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Oxytocics reverse the tocolytic effect of glyceryl trinitrate on the human uterus
British Journal of Obstetrics and Gynaecology February 2001, Vol. 108, pp. 164±168
Oxytocics reverse the tocolytic effect of glyceryl trinitrate on the human uterus L.C. Lau a, P.G. Adaikan a,*, S. Arulkumaran b, S.C. Ng a Objectives To investigate the effect of glyceryl trinitrate on isolated human pregnant uterine strips and whether the uterine relaxation induced by glyceryl trinitrate could be reversed by oxytocics used in current clinical practice. Design In vitro pharmacological study. Setting Department of Obstetrics & Gynaecology, National University of Singapore, National University Hospital. Participants Eighteen women who delivered by caesarean section at term. Methods Myometrial strips were preloaded with an initial tension of 1.5g in organ baths containing KrebsHenseleit solution which was aerated with oxygen in 5% carbon dioxide and maintained at 378C, pH 7.4. The effect of glyceryl trinitrate was studied in strips displaying regular spontaneous contractions. The ability of oxytocin, ergometrine or prostaglandin F2a to stimulate uterine contractions was assessed in strips where uterine activity was signi®cantly inhibited by glyceryl trinitrate. Results Glyceryl trinitrate reduced the amplitude and frequency of spontaneous uterine contractions in a concentration-dependent manner, although the sensitivity of the myometrial strips varied considerably from one specimen to another. The concentration of glyceryl trinitrate producing complete inhibition of myometrial contractions ranged from 44-705mM. In the presence of glyceryl trinitrate which markedly depressed spontaneous contractions, oxytocin (20mU/mL), ergometrine (6.15mM) and prostaglandin F2a (6.15mM) were capable of reversing the uterine activity to either higher than or the untreated level of contractility. Conclusions This study demonstrates that glyceryl trinitrate is a potent uterine relaxant in vitro and that the tocolytic effect could be reversed with ease by oxytocics.
INTRODUCTION The drugs currently used in the treatment of preterm labour include magnesium sulphate, calcium channel blockers, beta-adrenoceptor agonists and prostaglandin synthetase inhibitors. However, most of the available drugs lack uterine selectivity and have adverse side effects for the woman and the infant 1. Glyceryl trinitrate (10 mg patches transdermally) in an uncontrolled study was reported to be effective in inhibiting preterm labour and prolonging gestation 2. Similarly, intravenous infusion of glyceryl trinitrate in pregnant sheep inhibited active labour 3. Glyceryl trinitrate has been shown to be an relaxant of rat myometrium strips in vitro 4. The pharmacologi-
a
Department of Obstetrics and Gynaecology, National University Hospital, Lower Kent Ridge Road, Singapore 119074 b Academic Division of Obstetrics & Gynaecology, University of Nottingham, Derby City General Hospital, Derby, UK * Correspondence: Dr P. G. Adaikan, Department of Obstetrics and Gynaecology, National University Hospital, Lower Kent Ridge Road, Singapore 119074. q RCOG 2001 British Journal of Obstetrics and Gynaecology PII: S03 06-5456(00)0003 5-8
cally active principle of glyceryl trinitrate is nitric oxide, which is an important mediator of relaxation of various smooth muscles including vascular, gastrointestinal and urogenital muscles. Nitric oxide binds to the ferrohaeme centre and activates soluble guanylate cyclase which results in increased cGMP levels 5,6, that acts as the second messenger of muscle relaxation 7. Two reports on the effect of glyceryl trinitrate on human uterine muscle in vitro have appeared recently. Glyceryl trinitrate induced a dose-dependent inhibition of spontaneous as well as oxytocin-induced myometrial contractions 8,9. Beta-mimetic drugs such as terbutaline can be used for acute tocolysis in cases of fetal distress 10. If delivery is expected by caesarean section immediately after uterine relaxation induced by betamimetic drugs, postpartum haemorrhage may ensue, as oxytocic drugs are ineffective in reversing the tocolysis caused by beta-mimetic drugs. A beta-blocking drug needs to be given to reverse the action of beta-mimetic drug to allow the action of oxytocin 11. Administration of the beta-blocking drug may have its disadvantages. It will be of value if uterine relaxation induced by glyceryl trinitrate can be reversed by oxytocics used in current clinical practice. The purpose of this study was to investigate the effect of glyceryl trinitrate on isolated human pregnant uterine strips and whether the tocolysis induced www.bjog-elsevier.com
EFFECT OF GLYCERYL TRINITRATE ON THE UTERUS 165
by glyceryl trinitrate could be reversed by some common oxytocic drugs.
