Differential regulation of proliferation and differentiation in neural precursor cells by the Jak pathway.

Neuronal precursor cells (NPCs) are temporally regulated and have the ability to proliferate and differentiate into mature neurons, oligodendrocytes, and astrocytes in the presence of growth factors (GFs). In the present study, the role of the Jak pathway in brain development was investigated in NPCs derived from neurosphere cultures using Jak2 and Jak3 small interfering RNAs and specific inhibitors. Jak2 inhibition profoundly decreased NPC proliferation, preventing further differentiation into neurons and glial cells. However, Jak3 inhibition induced neuronal differentiation accompanied by neurite growth. This phenomenon was due to the Jak3 inhibition-mediated induction of neurogenin (Ngn)2 and NeuroD in NPCs. Jak3 inhibition induced NPCs to differentiate into scattered neurons and increased the expression of Tuj1, microtubule associated protein 2 (MAP2), Olig2, and neuroglial protein (NG)2, but decreased glial fibrillary acidic protein (GFAP) expression, with predominant neurogenesis/polydendrogenesis compared with astrogliogenesis. Therefore, Jak2 may be important for NPC proliferation and maintenance, whereas knocking-down of Jak3 signaling is essential for NPC differentiation into neurons and oligodendrocytes but does not lead to astrocyte differentiation. These results suggest that NPC proliferation and differentiation are differentially regulated by the Jak pathway.

Calpain-mediated N-cadherin proteolytic processing in brain injury.

Neural-cadherin (N-cadherin), a member of the classical cadherin family of transmembrane glycoproteins, mediates cellular recognition and cell-cell adhesion through calcium-dependent homophilic interactions and plays important roles in the development and maintenance of the nervous system. Metalloproteinase is known to cleave N-cadherin, which is further cleaved by gamma-secretase. The intracellular domain of N-cadherin interacts with beta-catenin, and beta-catenin stability is critical for cell-cell adhesion and cell survival. In the present study, we showed that N-cadherin is cleaved specifically by calpain, resulting in the generation of a novel 110 kDa fragment. The cleavage occurred in ischemic brain lesions and in vitro neural cells in the presence of NMDA and ionomycin, and was restored by calpain inhibitors but not matrix metalloproteinase or gamma-secretase inhibitors. Calpain directly cleaved N-cadherin in in vitro calpain assays, and calpain inhibitors prevented its cleavage in a dose-dependent manner. Using N-cadherin deletion mutants, we found that calpain cleavage sites exist in at least four regions of the cytoplasmic domain. Treatment with NMDA induced neuronal death, and it suppressed the expression of surface N-cadherin and the N-cadherin/beta-catenin interaction, effects that were prevented by calpain inhibitor. Furthermore, calpain-mediated N-cadherin cleavage significantly affected cell-cell adhesion, AKT signaling, the N-cadherin/beta-catenin interaction and the Wnt target gene expressions through the accumulation of nuclear beta-catenin.

Methanolic extract of onion (Allium cepa) attenuates ischemia/hypoxia-induced apoptosis in cardiomyocytes via antioxidant effect.

BACKGROUND: Although there is growing awareness of the beneficial potential of onion intake to lower the risk of cardiovascular disease, there is little information about the effect of onion on ischemic heart injury, one of the most common cardiovascular diseases. AIM OF THE STUDY: This study investigates the effect of the methanol-soluble extract of onion on ischemic injury in heart-derived H9c2 cells in vitro and in rat hearts in vivo. The underlying mechanism is also investigated. METHODS: To evaluate the effect of onion on ischemia-induced cell death, LDH release and TUNEL-positivity were assessed in H9c2 cells, and the infarct size was measured in a myocardial infarct model. To investigate the mechanism of the cardioprotection by onion, the reactive oxygen species (ROS) level and the mitochondrial membrane potential (DeltaPsi(m)) were measured using an imaging technique; the caspase-3 activity was assayed, and Western blotting was performed to examine cytochrome c release in H9c2 cells. RESULTS: The methanolic extract of onion had a preventive effect on ischemia/hypoxia-induced apoptotic death in H9c2 cells in vitro and in rat heart in vivo. The onion extract (0.05 g/ml) inhibited the elevation of the ROS, mitochondrial membrane depolarization, cytochrome c release and caspase-3 activation during hypoxia in H9c2 cells. In the in vivo rat myocardial infarction model, onion extract (10 g/kg) significantly reduced the infarct size, the apoptotic cell death of the heart and the plasma MDA level. CONCLUSION: In conclusion, the results of this study suggest that the methanolic extract of onion attenuates ischemia/hypoxia-induced apoptosis in heart-derived H9c2 cells in vitro and in rat hearts in vivo, through, at least in part, an antioxidant effect.

