ATP-sensitive and maxi potassium channels regulate BRL 37344-induced tocolysis in buffaloes-an in vitro study

Vipin Sharma, Sooraj V. Nair, Pooja Jaitley, Udayraj P. Nakade, Abhishek Sharma, Soumen Choudhury, Satish Kumar Garg
PII: S0093-691X(17)30529-0
DOI: 10.1016/j.theriogenology.2017.10.044 Reference: THE 14327

To appear in: Theriogenology

Received Date: 22 June 2017
Revised Date: 26 October 2017
Accepted Date: 30 October 2017
Please cite this article as: Sharma V, Nair SV, Jaitley P, Nakade UP, Sharma A, Choudhury S, Garg SK, ATP-sensitive and maxi potassium channels regulate BRL 37344-induced tocolysis in buffaloes-an in vitro study, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.10.044.

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 ATP-sensitive and maxi potassium channels regulate BRL 37344-inducedtocolysis in buffaloes- an in vitro study

Vipin Sharma, Sooraj V Nair, Pooja Jaitley, Udayraj P. Nakade, Abhishek Sharma,

Soumen Choudhury and Satish Kumar Garg

Smooth Muscle Pharmacology & Molecular Pharmacology Laboratory

Department of Veterinary Pharmacology & Toxicology

College of Veterinary Science and Animal Husbandry

U.P. Pandit Deen Dayal Upadhyaya pashu Chikitsa Vigyan Vishwavidyalaya Evam

Go-Anusandhan Sansthan, Mathura-281001


33 Cellular coupling of beta3-adrenoceptors (β3-ADR) to potassium channels in

34 myometrium is largely unknown. In vitro study was undertaken to unravel the presence of β3-

35 adrenergic receptors (ADR) and the role of K+-channels in mediating β3-ADR-induced

36 relaxation in isolated myometrial strips from cyclic non-pregnant water buffaloes. Isometric

37 tension was recorded in isolated myometrial strips using data acquisition system based

38 physiograph. Compared to SR 59230A, BRL 37344 was found to be more potent in inducing

39 β3-dependent myometrial relaxation which was significantly (p<0.05) inhibited in the

40 presence of β3 anagonist, SAR 150640. The immunoreactive protein to β3-ADR was also

41 detected in membrane fraction of myometrial protein. Further, incubation with BRL 37344

42 (10 µM) significantly (p<0.05) increased c-AMP accumulation (37.58 ± 9.52 pmol/mg

43 protein; n=4) in the myometrial strips compared to basal c-AMP level (16.85 ± 3.87 pmol/mg

44 protein; n=4). The concentration response curves (CRC) of BRL 37344 were significantly

45 (p<0.05) shifted towards right in the presence of KATP channels specific blocker,

46 glibenclamide (10 µM) and maxi K+-channels (BKCa) specific blocker, iberiotoxin (100 nM),

47 with decrease in both efficacy and potency compared to control. However, 4-aminopyridine

48 (4-AP), a specific blocker of voltage gated K+-channels (Kv), failed to alter the CRC of BRL

49 37344. Existence of immunoreactive protein to Kir6.1, α-subunit of BKCa and Kv1.1 channels

50 were also detected in the membrane fraction of myometrial protein. Based on the above

51 findings, it can be concluded that BRL 37344 is a potent stimulator of β3-adrenoceptors in

52 buffalo myometrium and besides mediating their effect through rise in c-AMP, they are

53 coupled to KATP and BKCa channels in inducing tocolytic effects.

54 Keywords: BRL 37344, KATP, BKCa, beta3-adrenoceptors, myometrium.


56 1. Introduction

57 Uterine contractility in non-pregnant uterus governs semen transport in the

58 reproductive tract as well as the movement and positioning of embryos in the uterine cavity

59 thus regulating implantation, gestation and parturition. The complex interaction between

60 different factors [1,2] regulate the uterine activity and abnormality in uterine activity leads to

61 implantation failure, spontaneous miscarriage, preterm birth and many other disorders [3]

62 Beta3-adrenoceptors (β3-ADR) are considered to be a potential target for anti-obesity

63 and anti-diabetic drugs due to their ability to stimulate lipolysis in white adipose tissue

64 (WAT) and lipolysis and thermogenesis in brown adipose tissue (BAT). Besides WAT and

65 BAT, the importance of β3-ADR has also been demonstrated in variety of mammalian tissues

66 including stomach, colon, ileum, gallbladder and brain [4, 5, 6, 7, 8] and also in myometrial

67 inflammation, apoptosis [9] and uterine remodelling [10]. Recently, involvement of β3-ADR

68 in regulation of myometrial activity in human [11,12], rats [13,14] and mice [15] has also

69 been documented and these studies have elucidated some species differences in β3-ADR

70 pharmacology.

71 Therapeutic use of selective beta2-adrenoceptor agonists is well accepted due to their

72 inhibitory nature in uterine and bronchial smooth muscles. β2-ADRs contribute to uterine

73 quiescence which is essential for development of fetus during gestation [16,17]. However,

74 clinical use of selective β2-ADR agonists (eg. ritodrine and salbutamol) in uterine disorders

75 [18, 19] is associated with maternal and foetal side effects as well as reduced efficacy of

76 these drugs [20, 21]. This reduced efficacy is due to the associated desensitization of β2-ADR

77 upon stimulation by agonist and thereby phosphorylation of serine and threonine residues at

78 C-terminal ends of the receptor protein by G-protein receptor kinase (GRK) [13, 22].

79 However, β3-ADR lacks this protein kinase-A (PK-A)-dependent phosphorylation site and

80 has fewer serine and threonine residues at C-terminal tail [23], thus making it a potential

81 therapeutic target for addressing challenges in animal reproduction including implantation

82 failure, spontaneous miscarriage, preterm birth etc.

83 Beta-adrenoceptors are coupled to Gs protein and mediate smooth muscle relaxation

84 via stimulating adenosine 3’5′ cyclic monophosphate (c-AMP) production [24]. In some cell

85 types, although the predominant beta-adrenergic receptor present is β3, but increase in c-AMP

86 primarily occurs through β1 and β2 adrenoceptors activation [25]; thus suggesting the possible

87 existence of some alternating signaling pathway in β3-ADR-mediated action on smooth

88 muscle. Role of potassium channels in mediating beta adrenoceptors-mediated action has

89 been demonstrated by several authors [26, 27, 28]. Recently, Hristov and co-workers [29]

90 have reported a functional link between maxi-potassium (BKCa) channels and β3-ADR via

91 ryanodine receptors of the sarcoplasmic reticulum to induce myogenic relaxation in rat

92 detrusor smooth muscle. Activation of the BKCa channels following stimulation of β3-ADR in

93 human uterine myocytes has also been reported [30]. However, functional presence of β3-

94 ADR and the underlining signaling mechanism(s) of β3-ADR activation along with their

95 cellular coupling with potassium channels is yet to be established in myometrium of any farm

96 animal species including buffaloes. Therefore, in view of the paucity of information on

