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Original Studies |
Maternal Health Research Centre (E.-C.C., J.T.F., R.S.), Endocrine Unit, John Hunter Hospital, Newcastle, N.S.W. 2310, Australia; and Department of Neurobiology (X.N.), Second Military Medical University, Shanghai 200433, P.R. China
Address all correspondence and requests for reprints to: Prof. Roger Smith, Maternal Health Research Centre, Endocrine Unit, Locked Bag 1, Hunter Regional Mail Centre, Newcastle, N.S.W. 2310, Australia. E-mail: mdrsm{at}mail.newcastle.edu.au
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Abstract |
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-L-arginine methyl ester (1 mmol/L;
P < 0.01) and the guanylate cyclase inhibitor,
LY83583 (1 µmol/L; P < 0.01). The inhibitory
effect of SNP was also blocked by LY83583. CRH messenger RNA content
did not change when the placental cells were incubated with SNP,
N-L-arginine methyl ester, and LY83583 for 6
and 24 h, and this was consistent with studies showing that total
CRH immunoreactivity (cells plus media) did not change in the presence
of SNP. These studies indicate that exogenous NO inhibits CRH
exocytosis, rather than biosynthesis, by human trophoblasts and that
endogenous NO has tonic inhibitory effects on CRH release by these
cells. The inhibitory effect of NO on basal and stimulated CRH release
by placental trophoblasts seems to be a guanylate cyclase-mediated
inhibition of exocytosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, nitric oxide (NO) has been postulated to be a paracrine regulator of CRH secretion within the placenta because endothelial NO synthase has been localized in placental syncytiotrophoblasts by immunocytochemistry (12, 13), and soluble guanylate cyclase (the target of NO) also has been purified from placenta (13). We demonstrated that NO inhibits CRH release from perfused human placenta in vitro and sodium nitroprusside (SNP), a NO-donating drug, inhibited CRH release from cultured placental syncytiotrophoblasts (10, 14). In other tissues, NO has variously been reported to stimulate or inhibit neurotransmitter and hormone release and synthesis (15, 16, 17). However, the effects of NO on CRH synthesis by placental cells are still unknown.
The purpose of this study was to investigate the role of the NO signaling pathway on CRH biosynthesis and secretion from human placental cells. We have studied the effect of NO donors, an NOS inhibitor, and a guanylate cyclase inhibitor on CRH protein and messenger RNA (mRNA) expression in cultured trophoblasts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was performed with the approval of the Newcastle John Hunter Hospital and University of Newcastle Human Ethics Committees. Human term placentas were obtained from normal pregnant women after spontaneous vaginal delivery or elective cesarean. Tissue was enzymatically dispersed, and cytotrophoblast cells were purified according to a slightly modified Kliman’s method (18). Briefly, cubes of villous tissue from the maternal surface were thoroughly rinsed with saline (4 C), dissected free from the connective tissue and blood vessels, and then immersed in DMEM (Life Technologies, Grand Island, NY) containing 0.1% BSA (Sigma Chemical Co., Castle Hill, NSW, Australia), 0.005% gentamycin (Sigma Chemical), and 0.01% streptomycin (Sigma Chemical). The tissue was coarsely minced with scissors and digested with 0.125% trypsin (Sigma Chemical) and 0.02% deoxyribonuclease-I (Sigma Chemical) in suspension medium thrice for 30 min each at 37 C. The cell suspension was centrifuged at 2300 x g for 10 min, and the cell pellet was resuspended in DMEM, then loaded onto a 5–70% discontinuous Percoll gradient and centrifuged to separate the different cell types. Cytotrophoblasts, which banded between the density markers of 1.049 and 1.062 g/mL, were collected, then washed, and suspended in DMEM containing 15% FCS (Life Technologies, Grand Island, NY), 0.005% gentamicin, and 0.01% streptomycin. Cells were counted, and viability was assessed by trypan blue exclusion. Live cells comprised more than 90% of the cytotrophoblast preparation. Cytotrophoblasts were plated into six-well plates at a cell density of 3x105 cells/cm2 and incubated at 37 C in a humidified atmosphere (95% air-5% CO2).
