Forgèt Mary A, Sisson Joseph H, Spurzem John R, Wyatt Todd A
Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, 985300 Nebraska Medical Center, Omaha, NE 68198-5300, USA.
Alcohol. 2003 Aug-Oct;31(1-2):31-8. doi: 10.1016/j.alcohol.2003.06.005.
Ethanol exposure in airway epithelium increases cyclic AMP (cAMP)-dependent protein kinase (PKA) activity. Activation of PKA and cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) has been shown to increase ciliary beat frequency (CBF) in bovine bronchial epithelial cells (BBECs). We have shown that biologically relevant concentrations of ethanol stimulate increases in CBF in a nitric oxide-dependent manner, mediated through elevated cAMP levels and subsequent PKA activation. This ethanol-driven rapid and transient increase in CBF occurs 15 to 30 min after exposure to 100 mM ethanol. However, after prolonged exposure to 100 mM ethanol (>/=6 h), CBF and the catalytic activity of PKA return to baseline levels. We hypothesize that cyclic nucleotide-dependent phosphodiesterase (PDE) activity attenuates the duration of ethanol-stimulated ciliary motility. The effect of ethanol on the PDE activity in BBECs was determined through direct assay of catalytic activity. When BBECs were incubated with 100 mM ethanol, significant increases in cAMP levels occurred within 1 h, with corresponding increases in PKA activity. Treatment of BBECs with 100 mM ethanol increased cAMP-PDE activity significantly by 4 h. 3-Isobutyl-1-methylxanthine, Ro 20-1724, and rolipram inhibited ethanol-stimulated cAMP-PDE activity. These agents inhibited ethanol-stimulated cAMP-PDE activity and increased the magnitude of ethanol-stimulated PKA activity observed under the same conditions. These findings support the idea that acute exposure (<6 h) to ethanol increases cAMP levels, and the associated increase in PKA activation is regulated by cAMP-dependent PDE, specifically PDE4. Other compensatory mechanisms however, may be responsible for the down-regulation of PKA, which occurs after chronic epithelial exposure (>/=6 h) to ethanol.
气道上皮细胞暴露于乙醇会增加环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）的活性。已表明，PKA和环磷酸鸟苷（cGMP）依赖性蛋白激酶（PKG）的激活可增加牛支气管上皮细胞（BBECs）的纤毛摆动频率（CBF）。我们已经表明，具有生物学相关性的乙醇浓度以一氧化氮依赖性方式刺激CBF增加，这是通过cAMP水平升高和随后的PKA激活介导的。在暴露于100 mM乙醇后15至30分钟，会出现这种由乙醇驱动的CBF快速短暂增加。然而，在长时间暴露于100 mM乙醇（≥6小时）后，CBF和PKA的催化活性会恢复到基线水平。我们推测，环核苷酸依赖性磷酸二酯酶（PDE）活性会减弱乙醇刺激的纤毛运动持续时间。通过直接测定催化活性来确定乙醇对BBECs中PDE活性的影响。当BBECs与100 mM乙醇一起孵育时，1小时内cAMP水平显著增加，PKA活性也相应增加。用100 mM乙醇处理BBECs 4小时后，cAMP-PDE活性显著增加。3-异丁基-1-甲基黄嘌呤、Ro 20-1724和咯利普兰可抑制乙醇刺激的cAMP-PDE活性。这些药物抑制了乙醇刺激的cAMP-PDE活性，并增加了在相同条件下观察到的乙醇刺激的PKA活性的幅度。这些发现支持了这样一种观点，即急性暴露（<6小时）于乙醇会增加cAMP水平，并且PKA激活的相关增加受cAMP依赖性PDE（特别是PDE4）调节。然而，其他补偿机制可能是慢性上皮暴露（≥6小时）于乙醇后PKA下调的原因。
