P31 Sulfide metabolism at low oxygen conditions

Abstract

Background Even in higher species sulfide is quickly metabolised and thus degraded by an ancient metabolic pathway linked to the mitochondrial respiratory chain [1] . Since this metabolic process is strictly aerobic, sulfide concentration is expected to increase at low oxygen, provided that the production rate remains constant. This mechanism has been suggested as an explanation of how sulfide acts as an oxygen sensor within cells [2] . However, one may argue that the efficiency of sulfide as an O 2 -sensor requires that the capacity of the cells to metabolise this molecule already decreases at oxygen concentrations still not affecting the aerobic cell metabolism. In fact, only in that way sulfide concentration would start rising, thus signaling the imminent hypoxia, before low oxygen per se becomes a seriously limiting factor for the cell. Therefore, in a series of preliminary experiments we quantified the sulfide metabolism in a stable cell line derived from alveolar macrophages (AMJ2-C11), which previously proved to efficiently metabolise sulfide under aerobic conditions [3] , at O 2 partial pressures approaching hypoxia from 0.8 kPa down to 0.4 and 0.1 kPa. Methods All Measurements were conducted using an O2K ® -Oxygraph together with a TIP-2K ® titration pump (Oroboros Instruments, Austria). For each O 2 -partial pressure we performed 6–8 separate experiments. Before performing the experiments we recorded several trasitions to anoxia in order to quantify the un-inhibited respiratory capacity close to anoxia under the O 2 -partial pressures of 0.1, 0.4, and 0.8 kPa. The inhibition of mitochondrial respiration was quantified in terms of the total amount of sulfide required to reduce the routine oxygen flux (JO2) to 50% by means of a continuous sulfide injection at a rate of 10 nM/s. A second titration pump was used to simultaneously maintain the oxygen concentration at the predefined level adopting a previously described technique [4] . Results Fig. 1 shows representative records of 3 anoxic transition experiments in AMJ2-C11 cells; the data suggest that the JO2 only moderately declines up to a O 2 -partial pressure of 0.1 kPa. In contrast, the total amount of sulfide required for achieving a 50%-inhibition of mitochondrial respiration was about 10% at 0.1 kPa when compared to that needed at 0.8 kPa (0.2 ± 0.03 μmol at 0.1 kPa vs. 1.3 ± 0.4 μmol at 0.4 kPa, and 2.2 ± 0.1 μmol at 0.8 kPa). This result suggests a much less efficient degradation of sulfide at the lower oxygen level. Fig.2 plots the injected amount of sulfide against the JO 2 at all 3 partial pressures. Conclusions Our preliminary data indicate that the capacity to metabolise sulfide is already affected at oxygen concentrations still not severily limiting cell respiration. This result further emphasize the potential role of sulfide as an oxygen sensor in the cell.