- Email: [email protected]
Pharmacological characterization of mare uterus motility with special reference to calcium antagonists and beta-2-adrenergic stimulants
Gen. Pharmac. Vol. 20, No. 4, pp. 513-518, 1989
0306-3623/89 $3.00+ 0.00 Copyright © 1989PergamonPress pie
Printed in Great Britain. All fights reserved
PHARMACOLOGICAL CHARACTERIZATION OF MARE UTERUS MOTILITY WITH SPECIAL REFERENCE TO CALCIUM ANTAGONISTS AND BETA-2-ADRENERGIC STIMULANTS G. CORUZZI, E. POLI, C. MONTANARIl and G. BERTACCINI* Institute of Pharmacology, Faculty of Medicine and Faculty of Veterinary, University of Parma, Italy and t National Health Service, Parma, Italy (Received 21 October 1988)
Abstract--l. Uterine motility was studied in vitro in the myometrial tissue obtained from pregnant and non-pregnant mares. 2. The spontaneous contractions of the preparations were not modified by tetrodotoxin, by antieholinergics, antiadrenergics, histamine H1 and H2 blockers, antiserotoninergic and opioid antagonists; but disappeared in Ca 2+ and Na + free medium. 3. ~2-adrenergic stimulants like salbutamol and hexoprenaline and the calcium channel blockers nifedipine and verapamil were effective inhibitors of the amplitude of phasic contractions (IDs0s for salbutamol and nifedipine were 7.7 nM and 14.6 nM, respectively in oestrus preparations). 4. The above data indicated that the mare myometrium contractility in vitro is very sensitive to the action of/~2 mimetic compounds and calcium antagonists; nifedipine, in particular, seems to be a very promising alternative to/~2 stimulants in the tocolytic therapy.
INTRODUCTION
Selective /~2-adrenoceptor stimulants are extensively used in both h u m a n and veterinary medicine in diseases characterized by smooth muscle deranged motility and they are the most effective drugs available to prevent abortion, to control parturition and reduce dystocia or uterine hypertony (Caritis, 1983; Kern and Schill, 1983; Andersson et aL, 1980; Zerobin and Kundig, 1987). Clinical toxicity studies showed that these drugs are reasonably safe in most species, however some untoward reactions may occur in women and in dogs, concerning cardiovascular system (tachyeardia, pulmonary edema, etc.) (Caritis, 1983; Ueberberg et al., 1976). Moreover a rapid desensitization to ~2-adrenergic agents has been reported both in vitro and in vivo in the long term therapy (Berg et al., 1985; Johansson and Andersson, 1981). Therefore new tocolytic agents, more effective and safer have been searched as an alternative to //2-sympathomimetic drugs. Preliminary studies on isolated myometrium from various animal species and humans indicated that drugs which negatively interfere with the intracellular availability of Ca 2+, could be considered as potential tocolytics (Abel and Hollingsworth, 1985; F o r m a n et al., 1979; Lirette et al., 1985). Indeed Ca 2+ entry blockers by impairing the transmembrane influx into cells (Fleckenstein, 1977) are effective relaxant agents both in vascular and extravascular smooth muscle. Preliminary clinical studies in humans showed that these drugs effectively inhibited uterine contractility
*Address correspondence to: Professor Giulio Bertaoeini, Institute of Pharmacology, via Gramsei 14, 43100 Parma, Italy
in preterm labor and that induced by spasmogenic agents like prostaglandins and oxytocin (Csapo et al., 1982). The present study was undertaken to investigate the action of the Ca z+ antagonists nifedipine, verapamil and diltiazem on the isolated myometrial strips from mare uterus, in comparison with the/~2-adrenergic stimulants salbutamol and hexoprenaline. METHODS
Fifty mares weighing 450-500 kg were used. The uterine horns were removed from animals in oestrus and dioestrus; a small percentage were obtained from pregnant animals (3-5 month gestation). After slaughtering the animals the uterine horns were rapidly removed, immediately transferred into an oxygenated 95% 02 and 5% CO2) nutrient fluid at 4°C and transported to the laboratory. Myometrial longitudinal strips (0.3 cm wide and 1.5 cm long) were dissected free of endometrium and serosa and set up in isolated organ baths (10ml) at 37°C. At least 4 preparations were obtained from each horn. The composition of the nutrient fluid was (raM): NaCI 113; KC14.7; MgSO4 1.2; CaCI2 1.9; KH2PO4 1.2; NaHCO 3 25; Glucose 11.5. Myometrial contractions were recorded by an isometric transducer connected with a pen recorder (Basile, Comerio) under a resting tension of 2 g. While most of the preparations were set up within 1 hr after the removal, some were stored at 4°C overnight and set up the following day; no significant differences were noted in spontaneous motility and in response to drugs between fresh and refrigerated preparations. In a first series of experiments the pattern of spontaneous motility was characterized, in the absence of any drug, by observation of the phasic movements for the whole experiment, in terms of frequency, amplitude and duration of the contractions. In other series of experiments, after an equilibration time of about 30 min the effect of different drugs on this motor activity was evaluated by administering single or cumulative concentrations of the drug considered. 513
G. CORUZZI et al.
514
Some experiments were performed in Ca2+-free medium obtained by simply removing Ca2+ ions or in Ca2+free-EGTA solution by adding 1 mM EGTA. Experiments in Na + free medium were carried out by replacing NaC1 with equimolar amount of choline chloride (Casteels, 1970).
Evaluation of data Significancelevels of the differences between groups were assessed by using Student's t-test or the analysis of variance. The potency of the inhibitory compounds was expressed by the IDs0 value, calculated for each compound from the individual concentration-response curves.
