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Pregnancy |
a Departments of Anesthesiology, College of Physicians & Surgeons, Columbia University, New York, New York 10032
b Universitätsklinikum Münster, 48129 Münster, Germany
c The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF) have been implicated in amniotic fluid infections and preterm and term labor. The underlying mechanisms are incompletely understood. In some smooth muscle cells, TNF affects function of the ß-adrenergic/adenylyl cyclase pathway. The present study was performed to examine the effects of chronic TNF exposure on adenylyl cyclase activity in cell cultures of human myometrium. Chronic TNF exposure led to a dose- and time-dependent increase in basal-, GTP-, NaF-, and forskolin-stimulated adenylyl cyclase (AC) activity. The increase in AC activity was not mediated by changes in the expression of the heterotrimeric G proteins Gs or Gi as determined by immunoblotting. In addition, increases in AC activity occurred in the presence of indomethacin, indicating that these changes were not provoked by TNF-induced changes in prostaglandin production. The present results suggest that TNF-induced increases in AC activity in human myometrial cells obtained from the lower uterine segment occur at the level of G-protein/AC interaction or at the level of the AC enzyme itself.
cyclic adenosine monophosphate, cytokines, female reproductive tract, immunology, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF) [2]. Interleukin-1 and interleukin-8 levels increase exponentially with gestational age in cervicovaginal fluids of nonlaboring women [3], and healthy women in preterm and term labor show elevated cytokine levels [4, 5]. It was therefore suggested that the release of inflammatory cytokines such as interleukin-1, interleukin-6, and TNF play a role in the regulation of parturition. Cytokines are involved in cervical ripening via an induction of matrix metalloproteinases [6]. They induce cyclooxygenase-2 (COX-2), with subsequent increases in the uterotonic prostaglandins PGF2 and PGE2 [7], whereas subcutaneous injection of interleukin-1 causes preterm delivery in mice [8].
Mechanisms for cytokine effects on uterine activity are not entirely understood. Indirect effects, especially those mediated through amniotic or myometrial prostaglandin production, are well described, but direct effects of cytokines on the myometrium are not well understood [9]. The myometrial ß-adrenergic/adenylyl cyclase pathway is one potential site for direct effects of cytokines. In other cell types, TNF increases adenylyl cyclase (AC) activity [10, 11] or enhances carbachol-mediated inhibition of AC [12]. Increases or decreases in AC activity would either decrease or increase myometrial tone by altering the accumulation of intracellular cAMP. In addition, acute exposure of myometrial strips to TNF does not alter uterine contractility [13]. We therefore questioned whether chronic TNF exposure of previously characterized human myometrial cells [14] affects AC activity and whether the observed effects result from prostaglandin production or changes in expression of the heterotrimeric G-proteins Gs or Gi.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell cultures of human myometrium were established from enzymatically dispersed biopsies taken at cesarean section after Institutional Review Board approval as previously described [14]. The cells were grown in high-glucose (4.5 g/L) Dulbecco modified Eagle medium and remained viable over several passages. The medium was supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. Cells were kept in a humidified atmosphere of 5% CO2 and 95% air at 37°C; the medium was replaced every 2–3 days. Passages 4–9 were used for experiments. The smooth muscle phenotype of the cultured cells was previously characterized [14].
TNF Treatment
After achieving confluence, cell culture flasks of identical passage were treated simultaneously with human recombinant TNF (10 ng/ml) for 12, 24, 48, or 72 h (n = 9 experiments) to establish a time-response relationship. Each experiment utilized cells obtained from a new set of flasks. All flasks were extensively washed and kept in serum-free medium for 72 h while TNF was added at the indicated times. In subsequent experiments, a dose-response relationship was established by exposing cells to 0.01, 0.1, 1.0, 10, or 100 ng/ml TNF for 48 h (n = 10 experiments). As TNF may exert some of its effects through increased prostaglandin production, we next sought to inhibit prostaglandin production by the addition of the cyclooxygenase inhibitor indomethacin in a further series of experiments (n = 8 experiments). In these experiments, cells were simultaneously incubated with the vehicle (200 µM sodium carbonate) or indomethacin (10-5 M) with or without TNF 10 ng/ml. Indomethacin was dissolved in Na2CO3 as described by Hata et al. [15]. Preliminary results showed that Na2CO3 alone had no effect on AC activity, whereas the alternative vehicles dimethyl sulfoxide and methanol confounded results of the assay by increasing AC activity.
