Molecular Human Reproduction, Vol. 5, No. 5, 487-494,
May 1999
© 1999 European Society of Human Reproduction and Embryology
Expression and functional analysis of endothelial nitric oxide synthase (eNOS) in human placenta
1 Department of Obstetrics–Gynecology, University of Ulm, Prittwitzstrasse 43, D-89075 Ulm, Germany and 2 Department of Endocrinology, St Bartholomew's Hospital, London, UK
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Abstract |
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We have investigated the expression and localization of endothelium-derived nitric oxide synthase (eNOS) and the effect of eNOS on placental human chorionic gonadotrophin (HCG) release. eNOS mRNA was found to be expressed in all tissues, with its expression significantly (P < 0.05) increased across gestation. Compared to normal term gestation, placentae from term pregnancies with fetal retardation, or maternal diabetes, but not with maternal hypertension, displayed significantly more (P < 0.05) eNOS mRNA. By immunocytochemistry, we found staining for eNOS in both the cyto- and syncytiotrophoblasts of first trimester and a loss of cytotrophoblast eNOS staining in term placentae, while syncytiotrophoblasts at term were strongly eNOS positive. Additional staining was found in endothelium surrounding the vascular tree. HCG was found to colocalize with eNOS in trophoblasts, but not in endothelia. When placental explants were perifused, exposure to the NOS substrate, the NO donor, l-arginine and trinitroglycerol evoked a prompt, albeit transient, increase of HCG release. The NOS inhibitor delayed, but did not block arginine-induced HCG release. Thus, eNOS is expressed in the human placenta at increasing levels during gestation with further increases during some pathological conditions. A role for NO in the acute endocrine modulation of the placenta is suggested by the colocalization of eNOS with HCG in human trophoblasts and the prompt secretion of HCG in response to agents which increase NO concentrations.
endothelial nitric oxide synthase/immunocytochemistry/perifusion/placenta/RT–PCR
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Introduction |
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Nitric oxide (NO) is now considered to be one of the key regulators of a variety of physiological functions. For example, when released by endothelial cells, it acts as a vasodilator, while additionally it inhibits platelet aggregation and endothelial adhesion. It is thus of considerable importance in the regulation of blood pressure (Ignarro, 1990
; Dawson et al., 1992; Nathan 1992; Garvey et al., 1994). NO has also been noted to play a critical role in neural transmission and in macrophage cytoxicity. For all such events, NO is generated within the cells; using L-arginine as substrate the NO synthase (NOS) catalyses oxidation to NO in the presence of other cofactors. In keeping with its wide spectrum of activities, NOS has been located in a variety of different tissues and cell types (Forstermann et al., 1991; Stuehr and Griffith, 1992). So far, two types of NOS have been distinguished on the basis of their regulatory/control mechanisms: a constitutive NOS (cNOS) regulated by calcium/calmodulin, and an inducible NOS (iNOS) principally controlled at the transcriptional level. Two cNOS isozymes differ in their DNA sequences and apparent molecular weights: neuronal NOS (nNOS) and endothelial NOS (eNOS). The sequence of iNOS is distinct from both constitutive isoenzymes (for review see Sessa, 1994).
One essential role for NOS is the regulation of human reproductive function. NO is involved in neuroendocrine regulation in that generation of NO is capable of releasing gonadotrophin-releasing hormone (GnRH) from hypothalamic neurons (Lopez et al., 1997). The importance of NOS in this tissue is further substantiated by the finding of adjacent expression of NOS to GnRH in closely apposed neurons (Grossman et al., 1994), or their colocalization in immortalized neurons (Bhat et al., 1995). Because GnRH is also an important neuroendocrine regulator in the placenta, we have speculated that NO and, by inference, NOS may also play a pivotal role in the human placenta. In fact, eNOS has been found to reside in the endothelium of placental vessels such as umbilical arteries and veins, chorionic arteries and veins, but also in the layer of the syncytiotrophoblasts (Myatt et al., 1997b). NO generated by NOS has been demonstrated to contribute to the regulation of vascular tone by counteracting the actions of vasoconstrictors (Myatt et al., 1992; Gonzalez et al., 1995). NOS in the placental villous vasculature also corresponds to the type III calcium-calmodulin-dependent endothelial isoform (Myatt et al., 1993a). However, no such NOS was found in the cytotrophoblast layer or in small fetal blood capillaries of term placenta (Myatt et al., 1993a), whereas eNOS activity was observed in the early placenta (Sahin-Toth et al., 1997). Whereas eNOS appears to be the most abundant isoform in early placenta (Sahin-Toth et al., 1997), other isoforms such as iNOS are predominantly expressed throughout pregnancy in the uterus (Ali et al., 1997).