97 existence of β3-ADR and its cross-talk with K-channels in modulating uterine physiology and

98 possible use of such information in addressing reproductive disorders in buffaloes (Bubalus

99 bubalis), present study was undertaken to elucidate the presence of β3-ADR and their cellular

100 coupling with BKCa and ATP sensitive potassium (KATP) channels in mediating uterine

101 relaxation in buffaloes employing functional and molecular biology methods.
102 2. Materials and Methods
103 2.1 Tissue source

104 Uteri along with ovaries were collected from nondescript adult cyclic buffaloes

105 (Bubalus bubalis), from the local abattoir immediately after slaughter and transported to

106 laboratory in chilled (4.0 ± 0.5◦C) Ringer-Locke solution (RLS) having pH of 7.4. Diestrous

107 stage uteri were selected based on the well developed projected (crowned) corpus luteum on

108 ovaries, genitalia with closed cervix and thick mucus [31]. The uteri were also cut open to

109 further rule out the possibility of any early pregnancy. All the experimental protocols were

110 followed as per the guidelines of Committee for the Purpose of Control and Supervision of

111 Experiments on Animals (CPCSEA) with the approval no. 110/IAEC/16/40/2.
112 2.2 Recording of isometric tension
113 Longitudinal strips (3 mm x 1 cm) of myometrium were prepared from mid-cornual

114 region of the uterus by carefully removing the endometrial tissues [28] and mounted in an

115 organ bath (Ugo Basile, Italy) of 10 ml capacity containing RLS maintained at 37.0 ± 0.5◦C

116 and continuously aerated with carbogen (95% O2 + 5% CO2) under a resting tension of 2 g.

117 During the equilibration period of 2 hrs, bath fluid was changed almost after every 10 min.

118 Isometric tension was recorded with the help of force transducer (Panlab, Spain) using Lab

119 Chart Pro V6.1.3 software (Powerlab, AD Instruments; Australia). The dosing interval for

120 BRL-37344 (a selective β3 agonist) and terbutaline (a selective β2 agonist) were kept 10 min

121 and 4 min, respectively based on our pilot studies to find out the time required for sub-

122 maximal concentration of these agonists to produce maximal relaxant effect on myometrial

123 spontaneity.
124 The concentration-dependent relaxant response to BRL-37344 was also recorded in

125 the presence of SR 59230A (a specific β3-adrenoceptor antagonist), glybenclamide (a

126 selective KATP channel blocker), iberiotoxin (a selective BKCa channel blocker), tetraethyl

127 ammonium (TEA; a non-specific blocker of BKCa and Kv channels) and 4-aminopyridine (4-

128 AP, a specific blocker of Kv channel). All these selective receptor antagonists or ion channel

129 blockers were incubated for 30 minutes prior to recording the response to agonist(s).
130 2.3 Estimation of c-AMP in myometrial tissues
131 Myometrial strips were incubated in a thermostatically (37 ± 0.5°C) controlled organ

132 bath containing Ringers Locke solution and bubbled continuously with carbogen. Following

133 equilibration, myometrial strips were exposed to 10 µM rolipram to inhibit phosphodiesterase

134 enzyme. After that the myometrial strips were incubated with either BRL 37344 (0.1 µM or

135 10 µM) alone or BRL 37344 (10 µM) in presence of SR 59230A (1 µ M) for 10 min and

136 immediately snap frozen and stored at -80°C until further use. The above concentration of

137 BRL 37344 was selected based on the EC50 and Rmax value of this agonist from our functional

138 experiments. Tissue samples treated similarly in all aspects except exposure to BRL 37344

139 were used to measure basal c-AMP level. The tissue samples from different groups were then

140 homogenized and myometrial c-AMP accumulation was determined using commercially

141 available ELISA kit (BlueGene, Shanghai, China) following manufacturer’s instructions. The

142 proteins contents in tissue homogenates were estimated by Bradford assay kit (Biorad, USA)

143 and c-AMP levels were expressed as pmol/mg protein.
144 2.4 Western blot analysis
145 Uteri were collected in ice-cold phosphate buffer saline (PBS) and the myometrial

146 strips prepared were quickly snap frozen in liquid nitrogen and stored at -80°C until further

147 use. Uterine membrane proteins were isolated with the help of commercially available native

148 membrane extraction kit (Calbiochem, USA) by following manufacturer’s instructions and

149 the total protein contents were estimated using Bradford assay kit (Biorad, USA).

150 Approximately 80 µg protein samples were loaded in the well after diluting in Laemmli

151 buffer and denatured at 100°C for 3 min in boiling water bath. Proteins were then separated

152 on 10% SDS polyacrylamide gel using mini-protean tetra cell (Biorad, USA) and

153 subsequently transferred onto a polyvinylidene difluoride (PVDF, 0.45 µm) membranes using

154 semidry transfer apparatus (Biorad, USA) at 10V for 30 min. The Bluelf pre-stained protein

155 ladder (Biochem Life Sciences, USA) was also run along with the samples in the gel to

156 determine the approximate molecular weight of the immunoreactive protein. The membranes

157 were then blocked for 2 h at room temperature in a blocking buffer containing 5% (w/v)

158 skimmed milk powder in TBS (pH 7.4) containing 0.05% Tween 20. After washing, the blots

159 were incubated overnight at 4°C with either goat polyclonal antibody against beta3ADR (sc-

160 1473; 1:200) or Kir 6.1 (sc-11225; 1:500) or maxi K+ channels (sc-14747; 1:500) or rabbit

161 polyclonal antibody against Kv1.1 (sc-25680; 1:500) diluted in TBS containing. The blot was

162 then washed with TBS-T for 3-4 times and incubated for 2 h at room temperature with

163 horseradish peroxidise (HRP) conjugated secondary antibodies viz. bovine anti-goat IgG (sc-

164 2352; for beta3-ADR, Kir6.1 and maxi K+-channels) or goat anti-rabbit (sc-2030; for Kv1.1

165 channels). These secondary antibodies were diluted at 1:500 dilutions in TBS. After three

166 washings with TBS-T for 30 min, membranes were developed using diaminobenzidine

167 (DAB, Sigma) solution and immune-reactive proteins were visualized.
168 2.5 Data analysis
169 The concentration–response curves were constructed by measuring the mean integral

170 tension (MIT) using Labchart pro v 6.1.3 software [32]. Only one tissue was used from each

171 animal and ‘n’ denotes the number of animals for each tension experiment. Isometric

172 developed tension (IDT) or amplitude values were obtained by measuring the mean

173 amplitude of all the contractions recorded over a period of time and frequency of contraction

174 was determined as the mean number of contractile cycles observed during the same period

175 [33]. Amplitude of the tonic contractions was measured from the baseline while phasic

176 contractions were measured by subtracting the average cyclic maxima from the mean integral

177 tension of contractions [34]. EC50 and Rmax (maximum relaxation) values were determined by

178 non linear regression analysis using Graph Pad Prism 4.0 (Graph Pad, La jolla, USA) and pD2

