Kazutaka Momose

Itch-Scratch Responses Induced by Lysophosphatidic Acid in Mice

The present investigation was conducted in order to determine whether lysophosphatidic acid (LPA) induces itch-scratch responses (ISRs) in mice. Intradermal administration of LPA induces ISRs; furthermore, the time course for LPA-induced ISRs was similar to that for histamine-induced responses. Comparative study of the pruritogenic activity revealed that histamine possessed a potent effect characterized by a dose-response relationship; however, prostaglandin D2 failed to induce this response. Pretreatment with ketotifen, a histamine H1 receptor antagonist, and capsaicin inhibited LPA-induced ISRs. Additionally, LPA-induced ISRs were abolished by Y-27632, an inhibitor of Rho-associated protein kinase (ROCK). These findings suggest that LPA-induced ISRs are attributable to histamine- and substance-P-mediated pathways. Moreover, the Rho/ROCK-mediated pathway may be involved.

PROTECTIVE EFFECTS OF TETRAHYDROBIOPTERIN AGAINST NITRIC OXIDE-INDUCED ENDOTHELIAL CELL DEATH

The purpose of this study was to examine whether tetrahydrobiopterin (BH4), one of the cofactors of nitric oxide (NO) synthase, attenuates NO-induced endothelial cell death. S-Nitroso-N-acetyl-DL-penicillamine (SNAP) was used as a NO donor. Endothelial cell death was assessed by the leakage of intracellular lactate dehydrogenase (LDH). Addition of SNAP to endothelial cells time- and concentration-dependently induced endothelial cell death. The SNAP-induced endothelial cell death was strongly reduced by the treatment with carboxy-PTIO, a NO scavenger, or catalase, but not with superoxide dismutase (SOD). Moreover, pretreatment with sepiapterin, a precursor of BH4, increased intracellular BH4 content, and strongly reduced the SNAP-induced endothelial cell death. Both the increase in BH4 content and the protective effects of sepiapterin were prevented by co-pretreatment with N-acetylserotonin (NAS), an inhibitor of BH4 synthesis. These findings suggest that the cytotoxicity of NO released from SNAP involves H2O2 production, and increase in intracellular BH4 content attenuates NO-induced endothelial cell death. Scavenging of H2O2 by BH4 may be at least one of the mechanisms by which BH4 reduces NO-induced endothelial cell death.

Sodium nitroprusside-induced apoptotic cellular death via production of hydrogen peroxide in murine neuroblastoma N1E-115 cells☆

Sodium nitroprusside is widely used in pharmacological studies as a potent vasodilator or a nitric oxide donor. The mechanisms of cellular death induced by sodium nitroprusside were investigated in murine neuroblastoma N1E-115 cells. Sodium nitroprusside reduced the cellular viability, and the DNA extracted from treated cells showed a ladder-like intranucleosomal fragmentation pattern, which is an indication of apoptosis. The DNA fragmentations were also visualized by in situ nick translation. The cellular death was attenuated by cycloheximide, indicating that ongoing protein synthesis was essential for the initiation of the degenerative response. However, other nitric oxide donors did not decrease the cellular viability. The nitric oxide scavenger, hemoglobin, had no effect on sodium nitroprusside-induced cellular death. Furthermore, sodium cyanide, which is formed by the metabolism of sodium nitroprusside, did not cause cellular death. On the other hand, hydrogen peroxide, another product of sodium nitroprusside metabolism, reduced the cellular viability and induced DNA fragmentation. In addition, the cell damage induced by sodium nitroprusside was enhanced by a medium without fetal bovine serum. In conclusion, we proposed that hydrogen peroxide is the important toxic species for induction of apoptosis in N1E-115 cells exposed to sodium nitroprusside.