METHODS Approval for this study was given by the ethic committee and informed consent was obtained from the participating women. Eighteen pregnant myometrium specimens were obtained from the lower segment during elective caesarean section at term. They were immediately immersed in chilled Krebs-Henseleit solution and transferred to the laboratory. The specimens were used within 24 hours of collection. Myometrial strips (n 67) of 2 £ 2 £ 10 mm were suspended in 10 mL thermostatically controlled organ baths (378C) containing Krebs-Henseleit solution, and aerated continuously with 95% O2 / 5% CO2 at pH 7.4. The Krebs-Henseleit solution (all in mM) consisted of sodium chloride 115, potassium chloride 4.7, calcium chloride 1.8, potassium dihydrogen orthophosphate 1.2, magnesium sulphate 1.2, glucose 8.5, sodium bicarbonate 22.1 and disodium EDTA 0.03. Uterine strips were loaded with an initial tension of 1.5g and left to equilibrate for at least 1 to 2 hour. Responses were recorded on a MacIntosh computer using MacLab analog to digital conversion and Chart software. When regular spontaneous contractions were established for about an hour, glyceryl trinitrate was added to the baths and uterine motility was recorded for 30 minutes. Oxytocin (20mU/mL), ergometrine (6.15mM) or prostaglandin F2a (6.15mM) were then administered and the effect on uterine muscle recorded for a further 30 minutes. Each uterine strip was subjected to only one experiment with glyceryl trinitrate and an oxytocic as signi®cant tachyphylaxis was observed with repeated doses of oxytocin, ergometrine and prostaglandin F2a . Uterine activity was quanti®ed by measuring the area under the contraction curves (which re¯ects the product of amplitude and frequency of the uterine contractions) for each control and treated period of 30 minutes. Parallel control strips (n 18 from 18 women) were also tested to monitor time-related changes in uterine activity. Data was expressed as means (SEM). The results were tested using multivariate ANOVA for repeated measures to compare the uterine activity before and after treatment with glyceryl trinitrate and oxytocics. Values were considered signi®cant at P , 0.05. Working concentrations of glyceryl trinitrate (Schwarzpharma, Monheim, Germany), synthetic oxytocin (Syntocinon, Sandoz, Switzerland), ergometrine maleate (DBL, Mulgrave, Australia) and prostaglandin F2a tromethamine salt (Pharmacia & Upjohn, Kalamazoo, USA) were prepared freshly on the day of experiment. q RCOG 2001 Br J Obstet Gynaecol 108, pp. 164±168
RESULTS Glyceryl trinitrate produced concentration-dependent inhibition of uterine activity as re¯ected by the reduction in the frequency and amplitude of contractions (Fig.1). Low concentrations of glyceryl trinitrate produced transient partial inhibition while higher concentrations were able to abolish the uterine contractions completely. In some tissues the concentration of glyceryl trinitrate producing complete inhibition ranged from 44 to 705mM. Although the spontaneous uterine activity in these strips was almost identical, the sensitivity of the myometrial strips to glyceryl trinitrate varied considerably from one specimen to another. The inhibitory effect of glyceryl trinitrate on spontaneously contracting myometrial strips was immediately reversed upon washing of the organ baths. The ability of oxytocics to contract uterine muscle in the presence of glyceryl trinitrate is shown in Figs. 2 & 3. Statistical analysis revealed that glyceryl trinitrate reduced uterine activity to a great extent (P , 0.001, Table 1). Oxytocin (20mU/mL) added to the bath 30 minutes after each addition of glyceryl trinitrate was capable of eliciting marked contractions (n 24 from 8 women); these responses which were 1.5- to 2.8-fold stronger than the spontaneous contractility recorded during the control period were statistically signi®cant compared with basal uterine activity (P , 0.001) (Table 1). In the presence of glyceryl trinitrate (88-352mM), equimolar concentrations of ergometrine (n 12 from four women) and prostaglandin F2a (n 13 from six women) resulted in a signi®cant increase in uterine motility (P 0.012 and P , 0.001 respectively); this was not signi®cant compared with control uterine activity (Table 1).
Fig. 1. A representative trace showing the concentration-dependent relaxant effect of glyceryl trinitrate (GTN) (cumulative bath concentration shown in mM) on spontaneous contractions in human pregnant myometrium in vitro. The contractile activity was maintained constant in the control experiment.
