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Differential Regulation of 11 ß-Hydroxysteroid Dehydrogenase Type 1 and 2 by Nitric Oxide in Cultured Human Placental Trophoblast and Chorionic Cell Preparation¹

Medical Research Council Group in Fetal and Neonatal Health and Development (K.S., J.R.G.C.), Departments of Physiology and Obstetrics and Gynecology, University of Toronto, Toronto, Ontario M5S 1A8, Canada; and Departments of Obstetrics and Gynaecology and Physiology, University of Western Ontario (K.Y.), Ontario, Canada

Address all correspondence and requests for reprints to: Kang Sun, Department of Physiology, University of Toronto, Faculty of Medicine, 1 King’s College Circle, Toronto, Ontario, M5S 1A8, Canada. E-mail: kang.sun{at}utoronto.ca

	Abstract

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Two types of 11 ß-hydroxysteroid dehydrogenase (11 ß-HSD) have been identified in different tissues. Type 1 has both oxidase and reductase activities interconverting cortisol and cortisone, whereas type 2 has only oxidase activity converting cortisol to cortisone. It has been proposed that placental 11 ß-HSD controls the passage of maternal glucocorticoids to the fetal circulation. However, little is known about the regulation of 11 ß-HSD in the human placenta and fetal membranes. We cultured human term placental trophoblast and chorionic trophoblast cells to examine effects of nitric oxide donors, sodium nitroprusside (SNP) and S-nitroso-N-acetyl penicillamine (SNAP), on the activity and messenger RNA (mRNA) expression of 11 ß-HSD. At 72 h of culture, placental trophoblast formed syncytial clumps that were cytokeratin positive and displayed mainly type 2 oxidase activity, although some type 1 reductase activity was detectable. Chorion preparations contain greater than 90% trophoblast cells as demonstrated by immunostaining for cytokeratin and less than 5% vimentin positive cells. Type 1 reductase activity predominated in the chorionic trophoblast cells with barely detectable type 1 or type 2 oxidase activity. Both SNP (1–400 µM) and SNAP (1 mM) inhibited placental 11 ß-HSD type 2 oxidase activity but not type 1 reductase activity either in placental or chorionic cells. An inhibitory effect on type 2 oxidase activity was reproduced in part by 8-bromo cGMP, blocked partially by the guanylate cyclase inhibitor LY83583 (1 µM), but not by an ADP-ribosylation inhibitor N, N'-hexamethylene-bis-acetamide (HMBG) (10 mM). SNP also suppressed the expression of type 2 mRNA in cultured placental trophoblast in a dose-dependent manner, and this effect was also blocked by LY83583. We conclude that human placental trophoblast possesses predominantly 11 ß-HSD type 2 oxidase activity, whereas chorionic cells possess mainly type 1 reductase activity under the culture conditions employed. Nitric oxide specifically attenuated 11 ß-HSD type 2 oxidase activity as well as its mRNA expression in the placental trophoblast. The effect was mediated at least partially through the cGMP pathway, although an alternative pathway other than ADP-ribosylation may exist.

	Introduction

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THE ENZYME 11 ß-hydroxysteroid dehydrogenase (11 ß-HSD) is responsible for the interconversion of glucocorticoids and their 11-keto metabolites. Two types of 11 ß-HSD have been identified. Type 1 11 ß-HSD possesses both oxidase and reductase activities and interconverts biologically active cortisol and inactive cortisone and requires NADP(H) as its cofactor. Type 2 11 ß-HSD acts as an exclusive oxidase converting cortisol to cortisone requiring NAD as cofactor (1, 2, 3). Importantly, the type 2 enzyme has a much higher affinity for its substrate than 11 ß-HSD type 1, with K_m values in the nanomolar range, whereas the K_m value of type 1 is in the micromolar range (4). 11 ß-HSD type 2 is discretely distributed with highest abundance in the kidney, pancreas, and placenta, whereas 11 ß-HSD type 1 is ubiquitously distributed (5, 6, 7, 8, 9). Type 1 11 ß-HSD messenger RNA (mRNA) and protein are present in human term placenta and fetal membranes, although our previous studies (10) showed that ir-11 ß-HSD type 1 predominates in chorion trophoblast, and placental intermediate trophoblasts with less ir-11 ß-HSD type 1 in decidual stromal cells, and negligible activity in amnion. The type 2 mRNA was only detectable in the placenta (10, 11). It has been proposed that the high affinity of placental 11 ß-HSD type 2 makes it more suited to regulate the amounts of maternal glucocorticoids passing into the fetal circulation (12). The level of cortisol in the fetal circulation is important for fetal organ maturation, but excessive cortisol could result in fetal growth retardation (12, 13, 14). The presence of 11 ß-HSD type 1 in the placenta and fetal membranes (10, 15) strongly suggests a possible coordinated interaction between type 1 and 2, but the direction of the reaction catalyzed by 11 ß-HSD type 1 in the human placenta and fetal membranes is not very well resolved.