Time-course experiment of basal CRH levels in cultured placental cytotrophoblasts
Basal CRH immunoreactivity (IR) levels in cytotrophoblasts cultured in DMEM containing 15% FCS, over 6 days, were determined by collecting samples daily. The culture media and cells were collected, extracted, and assayed for CRH IR.
Release studies
On day 3 after plating cells, the cells were washed twice with
suspension medium and preincubated with culture media for 1 h. KCl
(BDH Chemicals, Kilsyth, Victoria, Australia), SNP (Ajax chemicals,
Auburn, Australia), S-nitroso-N-acetyl-penicillamine (SNAP;
Sigma Chemical), L-arginine (L-ARG; Sigma Chemical),
N-nitro-L-ARG methyl ester (NAME; Sigma Chemical), and
LY83583 (ICN Chemicals, Seven Hills, NSW, Australia) were added to the
cells to achieve a final concentration of 45 mmol/L for KCl, 1–100
µmol/L for SNP and SNAP or 100 µmol/L for L-ARG, 1 mmol/L for NAME
and 1 µmol/L for LY83583 (these doses were determined by optimization
experiments). Each treatment was performed in triplicate wells and
repeated with five different placentas. Media and cells were collected
and stored at -20 C for CRH RIA after incubating for 1, 3, 6, 12, or
34 h with these agents.
CRH RIA
CRH IR in the culture media and cells was extracted using Vycor
glass extraction and acetic acid, respectively. Frozen culture medium
samples (2 mL) were thawed at room temperature and Vycor-extracted, as
previously described (19). CRH RIA was performed as previously
described (19). Human CRH1–41 (Sigma Chemical) was used as
the standard, and radioligand was prepared with the chloramine-T method
and purified by high pressure liquid chromatography. The anti-CRH
antibody Y2B0 (a gift from Prof. P. J.
Lowry, University of Reading, Reading, UK) does not cross-react with
human ß-lipotropin, 
-endorphin, ACTH, vasopressin and human
melanocyte-stimulating hormone. The assay sensitivity was 2.8 pg/L. The
concentrations of CRH IR in both cells and media were expressed as
pg/106 cells.
Total RNA preparation and Northern blot analysis
Total cellular RNA was isolated by the method of Chomczynski and Sacchi (20). Pooled placental cell cultures of three individual wells, each with 3x106 cells, constituted a single sample for these studies. The cells were treated for 6 or 24 h with vehicle, SNP (100 µmol/L), NAME (1 mmol/L), and LY83583 (1 µmol/L). RNA samples (15–20 µg/lane) were denatured and electrophoresed in 1.2% agarose containing 2.2 mol/L formaldehyde. Ethidium bromide-stained ribosomal RNA bands were visualized (under ultraviolet light) to ensure that RNA degradation had not occurred and that equal amounts of RNA had been loaded into each lane. After electrophoresis, RNA was transferred to a nylon membrane (Schleicher & Schuell, Keene, NH) by capillary blotting overnight, then cross-linked in an ultraviolet cross-linker and air dried. Completion and uniformity of transfer was assessed by determining transfer of 28S and 18S ribosomal RNA from the gel.
Hybridizations were performed using digoxigenin-labeled antisense complementary RNA probes. The riboprobe template for human CRH was constructed by subcloning the 480-nucleotide fragment of CRH gene that contains a portion of exon 1 (nucleotides 87–578, Ref.21) obtained by PCR on an 8-kb human CRH genomic clone (kindly provided by J. A. Majzoub, Harvard Medical School, Boston, MA) into plasmid pGEM-T Easy (Promega Co., Madison, WI). The forward primer is 5'-AATGGACAAGTCATAAGAAGAAGCC-3', and the reverse primer is 5'-ACGCAAAGTTGGTGGCG-3'. After digestion with NdeI (Boehringer Mannheim, Castle Hill, NSW, Australia), the recombinant plasmid was transcribed with T7 RNA polymerase to generate a complementary RNA probe using a digoxigenin labeling kit (Boehringer Mannheim). The human GAPDH (Clontech, Palo Alto, CA) complementary RNA probe was synthesized using T7 RNA polymerase. The membranes were prehybridized for 2 h and hybridized for 16 h in hybridization solution (Boehringer Mannheim) at 68 C. After washing with 2xSSC containing 0.1% SDS (twice at room temperature) and 0.1xSSC containing 0.1% SDS (twice at 68 C), the membranes were treated for detection using digoxigenin detection kits (Boehringer Mannheim). The amount of CRH mRNA in each lane was corrected for differences, based on the amount of GAPDH mRNA.
