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1. This study examines beta(1)-, beta(2)- and beta(3)-adrenoceptor (AR)-mediated responses, mRNA levels and radioligand binding in ileum from beta(3)-AR knock-out (-/-) (KO) and wild type (+/+) (FVB) mice. 2. In KO and FVB mice, SR59230A (100 nM) (beta(3)-AR antagonist) antagonized responses to (-)-isoprenaline in both KO and FVB mice. (-)-Isoprenaline mediated relaxation of ileum was antagonized weakly by ICI118551 (100 nM) (beta(2)-AR antagonist). Responses to (-)-isoprenaline were more strongly antagonized by CGP20712A (100 nM) (beta(1)-AR antagonist), propranolol (1 microM) (beta(1)-/beta(2)-AR antagonist), carvedilol (100 nM) (non-specific beta-AR antagonist), and CGP12177A (100 nM) (beta(1)-/beta(2)-AR antagonist) in ileum from KO than in FVB mice. 3. Responses to CL316243 (beta(3)-AR agonist) in ileum from FVB mice were antagonized by SR59230A (100 nM) but not by propranolol (1 microM) or carvedilol (100 nM). CL316243 was ineffective in relaxing ileum from KO mice. 4. CGP12177A had no agonist actions in ileum from either KO or FVB mice. 5. beta(1)-AR mRNA levels were increased 3 fold in ileum from KO compared to FVB mice. This was associated with an increased maximum number of beta(1)-/beta(2)-AR binding sites (B(max)). beta(2)-AR mRNA levels were unaffected while no beta(3)-AR mRNA was detected in KO mice. 6. In mouse ileum, beta(3)-ARs and to a lesser extent beta(1)-ARs are the predominant adrenoceptor subtypes mediating relaxation in ileum from FVB mice. In KO mice beta(1)-ARs functionally compensate for the lack of beta(3)-ARs, and this is associated with increased beta(1)-AR mRNA and levels of binding.

G protein-coupled receptors are the largest group of membrane proteins and are the targets for approximately 30% of drugs currently used therapeutically. These 7-transmembrane-spanning proteins continue to provide new opportunities to develop therapeutics based on emerging knowledge of their structure, signalling properties and interactions with other proteins. This themed issue of the British Journal of Pharmacology contains a series of papers that cover these issues and identify approaches that may determine future directions. Many of these papers contain material that was presented at the 5th International Molecular Pharmacology of G Protein-Coupled Receptors meeting held in Sydney Australia in late 2008.

Intracerebroventricular (i.c.v.) administration of the beta(3)-AR agonist BRL37344 causes dose dependent decreases in food intake in rats suggesting a role for beta(3)-AR in the central control of feeding. We have conducted experiments investigating the effects of i.c.v. administration of the selective beta(3)-AR agonist CL316243 on Fos expression to determine whether beta(3)-AR stimulation affects neurones within specific brain nuclei. Significantly higher numbers of Fos positive cells were found in the rats treated i.c.v. with CL316243 compared with control rats in the paraventricular hypothalamus, lateral hypothalamic area, ventromedial hypothalamic nucleus and dorsal hypothalamic area. Pre-treatment with the selective beta(3)-AR antagonist SR59230A resulted in a significant decrease in the number of Fos positive cells in all those areas compared with rats treated with CL316243 alone. These experiments demonstrate that i.c.v. administration of selective beta(3)-AR agonist causes neuronal activation in hypothalamic areas important in the central regulation of appetite via a beta(3)-AR mediated effect.

The ‘template-assembled synthetic protein’ (TASP) concept provides a simple and elegant approach for the preparation of analogues that retain key structural elements. We have synthesized TASP molecules containing the putative active site of relaxin, a peptide that has similar structural features to insulin but a markedly different biological role. Two types of chemoselective thiol ligation strategies (thioether and thiazolidine) were used and compared. The synthetic pendant peptides contain an essential region for bioactivity that is located in the α-helical region of the relaxin B-chain. Depending on whether the thioether or the thiazolidine chemistry was used to attach the peptides to the template, the reacting amino acid was placed either at the C-terminus or N-terminus, respectively, thus allowing the choice of orientation relative to the carrier molecule. The template molecule consists of a decapeptide with two proline–glycine turns and four evenly spaced lysine residues that were functionalized with the appropriate chemical moiety. This allowed reaction with the appropriately derivatized peptides in solution. To improve the template ligation step using the thioether approach, a pendant peptide C-terminal cysteamine residue was used to reduce potential steric hindrance during conjugation. The design of the peptides as well as the synthetic strategy resulted in the acquisition of mimetics showing weak non-competitive and weak competitive antagonist properties. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.

