Ken-ichi Yoshida

A Novel Mechanism of JNK1 Activation NUCLEAR TRANSLOCATION AND ACTIVATION OF JNK1 DURING ISCHEMIA AND REPERFUSION

Cytokines and various cellular stresses are known to activate c-Jun NH2-terminal kinase (JNK), which plays a role in conveying signals from the cytosol to the nucleus. Here we investigate the translocation and activation of JNK1 during ischemia and reperfusion in perfused rat heart. Ischemia induces the translocation of JNK1 from the cytosol fraction to the nuclear fraction in a time-dependent manner. Immunohistochemical observation also shows that JNK1 staining in the nucleus is enhanced after ischemia. During reperfusion after ischemia, further nuclear translocation of JNK1 is apparently inhibited. In contrast, JNK1 activity in the nuclear fraction does not increased during ischemia but increases significantly during reperfusion with a peak at 10 min of reperfusion. The activation of JNK1 is confirmed by the phosphorylation of endogenous c-Jun (Ser-73) with similar kinetics. The level of c-jun mRNA also increases during reperfusion but not during ischemia. Based on fractionation and immunohistochemical analyses, an upstream kinase for JNK1, SAPK/ERK kinase 1 (SEK1), is constantly present in both the nucleus and cytoplasm throughout ischemia and reperfusion, whereas an upstream kinase for mitogen-activated protein kinase, MAPK/ERK kinase 1, remains in the cytosol. Furthermore, phosphorylation at Thr-223 of SEK1, necessary for its activation, rapidly increases in the nuclear fraction during postischemic reperfusion. These findings demonstrate that JNK1 translocates to the nucleus during ischemia without activation and is then activated during reperfusion, probably by SEK1 in the nucleus.

Carbon Monoxide Protects Against Cardiac Ischemia-Reperfusion Injury In Vivo Via MAPK and Akt-eNOS Pathways

Background—Carbon monoxide (CO) is postulated to protect tissues against several types of injuries. We investigated the role of CO in amelioration of cardiac ischemia–reperfusion injury in vivo and the mechanisms involved in it. Methods and Results—Rats inhaled CO (250 ppm, 500 ppm, or 1000 ppm) for 24 hours in a chamber after myocardial ischemia–reperfusion induced by occluding the left anterior descending coronary artery for 30 minutes. Pre-exposure to 1000 ppm of CO significantly reduced the ratio of infarct areas to risk areas and suppressed the migration of macrophages and monocytes into infarct areas, and the expression of tumor necrosis factor (TNF)-&agr; in the heart; however, 250 ppm, 500 ppm of CO, or low barometric pressure hypoxia (0.5 atm) did not affect them. Exposure to 1000 ppm CO resulted in the activation of p38 mitogen-activated protein kinase (p38MAPK), protein kinase B&agr;(Akt), endothelial nitric oxide synthase (eNOS), and cyclic guanosine monophosphate (cGMP) in the myocardium. Inhibition of p38MAPK, PI3kinase, NO, and soluble guanylate cyclase with SB203580, wortmannin, N(G)-nitro-l-arginine methyl ester (l-NAME), and methylene blue, respectively, attenuated the cytoprotection by CO. Conclusion—CO has beneficial effects on cardiac ischemia–reperfusion injury; this effect is mediated by p38MAPK pathway and Akt–eNOS pathway, including production of cGMP.

ERK1/2 regulates intracellular ATP levels through alpha-enolase expression in cardiomyocytes exposed to ischemic hypoxia and reoxygenation.

Extracellular signal-regulated kinase 1/2 (ERK1/2) is known to function in cell survival in response to various stresses; however, the mechanism of cell survival by ERK1/2 remains poorly elucidated in ischemic heart. Here we applied functional proteomics by two-dimensional electrophoresis to identify a cellular target of ERK1/2 in response to ischemic hypoxia. Approximately 1500 spots were detected by Coomassie Brilliant Blue staining of a sample from unstimulated cells. The staining intensities of at least 50 spots increased at 6-h reoxygenation after 2-h ischemic hypoxia. Of the 50 spots that increased, at least 4 spots were inhibited in the presence of PD98059, a MEK inhibitor. A protein with a molecular mass of 52 kDa that is strongly induced by ERK1/2 activation in response to ischemic hypoxia and reoxygenation was identified as α-enolase, a rate-limiting enzyme in the glycolytic pathway, by liquid chromatography-mass spectrometry and amino acid sequencing. The expressions of the α-enolase mRNA and protein are inhibited during reoxygenation after ischemic hypoxia in the cells containing a dominant negative mutant of MEK1 and treated with a MEK inhibitor, PD98059, leading to a decrease in ATP levels. α-Enolase expression is also observed in rat heart subjected to ischemia-reperfusion. The induction of α-enolase by ERK1/2 appears to be mediated by c-Myc. The introduction of the α-enolase protein into the cells restores ATP levels and prevents cell death during ischemic hypoxia and reoxygenation in these cells. These results show that α-enolase expression by ERK1/2 participates in the production of ATP during reoxygenation after ischemic hypoxia, and a decrease in ATP induces apoptotic cell death. Furthermore, α-enolase improves the contractility of cardiomyocytes impaired by ischemic hypoxia. Our results reveal that ERK1/2 plays a role in the contractility of cardiomyocytes and cell survival through α-enolase expression during ischemic hypoxia and reoxygenation.

