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Physiologic role of nitric oxide in the maintenance of uterine quiescence in nonpregnant and pregnant sheep
Physiologic role of nitric oxide in the maintenance of uterine quiescence in nonpregnant and pregnant sheep Charles P. Mirabile, Jr, MD, G. Angela Massmann, MD, and Jorge P. Figueroa, MD, PhD Winston-Salem, North Carolina OBJECTIVE: This study evaluated the role of nitric oxide in the maintenance of uterine quiescence in nonpregnant and pregnant ewes. STUDY DESIGN: Sixteen ovariectomized nonpregnant and 10 pregnant (115 days’ gestation) chronically instrumented ewes were studied. Uterine contractility was assessed by electromyography and intrauterine pressure recordings. Nitric oxyde synthase inhibition was induced with nitro-L-arginine methyl ester or aminoguanidine (4.5 mg/kg per hour) given during estrogen replacement with 17β-estradiol (100 µg/d) or in late gestation. In the pregnant group we evaluated the ability of nitric oxide synthase inhibition to alter the responsiveness to oxytocin-induced uterine contractility. Blood pressure and common internal iliac artery blood flow were assessed to confirm nitric oxide synthase inhibition. In addition, the effects of the nitric oxide donor nitroglycerin and the cyclooxygenase inhibitor indomethacin were studied in nonpregnant sheep. The effect of nitric oxide in vitro on myometrial spontaneous and induced contractions was also studied. RESULTS: In nonpregnant estrogen-replaced sheep, nitric oxide synthase inhibition and nitroglycerin administration did not alter uterine contractility, despite significant changes in blood pressure. In contrast, indomethacin decreased electromyographic results to 70% of baseline after 1 hour and 47% after 2 hours. In pregnant ewes nitric oxide synthase inhibition failed to alter uterine contractility in response to oxytocin. These findings are in contrast to results of the in vitro study in which nitric oxide was shown to relax sheep myometrium. CONCLUSION: The absence of significant effects of nitric oxide synthase inhibition and nitric oxide donors on uterine contractility in vivo suggests that nitric oxide does not play a physiologic role in the regulation of uterine contractility in nonpregnant or pregnant ewes. (Am J Obstet Gynecol 2000;183:191-8.)
Key words: Nitric oxide, uterine quiescence, sheep, pregnancy, oxytocin, indomethacin, nitro-L-arginine methyl ester, L-NAME
Nitric oxide synthase (NOS) is a monooxygenase flavoprotein that uses L-arginine as substrate to produce nitric oxide (NO), a highly reactive messenger molecule. Three distinct NOS isoforms have been isolated and identified, 2 constitutive (type I NOS, or nNOS, and type III NOS, or eNOS) and 1 inducible (type II NOS or iNOS). All 3 isoforms are present in uterine tissues during pregnancy in all species investigated to date.1 Regardless of the particular isoform that is more abundant in a given species and the site at which it is present (endometrium and decidua or myometrium), uterine NOS expression or activity increases during pregnancy, and a decrease in decidual NOS expression before parturition From the Section on Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Wake Forest University School of Medicine. Received for publication August 27, 1999; revised November 23, 1999; accepted December 31, 1999. Reprint requests: Jorge Figueroa, MD, PhD, Department of Obstetrics and Gynecology, Section on Maternal-Fetal Medicine, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1066. Copyright © 2000 by Mosby, Inc. 0002-9378/2000 $12.00 + 0 6/1/105428 doi:10.1067/mob.2000.105428
has been observed by most but not all investigators.1 Previous work from our laboratory has shown that NOS activity is found in endometrium and myometrium of pregnant and nonpregnant sheep.2, 3 Furthermore, estrogen and pregnancy regulate the expression of nNOS in myometrium and that of eNOS in the vasculature in this species.1, 3, 4 Several in vitro studies have demonstrated the ability of NO donors to decrease spontaneous and induced myometrial contraction in a cyclic guanosine monophosphate (cGMP)–dependent and a cGMP-independent manner.5, 6 In these studies the effect was a reduction in either the frequency or the amplitude of myometrial contractions. However, responsiveness seems to vary if the tissue was obtained before, during, or after labor and with regard to the chemical nature of the NO donor.1 Compared with the data available from in vitro studies, the reports on in vivo effects of NO are fewer and the effects are widely variable. Many case reports support the ability of NO donors to relax the uterus in pregnant women. These examples include resolution of uterine contraction rings, retained placenta, and uterine inversion. Lees et al7 have even suggested that an NO donor 191
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A B C D E Fig 1. Sequential steps of uterine EMG analysis in pregnant sheep. A, Raw data. B, Band-pass–filtered data. C, Full-wave rectification of B. D, 1-minute summaries of power spectral analysis of C. E, Intrauterine pressure recordings.
can be used for tocolysis in preterm labor. Such findings, however, have yet to be confirmed in prospective, randomized clinical trials. Reports are that relatively large doses of the NO donors can decrease uterine contractility in pregnant rats,8 monkeys,9 and sheep.10 The questions that remain are whether a physiologic role exists for endogenous NO production as a modulator of uterine contractility and whether the in vivo response of the uterus to exogenous NO can be further defined. Therefore this study sought to evaluate the role of NO in the maintenance of uterine quiescence in pregnant and nonpregnant ewes in vivo. Material and methods Animal preparation and postoperative care. All procedures for housing, handling, surgical implantation of catheters, and postoperative management were approved by Wake Forest University’s Institutional Animal Care and Use Committee. Sixteen chronically instrumented, ovariectomized nonpregnant ewes and 10 chronically instrumented pregnant ewes at 115 days’ gestation were studied. Animals were brought into the laboratory and placed in metabolism cages with free access to food and water. Twenty-four hours before surgery, food and water were withheld. Sheep (average weight, 50 kg) were operated on while they were under halothane general anesthesia (1.5%-2% in oxygen 2 L/min) after premedication with 1 g ketamine intramuscularly. The surgery, which included laparotomy and hysterotomy, was performed for placement of vascular and intrauterine catheters and for removing the ovaries in nonpregnant sheep. Catheters were placed into the femoral vein, both femoral arteries, carotid artery, and jugular vein in adult animals. A Transonics flow probe (6-8 mm) was placed around the common internal iliac artery in both pregnant and nonpregnant sheep. For a more accurate assessment of uterine blood flow, the ovar-
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ian, middle sacral, and cervical-vaginal arteries were ligated with sutures. Multistranded, bipolar stainless steel wires were implanted 3 to 5 mm apart on at least 2 sites of the myometrial surface for electromyography (EMG) recordings.11 In the pregnant animals additional catheters were placed into the maternal uterine vein, amniotic cavity, and the fetal carotid artery and jugular vein. Perioperative antibiotics were given on days 0 through 4 (80 mg gentamicin, 1 g ampicillin through the adult jugular vein, 20 mg of gentamicin infused through the fetal jugular vein along with 60 mg gentamicin, and 1 g ampicillin intra-amniotically). All animals were allowed 5 days of postoperative recovery before the initiation of experimentation. In experiments involving pregnant animals, fetal blood gas values were within the normal range established for this laboratory before, during, and after all studies. Uterine contractility. Uterine contractility was assessed by EMG and intrauterine pressure (IUP) recordings. The analog signal was low-pass filtered at 50 Hz, and data were collected at a rate of 68 Hz. The EMG data were bandpass filtered at frequencies between 1.8 and 20 Hz and were further processed with Fast Fourier Transform and Power Spectrum Analysis. Such analysis provided numeric values with which uterine activity could be quantified. For comparison, the magnitude of the EMG signal was normalized for baseline activity, because magnitude is determined by electrode placement and varied between animals. Nonpregnant sheep studies. All 16 sheep received a continuous intravenous infusion of 17β-estradiol (100 µg/d). Estrogen replacement was given to increase NOS expression and elicit a regular contractile pattern.2, 11 Ewes were allocated to 2 different regimens to determine whether the length of estrogen replacement would affect the response to NOS inhibition. Twelve sheep were used for studying the effects of NOS inhibition on uterine contractile activity. Six ewes were studied after 48 hours of estrogen replacement, and the remaining 6 were studied after 72 hours of estrogen replacement. All 12 sheep received a 2-hour continuous infusion of the NOS inhibitor nitro-L-arginine methyl ester (L-NAME) (total dose, 9.0 mg/kg) given into the descending aorta. In 6 of these 12 sheep, the cyclooxygenase inhibitor indomethacin was administered on separate days. These 6 animals received a 2-hour intravenous indomethacin infusion (1 mg/kg per hour) at either 72 or 96 hours of estrogen replacement. A separate group of 4 nonpregnant ewes were treated with the NO donor nitroglycerin. These animals received a 2-hour nitroglycerin infusion (10 µg/kg per minute) at either 48 or 72 hours of estrogen replacement. Pregnant sheep studies. Ten chronically instrumented, late-gestation pregnant ewes were studied. In this group we evaluated the ability of L-NAME (n = 4) or aminoguanidine (n = 6) to alter uterine contractility. Oxytocin
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Table I. NOS inhibition in estrogen-replaced nonpregnant ovariectomized sheep
Control hour Hour 1 Hour 2 Statistical significance
Uterine EMG (fold increase) (n = 12)
Arterial blood pressure (mm Hg) (n = 10)
1.28 ± 0.1 1.30 ± 0.2 P > .09
77.3 ± 3 81.4 ± 3 86.8 ± 3* P < .05
Uterine blood flow (mL · min–1) (n = 6) 117.6 ± 32 94.8 ± 25 97.4 ± 27 P > .8
L-NAME
was given as a continuous infusion over 2 hours (total dose, 9 mg/kg). Mean ± SEM. *Statistically significant difference.
was used to establish a steady contractile pattern for study and overcome the inherent low frequency and lack of pattern of contractures. Each animal received oxytocin on 2 separate occasions at least 2 days apart, being treated with either NOS inhibitor or vehicle. In 2 sheep oxytocin was infused into the descending aorta in a dose-response fashion (6 sequentially increasing doses from 0.05 up through 3.84 ng/kg per minute, each infused over a 1-hour period as a 5-minute pulse every 15 minutes) for a total of 6 hours. Eight sheep received a continuous infusion at a rate of 0.15 ng/kg per minute for a total of 5 hours. The NOS inhibitors were given at the same dose (4.5 mg/kg per hour); however, on a molar basis the dose of aminoguanidine is twice that of L-NAME. The total dose of NOS inhibitor ranged from 9 mg/kg (L-NAME and aminoguanidine) in the continuous oxytocin paradigm to 31.5 mg/kg (L-NAME) in the pulsatile administration paradigm, depending on the overall duration of NOS inhibitor administration. In 2 of the pregnant ewes, on a separate day (removed from an oxytocin infusion) nitroglycerin was administered to assess the effects of exogenous NO (2-hour nitroglycerin infusion at 10 µg/kg per minute). In vitro contractility. Strips of longitudinal muscle were prepared from sheep uteri during pregnancy (115 days’ gestation) and immediately post partum. Strips (1 × 10 mm) were mounted in 20-mL organ chambers (Radnoti) filled with modified Krebs-Henseleit solution warmed at 37°C, gassed with 95% oxygen to 5% carbon dioxide, and containing 10-5-mol/L indomethacin. Isometric tension was measured continuously with a force transducer and collected with an A/D board (Data Translation) at 20 Hz with Asyst 4.0. Strips were stretched to their optimum length-tension relationship by repeated stimulation with a 40-mmol/L solution of potassium chloride. Before the start of the experiment, each chamber was washed 3 times and the strips were allowed to equilibrate for at least 60 minutes. After a 2-hour equilibration period, the effect of the NO donors (100 µmol/L) was evaluated on strips spontaneously contracting or contracted with either potassium chloride (40 mmol/L) or increasing doses of oxytocin (10-11-10-7 mol/L). Five structurally dif-
ferent NO donors were tested, as follows: sodium nitroprusside (SNP), nitroglycerin, S-nitrosyl-n-acetyl-D,L-penicillamine (SNAP), diethylenetriamine NO adduct (DETA/NO), and 3-morpholonosydnonimine (SIN-1). Contractility data were integrated over time, normalized for maximal contraction, and presented as mean ± SEM with n being the number of animals. Statistical analysis. All data are presented as mean ± SEM. The 1- or 2-way ANOVA and 2-sample t tests were used for statistical analysis where appropriate. Results Validation of methods. Analysis of EMG data, as described, provided numeric values with which electrical uterine activity could be quantified for comparison. In Fig 1 the first 4 tracings depict the progression from raw EMG data to filtered and analyzed data. The fourth and fifth tracings show the computer output value for the electrical activity and the recorded IUP, demonstrating the excellent correlation between electrical and mechanical activity with this form of analysis. Such correlation permits use of the electrical signal for assessment of contractility, thus avoiding the effects by which postural changes could compromise intra-amniotic pressure assessment. Effects of NOS inhibition Nonpregnant sheep. Continuous estrogen replacement with 17β-estradiol in ovariectomized nonpregnant ewes resulted in regular uterine contractile activity. No difference in estrogen-induced contractility was noted regardless of the duration of estrogen replacement, that is, 48 hours versus 72 hours; therefore data with respect to the effects of NOS inhibition at these 2 different times were pooled. The effects of NOS inhibition in sheep receiving continuous estrogen are shown in Table I. During the second hour of L-NAME administration, a significant and consistent increase in arterial blood pressure, when compared with baseline, was observed (P < .05). A decrease in common internal iliac blood flow was also observed; however, it did not reach statistical significance with the number of animals available for analysis (n = 6). Uterine EMG activity was not significantly increased by the 2-hour L-NAME infusion. Despite the large number of animals
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A
B
Fig 2. Representative raw EMG recording of effects of NOS inhibition on myometrial responsiveness to oxytocin in pregnant sheep 96 to 124. Oxytocin was given as 4 pulses per hour at doses indicated (nanograms per kilogram per minute). A, NOS vehicle control. B, Continuous infusion of L-NAME (4.5 mg/kg per hour) for 7 hours.
Table II. Effect of nitroglycerin in estrogen-replaced nonpregnant ovariectomized sheep
Hour 1 Hour 2 Statistical significance
Uterine EMG (fold change) (n = 4)
Mean arterial blood pressure (fold change) (n = 4)
1.0 ± 0.15 0.8 ± 0.16 P > .39
0.9 ± 0.02* 0.8 ± 0.02* P < .05
Nitroglycerin (10 µg/kg per minute) was administered as a constant infusion during 2 hours. Mean ± SEM. *Statistically significant difference.