1. This study examines the relationship between beta(3a)- and beta(3b)-adrenoceptor (AR) mRNA levels, beta(3)-AR binding and changes in ileum responses in mice treated with the beta(3)-AR agonist (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1, 3-benzodioxole-2,2-dicarboxylate (CL316243), or the beta(3)-AR antagonist 3-(2-ethylphenoxy)-1-[(1S)-1,2,3, 4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A), or dexamethasone or forskolin. 2. Levels of beta(3a)- and beta(3b)-AR mRNA and the maximum number of binding sites (B(max)) in ileum were unaffected following CL316243 treatment, although responses to CL316243 were reduced by 50% following 4 and 24 h treatment, indicating another desensitization mechanism not involving changes in receptor expression or number. beta(3a)-AR mRNA levels were reduced in both brown (BAT) and white adipose tissue (WAT) but beta(3b)-AR mRNA levels were significantly reduced only in WAT. Levels of beta(3a)- and beta(3b)-mRNA returned towards normal with continued treatment. 3. SR59230A treatment markedly increased beta(3)-AR mRN levels in ileum and BAT but not in WAT. The increase in beta(3)-AR mRNA levels in ileum was associated with increased B(max) levels in binding analysis and increased responses to CL316243, suggesting these as the cause of sensitization. 4. Treatment with forskolin (4 h) or dexamethasone (4 h) significantly reduced beta(3a)-AR mRNA levels in BAT and WAT but did not alter levels in ileum. Responses to CL316243 in ileum were unaffected by either treatment. 5. In summary, the beta(3)-AR is differently regulated in adipose tissue and ileum: Treatment with SR59230A increased beta(3)-AR number, mRNA and responsiveness in ileum, whereas treatment with CL316243 reduced responses without affecting beta(3)-AR number or mRNA levels.

The relaxin family peptide receptor 3 (RXFP3) is the cognate receptor for the neuropeptide relaxin-3. RXFP3 was tagged at the carboxy-terminus with a variant of the green fluorescent protein (GFP(2)) for use in receptor localization studies. RXFP3-GFP(2) was examined to ensure it retained binding and signaling properties similar to untagged RXFP3. Competition for [(125)I]INSL5/H3 relaxin chimera binding to RXFP3 and RXFP3-GFP(2) indicated that the carboxy-terminal tag did not affect receptor binding or receptor internalization. RXFP3-GFP(2) activated ERK1/2 with a similar potency to RXFP3 when transiently expressed in CHO-K1 or HEK293T cells, suggesting that the GFP(2) tag did not affect receptor function. This study demonstrated that addition of a carboxy-terminal fusion protein to RXFP3 did not alter the binding or signaling properties of RXFP3, making RXFP3-GFP(2) a useful tool for future receptor localization and trafficking studies.

The β3-adrenoceptor (AR) differs from the β1-AR and β2-ARs in having introns within and downstream of the coding block. This study demonstrates two splice variants of the mouse β3-AR which differ within the coding region. Reverse transcription/polymerase chain reaction with intron-spanning primers was used to demonstrate the splice variant of the mouse β3-adrenoceptor. The novel β3b-AR has 17 amino acids encoded by exon 2 (SSLLREPRHLYTCLGYP) which differ from the 13 in the known β3a-AR (RFDGYEGARPFPT). β3b-AR mRNA is differentially expressed in mouse tissues, with levels relative to β3a-AR mRNA highest in hypothalamus, cortex and white adipose tissue, and lower in ileum smooth muscle and brown adipose tissue. British Journal of Pharmacology (1999) 127, 1525–1531; doi:10.1038/sj.bjp.0702688

