Atypical beta-adrenoceptors mediating relaxation in the human colon: functional evidence for beta3-rather than beta4-adrenoceptors.

The aim of the present functional study was to assess the role of beta3-adrenoceptors in the light of recent findings suggesting the existence of a putative fourth beta-adrenoceptor in adipose and heart tissue. The effect of the non-conventional beta3-adrenoceptor partial agonist CGP12177A is resistant to the effect of the beta3-adrenoceptor antagonist SR59230A. Under isotonic conditions in circular muscle strips of human distal colon, the concentration-effect relationship of CGP12177A and SR59104A (beta3-adrenoceptor agonists), alone and in the presence of CGP20712A (beta1-adrenoceptor antagonist) ICI118551 (beta2-adrenoceptor antagonist) and SR59230A, all 0.1 microm was studied. CGP12177A concentration-dependently relaxed circular muscle strips (pEC50=6.16+/-0.05). This effect was left unchanged by beta1-/beta2-adrenoceptor blockade, but antagonised by SR59230A (pA2=8.12+/-0.02). SR59104A concentration-dependently relaxed circular muscle strips (pEC50=5.43+/-0.01), an effect that was not significantly affected by pretreatment with CGP20712A and ICI118551, but competitively antagonised by SR59230A (p KB=7.89). Isoprenaline-induced relaxations were antagonised by propranolol with a low pA2value (7.76+/-0.16). These results provide further evidence for the presence of functional beta3-adrenoceptors in the human colon, but do not support a role for an atypical beta-adrenoceptor distinct from the beta3-subtype.     

Ligand-directed signaling at the beta3-adrenoceptor produced by 3-(2-Ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A) relative to receptor agonists

This study examines signaling pathways activated by the mouse beta(3)-adrenoceptor (AR) expressed in Chinese hamster ovary cells at high (CHObeta(3)H) or low (CHObeta(3)L) levels. Functional responses included extracellular acidification rate (ECAR), cAMP accumulation, and p38 mitogen-activated protein kinase (MAPK) or extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation. (-)-Isoproterenol and the beta(3)-AR agonist (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-decarboxylate (CL316243) caused concentration-dependent increases in cAMP accumulation and ECAR in CHObeta(3)H and CHObeta(3)L cells. For cAMP accumulation, the beta(3)-AR ligand SR59230A was a partial agonist in CHObeta(3)H and an antagonist in CHObeta(3)L cells but for ECAR was an agonist at both expression levels. This suggested that SR59230A, which is normally regarded as an antagonist, can selectively activate pathways leading to ECAR. Examination of the pathways stimulated by (-)-isoproterenol, CL316243, and SR59230A for both ECAR and cAMP accumulation suggested that the cAMP pathway predominates in CHObeta(3)H cells, whereas p38 MAPK is a major contributor to ECAR in CHObeta(3)L cells and was the sole contributor to responses to SR59230A. Western blots of p38 MAPK and Erk1/2 phosphorylation confirmed that MAPKs are activated in CHObeta(3)H and CHObeta(3)L cells by CL316243 and SR59230A but that SR59230A has much higher efficacy. In addition, p38 MAPK phosphorylation displayed differences in drug potency and efficacy between CHObeta(3)H and CHObeta(3)L cells related to inhibition of the response by cAMP. Thus, CL316243 and SR59230A display reversed orders of efficacy for cAMP accumulation compared with Erk1/2 and p38 MAPK phosphorylation, providing a strong indication of ligand-directed signaling.     

beta(1)-Adrenoceptors compensate for beta(3)-adrenoceptors in ileum from beta(3)-adrenoceptor knock-out mice

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.     

Functional evidence of atypical beta 3-adrenoceptors in the human colon using the beta 3-selective adrenoceptor antagonist, SR 59230A.

