Hypoxia-induced Bax and Bcl-2 protein expression, caspase-9 activation, DNA fragmentation, and lipid peroxidation in mitochondria of the cerebral cortex of newborn piglets: the role of nitric oxide

The present study tests the hypothesis that cerebral hypoxia results in increased ratio of Bax/Bcl-2, activation of caspase-9, lipid peroxidation, and DNA fragmentation in mitochondria of the cerebral cortex of newborn piglets and that the inhibition of nitric oxide synthase by N-nitro-L-arginine during hypoxia will prevent the events leading to mitochondrial DNA fragmentation. To test this hypothesis, six piglets, 3-5 days old, were divided into three groups: normoxic (n=5), hypoxic (n=5), and hypoxic-nitric oxide synthase (n=4). Hypoxic animals were exposed to a FiO2 of 0.6 for 60 min. Nitric oxide synthase (40 mg/kg) was infused over 60 min prior to hypoxia. Tissue hypoxia was confirmed by measuring levels of ATP and phosphocreatine. Cerebral cortical tissue mitochondria were isolated and purified using a discontinuous ficoll gradient. Mitochondrial Bax and Bcl-2 proteins were determined by Western blot. Caspase-9 activity in mitochondria was determined spectro-fluorometrically using fluorogenic substrate for caspase-9. Fluorescent compounds, an index of mitochondrial membrane lipid peroxidation, were determined spectrofluorometrically. Mitochondrial DNA was isolated and separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. ATP levels (micromol/g brain) were 4.52+/-0.34 in normoxic, 1.18+/-0.29 in hypoxic (P<0.05) and 1.00+/-0.26 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic). Phosphocreatine levels (micromol/g brain) were 3.61+/-0.33 in normoxic, 0.70+/-0.20 in hypoxic (P<0.05 vs. normoxic) and 0.57+/-0.14 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic, P=NS vs. hypoxic). Bax density in mitochondrial membranes was 160+/-28 in normoxic and 324+/-65 in hypoxic (P<0.001 vs. normoxic). Bcl-2 density mitochondria was 96+/-18 in normoxic and 98+/-20 in hypoxic (P=NS vs. normoxic). Mitochondrial caspase-9 activity (nmol/mg protein/h) was 1.32+/-0.23 in normoxic and 2.25+/-0.24 in hypoxic (P<0.01 vs. normoxic). Levels of fluorescent compounds (microg of quinine sulfate/g protein) were 12.48+/-4.13 in normoxic and 37.92+/-7.62 in hypoxic (P=0.003 vs. normoxic). Densities (ODxmm2) of low molecular weight DNA fragments were 143+/-38 in normoxic, 365+/-152 in hypoxic, (P<0.05 vs. normoxic) and 163+/-25 in hypoxic-nitric oxide synthase animals (P<0.05 vs. hypoxic, P=NS vs. normoxic). The data demonstrate that hypoxia results in increased mitochondrial proapoptotic protein Bax, increased mitochondrial caspase-9 activity, increased mitochondrial lipid peroxidation, and increased fragmentation of DNA in mitochondria of the cerebral cortex of newborn piglets. The administration of a nitric oxide synthase inhibitor, nitric oxide synthase, prior to hypoxia prevented fragmentation of mitochondrial DNA, indicating that the hypoxia-induced mitochondrial DNA fragmentation is NO-mediated. We propose that NO free radicals generated during hypoxia lead to NO-mediated altered expression of Bax leading to increased ratio of pro-apoptotic/anti-apoptotic protein resulting in modification of mitochondrial membrane, and subsequently Ca2+-influx and fragmentation of mitochondrial DNA.     

Mechanism of Ca2+-influx and Ca2+/calmodulin-dependent protein kinase IV activity during in utero hypoxia in cerebral cortical neuronal nuclei of the guinea pig fetus at term

Previously we showed that following hypoxia there is an increase in nuclear Ca(2+)-influx and Ca(2+)/calmodulin-dependent protein kinase IV activity (CaMK IV) in the cerebral cortex of term guinea pig fetus. The present study tests the hypothesis that clonidine administration will prevent hypoxia-induced increased neuronal nuclear Ca(2+)-influx and increased CaMK IV activity, by blocking high-affinity Ca(2+)-ATPase. Studies were conducted in 18 pregnant guinea pigs at term, normoxia (Nx, n=6), hypoxia (Hx, n=6) and clonidine with Hx (Hx+Clo, n=6). The pregnant guinea pig was exposed to a decreased FiO(2) of 0.07 for 60 min. Clonidine, an imidazoline inhibitor of high-affinity Ca(2+)-ATPase, was administered 12.5 microg/kg IP 30 min prior to hypoxia. Hypoxia was determined biochemically by ATP and phosphocreatine (PCr) levels. Nuclei were isolated and ATP-dependent (45)Ca(2+)-influx was determined. CaMK IV activity was determined by (33)P-incorporation into syntide 2 for 2 min at 37 degrees C in a medium containing 50mM HEPES (pH 7.5), 2mM DTT, 40muM syntide 2, 0.2mM (33)P-ATP, 10mM magnesium acetate, 5 microM PKI 5-24, 2 microM PKC 19-36 inhibitor peptides, 1 microM microcystine LR, 200 microM sodium orthovanadate and either 1mM EGTA (for CaMK IV-independent activity) or 0.8mM CaCl(2) and 1mM calmodulin (for total activity). ATP (mumoles/gbrain) values were significantly different in the Nx (4.62+/-0.2), Hx (1.65+/-0.2, p<0.05 vs. Nx), and Hx+Clo (1.92+/-0.6, p<0.05 vs. Nx). PCr (mumoles/g brain) values in the Nx (3.9+/-0.1), Hx (1.10+/-0.3, p<0.05 vs. Nx), and Hx+Clo (1.14+/-0.3, p<0.05 vs. Nx). There was a significant difference between nuclear Ca(2+)-influx (pmoles/mg protein/min) in Nx (3.98+/-0.4), Hx (10.38+/-0.7, p<0.05 vs. Nx), and Hx+Clo (7.35+/-0.9, p<0.05 vs. Nx, p<0.05 vs. Hx), and CaM KIV (pmoles/mg protein/min) in Nx (1314.00+/-195.4), Hx (2315.14+/-148.5, p<0.05 vs. Nx), and Hx+Clo (1686.75+/-154.3, p<0.05 vs. Nx, p<0.05 vs. Hx). We conclude that the mechanism of hypoxia-induced increased nuclear Ca(2+)-influx is mediated by high-affinity Ca(2+)-ATPase and that CaMK IV activity is nuclear Ca(2+)-influx-dependent. We speculate that hypoxia-induced alteration of high-affinity Ca(2+)-ATPase is a key step that triggers nuclear Ca(2+)-influx, leading to CREB protein-mediated increased expression of apoptotic proteins and hypoxic neuronal death.     