Involvement of endogenous prostaglandin F2alpha on kainic acid-induced seizure activity through FP receptor: the mechanism of proconvulsant effects of COX-2 inhibitors.

COX-2 and prostaglandins (PGs) might play important roles in epilepsy. In kainic acid-induced seizures, the brain largely increases PGD(2), first from COX-1 and later COX-2-induced PGF(2alpha). Pre-treatment with COX-2 inhibitors such as indomethacin, nimesulide, and celecoxib is known to aggravate kainic acid (KA)-induced seizure activity. However it is not known whether the proconvulsant effect of those non-steroidal anti-inflammatory drugs (NSAIDs) is due to changes in endogenous prostaglandins (PGs), or what types of PGs are involved. The purpose of this study was to determine the effect of intracisternally administered PGs on KA-induced seizures aggravated by pre- or post-treatment with COX-2 inhibitors. Systemic KA injection (10 mg/kg i.p.) in mice evoked mild seizure activity within 15 min. PGs were administrated intracisternally 20 min prior to KA administration. COX inhibitors (indomethacin, nimesulide, and ketoprofen, 10 mg/kg i.p.) were injected 1 h before or 15 min after KA. An additional COX-2 inhibitor, celecoxib, was administered orally. Intracisternally administered PGF(2alpha) (700 ng), but not PGD(2) (700 ng) or PGE(2) (700 ng) completely alleviated KA-induced seizures potentiated by COX-2 inhibitors, and also reduced KA-induced hippocampal neuronal death aggravated by indomethacin. PGF(2alpha) alone did not affect KA-induced seizures. However, an FP receptor antagonist, AL 8810 (10 or 50 ng) which is an 11beta-fluoro analogue of PGF(2alpha) potentiated KA-induced seizure activity dose-dependently. In summary, pre- or post-treatment with COX-2 inhibitors aggravates KA-induced seizures, which suggests to change the endogenous PGF(2alpha). Seizure-induced PGF(2alpha) might act as an endogenous anticonvulsant through FP receptors.

Cadmium induces apoptotic cell death through p38 MAPK in brain microvessel endothelial cells.

Cadmium (Cd), an ubiquitous heavy metal, is known to be accumulated outside of the blood-brain barrier. In this study, we investigated whether Cd has cytotoxicity in mouse brain microvascular endothelial cells (bEnd.3). Results from the cell viability assay showed that Cd caused a remarkable decrease in cell viability in a dose-dependent manner. The cell death induced by Cd appeared to involve apoptosis, based on our results from annexin V staining, electron microscopy and TUNEL staining. And the cell death induced by Cd was inhibited by caspase inhibitor ZVAD-fmk. To further investigate the mechanism of the Cd-induced cell death, we examined the effects of selective inhibitors for mitogen activated protein kinase (MAPK) pathways on the cell death. The Cd-induced cell death was significantly inhibited by p38 MAPK inhibitor SB202190, but not by either, c-Jun N-terminal kinase (JNK) inhibitor SP600125 or extracellular signal-regulated kinase (ERK) inhibitor U0126. Phosphorylations of p38 MAPK, JNK and ERK were stimulated by treatment with CdCl(2). In summary, our results suggest that Cd can induce apoptotic cell death, at least in part, through the p38 MAPK pathway in brain microvascular endothelial cells.

Anti-hypertensive effect of the Dongchunghacho, Isaria sinclairii, in the spontaneously hypertensive rats.