Statistical analyses
Statistical analyses were carried out using Student’s paired and unpaired t tests. The values are expressed as the mean ± SEM. In some cases, the values are expressed as mean ± SD. In all cases, statistical significance is indicated by P < 0.05. Multiple comparisons were statistically compared by ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When trophoblasts were cultured for 6 days, basal CRH IR was readily detected in the culture media and cells collected each day. CRH IR concentrations in medium increased from day 1 and peaked at day 3 (data not shown), suggesting that there is basal CRH IR release into medium from cultured trophoblasts. During the period of culture, CRH IR concentration in cells also increased from day 1 and peaked at day 3. CRH IR was undetectable in cytotrophoblasts freshly isolated from the placenta (data not shown).
Effects of SNP and SNAP on basal and KCl-stimulated CRH secretion in placental cells
SNP (100 µmol/L), an NO donor, significantly decreased CRH IR
concentration in the medium by at least 50% (P <
0.01) and increased cellular CRH IR (P < 0.01; Fig. 1
), suggesting that SNP inhibited CRH
secretion into the media. In the presence of KCl (45 mmol/L), CRH IR
concentration in media increased markedly, whereas CRH IR in cells was
reduced. KCl-induced CRH IR release also was abolished by SNP
(P < 0.01; Fig. 1). Another NO donor, SNAP, also
inhibited CRH IR release from trophoblasts, but its action was less
potent than that of SNP (Fig. 2). In the
presence of hemoglobin (Hb, 20 µg/mL), an NO scavenger, basal CRH IR
release was increased and the SNP effect was reversed (Fig. 2).
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The competitive NOS inhibitor, NAME, caused a significant increase
of CRH release into the culture media, compared with basal CRH levels
(P < 0.01, Fig. 3). This
effect was blocked by administration of L-ARG. However, L-ARG did not
affect basal CRH release (Fig. 3).
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To determine whether soluble guanylate cyclase activation was
involved in the effect of SNP on CRH secretion, LY83583, a guanylate
cyclase inhibitor, was used either by itself or in combination with
SNP. LY83583 (1 µmol/L) increased basal CRH release into media
(P < 0.01) and also blocked the inhibition of CRH
release induced by SNP (Fig. 4).
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Northern blot analyses of total RNA, from both cultured
trophoblasts and placental tissues, showed that a predominant species
of 1.5-kb CRH mRNA was present. When cells were incubated with SNP (100
µmol/L) for 6 and 24 h, CRH mRNA levels did not change. In the
presence of NAME and LY83583, CRH mRNA content did not change (Fig. 5). This result is consistent with the
study of total CRH protein expression in cells in the presence of SNP.
When cultured trophoblasts were incubated with SNP for 1–24 h, total
CRH IR in cultures did not change (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO donors, SNP and SNAP, spontaneously produce NO and provide an exogenous source of NO. In the process of releasing NO, various breakdown products, including cyanide, are generated by NO donors. Although trophoblasts exposed to SNP at 3 mmol/L continued to exclude trypan blue (M. Read, personal communication), we further controlled for the effects of substrates other than NO by using two structurally unrelated NO donors that generate dissimilar breakdown products. Here, our data show that these two NO donors produced a similar inhibitory effect on CRH release, and these effects were reversed by an NO scavenger hemoglobin, strongly suggesting that NO inhibits placental CRH release. In vivo, NO is produced by NOS converting L-ARG into L-citrulline (22). The constitutive endothelial isoform of NOS has been identified in the syncytiotrophoblast layer of the placental villi (12, 13), as has CRH (7). This raises the possibility that a paracrine relationship could exist between NO and CRH in placenta. Using RT-PCR, we also found that cultured human trophoblasts also expressed endothelial NO synthase mRNA (X. Ni, unpublished data). To ascertain the role of endogenous NO on CRH, the NOS inhibitor, NAME, which blocks the conversion of L-ARG into citrulline, was used and found to increase basal CRH release into media. This effect was reversed by L-ARG, a competitive substrate of NO synthase. Furthermore, addition of hemoglobin not only reversed the effect of SNP on CRH release but also increased basal CRH secretion into the media. These data provide convincing evidence for a tonic inhibitory role of endogenous NO in the control of CRH release from trophoblasts.