Nitric oxide-mediated mechanism of neuronal nitric oxide synthase and inducible nitric oxide synthase expression during hypoxia in the cerebral cortex of newborn piglets

Previously, we have shown that hypoxia results in increased generation of nitric oxide free radicals in the cerebral cortex of newborn piglets that may be due to up-regulation of nitric oxide synthases, neuronal nitric oxide synthase and inducible nitric oxide synthase. The present study tests the hypothesis that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase in the cerebral cortex of newborn piglets and that the increased expression is nitric oxide-mediated. Newborn piglets, 2-4 days old, were divided to normoxic (n=4), hypoxic (n=4) and hypoxic-treated with 7-nitro-indazole-sodium salt, a selective neuronal nitric oxide synthase inhibitor (hypoxic-7-nitro-indazole-sodium salt, n=6, 1 mg/kg, 60 min prior to hypoxia). Piglets were anesthetized, ventilated and exposed to an FiO2 of 0.21 or 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine. The expression of neuronal nitric oxide synthase and inducible nitric oxide synthase was determined by Western blot using specific antibodies for neuronal nitric oxide synthase and inducible nitric oxide synthase. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and the protein band density expressed as absorbance (OD x mm(2)). The density of neuronal nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 41.56+/-4.27 in normoxic, 61.82+/-3.57 in hypoxic (P<0.05) and 47.80+/-1.56 in hypoxic-7-nitro-indazole-sodium salt groups (P=NS vs normoxic), respectively. Similarly, the density of inducible nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 105.21+/-9.09, 157.71+/-13.33 (P<0.05 vx normoxic), 117.84+/-10.32 (p=NS vx normoxic), respectively. The data show that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase proteins in the cerebral cortex of newborn piglets and that the hypoxia-induced increased expression is prevented by the administration of 7-nitro-indazole-sodium salt. Furthermore, the neuronal nitric oxide synthase inhibition prevented the inducible nitric oxide synthase expression for a period of 7 days after hypoxia. Since administration of 7-nitro-indazole-sodium salt prevents nitric oxide generation by inhibiting neuronal nitric oxide synthase, we conclude that the hypoxia-induced increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase is mediated by neuronal nitric oxide synthase derived nitric oxide. We speculate that during hypoxia nitric oxide-mediated up-regulation of nitric oxide synthases will continue the perpetual cycle of nitric oxide generation-->NOS up-regulation-->nitric oxide generation resulting in hypoxic neuronal death.     

Nitric oxide-mediated activation of extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of newborn piglets

Previous studies have shown that mitogen-activated protein kinases, such as extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK), mediate signal transduction from cell surface receptors to the nucleus and phosphorylate anti-apoptotic proteins thereby regulating programmed cell death. The present study tests the hypotheses that hypoxia activates ERK and JNK in neuronal nuclei of newborn piglets and the hypoxia-induced activation of ERK and JNK is mediated by nitric oxide (NO). Activated ERK and JNK were assessed by determining phosphorylated ERK and JNK using immunoblotting in six normoxic (Nx) and 10 hypoxic (Hx) and five N-nitro-L-arginine (a NOS inhibitor, 40 mg/kg,) -pretreated hypoxic (N-nitro-L-arginine [NNLA]-Hx) 3-5 day old piglets. Hypoxia was induced by decreasing inspired oxygen from 21% to 7% for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Cortical neuronal nuclei were isolated and the nuclear protein was analyzed for activated ERK and JNK using anti-phosphorylated ERK and JNK antibodies. Protein bands were detected using enhanced chemiluminescence method and analyzed by imaging densitometry. Protein density was expressed as absorbance ODxmm(2). ATP levels were 4.57+/-0.45 micromoles/g brain in the Nx group, 1.29+/-0.23 micromoles/g brain in the Hx group (P<0.05 vs Nx) and 1.50+/-0.14 micromoles/g brain in the NNLA-Hx group (P<0.05 vs Nx). PCr levels were 3.77+/-0.36 micromoles/g brain in the Nx group, 0.77+/-0.13 micromoles/g brain in the hypoxic group (P<0.05) and 1.02+/-0.24 in the NNLA-Hx group (P<0.05 vs Nx). Density of phosphorylated ERK protein was 170.5+/-53.7 ODxmm(2) in the Nx group as compared with 419.6+/-63.9 ODxmm(2) in the hypoxic group (P<0.001 vs Nx) and 270.0+/-28.7 in the NNLA-Hx group (P<0.002 vs Hx). Density of phosphorylated JNK protein was 172.8+/-42.8 ODxmm(2) in the normoxic group as compared with 364.6+/-60.1 ODxmm(2) in the Hx group (P<0.002) and 254.8+/-24.8 in the NNLA-Hx group (P<0.002 vs Hx). The data demonstrate increased phosphorylation of ERK and JNK during hypoxia indicating that hypoxia results in activation of ERK and JNK in neuronal nuclei of newborn piglets. The administration of NNLA, a NOS inhibitor, prevented the hypoxia-induced phosphorylation of ERK and JNK indicating that the hypoxia-induced activation of ERK and JNK in the cerebral cortical nuclei of newborn piglets is NO-mediated     

