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Original Studies |
Institutes of Pharmacology (G.T., P.N.) and Obstetrics and Gynecology (A.L., Fi.M., Fr.M., M.G.P., R.A., S.M.), Catholic University Medical School, Largo Francesco Vito 1-00168 Rome, Italy
Address all correspondence and requests for reprints to: Prof. Pierluigi Navarra, Institute of Pharmacology, Catholic University Medical School, Largo Francesco Vito 1-00168 Rome, Italy. E-mail pnavarra{at}rm.unicatt.it
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Abstract |
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Introduction |
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The secretion of CRH from trophoblast cells seems to be constitutive rather than regulated through exocytosis of storage vesicles, as occurs in the hypothalamus (7). In spite of this difference, the mechanisms controlling CRH release from the placenta are reminiscent of those acting at the hypothalamic level. For example, the inflammatory cytokine interleukin (IL)-1ß stimulates CRH release via the cyclooxygenase (COX) pathway in the hypothalamus (8), as well as the placenta (9). Furthermore, NO is able to inhibit the release of CRH from human trophoblasts (10), matching similar evidence concerning NO and CRH release from the hypothalamus (11).
Another gaseous neuromodulator, carbon monoxide (CO), has been shown to influence hypothalamic CRH release (12, 13). CO is normally produced during the hydrolysis of heme groups by the enzyme heme oxygenase (HO) (14); HO activity is constitutive in discrete regions and organs such as the brain, but it can be induced in virtually all cell types by various stimuli, including oxidative and heat-shock stress, disease states, and endotoxin challenges (14). Very recently, HO has been described in human placental cells (15, 16, 17). Because CO is involved in the control of CRH release from the hypothalamus, in the present study we sought to determine whether the HO-CO pathway is involved in the regulation of placental CRH as well. Furthermore, we have also investigated the possible involvement of COX in the HO-CO signal transduction process (18, 19).
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Materials and Methods |
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A total of 22 placentas were obtained, immediately after term delivery, from nonlaboring pregnant women (range, 21–44 yr; mean age, 32 ± 1.4 yr) undergoing cesarean section at term because of previous cesarean sections or breech delivery. Diabetic or hypertensive patients were excluded. Informed consent was obtained from all patients.
Trophoblast cells were isolated using a slight modification of the method of Nelson (20). The villous tissue was removed by sterile dissection from the center of different cotyledons (excluding chorionic and basal plates), washed repeatedly with 0.9% sodium chloride to remove blood from the intervillous space, and minced with scalpel blades. Approximately 15 g whole tissue villi were placed into a 50-mL polypropylene tube and incubated with an equal volume of Dispase (Becton Dickinson and Co. Labware, Bedford, MA) at 37 C for 15 min with constant shaking. The enzyme was inactivated by adding 200 µL 0.5-mol/L EDTA, and the tissue suspension was centrifuged at 200 x g for 5 min at 4 C. The supernatant was discarded, DMEM (Life Technologies, Inc., Grand Island, NY) was added to the digest to the 50-mL mark, and the suspension was mixed and incubated on ice for 20 min, during which time the villous fragments settled to the bottom of the tube. The supernatant containing trophoblast cells was transferred to another tube, filtering through a 100-µmol/L nylon mesh. This procedure was repeated once. The two supernatants containing trophoblast cells were pooled and centrifuged at 200 x g for 10 min at 4 C. Further purification of the trophoblast cell pellet was performed by Percoll density gradient centrifugation (21). The gradients were made from 45% to 15% Percoll (vol/vol) in 5% steps of 1.5 mL each, by dilutions of Percoll with calcium and magnesium-free Hanks’, and layered in a 15-mL conical polystyrene centrifuge tube. The cell pellet was resuspended in DMEM to a vol of 2 mL and then layered onto the Percoll gradients and centrifuged at 1850 x g for 30 min. Trophoblast cells were recovered with a Pasteur pipette in the density of the gradient between 1.084 and 1.062 g/mL. The cells were transferred to a fresh tube and centrifuged at 200 x g for 30 min at 4 C. The trophoblast cell pellet was washed with 25 mL DMEM and centrifuged again. The supernatant was aspirated and discarded. The cells were then resuspended in DMEM, counted on a hemocytometer, and checked for cell viability by trypan blue exclusion. Viability of the cells was greater than 90%. Using immunohistochemical staining techniques, approximately 95% of the cells stained for cytokeratin (a trophoblast marker). The cells were plated onto 24-well culture plates in DMEM, 10% FCS (1 x 106 cells/mL) and allowed to adhere for 24 h in a humidified incubator gassed with 95% air-5% CO2.
