replaced with fresh serum-free F12/DMEM, containing the test substances. After 30 min at 37C, the supernatants were collected and stored at 80C until PGE2 radioimmunoassay was performed.
Cell culture PGE2 radioimmunoassay Amnion-like WISH cells were obtained from the American Type Culture Collection (ATCC CCL-25) and maintained in the laboratory. Cells were grown at 37C in an atmosphere of 5 per cent CO2/95 per cent air, in a mixture of Ham F12 and Dulbecco Modified Eagle Medium (F12/DMEM) (1 : 1 vol/vol) supplemented with 10 per cent foetal bovine serum (10 per cent FBS), 30 g/ml gentamicin and 0.25 g/ml amphotericin B.
Incorporation of [3H]arachidonic acid The cells were seeded in 24-well plates in F12/DMEM containing 10 per cent foetal bovine serum until 80–90 per cent
The amount of PGE2 was assayed in the collected media by a specific RIA procedure according to the manufacturer’s protocol (Sigma-Aldrich). A specific antiserum for both PGE1 and PGE2 (cross reactions 100 per cent and 165 per cent respectively) was used. Label [3H]PGE2 (3nCi) was added to each tube. The level of PGE2 in each serially diluted sample was determined by comparison with a displacement standard curve (15–500 pg/0.1 ml). Incubation was performed for 90 min at 4C. Bound and free radioligands were separated by dextrancoated charcoal and centrifugation of tubes for 10 min at 2000g. Assay sensitivity was 15 pg/tube, and the intra-assay or inter-assay coefficients of variations were <10 per cent. Data were expressed as nanograms of PGE2 produced per 106 cells.
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Figure 1. Effect of two different oxytocin concentrations on [3H]AA release by perifused WISH cells. Duration of treatments is indicated by shaded areas. [3H]AA release, measured as dpm/fraction (one fraction=3 min), is expressed as percent with respect to basal release. Each point represents the mean of four independent experiments performed with different cell cultures, each run in parallel on four wells. *P<0.05 compared to normalized basal value.
Specimen preparation for scanning electron microscopy Samples for imaging were fixed with 2.5 per cent glutaraldehyde (EMS) buffered in 0.1 phosphate buffer, pH 7.2– 7.4. The samples were fixed for 2 h or longer at 4C. After fixation, the samples were rinsed three times with 0.1 phosphate buffer and post-fixed in 2 per cent (w/v) osmium tetroxide/0.1 phosphate buffer for 1 h at room temperature. After dehydratation in a graded ethanol series and critical point drying (Balzer CPD), the specimens were sputter coated with gold in EDWARDS sputter coating S 150 and examined in a Cambrige S 360 operating at 20 kV. Calculations and statistics Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Bonferroni’s test for AA release data and Dunnet’s test for dose-response curve of PGE2. Differences were considered statistically significant at P<0.05. RESULTS Release of [3H]arachidonic acid Time courses of radioactivity incorporated into WISH cell culture revealed that incorporation was maximal at 18 h (data
not shown). In a first series of experiments we measured AA output from WISH cells in the absence and presence of oxytocin, a hormone able to increase PGE2 output from these cells. We tested 10 7 and 10 6 oxytocin because these concentrations were the most efficacious on PGE2 release (Pavan et al., 2000). As shown in Figure 1, 10 7 oxytocin significantly increased AA release (190 per cent, peak value vs basal level); this stimulatory effect was less evident, but highly significant, at 10 6 oxytocin (160 per cent, peak value vs basal level). We thus concluded that our cell model is suitable for study of AA release. To evaluate the exogenous NO effect on AA release we used SNP, a classic NO donor. Since SNP was used at high concentrations to ensure optimal NO release, before the perifusion experiments we evaluated the possibile toxic effects of this compound, observing the cells by scanning electron microscopy before and after the treatments. As shown in Figure 2, the cells produced an epithelial-like monostratal organization. The monolayer culture showed homogeneously arranged cells, connected with close filopodial extension. When we treated the cells for 30 min with 10 2 SNP, no significant alteration in cellular organization was observed in the presence of either pseudoamniotic fluid or serum-free medium, which are the experimental conditions for measuring AA and PGE2 release, respectively. Indeed, in both conditions the rate of cell death was about 20–25 per cent, in both control conditions and in treated cells.
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Figure 2. Scanning electron micrography of WISH cells in a FBS free medium treated in absence (A) and in presence (B) of 10 2 SNP and in pseudoamniotic fluid without (C) and with (D) 10 2 SNP.
