Cardiac-specific overexpression of human beta2 adrenoceptors in mice exposes coupling to both Gs and Gi proteins

1. Left atrial strips from transgenic (TG4) mice with cardiac-specific overexpression ( approximately 200-fold) of the beta(2) adrenoceptor (beta(2)AR) were isolated, and their isometric force of contraction (F(c)) in response to electrical stimulation was measured. 2. The betaAR agonist isoprenaline elicited negative inotropic responses in all left atrial strips; in 6/11 preparations, it also had a small positive inotropic effect. This 'up-phase' was observed from 0.1 to 10 nM, with the 'down-phase' occurring at higher concentrations. Both phases were mediated by beta(2)AR, as shown by their sensitivity to the beta(2)AR antagonist ICI-118,551 (100 nM; pA(2) 8.60+/-0.07, 8.45+/-0.19, for 'up-phase' and 'down-phase,' respectively), but not the beta(1)AR antagonist CGP-20712A (100 nM). Conversely, nontransgenic littermate preparations responded to isoprenaline treatment solely by an increase in F(c), which was beta(1)AR-mediated. 3. Pretreatment of left atrial strips with either 10 nM isoprenaline or 1 mM 8-bromo-cAMP significantly attenuated the TG4 'up-phase', while having no effect on either the TG4 'down-phase' or the littermate controls' responses. B. pertussis toxin treatment of the animals prevented isoprenaline's negative inotropic effects in TG4 preparations, but had no effect in littermate controls. 4. The findings imply that the responses of TG4 left atrium to isoprenaline are because of beta(2)AR coupling to G(s) and G(i) proteins, consistent with the model of Daaka et al., in which protein kinase A phosphorylation of the beta(2)AR causes a switch from G(s) to G(i) protein coupling.     

Cardiostimulant and cardiodepressant effects through overexpressed human β<Subscript>2</Subscript>-adrenoceptors in murine heart: regional differences and functional role of β<Subscript>1</Subscript>-adrenoceptors

(-)-Isoprenaline enhances cardiac contractility through beta-adrenoceptors. However, in cardiac tissue from transgenic mice with a 200-400-fold cardiac overexpression of the human beta(2)-adrenoceptor (TG4) we observed a pronounced cardiodepression at high (-)-isoprenaline concentrations. Here, we investigated the functional role of the coexisting beta(1)-, beta(2)-, and beta(3)-adrenoceptor subtypes in several regions of the TG4 heart, and in particular their contribution to the negative inotropic effect. In paced TG4 left atria, (-)-isoprenaline produced bell-shaped concentration-effect curves increasing (-logEC(50)M=9.0) and decreasing (-logIC(50)M=6.4) contractile force. These effects were unaffected by the beta(1)-selective CGP 20712A (300 nM). The beta(2)-selective inverse agonist ICI 118,551 (30-1,000 nM) antagonised in surmountable manner both the positive and negative inotropic effects of (-)-isoprenaline with similar concentration-dependence, consistent with an exclusive mediation through beta(2)-adrenoceptors. The beta(3)-adrenoceptor-selective agonist BRL37344 (1 nM-10 microM) failed to produce significant inotropic effects in TG4 left atria. Subsequently, we measured left atrial action potentials accompanying the inotropic changes induced by (-)-isoprenaline. Action potentials tended to have shorter duration in left atria from TG4 mice than from non-transgenic littermate mice. However, (-)-isoprenaline prolonged the duration of 30% repolarisation in atria from non-transgenic littermate but not from TG4 mice, while 90% repolarisation was abbreviated in both groups of atria. Negative inotropic effects of (-)-isoprenaline were also observed in right ventricular preparations. Pertussis toxin-treatment of the mice abolished the negative inotropic effects in left atria and reduced cardiodepression in right ventricle, indicating an involvement of beta(2)-adrenoceptor coupling to PTX-sensitive G-proteins. In additional experiments, designed to study the native murine beta(1)-adrenoceptor function, we used the physiological beta(1)-adrenoceptor agonist (-)-noradrenaline. In the presence of 600 nM ICI 118,551 we failed to find a functional role of the beta(1)-adrenoceptors in left atria, and detected only a marginal contribution to the positive chronotropic effect in right atria. We also investigated the effects of the non-conventional partial agonist (-)-CGP 12177 (0.2 nM-6 microM), which in wild-type mice causes tachycardia through beta(1)-adrenoceptors. In TG4 right atria, however, (-)-CGP 12177-evoked tachycardia was resistant to blockade by CGP 20712A but antagonised by ICI 118,551, consistent with mediation through human beta(2)-adrenoceptors.The results from TG4 mice suggest that the positive and negative inotropic effects of (-)-isoprenaline are mediated through human overexpressed beta(2)-adrenoceptors coupled to G(s) protein and G(i) protein, respectively. The (-)-isoprenaline-evoked shortening of the atrial action potential combined with reduced responses of L-type Ca(2+) current may contribute to the negative inotropic effects. The function of murine cardiac beta(1)-adrenoceptors is suppressed by overexpressed human beta(2)-adrenoceptors.     