166 L.C. LAU ET AL.
showed that glyceryl trinitrate is a potent uterine muscle relaxant although there was marked variation in the concentrations of glyceryl trinitrate required to inhibit uterine contractions completely. This could be due to variations in enzyme levels or factors affecting the metabolism of glyceryl trinitrate to nitric oxide in individual myometrial strips in vitro, since glyceryl trinitrate requires bioconversion to nitric oxide in order to exert its pharmacological effect. The biochemical pathway(s) responsible for the formation of nitric oxide from glyceryl trinitrate are not well characterised, although one enzyme probably involved is cytochrome P450, as demonstrated in microsomes from rat liver 12 and cell lines of rat lung ®broblasts 13. While the nitric oxide derived from glyceryl trinitrate is suf®cient for the tocolytic effect, it appears that endogenous nitric oxide
Fig. 2. Representative recordings showing that oxytocin (20mU/mL), ergometrine (6.15mM) and PGF2a (6.15mM) effectively overcame the marked inhibitory effect of glyceryl trinitrate (GTN) on the spontaneously contracting human pregnant myometrium in vitro.
DISCUSSION This is the ®rst study in vitro on human myometrium showing the ease of reversibility by oxytocics of uterine relaxation induced by glyceryl trinitrate. The results
Fig. 3. Effect of glyceryl trinitrate (GTN) on uterine activity and the reversal of GTN-induced tocolysis by oxytocin (20mU/mL), ergometrine (6.15mM) and PGF2a (6.15mM) on pregnant human myometrium in vitro. Figs. in parentheses denote the number of experiments.
q RCOG 2001 Br J Obstet Gynaecol 108, pp. 164±168
EFFECT OF GLYCERYL TRINITRATE ON THE UTERUS 167 Table 1. Effect of oxytocics on glyceryl trinitrate (GTN)-induced tocolysis in pregnant human myometrial strips.Values are given as means (SEM) unless otherwise shown, of uterine activity measured by the area under the contraction curves in 30 minutes. Figs. in parentheses denote (number of experiments / number of patients). P-level of 0.05 or less is indicative of a signi®cant difference. (a) uterine activity during GTN treatment compared to control; (b) uterine activity after GTN and oxytocics compared to control; (c) uterine activity after GTN and oxytocics compared to that during GTN treatment. Uterine activity Oxytocics
GTN (mM)
Controls
Oxytocin: 20mU/mL (24/8)
44 88 176 705 88 176 176 352
2649 (657) 2543 (533) 1765 (430) 2656 (794) 2199 (486) 1763 (246) 1512 (290) 1972 (209)
Ergometrine: 6.15mM (12/6) PGF2a: 6.15mM (13/6)
GTN 1057 (812) 826 (486) 400 (98.5) 288 (167) 364 (151) 146 (75.2) 697 (264) 729 (140)
production may not exert a physiological effect on the sensitivity of myometrium to glyceryl trinitrate. The precursor of nitric oxide, L-arginine, and an inhibitor of nitric oxide synthase, Nv - NO-L-arginine, failed to affect the spontaneous contractions of the pregnant uterine muscle strips (data not shown). Besides glyceryl trinitrate, other nitric oxide donors such as sodium nitroprusside and diethylamine/nitric oxide have also been shown to inhibit human uterine contractions in vitro 8,14. The increased effectiveness of glyceryl trinitrate in vivo as a uterine relaxant is likely to be due to its conversion to nitric oxide by vascular smooth muscle cells and endothelial cells which are known to liberate nitric oxide from glyceryl trinitrate 15±17 . The prompt recovery of spontaneous activity observed in the present study after removal of glyceryl trinitrate from the bathing medium is characteristic of the short duration of action of nitric oxide. Indeed, clinically rapid uterine relaxation was attainable by a bolus injection of 50-200mg glyceryl trinitrate for manual removal of a retained placenta 9,18, atraumatic delivery during caesarean section 9, twin extraction 19,20and for the treatment of uterine inversion following vaginal delivery 21. In these case reports, uterine relaxation was achieved within 90 seconds, and lasted about one minute; the inhibitory effect on uterine contractility disappeared spontaneously without an antidote. The results of this study con®rm that tocolysis induced by glyceryl trinitrate can be reversed with ease