Clarithromycin inhibits myometrial contractions in isolated human myometrium independent of stimulus with use of oscillograph
Clarithromycin Inhibits Myometrial Contractions in Isolated
Human Myometrium Independent of Stimulus
Department of Obstetrics and Gynaecology, and 1Department of Pharmacology, Faculty ofMedicine, Firat University, Elazig, Turkey
Erythromycin has a well-known dual effect on the contractility of the gastrointestinal system and recently has also been
shown to inhibit contractions of the rat myometrium. The aim of the present study was to investigate the effects of
clarithromycin on oxytocin, prostaglandin F2α (PGF2α) and KCl-induced contractions of human myometrium in vitro
Myometrial strips were obtained from pregnant women undergoing elective Cesarean section and the strips were
suspended in a jacketed organ bath filled with Krebs solution at 37 oC (pH 7.4) and continuously aired with 95 %
oxygen and 5 % carbon dioxide. Isometric contractions were measured using a force displacement transducer.
Oxytocin, PGF2α, KCl and clarithromycin were applied to the tissue bath and the amplitude and frequency of
contractions were evaluated at 20-min intervals. Freidmann analysis of variance, Kruskal Wallis and Wilcoxon Rank
tests were used for statistical analysis of the data. Clarithromycin dose dependently inhibited the amplitude of
contractions independent of the stimulus. Pre-treatment with apamin prevented clarithromycin-induced effects on
amplitude and frequency of contractions. We conclude that the macrolide antibiotic clarithromycin may have a direct
inhibitory effect on contractions of human myometrium.
Myometrium • Clarithromycin • Oxytocin • Isometric contraction • Human
longitudinal smooth muscle of the guinea-pig smallintestine (Minocha et al.
and in bronchial smooth
It is well established that the macrolide antibiotic
muscle (Tamaoki et al.
1995). However, the exact
erythromycin has a dual effect on the contractility of
mechanism of inhibition induced by erythromycin is not
smooth muscle. In smooth muscles of the stomach and
known. Furthermore, a recent study has reported that
duodenum, it has a stimulatory effect that was suggested
erythromycin inhibits myometrial contractions of rat
to be mediated by motilin receptors (Peeters et al.
independent of stimulant (Granovsky-Grisaru et al.
Collard et al.
1999). It also has a direct inhibitory effect
1998). Since structural analogs of erythromycin are not
in guinea-pig and human gallbladder, the rat urinary
antibiotic but are prokinetic, the modulatory effect of
bladder smooth muscle (Nissan et al.
1999), in the
erythromycin on smooth muscles contractions is not
2002 Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
related to its antimicrobial activity (Omura et al.
Socio-demographic characteristics of sample
Clarithromycin is the new member of the macrolide
family of antibiotics with a broad spectrum of activity invitro
against clinically important gram-positive and gram-
negative aerobes and anaerobes. The modulatory effect ofclarithromycin on contractility of smooth muscle has
been poorly studied compared to erythromycin, with the
exception of one study evaluating its effect on
The aim of the present study was to investigate
whether clarithromycin has any similar effect as itsprogenitor, erythromycin, on myometrial contractility. Toour knowledge, our study shows for the first time the
The contractile activities were recorded using a Harvard
inhibitory effects of clarithromycin using human isolated
Universal Oscillograph (Harvard Apparatus Limited,
Kent, England). The myometrial strips were initiallyplaced under 2.0 g tension and a 90-min equilibration
period was allowed before the start of each experiment.
Most of the strips developed spontaneous contractions
The protocol of this study was approved by the
within 30 to 90 min and strips with no spontaneous
Firat University Local Ethics Committee for Research on
activity in this period were discarded. After development
Human Subjects and a written informed consent was
of spontaneous contractions 800 mU/l oxytocin, PGF2α
obtained from each subject donating a tissue sample.
(1 µM) or KCl (30 mM) was applied to amplify these
Patients undergoing elective Caesarean section, gestation
period of 37-42 weeks, in the Department of Obstetrics
The amplitude of contractions was evaluated at
and Gynecology at The Firat University Medical Center
20 min periods before (control) and after application of
were included in this study. Patients with medical
clarithromycin. The first minute of the control period was
problems, including diabetes, hyperthyroidism,
taken as the starting point for this comparison. The mean
hypertension, pre-eclampsia or connective tissue diseases
amplitude of the contractions under control conditions
were excluded from this study. None of the subjects were
and after application of clarithromycin were determined
under any regular medication. Socio-demographic
and compared. The frequencies of contractions were also
characteristics of sample donating patients are given in
evaluated at 20 min intervals before and after application
Table 1. Two myometrial strips were used from each
of clarithromycin. In some experiments, after
amplification of spontaneous contractions with agonists
A single full-thickness myometrial strip was
(oxytocin, PGF2α or KCl), apamin was applied for 5 min
removed from the upper margin of the lower uterine
segment incision at the time of the Caesarean section
The agents used in the present study included
after the infant had been delivered. The strip was than
clarithromycin lactobionate (Abbot Laboratories,
immediately placed in a Krebs’ solution. Small strips
intravenous injection, Wiesbaden, Germany), oxytocin
(10 x 2 x 2 mm) of myometrial tissue were cut from
(Synpitan iv, 5IU/ml, Deva, Istanbul, Turkey), PGF2α,
uterine samples. Each strip was placed in a 20 ml
KCl (Sigma) and apamin (Alomone Labs, Israel).