Pertussis toxin suppresses carbachol-evoked cardiodepression but does not modify cardiostimulation mediated through β1- and putative β4-adrenoceptors in mouse left atria: no evidence for β2- and β3-adrenoceptor function

Activation of beta1-, beta2-, beta 3- and putative beta4-adrenoceptors modifies cardiac function. These receptors are usually coupled to Gs protein, but beta2- and beta3-adrenoceptors could also couple to Gi/o proteins. The mouse heart is used increasingly for studies of genetically disrupted or overexpressed proteins, including beta-adrenoceptor subtypes. We therefore investigated in contracting mouse left atria (2 Hz, 37 degrees C) if inactivation of Gi/o proteins with pertussis toxin modifies or uncovers effects mediated through beta-adrenoceptor subtypes. The negative inotropic effects of carbachol in atria exposed to catecholamine or high calcium (6.8 mmol/l) were assumed to be mediated through activation of muscarinic receptors coupled to Gi/o. We report conditions under which incubation of left atria with 200 ng/ml pertussis toxin for 24 h nearly abolished the carbachol responses. Although it has been reported that muscarinic receptor-mediated cardiodepression has an obligatory contribution of nitric oxide, the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (0.1-1 mmol/l) did not modify the negative inotropic effects of carbachol, inconsistent with an involvement of nitric oxide. The positive inotropic effects of (-)-noradrenaline and (-)-adrenaline, mediated through beta1-adrenoceptors, were not affected by pertussis toxin. (-)-Adrenaline did not cause positive inotropic effects attributable to beta2-adrenoceptor-mediation, in the presence of CGP 20712A (300 nmol/l) to block beta1-adrenoceptors, in control atria or atria pretreated with pertussis toxin. The positive inotropic effects of (-)-CGP 12177 (1 micromol/l), a compound with agonist activity at the putative beta4-adrenoceptor, were unaffected by pertussis toxin. The beta3-adrenoceptor-selective agonist BRL 37344 (1 micromol/l), in the presence of (-)-propranolol (200 nmol/l), did not cause positive or negative inotropic effects in control and pertussis toxin-treated atria. In left atria obtained from mice injected with 150 microg/kg i.p. pertussis toxin which abolished carbachol-evoked cardiode-pression, the positive inotropic effects of (-)-adrenaline were antagonised by CGP 20712A. The beta2-adrenoceptor-selective antagonist ICI 118551 (50 nmol/l) did not cause additional blockade of the effects of high (-)-adrenaline concentrations in the presence of CGP 20712A, ruling out the involvement of beta2-adrenoceptors. The results with intraparenteral PTX validate our in vitro PTX method. We conclude that inhibition of murine Gi/o proteins does not alter atrial positive inotropic effects mediated through beta1- and putative beta4-adrenoceptors and does not reveal functional beta2- and beta3-adrenoceptors.     

β-ADRENOCEPTORS ON ENDOTHELIAL CELLS DO NOT INFLUENCE RELEASE OF RELAXING FACTOR IN DOG CORONARY ARTERIES

1. The aim of this study was to determine if stimulation of a beta-adrenoceptor on endothelial cells could release an endothelium-derived relaxing factor (EDRF) similar to that released by acetylcholine. 2. In dog coronary rings preconstricted with PGF2 alpha or serotonin, removal of the endothelium did not alter the relaxant responses to isoprenaline. However, in rings preconstricted with the thromboxane-mimetic U46619, removal of the endothelium enhanced the response to isoprenaline. 3. The vasorelaxant responses to isoprenaline in endothelium-denuded rings were inhibited in a concentration-dependent manner by the specific beta 1-adrenoceptor antagonist CGP-20712A (3-100 nmol/l), but were not altered by the beta 2-adrenoceptor antagonist ICI-118551 (30 nmol/l). 4. The vasorelaxant responses to isoprenaline in the presence of CGP-20712A (30 nmol/l) or ICI-118551 (30 nmol/l) were not impaired by removal of the endothelium. 5. Endothelium-dependent vasorelaxant responses to acetylcholine and bradykinin were not altered by isoprenaline (0.1 mumol/l) in the presence of CGP-20712A (30 nmol/l). 6. Therefore, the beta-adrenoceptors on dog coronary artery smooth muscle which produce relaxation are predominantly of the beta 1-subtype. Stimulation of any beta 2-adrenoceptors on the endothelium in dog coronary artery does not release EDRF, and does not modulate endothelium-dependent vasodilatation induced by other agents.     

