Mechanism of superoxide anion production by hepatic sinusoidal endothelial cells and Kupffer cells during short-term ethanol perfusion in the rat

Background/Aims: The aim of this study was to clarify the candidate cells for and the mechanism of superoxide anion (O-2 (.-) ) release into the hepatic sinusoids during short-term exposure to ethanol. Methods: The rat liver was perfused continuously with ethanol (a substrate for alcohol dehydrogenase) or tert -buthanol (not a substrate for alcohol dehydrogenase) for 20 min at a final concentration of 40 mM. In order to detect O-2 (.-) production, MCLA (2-methyl-6-[p -methoxyphenyl]-3,7-dihydroimidazo[1,2-a ]pyrazin-3-one), a Cypridina luciferin analogue, was simultaneously infused and MCLA-enhanced chemiluminescence was measured. The effects of gadolinium chloride (GdCL3 ) (a suppressor of Kupffer cells (KCs)), staurosporine (ST) (an inhibitor of serine-threonine kinases, including protein kinase C), diphenyleneiodonium chloride (DPI) (an inhibitor of NADPH oxidase), ibuprofen (IB) (an inhibitor of cyclooxygenase) and 4-methylpyrazole (4MP) (an inhibitor of ethanol metabolism) on the ethanol-induced chemiluminescence were also evaluated. Sites where O-2 (.-) could be released were determined by histochemical detection of nitro blue tetrazolium reduction. Results: Both ethanol and tert -buthanol rapidly caused O-2 (.-) release. GdCL3 suppressed the ethanol-induced O-2 (.-) release by 61%. Staurosporine and DPI, but neither IB nor 4-MP, also significantly inhibited the ethanol-induced O-2 (.-) release. In the histochemical examination, ethanol-stimulated liver showed blue formazan precipitate on both sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), whereas the GdCl3 -pretreated liver had the precipitate only on SECs. Conclusions: This study shows that ethanol itself stimulates both SECs and KCs to release O-2 (.-) via activation of NADPH oxidase probably involving protein kinase C (PKC).