Fig 3. Effects of NOS inhibition on myometrial responsiveness to oxytocin in pregnant sheep. Oxytocin was given as continuous infusion (0.15 ng/kg per minute) for 5 hours. Simultaneous administration of aminoguanidine (4.5 mg/kg per hour) during last 2 hours did not alter responsiveness to oxytocin (n = 6). Filled circles, Control; open triangles, NOS inhibition.
included in the analysis (n = 12), the increase in uterine EMG observed during the second hour of infusion did not reach statistical significance (Table I). Pregnant sheep. Sequentially increasing doses of oxytocin resulted in a progressive increase in both electrical and mechanical uterine activity. Fig 2 shows representative data of 1 of the 2 animals studied. During the incrementing dose pulsatile oxytocin administration paradigm (dashed bar), simultaneous infusion of L-NAME (B, hatched bar; total dose, 31.5 mg/kg) did not alter the uterine electrical activity pattern when compared with L-NAME vehicle (Fig 2, A). No effect was noted in IUP in either of these 2 animals; therefore, to address concerns regarding potential uterine pacing in response to a pulsatile infusion, the remaining studies were carried out according to the continuously infused oxytocin paradigm that is also a lower oxytocin dose. As shown in Fig 3, steady consistent uterine activity was established
after 1 hour of oxytocin infusion, and this pattern of activity was maintained for at least 4 hours of oxytocin infusion. Administration of the NOS inhibitor during the last 2 hours of oxytocin infusion did not significantly alter the myometrial response to oxytocin. Aminoguanidine failed to alter the pattern of the oxytocininduced uterine EMG activity, even though an increase in arterial blood pressure was observed in all animals (data not shown). In 2 pregnant sheep using L-NAME at a dose of 9 mg/kg per hour instead of aminoguanidine produced no effect on uterine EMG activity despite a more pronounced effect on blood pressure (Fig 4). Neither NOS inhibitor affected common internal iliac blood flow (data not shown). Effect of exogenous NO on uterine contractility. The administration of nitroglycerin at 10 µg/kg per minute resulted in a significant reduction in blood pressure, as expected. Table II shows that nitroglycerin, given in doses sufficient to produce a significant fall in arterial blood pressure, does not decrease uterine EMG activity in ovariectomized, estrogen-replaced nonpregnant sheep. To confirm that the effects of NO on uterine contractility are small, we compared them with those of prostaglandin synthesis inhibition. Indomethacin decreased the magnitude of the uterine EMG to 70% ±
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Fig 5. Effects of indomethacin on uterine contractility in estrogen-replaced (100 µg/d) nonpregnant ovariectomized sheep. Significant decrease in uterine electrical and mechanical activity was observed within 30 minutes of indomethacin infusion (1 mg/kg per hour).
Fig 4. Effects of NOS inhibition on myometrial responsiveness to oxytocin in pregnant sheep. Oxytocin was given as continuous infusion (0.15 ng/kg per minute). Continuous infusion of L-NAME (9 mg/kg per hour; total dose, 18 mg/kg) was started at the up arrow and maintained for 2 hours. Despite clear increase in arterial blood pressure, uterine electrical and mechanical indexes of contractility were not affected.
5% and 47% ± 7% during the first and second hours of administration, respectively (percentage of baseline activity; n = 6; P < .05). The effect of indomethacin present in the EMG and intrauterine pressure recording (Fig 5) is in significant contrast to the effects of the NO donor nitroglycerin (Fig 6). Similarly, in the late-gestation pregnant ewe, nitroglycerin decreased arterial blood pressure without affecting uterine EMG activity (data not shown). Effects of NO in vitro. In vitro studies of pregnant sheep myometrium demonstrated that NO is capable of inducing the relaxation of sheep myometrial strips. Fig 7 shows the effect of 5 chemically different NO donors (100 µmol/L) on potassium chloride–induced (40 mmol/L) myometrial tonic contraction. All NO donors used had similar potencies, a fast onset, and a duration of 15 to 30 minutes. This dose of SNP decreased potassium chloride–induced tonic contractions by almost 50% (Fig 8; P < .05; n = 5) Also, SNP at a concentration of 100 µmol/L decreased spontaneous rhythmic contractions to 31% ± 5% (percentage of baseline activity; data not shown; P < .05; n = 7) and decreased the sensitivity to oxytocin when given before administration of oxytocin (Fig 9; P < .05; n = 6).
Fig 6. Effects of nitroglycerin on uterine contractility in estrogen-replaced (100 µg/d) nonpregnant ovariectomized sheep. Although a significant decrease in arterial blood pressure was present, no effect of nitroglycerin (10 µg/kg per minute) on uterine electrical and mechanical activity was observed.
Comment The precise mechanism responsible for the maintenance of uterine quiescence remains unclear but appears to involve a complex cascade of events incorporating many intersecting pathways. The progression from uterine quiescence to active labor and, ultimately, parturition may be activated through either the appearance of stimulatory factors or the withdrawal of inhibitory ones or both. NO has been suggested as one such inhibiting factor whose withdrawal may increase uterine contractility, because it is capable of inducing uterine relaxation and NOS is found in the uteri of all species studied. To date, no report has directly addressed the physiologic role of uterine NO production as it pertains to the maintenance of uterine quiescence. All the evidence supporting a role for NO in the maintenance of uterine quiescence originates from in vitro pharmacologic experiments or the demonstration of NO synthetic capacity in
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Fig 8. Effect of SNP (100 µmol/L) on potassium chloride–induced (40 mmol/L) uterine contractions in vitro. Contractile force of preconstricted myometrial strips (filled bar) of pregnant sheep uterus was compared with force developed 5 minutes after strips were exposed to SNP (open bar). Significant decrease in contractile force was observed (asterisk, P < .05, by t test; n = 6). Fig 7. Relaxation effect of chemically different NO donors on potassium chloride–induced (40 mmol/L) myometrial contraction in vitro. Myometrial strips of pregnant sheep uterus were exposed to 100-µmol/L concentrations of NO donor. Significant decrease in force was observed with all NO donors tested.
uterine tissues.2, 6, 12 This study addresses, for the first time, the effects of NOS inhibition on direct indexes of uterine contractility in vivo. Reports in which time of delivery was used as the end point for evaluating the effects of NOS inhibition show that administration of L-NAME does not induce premature labor13 unless progesterone synthesis is also inhibited.14 Our data are thus the first to show, by means of direct indexes of uterine contractility, that NO does not play a significant physiologic role in the regulation of uterine contractility. Using 3 different experimental approaches, we failed to demonstrate that NO significantly influences uterine contractility in vivo. This is in contrast to our findings in vitro, in which a significant reduction in uterine contractions, whether spontaneous or induced, was elicited by NO donors. If it is assumed that a physiologic role for NO in uterine quiescence could be demonstrated in vivo, the estrogen-replaced nonpregnant ewe should have been ideally suited for such a task. We have previously shown that estrogen administration increases nNOS and eNOS expression in the nonpregnant ewe2, 4 and, as well, establishes a characteristic uterine contractility pattern.11 Inhibition of endogenous NO production under these conditions was expected to alter the uterine activity pattern if this hypothesis were indeed true, yet we found no
significant alteration in contractility in response to NOS inhibition in such a setting. Although there was an increase in EMG activity at the end of 2 hours of L-NAME administration, this increase did not reach statistical significance despite the large number of animals included in the analysis. This reflects the fact that in one third of the animals the change was in the opposite direction. In vivo studies in pregnant sheep also failed to demonstrate a physiologic role for NO in the regulation of uterine contractility. On the basis of the premise that uterine NOS expression is indeed increased during pregnancy, we expected that oxytocin-induced uterine contractility would be influenced by NOS inhibition. Oxytocin was chosen as an induction agent because of its ability to elicit a steady contractile response. This served to reduce interanimal variation with respect to the frequency and regularity of spontaneous contractures, yet in this paradigm, despite variations in both the dose and the rate of infusion, oxytocin-induced contractility was not significantly altered by NOS inhibition. We have shown previously the lack of isoform specificity for L-NAME and aminoguanidine to block NOS activity in sheep uteri in vitro because they exhibit a difference in potency of about 2-fold.2 In the current study we used the 2 different NOS inhibitors to rule out isoform selectivity in vivo. The inability to demonstrate a physiologic role for NO in the maintenance of uterine quiescence in both nonpregnant and pregnant sheep does not appear to be dose-related, as illustrated by the wide dose range used in the different paradigms (9-31.5 mg/kg). The total dose of NOS inhibitor infused is within the
