Background/Aims: The aim of this study was to clarify the candidate cells for and the mechanism of superoxide anion (O₂^·−) 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 O₂^·− 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 O₂^·− could be released were determined by histochemical detection of nitro blue tetrazolium reduction.

Results: Both ethanol and tert‐buthanol rapidly caused O₂^·− release. GdCL₃ suppressed the ethanol‐induced O₂^·− release by 61%. Staurosporine and DPI, but neither IB nor 4‐MP, also significantly inhibited the ethanol‐induced O₂^·− 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 O₂^·− via activation of NADPH oxidase probably involving protein kinase C (PKC).