by the aforementioned oxytocic compounds. This observation is signi®cant, in that preterm labour treated with glyceryl trinitrate patches are likely to respond to oxytocin or ergometrine, should these oxytocics be indicated for emergency obstetric manoeuvres. Furthermore, our in vitro results agree with the clinical observation that glyceryl trinitrate and oxytocin could be used to produce consecutive periods of uterine relaxation and contraction for uterine exploration and manual removal of a retained placenta 22. In another case report intravenous oxytocin and intramuscular ergotrate also succeeded in contracting q RCOG 2001 Br J Obstet Gynaecol 108, pp. 164±168
P GTN 1 oxytocics 7031 (1269) 5985 (1659) 4665 (1382) 3959 (924) 2257 (734) 1439 (238) 2245 (480) 1790 (348)
a
b
c
, 0.001
, 0.001
, 0.001
, 0.001
1.0
0.012
, 0.001
0.207
, 0.001
the uterus after intravenous nitroglycerin was administered for uterine relaxation of an inverted uterus 21. In contrast, oxytocic drugs are ineffective in reversing the tocolysis caused by beta-mimetic drugs. Therefore, our observation of reversal by oxytocics of relaxation of the uterus induced by glyceryl trinitrate would suggest that glyceryl trinitrate should be used for acute tocolysis in clinical obstetrics. Acknowledgements The authors would like to thank Ms. J. Tang, Ms. X.X. Chen and Ms. K. Veena for their skillful technical assistance and the Staff of Delivery Suites for their contribution to this study. References 1. Black RS, Flint S, Lees C, Campbell S. Preterm labour and delivery. Eur J Pediatr 1996;155(Suppl 2):S2±S7. 2. 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±1326. 3. Heymann MA, Bootstaylor B, Roman C, et al. Glyceryl trinitrate stops active labour in sheep. In: Moncada S, Feelisch M, Busse R, Higgs EA, editors. The Biology of Nitric Oxide Part 3 Physiological and Clinical Aspects. London: Portland Press, 1994:201±202. 4. Diamond J, Marshall J. A comparison of effects of various smooth muscle relaxants on the electrical and mechanical activity of rat uterus. J Pharmacol Exp Ther 1969;168:21±30. 5. Ignarro LJ, Adams JB, Horwitz PM, Wood KS. Activation of soluble guanylate cyclase by NO-hemoproteins involves NO-heme exchange. J Biol Chem 1986;261:4997±5002. 6. Gruetter CA, Gruetter DY, Lyon JE, Kadowitz PJ, Ignarro LJ. Relationship between cyclic guanosine 3':5'-monophosphate formation and relaxation of coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide: effects of methylene blue and methaemoglobin. J Pharmacol Exp Ther 1981;219:181±186. 7. Murad P. Cyclic guanosine monophosphate as a mediator of vasodilation. J Clin Invest 1986;78:1±5. 8. Norman JE, Ward LM, Martin W, et al. Effects of cGMP and the nitric oxide donors glyceryl trinitrate and sodium nitroprusside on contrac-
168 L.C. LAU ET AL.
9. 10. 11. 12.
13.
14.
15.
tions in vitro of isolated myometrial tissue from pregnant women. J Reprod Fertil 1997;110:249±254. Axemo P, Fu X, Lindberg B, Ulmsten U, Wessen A. Intravenous nitroglycerin for rapid uterine relaxation. Acta Obstet Gynecol Scand 1998;77:50±53. Ingermarrson I, Arulkumaran S, Ratnam SS. Single injection of terbutaline in term labour. I. Effect of fetal pH on cases with prolonged bradycardia. Am J Obstet Gynecol 1985;153:859±865. Andersson KE, Ingermarsson I, Persson CGA. Effects of terbutaline on human uterine motility at term. Acta Obstet Gynecol Scand 1974;53:1±8. Servent D, Delaforge M, Ducrocq C, Mansuy D, Lenfant M. Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: involvement of cytochrome P-450. Biochem Biophy Res Commun 1989;160:1210±1216. Schroder H, Machunsky C, Schror K. Cytochrome P-450 mediates bioactivation of organic nitrates in cultured cells. In: Moncada S, Marletta MA, Hibbs JB, Higgs EA, editors. The Biology of Nitric Oxide Part 2: Enzymology, Biochemistry and Immunology , London: Portland Press, 1992:38±40. Buhimschi I, Yallampalli C, Dong Y-L, Gar®eld RE. Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility during pregnancy. Am J Obstet Gynecol 1995;172:1577±1584. Benjamin N, Dulton JAE, Ritter JM. Human vascular smooth muscle cells inhibit platelet aggregation when incubated with glyceryl trini-
16. 17.
