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Research ArticleArticle

Pharmacological Inhibition of Protein Kinases in Intact Cells: Antagonism of Beta Adrenergic Receptor Ligand Binding by H-89 Reveals Limitations of Usefulness

Raymond B. Penn, Jean-Luc Parent, Alexey N. Pronin, Reynold A. Panettieri Jr. and Jeffrey L. Benovic
Journal of Pharmacology and Experimental Therapeutics February 1999, 288 (2) 428-437;
Raymond B. Penn
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Jean-Luc Parent
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Alexey N. Pronin
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Reynold A. Panettieri Jr.
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Jeffrey L. Benovic
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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

  • 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

  • ↵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
    • Received May 5, 1998.
    • Accepted August 25, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 288 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 288, Issue 2
1 Feb 1999
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Research ArticleArticle

Pharmacological Inhibition of Protein Kinases in Intact Cells: Antagonism of Beta Adrenergic Receptor Ligand Binding by H-89 Reveals Limitations of Usefulness

Raymond B. Penn, Jean-Luc Parent, Alexey N. Pronin, Reynold A. Panettieri and Jeffrey L. Benovic
Journal of Pharmacology and Experimental Therapeutics February 1, 1999, 288 (2) 428-437;

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Research ArticleArticle

Pharmacological Inhibition of Protein Kinases in Intact Cells: Antagonism of Beta Adrenergic Receptor Ligand Binding by H-89 Reveals Limitations of Usefulness

Raymond B. Penn, Jean-Luc Parent, Alexey N. Pronin, Reynold A. Panettieri and Jeffrey L. Benovic
Journal of Pharmacology and Experimental Therapeutics February 1, 1999, 288 (2) 428-437;
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