Functional and molecular approaches were used to characterize the β-AR subtypes mediating relaxation of rat ileal smooth muscle. In functional studies, (−)-isoprenaline relaxation was unchanged by CGP20712A (β1-AR antagonist) or ICI118551 (β2-AR antagonist) but shifted by propranolol (pKB=6.69). (±)-Cyanopindolol, {"type":"entrez-protein","attrs":{"text":"CGP12177","term_id":"877152897","term_text":"CGP12177"}}CGP12177 and ICID7114 did not cause relaxation but antagonized (−)-isoprenaline relaxation. BRL37344 (β3-AR agonist) caused biphasic relaxation. The high affinity component was shifted with low affinity by propranolol, (±)-cyanopindolol, tertatolol and alprenolol. {"type":"entrez-nucleotide","attrs":{"text":"CL316243","term_id":"44896132","term_text":"CL316243"}}CL316243 (β3-AR agonist) relaxation was unaffected by CGP20712A or ICI118551 but blocked by SR58894A (β3-AR antagonist; pA2=7.80). Enhanced relaxation after exposure to forskolin and pertussis toxin showed that β3-AR relaxation can be altered by manipulation of components of the adenylate cyclase signalling pathway. The β1-AR agonist RO363 relaxed the ileum (pEC50=6.18) and was blocked by CGP20712A. Relaxation by the β2-AR agonist zinterol (pEC50=5.71) was blocked by SR58894A but not by ICI118551. In rat ileum, β1-, β2- and β3-AR mRNA was detected. Comparison of tissues showed that β3-AR mRNA expression was greatest in WAT>colon=ileum>cerebral cortex>soleus; β1-AR mRNA was most abundant in cerebral cortex>WAT>ileum=colon>soleus; β2-AR mRNA was expressed in soleus>WAT>ileum=colon>cerebral cortex. These results show that β3-ARs are the predominant β-AR subtype mediating rat ileal relaxation while β1-ARs may produce a small relaxation. The β2-AR agonist zinterol produces relaxation through β3-ARs and there was no evidence for the involvement of β2-ARs in relaxation despite the detection of β2-AR mRNA. Keywords: β-adrenoceptors, β3-adrenoceptors, gastrointestinal smooth muscle, relaxation, rat ileum, messenger RNA Introduction Although the intestinal β-AR was originally described as a β1-AR (Lands et al., 1967) atypically low affinities of β-AR antagonists and propranolol-resistant relaxation responses observed in gastrointestinal smooth muscle preparations from several species indicated that other β-AR subtypes were involved in these responses (see Arch & Kaumann, 1993, for review). Atypical β-AR responses that were resistant to blockade by propranolol and selectively stimulated by a novel group of β-AR agonists were also described in adipocytes. The β3-AR was subsequently cloned and sequenced (Emorine et al., 1989) and found to share many of the pharmacological characteristics of the atypical β-AR. β3-ARs are highly expressed in adipose tissue and β3-AR agonists such as BRL 37344, SR 58611A and CL 316,243 are potent stimulants of lipolysis and thermogenesis, cause marked weight loss in obese animals, increase insulin sensitivity and improve glucose tolerance in diabetic animals. Whilst the human β3-AR is emerging as an exciting target for the development of selective stimulants as potential treatments for obesity and diabetes (Strosberg & Pietri-Rouxel, 1996), these same compounds may also be useful in the treatment of irritable bowel syndrome. β3-ARs are also highly expressed in regions of the gastrointestinal tract such as colon, ileum and stomach in many species including human (Berkowitz et al., 1995; Evans et al., 1996; Roberts et al., 1997). β3-AR mediated relaxation in gastrointestinal smooth muscle is resistant to blockade by propranolol and selective β1- and β2-AR antagonists such as CGP 20712A and ICI 118551 (for review see Manara et al., 1995). Earlier studies of gastrointestinal smooth muscle used non-selective β-AR or selective β1- or β2-AR agonists to characterize β-AR responses in various animal models. For example, studies using non-selective β-AR or selective β2-AR agonists in diabetic animal models have shown a marked reduction in β-AR responsiveness in the intestine (Ozturk et al., 1996). Decreased responses to the selective β2-AR agonist salbutamol are of particular note as the low potency of salbutamol strongly suggests that it is acting through β3-ARs rather than β2-ARs. In addition, a recent binding study mapping β-AR subtypes throughout the diabetic rat gastrointestinal tract used a concentration of the radioligand [125I]-CYP (20 pM) so low that it would label only 1.5% of β3-ARs in each sample (Yu & Ouyang, 1997). The findings from these studies therefore offer limited information on the changes in β-AR density and responsiveness as the tools/compounds used were not appropriate for examination of all three β-AR subtypes. It is necessary to clearly establish the role and contribution of each β-AR subtype to gastrointestinal relaxation and as selective compounds for each subtype are now available, a more comprehensive study is clearly possible. A highly selective β3-AR agonist, CL 316243, has been developed that has at least 10,000 fold selectivity for the β3-AR compared with the β1- and β2-ARs respectively (Dolan et al., 1994). More recently the first selective β3-AR antagonists were developed and these new pharmacological tools have enabled a clear demonstration of β3-AR mediated functional responses in rat colon and rat brown adipose tissue (Manara et al., 1996). Previous studies of the β3-AR in rat ileum have been carried out in the presence of β1- and β2-AR blockade and the relative contribution of β-AR subtypes has not been examined using agonists and antagonists selective for each of the three β-AR subtypes. Studies of functional responses of rat ileum relaxation (Growcott et al., 1993; Hoey et al., 1996) have shown that stimulation by selective β3-AR agonists produces a relaxation response in the presence of β1- and β2-AR blockade. The aim of this study therefore was to utilize selective β3-AR compounds as well as selective β1- and β2-AR agonists and antagonists to characterize the functional β-AR subtypes present in rat ileum and assess the relative contribution of these subtypes to β-AR mediated relaxation of the rat ileal smooth muscle. We have also measured mRNA levels of the three β-AR subtypes in ileal smooth muscle.

