Indi an Journal of Experimental Biology Vol. 42, September 2004, pp. 913-918

Evaluation of tocolytic efficacy of selective beta2 adrenoceptor agonists on buffalo uterus

Satish K Garg, K M Garg & M Sabir

Department of Pharmacology and Toxicology, U.P. Pandit Deen Dayal Upadhyaya Veterinary Science Un iversity and Cattle Research Institute, Mathura 28 1 001 , India

Received 25 July 2003: revised 3 1 May 2004

Present study was conducted on prostaglandin F2atptm (PGF2a), oxytocin , (OT), potassium chloride (KCI) and barium chloride (BaCI2) pre-contracted perimetria! uterine strips of dioestrus and pregnant buffaloes to evaluate the tocolytic effi­cacy or selective 132 adrenoceptor agonists-albuterol (salbutamol) and terbutaline. Cumul ati ve concentration-response curves or both the 132 adrenoceptor agoni sts were constructed and the mean effective concentrati on (EC50) values determined and compared statisticall y. Based on the comparative EC50 values in relaxing the pre-contracted uterine strips with different spasmogcns, the rank order potency of albuterol was found to be-- PGF2a > BaCI2 > OT > KCI on uterine strips from dioes­trus animal s, whi le OT> BaCI2> PGF2a >KCI on the uterine strips of pregnant buffaloes. The rank order potency of terbuta­Iine on uterine strips from dioestrus stage animals was- BaC I2 > OT > KCI > PGF2w while BaCI2 > PGF2a > KCI > OT on uterine tissues of pregnant animals . Thus, irrespective of the state of uterus, whether gravid or non-grav id, KCI-depolari zcd uterine ti ssues required comparat ive ly higher concentrations of albuterol or terbutaline to produce tocolytic effect. Hi gh concentrations of K+ in biophase may have interfered wi th the 13 2 adrenoceptor agonists-induced outward K+ current and hy­perpolari zation. From the results of present study, it was evident that selective 132 adrenergic agoni sts had good tocolytic ef­ficacy on the uterus or buffaloes. Further, indirectl y the possibility of existence and activation of Kca channels by selec ti ve 132 adrcnoceptor agoni sts in mediating tocolysis of buffalo myometrium can not be ruled out , however, detai led studies using specific Kca channel blockers are required for characteri zing the nature of such channels in buffalo uterus.

Keywords: {32 adrenoceptor agonists, Albuterol, Terbutaline, Buffaloes, Toco lyt ics.

Beta2 adrenoceptor agonists have been reported to ex hibit tocolytic effect in different spec ies of animals including buffaloes' and goats2

. Selective B2 adrener­gic receptor agonists-induced myometrium relaxa­tions has been postulated to be due to hyperpolariza­tion3. However, the pharmacodynamics of B2 adreno­ceptor agonists-induced tocolysis of buffalo myome­trium is yet to be elucidated . Therefore , the present investigation was undertaken to evaluate the tocolytic efficacy of selective B2 adrenoceptor agonists on iso­lated uterine stri ps of pregnant and dioestrus stage buffaloes pre-contracted with different spasmogens.

Materials and Methods The non-gravid and gravid uteri along with the

ovaries of non-descript breed buffaloes were collected in chilled Ringer-Locke solution in thermos flask from the local abattoir of Mathura. Immediately, the material was transported to the laboratory. Stage of the estrous cycle was assessed on the basis of thor-

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ough examination of the genital tract including ova­ries. Pregnancy was confirmed based on the presence of foetus in the uterus and the corpus luteum on the ovary of the epsi lateral side. The stage of pregnancy was determined foll ow ing the formula proposed by Soliman4 by measuring the curved crown versus ru mp length. Only, the uterine ti ssues from dioestrus and pregnant buffaloes were selected for detail ed tocolytic studies using selective B2 adrenergic receptor agoni sts.

