Polish Journal of Pharmacology Pol. J. Pharmacol., 2002, 54, 281–284 ISSN 1230-6002
Copyright © 2002 by Institute of Pharmacology Polish Academy of Sciences
SHORT COMMUNICATION
HOMOCYSTEINE-INDUCED AUGMENTATION OF SPONTANEOUS CONTRACTION OF ISOLATED GUINEA PIG MYOMETRIUM Ahmet Ayar1,#, Niyazi Tug2, Husnu Celik2, Mete Ozcan3, Oguz Ozcelik3 Firat (Euphrates) University, Faculty of Medicine, Departments of 1Pharmacology, 2Obstetrics and Gynaecology, 3 Biophysics, TR-23119 Elazig, Turkey
Homocysteine-induced augmentation of spontaneous contraction of isolated guinea pig myometrium. A. AYAR, N. TUG, H. CELIK, M. OZCAN, O. OZCELIK. Pol. J. Pharmacol., 2002, 54, 281–284. Recent studies have suggested association between the elevated homocysteine levels and pregnancy complications. The aim of this study was to investigate the effects of homocysteine on spontaneous contractions of myometrium isolated from pregnant guinea pig. Full-thickness myometrial strips were obtained from late-pregnant guinea pigs following decapitation, and suspended in an organ bath which was filled with physiological saline solution (pH 7.4), maintained at 37°C and continuously bubbled with 95% O2 and 5% CO2. After the observation of spontaneous contractions under one gram of resting tension, homocysteine (0.2, 0.5 and 1 mM) was added to the organ bath and effects of this agent on frequency and amplitude of contractions were evaluated in 20-min periods. One-way analysis of variance was used for statistical analysis of the data. Homocysteine caused increase in both frequency and amplitude of spontaneous contractions in a dose-dependent manner. The results from this in vitro study indicate that cardiovascular risk factor homocysteine has contracting pharmacological effects on guinea pig myometrium. Key words: homocysteine, myometrium, spontaneous contraction, guinea pig
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correspondence; e-mail:
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A. Ayar, N. Tug, H. Celik, M. Ozcan, O. Ozcelik
INTRODUCTION Homocysteine (Hcyt) is non-essential, non-protein forming, sulphur-containing small amino acid [14]. It is not contained in the diet, and it is produced by demethylation of methionine and under normal physiological conditions it is converted to methionine and cysteine by two metabolic pathways. In the most common re-methylation pathway with vitamin B12 acting as a cofactor and in the presence of methyl donor N-5-methyltetrahydrofolate, methionine synthase catalyses conversion of Hcyt back to methionine. In the alternative pathway with vitamin B6 participation, the cells convert Hcyt to cystathionine and then to cysteine [14]. Age, sex, race, renal insufficiency, vitamin B6 and B12 levels, genetic mutations and medications such as antifolate drugs are among the conditions that affect Hcyt levels and, therefore, should be considered in the interpretation of Hcyt levels. After 12 h of fasting, serum levels of 5–15 mM/l Hcyt are considered normal; while serum levels between 16 and 30 mM/l, 31–100 mM/l and > 100 mM/l are classified as moderately, intermediately and severely elevated, respectively [7]. Hyperhomocysteinemia is considered to be a risk factor for cardiovascular and cerebrovascular diseases, independent of the established risk factors such as heredity, hypertension, smoking, dislipidemia, diabetes, lack of physical activity and obesity [3]. Recent investigations have indicated that moderately elevated Hcyt levels are associated with pregnancy complications including preeclampsia, neural tube defects, spontaneous aborts, intrauterine growth restriction, and placental abruption [1, 9, 11, 13, 18]. Steegers-Theunissen et al. [15] noted the association between recurrent early pregnancy loss and hyperhomocysteinemia for the first time, and following studies have confirmed these findings [4, 18]. However, the mechanism(s) by which hyperhomocysteinemia causes negative pregnancy complications remain to be determined. The aim of this study was to investigate the effects of Hcyt on spontaneous contractility of myometrium isolated from pregnant guinea pig.
