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Nitric oxide synthase activities in human myometrium and villous trophoblast throughout pregnancy
Nitric Oxide Synthase Activities in Human Myometrium and Villous Trophoblast Throughout Pregnancy BRUCE RAMSAY, MBBS, MRCOG, SUREN R. SOORANNA,
PhD, AND
MARK R. JOHNSON, MBBS, MRCP, Phi3 Objective: To study the changes in nitric oxide synthase activities in human myometrium and trophoblast throughout pregnancy and around delivery. Methods: Samples of villous trophoblast were collected from women undergoing elective c-n delivery at term (n = 12) or voluntary termination of pregnancy in the first (n = 27) or second (n = 11) trimesters of pregnancy. Myometrial samples were obtained from nonpregnant women undergoing hysterectomy tn = 5) and pregnant women both before tn = 7) and after (n = 7) the onset of spontaneous labor at term. Nitric oxide synthase activity was quantified for homogenized samples using the L-citrulline assay in the presence and absence of calcium. Results: The highest levels of nitric oxide synthase activity were found in first-trimester villi (range 2-29 nmol Lcitrulline/minute/g protein), with a significant fall in activity in the third trimester (range 2-10 nmot Gcihulhne/minute/g protein; P < .OOl for both calcium-dependent and calciumindependent activity). Myometrial activities were relatively low compared with those in the trophoblast (O-2 nmol L-citrulline/minute/g protein), with no significant differences in calcium-dependent activities between subgroups. Myometrial calcium-independent activities were lower in pregnant than in nonpregnant women (P = .007), with those in labor having levels higher than those not in labor (P = .048). Conclusion: Levels of nitric oxide synthase activity are relatively high in villous trophoblaat, particularly during the first trimester. Although the contribution to total nitric oxide production in the uterus by myometrial nitric oxide synthase appears to be relatively small, nitric oxide produced by the trophoblast may play a role in maintaining uterine quiescence by a paracrine effect. Further work is needed to test this hypothesis and explore other possible roles for trophoblast-derived nitric oxide in early pregnancy. (Obstet Gynecol 1996;87:249-53)
From the Academic Department of Obstetrics and Gymecology, Chelsen and Westminster Hospital, London, United Kingdom.
VOL.
87, NO.
2, FEBRUARY
1996
Preterm delivery is the single most important contributor to perinatal morbidity and mortality in the developed world. Although many causes have been identified, in most cases the underlying mechanisms remain obscure. Our limited understanding is reflected in our inability to treat this condition effectively.’ However, there are two new pharmacologic approaches that show promise. The first, an oxytocin receptor antagonist, atosiban, is a logical development because it is known that myometrial sensitivity to oxytocin is enhanced in preterm labor.* The second, a nitric oxide donor drug, glyceryl trinitrate, is more speculative because little is known about the role of nitric oxide in pretenn labor. Reports so far have centered on clinical observations of its efficacy.3,4 In 1980, Furchgott and Zawadski5 discovered that vascular endothelium could induce vasodilation by the production of endothelium-derived relaxing factor. This factor was identified subsequently as nitric oxide,6 which is formed from its substrate, L-arginine, by the enzyme nitric oxide synthase. This enzyme exists in both calcium-dependent and calcium-independent isoforms.7 Animal’ and human’ studies have confirmed the important contribution that nitric oxide makes to the physiologic control of vascular tone and have also shown nitric oxide to be important in the regulation of platelet aggregation, neurotransmission, and immune responses.7 Currently, the role of nitric oxide in the control of myometrial contractility is defined less clearly. Others have shown that administration of a nitric oxide donor drug will abolish contractions in rat uterine smooth muscle both in vitro and in viv0.l’ Studies in vitro have shown a similar effect on human myometrium.” Nitric oxide-induced relaxation was less in samples of rat myometrium obtained at delivery than in those from the second-trimester, suggesting that nitric oxide may
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249
play a role in the complex control system that maintains uterine quiescence during pregnancy.‘2,‘3 There are no reports of the relationship between onset of *labor and nitric oxide production in human pregnancy. To investigate this, we measured nitric oxide synthase activities in human trophoblast throughout gestation and in myometrium obtained from nonpregnant and pregnant women before and after the onset of labor.