18. 19. 20. 21. 22.
trate: evidence for generation of nitric oxide. Br J Pharmacol 1991;102:847±850. Feelisch M, Kelm M. Biotransformation of organic nitrates to nitric oxide by vascular smooth muscle and endothelial cells. Biochem Biophy Res Commun 1991;180:286±293. Salvemini D, Mollace V, Pistelli A, Anggard E, Vane J. Metabolism of glyceryl trinitrate to nitric oxide by endothelial cells and smooth muscle cells and its induction by Escherichia coli lipopolysaccharide. Proc Natl Acad Sci USA 1992;89:982±986. DeSimone CA, Norris MC, Leighton BL. Intravenous nitroglycerin aids manual extraction of a retained placenta. Anaesthesiology 1990;73:787. Wessen A, Elowsson P, Axemo P, Lindberg B. The use of intravenous nitroglycerin for emergency cervico-uterine relaxation. Acta Anaesthesiol Scand 1995;39:847±849. Mayer DC, Weeks SK. Antepartum uterine relaxation with nitroglycerin at Caesarean delivery. Can J Anaesth 1992;39:166±169. Altabef KM, Spencer JT, Zinberg S. Intravenous nitroglycerin for uterine relaxation of an inverted uterus. Am J Obstet Gynecol 1992;166:1237±1238. Chan AS, Ananthanarayan C, Rolbin SH. Alternating nitroglycerin and syntocinon to facilitate uterine exploration and removal of an adherent placenta. Can J Anaesth 1995;42:335±337.
Accepted 17 June 2000
q RCOG 2001 Br J Obstet Gynaecol 108, pp. 164±168
Oxytocics reverse the tocolytic effect of glyceryl trinitrate on the human uterus L.C. Lau a, P.G. Adaikan a,*, S. Arulkumaran b, S.C. Ng a Objectives To investigate the effect of glyceryl trinitrate on isolated human pregnant uterine strips and whether the uterine relaxation induced by glyceryl trinitrate could be reversed by oxytocics used in current clinical practice. Design In vitro pharmacological study. Setting Department of Obstetrics & Gynaecology, National University of Singapore, National University Hospital. Participants Eighteen women who delivered by caesarean section at term. Methods Myometrial strips were preloaded with an initial tension of 1.5g in organ baths containing KrebsHenseleit solution which was aerated with oxygen in 5% carbon dioxide and maintained at 378C, pH 7.4. The effect of glyceryl trinitrate was studied in strips displaying regular spontaneous contractions. The ability of oxytocin, ergometrine or prostaglandin F2a to stimulate uterine contractions was assessed in strips where uterine activity was signi®cantly inhibited by glyceryl trinitrate. Results Glyceryl trinitrate reduced the amplitude and frequency of spontaneous uterine contractions in a concentration-dependent manner, although the sensitivity of the myometrial strips varied considerably from one specimen to another. The concentration of glyceryl trinitrate producing complete inhibition of myometrial contractions ranged from 44-705mM. In the presence of glyceryl trinitrate which markedly depressed spontaneous contractions, oxytocin (20mU/mL), ergometrine (6.15mM) and prostaglandin F2a (6.15mM) were capable of reversing the uterine activity to either higher than or the untreated level of contractility. Conclusions This study demonstrates that glyceryl trinitrate is a potent uterine relaxant in vitro and that the tocolytic effect could be reversed with ease by oxytocics.
