Indian Journal of Experimenlal Biology Vol. 43, September 2005, pp. 795-801 Effects of calcium channel blocker, mibefradil, and potassium channel opener, pinacidil, on the contractile response of mid-pregnant goat myometrium Gurupada Mandi, S N Sarkar", S K Mishra & V Raviprakash Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar 243 122, India Received 11 March 2005; revised 10 June 2005 The present study was undertaken to investigate the ill vitro influence of mibefradil, a calcium channel blocker, and pinacidil, a potassium channel opener, on pregnant goat myometrial spontaneous rhythmic contractility and contractions induced with the agonist, oxytocin. Longitudinal strips from the distal region of uterus, collected from goats at mid­ 8 10 gestation. were mounted in an organ bath for recording isometric contractions. Mibefradil (10- _10-4 M) or pinacidil (10- - 10-4 M), added cumulatively to the bath at an increment of 1 log unit, caused concentration-dependent inhibition of the spontaneous rhythmic cOnlractions of isolated uterine strips. The rhythmic contraction was, respectively, abolished at 100 and 10 IlM concenlrations of mibefradil and pinacidil. In a concentration-dependent manner, mibefradil (I and 10 1lM) anlagonized the contractions elicited with oxytocin 00-5_10-2 IU). Pretreatment of uterine strips with glibenclamide (10 11M), a selective KAT? channel bloc;ker, caused a rightward shift of the concentration-response curve of pinacidil with a concomitant decrease in its pD2 value. Pinacidil (0.3, 1 and 3 IJ-M), in a concentration-related manner, antagonized the oxytocin (10-5_10-2 IU)-induced contractile response. The inhibition of spontaneous rhythmic contractions and antagonism of oxytocin-induced contraction by mibefradil in the pregnanl goat myometrium may be related to the antagonism of 2 voltage-dependenl Ca .. channels, while by pinacidil suggests that KAT? channel could be a therapeutic target for tocolysis. Keywords: Calcium channel blocker. Mibefradil, Pinacidil, Potassium channel opener. Goat myometrium 2 4 Different adverse outcomes of pregnancy include through L-type Ca + channels . Several calcium premature labor and abortion. Preterm labor and delivery channel blockers have been shown to be potent is the most important clinical problem in obstetrics, inhibitors of spontaneous tension development by 5 6 occurring in 5-10% of all pregnancies]. It is well isolated uterine smooth muscles of rats and human . established that both preterm delivery and spontaneous Therefore, calcium channel blockers have the abortion are associated with an increased uterine potential for use in the clinical management of contractilitl. Pharmacologic inhibition of premature preterm labor. Mibefradil (Ro 40-5967), {IS, 2S)-2- labor remains the most effective way to delay the [2[[3-(2-benzirnidazolylpropyl] methylamino] ethyl]- 3 parturition and improve neonatal outcome . Drugs that 6-fl uoro-l ,2,3,4-tetrahydro-l-isopropyl-2-naphthyl­ have been used as tocolytic agents include /3-adrenergic methoxyacetate dihydrochloride}, a benzimidazolyl agonist (ritodrine), cyclooxygenase inhibitor tetraline derivative, is a recently introduced calcium (indomethacin), calcium channel blocker (nifedipine), channel antagonist. It is structurally different from oxytocin antagonist (atosiban) and magnesium sulphate. 2 several classical Ca .. channel antagonists, viz., . These agents are potent inhibitors of uterine contractions di Itiazem, nifedipine and veraparnil. It differs from and, therefore, they have potential for use in the clinical 2 other Ca + channel antagonists by its ability to management of preterm labor. 2 7 potently block T-type Ca .. channels . Mibefradil has In a recent study from our laboratory, it has been been shown to have antihypertensive, antianginal and shown that lipopolysaccharide, one of the causative 8 coronary vasodilatory activities • In addition to block agents of preterm labor increased rat myometrial 2 2 of T-type Ca .. channels, it also blocks vascular L-type contractility by increased influx of calcium (Ca .. ) 2 2 9 IO Ca + channels and inhibit intracellular Ca + release • • Wanner et at. II have demonstrated that mibefradil and 'ColTespondent author some of its analogues display a high affinity for E-mail: snsarkar@i vri.up.nic.in Phone: 91-581-2300291; Fax: 91-581-2300312 neuronal voltage-gated sodium channels. 