Akina Nara

Methamphetamine induces macropinocytosis in differentiated SH-SY5Y human neuroblastoma cells

Acute and chronic abuses of psychostimulant drugs such as methamphetamine (METH) have been known to cause cell death. In particular, neurotoxicity caused by such drugs is one of the most serious adverse events in humans. Although various effects on neuronal cells caused by METH have been studied, the cellular and molecular mechanisms of METH-induced neurotoxicity remain to be elucidated. To investigate the mechanism of METH-induced cytotoxicity, we studied cytological as well as biochemical changes in retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells. Marked cell death was observed with more than 7mM METH, although caspase-dependent apoptotic cell death was not observed with any concentration of METH treatment. The most prominent cytomorphological effect by METH was the formation of large cytoplasmic vacuoles which were not colocalized with either GFP-LC3 or HSP47-GFP, autophagosome and ER markers respectively. In contrast, many of these vacuoles incorporated large molecular weight FITC-dextran and were confirmed as macropinosomes. Our results indicate that METH-induced cytomorphological effects on RA-differentiated SH-SY5Y human neuroblastoma cells involve macropinocytosis and the hyperstimulation of this process may be involved in METH-caused cytotoxicity.

Hyperstimulation of macropinocytosis leads to lysosomal dysfunction during exposure to methamphetamine in SH-SY5Y cells

Although various cytotoxic effects on neuronal cells caused by methamphetamine (METH) have been investigated, the cellular and molecular mechanisms of METH-induced neurotoxicity remain to be elucidated. We previously reported that METH-induced cytomorphological effects on retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells involved macropinocytosis, which is an actin-dependent endocytic pathway. We also noted that hyperstimulation of this process might play an important role in the cytotoxicity of METH in neuronal cells. In this study, we investigated the molecular mechanisms as well as subsequent outcomes of macropinocytosis during METH treatment. It was found that macropinosomes formed upon exposure to METH were colocalized with constitutively active GFP-Ras (G12V) and GFP-Rac1 (Q61L). Furthermore, both Ras inhibitor, farnesylthiosalicylic acid (FTS), and Rac1 inhibitor, EHT1864, significantly inhibited the formation of macropinosomes, suggesting the involvement of these molecules. The expressions of lysosome-associated membrane proteins (lamps) gradually increased by METH in a time-dependent manner. In contrast, the proteolytic activation of cathepsin L, a marker of lysosomal function, was markedly suppressed by METH. METH-induced dysfunction of lysosomal enzyme as well as cell death was significantly attenuated by nocodazole, a microtube interfering reagent that inhibits the transport of vesicles, including macropinosome, to lysosomes. Our results indicate that METH has cytotoxic effects, at least in part, by inhibiting normal lysosomal function through Ras- and Rac1-mediated macropinocytosis in RA-differentiated SH-SY5Y cells.

β2-adrenergic receptor-dependent attenuation of hypoxic pulmonary vasoconstriction prevents progression of pulmonary arterial hypertension in intermittent hypoxic rats

In sleep apnea syndrome (SAS), intermittent hypoxia (IH) induces repeated episodes of hypoxic pulmonary vasoconstriction (HPV) during sleep, which presumably contribute to pulmonary arterial hypertension (PAH). However, the prevalence of PAH was low and severity is mostly mild in SAS patients, and mild or no right ventricular hypertrophy (RVH) was reported in IH-exposed animals. The question then arises as to why PAH is not a universal finding in SAS if repeated hypoxia of sufficient duration causes cycling HPV. In the present study, rats underwent IH at a rate of 3 min cycles of 4–21% O2 for 8 h/d for 6w. Assessment of diameter changes in small pulmonary arteries in response to acute hypoxia and drugs were performed using synchrotron radiation microangiography on anesthetized rats. In IH-rats, neither PAH nor RVH was observed and HPV was strongly reversed. Nadolol (a hydrophilic β1, 2-blocker) augmented the attenuated HPV to almost the same level as that in N-rats, but atenolol (a hydrophilic β1-blocker) had no effect on the HPV in IH. These β-blockers had almost no effect on the HPV in N-rats. Chronic administration of nadolol during 6 weeks of IH exposure induced PAH and RVH in IH-rats, but did not in N-rats. Meanwhile, atenolol had no effect on morphometric and hemodynamic changes in N and IH-rats. Protein expression of the β1-adrenergic receptor (AR) was down-regulated while that of β2AR was preserved in pulmonary arteries of IH-rats. Phosphorylation of p85 (chief component of phosphoinositide 3-kinase (PI3K)), protein kinase B (Akt), and endothelial nitric oxide synthase (eNOS) were abrogated by chronic administration of nadolol in the lung tissue of IH-rats. We conclude that IH-derived activation of β2AR in the pulmonary arteries attenuates the HPV, thereby preventing progression of IH-induced PAH. This protective effect may depend on the β2AR-Gi mediated PI3K/Akt/eNOS signaling pathway.

