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Background Cilia are finger-like motor-driven organelles, which propel inhaled particles and mucus from the lung and airways. We have previously shown that brief alcohol exposure stimulates ciliary motility through an endothelial nitric oxide synthase (eNOS)-dependent pathway localized in the ciliary metabolon. However, the signaling molecules of the ciliary metabolon involved in alcohol-triggered ciliary beat frequency (CBF) stimulation upstream of eNOS activation remain unknown. Methods We hypothesized that brief alcohol exposure alters threonine and serine phosphorylation of proteins involved in stimulating CBF. Two-dimensional electrophoresis indicated both increases and decreases in the serine and threonine phosphorylation states of several proteins. One of the proteins identified was heat shock protein 90 (HSP90), which undergoes increased threonine phosphorylation after brief alcohol exposure. Because HSP90 has been shown to associate with eNOS in lung tissue, we hypothesized that HSP90 is a key component in alcohol-triggered eNOS activation and that these 2 proteins co-localize within the ciliary metabolon. Results Immunofluorescence experiments demonstrate that eNOS and HSP90 co-localize within basal bodies of the ciliary metabolon and partially translocate to the axoneme upon brief alcohol exposure. Pretreatment with geldanamycin, which disrupts HSP90 chaperone functions, prevented eNOS-HSP90 association and prevented the translocation of eNOS from the ciliary metabolon to the axoneme. Functional cilia motility studies revealed that geldanamycin blocked alcohol-stimulated ciliary motility in bovine bronchial epithelial cells and mouse tracheal rings. Conclusions On the basis of the HSP90 localization with eNOS, alcohol activation of HSP90 phosphorylation, and geldanamycin's ability to inhibit HSP90-eNOS association, prevent eNOS translocation to the axoneme, and block alcohol-stimulated ciliary motility, we conclude that alcohol-induced cilia stimulation occurs through the increased association of HSP90 with eNOS. These data help further elucidate the mechanism through which brief alcohol exposure stimulates CBF.

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 (AICD) occurs through impairment of the 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 (ATP). Ciliary beat frequency (CBF), 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 (1-10 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 cGMPdependent 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 alcoholdriven 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.

Background One important contributor to pathologic effects on the liver associated with alcohol abuse is viral hepatitis, especially hepatitis C virus (HCV) infection. Alcohol consumption has been shown to be associated with more severe HCV infection and hepatitis. The mechanisms of the more severe viral infection of the liver are unclear, and studies have been hampered by the lack of an animal model of hepatotropic viral infections. Methods We have established a murine model system of viral hepatitis in which C57BL/6 mice are infected with murine cytomegalovirus, a herpesvirus that produces self-limiting hepatitis in immunocompetent mice. Mice were fed a liquid diet containing 36% ethanol-derived calories with a pair-feeding protocol. After infection with a sublethal dose of murine cytomegalovirus, the severity of liver infection was determined by measuring serum levels of alanine aminotransferase and by histological evaluation. Other parameters determined included the serum levels of cytokines, cytokine RNA in liver samples, and viral concentration in liver samples. Results Ethanol-fed mice showed more severe hepatitis in the later stages of the infection as compared with the hepatitis noted in control mice. The ethanol-fed mice did not control the virus replication in the liver, which was associated with a greater mononuclear cell inflammatory response, composed predominantly of cells with morphological characteristics of lymphocytes and macrophages. The early production of interferon γ, as well as production throughout the infection, was significantly lower in the ethanol-fed mice. The early production of interleukin 12 was also less in ethanol-fed mice. Conclusions The more severe hepatitis seen in the ethanol-fed mice is likely to be the result of an inability to control the growth of the virus, which is associated with a continued inflammatory response. The inability to control the virus in the liver may be related to the decreased production of interferon γ and interleukin 12.