Nitration is a mechanism of regulation of the NMDA receptor function during hypoxia

The present study tested the hypothesis that nitration is a mechanism of hypoxia-induced modification of the N-methyl-D-aspartate (NMDA) receptor. To test this hypothesis the effect of hypoxia on the nitration of the NR1, NR2A and NR2B subunits of the NMDA receptor was determined. Furthermore, the effect of administration of a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (NNLA) on the hypoxia-induced nitration of the NMDA receptor subunits as well as the NMDA receptor-mediated Ca2+ influx, an index of NMDA receptor-ion channel function, were determined in cortical synaptosomes. Studies were performed in newborn piglets divided into normoxic, hypoxic and hypoxic-NNLA groups. Hypoxia was induced by decreasing the FiO(2) to 0.07-0.09 for 60 min. Cerebral tissue hypoxia was confirmed by determining the levels of high energy phosphates ATP and phosphocreatine. Nitration of the NMDA receptor subunits was determined by immunoprecipitation using specific antibodies and western blot analysis. NMDA receptor-ion channel-mediated Ca2+ influx was determined using 45Ca2+. There was a significant increase in the nitrated NR1, NR2A and NR2B subunits following hypoxia: 104+/-11 vs. 275+/-18 optical density (OD)xmm(2) for NR1 (P<0.05), 212+/-36 vs. 421+/-16 ODxmm(2) for NR2A (P<0.05) and 246+/-44 vs. 360+/-26 ODxmm(2) for NR2B (P<0.05). This increase in nitrated NR1, NR2A and NR2B subunits of the NMDA receptor was prevented by the administration of NNLA prior to hypoxia (NR1 160+/-19, P=NS, NNLA vs. normoxic; NR2A 304+/-49, P=NS, NNLA vs. normoxic, and NR2B 274+/-19, P=NS, NNLA vs. normoxic). The increase in nitration of the NR1, NR2A and NR2B subunits of the NMDA receptor increased as a function of decreased cerebral high-energy phosphates, ATP and phosphocreatine, during hypoxia. Furthermore, NOS blockade prior to hypoxia resulted in prevention of the hypoxia-induced increase in NMDA receptor-mediated Ca2+ influx. Our results demonstrate that hypoxia results in increased nitration of the NMDA receptor subunits and that administration of an NOS inhibitor prior to hypoxia prevents the hypoxia-induced nitration of the NMDA receptor subunits as well as the hypoxia-induced increase in NMDA receptor-mediated Ca2+ influx. We conclude that nitration is a mechanism of modification of the NMDA receptor function during hypoxia in the newborn piglet brain.     

Effect of neuronal nitric oxide synthase inhibition on caspase-9 activity during hypoxia in the cerebral cortex of newborn piglets

Previous studies have shown that cerebral hypoxia results in increased activity of caspase-9, a key initiator of programmed cell death. We have also shown increased nitric oxide (NO) free radical generation during hypoxia in the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypoxia-induced increase in caspase-9 activity in the cerebral cortex of newborn piglets is mediated by NO derived from neuronal nitric oxide synthase (nNOS). To test this hypothesis, cytosolic caspase-9 activity was determined in 15 newborn piglets divided into three groups: normoxic (Nx, n=5), hypoxic (Hx, n=5), and Hx pretreated with 7-nitroindazole sodium salt (7-NINA), a selective nNOS inhibitor, 1mg/kg, i.p., 1h prior to hypoxia (Hx+7NI, n=5). The hypoxic piglets were exposed to an FiO(2) of 0.06 for 1h. Tissue hypoxia was documented by ATP and phosphocreatinine (PCr) levels. The cytosolic fraction was obtained from the cerebral cortical tissue following centrifugation at 100,000 x g for 1h and caspase-9 activity was assayed using Ac-Leu-Glu-His-Asp-amino-4-methyl coumarin, a specific fluorogenic substrate for caspase-9. Caspase-9 activity was determined spectroflourometrically at 460 nm using 380 nm as excitation wavelength. ATP levels (micromol/g brain) were 4.35+/-0.21 in the Nx 1.43+/-0.28 in the Hx (p<0.05 versus Nx), and 1.73+/-0.33 in the Hx+7-NINA group (p<0.05 versus Nx, p=NS versus Hx). PCr levels (micromol/g brain) were 3.80+/-0.26 in the Nx, 0.96+/-0.20 in the Hx (p<0.05 versus Nx), and 1.09+/-0.39 in the Hx+7 NINA group (p<0.05 versus Nx, p=NS versus Hx). Cytosolic caspase-9 activity (nmol/mg protein/h), increased from 1.27+/-0.15 in the Nx to 2.13+/-0.14 in the Hx (p<0.05 versus Nx) compared to 1.10+/-0.21 in the Hx+7-NINA group (p<0.05 versus Hx, p=NS versus Nx). Caspase-3 activity (nmol/mg protein/h) also increased from 9.39+/-0.73 in Nx to 18.94+/-3.64 in Hx (p<0.05 versus Nx) compared to 8.04+/-1.05 in the Hx+7-NINA group (p<0.05 versus Hx, p=NS versus Nx). The data show that administration of 7-NINA, an nNOS inhibitor, prevented the hypoxia-induced increase in caspase-9 activity that leads to increase in caspase-3 activity. Since nNOS inhibition blocked the increase in caspase-9 activity during hypoxia, we conclude that hypoxia-induced increase in caspase-9 activity is mediated by nNOS derived NO. We propose that the NO generated during hypoxia leads to activation of caspase-9 and results in initiation of caspase-cascade-dependent hypoxic neuronal death.     

