The aim of the present study was to investigate the role of hemoglobin autoxidation in the induction of endothelial heme oxygenase (HO), an inducible "stress" protein which is responsible for heme catabolism. Porcine aortic endothelial cells were incubated for six hours in the presence of 60 microM unmodified hemoglobin (HbA0), hemoglobin cross-linked between the alpha chains with bis-(3,5-dibromosalicyl) fumarate (alpha alpha Hb) or cyanomet-alpha alpha-hemoglobin (CNmet alpha alpha Hb). Microsomal HO content increased 4.1-fold in the presence of alpha alpha Hb, 2.7-fold with HbA0 and 1.8-fold with CNmet alpha alpha Hb over the control value. The rates of methemoglobin formation exhibited a linear relationship over the time of incubation (r = 0.94) and the apparent rate constant was 1.8-fold higher for alpha alpha Hb (0.023 h-1) than HbA0 (0.013 h-1). In addition, a linear relationship was obtained by plotting the rates of autoxidation of hemoglobins versus the HO activity (r = 0.99). When cells were incubated with 100% methemoglobin, HO activity increased 5.0-fold and 4.7-fold for HbA0 and alpha alpha Hb, respectively. Intracellular heme concentration, measured after 24 hours of incubation, was also significantly greater in the presence of alpha alpha Hb (52.6% over baseline) compared to HbA0 (10.8%) and CNmet alpha alpha Hb (15.3%) groups (p < 0.05). However, lactate dehydrogenase (LDH) release, measured as an index of endothelial cell injury, increased in all the hemoglobins examined: alpha alpha Hb, 33.8 +/- 1.1 U/l; HbA0, 38.5 +/- 3.5 U/l; CNmet alpha alpha Hb, 41.9 +/- 4.0 U/l; (control group, 19.4 +/- 2.8 U/l). We conclude that: 1) the higher rate of oxidation of alpha alpha Hb renders the molecule more susceptible to induce endothelial oxidative stress (HO induction); 2) the accelerated methemoglobin formation is directly correlated to intracellular HO content and endothelial heme uptake; 3) persistent cell injury suggests that other factors besides heme release may contribute to the hemoglobin-mediated cytotoxicity.