jacketed tissue bath containing Krebs’ solution at 37 0C
The results are expressed as means
and pH 7.4. The composition of the Krebs’ solution was
± S.D. All statistical analysis was performed using the
(in mmol/l): sodium chloride 121, potassium chloride 4.5,
statistical program SPSS for Windows (version 6.0.1,
sodium bicarbonate 15.5, sodium phosphate 1.2, calcium
chloride 2.5, magnesium chloride 1.2, and glucose 11.5.
The Krebs’ solution was constantly gassed with 95 % O2–
Wilcoxon Rank test. Figures were made using Origin
5 % C02. A silk thread was used to attach the myometrial
version 5.0 (Microcal Software Inc. Northampton, USA).
strips to a fixed hook and an isometric force displacementtransducer (Harvard Apparatus Limited, Kent, England).
Clarithromycin Inhibits Contractions of Isolated Human Myometrium 241
tracing showing the effectof 0.5 mM clarithromycinon oxytocin-induced myo-metrial contractile activityreported in grams oftension generated. Sponta-neous contractions werenoted during period pre-ceding 20-min time point;subsequently oxytocin andclarithromycin was ap-plied at points indicated bythe arrow.
dependent. 0.1 mM clarithromycin had no significant
A total of 300 myometrial strips from 150
effect on either parameter of agonist-induced contractions
patients were used in this study. 284 out of these strips
(Tabs. 2, 3, p>0.05). The inhibition of the amplitude and
developed spontaneous contractions (in 95 % cases).
increased frequency of contractions were significant after
Each dose of clarithromycin was tested on 30,
application of 0.2 mM clarithromycin (Tabs. 2, 3,
20, and 10 myometrial strips contracted by oxytocin,
The inhibitions of the amplitude of oxytocin-
Myometrial strips were contracted by application
induced contractions were 5 %, 15 %, and 44 % after
of oxytocin (800 mU/l), PGF2α (1 µM) and KCl (30 mM).
applications of 0.2 mM, 05 mM (Fig. 1) and 1 mM
The mean peak amplitude and frequency values after
clarithromycin, respectively (Tab. 2). The effects of 0.2
each agonist and the dose-dependent effects ofclarithromycin on these parameters are given in Table 2.
Dose-dependent effects of clarithromycin on peak amplitude of agonist-induced (oxytocin, PGF2α or KCl)
Cla: clarithromycin, PGF2
α: prostaglandin F2
α, KCl: potassium chloride. Data are presented as mean ± SD (each doseof clarithromycin was tested on n= 30 strips for oxytocin-induced, n= 30 strips for PGF2
α-induced and n= 10 strips forKCl-induced contractions, lactobionat was tested on n=7 strips for each agonist, apamin+ 1 mM clarithromycin was
tested on n=8 strips for each agonist), p<0.05 statistically significant.
mM, 0.5 mM, and 1 mM clarithromycin enhanced the
The effects of clarithromycin on the PGF2α-
frequency of oxytocin-induced contractions by 24 %,
induced contractions led to a 7 %, 12 % and 55 %
66 % and 79 %, respectively (Tab. 3).
decrease in amplitude (Tab. 2), while an increase of
29 %, 59 % or 83 % of frequency was found for 0.2, 0.5
investigate the possible role of Ca2+-activated K+
channels on the inhibitory action of clarithromycin on the
clarithromycin was applied to myometrium contracted
amplitude of agonist-induced contractions using apamin.
with KCl there was an inhibition in amplitude of 18 %,
For these experiments, 1 mM dose of clarithromycin were
36 % and 65 % (Tab. 2), and an increase of 12 %, 47 %
applied on oxytocin, PGF2α or KCl-induced contractions.
and 59 % in frequency of contractions (Tab. 3).
After pre-treatment with apamin (1 µM) for 5 min,
When lactobionat, the vehicle of clarithromycin,
application of clarithromycin had no significant effect on
was applied in comparable concentrations (1 mM), it had
amplitude and frequency of contractions independent of
no significant effect on either amplitude or frequency of
stimulant (n=8 for each agonist, Tabs. 2, 3).
agonist-induced contractions of myometrium (n= 7 foreach agonist, Tabs. 2, 3).
