To assess whether pregnancy might influence the functionality and expression of human myometrial beta(2)- and beta(3)-adrenoceptors (beta(2)- and beta(3)-AR), we performed functional, binding, Western blot, and molecular biology experiments in human nonpregnant and near-term pregnant myometrium. Inhibition of spontaneous contractions induced by a beta(3)-AR agonist, SR 59119A, was significantly greater in pregnant, compared with nonpregnant, myometrial strips (E'(max) = 61 +/- 5% vs. 44 +/- 5% for pregnant and nonpregnant myometrium, respectively), whereas salbutamol, a beta(2)-AR agonist, was significantly less efficient in pregnant, compared with nonpregnant, myometrium (E(max) = 29 +/- 4 vs. 54 +/- 8%). Although two populations of binding sites corresponding to beta(2)- and beta(3)-AR were identified in both nonpregnant and pregnant myometrium, we found a clear predominance of the beta(3)-AR subtype. Moreover, beta(3)-AR binding sites were up-regulated 2-fold in myometrium at the end of pregnancy. Both beta(2)- and beta(3)-AR mRNA were expressed in human nonpregnant and pregnant myometrium. Contrary to beta(2)-AR, the expression of the beta(3)-AR transcripts and immunoreactive proteins was increased in pregnant, compared with nonpregnant, myometrium. Such compelling data suggest a predominant role for beta(3)-AR in the regulation of human myometrium contractility, especially at the end of pregnancy, which might have important consequences for the clinical management of preterm labor.

1 In order to compare the b 2 -and b 3 -adrenoceptor (b-AR) desensitisation process in human nearterm myometrium, we examined the influence of a pretreatment of myometrial strips with either a b 2 -or a b 3 -AR agonist (salbutamol or SR 59119A, respectively, both at 10 mM, for 5 and 15 h) on the relaxation and the cyclic adenosine monophosphate (cAMP) production induced by these agonists. 2 To assess some of the mechanisms potentially implicated in the b-AR desensitisation process, we studied the influence of such treatment on the number of b 2 -and b 3 -AR binding sites, the b 2 -and b 3 -AR transcripts expression and the phosphodiesterase 4 (PDE4) activity. 3 Salbutamol, but not SR 59119A, concentration-response curve (CRC) was shifted by a 15 h salbutamol preincubation, with a significant difference in Àlog EC 20 values (6.3170.13 vs 5.5870.24, for control and 15 h salbutamol pretreatment, respectively, Po0.05). Neither salbutamol nor SR 59119A CRCs were modified after a 15 h preincubation with SR 59119A. 4 A 15 h exposure of myometrial strips to salbutamol significantly reduced the salbutamol-induced (0.6070.26 vs 1.5470.24 pmol mg À1 protein, Po0.05), but not the SR 59119A-induced, cAMP production. No decrease in cAMP production was observed after a 15 h SR 59119A exposure. 5 A 15 h salbutamol exposure of myometrial strips significantly reduced the b 2 -but not the b 3 -AR binding site density, whereas no decrease in the number of b 2 -and b 3 -AR binding sites was observed after a 15 h SR 59119A treatment. 6 Neither PDE4 activity nor the b 2 -and b 3 -AR mRNA expression levels were affected by salbutamol or SR 59119A treatments. 7 Our results indicate that b 3 -AR, but not b 2 -AR, are resistant to the agonist-induced desensitisation. In our model, b 2 -AR desensitisation is mediated by a decreased number of b 2 -AR that was not explained by transcriptional regulation of the receptor.

BACKGROUND AND PURPOSEMuscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs. EXPERIMENTAL APPROACHIn binding experiments with 3 H-rauwolscine, we studied the interactions of green mamba venom fractions with a2-adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, r-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human a2-adrenoceptors expressed in mammalian cells. KEY RESULTSr-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of 3 H-rauwolscine binding to the three a2-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on a2A-adrenoceptor demonstrated that r-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA2 values of 5.93 and 5.32 for yohimbine and r-Da1b, respectively. CONCLUSIONS AND IMPLICATIONSr-Da1b is the first toxin identified to specifically interact with a2-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of a2-adrenoceptor subtypes. AbbreviationsBRET, bioluminescence resonance energy transfer; EPA, 5-(N-ethyl-N-isopropyl)-amiloride; GPCR, G protein-coupled receptor; RBS, rat brain synaptosomes BJP British Journal of Pharmacology