Materials and Methods The study was approved by the hospital ethics committee and informed consent was obtained from all subjects. All pregnant women were healthy and carried singleton pregnancies, and those who had been pregnant previously all had uneventful spontaneous vaginal deliveries. Women with medical conditions or a history of dysfunctional labor or cesarean delivery were excluded. Placental villi were collected at the time of first- and second-trimester terminations of pregnancy and cesarean delivery before the onset of labor. The indication for cesarean was breech presentation of the fetus, with an otherwise uncomplicated obstetric history in each case. The gestational ages of first- and second-trimester pregnancies were estimated by ultrasonic measurement of fetal crown-rump length immediately before the procedure, and third-trimester pregnancies were dated from the onset of the last menstrual period (confirmed at a 20-week ultrasound scan). Placental villi were collected from 27 first- (mean gestation It_ standard deviation 9.8 t 1.4 weeks), 11 second- (17.6 t 2.5 weeks), and 12 third- (38.5 % 1.3 weeks) trimester pregnancies. Myometrial samples were obtained from nonpregnant women of childbearing age at the time of hysterectomy (n = 5) and from pregnant women at cesarean before (n = 7) and after (n = 7) the onset of labor. The indication for cesarean delivery was breech presentation for the pre-labor group and abnormal fetal heart rate (FHR) pattern in the labor group. All women in the labor group had gone into spontaneous labor at 37-40 weeks’ gestation and had been in established labor for at least 4 hours before delivery. Hysterectomies were all performed for dysfunctional uterine bleeding unresponsive to medication but not associated with structural abnormalities (eg, leiomyomas). All women undergoing hysterectomy were of proven fertility and delivered spontaneously per vaginam. Samples were taken from the upper edge of the transverse uterine incision at cesarean and from the anterior wall of the uterus after hysterectomy. They were weighed and frozen immediately in liquid nitrogen for storage at -70C for up to 4 weeks until analysis.
250
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For analysis, the tissue sample was mixed with five volumes of 0.25 mol/L sucrose containing 50 mmol/L Tris-HCl, pH 7.0, 1 mmol/L dithiothreitol, 1 mmol/L ethylenediaminetetra-acetic acid, 100 Fg/mL phenylmethylsulphonyl fluoride, 10 Fg/mL of soya-bean inhibitor, and 2 Fg/mL of apoprotinin, and was homogenized in four bursts of 15 seconds over 2 minutes at 4C. After centrifugation for 5 minutes at 11,000 X g, supernatants were kept on ice for no longer than 15 minutes before assay. Aliquots of supernatants were dispensed into 0.1 mol/L NaOH and kept at 4C for protein determination by the method of Lowry et a1.14 Nitric oxide synthase activity was measured by the activity method of Salter et a1.i5 Calcium-dependent was determined from the difference between the [‘4Clcitrulline produced from control samples and samples containing 1 mmol/L L-NMMA. Calcium-independent activity was determined from the difference between the [‘%Z] citrulline produced by the samples containing 1.5 mmol/L ethylene-bis(oxy-ethylenenitrilo~tetra-acetic acid (EGTA) with 1 mmol/L L-NMMA and those containing 1.5 mmol/L EGTA alone. Duplicate assays were performed for 16 minutes for myometrial samples and 8 minutes for trophoblast samples, with blanks at zero time points. These timings were based on our earlier experiments demonstrating the linearity of the assay together with the large ditIerence in activities between trophoblast and myometrial samples. The limit of detection of the assay was approximately 0.2 run01 of L-citrulline formed per minute per gram of tissue protein. Statistical comparison of groups was carried out using Km&al-Wallis analysis of variance test for multiple indices and MannWhitney U test when appropriate.
Results In the placental villi, both calcium-dependent and independent nitric oxide synthase activities were present in normal human villous tissue throughout pregnancy, with significantly higher levels of activity in the first trimester than in the third (P < .OOl for both isoforms) (Figures 1 and 2). In the myometrium, the nitric oxide synthase activity was much lower than in the trophoblast (range O-2 versus O-29 run01 L-citrulline/minute/g protein; P < .OOl). The calcium-dependent activities were statistically similar in all subgroups (Figure 3). The calcium-independent activities were significantly lower in the pregnant compared with the nonpregnant uterus (P = 007) (Figure 4). The lowest activities were seen in myometrial samples taken before the onset of labor, which were significantly less than samples taken after a period of labor 0’ = .048).
