Erythromycin inhibits prostaglandin F 2a -induced contractions of myometrium isolated from non-pregnant rats

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1 BJOG: an International Journal of Obstetrics and Gynaecology September 2002, Vol. 109, pp Erythromycin inhibits prostaglandin F 2a -induced contractions of myometrium isolated from non-pregnant rats Husnu Celik a, Ahmet Ayar b, *, Abdulkerim Baltaci c, Niyazi Tug a Objective The aim of this study was to investigate the effects of erythromycin on prostaglandin F 2a (PGF 2a )-induced contractions of isolated myometrial strips from non-pregnant rats. Design In vitro pharmacological study. Setting Firat University Faculty of Medicine. Sample Myometrium samples were taken from 55 adult Wistar rats. Methods Myometrial strips were isolated from mature, non-pregnant Wistar rats. Isometric contractions of these strips were induced with 1 AM PGF 2a. Effects of 0.01, 0.1, 0.2, 0.5 and 1 mm erythromycin on the frequency and amplitude of these PGF 2a -induced contractions were recorded. Main outcome measures The inhibition of prostaglandin F 2a -induced contractions in vitro. Results Application of 0.01 mm erythromycin had no effect on either amplitude or frequency of contractions. However, 0.1, 0.2, 0.5 and 1 mm erythromycin decreased the frequency and amplitude of PGF 2a -induced contractions. The inhibitory effect of erythromycin on amplitude was 27%, 38%, 54% and 83% (P < 0.05), and that on frequency was 10%, 16%, 32% and 61% (P < 0.05) at 0.1, 0.2, 0.5 and 1 mm concentrations, respectively. Conclusion The results of this study demonstrate that erythromycin inhibits PGF 2a -induced contractions in rat myometrium. Because PGF 2a -induced contractions have been suggested to be involved in the pathogenesis of primary dysmenorrhoea, effects of erythromycin in this clinical entity may present a new approach for the treatment. INTRODUCTION Erythromycin is a macrolid group antibiotic that was shown to have modulatory effects on smooth muscle contractility besides its antimicrobial function. While the motility of the stomach, duodenum and colon were enhanced via motilin receptors by erythromycin, contractions of urinary bladder, some parts of the small intestine and bronchial smooth muscle were inhibited by this agent in vitro 1 3. Erythromycin was shown to prolong gestation in idiopathic preterm labour when used as an adjunct to conventional tocolytics in obstetric literature 4. Additionally, oxytocin- and carbachol-induced contractions of pregnant rat myometrium and spontaneous and oxytocininduced contractions of pregnant human myometrium were shown to be inhibited by this agent 5 7. a Department of Obstetrics and Gynaecology, Faculty of Medicine, Fırat University, Elazıg, Turkey b Department of Pharmacology, Faculty of Medicine, Fırat University, Elazıg, Turkey c Department of Physiology, Faculty of Medicine, Selcuk University, Konya, Turkey * Correspondence: Dr A. Ayar, Department of Pharmacology, Faculty of Medicine, Fırat University (Tip Fak), Elazıg, Turkey. D RCOG 2002 BJOG: an International Journal of Obstetrics and Gynaecology PII: S (02) Prostaglandin-induced contractions play an important role in term and preterm labour in pregnancy and primary dysmenorrhoea in non-pregnant uterus In previous studies, erythromycin was shown to cause both inhibition of prostaglandin synthesis and direct inhibition on myometrial contraction 5,12. However, the effect of erythromycin on prostaglandin-induced myometrial contractions has not been studied. In this study, we aimed to investigate the effects of erythromycin on prostaglandin F 2a (PGF 2a )-induced contractions that was suggested to play an important role in the pathogenesis of primary dysmenorrhoea. METHODS The protocol of this study was approved by the Local Ethics Committee. A total of 55 Wistar rats, obtained from Fırat University, Medical Faculty Biomedical Unit, weighing g and with regular oestrous cycle were used in this study. Rats were given water and food ad libitum before and during experimentation. The rats were exposed to 12-hour light and 12-hour dark cycles at 24 F 2jC and 60 F 15% humidified conditions. Rats were sacrificed by cervical dislocation and both of their uterine horns were extracted through midline abdominal incision. The extracted uteri were taken into Krebs

