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CELLULAR AND MOLECULAR

Mechanisms Linking Adenosine A₁ Receptors and Extracellular Signal-Regulated Kinase 1/2 Activation in Human Trabecular Meshwork Cells

Hewitt Laboratory of the Ola B. Williams Glaucoma Center, Department of Ophthalmology, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina

This study was designed to evaluate the signaling pathways coupling adenosine A₁ receptors and extracellular signal-regulated kinase (ERK) 1 and 2 in human trabecular meshwork (HTM) cells. Studies were conducted using cultures of primary HTM cells and the HTM-3 cell line. Activation of ERK1/2, location of protein kinase C (PKC) isoforms, and matrix metalloproteinase (MMP) secretion were determined by Western blotting. In primary HTM cells and the HTM-3 cell line, administration of the A₁ agonist N⁶-cyclohexyladenosine (CHA) produced a concentration-dependent increase in ERK1/2 activation. This CHA-induced ERK activation was blocked by pretreatment with the A₁ receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine or pertussis toxin. Transfection with dominant negative N17 Ras produced only a small (31%) decline in CHA-induced ERK activation, and the response was not altered by pretreatment with the Src tyrosine kinase inhibitor, PP2 [3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-D] pyrimidin-4-amine], the phosphoinositide kinase-3 inhibitor, LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], or the A₃ receptor antagonist, MRS-1191 [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate]. Administration of CHA also induced the translocation of PKC from the cytosol to the membrane, and pretreatment with the phospholipase C (PLC) inhibitor, U73122 [GenBank] [1-[6-[[(17)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]-1H-pyrrole-2,5-dione], blocked ERK1/2 activation induced by CHA. Transfection of short interfering RNA targeting PKC blocked the CHA-induced ERK1/2 activation and the secretion of MMP-2. These results confirm the existence of functional adenosine A₁ receptors in the trabecular meshwork cells. These receptors are coupled to the activation of ERK1/2 through G_i/o proteins and dependent upon the upstream activation of PLC and PKC. These studies provide evidence that adenosine A₁ receptor agonists increase outflow facility through sequential activation of G_i/o > PLC > PKC > c-Raf > mitogen-activated protein kinase kinase > ERK1/2, leading to secretion of MMP-2.

Address correspondence to: Dr. Craig E. Crosson, Medical University of South Carolina, 167 Ashley Avenue, Charleston, SC 29425. E-mail: crossonc{at}musc.edu