Induction of β3-Adrenergic Receptor Functional Expression following Chronic Stimulation with Noradrenaline in Neonatal Rat Cardiomyocytes

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Abstract
This study aimed to characterize β3-adrenergic receptors (ARs) in rat neonatal cardiomyocytes using the noradrenaline (NOR) properties to modulate the expression and function of the three β-ARs. We assessed the effect of NOR (physiological nonselective agonist), isoprenaline (ISO, β-nonselective agonist), dobutamine (DOB, β1-selective agonist), and procaterol (PROC, β2-selective agonist) on cAMP accumulation using cardiomyocytes untreated or treated with 100 μM NOR for 24 h. The inhibition of forskolin-stimulated cAMP accumulation was determined using NOR, isoprenaline, and the β3-selective agonists 4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid (BRL 37344) and 5-[-2-([-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-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)-1H-imidazol-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 β1- and β2-AR stimulation and 1-(2-ethylphenoxy)-3-[[1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino-(2S)-2-propanol hydrochloride (SR 59230A) (β3-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 β1- and β2-ARs are down-regulated and that β2-AR functional activity was also abolished in cardiomyocytes exposed to chronic stimulation. β3-AR function was observed with NOR and ISO when β1-/β2-ARs were blocked and with both β3-selective agonists in NOR-treated cells only. This response was completely inhibited by SR 59230A and involved Gi protein. Furthermore, the results from functional studies agree well with those from expression experiments. In conclusion, these data provide strong evidence that β3-ARs are functionally up-regulated and coupled to Gi protein in rat neonatal cardiomyocytes following chronic exposure to NOR when β1- and β2-ARs are down-regulated.

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