Cellular Lysate Preparation
After pretreatment with TNF, cells were incubated in lysis buffer (10 mM Hepes, 2 mM EDTA, 100 µM phenylmethylsulfonyl fluoride, pH 8.0) for 30 min at 37°C and scraped off the flasks. Cellular lysates were prepared by homogenization with a motor-driven Teflon pestle (Dupont, Wilmington, DE) at 4°C using 30 strokes. Homogenates were centrifuged at 48 000 x g for 30 min at 4°C, and the pellet was resuspended in lysis buffer. Protein concentrations were determined with bicinchoninic acid, using bovine serum albumin as a standard [16].
AC Assay
The activity of AC was determined by measuring the conversion of [-32P]ATP to [32P]cAMP according to the method of Salomon et al. [17]. In TNF time-response experiments, AC activity was measured under basal conditions and after stimulation with GTP (10-5 M), isoproterenol (10-4 M) in the presence of GTP (10-5 M), NaF/AlCl3 (10 mM/100 µM), and forskolin (10-5 M). In brief, basal and stimulated AC activity was measured in triplicate samples at 30°C by adding 10 µl of cellular lysates to assay buffer in the presence of the respective effectors (total volume, 100 µl), resulting in a final assay buffer concentration of 0.5 mM 3-isobutyl-1-methylxanthine, 50 mM Hepes, 50 mM NaCl, 0.4 mM EGTA, 1 mM cAMP; 7 mM MgCl2, 0.1 mM ATP, 7 mM creatine phosphate, 50 units/ml creatine phosphokinase, 0.1 mg/ml BSA, 10 µCi/ml [-32P]ATP (specific activity, 800 Ci/mmol) (pH 8.0). The reaction was terminated after 20 min with the addition of 100 µl of stop buffer (50 mM Hepes, pH 7.5, 2% sodium dodecyl sulfate, 2 mM ATP, 0.5 mM cAMP, and 1 µCi/ml [3H]cAMP [specific activity, 25 Ci/mmol]). In addition, the samples were boiled for 3 min. The synthesis of [32P]cAMP was determined by sequential column chromatography over Dowex (Bio-Rad, Hercules, CA) and alumina [17]. Recovery rates of columns ranged from 75% to 95%. AC activity was corrected for protein concentration to exclude effects of TNF on cell proliferation. AC activity was obtained as picomoles of cAMP per milligram protein per 20 min.
Immunoblot Analysis
Immunoblot analysis was performed to determine if chronic TNF exposure induces changes in G protein expression of Gs or Gi. Cells grown in 75-cm2 flasks were serum starved for 24 h and subsequently pretreated with TNF (10 ng/ml) for 48 h in serum-free medium. Flasks were extensively washed and cells subjected to lysis buffer for 30 min at 37°C (10 mM Hepes, 2 mM EDTA, 100 µM phenylmethylsulfonyl fluoride, pH 8.0). Cellular lysates were prepared as described above and dissolved in sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 5% ß-mercaptoethanol). Twenty micrograms of protein from each sample was loaded onto 10% polyacrylamide gels and electrophoresed at room temperature at 80 mV in a tank buffer containing 25 mM Tris, 0.1% SDS, and 192 mM glycine. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes overnight at a constant voltage of 20 mV in transfer buffer (192 mM glycine, 25 mM Tris, 10% methanol). The PVDF membranes were washed twice in Tris-buffered saline (TBS; 20 mM Tris, 500 mM NaCl, pH 7.5) and incubated in TBS-T (TBS plus 0.1% Tween-20) containing 5% nonfat dry milk for 90 min at room temperature to block nonspecific binding. The PVDF membranes were incubated in primary antibodies against Gs or Gi-2 (dilutions 1:500 in TBS-T with 1% nonfat dry milk and 0.02% sodium azide) for 4–6 h at room temperature with gentle rocking. After two washes for 15 min with TBS-T, PVDF membranes were incubated with secondary donkey anti-rabbit IgG conjugated to horseradish peroxidase for 60 min at room temperature (dilution 1:3000 in TBS-T plus 1% nonfat dry milk) and again washed twice in TBS-T. Bound secondary antibody was detected using an enhanced chemiluminescence detection kit according to the manufacturer's protocol (ECL plus; Amersham-Pharmacia Biotech, Piscataway, NJ) and subsequent exposure to autoradiography film. Immunoblot intensities were quantified with Mac Bas 2.2 software (Fuji Medical Systems, Stamford, CT) after scanning the exposed films into a personal computer.
