Role of alpha-adrenergic receptors in the effect of the beta-adrenergic receptor ligands, CGP 12177, bupranolol, and SR 59230A, on the contraction of rat intrapulmonary artery

This study investigates the effect of the aryloxypropanolamines 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one (CGP 12177), bupranolol, and 3-(2-ethylphenoxy)-1[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate (SR 59230A) [commonly used as beta(3)- and/or atypical beta-adrenergic receptors (beta-AR) ligands] on the contractile function of rat intralobar pulmonary artery. Affinities of beta-AR ligands for alpha(1)-adrenergic receptors (alpha(1)-AR) were also evaluated using [(3)H]prazosin binding competition experiments performed in rat cortical membranes. In intralobar pulmonary artery, CGP 12177 did not modify the basal tone, but antagonized the contraction induced by the alpha(1)-AR agonist phenylephrine (PHE). In arteries precontracted with PHE, CGP 12177 elicited relaxation, whereas in those precontracted with prostaglandin F(2alpha) (PGF(2alpha)), it further enhanced contraction. CGP 12177 induced an increase in intracellular calcium concentration in pressurized arteries loaded with Fura PE-3 and precontracted with PGF(2alpha). In PGF(2alpha) precontracted arteries, phentolamine (an alpha-AR antagonist) and phenoxybenzamine (an irreversible alpha-AR antagonist) antagonized the contractile responses to PHE and CGP 12177. Both responses were also decreased by bupranolol and SR 59230A. Specific [(3)H]prazosin binding was displaced by CGP 12177, bupranolol, and SR 59230A with pK(i) values of 5.2, 5.7, and 6.6, respectively. In contrast, (+/-)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium (BRL 37344) and disodium 5-[(2R)-2-([(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243) (nonaryloxypropanolamines beta(3)-AR agonists) displayed very low affinity for [(3)H]prazosin binding sites (pK(i) values below 4). These data suggest that CGP 12177 exhibits partial agonist properties for alpha(1)-AR in rat pulmonary artery. They also show that bupranolol and SR 59230A exert an alpha(1)-AR antagonist effect. As a consequence, these aryloxypropanolamine compounds should be used with caution when investigating the role of beta(3)- and atypical beta-AR in the regulation of vascular tone.     

Autoradiographic localization and densitometric analysis of beta-1 and beta-2 adrenoceptors in the canine left anterior descending coronary artery

The location and proportions of beta-1 and beta-2 adrenoceptors in canine coronary arteries (0.5-2 mm) has been examined by autoradiography. X-ray film and nuclear emulsion-coated coverslips were exposed to sections of coronary artery previously incubated with [125I]iodocyanopindolol (50 pM) in the absence and presence of ICI 118,551 (70 nM) to block beta-2 adrenoceptors, CGP 20712A (100 nM) to block beta-1 adrenoceptors or (-)-propranolol (1 microM) to define nonspecific binding. The medial smooth muscle of the coronary artery had an even distribution of beta-1 adrenoceptors and two populations of beta-2 adrenoceptors, one evenly distributed and the other highly localized. Beta-2 adrenoceptors were also located on nerve tissue and in the adventitia. There was no evidence for localization of beta adrenoceptors on endothelial cells. Quantitative autoradiography was performed using computer-assisted image processing and the program AVID. The binding of [125I]cyanopindolol was saturable (KD = 50 pM) and competition binding curves with the beta-1 selective antagonist CGP 20712A and beta-2 selective antagonist ICI 118,551 showed beta-1 and beta-2 adrenoceptors in the proportions of 85:15% in both 0.5- and 2-mm arteries.     

Differential regulation of beta-1 and beta-2 adrenoceptors in guinea pig atrioventricular conducting system after chronic (-)-isoproterenol infusion

Quantitative autoradiography was used to determine the effects of chronic administration of (-)-isoproterenol (400 micrograms/kg/hr, 7 days) on the distribution and density of beta-1 and beta-2 adrenoceptors in guinea pig atrioventricular conducting system and surrounding regions. X-ray film was exposed to sections of heart labeled with (-)-[125I]-cyanopindolol in the absence or presence of ICI 118,551 (70 nM) to block beta-2 adrenoceptors, CGP 20712A (100 nM) to block beta-1 adrenoceptors or (-)-propranolol (1 microM) to define nonspecific binding. Quantitative autoradiography was performed using computer-assisted image processing and the AVID system. The proportions of beta-1 and beta-2 adrenoceptors were determined by the extent of inhibition of (-)-[125I]cyanopindolol binding by CGP 20712A (100 nM) together with equations which take into account the differing affinities of ligands for beta-1 and beta-2 adrenoceptors. In vehicle (1 mM HCl)-treated animals there was a higher density and proportion of beta-2 adrenoceptors in the atrioventricular node, bundle of His and right and left bundle branches than in myocardium. Chronic administration of (-)-isoproterenol produced marked loss of beta-2 adrenoceptor binding in all regions of the atrioventricular conducting system, surrounding myocardium, cardiac valves and the smooth muscle of the aorta accompanied by less pronounced effects on beta-1 adrenoceptors. Beta-1 adrenoceptors were significantly reduced only in the left bundle branch whereas there were trends toward a reduction in the right bundle branch, bundle of His and an increase in the atrioventricular node. There was no alteration in beta-1 adrenoceptor density in myocardium. Loss of beta adrenoceptor binding produced by (-)-isoproterenol was mainly confined to beta-2 adrenoceptors.     

Aryloxypropanolamine and catecholamine ligand interactions with the beta(1)-adrenergic receptor: evidence for interaction with distinct conformations of beta(1)-adrenergic receptors

Pharmacological responses to aryloxypropanolamines were examined in cells expressing rat or human beta(1)-adrenergic receptors (ARs) using adenylyl cyclase assays. The aryloxypropanolamines CGP 12177 and LY 362884, originally developed as beta(3)-AR agonists, were found to stimulate the beta(1)-AR. Interestingly, both CGP 12177 and LY 362884 exhibited an anomalous biphasic effect on beta(1)-AR. Low concentrations of either CGP 12177 or LY 362884 potently blocked isoproterenol-induced stimulation of beta(1)-AR, whereas higher concentrations of these compounds stimulated the beta(1)-AR. The unusual interaction of these aryloxypropanolamine ligands with the beta(1)-AR was further characterized using beta-AR antagonists. Activation of beta(1)-AR by CGP 12177 or LY 362884 was observed to be significantly more resistant to blockade by beta-AR antagonists compared with activation by catecholamines. These results suggest that catecholamines and aryloxypropanolamines interact with distinct active conformations of the beta(1)-AR: a state that is responsive to catecholamines and is blocked with high affinity by CGP 12177 and LY 362884, and a novel state that is activated by aryloxypropanolamines but is resistant to blockade by standard beta-AR antagonists. Moreover, dependence of antagonist affinity on agonist structure is unprecedented, and its implications on the use of beta-AR agonists such as CGP 12177 in receptor classification are discussed.     

Effects of milrinone on contractility and cyclic adenosine monophosphate production induced by beta1- and beta2-adrenergic receptor activation in human myocardium

BACKGROUND: Because milrinone is a widely used phosphodiesterase-3 (PDE3) inhibitor, it would be of interest to know whether it interacts with beta1- and beta2-adrenergic receptor (AR) agonists in human myocardium. OBJECTIVES: This in vitro study was conducted to test whether milrinone differentially regulates cyclic adenosine-3',5'-monophosphate (cAMP) production and to examine the effect of milrinone on the positive inotropic responses and cAMP production induced by activation of the beta1-AR with norepinephrine (NE) and activation of the beta2-AR with epinephrine (EPI) in human atrial myocardium. METHODS: Right atrial trabeculae were obtained from patients undergoing cardiac surgery for valve repair. Concentration-response curves for inotropic responses mediated through the beta1-AR (NE in the presence of the beta2-blocker ICI 118, 551) and the beta2-AR (EPI in the presence of the beta1-blocker CGP 20712A) were obtained in the absence and presence of milrinone 1 micromol/L. This concentration of milrinone was chosen because it corresponded to its 50% inhibitory concentration as a PDE3 inhibitor and its therapeutic plasma concentration. The production of cAMP induced by exposure to selective beta1- and beta2-AR stimulation was also measured in the absence and presence of milrinone. RESULTS: Right atrial tissue samples were obtained from 12 white patients (7 women, 5 men; mean [SE] age, 64.6 [6.3] years) undergoing cardiac surgery for valve repair (8 mitral, 4 aortic). The presence of milrinone was associated with leftward shifts in the concentration-response curves for both NE and EPI. cAMP production in myocardial tissue samples in the presence of milrinone was increased only with NE induction (mean [SEM], 745.0 [136.7] pmol/g in the absence of milrinone vs 1620.5 [372.3] pmol/g in the presence of milrinone; P < 0.05). CONCLUSIONS: In this preliminary study in human atrial myocardium, milrinone potentiated the contractile responses to both NE and EPI. However, only the effect of NE on tissue levels of cAMP was increased in the presence of milrinone.     