1. This study examined beta-adrenoceptor signalling in cardiac tissues following infusion of isoprenaline (400 microg kg(-1) h(-1)) or vehicle to rats for 14 days. 2. Isoprenaline infusion caused marked hypertrophy of atria and ventricles and reduced the resting rate of spontaneously beating right atria and the basal force of left atrial contraction. 3. In spontaneously beating right atria, concentration-response curves to isoprenaline and forskolin were shifted 7.9 and 3.2 fold to the right following treatment whereas responses to the cyclic AMP analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-cyclic monophosphorothioate were unchanged. 4. In electrically driven left atria, concentration-response curves to isoprenaline and forskolin were shifted 4 fold to the right and maximum responses reduced. Responses to dibutyryl cyclic AMP were shifted 3.2 fold to the right but those to Ca2+ were unchanged. 5. Inotropic responses of left and right ventricular papillary muscles to isoprenaline were abolished and markedly reduced respectively by isoprenaline treatment. Responses to forskolin were shifted 5 fold to the right. Responses to dibutyryl cyclic AMP were shifted to the right 3.2 and 2 fold in left and right ventricular papillary muscles. Responses to isobutyl methyl xanthine were shifted to the right 15.8 and 6.3 fold in left and right papillary muscles whereas those to Ca2+ were not significantly altered. 6. This study indicates differences in beta-adrenoceptor desensitization in different regions of the heart following chronic infusion of isoprenaline. Chronotropic responses showed impaired signalling between the receptor and adenylate cyclase whereas inotropic responses exhibited additional desensitization at the level of cyclic AMP dependent protein kinase.

The binding characteristics of the relaxin receptor in rat atria, uterus and cortex were studied using a [33P]-labelled human gene 2 relaxin (B33) and quantitative receptor autoradiography. The binding kinetics of [33P]-human gene 2 relaxin (B33) were investigated in slide-mounted rat atrial sections. The binding achieved equilibrium after 60 min incubation at room temperature (23+/-1 degrees C) and dissociated slowly. The association and dissociation rate constants were 4.31+/-0.34x10(8) M(-1) x min(-1) and 1.55+/-0.38x10(-3) min(-1) respectively. Thus, the kinetic dissociation constant was 3.46+/-0.59 pM. Binding was saturable to a single population of non-interacting sites throughout atria, in uterine myometrium and the 5th layer of cerebral cortex. The binding affinities (pK(D)) of [33P]-human gene 2 relaxin (B33) were 8.92+/-0.09 in atrial myocardium and 8.79+/-0.04 in cerebral cortex of male rats, and 8.79+/-0.10 in uterine myometrium. Receptor densities in the cerebral cortex and atria were higher than in uterine myometrium, indicating that relaxin also has important roles in non-reproductive tissues. In male rats, treatment with 17beta-oestradiol (20 microg in 0.1 ml sesame oil s.c., 18-24 h) significantly decreased the density of relaxin receptors in atria and cerebral cortex. Identical treatment in female rats had no significant effect in atria and cerebral cortex, but it significantly increased the density of relaxin receptors in uterine myometrium. Relaxin binding was competitively displaced by porcine and rat native relaxins. Porcine native relaxin binds to the relaxin receptor in male rat atria (8.90+/-0.02), and cerebral cortex (8.90+/-0.03) and uterine myometrium (8.89+/-0.03) with affinities not significantly different from human gene 2 (B33) relaxin. Nevertheless, rat relaxin binds to the receptors with affinities (8.35+/-0.09 in atria, 8.22+/-0.07 in cerebral cortex and 8.48+/-0.06 in uterine myometrium) significantly less than human gene 2 (B33) and porcine relaxins. Quantitative receptor autoradiography is the method of choice for measurement of affinities and densities of relaxin receptor in atria, uterine myometrium and cerebral cortex. High densities were found in all these tissues. 17beta-oestradiol treatment produced complex effects where it increased the densities of relaxin receptors in uterus but decreased those in atria and cerebral cortex of the male rats, and had no effect on the atria and cerebral cortex of the female rats.