The role of beta 3-adrenoceptors in human colonic circular smooth muscle was assessed in vitro by use of the beta 3-selective antagonist SR 59230A. Isoprenaline, in the presence of the selective beta-adrenoceptor antagonists CGP 20712A (beta 1) and ICI 118551 (beta 2), both at 0.1 microM, concentration-dependently relaxed the preparation (pEC50 = 5.22). This effect was potently and competitively antagonized by SR 59230A with a pA2 of 8.31, while its R,R enantiomer SR 59483A gave an apparent pKB of 6.21. Relaxation was likewise produced by CGP 12177A (pEC50 = 6.05), but not by BRL 37344. Although only one of these beta 3-selective agonists was effective, the remarkably high potency of SR 59230A as a stereospecific antagonist of non-beta 1 non-beta 2 relaxation of human colonic muscle by isoprenaline provides strong functional evidence of beta 3-adrenoceptors in that tissue.     

Characterization of beta-adrenoceptor mediated smooth muscle relaxation and the detection of mRNA for beta1-, beta2- and beta3-adrenoceptors in rat ileum.

1. Functional and molecular approaches were used to characterize the beta-AR subtypes mediating relaxation of rat ileal smooth muscle. 2. In functional studies, (-)-isoprenaline relaxation was unchanged by CGP20712A (beta1-AR antagonist) or ICI118551 (beta2-AR antagonist) but shifted by propranolol (pKB=6.69). (+/-)-Cyanopindolol, CGP12177 and ICID7114 did not cause relaxation but antagonized (-)-isoprenaline relaxation. 3. BRL37344 (beta3-AR agonist) caused biphasic relaxation. The high affinity component was shifted with low affinity by propranolol, (+/-)-cyanopindolol, tertatolol and alprenolol. CL316243 (beta3-AR agonist) relaxation was unaffected by CGP20712A or ICI118551 but blocked by SR58894A (beta3-AR antagonist; pA2 = 7.80). Enhanced relaxation after exposure to forskolin and pertussis toxin showed that beta3-AR relaxation can be altered by manipulation of components of the adenylate cyclase signalling pathway. 4. The beta-AR agonist RO363 relaxed the ileum (pEC50=6.18) and was blocked by CGP20712A. Relaxation by the beta2-AR agonist zinterol (pEC50=5.71) was blocked by SR58894A but not by ICI118551. 5. In rat ileum, beta1-, beta2- and beta3-AR mRNA was detected. Comparison of tissues showed that beta3-AR mRNA expression was greatest in WAT>colon=ileum >cerebral cortex>soleus; beta1-AR mRNA was most abundant in cerebral cortex > WAT > ileum = colon > soleus; beta2-AR mRNA was expressed in soleus > WAT > ileum = colon > cerebral cortex. 6. These results show that beta3-ARs are the predominant beta-AR subtype mediating rat ileal relaxation while beta1-ARs may produce a small relaxation. The beta2-AR agonist zinterol produces relaxation through beta3-ARs and there was no evidence for the involvement of beta2-ARs in relaxation despite the detection of beta2-AR mRNA.     

Direct demonstration of beta1- and evidence against beta2- and beta3-adrenoceptors, in smooth muscle cells of rat small mesenteric arteries