Recruitment of functionally active heart beta2-adrenoceptors in the initial phase of endotoxic shock in pithed rats

A supersensitivity of the beta-adrenoceptor-mediated chronotropic response has been demonstrated in atria isolated from rats subjected to septic shock. Our study was undertaken to investigate whether bacterial endotoxin/LPS affects the increase in heart rate induced by beta-adrenoceptor agonists in the rat also in vivo. In pithed and vagotomized rats, the nonselective beta-adrenoceptor agonist isoprenaline (0.05-0.15 nmol/kg) and agonists at the high- and low-affinity state of beta1-adrenoceptors, that is, prenalterol (0.3-3 nmol/kg) and (+/-)-4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazole-2-one (CGP 12177; 3-6 nmol/kg), respectively, and at beta2-adrenoceptors, that is, fenoterol (1-5 nmol/kg), increased heart rate by 50 to 60 beats/min. Administration of LPS (0.4, 1, and 1.5 mg/kg), under continuous infusion of vasopressin, dose-dependently amplified the chronotropic response to isoprenaline, prenalterol, and fenoterol (by 80%, 50%, and 100%, respectively) but not to CGP 12177. The beta2-adrenoceptor antagonist erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol (ICI 118551 0.1 mumol/kg) did not affect the chronotropic responses of isoprenaline, fenoterol, and prenalterol under non-endotoxic conditions, but abolished the potentiation of tachycardia produced by LPS (1.5 mg/kg). The beta1-adrenoceptor antagonist (+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]-phenoxy]propyl]-amino]ethoxy]-benzamide CGP 20712A; 0.1 mumol/kg almost completely reduced the chronotropic effects of isoprenaline, fenoterol, and prenalterol both in control rats and in animals exposed to LPS (1.5 mg/kg). We conclude that LPS sensitizes cardiac beta-adrenoceptors by recruiting functionally active beta2-adrenoceptors, but the amplification of tachycardia occurs only when both beta1- and beta2-adrenoceptors are concomitantly activated. The pithed rat may serve as a model to examine the beta-adrenoceptor supersensitivity in vivo.     

Autoradiographic localization of beta-1 and beta-2 adrenoceptors in guinea pig atrium and regions of the conducting system

[125I]Cyanopindolol binding to slide mounted guinea pig atrial sections was time-dependent (Ki = 2.4 X 10(8) M-1 min-1, K-1 = 1.2 X 10(-3) min-1) saturable (24-32 pM) and stereoselective with respect to the isomers of propranolol. Competition binding curves with the beta-1 selective antagonist CGP 20712A and beta-2 selective antagonist ICI 118,551 revealed the presence of beta-1 and beta-2 adrenoceptors in the proportions of 85 to 15%. X-ray film exposed to sections of guinea pig heart incubated previously with [125I]cyanopindolol with or without ICI 118,551 (70 nM) or CGP 20712A (100 nM) or (-)-propranolol (1 microM) showed a high density and even distribution of beta-1 adrenoceptors and a lower density and even distribution of beta-2 adrenoceptors over the myocardium. Beta-2 adrenoceptors also were located in the epicardium, the adventitia of the pulmonary artery, aorta, superior and inferior vena cava and the endothelium of the superior and inferior vena cava. Development of nuclear emulsion-coated coverslips confirmed these observations and, in addition, localized beta-2 adrenoceptors to nerve tissue and the aortic valve. In the papillary muscle and surrounding Purkinje cells there was a high density and even distribution of beta-1 and a much lower density of beta-2 adrenoceptors. The atrioventricular node, atrioventricular bundle and ventricular Purkinje system also contained both beta-1 and beta-2 adrenoceptors and in comparison with the surrounding myocardium, these tissues had a slightly greater density of beta-2 adrenoceptors.     