If eNOS is indeed an important regulator of endocrine function in the human placenta, it should also be found in trophoblasts containing the essential hormones supporting pregnancy. More specifically, eNOS should then be localized to those syncytiotrophoblasts which store the essential pregnancy hormone human chorionic gonadotrophin (HCG). In order to shed further light onto the role of eNOS as a placental endocrine regulator, we have investigated the expression, localization and function of eNOS in human placentae of different gestational ages, using a variety of complementary techniques. First, we evaluated eNOS gene expression in human placenta by reverse transcriptase–polymerase chain reaction (RT–PCR). Second, we assessed the presence of eNOS in tissue sections and cultured trophoblast cells by means of immunohistochemistry. Finally, by perifusion of placental explants and cell cultures we evaluated the influence of eNOS on HCG secretion. Our data point to an important role of NO on endocrine function of human placenta.
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Materials and methods |
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The experiments were conducted with human placentae of different gestational ages. Permission to use human material was obtained from the local Ethical Committee prior to the study.
Characterization of the used tissue preparations
Fresh human placentae of first to third trimester gestation were obtained immediately following spontaneous vaginal deliveries, Caesarean sections, or after therapeutic first trimester abortions. The placental tissue was placed on ice; fragments (5–40 g) were dissected from the placentae and adherent membranes were removed. Tissue blocks were then washed in physiological saline (0.9%) and subjected to further investigation.
Cell culture methods
Cultures of human trophoblasts were used for the detection of eNOS and for the ß-chain of HCG (ß-HCG) in individual cells by immunocytochemistry and for exposure to NO donors in an incubation system. A protocol previously described (Kliman et al., 1986) was adapted for this experiment. Following the mechanical and enzymatic dispersal of placental fragments (first to third trimesters), cells were washed and then separated according to their density in a Percoll density gradient (at 400 g for 10 min). The layer containing purified trophoblasts was then used. The cells were placed into cell culture dishes (35x10 mm; 5x105 cells per well). M199 (Gibco, Munich, Germany) supplemented with 20% fetal calf serum (Boehringer, Mannheim, Germany) was used as culture medium. Cultures were incubated at 37°C with 5% CO2 in a humidified atmosphere. Culture medium was changed every 24 h, and cells harvested after 3 days. Trophoblast cells adherent to the plastic were incubated with L-arginine (Calbiochem, Bad Soden, Germany) or the NO donor trinitroglycerol (Merck, Darmstadt, Germany) at final concentrations of 10–4 to 10–6 mol/l. Alternatively, cells were washed with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde for immunocytochemical analysis.
Immunocytochemistry
For these experiments, placental tissue blocks were flash-frozen in 2-methylbutane at –70°C, cut into 5 µm sections, and stored at –70°C until further use. Staining was performed as follows. All incubation steps were carried out at room temperature in a humid and dark chamber. Slides were equilibrated in PBS, and thereafter incubated with either primary rabbit anti eNOS (Calbiochem) or biotinylated rat anti-ß-HCG antibodies (Dianova, Hamburg, Germany), or a combination of both. Binding of anti-eNOS was developed using Texas Red-coupled goat anti-rabbit antibodies (Dianova). The biotinylated anti-ß-HCG was detected with fluorescein coupled avidin (Vector Laboratories, Wiesbaden, Germany). Nuclei were counterstained by 4,6-diamidino-2-phenylindol (DAPI; Sigma). The slides were finally coverslipped with mounting medium (Vectashield Mounting Medium, Vector Laboratories). The presence of eNOS and ß-HCG was visualized by means of a stereo fluorescence microscope (Zeiss, Oberkochen, Germany).