Obstetrics 6 Gynecology
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Trimester OFRsetpncy Figure 1. Calcium-dependent nitric oxide synthase activities in villous trophoblast throughout gestation.
Discussion We found nitric oxide synthase activity in myometrium and trophoblast throughout gestation. First-trimester trophoblast tissue had the highest activities, which fell with advancing gestational age to levels at term similar to those reported previously by other investigators.‘6,17
Figure 3. Calcium-dependent nitric oxide synthase activities in myometrium from nonpregnant and pregnant women before and after the onset of labor at term.
It is worth noting that the first-trimester levels of calcium-independent activity are as high as those reported previously only in pathophysiologic inflammatory states.‘8,‘9 Calcium-independent nitric oxide synthase usually is produced in response to cytokines and requires new protein synthesis to occur. This was first shown to occur
0
I
1
2
3
Trimesterof Pregeaecy Figure 2. Calcium-independent nitric oxide synthase activities in villous trophoblast throughout gestation.
VOL. 87, NO. 2, FEBRUARY
1996
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Figure 4. Calcium-dependent nitric oxide synthase activities in myometrium from nonpregnant and pregnant women before and after the onset of labor at term.
Ramsay
et al
Nitric
Oxide Synfhase
251
in macrophages in inflammatory conditions. During early pregnancy, the trophoblast is at its most active, invading deeply into the decidua and myometrium. The decidua is known to be infiltrated by lymphocyteszo and, indeed, the maternal response to implantation has been likened to an inflammatory process by several authors.21 The high levels of calcium-independent activity shown here may be a reflection of such a reaction. At implantation, it would also be advantageous for the trophoblast to suppress uterine activity and the maternal immune response whilst encouraging local vasodilation. A role for nitric oxide in the modulation of some or all of these processes would be consistent with the higher levels of nitric oxide synthase activity found in first-trimester villi. The subsequent decline in trophoblast-derived nitric oxide synthase activity, which occurred with advancing gestational age, took place against a background of decreasing uteroplacental resistance to blood flo~.~l This supports the notion of trophoblast-derived nitric oxide having other roles beyond simple vasodilation of the uteroplacental circulation. All the myometrial nitric oxide synthase activities were significantly lower than those from villous trophoblast, with calcium-independent activities showing some variation in relation to the onset of labor. The calcium-dependent activities may derive from the endothelium of blood vessels within the uterus. The calcium-independent activities were less in pregnant myometrium than in nonpregnant, with the lowest values being obtained before the onset of labor rather than during labor. The diIIerence in calcium-independent activities between the labor and nonlabor groups barely achieves statistical significance (P = .048). The small numbers of myometrial samples mean that this part of the study has limited statistical power compared with the trophoblast data (80 versus 95% power). In addition, the low levels of myometrial activity make intra-assay variation a significant factor. Although these data would suggest that nitric oxide production by the myometrium is unlikely to have a role in the maintenance of myometrial quiescence, there are several complicating factors to take into account. First, there are problems of study design inherent in observational studies of human pregnancy. Also, the myometrium from the labor subgroup may not have been truly “normal.” All the women in this group were undergoing cesarean delivery (after a period of spontaneous labor at term) for abnormal FHR. However, three of the seven also had an element of poor progress in the clinical decision for cesarean; therefore, the characteristics of myometrium taken from these women may have been abnormal. This point is difficult to answer because the only truly “normal” labors ending in cesarean occur
252
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Oxide Synthase
in the unusual circumstance of a woman already booked for surgery (eg, a primigravida with breech presentation) arriving at the hospital in spontaneous labor. The argument can be extended to cover the hysterectomy samples, too, because dysfunctional bleeding may also produce abnormal results. We attempted to minimize this problem by selecting women of proven fertility with uneventful past obstetric histories. Second, there may be enhanced responsiveness of the myometrium to nitric oxide during pregnancy in much the same way as there is to oxytocin. There is no information at present to support or refute this notion, but this question must be answered by future work. Finally, nitric oxide released by the high levels of trophoblast enzyme activity may exert a paracrine effect on the myometrium and thus contribute to the maintenance of uterine quiescence in this way. Support for this view has already come from animal studies that have shown a reduction in decidual nitric oxide synthase activity with advancing gestation, reaching a nadir on the day of delive$’ and paralleled by a decrease in myometrial guanosine 3’,5’,-cyclic monophosphate levels.23 It is possible that high levels of nitric oxide derived from the trophoblast influence the expression of the calcium-independent nitric oxide synthase in the myometrium by negative feedback, switching off the production of new messenger RNA, thus explaining the lower levels in pregnant specimens than in those taken at routine hysterectomy. At present, there is no direct evidence to support this notion. These data demonstrate appreciable amounts of nitric oxide synthase activity in the first-trimester villous trophoblast and suggest a physiologic role for nitric oxide in the early stages of pregnancy. The relatively low levels of nitric oxide synthase in myometrium and the decline in activity between nonpregnant and cesarean delivery samples make it unlikely that nitric oxide derived from the myometrium has any role in the maintenance of uterine quiescence, although it is still possible that trophoblast-derived nitric oxide may influence uterine contractility. Further work is needed to establish the role of nitric oxide produced by the trophoblast in early pregnancy, but possibilities include local vasodilation, control of trophoblastic invasion, and the prevention of fetal rejection.