MATERIALS and METHODS The protocol of this study was approved by Firat University Local Ethics Committee for Animal Experimentation. Guinea pigs were killed by decapitation at term (days 62–66 of pregnancy). The
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day on which a plug of semen was found in the vagina was designated day 1 of pregnancy and the length of pregnancy in our colony was 67 ± 1 days. Only one strip was used from each guinea pig. Full-thickness 3 × 0.2 mm myometrial strips were prepared and placed in a jacketed tissue bath containing physiological saline solution (PSS) that was maintained at 37oC, pH 7.4, and continuously bubbled with 95% O2 and 5% CO2. Composition of PSS was (in mM): NaCl 120; KCl 5; NaHCO3 25; KH2PO4 1; MgSO4 1.2; glucose 11 and CaCl2 2.5. Silk thread was used to attach the myometrial strips to a fixed hook, and an isometric force displacement transducer (Harvard Apparatus Limited, Kent, England) was applied to measure isometric tension. The contractile activities were recorded using a Harvard Universal Oscillograph (Harvard Apparatus Limited, Kent, England). The myometrial strips were initially placed under 1.0 g tension and a 60-min equilibration period was allowed before the start of each experiment. Most of the strips (14 out of 18) developed spontaneous contractions within this 60-min equilibration period and strips with no spontaneous activity in this period were discarded. The drug used in this study was homocysteine thiolactone (Sigma), it was dissolved in PSS. Different concentrations of Hcyt were added to the tissue bath cumulatively. The amplitude of contractions was evaluated in 20-min periods before (control) and after application of Hcyt. For these measurements, after 60-min equilibration period was completed, the first minute of the control period was taken as a starting point. Mean amplitude of the contractions in 0-20th min period (control period) and 20–40 min period (after application of Hcyt, drug period) were determined and comparisons were made within and between the groups. Data are given as means ± SEM. All statistical analyses were conducted using the statistical program SPSS for Windows ver. 6.0.1. (SPSS Inc., Chicago, Illinois). For statistical analysis of amplitude and frequency of contractions, Wilcoxon Rank and Kruskal Wallis analysis of variance tests were used. A level of p < 0.05 was accepted as indicating significant difference.
RESULTS Spontaneous contractions were observed in 14 out of 18 myometrial strips studied; 3 strips with-
Pol. J. Pharmacol., 2002, 54, 281–284
HOMOCYSTEINE CONTRACTS GUINEA PIG MYOMETRIUM
DISCUSSION Our present experimental data clearly demonstrates for the first time that Hcyt augments spontaneous contractions of the isolated myometrium from late pregnant guinea pig in a dose-dependent manner. This contracting effect of Hcyt is not dependent on the presence of extracellular Ca2+. The mechanism by which Hcyt is increasing spontaneous contractions is not clear and no attempt was made for clarifying the possible mechanism of action of Hcyt in this study. Since the effect was dose-dependent and relatively fast, we think that Hcyt has a direct effect on contractility rather than a nonspecific effect. Persistence of contracting effect of Hcyt in extracellular Ca2+-free PSS suggests that Ca2+ entry from extracellular space is not essential for this effect. The effect of Hcyt on extracellular matrix of rat vascular smooth muscle was determined to be dependent upon an increase in free intracellular Ca2+ and was not affected by removal of extracellular Ca2+ but was abolished when intracellular Ca2+ stores were depleted with thapsigargin [10]. In this study, Hcyt may augment spontaneous contractions by release of Ca2+ from intracellular stores. Another possibility is that an increase in the sensitivity of the contractile apparatus to Ca2+ may occur after Hcyt action, as in the case of the reaction of many smooth muscles [16], including uterus [6], to excitatory agonists. To our best knowledge there has been no study in the literature investigating the effects of Hcyt on
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out spontanous contractions in 60-min equilibration period and 1 strip with irregular spontanous contractions were excluded from the study. The frequency of spontanous contractions (number of contractions/20-min period) was 7.4 ± 0.4 (n = 10) under control conditions, and 7.5 ± 0.5 (p > 0.05, n = 10), 9.3 ± 0.5 (p < 0.05, n = 10) and 11.6 ± 0.6 (p < 0.05, n = 10) after application of 0.2 mM, 0.5 mM and 1 mM Hcyt, respectively (Fig. 1). The amplitude of contractions was 1.74 ± 0.05 g (n = 10) under control conditions and 1.82 ± 0.06 g (p > 0.05, n = 10), 2.06 ± 0.07 g (p < 0.05, n = 10) and 2.19 ± 0.09 g (p < 0.05, n = 10) after application of 0.2 mM, 0.5 mM and 1 mM Hcyt (Fig. 2). Hcyt similarly augmented spontaneous contractions of guinea pig myometrium when Ca2+ was removed from the extracellular solution. Using Ca2+-free physiological saline solution, we observed spontaneous contractions in 4 out of 6 strips studied. The frequencies of these contractions were 5 ± 0.9 (n = 4) under control conditions, and 5.1 ± 0.7 (p > 0.05, n = 4), 6.1 ± 0.8 (p > 0.05, n = 4) and 8.9 ± 0.9 (p < 0.05, n = 4) after application of 0.2 mM, 0.5 mM and 1 mM Hcyt, respectively (Fig. 1). Spontaneous contractions observed under Ca2+-free conditions had mean amplitude of 1.44 ± 0.08 g (n = 4) under control conditions, and 1.51 ± 0.09 g (p > 0.05, n = 4), 1.69 ± 0.07 g (p < 0.05, n = 4) and 1.81 ± 0.09 g (p < 0.01, n = 4) after application of 0.2 mM, 0.5 mM and 1 mM Hcyt, respectively (Fig. 2).