In-vitro perifusion experiments
A previously characterized in-vitro microperifusion system (Rossmanith et al., 1991; Szilagyi et al., 1993) was utilized and adapted for this experiment. Explants of term placentae (150 mg) were placed in perifusion chambers (100 µl volume) and continuously perifused (100 µl/min) for 12 h with medium M199 (Gibco). Five hours after the start of perifusion, either medium 199 alone (as control; n = 11) or medium containing the nitric oxide donor trinitroglycerol (Merck; n = 4) or L-arginine as substrate (Calbiochem; n = 8) at concentrations of 10–6 to 10–4 mol/l continued to perifuse the different perifusion chambers. In order to demonstrate receptor-specific actions of NO donors, the antagonist NG-nitro-L-arginine-methyl-ester (L-NAME; Calbiochem; n = 3) was added at equimolar concentrations to the perifusion medium that contained L-arginine. The effluent was collected at 10 min intervals into ice-chilled tubes. The effluent fractions were then assayed for HCG in a two-step sandwich immunoassay (Enzymun-Test HCG; Boehringer).
RT-PCR reaction
Total RNA was extracted from placental fragments (100–200 mg) using TRIzol (Gibco). The mRNA was reversely transcribed with oligo(dT) primers using a commercial kit (Invitrogen, Leek, Netherlands). A multiplex PCR was set up using primers for eNOS and for glyceraldehyde-3-phosphate dehydrogenase primers (GAPDH), an established `housekeeping' gene (Markvardsen et al., 1995). The 50 µl PCR reaction mix contained the reverse transcription reaction mix, 1 mM dNTP mixture, 1 U Taq-polymerase and 1x reaction buffer containing 1.5 mM magnesium chloride (Perkin–Elmer, Weiterstadt, Germany). Primers were added at 0.4 µM each for eNOS (sense: 5'-AAG ATC TCC GCC TCG CTC A-3'; antisense: 5'-GCT GTT GAA GCG GAT CTT A-3') and at 0.28 µM each for GAPDH (sense: 5'-GGA GTC AAC GGA TTT GGT-3'; antisense: 5'-GTG ATG GGA TTT CCA TTG AT-3'). We performed 35 amplification cycles after 4 min of initial denaturation at 94°C, followed by cycles for denaturation (1 min at 94°C), annealing (1 min at 60°C), and extension (2 min at 72°C). Final extension was allowed for 10 min at 72°C. Reactions were run in a thermal minicycler (Biozym, Oldendorf, Germany). We additionally confirmed that the intensities of the resulting bands after electrophoresis had not reached the plateau phase of amplification. The expected band lengths for eNOS were 336 bp and for GAPDH 206 bp, respectively. Restriction enzyme digest using Ava II and Bsmf I produced bands of the predicted size for eNOS. In addition, amplified products were sequenced, and the sequences were found to be identical to those published. PCR products were separated on an 8% acrylamide gel and made visible with ethidium bromide. The intensity of the stained products was assessed by densitometry. Gels were scanned with a digital camera (Intas, Göttingen, Germany). Band intensities were evaluated and compared by use of the Imagemaster program (Pharmacia, Freiburg, Germany). The quantification and validation of this technique has been previously published (Dahia et al., 1997)
eNOS expression was assessed in placentae of first (n = 4) and third trimester gestations (n = 8), but also in term placentae of pathological gestations [maternal hypertension (n = 3), fetal growth retardation (n = 3), and maternal diabetes (n = 2)]. Human umbilical cord preparations with established high eNOS contents served as positive controls; cultured granulosa cells (after 4 days in culture) had previously been shown to be devoid of eNOS mRNA (S.Wolfahrt and W.G.Rossmanith, unpublished). Data sets were evaluated for differences by Wilcoxon–Mann–Whitney rank test; P < 0.05 was considered to be statistically significant.