179 value (potency) was calculated as – log of EC50.
180 Results are expressed as mean ± SEM. Multiple mean values were analyzed using

181 two-way ANOVA followed by Bonferroni post hoc test to compare between different

182 treatments while student’s ‘t’ test was used to compare only between the two groups and one-

183 way ANOVA followed by Tukeys post-hoc tests for more than two groups. Statistical

184 comparison of c-AMP level between different groups was done using one-way ANOVA

185 followed by Newman-Keuls multiple comparison test.
186 2.6 Chemicals
187 (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]

188 acetic acid sodium hydrate (BRL37344), terbutaline, 3-(2-Ethylphenoxy)-1-[[(1S)-1,2,3,4-

189 tetrahydronaphth-1-yl]amino]-(2S)-2-propanol oxalate (SR 59230A), isoprenaline,

190 glibenclamide, tetraethyl ammonium (TEA), iberiotoxin and 4-aminopyridine (4-AP) were

191 purchased from Sigma-Aldrich (USA). SAR 150640 (ethyl-4-{trans-4-[((2S)-2-hydroxy-3-

192 {4-hydroxy- 3[(methylsulfonyl)amino]-phenoxy}propyl)amino]cyclohexyl}- benzoate

193 hydrochloride was a gift from Sanofi Aventis. All other chemicals were of analytical grade

194 BRL-37344, SAR150640, SR 59230A and glibenclamide were dissolved in DMSO while rest

195 of the chemicals were dissolved in tripled distilled water. The pH of 4-AP was adjusted to 7.4

196 using 0.1N HCl. Further dilutions of the required concentrations were made in freshly

197 prepared RLS on the day of use. The vehicle at its final concentrations did not influence

198 myometrial activity.
199 3. Results

200 3.1 Comparative potency of different beta adrenoceptor agonists on myometrial spontaneity

201 Representative physiographic recordings illustrated in Fig.1A and B show the

202 comparative concentration-dependent inhibitory effects of terbutaline (specific beta2 agonist)

203 and BRL 37344 (specific beta3 agonist) on spontaneity in isolated myometrial strips of non-

204 pregnant buffaloes. The basal mean integral tension (MIT) of myometrial spontaneity was

205 1.95 ± 0.25 g (n=20). As shown in the Table.1, the maximum relaxation (Rmax) induced by all

206 these agonists was almost similar, however, potency of different agonists differed and order

207 of the potency was found to be isoprenaline > albuterol > BRL 37344 > terbutaline> SAR

208 150640. Among the beta3 agonists, BRL 37344 was found to be more potent than SAR

209 150640 (Table1). Therefore further studies to investigate the mechanistic pathway(s) of β3-

210 dependent relaxation in buffalo myometrium were carried out using BRL 37344 only.

211 Comparison of the effect of terbutaline and BRL 37344 on amplitude, frequency and

212 basal tone in myometrial strips of buffaloes revealed that BRL 37344 at higher concentrations

213 significantly reduced the basal tone (Fig.1C), albeit, it was not much altered by terbutaline.

214 Compared to terbutaline, BRL 37344 significantly reduced the amplitude (Fig.1D) and

215 phasicity (Fig. 1E) of myometrial spontaneity, but there was no significant difference

216 between the effect of terbutaline and BRL 37344 on frequency of contraction (Fig. 1F).

217 Effect of SR 59230A on BRL 37344-induced myometrial relaxation
218 BRL 37344 produced concentration-dependent (0.1 nM to 10 µM) relaxant effect on

219 isolated myometrial strips of buffaloes and the maximum effect (Rmax) was attained at 10 µM

220 concentration (Fig. 1A). As depicted in Fig.2A, in the presence of a specific β3-ADR

221 antagonist SR 59230A (1 µM), the concentration response curve (CRC) of BRL 37344 was

222 significantly (P<0.05) shifted towards right with significant (P<0.05) decrease in both

223 potency (7.22 ± 0.094 vs 5.49 ± 0.22; n=7) and efficacy (98.05 ± 2.52 % vs 57.19 ± 8.25%;

224 n=7) compared to control i.e. the response to BRL 37344 alone.

225 The immunoreactive protein to β3-ADR was detected with approximate molecular weight

226 of 40 kDa in the membrane fraction of buffalo myometrium (Fig. 2B).

227 3.2 Effect of BRL 37344 on c-AMP accumulation in buffalo myometrium
228 As shown in the Fig.3, the basal c-AMP level in myometrial strips of non-pregnant

229 buffaloes was 16.85 ± 3.87 pmol/mg protein (n=4). Although lower concentration (0.1 µM)

230 of BRL 37344 failed to alter basal c-AMP level, but at higher concentration (10 µM), BRL

231 37344 significantly (P<0.05) increased the c-AMP accumulation (37.58 ± 9.52 pmol/mg

232 protein; n=4) in the myometrium. This rise in c-AMP accumulation in the myometrium was

233 significantly reduced to basal level (17.06 ±0.64 pmol/mg protein, n=4) following concurrent

234 exposure to SR 59230A (1 µM) + BRL 37344 (10 µM).
235 3.3 Effect of glibenclamide on BRL 37344-induced myometrial relaxatio.n

236 To explore the possible involvement of KATP channel in beta3-induced myometrial

237 relaxation, glibenclamide (10 µM), a selective KATP channel blocker, was used. As shown in

238 Fig.4B, in the presence of glibenclamide (10µM), BRL37344-induced (0.1 nM to 10 µM)

239 relaxant effect was significantly inhibited. In presence of glibenclamide, compared to control,

240 the concentration response curve of BRL 37344 was significantly (P<0.05) shifted towards

241 right (Fig.4C) with decrease in potency (6.76 ±0.09 vs 7.22 ± 0.09; n=7) and efficacy (77.56

242 ± 4.39 vs 98.05 ± 2.52, n=7).

243 Existence to immunoreactive protein to Kir6.1, a regulatory protein of KATP channels,

244 was also detected in the membrane fraction of buffalo myometrium as shown in the

245 immonoblot (Fig.4D) with a molecular weight of 51 kDa.
246 3.4 Effect of tetraethyl ammonium on BRL 37344-induced myometrial relaxation

247 To explore the possible involvement of BKCa and Kv channels channels in beta3-

248 induced myometrial relaxation,, the relaxant response of BRL 37344 was recorded in

249 presence of tetraethyl ammonium (TEA, 1 mM), a nonspecific blocker of BKCa and Kv

250 channels. Following exposure to TEA, there was immediate change in myometrial

251 spontaneity followed by regaining of normal spontaneity. As shown in Fig.5A, the relaxant

252 effect of BRL 37344 was inhibited in presence of TEA (1mM) and there was significant

253 (P<0.05) rightward shift of the CRC of BRL 37344 with decrease in efficacy (77.03 ± 4.44 vs

254 98.05 ± 2.52; n=7) but without any significant change in potency (7.11 ± 0.10 vs 7.22 ± 0.09;

255 n=7) compared to control.
256 3.5 Effect of iberiotoxin on BRL 37344-induced myometrial relaxation
257 To assess the involvement of big conductance calcium-dependent potassium channels

258 or maxi potassium channels (BKCa) in regulating BRL 37344-induced myometrial relaxation,

259 iberiotoxin (100 nM), a specific blocker of BKCa channels, was used. As shown in Fig.5C,

260 following exposure to iberiotoxin, the myometrial spontaneity was changed as evidenced by

261 the change in frequency of myometrial rhythmicity. Further, in the presence of iberiotoxin,

262 BRL 37344-induced myometrial relaxation was inhibited (Fig.5B) and the CRC of BRL

263 37344 was significantly (P<0.05) shifted towards right (Fig.5C) with decrease in efficacy

264 (84.86 ± 6.09 % vs 98.05 ± 2.52 %) and potency (6.80 ± 0.08 vs 7.22 ± 0.09) compared to

265 control.

