2.13. Cell stimulation
VSMC were stimulated with ethanol 50 mM for 1 min. The concentration and period of exposition to ethanol were based in previous studies [5] and [9]. Superoxide anion production was measured with the use of lucigenin-enhanced chemiluminescence as described previously [8]. In another set of experiments O2− production was measured in response to ethanol (50 mM, 1 min), after pre-incubation of VSMC with apocynin (NAD(P)H oxidase inhibitor, 10 μM), tiron (O2− scavenger, 10 μM) or vitamin C (100 μM). Vitamin C was incubated for 24 h [23], while the other inhibitors were incubated for 30 min. Superoxide anion production is expressed as percentage of increase from baseline values in vehicle-treated cells.