Experimental protocol
After 24 h of preincubation, the medium was removed, and cells were treated for 24 h with serum-free DMEM conditioned with 80 UI/mL, BSA 0.1%, with or without test substances. The HO antagonist was added 30 min before hemin. At the end of experiments, incubation media were removed, centrifuged per 5 min at 1,000 rpm, and the supernatants collected and stored at -35 C until assayed for CRH and PGE2 immunoreactivities. In experiments for the measurement of intracellular CRH content, incubation media were removed and treated as described above, and cells were resuspended in 1 mL Tris 50 mmol/L and sonicated for 30 sec with a Labsonic sonicator (B. Braun Biotech International, Melsungen, Germany). The samples were then centrifuged at 20,000 rpm per 25 min., and the supernatants were collected and assayed for CRH immunoreactivity.
Analytical methods
CRH assay. CRH was measured by RIA as previously described (8), with modifications. A CRH antiserum, kindly provided by Prof. Renato Bernardini, and (2-[125I]-iodohystidyl32)-CRH (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) were used. The lower detection limit of the assay was 1 pg/tube (200 µL sample vol), with intra- and interassay coefficients of variation of 5% and 10%, respectively.
PGE2 assay. The method has been described in detail elsewhere (18). The detection limit of the assay was 2 pg/tube (50 µL sample vol), with an EC50 of 28 pg/tube.
Drugs
Hemin HCl, cobalt chloride (CoCl2), biliverdin (BV), bilirubin (BR), and indomethacin (INDO), as free bases, were purchased from Sigma-Aldrich Corp. Ltd. (Milan, Italy). Tin-mesoporphyrin 9 (SnMP9) was obtained from Affiniti Research Products Ltd. (Mamhead Castle, Mamhead, Exeter, UK).
Hemin, BR, and SnMP9 were dissolved in 0.1 mol/L NaOH; BV and INDO were dissolved in absolute methanol and ethanol, respectively. All substances were further diluted in incubation media to obtain working solutions. The latter had a final pH of 7.4. All drugs tested were found to produce no shift in the standard curves of the assays.
Statistical analysis
All results are means ± SEM. Data were analyzed by one-way ANOVA or two-way ANOVA (where appropriate) and post hoc Newman-Keul test for multiple comparisons among group means, using a Prism computer program (GraphPad Software, Inc., San Diego, CA). Differences were considered statistically significant if P was less than 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Under these conditions, hemin induced a concentration-dependent
increase in CRH release; in spite of large interexperimental variations
(Table 1
), the highest concentration
of hemin used, 10 µmol/L, was always able to induce statistically
significant increases (Table 1; Figs. 1, 2B, and 5B), whereas increases induced by 0.1 µmol/L hemin attained
statistical significance in most, but not all of the experiments (Table 1, Fig. 2A). To ascertain whether such
increased release reflected an increase in CRH biosynthesis, in
subsequent experiments we carried out the simultaneous measurement of
intracellular and released CRH levels, under basal conditions and after
the addition of 10 µmol/L hemin. The latter did not modify
intracellular CRH content, compared with controls, in spite of
increased CRH secretion, suggesting that the porphyrin acts primarily
via the modulation of CRH exocytosis (Fig. 1).
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, A and B, respectively). However, the stable
HO end-products, BV and BR, failed to mimic the effect of hemin (data
not shown), thus suggesting that CO mediates the effect of hemin on CRH
release in this experimental paradigm.