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Results |
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RT–PCR reaction
Figure 1
displays a representative PCR experiment with amplification products of eNOS obtained from first and third trimester placentae. The presence of eNOS mRNA in human placenta was demonstrated by the 336 bp band. PCR products for eNOS appeared to be more prominent in the third trimester than in first trimester placentae. To quantify these apparent differences in eNOS expression between placentae of different gestational ages, eNOS and GAPDH mRNA were reversely transcribed and amplified in the same tube (multiplex RT–PCR). The ratio between the intensities of the eNOS and GAPDH bands were determined and are summarized in Figure 2. eNOS expression was found in all placentae investigated, but varied between placentae of different gestational ages. In particular, placental eNOS expression increased from the first (mean ratio 0.045 ± 0.02) to third trimester placentae (mean ratio 1.3 ± 1.2). eNOS expression was evaluated in placentae of pathological gestations. Any differences in the eNOS gene expression levels could not be observed between placentae of hypertensive pregnancies (mean 0.78 ± 0.16) and those of normal term gestations. Placentae obtained from pregnancies with growth-retarded fetuses or with maternal diabetes showed significantly (P < 0.05) higher ratios (fetal retardation: 6.1 ± 2.3, diabetes: 3.3 ± 1.1) compared with normal placentae. Umbilical cord showed a very high eNOS expression (9.8), whereas we confirmed that cultured granulosa cells showed none.
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); granulosa cells (
)]. The numbers of symbols are not equal to the numbers of placentae mentioned in the text for the first and third trimester because some pairs of values were superimposed on one another (one pair first trimester, two pairs third trimester). The difference between first versus third trimester placentae and between normal third trimester placentae versus those from gestations with growth retardations or diabetes were significant (P < 0.05) as determined by the Mann–Whitney U-test.Immunocytochemistry
The eNOS protein was detected in placental cryosections at all investigated gestational ages (Figures 3 and 4
). Simultaneous localization of the eNOS and of ß-HCG proteins was indicated by concomitant red and green immunofluorescence. Nuclei were identified by a blue fluorochrome (DAPI staining). In cultured trophoblasts, both eNOS and ß-HCG were found within the cytoplasm close to the nuclei (not shown). No staining signals were detected in the negative control experiments omitting the specific antibody (Figures 3D and 4D). This confirmed that the methodology used was sensitive and specific for the detection of eNOS. In first trimester placental cryosections cytotrophoblasts stained brightly for eNOS while dendrite-like pseudopodia of syncytiotrophoblasts were less intensively stained (Figure 3A). In tissue sections of third trimester placentae, eNOS was found in syncytiotrophoblasts, additionally in endothelial cells surrounding blood vessels, but not in the cytotrophoblasts (Figure 4A). eNOS was detected in all investigated areas of the placental cotyledon, without any preference for central or peripheral sites. No differences were observed in eNOS staining in placentae from spontaneous deliveries compared to those from Caesarean sections.
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ß-HCG was found in both layers of cytotrophoblast and syncytiotrophoblast cells (Figures 3B and 4B
) with no differences between different gestational ages. Colocalization of eNOS and ß-HCG was indicated by orange immunofluorescence. These experiments revealed that early in gestation cytotrophoblasts were found positive for eNOS besides syncytiotrophoblasts, while at term placental endothelium and the syncytiotrophoblasts are still positive, whereas cytotrophoblasts no longer contain the enzyme (Figures 3C and 4C).
In-vitro perifusion experiments
To study whether and by which dynamics eNOS regulates HCG release, we employed in-vitro perifusion. Placental explants were perifused for 24 h and, after snap freezing, subjected to immunocytochemistry. Effluates were analysed for HCG. In control experiments, following the equilibration periods, HCG concentrations in the perifusate fractions tended to decline, but were clearly detectable throughout perifusion in all placentae investigated (Figure 5A). In response to perifusion with L-arginine at 10–4 and 10–6 mol/l, a highly reproducible increase (~180% above levels before stimulus) in the HCG release rates was observed within 20 min in all placentae tested (Figure 5B). However, this HCG increase was transient, and HCG release rates returned to basal levels after 30 min, in spite of the fact that the tissue continued to be exposed to L-arginine. No further increases in the HCG levels could be detected, even when arginine was taken from the perifusion medium and re-added later on (not shown). Comparably timed increases of the HCG concentrations in the effluent fractions were observed with 10–4 or 10–6 mol/l of the NO donor glycerol trinitrate (Figure 5C). Addition of trinitroglycerol to the perifusion medium resulted in a less pronounced HCG increase (~125% above basal values) within 20 min after exposure. Following the pattern, this HCG increase was also transient, and HCG effluent concentrations returned to basal levels within 30 min (Figure 5C). When the NOS antagonist L-NAME was added together with L-arginine, the peak response of HCG to L-arginine was markedly blunted, but the increase in HCG release was prolonged, compared to perifusions in the presence of L-arginine alone (Figure 5D). Maximal HCG release was observed 40–50 min after addition of medium containing equimolar concentrations of stimulus and inhibitor. Thus, the NOS inhibitor did not completely inhibit L-arginine-induced effects in placental explants. Endothelial NOS remained detectable by immunocytochemistry after the perifusion period (results not shown), confirming the preservation of this protein and the functional integrity of the placental explants throughout our investigations.