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range shown by other investigators to inhibit both inducible and constitutive NOS in sheep in vivo.15, 16 Of all NOS isoforms, the iNOS isoform is the one most commonly reported to decrease in those species in which NOS has been found to decline before parturition.1 By association, it would imply that this is the isoform that participates in the regulation of uterine contractility1, 17; however, it is unlikely that this is the case in sheep.3 There is very little calcium and calmodulin–independent NOS activity in nonpregnant and pregnant sheep.2, 3 In addition, sheep and goats respond to macrophage activation, in vivo by bacterial infection or in vitro with the use of bacterial products with a very weak iNOS change compared with other markers of macrophage activation.18 Inhibition of iNOS in healthy and septic sheep is accompanied by similar changes in arterial blood pressure, reiterating the lack of selectivity of most NOS inhibitors.16 An increase in arterial blood pressure is considered evidence of successful inhibition of the NOS-NO system in sheep.16, 19 We observed a 10 mm Hg increase in mean arterial blood pressure in nonpregnant sheep receiving L-NAME, which is the same magnitude of change as reported by others in this species.16, 19 Doses of L-NAME as high as 25 mg/kg do not increase blood pressure any further.19 We used L-NAME in doses as high as 31 mg/kg in the oxytocin pulsatile administration paradigm; thus we are confident that the dose of the NOS inhibitors used was sufficient for accomplishing the desired goal. Nevertheless, we attempted to measure arterial-venous differences in nitrite-nitrate concentration across the uterus. We found that, as previously described,20 in the ewe’s serum the nitrite-nitrate concentrations are low (<15 mmol/L) with a long half-life, making accurate assessment of arterial-venous differences nonfeasible. Furthermore, inhibition of NOS is not associated with a commensurate change in nitrite and nitrate concentrations and blood pressure.19, 21 Doses of L-NAME capable of increasing blood pressure do not change nitrite and nitrate concentration levels in humans. In sheep a significant fall in nitrite and nitrate concentrations is observed only after 3 hours of L-NAME.19, 21 Weiner et al22 have shown that in pregnant guinea pigs there is no correlation between uterine NOS activity and uterine cGMP content. In addition, recent evidence suggests that cGMP does not play an important role in the control of uterine contractility.23 We elected not to measure cGMP plasma concentration, because findings would not be confirmatory of NOS inhibition. The rationale for the use of nitroglycerin was that if pharmacologic doses of NO decrease uterine contractility in vivo and NOS inhibition is ineffective, it would suggest that the NOS synthesis capacity of the uterus does not provide sufficient NO to relax the myometrium. However, despite a pronounced fall in arterial blood pressure indicative of NO effects in vascular smooth muscle, this NO donor failed to significantly decrease uterine
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Fig 9. Effect of SNP (100 µmol/L) on oxytocin-induced uterine contractions in vitro. Contractile force of vehicle-treated (filled circles) myometrial strips of pregnant sheep uterus was compared with force developed by strips preexposed to SNP (open circles). Significant decrease in contractile force was observed at low oxytocin concentrations (asterisk, P < .05, by analysis of variance; n = 6).
contractility. This is in agreement with reports showing that in vitro the vascular smooth muscle is as much as 100 times more sensitive to the effects of NO as compared with myometrium.24 The reported effects of NO donors on uterine contractility in vivo vary, depending on species and stage of pregnancy. Buhimschi et al8 reported contrasting effects of an NO donor on pregnant rat uterine contractility in vitro versus in vivo. This investigation included in vivo evaluation of an intraperitoneal administration of the NO donor DETA/NO in both preterm, antiprogesterone-induced labor and spontaneous term labor. It is interesting that, in contrast with previous in vitro studies,6 the authors found in vivo that uterine contractility decreased more effectively in NO donors during delivery. Although this study demonstrates that high dosages of exogenously provided NO may alter uterine contractility, no proof of a physiologic role for NO as it pertains to uterine contractility was revealed. Unfortunately, the authors did not provide data regarding the pharmacologic effects of NO on blood pressure and rat pup outcome. Jennings et al9 reported that the NO donor SNAP was found to suppress surgically induced uterine activity in the rhesus monkey. The effects of SNAP on uterine contractions were dose dependent but at the expense of significant hemodynamic changes. The authors also reported that in a pilot study neither NOS substrate nor competitive inhibitors demonstrated any consistent effect on uterine contractility in this species.
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Surprisingly, nitroglycerin (administered up to 200 times the dose used in our study) had little effect on either uterine contractility or maternal hemodynamics. Perhaps this may be explained in that nitroglycerin requires enzymatic release of NO whereas SNAP does not. The lack of effect of nitroglycerin in the current study is at variance with the study of Heymann et al,10 in which nitroglycerin administered at the same dose used in our current study was shown to suppress spontaneous term labor in sheep. Although this was a non–peer-reviewed report, the discrepancy may be explained by the different functional status of the uterus in the 2 studies (labor vs nonlabor), as has been reported to be the case in the rat.6, 8 Results presented within our study demonstrating the effects of prostaglandin synthesis inhibition on both electrical and mechanical uterine activity in nonpregnant sheep are particularly revealing. We interpret the indomethacin-induced reduction in uterine contractility as confirmatory evidence that the effects of NO on contractility are comparatively small and, by themselves, are of no physiologic relevance. This view is in keeping with data obtained in the pregnant rat in which the effects of NO on timing of parturition can be unmasked by inhibiting progesterone synthesis. In the rat NOS inhibition was found to potentiate the ability of an antiprogesterone to induce preterm labor.14 Also, NO prevents prostaglandin-induced preterm labor by blunting the fall in serum progesterone.25 In both reports the effects of NO appear to be related to alterations in serum progesterone, and a direct effect on uterine contractility was neither evaluated nor demonstrated in vivo. In conclusion, to date no in vivo animal or human study has provided evidence for a physiologic role of NO in the maintenance of uterine quiescence. With 3 different experimental approaches a role for endogenous NO production as it relates to uterine contractility also could not be demonstrated in the current study. Despite clinical reports of uterine relaxation in response to NO donors, the effectiveness of exogenous NO in treating premature labor has yet to be confirmed in animal or human trials. REFERENCES
1. Sladek SM, Magness RR, Conrad KP. Nitric oxide and pregnancy. Am J Physiol 1997;272:R441-63. 2. Figueroa JP, Massmann GA. Estrogen increases nitric oxide synthase in the uterus of nonpregnant sheep. Am J Obstet Gynecol 1995;173:1539-45. 3. Massmann GA, Zhang J, Figueroa JP. Functional and molecular characterization of nitric oxide synthase in the endometrium and myometrium of pregnant sheep during the last third of gestation. Am J Obstet Gynecol 1999;181:116-25. 4. Zhang J, Massmann GA, Mirabile CP, Figueroa JP. Nonpregnant sheep uterine type I and type II nitric oxide synthase expression is differentially regulated by estrogen. Biol Reprod 1999;60:1198-203. 5. Kuenzli KA, Bradley ME, Buxton IL. Cyclic GMP-independent effects of nitric oxide on guinea-pig uterine contractility. Br J Pharmacol 1996;119:737-43. 6. Yallampalli C, Garfield RE, Byam-Smith M. Nitric oxide inhibits uterine contractility during pregnancy but not during delivery. Endocrinology 1993;133:1899-902.