Drugs The following drugs were used: atropine, phentolamine, methysergide, pyrilamine, theophylline and forskolin (Sigma); famotidine and diltiazem (Sigma Tau); tetrodotoxin (Sankyo); nifedipine (Bayer, Leverkusen); verapamil (Knoll); salbutamol (Glaxo); hexoprenaline (Byk Gulden); indomethacin (Chiesi); choline chloride (Carlo Erba). RESULTS
Characteristics of the spontaneous motility of mare uterus Approximately 98% of the preparations from oestrus and dioestrus uteri exhibited a spontaneous motor activity, which was evident after 15-60 min and lasted from 6 to 12 hr. Conversely only 30% of the strips removed from pregnant uteri showed spontaneous contractions. Some examples of the different pattern of motility are shown in Fig. 1. No significant differences were found between uterine strips in different periods of oestrus cycle with respect to frequency and amplitude of contractions; on the contrary pregnant uteri endowed with spontaneous motility showed a significantly higher amplitude of contractions, with longer interval periods and latency (Table 1).
Table I. Spontaneousmotility of myometrial longitudinalstrips from mare uterus Latency Amplitude Interval* (rain)
Oestrus Dioestrus Pregnant
57.9 + 5.8 56.1 + 3.9 135.2+ 23.9a
(g)
3.4 + 0.3 3.9 + 0.4 4.7 _+0.7b
(rain)
5.9 + 0.4 6.7 + 0.6 8.7 +_1.0b
n
38 63 20
*Interval between two subsequent contractions; asignificantly different (P < 0.05) from oestrus and dioestrus uteri; bsignificantly different from oestrus uteri. Mean values + SEM. n = number of preparations.
Effect of tetrodotoxin, indomethacin and membrane receptor antagonists on the spontaneous phasic contractions. This motility was not affected by tetrodotoxin (1 gM). Similarly ineffective were a series of membrane receptor antagonists, like the alpha blocking agent phentolamine (1 #M), the histamine HI receptor antagonist pyrilamine (10 # M); the new H2 receptor antagonist famotidine (10#M) and the 5HT receptor antagonist methysergide (10 #M). Also the cyclooxygenase inhibitor indomethacin was ineffective up to 10#M; very high concentrations (30-100 juM) caused a consistent inhibition probably due to non specific effects. The effect of the antimuscarinic compound atropine (0.01-0.1/zM) was erratic: sometimes it was ineffective, whereas in some strips a 100% inhibition was observed.
Effect of compounds interfering in the cyclic A M P metabolism on the spontaneous contractions The new compound forskolin, recently described as a specific activator of the adenylate cyclase in different tissues (Seamon and Daly, 1983), caused a concentration-dependent inhibition of the spontaneous contractions starting from 10nM; 0 . 1 g M b
¢
e 10 min
Fig. 1. Different patterns of spontaneous motility of myometrial strips removed from oestrus (a, b), dioestrus (c, d) and pregnant (e, f) mare uteri. Tension of the transducer in g; time in rain.
Horse uterus motility
515
forskolin caused the disappearance of any activity. In the same experimental conditions theophylline was ineffective up to 1 #M. The administration of//z-adrenoceptor stimulants, salbutamol and hexoprenaline, caused a concentration dependent inhibition of the phasic contractions in the range of concentrations 1-1000 nM (Fig. 2). ICs0 for salbutamol and hexoprenaline in oestrus uteri were 7.70 + 1.44 nM and 43.4 + 6.5 nM, respectively. In some preparations from pregnant uteri (about 20%), a 100% inhibition was not obtained after administration of maximal concentrations of hexoprenaline.
dependent on the Ca 2+ content, as shown in Fig. 5, in which a comparison was made with maximal concentration of nifedipine; in the case of nifedipine a transient increase in frequency was often observed before the activity was abolished. No consistent effect on basal tone was observed. The inhibition induced by removal of calcium ions was counteracted by an excess of calcium in the medium (data not shown). Removing Na + from the bathing medium strongly reduced both the spontaneous phasic contractions and the response to the stimulants (data not shown).
Effect of the calcium channel blockers nifedipine, verapamil and diltiazem on the spontaneous contractions A strong inhibitory effect was observed after administration of nifedipine, verapamil and diltiazem (Fig. 3). It may be observed from the figure the high potency of nifedipine which had a threshold concentration as low as 0.1 nM. The IDs0 values for the three calcium channel blockers examined are reported in Table 2; nifedipine was by far the most potent drug, whereas verapamil and diltiazem were approximately equipotent. In comparison with the effect of fl2adrenergic compounds, the inhibition induced by Ca 2÷ entry blockers was more reliable, longer lasting and more efficacious in pregnant uteri preparations (100% inhibition); moreover, the inhibitory effect of Calcium antagonists was not easily reversed after washing. An example of the different time course of the response to nifedipine, hexoprenaline and salbutamol in dioestrus myometrial strips is shown in Fig. 4. The inhibition induced by Calcium antagonists was not significantly different when considering oestrus, dioestrus and pregnant uterus preparations.
Our experiments showed that isolated mare uterus is endowed with remarkable and long lasting (about 6-8 hr) spontaneous motility, which was present in oestrus and dioestrus uterus preparations, though with some qualitative differences. Surprisingly also pregnant uterus strips may present a typical pattern of phasic contractions, which, however, was evident in a small percentage (about 30%) of the preparations examined. Neuronal mechanisms do not seem to be involved in maintaining a phasic motility /n vitro since the neuronal blocker tetrodoxin was unable to modify it. In our experimental conditions also the involvement of the most common endogenous stimudatory mediators could be excluded when considering the lack of effect of the respective receptor antagonists (antihistaminics, alpha-blocking agents, antiserotoninergic compounds, etc.). Even prostaglandins seem to have a minor role since indomethacin at the concentrations normally used to block PGs synthesis did not significantly modify the spontaneous contractions. The erratic effect of atropine suggests that, at least in some cases, the cholinergic drive was partly responsible for the observed motility. It seems reasonable to conclude that the spontaneous contractions of mare uterus in vitro are myogenic in nature, as reported for other animal species (Ruegg, 1971); experiments in
Effect of changes in the ionic content of the medium on the spontaneous motility Experiments performed in Ca 2+ free medium showed that the spontaneous motor activity is highly lOO.
