Accumulating evidence shows that hydrogen sulfide (H₂S) has physiological roles in various tissues and organs, including the regulation of neuronal activity, vascular tension, a release of insulin, and protection of the heart, kidney, and brain from ischemic insult. H₂S is produced from l-cysteine by pyridoxal 5'-phosphate (PLP)-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). 3-Mercaptopyruvate sulfurtransferase (3MST) is the third H₂S-producing enzyme, and its substrate 3-mercaptopyruvate (3MP) is provided from l-cysteine and α-ketoglutarate (α-KG) by a PLP-dependent cysteine aminotransferase (CAT). An additional pathway for the production of H₂S from d-cysteine metabolized by d-amino acid oxidase (DAO) together with 3MST has been identified. Recent studies have shown that hydrogen polysulfides (H₂S_n) have been found to stimulate transient receptor potential ankyrin1 (TRPA1) channel, much more potently than does H₂S. 3MST produces cysteine-persulfide (CysSSH) and its glutathione counterpart (GSSH), potential redox regulators, together with the potential signaling molecules H₂S_n. In addition, the interaction between H₂S and nitric oxide (NO) also generates H₂S_n. These observations provide new insights into the production and physiological roles of these molecules.

Modification of protein thiol is one of the most important post-translational modifications and it occurs depending on the redox state in cells. Protein S-nitrosylation is NO (nitric oxide)-dependent modification of protein thiols and is crucial for regulation of cellular functions such as transcription, protein expression, and signal transduction. Maleimide reagents are generally used to assess the redox status of the thiols in a protein of interest. The maleimides AMS and polyethylene glycol-maleimide (PEG-Mal) have generally been used to distinguish between the reduced and oxidized states of proteins. We have introduced a photocleavable group between the PEG and the maleimide moiety and designated these molecules as PEG-PCMal. When a PEG-PCMal-labeled protein is separated by SDS-PAGE and subsequently irradiated with UV on the polyacrylamide gel, the PEG moiety is removed from the protein. In this study, we tried analysis of protein S-nitrosylation using a new maleimide reagent PEG-PCMal.

In our classical knowledge, redox molecules, including reactive oxygen species (ROS), nitric oxide (NO) and hydrogen sulfide, are considered to be generated as byproducts of aerobic metabolism and act as harmful oxidants of macromolecules, such as proteins and lipids. On the other hands, recently, expressions of enzymes producing redox molecules are identified and reported to be expressed in wide range of tissues, including brain. Moreover, activities of some of these enzymes are revealed to be regulated by physiological signals (e.g. calcium). These observations suggest that redox molecules act as physiological messengers and have biological functions. Actually, recent studies indicate possible involvement of redox signals in functional modification of proteins essential for synaptic plasticity in cultured cells and acute slice preparations. For example, S-nitrosylation of type 1 ryanodine receptor, an intracellular calcium-release channel, is revealed to be essential for NO-induced calcium release (NICR) and synaptic plasticity in cerebellar Purkinje cells. Further studies on mutant animals deficient in redox-modification site may clarify essential role of redox signals in brain functions in vivo.

Since the discovery of nitric oxide (NO) as gaseous signaling molecule, two other gaseous mediators, carbon monoxide (CO) and hydrogen sulfide (H₂S) have been found to be also involved in many physiological and pathophysiological functions. This review will briefly summarize our recent progress in the pathophysiology of NO and H₂S. In the photoreceptor cells, the level of intracellular Ca²⁺ is kept relatively low by H₂S. Intraperitoneal injection of H₂S donor to mice protected photoreceptor cells from light-induced retinal degeneration caused by oxidative stress and elevation of intracellular Ca²⁺. Another gaseous mediator NO induces Ca²⁺ release from the endoplasmic reticulum via S-nitrosylated type 1 ryanodine receptor (RyR1) Ca²⁺ release channel. NO-induced Ca²⁺ release (NICR) was abolished in primary cultured neurons from the knock-in mice, in which the S-nitrosylation site Cys-3636 of RyR1 was replaced by Ala (Ryr1^C3636A). The neurons in hippocampal CA3 region of Ryr1^C3636A mice were protected against seizure-induced neuronal cell death. The result indicates that NICR is critical for status epilepticus-induced neurodegeneration. The developments in the pathophysiology of gaseous mediators in the central nervous system will provide a better pharmacological advances for the treatment of neurodegenerative diseases.

The nigrostriatal dopaminergic pathway is implicated with Parkinson’s disease. Elucidation of this projection mechanism is not only important for considering developmental brain formation, but also contributes to the development of a therapy for regenerating the lost neural circuit. Although several axon guidance cues have been reported to induce dopaminergic axons from the substantia nigra to the striatum, the mechanisms by which the dopaminergic axons extend in the striatum remain unclear. An excellent culture system is necessary for studying the formation process of a neural circuit. Therefore, we tried to establish an in vitro model for the quantitative analysis of dopaminergic innervation of striatal neurons using primary dissociated cells. Mesencephalic cells prepared from rat embryos were seeded on the opposite side to striatal cells with the isolation wall in between. When the isolation wall was removed, the dopaminergic axons extended toward the striatal cell region and formed synapses with striatal neurons. The dopaminergic innervation of striatal neurons was suppressed by inhibiting integrin α5β1 expressed on dopaminergic neurons. Furthermore, dopaminergic neurons overexpressing integrin α5 exhibited a longer neurite outgrowth on striatal cells than normal dopaminergic neurons did. Because this evaluation system using dissociated cell culture has relatively high throughput and is easy to be pharmacologically and genetically manipulated, it is considered to be a useful tool in the study of neural circuit formation. In addition, as a result, we found integrin α5β1 as a molecule promoting striatal innervation by dopaminergic neuron, which is expected to contribute to regeneration of the nigrostriatal dopaminergic projection.

ALLESAGA_® TAPE is the first transdermal drug delivery system of emedastine difumarate, as a second-generation antihistamine, for allergic rhinitis. It has been suggested that the efficacy of emedastine difumarate in allergic rhinitis is mediated through a combination of chemical mediator release inhibitory effects and eosinophil chemotaxis inhibitory effects, in addition to a strong anti-histaminic effect. In the pharmacological evaluation on histamine-induced vascular hyperpermeability in rats, ALLESAGA_® TAPE showed an anti-histaminic effect in a dose-dependent manner and exhibited a long-lasting anti-histaminic effect until 24 hours after administration. In the patients having allergic nasal symptoms, the plasma concentration of emedastine reached at steady-state within 7 days after multiple administration of ALLESAGA_® TAPE. ALLESAGA_® TAPE was effective for treating seasonal allergic rhinitis with sustained action throughout the day. Long-term application of ALLESAGA_® TAPE raised no safety concerns in patients with perennial allergic rhinitis. Plasma drug concentrations showed little change over a long period and there was no decrease of efficacy. Based on the above results, ALLESAGA_® TAPE is possible to provide a new option for the treatment of allergic rhinitis.