266 Existence to immunoreactive protein to α-subunit of BKCa was also detected in the

267 membrane fraction of buffalo myometrium as shown in the immonoblot (Fig.5D) with a

268 molecular weight of 63 kDa.
269 3.6 Effect of 4-aminopyridine (4-AP) on BRL 37344-induced myometrial relaxation

270 To rule out possible involvement of voltage-gated potassium channels (Kv) in

271 mediating β3-ADR-induced myometrial relaxation, 4-aminopyrdine (4-AP), a Kv channels

272 blocker, was used. Fig. 6A shows the representative physiograph tracings of relaxant effect of

273 BRL 37344 alone and in presence of 4-AP. Following exposure to 4-AP, there was

274 immediate change in the myometrial rhythmicity as evidenced by the increase in amplitude

275 and frequency compared to basal spontaneity. Further as shown in Fig.6B, the CRC of BRL

276 37344 in presence of 4-AP almost overlapped over the CRC of BRL 37344 alone. The pD2

277 and Rmax values of BRL-37344 in the absence and presence of 4-AP were 7.22 ± 0.09 vs 7.43

278 ± 0.13 and 98.05 ± 2.52 % vs 95.10 ± 2.61 %, respectively.
279 Existence to immunoreactive protein to Kv1.1 was also detected in the membrane

280 fraction of buffalo myometrium as shown in the immonoblot (Fig. 6C) with a approximate

281 molecular weight of 57-59 kDa.
282 4. Discussion:

283 The major findings of the present study were i) BRL 37344, a beta3-adrenoceptor (β3-

284 ADR) agonist, produced concentration-dependent relaxant effect in buffalo myometrium

285 which was sensitive to blockade by SR 59230A and dependent on c-AMP production ii) the

286 immunoreactive proteins to β3-ADR was present in the membrane fraction of buffalo

287 myometrium iii) the concentration response curves BRL 37344 were significantly shifted

288 towards right in presence of glibenclamide, tetraethylammonium or iberiotoxin, however, 4-

289 AP failed to exert any significant alterations in BRL 37344-induced tocolytic effect. The

290 pharmacological implications of these findings are discussed hereunder.

291 4.1 Beta3-adrenoceptor (β3-ADR) mediates tocolysis in buffaloes: A functional and molecular

292 evidence

293 Existence of beta3-ADR has been demonstrated in myometrium of rat [14] mouse [15] and

294 human [35, 36, 37, 22]. However, such information is lacking in farm animals including

295 buffaloes. Thus as per our first objective, we investigated the presence of β3-ADR in buffalo

296 myometrium and compared the efficacy and potency of different agonists in inducing

297 myometrial relaxation. Both SAR 150640 and BRL 37344 produced concentration-dependent

298 relaxation with almost equal efficacy (116.63 ± 9.19 % vs 98.05 ± 2.52 %), albeit, BRL

299 37344 was found to be more potent (4.36 ± 0.16 vs 7.22 ± 0.09). The rightward shifts of the

300 CRCs of SAR 150640 and BRL 37344 in the presence of selective β3 antagonist SR 59230A

301 [11] indicates the presence of β3-ADR in buffalo myometrium; and also selectivity of these

302 compounds towards β3-ADR as has also been reported earlier [38, 39, 40]. Presence of

303 protein band of ~40 kDa immunoreactive to β3 antibody in the membrane fraction of

304 myometrium (Fig. 2B) also substantiate our functional study based evidence about the

305 molecular existence of β3-ADR in buffalo mymetrium. Similar molecular weight of

306 immunoreactive protein has also been reported in the myometrium of nonpregnant women

307 [22].
308 The potential role c-AMP as a second messenger in BRL 37344-induced myometrial

309 relaxation in the present study is evident from significant increase in accumulation of c-AMP

310 following exposure to 10 µM BRL 37344. This rise in the c-AMP level was concentration-

311 dependent and selective to β3 activation as the c-AMP accumulation in the myometrium was

312 found to be almost comparable to that with the basal level in the presence of β3 antagonist,

313 SR59230A. Our findings are in agreement with the observations of Deng and co-workers [24]

314 who have also reported that stimulation of β3-ADR resulted in increase in level of cellular c-

315 AMP via activation of Gs protein in mouse brown adipocytes. But contrary to the

316 myometrium, relaxation of rat ileal smooth muscle by β3-ADR was not accompanied by an

317 increase in c-AMP (S.J. Roberts, unpublished data). Again, the stimulation of cyclic AMP by

318 BRL 37344 in isolated adipocytes is limited by coupling of the β3-ADR to Gi [41].

319 Compared to beta3 activation, stimulation of beta2-ADR by albuterol and terbutaline

320 produced more potent tocolytic effects (Table 1) which may be attributed to the differential

321 expression and density of these receptors in this tissue [15]. Although the maximum

322 relaxation produced either by beta3 or beta2-adrenoceptors stimulation was statistically

323 similar (Table.1), but these values are comparatively higher than those reported by Bardou

324 and co-workers [22] in non-pregnant human myometrium (44 ± 5% and 54 ± 8%,

325 respectively) suggesting their potential involvement in producing myometrial relaxation in

326 buffaloes.