Mechanism of caspase-9 activation during hypoxia in the cerebral cortex of newborn piglets: the role of Src kinase

We have previously shown that hypoxia results in increased activation of caspase-9 in the cerebral cortex of newborn piglets. The present study tests the hypothesis that the increased activation of caspase-9 during hypoxia is mediated by Src kinase. To test this hypothesis a highly selective Src kinase inhibitor PP2 [IC(50) 5 nm] was administered to prevent caspase-9 activation during hypoxia. Cytosolic fraction from the cerebral cortical tissue was isolated and the activation of caspase-9 was documented by the expression of active caspase-9 and the activity of caspase-9 and caspase-3. Piglets were divided into: normoxic (Nx, n=5), hypoxic (Hx, n=5) and hypoxic-treated with Src inhibitor (Hx-PP2). Hypoxia was induced by decreasing FiO(2) to 0.07 for 60 min. PP2 was administered (0.4 mg/kg, i.v.) 30 min prior to hypoxia. ATP and phosphocreatine (PCr) levels were determined to document cerebral tissue hypoxia. Activity of caspase-9 and caspase-3 were determined spectrofluorometrically using specific fluorogenic substrates. Expression of active caspase-9 was determined by Western blot using active caspase-9 antibody. Caspase-9 activity (nmoles/mg protein/h) was 1.40±0.12 in Nx, 2.12±0.11 in Hx (p<0.05 vs Nx) and 1.61±0.14 in Hx-PP2 (p<0.05 vs Hx). Active caspase-9 expression (OD×mm(2)) was 42.3±8.3 in Nx, 78.9±11.0 in Hx (p<0.05 vs Nx) and 41.2±7.6 in Hx-PP2 (p<0.05 vs Hx). Caspase-3 activity (nmoles/mg protein/h) was 4.11±0.1 in Nx, 6.51±0.1 in Hx (p<0.05 vs Nx) and 4.57±0.7 in Hx+PP2 (p<0.05 vs Hx). Active caspase-3 expression (OD×mm(2)) was 392.1±23.1 in Nx, 645.0±90.3 in Hx (p<0.05 vs Nx) and 329.7±51.5 in Hx-PP2 (p<0.05 vs Hx). The data show that pretreatment with Src kinase inhibitor prevents the hypoxia-induced increased expression of active caspase-9 and the activity of caspase-9. Src kinase inhibitor also prevented the hypoxia-induced increased activation of caspase-3, a consequence of caspase-9 activation. We conclude that the hypoxia-induced activation of caspase-9 is mediated by Src kinase. We propose Src kinase-dependent tyrosine phosphorylation (Tyr(154)) in the active site domain of caspase-9 is a potential mechanism of caspase-9 activation in the hypoxic brain.     

Activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of guinea pig fetus at term: role of nitric oxide

Previously we have shown that cerebral tissue hypoxia results in generation of nitric oxide (NO) free radicals as well as increased expression of mitogen-activated protein kinase like extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK). The present study tested the hypothesis that administration of l-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, prior to hypoxia prevents the hypoxia-induced activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) and in the cerebral cortex of the term guinea pig fetus. To test this hypothesis normoxic (Nx, n=6), hypoxic (Hx, n=7) and hypoxic pretreated with l-NAME (Hx+L-NAME, n=6) guinea pig fetuses at 60 days gestation were studied to determine the phosphorylated p38, ERK and JNK. Hypoxia was induced by exposing pregnant guinea pigs to FiO2 of 0.07 for 1h. l-NAME (30mg/kg i.p.) was administered to pregnant mothers 60min prior to hypoxia. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Neuronal nuclei were isolated, purified and proteins separated using 12% SDS-PAGE, and then probed with specific phosphorylated ERK, JNK and p38 antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and expressed as absorbance (ODxmm2). The relative level of p-p38 was 51.41+/-9.80 (Nx), 173.67+/-3.63 (Hx), 58.56+/-3.40 (Hx+L-NAME), p<0.05 vs. Hx. The relative level p-ERK was 44.91+/-4.20 (Nx), 135.12+/-17.02 (Hx), 58.37+/-9.5 (Hx+L-NAME), p<0.05 vs. Hx. The relative level of p-JNK was 34.86+/-6.77 (Nx), 97.36+/-19.24 (Hx), 46.65+/-12.81 (Hx+L-NAME), p<0.05 vs. Hx. The data show that administration of l-NAME prior to hypoxia decreased the relative level of phosphorylated p38, ERK and JNK at term gestation. Since a NOS inhibitor prevented the hypoxia-induced phosphorylation of p38, ERK and JNK, we conclude that the hypoxia-induced activation of p38, ERK and JNK in the cerebral cortical nuclei of guinea pig fetus at term is NO-mediated. We speculate that NO-mediated modification of cysteine residue leading to inhibition of MAP kinase phosphatases results in increased activation of p38, ERK and JNK in the guinea pig fetus at term.     