DISCUSSION
100.
T. 0
\
E g 8 © dl
50
.g i*
o I
I
I
I
I
I
I
I
I
I
I
I
I
I
11
10
9
8
7
6
5
11
10
9
8
7
6
5
- Log
[$111butamol]
- Log
J'Hexoprenallne]
Fig. 2. Isolated longitudinal strips from oestrus (O), dioestrus (O) and pregnant (&) mare uteri. Inhibitory effect of p:-adrenoceptor stimulants on the height of spontaneous contractions, taken as 100. Values are mean _+SEM from 6 experiments.
516
G. CORUZZI et al. 100 -
100-
50-
50-
O-
I
I
11
10
. tog
w
I
i
i
i
i
i
i
i
I
i
9
8
7
6
5
9
8
7
6
5
4
[.IFEDIPINt]
-Log
[VERAPAMIL]
Fig. 3. Isolated longitudinal strips from oestrus (A), dioestrus (11) and pregnant (&) mare uteri. Inhibitory effect of calcium entry blockers on the height of spontaneous contractions, taken as 100. Values are mean + SEM from 6~ experiments. Na ÷- and Ca2+-free medium (which caused the disappearance of the motility) pointed out the importance of these ions for the spontaneous spike generation in the uterine smooth muscle, as observed in other animal models (Casteels, 1970). The only compounds which markedly affected the uterine motility, were the fl2-adrenoceptor stimulants, the Ca 2+ channel blockers and the adenylate cyclase activator forskolin. The strong inhibitory effect of the adrenergic drugs is not surprising taking into account that the uterus of different animal species, including humans, contains a great number of fl2-andrenoceptors, which, when stimulated, cause smooth muscle relaxation. The receptor activation brings about an increase in the intracellular levels of cyclic AMP, which is considered the intracellular mediator of these drugs at different levels. The importance of cyclic AMP is emphasized by the results obtained with forskolin, which has been shown to increase cAMP production (by direct adenylate cyclase activation) also in the uterine smooth muscle (Marshall and Fain, 1985). More interesting and new appears the effect of Ca 2÷ channel blockers, described in the early experiments as drugs acting essentially at cardiovascular level and employed at present mainly in the therapy of cardiovascular diseases (Nayler and Horowitz, 1983; Opie, 1984). The strong inhibitory effect observed in the mare uterus confirmed previous findings obtained in rats, rabbits, ewes and humans (Abel and Hollingsworth, 1985; Lirette et al., 1985; Forman et al., 1979; Golichowski et al., 1985; Ballejo et al., 1986). Recent experiments revealed that many extravascular smooth muscle tissues are very sensitive
to low concentrations of these drugs (Berger and McCallum, 1982; Barone et al., 1986; Bertaccini and Coruzzi, 1987; Coruzzi and Poli, 1987; Poli et al., 1987). Indeed Calcium antagonists have been successfully tested in different pathological hypermotility disorders, like achalasia, irritable bowel syndrome, etc. The possibility of employing selective Ca 2+ channel blockers as tocolytics may open new horizons in the obstetric clinical practice. Preliminary studies performed in pregnant animals (Lirette et al., 1985) and in humans (Csapo et al., 1982) reported a very NIF
HEX
10 "7
10 .7
SALB
lOmin ! I
10 .7
¢
Table 2. Relative potencies of some calcium entry blockers on the spontaneous mare uterus motility Nifedipine (nM) Verapamil
(vM)
Diltiazem (I~M)
Oestrus
Dioestrus
Pregnant
14.60 (3.3)
55.8 (16.1)
6.70 (2.8)
1.53 (0.87)
1.89 (0.72)
1.66 (0.8)
1.56 (0.80)
1.80 (0.86)
0.47 (0.22)
Values refer to mean IDs0s + SEM (in parenthesis) from 6 to 8 observations.
Fig. 4. I s o l a t e d m y o m e t r i a l strips f r o m d i o e s t r u s uteri. Single e x p e r i m e n t s h o w i n g the different d u r a t i o n o f the i n h i b i t o r y a c t i o n i n d u c e d b y nifedipine ( N I F ) , hexopre-
naline (HEX) and salbutamol (SALB). Drugs were administered in molar concentrations. Tension of the transducer in g; time in rain.
Horse uterus motility Ca';Iree
517
Acknowledgement--Tlus study was supported by the M.P.I.
Roma, Italy. REFERENCES
NIF
10 "s
~'
lomln NIF
10 "'r
'
'
Fig. 5. Isolated myometrial strips from oestrus uteri. Single experiment showing the effect of calcium removal from the bathing medium on the spontaneous phasic contractions. Nifedipine (NIF), administered in molar concentrations, was reported for comparison. Tension of the transducer in grams; time in rain. satisfactory tocolytic effect after administration of the dihydropyridines nifedipine and nicardipine, without significant hemodynamic alterations. However, the presence of more or less pronounced hemodynamic changes, reported in other studies, suggests that new compounds of the family in which the dissociation between vascular and extravascular activity is more pronounced, are awaited with great interest.