1 Recent evidence supports additional subtypes of vasodilator beta-adrenoceptor (beta-AR) besides the 'classical' beta(2). The aim of this study was to investigate the distribution of beta-ARs in the wall of rat mesenteric resistance artery (MRA), to establish the relative roles of beta-ARs in smooth muscle and other cell types in mediating vasodilatation and to analyse this in relation to the functional pharmacology. 2 We first examined the vasodilator beta-AR subtype using 'subtype-selective' agonists against the, commonly employed, phenylephrine-induced tone. Concentration-related relaxation was produced by isoprenaline (pEC(50): 7.70+/-0.1) (beta(1) and beta(2)). Salbutamol (beta(2)), BRL 37344 (beta(3)) and CGP 12177 (atypical beta) caused relaxation but were 144, 100 and 263 times less potent than isoprenaline; the 'beta(3)-adrenoceptor agonist' CL 316243 was ineffective. 3 In arteries precontracted with 5-HT or U 46619, isoprenaline produced concentration-related relaxation but salbutamol, BRL 37344, CGP 12177 and CL 316243 did not. SR 59230A, CGP 12177 and BRL 37344 caused a parallel rightward shift in the concentration-response curve to phenylephrine indicating competitive alpha(1)-AR antagonism, explaining the false-positive 'vasodilator' action against phenylephrine-induced tone. Endothelial denudation but not L-NAME slightly attenuated isoprenaline-mediated vasodilatation in phenylephrine and U 46619 precontracted MRA. 4 The beta-AR fluorescent ligand BODIPY TMR-CGP 12177 behaved as an irreversible beta(1)-AR antagonist in MRA and bound to the surface and inside vascular smooth muscle cells in intact vascular wall. Beta-ARs in smooth muscle cells were observed in a perinuclear location, consistent with the location of Golgi and endoplasmic reticulum. 5 Binding of BODIPY TMR-CGP 12177 was inhibited by BAAM (1 microM) in all three vascular tunics, confirming the presence of beta-ARs in adventitia, media and intima. Binding in adventitia was observed in both neuronal and non-neuronal cell types. Lack of co-localisation with a fluorescent ligand for alpha-ARs confirms the selectivity of BODIPY TMR-CGP 12177 for beta-ARs over alpha-ARs. 6 Our results support the presence of functional vasodilator beta(1)-ARs and show that they are mainly located in smooth muscle cells. Furthermore, we have demonstrated, for the first time, the usefulness of BODIPY TMR-CGP 12177 for identifying beta-AR distribution in the 'living' vascular wall.     

Impairment of the low-affinity state beta1-adrenoceptor-induced relaxation in spontaneously hypertensive rats

1 In hypertension, a decrease of the vascular beta-adrenergic relaxation has been described. However, the specific involvement of each beta-adrenoceptor (beta-AR) subtype, in particular the low-affinity state of beta1-AR, has not yet been evaluated. We investigated whether the low-affinity state of beta1-AR-induced relaxation was impaired in Spontaneously Hypertensive Rats (SHR). 2 The relaxant responses to CGP 12177 and cyanopindolol, low-affinity state beta1-AR agonists (with beta1-/beta2-AR antagonistic and partial beta3-AR agonistic properties) were evaluated on thoracic aortic rings isolated from 12-weeks-old Wistar Kyoto rats (WKY) and SHR. 3 In WKY, CGP 12177 and cyanopindolol produced an endothelium and nitric oxide (NO)-independent relaxation. CGP 12177-induced endothelium-independent relaxation was not modified either by beta1-, beta2-AR (nadolol) or beta3-AR (L-748337 or SR 59230A) antagonists but was significantly reduced by high concentrations of CGP 20712A (P<0.05). This relaxation was also reduced by adenylyl cyclase inhibitors, SQ 22536 or MDL 12330A. 4 In SHR, CGP 12177 produced mainly an endothelium and NO-dependent relaxation. This effect was not modified by nadolol, but was strongly reduced by beta3-AR blockade. Endothelium-independent relaxation to CGP 12177 was not altered by adenylyl cyclase inhibition, but was amplified in preparations from pertussis toxin-pretreated SHR. 5 The immunohistochemical analysis revealed an upregulation of beta3-AR in the endothelial layer of SHR aorta, whereas the beta3-AR-induced relaxation was not modified. 6 In conclusion, we demonstrated an impaired low-affinity state of the beta1-AR-induced relaxation and an upregulation of the beta3-AR in hypertension. Some clinical implications of those findings are discussed.     