Dose-dependent effects of clarithromycin on frequency of agonist-induced (oxytocin, PGF2α or KCl)
Cla: clarithromycin, PGF2
α: prostaglandin F2
α, KCl: potassium chloride. Data are presented as mean ± SD (each doseof clarithromycin was tested on n= 30 strips for oxytocin-induced, n= 30 strips for PGF2
α-induced and n= 10 strips forKCl-induced contractions, lactobionat was tested on n=7 strips for each agonist, apamin+1 mM clarithromycin was
tested on n=8 strips for each agonist). Frequency reflects number of contractions in 20 minute-periods. p<0.05
reports about inhibitory effects of neomycin, gentamycinand clindamycin on myometrial contractions induced by
The data presented in this study demonstrate the
oxytocin, KCl or aluminium fluoride (Phillipe 1994,
ability of clarithromycin to inhibit oxytocin, PGF2α, and
KCl-induced contractile activity of uterine tissues from
The finding of inhibition at peak amplitude of
pregnant women at term. However, clarithromycin also
myometrial contractions by clarithromycin is consistent
caused an increase in frequency of contractions
with a result of the previous study where other macrolide
independently of stimulant. The inhibitory effect of
erythromycin was used (Granovsky-Grisaru et al.
clarithromycin was dose dependent, significant inhibition
However, contrary to erythromycin, clarithromycin
on peak amplitude started after 0.2 mM whereas 0.1 mM
increased the frequency of contractions. This may be due
had no significant effect either on amplitude or
to species differences but there may also be a mechanistic
difference involved. Similar to clarithromycin,
Previous studies have shown inhibitory effects
erythromycin inhibited the amplitude but increased the
of antibiotics on myometrial contractions. The finding of
frequency of oxytocin-induced contractions in isolated
the inhibitory effect of erythromycin on rat myometrial
pregnant human myometrium (Celik et al.
contractions by Granovsky-Grisaru et al.
(1998) was the
Another possible explanation of the different effects of
starting point of the present study. Similarly, there are
these two macrolides could be due to the different uterus
Clarithromycin Inhibits Contractions of Isolated Human Myometrium 243
parts being studied. We obtained myometrial samples
role in modulation of uterine contractility (Anwer et al.
from the lower uterine segment whereas Granovsky-
Grisaru et al.
(1991) used the uterine horns. However, the
We have no explanation for the increase in
explanation by anatomical difference is not very
frequency of contractions after clarithromycin
convincing because there were no significant differences
application. This could be the result of increased
in the contractile rate and force production produced by
pacemaker activity that is considered to be related with
myometrium from the upper and lower segments when
T-type Ca2+ channels (Wray 1993). But the amplitude of
biopsies were obtained from women undergoing classical
contractions gradually decreased (Fig. 1). Since apamin
Caesarean section (Luckas et al.
prevented the inhibitory effect of clarithromycin, it seems
The inhibitory activity of clarithromycin in the
likely that this is related to the increase in frequency and
uterine tissue may be the result of activation of adenylate
activation of Ca2+-activated K+ channels.
cyclase, but also from other effects such as the opening of
Clarithromycin may be activating the T-type calcium
potassium channels and a decrease of intracellular free
channels and the resultant increase in free intracellular
Ca2+ levels (Wray 1993). Although no direct evidence is
Ca2+ may in turn activate the Ca2+-activated K+ channels.
available, it is also possible that clarithromycin may
The inhibitory effects of clarithromycin on the
inhibit Ca2+ entry through the L-type calcium channels
contractility found in this study made us to consider the
and thereby cause inhibition of amplitude of agonist-
possibility that this could be used in the treatment of
induced myometrial contractions (Kaya et al.
preterm labour. There are some limitations regarding this
Sanborn 2001). Inhibition of KCl-induced contractions
point. Firstly, the dose of clarithromycin used in this
by clarithromycin provides evidence for the latter
study is higher than the levels achieved after its
therapeutic administration (its peak plasma concentration
is 2.4 µg/ml after a 500-mg oral dose) (Cheng et al.
contractions induced by oxytocin, PGF2α, and KCl
1998). Secondly, the use of clarithromycin in pregnancy
suggesting that the effect of clarithromycin is
has not been studied thoroughly and available data on
independent of the stimulant. High concentrations of KCl
clarithromycin are too limited for recommendation its use
induce contractions by membrane depolarization and a
during pregnancy. It was found in a study performed on a
subsequent increase in free intracellular Ca2+
restricted population of patients that clarithromycin
concentration resulting from influx via
increased spontaneous miscarriage in the first trimester
channels (Wray 1993, Trujillo et al.
without any evidence of teratogenic actions (Einarson et
causes increase in intracellular free Ca2+ mediated by
1998). Further studies need to be performed in order
inositol 1,4,5-triphosphate (IP3) formation while
to determine the effects of clarithromycin in this respect
prostaglandins increase intracellular free Ca2+ via voltage
and relation to the gestational age should be addressed.
or receptor-operated Ca2+ channels (Wray 1993). The
There is no report in the literature about the effects of
only evidence relating the mechanism underlying the
clarithromycin on the contractility of smooth muscles.
inhibitory effect of clarithromycin was that the specific
In conclusion, we have shown for the first time
Ca2+-activated K+ channel blocker apamin prevented the
the inhibitory effects of clarithromycin on agonist-
inhibitory effect of clarithromycin on oxytocin-induced
induced contractions of the isolated myometrium from
contractions suggesting involvement of Ca2+-activated K+
channels. These channels are reported to play important
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Dr. Ahmet AYAR, Firat University, Faculty of Medicine (TIP FAK), Department of Pharmacology, Tr-23119, Elazig,
Turkey, Fax: + 90 424 237 91 38, e-mail: email@example.com
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