2 ERYTHROMYCIN INHIBITS CONTRACTIONS OF RAT MYOMETRIUM 1037 Table 1. Effects of 0.01, 0.1, 0.2, 0.5 and 1 mm erythromycin and 1 mm lactobionate (vehicle) on the amplitude (g) of isolated rat myometrial strip contractions induced with 1 AM PGF 2a. Values are expressed as mean [SD]. Spontaneous PGF 2a Erythromycin (mm) % Inhibition n P 3.31 [0.96] 3.77 [0.32] y 3.68 [0.30] a (0.01) 3 25 > [0.91] 3.91 [0.24] y 2.85 [0.14] b (0.1) <0.02* 3.33 [0.78] 3.87 [0.18] y 2.42 [0.18] c (0.2) <0.02* 3.31 [0.87] 3.86 [0.27] y 1.77 [0.68] d (0.5) <0.02* 3.42 [0.77] 3.97 [0.23] y 0.68 [0.23] e (1) <0.02* 3.44 [0.86] 3.94 [0.31] y 3.88 [0.26] a (LB) 2 15 >0.5 mm: millimolar, AM: micromolar, g: gram, LB: lactobionate (vehicle of erythromycin). Different letter symbols (a, b, c, d, e) in the erythromycin column indicate statistically significant differences between the mean data presented in the same column. * P < 0.05 as compared with mean amplitude of its respective PGF 2a -induced contraction period. y P < 0.05 as compared with mean amplitude of its respective spontaneous contraction period. solution (potassium chloride 5.9 mmol/l, sodium chloride mmol/l, glucose 11.5 mmol/l, sodium phosphate 1.5 mmol/l, sodium bicarbonate 14.0 mmol/l, calcium chloride 2.5 mmol/l and magnesium sulphate 1.2 mmol/l) immediately. Two longitudinally directed full thickness muscle strips of approximately 15 2 mm size from anti-mesenteric site of each horn was obtained. Only one strip from each animal was used for the same experimental protocol so that the other strips from the same animal were used for either controls, for different doses of erythromycin or PGE-induced contractions. The muscle strips were used immediately or within 1 hour of sectioning. Each strip was placed into a 20-mL jacketed tissue bath containing Krebs solution at 37jC and ph 7.4 and gassed with 95% O 2 5% CO 2. 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 used to measure the isometric tension. The contractile activities were recorded using a Harvard Universal Oscillograph (Harvard Apparatus Limited). The myometrial strips were equilibrated under 0.5 g of resting tension for 30 minutes and spontaneous contractions were observed. Strips with no spontaneous activity during this equilibration period and strips with irregular spontaneous contractions were removed from the study. Spontaneous contractions were observed in 199 out of 220 total strips. After manifestation of spontaneous contractions, PGF 2a (1 or 0.5 AM for some experiments) or PGE 2 (10 AM) was added to the organ bath and the response was recorded for 10 minutes; 0.01, 0.1, 0.2, 0.5, 1 mm erythromycin, or 1 mm lactobionate as a placebo (vehicle of erythromycin) was applied to the bath and effects on frequency and amplitude were evaluated in the following 10-minute period. Each concentration of erythromycin was studied in 25 strips for PGF 2a -induced, and lactobionate was studied in 15 strips. Drugs used in this study were erythromycin (erythromycin lactobionate, Abbott Laboratories, intravenous injection, Wiesbaden, Germany), vehicle of our erythromycin lactobionate, and PGF 2a, PGE 2 (Sigma Deishofen, Germany). For the measurements, the first minute of the control period was taken as the starting point. Mean amplitude and frequency of the contractions between 0th and 10th minutes (control period) and 10th and 20th minutes (drug period) were determined and comparisons were made. In additional series of experiments, the effects of same erythromycin concentrations were examined on PGE 2 - induced contraction. The effects of the same erythromycin concentrations were examined on myometrial contraction induced by a half dose of PGF 2a. The time of the inhibition Table 2. Effects of 0.01, 0.1, 0.2, 0.5 and 1 mm erythromycin and 1 mm lactobionate on the frequency (number/10 minutes) of isolated rat myometrial strip contractions induced with 1 AM PGF 2a. Values are expressed as mean [SD]. Spontaneous PGF 2a Erythromycin (mm) % Inhibition n P 8.1 [0.56] 11.4 [0.7] y 11.3 [0.3] a (0.01) 1 25 > [0.63] 11.3 [0.5] y 10.2 [0.5] b (0.1) <0.05* 7.9 [0.47] 10.7 [1] y 9 [0.4] c (0.2) <0.05* 7.8 [0.74] 10.8 [0.8] y 7.4 [1.1] d (0.5) <0.02* 8.4 [0.86] 11.2 [0.9] y 4.4 [0.8] e (1) <0.02* 8.2 [0.49] 11.1 [0.7] y 10.8 [0.9] a (LB) 3 15 >0.5 mm: millimolar, AM: micromolar, g: gram, LB: lactobionate (vehicle of erythromycin). Different letter symbols (a, b, c, d, e) in the erythromycin column are indicating statistically significant differences between the mean data presented in the same column. * P < 0.05 as compared with mean frequency of its respective PGF 2a -induced contraction period. y P < 0.05 as compared with mean frequency of its respective spontaneous contraction period.

