Yoshinori Mikami

Development of a Highly Selective Fluorescence Probe for Hydrogen Sulfide

Hydrogen sulfide (H(2)S) has recently been identified as a biological response modifier. Here, we report the design and synthesis of a novel fluorescence probe for H(2)S, HSip-1, utilizing azamacrocyclic copper(II) ion complex chemistry to control the fluorescence. HSip-1 showed high selectivity and high sensitivity for H(2)S, and its potential for biological applications was confirmed by employing it for fluorescence imaging of H(2)S in live cells.

Vascular endothelium expresses 3-mercaptopyruvate sulfurtransferase and produces hydrogen sulfide.

Hydrogen sulfide (H(2)S) has been recognized as a smooth muscle relaxant. Cystathionine gamma-lyase, which is localized to smooth muscle, is thought to be the major H(2)S-producing enzyme in the thoracic aorta. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT) are localized to vascular endothelium in the thoracic aorta and produce H(2)S. Both 3MST and CAT were localized to endothelium. Lysates of vascular endothelial cells produced H(2)S from cysteine and alpha-ketoglutarate. The present study provides a new insight into the production and release of H(2)S as a smooth muscle relaxant from vascular endothelium.

Polysulfides are possible H2S-derived signaling molecules in rat brain

Accumulating evidence shows that hydrogen sulfide (H2S) has a variety of physiological functions. H2S is produced from cysteine by 3 sulfurtransferases. H2S, in turn, generates polysulfides, the functions of which are not well understood. H2S induces Ca2+ influx in astrocytes, a type of glia. However, the receptor that mediates the response has not been identified. Here, we have shown that polysulfides induce Ca2+ influx by activating transient receptor potential (TRP)A1 channels in rat astrocytes (EC50 91 nM, Hill coefficient value 1.77±0.26) and that the maximum response was induced at 0.5 μM, which is 1/320 of the concentration of H2S required to achieve a response of similar magnitude (160 μM, EC50 116 μM). TRPA1‐selective agonists, allyl isothiocyanate and cinnamaldehyde, induced Ca2+ influx, and responses to polysulfides were suppressed by TRPA1‐selective inhibitors, HC‐030031 and AP‐18, as well as by siRNAs selective to TRPA1. The present study suggests that polysulfides are possible H2S‐derived signaling molecules that stimulate TRP channels in the brain.—Kimura, Y., Mikami, Y., Osumi, K., Tsugane, M., Oka, J., Kimura, H. Polysulfides are possible H2S‐derived signaling molecules in rat brain. FASEB J. 27, 2451–2457 (2013). www.fasebj.org

Thioredoxin and dihydrolipoic acid are required for 3-mercaptopyruvate sulfurtransferase to produce hydrogen sulfide.

H2S (hydrogen sulfide) has recently been recognized as a signalling molecule as well as a cytoprotectant. We recently demonstrated that 3MST (3-mercaptopyruvate sulfurtransferase) produces H2S from 3MP (3-mercaptopyruvate). Although a reducing substance is required for an intermediate persulfide at the active site of 3MST to release H2S, the substance has not been identified. In the present study we show that Trx (thioredoxin) and DHLA (dihydrolipoic acid) associate with 3MST to release H2S. Other reducing substances, such as NADPH, NADH, GSH, cysteine and CoA, did not have any effect on the reaction. We also show that 3MST produces H2S from thiosulfate. The present study provides a new insight into a mechanism for the production of H2S by 3MST.

Hydrogen sulfide protects the retina from light-induced degeneration by the modulation of Ca2+ influx

Background: Hydrogen sulfide (H2S) has been recognized as a signaling molecule as well as a cytoprotectant. Results: Ca2+ regulates the 3-mercaptopyruvate sulfurtransferase/cysteine aminotransferase pathway to produce H2S production. H2S, in turn, regulates Ca2+ influx and protects retinal neurons from light-induced degeneration. Conclusion: H2S regulates Ca2+ levels and protects retinal neurons. Significance: It provides a possible role of H2S and its therapeutic application in the retina. Hydrogen sulfide (H2S) has recently been recognized as a signaling molecule as well as a cytoprotectant. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are well-known as H2S-producing enzymes. We recently demonstrated that 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase (CAT) produces H2S in the brain and in vascular endothelium. However, the cellular distribution and regulation of these enzymes are not well understood. Here we show that 3MST and CAT are localized to retinal neurons and that the production of H2S is regulated by Ca2+; H2S, in turn, regulates Ca2+ influx into photoreceptor cells by activating vacuolar type H+-ATPase (V-ATPase). We also show that H2S protects retinal neurons from light-induced degeneration. The excessive levels of light exposure deteriorated photoreceptor cells and increased the number of TUNEL- and 8-hydroxy-2′-deoxyguanosine (8-OHdG)-positive cells. Degeneration was greatly suppressed in the retina of mice administered with NaHS, a donor of H2S. The present study provides a new insight into the regulation of H2S production and the modulation of the retinal transmission by H2S. It also shows a cytoprotective effect of H2S on retinal neurons and provides a basis for the therapeutic target for retinal degeneration.