Several studies have demonstrated that 11 ß-HSD type 1 and 2 are regulated by sex steroids and the cAMP pathway (16, 17, 18). However, no studies have addressed the regulation of 11 ß-HSD by nitric oxide (NO), an important messenger in the human placenta and fetal membranes. The vascular endothelium and syncytiotrophoblast of the placenta, the fetal membranes and myometrium are able to synthesize NO (19, 20). In pregnancy, NO may contribute to the local regulation of placental hormones, for example inhibition of CRH release (21), as well as the maintenance of myometrial quiescence (22). It may also affect basal vascular tone in the fetal-placental circulation (23). Glucocorticoids exert positive feedback on CRH production in the placenta (24, 25). They can also induce hypertension through either a direct effect on blood vessels or by binding to the mineralocorticoid receptor (12, 26), in addition to the their effects on fetal organ maturation and growth retardation. Therefore, both NO and glucocorticoids are involved in the regulation of vascular tone and CRH release. We hypothesized that NO might also regulate the enzyme 11 ß-HSD, which in turn could influence local levels of bioactive glucocorticoid in the placenta, fetal membranes and myometrium, as well as the passage of maternal glucocorticoids into the fetal circulation. Therefore, we examined the catalytic properties, and determined the effects of different NO donors, on 11 ß-HSD type 1 and 2 activities or mRNA expression in cultured human placental trophoblast and chorionic trophoblast cells prepared from patients in late gestation.

	Materials and Methods

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Placental trophoblast and chorionic cell cultures
Placental trophoblast and chorionic trophoblast cells were prepared using a modification of the method of Kliman (27), as described previously (21). Briefly, term human placentae (n = 21) and chorion tissue (n = 8) were obtained from normal women after elective Cesarean section at term. Several aliquots of tissue were removed randomly from the maternal side of the placenta, pooled, and digested with 0.125% trypsin (Sigma Chemical Co., St Louis, MO) and 0.02% deoxyribonuclease-I (Sigma) in DMEM (GIBCO, Grand Island, NY) containing 0.1% BSA, 0.005% gentamycin, and 0.01% streptomycin, three times for 30 min each time. The chorion with adherent decidua was peeled off amnion and digested three times for 60 min each time with the same digestion medium, except that 0.2% collagenase (Sigma) was used instead of deoxyribonuclease-I. The placental or chorion/decidual cells were loaded onto a 5–75% Percoll (Sigma) gradient at step increments of 5% Percoll, and centrifuged at 37 C and 2500 x g for 20 min to separate different cell types. Cytotrophoblasts between the density markers of 1.049 and 1.062 g/ml were collected and plated at a density of 10⁶ cells/ml·well in DMEM culture medium containing 10% FCS (GIBCO). The cells were cultured for three days at 37 C in 5% CO₂ and 95% O₂ before experimentation. Under these conditions, the placental cells aggregate to form a syncytium, whereas the chorionic trophoblast cells form clumps or remained as single cells. Purity of the cell preparation was assessed at the end of the experiment by immunohistochemistry (IHC) (28). Representative cultures were stained to determine the proportion of cytokeratin or vimentin positive cells; cells were counterstained with Carazzi’s hematoxylin.

Preparation of ³H-cortisol and ³H-cortisone
³H-cortisol (specific activity: 64.0 Ci/mmol) (Amersham Life Science, Buckinghamshire, UK) was purified by thin layer chromatography (TLC) in the solvent system chloroform/ethanol (95/5, vol/vol) for each separate measurement of 11 ß-HSD activity. ³H-cortisone was prepared by oxidizing ³H-cortisol with chromium trioxide (29) and was purified by TLC before it was used for the enzyme activity assay.