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7. Lees C, Campbell S, Jauniaux E, Brown R, Ramsay B, Gibb D, et al. Arrest of preterm labour and prolongation of gestation with glyceryl trinitrate, a nitric oxide donor. Lancet 1994; 343:1325-6. 8. Buhimschi C, Buhimschi I, Yallampalli C, Chwalisz K, Garfield RE. Contrasting effects of diethylenetriamine nitric oxide, a spontaneously releasing nitric oxide donor, on pregnant rat uterine contractility in vitro versus in vivo. Am J Obstet Gynecol 1997;177:690-701. 9. Jennings RW, MacGilliray TE, Harrison MR. Nitric oxide inhibits preterm labor in the Rhesus monkey. J Matern Fetal Med 1993;2:170-5. 10. Heymann MA, Bootstaylor BS, Roman C, Parer JT. Glyceryl trinitrate stops active labor in sheep. In: Moncada S, Feelisch M, Busse R, Higgs EA, editors. The biology of nitric oxide. London: Portland Press; 1993. p. 201-3. 11. Massmann GA, Figueroa JP, Nathanielsz PW. Further characterization of the electromyographic activity of the myometrium and mesometrium in non-pregnant sheep under estrogen supplementation. Biol Reprod 1991;45:605-10. 12. Yallampalli C, Izumi H, Byam-Smith M, Garfield RE. An L-arginine–nitric oxide–cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pregnancy. Am J Obstet Gynecol 1994;170:175-85. 13. Buhimschi I, Yallampalli C, Chwalisz K, Garfield RE. Pre-eclampsia-like conditions produced by nitric oxide inhibition: effects of L-arginine, D-arginine and steroid hormones. Hum Reprod 1995;10:2723-30. 14. Yallampalli C, Buhimschi I, Chwalisz K, Garfield RE, Dong YL. Preterm birth in rats produced by the synergistic action of a nitric oxide inhibitor (NG-nitro-L-arginine methyl ester) and an antiprogestin (onapristone). Am J Obstet Gynecol 1996;175:207-12. 15. Bone HG, Waurick R, Van Aken H, Booke M, Prien T, Meyer J. Comparison of the haemodynamic effects of nitric oxide synthase inhibition and nitric oxide scavenging in endotoxaemic sheep. Intensive Care Med 1998;24:48-54. 16. Booke M, Hinder F, McGuire R, Traber LD, Traber DL. Selective inhibition of inducible nitric oxide synthase: effects on hemodynamics and regional blood flow in healthy and septic sheep. Crit Care Med 1999;27:162-7. 17. Bansal RK, Goldsmith PC, He Y, Zaloudek CJ, Ecker JL, Riemer RK. A decline in myometrial nitric oxide synthase expression is associated with labor and delivery. J Clin Invest 1997;99:2502-8. 18. Jungi TW, Pfister H, Sager H, Fatzer R, Vandevelde M, Zurbriggen A. Comparison of inducible nitric oxide synthase expression in the brains of Listeria monocytogenes–infected cattle, sheep, and goats and in macrophages stimulated in vitro. Infect Immun 1997;65:5279-88. 19. Allman KG, Stoddart AP, Kennedy MM, Young JD. L-Arginine augments nitric oxide production and mesenteric blood flow in ovine endotoxemia. Am J Physiol 1996;271:H1296-301. 20. Yang DS, Lang U, Greenberg SG, Myatt L, Clark KE. Elevation of nitrate levels in pregnant ewes and their fetuses. Am J Obstet Gynecol 1996;174:573-7. 21. Avontuur JA, Stam TC, Jongen-Lavrencic M, van Amsterdam JG, Eggermont AM, Bruining HA. Effect of L-NAME, an inhibitor of nitric oxide synthesis, on plasma levels of IL-6, IL-8, TNF alpha and nitrite/nitrate in human septic shock. Intensive Care Med 1998;24:673-9. 22. Weiner CP, Knowles RG, Nelson SE, Stegink LD. Pregnancy increases guanosine 3´,5´-monophosphate in the myometrium independent of nitric oxide synthesis. Endocrinology 1994;136:2473-8. 23. Hennan JK, Diamond J. Evidence that spontaneous contractile activity in the rat myometrium is not inhibited by NO-mediated increases in tissue levels of cyclic GMP. Br J Pharmacol 1998; 123:959-67. 24. Kreye VA, Baron GD, Luth JB, Schmidt-Gayk H. Mode of action of sodium nitroprusside on vascular smooth muscle. Naunyn Schmiedebergs Arch Pharmacol 1975;288:381-402. 25. Dong YL, Dai BS, Singh P, Yallampalli C. Involvement of nitric oxide pathway in prostaglandin Fα-induced preterm labor in rats. Am J Obstet Gynecol 1997;177:907-17.
Key words: Nitric oxide, uterine quiescence, sheep, pregnancy, oxytocin, indomethacin, nitro-L-arginine methyl ester, L-NAME
Nitric oxide synthase (NOS) is a monooxygenase flavoprotein that uses L-arginine as substrate to produce nitric oxide (NO), a highly reactive messenger molecule. Three distinct NOS isoforms have been isolated and identified, 2 constitutive (type I NOS, or nNOS, and type III NOS, or eNOS) and 1 inducible (type II NOS or iNOS). All 3 isoforms are present in uterine tissues during pregnancy in all species investigated to date.1 Regardless of the particular isoform that is more abundant in a given species and the site at which it is present (endometrium and decidua or myometrium), uterine NOS expression or activity increases during pregnancy, and a decrease in decidual NOS expression before parturition From the Section on Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Wake Forest University School of Medicine. Received for publication August 27, 1999; revised November 23, 1999; accepted December 31, 1999. Reprint requests: Jorge Figueroa, MD, PhD, Department of Obstetrics and Gynecology, Section on Maternal-Fetal Medicine, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1066. Copyright © 2000 by Mosby, Inc. 0002-9378/2000 $12.00 + 0 6/1/105428 doi:10.1067/mob.2000.105428
has been observed by most but not all investigators.1 Previous work from our laboratory has shown that NOS activity is found in endometrium and myometrium of pregnant and nonpregnant sheep.2, 3 Furthermore, estrogen and pregnancy regulate the expression of nNOS in myometrium and that of eNOS in the vasculature in this species.1, 3, 4 Several in vitro studies have demonstrated the ability of NO donors to decrease spontaneous and induced myometrial contraction in a cyclic guanosine monophosphate (cGMP)–dependent and a cGMP-independent manner.5, 6 In these studies the effect was a reduction in either the frequency or the amplitude of myometrial contractions. However, responsiveness seems to vary if the tissue was obtained before, during, or after labor and with regard to the chemical nature of the NO donor.1 Compared with the data available from in vitro studies, the reports on in vivo effects of NO are fewer and the effects are widely variable. Many case reports support the ability of NO donors to relax the uterus in pregnant women. These examples include resolution of uterine contraction rings, retained placenta, and uterine inversion. Lees et al7 have even suggested that an NO donor 191
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A B C D E Fig 1. Sequential steps of uterine EMG analysis in pregnant sheep. A, Raw data. B, Band-pass–filtered data. C, Full-wave rectification of B. D, 1-minute summaries of power spectral analysis of C. E, Intrauterine pressure recordings.