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Isolated trophoblasts after 3 days in culture did not show modulated HCG release in response to addition of L-arginine or trinitroglycerol at concentrations between 10–4 and 10–6 mol/l at any time investigated following the start of the incubation experiments (data not shown).
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Discussion |
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Whenever a role for NO in endocrine function has been postulated, the key enzyme NOS has been sought in endocrine tissue. The present study investigated the localization of eNOS in human placenta and its function in relation to HCG release. Using complementary methodologies such as molecular, immunohistochemical, and cell perifusion and culture techniques, we have demonstrated eNOS gene expression in human placentae of different gestational ages and pathologies. To our knowledge, this is the first demonstration that colocalizes eNOS and HCG to human cyto- and syncytiotrophoblasts. The notion that eNOS may have a function in placental HCG release was substantiated by our observation that the generation of NO regulated HCG release from trophoblasts in vitro. In fact, perifusion with NO donors provoked a release surge in HCG from placental explants, whereas incubation of cultured trophoblasts with NO donors did not, suggesting that NO may play a role in the acute endocrine modulation of the placenta. The functional significance of placental HCG release in later pregnancies and of its modulation by NO is not known. However, HCG synthesis and release appears to be the key element of neuroendocrine hormonal regulators in placenta. Thus, HCG and its regulation by NO are supposed to be essential for placental development and fetal survival (compare Petraglia et al., 1996
).
Our immunohistochemical data on placental sections demonstrated the presence of eNOS in both the vascular endothelium and trophoblasts at varying degrees throughout gestation. Specifically, early in gestation the cytotrophoblast layer was clearly stained. At term, the endothelium of all fetal blood vessels and the syncytiotrophoblasts were eNOS positive, while cytotrophoblasts had become negative. These data confirm earlier observations on the localization of eNOS in the syncytiotrophoblast layer and the endothelium of larger fetal vessels (Eis et al., 1992; Springall et al., 1992; Conrad et al., 1993; Myatt et al., 1993a, 1997b; Buttery et al., 1994; Ghabour, 1995). They extend these former reports on placental eNOS localization: the cytotrophoblasts are stained in early placentae, and small capillaries are clearly positive at term.
It is important to note that eNOS gene expression in human placenta was found to increase with advancing gestation. This finding is compatible with the immunological staining for the eNOS peptide, which we found to be increased in placental syncytiotrophoblasts with gestational age. It thus appears that there is enhanced biosynthetic demand for the production of NO in the growing human placenta. By RT–PCR we only globally assessed eNOS expression in human placenta. As demonstrated by our immunofluorescence findings, eNOS is located in placental trophoblasts, but is also found in the endothelial layer of large and small vessels. Assuming that localization of the eNOS peptide reflects the distribution of the eNOS gene message, increased eNOS expression during gestation may then represent enhanced transcriptional activity in the vascular tree. Therefore, changes in eNOS gene expression across gestation may pertain to all functions of eNOS in human placenta, and eNOS may possibly help to maintain pregnancy by controlling both endocrine function and vascular tone.
Hypertensive gestations are characterized by compromised endocrine and vascular function of the corresponding placentae; however, we did not observe a substantial loss of eNOS expression in placentae of hypertensive term gestations, compared with normal term gestations. These data, albeit in small numbers and essentially descriptive, suggest that the regulatory cascade initiated by eNOS is still functionally intact and may be instrumental in compensating for functional placental compromise characteristic of hypertensive gestations. Conversely, the small number of placentae of diabetic gestations and of growth-retarded fetuses showed increased eNOS expression levels in our investigation, probably indicating higher eNOS activity within the vascular tree and also in placental trophoblasts during these pregnancies. These findings appear to contrast previous ones (Morris et al., 1995) who reported lower eNOS activity in gestations complicated by fetal growth retardation. Our analysis assessed the eNOS gene expression levels, and thus any change in placental eNOS activity during physiological and pathological pregnancy can only be deduced with caution. Placental eNOS gene expression may represent changes in the NOS activity required to cover the increasing demand during gestation; conversely, changes in the eNOS expression during pathological pregnancies may indicate that the eNOS activity needs to adapt to these altered conditions.