Physiological antagonism between ventricular beta 1-adrenoceptors and alpha 1-adrenoceptors but no evidence for beta 2- and beta 3-adrenoceptor function in murine heart

1. Murine left atrium lacks inotropic beta(2)-adrenoceptor function. We investigated whether beta(2)-adrenoceptors are involved in the cardiostimulant effects of (-)-adrenaline on spontaneously beating right atria and paced right ventricular myocardium of C57BL6 mice. We also studied a negative inotropic effect of (-)-adrenaline. 2. Sinoatrial tachycardia, evoked by (-)-adrenaline was resistant to blockade by beta(2)-selective ICI 118,551 (50 nM) but antagonized by beta(1)-selective CGP 20712A (300 nM). This pattern was unaffected by pretreatment with pertussis toxin (PTX, 600 microg kg(-1) i.p. 24 h) which reversed carbachol-evoked bradycardia to tachycardia. 3. Increases of ventricular force by (-)-adrenaline and (-)-noradrenaline were not blocked by ICI 118,551 but antagonized by CGP 20712A. 4. Under blockade of beta-adrenoceptors, (-)-adrenaline and (-)-noradrenaline depressed ventricular force (-logIC(50)M=7.7 and 6.9). The cardiodepressant effects of (-)-adrenaline were antagonized by phentolamine (1 microM) and prazosin (1 microM) but not by (-)-bupranolol (1 microM). Prazosin potentiated the positive inotropic effects of (-)-adrenaline (in the absence of beta-blockers) from -logEC(50)M=6.2 - 6.8. 5. PTX-treatment reduced carbachol-evoked depression of ventricular force in the presence of high catecholamine concentrations. Inhibition of ventricular function of G(i) protein was verified by 82% reduction of in vitro ADP-ribosylation. PTX-treatment tended to increase the positive inotropic potency of (-)-adrenaline under all conditions investigated, including the presence of ICI 118,551. 6. (-)-Adrenaline causes murine cardiostimulation through beta(1)-adrenoceptors but not through beta(2)-adrenoceptors. The negative inotropic effects of (-)-adrenaline are mediated through ventricular alpha(1)-adrenoceptors but not through beta(3)-adrenoceptors. Both G(i) protein and alpha(1)-adrenoceptors restrain (-)-adrenaline-evoked increases in right ventricular force mediated through beta(1)-adrenoceptors.     

The role of beta(3)-adrenoceptors in mediating relaxation of porcine detrusor muscle.

1. beta-adrenoceptors mediate relaxation of bladder detrusor smooth muscle. This study investigates the contribution of beta(3)-adrenoceptors to relaxation of the pig urinary bladder. 2. Cell membranes were prepared from detrusor muscle of the pig bladder dome and competition experiments with [(3)H]-dihydroalprenolol (DHA), a non-selective beta-adrenoceptor antagonist was used as a specific radioligand to determine the presence of beta-adrenoceptor subtypes. In functional experiments, isolated detrusor muscle strips were used to determine the potency of agonists and the affinity of antagonists. 3. In competition binding experiments, CGP20712A (beta(1)-adrenoceptor selective) displaced [(3)H]-DHA from a single binding site with a low affinity. In contrast, displacement data for ICI 118551 (beta(2)-adrenoceptor antagonist) and SR59230A (beta(3)-adrenoceptor antagonist) best fitted a two-site model suggesting a predominant (70%) population of beta(3)-adrenoceptors. 4. In functional studies, isoprenaline and salbutamol (beta(2)-adrenoceptor agonist) relaxed KCl precontracted muscle strips with high potency (pEC(50) 7.7 and 7.2, respectively), whilst CGP12177 and BRL37344 (beta(3)-adrenoceptor agonists) had low potency and were partial agonists. CGP20712A and atenolol (beta(1)-adrenoceptor antagonists) antagonised responses with a low affinity. ICI118551 antagonized responses to isoprenaline and salbutamol with a high affinity (pK(B)=7.8 and 8.7, respectively), but the Schild slopes were low suggesting that responses were mediated by more than one beta-adrenoceptor. The Schild plot for SR59230A was biphasic, apparent pK(B) values for 3 - 10 nM SR59230A being 8.6 and those for 30 nM - 1 microM being 7.7. 5. These data suggest that beta(3)-adrenoceptors are the predominant beta-adrenoceptor subtype present in the pig bladder and that beta-adrenoceptor mediated responses of this tissue are mediated via both the beta(2)- and beta(3)-adrenoceptor subtypes.     

