PT  - JOURNAL ARTICLE
AU  - S. Husain
AU  - T. W. Shearer
AU  - C. E. Crosson
TI  - Mechanisms Linking Adenosine A<sub>1</sub> Receptors and Extracellular Signal-Regulated Kinase 1/2 Activation in Human Trabecular Meshwork Cells
AID  - 10.1124/jpet.106.110981
DP  - 2007 Jan 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 258--265
VI  - 320
IP  - 1
4099  - http://jpet.aspetjournals.org/content/320/1/258.short
4100  - http://jpet.aspetjournals.org/content/320/1/258.full
SO  - J Pharmacol Exp Ther2007 Jan 01; 320
AB  - 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 PKCα from the cytosol to the membrane, and pretreatment with the phospholipase C (PLC) inhibitor, U73122 [1-[6-[[(17β)-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 PKCα 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 Gi/o proteins and dependent upon the upstream activation of PLC and PKCα. These studies provide evidence that adenosine A1 receptor agonists increase outflow facility through sequential activation of Gi/o > PLC > PKCα > c-Raf > mitogen-activated protein kinase kinase > ERK1/2, leading to secretion of MMP-2. The American Society for Pharmacology and Experimental Therapeutics