The primary structure of ovine Leydig cell insulin-like peptide (Ley I-L) was recently deduced from the corresponding cDNA sequence. It consists of two peptide chains and three disulphide bonds in an arrangement similar to both relaxin and insulin. As in relaxin B-chain, an Arg-X-X-X-Arg sequence exists within the Ley I-L B-chain although it is located four residues towards the C-terminus from the corresponding position within relaxin. This sequence of amino acids is known to be essential for relaxin biological activity and its presence in Ley I-L suggested that the peptide might possess a relaxin-like function. Ovine Ley I-L was assembled by Fmoc-solid-phase synthesis of the separate chains followed by their combination in solution at high pH. The purity and identity of the chain-combined peptide was confirmed by chemical characterization including mass spectrometry. At physiological concentrations, the peptide was shown not to possess relaxin-like activity in the rat isolated atrial chronotropic and inotropic assay. This strongly suggests that Ley I-L is not a relaxin in the sheep. In order to explore further a possible structural relationship between Ley I-L and relaxin, we prepared a synthetic analogue of ovine Ley I-L containing a single replacement of B-chain residue 12, His, with Arg. This was found to possess significant relaxin-like chronotropic and inotropic activity demonstrating that the tertiary structure of Ley I-L is similar to that of relaxin and highlighting the key requirement for the five-residue sequence, Arg-X-X-X-Arg, to be present in position B12-16 for characteristic relaxin activity.

The objectives of this study were to determine whether leptin synthesis is regulated by the sympathetic nervous system and if so whether beta-adrenergic receptors mediate this effect. We show that sympathetic blockade by reserpine increases leptin mRNA levels in brown but not white adipose tissue, while acute cold-exposure decreases leptin expression 10-fold in brown adipose tissue and 2-fold in white adipose tissue. The cold-induced reduction in leptin mRNA can be prevented by a combination of propranolol and SR 59230A but not by either antagonist alone, indicating that beta3-adrenergic receptors and classical beta1/beta2-adrenergic receptors both mediate responses to sympathetic stimulation. Circulating leptin levels reflect synthesis in white adipose tissue but not in brown adipose tissue.

β-Adrenoceptors are one of the most widely studied groups of G-protein-coupled receptors but continue to provide surprises and insights that have general relevance to pharmacology. Atypical pharmacologies have been described for ligands formerly (and still) described as antagonists acting at β1-, β2- and β3-adrenoceptors that involve ligand-directed signalling and possibly allosteric interactions at the receptors. In the article by Ngala et al., in this issue of the Br J Pharmacol, another example of atypical interactions with β-adrenoceptors is described, this time for agonists. Some of the responses to BRL37344 and clenbuterol can be explained in terms of actions at β2-adrenoceptors, whereas others such as the increased glucose uptake and palmitate oxidation observed with pM concentrations of BRL37344 may involve interactions with other (possibly allosteric) sites. Atypical pharmacologies of ligands acting at β-adrenoceptors have already indicated new ways in which ligands can interact with G-protein-coupled receptors and these mechanisms are likely to have important consequences for future drug development.

The mitochondrial uncoupling protein UCP-1 uncouples respiration from ATP synthesis in brown adipose tissue (BAT) and thus energy is dissipated as heat. Recently two further isoforms have been identified which may play a similar role in other tissues. We have determined the effects of the rodent-selective beta3-adrenoceptor (beta3-AR) agonist BRL 35135, on beta3-AR and UCP mRNA levels in tissues from lean and obese (fa/fa) Zucker rats. beta3-AR mRNA levels were reduced in fa/fa white (WAT) and brown (BAT) adipose tissue relative to levels in lean littermates. BRL 35135 treatment increased expression levels of beta3-AR mRNA in both genotypes. UCP-2 and UCP-3 mRNA levels in BAT, WAT and skeletal muscle were reduced by 2-3 fold in the fa/fa rats relative to the lean rats. We confirm that BRL 35135 increases BAT UCP-1 mRNA in lean rats, and find that BAT UCP-3 mRNA was reduced 3.2 fold, with no changes in UCP-2 expression. In WAT BRL 35135 increased UCP-2 and UCP-3 expression 2-3 fold in both lean and fa/fa rats. In lean rats, skeletal muscle UCP-3 mRNA was increased 2.3 fold by BRL 35135 whereas UCP-2 was reduced by 2.2 fold. BRL 35135 had no effects on UCP-2 and UCP-3 expression in skeletal muscle of the fa/fa rats. Our results demonstrate that mechanisms regulating UCP isoform synthesis in fa/fa rats are impaired and that WAT could be involved in the thermogenic response of BRL 35135.

This study investigated the effects of myocardial infarction (MI)-induced cardiac failure and treatment with an angiotensin-converting enzyme (ACE) inhibitor perindopril (2 mg/kg/day) on rat beta-adrenoceptor (beta-ar) subtypes in anatomically defined regions of infarcted left ventricular (LV) free wall and noninfarcted tissue from right ventricle (RV) by using autoradiography. After 5 weeks of MI, rats with large MI size (>42%) had developed cardiac failure and beta1-ars were significantly decreased (-59%; p < 0.01) in the border region of the infarcted LV and almost abolished in the infarcted area (-90%; p < 0.005) compared with normal LV from sham-operated controls. The beta-ar changes were not found in the noninfarcted area of the same LV or in RV. MI did not significantly alter the number of beta2-ar subtypes in any region of the ventricles. Perindopril treatment for 4 weeks reduced mean cardiac region weights but did not affect beta-ar density in any cardiac region in either sham-operated or MI rats. These results indicate that cardiac failure due to MI causes significant downregulation of beta1-ars only in border and infarcted regions of rat LV and no change in beta2-ar in any area. It also suggests that the improved response of the infarcted rat heart to isoprenaline stimulation after ACE inhibitors does not result from changes in the numbers of cardiac beta-ars.