3 1038 H. CELIK ET AL. Fig. 1. Representative tracing showing the effects of 0.5 mm erythromycin on isolated rat myometrial strip contractions induced with 1 AM PGF 2a. (g: gram, min: minute, mm: millimolar, AM: micromolar, E: erythromycin) period was controlled for each of the erythromycin concentrations in a few randomised experiments. The results are expressed as mean [SD] and as percentages. All statistical analyses were done using the statistical program, SPSS for windows version (SPSS, Chicago, Illinois). Statistical analysis was performed by repeated measures analysis of variance with the use of the Tukey s post hoc test. Data were found to be normally distributed. P < 0.05 was accepted as significant. RESULTS Spontaneous contractions were observed in 199 out of 220 myometrial strips used in this study; four strips with non-regular contractions and the remaining 17 strips with no spontaneous activity were removed from the study. Under our experimental conditions, once the spontaneous contractions were manifested, they were stable for at least 10 hours and during this period amplitude and frequency of spontaneous contractions did not decline in any of the strips studied (n ¼ 5). After manifestation of spontaneous contractions in the 30-minute equilibration period, application of PGF 2a (1 AM) significantly augmented the amplitude and frequency of spontaneous contractions (Tables 1 and 2, P < 0.05). Effects of 0.01, 0.1, 0.2, 0.05 and 1 mm erythromycin and 1 mm lactobionate on amplitude of PGF 2a -induced contractions are given in Table 1 and Figs 1 and 2. There was no significant difference in amplitudes of contractions before drug application between the groups (P ¼ 0.55). Erythromycin produced significant inhibition on the amplitudes of contractions in all doses compared with the placebo and the control period, except in the 0.01 mm concentration. Effects of 0.01, 0.1, 0.2, 0.05 and 1 mm erythromycin and 1 mm lactobionate on the frequency of PGF 2a -induced contractions are given in Table 2 and Figs 1 and 2. There was no significant difference in frequency of contractions before drug application between the groups (P ¼ 0.55). Erythromycin produced significant inhibition on the frequency of contractions in all doses compared with the control period and the placebo, except for the 0.01 mm concentration. It was observed that inhibitory effects of erythromycin on amplitude and frequency of PGF 2a -induced contractions Fig. 2. Representative tracing showing the effects of 1 mm erythromycin on isolated rat myometrial strip contractions induced with 1 AM PGF 2a. (g: gram, min: minute, mm: millimolar, AM: micromolar, E: erythromycin)

4 ERYTHROMYCIN INHIBITS CONTRACTIONS OF RAT MYOMETRIUM 1039 were dose dependent when postdrug results of groups were compared (Tables 1 and 2, Figs 1 and 2). In additional series of experiments, it was observed that erythromycin also inhibited amplitude and frequency of PGE 2 -induced contractions similar to those of PGF 2a. The inhibition rate in amplitude of PGE 2 -induced contractions by erythromycin was 2%, 24%, 33%, 51% and 86% for 0.01, 0.1, 0.2, 0.5 and 1 mm erythromycin, respectively (n ¼ 5). Application of 0.01, 0.1, 0.2, 0.5 and 1 mm erythromycin resulted in 3%, 13%, 18%, 37% and 65% inhibition of frequency of PGE 2 -induced contractions (n ¼ 5). When the dose of PGF 2a reduced by 50% (0.5 AM), the inhibitory effects of erythromycin on amplitude and frequency of PGF 2a -induced contractions were increased significantly (n ¼ 5, data not shown). The dose-dependent inhibition by erythromycin was continued at least for a 2-hour study period in the presence of erythromycin (one experiment for each dose, data not shown). Only partial recovery was observed when three consecutive washouts of erythromycin were performed in 5-minute intervals after the end of a 10-minute drug period (n ¼ 5, for each dose of 0.1, 0.2, 0.5 and 1 mm erythromycin). These strips were still able to respond to PGF 2a and KCl when applied 30 minutes after washout of erythromycin (n ¼ 5). DISCUSSION The results of this study show that erythromycin inhibits amplitude and frequency of PGF 2a -induced contractions of rat myometrium in a dose-dependent manner. This is the first preliminary study to show that erythromycin leads to a decrease in the amplitude and frequency of PGF 2a -induced contractions. Primary dysmenorrhoea is known as cramping pain in the lower abdomen occurring just before or during menstruation, in the absence of other diseases such as endometriosis 10. Uterine hypertonicity and contractions secondary to the increase in endometrial prostaglandin synthesis have been suggested to play an important role in the pathogenesis of this clinical entity. Current management guidelines are based on the inhibition of this increased synthesis of prostaglandin and related uterine contractions 10,13,14. Our study may be considered as an in vitro model of primary dysmenorrhoea despite the fact that excess prostaglandin synthesis is not the only responsible mechanism in primary dysmenorrhoea, which is also suggested as a partial psychosomatic disorder. PGF 2a was used in our experiments, and was shown to increase myometrial contractility causing pelvic pain 15,16. In our