We have assessed the functional relevance of eNOS in the human placenta and have found, for the first time, modulation of endocrine secretion by changes in the eNOS activity within this organ. In fact, a surge in HCG release from perifused placental explants could be evoked by medium containing the NO substrate L-arginine and the NO donor trinitroglycerol (Ignarro et al., 1991; Jorens et al., 1993). The observed effects on HCG release were more pronounced with perifusion in medium containing L-arginine than trinitroglycerol. This difference in the response magnitude to NO donors may reflect variation in the metabolic rates for these substances. L-Arginine is readily metabolized (Jorens et al., 1993), while the metabolism of trinitroglycerol is less rapid. In all placentae investigated, we observed a prompt but transient increase in HCG release in response to NO donors, while this increase was not observed during control conditions. HCG release from human placentae has been found to occur in secretory episodes or pulses (Szilagyi et al., 1992). These HCG secretory episodes may be masked, as they are averaged by the relatively long sampling intervals for the perifusion effluent (Rossmanith et al., 1991). An alternative interpretation is that the acutely releasable store of HCG within the placenta may be depleted, since the amount of HCG released may exceed the capacity of the trophoblasts to re-synthesize. When placental explants were repeatedly exposed to NO donors during prolonged perifusion periods, no additional secretory peaks could be observed, suggesting a complete depletion of the releasable pools of HCG. Addition of equimolar concentrations of the eNOS inhibitor L-NAME to medium containing L-arginine attenuated, but did not abolish the HCG surge, although the increase in HCG release was of longer duration. The mechanisms responsible for this change remain speculative at present, but this finding may represent receptor binding competition, or alternatively, partial degradation of the inhibitor during the perifusion period. L-NAME is a specific eNOS inhibitor which does not affect other placental NO synthases such as iNOS (Myatt et al., 1997a). The lack of a second peak in the HCG release in response to L-arginine alone argues against the involvement of iNOS, since the HCG responses to L-arginine alone and to L-arginine plus L-NAME are of comparable magnitude. Thus, an effect of iNOS on HCG release, if any, should not have been obscured by that observed through eNOS.
In the hypothalamus, NO is able to release GnRH from GnRH-containing neurons, and inhibitors of NOS activity may prevent this (Lopez et al., 1997). By analogy with this finding, we speculate that GnRH may be the essential factor that is released from human trophoblasts in response to NO. In turn, GnRH may stimulate HCG secretion from human trophoblasts. In support of this notion is the observation of GnRH gene expression and synthesis in individual trophoblasts (Wolfahrt et al., 1998). GnRH has also been found to stimulate HCG release from human placentae of different gestational ages (Szilagyi et al., 1992; Petraglia et al., 1996) Although not directly evaluated by the current investigation, we suggest that the effects of NO on HCG secretion may be mediated by increased secretion of GnRH from identical trophoblasts, and this speculation requires experimental verification.
In conclusion, we have provided evidence for eNOS gene expression and transcription in human placenta. In addition, an important role for eNOS in the regulation of placental HCG release was suggested by the demonstration of colocalization of eNOS and HCG in individual trophoblasts of different gestational ages, and by the prompt but transient release of HCG mediated by NO. These combined findings allow us to speculate on a role for NO in the acute endocrine regulation of the placenta. We propose that eNOS, besides its role in placental vasoregulation, may be essential for the acute adaptation of the human placenta to metabolic or endocrine demands.
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Acknowledgments |
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The authors gratefully acknowledge the help of all collaborators in the endocrinological laboratories in conducting parts of this study. W.G.R. is recipient of a Deutsche Forschungsgemeinschaft grant (DFG Ro 657/6-2).
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3 To whom correspondence should be addressed
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References |
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Submitted on September 1, 1998; accepted on February 11, 1999.
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