SUMMARY The spontaneous motility of mare uterus in vitro was studied on myometrial strips removed from animals in different periods of the oestrus cycle and during pregnancy. A series of drugs with different mechanism of action were tested on uterine spontaneous contractions. Tetrodotoxin, membrane receptor antagonists, like alpha blocking agents, antihistaminics antiserotoninergic drugs and the prostaglandin synthesis inhibitor, indomethacin, were ineffective. On the contrary t2 selective adrenoceptor stimulants, like salbutamol and hexoprenaline caused a concentration-dependent reduction of the amplitude of spontaneous contraction up to a complete abolition. ICso values were in the range of nM concentrations. A marked inhibition of the spontaneous motility was obtained also with the Calcium channel blockers nifedipine, diltiazem and verapamil which had a more prolonged effect in comparison with fl2adrenergic drugs. Nifedipine was the most potent compound (IDs0 = 14.6 nM). No significant differences were found in the inhibitory effect of these drugs between preparations removed from oestrus, dioestrus and pregnant uteri. From the above data it was concluded the isolated uterus from mares is very sensitive to the inhibitory action of Calcium antagonists; in particular nifedipine, could be very promising alternative to the use of 82 sympathomimetic drugs in the treatment of diseases characterized by uterine hypermotility.
Abel M. H. and Hollingsworth M. (1985) The potencies and selectivities of four calcium antagonists as inhibitors of uterine contractions in the rat in vivo. 8r. J. Pharmac. 85, 263-269. Andersson K. E., Bengtsson L. P., Gustafson I. and Ingemarsson I. (1975) The relaxing effect of terbutaline on the human uterus during term labor. Am. J. Obst. Gynecol. 121, 602-609. Ballejo G., Calixto J. B. and Medeiros Y. S. (1986) "In vitro" effects of calcium entry blockers, chlorpromazine and fenoterol upon human pregnant myometrium contractility. Br. J. Pharmac. 89, 515-523. Barone F. C., White R. F., Ormsbee III. and Wasserman M. A. (1986) Effects of Calcium channel entry blockers nifedipine and nilvadipine on colonic motor activity. J. Pharmac. exp. Ther. 237, 99-106. Berg G., Andersson R. G. G. and Ryden G. (1985) ~-adrenergic receptors in human myometrium during pregnancy: changes in the number of receptors after ~-mimetic treatment. Am. J. Obstet. Gynecol. 151, 392-396. Berger K. and McCallum R. W. (1982) Nifedipine in the treatment of achalasia. Ann. Int. Med. 96, 61-62. Bertaccini G. and Coruzzi G. (1987) Calcium antagonists and the GI tract. GI Reports 5, 1-7. Caritis S. N. (1983) Treatment of preterm labour. A review of the therapeutic options. Drugs 26, 243-261. Casteels R. (1970) The relation between the membrane potential and the ion distribution in smooth muscle cells In: Smooth muscle (Edited by Bulbring E., Brading A., Jones A. W. and Tomita T.) pp. 70-99. Edward Arnold London. Coruzzi G. and Poli E. (1987) Changes in duodenal contractility induced by "Calcium antagonists" with different modes of action. Gen. Pharmac. 18, 69-74. Csapo A. I., Purl C. P., Tarro S. and Henzl M. R. 0982) Deactivation of the uterus during normal and premature labor by the calcium antagonist nicardipine Am. J. Obstet. Gynecol. 142, 483-491. Fleckenstein A. (1977) Specific pharmacology of calcium in myocardium, cardiac pacemakers and vascular smooth muscle. Ann. Rev. Pharmac. Toxicol. 17, 149166. Forman A., Andersson K-E., Persson C. G. A. and Ulmsten U. (1979) Relaxant effects of nifedipine on isolated, human myometrium. Acta Pharmac. Toxicol. 45, 81-86.
Golichowski A. M., Hathaway D. R., Fineberg N. and Peleg D. 0985) Tocolytic and hemodynamic effects of nifedipine in the ewe. Am. J. Obst. Gynecol. 151, 1134-1140.
Kern O. and Schill H. (1983) Pharmacology of uterine motility and relaxation. In Veterinary Pharmacology and Toxicology (Edited by Ruckebush Y.). pp. 203-211. MTP Press, Boston. Johansson S. R. M. and Andersson R. G. G. (1981) Mechanism of beta adrenergic desensitization in rat myometrium. Acta Pharmac. ToxicoL 49, 241-247. Lirette M., Holbrook H. and Katz M. (1985) Effect of nicardipine HC1 on prematurely induced uterine activity in the pregnant rabbit. Obst Gynecol. 65, 31-36. Marshall J. and Fain J. N. (1985) Effects of forskolin and isoproterenol on cyclic AMP and tension in the myometrium. Eur. J. Pharmac. 107, 25-34. Nayler W. G. and Horowitz J. D. (1983) Calcium antagonists: a new class of drugs. Pharmac. Ther., 20, 203-262. Opie L. H. (1984) Calcium antagonists and cardiovascular disease. Raven Press, New York.
518
G. CORUZZIet al.
Poli E., Rusagara J., Merialdi A. and Coruzzi G. (1987) Effect of calcium antagonists on the human uterus "in vitro". Acta Chit. Austr. 19, (Suppl 70) 8-10. Ruegg J. C. (1971) Smooth muscle tone. Physiol. Rev. 51, 201-248. Seamon K. B. and Daly J. W. (1983) Forskolin,cyclicA M P and cellularphysiology. Trends Pharmac. Sci. 4, 120-123.
Ueberberg H., Bauer M., Eckenfelds A., Lehmann H., Pappritz G. and Serbedija R. (1976) TierexperimenteUe Untersuchungen zur Vertraglichkeit yon NAB 365 (Clenbuterol). Arzneimittel Forsch. 26, 1420-1427. Zerobin K. and Kundig H. (1980) The control of myometrial functions during parturition with a //2 mimetic compound, Planipart. Theriogenology 14, 21-35.