The effect of exogenous and endogenous NO on [3H]AA release from perifiused WISH cells is reported. As shown in Figure 3A, SNP at concentrations below 10 3 was ineffective while at higher doses the NO donor dose-dependently and greatly increased AA release, reaching the maximum (450 per cent, peak value vs basal level) at 10 2 . This effect was counteracted by 1,1,1-trifluoromethyl-6,9,12,15heicosatetraen-2-one (AACOCF3), a specific cytosolic PLA2 inhibitor (Street et al., 1993). The drug at 10 5 , per se ineffective on basal release, completely blocked the effect of a second pulse of SNP (10 2 ) (Figure 3B). The effect of 10 4 and 10 3 -arginine on AA release is shown in Figure 3C. These concentrations caused a significant increase in AA output (150 per cent and 210 per cent, peak value vs basal level, respectively). -nitroarginine methyl esther (L-NAME), a competitive inhibitor of NOS, instead of counteracting -arginine response, paradoxically potentiated it (data not shown). In order to examine the mechanisms of NO involved in AA mobilization, WISH cells were perifused with drug able to
affect the cGMP pathway, since the soluble isoform of guanylyl cyclase is a known target of NO action. In Figure 4A it is shown that 3-(5 -Hydroxymethyl-2 -furyl)-1-benzyl indazole (YC-1), an activator of soluble guanylyl cyclase at 10 6 and 10 5 significantly increased AA release (170 per cent and 340 per cent, peak value vs basal level, respectively). In addition the treatment with N2,2 -O-dibutyrylguanosine-3 ,5 cyclic monophosphate (db-cGMP), a membrane-permeable, non-hydrolyzable analogue of cGMP, at 10 6 and 10 5 significantly enhanced AA output by 150 per cent and 185 per cent (peak value vs basal level), respectively (Figure 4B). To provide further evidence of the involvement of guanylyl cyclase in this response, WISH cells were perifused with the optimal dose of 10 5 1H-[1,2,4]oxadiazolo[4,3-] quinoxalin-1-one (ODQ), an inhibitor of the soluble form of the cyclic GMP producing enzyme. As shown in Figure 4C, the drug was able to completely counteract the effect of SNP when the NO donor was applied at 10 3 . A further putative inhibitor of guanylyl cyclase, methylene blue, was tested. However, instead of the espected inhibition, a
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Figure 3. Effect of exogenous and endogenous NO on [3H]AA release by WISH cells. A: dose response effect of SNP. B: effect of the AACOCF3 (in frame) on SNP-induced [3H]AA output. C: effect of two different -arginine concentrations on [3H]AA release. Shaded areas represent administration of the substances different from AACOCF3. [3H]AA release, measured as dpm/fraction, is expressed as percent with respect to basal release. Each point represents the mean of four independent experiments performed with different cell cultures, each run in parallel on four wells. *P<0.05 compared to normalized basal value.
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Figure 4. Influence of cGMP pathway modulators on [3H]AA release from WISH cells. A: effect of guanylyl cyclase activator YC-1. B: effect of two different db-cGMP concentrations. C: effect of guanylyl cyclase inhibitor ODQ (in frame) on SNP-induced [3H]AA release. Shaded areas represent administration of the substances different from ODQ. [3H]AA release, measured as dpm/fraction, is expressed as percent with respect to basal release. Each point represents the mean of four independent experiments performed with different cell cultures, each run in parallel on four wells. *P<0.05 compared to normalized basal value. P<0.05 vs value obtained with 510 3 SNP.
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Figure 5. Concentration-dependent enhancement of PGE2 release by WISH cells treated with SNP. Values are mean of four independent determinations. *P<0.05 compared to basal value.
large increase of the basal AA release was observed (data not shown).
PGE2 output by WISH cells In order to evaluate the fate of AA released by NO stimulus, we tested the effect of SNP concentrations ranging from 10 4 to 10 2 on PGE2 synthesis. The donor increased PGE2 production, dose-dependently, even if only slightly, with a maximal effect (140 per cent with respect to basal value) at 510 3 , while for higher concentrations no further increase was observed (Figure 5).