Differential expression of VAMP2/synaptobrevin-2 after antidepressant and electroconvulsive treatment in rat frontal cortex

The biological basis for the therapeutic mechanisms of depression is still unknown. We have previously performed expressed-sequence tag (EST) analysis to identify some molecular machinery responsible for antidepressant effect. Then, we developed our original cDNA microarray, on which cDNA fragments identified as antidepressant-related genes/ESTs were spotted. In this study, with this microarray followed by Western blot analysis, we have demonstrated the induction of vesicle-associated membrane protein 2(VAMP2/synaptobrevin-2) in rat frontal cortex not only after chronic antidepressant treatment, but also after repeated electroconvulsive treatment. On the other hand, expression of SNAP-25 and syntaxin-1 was not changed by these treatments. These components make a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex with VAMP2 and mediate the synaptic vesicle docking/fusion machinery. In conclusion, it is suggested that VAMP2/synaptobrevin-2 plays important roles in the antidepressant effects. Our results may contribute to a novel model for the therapeutic mechanism of depression and new molecular targets for the development of therapeutic agents.

Reduction by NG‐nitro‐L‐arginine of H2O2‐induced endothelial cell injury

1 The effects of three analogues of NG‐nitro‐L‐arginine (L‐NOARG) and NG‐monomethyl‐L‐arginine (L‐NMMA), inhibitors of nitric oxide (NO) synthase, on hydrogen peroxide (H2O2)‐induced endothelial cell injury were studied. 2 Endothelial cell injury was assessed by measuring the release of intracellular lactate dehydrogenase (LDH) and 51Cr. 3 Addition of H2O2 (250‐1,000 μm) to endothelial cells induced the release of LDH dose‐dependently. The release of LDH was reduced by pretreatment with NG‐nitro‐L‐arginine methyl ester (L‐NAME, 10−4‐4times 10−3m), L‐NOARG (10−4‐4 × 10−3 m) and NG‐nitro‐L‐arginine benzyl ester (L‐NABE, 10−4‐4times 10−3m), inhibitors of NO synthase. 4 L‐NOARG analogues also reduced H2O2‐induced 51Cr release from endothelial cells, while L‐NMMA had no effect. 5 The protective effect of L‐NAME was not reversed by addition of L‐arginine (L‐Arg, 1‐10mM). 6 Both L‐NAME and L‐NMMA completely inhibited L‐Arg metabolism to L‐citrulline coupled with NO synthesis. 7 These findings suggest that L‐NOARG analogues but not L‐NMMA reduced H2O2‐induced endothelial cell injury, and that these effects may not be related to inhibition of NO production.

Induction of cysteine string protein after chronic antidepressant treatment in rat frontal cortex

We have previously identified 204 partial cDNA fragments (ADRG1-204) as antidepressant related genes/expressed sequence tags. Then, we developed our original cDNA microarrays, on which the 194 clones out of ADRG1-204 were spotted. With this ADRG microarray, we found that the expression of a spot, ADRG55, which representing cysteine string protein (CSP), was significantly increased in rat brain after chronic treatment with a selective serotonin reuptake inhibitor, sertraline. In the present study, reverse transcription-polymerase chain reaction analysis confirmed the induction of CSP at mRNA levels in rat frontal cortex after chronic treatment with two different classes of antidepressants, imipramine or sertraline. Western blot analysis also revealed that CSP-immunoreactivity was increased after antidepressant treatment. In conclusion, our data suggest that CSP is one of the common functional molecules induced after chronic antidepressant treatment.

Lysophosphatidic acid sensitizes mechanical stress-induced Ca2+ mobilization in cultured human lung epithelial cells

We conformed that lysophosphatidic acid (LPA), which is known to be released from activated platelets, sensitizes response in cytosolic free Ca2+ concentration ([Ca2+]i) to mechanical stimulation in cultured epithelial cells (REPF-LC-AI cells) from human lung carcinoma. [Ca2+]i was transiently increased by spritzing of bath solution onto cells as mechanical stimulation in the presence of LPA with concentration-dependent manner (10-100 nM). The transient increase induced by the mechanical stimulation in the presence of LPA was inhibited by 10 microM Ga3+ or removing extracellular Ca2+, but not by 10 microM nicaridipine, suggesting that LPA sensitizes mechanical stimulation-induced Ca2+ influx through stretch-activated ion channels. Phosphatidic acid (1 microM), but not lysophosphatidycholine (10 microM), histamine (100 nM), bradykinin (10 nM), nor ionomycin (100 nM), caused the same effect as that of LPA. This effect was observed in confluent cells, but not in subconfluent cells. These results show that LPA sensitizes mechanoreceptor-linked response in human lung epithelial cells, suggesting a possibility that LPA affects lung function, in particular, during pathological state.

