Pharmacological Inhibition of Protein Kinases in Intact Cells: Antagonism of Beta Adrenergic Receptor Ligand Binding by H-89 Reveals Limitations of Usefulness1
- Raymond B. Penn1,
- Jean-Luc Parent1,
- Alexey N. Pronin1,
- Reynold A. Panettieri, Jr.2 and
- Jeffrey L. Benovic1
- 1Department 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 2Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (R.A.P.)
Abstract
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.
Footnotes
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Send reprint requests to: Raymond B. Penn, Ph.D., Thomas Jefferson University, Kimmel Cancer Institute, Room 930 B.L.S.B., 233 S. 10th St., Philadelphia, PA 19107. E-mail:rpenn{at}lac.jci.tju.edu
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↵1 J.-L.P. is the recipient of a postdoctoral fellowship from the Medical Research Council of Canada. J.L.B. is the recipient of an American Heart Association Established Investigator Award. R.A.P. is the recipient of a Career Investigator Award from the ALA. This work was supported in part by National Institutes of Health Grants HL58506, GM44944, and HL55301.
- Abbreviations:
- AR
- adrenergic receptor
- DMEM
- Dulbecco’s modified Eagle’s medium
- DMSO
- dimethyl sulfoxide
- FSK
- forskolin
- GPR
- G protein-coupled receptor
- GRK
- G protein-coupled receptor kinase
- HASM
- human airway smooth muscle
- HEK
- human embryonic kidney
- IBMX
- isobutylmethylxanthine
- IPIN
- iodopindolol
- ISO
- isoproterenol
- mAchR
- muscarinic acetylcholine receptor
- NMS
- scopolamine methylchloride
- PBS
- phosphate-buffered saline
- PGE2
- prostaglandin E2
- PKA
- cAMP-dependent protein kinase
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- PAGE
- polyacrylamide gel electrophoresis
- TXA2αR
- thromboxane A2α receptor
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- Received May 5, 1998.
- Accepted August 25, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