References 1. The Canadian preterm labour investigation group. Treatment of preterm labor with a beta-adrenergic agonist, ritodrine. N Engl J Med 1992;327:308-12. 2. Takahashi K, Diamond F, Bieniarz J, Yen H, Burd L. Uterine contractility and oxytocin sensitivity in preterm, term, and postterm pregnancy. Am J Obstet Gynecol 1980;136:774-9.
Obstetrics 6 Gynecology
3. Greenspoon JS, Koviacic A. A breech extraction facilitated by glyceryl trinitrate spray. Lancet 1991;338:124-5. 4. Lees C, Campbell S, Jauniaux E, et al. Arrest of preterm labour and prolongation of gestation with glyceryl trinitrate, a nitric oxide donor. Lancet 1994;343:1325-6. 5. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 19&?0;288:373-6. 6. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium derived relaxing factor. Nature 1987;327:524-6. 7. Moncada S, Palmer RM, Hlggs EA. Nitric oxide: Physiology, pathophysiology and pharmacology. Pharmacol Rev 1991;43:10942. 8. Rees DD, Palmer RMJ, Moncada S. The role of endothelium derived nitric oxide in the regulation of blood flow. Proc Nat1 Acad Sci U S A 1989;86:3375-8. 9. Valiance P, Collier J, Moncada S. Effects of endothelium derived nitric oxide on peripheral arteriolar tone in man. Lancet 1989;ii: 997-looo. 10. Natuzzi ES, Ursell PC, Harrison M, Buscher C, Reimer RK. Nitric oxide synthase activity in the pregnant uterus decreases at parturition. Biochem Biophys Res Comm 1993;194:1-8. 11. Yallampalli C, Garfield RE, Byam-Smith M. Nitric oxide inhibits uterine contractility during pregnancy but not during delivery. Endocrinol 1993;133:1%99-902. 12. Izurni H, Yallampalli C, Garfield RE. Gestational changes in L-arginine induced relaxation of pregnant rat and human myometrial smooth muscle. Am J Obstet Gynecol 1993;169:1327-37, 13. Yallampalli C, lzumi H, Byam-Smith M, Garfield RE. An Larginine-nitric oxide-cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pregnancy. Am J Obstet Gynecol 1994;170:175-85. 14. Lowry OH, Rosebrough NI, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;210:26575. 15. Salter M, Knowles RG, Moncada S. Widespread tissue distribution, species and changes in activity of Ca’+dependent and Ca*+independent nitric oxide synthases. FEBS Lett 1991;291:145-9. 16. Conrad KP, Vill M, McGuire PG, Dal1 WG, Davis AK. Expression
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1996
17.
18. 19. 20.
21.
22.
23.
of nhric oxide synthase by syncytiotrophoblast in human placental villi. FASEB J 1993;7:1269-76. Myatt L, Brockman DE, Langdon G, Pollock JS. Constitutive calcium-dependent &form of nitric oxide synthase in the human placental villous vascular tree. Placenta 1993;14:373-83. De Belder AJ, Radomski MW, Why HJF, et al. Nitric oxide synthase activities in human myocardium. Lancet 1993;341:84-5. Hamid A, Springall DR, Riveros-Moreno V, et al. Induction of nitric oxide synthase in asthma. Lancet 1993;342:1510-3. King A, Loke YW. Uterine large granular lymphocytes: A possible role in embryonic implantation? Am J Obstet Gynecol 1990;162: 308-10. Jauniaux E, Burton GJ, Moscoso GJ, Hustin J. Development of the early human placenta: A morphometric study. Placenta 1991;12: 269-76. Sladek SM, Regenstein AC, Lykins D, Roberts JM. Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy. Am J Obstet Gynecol 1993;169:1285-91. Kishikawa T. Alterations in the properties of the rat myometrium during gestation and postpartum. Jpn J Physiol 1981;31:515-36.
Address reprint requests to: Bruce Ramsay, MBBS, MRCOG Academic Department of Obstetrics and Gymecology Chelsea and Westminster Hospital 369 Fulhm Raad London SWlO 9NH United Kingdom
Receiwd April 11, 1995. Receivedin revisedform September28,199s. Accepted October 17,199s. Copyright 0 1996 by The American College of Obstetricians Gynecologists. Published by Elsevier Science Inc.
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and
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PhD, AND
MARK R. JOHNSON, MBBS, MRCP, Phi3 Objective: To study the changes in nitric oxide synthase activities in human myometrium and trophoblast throughout pregnancy and around delivery. Methods: Samples of villous trophoblast were collected from women undergoing elective c-n delivery at term (n = 12) or voluntary termination of pregnancy in the first (n = 27) or second (n = 11) trimesters of pregnancy. Myometrial samples were obtained from nonpregnant women undergoing hysterectomy tn = 5) and pregnant women both before tn = 7) and after (n = 7) the onset of spontaneous labor at term. Nitric oxide synthase activity was quantified for homogenized samples using the L-citrulline assay in the presence and absence of calcium. Results: The highest levels of nitric oxide synthase activity were found in first-trimester villi (range 2-29 nmol Lcitrulline/minute/g protein), with a significant fall in activity in the third trimester (range 2-10 nmot Gcihulhne/minute/g protein; P < .OOl for both calcium-dependent and calciumindependent activity). Myometrial activities were relatively low compared with those in the trophoblast (O-2 nmol L-citrulline/minute/g protein), with no significant differences in calcium-dependent activities between subgroups. Myometrial calcium-independent activities were lower in pregnant than in nonpregnant women (P = .007), with those in labor having levels higher than those not in labor (P = .048). Conclusion: Levels of nitric oxide synthase activity are relatively high in villous trophoblaat, particularly during the first trimester. Although the contribution to total nitric oxide production in the uterus by myometrial nitric oxide synthase appears to be relatively small, nitric oxide produced by the trophoblast may play a role in maintaining uterine quiescence by a paracrine effect. Further work is needed to test this hypothesis and explore other possible roles for trophoblast-derived nitric oxide in early pregnancy. (Obstet Gynecol 1996;87:249-53)
From the Academic Department of Obstetrics and Gymecology, Chelsen and Westminster Hospital, London, United Kingdom.
VOL.
87, NO.
2, FEBRUARY
1996
Preterm delivery is the single most important contributor to perinatal morbidity and mortality in the developed world. Although many causes have been identified, in most cases the underlying mechanisms remain obscure. Our limited understanding is reflected in our inability to treat this condition effectively.’ However, there are two new pharmacologic approaches that show promise. The first, an oxytocin receptor antagonist, atosiban, is a logical development because it is known that myometrial sensitivity to oxytocin is enhanced in preterm labor.* The second, a nitric oxide donor drug, glyceryl trinitrate, is more speculative because little is known about the role of nitric oxide in pretenn labor. Reports so far have centered on clinical observations of its efficacy.3,4 In 1980, Furchgott and Zawadski5 discovered that vascular endothelium could induce vasodilation by the production of endothelium-derived relaxing factor. This factor was identified subsequently as nitric oxide,6 which is formed from its substrate, L-arginine, by the enzyme nitric oxide synthase. This enzyme exists in both calcium-dependent and calcium-independent isoforms.7 Animal’ and human’ studies have confirmed the important contribution that nitric oxide makes to the physiologic control of vascular tone and have also shown nitric oxide to be important in the regulation of platelet aggregation, neurotransmission, and immune responses.7 Currently, the role of nitric oxide in the control of myometrial contractility is defined less clearly. Others have shown that administration of a nitric oxide donor drug will abolish contractions in rat uterine smooth muscle both in vitro and in viv0.l’ Studies in vitro have shown a similar effect on human myometrium.” Nitric oxide-induced relaxation was less in samples of rat myometrium obtained at delivery than in those from the second-trimester, suggesting that nitric oxide may
0029-7844/96/$15.00 SSDI 0029-7844(95MlO391-6
249
play a role in the complex control system that maintains uterine quiescence during pregnancy.‘2,‘3 There are no reports of the relationship between onset of *labor and nitric oxide production in human pregnancy. To investigate this, we measured nitric oxide synthase activities in human trophoblast throughout gestation and in myometrium obtained from nonpregnant and pregnant women before and after the onset of labor.