We have previously shown that hemin is able to increase COX activity
through a HO-dependent mechanism (18, 19). Here, we have
investigated whether the COX pathway is also involved in the cascade of
events linking HO to the release of CRH, because PGs E2 and F2
have
been reported to stimulate CRH release from placental cell cultures
(22). Indeed, hemin elicited a concentration-dependent
increase in PGE2 release from placental cells in our model, which
was statistically significant at 10 µmol/L (Fig. 3A). This effect of hemin, however, did
not seem to depend on its degradation by HO, because it was not
counteracted by HO inhibition with SnMP9 (Fig. 3B). Nevertheless, the
possibility of a dual mechanism of action for hemin, depending on both
COX and HO activation, could not be completely ruled out. To clarify
this point, we used a pure inducer of the inducible HO isoform
CoCl2 (23). The latter also produced
significant increases in CRH secretion (Fig. 4A), but it had no stimulatory effect
and, in fact, inhibited PGE2 release at the higher concentration tested
(Fig. 4B). Further evidence against the involvement of COX came from
experiments carried out in the presence of a nonselective COX
inhibitor, INDO. Although the latter was able to abolish both basal and
hemin-stimulated PGE2 release (Fig. 5A),
it did not counteract, and even tended to potentiate, the effect of
hemin on CRH secretion (Fig. 5B). Moreover, INDO showed an intrinsic
stimulatory activity, suggesting that the overall effect of prostanoids
in this paradigm is inhibitory in nature. Indeed, 24-h incubations in
the presence of either PGE2 or PGF2, both given at 1 µg/mL, were
associated with reduced CRH release; such reduction was statistically
significant with PGF2 treatments (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, modulation of HO activity was obtained using
substrates and inducers, as well as specific inhibitors of the enzyme;
these treatments were consistently associated with significant changes
in CRH release, suggesting that a functional link exists between the
HO-CO system and placental CRH production. Increased HO activity was
achieved by supplementing the incubation media with the physiological
HO substrate, hemin, which normally is not detectable in tissues in
free form (14). Although the above mechanism mostly
accounts for increased HO activity in short-term experiments, hemin
also strongly induces HO-1 expression (24); therefore, in
prolonged incubation experiments (such as those carried out in this
study), hemin can increase HO activity via a dual mechanism,
i.e. acting both as a substrate and an inducer of the
enzyme. Besides, free hemin can be incorporated as a prosthetic group
into various hemoproteins; if these are enzymes in nature, as in the
case of COX (25), their activity will be enhanced
accordingly. This mechanism most probably accounts for HO-independent
activation of COX elicited by hemin in the present study. Thus, though
hemin is widely used as a CO-donor, its lack of specificity may
confound the interpretation of experimental results. Provided that
incubation experiments last long enough to allow gene expression and
new protein synthesis, CoCl2 can also be used to
induce HO-1 (23). This salt possesses a simpler mechanism
of action, compared with hemin: it is not a HO substrate, nor does it
act as a prosthetic group. In the present study, this tool was very
useful to clarify that HO activation is able per se to
stimulate CRH secretion, in a manner independent of prostanoid
production. The latter concept was further reinforced by the results of
INDO experiments, showing that the inhibition of COX, regardless of the
COX isoform involved, does not antagonize the effect of hemin on CRH
release. Surprisingly, we found that the marked inhibition of PG
synthesis caused by INDO was associated to increased CRH release; these
findings are in contrast with previous observations by Petraglia and
colleagues (22) showing that both PGE2 and PGF2
stimulate CRH secretion from placenta in 3-h incubation experiments. In
all probability, the reason for such apparent discrepancy lies on the
different length of experiments. Indeed, in the present study, the same
prostanoids were able to reduce CRH release, and PGF2 in a
significant manner, if incubation was prolonged to 24 h. In any
case, regardless of whether prostanoids stimulate or inhibit CRH
secretion from human trophoblast cells, it can be reasonably excluded
that they play any role in mediating stimulation of CRH release induced
by the HO-CO system.
Porphyrins with transition metals substituting iron at the center of the heme ring are able to inhibit HO, because they compete with heme for specific binding sites, causing a block, or at least a strong interference, in enzyme activity (26). At present, preferred inhibitors are the tin compounds, because of their specificity and selectivity of action (27). In this study, SnMP9 was able to completely antagonize the stimulatory action of hemin on CRH release; the porphyrin had no effect per se on basal CRH secretion, suggesting that, if endogenous basal CO production occurs in this paradigm, it does not seem to significantly influence peptide release.