1. Levels of beta3-adrenoceptor (AR) mRNA were compared using reverse transcription-polymerase chain reaction (RT PCR) in white adipose tissue (WAT), brown adipose tissue (BAT), ileum and colon from genetically obese (ob/ob) and lean (+/+) C57BL/6J mice. Functional responses to the beta3-AR agonist CL 316243 were also characterized in ileal longitudinal smooth muscle from obese and lean mice. 2. Beta3-AR mRNA levels were significantly higher in WAT (100+/-16%) and BAT (100+/-13%) from lean compared to WAT (21.0+/-0.9%; n=4; P

The receptors for relaxin in the rat atria and uterus were investigated and compared by use of a series of synthetic and native relaxin analogues. The assays used were the positive chronotropic and inotropic effects in rat spontaneously beating, isolated right atrium and electrically driven left atrium and the relaxation of K+ precontracted uterine smooth muscle. Relaxin analogues with an intact A- and B-chain were active in producing powerful chronotropic and inotropic effects in the rat isolated atria at nanomolar concentrations. Single-chain analogues and structural homologues of relaxin such as human insulin and sheep insulin-like growth factor I had no agonist action and did not antagonize the effect of the B29 form of human gene 2 relaxin. Shortening the B-chain carboxyl terminal of human gene 1 (B2–29) relaxin to B2–26 reduced the activity of the peptide and removal of another 2 amino acid residues (B2–24) abolished the activity. This suggests that the B-chain length may be important for determination of the activity of relaxin. More detailed studies are needed to determine the effect of progressive amino acid removal on the structure and the bioactivity of relaxin. Porcine prorelaxin was as active as porcine relaxin on a molar basis, suggesting that the presence of the intact C-peptide did not affect the binding of the prorelaxin to the receptor to produce functional responses. Relaxin caused relaxation of uterine longitudinal and circular smooth muscle precontracted with 40 mM K+. The pEC50 values for human gene 2 and porcine relaxins were lower than those in the atrial assay, but rat relaxin had similar pEC50 values in both atrial and uterine assays. Rat relaxin was significantly less potent than either human gene 2 or porcine relaxin in the atrial assay, but in the uterine assay they were equipotent. The results suggest that the relaxin receptor or the signalling pathway in rat atria may differ from that in the uterus. British Journal of Pharmacology (1998) 123, 762–770; doi:10.1038/sj.bjp.0701659

High use of CPAP increased weight more than high use sham (1.4 kg; 0.04—2.9, p = 0.05) and more than low CPAP use (1.6 kg; 0.4—2.8, p = 0.01). There was no difference between high and low sham users (0.36 kg;−1.0 to 1.7, NS). Neither treatment alone, CPAP dose alone nor the combined effect of both influenced glucose, insulin or insulin resistance. Conclusion: High use CPAP increases weight compared to high use sham and low use CPAP without any subsequent changes in metabolic dysfunction. It is possible an increase in lean muscle mass rather than fat is the cause of the weight gain which may not necessarily have a detrimental effect on health.

1. Relaxation of carbachol pre-contracted human colonic muscle to (-)-isoprenaline was examined in circular, longitudinal and taenia coli preparations to determine the beta-adrenoceptor subtypes involved. beta 1-, beta 2- and beta 3-Adrenoceptor mRNAs were also measured in colonic muscle and mucosa. 2. (-)-isoprenaline caused relaxation of longitudinal smooth muscle preparations with pEC50-7.39 +/- 0.12, and this response was inhibited by both propranolol (0.1 microM, pKB 8.55 +/- 0.12) and the selective beta 1-antagonist, CGP 20712A (0.1 microM, pKB 8.80 +/- 0.20), while the selective beta 2-antagonist, ICI 118551 (0.1 microM) failed to inhibit isoprenaline relaxation consistently. 3. (-)-Isoprenaline caused relaxation of taenia coli with a pEC50 of 6.70 +/- 0.17. Propranolol (0.1 microM). CGP 20712A (0.1 microM) and ICI 118551 (0.1 microM) inhibited the isoprenaline response with similar low affinities (pKB values 7.93, 7.71 and 7.54, respectively). Carbachol pre-contracted circular smooth muscle preparations failed to relax consistently to isoprenaline and these responses were not characterized. 4. beta 1- and beta 2-Adrenoceptor mRNAs were present in circular longitudinal muscle samples and taenia coli samples, and lower levels were detected in mucosa. beta 3-mRNA was also present in both muscle preparations but was not detected in human colonic mucosa. 5. In summary, beta 1-adrenoceptors are the predominant subtype mediating isoprenaline-induced relaxation of the thin longitudinal smooth muscle of human colon, while beta 1-receptors do not appear to be involved in these responses. However, beta 3-adrenoceptors may play a role in relaxation of the taenia coli as conventional antagonist affinities are low. beta 3-Adrenoceptor mRNA was present in taenia coli and circular/longitudinal smooth muscle but absent from human colonic mucosa.