Materials and Methods The study was approved by the hospital ethics committee and informed consent was obtained from all subjects. All pregnant women were healthy and carried singleton pregnancies, and those who had been pregnant previously all had uneventful spontaneous vaginal deliveries. Women with medical conditions or a history of dysfunctional labor or cesarean delivery were excluded. Placental villi were collected at the time of first- and second-trimester terminations of pregnancy and cesarean delivery before the onset of labor. The indication for cesarean was breech presentation of the fetus, with an otherwise uncomplicated obstetric history in each case. The gestational ages of first- and second-trimester pregnancies were estimated by ultrasonic measurement of fetal crown-rump length immediately before the procedure, and third-trimester pregnancies were dated from the onset of the last menstrual period (confirmed at a 20-week ultrasound scan). Placental villi were collected from 27 first- (mean gestation It_ standard deviation 9.8 t 1.4 weeks), 11 second- (17.6 t 2.5 weeks), and 12 third- (38.5 % 1.3 weeks) trimester pregnancies. Myometrial samples were obtained from nonpregnant women of childbearing age at the time of hysterectomy (n = 5) and from pregnant women at cesarean before (n = 7) and after (n = 7) the onset of labor. The indication for cesarean delivery was breech presentation for the pre-labor group and abnormal fetal heart rate (FHR) pattern in the labor group. All women in the labor group had gone into spontaneous labor at 37-40 weeks’ gestation and had been in established labor for at least 4 hours before delivery. Hysterectomies were all performed for dysfunctional uterine bleeding unresponsive to medication but not associated with structural abnormalities (eg, leiomyomas). All women undergoing hysterectomy were of proven fertility and delivered spontaneously per vaginam. Samples were taken from the upper edge of the transverse uterine incision at cesarean and from the anterior wall of the uterus after hysterectomy. They were weighed and frozen immediately in liquid nitrogen for storage at -70C for up to 4 weeks until analysis.
250
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et al
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For analysis, the tissue sample was mixed with five volumes of 0.25 mol/L sucrose containing 50 mmol/L Tris-HCl, pH 7.0, 1 mmol/L dithiothreitol, 1 mmol/L ethylenediaminetetra-acetic acid, 100 Fg/mL phenylmethylsulphonyl fluoride, 10 Fg/mL of soya-bean inhibitor, and 2 Fg/mL of apoprotinin, and was homogenized in four bursts of 15 seconds over 2 minutes at 4C. After centrifugation for 5 minutes at 11,000 X g, supernatants were kept on ice for no longer than 15 minutes before assay. Aliquots of supernatants were dispensed into 0.1 mol/L NaOH and kept at 4C for protein determination by the method of Lowry et a1.14 Nitric oxide synthase activity was measured by the activity method of Salter et a1.i5 Calcium-dependent was determined from the difference between the [‘4Clcitrulline produced from control samples and samples containing 1 mmol/L L-NMMA. Calcium-independent activity was determined from the difference between the [‘%Z] citrulline produced by the samples containing 1.5 mmol/L ethylene-bis(oxy-ethylenenitrilo~tetra-acetic acid (EGTA) with 1 mmol/L L-NMMA and those containing 1.5 mmol/L EGTA alone. Duplicate assays were performed for 16 minutes for myometrial samples and 8 minutes for trophoblast samples, with blanks at zero time points. These timings were based on our earlier experiments demonstrating the linearity of the assay together with the large ditIerence in activities between trophoblast and myometrial samples. The limit of detection of the assay was approximately 0.2 run01 of L-citrulline formed per minute per gram of tissue protein. Statistical comparison of groups was carried out using Km&al-Wallis analysis of variance test for multiple indices and MannWhitney U test when appropriate.