Phosphodiesterase-4 blunts inotropism and arrhythmias but not sinoatrial tachycardia of (-)-adrenaline mediated through mouse cardiac beta(1)-adrenoceptors

BACKGROUND AND PURPOSE: beta(1) and beta(2)-adrenoceptors coexist in murine heart but beta(2)-adrenoceptor-mediated effects have not been detected in atrial and ventricular tissues, possibly due to marked phosphodiesterase (PDE) activity. We investigated the influence of the PDE3 inhibitor cilostamide and PDE4 inhibitor rolipram on the effects of (-)-adrenaline in three regions of murine heart. EXPERIMENTAL APPROACH:(-)-Adrenaline-evoked cardiostimulation was compared on sinoatrial beating rate, left atrial and right ventricular contractile force in isolated tissues from 129SvxC57B1/6 cross mice. Ventricular arrhythmic contractions were also assessed. KEY RESULTS: Both rolipram (1 microM) and cilostamide (300 nM) caused transient sinoatrial tachycardia but neither enhanced the chronotropic potency of (-)-adrenaline. Rolipram potentiated 19-fold (left atrium) and 7-fold (right ventricle) the inotropic effects of (-)-adrenaline. (-)-Adrenaline elicited concentration-dependent ventricular arrhythmias that were potentiated by rolipram. All effects of (-)-adrenaline were antagonized by the beta(1)-adrenoceptor-selective antagonist CGP20712A (300 nM). Cilostamide (300 nM) did not increase the chronotropic and inotropic potencies of (-)-adrenaline, but administered jointly with rolipram in the presence of CGP20712A, uncovered left atrial inotropic effects of (-)-adrenaline that were prevented by the beta(2)-adrenoceptor-selective antagonist ICI118551.CONCLUSIONS AND IMPLICATIONS: PDE4 blunts the beta(1)-adrenoceptor-mediated effects of (-)-adrenaline in left atrium and right ventricle but not in sinoatrial node. Both PDE3 and PDE4 reduce basal sinoatrial rate in a compartment distinct from the beta(1)-adrenoceptor compartment. PDE3 and PDE4, acting in concert, prevent left atrial beta(2)-adrenoceptor-mediated inotropy. PDE4 partially protects the right ventricle against (-)-adrenaline-evoked arrhythmias.     

Mediation of the positive chronotropic effect of CGP 12177 and cyanopindolol in the pithed rat by atypical beta-adrenoceptors, different from beta 3-adrenoceptors.

1. The influence of beta 1-, beta 2-, and beta 3-adrenoceptor agonists and of CGP 12177 and cyanopindolol on heart rate and diastolic blood pressure was studied in the pithed rat. 2. The beta 1-adrenoceptor agonist, prenalterol, increased heart rate and the beta 2-adrenoceptor agonist, fenoterol, caused a fall in blood pressure. The effect of prenalterol was antagonized by the beta 1-adrenoceptor antagonist, CGP 20712 0.1 mumol kg-1 and the action of fenoterol was attenuated by the beta 2-adrenoceptor antagonist, ICI 118551 0.1 mumol kg-1. Both effects were markedly diminished by the non-selective beta-adrenoceptor antagonist, bupranolol 0.1 mumol kg-1. 3. The non-selective beta-adrenoceptor agonist, isoprenaline, three beta 3-agonists as well as CGP 12177 and cyanopindolol elicited a positive chronotropic effect, exhibiting the following pED delta 60 values (negative log values of the doses increasing heart rate by 60 beats min-1): isoprenaline 10.4, CGP 12177 8.3, cyanopindolol 7.2, BRL 37344 6.9, ZD 2079 5.2 and CL 316243 < 5. 4. CGP 20712 0.1 mumol kg-1, given together with ICI 118551 0.1 mumol kg-1, markedly attenuated the positive chronotropic effect of isoprenaline, BRL 37344, ZD 2079 and CL 316243 without affecting the increase in heart rate produced by CGP 12177 and cyanopindolol. 5. The positive chronotropic effect of CGP 12177 and cyanopindolol was attenuated by CGP 20712, 1 and 10 mumol kg-1 and bupranolol, 10 mumol kg-1 but was not affected by ICI 118551, 10 mumol kg-1. The effect of CGP 12177 was also not changed by BRL 37344 1 mumol kg-1, ZD 2079 10 mumol kg-1, CL 316243 10 mumol kg-1, the alpha 1-adrenoceptor antagonist, prazosin 1 mumol kg-1 and the 5-hydroxytryptamine 5-HT2A receptor antagonist, ketanserin 3 mumol kg-1. 6. CGP 12177 0.002 mumol kg-1 and cyanopindolol 0.003 mumol kg-1 shifted to the right the dose-response curve of prenalterol for its positive chronotropic effect. The -log values of the doses causing a twofold shift to the right were 9.6 and 9.5, respectively. 7. Isoprenaline 0.00001-0.001 mumol kg-1, BRL 37344 0.01-1 mumol kg-1 and CGP 12177 0.1 mumol kg-1 caused a fall in diastolic blood pressure which was markedly attenuated by combined administration of CGP 20712 and ICI 118551, 0.1 mumol kg-1 each. 8. CGP 12177 0.01 and 0.1 mumol kg-1 and cyanopindolol 1 mumol kg-1 elicited an increase in diastolic blood pressure. CGP 20712, ICI 118551, bupranolol and, in the case of CGP 12177, also BRL 37344, ZD 2079, CL 316243, prazosin and ketanserin did not influence this effect. 9. In conclusion, the positive chronotropic effect of CGP 12177 and cyanopindolol is not mediated via beta 1-, beta 2-, beta 3-, alpha 1-adrenoceptors or 5-HT2A receptors. This effect may involve atypical beta-adrenoceptors, similar or identical to those described by Kaumann (1989) in isolated heart preparations.     

