Cerebral vasospasm is a persistent arterial narrowing typically observed during the 3 – 14 days after subarachnoid hemorrhage (SAH). Vasospasm is frequently associated with ischemic neurological deficits or even death, resulting in a poor prognosis for patients with SAH. However, the mechanism underlying cerebral vasospasm remains elusive, and no effective therapeutic strategies have been established. A large amount of thrombin is produced during SAH. Recent investigations have uncovered a key role of the thrombin receptor in the pathogenesis of cerebral vasospasm. Thrombin has little contractile effect in the normal cerebral artery, but it induces an enhanced and prolonged contraction after SAH, owing to the up-regulation of thrombin receptor PAR1 (proteinase-activated receptor 1) and the impairment of receptor desensitization in arterial smooth muscle. Thrombin-mediated activation of PAR1 is an irreversible process, as it is initiated by the proteolytic removal of the N-terminal region. Since the mechanism of receptor desensitization is impaired after SAH, the thrombin-induced contraction irreversibly persists even after terminating thrombin stimulation. Intrathecal administration of a PAR1 antagonist prevents the PAR1 up-regulation and the increased reactivity to thrombin. PAR1 is suggested to play a key role in cerebral vasospasm and may be useful as a therapeutic target for prevention and treatment of cerebral vasospasm.
NADPH oxidase is a superoxide (O2−)-generating enzyme first identified in phagocytes that shows bactericidal activities. It has been reported that O2− is also produced in an NADPH-dependent manner in non-phagocytes. In the last decade, non-phagocyte-type NADPH oxidases have been identified, and the catalytic subunit NOX family has been found to be composed of five homologs, NOX1 to NOX5, and two related enzymes, DUOX1 and DUOX2. These NOX proteins have distinct features in dependency on other components for maximal enzymatic activity, tissue distribution, expressional regulation, and physiological functions. This review summarized the distinct characteristics of NOX family proteins, especially focusing on their functions and mechanisms of their expressional regulation.
The hepatoprotective effects of sarmentosin-containing extracts of Sedum sarmentosum (SS) in D-galactosamine (D-GalN) / lipopolysaccharide (LPS)–induced fulminant hepatic failure mouse model. Pretreatment with SS markedly protected mice from lethal liver injury, which has known to be associated with an abrupt elevation of serum tumor necrosis factor (TNF)-α level. Indeed, SS significantly blocked the elevation of TNF-α and alanine aminotransferase and aspartate aminotransferase as well. SS also remarkably reduced number of apoptotic hepatocytes and DNA fragmentation in the liver, which correlated with blockade of caspase-3 activation. In addition, SS suppressed the increased expression of toll-like receptor 4 (TLR4). The activation of c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 induced by D-GalN/LPS was also significantly suppressed by SS treatment. Furthermore, SSsignificantly inhibited the activation of nuclear factor-κB. In RAW 264.7 cells stimulated with LPS, TNF-α release and TLR4 expression was suppressed by SS pretreatment, which was in line with in vivo results. These findings suggested that SS prevents D-GalN/LPS–induced fulminant hepatic failure, and this protection is likely associated with its anti-apoptotic activity and the down-regulation of mitogen activated protein kinase activity associated at least in part with suppressing the transcription of LPS receptors.
Genistein is a naturally occurring plant-derived phytoestrogen, present in the human diet, known to possess some beneficial effects. The present study investigated the effect of genistein on neuroprotection evaluated through electroencephalographic and behavioural correlates in a model of global cerebral ischemia in gerbils. Over the dose range tested, genistein (3 and 10 mg/kg), given 5 min after recirculation antagonized the ischemia-induced electroencephalographic total spectral power decrease 7 days after ischemia; fully prevented ischemia-induced hyperlocomotion evaluated 1 day after ischemia; reversed ischemia-induced memory impairment evaluated through both nest building behaviour and object recognition test; decreased malondialdehyde overproduction in the brain, evaluated 7 days after reperfusion; and fully promoted the survival of pyramidal cells in the CA1 hippocampal subfield. The selective antagonist for estrogen receptor–β (ERβ), 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) given 30 min before carotid occlusion, fully prevented the neuroprotective effect of genistein at the dose of 3 mg/kg. These results demonstrate the neuroprotective effect of genistein through the activation of ERβ and provide further grounds for the growing interest concerning the true potential of phytoestrogens as compounds to beneficially affect brain injury without having the disadvantages of estrogens.