The mode of inotropic action of ciguatoxin on guinea‐pig cardiac muscle

1 Ciguatoxin (CTX) caused a dose‐dependent increase in the contractile force of the guinea‐pig isolated left atria at concentrations ranging from 0.1 to 10 ng ml−1 with the ED50 value of 0.5 ng ml−1. 2 In the atria, tetrodotoxin (5 × 10−7 m) inhibited markedly the inotropic action of CTX. The inotropic effect of CTX at low concentrations was abolished by practolol (10−5 m) and reserpine (2 mg kg−1 daily, for 3 days), whereas that of CTX at high concentrations was partially inhibited by both drugs. 3 In single atrial cells, CTX (3 ng ml−1) produced a marked increase in the amplitude of longitudinal contractions. 4 CTX (3 ng ml−1) caused marked prolongation in the falling phase of action potentials of atrial strips without affecting the maximum rate of rise of action potentials and membrane resting potentials. The effect of CTX on action potentials was abolished by tetrodotoxin (10−6m). 5 The whole‐cell patch‐clamp experiments on myocytes revealed that CTX (20 ng ml−1) shifted the current‐voltage curve of Na inward currents by 40 mV in the negative direction. CTX caused a small sustained Na inward current even at resting membrane potentials. 6 These results suggest that the inotropic action of lower concentrations of CTX is primarily due to an indirect action via noradrenaline release, whereas that of higher concentrations is caused not only by an indirect action but also by a direct action on voltage‐dependent Na channels of cardiac muscle. It is also suggested that CTX activates cardiac muscle Na channels by modifying the voltage‐dependence of channel activation to increase Na inward currents, thus producing cardiotonic actions.

Expression of Ndrg2 in the rat frontal cortex after antidepressant and electroconvulsive treatment

Although the therapeutic action of antidepressants most likely involves the regulation of serotonergic and noradrenergic signal transduction, no consensus has been reached concerning their precise molecular or cellular mechanisms of action. In the present study, we demonstrated that chronic treatment with a tricyclic antidepressant (imipramine) and a selective serotonin reuptake inhibitor (sertraline) reduced the expression of Ndrg2 mRNA and protein in the rat frontal cortex. Ndrg2 is a member of the N-Myc downstream-regulated genes. Interestingly, repeated ECT also significantly decreased Ndrg2 expression in this region of the brain. These data suggest that Ndrg2 may be a common functional molecule that is decreased after antidepressant treatment and ECT. Although, the functional role of Ndrg2 in the central nervous system remains unclear, our findings suggest that Ndrg2 may be associated with treatment-induced adaptive neural plasticity in the brain, a chronic target of antidepressant action. In conclusion, we have identified Ndrg2 as a candidate target molecule of antidepressants and ECT.

The requirement for and mobilization of calcium during induction by sodium ascorbate and by hydrogen peroxide of cell death

The requirement for and mobilization of Ca2+ ions during induction of cell death by sodium ascorbate were compared with those during induction of cell death by hydrogen peroxide. When HL-60 cells were incubated with sodium ascorbate, a rapid increase in the intracellular concentration of Ca2+ ions and subsequent apoptotic cell death, characterized by cell shrinkage, nuclear fragmentation and cleavage of internucleosomal DNA to yield fragments that were multiples of 180-200 base pairs, were induced. However, these effects of sodium ascorbate were significantly reduced in Ca2+-depleted medium. By contrast, hydrogen peroxide induced similar apoptosis associated phenomena in the presence and in the absence of extracellular Ca2+ ions. The intracellular concentration of the reduced form of glutathione was not significantly affected and glutathione disulfide was undetectable during the early stages of apoptosis. These data suggest that sodium ascorbate and hydrogen peroxide initiate cell death by different mechanisms.