Ethanol Stimulates Ciliary Beating by Dual Cyclic Nucleotide Kinase Activation in Bovine Bronchial Epithelial Cells

Abstract

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).

Desensitization of PKA‐Stimulated Ciliary Beat Frequency in an Ethanol‐Fed Rat Model of Cigarette Smoke Exposure

“…We observed that ethanol acutely elevates ciliary motility in an in vitro cell culture model. In this model, ethanol requires an adenosine 3':5'-cyclic monophosphate (cAMP) and nitric oxide-dependent signaling pathway (Sisson et al, 1999;Wyatt et al, 2003). This follows the observation that either cAMP or cyclic guanosine monophosphate (cGMP) elevations result in increased cilia beating (Wyatt et al, 1998).…”

Alcohol and airways function in health and disease

“…These investigators found that CBF was stimulated by low concentrations of alcohol (0.01-0.1% or ≈ 2-20 mM), not changed by modest concentrations of alcohol (0.5-1.0% or ≈ 100-200 mM) and slowed at higher concentrations of alcohol (2% or ≈ 400 mM). Subsequent studies in bovine tracheal ciliated cells established that alcohol stimulation of CBF was dependent on nitric oxide production in airway epithelium (Sisson, 1995) and required the dual activation of both cyclic AMP-and cyclic GMP-dependent protein kinases, PKA and PKG, in ciliated cells (Sisson et al, 1999;Wyatt et al, 2003). This transient alcohol stimulation effect on cilia was recapitulated in vivo in alcohol-fed rats (Wyatt et al, 2004).…”

“…The effects of alcohol on mucociliary function (also see Figure 1) (Maurer and Liebman, 1988;Sisson, 1995;Sisson et al, 1999;Wyatt et al, 2003;Wyatt et al, 2004) Cilia kinases desensitized • prolonged exposure to alcohol slows cilia beating Wyatt et al, 2004) Clearance slowed • in vivo after intraperitoneal injection of alcohol (Laurenzi and Guarneri, 1966) Cilia motility desensitized • prolonged exposure to alcohol desensitizes cilia to betaagonists (Wyatt and Sisson, 2001) Atopic patients wheeze from alcohol congeners but not pure alcohol (Breslin et al, 1973) Alcohol ingestion improves vital capacity (Herxheimer and Stresemann, 1963) Nebulized alcohol triggers coughing and reduction in airflow in healthy volunteers (Zuskin et al, 1981) Alcohol triggers an immediate irritant response and causes a slow, bronchodilator effect (Ayres et al, 1982) Sulfur dioxide in some wines cause wheezing in asthmatics (Dahl et al, 1986) Alcohol administered IV acts as a bronchodilator (Brown, 1947) Acetaldehyde causes "alcohol-induced bronchial asthma" in some Asians (Shimoda et al, 1996) Alcohol administered IV acts as a concentration-dependent bronchodilator (Ayres and Clark, 1983b) Aldehyde dehydrogenase type-2 (ALHD2) deficiency identified as cause of asthma some Asians (Matsuse et al, 2001)…”

Inhibition of Protein Kinase C Epsilon Causes Ciliated Bovine Bronchial Cell Detachment

“…Primary bovine bronchial epithelial cells (BBECs) were prepared from bovine lung as described previously [18]. Briefly, bronchi were dissected from the lung and digested overnight at 4°C in 0.1% bacterial protease type IV in minimum essential medium (M199 with Earl's salts; Gibco, Carlsbad, CA).…”

“…PKCε activity was determined in crude whole cell cytosolic and particulate fractions from BBECs similar to methods described previously [18,19]. Cell monolayers were flash-frozen in liquid N 2 in cell lysis buffer containing 50 mM Tris-HCl (pH 7.4) and 1 μg/mL each of leupeptin A, phenylmethyl-sulphonylfluoride, and aprotinin.…”