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CELLULAR AND MOLECULAR

Evidence for Multiple P2Y Receptors in Trabecular Meshwork Cells

Departments of Ophthalmology (C.E.C., P.W.Y., A.N.B., S.H.), MUSC-Hewitt Laboratory of the Ola B. Williams Glaucoma Center, and Medicine (Y.V.M.), Medical University of South Carolina, Charleston, South Carolina

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.

Address correspondence to: Dr. Craig E. Crosson, Storm Eye Institute, 167 Ashley Ave., Charleston, SC 29425. E-mail: crossonc{at}musc.edu

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