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Identification of novel competing β2AR phospho-extracellular signal regulated kinase 1/2 signaling pathways in human trabecular meshwork cells



Identification of novel competing β2AR phospho-extracellular signal regulated kinase 1/2 signaling pathways in human trabecular meshwork cells



Journal of Ocular Pharmacology and Therapeutics 28(1): 17-25



β-Adrenergic receptor (βAR) agonists reduce intraocular pressure (IOP) by increasing aqueous outflow through the trabecular meshwork (TM). However, although this effect is well established, the specific signaling mechanisms responsible are less clear. To address this, the current study examined βAR signaling in primary human trabecular meshwork cells (HTMCs), specifically, focusing on the effect of βAR activation on the extracellular signal regulated kinases 1/2 (ERK). HTMCs were cultured and assessed for βAR expression by both immunofluorescence and reverse-transcription-polymerase chain reaction. The effect of βAR activation on ERK phosphorylation was examined in these cells by In-Cell Western™ analysis. Pharmacological approaches were used to characterize the mechanism of the βAR effect on ERK. Treatment of HTMCs with the nonselective βAR agonist, isoproterenol (ISO), decreased the basal phospho-ERK (pERK) level, through actions at the β2AR. The response was insensitive to pertussis toxin (PTx), but pretreatment with cholera toxin (CTx) resulted in a reversal of the response, such that ISO treatment instead increased pERK, thus implicating Gα(s) in the inhibitory pERK response. The adenylyl cyclase activator, forskolin, also decreased pERK, suggesting the involvement of adenylyl cyclase and cAMP, whereas a protein kinase A (PKA) inhibitor, H-89, blocked both ISO and forskolin-mediated pERK inhibition in HTMCs. Finally, a closer examination of the pERK increase generated in the presence of CTx demonstrated that it was also insensitive to PTx, and appeared to have differing rank orders of efficacy for various βAR agonists compared with the inhibitory pERK pathway in HTMCs. A novel β(2)AR-signaling pathway leading to a decrease in pERK, which was dependent on Gα(s), cAMP, and PKA, was identified in HTMCs. A competing β(2)AR signaling pathway resulting in increased pERK was also identified. Since βAR effects on aqueous humor (AH) outflow have been linked to cAMP, and inhibition of ERK in TM cells has recently been suggested as increasing AH outflow, our findings suggest that the inhibitory β(2)AR-pERK pathway likely represents the mechanism by which βAR agonists decrease IOP. The presence of a competing β(2)AR-ERK activation pathway in the same cells suggests that this is an ideal system for the development and validation of functionally selective β(2)AR agonists.

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Accession: 053668649

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PMID: 22010988

DOI: 10.1089/jop.2011.0016


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