Pulmonary arterial hypertension in rats due to age-related arginase activation in intermittent hypoxia

Pulmonary arterial hypertension (PAH) is prevalent in patients with obstructive sleep apnea syndrome (OSAS). Aging induces arginase activation and reduces nitric oxide (NO) production in the arteries. Intermittent hypoxia (IH), conferred by cycles of brief hypoxia and normoxia, contributes to OSAS pathogenesis. Here, we studied the role of arginase and aging in the pathogenesis of PAH in adult (9-mo-old) and young (2-mo-old) male Sprague-Dawley rats subjected to IH or normoxia for 4 weeks and analyzed them with a pressure-volume catheter inserted into the right ventricle (RV) and by pulsed Doppler echocardiography. Western blot analysis was conducted on arginase, NO synthase isoforms, and nitrotyrosine. IH induced PAH, as shown by increased RV systolic pressure and RV hypertrophy, in adult rats but not in young rats. IH increased expression levels of arginase I and II proteins in the adult rats. IH also increased arginase I expression in the pulmonary artery endothelium and arginase II in the pulmonary artery adventitia. Furthermore, IH reduced pulmonary levels of nitrate and nitrite but increased nitrotyrosine levels in adult rats. An arginase inhibitor (N(ω)-hydroxy-nor-1-arginine) prevented IH-induced PAH and normalized nitrite and nitrate levels in adult rats. IH induced arginase up-regulation and PAH in adult rats, but not in young rats, through reduced NO production. Our findings suggest that arginase inhibition prevents or reverses PAH.

Pulmonary Macrophages Attenuate Hypoxic Pulmonary Vasoconstriction via β3AR/iNOS Pathway in Rats Exposed to Chronic Intermittent Hypoxia.

Chronic intermittent hypoxia (IH) induces activation of the sympathoadrenal system, which plays a pivotal role in attenuating hypoxic pulmonary vasoconstriction (HPV) via central β1-adrenergic receptors (AR) (brain) and peripheral β2AR (pulmonary arteries). Prolonged hypercatecholemia has been shown to upregulate β3AR. However, the relationship between IH and β3AR in the modification of HPV is unknown. It has been observed that chronic stimulation of β3AR upregulates inducible nitric oxide synthase (iNOS) in cardiomyocytes and that IH exposure causes expression of iNOS in RAW264.7 macrophages. iNOS has been shown to have the ability to dilate pulmonary vessels. Hence, we hypothesized that chronic IH activates β3AR/iNOS signaling in pulmonary macrophages, leading to the promotion of NO secretion and attenuated HPV. Sprague-Dawley rats were exposed to IH (3-min periods of 4–21% O2) for 8 h/d for 6 weeks. The urinary catecholamine concentrations of IH rats were high compared with those of controls, indicating activation of the sympathoadrenal system following chronic IH. Interestingly, chronic IH induced the migration of circulating monocytes into the lungs and the predominant increase in the number of pro-inflammatory pulmonary macrophages. In these macrophages, both β3AR and iNOS were upregulated and stimulation of the β3AR/iNOS pathway in vitro caused them to promote NO secretion. Furthermore, in vivo synchrotron radiation microangiography showed that HPV was significantly attenuated in IH rats and the attenuated HPV was fully restored by blockade of β3AR/iNOS pathway or depletion of pulmonary macrophages. These results suggest that circulating monocyte-derived pulmonary macrophages attenuate HPV via activation of β3AR/iNOS signaling in chronic IH.

Critical roles of Rho-associated kinase in membrane blebbing and mitochondrial pathway of apoptosis caused by 1-butanol

Alcohols are widely used as industrial solvents and chemical intermediates but can cause serious damage to human health. Nevertheless, few studies have addressed the molecular mechanisms underlying the cytotoxicity of industrial alcohols, with the notable exception of ethanol. The goal of our current study is to elucidate the molecular mechanism of cytotoxicity caused by primary alcohols containing longer carbon chains than ethanol. We find that 1-butanol induces morphological changes in H9c2 cardiomyoblastoma including nuclear condensation and membrane blebbing, both of which are features of apoptotic response. Moreover, a decrease in the mitochondrial membrane potential, the cytosolic release of cytochrome c, and the activation of caspase 9 and 3 was observed, thus revealing the activation of the mitochondrial apoptotic pathway by 1-butanol. The addition of Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed the membrane blebbing and mitochondrial apoptotic pathway. In comparison z-VAD-fmk, a pan-caspase inhibitor, did not inhibit membrane blebbing but did prevent cell death following exposure to 1-butanol. These results indicate that mitochondrial pathway of apoptosis and membrane blebbing are parallel phenomena that occur downstream of ROCK. This kinase thus plays an essential role in 1-butanol cytotoxicity and subsequent cell death in H9c2 cells.

