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Intracellular events in glutamate induced death of HT4 neuronal cells
REGULATION OF ENDOTHELIAL HEME OXYGENASE ACTIVITY DURING HYPOXIA DEPENDS ON INTRACELLULAR IRON. Southern
Illinois
University
School
of
or oxidative stress occurring during ischemia/ reperfusion episodes modulates vascular endothelial cell function. We have studied heme oxygenase (HO) activity in bovine aortic endothelial cells (BAEC) subjected to hypoxia/ Hypoxia
reoxygenation (H/R), and its dependence on intracellular chelatable iron. Total HO activity was induced 2-3 fold by hypoxia (10 hrs), and continued to increase during the reoxygenation phase up to 4.5 fold. HO-l protein levels were strongly induced by hypoxia from undetectable levels in control cultures, and remained elevated at least 8 hrs post-reoxygenation. Endothelial nitric oxide synthase (eNOS) protein levels were not affected by the hypoxia, and increased slightly during the reoxygenation. Addition of the Fe(II1) chelator desferrioxamine (DFO) or the Fe(II) chelator
o-phenanthroline during the hypoxia alone, or the entire H/R treatment, inhibited the induction of HO activity and HO-1 a protein. However, DFO had no effect, and o-phenanthroline partial effect, on the induction of HO activity by H/R when added during the reoxygenation stage only. Loading of BAEC with iron (III) as 8-hydroxyquionoline/ ferric ammonium citrate potentiated the activation of the HO-1 gene by hypoxia and H/R. These results suggest that chelatable iron participates in regulating HO activity during hypoxia.
11861 INDUCED
Roy, Savitn Kknnnn, Lester Packer. and University of Kuop~o, FINLAND.
Glutamate (G)-induced oxidative stress and toxicity has been observed in primary neuronal cells, tissue slices as well as mouse hippocampal HT cell lines. G treatment of HT4 cells caused DNA fragmentation and death after 12 h. G lowered [GSH]i, increased (oxidantli and caused initial decrease in mitochondrial membrane potential (MMP). [Oxidantli level peaked at 6 h and was associated with increased [Ca2+]i and loss of outer mitochondrial membrane (OMM) integrity. Following 8 h of G, enhanced loss of OMM integrity was associated with mitochondrial swelling and further increase in [Ca2+]i. Bcl-2 overexpression did not influence G-induced changes in [Ca2+]i, MMP or toxicity. G treated HT4 cells were completely protected by cycloheximide suggesting that protein synthesis is required to render the death pathway functional. Also, G-induced cell death was completely inhibited when mitochondrial inner membrane Ca2+ uniporter was blocked by ruthenium red (RR) showing that Ca2c entry into the mitochondria via the uniporter is a crucial step in the death pathway. Examination of signal transduction pathways revealed that protein tyrosine phosphorylation processes play a central role in the execution of death. Activation of c-src kinase and phosphorylation of ERK were observed in response to G treatment. Active src, but not kinase-dead protein, transfected cells were sensitized to G-induced death. Nanomolar amounts (lo-25% of values found
in plasma of supplemented humans) of alpha-tocotrienol, but not a-tocopherol, blocked G-induced death by suppressing early activation of c-src kinase. a-T3 did not influence activity of recombinant c-src kinase suggesting that its mechanism of action in cells includes prevention of G-induced reorganization of SH domains.
S66
Akt is a serine/threonine kinase regulated by the phosphorylation state of phosphatidylinositol (PI) in the plasma membrane. Akt is a member of a PI 3-kinase (PI 3-K)-dependent signal transduction pathway that is activated in response to mitogenic and survival factors. Phosphorylation of PI-4,5bisphosphate at the 3 position by?1 3-K results in the recruitment of Akt to the membrane followed by its phosphorylation at Thr308 and Ser473. An antibody directed against phosphorylated Ser473 was used to assay Akt phosphorylation in serum-starved primary rat astrocytes treated with epidennal growth factor (EGF) or with Hz02 by Western blotting. Akt phosphorylation was induced upon addition of EGF or H,O,. This induction was abrogated by preincubation of cells with wortmannin, a specific PI 3-K inhibitor. We propose a model in which the active site of phosphatases such as PTEN, a PI 3-phosphatase, that negatively regulate the PI 3-K/Akt pathway are oxidized by H,02 leading to inactivation of the enzyme. Data are presented to support this notion. Supported by NIH grant NS35747 and the Oklahoma Center for the Advancement of Science and Technology.
11871
INTRACELLULAR EVENTS IN GLUTAMATE DEATH OF HT4 NEURONAL CELLS Wwzdan K. Sen.Orm Tirosh, Snshwatl Lawrcncc Berkeley Nntionnl Laboratory
ACTIVATION OF THE PI 3-WAKT PATHWAY IN RAT PRIMARY ASTROCYTES BY EPIDERMAL GROWTH FACTOR AND H,OZ. Scott Salsman, Nicole Felts, Kelly Williamson, Robert A. Floyd and Kenneth Hensley, Department of Free Radical Biology and Aging, Oklahoma Medical Research Foundation, Oklahoma City, OK 73 104.
OXYGEN
INDUCTION OF C-JUN-N-TERMINAL KINASE (JNK, AND APOPTOSIS IN LUNG EPITHELIAL CELLS EXPOSED TO
.NOZ
,
RP Soulfnnnkrs. WM Blny, I-IL Berr NH Heintz, ‘D Hemenmny, nnd YMW lnnssen-Heinmger. De@ of Potho ro&y nnd ‘Cmil Engimemg, University
VT, Bwi,n$$ur1, VT.
of
Nitrogen dioxide (.NOZ) is a free radical gas that exists as a component of air pollution and is associated with epithelial injury, inflammation and the aggravation of asthma. To elucidate the signaling events that may be involved in the mechanisms of injury, we determined whether the stress activated protein kinase, JNK of the family of mitogen activated protein kinases is activated in response to .N02. Rat alveolar type II epithelial cells (RLE) were exposed to .NOZ (1 ppm 5 ppm) for 4 hours in an air-liquid interface and then JNK activity and apoptosis were evaluated. Following exposure of log-phase cells to .N02. a striking induction of auootosis (10 ii 30 fold increases over sham c&trols) was observed. In contrast, confluent cultures were refractory to undergoing apoptosis by .N02. Using ONOO- or the ONOO- generator, 3-morpholinosydnonimine we also observed a selective induction of apoptosis in log-phase cultures. Using kinase activity assays, a 5 - 25 fold activation of the stress-responsive JNK pathway was observed in log-phase cultures with no change in confluent cultures. The appearance of JNK activation was consistent wth the kinetics of .N02 induced apoptosis, suggesting a link between JNK activation and apoptosis following exposure to nitrating agents. In addition, C57/816 mice were exposed to 5 or 50 ppm of .N02. for 6 hours. Ribonuclease protection assays from mouse lungs exposed to .N02 revealed increases from 2 to 12 fold in mRNA for fos-jun family protooncogenes and a variety of genes involved in apoptosis, including Fas and Fas ligand. Our results suggest that exposure to nitrating agents such as .N02 can cause the activation of cell signaling cascades which may be linked to apoptosis in lung epithelium.
’ 9
9
Illinois
University
School
of
or oxidative stress occurring during ischemia/ reperfusion episodes modulates vascular endothelial cell function. We have studied heme oxygenase (HO) activity in bovine aortic endothelial cells (BAEC) subjected to hypoxia/ Hypoxia
reoxygenation (H/R), and its dependence on intracellular chelatable iron. Total HO activity was induced 2-3 fold by hypoxia (10 hrs), and continued to increase during the reoxygenation phase up to 4.5 fold. HO-l protein levels were strongly induced by hypoxia from undetectable levels in control cultures, and remained elevated at least 8 hrs post-reoxygenation. Endothelial nitric oxide synthase (eNOS) protein levels were not affected by the hypoxia, and increased slightly during the reoxygenation. Addition of the Fe(II1) chelator desferrioxamine (DFO) or the Fe(II) chelator
o-phenanthroline during the hypoxia alone, or the entire H/R treatment, inhibited the induction of HO activity and HO-1 a protein. However, DFO had no effect, and o-phenanthroline partial effect, on the induction of HO activity by H/R when added during the reoxygenation stage only. Loading of BAEC with iron (III) as 8-hydroxyquionoline/ ferric ammonium citrate potentiated the activation of the HO-1 gene by hypoxia and H/R. These results suggest that chelatable iron participates in regulating HO activity during hypoxia.
11861 INDUCED
Roy, Savitn Kknnnn, Lester Packer. and University of Kuop~o, FINLAND.