Phosphorylation of caspase-9 in the cytosolic fraction of the cerebral cortex of newborn piglets following hypoxia

We have previously shown that hypoxia leads to increased expression and increased activity of caspase-9 in the cerebral cortex of newborn piglets. Previous studies have demonstrated the importance of caspase-9 in the initiation of the apoptotic cascade, however, the mechanism of caspase-9 activation is not well understood. Experiments were conducted on newborn piglets 2-3 days of age that were anesthetized and mechanically ventilated. Hypoxia was induced by lowering the FiO(2) to 0.05-0.07 x 1h, and was confirmed biochemically by demonstrating decreased levels of ATP and PCr in the hypoxic groups in comparison with the normoxic group. The ATP level was 1.99+/-0.66 in the hypoxic group versus 4.10+/-0.19 in the normoxic group, P<0.05, and the PCr value was 0.68+/-0.14 in the hypoxic group, compared to 2.98+/-0.39 in the normoxic group, P<0.05. The cytosol of the neuronal nuclei from the cerebral cortex was probed with anti-phosphorylated Ser(196) caspase-9 antibody, using Western blot analysis. Protein bands were analyzed using image densitometry. In both the hypoxic and normoxic samples, protein bands were demonstrated just above the 50 kDa marker. Phosphorylated caspase-9 expression in OD x mm(2) was 43.85+/-8.4 in the normoxic group and 67.6+/-9.88 in the hypoxic group, P<0.05. The results of this study demonstrate that caspase-9, a key protein in hypoxia induced apoptosis, is phosphorylated at the Ser(196) site during hypoxia. The results demonstrate that hypoxia results in a post-translational modification of caspase-9 at Ser(196), which may alter the activity of caspase-9 in the hypoxic newborn brain.     

Effect of hypoxia on the expression and activity of mitogen-activated protein (MAP) kinase-phosphatase-1 (MKP-1) and MKP-3 in neuronal nuclei of newborn piglets: the role of nitric oxide

Mitogen-activated protein kinase-1 (MAPK-1) and MAPK-3 regulate survival and programmed cell death of neurons under stress conditions. The activity of MAPK-1 and MAPK-3 is regulated by dual specificity phosphatases: MKP-1 and MKP-3. In previous studies, we have shown that cerebral hypoxia results in increased activation of MAPK-1 and MAPK-3. Furthermore, we have shown that the hypoxia-induced activation of MAPK is nitric oxide (NO)-mediated. The present study tested the hypothesis that hypoxia results in altered expression and activity of MKP-1 and MKP-3 in neuronal nuclei and the administration of 7-nitro-indazole (7-NINA; 1 mg/kg, 60 min prior to hypoxia), a selective nNOS inhibitor, will prevent the hypoxia-induced alteration in the expression and activity of MKP-1 and MKP-3. To test this hypothesis expression and activity of MKP-1 and MKP-3 were determined in neuronal nuclei of normoxic (Nx; n=5), hypoxic (Hx; n=5) and 7-NINA-pretreated-hypoxic (7-NINA-Hx; n=5). Hypoxia was achieved by exposing the animals to an FiO2 of 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine levels. Neuronal nuclei were isolated using discontinuous sucrose gradient centrifugation and purified. Nuclear proteins were analyzed by Western blot using specific antibodies for MKP-1 and MKP-3 (Santa Cruz, CA, USA). The protein band density was determined by imaging densitometry and expressed as OD x mm2. The density of MKP-1 was 61.57+/-5.68, 155.86+/-44.02 and 69.88+/-25.54 in the Nx, Hx and 7-NINA-Hx groups, respectively (P<0.05, ANOVA). Similarly, the density of MKP-3 was 66.46+/-5.88, 172.04+/-33.10 and 116.88+/-14.66 in the Nx, Hx and 7-NINA-Hx groups, respectively (P<0.05, ANOVA). The data show an increased expression of MKP-1 and MKP-3 during hypoxia in neuronal nuclei of newborn piglets and the administration of 7-NINA, an nNOS inhibitor, prevented the hypoxia-induced increased expression of MKP-1 and MKP-3. The activity of MKP-1 (pmol/min) was 176.17+/-16.95 in Nx, 97.56+/-10.64 in Hx and 130+/-14.42 in the 7-NINA-Hx groups, respectively (P<0.05, ANOVA). Similarly the activity of MKP-3 was 104.11+/-12.17 in Nx, 36.29+/-16.88 in Hx and 77.89+/-20.18 in the 7-NINA groups, respectively (P<0.05, ANOVA). The results demonstrate that cerebral hypoxia results in increased expression of MKP-1 and MKP-3 expression that was prevented by the administration of 7-NINA. In contrast, hypoxia resulted in decreased activity of MKP-1 and MKP-3 that was prevented by the administration of a nNOS inhibitor. We conclude that hypoxia-induced decrease in MKP-1 and MKP-3 activity is not due to altered expression but due to NO-mediated modification of the cysteine residue at the active site of these dual specificity phosphatases, a mechanism of their inactivation that leads to activation of MAP kinases.     

Phosphorylation of cAMP response element binding (CREB) protein during hypoxia in cerebral cortex of newborn piglets and the effect of nitric oxide synthase inhibition

Previous studies have shown that hypoxia results in increased phosphorylation of CREB protein that mediates gene expression including that of the pro-apoptotic gene bax. We also have shown that hypoxia-induced expression of Bax protein is prevented by blocking nitric oxide synthase (NOS). The present study tests the hypothesis that inhibition of NOS by N-nitro-L-arginine (NNLA) will prevent the hypoxia-induced increased phosphorylation of CREB protein in neuronal nuclei of newborn piglets. To test this hypothesis, phosphorylation of CREB protein was assessed by immunoblotting neuronal nuclear proteins from five normoxic (Nx), 10 hypoxic (Hx) and five Hx-NNLA-treated 3-5-day-old piglets. NNLA (40 mg/kg) or saline was infused over 60 min prior to induction of hypoxia. Hypoxia was achieved by reducing the FiO(2) (0.15 to 0.05) for 60 min and documented biochemically by ATP and phosphocreatine (PCr) levels. Neuronal nuclei were isolated using discontinuous sucrose gradient centrifugation and purified. Nuclear proteins were separated on 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, reacted with anti-phosphorylated CREB protein antibody and conjugated with horseradish peroxidase antibody. Protein bands were detected using the enhanced chemiluminescence method and quantitated by imaging densitometry. Protein density was expressed as absorbance (OD)xmm(2). ATP levels (micromol/g brain) were 4.3+/-0.6 in the Nx group, 1.3+/-0.5 in the Hx group (P<0.001) and 1.1+/-0.2 in the Hx-NNLA group (P<0.001 vs. Nx and Hx). Similarly, PCr levels (micromol/g brain) were 3.8+/-0.6 in the Nx group, 0.7+/-0.2 in the Hx group (P<0.001) and 0.6+/-0.1 in the Hx-NNLA group (P<0.001 vs. Nx and Hx). Density of phosphorylated CREB protein (ODxmm(2)) was 134.2+/-52.4 in the Nx group compared to 746.0+/-76.8 in the Hx group (P<0.05) and 491.1+/-40.9 in the Hx-NNLA group (P<0.05 Hx).The data show that NOS inhibition attenuates the hypoxia-induced increase in CREB protein phosphorylation in the cerebral cortex of newborn piglets.     

