PT  - JOURNAL ARTICLE
AU  - Raymond B. Penn
AU  - Jean-Luc Parent
AU  - Alexey N. Pronin
AU  - Reynold A. Panettieri, Jr.
AU  - Jeffrey L. Benovic
TI  - Pharmacological Inhibition of Protein Kinases in Intact Cells: Antagonism of <em>Beta</em> Adrenergic Receptor Ligand Binding by H-89 Reveals Limitations of Usefulness
DP  - 1999 Feb 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 428--437
VI  - 288
IP  - 2
4099  - http://jpet.aspetjournals.org/content/288/2/428.short
4100  - http://jpet.aspetjournals.org/content/288/2/428.full
SO  - J Pharmacol Exp Ther1999 Feb 01; 288
AB  - The use of pharmacological inhibitors of protein kinases represents a potentially powerful tool in dissecting the regulatory features of intracellular signaling pathways. However, although the in vitro potency, selectivity, and efficacy of numerous kinase inhibitors have been characterized, little is 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 thebeta-2 adrenergic receptor (AR) in human airway cells, we observed a seemingly profound capacity of the isoquinoline H-89, a potent and widely used PKA inhibitor, to attenuate agonist-mediated desensitization of the beta-2 AR. Although additional experiments identified H-89 as an effective inhibitor of intracellular PKA, extended analysis of the compound determined the principal effect of H-89 was via its action as a beta-2 AR antagonist. Pretreatment with or the acute addition of H-89 significantly attenuated isoproterenol-stimulated cAMP accumulation. In cells pretreated with H-89 and then washed extensively, the subsequent dose-dependent response to isoproterenol suggestedbeta-2 AR antagonism by retained H-89. Competition binding of [125I]iodopindolol establishedKi values of ∼180 nM 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 beta-2 AR and beta-1 AR, although a weak antagonism (Ki values of ∼10 μM or greater) of other G protein-coupled receptors was observed. Results from additional pharmacological and biochemical analyses of various protein kinase inhibitors further established the need for careful characterization of pharmacological inhibitors when used in intact cell models. The American Society for Pharmacology and Experimental Therapeutics