A piece of uterus was cut longitudina lly from the mid-cornual region. Perimetria! strips of about 0.5 x 3.0 em prepared by carefully removing the endo- and myometrial tissues. The ti ssue-strips were mounted in an organ bath containing continuously aerated Ringer­Locke solution (10 ml) having a pH of 7.4 and main­tained at 37°±0.5°C. The ti ssues were allowed to equilibrate under a constant tension of 2g for 1 to 1.5 hr. During the equilibration period, the perfusion fluid was changed every 15 min. The responses of tissues to various chemicals/agents were recorded on a ky­mograph assembly with the help of an isotonic si mple straw lever (magnification x 10).

914 I DIAN J EXP BIOL, SEPTEMBER 2004

For quantitative tocolytic studies, cumulative con­centration response curves of ~ 2 adrenoceptor ago­ni sts, namely- albuterol (salbutamol) and terbutaline were constructed on prostaglandin F2a (0.8 ~tM), oxy­tocin (0.004 TIJ/ml), potass ium chloride (30 mM) and barium chloride (200 ~tM) pre-contrac ted perimetria] strips. A minimum time period of 1.0 to 1.5 hr was al lowed to elapse between the recording of two suc­cessive concentration responst.:-curves and the perfu­sion fluid was changed every 10-15 min .

The responses to each concentration of the agoni st aga inst different spasmogens were plotted 1 as per cent of the max imum relaxation response versus molar concentration of the drug. The median effec tive con­centration (EC50) of each agonist aga inst different spasmogens(s) was calculated from the cumulative­concentration response curves and the 95 per cent confidence limits of EC50 were determined. The data were subj ected to stati stical analysis to determine the sign ificant differences5

.

Results Effect of albuterol and terbutaline on PGF2a­

precontratced uterine tissues- The isolated uterine strips of dioestrus and pregnant buffaloes pre­contracted with PGF2a (0.8 ~tM) were effectively re­laxed in a concentration-dependent manner by albute­rol and terbutaline. The minimum threshold concen­tration (MTC), the max imum concentrations required to induce max imal relaxation along with the cumula­ti ve dose response curves (CDRCs) of albuterol and

terbutaline are shown in Fig. 1A. The EC50 va lu along with confidence limits of alburerol and terbUJ line against PGF2a are presented in Table I.

Effect of albuterol and terbutaline on oxytoc1 precontracted uterine tissues- Both albuterol a terbutaline relaxed oxytoc in (0.004 IU/ml) p1 contracted isolated di oestrus and pregnant buffalo myometrium in a concentration-dependent mann1 The CDRCs showing the MTC and the concentratio of albuterol or terbutaline required to induce maxirr relaxation are shown in Fig. lB. The EC50 values wi 95 per cent confidence limits of albu terol and terbul line are summari zed in Table 1.

Effect of albuterol and terbutaline on potassil, chloride-precontracted uterine tissues- Albute1 and terbutaline relaxed the potass ium chlori, (30 mM) pre-contracted uterine ti ss ues from both t dioestrus and pregnant buffaloes in a concentratio dependent manner. The MTC and the max imal co centrations required to induce maxi mal relaxati1 along with the CDRCs of albuterol and terbutaline a shown in Fig. 2A. The EC50 va lues with 95 per ce confidence limits of these agoni sts are presented Table 1.

Effect of albuterol and terbutaline on barium chi ride-precontracted uterine tissues--Barium chlori1 (200 ~ pre-contracted uterine tissues from both tl dioestrus and pregnant buffaloes were effec ti vely r !axed in a concentration-dependent manner by albut rol and terbutaline. The CDRCs of these agoni s showing the MTC and the concentrations required

Tab le 1- EC50 values of albuterol and terbutaline (M) against different spasmogens on the iso lated uterine strips of dioestrus and pregnant buffaloes. [Values are mean±SE of 4-9 replicates]

EC50 values of albuterol (M) EC5o values of terbutaline (M) Spasmogen(conc in Pregnant Dioestrus Pregnant Dioestrus

bath lluid/ml )

