Haruki Yamakawa

Inhibition of Neutral Sphingomyelinase Activation and Ceramide Formation by Glutathione in Hypoxic PC12 Cell Death

Abstract : Reduced glutathione (GSH) and N‐acetylcysteine (NAC), but not other antioxidative or reducing agents, were found to inhibit cell death, both apoptosis and necrosis, induced by hypoxia in naive and nerve growth factor‐differentiated PC12 cells. The level of intracellular total GSH decreased time‐dependently during hypoxia, but exogenously added GSH prevented such a decrease in GSH. Pretreatment of cells with exogenous GSH or NAC resulted in inhibition of both neutral sphingomyelinase (SMase) activation and ceramide formation during hypoxia. In the in vitro assay system, neutral SMase activity was inhibited dose‐dependently by GSH and NAC. Activation of caspase‐3 induced by hypoxia was also inhibited by either GSH or NAC. NAC but not GSH inhibited caspase‐3 activation induced by C2‐ceramide. These results suggest that GSH protects cells from hypoxic injury by direct inhibition of neutral SMase activity and ceramide formation, resulting in inhibition of caspase‐3 activation, and that NAC exerts an additional inhibitory effect(s) downstream of ceramide.

Cell permeable ROS scavengers, Tiron and Tempol, rescue PC12 cell death caused by pyrogallol or hypoxia/reoxygenation

The role of superoxide anion (O(2)*-) in neuronal cell injury induced by reactive oxygen species (ROS) was examined in PC12 cells using pyrogallol (1,2,3-benzenetrior), a donor to release O(2)*-. Pyrogallol induced PC12 cell death at concentrations, which evidently increased intracellular O(2)*-, as assessed by O(2)(*-)-sensitive fluorescent precursor hydroethidine (HEt). Caspase inhibitors, Z-VAD-FMK and Z-Asp-CH(2)-DCB, failed to protect cells from injury caused by elevation of intracellular O(2)*-, although these inhibitors had effects on hypoxia- or hydrogen peroxide (H(2)O(2))-induced PC12 cell death. Two known O(2)*- scavengers, Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid) and Tempol (4-hydroxy-2,2,6,6-tetramethylpiperydine-1-oxyl) rescued PC12 cells from pyrogallol-induced cell death. Hypoxia/reoxygenation injury of PC12 cells was also blocked by Tiron and Tempol. Further understanding of the underlying mechanism of the protective effects of these radical scavengers reducing intracellular O(2)*- on neuronal cell death may lead to development of new therapeutic treatments for hypoxic/ischemic brain injury.

Influence of Bax or Bcl-2 overexpression on the ceramide-dependent apoptotic pathway in glioma cells

Ceramide has recently been regarded as a potential mediator of apoptosis. In the present study, the effects of Bcl-2 and Bax on the ceramide-mediated apoptotic pathways were examined in glioma cells overexpressing Bcl-2 or Bax. Etoposide, cisplatin and tumor necrosis factor-α induced apoptosis of C6 rat glioma cells which was associated with ceramide formation due to activation of neutral sphingomyelinase, followed by release of mitochondrial cytochrome c into the cytosol and activation of caspases-9 and -3. The growth of C6 cells stably overexpressing either Bcl-2 or Bax was almost equal to that of the vector-transfected cells. Bax overexpression enhanced etoposide-induced apoptosis through acceleration of cytochrome c release and caspases activation. However, Bax had no effect on ceramide formation. Similar findings were obtained in C6 cells and U87-MG human glioblastoma cells which were transiently overexpressed with Bax. In contrast, Bcl-2 overexpression resulted in a retardation of the apoptotic process via prevention of cytochrome c release and caspases activation, and ceramide formation was also blocked when Bcl-2 was highly overexpressed in glioma cells. In addition, transient overexpression of Bcl-xL also exerted inhibitory effects on ceramide formation and apoptotic cell death induced by etoposide. These results indicate that Bax promotes apoptosis regardless of ceramide formation and that Bcl-2 or Bcl-xL prevents ceramide formation by repressing neutral sphingomyelinase as well as ceramide-induced cytochrome c release.