Materials
Cell culture reagents were obtained from GIBCO BRL (Grand Island, NY). The BCA protein assay reagent was obtained from Pierce Chemical (Rockford, IL). [-32P]ATP, [3H]cAMP, Gs, and Gi-2 primary antibodies were obtained from New England Nuclear (Boston, MA). The secondary antibody was purchased from Amersham Pharmacia Biotech. The PVDF membranes were from Bio-Rad. All other chemicals were obtained from Sigma (St. Louis, MO).
Statistics
Statistical analysis was performed using repeated measures of ANOVA, followed by Bonferroni posttest comparison using Prism 3.0 software (GraphPad, San Diego, CA). Immunoblots were analyzed with a two-tailed paired Student t-test. Data are presented as mean ± SEM; P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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AC activity was 54 ± 10 pmol cAMP (mg protein)-1 (20 min)-1 at baseline and increased significantly during stimulation with GTP (10-5 M), NaF (10 mM), and forskolin (10-5 M) (P < 0.05), indicating a functional pathway for adenylyl cyclase activation in these cultured human myometrial cells. The resulting AC activities in the presence of these agonists were 93 ± 24, 639 ± 155, and 828 ± 176 pmol cAMP (mg protein)-1 (20 min)-1, respectively. Stimulation of AC activity with isoproterenol in the presence of GTP did not lead to an increase over AC activity in the presence of GTP alone (94 ± 23 versus 93 ± 24 pmol cAMP (mg protein)-1 (20 min)-1) (P > 0.05).
Time-Response Relationship
Incubation of human myometrial cells with TNF over time periods of 12, 24, 48, and 72 h resulted in significant increases in basal and agonist-stimulated AC activities after 48 and 72 h, whereas TNF exposure times of 12 and 24 h did not increase activity. In cells treated with TNF (10 ng/ml), basal AC activity increased 2-fold after 48 h (P < 0.001; 9 experiments), which was not further increased after longer incubation periods. The GTP-stimulated AC activity was nearly tripled after 48 and 72 h of TNF exposure (P < 0.01). Similar increases in AC activity were observed in cells stimulated with NaF or forskolin in the presence of TNF (P < 0.05; Fig. 1).
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Dose-Response Relationship
Myometrial cells were pretreated with TNF in doses ranging from 0.01 to 100 ng/ml for 48 h to establish a dose-response relationship. In these experiments, basal and agonist-stimulated AC activities increased significantly with TNF doses as low as 1 ng/ml, whereas lower doses of TNF did not increase AC activity. Basal AC activity was increased twofold in the presence of 1 ng/ml TNF without further increases with higher doses of TNF (P < 0.05 versus controls; n = 10 experiments). The GTP-stimulated AC activity was 1.8-, 2.1-, and 2.2-fold with doses of 1, 10, and 100 ng/ml TNF, respectively (P < 0.05 versus controls). In these experiments, forskolin-stimulated AC activity increased by 39%, 55%, and 56% at the indicated doses of TNF (P < 0.05 versus controls; Fig. 2).
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Coincubation with Indomethacin
Human myometrial cell cultures were coincubated with TNF (10 ng/ml) and indomethacin (10-5 M) for 48 h to evaluate whether the effects of TNF on AC were dependent on TNF-induced prostaglandin production. The vehicle Na2CO3 (200 µM) or indomethacin alone did not significantly increase AC activity, yielding comparably low basal activities in controls, controls treated with vehicle, and controls treated with indomethacin (P > 0.05; n = 8 experiments). The TNF-induced increases in basal AC activity were not attenuated by coincubation with indomethacin. Basal AC activities were increased 2-fold in the absence of indomethacin and 1.8-fold in the presence of indomethacin (P < 0.05 compared with controls). In addition, pretreatment with indomethacin did not inhibit TNF induced increases in GTP- and forskolin-stimulated AC activity (P < 0.05 versus controls in the absence and presence of indomethacin; Fig. 3).