PGF2a (0.8 pM) 6.31 x i0·13 Aa 3.95x l0·14 Ba 3.00x i0.13 Aa 2.30x Hr 12 Ba

(4.80x 10. 13 -7.92x 10·13) (3 .29x 10"14 -4.76x 10·14) (9.0x l o-14 -7 .46x I o-13) ( 1.42x 1 o-12 - 3.7 1 x 1 o-12) Oxytocin 2.51 x !0-13 Ah 1.78x l0·13 Ah 1.28x I o-IO Ah 5.62x Jo·IJBh

(0.004 IU/ ml) (2. 15x I 0"13 -2.94x 10"13) ( 1.32x 10·13 -2.29x 10-13) (6.0x l 0"11-2.70x l 0"10) (3.60x I o- 13 -7 .90x 10"13)

KCI (30 mM) 3.92x 10-12 Ac 1.37x 10-12 Be 2.05x l0-11 Ac 1.97x 10·12 Ba

(3.39x I 0"12 -4.55x I o-12) (9 .36x 10"13 -2.0x I o-12) ( 1.23x 10"11 -3.42x I 0"11 ) (8.59x:0- 13 -4.53x I o-12 )

BaC I2 (200 11M) 5.54x 10"13 Aa 7.98x i0-14 8J 1.83x 10·13 Aa 9.80x i0.14Ac

(4.50x 10"13 -6.82x 10·13) (5.08x 10·14 -1 .25x 10·13) ( 1.20x 10"13 -2.78x 10"13) (7.45x I 0"14 -1 .29x 10-13)

Data in parentheses indicate the confidence limits of EC50 values Means in a row with different capital superscri pts indicate significant difference between the stages of uterus Means in a column with different small superscripts indicate significant difference between the spasmogens

GA RG er a/. : TOCOL YTIC EFFICACY OF BET A2 ADR ENOCEPTOR AGON ISTS 915

induce max ima l relaxat ion are shown in F ig. 2B. The EC50 va lues with 95 per cent confidence limits of al­buterol and terbutaline are presented in Table l.

Comparative assessment of the tocolytic efficacy of albuterol and terbutaline

Albuterol-The overa ll comparative assessme nt of the EC50 va lues of albutero l (Table 1) on the isolated uterine strips fro m the d ioestrus and pregnant buffa ­loes, pre-contracted wi th different spasmogens, re­vealed that irres pecti ve of the state of uterus, whether

100

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0 +----r--.------,--..., -14 -13 -12 -11 -10

grav id or non-grav id, KC I-depolarized ti ssues re­quired the hi ghest concentration of albutero l fo r re­lax ing the max imally contracted ti ss ues by 50 per cent. Further, compared to the uteri of pregnant buf­fa loes, the uteri of d ioes trus stage animals were more sensiti ve to the re laxant effect of albuterol irrespective of the spasmogens(s) used except in case of OT­precontracted ti ssues where the sensitivity of uteri of both the stages di e; oat differ signi ficantly.

Terbutaline- Comparati ve assess ment of EC5o va lues of terbutaline (Table 1) on the d ioestrus and

100 l5 __ o i5" -

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Albuterol Terbutaline

Concentration (M) Fi g. l~umul ative concentration response-curves o f albuterol and terbutaline against (A) PGF2• 1011" (0 .8 JlM) and (B) oxytocin (0.004 IU/ml ) pre-contracted isolated uterine stri ps of dioestru s and pregnant buffaloes. [Vertical bars denote theSE of mean values (n=4-6)]

916 IND IAN J EXP BIOL, SEPTEMBER 2004

pregnant buffaloes uterine strips pre-contracted with different spasmogens revealed that irrespective of the stage of uterus, whether gravid or non-gravid, BaCI2-

depolarized tissues required comparatively lowest concentration of terbutal ine and there was no signifi­cant d ifference in the sensitivity of uteri of both the stages depolarized with BaCI2 to terbuta line-induced

100 - · -o- · - Dioestrus

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relaxation .. The uteri of pregnant buffaloes, compare to those of dioestrus animals, pre-contracted wi1 oxytocin and KCI were significantly less sensitive · terbuta line-induced toco lysis. On the contrary, uterir strips of pregnant an imals pre-contracted with PGF were significantly more sensitive to the re laxant effe of terbutaline.