model, erythromycin decreased the amplitude and frequency of PGF 2a -induced contractions in a dosedependent manner. There are a limited number of studies evaluating the effects of erythromycin on myometrial contractility. In a study performed by Granovsky-Grisaru et al. 5, erythromycin inhibited oxytocin-induced contractions in isolated pregnant rat myometrium. That inhibition was 41% and 78% on amplitudes and 30% and 62% on frequencies in 0.5 and 1 mm concentrations, respectively 5. In our study, the effect of erythromycin on the frequency of PGF 2a -induced contractions was comparable, whereas the inhibition on amplitudes was higher. This difference may simply reflect the use of PGF 2a instead of oxytocin. Both the oxytocin-induced and the prostaglandininduced contractions come about as a result of an increase in free cytoplasmic Ca 2+. Prostaglandins promote influx of Ca 2+ into the cells as well as liberating Ca 2+ from intracellular stores similar to oxytocin that has also been shown to cause both influx from the extracellular space and release of Ca 2+ from the sarcoplasmic reticulum 17,18.In smooth muscles other than uteri, erythromycin was shown to inhibit Ca 2+ influx from the extracellular space into the cytoplasm and to inhibit contractions 19,20. Because oxytocin and PGF 2a have similar effects on intracellular free Ca 2+ profile in the myometrium, the higher inhibition of PGF 2a -induced contractions by erythromycin may be due to its direct effect on PG synthesis 12. Oropeza et al. 21 suggested that different regions of the uterus display different contractile responses to PGF 2a in different phases of the oestrous cycle. However, in our study, there were no significant differences in myometrial strips (which were taken from randomly chosen rats without knowing oestrous phase and also uterus regions) contractile response to PGF 2a. All of the strips showed similar contractility response to PGF 2a. The dose of erythromycin used in the present study is higher than its usual therapeutic doses in adults. It is well known that the concentration of PGF 2a is significantly higher in dysmenorrhoeic women than in normal subjects; ranging from being just significantly higher to several times higher than levels seen in normal subjects 22. Therapeutic doses of erythromycin may be effective in dysmenorrhoea cases with only mildly elevated prostaglandin levels as decreasing the dose of PGF 2a resulted in increased inhibition on both frequency and amplitude of contractions by the same erythromycin doses (data not shown). It has been demonstrated that the macrolide antibiotic erythromycin has a dual effect on the contractility of smooth muscle and because structural analogs of erythromycin is not antibiotic but are prokinetic the modulatory effects of erythromycin on smooth muscle contraction is not related to its antimicrobial activity 23. In the smooth muscle of the stomach, duodenum, it has a stimulatory effect suggested to be mediated by motilin receptors 24,25. It also has a direct inhibitory effect in the guinea pig and human gallbladder, in the rat urinary bladder smooth muscle 3,in the longitudinal smooth muscle of the guinea pig small intestine 19 and in the rat and human myometrium 5,7. The exact mechanism of inhibition of smooth muscle contraction by erythromycin is not known but some studies are

5 1040 H. CELIK ET AL. suggesting a direct effect on smooth muscle 19 as well as a suggestive effect on intracellular calcium concentration 5. In this preliminary study, only the direct inhibitory effect of erythromycin on myometrial contractions was investigated. Additionally, it is suggested that erythromycin inhibits prostaglandin synthesis 12. The findings are a partial recovery of erythromycin-induced inhibition after washout and the responsiveness to the PGF 2a and KCl, suggesting that the excitability and the contractile apparatus of the tissue remain undamaged following erythromycin-induced inhibition. Because the spontaneous contractions were stable up to 10 hours, and only a modest amount of inhibition was observed with vehicle and 0.01 mm erythromycin, the decrease of amplitude and frequency of PGF 2a -induced contractions cannot be due to time-dependent decay in the tissue activity. According to the knowledge above, erythromycin may be useful in the treatment of dysmenorrhoea. At least, it may also be useful to consider those effects of erythromycin (i.e. inhibition of PGF 2a -induced contractions) when antibiotic treatment is chosen in women with primary dysmenorrhoea for any reason. However, it is also important to mention that to eliminate the differences between species, similar studies in human myometrium should be done. Importantly, to support our findings, it is necessary to perform clinical studies. References 1. Kaufman HS, Ahrendt SA, Pitt HA, Lillemoe KD. The effect of erythromycin on motility of the duodenum, sphincter of oddi, and gallbladder in the prairie dog. Surgery 1993;114: Frederick H, Weber Jr, Robert D, Rirhards MD, Rirhard W, McCallum D. Erythromycin: a motilin agonist and gastrointestinal prokinetic agent. Am J Gastroenterol 1993;88: Nissan A, Maudlej N, Beglaibter N, Haskel Y, Freund HR, Hanani M. 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