P2 High turnover rates for hydrogen sulfide in murine tissues

Abstract

Hydrogen sulfide (H 2 S) is a biologically active metabolite, which influences many physiological processes. While H 2 S can be produced and degraded in different mammalian tissues, the kinetics of its turnover, which determine its steady state levels, have not been well studied. We have assessed the rates of H 2 S production in murine liver, kidney, and brain homogenates at physiologically relevant conditions. We have also studied the kinetics of H 2 S clearance by liver, kidney, and brain homogenates under aerobic and anaerobic conditions. Organs were obtained from 2 month old BALB/C males. Kinetics of H 2 S production and clearance were studied in tissue homogenates diluted 1:9 with 100 mM HEPES buffer, pH 7.4. Production of H 2 S was studied under anaerobic conditions at 37 °C and cysteine concentration from 0 to 5.0 mM. Kinetics of H 2 S clearance was studied at 25 °C under both anaerobic and aerobic conditions with initial H 2 S concentration of 20 ppm. Concentration of H 2 S was determined using gas chromatography with DB-1 column (30 m, 0.32 mm, 1.0 μm) and 355 Sulfur Chemiluminescence Detector (Agilent). We found that the rate of H 2 S production by tissue homogenates is considerably higher than background rates observed in the absence of exogenous substrates. At physiological cysteine concentration of 100 μM our estimations for the rate of H 2 S production (mean ± SD) in mouse liver and brain are 484 ± 271 μmol/h kg ( n = 7) and 29 ± 7 μmol/h kg ( n = 4) respectively. In kidney the rate of H 2 S production of 104 ± 44 μmol/h kg ( n = 3) was obtained at 0.5 mM cysteine. In the absence of cysteine H 2 S added to homogenate decays exponentially with time and, as expected, the decay is significantly faster under aerobic conditions. The half-life for H 2 S under aerobic conditions is 2.0, 2.8, and 10.0 min with liver, kidney and brain homogenate, respectively. Western-blot analysis of the sulfur dioxygenase, ETHE1, involved in H 2 S catabolism, demonstrates higher protein levels in liver and kidney versus brain. At pH 5.8 the rate of H 2 S production decreases dramatically and becomes negligible at physiologically relevant cysteine levels, while the rate of H 2 S clearance decreases only slightly. By combining experimental and simulation approaches, we demonstrate high rates of tissue H 2 S turnover and provide estimates of in vivo steady-state H 2 S levels which are 29, 8.7, and 18 nmoles per kg tissue in mouse liver, kidney, and brain respectively. Our study reveals that mammalian tissues maintain a high metabolic flux of sulfur through H 2 S, providing the possibility for rapid regulation of H 2 S levels.