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Research ArticleCELLULAR AND MOLECULAR

Evidence for Multiple P2Y Receptors in Trabecular Meshwork Cells

Craig E. Crosson, Phillip W. Yates, Aruna N. Bhat, Yurii V. Mukhin and Shahid Husain
Journal of Pharmacology and Experimental Therapeutics May 2004, 309 (2) 484-489; DOI: https://doi.org/10.1124/jpet.103.060319
Craig E. Crosson
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Phillip W. Yates
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Aruna N. Bhat
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Yurii V. Mukhin
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Shahid Husain
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Abstract

The purpose of this study was to determine whether functional purinergic P2 receptors are present in trabecular meshwork cells. The human trabecular cell line HTM-3 and cultured bovine trabecular cells were used to assess the effects of P2 agonists on intracellular Ca2+ levels, extracellular signal-regulated kinase (ERK1/2) activation, and P2Y receptor expression. ATP, UTP, ADP, and 2-methyl-thio-adenosine triphosphate (2-MeS-ATP) each produced a concentration-dependent increase in intracellular Ca2+ in bovine trabecular cells and the HTM-3 cell line. The addition of UDP did not produce any detectable rise in intracellular Ca2+. Pretreatment with the P2Y1 receptor antagonist 2′-deoxy-N6-methyladenosine-3′,5′-diphosphate (MRS-2179) blocked the ADP- and 2-MeS-ATP-induced rise in intracellular Ca2+. However, the ATP- or UTP-induced rise in intracellular Ca2+ was not inhibited by MRS-2179 pretreatment. The addition of ADP, 2-MeS-ATP, ATP, or UTP were also found to activate the ERK1/2 signaling pathway. This activation of ERK1/2 was blocked by pretreatment with the mitogen-activated protein kinase kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U-0126) or the protein kinase C inhibitor chelerythrine chloride, but not by MRS-2179. Analysis of mRNA from HTM-3 cells by reverse transcription-polymerase chain reaction revealed the expression of P2Y1, P2Y4, and P2Y11 receptor subtypes. These data demonstrate that multiple P2Y receptors are present in trabecular cells. Our results are consistent with the idea that the mobilization of intracellular Ca2+results from the activation of P2Y1 and P2Y4 receptors, whereas the activation of the ERK1/2 pathway results from the activation of P2Y4 receptors alone. However, a role for the P2Y11 receptors in mobilization of Ca2+, or activation of the ERK1/2 pathway, cannot be discounted.

Footnotes

  • This study was supported in part by National Eye Institute Grants EY-09741 and EY-014793 (to C.E.C.), shared equipment grant S-10-RR-13005 (to John Raymond), and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness, New York, NY.

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

  • DOI: 10.1124/jpet.103.060319.

  • ABBREVIATIONS: IOP, intraocular pressure; ERK, extracellular signal-regulated kinase; DMEM, Dulbecco's modified Eagle's medium; 2-Mes-ATP, 2-methyl-thio-adenosine triphosphate; PPADS, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid; 8-SPT, 8-sulfophenylthephylline; AM, acetoxymethyl ester; FBS, fetal bovine serum; PCR, polymerase chain reaction; MEK, mitogen-activated protein kinase kinase; PKC, protein kinase C; RT-PCR, reverse transcription-polymerase chain reaction; RT, reverse transcription; MRS-2179, 2′-deoxy-N6-methyladenosine-3′,5′-diphosphate; U-0126, 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene.

    • Received September 19, 2003.
    • Accepted January 16, 2004.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 309 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 309, Issue 2
1 May 2004
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Research ArticleCELLULAR AND MOLECULAR

Evidence for Multiple P2Y Receptors in Trabecular Meshwork Cells

Craig E. Crosson, Phillip W. Yates, Aruna N. Bhat, Yurii V. Mukhin and Shahid Husain
Journal of Pharmacology and Experimental Therapeutics May 1, 2004, 309 (2) 484-489; DOI: https://doi.org/10.1124/jpet.103.060319

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Research ArticleCELLULAR AND MOLECULAR

Evidence for Multiple P2Y Receptors in Trabecular Meshwork Cells

Craig E. Crosson, Phillip W. Yates, Aruna N. Bhat, Yurii V. Mukhin and Shahid Husain
Journal of Pharmacology and Experimental Therapeutics May 1, 2004, 309 (2) 484-489; DOI: https://doi.org/10.1124/jpet.103.060319
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