Atypical beta-adrenoceptor subtypes mediate relaxations of rabbit corpus cavernosum

This study was performed to characterize the beta-adrenoceptor population in rabbit isolated corpus cavernosum (RbCC) by using nonselective and selective beta-adrenoceptor agonists and antagonists in functional assays. Metaproterenol, ritodrine, fenoterol, and 8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(rho-methoxyphenyl)-1-methylethyl]amino]ethyl]carbostyril (TA 2005) (3-100 nmol each) dose dependently relaxed the RbCC preparations. These relaxations were markedly reduced by N(omega)-nitro-L-arginine methyl ester (L-NAME; 10 microM) and 1H-[1,2,4]-oxadiazolo-[4,3,-a]quinoxalin-1-one (ODQ) (10 microM), whereas the adenylyl cyclase inhibitor SQ 22,536 [9-(2-tetrahydrofuryl) adenine] (10 microM) had no effect. In contrast, neither L-NAME nor ODQ affected the isoproterenol-induced RbCC relaxations, but SQ 22,536 abolished this response. Sildenafil (1 microM) significantly potentiated the relaxations induced by beta(2)-agonists without affecting the isoproterenol-evoked relaxations. Rolipram (10 microM) enhanced the relaxations elicited by isoproterenol but had no effect on those induced by the selective beta(2) agonists. Propranolol and (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118,551) determined a rightward shift in the concentration-response curves to isoproterenol in a noncompetitive manner with a reduction of maximum response at the highest antagonist concentration, with the slope values significantly different from unity. Propranolol and ICI 118,551 had no effect on the relaxations elicited by fenoterol, TA 2005, metaproterenol, and ritodrine. Atenolol and 1-[2-((3-carbamoyl-4-hydroxy)phenoxy) ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)-phenoxy]-2-propanol methanesulfonate (CGP 20712A) (0.1-10 microM) failed to affect the relaxations induced by all tested beta-adrenoceptor agonists. Our study revealed the existence of two atypical beta-adrenoceptors in the rabbit erectile tissue. Isoproterenol relaxes the rabbit cavernosal tissue by activating atypical beta-adrenoceptors coupled to adenylyl cyclase pathway, whereas the selective beta(2)-adrenoceptor agonists relax the RbCC tissue through another atypical beta-adrenoceptor subtype coupled to nitric oxide release from the sinusoidal endothelium.     

Enhanced inhibition of L-type Ca2+ current by beta3-adrenergic stimulation in failing rat heart

beta3-adrenergic receptors (AR) have recently been identified in mammalian hearts and shown to be up-regulated in heart failure (HF). beta3-AR stimulation reduces inotropic response associated with an inhibition of L-type Ca2+ channels in normal hearts; however, the effects of beta3-AR activation on Ca2+ channel in HF remain unknown. We compared the effects of beta(3)-AR activation on L-type Ca2+ current (ICa,L) in isolated left ventricular myocytes obtained from normal and age-matched rats with isoproterenol (ISO)-induced HF (4 months after 340 mg/kg s.c. for 2 days). ICa,L was measured using whole-cell voltage clamp and perforated-patch recording techniques. In normal myocytes, superfusion of 4-[-[2-hydroxy-(3-chlorophenyl)ethylamino]propyl]phenoxyacetate (BRL-37,344; BRL), a beta3-AR agonist, caused a dose-dependent decrease in ICa,L with maximal inhibition (21%, 1.1 +/- 0.2 versus 1.4 +/- 0.1 nA) (p < 0.01) at 10(-7) M. In HF myocytes, the same concentration of BRL produced a proportionately greater inhibition (31%) in ICa,L (1.1 +/- 0.2 versus 1.6 +/- 0.2 nA) (p < 0.05). A similar inhibition of ICa,L was also observed with ISO (10(-7) M) in the presence of a beta1- and beta2-AR antagonist, nadolol (10(-5) M). Inhibition was abolished by the beta3-AR antagonist (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L-748,337; 10(-6) M), but not by nadolol. The inhibitory effect of BRL was attenuated by a nitric-oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (10(-4) M), and was prevented by the incubation of myocytes with pertussis toxin (PTX; 2 microg/ml, 36 degrees C, 6 h). In conclusion, beta3-AR activation inhibits L-type Ca2+ channel in both normal and HF myocytes. In HF, beta3-AR stimulation-induced inhibition of Ca2+ channel is enhanced. These effects are likely coupled with PTX-sensitive G-protein and partially mediated through a NOS-dependent pathway.     

