Chronic ethanol exposure stimulates endothelial cell nitric oxide production through PI-3 kinase-and hsp90-dependent mechanisms.
BACKGROUND
Chronic ethanol (EtOH) ingestion increases the incidence of the Acute Respiratory Distress Syndrome (ARDS), a severe form of acute lung injury characterized by endothelial and epithelial barrier dysfunction. The regulated production of nitric oxide (NO) by the endothelium plays a central role in normal vascular function, and alterations in NO production have been implicated in barrier dysfunction. Although previous reports examined the impact of acute EtOH stimulation on endothelial NO production, this study extends those observations to clarify mechanisms of chronic EtOH-mediated alterations in endothelial nitric oxide synthase (eNOS) expression and NO production.


METHODS
Porcine pulmonary artery endothelial cells (PAEC) were treated with EtOH (0.04-0.16%, w/v) for 72 hr in sealed chambers to prevent evaporation. NO release and eNOS expression were determined to examine the effect of chronic EtOH stimulation on endothelial NO metabolism.


RESULTS
While there was no change in the extent of phosphorylated eNOS at ser, chronic EtOH stimulation caused dose-dependent increases in NO production and increased eNOS expression, effects that were attenuated by the transcriptional inhibitor, alpha-amanitin (AA), and wortmannin, a specific phosphatidylinositol 3 kinase (PI3 K) inhibitor. EtOH stimulation also increased eNOS interaction with heat shock protein (hsp90), a molecular chaperone known to enhance eNOS activity. Geldanamycin, an hsp90 inhibitor, attenuated chronic EtOH-mediated increases in NO production.


CONCLUSIONS
These results indicate that chronic EtOH exposure increases endothelial NO production by increasing eNOS protein levels through PI3 K-dependent up regulation of eNOS gene transcription and by increasing interactions between eNOS and hsp90. These findings clarify mechanisms by which chronic EtOH stimulation modulates vascular endothelial function and suggest new targets for investigation and intervention in EtOH-induced alterations in susceptibility to lung injury.
Polikandriotis
John A
JA
Department of Medicine, Atlanta Veterans Affairs and Emory University Medical Centers, Atlanta, GA 30033, USA. jpolika@emory.edu
Rupnow
Heidi L
HL
Hart
C Michael
CM
D000818:Animals; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011651:Pulmonary Artery / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D012128:Respiratory Distress Syndrome / Q000378:metabolism; D013552:Swine
D000431:Ethanol; D018841:HSP90 Heat-Shock Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases