Heme oxygenase attenuates angiotensin II-mediated superoxide production in cultured mouse thick ascending loop of Henle cells

Heme oxygenase (HO)-1 induction can attenuate the development of angiotensin II (ANG II)-dependent hypertension. However, the mechanism by which HO-1 lowers blood pressure is not clear. The goal of this study was to test the hypothesis that induction of HO-1 can reduce the ANG II-mediated increase in superoxide production in cultured thick ascending loop of Henle (TALH) cells. Studies were performed on an immortalized cell line of mouse TALH (mTALH) cells. HO-1 was induced in cultured mTALH cells by treatment with cobalt protoporphyrin (CoPP, 10 mu M) or hemin (50 mu M) or by transfection with a plasmid containing the human HO-1 isoform. Treatment of mTALH cells with 10(-9) M ANG II increased dihydroethidium (DHE) fluorescence (an index of superoxide levels) from 35.5 +/- 5 to 136 +/- 18 relative fluorescence units (RFU)/mu m(2). Induction of HO-1 via CoPP, hemin, or overexpression of the human HO-1 isoform significantly reduced ANG II-induced DHE fluorescence to 64 +/- 5, 64 +/- 8, and 41 +/- 4 RFU/mu m(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 mu M) before exposure to ANG II. DHE fluorescence averaged 80 +/- 7 RFU/mu m(2) after incubation with ANG II and was significantly decreased to 55 +/- 7 and 53 +/- 4 RFU/mu 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.