Evidence against beta 3-adrenoceptors or low affinity state of beta 1-adrenoceptors mediating relaxation in rat isolated aorta

1 The presence of beta(3)-adrenoceptors and the low affinity state of the beta(1)-adrenoceptor (formerly "putative beta(4)-adrenoceptor") was investigated in ring preparations of rat isolated aorta preconstricted with phenylephrine or prostaglandin F(2alpha) (PGF(2alpha)). Relaxant responses to isoprenaline, selective beta(3)-adrenoceptor agonists (BRL 37344, SR 58611A, CL 316243) and non-conventional partial agonists (CGP 12177A, cyanopindolol, pindolol) were obtained. 2 In phenylephrine-constricted, but not PGF(2alpha)-constricted rings, relaxations to isoprenaline showed a propranolol-resistant component. 3 In phenylephrine-constricted rings, relaxations to BRL 37344 (pEC(50), 4.64) and SR 58611A (pEC(50), 4.94) were not antagonized by the selective beta(3)-adrenoceptor antagonist SR 59230A (< or =1 microM). CL 316243 (< or =100 microM) failed to produce relaxation. In PGF(2alpha)-constricted rings only SR 58611A produced relaxation, which was not affected by SR 59230A (< or =3 microM). 4 Non-conventional partial agonists produced relaxation in phenylephrine-constricted but not PGF(2alpha)-constricted rings. The relaxation to CGP 12177A was unaffected by SR 59230A (< or =1 microM) or by CGP 20712A (10 microM), reported to block the low affinity state of the beta(1)-adrenoceptor. 5 beta-adrenoceptor antagonists also produced relaxation in phenylephrine-constricted rings with an order of potency of (pEC(50) values): bupranolol (5.5) approximately 38;SR 59230A (5.47) approximately 38;cyanopindolol (5.47)>pindolol (5.30)>alprenolol (5.10)>propranolol (4.83)>ICI 118551 (4.60)>CGP 12177A (4.38) approximately 38;CGP 20712A (4.35). Bupranolol (100 microM), alprenolol (30 microM), propranolol (100 microM) and SR 59230A (10 microM) produced no relaxation in PGF(2alpha)-constricted rings. 6 These results provide no evidence for the presence of functional beta(3)-adrenoceptors or the low affinity state of the beta(1)-adrenoceptor in rat aorta.     

Stimulation of beta(3)-adrenoceptors causes phosphorylation of p38 mitogen-activated protein kinase via a stimulatory G protein-dependent pathway in 3T3-L1 adipocytes

1. This study deals with phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) via beta(3)-adrenoceptor (AR) and the signal transduction pathway in 3T3-L1 adipocytes. 2. beta(3)-AR agonist BRL37344A (10 nM) caused phosphorylation and activation of p38 MAPK in 3T3-L1 adipocytes but not in fibroblasts. BRL37344A and also the other beta(3)-AR agonists, CGP12177A and SR58611A, caused p38 MAPK phosphorylation in dose-dependent manners. 3. The p38 MAPK phosphorylations by BRL37344A (10 nM), CGP12177A (100 nM), and SR58611A (10 nM) were not antagonized by beta(1)- and beta(2)-ARs antagonist 1-propranolol (100 nM) but blocked by beta(3)-AR antagonist SR59230A (10 microM), suggesting the phosphorylation was caused via beta(3)-AR. 4. The phosphorylations of p38 MAPK were completely abolished by treatment with cholera toxin (CTX) but not pertussis toxin (100 ng ml(-1), 24 h). Activation of Gs by CTX (100 ng ml(-1)) and adenylyl cyclase by forskolin mimicked p38 MAPK phosphorylation. 5. p38 MAPK phosphorylation by BRL37344A was reduced to almost 50% by cyclic AMP-dependent protein kinase (PKA) inhibitors such as H89 (10 microM) and PKI (10 microM). A src-family tyrosine kinases inhibitor PP2 (1 microM) also halved the p38 MAPK phosphorylation. Combined use of H89 (10 microM) and PP2 (10 microM) did not bring about further inhibition. 6. These results suggest that beta(3)-AR caused phosphorylation of p38 MAPK via Gs protein and partly through a pathway involving PKA and src-family kinase(s), although the contribution of the unidentified pathway remains to be clarified.