100

80

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0 -14 -12 .:.... 10 - 8 -6

100

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0+--------.---------r----

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Albuterol Terbutaline

Concentration (M) Fig. 2---Cumulativc concentration response-curves of albuterol and terbutaline against; (A) KC I (30 mM) and (B) BaCI 2 (200 pM pre-contracted isolated uterine strips of dioestrus and pregnant buffaloes. [Vertical bars denote theSE of mean values (n=S-9)]

GARG eta/. : TOCOL YTIC EFFICACY OF BETA2 ADRENOCEPTOR AGONISTS 91 7

Rank order pate/ley of albuterol and terbutaline agai!lst different spasmogens- Based on the com­parative EC50 values of albuterol and terbutaline on the uterine strips from the pregnant buffaloes, the rank order potency of albuterol was OT > BaCh> PGF2a > KCl while tha t of terbutaline was BaCh > PGF2a > KCI > OT. Pharmacological response of uterine strips of the dioestrus stage animals was different from that of the pregnant animals as the rank order potency of albuterol on dioestrus stage uterus was PGF2a > BaCh> OT > KCI and that of terbutaline was BaCI2 > OT > KCI > PGF2a . Uterine strips of pregnant animals pre-contracted with oxytocin were most sensitive to albuterol and least to terbutaline, while dioestrus stage animals uteri pre-contracted with PGF2a were most sensitive to albuterol and almost least to terbutaline.

Discussion Studies on the isolated uterine strips from both the

di oestrus and pregnant buffaloes revealed that PGF2a, OT, KCI or BaCh pre-contracted tissues were effec­tively relaxed in a concentration-dependent manner by albuterol and terbutaline, thus, convincingly sug­gesting that both albuterol and terbutaline are prom­ising tocolytic for buffaloes as has been reported in rats6

·7 and mares8

· Therapeutic tocolytic efficacy of certain B2 adrenoceptor agonists has already been proved in certain species of animals in veterinary medicine9

-12.

Critical evaluation of the rank order potency of al­buterol against different spasmogens, both on the di o­estrus and pregnant animal uterine strips revealed that irrespective of the state of uterus, KCI-depolarized ti ssues required the highest concentrations of albute­rol to induce uterine relaxation. However, the rank order potency of terbutaline was slightly different from that of albuterol as in the rank order, KCI­depolarized tissues were at third position instead of fourth .

Overall comparison of the tocolytic efficacy of al­buterol and terbutaline suggested that dioestrus uter­ine strips were equi-responsive or slightly to signifi­cantly more sensitive irrespective of the spasmogens used except that of PGF2a, wherein uterine strips from pregnant buffaloes were more sensitive to the relaxant effect of terbutaline. Further, the uterine strips of pregnant animals pre-contracted with OT were more responsive to albuterol, while BaCh- or PGF2a­precontracted tissues were sensitive to terbutaline. The observations on the differences in the sensitivity of uteri of pregnant and dioestrus animals to the to-

colytic efficacy of these B2 adrenomimetics indicate that in addition to the selective B2 adrenergic receptor, involvement some other subtle cellular or molecular mechanisms13-15 may be influencing the tocolytic effi ­cacy in buffaloes.

Beta adrenoceptor agonists relax smooth muscles by increasing the intracellular levels of cyclic AMP16

and/or increasing K+ conductance15. The latter process causes hyperpolarization thus, resulting in inhibition of voltage-operated calcium channels and decrease in calmodulin-activation . Isoproterenol-induced relaxa­tion of rat myometrium is due to its hyperpolari zing effect which is associated with an increase in K+ con­ductance3' 13. Isoproterenol-induced myometri al re­laxation in rats has been reported to be mediated via outward K+ current through Ca2+-acti vated K+ chan­nel s17 and thus suggested that isoproterenol-induced tocolytic effect may also be due to outward K+ current and hyperpolarization of myometrium.