0306-3623/89 $3.00+ 0.00 Copyright © 1989PergamonPress pie
Printed in Great Britain. All fights reserved
PHARMACOLOGICAL CHARACTERIZATION OF MARE UTERUS MOTILITY WITH SPECIAL REFERENCE TO CALCIUM ANTAGONISTS AND BETA-2-ADRENERGIC STIMULANTS G. CORUZZI, E. POLI, C. MONTANARIl and G. BERTACCINI* Institute of Pharmacology, Faculty of Medicine and Faculty of Veterinary, University of Parma, Italy and t National Health Service, Parma, Italy (Received 21 October 1988)
Abstract--l. Uterine motility was studied in vitro in the myometrial tissue obtained from pregnant and non-pregnant mares. 2. The spontaneous contractions of the preparations were not modified by tetrodotoxin, by antieholinergics, antiadrenergics, histamine H1 and H2 blockers, antiserotoninergic and opioid antagonists; but disappeared in Ca 2+ and Na + free medium. 3. ~2-adrenergic stimulants like salbutamol and hexoprenaline and the calcium channel blockers nifedipine and verapamil were effective inhibitors of the amplitude of phasic contractions (IDs0s for salbutamol and nifedipine were 7.7 nM and 14.6 nM, respectively in oestrus preparations). 4. The above data indicated that the mare myometrium contractility in vitro is very sensitive to the action of/~2 mimetic compounds and calcium antagonists; nifedipine, in particular, seems to be a very promising alternative to/~2 stimulants in the tocolytic therapy.
INTRODUCTION
Selective /~2-adrenoceptor stimulants are extensively used in both h u m a n and veterinary medicine in diseases characterized by smooth muscle deranged motility and they are the most effective drugs available to prevent abortion, to control parturition and reduce dystocia or uterine hypertony (Caritis, 1983; Kern and Schill, 1983; Andersson et aL, 1980; Zerobin and Kundig, 1987). Clinical toxicity studies showed that these drugs are reasonably safe in most species, however some untoward reactions may occur in women and in dogs, concerning cardiovascular system (tachyeardia, pulmonary edema, etc.) (Caritis, 1983; Ueberberg et al., 1976). Moreover a rapid desensitization to ~2-adrenergic agents has been reported both in vitro and in vivo in the long term therapy (Berg et al., 1985; Johansson and Andersson, 1981). Therefore new tocolytic agents, more effective and safer have been searched as an alternative to //2-sympathomimetic drugs. Preliminary studies on isolated myometrium from various animal species and humans indicated that drugs which negatively interfere with the intracellular availability of Ca 2+, could be considered as potential tocolytics (Abel and Hollingsworth, 1985; F o r m a n et al., 1979; Lirette et al., 1985). Indeed Ca 2+ entry blockers by impairing the transmembrane influx into cells (Fleckenstein, 1977) are effective relaxant agents both in vascular and extravascular smooth muscle. Preliminary clinical studies in humans showed that these drugs effectively inhibited uterine contractility
*Address correspondence to: Professor Giulio Bertaoeini, Institute of Pharmacology, via Gramsei 14, 43100 Parma, Italy
in preterm labor and that induced by spasmogenic agents like prostaglandins and oxytocin (Csapo et al., 1982). The present study was undertaken to investigate the action of the Ca z+ antagonists nifedipine, verapamil and diltiazem on the isolated myometrial strips from mare uterus, in comparison with the/~2-adrenergic stimulants salbutamol and hexoprenaline. METHODS
Fifty mares weighing 450-500 kg were used. The uterine horns were removed from animals in oestrus and dioestrus; a small percentage were obtained from pregnant animals (3-5 month gestation). After slaughtering the animals the uterine horns were rapidly removed, immediately transferred into an oxygenated 95% 02 and 5% CO2) nutrient fluid at 4°C and transported to the laboratory. Myometrial longitudinal strips (0.3 cm wide and 1.5 cm long) were dissected free of endometrium and serosa and set up in isolated organ baths (10ml) at 37°C. At least 4 preparations were obtained from each horn. The composition of the nutrient fluid was (raM): NaCI 113; KC14.7; MgSO4 1.2; CaCI2 1.9; KH2PO4 1.2; NaHCO 3 25; Glucose 11.5. Myometrial contractions were recorded by an isometric transducer connected with a pen recorder (Basile, Comerio) under a resting tension of 2 g. While most of the preparations were set up within 1 hr after the removal, some were stored at 4°C overnight and set up the following day; no significant differences were noted in spontaneous motility and in response to drugs between fresh and refrigerated preparations. In a first series of experiments the pattern of spontaneous motility was characterized, in the absence of any drug, by observation of the phasic movements for the whole experiment, in terms of frequency, amplitude and duration of the contractions. In other series of experiments, after an equilibration time of about 30 min the effect of different drugs on this motor activity was evaluated by administering single or cumulative concentrations of the drug considered. 513
G. CORUZZI et al.
514
Some experiments were performed in Ca2+-free medium obtained by simply removing Ca2+ ions or in Ca2+free-EGTA solution by adding 1 mM EGTA. Experiments in Na + free medium were carried out by replacing NaC1 with equimolar amount of choline chloride (Casteels, 1970).
Evaluation of data Significancelevels of the differences between groups were assessed by using Student's t-test or the analysis of variance. The potency of the inhibitory compounds was expressed by the IDs0 value, calculated for each compound from the individual concentration-response curves.