GW427353 (solabegron), a novel, selective beta3-adrenergic receptor agonist, evokes bladder relaxation and increases micturition reflex threshold in the dog

Functional studies have demonstrated that adrenoceptor agonist-evoked relaxation is mediated primarily by beta3-adrenergic receptors (ARs) in human bladder. Thus, the use of selective beta3-AR agonists in the pharmacological treatment of overactive bladder is being explored. The present studies investigated the effects of a novel selective beta3-AR agonist, (R)-3'-[[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]ethyl]amino]-[1,1'-biphenyl]-3-carboxylic acid (GW427353; solabegron) on bladder function in the dog using in vitro and in vivo techniques. GW427353 stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human beta3-AR, with an EC50 value of 22 +/- 6 nM and an intrinsic activity 90% of isoproterenol. At concentrations of 10,000 nM, GW427353 produced a minimal response in cells expressing either beta1-ARs or beta2-ARs (maximum response <10% of that to isoproterenol). In dog isolated bladder strips, GW427353 evoked relaxation that was attenuated by the nonselective beta-AR antagonist bupranolol and 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol (SR59230A) (reported to have beta3-AR antagonist activity). The relaxation was unaffected by atenolol, a selective beta1-AR antagonist, or (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118551), a selective beta2-AR antagonist. GW427353 increased the volume required to evoke micturition in the anesthetized dog following acetic acid-evoked bladder irritation, without affecting the ability of the bladder to void. GW427353-evoked effects on bladder parameters in vivo were inhibited by bupranolol. The present study demonstrates that selective activation of beta3-AR with GW427353 evokes bladder relaxation and facilitates bladder storage mechanisms in the dog.     

Pharmacological characterization of chick and frog beta adrenergic receptors in primary cultures of myocardial cells.

In membrane preparations derived from primary cultures of chick myocardial cells, beta adrenergic receptors modeled for a single low-affinity site for both betaxolol (beta-1-selective) and ICI 118551 (beta-2-selective) displacement of [125I]iodocyanopindolol (ICYP), indicating that the chick beta receptor is pharmacologically distinct from both mammalian beta-1 and beta-2 adrenergic receptors with respect to these antagonists. However, the highly beta-1-selective compound CGP 20712A was able to distinguish two binding sites on ICYP competition curves, a high-affinity "beta-1 site" (75%) and a low-affinity "beta-2 site" (25%). Also, in chick heart cell membranes the relative ability of agonists to displace ICYP produced a profile typical of beta-1 adrenergic receptors with a rank order of potency or efficacy of: isoproterenol greater than epinephrine = norephinephrine. When agonist-mediated adenylyl cyclase stimulation was assessed the order of potency was slightly different, isoproterenol greater than epinephrine greater than or equal to norepinephrine. Additionally, antagonism of isoproterenol stimulation of adenylyl cyclase by CGP 20712A yielded a Kb value (1.16 +/- 0.35 x 10(-7) M) intermediate between the high and low-affinity binding sites of CGP 20712A, suggesting that the low-affinity site is coupled to adenylyl cyclase. In membrane preparations of frog myocardial cells, ICYP/antagonist competition curves modeled for a mixed population of receptors, with subtype percentages varying from 50:50 beta-1:beta-2 to 100% beta-2 depending on the specific antagonist used and the individual cell preparation. For ICYP/agonist competition binding experiments the relative ability to displace ICYP was isoproterenol greater than epinephrine = norepinephrine, a profile typical of beta-1 adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of bucindolol and carvedilol on noradenaline-induced hypertrophic response in ventricular cardiomyocytes of adult rats