327 4.2 Beta3-adrenoceptors are coupled to KATP and BKCa channels to mediate tocolysis

328 Although existence of β3-ADR has been reported in myometrium of some species but

329 involvement of different potassium channels in mediating β3-ADR regulated tocolysis is yet

330 to be established. As BRL 37344 was found to be more potent agonist of β3-ADR compared

331 to SAR 150640 in our study, therefore, we used BRL 37344 to examine the interaction

332 between β3-ADR and different potassium channels to produce tocolytic effect.
333 Potassium channels are widely distributed virtually in all the cell types and are

334 involved in dampening the cellular excitability by maintaining the cell membrane potential

335 close to the reversal potential of K+. Thus the outward K+ current induces hyperpolarization

336 or repolarization and opposes depolarization to occur and thereby reducing the generation of

337 contraction [42]. ATP-sensitive K+-channels (KATP) have been reported to mediate relaxation

338 phase of spontaneous contractility in myometrium [43] and are involved in mediating β-

339 adrenoceptor agonists-induced myometrial relaxation in human [44, 45]. We have also

340 reported earlier that these channels are functionally present in buffalo myometrium and

341 regulate albuterol-induced tocolytic effect [28]. These channels are comprised of an inwardly

342 rectifying K+ channel (Kir) and a modulatory sulphonylurea receptor subunit (SUR)

343 responsible for ATP sensitivity and pharmacological actions [46, 47, 48]. Among others,

344 Kir6.1 seems to be the predominant subunit in the myometrium [49]. In support to our

345 previous report regarding the functional presence of KATP channels [28], here we provide the

346 molecular evidence about the existence of the protein immunoreactive to Kir6.1 in the

347 membrane fraction of myometrium of non-pregnant buffaloes. Significant rightward shift of

348 the CRC of BRL 37344 in the presence of glibenclamide with reduction in both potency and

349 efficacy suggests the potential role of these channels in beta3-mediated uterine relaxation.

350 Since KATP channels appear to be intimately linked to the metabolic state of the cell, further

351 studies are warranted to unravel how these channels are coupled to β3-ADR during different

352 metabolic stages of myometrium.

353 Maxi potassium channels (BKCa) are reported to be richly endowed in uteri of non-

354 pregnant [50] and pregnant women [51, 52], pregnant rat [53], rabbit and pigs [54] and

355 function as negative feedback regulators of the intracellular Ca+2 [55]. Recently, we have also

356 reported the functional presence of BKCa channels and their involvement in β2-mediated

357 tocolysis in buffaloes [28, 31]. Change in the basal tone and frequency of spontaneity

358 following exposure to iberiotoxin observed in the present study (Fig. 5B) further indicates the

359 functional role of maxi potassium channels in regulation of myometrial spontaneity. Though

360 the immunoreactive protein of BKCa channels has already been reported in total protein

361 isolated from buffalo uterus from our laboratory [31], in the present study by refining our

362 isolation procedure of the myometrial protein, we have observed the presence of these

363 proteins in membrane fraction of the uterus, a location which is more logical for its

364 interaction with GPCR. The significant rightward shift of the CRC of BRL 37344 in the

365 presence of TEA (a non-specific BKCa channels blocker) and iberiotoxin (more specific BKCa

366 channels blocker) provide further evidence that BKCa channels regulate β3-ADR-mediated

367 tocolytic effect in buffaloes. Doheny and co-workers [30] have also reported the functional

368 coupling of β3-ADR with BKCa channels in human uterine myocytes. In rat myometrial cells

369 also, the relaxant action of isoproterenol, a non-selective β-ADR agonist which acts on all the

370 three subtypes of β-ADR, is regulated by Ca 2+-dependent K+-channels [51]. Sensitivity of

371 BKCa channels to calcium differs with the stages of myometrium (gravid versus nongravid).

372 Since myometrial BKCa channels are present in the caveolae and clustering of receptors and

373 channels within these signalling structures may be one mechanism for receptor-channel

374 interactions [56], therefore, further studies are warranted on these aspects especially on the

375 uteri of pregnant buffaloes.
376 The finding that, tetraethylammonium significantly shifted the CRC of BRL 37344

377 towards right with decrease in efficacy but without any change in potency also suggests the

378 possibility of involvement of both BKCa and voltage-gated potassium (Kv) channels in

379 modulating β3-ADR-dependent relaxation. Thus to rule out the involvement of Kv channels,

380 the relaxant response of β3 agonist was studied in presence of 4-AP, a more specific blocker

381 of Kv channels. The sudden risein the base line, and increase in amplitude and frequency of

382 myometrial spontaneity following blockade of Kv channels by 4-AP (Fig. 6A) suggests their

383 functional role in regulating the spontaneity in myometrial strips of non-pregnant buffaloes.

384 Existence of immunoreactive protein to Kv1.1 in the membrane fraction of buffalo uterus

385 (Fig. 6C) further substantiated the presence of KV channels in buffalo myometrium. Although

386 role of KV channels in regulating the resting membrane potential in uterine myocytes and

387 maintenance of quiescence state has been documented by several researchers [57, 58, 59] but

388 in the present study, the CRC of BRL37344 in the presence of 4-AP overlapped over that of

389 BRL37344 alone. Therefore, it rules out the possible involvement of KV channels in β3-ADR-

390 induced relaxation in buffalo myometrium.

391 Taken together, our functional and molecular study results on isolated myometrial

392 strips of buffaloes evidently reveal the presence of β3-adrenoceptors in buffalo myometrium

393 and these are coupled to KATP and BKCa channels, these together have potential role in

394 regulating myogenic spontaneity and tocolytic effect. However, further, studies are warranted

395 on uteri of pregnant buffaloes to elucidate their functional role in uterine quiescence, foetal-

396 implantation, maintenance of pregnancy, and preterm labour or using these selective

397 receptors or ion channels as specific drug targets.
398 Acknowledgements

399 This research work was supported by Indian Council of Agricultural Research, New

400 Delhi under Niche Area of Excellence Programme (Grant No. 10 (10)/2012-EPD dated 23rd

401 march 2012) to Department of Veterinary Pharmacology and Toxicology, DUVASU,

402 Mathura, India. Financial assistance by ICAR is thankful acknowledged.

403 Conflict of interest

404 None of the authors have any financial or personal relationships that could

405 inappropriately influence or bias the content of the paper.