Drugs The following drugs were used: atropine, phentolamine, methysergide, pyrilamine, theophylline and forskolin (Sigma); famotidine and diltiazem (Sigma Tau); tetrodotoxin (Sankyo); nifedipine (Bayer, Leverkusen); verapamil (Knoll); salbutamol (Glaxo); hexoprenaline (Byk Gulden); indomethacin (Chiesi); choline chloride (Carlo Erba). RESULTS
Characteristics of the spontaneous motility of mare uterus Approximately 98% of the preparations from oestrus and dioestrus uteri exhibited a spontaneous motor activity, which was evident after 15-60 min and lasted from 6 to 12 hr. Conversely only 30% of the strips removed from pregnant uteri showed spontaneous contractions. Some examples of the different pattern of motility are shown in Fig. 1. No significant differences were found between uterine strips in different periods of oestrus cycle with respect to frequency and amplitude of contractions; on the contrary pregnant uteri endowed with spontaneous motility showed a significantly higher amplitude of contractions, with longer interval periods and latency (Table 1).
Table I. Spontaneousmotility of myometrial longitudinalstrips from mare uterus Latency Amplitude Interval* (rain)
Oestrus Dioestrus Pregnant
57.9 + 5.8 56.1 + 3.9 135.2+ 23.9a
(g)
3.4 + 0.3 3.9 + 0.4 4.7 _+0.7b
(rain)
5.9 + 0.4 6.7 + 0.6 8.7 +_1.0b
n
38 63 20
*Interval between two subsequent contractions; asignificantly different (P < 0.05) from oestrus and dioestrus uteri; bsignificantly different from oestrus uteri. Mean values + SEM. n = number of preparations.
Effect of tetrodotoxin, indomethacin and membrane receptor antagonists on the spontaneous phasic contractions. This motility was not affected by tetrodotoxin (1 gM). Similarly ineffective were a series of membrane receptor antagonists, like the alpha blocking agent phentolamine (1 #M), the histamine HI receptor antagonist pyrilamine (10 # M); the new H2 receptor antagonist famotidine (10#M) and the 5HT receptor antagonist methysergide (10 #M). Also the cyclooxygenase inhibitor indomethacin was ineffective up to 10#M; very high concentrations (30-100 juM) caused a consistent inhibition probably due to non specific effects. The effect of the antimuscarinic compound atropine (0.01-0.1/zM) was erratic: sometimes it was ineffective, whereas in some strips a 100% inhibition was observed.
Effect of compounds interfering in the cyclic A M P metabolism on the spontaneous contractions The new compound forskolin, recently described as a specific activator of the adenylate cyclase in different tissues (Seamon and Daly, 1983), caused a concentration-dependent inhibition of the spontaneous contractions starting from 10nM; 0 . 1 g M b
¢
e 10 min
Fig. 1. Different patterns of spontaneous motility of myometrial strips removed from oestrus (a, b), dioestrus (c, d) and pregnant (e, f) mare uteri. Tension of the transducer in g; time in rain.
Horse uterus motility
515
forskolin caused the disappearance of any activity. In the same experimental conditions theophylline was ineffective up to 1 #M. The administration of//z-adrenoceptor stimulants, salbutamol and hexoprenaline, caused a concentration dependent inhibition of the phasic contractions in the range of concentrations 1-1000 nM (Fig. 2). ICs0 for salbutamol and hexoprenaline in oestrus uteri were 7.70 + 1.44 nM and 43.4 + 6.5 nM, respectively. In some preparations from pregnant uteri (about 20%), a 100% inhibition was not obtained after administration of maximal concentrations of hexoprenaline.
dependent on the Ca 2+ content, as shown in Fig. 5, in which a comparison was made with maximal concentration of nifedipine; in the case of nifedipine a transient increase in frequency was often observed before the activity was abolished. No consistent effect on basal tone was observed. The inhibition induced by removal of calcium ions was counteracted by an excess of calcium in the medium (data not shown). Removing Na + from the bathing medium strongly reduced both the spontaneous phasic contractions and the response to the stimulants (data not shown).
Effect of the calcium channel blockers nifedipine, verapamil and diltiazem on the spontaneous contractions A strong inhibitory effect was observed after administration of nifedipine, verapamil and diltiazem (Fig. 3). It may be observed from the figure the high potency of nifedipine which had a threshold concentration as low as 0.1 nM. The IDs0 values for the three calcium channel blockers examined are reported in Table 2; nifedipine was by far the most potent drug, whereas verapamil and diltiazem were approximately equipotent. In comparison with the effect of fl2adrenergic compounds, the inhibition induced by Ca 2÷ entry blockers was more reliable, longer lasting and more efficacious in pregnant uteri preparations (100% inhibition); moreover, the inhibitory effect of Calcium antagonists was not easily reversed after washing. An example of the different time course of the response to nifedipine, hexoprenaline and salbutamol in dioestrus myometrial strips is shown in Fig. 4. The inhibition induced by Calcium antagonists was not significantly different when considering oestrus, dioestrus and pregnant uterus preparations.
Our experiments showed that isolated mare uterus is endowed with remarkable and long lasting (about 6-8 hr) spontaneous motility, which was present in oestrus and dioestrus uterus preparations, though with some qualitative differences. Surprisingly also pregnant uterus strips may present a typical pattern of phasic contractions, which, however, was evident in a small percentage (about 30%) of the preparations examined. Neuronal mechanisms do not seem to be involved in maintaining a phasic motility /n vitro since the neuronal blocker tetrodoxin was unable to modify it. In our experimental conditions also the involvement of the most common endogenous stimudatory mediators could be excluded when considering the lack of effect of the respective receptor antagonists (antihistaminics, alpha-blocking agents, antiserotoninergic compounds, etc.). Even prostaglandins seem to have a minor role since indomethacin at the concentrations normally used to block PGs synthesis did not significantly modify the spontaneous contractions. The erratic effect of atropine suggests that, at least in some cases, the cholinergic drive was partly responsible for the observed motility. It seems reasonable to conclude that the spontaneous contractions of mare uterus in vitro are myogenic in nature, as reported for other animal species (Ruegg, 1971); experiments in
Effect of changes in the ionic content of the medium on the spontaneous motility Experiments performed in Ca 2+ free medium showed that the spontaneous motor activity is highly lOO.