INTRODUCTION The drugs currently used in the treatment of preterm labour include magnesium sulphate, calcium channel blockers, beta-adrenoceptor agonists and prostaglandin synthetase inhibitors. However, most of the available drugs lack uterine selectivity and have adverse side effects for the woman and the infant 1. Glyceryl trinitrate (10 mg patches transdermally) in an uncontrolled study was reported to be effective in inhibiting preterm labour and prolonging gestation 2. Similarly, intravenous infusion of glyceryl trinitrate in pregnant sheep inhibited active labour 3. Glyceryl trinitrate has been shown to be an relaxant of rat myometrium strips in vitro 4. The pharmacologi-
a
Department of Obstetrics and Gynaecology, National University Hospital, Lower Kent Ridge Road, Singapore 119074 b Academic Division of Obstetrics & Gynaecology, University of Nottingham, Derby City General Hospital, Derby, UK * Correspondence: Dr P. G. Adaikan, Department of Obstetrics and Gynaecology, National University Hospital, Lower Kent Ridge Road, Singapore 119074. q RCOG 2001 British Journal of Obstetrics and Gynaecology PII: S03 06-5456(00)0003 5-8
cally active principle of glyceryl trinitrate is nitric oxide, which is an important mediator of relaxation of various smooth muscles including vascular, gastrointestinal and urogenital muscles. Nitric oxide binds to the ferrohaeme centre and activates soluble guanylate cyclase which results in increased cGMP levels 5,6, that acts as the second messenger of muscle relaxation 7. Two reports on the effect of glyceryl trinitrate on human uterine muscle in vitro have appeared recently. Glyceryl trinitrate induced a dose-dependent inhibition of spontaneous as well as oxytocin-induced myometrial contractions 8,9. Beta-mimetic drugs such as terbutaline can be used for acute tocolysis in cases of fetal distress 10. If delivery is expected by caesarean section immediately after uterine relaxation induced by betamimetic drugs, postpartum haemorrhage may ensue, as oxytocic drugs are ineffective in reversing the tocolysis caused by beta-mimetic drugs. A beta-blocking drug needs to be given to reverse the action of beta-mimetic drug to allow the action of oxytocin 11. Administration of the beta-blocking drug may have its disadvantages. It will be of value if uterine relaxation induced by glyceryl trinitrate can be reversed by oxytocics used in current clinical practice. The purpose of this study was to investigate the effect of glyceryl trinitrate on isolated human pregnant uterine strips and whether the tocolysis induced www.bjog-elsevier.com
EFFECT OF GLYCERYL TRINITRATE ON THE UTERUS 165
by glyceryl trinitrate could be reversed by some common oxytocic drugs.
METHODS Approval for this study was given by the ethic committee and informed consent was obtained from the participating women. Eighteen pregnant myometrium specimens were obtained from the lower segment during elective caesarean section at term. They were immediately immersed in chilled Krebs-Henseleit solution and transferred to the laboratory. The specimens were used within 24 hours of collection. Myometrial strips (n 67) of 2 £ 2 £ 10 mm were suspended in 10 mL thermostatically controlled organ baths (378C) containing Krebs-Henseleit solution, and aerated continuously with 95% O2 / 5% CO2 at pH 7.4. The Krebs-Henseleit solution (all in mM) consisted of sodium chloride 115, potassium chloride 4.7, calcium chloride 1.8, potassium dihydrogen orthophosphate 1.2, magnesium sulphate 1.2, glucose 8.5, sodium bicarbonate 22.1 and disodium EDTA 0.03. Uterine strips were loaded with an initial tension of 1.5g and left to equilibrate for at least 1 to 2 hour. Responses were recorded on a MacIntosh computer using MacLab analog to digital conversion and Chart software. When regular spontaneous contractions were established for about an hour, glyceryl trinitrate was added to the baths and uterine motility was recorded for 30 minutes. Oxytocin (20mU/mL), ergometrine (6.15mM) or prostaglandin F2a (6.15mM) were then administered and the effect on uterine muscle recorded for a further 30 minutes. Each uterine strip was subjected to only one experiment with glyceryl trinitrate and an oxytocic as signi®cant tachyphylaxis was observed with repeated doses of oxytocin, ergometrine and prostaglandin F2a . Uterine activity was quanti®ed by measuring the area under the contraction curves (which re¯ects the product of amplitude and frequency of the uterine contractions) for each control and treated period of 30 minutes. Parallel control strips (n 18 from 18 women) were also tested to monitor time-related changes in uterine activity. Data was expressed as means (SEM). The results were tested using multivariate ANOVA for repeated measures to compare the uterine activity before and after treatment with glyceryl trinitrate and oxytocics. Values were considered signi®cant at P , 0.05. Working concentrations of glyceryl trinitrate (Schwarzpharma, Monheim, Germany), synthetic oxytocin (Syntocinon, Sandoz, Switzerland), ergometrine maleate (DBL, Mulgrave, Australia) and prostaglandin F2a tromethamine salt (Pharmacia & Upjohn, Kalamazoo, USA) were prepared freshly on the day of experiment. q RCOG 2001 Br J Obstet Gynaecol 108, pp. 164±168
RESULTS Glyceryl trinitrate produced concentration-dependent inhibition of uterine activity as re¯ected by the reduction in the frequency and amplitude of contractions (Fig.1). Low concentrations of glyceryl trinitrate produced transient partial inhibition while higher concentrations were able to abolish the uterine contractions completely. In some tissues the concentration of glyceryl trinitrate producing complete inhibition ranged from 44 to 705mM. Although the spontaneous uterine activity in these strips was almost identical, the sensitivity of the myometrial strips to glyceryl trinitrate varied considerably from one specimen to another. The inhibitory effect of glyceryl trinitrate on spontaneously contracting myometrial strips was immediately reversed upon washing of the organ baths. The ability of oxytocics to contract uterine muscle in the presence of glyceryl trinitrate is shown in Figs. 2 & 3. Statistical analysis revealed that glyceryl trinitrate reduced uterine activity to a great extent (P , 0.001, Table 1). Oxytocin (20mU/mL) added to the bath 30 minutes after each addition of glyceryl trinitrate was capable of eliciting marked contractions (n 24 from 8 women); these responses which were 1.5- to 2.8-fold stronger than the spontaneous contractility recorded during the control period were statistically signi®cant compared with basal uterine activity (P , 0.001) (Table 1). In the presence of glyceryl trinitrate (88-352mM), equimolar concentrations of ergometrine (n 12 from four women) and prostaglandin F2a (n 13 from six women) resulted in a signi®cant increase in uterine motility (P 0.012 and P , 0.001 respectively); this was not signi®cant compared with control uterine activity (Table 1).
