H₂S production by three enzymes has been studied extensively; these enzymes are cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST; Stipanuk and Beck, 1982; Chiku et al., 2009; Shibuya et al., 2009a,b; Singh et al., 2009) (Figure 1). CBS and CSE metabolize cysteine and/or homocysteine to release H₂S, whereas 3MST produces H₂S from 3-mercaptopyruvate (3MP), which is produced by the action of cysteine aminotransferase (CAT) on cysteine and α-ketoglutarate (Cooper, 1983; Shibuya et al., 2009a,b). 3MST requires cofactors to reduce a persulfide intermediate generated between a cysteine residue of 3MST and a sulfide provided by 3MP. We recently found that thioredoxin and dihydrolipoic acid (DHLA) are endogenous reducing cofactors that facilitate H₂S release from 3MST (Mikami et al., 2011a). We also found that the 3MST/CAT pathway is regulated by Ca²⁺ (Mikami et al., 2011b; Mikami and Kimura, 2012) and that the activity of CBS is enhanced by S-adenosyl methionine (SAM; Abe and Kimura, 1996). Thus, the physiological stimuli that alter the intracellular levels of Ca²⁺ and SAM should be investigated in order to clarify the regulation of H₂S production.

H₂S is metabolized in the mitochondria, via the sulfide oxidation pathway (Hildebrandt and Grieshaber, 2008). The first step is catalyzed by a membrane-bound sulfide:quinone oxidoreductase (SQR), which oxidizes H₂S to persulfide. SQRs were initially identified in invertebrates, and their mammalian counterpart SQRs were later discovered (Theissen et al., 2003; Theissen and Martin, 2008). In the presence of O₂ and water, sulfur dioxygenase oxidizes persulfide to sulfite, which is combined with another persulfide molecule by sulfur transferase rhodanese in order to producing thiosulfate (Figure 1). Because H₂S consumption by a dioxygenase is high in the presence of O₂ (Furne et al., 2008), high H₂S production is offset by rapid H₂S clearance under aerobic conditions, accounting for very low basal levels of H₂S. Small deviations in the rates of H₂S production and clearance may lead to rapid and several-fold changes in the H₂S levels (Vitvitsky et al., 2012).

In addition to enzymatic regulation of H₂S levels, H₂S may be stored in proteins as bound sulfane sulfur that is divalent sulfur bound mostly to sulfur of cysteine residues. (Ishigami et al., 2009) (Figure 1). Exogenously applied H₂S is absorbed and stored as bound sulfane sulfur, and the rate of absorption varies according to tissue types. Cells expressing 3MST and CAT have higher levels of bound sulfane sulfur than control cells (Shibuya et al., 2009a,b). In contrast, cells expressing 3MST mutants – that lack the ability to produce H₂S – retain control-like levels of bound sulfane sulfur. Therefore, H₂S produced by enzymes is stored in cells as bound sulfane sulfur. Bound sulfane sulfur releases H₂S under reducing conditions. In the presence of major cellular reducing substances, glutathione, cysteine, and dihydrolipoic acid (DHLA) at their physiologic concentrations, H₂S is released from lysates of cultured neurons and astrocytes at pH 8.0–8.4 (Ishigami et al., 2009; Mikami et al., 2011a). When neurons are excited, sodium ions enter and potassium ions exit from cells, resulting in high potassium concentrations in the extracellular environment, which depolarizes the membrane of surrounding astrocytes. To recover from depolarization, Na+/HCO3− cotransporters are activated in astrocytes (Brookes and Turner, 1994). The entrance of HCO3− causes alkalinization. Approximately 10% of the astrocytes shifted their intracellular pH to 8.4, which can induce bound sulfane sulfur to release H₂S (Ishigami et al., 2009).

The concentrations of H₂S can vary locally in restricted areas of cells, while the reported values are those averaged over whole tissues, using tissue homogenates or blood samples (Furne et al., 2008; Ishigami et al., 2009; Wintner et al., 2010). Such local variations in H₂S concentrations must be determined. Recently developed H₂S-sensitive fluorescence probes are able to estimate local H₂S levels in live cells (Lippert et al., 2011; Liu et al., 2011; Peng et al., 2011; Qian et al., 2011; Sasakura et al., 2011). However, the probes bind to H₂S irreversibly; the detectable limit for H₂S is approximately 5–30 μM, i.e., values higher than the basal or steady state concentrations; and the time to reach maximum sensitivity is between 3 min and 1 h. The development of probes able to detect rapid changes in H₂S concentrations is awaited.

The metabolic balance between the production and clearance of H₂S has a considerable effect on its endogenous concentrations. In addition, because of bound sulfane sulfur, which releases and absorbs H₂S, the H₂S concentrations change more rapidly and extensively. The discrepancy between the endogenous basal concentrations and the effective-concentrations of exogenously applied H₂S has been discussed frequently. Some effects of H₂S may be elicited by its entry into cells through the plasma membrane. However, the rate of permeation and how freely and rapidly H₂S diffuses within the cytosol have not been determined. We also need to clarify the H₂S concentrations reached when cells are stimulated and the associated mechanism.

In conclusion, the basal or steady state levels of H₂S have been re-evaluated and found to be much lower than those previously reported. There is a difference between the steady state- and the effective-concentrations of H₂S. Therefore, it is urgent to determine the local concentrations of H₂S achieved when cells are stimulated.