Pharmacological inhibition of protein kinases in intact cells: Antagonism of beta adrenergic receptor ligand binding by H-89 reveals limitations of usefulness

The use of pharmacological inhibitors of protein kinases represents a poten tially powerful tool in dissecting the regulatory features of intracellular signaling pathways. However, although the in vitro potency, selectivity, a nd efficacy of numerous kinase inhibitors have been characterized, little i s known regarding the usefulness of these compounds as inhibitors in intact cells. In attempting to characterize the role of protein kinase A (PKA) in regulating the beta-2 adrenergic receptor (AR) in human airway cells, we o bserved a seemingly profound capacity of the isoquinoline H-89, a potent an d widely used PKA inhibitor, to attenuate agonist-mediated desensitization of the beta-2 AR. Although additional experiments identified H-89 as an eff ective inhibitor of intracellular PKA, extended analysis of the compound de termined the principal effect of H-89 was via its action as a beta-2 AR ant agonist. Pretreatment with or the acute addition of H-89 significantly atte nuated isoproterenol-stimulated cAMP accumulation. In cells pretreated with H-89 and then washed extensively, the subsequent dose-dependent response t o isoproterenol suggested beta-2 AR antagonism by retained H-89. Competitio n binding of [I-125]iodopindolol established K-i values of similar to 180 n M and 350 nM for H-89 antagonism of beta-2 AR and beta-1 AR, respectively. Additional receptor binding studies suggest selectivity of H-89 for the bet a-2 AR and beta-1 AR, although a weak antagonism (K-i values of similar to 10 mu M or greater) of other G protein-coupled receptors was observed. Resu lts from additional pharmacological and biochemical analyses of various pro tein kinase inhibitors further established the need for careful characteriz ation of pharmacological inhibitors when used in intact cell models.