Fig. 1. A representative trace showing the concentration-dependent relaxant effect of glyceryl trinitrate (GTN) (cumulative bath concentration shown in mM) on spontaneous contractions in human pregnant myometrium in vitro. The contractile activity was maintained constant in the control experiment.
166 L.C. LAU ET AL.
showed that glyceryl trinitrate is a potent uterine muscle relaxant although there was marked variation in the concentrations of glyceryl trinitrate required to inhibit uterine contractions completely. This could be due to variations in enzyme levels or factors affecting the metabolism of glyceryl trinitrate to nitric oxide in individual myometrial strips in vitro, since glyceryl trinitrate requires bioconversion to nitric oxide in order to exert its pharmacological effect. The biochemical pathway(s) responsible for the formation of nitric oxide from glyceryl trinitrate are not well characterised, although one enzyme probably involved is cytochrome P450, as demonstrated in microsomes from rat liver 12 and cell lines of rat lung ®broblasts 13. While the nitric oxide derived from glyceryl trinitrate is suf®cient for the tocolytic effect, it appears that endogenous nitric oxide
Fig. 2. Representative recordings showing that oxytocin (20mU/mL), ergometrine (6.15mM) and PGF2a (6.15mM) effectively overcame the marked inhibitory effect of glyceryl trinitrate (GTN) on the spontaneously contracting human pregnant myometrium in vitro.
DISCUSSION This is the ®rst study in vitro on human myometrium showing the ease of reversibility by oxytocics of uterine relaxation induced by glyceryl trinitrate. The results
Fig. 3. Effect of glyceryl trinitrate (GTN) on uterine activity and the reversal of GTN-induced tocolysis by oxytocin (20mU/mL), ergometrine (6.15mM) and PGF2a (6.15mM) on pregnant human myometrium in vitro. Figs. in parentheses denote the number of experiments.
q RCOG 2001 Br J Obstet Gynaecol 108, pp. 164±168
EFFECT OF GLYCERYL TRINITRATE ON THE UTERUS 167 Table 1. Effect of oxytocics on glyceryl trinitrate (GTN)-induced tocolysis in pregnant human myometrial strips.Values are given as means (SEM) unless otherwise shown, of uterine activity measured by the area under the contraction curves in 30 minutes. Figs. in parentheses denote (number of experiments / number of patients). P-level of 0.05 or less is indicative of a signi®cant difference. (a) uterine activity during GTN treatment compared to control; (b) uterine activity after GTN and oxytocics compared to control; (c) uterine activity after GTN and oxytocics compared to that during GTN treatment. Uterine activity Oxytocics
GTN (mM)
Controls
Oxytocin: 20mU/mL (24/8)
44 88 176 705 88 176 176 352
2649 (657) 2543 (533) 1765 (430) 2656 (794) 2199 (486) 1763 (246) 1512 (290) 1972 (209)
Ergometrine: 6.15mM (12/6) PGF2a: 6.15mM (13/6)
GTN 1057 (812) 826 (486) 400 (98.5) 288 (167) 364 (151) 146 (75.2) 697 (264) 729 (140)
production may not exert a physiological effect on the sensitivity of myometrium to glyceryl trinitrate. The precursor of nitric oxide, L-arginine, and an inhibitor of nitric oxide synthase, Nv - NO-L-arginine, failed to affect the spontaneous contractions of the pregnant uterine muscle strips (data not shown). Besides glyceryl trinitrate, other nitric oxide donors such as sodium nitroprusside and diethylamine/nitric oxide have also been shown to inhibit human uterine contractions in vitro 8,14. The increased effectiveness of glyceryl trinitrate in vivo as a uterine relaxant is likely to be due to its conversion to nitric oxide by vascular smooth muscle cells and endothelial cells which are known to liberate nitric oxide from glyceryl trinitrate 15±17 . The prompt recovery