796 INDIAN J EXP BIOL, SEPTEMBER 2005 Rat uterine myometrium has both fast sodium Tissue source - Uterus from goats in mid­ 2 (Na+) channels and slow L-type Ca + channels, but no pregnancy was collected from the local 2 l2 T-type Ca + channels , while that of pregnant human slaughterhouse. The stage of gestation was myometrium expresses both L- and T-type Ca2+ determined from foetal parameters such as size of the channels 13. It is well established that mechanical foetus, stage of organogenesis and crown to rump activity of uterus is dependent on the presence of length. The uterus was collected in chilled modified 2 extracellular Ca + (Ref. 14). Therefore, it can be Ringer-Locke physiological salt solution of the predicted that development of tension in the uterus following composition (nmolell): NaCl, 136.9; KCI, 2 would be prevented with Ca + channel blockers. 5.6; NaHC03, 11.9; CaCI 2• 2H20, 2.2 and glucose, Recently, we have reported the inhibitory activity of 5.6; pH 7.4. The solution contained penicillin (100 mibefradil on uterine myometrial contractility in both Units/ml) and streptomycin (100 Ilg/ml) and was lS pregnant and nonpregnant rats and attributed this transported to the laboratory for experimental use. 2 effect of mibefradil to the block of L-type Ca + Recording of isometric contractions - Approxi­ 2 channels and inhibition of intracellular Ca + release. mately 3xlO mm longitudinal strips were dissected ATP-sensitive potassium channels (KATP) are out from the mid-corunal region of the uteri. Each present in vascular and non-vascular smooth muscle strip was mounted in a thermostatically controlled cells 16. 17. They are activated by a chemically diverse organ bath of 20 rnl capacity, containing Ringer group of agents named potassium channel opening Locke modified solution and bubbled continuously drugs and exemplified by pinacidil, levcromakalim, l6 with room air at 37"±0.5"C under a resting tension of aprikalim, nicorandil and diazoxide , which produce 1 g. The mounted uterine strip was equilibrated for a membrane hyperpolarization and the consequent period of 1 hr with the change of bathing fluid every closure of voltage-dependent calcium channels 15 min. Isometric contractions were recorded by a resulting in smooth muscle relaxation. Using RT-PCR force transducer (Model: MLT0202/D, Powerlab, AD technique, mRNAs for Kir 6.1 and SUR 2B Instruments, Castle Hill, Australia) connected to a (molecular constituents of KATP channels) have been l8 personal computer using the Chart v4.1.2 software detected in rat myometrium . Pinacidil has been programme (Powerlab). reported to produce uterine relaxant effect both in 2 Recording of spontaneous rhythmic contractions - ­ rats 19 .20 and humans 1.22. After equilibration for a peliod of 1 hI' in the modified A search of literature shows that there is no report Ringer-Locke solution, spontaneous rhythmic available on the effects of pinacidil and mibefradil on contractions of the myometrium were recorded. When the uterus of pregnant farm animals or human beings. the rhythmic contractions attained equilibrium both the Further, uterine KATP channels have not been frequency (contraction/min) and amplitude (g) were characterized either in goats or in other farm animals. determined and average was calculated. It is essential for the well being of the fetus and the Sensitivity of spontaneous rhythmic contractions to generation of rhythmic contrdctions in labor that mibefradil and pinacidil - After equilibration, uterine myometrium should be able to relax fully spontaneous rhythmic contractions of the during each contraction-relaxation cycle. The aim of myometrium was recorded in the absence and the present study was to examine whether mibefradil presence of mibefradil or pinacidil. When and pinacidil induce a relaxant effect on the spontaneous rhythmic contractions reached a steady contractility of uterus obtained from pregnant goat as state, mibefradil (10-8_10-4 M) or pinacidil (10- 10_10-4 a model for farm animals, especially ruminants. M) was added to the bath at an increment of 1 log unit in a cumulative pattern. A contact period of 15 min Materials and Methods was maintained at each concentration of mibefradil to Chemicals - Mibefradil was obtained as a gift produce its maximal effect. Effects of mibefradil and from Dr P I Aaronson, London. Pinacidil, pinacidil on the spontaneous rhythmic contractions glibenclamide and oxytocin were procured from were measured considering the control rhythmic Sigma-Aldrich, Hoechest and Novartis (Mumbai, contractions as 100%. The drug concentration that India), respectively. All other chemicals were of produced 50% inhibition (ICso) was determined from analytical grade. All chemkals were diluted in triple the linear regression of log concentration-response distilled water. curve. MANDl et al.: EFFECTS OF MIBEFRADIL AND PINAClDIL ON GOAT MYOMETRIUM 797 Effect of pinacidil on glibenclamide-treated uterine contractions were 0.93±0.09 g (n = 17) and 4.70±0.33 strips - Separate tissues were used to study the effect per min (n=17), respectively. of selective KATP channel