can be used for tocolysis in preterm labor. Such findings, however, have yet to be confirmed in prospective, randomized clinical trials. Reports are that relatively large doses of the NO donors can decrease uterine contractility in pregnant rats,8 monkeys,9 and sheep.10 The questions that remain are whether a physiologic role exists for endogenous NO production as a modulator of uterine contractility and whether the in vivo response of the uterus to exogenous NO can be further defined. Therefore this study sought to evaluate the role of NO in the maintenance of uterine quiescence in pregnant and nonpregnant ewes in vivo. Material and methods Animal preparation and postoperative care. All procedures for housing, handling, surgical implantation of catheters, and postoperative management were approved by Wake Forest University’s Institutional Animal Care and Use Committee. Sixteen chronically instrumented, ovariectomized nonpregnant ewes and 10 chronically instrumented pregnant ewes at 115 days’ gestation were studied. Animals were brought into the laboratory and placed in metabolism cages with free access to food and water. Twenty-four hours before surgery, food and water were withheld. Sheep (average weight, 50 kg) were operated on while they were under halothane general anesthesia (1.5%-2% in oxygen 2 L/min) after premedication with 1 g ketamine intramuscularly. The surgery, which included laparotomy and hysterotomy, was performed for placement of vascular and intrauterine catheters and for removing the ovaries in nonpregnant sheep. Catheters were placed into the femoral vein, both femoral arteries, carotid artery, and jugular vein in adult animals. A Transonics flow probe (6-8 mm) was placed around the common internal iliac artery in both pregnant and nonpregnant sheep. For a more accurate assessment of uterine blood flow, the ovar-
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ian, middle sacral, and cervical-vaginal arteries were ligated with sutures. Multistranded, bipolar stainless steel wires were implanted 3 to 5 mm apart on at least 2 sites of the myometrial surface for electromyography (EMG) recordings.11 In the pregnant animals additional catheters were placed into the maternal uterine vein, amniotic cavity, and the fetal carotid artery and jugular vein. Perioperative antibiotics were given on days 0 through 4 (80 mg gentamicin, 1 g ampicillin through the adult jugular vein, 20 mg of gentamicin infused through the fetal jugular vein along with 60 mg gentamicin, and 1 g ampicillin intra-amniotically). All animals were allowed 5 days of postoperative recovery before the initiation of experimentation. In experiments involving pregnant animals, fetal blood gas values were within the normal range established for this laboratory before, during, and after all studies. Uterine contractility. Uterine contractility was assessed by EMG and intrauterine pressure (IUP) recordings. The analog signal was low-pass filtered at 50 Hz, and data were collected at a rate of 68 Hz. The EMG data were bandpass filtered at frequencies between 1.8 and 20 Hz and were further processed with Fast Fourier Transform and Power Spectrum Analysis. Such analysis provided numeric values with which uterine activity could be quantified. For comparison, the magnitude of the EMG signal was normalized for baseline activity, because magnitude is determined by electrode placement and varied between animals. Nonpregnant sheep studies. All 16 sheep received a continuous intravenous infusion of 17β-estradiol (100 µg/d). Estrogen replacement was given to increase NOS expression and elicit a regular contractile pattern.2, 11 Ewes were allocated to 2 different regimens to determine whether the length of estrogen replacement would affect the response to NOS inhibition. Twelve sheep were used for studying the effects of NOS inhibition on uterine contractile activity. Six ewes were studied after 48 hours of estrogen replacement, and the remaining 6 were studied after 72 hours of estrogen replacement. All 12 sheep received a 2-hour continuous infusion of the NOS inhibitor nitro-L-arginine methyl ester (L-NAME) (total dose, 9.0 mg/kg) given into the descending aorta. In 6 of these 12 sheep, the cyclooxygenase inhibitor indomethacin was administered on separate days. These 6 animals received a 2-hour intravenous indomethacin infusion (1 mg/kg per hour) at either 72 or 96 hours of estrogen replacement. A separate group of 4 nonpregnant ewes were treated with the NO donor nitroglycerin. These animals received a 2-hour nitroglycerin infusion (10 µg/kg per minute) at either 48 or 72 hours of estrogen replacement. Pregnant sheep studies. Ten chronically instrumented, late-gestation pregnant ewes were studied. In this group we evaluated the ability of L-NAME (n = 4) or aminoguanidine (n = 6) to alter uterine contractility. Oxytocin
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Table I. NOS inhibition in estrogen-replaced nonpregnant ovariectomized sheep
Control hour Hour 1 Hour 2 Statistical significance
Uterine EMG (fold increase) (n = 12)
Arterial blood pressure (mm Hg) (n = 10)
1.28 ± 0.1 1.30 ± 0.2 P > .09
77.3 ± 3 81.4 ± 3 86.8 ± 3* P < .05
Uterine blood flow (mL · min–1) (n = 6) 117.6 ± 32 94.8 ± 25 97.4 ± 27 P > .8
L-NAME
was given as a continuous infusion over 2 hours (total dose, 9 mg/kg). Mean ± SEM. *Statistically significant difference.
was used to establish a steady contractile pattern for study and overcome the inherent low frequency and lack of pattern of contractures. Each animal received oxytocin on 2 separate occasions at least 2 days apart, being treated with either NOS inhibitor or vehicle. In 2 sheep oxytocin was infused into the descending aorta in a dose-response fashion (6 sequentially increasing doses from 0.05 up through 3.84 ng/kg per minute, each infused over a 1-hour period as a 5-minute pulse every 15 minutes) for a total of 6 hours. Eight sheep received a continuous infusion at a rate of 0.15 ng/kg per minute for a total of 5 hours. The NOS inhibitors were given at the same dose (4.5 mg/kg per hour); however, on a molar basis the dose of aminoguanidine is twice that of L-NAME. The total dose of NOS inhibitor ranged from 9 mg/kg (L-NAME and aminoguanidine) in the continuous oxytocin paradigm to 31.5 mg/kg (L-NAME) in the pulsatile administration paradigm, depending on the overall duration of NOS inhibitor administration. In 2 of the pregnant ewes, on a separate day (removed from an oxytocin infusion) nitroglycerin was administered to assess the effects of exogenous NO (2-hour nitroglycerin infusion at 10 µg/kg per minute). In vitro contractility. Strips of longitudinal muscle were prepared from sheep uteri during pregnancy (115 days’ gestation) and immediately post partum. Strips (1 × 10 mm) were mounted in 20-mL organ chambers (Radnoti) filled with modified Krebs-Henseleit solution warmed at 37°C, gassed with 95% oxygen to 5% carbon dioxide, and containing 10-5-mol/L indomethacin. Isometric tension was measured continuously with a force transducer and collected with an A/D board (Data Translation) at 20 Hz with Asyst 4.0. Strips were stretched to their optimum length-tension relationship by repeated stimulation with a 40-mmol/L solution of potassium chloride. Before the start of the experiment, each chamber was washed 3 times and the strips were allowed to equilibrate for at least 60 minutes. After a 2-hour equilibration period, the effect of the NO donors (100 µmol/L) was evaluated on strips spontaneously contracting or contracted with either potassium chloride (40 mmol/L) or increasing doses of oxytocin (10-11-10-7 mol/L). Five structurally dif-
ferent NO donors were tested, as follows: sodium nitroprusside (SNP), nitroglycerin, S-nitrosyl-n-acetyl-D,L-penicillamine (SNAP), diethylenetriamine NO adduct (DETA/NO), and 3-morpholonosydnonimine (SIN-1). Contractility data were integrated over time, normalized for maximal contraction, and presented as mean ± SEM with n being the number of animals. Statistical analysis. All data are presented as mean ± SEM. The 1- or 2-way ANOVA and 2-sample t tests were used for statistical analysis where appropriate. Results Validation of methods. Analysis of EMG data, as described, provided numeric values with which electrical uterine activity could be quantified for comparison. In Fig 1 the first 4 tracings depict the progression from raw EMG data to filtered and analyzed data. The fourth and fifth tracings show the computer output value for the electrical activity and the recorded IUP, demonstrating the excellent correlation between electrical and mechanical activity with this form of analysis. Such correlation permits use of the electrical signal for assessment of contractility, thus avoiding the effects by which postural changes could compromise intra-amniotic pressure assessment. Effects of NOS inhibition Nonpregnant sheep. Continuous estrogen replacement with 17β-estradiol in ovariectomized nonpregnant ewes resulted in regular uterine contractile activity. No difference in estrogen-induced contractility was noted regardless of the duration of estrogen replacement, that is, 48 hours versus 72 hours; therefore data with respect to the effects of NOS inhibition at these 2 different times were pooled. The effects of NOS inhibition in sheep receiving continuous estrogen are shown in Table I. During the second hour of L-NAME administration, a significant and consistent increase in arterial blood pressure, when compared with baseline, was observed (P < .05). A decrease in common internal iliac blood flow was also observed; however, it did not reach statistical significance with the number of animals available for analysis (n = 6). Uterine EMG activity was not significantly increased by the 2-hour L-NAME infusion. Despite the large number of animals
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A
B
Fig 2. Representative raw EMG recording of effects of NOS inhibition on myometrial responsiveness to oxytocin in pregnant sheep 96 to 124. Oxytocin was given as 4 pulses per hour at doses indicated (nanograms per kilogram per minute). A, NOS vehicle control. B, Continuous infusion of L-NAME (4.5 mg/kg per hour) for 7 hours.
