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Proteomic Analysis of Bovine Axonemes Exposed to Acute Alcohol: Role of Endothelial Nitric Oxide Synthase and Heat Shock Protein 90 in Cilia Stimulation
Author(s) -
Simet Samantha M.,
Pavlik Jacqueline A.,
Sisson Joseph H.
Publication year - 2013
Publication title -
alcoholism: clinical and experimental research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.267
H-Index - 153
eISSN - 1530-0277
pISSN - 0145-6008
DOI - 10.1111/acer.12014
Subject(s) - geldanamycin , axoneme , microbiology and biotechnology , heat shock protein , endothelial nos , motility , cilium , nitric oxide synthase , phosphorylation , biology , nitric oxide , hsp90 , chemistry , enos , biochemistry , endocrinology , gene , flagellum
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 (e NOS )‐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 e NOS 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 ( HSP 90), which undergoes increased threonine phosphorylation after brief alcohol exposure. Because HSP 90 has been shown to associate with e NOS in lung tissue, we hypothesized that HSP 90 is a key component in alcohol‐triggered e NOS activation and that these 2 proteins co‐localize within the ciliary metabolon. Results Immunofluorescence experiments demonstrate that e NOS and HSP 90 co‐localize within basal bodies of the ciliary metabolon and partially translocate to the axoneme upon brief alcohol exposure. Pretreatment with geldanamycin, which disrupts HSP 90 chaperone functions, prevented e NOS ‐ HSP 90 association and prevented the translocation of e NOS 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 HSP 90 localization with e NOS , alcohol activation of HSP 90 phosphorylation, and geldanamycin's ability to inhibit HSP 90‐e NOS association, prevent e NOS translocation to the axoneme, and block alcohol‐stimulated ciliary motility, we conclude that alcohol‐induced cilia stimulation occurs through the increased association of HSP 90 with e NOS . These data help further elucidate the mechanism through which brief alcohol exposure stimulates CBF .