In adult rat ventricular cardiomyocytes, noradrenaline exerts dual effects on protein synthesis: increases via alpha(1)-adrenoceptors and decreases via beta(1)-adrenoceptors. Carvedilol and bucindolol are beta-blockers with additional alpha(1)-adrenoceptor blocking activities. We studied the effects of carvedilol and bucindolol on noradrenaline-induced protein synthesis (assessed by [(3)H]phenylalanine incorporation) in adult rat ventricular cardiomyocytes. Radioligand binding studies with [(125)I]iodocyanopindolol and [(3)H]prazosin revealed that carvedilol had a much higher affinity to alpha(1)-adrenoceptors than bucindolol (beta(1)-/alpha(1)-adrenoceptor ratio for carvedilol, 1:2.7; for bucindolol, 1:43). Noradrenaline-evoked increases in protein synthesis were enhanced by propranolol (1 microM) and beta(1)-adrenoceptor-selective antagonists bisoprolol (1 microM) and CGP 20712A [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)-ethyl-amino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propranol methanesulfonate] (300 nM). Carvedilol (100 pM-10 microM) inhibited 1 microM noradrenaline-induced increase in protein synthesis with monophasic concentration-inhibition curves independent of whether CGP 20712A was present or not; K(i) values for carvedilol were 5 to 6 nM. In contrast, bucindolol (100 pM-10 microM) inhibited l microM noradrenaline-induced increase in protein synthesis with a bell-shaped concentration-inhibition curve; it increased noradrenaline-induced protein synthesis at 10 nM, although at concentrations >100 nM it was inhibited. In the presence of 300 nM CGP 20712A or 1 microM propranolol, however, bucindolol inhibited 1 microM noradrenaline-induced increase in protein synthesis with monophasic concentration-inhibition curves; K(i) values were 40 to 75 nM. On the other hand, both carvedilol and bucindolol inhibited 1 microM phenylephrine-induced protein synthesis with monophasic concentration-inhibition curves; K(i) values were 4 (carvedilol) and 45 nM (bucindolol). These results indicate that, at low (beta-adrenoceptor blocking) concentrations, bucindolol can enhance noradrenaline-induced protein synthesis whereas it is inhibited by carvedilol.     

Characterization of beta-1 and beta-2 adrenoceptors in guinea pig atrium: functional and receptor binding studies

The selective beta-2 adrenoceptor agonist procaterol produced positive inotropic and chronotropic responses over a concentration range of 1 nM to 0.1 mM in spontaneously beating right atria and in three of seven electrically driven left atria. The pD2 values (right atria, 7.30; left atria, 7.18) were midway between its known affinities at beta-1 and beta-2 adrenoceptors and are evidence that positive inotropic and chronotropic responses involve a minor beta-2 adrenoceptor component. The pKB values for procaterol against (-)-isoproterenol in the right atria (5.59) and left atria (5.29) were consistent with its affinity for beta-1 adrenoceptors and suggest that these are responsible primarily for positive inotropic and chronotropic responses. Receptor binding studies in right atrial homogenates showed that [125I]cyanopindolol binding was saturable (KD = 36.2 pM, maximal density of binding sites = 49.2 fmol mg-1 protein) and stereoselective with respect to the isomers of propranolol. Competition binding curves for the beta-1 adrenoceptor antagonist CGP 20712A and beta-2 selective antagonist ICI 118,551 against [125I]cyanopindolol binding were resolved into two components using iterative curve fitting techniques. Binding sites with the characteristics of beta-1 and beta-2 adrenoceptors were present in the proportions of approximately 75 to 25%. These studies indicate either that the beta-1 adrenoceptor is coupled more efficiently to the positive inotropic and chronotropic response than the beta-2 adrenoceptor or that a proportion of the beta-2 adrenoceptors subserve other functions.     