CO shares with NO the ability to activate soluble guanylyl cyclase (sGC), resulting in increased cyclic guanosine monophosphate production (14). However, activation of this pathway by NO was clearly associated with the inhibition of CRH release in human trophoblast cells (10, 28, 29). Under certain conditions, NO and CO may elicit opposite biological responses; this was shown by us in an in vitro paradigm involving acute incubations of rat hypothalamic explants (19). In that study, the ability of CO to reduce IL-1ß release from the hypothalamus was associated with the activation of COX and could be counteracted by blocking PG production. This does not seem to be the case in the present paradigm; in fact, we have clearly shown that the increase in PG production induced by hemin does not correlate with the effect of porphyrin on CRH release. The apparent discrepancies between the effects of NO and CO might reflect differences in experimental conditions. In fact, previous studies, looking at the effect of NO on trophoblasts, involved relatively short (3 h) incubation times; similar to CO, NO was able to modulate CRH exocytosis, with no apparent change in the rate of synthesis (10). On the contrary, in the present study, we adopted longer incubation times, because the sensitivity of our assay did not allow us to consistently measure the amounts of CRH released after 3 h of incubation. It is conceivable that, given such prolonged incubations, a number of mechanisms might have been secondarily activated that eventually result in increased CRH release; for example, CO might accumulate into the system causing NO synthase inhibition (14), which fact would, in turn, relieve the inhibitory action of NO on CRH release. In addition, the possibility should be taken into account that CO may act independently of sGC, as well as any other hemoprotein. In fact, Otterbein and colleagues (30) have recently demonstrated that CO exerts potent antiinflammatory activities through a still-poorly-understood mechanism involving the mitogen-activated protein kinase pathway (30).
The present finding of a functional link between placental HO-CO and
CRH opens interesting new avenues in the interpretation of certain
pathophysiological conditions in obstetrics. Constant levels of CO are
generated by constitutive HO activity during pregnancy, but the total
amount of gas produced within the placenta might increase with time,
because HO-1 is exquisitely sensitive to factors that normally increase
under this condition, namely human chorionic gonadotrophin and
progesterone (31, 32); indeed, at least one group has
reported that HO-1 expression and protein synthesis increase steadily
during pregnancy (17). Thus, based on the present
observations, placental CO may be among the factors which, under
physiological conditions, regulate the normal increase in placental CRH
production during pregnancy. Apart from mere physiological
considerations, the recent findings that the lack of HO-1 gene
expression is associated, both in humans and mice (33, 34), with an increased inflammatory state, has led to the notion
that endogenous CO exerts protective actions against oxidative
stress-mediated disorders. Seemingly, various mechanisms are involved
in this phenomenon; Otterbein and colleagues (30) have
recently shown that CO down-regulates endotoxin-induced production of
proinflammatory cytokines [including IL-1ß and tumor necrosis factor
(TNF)] from activated macrophages. Similarly, the activation of
HO-1 induced by hemin significantly protects against the damage of
villous placental explants caused by TNF (17). In our
opinion, most important in CO-driven defense mechanisms is the action
of the gas on vascular beds. CO has general vasodilating properties,
mimicking those of NO, which are mostly mediated by an
endothelium-dependent mechanism (35). Hypoxia potently
stimulates HO-1 expression (14); CO produced by vascular
smooth muscle cells under hypoxic conditions was found to down-regulate
the expression of endothelin-1 from endothelial cells in
vitro (36), suggesting for CO both direct and
indirect relaxing mechanisms; this feature of the gas may become
crucial in abnormal pregnancies associated with increased local
vascular resistance, such as pregnancy-induced hypertension and
intrauterine growth retardation. Interestingly, the latter conditions
are also consistently associated with significantly higher CRH levels
(2, 3). Such elevated amounts of circulating CRH may
access the myometrium, where (apart from influencing contractility and
regulating the timing of parturition) they cause vasodilatation
mediated by the NO-sGC pathway (6). CRH messenger RNA and
receptor have been also found expressed in human umbilical vein
endothelial cells (37). Thus, the emerging picture
describes a complex vasorelaxing action for CO in the placental
circulation, both direct and mediated by increased CRH production,
which, in turn, activates the NO pathway. The above model would seem
redundant if we consider that both CO and NO induce vasorelaxation by
stimulating endothelial cyclic guanosine monophosphate production, and
NO is a more efficient sGC activator, compared with CO
(38). However, there is evidence that nitrergic
vasorelaxing tone is impaired under the same hypoxic conditions
(39, 40) that, incidentally, stimulate CO production. In
these cases, CO might take over as the primary vasorelaxing factor in
the fetal-placental circulation.
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Footnotes |
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Received April 28, 2000.
Revised July 25, 2000.
Revised August 29, 2000.
Accepted September 8, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induced cytotoxicity and promotes vessel
relaxation (Abstract). J Soc Gynecol Invest. 7(Suppl 1):388.
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