The present study examined the effect of the methanol extract of Isaria sinclairii, a kind of Donchunghacho (Tochukaso), on blood pressure in spontaneously hypertensive rats (SHR). Blood pressure and heart rate were measured after treatment with the methanol extract of I. sinclairii by the indirect tail-cuff method and the direct in vivo model. Starting at 12 weeks of age, male SHR were treated with the extracts for 2 or 4 weeks. We found that, when compared to untreated control SHR, oral treatment with I. sinclairii methanol extract (30 mg/kg/day) remarkably decreased systolic blood pressure from 200 to 112 mmHg and decreased diastolic blood pressure from 114 to 88 mmHg. Furthermore, efficacy of methanol extract of I. sinclairii was superior to captopril (30 mg/kg/mL, positive control), an angiotensin-converting enzyme inhibitor, with a lowering effect that dropped systolic blood pressure from 201 to 130 mmHg and diastolic blood pressure from 102 to 92 mmHg. However, in normal Wistar Kyoto rats, I. sinclairii methanol extract did not significantly change the normal blood pressure, suggesting that this type of Dongchunghacho has a selective effect against hypertension. Therefore, methanol extract of I. sinclairii may be used as an anti-hypertensive food/agent. Furthermore, this extract also has multiple actions such as No production in endothelial cells, inhibiting thrombin-induced blood coagulation by thrombin and mildly decreasing in prostaglandin E2 levels in cultured macrophage cells, all of which might contribute to protection against atherogenesis and thrombus formation. HPLC and MS analysis of methanol extract of I. sinclairii revealed the presence of adenosine.

Effects of DK-002, a synthesized (6aS,cis)-9,10-Dimethoxy-7,11b-dihydro-indeno[2,1-c]chromene-3,6a-diol, on platelet activity.

In the present study, the mechanism of antiplatelet activity of DK-002, a synthesized (6aS,cis)-9,10-Dimethoxy-7,11b-dihydro-indeno[2,1-c]chromene-3,6a-diol, was investigated. DK-002 inhibited the thrombin, collagen, and ADP-induced rat platelet aggregation in a concentration-dependent manner, with IC50 values of 120, 27, and 47 microM, respectively. DK-002 also inhibited thrombin-induced dense granule secretion, thromboxane A2 synthesis, and [Ca2+]i elevation in platelets. DK-002 did not show any significant effect on ADP-induced inhibition of cyclic AMP elevation by prostaglandin E1, but DK-002 was confirmed to inhibit ADP-induced [Ca2+]i elevation and shape change. DK-002 inhibited 4-bromo-A23187-induced [Ca2+]i elevation in the presence of creatine phosphate/creatine phosphokinase (CP/CPK, a ADP scavenging system) and indomethacin (a specific inhibitor of cyclooxygenase). DK-002 also inhibited Ca2+ mobilization in thrombin- or 4-bromo-A23187-stimulated platelets through its inhibitory effects on both Ca2+ release from intracellular stores and Ca2+ influx, in the presence of CP/CPK and indomethacin. Taken together, the present study shows that DK-002 has inhibitory effects on stimulation of platelets, and suggests that its antiplatelet activity might be related to the inhibitory mechanism on Ca2+ mobilization in stimulated platelets.

COX-2 is associated with cadmium-induced ICAM-1 expression in cerebrovascular endothelial cells.

In order to get insight into the mechanism of cadmium (Cd)-induced brain injury, we investigated the effects of Cd on the induction of COX-2 and ICAM-1 in bEnd.3 mouse brain endothelial cells (EC). Cd stimulated PGE(2) release in a time and dose dependent manner, which was accompanied by increase of COX-2 expression. The thiol-reducing antioxidant N-acetylcyteine attenuated Cd-induced PGE(2) production and COX-2 expression. Cd increased phosphorylation of p38 MAPK, but not of JNK and ERK1/2. A blockade of p38 MAPK pathway abrogated Cd-induced COX-2 expression and PGE(2) production. Cd-induced ICAM-1 expression and leukocyte-EC adhesion were diminished by non-steroidal anti-inflammatory drugs such as indomethacin and NS-398, which was reversed by addition of PGE(2). Together, these data suggest that Cd induces COX-2 expression through the activation of p38 MAPK, an oxidative stress-sensitive cellular signaling molecule, and induction of COX-2 is associated with ICAM-1 expression in brain endothelial cells following Cd exposure.