of spontaneous activity observed in the present study after removal of glyceryl trinitrate from the bathing medium is characteristic of the short duration of action of nitric oxide. Indeed, clinically rapid uterine relaxation was attainable by a bolus injection of 50-200mg glyceryl trinitrate for manual removal of a retained placenta 9,18, atraumatic delivery during caesarean section 9, twin extraction 19,20and for the treatment of uterine inversion following vaginal delivery 21. In these case reports, uterine relaxation was achieved within 90 seconds, and lasted about one minute; the inhibitory effect on uterine contractility disappeared spontaneously without an antidote. The results of this study con®rm that tocolysis induced by glyceryl trinitrate can be reversed with ease by the aforementioned oxytocic compounds. This observation is signi®cant, in that preterm labour treated with glyceryl trinitrate patches are likely to respond to oxytocin or ergometrine, should these oxytocics be indicated for emergency obstetric manoeuvres. Furthermore, our in vitro results agree with the clinical observation that glyceryl trinitrate and oxytocin could be used to produce consecutive periods of uterine relaxation and contraction for uterine exploration and manual removal of a retained placenta 22. In another case report intravenous oxytocin and intramuscular ergotrate also succeeded in contracting q RCOG 2001 Br J Obstet Gynaecol 108, pp. 164±168
P GTN 1 oxytocics 7031 (1269) 5985 (1659) 4665 (1382) 3959 (924) 2257 (734) 1439 (238) 2245 (480) 1790 (348)
a
b
c
, 0.001
, 0.001
, 0.001
, 0.001
1.0
0.012
, 0.001
0.207
, 0.001
the uterus after intravenous nitroglycerin was administered for uterine relaxation of an inverted uterus 21. In contrast, oxytocic drugs are ineffective in reversing the tocolysis caused by beta-mimetic drugs. Therefore, our observation of reversal by oxytocics of relaxation of the uterus induced by glyceryl trinitrate would suggest that glyceryl trinitrate should be used for acute tocolysis in clinical obstetrics. Acknowledgements The authors would like to thank Ms. J. Tang, Ms. X.X. Chen and Ms. K. Veena for their skillful technical assistance and the Staff of Delivery Suites for their contribution to this study. References 1. Black RS, Flint S, Lees C, Campbell S. Preterm labour and delivery. Eur J Pediatr 1996;155(Suppl 2):S2±S7. 2. 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±1326. 3. Heymann MA, Bootstaylor B, Roman C, et al. Glyceryl trinitrate stops active labour in sheep. In: Moncada S, Feelisch M, Busse R, Higgs EA, editors. The Biology of Nitric Oxide Part 3 Physiological and Clinical Aspects. London: Portland Press, 1994:201±202. 4. Diamond J, Marshall J. A comparison of effects of various smooth muscle relaxants on the electrical and mechanical activity of rat uterus. J Pharmacol Exp Ther 1969;168:21±30. 5. Ignarro LJ, Adams JB, Horwitz PM, Wood KS. Activation of soluble guanylate cyclase by NO-hemoproteins involves NO-heme exchange. J Biol Chem 1986;261:4997±5002. 6. Gruetter CA, Gruetter DY, Lyon JE, Kadowitz PJ, Ignarro LJ. Relationship between cyclic guanosine 3':5'-monophosphate formation and relaxation of coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide: effects of methylene blue and methaemoglobin. J Pharmacol Exp Ther 1981;219:181±186. 7. Murad P. Cyclic guanosine monophosphate as a mediator of vasodilation. J Clin Invest 1986;78:1±5. 8. Norman JE, Ward LM, Martin W, et al. Effects of cGMP and the nitric oxide donors glyceryl trinitrate and sodium nitroprusside on contrac-
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Accepted 17 June 2000
q RCOG 2001 Br J Obstet Gynaecol 108, pp. 164±168