Treatment of cells and 11 ß-HSD activity assay
On the day of an experiment, the cells were washed with FCS free culture medium (pH 7.4) and preincubated in the same medium for 1 h. 11 ß-HSD type 2 oxidase activity was assayed using 100 nM cortisol containing 200,000 cpm ³H-cortisol; 11 ß-HSD type 1 oxidase and reductase activities were measured using 1 µM cortisol containing 200,000 cpm ³H-cortisol or 1 µM cortisone containing 200,000 cpm ³H-cortisone, respectively. The assays were performed in the absence and presence of NO donors: sodium nitroprusside (SNP) (Sigma), S-nitroso-N-acetylpenicillamine (SNAP) (Sigma); soluble guanylate cyclase inhibitor: LY83583 (Sigma); 8-bromo-guanosine-3'5'-cyclic monophosphate (8-br-cGMP) (ICN) or ADP-ribosylation inhibitor: N, N'-hexamethylene-bis-acetamide (HMBA) (Sigma). Each treatment was performed in duplicate or triplicate for each preparation of cells. Media were collected after 24 h incubation at 37 C with the drugs. For representative cultures, cells were washed twice with culture medium and lysed with 0.5% Triton X-100. The cells were then scraped off the plate with a rubber policeman and collected for the determination of intracellular uptake of cortisol and cortisone. A time course study of both type 1 and 2 activities was conducted at 1, 2, 4, 8, 20, and 24 h incubation. Cell viability before and after drug treatment was examined by trypan blue exclusion study.

To measure conversions, a mixture of cortisol and cortisone (40 µg each) was added to the collected medium to allow localization of the steroids when subjected to TLC. Steroids in the media and cells were extracted with ethyl acetate. The extract from the medium was dried, reconstituted with ethyl acetate (100 µl) and applied to the TLC plate (Fisher Scientific). Cortisol and cortisone were separated in the solvent system chloroform/ethanol (95/5, vol/vol). Steroids were visualized under UV light, scraped off, and extracted with ethyl acetate. The solvent was dried, scintillation fluid added, and the radioactivity was counted in a liquid scintillation counter. The recovery rate of ³H-cortisol or ³H-cortisone was measured by adding ³H-cortisol or ³H-cortisone to culture medium and processing in the samples in identical fashion. The mean recovery rates of ³H-cortisol and ³H-cortisone were 86.7 ± 5.3%, 88.3 ± 4.3%, respectively. The 11 ß-HSD type 1 and 2 oxidase activity was expressed as the percent of cortisone formed from cortisol, whereas 11 ß-HSD type 1 reductase activity was expressed as the percent of cortisol formed from cortisone. Extracts of intracellular cortisol and cortisone was dried and counted for the determination of intracellular uptake of cortisol and cortisone.

Kinetic analysis
Kinetic analysis was performed as described previously (30) for the respective oxidase and reductase activities. Conversion assays were conducted using a fixed number of cells (10⁶ cells) and reaction time (3.5 h for placental cells, 8 h for chorionic cells), but with varying amounts of substrates. The conditions were chosen so that the initial velocity was linear with the reaction time. In all cases, the conversion in the blank well (no cells) was subtracted from that in the experimental wells before analysis. The data were plotted as a straight line of s/v against s according to the Michaelis-Menten Equation, and the K_m and V_max values were calculated from the intercepts of these plots.

RNA extraction and Northern blot analysis
Total cellular RNA was extracted with lithium chloride/urea from placental trophoblast that had been treated with SNP and/or LY83583 for 24 h (31), except that the cells were grown in culture dishes at a density of 15 x 10⁶ cells/dish. After treatment of cells, the cells were washed with PBS, and then 3 M lithium chloride/6 M urea was added and the cells scraped off the dish with a rubber policeman. The cells were stored at -80 C for later extraction of total RNA. Northern blot hybridization was carried out using specific human 11ß-HSD type 1 (32) and two complementary DNAs (cDNAs) (courtesy of Dr. A. L. Albiston) (7). A cDNA for mouse 18S ribosome RNA (rRNA) was used as an internal standard to determine the relative amounts of RNA loaded into each well and the transfer efficiency.

The autoradiographs were scanned using a densitometer (Ultrascan XL LKB 222—2-, LKB Produkter, Bromma, Sweden) to determine the relative optical densities of 11ß-HSD type 2 and 18S rRNA hybridization signals. For each RNA sample, the signal for the transcripts was measured within the linear range of the densitometer, and the ratio of 11ß-HSD type 2 mRNA signals to 18S rRNA was calculated.

Statistical analysis
All data are shown as mean ± SEM Student’s t test; linear regression analysis or two-way ANOVA test were used to assess statistical differences. Statistical significance was set at P < 0.05.

