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Vol. 288, Issue 2, 428-437, February 1999
Department of Microbiology and Immunology, Kimmel Cancer Institute,
Thomas Jefferson University, Philadelphia, Pennsylvania (R.B.P.,
J.-L.P., A.N.P., J.L.B.); and
Department of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania (R.A.P.)
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 the
beta-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 suggested
beta-2 AR antagonism by retained H-89. Competition
binding of [125I]iodopindolol established
Ki 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.
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