Translocation of protein kinase C-α, δ and ϵ isoforms in ischemic rat heart

To explore the spatial and temporal localization of PKC isoforms during ischemia, we quantified PKC isoforms in the subcellular fractions in perfused rat heart by immunoblotting using specific antibodies against PKC isoforms. PKCs-alpha and epsilon translocated from the 100000 x g supernatant (S, cytosolic) fraction to the 1000 x g pellet (PI, nucleus-myofibril) and the 1000-100000 x g pellet (P2, membrane) fractions during 5-40 min of ischemia. PKC-delta redistributed from the P2 to the S fraction. A 50-kDa fragment of PKC-alpha appeared during ischemia possibly through calpain action. Immunohistochemical observations showed the different localizations of PKC-alpha, delta, and epsilon in the myocytes. The PKC assay displayed high basal levels of Ca(2+)-independent PKC, the activation of Ca(2+)-dependent PKC in the P1 and P2 fractions, and the activation of Ca(2+)-independent PKC in the P1 fraction after 20 min of ischemia. These observations show that ischemia induces different patterns of translocation of the three PKC isoforms, suggesting differences in their roles.

Nuclear translocation of PKCζ during ischemia and its inhibition by wortmannin, an inhibitor of phosphatidylinositol 3-kinase

Protein kinase Cζ (PKCζ), a member of the atypical PKC subgroup, is insensitive to Ca2+, diacylglycerol, and phorbol esters, but is activated by phospholipids such as phosphatidylinositol‐3,4,5‐triphosphate, a product of phosphatidylinositol 3‐kinase (PI3‐kinase). Here we show that PKCζ translocates from the cytosol to the 1000×g pellet (nuclear‐myofibrillar) fraction during ischemia for 40 min in Langendorff‐perfused rat hearts. In addition, immunohistochemical observation shows that ischemia induces the translocation of PKCζ to the nucleus. The nuclear translocation during ischemia is inhibited in a dose‐dependent manner by wortmannin (10−9–10−7 M), an inhibitor of PI3‐kinase.

Calpain activity alters in rat myocardial subfractions after ischemia or reperfusion.

To examine whether calpain is activated during ischemic or reperfusion injury, we measured calpain activity of the subfractions of rat myocardia after global ischemia for 60 min or the ischemia followed by 30 min reperfusion by the Langendorff procedure. The myocardial homogenate was fractionated into 600 x g, 10,000 x g and 100,000 x g pellet fractions as well as 10,000 x g supernatant fraction. The supernatant fraction was further subjected to DEAE cellulose and phenyl-Sepharose chromatographies to separate mu- and m-calpains. The m-calpain activity of the DEAE fractions after global ischemia for 60 min was higher but that after ischemia-reperfusion was lower than that of the control. On the other hand, the ischemia-reperfusion but not ischemia by itself raised the calpain activity of the phenyl-Sepharose fraction (mu-calpain) and the 10,000 x g pellet measured at 100 microM and 5 mM Ca2+. Treatment with verapamil but not with ryanodine during ischemia attenuated the increase in m-calpain activity. A dot-blotting analysis of calpain antigenicity showed a decrease in soluble but no change in the particulate fractions after ischemia-reperfusion. An immunoblotting technique did not detect proteolysis of the calpain 80-kDa subunit. These observations suggest that calpain is activated by Ca2+ influx during ischemia and reperfusion without gross changes in its amount. Some unknown processes other than translocation or autolysis are thought to be involved in the alterations.

Biochemical blood markers and sampling sites in forensic autopsy

Forensic pathologists often hesitate to use biochemical blood markers due to the risk of large postmortem changes and deviations from healthy subjects. Biochemical analyses of postmortem blood, if possible, may help to evaluate pathological status and determining the cause of death in forensic diagnosis, for example, in sudden unexpected death without obvious cause, or young adults with no apparent cause of death or antemortem information. Even commercially available biochemical markers were re-evaluated in the blood samples of 164 forensic autopsy cases. Biochemical markers examined were HbA1c, fructosamine, blood nitrogen urea (BUN), creatinine, total protein, total bilirubin, gamma-glutamyl transpeptidase (gamma-GTP), triglyceride, total cholesterol, C-reactive protein (CRP) and pseudocholine esterase (pChE). We collected cardiac blood (left cardiac blood and right cardiac blood) and peripheral blood (femoral vein blood) to clarify the differences in measured values by sampling site. The measured values were analyzed in relation to postmortem interval, etiology of death and sampling sites. Of all eleven markers, HbA1c is the most useful and reliable because of its negligible postmortem changes and small deviation from healthy subjects. Total bilirubin, BUN, CRP and total cholesterol can be useful if we set appropriate limit ranges and pay attention to the interpretation. For the evaluation of changes due to postmortem intervals, none of the markers except for triglyceride showed significant changes up to three days postmortem. As for sampling sites, femoral vein blood is generally recommended considering postmortem changes, but left cardiac blood was suitable for creatinine, pChE, and total cholesterol. For clinical forensic diagnosis of biochemical blood markers, we must determine the forensic abnormal value after collecting more cases by known causes with more information about the population.