Axonal transport plays a crucial role in neuronal morphogenesis, survival, and function. Despite its importance, however, the molecular mechanisms of axonal transport remain mostly unknown because a simple and quantitative assay system for axonal transport has been lacking. In order to better characterize the molecular mechanisms involved in axonal transport, we here developed a computer-assisted monitoring system. Using lipophilic fluorochrome chloromethylbenzamido dialkylcarbocyanine (CM-DiI) as a labeling dye, we have successfully labeled membranous organelles in cultured chick dorsal root ganglia neurons. We confirmed that sodium azide, an ATPase inhibitor, and nocodazole, a microtubule-destabilizing agent, markedly suppressed anterograde and retrograde axonal transport of CM-DiI–labeled particles. We further tested the effects of several anti-neoplastic drugs on axonal transport. Paclitaxel, vincristine, cisplatin, and oxaliplatin, all of which are known to be neurotoxic and to cause neurological symptoms, suppressed anterograde and retrograde axonal transport. Another series of anti-neoplastic drugs, including methotrexate and 5-fluorouracil, did not affect the axonal transport. This is the first report of an automated monitoring system for axonal transport. This system will be useful for toxicity assays, characterizing axonal transport, or screening drugs that may modify neuronal functions.
We recently reported endothelium-dependent relaxation caused by nucleotides in the non-human primate cerebral artery. Here, we investigated the endothelium-dependent, nitric oxide- and prostanoid-independent relaxation induced by 2-methylthio-ADP (2MeSADP) in monkey cerebral artery. Mechanical responses of isolated monkey cerebral arteries to the agents were isometrically recorded. In endothelium-intact arterial strips treated with indomethacin plus NG-nitro-L-arginine and partially contracted with prostaglandin F2α, 2MeSADP (1 nM – 10 μM) induced concentration-dependent relaxation that was abolished by removal of endothelium but was not influenced by either carboxy PTIO or 18α-glycyrrhetinic acid. The 2MeSADP-induced relaxation was inhibited by MRS2179 and U73122. The relaxation was markedly suppressed by exposure of the strips to high K+ media, but was not affected by glibenclamide. Combination of charybdotoxin plus apamin markedly suppressed the relaxation, whereas iberiotoxin partially attenuated it. Relaxation induced by 2MeSADP was inhibited by arachidonyl trifluoromethyl ketone, ketoconazole, and 14,15-epoxyeicosa-5(Z)-enoic acid. The inhibitors that affected the 2MeSADP-induced relaxation did not influence relaxation caused by sodium nitroprusside or forskolin. These findings indicate that 2MeSADP elicits ‘endothelium-derived hyperpolarizing factor (EDHF)-type’ relaxation via stimulation of endothelial P2Y1 receptors in monkey cerebral artery. Furthermore, phospholipase A2, cytochrome P450–derived epoxyeicosatrienoic acids and Ca2+-activated K+ channels appear to be involved in the relaxation.
We performed this study to elucidate whether a newly developed liposome-encapsulated hemoglobin, TRM-645 (TRM), can prevent cerebral dysfunction resulting from acute ischemic stroke when used as an oxygen carrier. Hippocampal long-term potentiation (LTP) in the perforant path–dentate gyrus synapses and anxiety-related behaviors in the elevated plus-maze test were evaluated as indices of cerebral functional outcomes in the rat with two-vessel occlusion (2VO), which was induced by 10-min clamping of bilateral common carotid arteries. Saline or TRM (hemoglobin concentration of 6 g/dl: 2.5 or 5 ml/kg) was administered via the tail vein immediately after ischemic insult. Hippocampal LTP formation was markedly impaired and the open arm durations in the elevated plus-maze decreased significantly 4 days after 2VO, compared to those of sham-operated (control) rats, suggesting the hippocampal synaptic dysfunction and anxiogenic properties in 2VO rats. TRM (5 ml/kg) restored the hippocampal LTP formation and normalized the anxiety-related behavior. TRM also improved the decreased tissue oxygen partial pressure in the 2VO rat hippocampus, possibly due to oxygen delivery to ischemic regions. Liposome-encapsulated hemoglobin TRM might have therapeutic potentials for protecting the brain from neurological complications associated with acute ischemic stroke, as a promising blood substitute for oxygen therapy.
Large-conductance Ca2+-activated K+ (BK) channel regulates action potential firing in pancreatic β-cells. We cloned novel spliced variants of the BK-channel β2-subunit (BKβ2b), which consisted of 36 amino acids including the N-terminal in the original human BKβ2 (BKβ2a), from human and rodent pancreas. Real-time PCR analysis showed the abundant expression of BKβ2b transcripts in human and rodent pancreas and also in the RINm5f insulinoma cell line. In addition, up-regulation of both BK-channel α-subunit (BKα) and BKβ2b transcripts was observed in pancreas tissues from diabetes mellitus patients. In HEK293 cells co-expressing BKα and BKβ2b, the inactivation of BK-channel currents, which is typical for BKα + BKβ2a, was not observed, and electrophysiological and pharmacological properties of BKα + BKβ2b were almost identical to those of BKα alone. In HEK293 cells stably expressing BKα, the transient co-expression of yellow fluorescence protein (YFP)-tagged BKβ2a proteins resulted in their distribution along the cell membrane. In contrast, the co-expression of YFP-tagged BKβ2b with BKα showed diffusely distributed fluorescence signals throughout the cell body. Taken together, the predominant splicing of BKβ2b versus that of BKβ2a presumably enhances the contribution of BK channels to membrane potential and may possibly be a factor modulating insulin secretion in a suppressive manner in pancreatic β-cells.
Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is susceptible to aggregation that induces apoptotic cell death. Congo red is widely used as a histological dye for amyloid detection. Recent evidence has revealed that Congo red has the property to inhibit amyloid oligomers and fibril formation of misfolded proteins. In the present study, we examine whether Congo red inhibits aggregate formation and cytotoxicity of mutant γPKC. Congo red likely inhibits aggregate formation of mutant γPKC – green fluorescent protein (GFP) without affecting its expression level in SH-SY5Y cells. Congo red counteracts the insolubilization of recombinant mutant γPKC, suggesting that the dye inhibits aggregation of mutant γPKC by a direct mechanism. Congo red also inhibits aggregation and oligomerization of mutant γPKC-GFP in primary cultured cerebellar Purkinje cells. Moreover, the dye reverses the improper development of dendrites and inhibits apoptotic cell death in Purkinje cells that express mutant γPKC-GFP. These results indicate that amyloid-inhibiting compounds like Congo red may be novel therapeutics for SCA14. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10170FP]
Several reports have shown that some dopamine receptor ligands modulate the ischemia–reperfusion injury in animal models; however, its underling mechanisms are still unclear. In this study, we sought to establish an in vitro experimental model of hypoxia/reoxygenation (H/R) using HT22 cells that originated from mouse hippocampal neurons and to examine protective the effect of dopamine-receptor ligands against H/R-induced cell injury. The treatment with hypoxia for 18 h followed by reoxygenation for 6 h induced the elevation of intracellular reactive oxygen species (ROS) and reduction of mitochondrial membrane potential; however, lactate dehydrogenase (LDH) release was not changed at this time point. LDH release was increased after reoxygenation for 18 h and longer, and this increase in LDH release was suppressed by dopamine receptor agonists such as apomorphine and apocodeine. The suppressive effects of these agonists were reversibly inhibited by L750667, a D4-receptor antagonist but not by D2- or D3-receptor antagonists. In addition, PD168077, a selective dopamine D4–receptor agonist, also protected against H/R-induced cell death. These results suggest that H/R causes oxidative stress–induced cell death and that the activation of dopamine D4 receptors protects against H/R-induced cell death in HT22 cells.
We performed this study to determine if curcumin affects pro-inflammatory responses to activation of proteinase-activated receptor-2 (PAR2) in human pulmonary adenocarcinoma A549 cells. Curcumin completely inhibited the PAR2-triggered prostaglandin E2 (PGE2) production, but notably not interleukin-8 release. Cyclooxygenase-2 (COX-2) upregulation, but not its upstream activation of mitogen-activated protein kinases, caused by PAR2 stimulation was partially inhibited by curcumin. Curcumin inhibited the PAR2-triggered phosphorylation of I-κB, an indicator for nuclear factor-κB (NF-κB) activation, and also its upstream signal Akt, which is known to contribute to PAR2-triggered PGE2 formation, but not COX-2 upregulation. Collectively, curcumin inhibits the PAR2-triggered PGE2 production by suppressing COX-2 upregulation and Akt/NF-κB signals in A549 cells.
Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diseases such as neurodegenerative disease. In the present study, we established the ER stress–resistant SH-SY5Y cell line, and through microarray analysis, we found that TEK/Tie2 expression is up-regulated in this cell line. Moreover, we found that TEK/Tie2 expression was markedly decreased in ER-stressed cells. The effect was time-dependent (2 – 24 h), which began to decrease from 2-h time point. Our findings suggest that TEK/Tie2 expression is involved in cell survival, whereas when severe ER stress occurs, TEK/Tie2 expression is down-regulated, resulting in cell death.
In the present study, we investigated whether ethanol physical dependence causes changes of serotonin transporter (SERT) expression in the brain. SERT expression increased in the cingulate cortex, nucleus accumbens, hippocampal CA1-CA3 layers, and mediodorsal nucleus of the thalamus and decreased in the basolateral nucleus of the amygdala. In addition, chronic ethanol treatment increased SERT mRNA in the dorsal raphe nucleus from which serotonergic neurons originate, although no SERT mRNA was detected in the regions where SERT protein increased. These findings suggest that alteration of SERT levels in the brain may be related to emotional changes observed in ethanol physical dependence.