Activation of protein kinase C-delta attenuates kainate-induced cell death of cortical neurons.

We investigated the role of individual protein kinase C (PKC) isoforms during kainate toxicity in cortical neurons. Treatment with 50 microM kainate induced isoform-specific activation of PKC-delta according to the translocation from the soluble to the particulate fraction, while it caused remarkable decreases in PKC alpha, beta, epsilon and zeta in both fractions. Kainate-induced neuronal death was significantly increased by pharmacological inhibition of PKC-delta with rottlerin, suggesting a protective role of PKC-delta against kainate toxicity. A PKC activator phorbol 12-myristate 13-acetate remarkably attenuated the kainate-induced neuronal death. Although phorbol 12-myristate 13-acetate activates PKC-epsilon and PKC-delta, the protective effect of phorbol 12-myristate 13-acetate was almost completely abolished by rottlerin, but not by epsilonV1-2. These results suggest that activation of PKC-delta attenuates the kainate-induced cell death of cortical neurons.

Effects of sabiporide, a specific Na+/H+ exchanger inhibitor, on neuronal cell death and brain ischemia.

We investigated the effects of an Na(+)/H(+) exchanger inhibitor, sabiporide, on excitotoxicity in cultured neuronal cells and in vivo. Sabiporide attenuated glutamate- or NMDA (N-methyl-d-aspartic acid)-induced neuronal cell death. Sabiporide also reduced glutamate or NMDA-induced increase in [Ca(2+)](i). In in vivo brain ischemia model, sabiporide produced protective effects, decreasing the infarct size and edema volume. Our results suggest that sabiporide elicits neuroprotective effect both in vitro and in vivo.

KR-32570, a novel Na+/H+ exchanger-1 inhibitor, attenuates hypoxia-induced cell death through inhibition of intracellular Ca2+ overload and mitochondrial death pathway in H9c2 cells.

A novel Na+/H+ exchanger-1 (NHE-1) inhibitor [5-(2-methoxy-5-chloro-5-phenyl)furan-2-ylcarbonyl]guanidine (KR-32570) has been previously demonstrated to elicit cardioprotective effect against ischemic injury in rat heart. In the present study, we examined the effects of KR-32570 on cell death induced by hypoxic insult in heart-derived H9c2 cells. Treatment with KR-32570 (1-10 microM) significantly reduced hypoxia-induced necrotic cell death (lactate dehydrogenase release) and apoptotic cell death (TUNEL-positivity, caspase-3 activity). KR-32570 also decreased the cytosolic and mitochondrial Ca2+ overload induced by hypoxia. Inhibition of mitochondrial Ca2+ overload by ruthenium red mimicked the anti-apoptotic effect of KR-32570. In addition, KR-32570 significantly recovered the large reduction in mitochondrial membrane potential (delta psi(m)) and cytochrome c release induced by hypoxia. Taken together, our results suggest that a new NHE-1 inhibitor KR-32570 elicits potent cardioprotective effects in H9c2 cells, and its effects may be mediated by inhibition of intracellular Ca2+ overload and mitochondrial death pathway during hypoxia.

Antiplatelet activity of BRX-018, (6aS,cis)-malonic acid 3-acetoxy-6a9-bis-(2-methoxycarbonyl-acetoxy)-6,6a,7,11b-tetrahydro-indeno[2,1-c]chromen-10-yl ester methylester.

