Evaluation of β-adrenergic receptor subtypes in the human prostate cancer cell line-LNCaP

The present study was undertaken to determine the effects of catecholamines, agonists, and antagonists of β-adrenergic receptors (AR) in the LNCaP cell line. Changes in cellular cyclic adenosine-3′,5′-monophosphate (cAMP) levels were quantified by the use of a 6 cAMP response element (CRE)-luciferase reporter gene assay. LNCaP cells were transiently transfected with this gene construct, incubated in 96-well microtiter plates for 24 hr, and then treated with β-AR agonists and/or antagonists for 4 hr. The rank order of potency for catecholamines and known β-AR agonists was terbutaline (3.31 nM)> isoproterenol (8.31 nM)≥ fenoterol (15 nM)= epinephrine (16.2 nM)> norepinephrine (77.5 nM)> BRL-37344 [( R ∗, R ∗)-(±)4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxy acetic acid, sodium salt] (1000 nM)> dobutamine (1770 nM)> CGP 12177 (4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2 H-benzimidazole-2-one hydrochloride) (inactive). The non-selective β 1-/-β 2-AR antagonists; propranolol and CGP 12177, at 10 −7 M, inhibited luciferase activity induced by these agonists by 80–96%. Propranolol blocked isoproterenol-induced luciferase responses in a competitive manner ( K B =1.4 nM). In addition, isoproterenol-activated luciferase expression was blocked more potently by ICI 118,551 [(±)-1-[2,3-(dihydro-7-methyl-1 H-inden-4-yl)oxy]-3-[(1-methylethy) amino]-2-butanol], a β 2-AR antagonist than by ICI 89,406 [(±)- N-[2-[3-(2-cyanophenoxy-)]-2-hydroxypropylamino]ethyl- N-phenylurea], a β 1-AR antagonist, giving K B values of 1.07 and 161 nM, respectively. These results suggest that the β 2-AR is the major subtype mediating catecholamine-induced cAMP changes in LNCaP cells.