2 , respectively. To determine which metabolite of HO-1 is responsible for reducing ANG II-mediated increases in superoxide production in mTALH cells, cells were preincubated with bilirubin or carbon monoxide (CO)-releasing molecule (CORM)-A1 (each at 100 M) before exposure to ANG II. DHE fluorescence averaged 80 Ϯ 7 RFU/m 2 after incubation with ANG II and was significantly decreased to 55 Ϯ 7 and 53 Ϯ 4 RFU/m 2 after pretreatment with bilirubin and CORM-A1. These results demonstrate that induction of HO-1 in mTALH cells reduces the levels of ANG II-mediated superoxide production through the production of both bilirubin and CO. bilirubin; carbon monoxide; carbon monoxide-releasing molecule A1RECENT STUDIES HAVE DEMONSTRATED the importance of superoxide generation in the development and maintenance of angiotensin II (ANG II)-dependent hypertension (1,14,35). ANG II can increase the production of superoxide by direct activation of NADPH oxidase (6, 7). In the kidney, ANG II-mediated increases in superoxide production have been demonstrated to mediate renal vasoconstriction, cortical hypoxia, and reduced efficiency of O 2 usage for sodium transport (10, 13, 31). Increased superoxide production in the thick ascending loop of Henle (TALH) has been reported to stimulate sodium reabsorption by decreasing the levels of nitric oxide (NO) (19,20).ANG II-mediated increases in superoxide production in the TALH have also been reported to limit NO availability to vasa recta, which could lead to decreases in medullary blood flow, blunting of pressure natriuresis, and hypertension (16). In line with this hypothesis, enhanced expression of NADPH oxidase and increased superoxide production in the medulla have been demonstrated to cause hypertension in normal rats and may contribute to salt-sensitive hypertension in Dahl salt-sensitive rats (17,26).Heme oxygenase (HO)-1 is an important cellular defense protein that is induced by a variety of stimulants including hypoxia and heavy metals. In the rat kidney, HO-1 is mainly expressed in the renal medulla, where it plays an important role in maintaining the blood flow to the renal medulla (36). Increases in HO-1 levels have also been shown to be protective against oxidant damage in tissues such as brain and liver (2, 9). In agreement with these observations, induction of HO-1 in TALH cells was found to limit ANG II-induced oxidative damage (21).While increases in HO-1 levels have been found to limit oxidative damage, the mechanism responsible for this protection is unknown. HO-1 metabolites carbon monoxide (CO) and bilirubin have antioxidant properties that may protect cells against excessive superoxide production. Bilirubin is an antioxidant molecule that can decrease superoxide production by direct actions as well as inhibition of NAD(P)H oxidase activity (11,12,24). CO also can inhibit NAD(P)H oxidase by interactions with the heme-containing subunits (25). We previously reported (29) that induction of HO-1 was associated with a decrease in ANG II-mediated superoxide production in ...