DISCUSSION The contractility of the uterus during pregnancy and labour is modulated by a number of agents, among which prostaglandins and nitric oxide. It is generally accepted that PGs, produced by both uterus and foetal membranes during pregnancy and in higher amounts during labour, play a significant role in parturition (Skinner and Challis, 1984). NO, on the other hand, has been reported to induce uterine relaxation during pregnancy in both animals and humans. This response is achieved through the production of cGMP generated from the
interaction of NO with the haem moiety of soluble guanylyl cyclase (Ledingham et al., 2000). The radical has moreover been shown to act also on COX, and a possible interaction between the two pathways seems to be involved in the regulation of uterine activity during pregnancy and labour in the rat. Indeed, in this animal model it has been clearly demonstrated that NO stimulates COX whereas, by means of a sort of negative feedback, the enzyme products PGE2 and PGF2alpha inhibit both NO production and NOS expression (Dong and Yallampally, 1996; Dong et al., 1997). Inhibition of NOS by COX products has in fact been observed also in other systems (Tetsuka et al., 1994), and a biphasic effect of PGE2 on NO production has been shown in macrophage cell line J774 (Milano et al., 1995). The action of NO on COX can be mediated in two ways: either by a cGMP-dependent or -independent mechanism. In the first case, the nucleotide influences the transcriptional processes of the COX gene (Tetsuka et al., 1996), whereas in the second, NO can act at the haem level or as peroxynitrite both on the haem and on the cysteine or tyrosine residues of the enzyme. The consequence of these interactions can be either stimulatory or inhibitory, depending on the experimental model (Goodman et al., 1999). We studied the action of NO on the AA pathway in human amnion-like WISH cells, considered a good model for studying gestational prostanoid metabolism. A criticism recently raised against the utilization of this cellular model is that WISH cells
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show the HeLa genotype due to contamination in culture (Kniss et al., 2002). However, in our experience the modulation of AA metabolism in these cells is comparable to that observed in amnion tissue (Buzzi et al., 1999; Pavan et al., 2000; Biondi et al. 2001). The data here reported demonstrate that exogenous NO induces a large increase of AA release. Since treatment with a selective PLA2 inhibitor completely counteracts SNP-induced AA output, it must be concluded that the enzyme mediates the stimulatory NO actions. In order to verify if this NO action is mediated by soluble guanylyl cyclase, we tested the direct addition of cGMP as well as that of the enzyme activator or inhibitor. Addition of db-cGMP or the activator YC-1, both of which are supposed to mimic NO action, significantly enhances AA output. On the contrary, in other systems, both stimulation and inhibition of phospholipase A2 (PLA2) by exogenous NO have been reported as independent of cyclic GMP-mediated signal transduction (Rupprecht et al., 1999, Thang et al., 2000). In the case of guanylyl cyclase inhibitors, we found that ODQ was able to inhibit SNP-induced AA release, whereas surprisingly methylene blue, that is considered a classic inhibitor of the enzyme, greatly enhanced its basal output. In this context, it has been shown that, in guinea pig airway smooth muscle, methylene blue either fails to modify or only partially inhibits both cyclic GMP accumulation and the relaxant responses induced by SNP (Hwang et al., 1998). On the basis of the above results, we can affirm that methylene blue does not exhibit the behavior of a general soluble guanylyl cyclase inhibitor. As for endogenous NO production, we found that the stimulatory action of -arginine on AA release instead of being
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counteracted by the competitive inhibitor of NOS, L-NAME, was enhanced. However, it has recently been reported that different -arginine analogs can be also a source for nonenzymatically produced NO both in vitro and ex vivo (Moroz et al., 1998). Our data also show that optimal oxytocin concentrations in increasing PGE2 output from WISH cells (Pavan et al., 2000) are able to increase AA release by the same magnitude, while the stimulatory effect of exogenous NO is strong on AA but weak on PGE2 output. As for foetal membranes, it has been reported that NO exerts a strong stimulatory action on PGE2 release. However, since these results were obtained from a complex of amnion and chorion-decidua (Ticconi et al., 1996), it is not possible to establish the contribution of amnion cells to prostaglandin release. With respect to WISH cells, it can therefore be supposed that the AA released by oxytocin is completely converted to PGE2 production. On the contrary the slight increase of PGE2 produced by NO, in spite of the wide availability of the substrate, could represent the expression of different actions of the radical on enzymes downstream to PLA2, other than COX. At this regard, it has recently been demonstrated that NO stimulates the constitutive form of COX (COX-1), but inhibits the inducible form of the enzyme (COX-2) in macrophages and in COX-1- or COX-2-deficient cells (Clancy et al., 2000). Therefore no conclusions can be drawn until NO influences on all the enzymes of the AA cascade will be investigated. In summary, our finding of a strong stimulation of arachidonic acid output coupled with a weak release of PGE2 may represent one aspect of NO protective function, which is open to investigation and debate.
ACKNOWLEDGEMENTS This work was supported by Grants from Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (PRIN 2000) and from University of Ferrara (ex 60 per cent).
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