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Research ArticleCellular and Molecular

Adenylate Cyclase/cAMP/Protein Kinase A Signaling Pathway Inhibits Endothelin Type A Receptor-Operated Ca2+ Entry Mediated via Transient Receptor Potential Canonical 6 Channels

Takahiro Horinouchi, Tsunaki Higa, Hiroyuki Aoyagi, Tadashi Nishiya, Koji Terada and Soichi Miwa
Journal of Pharmacology and Experimental Therapeutics January 2012, 340 (1) 143-151; DOI: https://doi.org/10.1124/jpet.111.187500
Takahiro Horinouchi
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Tsunaki Higa
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Hiroyuki Aoyagi
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Tadashi Nishiya
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Koji Terada
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Soichi Miwa
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Abstract

Receptor-operated Ca2+ entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca2+ concentration triggered by the activation of Gq protein-coupled receptors. TRPC6 is phosphorylated by various protein kinases including protein kinase A (PKA). However, the regulation of TRPC6 activity by PKA is still controversial. The purpose of this study was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of Gq protein-coupled endothelin type A receptor (ETAR)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ETAR and TRPC6 (wild type) or its mutants possessing a single point mutation of putative phosphorylation sites for PKA were used to analyze ROCE and amino acids responsible for PKA-mediated phosphorylation of TRPC6. Ca2+ measurements with thapsigargin-induced Ca2+-depletion/Ca2+-restoration protocol to estimate ROCE showed that the stimulation of ETAR induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. The ROCE was inhibited by forskolin and papaverine to activate the cAMP/PKA pathway, whereas it was potentiated by Rp-8-bromoadenosine-cAMP sodium salt, a PKA inhibitor. The inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 (TRPC6S28A) but not Thr69 (TRPC6T69A) of TRPC6 with alanine. In vitro kinase assay with Phos-tag biotin to determine the phosphorylation level of TRPC6 revealed that wild-type and mutant (TRPC6S28A and TRPC6T69A) TRPC6 proteins were phosphorylated by PKA, but the phosphorylation level of these mutants was lower (approximately 50%) than that of wild type. These results suggest that TRPC6 is negatively regulated by the PKA-mediated phosphorylation of Ser28 but not Thr69.

Footnotes

  • This study was supported in part by Grants-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science [grant 21790236] (to T. Horinouchi); Grants-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science [grant 21390068] (to S.M.); and grants from the Smoking Research Foundation of Japan (to S.M.), the Mitsubishi Pharma Research Foundation (to T. Horinouchi), the Pharmacological Research Foundation, Tokyo (to T. Horinouchi), the Shimabara Science Promotion Foundation (to T. Horinouchi), and Actelion Pharmaceuticals Japan Ltd. (to T. Horinouchi).

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    http://dx.doi.org/10.1124/jpet.111.187500.

  • ↵Embedded Image The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    TRPC
    transient receptor potential canonical
    AC
    adenylate cyclase
    BSA
    bovine serum albumin
    [Ca2+]i
    intracellular free Ca2+ concentration
    ECL
    enhanced chemiluminescence
    EPAC
    exchange protein activated by cAMP
    ER
    endoplasmic reticulum
    ET-1
    endothelin-1
    ETAR
    endothelin type A receptor
    fura-2/AM
    fura-2/acetoxymethyl ester
    GFP
    green fluorescent protein
    GqPCR
    Gq protein-coupled receptor
    HA
    hemagglutinin
    HEK293
    human embryonic kidney 293
    HRP
    horseradish peroxidase
    IB
    immunoblotting
    IP
    immunoprecipitation
    IPAH
    idiopathic pulmonary arterial hypertension
    PDE
    phosphodiesterase
    PKA
    protein kinase A
    PKG
    protein kinase G
    ROCC
    receptor-operated Ca2+ channel
    ROCE
    receptor-operated Ca2+ entry
    Rp-8-Br-cAMP
    Rp-8-bromoadenosine-cAMP sodium salt
    SA
    streptavidine
    SOCE
    store-operated Ca2+ entry
    SQ-22,536
    9-(tetrahydro-2-furanyl)-9H-purin-6-amine
    TG
    thapsigargin
    WT
    wild type.

  • Received August 31, 2011.
  • Accepted October 13, 2011.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 340 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 340, Issue 1
1 Jan 2012
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Research ArticleCellular and Molecular

PKA-Mediated Inhibition of ETAR-Induced Ca2+ Entry via TRPC6

Takahiro Horinouchi, Tsunaki Higa, Hiroyuki Aoyagi, Tadashi Nishiya, Koji Terada and Soichi Miwa
Journal of Pharmacology and Experimental Therapeutics January 1, 2012, 340 (1) 143-151; DOI: https://doi.org/10.1124/jpet.111.187500

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Research ArticleCellular and Molecular

PKA-Mediated Inhibition of ETAR-Induced Ca2+ Entry via TRPC6

Takahiro Horinouchi, Tsunaki Higa, Hiroyuki Aoyagi, Tadashi Nishiya, Koji Terada and Soichi Miwa
Journal of Pharmacology and Experimental Therapeutics January 1, 2012, 340 (1) 143-151; DOI: https://doi.org/10.1124/jpet.111.187500
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