Lethal pulmonary air embolism caused by the removal of a double-lumen hemodialysis catheter

Pulmonary air embolisms due to the removal of a central venous catheter are rare, but catheter removal is known to be a high risk factor for air embolism. In particular, the removal of a large catheter, such as a double-lumen hemodialysis catheter, can allow a large amount of air to enter into the bloodstream, which often results in sudden death. So, during catheter removal, special care should be taken to prevent air from entering blood vessels, for example, to ensure that the patients head is tilted downward, that they have inhaled and are holding their breath, and that a covering gauze and inert ointment have been applied to the exit site. We report a lethal case of pulmonary air embolism caused by the removal of a double-lumen catheter from the right internal jugular vein of a patient who was sitting up and had not been instructed to hold their breath.

Death due to blood transfusion-induced anaphylactic shock : A case report

In medical practice, many clinical accidents due to blood transfusion reactions have been reported, among which, nonhemolytic transfusion reactions (NHTRs) have been mainly reported in recent years. NHTRs induce reactions such as anaphylactic shock and transfusion-related acute lung injury (TRALI); however, few forensic autopsy case reports with blood transfusion reactions including anaphylactic shock have been published. A marker for anaphylactic shock is serum tryptase, which indicates systematic mast cell activation in living patients. In forensic medicine, serum tryptase has been used in the postmortem diagnosis of anaphylactic shock. In this autopsy case report, the blood tryptase level was elevated to 49.0 ng/mL (reference standard level <13.5 ng/mL). When considered comprehensively with autopsy findings and blood typing, we concluded that this patient was suspected to have suffered from anaphylactic shock as a result of blood transfusion.

Sequence analysis for HV I region of mitochondrial DNA using WGA (Whole Genome Amplification) method

The hypervariable (HV) region in mitochondrial DNA (mtDNA) has been studied for forensic identification, DNA analysis of mother-child relationship and comparison between ethnic groups. Meanwhile, the WGA (Whole Genome Amplification) method is a technique to amplify genome DNA, and it may be useful for analyzing degraded or limited DNA. However, we need to make a comparative study to confirm whether or not amplified DNAs have the same sequence as the original DNA because the amplification procedure may have some influence on sequencing. We sequenced mtDNA HV I region using amplified human DNAs by WGA method and original human DNAs, and compared the results. DNAs were extracted from four tissue samples from each of 10 healthy volunteers: blood, oral mucosa, nail and hair shaft. WGA was then performed and sequences were analyzed using the amplified samples. After sequencing of the PCR products, the results were compared with the Anderson reference sequence. For each individual, DNA sequences of mtDNA HV I region from the four tissues were the same and WGA amplified samples also showed identical results. Although the WGA procedure may have little influence on the data, we still have to investigate the effectiveness and technical limitation of the method.

Evaluations of lipid peroxidation and inflammation in short-term glycerol-induced acute kidney injury in rats.

Rhabdomyolysis is characterised by acute kidney injury (AKI) resulting from skeletal muscle injury. Lipid peroxidation‐mediated oxidant injury and pro‐inflammatory cytokine‐mediated inflammatory response play critical roles in the pathogenesis of rhabdomyolysis‐induced AKI. The present study aimed to investigate the short‐term effects of both lipid peroxidation and inflammatory responses on rhabdomyolysis‐induced AKI in a rat model of glycerol‐induced rhabdomyolysis. Rhabdomyolysis was induced by the intramuscular injection of 50% glycerol in saline (10 mL/kg) into the hind limbs of rats. Rats were killed 1 or 3 hours after glycerol injection. Time‐dependent increases in serum biochemical parameters, including blood urea nitrogen, creatinine, lactate dehydrogenase and creatine phosphokinase levels, were observed 1 hour after glycerol injection. In kidneys, glycerol injection resulted in histopathological changes such as renal tubular injury and renal tubular myoglobin deposition. Levels of Nε‐(hexanoyl)lysine‐modified, 4‐hydroxy‐2‐nonenal‐modified, and nitrotyrosine‐modified proteins in rat kidneys were unaltered at 1 hour after glycerol injection, but increased significantly at 3 hours. Increases in renal nitric oxide production and the expression levels of inducible nitric oxide synthase, interleukin‐6 and tumour necrosis factor‐α in the renal parenchyma were observed at 1 hour after glycerol injection and plateaued at 3 hours. Our findings suggest that the pro‐inflammatory cytokine‐mediated inflammatory response may cause rhabdomyolysis‐induced AKI very shortly after glycerol injection, and lipid peroxidation‐mediated oxidant injury may promote the development of these pathophysiological processes.