	Results

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Characterization of cell type
At 72 h of culture, placental trophoblasts formed syncytial clumps that were predominantly cytokeratin positive. Most of the chorionic trophoblasts also formed clumps, although others remained as single cells. Both of these cell types were cytokeratin positive, suggesting that these cultures were predominantly (>90%) chorion-derived cells, rather than decidual stromal cells that are cytokeratin negative (Fig. 1). Trypan blue exclusion study also showed that the percentage of viability of the cells before and after treatment were both more than 95%.

View larger version (130K): [in this window] [in a new window]   Figure 1. Immunohistochemical staining for cytokeratin and vimentin in fetal membranes, cultured term placental trophoblast and chorionic trophoblast 72 h after culture. Brown color indicates positive staining. a, Amnion; c, chorion; d, decidua. Magnification: 10 x 10.

View larger version (13K): [in this window] [in a new window]   Figure 2. Plots according to the Michaelis-Menten equation for determining kinetic parameters. Data from a representative experiment for conversion of cortisol to cortisone in placental syncytiotrophoblast (a) and conversion of cortisone to cortisol in chorionic cells (b).

View larger version (15K): [in this window] [in a new window]   Figure 3. a, Time courses of the conversion of cortisol (100 nM) to cortisone (solid line) and cortisone (1 µM) to cortisol (dotted line) in cultured human term placental trophoblast. The conversion of cortisol to cortisone increased linearly over 24 h incubation (r = 0.964, n = 4). b, Time courses of the conversion of cortisol (100 nM: solid line; 1 µM: dashed line) to cortisone and the conversion of cortisone (1 µM) to cortisol (dotted line) in cultured human term chorionic trophoblast. The conversion of cortisone to cortisol is linearly increased over 24 h incubation (r = 0.992, n = 4).

Effects of SNP and SNAP on 11 ß-HSD oxidase and reductase activities
SNP, an NO donor, potently inhibited the conversion of cortisol (100 nM) to cortisone in cultured placental trophoblast (n = 7) in a dose-dependent manner (1–400 µM) (P < 0.01, Fig. 4a). The same doses of SNP did not affect the conversion of cortisone (1 µM) to cortisol either in the placental (n = 3) or chorionic trophoblasts (n = 5) (P > 0.05, Fig. 5, a and b). LY83583 (1 µM), an inhibitor of soluble guanylyl cyclase, partially blocked the inhibitory effect of SNP on the placental conversion of cortisol to cortisone (n = 7, P < 0.01, Fig. 4a). HMBG (10 mM), an inhibitor of ADP-ribosylation, did not affect the inhibitory influence of SNP on the conversion of cortisol to cortisone by placental trophoblast (P > 0.05, n = 3, Fig. 4b). Another donor of NO, SNAP (1 mM), also significantly inhibited the conversion of cortisol (100 nM) to cortisone in the placental syncytiotrophoblast (P < 0.01), an effect that was partially blocked by LY83583 (n = 4, P < 0.05). SNAP did not affect the conversion of cortisone (1 µM) to cortisol in the chorionic cell preparations (P > 0.05, n = 3, Fig. 6). 8-br-cGMP (100– 400 µM), a synthetic analog of cGMP also inhibited the conversion of cortisol (100 nM) to cortisone in the placental syncytiotrophoblast (P < 0.05 or 0.01, n = 3, Fig. 7).

View larger version (26K): [in this window] [in a new window]   Figure 4. a, SNP inhibited the conversion of cortisol (100 nM) to cortisone in a dose-dependent manner in cultured human term placental trophoblast. LY83583 (1 µM), a guanylate cyclase inhibitor, partially blocked the inhibitory effect of SNP (two way ANOVA test, P < 0.01, n = 7); b, the ADP-ribosylation inhibitor, N,N'-hexamethylene-bis-acetamide (HMBA) (10 mM), did not affect the inhibitory effect of SNP on the conversion of cortisol (100 nM) to cortisone in the cultured human term placental trophoblast (two-way ANOVA test, P > 0.05, n = 3).

View larger version (21K): [in this window] [in a new window]   Figure 5. SNP did not affect the conversion of cortisone (1 µM) to cortisol either in the cultured human term placental trophoblast (Fig. 4a, n = 3) or in the cultured human term chorionic trophoblast (Fig. 4b, n = 5). (ANOVA test, P > 0.05).