406 406

407 References

408 [1] Kitazawa T, Hatakeyama H, Cao J, Taneike T. Pregnancy-associated changes in
409 responsiveness of the porcine myometrium to bioactive substances. Eur J Pharmacol
410 2003; 469(1-3): 135-144.
411 [2] Markiewicz W, Kamińska K, Bogacki M, Maślanka T, Jaroszewski J. Participation of
412 analogues of lysophosphatidic acid (LPA): oleoyl-sn-glycero-3-phosphate (L-alpha-
413 LPA) and 1-oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT) in uterine smooth
414 muscle contractility of the pregnant pigs. Pol J Vet Sci 2012; 15(4): 635-643.
415 [3] Bulletti C, Ziegler DD, Setti P L, Cicinelli E, Polli V, Carlo F. The patterns of uterine
416 contractility in normal menstruating women. From Physiology to Pathology. Ann N Y
417 Acad Sci 2004; 1034: 64–83.
418 [4] Krief S, Lonnqvist F, Raimbault S, Baude B, Van Spronsen A, Arner P, Strosberg AD,
419 Ricquier D, Emorine LJ. Tissue distribution of b3-adrenergic receptor mRNA in man. J
420 Clin Invest 1993; 91: 344-349.
421 [5] Summers RJ, Papaioannou M, Harris S, Evans BA Expression of b3-adrenoceptor
422 mRNA in rat brain. Br J Pharmacol 1995; 116: 2547-2548.
423 [6] Evans BA, Papaioannou M, Bonazzi VR, Summers RJ Expression of β3- adrenoceptor
424 mRNA in rat tissues. Br J Pharmacol 1996; 117: 210 -216.
425 [7] Manara L, Badone D, Baroni M, Boccardi G, Cecchi R, Croci T, Giudice A, Guzzi U,
426 Landi M, Lefur G Functional identification of rat atypical b-adrenoceptors by the first
427 b3-selective antagonists, aryloxypropanolaminote-tralins. Br J Pharmacol 1996; 117:
428 435-442.
429 [8] Roberts SJ, Papaioannou M, Evans BA, Summers RJ. Functional and molecular
430 evidence for b1-, b2- and b3-adrenoceptors in human colon. Br J Pharmacol 1997; 120:
431 1527-1535.
432 [9] Lirussi F, Rakotoniaina Z, Madani S, Goirand F, Breuiller-Fou M, Leroy M, Sagot P,
433 Morrison JJ, Dumas M, Bardou M. ADRB3 adrenergic receptor is a key regulator of
434 human myometrial apoptosis and inflammation during chorioamnionitis. Biol Reprod
435 2008; 78: 497-505.
436 [10] Lirussi F, O’Brien M, Wendremaire M, Goirand F, Sagot P, Dumas M, Morrison JJ,
437 Bardou M SAR150640, a selective b3-adrenoceptor agonist, prevents human
438 myometrial remodelling and activation of matrix metalloproteinase in an in vitro model
439 of chorioamnionitis. Br J Pharmacol 2010;159: 1354–1366.
440 [11] Bardou M, Loustalot C, Cortijo J, Simon B, Naline E, Dumas M, Esteve S, Croci T,
441 Chalon P, Frydman R, Sagot P, Manara L, Morcillo EJ, Advenier C. Functional,
442 biochemical and molecular biological evidence for a possible beta(3)-adrenoceptor in
443 human near-term myometrium. Br J Pharmacol 2000; 130: 1960–1966.
444 [12] Pedzińska-Betiuk A, Modzelewska B, Jóźwik M, Jóźwik M, Kostrzewska A.
445 Differences in the effects of beta2- and beta3-adrenoceptor agonists on spontaneous
446 contractions of human nonpregnant myometrium. Ginekol Pol 2011; 82: 918-924.
447 [13] Yurtcu N, Cetin A, Karadas B, Gonca Imir A, Kaya T, Erselcan T, Bagcivan I, Cetin M
448 Comparison of effects of formoterol and BRL 37344 on isolated term-pregnant rat
449 myometrial strips in vitro. Eur J Pharmacol 2006; 530: 263–269.

Minorics R, Gaspar R, Gal A, Klukovits A, Falkay G Progesterone decreases the relaxing effect of the b3-adrenergic receptor agonist BRL37344 in the pregnant rat myometrium. Reproduction 2009; 138: 383–390.
Parida S, Singh TU, Prakash VR, Mishra SK. Molecular and functional characteristics of b3-adrenoceptors in late pregnant mouse uterus: A comparison with b2- adrenoceptors. Eur J Pharmacol 2013; 700: 74–79.
Engstrøm T, Bratholm P, Vilhardt H, Christensen NJ Effect of pregnancy on rat myometrial beta 2-adrenoceptor mRNA and isoproterenol-induced relaxation of isolated uterine strips. J Endocrinol 1997; 153: 393–399.
Chanrachakul B, Matharoo-Ball B, Turner A, Robinson G, Broughton-Pipkin F, Arulkumaran S, Khan RN. Reduced expression of immunoreactive beta2-adrenergic receptor protein in human myometrium with labor. J Clin Endocrinol Metab 2003; 88: 4997–5001.
de Heus R, Mol BW, Erwich JJ, van Geijn HP, Gyselaers WJ, Hanssens M, H¨armark L, van Holsbeke CD, Duvekot JJ, Schobben FF, Wolf H, Visser GH. Adverse drug reactions to tocolytic treatment for preterm labour: prospective cohort study. BMJ 2009; 338: b744.
Motazedian S, Ghaffarpasand F, Mojtahedi K & Asadi N Terbutaline versus salbutamol for suppression of preterm labor: a randomized clinical trial. Ann Saudi Med 2010; 30: 370–375.
Lye SJ, Dayes BA, Freitag CL, Brooks J, Casper RF. Failure of ritodrine to prevent preterm labor in the sheep. Am J Obstet Gynecol 1992; 167: 1399–1408.
Berkman ND, Thorp Jr JM, Lohr KN, Carey TS, Hartmann KE, Gavin NI, Hasselblad V, Idicula AE Tocolytic treatment for the management of preterm labor: a review of the evidence. Am J Obstet Gynecol 2003; 188: 1648–1659.
Bardou M, Rouget C, Breuiller-Fouche M, Loustalot C, Naline E, Sagot P, Frydman R, Morcillo EJ, Advenier C, Leroy MJ, Morrison JJ. Is the beta3-adrenoceptor (ADRB3) a potential target for uterorelaxant drugs? BMC Pregnancy Childbirth 2007; 7 (Suppl 1): S14.
Strosberg AD. Structure, function, and regulation of adrenergic receptors. Protein Science 1993; 2: 1198–1209.
Deng C, Moinat M, Curtis L, Nadakal A, Preitner F, Boss O, Assimacopoulos-Jeannet F, Seydoux J, Giacobino J Effects of β-adrenoceptor subtype stimulation on obese gene messenger ribonucleic acid and on leptin secretion in mouse brown adipocytes differentiated in culture. Endocrinol 1997; 138: 548 -552.
Jockers R, Issad T, Zilberfarb V, De Coppet P, Mar-Ullo S, Strosberg AD Desensitization of the b- adrenergic response in human brown adipocytes. Endocrinol 1998; 139: 2676 -2684.
Ferro A. β-Adrenoceptors and potassium channels. Naunyn Schmiedebergs Arch Pharmacol 2006; 373: 183–185.
Petkov GV, Nelson MT. Differential regulation of Ca2+-activated K+ channels by beta- adrenoceptors in guinea pig urinary bladder smooth muscle. Am J Physiol Cell Physiol 2005; 288: C1255–C1263.