Determination of beta-adrenoceptor subtype on rat isolated ventricular myocytes by use of highly selective beta-antagonists.

1. The relative proportions of beta 1- and beta 2-adrenoceptors were determined by radioligand binding studies in three different rat myocardial preparations: membranes prepared from rat ventricle (ventricular membranes), membranes prepared from rat isolated ventricular myocytes (myocyte membranes), and myocytes isolated from rat ventricle (myocytes). 2. Competition experiments using CGP 20712A or ICI 118,551 with [125I]-iodocyanopindolol ([125I]-ICYP) revealed high- and low-affinity binding sites in ventricular membranes. The concentration at which each beta-antagonist occupied 100% of its high-affinity binding sites was 300 nM for CGP 20712A (beta 1-adrenoceptor) and 50 nM for ICI 118,551 (beta 2-adrenoceptor). 3. The density of high-affinity (beta 1-adrenoceptor) and low-affinity (beta 2-adrenoceptor) binding sites for CGP 20712A was measured by a saturation experiment using [125I]-ICYP in the presence and absence of 300 nM CGP 20712A. In ventricular membranes, the proportions of high-affinity and low-affinity binding sites for CGP 20712A were 73% and 27%, respectively, whereas in myocyte membranes, the corresponding figures were 90% and 10%, respectively. The density of low-affinity binding sites for CGP 20712A in ventricular membranes, defined as [125I]-ICYP-specific binding in the presence of 300 nM CGP 20712A, was decreased by addition of 50 nM ICI 118,551, whereas that in myocyte membranes was not affected. 4. In myocytes, specific binding of [125I]-ICYP and [3H]-CGP 12177 was not detected by saturation experiments performed in the presence of 300 nM CGP 20712A.(ABSTRACT TRUNCATED AT 250 WORDS)     

CGP 20712 A: a useful tool for quantitating beta 1- and beta 2-adrenoceptors

CGP 20712 A (1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3- [4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-2-propanol methanesulfonate), a specific beta 1-adrenoceptor antagonist, was tested for resolution of beta 1- and beta 2-adrenoceptors in an in vitro [3H]dihydroalprenolol ([3H]DHA) binding assay. Competition experiments, using rat neocortical and cerebellar membranes, yielded two dissimilar concentration-effect curves. A distinct biphasic curve was evident for neocortex, with a plateau at 100 nM CGP 20712 A (60% [3H]DHA displacement). This plateau indicated a differentiation between beta 1- and beta 2-adrenoceptors; the ratio of IC50-beta 2 to IC50-beta 1 was approximately 10,000. In contrast, only a monophasic curve was obtained for cerebellum. CGP 20712 A is a useful tool for estimating percentages of beta 1- and beta 2-adrenoceptors in a given tissue.     

Overexpression of beta 1-adrenoceptors in adult rat ventricular myocytes enhances CGP 12177A cardiostimulation: implications for 'putative' beta 4-adrenoceptor pharmacology

1. CGP 12177A mediates cardiostimulation by activation of the 'putative' beta(4)-adrenoceptor; however, it has recently been reported that disruption of the beta(1)-adrenoceptor gene abolishes this effect. We have adenovirally overexpressed beta(1)-adrenoceptors in isolated, cultured adult rat ventricular cardiomyocytes and observed the inotropic potency of isoprenaline and CGP 12177A (in the presence of 1 microm propranolol). 2. Isoprenaline was a full inotropic agonist at rat ventricular myocytes (pD(2) 7.69+/-0.12). CGP 12177A was a nonconventional partial agonist (pD(2) 6.34+/-0.09), increasing inotropy and lusitropy, with an intrinsic activity of 0.34 and antagonised by bupranolol. 3. beta(1)-adrenoceptor overexpression enhanced the inotropic potency of isoprenaline by 11.7-fold (pD(2) 8.76+/-0.14) and CGP 12177A by 5.9-fold (7.11+/-0.10), respectively. Green fluorescent protein (GFP) overexpression did not alter the potency of isoprenaline or CGP 12177A (pD(2) 7.41+/-0.24 and pD(2) 6.60+/-0.50, respectively). 4. The cardiostimulant effects of CGP 12177A were enhanced by IBMX (phosphodiesterase inhibitor) and decreased by Rp-cAMPS (cAMP antagonist). CGP 12177A also increased cAMP levels. CGP 12177A but not isoprenaline initiated arrhythmias at lower concentrations following beta(1)-adrenoceptor overexpression. 5. (125)I-Cyanopindolol saturation binding in Adv.beta(1) myocytes demonstrated approximately 18-fold increase in beta(1)-adrenoceptors. (3)H-CGP 12177A saturation binding, in the presence of propranolol, increased approximately 5-fold following overexpression of beta(1)-adrenoceptors. 6. This study demonstrates enhanced cardiostimulation by CGP 12177A (in the presence of propranolol) in rat ventricular myocytes overexpressing beta(1)-adrenoceptors, mediated by a Gs/cAMP signalling pathway. 'Putative' beta(4)-adrenoceptor pharmacology appears to be mediated by activation of a novel affinity state of the beta(1)-adrenoceptor.     

