Hisayuki Ohata

Ndrg2 promotes neurite outgrowth of NGF-differentiated PC12 cells

Ndrg2 is a member of the N-myc downstream-regulated genes. Thus far, two different isoforms of rat Ndrg2 protein, Ndrg2S and Ndrg2L, have been identified. Recently, we have identified rat Ndrg2 as a novel target molecule of antidepressants and ECT. The functional role of Ndrg2 in the central nervous system, however, remains unclear. In the present study, we examined the expression of endogenous Ndrg2, cellular localization of transfected Ndrg2 protein, and morphological changes resulting from overexpression of Ndrg2 in NGF-differentiated PC12 cells. Neurites began to sprout 1-2 days after exposure to NGF; subsequent neurite growth continued for 5 days. During this time, we evaluated Ndrg2 mRNA expression by real-time quantitative PCR and found that expression significantly increased in a time-dependent manner. Interestingly, V5-conjugated Ndrg2S and Ndrg2L proteins expressed in NGF-differentiated PC12 specifically localized to cell surface membranes and growth cones. Moreover, Ndrg2S and Ndrg2L overexpression promoted neurite elongation in NGF-differentiated PC12 cells. In conclusion, our findings offer novel insights into the physiological roles of Ndrg2 in the central nervous system.

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.

m-Calpain Induction in Vascular Endothelial Cells on Human and Mouse Atheromas and Its Roles in VE-Cadherin Disorganization and Atherosclerosis

Background— Although dysfunction of VE-cadherin–mediated adherence junctions in vascular endothelial cells (ECs) is thought to be one of the initial steps of atherosclerosis, little is known regarding how VE-cadherin is disrupted during atherogenic development. This study focused on the role of calpain, an intracellular cysteine protease, in the proteolytic disorganization of VE-cadherin and subsequent progression of atherosclerosis. Methods and Results— Increased expression of m-calpain was observed in aortic ECs in atherosclerotic lesions in humans and low-density lipoprotein receptor–deficient (ldlr−/−) mice. Furthermore, proteolytic disorganization of VE-cadherin was shown in aortic ECs in ldlr−/− and apolipoprotein E–deficient (apoE−/−) mice. Long-term administration of calpain inhibitors into these mice attenuated atherosclerotic lesion development and proinflammatory responses, as well as VE-cadherin disorganization, without normalization of plasma lipid profiles. Furthermore, in vivo transfection of m-calpain siRNA to ldlr−/− mice prevented disorganization of VE-cadherin and proatherogenic hyperpermeability in aortic ECs. Treatment of cultured ECs with oxidized LDL, lysophosphatidylcholine, or LDL pretreated with secreted phospholipase A2 led to the induction of m-calpain but not of &mgr;-calpain, thereby eliciting selective m-calpain overactivation. These data suggest that lysophosphatidylcholine-induced m-calpain directly cleaves a juxtamembrane region of VE-cadherin, resulting in dissociation of &bgr;-catenin from the VE-cadherin complex, disorganization of adherence junctions, and hyperpermeability in ECs. Conclusions— Subtype-selective induction of m-calpain in aortic ECs during atherosclerotic progression is associated with proteolytic disorganization of VE-cadherin and proatherogenic hyperpermeability in cells. Thus, a strategy to selectively inhibit m-calpain may be useful for the therapeutic treatment of patients with atherosclerosis.

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.

Distinct Effects of Tissue-Type Plasminogen Activator and SMTP-7 on Cerebrovascular Inflammation Following Thrombolytic Reperfusion

Background and Purpose— Thrombolysis therapy using tissue-type plasminogen activator (t-PA) is occasionally accompanied by harmful outcomes, including intracerebral hemorrhage. We have reported that Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a candidate thrombolytic drug, has excellent therapeutic effect on cerebral infarction in embolic stroke model in mice; however, little is known regarding whether this agent influences cerebrovascular inflammation following thrombolytic reperfusion. The current study aimed to compare the effects of recombinant t-PA (rt-PA) and SMTP-7 on cerebrovascular inflammation. Methods— The impact of rt-PA- and SMTP-7-induced thrombolytic reperfusion on leukocyte dynamics was investigated in a photochemically induced thrombotic middle cerebral artery occlusion (tMCAo) model in mice. Results— Both rt-PA and SMTP-7 administration in tMCAo mice (each 10 mg/kg) resulted in thrombolytic reperfusion. The SMTP-7-administered mice showed relatively mild rolling and attachment of leukocytes to the vascular wall in the middle cerebral vein, with weak peroxynitrite reactions and proinflammatory gene expression (IL-1&bgr;, TNF-&agr;, ICAM-1, and VCAM-1); thus, a small infarct volume compared with rt-PA-administered mice. In vitro study suggested that rt-PA at 20 &mgr;g/mL, but not SMTP-7 at a similar concentration, promotes cytokine-induced reactive oxygen species generation in cultured endothelial cells; moreover, SMTP-7 suppressed cytokine-induced VCAM-1 induction in the cells and leukocyte/ endothelial cell adhesions. Conclusions— Relatively mild cerebrovascular inflammation and cerebral infarction in the SMTP-7 mice, compared with in rt-PA mice, is thought to be caused at least in part by direct antioxidative actions of SMTP-7 in ECs.

[29] Confocal imaging of Ca2+, pH, electrical potential, and membrane permeability in single living cells

Publisher Summary Responses of single cells to stimuli are often heterogeneous. Bulk measurements by conventional biochemical and physiological techniques may fail to represent accurately the magnitude and time course of individual cell changes. Spatial heterogeneity of responses within single cells also occurs. For this reason, microscopic techniques with good three-dimensional resolution are needed to study individual cells as they respond to imposed stimuli and stresses. Increasingly, confocal microscopy of parameter-specific fluorophores is permitting direct observation of single-cell physiology with high spatial and temporal resolution. Confocal microscopy has become an essential tool to study the physiology of single living cells. As more parameter-indicating fluorophores are discovered, the range of applications of confocal microscopy can only increase. Uniquely, confocal microscopy permits observation of the physiology and metabolism of single organelles inside cells, as illustrated here for measurements of Ca 2+ , pH, ΔΨ, and membrane permeability of mitochondria. Overall, the impact of confocal microscopy on experimental cell physiology may someday compete with that of single-cell electrical recording.

Measurement of electrical potential, pH, and free calcium ion concentration in mitochondria of living cells by laser scanning confocal microscopy.

Publisher Summary The improvement in the resolution of confocal microscopy over conventional microscopy is comparable to that of magnetic resonance imaging over conventional radiography. Confocal microscopy is increasingly an essential analytical tool for studying the structure and physiology of living cells. The examples discussed in this chapter illustrate the use of confocal microscopy to image the intracellular distribution of pH, Ca 2+ , and electrical potential inside single living cells. Because many ion-indicating fluorophores distribute into both mitochondria and cytosol, ions can be measured individually in both compartments. By comparing mitochondrial and cytosolic pH and electrical potential, total mitochondrial proton-motive force can also be estimated. By using stable fluorophores and low levels of illumination, hundreds of images of living cells can be collected with negligible phototoxicity or photobleaching. Application of confocal microscopy promises to provide unique insights into mitochondrial function inside single living cells.

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.

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.