ATP and cytochrome c-dependent activation of caspase-9 during hypoxia in the cerebral cortex of newborn piglets

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, in the cytosolic fraction of the cerebral cortex of newborn piglets. The present study examines the mechanism of caspase-9 activation during hypoxia and tests the hypothesis that the ATP and cytochrome c-dependent activation of caspase-9 increases in the cytosol of the cerebral cortex of newborn piglets. Newborn piglets were divided into normoxic (Nx, n=4), and hypoxic (Hx, n=4) groups. Anesthetized, ventilated animals were exposed to an FiO(2) of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Cytosolic fraction was isolated and passed through a G25-Sephadex column to remove endogenous ATP and cytochrome c. Fractions were collected and protein determined by UV spectrophotometry at 280 nm. Eluted high-molecular weight samples from normoxic and hypoxic animals were divided into four subgroups: subgroup 1 (control), incubated without added ATP and cytochrome c; subgroup 2, incubated with added ATP; subgroup 3, incubated with added cytochrome c; and subgroup 4, incubated with added ATP and cytochrome c. The incubation was carried out at 37 degrees C for 30 min. Following incubation, the protein was separated by 12% SDS-PAGE and active caspase-9 was detected using specific active caspase-9 antibody. Protein bands were detected by enhanced chemiluminescence. Protein density was determined by imaging densitometry and expressed as absorbance (OD x mm(2)). ATP (mumol/g brain) level was 4.7 +/- 0.18 in normoxic, as compared to 1.53 +/- 0.16 in hypoxic (p < 0.05 vs. Nx). PCr (mumol/g brain) level was 4.03 +/- 0.11 in the normoxic and 1.1 +/- 0.3 in the hypoxic brain (p < 0.05 vs. Nx). In the normoxic preparations, active caspase-9 density increased by 9, 4 and 20% in the presence of ATP, cytochrome c and ATP+cytochrome c, respectively. In the hypoxic preparations, active caspase-9 density increased by 30, 45 and 60% in the presence of ATP, cytochrome c and ATP+cytochrome c, respectively. These results show that incubation with ATP, cytochrome c and ATP+cytochrome c result in a significantly increased activation of caspase-9 in the hypoxic group (p < 0.05). We conclude that the ATP and cytochrome c dependent activation of caspase-9 is increased during hypoxia. We propose that the ATP and cytochrome c sites of apoptotic protease activating factor I that mediate caspase-9 activation are modified during hypoxia.     

Mitogen activated protein kinase (MAPK) pathway regulates heme oxygenase-1 gene expression by hypoxia in vascular cells

Hypoxia induces the stress protein heme oxygenase-1 (HO-1), which participates in cellular adaptation. The molecular pathways that regulate ho-1 gene expression under hypoxia may involve mitogen activated protein kinase (MAPK) signaling and reactive oxygen. Hypoxia (8 h) increased HO-1 mRNA in rat pulmonary aortic endothelial cells (PAEC), and also activated both extracellular signal-regulated kinase 1 (ERK1)/ERK2 and p38 MAPK pathways. The role of these kinases in hypoxia-induced ho-1 gene expression was examined using chemical inhibitors of these pathways. Surprisingly, SB203580, an inhibitor of p38 MAPK, and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK1), strongly enhanced hypoxia-induced HO-1 mRNA expression in PAEC. UO126, a MEK1/2 inhibitor, enhanced HO-1 expression in PAEC under normoxia, but not hypoxia. Diphenylene iodonium, an inhibitor of NADPH oxidase, also induced the expression of HO-1 in PAEC under both normoxia and hypoxia. Similar results were observed in aortic vascular smooth muscle cells. Furthermore, hypoxia induced activator protein (AP-1) DNA-binding activity in PAEC. Pretreatment with SB203580 and PD98059 enhanced AP-1 binding activity under hypoxia in PAEC; UO126 stimulated AP-1 binding under normoxia, whereas diphenylene iodonium stimulated AP-1 binding under normoxia and hypoxia. These results suggest a relationship between MAPK and hypoxic regulation of ho-1 in vascular cells, involving AP-1.     