493 [28] Choudhury S, Garg SK, Singh TU, Mishra SK. Cellular coupling of potassium channels
494 with 2-adrenoceptors in mediating myometrial relaxation in buffaloes (Bubalus
495 bubalis). J Vet Pharmacol Ther 2010; 33: 22–27.
496 [29] Hristov KL, Cui X, Brown SM, Liu L, Kellett WF, Petkov GV. Stimulation of beta3-
497 adrenoceptors relaxes rat urinary bladder smooth muscle via activation of the large-
498 conductance Ca2+-activated K+ channels. Am J Physiol Cell Physiol 2008; 295: C1344–
499 C1353.
500 [30] Doheny HC, Lynch CM, Smith TJ, Morrison JJ. Functional coupling of β3-
501 adrenoceptors and large conductance calcium-activated potassium channels in human
502 uterine myocytes. J Clin Endocrinol Metab 2005; 90: 5786– 5796.
503 [31] Choudhury S, Garg SK, Singh TU, Mishra SK. Functional and molecular
504 characterization of maxi K+-channels (BKCa) in buffalo myometrium. Anim Reprod
505 Sci 2011; 126: 173–178.
506 [32] Aaronson PI, Sarwar U, Gin S, Rockenbauch U, Connolly M, Tillet A, Watson S, Liu
507 B, Tribe RM. A role for voltage-gated, but not Ca2+-activated, K+ channels in regulating
508 spontaneous contractile activity in myometrium from virgin and pregnant rats. Br J
509 Pharmacol 2006; 147(7): 815-824.
510 [33] Chaud MA, Franchi AM, Beron de Astrada M, Gimeno MF. Role of nitric oxide on
511 oxytocin evoked contractions and prostaglandin synthesis in isolated pregnant rat
512 uterus. Prostaglandins Other Lipid Mediat 1997; 57(3): 323-329.
513 [34] Sharma A, Nakade UP, Choudhury S, Garg SK. Functional involvement of protein
514 kinase C, Rho-kinase and TRPC3 decreases while PLC increases with advancement of
515 pregnancy in mediating oxytocin-induced myometrial contractions in water buffaloes
516 (Bubalus bubalis). Theriogenology 2016; 92:176-189.
517 [35] Andersson RG, Berg G, Johansson SR, Ryden G. Effects of non-selective and selective
518 beta-adrenergic agonists on spontaneous contractions and cyclic AMP levels in
519 myometrial strips from pregnant women. Gynecol Obstet Invest 1980; 11: 268–293.
520 [36] Berg G, Andersson RG, Ryden G. Beta-adrenergic receptors in human myometrium
521 during pregnancy: changes in the number of receptors after beta-mimetic treatment. Am
522 J Obstet Gynecol 1985; 151: 392–396.
523 [37] Hakak Y, Shrestha D, Goegel MC, Behan DP, Chalmers DT. Global analysis of G-
524 protein-coupled receptor signaling in human tissues. FEBS Letters 2003; 550: 11–17.
525 [38] Dennedy MC, Friel AM, Gardeil F, Morrison JJ. β3 versus β2 adrenergic agonists and
526 preterm labour: in vitro uterine relaxation effects. BJOG 2001; 108: 605–609.
527 [39] Dennedy MC, Houlihan DD, McMillan H, Morrison JJ. Beta2- and beta3-
528 adrenoreceptor agonists: human myometrial selectivity and effects on umbilical artery
529 tone. Am J Obstet Gynecol 2002; 187: 641-647.
530 [40] Croci T, Cecchi R, Marini P, Rouget C, Viviani N, Germain G, Guagnini F, Fradin Y,
531 Descamps L, Pascal M, Advenier C, Breuiller-Fouche M, Leroy M, Bardou M. In vitro
532 and in vivo pharmacological characterization of ethyl-4-{trans-4-[((2S)-2-hydroxy-3-
533 {4-hydroxy-3[(methylsulfonyl)amino]-phenoxy}propyl) amino]cyclohexyl} benzoate
534 hydrochloride (SAR150640), a new potent and selective human b3-adrenoceptor
535 agonist for the treatment of preterm labor. J Pharmacol Exp Ther 2007; 321:1118–
536 1126.

537 [41] Chaudhry A, Mackenzie RG, Georgic LM, Granne-Man JG. Differential interaction of
538 b1- and b3- adrenergic receptors with Gi in rat adipocytes. Cell Signal. 1994; 6: 457-
539 465.
540 [42] Khan RN, Matharoo-Ball B, Arulkumaran S. Potassium channels in human
541 myometrium. Exp Physiol 2001;86(2): 255–264
542 [43] Kafali H, Kaya T, Gursoy S, Bagcivan I, Karadas B, Sarioglu Y. The role of K(+)
543 channels on the inhibitor effect of sevoflurane in pregnant rat myometrium. Anesth
544 Analg 2002; 94: 174–178.
545 [44] Hamada Y, Nakaya Y, Hamada SI, Kamada M, Aono T. Activation of K+ channels by
546 ritordine hydrochloride in uterine smooth muscle cells from pregnant women. Eur J
547 Pharmacol 1994; 288: 45–51.
548 [45] Okawa T, Longo M, Vedernikov YP, Chwalisz K, Saade GR, Garfield RE. Role of
549 nucleotide cyclases in the inhibition of pregnant rat uterine contractions by the openers
550 of potassium channels. Am J Obstet Gynecol 2000; 182: 913–918.
551 [46] Inagaki N, Tsuura Y, Namba N, Masuda K, Gonoi T, Horie M, Seino Y, Mizuta M,
552 Seino S. Cloning and functional characterization of a novel ATP-sensitive potassium
553 channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary,
554 skeletal muscle, and heart. J Biol Chem 1995; 270: 5691-5694.
555 [47] Isomoto S, Kondo C, Yamada M, Matsumoto S, Higashiguchi O, Horio Y, Matsuzawa
556 Y, Kurachi YA. A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth
557 muscle type ATP-sensitive K1 channel. J Biol Chem 1996; 271: 24321–24324.
558 [48] Aguilar-Bryan L, Clement JP 4th, Gonzalez G, Kunjilwar K, Babenko A, Bryan J.
559 Toward understanding the assembly and structure of KATP channels. Physiol Rev
560 1998; 78: 227–245.
561 [49] Curley M, Cairns MT, Friel AM, McMeel OM, Morrison JJ, Smith TJ. Expression of
562 mRNA transcripts for ATP-sensitive potassium channels in human myometrium. Mol
563 Hum Reprod 2002; 8: 941-945.
564 [50] Tritthart HA, Mahnert W, Fleischhacker A, Adelwohrer N. Potassium channels and
565 modulating factors of channel functions in the human myometrium. Z Kardiol 1991;
566 80: 29–33.
567 [51] Anwer K, Toro L, Oberti C, Stefani C, Sanborn BM. Ca+2- activated K+-channels in
568 pregnant rat myometrium: modulation by β-adrenergic agent. Am J Physiol 1992; 263:
569 C1049– C1056.
570 [52] Khan RN, Smith SK, Morrison JJ, Ashford ML. Properties of large conductance
571 potassium channels in human myometrium during pregnancy and labor. Proc Biol Sci
572 1993; 251: 9–13.
573 [53] Kihira M, Matsuzawa K, Tokuno H, Tomita T. Effects of calmodulin antagonists on
574 calcium-activated potassium channels in pregnant rat myometrium. Br J Pharmacol
575 1990; 100: 353-359
576 [54] Toro L, Ramos-Franco J, Stefani E. GTP-dependent regulation of myometrial KCa
577 channels incorporated into lipid bilayers. J Gen Physiol 1990; 96: 373–394.
578 [55] Zhou XB, Wang GX, Ruth P, Huneke B, Korth M. BK(Ca) channel activation by
579 membrane-associated c-GMP kinase may contribute to uterine quiescence in
580 pregnancy. Am J Physiol Cell Physiol 2002; 279: C1751–C1759.