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Immunoblot Analysis
Immunoblot analysis was performed to investigate whether changes in adenylyl cyclase activity induced by TNF were due to changes in protein expression of heterotrimeric G protein -subunits. Pretreatment of cells with 10 ng/ml TNF for 48 h did not significantly alter the expression of Gi-2 or Gs (Fig. 4). Gi-2 band intensities were 113.1 ± 16.8% of controls (P = 0.83, n = 8 experiments), and Gs band intensities were 96.6 ± 16.5 % of controls (P = 0.42).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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increased AC activity in cultured cells obtained from term myometrium in a dose- and time-dependent manner (Figs. 1 and 2). Enhanced AC activity was observed under basal conditions as well as in the presence of GTP, NaF, and forskolin, indicating that TNF-induced changes occurred either at the level of the AC enzyme or at the level of AC/G-protein interaction.
The TNF-induced increases in basal and agonist-stimulated AC activity occurred with concentrations as low as 1.0 ng/ml. These concentrations are similar to those measured in women with chorioamnionitis [18] and suggest that our observations may be clinically important. Using myometrial cell cultures obtained at hysterectomy, Hertelendy et al. [9] demonstrated an increased cAMP production after chronic TNF or interleukin-1 exposure. This effect, however, required much higher concentrations of TNF and may indicate that cells derived from myometrium at term are more susceptible to the stimulatory effects of cytokines than those derived from nonpregnant uterus.
Although the myometrial cells used in this study were obtained at term, steroid hormones were not added to the culture media to imitate the hormonal milieu of pregnancy. Steroid hormones control uterine responsiveness to stimulation through several mechanisms. They modulate G-protein coupling to their effector targets, increase cAMP activity, and inhibit cAMP degradation by phosphodiesterases [19]. Whereas progesterone enhances cAMP accumulation after ß-adrenergic stimulation [20], estrogen abolishes ß-adrenergic cAMP accumulation in rabbit uterus and decreases Gs [21]. In pregnancy, maximal AC activity increases selectively after stimulation with Gpp(NH)p and fluoride, whereas basal-, forskolin-, and manganese-stimulated AC activity remains unchanged [22]. This suggests an increase in pregnancy-related Gs coupling to the AC enzyme and indicates that cells used in the present study resembled myometrium at term. Estradiol and progesterone modify second messenger generation in myometrial cells in vitro by increasing the accumulation of cAMP and inositol 1,4,5-triphosphate [23]. In porcine myometrial cells obtained from ovarectomized nonpregnant animals, Kisielewska et al. [23] showed that these cells only grew confluent and displayed a typical hillock pattern if animals and cell cultures were treated with steroid hormones. In contrast, cells in the present study were obtained from pregnant women at term and, although the cells were not grown in an environment supplemented with steroid hormones, they displayed a pattern characteristic of myometrial cells as described. Although the omission of steroid hormones in the culture media may have influenced the present results, these should have been influenced in the same direction in both control and TNF-treated cells. In addition to hormonal changes, cells in culture may undergo phenotypic changes that may resemble cells of midpregnancy or the nonpregnant state. In ovine myometrial cell cultures, Vallet-Strouve et al. [19] observed an increase in phosphodiesterase activity and diminished cAMP accumulation with increasing number of cell passages. This effect became apparent after several passages, and the use of cells from passages greater than 10 were avoided in the present study.
