Kinase experiment:

One hour prior to assay, the growth media are removed from the wells and replaced with 50 uL of Hanks’balanced salt solution that also contained 0.5 mM of MgCl2o6H2O, 0.4 mM of MgSO4o7H2O, 20 mM of N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid (HEPES), 1.2 mM of 3-isobutyl-1-methylxanthine (IBMX), 0.95 mM of CaCl2, and 0.05% of BSA. Each plate is placed in a 37℃ shaking water bath for dose-response studies. In one study, various doses of isoproterenol (10-9-10-5 M) and β1- and β2-receptor-selective partial agonists (tazolol, prenalterol, salbutamol, and terbutaline, 10-6 and 10-5 M, respectively) are added (5 wells/dose/plate) and incubated for 10 min. In another study, the cells are stimulated with 10 μM isoproterenol in the presence or absence of various doses of β-adrenoceptor antagonists. The incubations are terminated after 10 min by the addition of 100 μL of 10% trichloroacetic acid (TCA) (final TCA concentration of 5%). TCA is removed twice by extraction with H20-saturated ether, and samples are dried at 80℃ overnight, prior to resuspension in 50 mM of sodium acetate buffer. The CAMP content is measured with a radioimmunoassay kit.

ICI 118,551 (hydrochloride) is a highly selective β2 adrenergic receptor antagonist, with Kis of 0.7, 49.5 and 611 nM for β2, β1 and β3 receptors, respectively.
ICI 118551 inhibits cAMP accumulation with IC50 of 1.7 μM in IMCD cells[1]. ICI 118551 (10 μM) induces a prominent vasorelaxation of norepinephrine (NE)-precontracted PA but not AO[2]. In failing human heart, ICI 118551 has significant effects on beat duration, with time-to-peak contraction and time-to-90% relaxation reduced compared with basal contraction. Negative Inotropic Effect of ICI 118551 Is Not cAMP-Related. Overexpression of β2AR in rabbit myocytes enhances negative inotropic effects of ICI 118551[3].
Reference:
[1]. Yasuda G, et al. The beta 1- and beta 2-adrenoceptor subtypes in cultured rat inner medullary collecting duct cells. Am J Physiol. 1996 Sep;271(3 Pt 2):F762-9.
[2]. Wenzel D, et al. beta(2)-adrenoceptor antagonist ICI 118,551 decreases pulmonary vascular tone in mice via a G(i/o) protein/nitric oxide-coupled pathway. Hypertension. 2009 Jul;54(1):157-63.
[3]. Gong H, et al. Specific beta(2)AR blocker ICI 118,551 actively decreases contraction through a G(i)-coupled form of the beta(2)AR in myocytes from failing human heart. Circulation. 2002 May 28;105(21):2497-503.
[4]. Hoffmann C, et al. Comparative pharmacology of human beta-adrenergic receptor subtypes--characterization of stably transfected receptors in CHO cells. Naunyn Schmiedebergs Arch Pharmacol. 2004 Feb;369(2):151-9.