581 [56] Brainard AM, Miller AJ, Martens JR, England SK. Maxi-K channels localize to
582 caveolae in human myometrium: a role for an actin-channel-caveolin complex in the
583 regulation of myometrial smooth muscle K+ current. Am J Physiol Cell Physiol 2005;
584 289: C49–C57.
585 [57] Lundgren DW, Moore JJ, Chang SM, Collins PL, Chang AS. Gestational changes in
586 the uterine expression of an inwardly rectifying K+ channel, ROMK. Proc Soc Exp
587 Biol Med 1997; 216: 57–64.
588 [58] Erulkar SD, Ludmir J, Ger B, Nori RD. Expression of different potassium channels in
589 cells isolated from human myometrium and leiomyomas. Am J Obstet Gynecol 1993;
590 68: 1628-1639.
591 [59] Knock GA, Smirnov SV, Aaronson PI. Voltage-gated K currents in freshly isolated
592 myocytes of the pregnant human myometrium. J Physiol 1999; 518.3: 769-781

598 Table 1: Comparative potency (pD2) and efficacy (Rmax) of different beta-adrenoceptor
599 agonists on isolated myometrial strips of non-pregnant buffaloes (Bubalus bubalis)

Agonists pD2 Rmax (%)
Isoprenaline (n=7) 9.14 ± 0.04 96.46 ± 1.18
SAR 150640 (n=6) 4.36 ± 0.16 116. 63 ± 9.19
BRL 37344(n=7) 7.22 ± 0.09* 98.05 ± 2.52
Terbutaline (n=5) 6.21 ± 0.13 97.74 ± 1.30
Albuterol (n=6)# 8.55 ± 0.12 101.10 ± 6.30
600 Data are expressed as mean ± SEM. ‘n’ represents number of animals used for isolation of
601 tissue sample. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test.
602 * P< 0.05 vs SAR 150640.
603 # Choudhury et al., 2010

605 Figure Legends:
607 Fig.1.: Effect of BRL-37344 and terbutaline on isolated myometrial strips from non- 608 pregnant buffaloes. Representative physiograph recordings (1A-B) showing the effect of 609 BRL-37344 (0.1 nM -10 µM) and terbutaline (0.1 nM-10 µM) on myometrial spontaneity. 610 Comparative cumulative concentration response curves (CRCs) showing the effect of BRL- 611 37344 and terbutaline on tonicity (1C), amplitude(1D), phasicity (1E) and frequency (1F), 612 respectively, of myogenic spontaneity on non-pregnant buffalo myometrium. Vertical bars 613 represent SEM. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc 614 tests. *P<0.05 vs BRL 37344 alone.
615 Fig.2.: Comparative cumulative concentration response curves (2A) showing the effect of 616 BRL-37344 in the absence and presence of SR 59230A (1 µM), a β3-adrenoceptor specific 617 antagonist, on isolated myometrial strips from non-pregnant buffaloes. Representative 618 Western Blot image (2B) of beta3-adrenoceptor (β3-ADR) protein (~40kDa) in membrane 619 fraction of uterine homogenates from non-pregnant buffaloes. Vertical bars represent SEM. 620 Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc tests. *P<0.05 vs 621 BRL 37344 alone. Lane M: marker; Lane L-1: β3-ADR.
622 Fig.3.: Bar diagrams showing the effect of BRL 37344 on c-AMP accumulation in 623 buffalo myometrium. BRL 37344 (10 µM) significantly increased c-AMP accumulation in 624 buffalo myometrium which was reversed to almost basal c-AMP level in the presence of SR 625 59230A (1 µM). Vertical bars represent SEM. Data were analyzed by one-way ANOVA 626 followed by Newman-Keuls multiple comparison test. * P<0.05 vs basal c-AMP level; 627 #P<0.05 vs BRL 37344 (10 µM) alone.

629 Fig.4.: Role of ATP-sensitive K+-channels (KATP) in mediating BRL-37344-induced 630 myometrial relaxation on non-pregnant buffaloes. Representative physiograph recordings 631 (4A-B) showing the effect of BRL-37344 (0.1 nM -10 µM) in the absence and presence of 632 glibenclamide (10 µM), a specific blocker of KATP channels, on myometrial spontaneity. 633 Comparative cumulative concentration response curves (4C) showing the effect of BRL- 634 37344 in the absence and presence of glibenclamide (10 µM) on myometrial spontaneity. 635 Representative Western Blot image (4D) of Kir6.1 regulatory subunit of KATP channels 636 protein (~51kDa) in membrane fraction of uterine homogenates from non-pregnant buffaloes. 637 Vertical bars represent SEM. Data were analyzed by two-way ANOVA followed by 638 Bonferroni post-hoc tests. *P<0.05 vs BRL 37344 alone. Lane M: marker; Lane NP: Non- 639 pregnant.
640 Fig.5.: Role of maxi-potassium channels (BKCa) in mediating BRL-37344-induced 641 myometrial relaxation on non-pregnant buffaloes. Comparative cumulative concentration 642 response curves showing the effect of BRL-37344 in the absence and presence of tetraethyl 643 ammonium (TEA: 1mM), a non-specific blocker of BKCa and Kv channels (5A) and 644 iberiotoxin (100 nM), a specific blocker of BKCa channels (5C), on myometrial spontaneity. 645 Representative physiograph recordings (5B) showing the effect of BRL-37344 (0.1 nM -10 646 µM) in the presence of iberiotoxin (100 nM) on myometrial spontaneity. Representative 647 Western Blot image (5D) of α-subunit of BKCa channels protein (~63kDa) in membrane

648 fraction of uterine homogenates from non-pregnant buffaloes. Vertical bars represent SEM. 649 Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc tests. *P<0.05 vs 650 BRL 37344 alone. Lane M: marker; Lane NP: Non-pregnant.
651 Fig.6.: Role of voltage gated K+-channels (KV) in mediating BRL-37344-induced 652 myometrial relaxation on non-pregnant buffaloes. Representative physiograph recordings 653 (6A) showing the effect of BRL-37344 (0.1 nM -10 µM) in the presence of 4-aminopyridine 654 (4-AP; 1mM), a specific blocker of KV channels, on myometrial spontaneity. Comparative 655 cumulative concentration response curves (6B) showing the effect of BRL-37344 in the 656 absence and presence of 4-AP (1 mM) on myometrial spontaneity. Representative Western 657 Blot image (6C) of KV1.1 channels protein (~57-59 kDa) in membrane fraction of uterine 658 homogenates from non-pregnant buffaloes. Vertical bars represent SEM. Data  SR59230A were analyzed 659 by two-way ANOVA followed by Bonferroni post-hoc tests. *P<0.05 vs BRL 37344 alone. 660 Lane M: marker; Lane NP: Non-pregnant.