Table II. Effect of nitroglycerin in estrogen-replaced nonpregnant ovariectomized sheep
Hour 1 Hour 2 Statistical significance
Uterine EMG (fold change) (n = 4)
Mean arterial blood pressure (fold change) (n = 4)
1.0 ± 0.15 0.8 ± 0.16 P > .39
0.9 ± 0.02* 0.8 ± 0.02* P < .05
Nitroglycerin (10 µg/kg per minute) was administered as a constant infusion during 2 hours. Mean ± SEM. *Statistically significant difference.
Fig 3. Effects of NOS inhibition on myometrial responsiveness to oxytocin in pregnant sheep. Oxytocin was given as continuous infusion (0.15 ng/kg per minute) for 5 hours. Simultaneous administration of aminoguanidine (4.5 mg/kg per hour) during last 2 hours did not alter responsiveness to oxytocin (n = 6). Filled circles, Control; open triangles, NOS inhibition.
included in the analysis (n = 12), the increase in uterine EMG observed during the second hour of infusion did not reach statistical significance (Table I). Pregnant sheep. Sequentially increasing doses of oxytocin resulted in a progressive increase in both electrical and mechanical uterine activity. Fig 2 shows representative data of 1 of the 2 animals studied. During the incrementing dose pulsatile oxytocin administration paradigm (dashed bar), simultaneous infusion of L-NAME (B, hatched bar; total dose, 31.5 mg/kg) did not alter the uterine electrical activity pattern when compared with L-NAME vehicle (Fig 2, A). No effect was noted in IUP in either of these 2 animals; therefore, to address concerns regarding potential uterine pacing in response to a pulsatile infusion, the remaining studies were carried out according to the continuously infused oxytocin paradigm that is also a lower oxytocin dose. As shown in Fig 3, steady consistent uterine activity was established
after 1 hour of oxytocin infusion, and this pattern of activity was maintained for at least 4 hours of oxytocin infusion. Administration of the NOS inhibitor during the last 2 hours of oxytocin infusion did not significantly alter the myometrial response to oxytocin. Aminoguanidine failed to alter the pattern of the oxytocininduced uterine EMG activity, even though an increase in arterial blood pressure was observed in all animals (data not shown). In 2 pregnant sheep using L-NAME at a dose of 9 mg/kg per hour instead of aminoguanidine produced no effect on uterine EMG activity despite a more pronounced effect on blood pressure (Fig 4). Neither NOS inhibitor affected common internal iliac blood flow (data not shown). Effect of exogenous NO on uterine contractility. The administration of nitroglycerin at 10 µg/kg per minute resulted in a significant reduction in blood pressure, as expected. Table II shows that nitroglycerin, given in doses sufficient to produce a significant fall in arterial blood pressure, does not decrease uterine EMG activity in ovariectomized, estrogen-replaced nonpregnant sheep. To confirm that the effects of NO on uterine contractility are small, we compared them with those of prostaglandin synthesis inhibition. Indomethacin decreased the magnitude of the uterine EMG to 70% ±
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Fig 5. Effects of indomethacin on uterine contractility in estrogen-replaced (100 µg/d) nonpregnant ovariectomized sheep. Significant decrease in uterine electrical and mechanical activity was observed within 30 minutes of indomethacin infusion (1 mg/kg per hour).
Fig 4. Effects of NOS inhibition on myometrial responsiveness to oxytocin in pregnant sheep. Oxytocin was given as continuous infusion (0.15 ng/kg per minute). Continuous infusion of L-NAME (9 mg/kg per hour; total dose, 18 mg/kg) was started at the up arrow and maintained for 2 hours. Despite clear increase in arterial blood pressure, uterine electrical and mechanical indexes of contractility were not affected.
5% and 47% ± 7% during the first and second hours of administration, respectively (percentage of baseline activity; n = 6; P < .05). The effect of indomethacin present in the EMG and intrauterine pressure recording (Fig 5) is in significant contrast to the effects of the NO donor nitroglycerin (Fig 6). Similarly, in the late-gestation pregnant ewe, nitroglycerin decreased arterial blood pressure without affecting uterine EMG activity (data not shown). Effects of NO in vitro. In vitro studies of pregnant sheep myometrium demonstrated that NO is capable of inducing the relaxation of sheep myometrial strips. Fig 7 shows the effect of 5 chemically different NO donors (100 µmol/L) on potassium chloride–induced (40 mmol/L) myometrial tonic contraction. All NO donors used had similar potencies, a fast onset, and a duration of 15 to 30 minutes. This dose of SNP decreased potassium chloride–induced tonic contractions by almost 50% (Fig 8; P < .05; n = 5) Also, SNP at a concentration of 100 µmol/L decreased spontaneous rhythmic contractions to 31% ± 5% (percentage of baseline activity; data not shown; P < .05; n = 7) and decreased the sensitivity to oxytocin when given before administration of oxytocin (Fig 9; P < .05; n = 6).
Fig 6. Effects of nitroglycerin on uterine contractility in estrogen-replaced (100 µg/d) nonpregnant ovariectomized sheep. Although a significant decrease in arterial blood pressure was present, no effect of nitroglycerin (10 µg/kg per minute) on uterine electrical and mechanical activity was observed.
Comment The precise mechanism responsible for the maintenance of uterine quiescence remains unclear but appears to involve a complex cascade of events incorporating many intersecting pathways. The progression from uterine quiescence to active labor and, ultimately, parturition may be activated through either the appearance of stimulatory factors or the withdrawal of inhibitory ones or both. NO has been suggested as one such inhibiting factor whose withdrawal may increase uterine contractility, because it is capable of inducing uterine relaxation and NOS is found in the uteri of all species studied. To date, no report has directly addressed the physiologic role of uterine NO production as it pertains to the maintenance of uterine quiescence. All the evidence supporting a role for NO in the maintenance of uterine quiescence originates from in vitro pharmacologic experiments or the demonstration of NO synthetic capacity in
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Fig 8. Effect of SNP (100 µmol/L) on potassium chloride–induced (40 mmol/L) uterine contractions in vitro. Contractile force of preconstricted myometrial strips (filled bar) of pregnant sheep uterus was compared with force developed 5 minutes after strips were exposed to SNP (open bar). Significant decrease in contractile force was observed (asterisk, P < .05, by t test; n = 6). Fig 7. Relaxation effect of chemically different NO donors on potassium chloride–induced (40 mmol/L) myometrial contraction in vitro. Myometrial strips of pregnant sheep uterus were exposed to 100-µmol/L concentrations of NO donor. Significant decrease in force was observed with all NO donors tested.
