Alcohol stimulates ciliary motility of isolated airway axonemes through a nitric oxide, cyclase, and cyclic nucleotide-dependent kinase mechanism.
BACKGROUND
Lung mucociliary clearance provides the first line of defense from lung infections and is impaired in individuals who consume heavy amounts of alcohol. Previous studies have demonstrated that this alcohol-induced ciliary dysfunction occurs through impairment of nitric oxide (NO) and cyclic nucleotide-dependent kinase-signaling pathways in lung airway ciliated epithelial cells. Recent studies have established that all key elements of this alcohol-driven signaling pathway co-localize to the apical surface of the ciliated cells with the basal bodies. These findings led us to hypothesize that alcohol activates the cilia stimulation pathway at the organelle level. To test this hypothesis we performed experiments exposing isolated demembranated cilia (isolated axonemes) to alcohol and studied the effect of alcohol-stimulated ciliary motility on the pathways involved with isolated axoneme activation.


METHODS
Isolated demembranated cilia were prepared from bovine trachea and activated with adenosine triphosphate. Ciliary beat frequency, NO production, adenylyl and guanylyl cyclase activities, cAMP- and cGMP-dependent kinase activities were measured following exposure to biologically relevant concentrations of alcohol.


RESULTS
Alcohol rapidly stimulated axoneme beating 40% above baseline at very low concentrations of alcohol (1 to 10 mM). This activation was specific to ethanol, required the synthesis of NO, the activation of soluble adenylyl cyclase (sAC), and the activation of both cAMP- and cGMP-dependent kinases (PKA and PKG), all of which were present in the isolated organelle preparation.


CONCLUSIONS
Alcohol rapidly and sequentially activates the eNOS-->NO-->GC-->cGMP-->PKG and sAC-->cAMP--> PKA dual signaling pathways in isolated airway axonemes. These findings indicate a direct effect of alcohol on airway cilia organelle function and fully recapitulate the alcohol-driven activation of cilia known to exist in vivo and in intact lung ciliated cells in vitro following brief moderate alcohol exposure. Furthermore, these findings indicate that airway cilia are exquisitely sensitive to the effects of alcohol and substantiate a key role for alcohol in the alterations of mucociliary clearance associated with even low levels of alcohol intake. We speculate that this same axoneme-based alcohol activation pathway is down regulated following long-term high alcohol exposure and that the isolated axoneme preparation provides an excellent model for studying the mechanism of alcohol-mediated cilia dysfunction.
Sisson
Joseph H
JH
Department of Internal Medicine, University of Nebraska Medical Center, Pulmonary, Critical Care, Sleep and Allergy Section, Omaha, Nebraska 68198-5300, USA. jsisson@unmc.edu
Pavlik
Jacqueline A
JA
Wyatt
Todd A
TA
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D054468:Axoneme / Q000187:drug effects* / Q000502:physiology; D002417:Cattle; D002923:Cilia / Q000187:drug effects* / Q000502:physiology; D002925:Ciliary Motility Disorders / Q000378:metabolism / Q000503:physiopathology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D017867:Cyclic Nucleotide-Regulated Protein Kinases / Q000378:metabolism*; D004195:Disease Models, Animal; D000431:Ethanol / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014132:Trachea / Q000187:drug effects* / Q000502:physiology
D009569:Nitric Oxide; D000431:Ethanol; D017867:Cyclic Nucleotide-Regulated Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase