Because alveolar macrophages generate and release reactive oxygen metabolites but also contain antioxidative enzymes, they have the potential of either damaging or protecting tissues. We investigated the relative role of the hydrogen peroxide (H2O2)-scavenging antioxidative enzymes in H2O2 disposal and cell protection using freshly isolated (5 h ex vivo) and overnight (24 h ex vivo) cultured human alveolar macrophages. Cell protection was assessed on the basis of maintenance of cellular high-energy phosphates, leakage of intact nucleotides into the extracellular medium, and appearance of the nucleotide catabolic products xanthine, hypoxanthine, and uric acid. To investigate the relative importance of catalase and the glutathione redox cycle, the experiments were conducted in cells pretreated with amino-triazole (ATZ) to inactivate catalase or with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inactivate glutathione reductase. Catalase, glutathione peroxidase, and glutathione reductase activities did not change significantly during overnight culture of the cells. Both freshly isolated and cultured cells consumed exogenous H2O2 mainly by the catalase-dependent pathway. When the cells were exposed to H2O2 (100 microM), catalase and the glutathione redox cycle equally participated in maintaining cellular high-energy nucleotides. However, when cultured cells were exposed to formylated peptide (FMLP) (10(-7) M), the glutathione redox cycle was responsible for the maintenance of high-energy nucleotides. Furthermore, in both exposures, the glutathione redox cycle was more important in maintaining cell membrane integrity and preventing nucleotide leakage from the cells. Immunocytochemical labeling showed that catalase was primarily localized in the peroxisomal compartment of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)