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Fig. 1. Dose-dependent effects of homocysteine on frequency of spontaneous contractions of guinea pig myometrium. The data are presented as means ± SEM, * p < 0.05 control vs. corresponding dose of homocysteine. PSS – physiological saline solution, £ – Ca2+-free PSS, ¢ – ordinary PSS (containing 2.5 mM/l Ca2+)
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Fig. 2. Dose-dependent effects of homocysteine on amplitude of spontaneous contractions of guinea pig myometrium. The data are presented as means ± SEM, * p < 0.05 control vs. corresponding dose of homocysteine. PSS – physiological saline solution, £ – Ca2+-free PSS, ¢ – ordinary PSS (containing 2.5 mM/l Ca2+)
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contractility of guinea pig myometrium. Application of 3-deazaadenosine plus homocysteine thiolactone dose-dependently inhibited acetylcholine and high KCl-induced contractions in rat uterine segments, and this effect was attenuated by L-methionine [5]. In contrast to our study, it was found no effect of homocysteine thiolactone on contractility of rat myometrium [5]. This may be due to the use of different species. Hcyt was shown to stimulate contractility in several smooth muscles by interfering with endothelium-dependent relaxation [8, 17]. Hcyt was demonstrated to cause vasoconstriction in human umbilical artery by the suggested interaction with bioavailable NO [8, 12]. The effect of Hcyt was reported to be dependent on the presence of endothelium, and pretreatment with melatonin significantly prevented the vasoconstriction induced by Hcyt. Endometrium was present in the full-thickness myometria used in the present study, and an increase in contractility caused by Hcyt action could be due to a decrease in bioavailable nitric oxide. Myometria from guinea pigs were used in the present study most closely resemble those of humans because of their oestrogen and progesterone profiles during pregnancy and labor [2]. In conclusion, results from this preliminary study demonstrate that Hcyt dose-dependently increased contractility assessed by measuring isometric contractions of uterine strips hung in standard muscle baths. These data support the finding that there is an association between elevated Hcyt levels and pregnancy complications especially spontaneous abortions. Further studies using human tissue samples are needed for clarifying this hypothesis. REFERENCES 1. Aubard Y., Darodes N., Cantaloube M.: Hyperhomocysteinemia and pregnancy – review of our present understanding and therapeutic implications. Eur. J. Obstet. Gyn. Reprod. Biol., 2000, 93, 157–165. 2. Bedford C.A., Challis J.R.G., Harrison F.A., Heap R.B.: The role of oestrogens and progesterone in the onset of parturition in various species. J. Reprod. Fertil., 1972, 16, 1–23. 3. Chambers J.C., Seddon M.D.I., Shah S., Kooner J.S.: Homocysteine – a novel risk factor for vascular disease. J. R. Soc. Med., 2001, 94, 10–13. 4. Coumans A.B., Huijgens P.C., Jakobs C., Schats R., de Vries J.I., van Pampus M.G., Dekker G.A.: Haemostatic and metabolic abnormalities in women with unexplained recurrent abortion. Hum. Reprod., 1999, 14, 211–214.
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