Chlorogenic acid, a polyphenol in coffee, protects neurons against glutamate neurotoxicity.

AIMS The present study has been designed to explore the molecular mechanism of chlorogenic acid (CGA) in the protective effect against glutamate-induced neuronal cell death. MAIN METHODS Cortical neurons in primary culture were exposed to 300 μM l-glutamic acid or vehicle, with or without 10 μM CGA or 10 μM MK-801. After 16 h, primary cultures were stained with propidium iodide (PI)/Hoechst or calcein. Double-staining with PI and Hoechst was performed to confirm whether cell death induced by glutamate was apoptotic. In addition, intracellular concentrations of Ca(2+) were observed using Ca(2+) indicator fura-2. KEY FINDINGS We investigated the protective effects of CGA on glutamate-induced neuronal cell death using primary cultures of mouse cerebral cortex because the release of glutamate during brain ischemia triggers death of neurons. Glutamate-induced neuronal cell death was inhibited by treatment with CGA. In addition, CGA prevented the increase in intracellular concentrations of Ca(2+) caused by the addition of glutamate to cultured neurons. On the other hand, there was little effect of CGA on cell death induced by nitric oxide, which is downstream of the ischemic neuronal cell death. Our results suggested that the polyphenol CGA in coffee protects neurons from glutamate neurotoxicity by regulating Ca(2+) entry into neurons. SIGNIFICANCE CGA in coffee may have clinical benefits for neurodegenerative diseases such as ischemic stroke.

Protein tyrosine phosphatase σ regulates the synapse number of zebrafish olfactory sensory neurons.

J. Neurochem. (2011) 119, 532–543.

Nitric Oxide-induced Activation of the Type 1 Ryanodine Receptor Is Critical for Epileptic Seizure-induced Neuronal Cell Death

Status epilepticus (SE) is a life-threatening emergency that can cause neurodegeneration with debilitating neurological disorders. However, the mechanism by which convulsive SE results in neurodegeneration is not fully understood. It has been shown that epileptic seizures produce markedly increased levels of nitric oxide (NO) in the brain, and that NO induces Ca2 + release from the endoplasmic reticulum via the type 1 ryanodine receptor (RyR1), which occurs through S-nitrosylation of the intracellular Ca2 + release channel. Here, we show that through genetic silencing of NO-induced activation of the RyR1 intracellular Ca2 + release channel, neurons were rescued from seizure-dependent cell death. Furthermore, dantrolene, an inhibitor of RyR1, was protective against neurodegeneration caused by SE. These results demonstrate that NO-induced Ca2 + release via RyR is involved in SE-induced neurodegeneration, and provide a rationale for the use of RyR1 inhibitors for the prevention of brain damage following SE.

A mechanism of retinal protection from light-induced degeneration by hydrogen sulfide

Since our initial demonstrations that hydrogen sulfide (H2S) may function as a neuromodulator in the brain and a smooth muscle relaxant in the vascular system, accumulating evidence shows that H2S may function as a signaling molecule. We and others also found that H2S has a cytoprotective effect. Because H2S is well-known toxic gas, a cytoprotective role has been overlooked. H2S protects neurons from oxidative stress. It also protects cardiac muscle from ischemia-reperfusion injury. The finding led to the application of H2S to the bypass surgery patients in Phase II clinical trial. Cystathionine β–synthase (CBS) and cystathionine γ–lyase (CSE) are well known as H2S-producing enzymes. We recently demonstrated that the other H2S-producing enzyme, 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase (CAT) is localized to neurons in the brain and to the vascular endothelium. However, the regulation of H2S production by 3MST/CAT pathway had not been well understood. The present study shows that H2S production by 3MST/CAT pathway is regulated by Ca2+ and that H2S protects retinal photoreceptor cells from light induced degeneration by suppressing excessive Ca2+ influx caused by intense light.

Identification and characterization of a novel zebrafish semaphorin.

The semaphorin gene family contains numerous secreted and transmembrane proteins. Some of them function as the repulsive and attractive axon guidance molecules during development. Herein, we report the cloning and characterization of a novel member of zebrafish semaphorin gene, semaphorin 6E (sema6E). Sema6E is expressed predominantly in the nervous system during embryogenesis. Results also show that Sema6E binds Plexin-A1, but not other Plexins. Sema6E chemorepels not only dorsal root ganglion axons but also sympathetic axons. Therefore, Sema6E might utilize Plexin-A1 as a receptor to repel axons of the specific types during development.