Hydrogen sulfide (H2S), which is well known as a toxic gas, is produced endogenously from L-cysteine in mammalian tissues. H2S is present at relatively high levels in the brain, suggesting that it has a physiological function. Two other gases, nitric oxide and carbon monoxide, are also endogenously produced and have been proposed as neuronal messengers in the brain. In this work we show the following: (1) an H2S-producing enzyme, cystathionine beta-synthase (CBS), is highly expressed in the hippocampus; (2) CBS inhibitors hydroxylamine and amino-oxyacetate suppress the production of brain H2S; and (3) a CBS activator, S-adenosyl-L-methionine, enhances H2S production, indicating that CBS contributes to the production of endogenous H2S. We also show that physiological concentrations of H2S selectively enhance NMDA receptor-mediated responses and facilitate the induction of hippocampal long-term potentiation. These observations suggest that endogenous H2S functions as a neuromodulator in the brain.

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The recent discovery that hydrogen sulfide (H2S) is an endogenously produced gaseous second messenger capable of modulating many physiological processes, much like nitric oxide, prompted us to investigate the potential of H2S as a cardioprotective agent. In the current study, we demonstrate that the delivery of H2S at the time of reperfusion limits infarct size and preserves left ventricular (LV) function in an in vivo model of myocardial ischemiareperfusion (MI-R). This observed cytoprotection is associated with an inhibition of myocardial inflammation and a preservation of both mitochondrial structure and function after I-R injury. Additionally, we show that modulation of endogenously produced H2S by cardiac-specific overexpression of cystathionine ␥-lyase (␣-MHC-CGL-Tg mouse) significantly limits the extent of injury. These findings demonstrate that H2S may be of value in cytoprotection during the evolution of myocardial infarction and that either administration of H2S or the modulation of endogenous production may be of clinical benefit in ischemic disorders.

Hydrogen sulfide (H(2)S) is a synaptic modulator as well as a neuroprotectant. Currently, pyridoxal-5'-phosphate (PLP)-dependent cystathionine beta-synthase (CBS) is thought to be the major H(2)S-producing enzyme in the brain. We recently found that brain homogenates of CBS-knockout mice, even in the absence of PLP, produce H(2)S at levels similar to those of wild-type mice, suggesting the presence of another H(2)S-producing enzyme. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) in combination with cysteine aminotransferase (CAT) produces H(2)S from cysteine. In addition, 3MST is localized to neurons, and the levels of bound sulfane sulfur, the precursor of H(2)S, are greatly increased in the cells expressing 3MST and CAT but not increased in cells expressing functionally defective mutant enzymes. These data present a new perspective on H(2)S production and storage in the brain.

3-(4,5-Dimethylthiazol-2-yl) -2‚5-diphenyltetrazolium bromide (MIT) reduction is one of the most frequently used methods for measuring cell proliferation and neural cytotoxicity. lt is widely assumed that MIT is reduced by active mitochondria in living cells. By using isolated mitochondria from rat brain and B12 cells, we indeed found that malate, glutamate, and succinate support MIT reduction by isolated mitochondria. However, the data presented in this study do not support the exclusive role of mitochondria in MIT reduction by intact cells. Using a variety of approaches, we found that MIT reduction by B12 cells is confined to intracellular vesicles that later give rise to the needle-like MIT formazan at the cell surface. Some of these vesicles were identified as endosomes or lysosomes. In addition, MIT was found to be membrane impermeable. These and other results suggest that MIT is taken up by cells through endocytosis and that reduced MIT formazan accumulates in the endosomal/lysosomal compartment and is then transported to the cell surface through exocytosis. Key Words: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-Endocytosis-Exocytosis-Oxidoreductase-Viability assay. J. Neurochem. 69, 581 -593 (1997).Tetrazolium salts are a large group of heterocyclic organic compounds that form highly colored and often insoluble formazans after reduction. First prepared in 1894, these compounds have been used widely as indicators of both biological redox systems and viability (Altman, 1976). 3-. (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is a monotetrazohum salt, the reduction of which is one of the most frequently used methods for measuring cell proliferation and cytotoxicity (Mosmann, 1983).Despite the widespread use of the MTT assay as a measure of cell viability and proliferation, the mechanism of cellular MTT reduction is poorly understood. An earlier study by Slater et al. (1963), using respiratory chain inhibitors, showed that succinate-dependent MTT reduction by rat liver homogenates occurred at two sites along the mitochondrial electron transport chain (coenzyme Q and cytochrome c; Fig. 1). Although this finding has often been taken as evidence that MTT is reduced by active mitochondria in viable cells, definitive proof for the association of mitochondna with MTT reduction in intact cells has been lacking because many nonmitochondrial dehydrogenases or fiavin oxidases can also reduce MTT (Altman, 1976;Burdon et al., 1993). Without a clear understanding of the site and the enzymatic system involved in cellular MTT reduction, it has been difficult to explain the discrepancies between the MTT assay and other measures of cell growth and viability (Jabber et al., 1989;Berridge and Tan, 1993), and the effect of extracellular D-glucose concentration and cellular pyridine nucleotide concentration on cellular MTT reduction (Vistica et al., 1991). Nikkhah et al. (1992) and Shearman et al. (1995) found that MTT formazan was deposited intracellularly in a granular form with a ...