Sympatho-adrenal involvement in methamphetamine-induced hyperthermia through skeletal muscle hypermetabolism.

We investigated the involvement of the sympatho-adrenal axis in the hyperthermia induced by methamphetamine by using a biotelemetric system. The intraperitoneal injection of methamphetamine (1 mg/kg) induced hyperthermia preceded by an increase in oxygen consumption in freely moving rats. The hyperthermic effect of methamphetamine was completely blocked by chemical sympathectomy with 6-hydroxydopamine (50 mg/kg, i.p.). Adrenalectomy, but not adrenal demedullation, prevented the hyperthermia. In adrenalectomized rats, dexamethasone supplementation (0.5 mg/kg, s.c.) restored the methamphetamine-induced hyperthermia. Furthermore, dantrolene (1 or 2 mg/kg, i.v.), which blocks Ca2+ release from the sarcoplasmic reticulum in skeletal muscle, attenuated the hyperthermia. These results suggest that methamphetamine stimulates norepinephrine release from sympathetic nerve terminals, which then enhances thermogenesis in skeletal muscle under the permissive action of glucocorticoids.

Adaptive HNE-Nrf2-HO-1 pathway against oxidative stress is associated with acute gastric mucosal lesions

Disturbance of the microcirculation and generation of reactive oxygen species are crucial in producing acute gastric mucosal lesions (AGML). To understand the protective mechanism against mucosal injury and oxidative stress in the stomach, we investigated sequential expression and localization of a product of lipid peroxidation and a chemical mediator of the oxidative response array, 4-hydroxynonenal (HNE), transcriptional factor, NF-E2-related factor (Nrf2), and the inducible heme oxygenase (HO-1) in the injured stomach. AGML was produced by intragastric administration of 0.6 N HCl in male rats. Expression and localization of HNE, Nrf2, and HO-1 were investigated by Western blotting, immunohistochemistry, real-time RT-PCR, and in situ hybridization histochemistry. Mucosal lesions and expression of HNE and HO-1 were assessed by prior treatment with the PGI2 analog beraprast or after sensory denervation by pretreatment with capsaicin. Mucosal lesions were assessed by prior treatment with a HO-1 inhibitor, zinc protoporphyrin (ZnPP). After AGML, increased generation of HNE was observed in the injured mucosa and the surrounding submucosa, followed by nuclear translocation of Nrf2 and upregulation of HO-1 in the macrophages located in the margin of the injured mucosa and in the submucosa. Pretreatment with beraprost attenuated AGML and downregulated the expression of HNE and HO-1, while sensory denervation aggravated AGML and upregulated the expression of HNE and HO-1. Pretreatment with ZnPP also aggravated AGML. The sequential HNE-Nrf2-HO-1 pathway in the gastric mucosal cells and the macrophages is involved in an adaptive mechanism against oxidative stress after AGML.

Postischemic Reperfusion Induces α‐Fodrin Proteolysis by m‐Calpain in the Synaptosome and Nucleus in Rat Brain

Abstract: A membrane cytoskeletal protein, fodrin, is a substrate for a Ca2+‐dependent protease, calpain. It remains unknown whether μ‐calpain or m‐calpain is involved in the proteolysis of either α‐ or β‐fodrin and in what subcellular localization during ischemia and reperfusion of the brain. To address these issues, we examined the distribution of fodrin and calpain and the activities of calpain and calpastatin (endogenous calpain inhibitor) in the same subcellular fractions. Rat forebrain was subjected to ischemia by a combination of occlusion of both carotid arteries and systemic hypotension, whereas reperfusion was induced by releasing the occlusion. Immunoblotting, activity measurement, and casein zymography did not detect the presence of μ‐calpain or a significant change of m‐calpain level after ischemia or reperfusion. However, casein zymography revealed a unique Ca2+‐dependent protease that was eluted with both 0.18 and 0.40 M NaCl from a DEAE‐cellulose column. α‐ and β‐fodrins and m‐calpain were found to be rich in the synaptosomal, nuclear, and cytosolic subfractions by immunoblotting analysis. Reperfusion (60 min) following ischemia (30 min) induced selective proteolysis of α‐fodrin, which was inhibited by a calpain inhibitor, acetylleucylleucylnorleucinal (400 µM, 1 ml, i.v.). The μ‐calpain‐specific fragment of β‐fodrin was not generated during ischemia‐reperfusion, supporting the possibility of the involvement of m‐calpain rather than μ‐calpain in the α‐fodrin proteolysis.