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.

Fibrosis is a hallmark of chronic kidney disease, for which there is currently no effective cure. The hormone relaxin is emerging as an effective antifibrotic therapy; however, its mechanism of action is poorly understood. Recent studies have shown that relaxin disrupts the profibrotic actions of transforming growth factor-β1 (TGF-β1) by its cognate receptor, relaxin family peptide receptor 1 (RXFP1), extracellular signal–regulated kinase phosphorylation, and a neuronal nitric oxide synthase–dependent pathway to abrogate Smad2 phosphorylation. Since angiotensin II also inhibits TGF-β1 activity through its AT2 receptor (AT 2 R), we investigated the extent to which relaxin interacts with the AT 2 R. The effects of the AT 2 R antagonist, PD123319, on relaxin activity were examined in primary rat kidney myofibroblasts, and in kidney tissue from relaxin-treated male wild-type and AT 2 R-knockout mice subjected to unilateral ureteric obstruction. Relaxin's antifibrotic actions were significantly blocked by PD123319 in vitro and in vivo , or when relaxin was administered to AT 2 R-knockout mice. While heterodimer complexes were formed between RXFP1 and AT 2 Rs independent of ligand binding, relaxin did not directly bind to AT 2 Rs but signaled through RXFP1-AT 2 R heterodimers to induce its antifibrotic actions. These findings highlight a hitherto unrecognized interaction that may be targeted to control fibrosis progression.

The capacity of G protein-coupled receptors (GPCRs) to activate multiple G protein isoforms and additional effectors such as β-arrestins has become a well-established paradigm and provides the basis for developing drugs that preferentially activate beneficial signalling pathways. There are many published examples of ligand-directed signalling, and recent studies have provided direct evidence that different agonists stabilise distinct GPCR conformations. This field is rapidly evolving, but a key question is whether signalling bias observed in heterologous cell expression systems can be translated to physiological systems of therapeutic relevance. The paper by Ngala et al. in this issue of the journal addresses the capacity of agonists acting at the β2-adrenoceptor to engender signalling bias in relation to glucose uptake in isolated skeletal muscle, an area of considerable potential interest in targeting insulin-independent pathways for the treatment of type 2 diabetes. The authors show that clenbuterol and BRL37344 have opposite effects on glucose uptake, despite both having agonist actions at β2-adrenoceptors. This study underlines some of the obstacles associated with studies in a complex physiological system but nonetheless highlights the need to consider signalling bias in the relevant target tissue when developing novel drugs.

1. This study examines the expression of beta 3-adrenoceptor messenger RNA (beta 3-AR mRNA) in rat tissues to allow comparison with atypical beta-adrenoceptors determined by functional and radioligand binding techniques. 2. A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of beta 3-AR mRNA in rat tissues. 3. Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute beta 3-AR mRNA. 4. The beta 3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the beta 3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of beta 3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of beta 3-AR. 5. Expression of adipsin in rat skeletal muscles coupled to very low levels of beta 3-AR mRNA indicates that the observed beta 3-AR may be due to the presence of intrinsic fat. beta 3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical beta-AR demonstrated by functional and/or binding studies in muscle and in heart is not the beta 3-AR. 6. By use of two different sets of primers for amplification of beta 3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7. The detection of beta 3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility beta 3-AR may subserve other functions in the gastrointestinal tract. The absence of beta 3-AR mRNA in rat heart or its presence with adipsin in skeletal muscle suggests that atypical beta-adrenoceptor responses in heart and skeletal muscle are unlikely to be mediated by beta 3-AR.