Results In the placental villi, both calcium-dependent and independent nitric oxide synthase activities were present in normal human villous tissue throughout pregnancy, with significantly higher levels of activity in the first trimester than in the third (P < .OOl for both isoforms) (Figures 1 and 2). In the myometrium, the nitric oxide synthase activity was much lower than in the trophoblast (range O-2 versus O-29 run01 L-citrulline/minute/g protein; P < .OOl). The calcium-dependent activities were statistically similar in all subgroups (Figure 3). The calcium-independent activities were significantly lower in the pregnant compared with the nonpregnant uterus (P = 007) (Figure 4). The lowest activities were seen in myometrial samples taken before the onset of labor, which were significantly less than samples taken after a period of labor 0’ = .048).
Obstetrics 6 Gynecology
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0 a
0
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0.
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Trimester OFRsetpncy Figure 1. Calcium-dependent nitric oxide synthase activities in villous trophoblast throughout gestation.
Discussion We found nitric oxide synthase activity in myometrium and trophoblast throughout gestation. First-trimester trophoblast tissue had the highest activities, which fell with advancing gestational age to levels at term similar to those reported previously by other investigators.‘6,17
Figure 3. Calcium-dependent nitric oxide synthase activities in myometrium from nonpregnant and pregnant women before and after the onset of labor at term.
It is worth noting that the first-trimester levels of calcium-independent activity are as high as those reported previously only in pathophysiologic inflammatory states.‘8,‘9 Calcium-independent nitric oxide synthase usually is produced in response to cytokines and requires new protein synthesis to occur. This was first shown to occur
0
I
1
2
3
Trimesterof Pregeaecy Figure 2. Calcium-independent nitric oxide synthase activities in villous trophoblast throughout gestation.
VOL. 87, NO. 2, FEBRUARY
1996
l
0
0 O-
,
L&or
-
$ I
I
I
Nonplrgwl
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Figure 4. Calcium-dependent nitric oxide synthase activities in myometrium from nonpregnant and pregnant women before and after the onset of labor at term.
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et al
Nitric
Oxide Synfhase
251
in macrophages in inflammatory conditions. During early pregnancy, the trophoblast is at its most active, invading deeply into the decidua and myometrium. The decidua is known to be infiltrated by lymphocyteszo and, indeed, the maternal response to implantation has been likened to an inflammatory process by several authors.21 The high levels of calcium-independent activity shown here may be a reflection of such a reaction. At implantation, it would also be advantageous for the trophoblast to suppress uterine activity and the maternal immune response whilst encouraging local vasodilation. A role for nitric oxide in the modulation of some or all of these processes would be consistent with the higher levels of nitric oxide synthase activity found in first-trimester villi. The subsequent decline in trophoblast-derived nitric oxide synthase activity, which occurred with advancing gestational age, took place against a background of decreasing uteroplacental resistance to blood flo~.~l This supports the notion of trophoblast-derived nitric oxide having other roles beyond simple vasodilation of the uteroplacental circulation. All the myometrial nitric oxide synthase activities were significantly lower than those from villous trophoblast, with calcium-independent activities showing some variation in relation to the onset of labor. The calcium-dependent activities may derive from the endothelium of blood vessels within the uterus. The calcium-independent activities were less in pregnant myometrium than in nonpregnant, with the lowest values being obtained before the onset of labor rather than during labor. The diIIerence in calcium-independent activities between the labor and nonlabor groups barely achieves statistical significance (P = .048). The small numbers of myometrial samples mean that this part of the study has limited statistical power compared with the trophoblast data (80 versus 95% power). In addition, the low levels of myometrial activity make intra-assay variation a significant factor. Although these data would suggest that nitric oxide production by the myometrium is unlikely to have a role in the maintenance of myometrial quiescence, there are several complicating factors to take into account. First, there are problems of study design inherent in observational studies of human pregnancy. Also, the myometrium from the labor subgroup may not have been truly “normal.” All the women in this group were undergoing cesarean delivery (after a period of spontaneous labor at term) for abnormal FHR. However, three of the seven also had an element of poor progress in the clinical decision for cesarean; therefore, the characteristics of myometrium taken from these women may have been abnormal. This point is difficult to answer because the only truly “normal” labors ending in cesarean occur