Inhibitory effects of SR 58611A on canine colonic motility: evidence for a role of beta 3-adrenoceptors.

1. In order to clarify whether atypical or beta 3-adrenoceptors can modulate canine colonic motility in vivo, we studied the effects of SR 58611A (a selective agonist for atypical beta-adrenoceptors) alone and after pretreatment with beta-adrenoceptor antagonists on colonic motility in the conscious dog. The gastrocolonic response (postprandial increase in motility) was monitored by means of electrodes and strain-gauge force transducers chronically implanted along the distal colon. In some experiments, heart rate was also measured. The possible role of beta 3-adrenoceptors in mediating the effects of SR 58611A was also tested in vitro in circular muscle strips taken from the canine distal colon. 2. Intravenous infusion of SR 58611A, ritodrine or isoprenaline at doses inducing the same degree of tachycardia inhibited the gastrocolonic response to a different extent, with SR 58611A and ritodrine being more effective than isoprenaline. 3. In a dose-response study, SR 58611A was more potent in inhibiting colonic motility than in inducing tachycardia: the ED35 values for inhibition of colonic motility and induction of tachycardia were 23 and 156 micrograms kg-1, i.v., respectively. 4. The inhibitory effect of SR 58611A 100 micrograms kg-1, i.v., on the gastrocolonic response was reversed by alprenolol (non-selective beta-adrenoceptor antagonist), but resistant to CGP 20712A (beta 1-adrenoceptor antagonist) or ICI 118551 (beta 2-adrenoceptor antagonist). 5. In vitro, SR 58611A concentration-dependently relaxed circular muscle strips, an effect that was competitively antagonized by alprenolol with a pA2 value of 7.1, but resistant to CGP 20712A (100 nM), ICI 118551 (100 nM) or tetrodotoxin (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of beta 1- and beta 2-adrenoceptors in rat skeletal muscles.

Binding studies with (-)-[125I]cyanopindolol (ICYP) were conducted to characterize beta-adrenoceptors in plantaris and soleus muscles of rats (male, 250-300 g). The distribution of beta 1- and beta 2-adrenoceptors in different muscle fiber types, identified in serial sections by succinic dehydrogenase (SDH) staining, was studied by autoradiography. The densities of binding sites (Bmax, fmol/mg protein) were 5.4 +/- 0.9 (mean +/- SEM) in plantaris and 11.5 +/- 2.0 in soleus muscle. In plantaris muscle, monophasic competition curves were observed when binding experiments were performed using CGP20712A (50 pM to 0.5 mM), a beta 1-adrenoceptor selective antagonist, or ICI 118,551 (50 pM to 20 microM), a beta 2-adrenoceptor selective antagonist, to compete for ICYP binding. Analysis with LIGAND revealed a single binding site with a KD value of 2.41 +/- 0.56 nM (mean +/- SEM) for ICI 118,551 and 8.93 +/- 3.00 microM for CGP 20712A, indicating the presence of a homogeneous population of beta 2-adrenoceptors. In soleus muscle, competition curves were biphasic with 16-21% beta 1-adrenoceptors. Autoradiographic studies supported the findings from binding studies with membrane homogenates. The ICYP binding pattern was associated closely with the muscle fiber types identified by SDH staining. Propranolol-resistant binding sites were observed, and these sites were associated with muscle fibers positive to SDH staining.     

An inhibitory effect of isoprenaline on stimulation-induced noradrenaline release from rat atria.