As increases in AC activity and cAMP production are the principal mechanisms of smooth muscle relaxation and uterine quiescence during pregnancy, the results of the present study appear at first to contradict the current concept of an involvement of proinflammatory cytokines in preterm and term labor [3–5]. However, sensitivity to TNF may differ depending on the origin of myometrial cells, e.g., fundus versus lower uterine segment or cervix. Myometrial tissues from the lower uterine segment, the origin of the myometrial cell cultures used in the present study, may be more susceptible to relaxation to facilitate the passage of the neonate, whereas expulsive forces mainly arise from the fundus. This is evidenced by nonhomogenous receptor distribution for oxytocin [24] and PGE2 [25], with lower receptor concentrations in the cervix versus the fundus. Similar to the results obtained with TNF, IL-1, which is released during preterm premature rupture of membranes, enhances NO production in myometrial cells obtained from the lower uterine segment at cesarean section [26]. An increase in NO production in the presence of IL-1 would promote relaxation of the lower uterine segment.
Effects of TNF on myometrial AC activity required greater than 24 h of exposure (Fig. 1) and confirmed previous data that demonstrated that prolonged exposure was required for effects of TNF on cAMP accumulation in nonpregnant myometrial cells [9]. The authors reported a maximum response after 18 h. Effects of TNF on AC activity with a maximal response after 48 h suggest de novo protein synthesis as a potential underlying mechanism. In support of this hypothesis, TNF is a potent stimulator of arachidonic acid release [27] and induces a dose-dependent increase in COX-2 expression in amnion cells with a sustained increase in PGE2 levels that is maintained for 48 h [7]. While PGF2 is a potent contractile agonist, the effects of PGE2 on uterine contractility are diverse. PGE2 exerts its effects through activation of prostaglandin EP1–4 receptors; these receptors couple through heterotrimeric G-proteins and generate contractile as well as relaxant responses, depending on the respective receptor distribution [28].
Although TNF is capable of stimulating prostaglandin synthesis, this did not appear to be the mechanism for the observed effects of TNF in the present study. Changes in AC activity were independent of prostaglandin production, as indirectly shown by the inability of indomethacin to prevent changes in AC activity caused by TNF treatment (Fig. 3). Although we did not measure prostaglandin levels in response to indomethacin directly, the dose and time-course of indomethacin used in the present study has previously been demonstrated to completely inhibit prostaglandin production in the presence of TNF in several cell types [7, 11], and lower doses of indomethacin (10-8–10-6 M) prevent prostaglandin production in cell cultures of rabbit colonic epithelium [15] and human amnion [29]. Indomethacin inhibits lipopolysaccharide-induced preterm labor in rats in a dose-dependent fashion and abolishes uterine and ovarian prostaglandin production [30]. In human myometrial cell cultures, IL-1-induced increases in cAMP are maintained in the presence of cycloheximide, which completely inhibited PGE2 accumulation, further suggesting that prostaglandins are not involved in cytokine-induced activation of the AC enzyme [9]. Their results in the presence of cycloheximide also demonstrate that stimulation of cAMP production by IL-1 occurs independent of de novo protein synthesis. In support of this hypothesis, Pascual et al. [31] recently demonstrated that IL-1ß and TNF induce AC sensitization independently of COX-2 and prostaglandin induction in airway smooth muscle cells, whereas inhibition of cell growth and desensitization of G protein-coupled receptors required the induction of COX-2 and was inhibited in the presence of indomethacin. Moreover, chronic IL-1ß-induced AC sensitization required new protein synthesis [32]. Sensitization of AC isoform VI activity in cultured human airway smooth muscle cells has also been demonstrated with chronic exposure to rhinovirus or agonists that classically couple through Gi proteins, effects that were blocked by inhibition of protein kinase C or pertussis toxin, respectively [32, 33]. Thus, the differential effects of TNF on AC sensitization are mediated by distinct pathways and raise the possibility that different isoforms of AC become sensitized via distinct signaling pathways.