Brazilin (7,11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10 (6H)-tetrol), the major component of Caesalpinia sappan L., was reported to show antiplatelet activity through the inhibition of phospholipase A2 (PLA2) activity and the increase in intracellular free Ca2+ concentration ([Ca2+]i). To search more potential antiplatelet agent, brazilin derivatives were synthesized and examined for their effects on the platelet aggregation. Among those compounds, BRX-018, (6aS,cis)-Malonic acid 3-acetoxy-6a9-bis-(2-methoxycarbonyl-acetoxy)-6,6a,7,11b-tetrahydro-indeno[2,1-c]chromen-10-yl ester methylester, was confirmed as one of the potential antiplatelet agents. In the present study, we investigated the antiplatelet mechanism of BRX-018. BRX-018 inhibited the thrombin-, collagen-, and ADP-induced rat platelet aggregation in a concentration-dependent manner, with IC50 values of 35, 15, and 25 microM, respectively. BRX-018 also inhibited thrombin-induced dense granule secretion, thromboxane A2 (TXA2) synthesis, and [Ca2+]i elevation in platelets. BRX-018 was also found to inhibit A23187-induced [Ca2+]i and aggregation in the presence of apyrase (ADP scavenger) but not in the presence of both apyrase and indomethacin (a specific inhibitor of cyclooxygenase, COX). Although BRX-018 significantly inhibited arachidonic acid (AA)-induced aggregation and TXA2 synthesis, it had no significant inhibitory effect on cyclooxygenase activity in vitro. In contrast, BRX-018 inhibited the activity of purified PLA2. Dixon plot showed that this inhibition was mixed type with an inhibition constant of Ki=23 microM. Taken together, the present study suggests that BRX-018 may be a promising antiplatelet agent and that its antiplatelet activity may be based on the inhibitory mechanisms on TXA2 synthesis in stimulated platelets.

Protective effect of KR-31378 on oxidative stress in cardiac myocytes.

In this study, we investigated whether a novel anti-ischemic KATP opener KR-31378 [(2S,3S,4R)-N"-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methly-2-dimethoxymethly-2H-benzopyran-4-yl)-N'-benzylguanidine] has protective effect against oxidative stress-induced death in heart-derived H9c2 cells. Cell death was induced by BSO, butionine sulfoximine, which inhibits GSH synthesis and subsequently increases reactive oxygen species (ROS) level. Cell death was quantitatively determined by measuring lactate dehydrogenase (LDH) activity and stained by Hoechst 33258. BSO-induced ROS production and mitochondrial membrane potential (MMP) were measured using 2',7'-dichlorofluorescein diacetate oxidation and rhodamine 123, respectively. Both the LDH release and the ROS elevation induced by treatment of H9c2 cells with 10 mM BSO, were significantly decreased by KR-31378. These protective effect and antioxidant effect of KR-31378 appeared to be independent on KATP channel opening. Cells exposed to BSO showed an early reduction in MMP, and this reduction in MMP was significantly reversed by treatment with KR-31378. Caspase-3 activity in BSO treated H9c2 cells was remarkably increased, and this increased caspase-3 activity was significantly reversed by KR-31378. In conclusion, our results suggest that KR-31378 can produce cardioprotective effect against oxidative stress-induced cell death through antioxidant mechanism.

Fructose-1,6-diphosphate attenuates prostaglandin E2 production and cyclo-oxygenase-2 expression in UVB-irradiated HaCaT keratinocytes.

1. Fructose-1,6-diphosphate (FDP), a glycolytic metabolite, is reported to ameliorate inflammation and inhibit the nitric oxide production in murine macrophages stimulated with endotoxin. It is also reported that FDP has cytoprotective effects against hypoxia or ischaemia/reperfusion injury in brain and heart. However, underlying mechanisms of its various biological activities are not completely understood. 2. In this study, we examined the effects of FDP on UVB-induced prostaglandin production in HaCaT keratinocytes. 3. Ultraviolet B (UVB, 280-320 nm) irradiation (30 mJ cm(-2)) increased prostaglandin E(2)(PGE(2)) production, which was significantly decreased by FDP in a concentration dependent manner. NS-398, a cyclo-oxygenase-2 (COX-2) selective inhibitor completely inhibited UVB-induced PGE(2) production showing that COX-2 activity is responsible for the increase in PGE(2) production under our experimental conditions. 4. UVB irradiation increased total COX activity and COX-2 mRNA in HaCaT keratinocytes, which were significantly blocked by FDP in a concentration dependent manner. 5. N-acetylcysteine (NAC) significantly attenuated UVB-induced PGE(2) production, COX activity and COX-2 mRNA expression indicating oxidative components might contribute to these events. 6. FDP reduced UVB-induced increase in cellular reactive oxygen species (ROS) level although it did not show direct radical scavenging effect in the experiment using 1,1-diphenyl-2picrylhydrazil (DPPH). FDP preserved the cellular antioxidant capacity including catalase activity and GSH content after irradiation. 7. Our data obtained hitherto suggest that FDP may have a protective role in UVB-injured keratinocyte by attenuating PGE(2) production and COX-2 expression, which are possibly through blocking intracellular ROS accumulation.

