|Matallo J, Reich V, Calzia E, Radermacher P, Groeger M (2010) Interactions between sulfide and mitochondrial respiration in cultured murine alveolar macrophage. Abstract. MiP2010.|
Link: Abstracts Session 5
Inhabitants of sulfide-rich environments like the ribbed mussel Geukensia demissa  or the lugworm Arenicola marina  are capable of using sulfide as a substrate for mitochondrial respiration. Actually, this capacity seems to be maintained even in mammalian and also human cells . In these species, however, increasing the sulfide concentration will inhibit cytochrome c oxidase (CIV) and consequently mitochondrial respiration. Thus, in mitochondria from mammalians, sulfide-consumption may be regarded as part of the metabolism of sulphur-containing amino-acids rather than an energy providing pathway. Nevertheless, it may be a way to control the toxicity of sulfide at low concentrations. Therefore, given the actual interest in the role of sulfide as a potential pharmacologic agent , we quantified in vitro the inhibition of mitochondrial respiration by sulfide in cultured murine alveolar macrophages paying particular attention at the influence of temperature and metabolic activity of the cells.
• Keywords: Hydrogen sulfide, Lung, Murine alveolar macrophages
• O2k-Network Lab: DE Ulm Radermacher P
Labels: MiParea: Respiration
Tissue;cell: Lung;gill, Macrophage-derived Preparation: Permeabilized cells Enzyme: Complex I, Complex II;succinate dehydrogenase, Complex IV;cytochrome c oxidase Regulation: Flux control, Inhibitor, Temperature Coupling state: OXPHOS, ET Pathway: N, S HRR: Oxygraph-2k, TIP2k
Sulfide-inhibition was measured at 25 and 37 °C using an Oroboros Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) by titrating Na2S in steps of 1, 2, 4, 8, 16, 32, and 64 µM after maximum stimulation of mitochondrial respiration achieved by addition of substrates for Complex I (CI; pyruvate 10 mM, malate 5 mM, and glutamate 10 mM) and CII (succinate 10 mM) in cells previously uncoupled by 1 µM FCCP. In separate experiments, the Na2S-titration was performed after blocking CI and CIII by rotenone (0.5 µM) and antimycin A (5 µM) and stimulating CIV by addition of ascorbate (2 mM) and TMPD (0.5 mM). An analogous titration protocol was repeated at 37 °C replacing Na2S with sodium azide (Azd) in 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6 mM-steps as a CIV-inhibitor that does not undergo any metabolism at the mitochondrial level. To quantify the effects of sulfide metabolism the inhibition of fully stimulated mitochondrial respiration and after selective CIV stimulation was compared to calculate an activity threshold of the CIV (i.e. the percentage of CIV activity needed to be reduced to observe an effect on mitochondrial respiration ) under the different conditions. Furthermore, the role of mitochondrial sulfide-metabolism was studied in additional experiments by titrating Na2S at slow (0.01 µM/s) and fast speed (0.1 µM/s) by means of the Oroboros Titration-injection pump (TIP2k) in cells with fully stimulated mitochondrial respiration and those respiring at routine level to obtain the amount of Na2S required for 50% inhibition of CIV.
All data are median [quartiles]. CIV activity threshold determined by stepwise inhibitor-titrations was 19 (-7;33)% for Azd and 81 (77; 82)% and 87 (86; 90)% for Na2S at 37 and 25 °C respectively (P<0.05 Azd vs. Na2S). Under continuous Na2S-injection we observed an initial increase O2-flux as indicator of mitochondrial sulfide-metabolism. 50% inhibition of mitochondrial respiration was reached at concentrations of 11.1 [10.2; 11.5] µM at high and 22.2 [18.5; 26.5] µM at low injection rate (P<0.05). At high injection rate, 50% inhibition was achieved at 20.6 [17.8; 21.4] µM, i.e. at a statistically significantly higher concentration when compared to the uncoupled state (P<0.05), in cells at routine respiration. In contrast, at 25 °C the Na2S concentration needed to achieve 50% inhibition was 10.6 [9.5; 11.6] µM, i.e. statistically not different from 37°C in uncoupled cells.
The CIV activity threshold determined using Na2S as inhibitor is excessively high when compared to Azd and therefore likely to be an artefact resulting from the partial consumption of sulfide by the mitochondrial respiratory system. This effect is further confirmed by the apparent difference of the Na2S concentrations needed to inhibit CIV depending on the injection rate. Thus, our data confirm that in murine alveolar macrophages sulfide is also partially oxidised at the level of the convergent electron transfer at the Q-junction [2,6] (further experiments performed on alveolar macrophages but actually not presented here indeed show in more details the presence of this mechanism). In view of the potential use of sulfide as a pharmacologic agent, these data show that exact predicting its therapeutic concentration as well as its toxic dose, respectively, may be very difficult to achieve, especially in-vivo, due to these complex interactions at mitochondrial level. In fact, even at low injection rates inhibition of mitochondrial respiration occurred rapidly once a threshold was reached. Furthermore, we show that the toxicity may depend on the actual mitochondrial respiratory activity. Therefore, direct and affordable in-vivo measurements of sulfide levels may be recommended to allow a sufficiently safe medical use of this compound.
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