急性缺氧肺动脉平滑肌细胞线粒体活性氧变化的观察

 目的:观察急性缺氧时大鼠肺动脉平滑肌细胞（PASMCs）线粒体活性氧（ROS）的变化。方法:分离并培养大鼠PASMCs,常氧（35 ℃、5% CO2、21% O2、74% N2）和急性缺氧(35 ℃、5% CO2、1% O2、94% N2)条件下,利用分子探针chloromethyl dichlorodihydrofluorescein diacetate (CM-H2DCF/DA) 和RedoxSensor Red CC-1通过激光共聚焦显微镜来检测细胞内ROS的生成量;直接分离线粒体,用线粒体电子传递链（ETC）复合物抑制剂通过荧光分光光度计检测线粒体ROS及其生成的具体位点。结果:急性缺氧细胞内ROS生成明显增加,其中缺氧组H2O2较常氧组增加3.35倍 (P<0.01),而H2O2 及O-·2较常氧组增加1.61倍（P<0.01）。与缺氧组比,用线粒体ETC复合物I抑制剂MPP、复合物II抑制剂NPA和TTFA及复合物III前泛半醌位点抑制剂myxothiazol都能显著降低缺氧时PASMCs胞内ROS的生成量（分别降低60%、73%、75%和61%,P<0.01）;而复合物III后泛半醌位点抑制剂antimycin A及复合物IV 抑制剂NaN3对ROS的生成无明显影响（升高13%和9.1%,P>0.05）。 直接检测线粒体ROS与测定细胞内ROS结果一致。结论: 急性缺氧PASMCs线粒体ROS（主要是H2O2）的生成量明显增加;其生成位点主要是线粒体ETC复合物Ⅰ、Ⅱ及III前泛半醌位点,而与复合物III后泛半醌位点和IV位点无明显关系。     

ATP and cytochrome c-dependent inhibition of caspase-9 activity in the cerebral cortex of newborn piglets

The present study investigates the mechanism of activation of caspase-9 during hypoxia and tests the hypothesis that ATP and cytochrome c regulate the activity of caspase-9 in the cerebral cortex of newborn piglets. Cerebral tissue hypoxia was documented by decreased levels of high energy phosphates, ATP and phosphocreatine (PCr). Cytosolic fractions were prepared from cerebral cortices and passed through a G50 column, to remove endogenous ATP and cytochrome c. Caspase-9 activity was determined spectrofluorometrically using a specific fluorogenic substrate for caspase-9 at increasing concentrations of ATP (0-1.0 mM) or cytochrome c (0-3.0 microM). Caspase-9 activity (nmol/mg protein/h) was 1.26 +/- 0.15 in the normoxic and 2.13 +/- 0.14 in the hypoxic group (P < 0.05). The enzyme activity was inhibited by ATP or cytochrome c in both normoxic and hypoxic groups. The IC50 for ATP and cytochrome c increased 5-fold and 1.5-fold, respectively, following hypoxia, suggesting a hypoxia-induced modification of the ATP and cytochrome binding sites. The data demonstrate that ATP (1 mM) and cytochrome c (3.0 microM) inhibit caspase-9 activity by approximately 70%. On the basis of these observations, we propose a new and novel concept that the caspase-9 activity remains inhibited under the normoxic conditions and during hypoxia the decrease in ATP and decreases in the affinity for ATP and cytochrome c release the inhibitory block to activate the enzyme. Results of ATP- and cytochrome c-dependent inhibition of purified caspase-9 human recombinant show that the inhibitory effect by ATP and cytochrome c does not require Apaf-1. To our knowledge, this is a completely new concept and a new mechanism of regulation of caspase-9 activity that may lead to hypoxia-induced programmed cell death.     

Mg2+-dependent modification of the N-methyl-D-aspartate receptor following graded hypoxia in the cerebral cortex of newborn piglets.

The present study tests the hypothesis that Mg2+ modification of N-methyl-D-aspartate receptor ion channel opening is altered during hypoxia and correlates with the progressive decrease in cerebral energy metabolism induced by hypoxia. Studies were performed in five normoxic and nine hypoxic ventilated piglets. In the hypoxic group, varying degrees of cerebral energy metabolism were achieved by administration of different fractions of inspired oxygen (FiO2) (5-9%) for varying durations of time and were documented by cortical tissue phosphocreatine levels. [3H]Dizocilpine maleate binding was performed with increasing concentrations of MgSO4 from 0.01 to 15 mM in cortical P2 membrane fractions. Mg2+ percentage activation and Mg2+ 50% inhibitory concentrations (IC50) were determined. The mean +/- S.D. phosphocreatine value was 3.0 +/- 1.3 micromol/g brain in the normoxic group and 1.4 +/- 1.0 micromol/g brain in the hypoxic group (P < 0.01). Low concentrations of Mg2+ (0.01-1 mM) increased [3H]dizocilpine maleate binding in the normoxic group (to 137 +/- 26% of baseline), significantly greater than in the hypoxic group (109 +/- 13%, P < 0.05). Receptor activation correlated with brain tissue levels of phosphocreatine, with percentage maximal activation decreasing linearly as phosphocreatine levels decreased (r=0.7). Higher levels of Mg2+ (1.5-15 mM) caused inhibition of [3H]dizocilpine maleate binding, with IC50 levels significantly higher in the normoxic group (3.2 +/- 1.1 mM) than in the hypoxic group (1.9 +/- 0.4 mM). Mg2+ IC50 values decreased in a linear fashion as phosphocreatine values decreased (r=0.9). The data demonstrate that, as brain cell energy metabolism decreases during hypoxia, maximal receptor activation by low levels of Mg2+ decreases and receptor inhibition by high levels of Mg2+ increases in a linear fashion. We speculate that, during hypoxia, dephosphorylation of the ion channel of the N-methyl-D-aspartate receptor increases Mg2+ blockade of the receptor by increasing Mg2+ accessibility to its binding site and that receptor modification may be initiated by subtle decreases in cortical oxygenation in the newborn brain.     