Glutamate (G)-induced oxidative stress and toxicity has been observed in primary neuronal cells, tissue slices as well as mouse hippocampal HT cell lines. G treatment of HT4 cells caused DNA fragmentation and death after 12 h. G lowered [GSH]i, increased (oxidantli and caused initial decrease in mitochondrial membrane potential (MMP). [Oxidantli level peaked at 6 h and was associated with increased [Ca2+]i and loss of outer mitochondrial membrane (OMM) integrity. Following 8 h of G, enhanced loss of OMM integrity was associated with mitochondrial swelling and further increase in [Ca2+]i. Bcl-2 overexpression did not influence G-induced changes in [Ca2+]i, MMP or toxicity. G treated HT4 cells were completely protected by cycloheximide suggesting that protein synthesis is required to render the death pathway functional. Also, G-induced cell death was completely inhibited when mitochondrial inner membrane Ca2+ uniporter was blocked by ruthenium red (RR) showing that Ca2c entry into the mitochondria via the uniporter is a crucial step in the death pathway. Examination of signal transduction pathways revealed that protein tyrosine phosphorylation processes play a central role in the execution of death. Activation of c-src kinase and phosphorylation of ERK were observed in response to G treatment. Active src, but not kinase-dead protein, transfected cells were sensitized to G-induced death. Nanomolar amounts (lo-25% of values found
in plasma of supplemented humans) of alpha-tocotrienol, but not a-tocopherol, blocked G-induced death by suppressing early activation of c-src kinase. a-T3 did not influence activity of recombinant c-src kinase suggesting that its mechanism of action in cells includes prevention of G-induced reorganization of SH domains.
S66
Akt is a serine/threonine kinase regulated by the phosphorylation state of phosphatidylinositol (PI) in the plasma membrane. Akt is a member of a PI 3-kinase (PI 3-K)-dependent signal transduction pathway that is activated in response to mitogenic and survival factors. Phosphorylation of PI-4,5bisphosphate at the 3 position by?1 3-K results in the recruitment of Akt to the membrane followed by its phosphorylation at Thr308 and Ser473. An antibody directed against phosphorylated Ser473 was used to assay Akt phosphorylation in serum-starved primary rat astrocytes treated with epidennal growth factor (EGF) or with Hz02 by Western blotting. Akt phosphorylation was induced upon addition of EGF or H,O,. This induction was abrogated by preincubation of cells with wortmannin, a specific PI 3-K inhibitor. We propose a model in which the active site of phosphatases such as PTEN, a PI 3-phosphatase, that negatively regulate the PI 3-K/Akt pathway are oxidized by H,02 leading to inactivation of the enzyme. Data are presented to support this notion. Supported by NIH grant NS35747 and the Oklahoma Center for the Advancement of Science and Technology.
11871
INTRACELLULAR EVENTS IN GLUTAMATE DEATH OF HT4 NEURONAL CELLS Wwzdan K. Sen.Orm Tirosh, Snshwatl Lawrcncc Berkeley Nntionnl Laboratory
ACTIVATION OF THE PI 3-WAKT PATHWAY IN RAT PRIMARY ASTROCYTES BY EPIDERMAL GROWTH FACTOR AND H,OZ. Scott Salsman, Nicole Felts, Kelly Williamson, Robert A. Floyd and Kenneth Hensley, Department of Free Radical Biology and Aging, Oklahoma Medical Research Foundation, Oklahoma City, OK 73 104.
OXYGEN
INDUCTION OF C-JUN-N-TERMINAL KINASE (JNK, AND APOPTOSIS IN LUNG EPITHELIAL CELLS EXPOSED TO
.NOZ
,
RP Soulfnnnkrs. WM Blny, I-IL Berr NH Heintz, ‘D Hemenmny, nnd YMW lnnssen-Heinmger. De@ of Potho ro&y nnd ‘Cmil Engimemg, University
VT, Bwi,n$$ur1, VT.
of
Nitrogen dioxide (.NOZ) is a free radical gas that exists as a component of air pollution and is associated with epithelial injury, inflammation and the aggravation of asthma. To elucidate the signaling events that may be involved in the mechanisms of injury, we determined whether the stress activated protein kinase, JNK of the family of mitogen activated protein kinases is activated in response to .N02. Rat alveolar type II epithelial cells (RLE) were exposed to .NOZ (1 ppm 5 ppm) for 4 hours in an air-liquid interface and then JNK activity and apoptosis were evaluated. Following exposure of log-phase cells to .N02. a striking induction of auootosis (10 ii 30 fold increases over sham c&trols) was observed. In contrast, confluent cultures were refractory to undergoing apoptosis by .N02. Using ONOO- or the ONOO- generator, 3-morpholinosydnonimine we also observed a selective induction of apoptosis in log-phase cultures. Using kinase activity assays, a 5 - 25 fold activation of the stress-responsive JNK pathway was observed in log-phase cultures with no change in confluent cultures. The appearance of JNK activation was consistent wth the kinetics of .N02 induced apoptosis, suggesting a link between JNK activation and apoptosis following exposure to nitrating agents. In addition, C57/816 mice were exposed to 5 or 50 ppm of .N02. for 6 hours. Ribonuclease protection assays from mouse lungs exposed to .N02 revealed increases from 2 to 12 fold in mRNA for fos-jun family protooncogenes and a variety of genes involved in apoptosis, including Fas and Fas ligand. Our results suggest that exposure to nitrating agents such as .N02 can cause the activation of cell signaling cascades which may be linked to apoptosis in lung epithelium.
’ 9
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