Isoprenaline (0.1 microM), in the presence of phentolamine (1 microM) to block autoinhibitory alpha-adrenoceptors, significantly increased the efflux of radioactivity produced by field stimulation (2 Hz for 60 s) from rat isolated atria in which the noradrenergic transmitter stores had been labelled with [3H]noradrenaline. This facilitatory effect of isoprenaline on noradrenergic transmission was not affected by the selective beta 1-adrenoceptor antagonist CGP 20712A (0.3 microM). However, the selective beta 2-adrenoceptor antagonist ICI 118551 (0.1 microM) not only abolished the facilitatory effect of isoprenaline but reversed it to an inhibitory effect, indicating that the prejunctional beta-adrenoceptors subserving facilitation of noradrenergic transmission in rat atria are of the beta 2-subtype. The inhibitory effect of isoprenaline that was revealed by blockade of beta 2-adrenoceptors was abolished by atenolol (3 microM) in a concentration which markedly reduced the effect of isoprenaline on the rate of atrial beating. This finding suggests that activation of beta 1-adrenoceptors on atrial myocytes by isoprenaline may have resulted in release of one or more substance(s) which inhibited stimulation-induced release of noradrenaline, presumably by activating prejunctional receptors. The inhibitory effect of isoprenaline on noradrenergic transmission was not affected by the prostaglandin synthesis inhibitor indomethacin (10 microM) suggesting that prostaglandins were not involved.     

Beta-adrenoceptor blocking effects of a selective beta 2-agonist, mabuterol, on the isolated, blood-perfused right atrium of the dog.

1. Effects of (+/-)-1-(4-amino-3-chloro-5-trifluoromethyl-phenyl)-2-tert.- butylamino-ethanol hydrochloride (mabuterol) on pacemaker activity and atrial contractility were investigated in the isolated and blood-perfused right atrium of the dog. 2. Mabuterol, injected into the sinus node artery of the isolated atrium, dose-dependently increased atrial rate and contractile force at doses of 0.01-10 nmol but the responses to over 10 nmol of mabuterol gradually decreased and mabuterol at higher doses induced biphasic cardiac responses, i.e., negative followed by positive cardiac responses. 3. The maximal increases in atrial rate and contractile force induced by mabuterol were 41.4% and 12.9%, respectively, of the maximal chronotropic and inotropic effects of isoprenaline. 4. Positive chronotropic and inotropic responses to mabuterol were dose-dependently inhibited by a selective beta 2-adrenoceptor antagonist, ICI 118,551. These responses were only slightly attenuated by atenolol. 5. Mabuterol (1-300 nmol) dose-dependently inhibited both dobutamine- and procaterol-induced positive chronotropic and inotropic responses. 6. These results indicate that mabuterol causes weak positive chronotropic and inotropic effects on the perfused canine right atrium by activating beta 2-adrenoceptors, and that higher concentrations non-selectively block both beta 1-and beta 2-adrenoceptors.     

Functional beta1- and beta2-adrenoceptors in the left and right atrium of pre-hypertensive rats

There is a small increase in the functional beta2-adrenoceptor response on the spontaneously hypertensive rat (SHR) left atrium in the early stages of hypertension. In the present study, the functional beta1- and beta2-adrenoceptors of the left and right atrium in SHR pre-hypertension and age-matched (5-week-old) Wistar Kyoto (WKY) rats were characterized. Contractility methods with isoprenaline, T-0509 (a selective beta1-adrenoceptor agonist) and procaterol (a selective beta2-adrenoceptor agonist) were used. At 5 weeks, the SHRs were pre-hypertensive. Isoprenaline was more potent on the left atrium of 5-week-old SHRs than WKY rats. Bisoprolol, a selective beta1-adrenoceptor antagonist, was more potent against isoprenaline and T-0509 on the SHR than WKY rat left atrium. ICI 118,551, a selective beta(2)-adrenoceptor antagonist, was more potent against procaterol and T-0509 on the SHR than WKY rat left atrium. The results with bisoprolol and ICI 118,551 suggest that there are more functional beta(1)- and beta(2)-adrenoceptors on the left atrium of 5-week-old SHRs than WKY rats. Isoprenaline, T-0509 and procaterol were equipotent on the right atrium of 5-week-old WKY rats and SHRs. Bisoprolol was more potent against isoprenaline, T-0509 and procaterol on the SHR than WKY rat right atrium. ICI 118,551 was more potent against T-0509, but not isoprenaline and procaterol, on the SHR than WKY rat left atrium. This suggests there are more functional beta1-adrenoceptors, and probably more functional beta2-adrenoceptors, on the right atrium of 5-week-old SHRs than WKY rats. These functional differences in beta1- and beta2-adrenoceptor-mediated responses of the left and right atria of pre-hypertensive SHRs cannot be caused by hypertension, and may be associated with the onset of hypertension.     

