Department of Pharmacology, Dalhousie University, Halifax, Canada.
J Ocul Pharmacol Ther. 2012 Feb;28(1):17-25. doi: 10.1089/jop.2011.0016. Epub 2011 Oct 19.
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.
β-肾上腺素能受体（βAR）激动剂通过增加小梁网（TM）中的房水流出来降低眼内压（IOP）。然而，尽管这种作用已经得到很好的证实，但负责的特定信号机制尚不清楚。为了解决这个问题，目前的研究检查了原代人眼小梁细胞（HTMC）中的βAR 信号，特别是关注βAR 激活对细胞外信号调节激酶 1/2（ERK）的影响。
培养 HTMC 并通过免疫荧光和逆转录聚合酶链反应评估 βAR 表达。通过 In-Cell Western™分析检查βAR 激活对 ERK 磷酸化的影响。使用药理学方法来表征βAR 对 ERK 的作用机制。
用非选择性βAR 激动剂异丙肾上腺素（ISO）处理 HTMC 可通过β2AR 降低基础磷酸化 ERK（pERK）水平。该反应对百日咳毒素（PTx）不敏感，但霍乱毒素（CTx）预处理导致反应逆转，使 ISO 处理反而增加了 pERK，从而表明 Gα（s）参与了抑制性 pERK 反应。腺苷酸环化酶激活剂 forskolin 也降低了 pERK，表明涉及腺苷酸环化酶和 cAMP，而蛋白激酶 A（PKA）抑制剂 H-89 阻断了 HTMC 中 ISO 和 forskolin 介导的 pERK 抑制。最后，对 CTx 存在下产生的 pERK 增加进行更仔细的检查表明，它对 PTx 也不敏感，并且与 HTMC 中抑制性 pERK 途径相比，它似乎对各种βAR 激动剂具有不同的效力顺序。
在 HTMC 中鉴定了一种导致 pERK 降低的新型β（2）AR 信号通路，该信号通路依赖于 Gα（s）、cAMP 和 PKA。还鉴定了一种导致 pERK 增加的竞争性β（2）AR 信号通路。由于βAR 对房水（AH）流出的影响与 cAMP 有关，并且最近有人提出抑制 TM 细胞中的 ERK 可增加 AH 流出，因此我们的研究结果表明，抑制性β（2）AR-pERK 途径可能是βAR 激动剂降低 IOP 的机制。同一细胞中存在竞争性β（2）AR-ERK 激活途径表明，这是开发和验证功能选择性β（2）AR 激动剂的理想系统。