Potassium chloride (30mM)-depolarized uterine preparations required comparatively hi gher concen­trations of albuterol and terbutaline to induce relaxa­tion. Presence of high concentrations of K+ in the bio­phase and/or the perfusion fluid may have interfered with the albuterol or terbutaline-induced outward K+ current and thus the higher concentrations of these selective B2 adrenomimetics might have been required for relaxing the KCI-precontracted uterine tissues. Thus, the EC50 values of albuterol and terbutaline against KCI-depolarized ti ssues were significant ly higher compared to those against other spasmogens. Ishine et a/. 18 have also observed that high K+ con­centration in the medium significantly inhibit isopro­terenol-induced relaxation. Based on the similari ty of our observations in the present study with the data available in the literature, indirectly the possibility of existence of Kca channels in buffalo myometrium and their activation by B2 adrenergic receptor agonists can not be ruled out. Anwer et a/. 11 have also reported the existence of prominent Kca channels in the pregnant rat myometrium which are regulated by B2 adrenergic receptor stimulation. However, to establish the exis­tence and investigate the properties of Kca channels in the uterus of buffaloes, fUither detailed studies using whole cell current recording or specific Kca channels antagonists are required .

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fect of beta2 adrenoceplor agonist in buffalo uteru s. Indian J Pharmacal, 28 (1996) 40.

918 INDIAN J EXP BIOL, SEPTEMBER 2004

2 Garg S K, Shah M A A & Garg K M, Tocolytic effect of se­lccti ve bcta2 adrcnoceptor agonists on isolated gravid utems or goats (Capra hircus) . J Vet Phannacol Toxicol, 2 (2002) 82.

3 Torok I & Herczeg J, Bctamimetic effects on the electro­mechanical characteri stics of the pregnant and postpartum myometrium in vitro. /leta Physio/1/ung, 65 ( 1985) 335.

4 Soliman M K. Studies on the physiological chemi stry of the allantoic and amniotic fluids of buffaloes at various periods or pregnancy. Indian Vet J, 52 ( 1975) I 06.

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7 Sharma S & Gupta U. Delay in ex perimentall y-induced pre­term labour in rats with calcium channel blockers and salbu­tamol. Indian J Exp /Jiol, 32 ( 1994) I 09.

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13 Kroeger E A & Marshall J M, Beta-adrene rgic effects on r myometrium, mechanism of hyperpolari zation . !1111 J Physii 225 (1973) 1339.

14 Ovcrweg N I A & Schiff J D, Two mechanisms of isoprot reno! inhibition of smooth mu sc les, Eur .I Phar111aco/. ' ( 1978)23 1.

IS Bulbring E & Tomita T, Suppression of spontaneous spil generation by catecholamincs in the s~< lOOth muscle of tl guinea pi g tacnea co li. Proc Noy{i/ Soc London, 172 ( 196' 103 .

16 lnatomi N. Takayanagi I. Uchida M & Takagi K, lntraccl li far cyclic AMP leve l and intestin al smooth muscle relax tion . Eur J Phar111acol. 26 ( 1978) 73.

17 Anwer 1-1 , Toro L, Obcrti C, Stefani E & Sanborn BM . Ca activated K+ channels in pregnant rat myometrium: Modul lion by a beta-adrenergic agent. 1!111 .1 Physiol. 263 ( 199: C I049.

18 !shine T, Miyauchi Y & Uchida M K, Ca2•-indepcndcnt n laxation mediated by beta adrenoceptors HI C}•-indepcndcr contraction of uteri ne smooth muscle . .I Phannaco/ £1 Th er, 266 ( 1993) 367.