The major site of action of TNF in the present study was not the ß-adrenergic receptor. Although isoproterenol stimulated AC activity in the presence of GTP, the resultant increase did not surmount AC activity in the presence of GTP alone. This low responsiveness of AC to isoproterenol may indicate desensitization and uncoupling of the ß-adrenergic receptor at term [34] and has also been observed in human myometrial tissue at term [35] and in rabbit [36] and rat myometrial cells [37]. It was recently demonstrated that ß-adrenergic receptors, G proteins, and AC are colocalized in caveolar microdomains of the membrane and that this colocalization enhances coupling efficiency [38]. It is unlikely that disruption of these caveoli during membrane preparation and homogenization have resulted in the loss of responses to stimulation with isoproterenol in the present study, as preliminary experiments in whole cells showed no isoproterenol-induced increase in AC activity compared with baseline values (data not shown). However, Litime et al. [35] demonstrated the presence of isoproterenol-induced AC activity at midpregnancy that was absent in myometrial tissues obtained at term, which would provide another likely explanation for the observed lack of response to isoproterenol in myometrial cells obtained at term. In that study, the response to isoproterenol could be restored by inhibition of Gi proteins, suggesting an underlying inhibition of the ß-adrenergic stimulatory pathway at term. In other cell types, inhibition of Gi proteins does not enhance ß-adrenergic stimulation of cAMP generation [38]. Another possible explanation for the lack of effect of isoproterenol to stimulate AC activity is the low density of ß-adrenergic receptors in human myometrium. Gsell et al. [39] reported a density of 5.8 and 6.2 fmol/mg in nonpregnant and pregnant myometrium, respectively, and were also unable to demonstrate any effects of isoproterenol on AC activity. In addition, these authors suggested that increases in AC activity during pregnancy are due to alterations in the AC and not due to enhanced Gs-protein expression. Our results confirm these previous results and indicate that subculturing myometrial cells over several passages did not change their physiological properties in the absence of hormone supplementation compared with fresh myometrial tissue.
TNF may affect AC activity by acting on heterotrimeric G proteins since both GTP- and fluoride-induced AC activities were enhanced by TNF exposure. TNF alters AC activity by influencing the expression of heterotrimeric G proteins in airway smooth muscle cells [12]. Effects of TNF in myometrial cells appear to differ, as neither increased expression of Gs nor decreased expression of Gi could account for increases in AC activity (Fig. 4). This is further supported by the observation that IL-1 induces changes in cAMP production in the presence of cycloheximide [9]. Regulation of AC activity independent of de novo protein synthesis was also observed in rat uterine cells treated with estrogen [40]. Thus, if TNF affected heterotrimeric G proteins in this study, a more likely mechanism would be an alteration of G protein function or Gs/adenylyl cyclase interaction. Although TNF may act through a decrease in Gi-mediated AC inhibition, pertussis toxin only minimally inhibited AC sensitization in airway smooth muscle cells [33] and had no effect on IL-1-induced cAMP accumulation in uterine smooth muscle cells [9].
The ability of TNF to potentiate basal and forskolin-stimulated adenylyl cyclase activity suggests that one site of action may be the AC enzyme. Improved function, increased total quantity, or changes in expression of AC isoforms might occur. At least nine isoforms of membrane-bound mammalian AC have been identified, of which most are expressed in human myometrium [41, 42]. Changes in relative quantities of myometrial adenylyl cyclase isoforms are possible. During pregnancy, a relative shift in AC isoform levels toward a dominance of those regulated by G protein ß
subunits and PKC has been observed [42]. It is currently unknown whether TNF is capable of inducing changes in AC isoform expression, but this would provide one mechanism that might alter total adenylyl cyclase activity. For example, TNF might stimulate synthesis of isoforms other than type IX, which is not responsive to forskolin. However, other cytokines may not have similar effects, as cycloheximide did not abolish the effects of interleukin-1 on AC activity in myometrial cells [9].
In summary, this study demonstrates that chronic exposure to the inflammatory cytokine TNF results in increased adenylyl cyclase activity in human myometrial cells obtained from the lower uterine segment. Increased cAMP synthesis likely results from increased enzyme activity at the level of the adenylyl cyclase enzyme itself or from enhanced Gs protein-adenylyl cyclase coupling. The results of this study are consistent with the speculation that TNF effects at the lower uterine segment and cervix are mediated by increases in AC activity, favoring relaxation and thus facilitating fetal passage.
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FOOTNOTES |
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2 Correspondence: Charles W. Emala, Department of Anesthesiology, College of Physicians & Surgeons of Columbia University, P & S Box 46, 630 West 168th Street, New York, NY 10032. FAX: 212 305 8287; e-mail: cwe5{at}columbia.edu
Received: 11 February 2002.
First decision: 6 March 2002.
Accepted: 3 September 2002.
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