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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02914

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Inhibition of cellular respiration by endogenously produced carbon monoxide

Gabriela D'Amico^*, Francis Lam^*, Thilo Hagen and Salvador Moncada

Wolfson Institute for Biomedical Research, University College London, Gower Street, London, WC1E 6BT, UK

View larger version (18K): [in a new window]   Fig. 1. Inhibition of cellular respiration by exogenous CO. (A) (i) Tracing of O2 consumption of control HEK293 cells. (ii) Tracing of O2 consumption of cells treated with 20 µM CO. (iii) Tracing of O2 consumption showing the effect of 5 µM DEANO (addition indicated by arrow). The rate of respiration recovers after the NO is degraded. Insert: the slopes obtained from the Lineweaver-Burk plot were plotted against [CO] (secondary plot). The points plotted were obtained from the means of at least three independent experiments. The Ki of CO on respiration calculated by interpolation to the x-axis was 1.44 µM. (B) The rates of respiration of HEK293 cells were measured 0, 10, 20 and 30 minutes after the addition of 20 µM CO, n=3. Tracings are representative of at least three independent experiments. *P<0.01 compared with control.

View larger version (8K): [in a new window]   Fig. 2. Effect of hypoxia (1% O2 for 10 minutes) on the inhibition of cellular respiration by exogenous CO in HEK293 cells. (i) Tracing of O2 consumption of control cells incubated in hypoxia. (ii) Tracing of O2 consumption of cells treated with 20 µM CO under ambient O2 concentrations. (iii) Tracing of O2 consumption of cells treated with 20 µM CO in hypoxia. No difference was observed between untreated control cells kept under ambient O2 concentrations and those incubated in hypoxia. The Ki of CO for cells in hypoxia was calculated to be 0.35 µM. Tracings are representative of at least three independent experiments.

View larger version (31K): [in a new window]   Fig. 3. Induced HEK293-HO-1 cells expressed HO-1 protein and exhibited a decreased rate of respiration. (A) HEK293-HO-1 cells were induced to express HO-1 protein by treatment with 1 µg/ml tetracycline (Tet) for 24 hours. The expression was compared with that of Hep3B cells treated with known inducers of HO-1; LPS (1 µg/ml and 50 µg/ml), CoCl2 (200 µM) and haemin (10 µM). (B) The respiration of induced HEK293-HO-1 cells incubated under ambient O2 concentrations (ii) and hypoxic conditions (iii) was decreased compared with that of non-induced cells (i). Tracings and western blots are representative of at least three independent experiments.

View larger version (25K): [in a new window]   Fig. 4. The expression and activity of HO-1 protein in HEK293-HO-1 cells is not altered by incubation under hypoxic conditions. (A) Western blot of cell extracts from induced (24 hours) and non-induced cells incubated under hypoxic (1% O2) and ambient (21% O2) conditions; Tet, tetracycline. (B) HO-1 activity is not affected by incubation under hypoxic or ambient conditions. (C) The concentrations of bilirubin measured in the culture medium. n=3. *P<0.05 compared with control at 21% O2; **P<0.01 compared with control at 1% O2.

View larger version (36K): [in a new window]   Fig. 5. Endogenously produced CO does not activate the p38 MAPK pathway in HEK293-HO-1 cells. (A) Phosphorylation of p38 (phospho-p38) was observed following treatment with GSNO (1 mM for 8 hours) and anisomycin (25 µg/ml for 20 minutes). (B) p38 phosphorylation was not observed in our induced HEK293-HO-1 cells after 24 hours in either ambient or hypoxic conditions; Tet, tetracycline. Blots are representative of at least three independent experiments.

View larger version (51K): [in a new window]   Fig. 6. Effect of activation of RAW264.7 cells on the expression of HO-1, iNOS and the rate of respiration. (A) Cells that were activated for 12 hours in the presence or absence of L-NIO (500 µM) under ambient O2 or hypoxic conditions expressed HO-1 and iNOS protein. Hypoxia itself induced HO-1 but not iNOS. (B) The respiration of activated cells was almost completely inhibited. Cells activated in the presence of L-NIO did not synthesise NO but still exhibited an inhibition of respiration that was increased by hypoxic incubation. n=3-4. **P<0.01 compared with control; *P<0.01 compared with respective L-NIO-treated controls; $P<0.01 compared with L-NIO-treated cells maintained at 21% O2; #P<0.01 compared with cells activated in the presence of L-NIO at 21% O2.

View larger version (13K): [in a new window]   Fig. 7. The synthesis of NO is inhibited by hypoxia. (A) The production of NO by RAW264.7 cells activated for 24 hours in hypoxia was inhibited by 85%, as determined by measurement of its metabolite nitrite (NO2-). (B) The synthesis of NO decreased from a concentration of 2.3 µM at 100 µM O2 to 0.3 µM at 10 µM O2 (indicated by dashed arrows). The Km of iNOS for O2 in RAW264.7 cells was between 20 and 30 µM (indicated by the dotted arrows). n=3. *P<0.01 compared with cells activated at 1% O2.