Department of Internal Medicine, Pulmonary, Critical Care, Sleep, & Allergy Division, University of Nebraska Medical Center, Omaha, Nebraska 68198-5910, USA.
Am J Respir Cell Mol Biol. 2010 Nov;43(5):539-45. doi: 10.1165/rcmb.2009-0252OC. Epub 2009 Nov 20.
Airway smooth muscle cells are the main effector cells involved in airway narrowing and have been used to study the signaling pathways involved in asthma-induced airway constriction. Our previous studies demonstrated that ethanol administration to mice attenuated methacholine-stimulated increases in airway responsiveness. Because ethanol administration attenuates airway responsiveness in mice, we hypothesized that ethanol directly blunts the ability of cultured airway smooth muscle cells to shorten. To test this hypothesis, we measured changes in the size of cultured rat airway smooth muscle (RASM) cells exposed to ethanol (100 mM) after treatment with methacholine. Ethanol markedly attenuated methacholine-stimulated cell shortening (methacholine-stimulated length change = 8.3 ± 1.2% for ethanol versus 43.9 ± 1.5% for control; P < 0.001). Ethanol-induced inhibition of methacholine-stimulated cell shortening was reversible 24 hours after removal of alcohol. To determine if ethanol acts through a cGMP-dependent pathway, incubation with ethanol for as little as 15 minutes produced a doubling of cGMP-dependent protein kinase (PKG) activity. Furthermore, treatment with the PKG antagonist analog Rp-8Br-cGMPS (10 μM) inhibited ethanol-induced kinase activation when compared with control-treated cells. In contrast to the effect of ethanol on PKG, ethanol pretreatment did not activate a cAMP-dependent protein kinase. These data demonstrate that brief ethanol exposure reversibly prevents methacholine-stimulated RASM cell contraction. In addition, it appears that this effect is the result of activation of the cGMP/PKG kinase pathway. These findings implicate a direct effect of ethanol on airway smooth muscle cells as the basis for in vivo ethanol effects.
气道平滑肌细胞是参与气道狭窄的主要效应细胞，已被用于研究哮喘引起的气道收缩涉及的信号通路。我们之前的研究表明，给小鼠给予乙醇可减弱乙酰甲胆碱刺激引起的气道反应性增加。由于乙醇给药可减弱小鼠的气道反应性，我们假设乙醇直接削弱培养的气道平滑肌细胞缩短的能力。为了验证这一假设，我们测量了暴露于乙醇（100mM）的培养大鼠气道平滑肌（RASM）细胞在乙酰甲胆碱处理后大小的变化。乙醇显著减弱了乙酰甲胆碱刺激的细胞缩短（乙醇刺激的长度变化为 8.3±1.2％，而对照为 43.9±1.5％；P<0.001）。乙醇诱导的对乙酰甲胆碱刺激的细胞缩短的抑制在酒精去除 24 小时后是可逆的。为了确定乙醇是否通过 cGMP 依赖性途径起作用，与乙醇孵育仅 15 分钟即可使 cGMP 依赖性蛋白激酶（PKG）活性增加一倍。此外，与对照处理的细胞相比，用 PKG 拮抗剂类似物 Rp-8Br-cGMPS（10μM）处理可抑制乙醇诱导的激酶激活。与乙醇对 PKG 的作用相反，乙醇预处理不会激活 cAMP 依赖性蛋白激酶。这些数据表明，短暂的乙醇暴露可可逆地阻止乙酰甲胆碱刺激的 RASM 细胞收缩。此外，似乎这种作用是 cGMP/PKG 激酶途径激活的结果。这些发现表明，乙醇对气道平滑肌细胞的直接作用是体内乙醇作用的基础。