View larger version (11K): [in this window] [in a new window]   Figure 6. SNAP, 1 mM) inhibited the conversion of cortisol (100 nM) to cortisone in cultured human term placental trophoblast (t test, ** P < 0.01 vs. control, n = 4) after 8 h incubation, but not the conversion of cortisone (1 µM) to cortisol in the chorionic cells (P > 0.05, n = 3). LY83583 (1 µM), a guanylate cyclase inhibitor, partially blocked the inhibitory effect of SNAP (t test, * P < 0.05 vs. control; #, P < 0.05 vs. SNAP).

View larger version (11K): [in this window] [in a new window]   Figure 7. Synthetic cGMP analog, 8-br-cGMP, inhibited the conversion of cortisol (100 nM) to cortisone in cultured human placental syncytiotrophoblast (t test, * P < 0.05; **, P < 0.01 vs. control, n = 3)

View larger version (27K): [in this window] [in a new window]   Figure 8. SNP suppressed the expression of 11ß-HSD type 2 mRNA in the cultured placental syncytiotrophoblast in a dose-dependent manner (1–100 µM). The effect of SNP (100 µM) was blocked by LY83583 (1 µM), a guanylyl cyclase inhibitor. Top panel, Representative autoradiogram of Northern blot of 11ß-HSD type 2 mRNA expression in cultured human placental syncytiotrophoblast. Total RNA (20 µg) was loaded per lane. Exposure time was 3 weeks for 11ß-HSD type 2; 2 h for 18S rRNA. Lower panel, Ratios of 11ß-HSD type 2 mRNA to 18S rRNA (t test, * P < 0.05, ** P < 0.01 vs. control, n = 3).

	Discussion

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We used immunostaining to characterize the cells present at the conclusion of the culture period. The placental trophoblast aggregated to form an apparent syncytium that was cytokeratin positive. Chorionic cells also formed clumps and remained as isolated cells; a characteristic that appears to depend on the initial plating density. These cells had been partially purified by Percoll density gradient before culture and were >90% cytokeratin positive. Our previous work using IHC demonstrated that immunoreactive 11 ß-HSD type 1 was present intensely in the chorionic trophoblast and abundant 11 ß-HSD type 1 mRNA was observed in the chorion/decidual tissues as well (10). We have therefore assumed the activity of these cultures to be predominantly chorion (trophoblast) cells but certainly recognize that there may be some contamination with decidual cells. Lopez Bernal et al. demonstrated that both 11 ß-HSD dehydrogenase and reductase activities was found in the human choriodecidual tissues with the dehydrogenase activity predominating. Their work also suggested that the activity in the choriodecidua was of decidual, rather than chorionic, origin (36). Recently, Arcuri et al. reported both 11 ß-HSD type 1 and 2 to be present in endometrial stromal cells; expression of the enzyme was were enhanced during decidualization (37). Therefore, the observed 11 ß-HSD type 1 reductase activity in our chorionic cell culture could be contributed in part by decidual cells, but this seems unlikely as a primary source because a predominant reductase activity was observe in the chorion cell, which is in contrast to the predominately oxidase activity reported for the decidual cell. Additional studies will be required to resolve this issue. In vivo, the close proximity of chorion trophoblast and decidual stromal cells suggests close functional interaction. Although the affinity of 11 ß-HSD type 1 is too low to exclude maternal glucocorticoids from the fetus effectively, the unique cortisol regenerating function of 11 ß-HSD type 1 could also provide a coordinated mechanism whereby the amount of maternal glucocorticoids reaching the fetus is precisely controlled. However, the mechanism by which the placental syncytiotrophoblast and chorionic trophoblast, the cells of same origin, express different types of 11 ß-HSD is unknown at present.

In this study, the percent recoveries of ³H-cortisol and ³H-cortisone were 86.7% and 88.3%, respectively, suggesting that very little ³H-steroid was taken up by the cells. In addition, we found that only 0.33% of total ³H-steroid was taken up by the cells, which further suggests that the determination of conversion of cortisol and cortisone in the medium reflected the intracellular enzyme activity.