252
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in the unusual circumstance of a woman already booked for surgery (eg, a primigravida with breech presentation) arriving at the hospital in spontaneous labor. The argument can be extended to cover the hysterectomy samples, too, because dysfunctional bleeding may also produce abnormal results. We attempted to minimize this problem by selecting women of proven fertility with uneventful past obstetric histories. Second, there may be enhanced responsiveness of the myometrium to nitric oxide during pregnancy in much the same way as there is to oxytocin. There is no information at present to support or refute this notion, but this question must be answered by future work. Finally, nitric oxide released by the high levels of trophoblast enzyme activity may exert a paracrine effect on the myometrium and thus contribute to the maintenance of uterine quiescence in this way. Support for this view has already come from animal studies that have shown a reduction in decidual nitric oxide synthase activity with advancing gestation, reaching a nadir on the day of delive$’ and paralleled by a decrease in myometrial guanosine 3’,5’,-cyclic monophosphate levels.23 It is possible that high levels of nitric oxide derived from the trophoblast influence the expression of the calcium-independent nitric oxide synthase in the myometrium by negative feedback, switching off the production of new messenger RNA, thus explaining the lower levels in pregnant specimens than in those taken at routine hysterectomy. At present, there is no direct evidence to support this notion. These data demonstrate appreciable amounts of nitric oxide synthase activity in the first-trimester villous trophoblast and suggest a physiologic role for nitric oxide in the early stages of pregnancy. The relatively low levels of nitric oxide synthase in myometrium and the decline in activity between nonpregnant and cesarean delivery samples make it unlikely that nitric oxide derived from the myometrium has any role in the maintenance of uterine quiescence, although it is still possible that trophoblast-derived nitric oxide may influence uterine contractility. Further work is needed to establish the role of nitric oxide produced by the trophoblast in early pregnancy, but possibilities include local vasodilation, control of trophoblastic invasion, and the prevention of fetal rejection.
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3. Greenspoon JS, Koviacic A. A breech extraction facilitated by glyceryl trinitrate spray. Lancet 1991;338:124-5. 4. Lees C, Campbell S, Jauniaux E, et al. Arrest of preterm labour and prolongation of gestation with glyceryl trinitrate, a nitric oxide donor. Lancet 1994;343:1325-6. 5. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 19&?0;288:373-6. 6. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium derived relaxing factor. Nature 1987;327:524-6. 7. Moncada S, Palmer RM, Hlggs EA. Nitric oxide: Physiology, pathophysiology and pharmacology. Pharmacol Rev 1991;43:10942. 8. Rees DD, Palmer RMJ, Moncada S. The role of endothelium derived nitric oxide in the regulation of blood flow. Proc Nat1 Acad Sci U S A 1989;86:3375-8. 9. Valiance P, Collier J, Moncada S. Effects of endothelium derived nitric oxide on peripheral arteriolar tone in man. Lancet 1989;ii: 997-looo. 10. Natuzzi ES, Ursell PC, Harrison M, Buscher C, Reimer RK. Nitric oxide synthase activity in the pregnant uterus decreases at parturition. Biochem Biophys Res Comm 1993;194:1-8. 11. Yallampalli C, Garfield RE, Byam-Smith M. Nitric oxide inhibits uterine contractility during pregnancy but not during delivery. Endocrinol 1993;133:1%99-902. 12. Izurni H, Yallampalli C, Garfield RE. Gestational changes in L-arginine induced relaxation of pregnant rat and human myometrial smooth muscle. Am J Obstet Gynecol 1993;169:1327-37, 13. Yallampalli C, lzumi H, Byam-Smith M, Garfield RE. An Larginine-nitric oxide-cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pregnancy. Am J Obstet Gynecol 1994;170:175-85. 14. Lowry OH, Rosebrough NI, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;210:26575. 15. Salter M, Knowles RG, Moncada S. Widespread tissue distribution, species and changes in activity of Ca’+dependent and Ca*+independent nitric oxide synthases. FEBS Lett 1991;291:145-9. 16. Conrad KP, Vill M, McGuire PG, Dal1 WG, Davis AK. Expression
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Address reprint requests to: Bruce Ramsay, MBBS, MRCOG Academic Department of Obstetrics and Gymecology Chelsea and Westminster Hospital 369 Fulhm Raad London SWlO 9NH United Kingdom
Receiwd April 11, 1995. Receivedin revisedform September28,199s. Accepted October 17,199s. Copyright 0 1996 by The American College of Obstetricians Gynecologists. Published by Elsevier Science Inc.
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