beta3-adrenoceptors in the cardiovascular system: putative roles in human pathologies

The sympathetic nervous system is central for the neurohumoral regulation of the cardiovascular system and is largely involved in many cardiovascular diseases affecting millions of people around the world. It is classically admitted that beta-adrenoceptors (beta-AR) of the beta1 and beta2 subtypes mediate the effects of catecholamines on the force of contraction of cardiac muscle, and on the relaxation of vascular smooth muscle. However, the molecular characterization in 1989 of a third beta-AR subtype, beta3, and later its identification in human heart has changed the classically admitted paradigm on the regulation of heart function by the beta-adrenergic system. In blood vessels, beta3-AR, like beta1 and beta2, produced a relaxation. But at the present time, the physiological role of beta3-AR is not clearly identified. Thus, the purpose of this review is to summarize the pharmacological and molecular evidence supporting the functional roles of beta3-AR in cardiovascular tissues of various species, including humans. In addition, this review discusses the potential role of beta3-AR in several cardiovascular diseases and emphasizes their putative involvement as new therapeutic targets.     

In vitro and in vivo pharmacological characterization of ethyl-4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl)amino]-phenoxy]propyl) amino]cyclohexyl]benzoate hydrochloride (SAR150640), a new potent and selective human beta3-adrenoceptor agonist for the treatment of preterm labor

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

Evidence for a noradrenergic innervation to "atypical" beta adrenoceptors (or putative beta-3 adrenoceptors) in the ileum of guinea pig.

Indirect evidence suggests that beta-1 adrenoceptors in the guinea pig ileum are innervated but it has not been determined whether "atypical" beta adrenoceptors also receive a postganglionic sympathetic innervation. To answer this question, experiments were undertaken using electrical stimulation of para-arterial sympathetic neurons to evoke relaxation in isolated segments of guinea pig ileum. Tension was developed in the ileal segments by either transmural electrical field stimulation to evoke the cholinergic "twitch" response, or by histamine to produce a steady-state contracture. Para-arterial sympathetic nerve stimulation evoked a frequency-dependent inhibition of the twitch response which was blocked by guanethidine and restored by dexamphetamine, indicating typical noradrenergic transmission. In preparations contracted with histamine and pretreated with benextramine to block alpha adrenoceptors, para-arterial sympathetic nerve stimulation evoked frequency-dependent relaxations which were reduced in magnitude but not abolished by the following beta adrenoceptor antagonists: bromoacetylalprenololmenthane (1 microM) or a combination of ICI 118,551 (0.3 microM) and CGP 20712A (0.1 microM). Remaining responses were blocked by compounds exhibiting affinity for atypical beta adrenoceptors, (-)-alprenolol (3 microM) and nadolol (300 microM), as well as the agonist (-)-isoproterenol (10 microM; to saturate the atypical beta adrenoceptor). However, relaxations to papaverine were unaffected by these treatments. Experiments revealed that potential cotransmitters (ATP, neuropeptide Y and somatostatin) do not appear to play a detectable role in relaxations produced by para-arterial sympathetic nerve stimulation. The results demonstrate, for the first time, that atypical beta adrenoceptors in guinea pig ileum receive a noradrenergic innervation.     

beta3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current

beta3-adrenergic receptor (beta3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of beta3-AR in the human atrium. Unexpectedly, beta3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (I Ca,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I(Ca,L) and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective beta3-AR agonists, SR58611 and BRL37344, and a beta3-AR partial agonist, CGP12177, stimulated I(Ca,L) in HAMs with nanomolar potency and a 60%-90% efficacy compared with isoprenaline. The beta3-AR agonists also increased contractility but with a much lower efficacy (approximately 10%) than isoprenaline. The beta3-AR antagonist L-748,337, beta1-/beta2-AR antagonist nadolol, and beta1-/beta2-/beta3-AR antagonist bupranolol were used to confirm the involvement of beta3-ARs (and not beta1-/beta2-ARs) in these effects. The beta3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I(Ca,L) stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, beta3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.