Hypoxia-induced Ca2+-influx in cerebral cortical neuronal nuclei of newborn piglets

The present study was designed to investigate the effect of hypoxia on nuclear calcium-influx in the cerebral cortex of newborn piglets. Anesthetized and ventilated newborn piglets divided into normoxic (n=4) and hypoxic groups with varying degrees of tissue hypoxia (n=10) were studied. Nuclear Ca(2+)-influx was determined using (45)Ca(2+) and plotted against ATP and phosphocreatine levels. The plots were analyzed by non-linear regression (exponential) analysis that showed a curvilinear relationship (r=0.92 for ATP and r=0.88 for phosphocreatine). These data suggest a threshold at which there is a sudden increase in the nuclear calcium-influx that then continues to increase with further decrease in the ATP and phosphocreatine levels. The results demonstrate an increase in the nuclear Ca(2+)-influx during hypoxia in newborn piglets and that this increase correlates in a curvilinear fashion with the increase in the degree of cerebral tissue hypoxia. We propose that the hypoxia-induced increase in intranuclear Ca(2+) is due to altered nuclear membrane Ca(2+)-influx mechanisms and will lead to Ca(2+)-mediated alteration of apoptotic gene expression as well as Ca(2+)-dependent activation of endonucleases that result in DNA fragmentation and subsequent programmed neuronal cell death.     

Mechanisms of expression of apoptotic protease activating factor-1 (Apaf-1) in nuclear, mitochondrial and cytosolic fractions of the cerebral cortex of newborn piglets

Apoptotic protease activating factor-1 (Apaf-1) is a critical regulator of apoptosis and a crucial part of the apoptosome that is assembled in response to several cellular stresses like hypoxia. We have previously shown that hypoxia results in increased influx of nuclear Ca(2+) and increased expression of nuclear apoptotic proteins. The present study investigates that Apaf-1 is expressed during hypoxia in the cerebral cortex of newborn piglets and that administration of clonidine prevents the hypoxia induced increase expression of Apaf-1. Studies were conducted in 19 newborn piglets, 6 normoxic (Nx), 7 hypoxic (Hx FiO(2) of 0.05-0.07 for 1h) and 6 clonidine-treated hypoxic (Hx-Clo) piglets. Tissue hypoxia was confirmed biochemically by determining the levels of high energy phosphates ATP and phosphocreatine (PCr). Neuronal nuclei, mitochondrial membranes and cytosolic fractions were isolated and separated by 12% SDS-PAGE and probed with specific antibodies to Apaf-1. The expression of Apaf-1 in neuronal nuclei was 48.86+/-5.27 in Nx, 108.43+/-6.37 in Hx and 78.53+/-7.00 in Hx-Clo. The Apaf-1 expression of in mitochondrial fraction was 72.73+/-11.76 in Nx, 132.27+/-16.15 in Hx and 85.17+/-5.64 in Hx-Clo. Similarly, the expression of Apaf-1 in cytosolic fraction was 86.79+/-6.97 in Nx, 193.95+/-15.41 in Hx and 111.07+/-7.91 in Hx-Clo. In summary, the results show that hypoxia results in increased expression of Apaf-1 proteins in neuronal nuclear, mitochondrial and cytosolic fractions. Administration of a high affinity Ca(2+)-ATPase, prevented the hypoxia induced increased expression of Apaf-1 protein, suggesting that the hypoxia-induced increased expression of Apaf-1 proteins is nuclear Ca(2+)-influx mediated. We conclude that cerebral hypoxia-induced increase in Apaf-1 protein will lead to increased activation of procaspase-9 to caspase-9 in the cytosolic compartment leading to a cascade of hypoxic neuronal death.     

低氧诱导肌成纤维细胞生成并通过ERK1/2途径促进Ⅰ型胶原的表达

目的：探讨低氧能否激活成纤维细胞转化为肌成纤维细胞,以及低氧环境对肾皮质肌成纤维细胞表达Ⅰ型胶原（Col-Ⅰ）的影响及其可能的信号通路。方法：（1）采用正常大鼠肾成纤维细胞系（NRK-49F），应用Western blotting方法检测低氧诱导因子-1α(HIF-1α)的表达;比较低氧和正常氧条件下α-平滑肌肌动蛋白(α-SMA)的蛋白水平。（2）原代培养正常大鼠肾皮质肌成纤维细胞，Western blotting方法检测低氧和正常氧条件下，HIF-1α和Ⅰ型胶原蛋白水平以及ERK1/2的活化及其特异阻断剂 PD98059的阻断效应；RT-PCR方法检测Ⅰ型胶原mRNA表达水平；细胞免疫化学法检测HIF-1α胞内表达部位的变化；明胶酶谱法检测细胞上清中基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP -9）的活性。结果：（1）低氧刺激6 h，NRK-49F细胞和肌成纤维细胞胞内和核内均有HIF-1α蛋白表达，核内更明显。（2）低氧培养12 h,NRK-49F细胞表达α-SMA蛋白明显增高，是正常氧组的187%±32%（P<0.05），验证了低氧可引起成纤维细胞表型转化。（3）低氧刺激原代培养的正常大鼠肾皮质肌成纤维细胞6 h、12 h上清中Ⅰ型胶原蛋白水平增加，分别为正常氧组的171%±27%（P<0.05）和256%±61% （P<0.05）；低氧刺激4 h、6 h，Ⅰ型胶原mRNA表达增加，为正常氧组的189%±28%（P<0.05）和221%±44%（P<0.05）。（4）低氧刺激肌成纤维细胞6 h、12 h、24 h，培养上清液中MMP-9、MMP-2活性无明显变化。（5）低氧刺激肌成纤维细胞15 min 即可使ERK1/2活化，阻断实验显示，PD98059可以使低氧12 h引起Ⅰ型胶原增加（低氧刺激组为正常氧对照组的273%±51%，P<0.05)显著减少（PD98059 +低氧组为正常氧组的108%±19%，P>0.05)。结论： 低氧促肾纤维化可能与其诱导肾成纤维细胞转化为肌成纤维细胞并经ERK1/2途径增加Ⅰ型胶原蛋白的表达有关。