Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological interest.

Hydrogen sulfide (H(2)S) is an endogenously generated gas that can also be administered exogenously. It modulates physiological functions and has reported cytoprotective effects. To evaluate a possible antioxidant role, we investigated the reactivity of hydrogen sulfide with several one- and two-electron oxidants. The rate constant of the direct reaction with peroxynitrite was (4.8±1.4)×10(3)M(-1) s(-1) (pH 7.4, 37°C). At low hydrogen sulfide concentrations, oxidation by peroxynitrite led to oxygen consumption, consistent with a one-electron oxidation that initiated a radical chain reaction. Accordingly, pulse radiolysis studies indicated that hydrogen sulfide reacted with nitrogen dioxide at (3.0±0.3)×10(6)M(-1) s(-1) at pH 6 and (1.2±0.1)×10(7)M(-1) s(-1) at pH 7.5 (25°C). The reactions of hydrogen sulfide with hydrogen peroxide, hypochlorite, and taurine chloramine had rate constants of 0.73±0.03, (8±3)×10(7), and 303±27M(-1) s(-1), respectively (pH 7.4, 37°C). The reactivity of hydrogen sulfide was compared to that of low-molecular-weight thiols such as cysteine and glutathione. Considering the low tissue concentrations of endogenous hydrogen sulfide, direct reactions with oxidants probably cannot completely account for its protective effects.

Hydrogen sulfide is a reversible inhibitor of the NADH oxidase activity of synaptic plasma membranes.

Hydrogen sulfide is now accepted as a neuromodulator, which can be involved in neuronal defence against oxidative stress insults in the brain. In this work we show that concentrations of H(2)S within the physiological range reported in the brain produce a reversible inhibition of the NADH oxidase activity and coupled superoxide anion production by synaptic plasma membranes from rat brain. At physiological pH 7 the concentration of H(2)S needed for 50% inhibition of the NADH oxidase activity is 5+/-1 microM, which is within the low range of the reported physiological H(2)S concentrations. Thus, the NADH oxidase activity of the neuronal plasma membrane can act as a sensor of local H(2)S depletion in neurones. H(2)S inhibition of the NADH oxidase activity of the neuronal plasma membrane can be accounted for direct reduction by H(2)S of cytochrome b(5). However, H(2)S fails to afford a significant protection against the inhibition of this activity by peroxynitrite. In conclusion, our results point out that H(2)S is more potent as inhibitor of reactive oxygen species formation than as a sacrificial antioxidant.

Regionalization of plasma membrane-bound flavoproteins of cerebellar granule neurons in culture by fluorescence energy transfer imaging.

Flavoproteins are components of plasma membrane redox chains, which have been suggested to play major roles in neuronal activity and survival. We found that the red/orange autofluorescence of mature primary cultures of cerebellar granule neurons (8-9 days in vitro) was largely quenched by millimolar concentrations of dithionite added to the extracellular medium, and pointed out that nearly 50% of this autofluorescence was due to plasma membrane-bound flavoproteins. We report in this work that the lipophilic neuronal plasma membrane markers N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)-pyridinium dibromide (RH-414) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64) can form fluorescence energy transfer donor-acceptor pairs with flavoproteins with calculated R (0) values between 3.7 and 4.2 nm. The quantification of the efficiency of fluorescence energy transfer with different concentrations of acceptor dyes has been worked out with re-suspended neurons. Using quantitative images of the neurons in culture, acquired with a CCD camera attached to an epifluorescence microscope, regionalization of the plasma membrane-bound flavoproteins of cerebellar granule neurons has been achieved from the quenching by dithionite of the fluorescence of the acceptor dye. The results unraveled that plasma membrane-bound flavoproteins are largely enriched in interneuronal contact sites forming clusters of 0.5-1 microm diameter size, which appears largely regionalized in the neuron's cell body.