Greater inotropic and cyclic AMP responses evoked by noradrenaline through Arg389 beta 1-adrenoceptors versus Gly389 beta 1-adrenoceptors in isolated human atrial myocardium

1. We studied the biochemical and contractile responses of isolated human myocardial tissue expressing native receptor variants of the 389G>R beta(1)-adrenoceptor polymorphism. 2. Right atrial appendage was obtained from homozygous RR patients (n=37) and homozygous GG patients (n=17) undergoing elective cardiac surgery. The positive inotropic effect of noradrenaline in these tissues, mediated through beta(1)-adrenoceptors, was studied using electrically stimulated (1 Hz) atrial strips, as well as the effects of noradrenaline on cyclic AMP levels and cyclic AMP-dependent protein kinase. 3. Tissue from RR homozygotes (n=14) showed significantly increased inotropic potency to noradrenaline (-log EC(50), M=6.92+/-0.12) compared to GG homozygotes (n=8, -log EC(50), M=6.36+/-0.11, P<0.005). This difference was not dependent on tissue basal force. 4. Tissue cyclic AMP levels (pmol mg(-1)) were also greater in RR homozygotes (basal 34.8+/-3.7 n=12, 300 nM noradrenaline 41.4+/-7.6 n=9, 30 micro M noradrenaline 45.2+/-3.2 n=22, 0.2 mM isoprenaline 48.3+/-4.2 n=16) compared to GG homozygotes (basal 30.7+/-4.4 n=5, 300 nM noradrenaline 32.6+/-6.92 n=5, 30 micro M noradrenaline 38.1+/-3.1 n=8, 0.2 mM isoprenaline 42.6+/-5.2 n=6, P=0.007). There were no differences between the variants in terms of cyclic AMP-dependent protein kinase activity. 5. These data provide the first evidence that enhanced G-protein coupling of the R389 beta(1)-adrenoceptor variant reported in rodent fibroblast expression systems is also present in native human receptors. The functional consequence of this is to significantly alter the inotropic potency of beta(1)-adrenoceptor activation depending on its genotype at the 389 position.     

Comparison between CL-316243- and CGP-12177A-induced relaxations in isolated canine ureter

We compared the effects of CL-316243, a selective beta(3)-adrenoceptor agonist, and CGP-12177A, a nonconventional partial beta-adrenoceptor agonist, on the KCl-induced contraction in the isolated canine ureter. CL-316243 concentration dependently relaxed the ureteral contraction, the pD(2) value being 7.75 +/- 0.11. This relaxation was competitively antagonized by the selective beta(3)-adrenoceptor antagonist SR58894A and by the nonselective beta-adrenoceptor antagonist bupranolol, their pA(2) values being 7.08 +/- 0.08 and 6.43 +/- 0.09, respectively. CGP-12177A concentration dependently reduced the KCl-induced contraction, the pD(2) value being 6.30 +/- 0.25. Even at 1 x 10(-5) mol/l, CGP-20712A (a selective beta(1)- adrenoceptor antagonist) did not shift the concentration-response curves for CL-316243 or CGP-12177A. SR58894A did not induce a parallel rightward shift in the concentration-response curve for CGP-12177A, but bupranolol did produce such a shift, pA(2) and slope values in the Schild plot being 7.15 +/- 0.77 and 0.60 +/- 0.15, respectively. Hence, the competition characteristics for SR58894A and bupranolol differed between the CL- 316243-induced and CGP-12177A-induced relaxations. Our results suggest that CGP-12177A produces ureteral relaxation in the dog via an atypical beta-adrenoceptor (possibly, an atypical site/state of the beta(3)-adrenoceptor) as well as via the typical beta(3)-adrenoceptor.     

Negative inotropic responses of the isolated heart of the rat to isoprenaline

The isolated heart of the rat perfused at 38 degrees C responded to isoprenaline with an initial positive inotropism followed by a bi-phasic negative inotropic effect. The latter was associated with the simultaneous biphasic rate increase. On reduction of the perfusion temperature to 20 degrees C, isoprenaline no longer increased the rate but a negative inotropic response remained. Blockade of this by practolol suggested that it was mediated via beta(1)-adrenoceptors.     

Beta 2-adrenoceptors in guinea-pig atria

The occurrence of beta 2-adrenoceptors in the isolated, spontaneously beating right atrium and the electrically driven left atrium of the guinea-pig was studied. Isoprenaline was used as reference compound and procaterol as selective beta 2-agonist. Cumulative concentration response (C/R) curves were obtained with the agonists. The C/R curve of procaterol was biphasic in both preparations. Compared with isoprenaline, procaterol was a partial agonist, with a mean maximum response of 0.78 +/- 0.04 in the right atrium and 0.29 +/- 0.05 in the left atrium. The beta 2-selective antagonist ICI 118,551, 10(-7) mol litre-1, caused a small but significant shift of the C/R curve of isoprenaline to a higher concentration range in both preparations. The same concentration of the beta 2-blocker changed the shape of the C/R curve of procaterol from biphasic to monophasic by blocking the responses to low concentrations of procaterol. Practolol, a beta 1-selective antagonist, 10(-6) mol litre-1, gave a highly significant shift of the C/R curve of isoprenaline to a higher concentration range in both preparations but had no effect on the responses to low concentrations of procaterol. The effect of practolol on the responses to high concentrations of procaterol is discussed. We conclude that the guinea-pig atria may contain beta 2-adrenoceptors mediating positive chronotropic and inotropic effects.