Wyatt Todd A, Forgèt Mary A, Sisson Joseph H
Research Service, Department of Veterans Affairs Medical Center, Omaha, Nebraska 68198, USA.
Am J Pathol. 2003 Sep;163(3):1157-66. doi: 10.1016/S0002-9440(10)63475-X.
Previously, we have shown that ethanol (EtOH) stimulates a rapid increase in the ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBECs) via the activation of PKA. We have also shown that inhibitors of nitric oxide synthase block EtOH-stimulated increases in CBF. We hypothesize that EtOH acutely stimulates CBF via the activation of both PKA and PKG pathways. Using chemiluminescence detection of nitric oxide (NO), we directly measured increases in NO production in BBECs treated with 100 mmol/L of EtOH beginning at 25 minutes. Pretreatment of BBECs with guanylyl cyclase inhibitors, ODQ or LY83583, resulted in the inhibition of EtOH-stimulated CBF. Low concentrations (1 nmol/L) of cyclic nucleotide analogues do not stimulate CBF increases. However, a combination of both 1 nmol/L of 8Br-cAMP and 8Br-cGMP stimulates a significant increase over baseline CBF. This effect could be blocked by pretreating BBECs with inhibitors of either PKA or PKG. Very high concentrations of either 8Br-cAMP or 8Br-cGMP (> or =100 micromol/L) were required to cross-activate both PKA and PKG. This suggests that cross-activation of PKA by cGMP is not occurring at the concentrations (1 nmol/L) capable of stimulating CBF. 8-pCPT-cGMPS, an antagonist analogue to cGMP, blocked EtOH-stimulated PKA activity increases. These data support that EtOH-stimulated increases in CBF require the dual activation of both PKA (via cAMP) and PKG (via NO).
此前，我们已经表明，乙醇（EtOH）通过激活蛋白激酶A（PKA）刺激牛支气管上皮细胞（BBECs）的纤毛摆动频率（CBF）迅速增加。我们还表明，一氧化氮合酶抑制剂可阻断乙醇刺激的CBF增加。我们假设乙醇通过激活PKA和蛋白激酶G（PKG）途径急性刺激CBF。使用一氧化氮（NO）的化学发光检测，我们直接测量了从25分钟开始用100 mmol/L乙醇处理的BBECs中NO产生的增加。用鸟苷酸环化酶抑制剂ODQ或LY83583预处理BBECs会导致乙醇刺激的CBF受到抑制。低浓度（1 nmol/L）的环核苷酸类似物不会刺激CBF增加。然而，1 nmol/L的8-溴腺苷-3',5'-环磷酸（8Br-cAMP）和8-溴鸟苷-3',5'-环磷酸（8Br-cGMP）的组合刺激CBF比基线有显著增加。这种效应可以通过用PKA或PKG抑制剂预处理BBECs来阻断。需要非常高浓度的8Br-cAMP或8Br-cGMP（≥100 μmol/L）才能交叉激活PKA和PKG。这表明在能够刺激CBF的浓度（1 nmol/L）下，cGMP不会发生对PKA的交叉激活。8-对氯苯硫基鸟苷-3',5'-环磷酸酯（8-pCPT-cGMPS），一种cGMP的拮抗剂类似物，阻断了乙醇刺激的PKA活性增加。这些数据支持乙醇刺激的CBF增加需要PKA（通过cAMP）和PKG（通过NO）的双重激活。