DISCUSSION
100.
T. 0
\
E g 8 © dl
50
.g i*
o I
I
I
I
I
I
I
I
I
I
I
I
I
I
11
10
9
8
7
6
5
11
10
9
8
7
6
5
- Log
[$111butamol]
- Log
J'Hexoprenallne]
Fig. 2. Isolated longitudinal strips from oestrus (O), dioestrus (O) and pregnant (&) mare uteri. Inhibitory effect of p:-adrenoceptor stimulants on the height of spontaneous contractions, taken as 100. Values are mean _+SEM from 6 experiments.
516
G. CORUZZI et al. 100 -
100-
50-
50-
O-
I
I
11
10
. tog
w
I
i
i
i
i
i
i
i
I
i
9
8
7
6
5
9
8
7
6
5
4
[.IFEDIPINt]
-Log
[VERAPAMIL]
Fig. 3. Isolated longitudinal strips from oestrus (A), dioestrus (11) and pregnant (&) mare uteri. Inhibitory effect of calcium entry blockers on the height of spontaneous contractions, taken as 100. Values are mean + SEM from 6~ experiments. Na ÷- and Ca2+-free medium (which caused the disappearance of the motility) pointed out the importance of these ions for the spontaneous spike generation in the uterine smooth muscle, as observed in other animal models (Casteels, 1970). The only compounds which markedly affected the uterine motility, were the fl2-adrenoceptor stimulants, the Ca 2+ channel blockers and the adenylate cyclase activator forskolin. The strong inhibitory effect of the adrenergic drugs is not surprising taking into account that the uterus of different animal species, including humans, contains a great number of fl2-andrenoceptors, which, when stimulated, cause smooth muscle relaxation. The receptor activation brings about an increase in the intracellular levels of cyclic AMP, which is considered the intracellular mediator of these drugs at different levels. The importance of cyclic AMP is emphasized by the results obtained with forskolin, which has been shown to increase cAMP production (by direct adenylate cyclase activation) also in the uterine smooth muscle (Marshall and Fain, 1985). More interesting and new appears the effect of Ca 2÷ channel blockers, described in the early experiments as drugs acting essentially at cardiovascular level and employed at present mainly in the therapy of cardiovascular diseases (Nayler and Horowitz, 1983; Opie, 1984). The strong inhibitory effect observed in the mare uterus confirmed previous findings obtained in rats, rabbits, ewes and humans (Abel and Hollingsworth, 1985; Lirette et al., 1985; Forman et al., 1979; Golichowski et al., 1985; Ballejo et al., 1986). Recent experiments revealed that many extravascular smooth muscle tissues are very sensitive
to low concentrations of these drugs (Berger and McCallum, 1982; Barone et al., 1986; Bertaccini and Coruzzi, 1987; Coruzzi and Poli, 1987; Poli et al., 1987). Indeed Calcium antagonists have been successfully tested in different pathological hypermotility disorders, like achalasia, irritable bowel syndrome, etc. The possibility of employing selective Ca 2+ channel blockers as tocolytics may open new horizons in the obstetric clinical practice. Preliminary studies performed in pregnant animals (Lirette et al., 1985) and in humans (Csapo et al., 1982) reported a very NIF
HEX
10 "7
10 .7
SALB
lOmin ! I
10 .7
¢
Table 2. Relative potencies of some calcium entry blockers on the spontaneous mare uterus motility Nifedipine (nM) Verapamil
(vM)
Diltiazem (I~M)
Oestrus
Dioestrus
Pregnant
14.60 (3.3)
55.8 (16.1)
6.70 (2.8)
1.53 (0.87)
1.89 (0.72)
1.66 (0.8)
1.56 (0.80)
1.80 (0.86)
0.47 (0.22)
Values refer to mean IDs0s + SEM (in parenthesis) from 6 to 8 observations.
Fig. 4. I s o l a t e d m y o m e t r i a l strips f r o m d i o e s t r u s uteri. Single e x p e r i m e n t s h o w i n g the different d u r a t i o n o f the i n h i b i t o r y a c t i o n i n d u c e d b y nifedipine ( N I F ) , hexopre-
naline (HEX) and salbutamol (SALB). Drugs were administered in molar concentrations. Tension of the transducer in g; time in rain.
Horse uterus motility Ca';Iree
517
Acknowledgement--Tlus study was supported by the M.P.I.
Roma, Italy. REFERENCES
NIF
10 "s
~'
lomln NIF
10 "'r
'
'
Fig. 5. Isolated myometrial strips from oestrus uteri. Single experiment showing the effect of calcium removal from the bathing medium on the spontaneous phasic contractions. Nifedipine (NIF), administered in molar concentrations, was reported for comparison. Tension of the transducer in grams; time in rain. satisfactory tocolytic effect after administration of the dihydropyridines nifedipine and nicardipine, without significant hemodynamic alterations. However, the presence of more or less pronounced hemodynamic changes, reported in other studies, suggests that new compounds of the family in which the dissociation between vascular and extravascular activity is more pronounced, are awaited with great interest.