NO donors, both SNP and SNAP, inhibited 11 ß-HSD type 2 oxidase activity in the placental trophoblast but did not affect 11 ß-HSD type 1 reductase activity, suggesting that the inhibitory effect of NO is specific for type 2 activity. Furthermore, SNP also reduced the levels of type 2 mRNA in the placental syncytiotrophoblast, indicating that NO may affect transcriptional regulation of the 11 ß-HSD type 2 gene. We accept, however, that the present studies do not exclude an effect of the NO donor on RNA stability. NO formed from L-arginine by NO synthase (NOS), can act in a paracrine manner and as an intracellular messenger. The main pathway of NO effects is stimulation of the soluble guanylyl cyclase (GC-S) (38). GC-S forms guanosine cyclic 3',5'-monophosphate (cGMP), which in turn specifically regulates protein phosphorylation, ion channel conductivity, and phosphodiesterase activity (38). In the present study, we found that the soluble guanylyl cyclase inhibitor, LY83583, partially blocked the inhibitory effect of SNP and SNAP on 11 ß-HSD type 2, suggesting that the effect of NO on 11 ß-HSD type 2 is mediated in part through the cGMP pathway. In an earlier study, we also demonstrated that similar doses of SNP stimulated the accumulation of cGMP in cultured human placental trophoblast (21). The synthetic cGMP analog, 8-br-cGMP, also mimicked the inhibitory effect of NO donors on placental 11 ß-HSD type 2 activity further suggesting a cGMP-mediated effect of NO on placental 11 ß-HSD type 2. On the other hand, as 8-br-cGMP was less potent than the NO donor, and the guanylyl cyclase inhibitor, LY83583, could only partially blocked the effect of NO, we propose that there may exist alternative pathways interacting with cGMP pathway resulting in more pronounced inhibition with NO donors.

When NO reaches high levels, it has been shown to have inhibitory effects on protein activities through ADP-ribosylation (38, 39). Because the ADP-ribosylation inhibitor, HMBG, was unable to block the effect of NO on 11 ß-HSD type 2 in this study, it is unlikely that this pathway plays a role in NO-induced inhibition of 11 ß-HSD type 2. Furthermore, it is also unlikely that the inhibitory effects of SNP and SNAP are a result of toxic effects on the cells, as the effects are specific on 11 ß-HSD type 2 activity, but not 11 ß-HSD type 1 reductase activity in the same cell type. The cells were more than 95% viable before and after drug treatment, which also excludes the possibility of toxic effects of the drugs tested in this study. Moreover, it has been recently reported that NO influences glyceraldehyde-3-phophate dehydrogenase (GADPH) activity by covalent linkage of NAD⁺ to the enzyme through ADP-ribosylation (40), which in turn would decrease the NAD⁺ available for steroid dehydrogenation. This may contribute in part to the inhibitory effect of NO on 11 ß-HSD type 2 activity. It is unlikely, however, to be the primary cause of the inhibition because SNP also suppressed the expression of 11 ß-HSD type 2 mRNA. In addition, the guanylyl cyclase inhibitor, LY83583, partially reversed the inhibitory effect of NO on 11 ß-HSD type 2 activity. Moreover, the inhibitory effect of NO was not affected by ADP-ribosylation inhibitor. We have interpreted our results to suggest that the specific inhibitory effect of NO on 11 ß-HSD type 2 is at least partially mediated by cGMP, although other pathways rather than ADP-ribosylation could also exist.

NO synthase is present in umbilical arteries and veins, myometrium, fetal membranes, and placental syncytiotrophoblast (19, 20). NO generated in the feto-placental circulation contributes to the local regulation of activities such as inhibition of CRH release, relaxation of basal vascular tone, and myometrial quiescence during pregnancy (21, 22, 23, 41). On the contrary, glucocorticoids induce hypertension either through a direct effect on the blood vessel or by binding to the minerolocorticoid receptor (12, 26), with positive feedback on the production of CRH and prostaglandins in trophoblast-derived cells in a manner that might facilitate myometrial contraction (24, 25, 42). The inhibitory effect of NO on 11 ß-HSD type 2 activity could result in more bioactive cortisol in the placenta. In turn, this could balance or override the effects of NO on the fetal-placental circulation, CRH production, and myometrial quiescence.

In conclusion, the results presented in this paper show that 11 ß-HSD type 2 activity predominates in cultured placental trophoblast, whereas 11 ß-HSD type 1 predominates in the chorion, mainly as a reductase. NO specifically inhibits placental 11 ß-HSD type 2 activity and its mRNA expression, but not 11 ß-HSD type 1 reductase activity. This effect is mediated at least partially through the cGMP pathway, although an alternative pathway not involving ADP-ribosylation may exist.

	Acknowledgments

 
We are grateful to Ms. Lindsay M. Mcwhirter for collecting human placentae.

	Footnotes

 
¹ This work was supported by operating grants from the Canadian Medical Research Council [Group Grant: Fetal and Neonatal Health and Development (to J.R.G.C.); MT12100 (to K.Y.)].

Received April 21, 1997.

	References

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