1. The effect of long-term treatment with the beta-adrenoceptor antagonists (--)-tertatolol and (--)-propranolol was studied. Sprague-Dawley rats were treated with either (--)-tertatolol (50 micrograms kg-1 hr-1), (--)-propranolol (250 micrograms kg-1 hr-1) or vehicle (1 mM HCl) for 14 days with osmotic minipumps implanted subcutaneously. 2. The mean daily systolic blood pressure and heart rate of rats treated with either (--)-tertatolol (108 +/- 1 mmHg/330 +/- 3 bpm) or (--)-propranolol (103 +/- 1 mmHg/330 +/- 2 bpm) were lower than in the control (126 +/- 1 mmHg/405 +/- 3 bpm, P0.001, n = 8-10) indicating the effectiveness of drug delivery. 3. Autoradiographic studies in areas of heart, lung and skin showed that beta-adrenoceptor populations were not significantly affected by the drug treatment (all regions P0.05). Nevertheless, the receptor population in the homogenates of (--)-tertatolol treated lung were halved (194 +/- 28 fmol mg protein-1 compared with a control value of 388 +/- 54 fmol mg protein-1, P0.01, n = 6). 4. In the presence of CGP 20712A, the left atrial inotropic and right atrial chronotropic responsiveness to (--)-isoprenaline were hypersensitive in both (--)-tertatolol and (--)-propranolol-treated groups (P0.005, ANCOVA). 5. (--)-Propranolol treated left ventricular free wall had lower basal [3H]-forskolin binding to adenylate cyclase (14.45 +/- 1.20 fmol mg protein-1 compared with a control value of 18.91 +/- 0.78 fmol mg protein-1, P = 0.01, n = 6). (--)-Tertatolol treatment had no effect on the basal binding. In the presence of the G-protein activators NaF and Gpp(NH)p, the enhancement of [3H]-forskolin binding did not differ between control and the drug treated groups. 6. Chronic (--)-tertatolol or (--)-propranolol treatment therefore did not produce an increase in receptors in heart, lung or skin but the beta-adrenoceptor-mediated responses were enhanced. In addition, [3H]-forskolin binding did not increase suggesting that the hypersensitivity was not due to changes in the number of receptors or adenylate cyclase. Hypersensitivity following beta-adrenoreceptor antagonist administration may therefore involve enhanced coupling of receptors to G-proteins.

Phenotyping of Gprc6a KO mice has shown that this promiscuous class C G protein coupled receptor is variously involved in regulation of metabolism, inflammation and endocrine function. Such effects are described as mediated by extracellular calcium, L-amino acids, the bone-derived peptide osteocalcin (OCN) and the male hormone testosterone, introducing the concept of a bone-energy-metabolism-reproduction functional crosstalk mediated by GPRC6A. However, whilst the calcium and L-amino acid-sensing properties of GPRC6A are well established, verification of activity of osteocalcin at both human and mouse GPRC6A in vitro has proven somewhat elusive. This study characterises the in vitro pharmacology of mouse GPRC6A in response to its putative ligands in both recombinant and endogenous GPRC6A-expressing cells. Using cell signalling, and glucagon-like peptide (GLP)-1 and insulin release assays, our results confirm that basic L-amino acids act as agonists of the murine GPRC6A receptor in both recombinant cells and immortalised entero-endocrine and pancreatic β-cells. In contrast, our studies do not support a role for OCN as a direct ligand for mouse GPRC6A, suggesting that the reported in vivo effects of OCN that require GPRC6A may be indirect, rather than via direct activation of the receptor.

Caveolins act as scaffold proteins in multiprotein complexes and have been implicated in signaling by G protein-coupled receptors. Studies using knock-out mice suggest that β(3)-adrenoceptor (β(3)-AR) signaling is dependent on caveolin-1; however, it is not known whether caveolin-1 is associated with the β(3)-AR or solely with downstream signaling proteins. We have addressed this question by examining the impact of membrane rafts and caveolin-1 on the differential signaling of mouse β(3a)- and β(3b)-AR isoforms that diverge at the distal C terminus. Only the β(3b)-AR promotes pertussis toxin (PTX)-sensitive cAMP accumulation. When cells expressing the β(3a)-AR were treated with filipin III to disrupt membrane rafts or transfected with caveolin-1 siRNA, the cyclic AMP response to the β(3)-AR agonist CL316243 became PTX-sensitive, suggesting Gα(i/o) coupling. The β(3a)-AR C terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant β(3a)-ARs (F389A/Y392A/F398A or P384S/F389A) promoted PTX-sensitive cAMP responses, and in situ proximity assays demonstrated an association between caveolin-1 and the wild type β(3a)-AR but not the mutant receptors. In membrane preparations, the β(3b)-AR activated Gα(o) and mediated PTX-sensitive cAMP responses, whereas the β(3a)-AR did not activate Gα(i/o) proteins. The endogenous β(3a)-AR displayed Gα(i/o) coupling in brown adipocytes from caveolin-1 knock-out mice or in wild type adipocytes treated with filipin III. Our studies indicate that interaction of the β(3a)-AR with caveolin inhibits coupling to Gα(i/o) proteins and suggest that signaling is modulated by a raft-enriched complex containing the β(3a)-AR, caveolin-1, Gα(s), and adenylyl cyclase.