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1 Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Our previous results suggest that microvascular leakage induced, in the guinea-pig airways, by intra-oesophageal hydrochloric acid (HCl) infusion was mainly dependent on the release of tachykinins. Nociceptin, an endogenous ligand of the opioid receptor NOP, has been shown to inhibit bronchoconstriction and cough in guinea-pig or cat by inhibiting tachykinin release. 2 The purpose of this study was to investigate the effects of nociceptin on the intra-oesophageal HClinduced airway microvascular leakage evaluated by Evans blue dye extravasation measurement in anaesthetised guinea-pigs pretreated with propranolol, atropine and phosphoramidon. 3 Infusion of intra-oesophageal HCl led to a significant increase in plasma extravasation in the main bronchi and trachea. This increase was abolished when animals underwent a bilateral vagotomy. Lys 15 ]N/OFQ mimicked the inhibitory effect of nociceptin, but at a 10-fold lower dose (3 mg kg À1 i.v). The NOP receptor antagonist J-113397 had no effect on plasma protein extravasation by itself, but was able to block the inhibitory effect of nociceptin. 5 Morphine (1 mg kg À1 ) had a similar inhibitory effect as that of nociceptin. Naloxone pretreatment abolished the effect of morphine, but was enable to block the inhibitory effect of nociceptin. 6 Under similar conditions, nociceptin, in the previous range of concentration, was unable to counteract the airway microvascular leakage induced by substance P (SP). 7 These results suggest that airway plasma extravasation induced by intra-oesophageal HCl instillation might be inhibited by specific stimulation of the NOP receptor with nociceptin. Nociceptin is likely to act at a pre-junctional level, by inhibiting tachykinin release, since it was unable to prevent SP-induced airway plasma extravasation.

Ethyl-4-{trans-4-[((2S)-2-hydroxy-3-{4-hydroxy-3[(methylsulfonyl)amino] phenoxy}propyl) amino]cyclohexyl}benzoate hydrochloride (SAR150640) was characterized as a new potent and selective ␤ 3 -adrenoceptor agonist for the treatment of preterm labor. SAR150640 and its major metabolite, the corresponding acid 4-{trans-4-[((2S)-2-hydroxy-3-{4-hydroxy-3[(methylsulfonyl) amino] phenoxy}propyl)amino]cyclohexyl}benzoic acid (SSR500400), showed high affinity for ␤ 3 -adrenoceptors (K i ϭ 73 and 358 nM) and greater potency than (Ϫ)-isoproterenol in increasing cAMP production in membrane preparations from human neuroblastoma cells (SKNMC), which express native ␤ 3 -adrenoceptors (pEC 50 ϭ 6.5, 6.2, and 5.1, respectively). SAR150640 and SSR500400 also increased cAMP production in membrane preparations from human uterine smooth muscle cells (UtSMC), which also express native ␤ 3 -adrenoceptors (pEC 50 ϭ 7.7 and 7.7, respectively). In these cells, SAR150640 dose-dependently inhibited oxytocin-induced intracellular Ca 2ϩ mobilization and extracellular signal-regulated kinase 1/2 phosphorylation. SAR150640 and SSR500400 had no ␤ 1 -or ␤ 2 -agonist or antagonist activity in guinea pig atrium and trachea, or in human isolated atrium and bronchus preparations. Both compounds concentration-dependently inhibited spontaneous contractions in human near-term myometrial strips, with greater potency than salbutamol and 4-[3-[(1,1-dimethylethyl)-amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP12177) (pIC 50 ϭ 6.4, 6.8, 5.9, and 5.8, respectively), but with similar potency to (Ϫ)-isoproterenol and atosiban (oxytocin/vasopressin V 1 a receptor antagonist). SAR150640 also inhibited the contractions induced by oxytocin and prostaglandin F 2␣ . In vivo, after intravenous administration, SAR150640 (1and 6 mg/kg), but not atosiban (6 mg/kg), dosedependently inhibited myometrial contractions in conscious unrestrained female cynomolgus monkeys, with no significant effects on heart rate or blood pressure. In contrast, salbutamol (50 and 250 g/kg) had no inhibitory effect on uterine contractions, but it dose-dependently increased heart rate. These findings indicate a potential for the therapeutic use of SAR150640 in mammals during preterm labor.Premature labor and its consequence, preterm delivery, defined by the World Health Organization as gestational age at birth of less than 37 complete gestational weeks, are major Article, publication date, and citation information can be found at