Induction of beta 3-adrenergic receptor functional expression following chronic stimulation with noradrenaline in neonatal rat cardiomyocytes

This study aimed to characterize beta(3)-adrenergic receptors ( ARs) in rat neonatal cardiomyocytes using the noradrenaline ( NOR) properties to modulate the expression and function of the three beta-ARs. We assessed the effect of NOR ( physiological nonselective agonist), isoprenaline ( ISO, beta-nonselective agonist), dobutamine ( DOB, beta(1)-selective agonist), and procaterol ( PROC, beta(2)-selective agonist) on cAMP accumulation using cardiomyocytes untreated or treated with 100 mu M NOR for 24 h. The inhibition of forskolin-stimulated cAMP accumulation was determined using NOR, isoprenaline, and the beta(3)-selective agonists 4-[ 2-[( 2-( 3-chlorophenyl-2-hydroxyethyl) amino] propyl] phenoxyacetic acid ( BRL 37344) and 5-[-2-([-2-( 3chlorophenyl)-2- hydroxyethyl] amino) propyl]-1,3-benzodioxole2,2- dicarboxylate ( CL 316243). The experiments were performed in the absence or presence of propranolol or 2-hydroxy-5-[ 2-[[ 2-hydroxy-3-[ 4-[ 1-methyl-4-( trifluoromethyl)-1-Himidazol-2-yl] phenoxy] propyl] amino] ethoxy]-benzamide methanesulfonate ( CGP 20712A) and/ or 1-[ 2,3-(dihydro-7-methyl-1H- inden-4-yl) oxy]-3-[( 1-methylethyl) amino]-2-butanol hydrochloride ( ICI 118551) to inhibit beta(1)-and beta(2)-AR stimulation and 1-( 2-ethylphenoxy)-3-[[ 1S)- 1,2,3,4- tetrahydro-1-naphthalenyl] amino-( 2S)-2-propanol hydrochloride ( SR 59230A) ( beta(3)-selective antagonist). In addition, the level of the three subtypes was determined by reverse transcription polymerase chain reaction and Western blotting. NOR pretreatment decreased the activation of cAMP induced by NOR, isoprenaline, and DOB, whereas PROC response was abolished. The inhibition of NOR response by CGP 20712A or ICI 118551 demonstrated that beta(1)- and beta(2)-ARs are down-regulated and that beta(2)-AR functional activity was also abolished in cardiomyocytes exposed to chronic stimulation. beta(3)-AR function was observed with NOR and ISO when beta(1)-/beta(2)-ARs were blocked and with both beta(3)-selective agonists in NOR-treated cells only. This response was completely inhibited by SR 59230A and involved G(i) protein. Furthermore, the results from functional studies agree well with those from expression experiments. In conclusion, these data provide strong evidence that beta(3)-ARs are functionally upregulated and coupled to G(i) protein in rat neonatal cardiomyocytes following chronic exposure to NOR when beta(1)- and beta(2)-ARs are down-regulated.