Antithrombotic effect of onion in streptozotocin-induced diabetic rat.

In the present study, we investigated whether onion has antithrombotic effect in streptozotocin (STZ)-induced diabetic rats. In diabetic rats, serum thromboxane B(2) (TXB(2)) level was elevated compared to that in normal, and this elevation in diabetes was significantly inhibited by treatment with onion (0.5 g/ml/kg/day, i.p.) for 4 weeks. In normal rats, the serum TXB(2) level remained unaltered after the treatment with onion. To investigate in vitro effect of onion, we examined its effect on TXB(2) formation, platelet aggregation and arachidonic acid (AA)-release in platelets from diabetic and normal rats. Onion showed a significant inhibitory effect on collagen- or AA-induced TXB(2) formation with greater potency in diabetic platelets than in normal. Similarly, more potent inhibitory effects of onion in diabetes were observed in collagen- or AA-induced platelet aggregation and collagen-induced AA release response. In conclusion, these results suggest that onion can produce more beneficial antithrombotic effect in diabetes.

Neuroprotection by fructose-1,6-bisphosphate involves ROS alterations via p38 MAPK/ERK.

Fructose-1,6-bisphosphate (FBP) is a glucose metabolism intermediate that shows a neuroprotective action in animal models of ischemia and other injuries. The intracellular mechanism of FBP on neuroprotection has not been previously defined. Here, we examined whether FBP has a neuroprotective effect against excitotoxicity, and whether it affects the production of reactive oxygen species (ROS), which are involved in the MAPK pathway in cortical neurons. FBP prevented neuronal death in a dose-dependent manner following 24 h of treatment with the excitotoxin, NMDA. After 8 h of NMDA treatment, we observed FBP-induced inhibition of the production of intracellular ROS, and at the earlier time FBP suppressed NMDA-induced p-p38 and p-ERK expression. In addition, MAPK inhibitors reduced NMDA-induced excitotoxicity and also ROS production. Taken together, our results suggest that the neuroprotective effects of FBP could be explained by down-regulation of free radical production through the p38MAPK/ERK pathway.

Effects of peroxisome proliferator-activated receptor agonists on LPS-induced neuronal death in mixed cortical neurons: associated with iNOS and COX-2.

In neurodegenerative disease, the use of non-steroidal anti-inflammatory drugs (NSAIDs) has been regarded as beneficial. The NSAID, an inhibitor of cyclooxygenase (COX), has been also suggested as a ligand of the peroxisome proliferator-activated receptor (PPAR). In cortical neuron-glial co-cultures, we examined the effect of PPAR agonists on lipopolysaccharide(LPS)-induced neuronal death, which has been known to be NO-dependent. LPS induced iNOS expression and the release of nitric oxide in microglia, and COX-2 expression in neurons. PPAR-gamma agonists such as 15d-PGJ(2), ciglitazone and troglitazone prevented LPS-induced neuronal death and abolished LPS-induced NO and PGE(2) release, however PPAR-alpha agonists such as clofibrate and WY14,643 did not produce the same results. PPAR-gamma agonists also reduced LPS-induced iNOS and COX-2 expression, which suggested by interfering with the NF-kappaB signal pathway.

KR-31378, a novel benzopyran analog, attenuates hypoxia-induced cell death via mitochondrial KATP channel and protein kinase C-epsilon in heart-derived H9c2 cells.