Spinal injuries in snowboarders: risk of jumping as an integral part of snowboarding

BACKGROUND The purpose of this study was to clarify the occurrence rate and characteristics of spinal injuries caused by snowboarding that were sustained at the Okumino skiing area in Gifu Prefecture, Japan, from 1988 to 2000. METHODS This study was a retrospective review of 13,490 cases of snowboard- or ski-related injury treated at Sumi Memorial Hospital over this period. RESULTS A total of 7,188 patients sustained snowboard-related injuries, and 238 of these had spinal injuries caused by snowboarding (3.3%), whereas 6,302 patients sustained ski-related injuries, and 86 of these had spinal injuries caused by skiing (1.4%). Although there were no significant differences in the difficulty of slope, location of vertebral fracture, or spinal cord injury between snowboarders and skiers, the incidence of transverse process fractures was significantly higher in snowboard-related than in skiing-related injury (p < 0.05). In addition, there was a significantly higher incidence of spinal injury among beginner snowboarders than among beginner skiers (p = 0.04). Furthermore, intermediate or expert snowboarders were more likely to be injured because of jumping than beginners (p < 0.001), whereas about 70% of spinal injuries caused by skiing resulted from a simple fall. CONCLUSION Spinal injuries sustained while snowboarding are increasing considerably in incidence and are characterized as complex injuries. We must educate young snowboarders of the risk of this sport, to prevent these serious injuries.

p53 regulates ceramide formation by neutral sphingomyelinase through reactive oxygen species in human glioma cells

The present study was designed to elucidate the relationship between p53 and ceramide, both of which are involved in apoptotic signaling. Treatment of human glioma cells with etoposide caused apoptosis only in cells expressing functional p53. p53 activation was followed by the formation of reactive oxygen species (ROS), superoxide anion (O2−•) measured by hydroethidium oxidation into ethidium and hydrogen peroxide (H2O2) measured by oxidation of 2′,7′-dichlorofluorescin (DCFH) into 2′,7′-dichlorofluorescein (DCF), which was accompanied with ceramide generation through the activation of neutral, but not acid, sphingomyelinase. Superoxide dismutase (SOD), a selective antioxidant for O2−•, had no effects on p53 expression but inhibited ceramide generation and apoptotic cell death caused by etoposide. However, catalase, a specific antioxidant for H2O2, only weakly inhibited and sodium formate, a hydroxyl radical (• OH) scavenger, unaffected etoposide-induced apoptosis. Like etoposide-induced cell death, treatment of glioma cells with the O2−•-releasing agent, pyrogallol, induced typical apoptosis and ceramide generation even in the presence of catalase. In contrast, human glioma cells lacking functional p53, either due to mutation or the expression of E6 protein of human papillomavirus, were highly resistant to etoposide and exhibited no significant change in the ceramide level. Moreover, expression of functional p53 protein in glioma cells expressing mutant p53 using a temperature-sensitive human p53Val138 induced ceramide accumulation by the activation of neutral sphingomyelinase which was dependent on the generation of O2−•. Taken together, these results suggest that p53 may modulate ceramide generation by activation of neutral sphingomyelinase through the formation of O2−•, but not its downstream compounds H2O2 or • OH.

Venous drainage patterns in perimesencephalic nonaneurysmal subarachnoid hemorrhage

OBJECTIVE The precise etiology of perimesencephalic nonaneurysmal subarachnoid hemorrhage (P-SAH) has not yet been determined. We decided to compare the venograms of patients with P-SAH with those of patients with aneurysmal SAH (A-SAH) to examine the relationship between P-SAH and venous drainage patterns. METHODS We retrospectively studied 18 patients with P-SAH during the past 10 years and 112 patients with ruptured A-SAH during the past 4 years by reevaluating their venograms for possible abnormalities in venous structures, particularly focusing on the basal vein of Rosenthal (BVR). Anatomical variants were classified into three types according to the drainage pathway. RESULTS The location and drainage pathway of the BVR proved to be a significantly more primitive configuration in patients with P-SAH than in those with A-SAH (P<0.05). On the other hand, physical action including components of the Valsalva maneuver were the cause of nine cases of P-SAH (69.2%) in this case profile. The occurrence rate was significantly higher in the P-SAH group than in the A-SAH group (14.3%) (p<0.05). CONCLUSION Our data suggest that failure of longitudinal anastomoses between the primary primitive veins as well as excessive strenuous exertion including components of the Valsalva maneuver plays an important predisposing role in the etiology of P-SAH.