SUMMARY The spontaneous motility of mare uterus in vitro was studied on myometrial strips removed from animals in different periods of the oestrus cycle and during pregnancy. A series of drugs with different mechanism of action were tested on uterine spontaneous contractions. Tetrodotoxin, membrane receptor antagonists, like alpha blocking agents, antihistaminics antiserotoninergic drugs and the prostaglandin synthesis inhibitor, indomethacin, were ineffective. On the contrary t2 selective adrenoceptor stimulants, like salbutamol and hexoprenaline caused a concentration-dependent reduction of the amplitude of spontaneous contraction up to a complete abolition. ICso values were in the range of nM concentrations. A marked inhibition of the spontaneous motility was obtained also with the Calcium channel blockers nifedipine, diltiazem and verapamil which had a more prolonged effect in comparison with fl2adrenergic drugs. Nifedipine was the most potent compound (IDs0 = 14.6 nM). No significant differences were found in the inhibitory effect of these drugs between preparations removed from oestrus, dioestrus and pregnant uteri. From the above data it was concluded the isolated uterus from mares is very sensitive to the inhibitory action of Calcium antagonists; in particular nifedipine, could be very promising alternative to the use of 82 sympathomimetic drugs in the treatment of diseases characterized by uterine hypermotility.
Abel M. H. and Hollingsworth M. (1985) The potencies and selectivities of four calcium antagonists as inhibitors of uterine contractions in the rat in vivo. 8r. J. Pharmac. 85, 263-269. Andersson K. E., Bengtsson L. P., Gustafson I. and Ingemarsson I. (1975) The relaxing effect of terbutaline on the human uterus during term labor. Am. J. Obst. Gynecol. 121, 602-609. Ballejo G., Calixto J. B. and Medeiros Y. S. (1986) "In vitro" effects of calcium entry blockers, chlorpromazine and fenoterol upon human pregnant myometrium contractility. Br. J. Pharmac. 89, 515-523. Barone F. C., White R. F., Ormsbee III. and Wasserman M. A. (1986) Effects of Calcium channel entry blockers nifedipine and nilvadipine on colonic motor activity. J. Pharmac. exp. Ther. 237, 99-106. Berg G., Andersson R. G. G. and Ryden G. (1985) ~-adrenergic receptors in human myometrium during pregnancy: changes in the number of receptors after ~-mimetic treatment. Am. J. Obstet. Gynecol. 151, 392-396. Berger K. and McCallum R. W. (1982) Nifedipine in the treatment of achalasia. Ann. Int. Med. 96, 61-62. Bertaccini G. and Coruzzi G. (1987) Calcium antagonists and the GI tract. GI Reports 5, 1-7. Caritis S. N. (1983) Treatment of preterm labour. A review of the therapeutic options. Drugs 26, 243-261. Casteels R. (1970) The relation between the membrane potential and the ion distribution in smooth muscle cells In: Smooth muscle (Edited by Bulbring E., Brading A., Jones A. W. and Tomita T.) pp. 70-99. Edward Arnold London. Coruzzi G. and Poli E. (1987) Changes in duodenal contractility induced by "Calcium antagonists" with different modes of action. Gen. Pharmac. 18, 69-74. Csapo A. I., Purl C. P., Tarro S. and Henzl M. R. 0982) Deactivation of the uterus during normal and premature labor by the calcium antagonist nicardipine Am. J. Obstet. Gynecol. 142, 483-491. Fleckenstein A. (1977) Specific pharmacology of calcium in myocardium, cardiac pacemakers and vascular smooth muscle. Ann. Rev. Pharmac. Toxicol. 17, 149166. Forman A., Andersson K-E., Persson C. G. A. and Ulmsten U. (1979) Relaxant effects of nifedipine on isolated, human myometrium. Acta Pharmac. Toxicol. 45, 81-86.
Golichowski A. M., Hathaway D. R., Fineberg N. and Peleg D. 0985) Tocolytic and hemodynamic effects of nifedipine in the ewe. Am. J. Obst. Gynecol. 151, 1134-1140.
Kern O. and Schill H. (1983) Pharmacology of uterine motility and relaxation. In Veterinary Pharmacology and Toxicology (Edited by Ruckebush Y.). pp. 203-211. MTP Press, Boston. Johansson S. R. M. and Andersson R. G. G. (1981) Mechanism of beta adrenergic desensitization in rat myometrium. Acta Pharmac. ToxicoL 49, 241-247. Lirette M., Holbrook H. and Katz M. (1985) Effect of nicardipine HC1 on prematurely induced uterine activity in the pregnant rabbit. Obst Gynecol. 65, 31-36. Marshall J. and Fain J. N. (1985) Effects of forskolin and isoproterenol on cyclic AMP and tension in the myometrium. Eur. J. Pharmac. 107, 25-34. Nayler W. G. and Horowitz J. D. (1983) Calcium antagonists: a new class of drugs. Pharmac. Ther., 20, 203-262. Opie L. H. (1984) Calcium antagonists and cardiovascular disease. Raven Press, New York.
518
G. CORUZZIet al.
Poli E., Rusagara J., Merialdi A. and Coruzzi G. (1987) Effect of calcium antagonists on the human uterus "in vitro". Acta Chit. Austr. 19, (Suppl 70) 8-10. Ruegg J. C. (1971) Smooth muscle tone. Physiol. Rev. 51, 201-248. Seamon K. B. and Daly J. W. (1983) Forskolin,cyclicA M P and cellularphysiology. Trends Pharmac. Sci. 4, 120-123.
Ueberberg H., Bauer M., Eckenfelds A., Lehmann H., Pappritz G. and Serbedija R. (1976) TierexperimenteUe Untersuchungen zur Vertraglichkeit yon NAB 365 (Clenbuterol). Arzneimittel Forsch. 26, 1420-1427. Zerobin K. and Kundig H. (1980) The control of myometrial functions during parturition with a //2 mimetic compound, Planipart. Theriogenology 14, 21-35.