A novel compound KR-31378 [(2S,3S,4R)-N''-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methly-2-dimethoxy-methly-2H-benzo-pyran-4-yl)-N-benzylguanidine] has been demonstrated as an anti-ischemic agent in rat heart and brain. Here, we report the effects of this compound on hypoxia-induced cell death and possible signaling pathways in heart-derived H9c2 cells. Treatment with KR-31378 (3-30 microM) 1 h before and during hypoxia significantly reduced hypoxia-induced cell death in a concentration-dependent manner. In addition, increase in hypoxia-induced transferase UTP nick end labeling (TUNEL)-positive cells was reduced by KR-31378, suggesting its antiapoptotic potential in H9c2 cells. The protective effect conferred by KR-31378 (10 microM) was abolished by cotreatment with 5-hydroxydecanoate (5HD), a specific blocker of the mitochondrial KATP (mtKATP) channel, but not by HMR-1883 (1-[[5-[2-(5-chloro-o-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-methylthiourea), a specific blocker of the sarcolemmal KATP channel. We observed that the treatment with KR-31378 could increase the expression of protein kinase C (PKC)-epsilon protein, but not other PKC isotypes (-alpha, -beta, -delta, -zeta), in the particulate fraction. This increased level of PKC-epsilon was sustained during the hypoxic period up to 8 h. In addition, our results showed that treatment with KR-31378 induced the expression of PKC-epsilon mRNA as early as 15 min after the treatment. A specific inhibitor for PKC-epsilon isoform, epsilonV1-2, completely blocked the protective effect of KR-31378 against hypoxia-induced cell death. In conclusion, our results suggest that KR-31378 can protect cultured H9c2 cells from hypoxia-induced death via the mtKATP channel and PKC-epsilon.

Role of PKC-delta during hypoxia in heart-derived H9c2 cells.

In the present study, we investigated the role of protein kinase C (PKC) isoforms during hypoxia in heart-derived H9c2 cells. Hypoxia caused a rapid translocation of PKC-delta from soluble to particulate fraction and a downregulation of PKC-epsilon and PKC-zeta, whereas PKC-alpha and PKC-beta I remained unaltered. When H9c2 cells were pretreated with PKC-delta inhibitor rottlerin (3 microM), hypoxia-induced apoptotic and necrotic cell death were significantly increased. Hypoxic insult also caused an activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK with no change in c-Jun NH(2)-terminal protein kinase (JNK) phosphorylation. Hypoxia-induced cell death was increased by treatment with ERK1/2 inhibitor U0126 (10 microM), but attenuated by p38 MAPK inhibitor SB202190 (10 microM). Treatment with rottlerin completely blocked the hypoxia-induced ERK phosphorylation, whereas it significantly increased p38 MAPK phosphorylation. The hypoxia-induced translocation of PKC-delta was not altered by U0126 and/or SB202190. From these results, it is suggested that hypoxia causes a rapid translocation of PKC-delta and subsequently ERK activation and p38 inactivation, rendering H9c2 cells resistant to hypoxia-induced cell death.

High-glucose induced protective effect against hypoxic injury is associated with maintenance of mitochondrial membrane potential.

Our previous report has showed that the treatment of 48 h with 22 mM glucose prevents hypoxia-induced cardiac cell death. In the present study, we investigated whether high glucose affects the mitochondrial death pathway during hypoxia, and if it does, what relates to the high glucose induced cardioprotection. Heart-derived H9c2 cells were incubated in low (5.5 mM) or high (22 mM) glucose medium for 48 h, then transferred to a normoxic or hypoxic condition. The hypoxia-induced reduction of mitochondrial redox potential, assessed by MTT assay, was inhibited in high glucose treated cells. The mitochondrial membrane potential was significantly decreased by hypoxia in low glucose treated cells, but not in high glucose treated cells. The hypoxia-induced cytoplasmic accumulation of cytochrome c, released from the mitochondria, was blocked by a treatment of high glucose. High glucose did not induce the expression of an antiapoptotic protein Bcl-2, nor did it reduce a proapoptotic protein Bax, but it did inhibit a hypoxia-induced downregulation of Bcl-2. The cellular ATP contents were not changed by the treatment of high glucose for 48 h, and the hypoxia-induced decline of intracellular ATP level was observed in high glucose treated cells and in low glucose. A glycolytic inhibitor, 2-deoxyglucose, did not reverse the high glucose induced reduction of LDH release. The elevation of [ROS](i) induced by hypoxia was inhibited in high glucose treated cells. These results suggest that high glucose induced cardioprotection may be accounted for in part by the preservation of MMP and the maintenance of a basal level of [ROS](i) during hypoxia.