Intracanalicular aneurysm at the meatal loop of the distal anterior inferior cerebellar artery: a case report and review of the literature

BACKGROUND Distal aneurysms of the anterior inferior cerebellar artery (AICA) are rare. Most of the reported cases have been located near the internal auditory meatus. Among these cases, only six located in the internal auditory meatus have been reported in the literature. METHODS A 64-year-old female presented with sudden onset of severe headache. Computed tomography (CT) revealed moderate subarachnoid hemorrhage and Gd-DTPA enhanced magnetic resonance imaging (MRI) showed a small high-intensity mass at the right cerebellopontine angle. Although initial digital subtraction angiography (DSA) showed no vascular abnormalities, repeated DSA disclosed a saccular aneurysm at the top of the meatal loop of the right AICA. The patient underwent a suboccipital craniectomy on the 18th day after the hemorrhage RESULTS . In this case, the aneurysm was completely buried in the internal auditory meatus. After unroofing the meatus, the aneurysm was successfully clipped. After 3 months of hospitalization, the patient was discharged with right-sided deafness, partial facial palsy, and no other complications. CONCLUSIONS We discuss some of the clinical features and pitfalls in the surgical management of intracanalicular AICA aneurysms and review previous reports of similar cases.

Crucial role of calpain in hypoxic PC12 cell death: Calpain, but not caspases, mediates degradation of cytoskeletal proteins and protein kinase C-α and-δ

Abstract Ca2+ influx is one of the main causative events in hypoxic PC12 cell death, because an extracellular Ca2+ chelator, ethylene glycol bis (2-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA) inhibited and Ca2+ ionophore A23187 mimicked the hypoxic cell death. The hypoxic cell death was markedly prevented by a broad spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) as well as a calpain inhibitor, calpeptin, as assessed by nuclear staining with Hoechst 33258 and lactate dehydrogenase release. The processing of procaspase-3 was inhibited by z-VAD-FMK, but not by calpeptin. In contrast, z-VAD-FMK failed to block the proteolytic cleavage of fodrin-α, a preferential substrate for calpain. On the other hand, degradation of actin and fodrin-α was prevented by calpeptin but not by z-VAD-FMK. In addition, not only protein kinase C (PKC)-α but also PKC-δ were cleaved to generate ~46 kDa fragments. The PKC fragmentation was inhibited by calpeptin but not by z-VAD-FMK. These findings suggest that the extracellular Ca2+ influx induced by hypoxic stress activates calpain, resulting in the degradation of cytoskeletal proteins and generation of PKC fragments almost independently of caspase activation. Therefore, calpain may play an important role in hypoxic PC12 cell death. [Neurol Res 2001; 23: 522-530]

Increased phospholipase D2 activity during hypoxia-induced death of PC12 cells: its possible anti-apoptotic role.

During hypoxic incubation (1% O2) of PC12 cells, the PLD activity was transiently increased within 12 h, followed by a gradual decrease. In the in vitro assay, the increased PLD activity was independent of GTPγS required for PLD1 or of oleic acid for PLDOA, suggesting the activation of PLD2. The level of PLD2 protein showed no change up to 12 h but a gradual decrease after 24 h. Pretreatment of cells with S. chromofuscus PLD resulted in inhibition of hypoxia-induced apoptotic cell death. In contrast, 1-butanol, but not 2-butanol, potentiated cell death. Moreover, the number of apoptotic cells significantly reduced in PC12 cells over-expressing PLD2. These results raise the possibility that PLD2 activation may play an anti-apoptotic role in hypoxia-induced cell death.

Analysis of phospholipase C gene in patients with subarachnoid hemorrhage due to ruptured intracranial saccular aneurysm

This study is designed to determine whether patients with aneurysmal subarachnoid hemorrhage have mutations in the phospholipase C-delta 1 (PLC-delta 1) gene, which was identified as a gene responsible for hypertension in spontaneously hypertensive rats. Seventy-two cases (31 male and 41 female) with intracranial saccular aneurysms were analyzed. The mean age was 60.1 +/- 11.5 years (mean +/- SD) (range 24-85 years). There were 35 patients (48.6%) with hypertension, 5 (6.9%) with diabetes mellitus, 12 (16.7%) with hyperlipidemia, 8 (11.1%) with ischemic heart disease, and 25 (34.7%) who were active smokers. The location of aneurysm was distributed as follows: 33 (33%) were at anterior cerebral artery, 23 (23%) were at middle cerebral artery, 28 (28%) were at internal carotid artery, and 16 (16%) were at vertebro-basilar artery. Six patients (8.3%) had a family history of intracranial aneurysms. There were 20 patients (27.8%) with multiple aneurysms, and 8 patients (11.1%) with a large or giant aneurysm. The four regions of PLC-delta 1 gene (bases 1099-1271, 1254-1401, 1343-1481, and 1882-2023) where genetic mutations were found in spontaneously hypertensive rats, were screened by PCR-SSCP analysis and their nucleotide sequences of all patients were determined. However, no mutations were detected in all patients. These results suggest that mutations of PLC-delta 1 gene previously implicated in hypertensive factor in rats may not be the case with human patients and therefore may be poorly related with aneurysmal subarachnoid hemorrhage.