There are few studies on the long-term action of NO on hormone synthesis and secretion. Belsham et al. (17) reported that NO acted through a cyclic guanosine monophosphate (cGMP)-signaling pathway to repress GnRH gene expression in a hypothalamic cell line after 4 h treatment. Tan et al. (23) also found that SNP treatment for 4 h caused a decrease in POMC mRNA expression, but no change in ß-endorphin IR in AtT-20 cells. However, our study showed here that SNP treatment up to 24 h did not change the CRH peptide and mRNA content. Furthermore the competitive NOS inhibitor, NAME, and the guanylate cyclase inhibitor, LY83583, did not affect CRH mRNA levels in trophoblasts. These results indicate that in placental trophoblasts, NO specifically inhibits CRH release but not synthesis.
Our data indicate that the NO donor, SNP, inhibits CRH exocytosis from cultured placental cells, and this effect is mediated by the guanylate cyclase-cGMP pathway. In the central nervous system, it is well described that NO modulates neurotransmitter release by regulating synaptic vesicle release (24). In peripheral tissues, NO also has been found to inhibit exocytosis of insulin secretory granules from rat pancreatic islet cells (16), and it is implicated in the inhibition of histamine exocytosis induced by relaxin from mast cells (25). Thus, NO may inhibit CRH secretion from cells by inhibiting the exocytosis of secretory granules. Our results support this hypothesis by showing that SNP decreased CRH IR concentration in culture media but increased its concentration in cells. High potassium is known to induce depolarization of the cell membrane, which opens voltage-gated calcium channels and increases intracellular calcium concentrations, thus inducing exocytosis of secretory granules or vesicles from neurons and endocrine cells (26). SNP also increased CRH concentrations in potassium-treated cells, suggesting that SNP abolished potassium-stimulated CRH release through its inhibition of exocytosis.
NO activates soluble guanylate cyclase and stimulates the production of cGMP. Presumably, this cGMP may cause a decrease in intracellular free calcium in cells, which may block depolarization of the cell membrane associated with release of the hormones or neurotransmitters from storage vesicles (24, 27, 28). Our previous study demonstrated that there was a concentration-dependent intracellular accumulation of cGMP in response to SNP (10). In this study, we found that LY83583 increased CRH release and blocked the inhibitory effect of SNP on CRH secretion into medium, suggesting that NO inhibits CRH exocytosis through its activation of guanylate cyclase.
Recent studies strongly suggest a role for placental CRH in the timing of parturition and as a trigger for labor in humans (6). Giles et al. (29) reported that NOS activity was significantly lower in placentas from women with abnormal umbilical artery flow velocity waveforms, compared with those with normal umbilical artery flow. It is possible that elevated CRH levels in maternal and umbilical cord plasma associated with preterm deliveries (4, 5), intrauterine growth retardation, and preeclampsia may be the result of deficient NO. If placental NOS activity was impaired, and placental CRH release into maternal circulation is abnormally increased, then premature delivery may result.
In conclusion, the current study shows that not only exogenous, but also endogenous NO, inhibits CRH release from placental trophoblasts. In placenta, NO specifically inhibits CRH release rather than synthesis, and this effect seems to be a guanylate cyclase-mediated inhibition of exocytosis.
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Acknowledgments |
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Footnotes |
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Received June 30, 1997.
Revised August 1, 1997.
Accepted August 20, 1997.
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