uterine tissues.2, 6, 12 This study addresses, for the first time, the effects of NOS inhibition on direct indexes of uterine contractility in vivo. Reports in which time of delivery was used as the end point for evaluating the effects of NOS inhibition show that administration of L-NAME does not induce premature labor13 unless progesterone synthesis is also inhibited.14 Our data are thus the first to show, by means of direct indexes of uterine contractility, that NO does not play a significant physiologic role in the regulation of uterine contractility. Using 3 different experimental approaches, we failed to demonstrate that NO significantly influences uterine contractility in vivo. This is in contrast to our findings in vitro, in which a significant reduction in uterine contractions, whether spontaneous or induced, was elicited by NO donors. If it is assumed that a physiologic role for NO in uterine quiescence could be demonstrated in vivo, the estrogen-replaced nonpregnant ewe should have been ideally suited for such a task. We have previously shown that estrogen administration increases nNOS and eNOS expression in the nonpregnant ewe2, 4 and, as well, establishes a characteristic uterine contractility pattern.11 Inhibition of endogenous NO production under these conditions was expected to alter the uterine activity pattern if this hypothesis were indeed true, yet we found no
significant alteration in contractility in response to NOS inhibition in such a setting. Although there was an increase in EMG activity at the end of 2 hours of L-NAME administration, this increase did not reach statistical significance despite the large number of animals included in the analysis. This reflects the fact that in one third of the animals the change was in the opposite direction. In vivo studies in pregnant sheep also failed to demonstrate a physiologic role for NO in the regulation of uterine contractility. On the basis of the premise that uterine NOS expression is indeed increased during pregnancy, we expected that oxytocin-induced uterine contractility would be influenced by NOS inhibition. Oxytocin was chosen as an induction agent because of its ability to elicit a steady contractile response. This served to reduce interanimal variation with respect to the frequency and regularity of spontaneous contractures, yet in this paradigm, despite variations in both the dose and the rate of infusion, oxytocin-induced contractility was not significantly altered by NOS inhibition. We have shown previously the lack of isoform specificity for L-NAME and aminoguanidine to block NOS activity in sheep uteri in vitro because they exhibit a difference in potency of about 2-fold.2 In the current study we used the 2 different NOS inhibitors to rule out isoform selectivity in vivo. The inability to demonstrate a physiologic role for NO in the maintenance of uterine quiescence in both nonpregnant and pregnant sheep does not appear to be dose-related, as illustrated by the wide dose range used in the different paradigms (9-31.5 mg/kg). The total dose of NOS inhibitor infused is within the
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range shown by other investigators to inhibit both inducible and constitutive NOS in sheep in vivo.15, 16 Of all NOS isoforms, the iNOS isoform is the one most commonly reported to decrease in those species in which NOS has been found to decline before parturition.1 By association, it would imply that this is the isoform that participates in the regulation of uterine contractility1, 17; however, it is unlikely that this is the case in sheep.3 There is very little calcium and calmodulin–independent NOS activity in nonpregnant and pregnant sheep.2, 3 In addition, sheep and goats respond to macrophage activation, in vivo by bacterial infection or in vitro with the use of bacterial products with a very weak iNOS change compared with other markers of macrophage activation.18 Inhibition of iNOS in healthy and septic sheep is accompanied by similar changes in arterial blood pressure, reiterating the lack of selectivity of most NOS inhibitors.16 An increase in arterial blood pressure is considered evidence of successful inhibition of the NOS-NO system in sheep.16, 19 We observed a 10 mm Hg increase in mean arterial blood pressure in nonpregnant sheep receiving L-NAME, which is the same magnitude of change as reported by others in this species.16, 19 Doses of L-NAME as high as 25 mg/kg do not increase blood pressure any further.19 We used L-NAME in doses as high as 31 mg/kg in the oxytocin pulsatile administration paradigm; thus we are confident that the dose of the NOS inhibitors used was sufficient for accomplishing the desired goal. Nevertheless, we attempted to measure arterial-venous differences in nitrite-nitrate concentration across the uterus. We found that, as previously described,20 in the ewe’s serum the nitrite-nitrate concentrations are low (<15 mmol/L) with a long half-life, making accurate assessment of arterial-venous differences nonfeasible. Furthermore, inhibition of NOS is not associated with a commensurate change in nitrite and nitrate concentrations and blood pressure.19, 21 Doses of L-NAME capable of increasing blood pressure do not change nitrite and nitrate concentration levels in humans. In sheep a significant fall in nitrite and nitrate concentrations is observed only after 3 hours of L-NAME.19, 21 Weiner et al22 have shown that in pregnant guinea pigs there is no correlation between uterine NOS activity and uterine cGMP content. In addition, recent evidence suggests that cGMP does not play an important role in the control of uterine contractility.23 We elected not to measure cGMP plasma concentration, because findings would not be confirmatory of NOS inhibition. The rationale for the use of nitroglycerin was that if pharmacologic doses of NO decrease uterine contractility in vivo and NOS inhibition is ineffective, it would suggest that the NOS synthesis capacity of the uterus does not provide sufficient NO to relax the myometrium. However, despite a pronounced fall in arterial blood pressure indicative of NO effects in vascular smooth muscle, this NO donor failed to significantly decrease uterine
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Fig 9. Effect of SNP (100 µmol/L) on oxytocin-induced uterine contractions in vitro. Contractile force of vehicle-treated (filled circles) myometrial strips of pregnant sheep uterus was compared with force developed by strips preexposed to SNP (open circles). Significant decrease in contractile force was observed at low oxytocin concentrations (asterisk, P < .05, by analysis of variance; n = 6).
contractility. This is in agreement with reports showing that in vitro the vascular smooth muscle is as much as 100 times more sensitive to the effects of NO as compared with myometrium.24 The reported effects of NO donors on uterine contractility in vivo vary, depending on species and stage of pregnancy. Buhimschi et al8 reported contrasting effects of an NO donor on pregnant rat uterine contractility in vitro versus in vivo. This investigation included in vivo evaluation of an intraperitoneal administration of the NO donor DETA/NO in both preterm, antiprogesterone-induced labor and spontaneous term labor. It is interesting that, in contrast with previous in vitro studies,6 the authors found in vivo that uterine contractility decreased more effectively in NO donors during delivery. Although this study demonstrates that high dosages of exogenously provided NO may alter uterine contractility, no proof of a physiologic role for NO as it pertains to uterine contractility was revealed. Unfortunately, the authors did not provide data regarding the pharmacologic effects of NO on blood pressure and rat pup outcome. Jennings et al9 reported that the NO donor SNAP was found to suppress surgically induced uterine activity in the rhesus monkey. The effects of SNAP on uterine contractions were dose dependent but at the expense of significant hemodynamic changes. The authors also reported that in a pilot study neither NOS substrate nor competitive inhibitors demonstrated any consistent effect on uterine contractility in this species.
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Surprisingly, nitroglycerin (administered up to 200 times the dose used in our study) had little effect on either uterine contractility or maternal hemodynamics. Perhaps this may be explained in that nitroglycerin requires enzymatic release of NO whereas SNAP does not. The lack of effect of nitroglycerin in the current study is at variance with the study of Heymann et al,10 in which nitroglycerin administered at the same dose used in our current study was shown to suppress spontaneous term labor in sheep. Although this was a non–peer-reviewed report, the discrepancy may be explained by the different functional status of the uterus in the 2 studies (labor vs nonlabor), as has been reported to be the case in the rat.6, 8 Results presented within our study demonstrating the effects of prostaglandin synthesis inhibition on both electrical and mechanical uterine activity in nonpregnant sheep are particularly revealing. We interpret the indomethacin-induced reduction in uterine contractility as confirmatory evidence that the effects of NO on contractility are comparatively small and, by themselves, are of no physiologic relevance. This view is in keeping with data obtained in the pregnant rat in which the effects of NO on timing of parturition can be unmasked by inhibiting progesterone synthesis. In the rat NOS inhibition was found to potentiate the ability of an antiprogesterone to induce preterm labor.14 Also, NO prevents prostaglandin-induced preterm labor by blunting the fall in serum progesterone.25 In both reports the effects of NO appear to be related to alterations in serum progesterone, and a direct effect on uterine contractility was neither evaluated nor demonstrated in vivo. In conclusion, to date no in vivo animal or human study has provided evidence for a physiologic role of NO in the maintenance of uterine quiescence. With 3 different experimental approaches a role for endogenous NO production as it relates to uterine contractility also could not be demonstrated in the current study. Despite clinical reports of uterine relaxation in response to NO donors, the effectiveness of exogenous NO in treating premature labor has yet to be confirmed in animal or human trials. REFERENCES
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