Mechanism of CaM kinase IV activation during hypoxia in neuronal nuclei of the cerebral cortex of newborn piglets: the role of Src kinase.

The present study aims to investigate the mechanism of CaM kinase IV activation during hypoxia and tests the hypothesis that hypoxia-induced increased activity of CaM kinase IV is due to Src kinase mediated increased tyrosine phosphorylation of calmodulin and CaM kinase IV in neuronal nuclei of the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, F(i)O(2) of 0.07 for 1 h, n = 5) and hypoxic-pretreated with Src kinase inhibitor PP2 (Hx-Srci, n = 5) groups. Src inhibitor was administered (1.0 mg/kg, I.V.) 30 min prior to hypoxia. Neuronal nuclei were isolated and purified, and tyrosine phosphorylation of calmodulin (Tyr(99)) and CaM kinase IV determined by Western blot using anti-phospho-(pTyr(99))-calmodulin, anti-pTyrosine and anti-CaM kinase IV antibodies. The activity of CaM kinase IV and its consequence the phosphorylation of CREB protein at Ser(133) were determined. Hypoxia resulted in increased tyrosine phosphorylation of calmodulin at Tyr(99), tyrosine phosphorylation of CaM kinase IV, activity of CaM kinase IV and phosphorylation of CREB protein at Ser(133). The data show that administration of Src kinase inhibitor PP2 prevented the hypoxia-induced increased tyrosine phosphorylation of calmodulin (Tyr(99)) and tyrosine phosphorylation of CaM.kinase IV as well as the activity of CaM kinase IV and CREB phosphorylation at Ser(133). We conclude that the mechanism of hypoxia-induced increased activation of CaM kinase IV is mediated by Src kinase-dependent tyrosine phosphorylation of the enzyme and its activator calmodulin. We propose that Tyr(99) phosphorylated calmodulin, as compared to non-phosphorylated, binds with a higher affinity at the calmodulin binding site (rich in basic amino acids) of CaM kinase IV leading to increased activation of CaM kinase IV. Similarly, tyrosine phosphorylated CaM kinase IV binds its substrate with a higher affinity and thus increased tyrosine phosphorylation leads to increased activation of CaM kinase IV resulting in increased CREB phosphorylation that triggers increased transcription of proapoptotic proteins that initiate hypoxic neuronal death.

Brain tissue energy dependence of CaM kinase IV cascade activation during hypoxia in the cerebral cortex of newborn piglets.

The present study aims to investigate the dependence of CaM kinase IV cascade activation during hypoxia and tests the hypothesis that hypoxia-induced tyrosine phosphorylation of CaM and CaM kinase IV, activation of CaM kinase IV and phosphorylation of CREB protein during hypoxia increases as a function of increase in cerebral tissue hypoxia as measured by decrease in tissue ATP and phosphocreatine (PCr). 3-5 days old newborn piglets were divided into normoxic (Nx, FiO2 of 0.21 for 1h) and hypoxic (Hx, FiO2 of 0.07 for 1h) groups. Cerebral tissue hypoxia was documented by determining the levels of high energy phosphates ATP and phosphocreatine (PCr). Cerebral cortical neuronal nuclei were isolated and purified, and tyrosine phosphorylation of calmodulin (Tyr99), the activator of CaM kinase IV, and CaM kinase IV determined by Western blot using anti-phospho-(pTyr99)-calmodulin, anti-pTyrosine and anti-CaM kinase IV antibodies. The activity of CaM kinase IV and its consequence the phosphorylation of CREB protein at Ser¹³³ were determined. The levels of ATP (µmole/g brain) ranged from 3.48 to 5.28 in Nx, and 0.41 to 2.26 in Hx. The levels of PCr (µmole/g brain) ranged from 2.46 to 3.91 in Nx and 0.72 to 1.20 in Hx. The pTyr99 calmodulin (OD x mm²) ranged from 20.35 to 54.47.60 in Nx, and 84.52 to 181.42 in Hx (r²=0.5309 vs ATP and r²=0.6899 vs PCr). Expression of tyrosine phosphorylated CaM kinase IV ranged from 32.86 to 82.46 in Nx and 96.70 to 131.62 in Hx (r²=0.5132 vs ATP and r²=0.4335 vs PCr). The activity of CaM kinase IV (pmole/mg protein/min) ranged from 1263 to 3448 in Nx and 3767 to 6633 in Hx (r²=0.7113 vs ATP and r²=0.6182 vs PCr). The expression of p-CREB at Ser¹³³ ranged from 44.26 to 70.28 in Nx and 82.70 to 182.86 in Hx (r²=0.6621 vs ATP and r²=0.5485 vs PCr). The data show that hypoxia results in increased tyrosine phosphorylation of calmodulin (Tyr99), increased tyrosine phosphorylation of CaM kinase IV, increased activity of CaM kinase IV and increased phosphorylation of CREB at Ser¹³³ as an inverse function of cerebral concentration of high energy phosphates, ATP and PCr. We conclude that the hypoxia-induced increased activation of CaM kinase IV cascade increases with the increase in the degree of cerebral tissue hypoxia as measured by cerebral tissue high energy phosphates in a curvilinear manner. The tyrosine kinases (Src kinase and EGFR kinase) mediated activation of CaM kinase IV cascade potentially results in increased CREB phosphorylation that triggers transcription of proapoptotic proteins during hypoxia.

Hypoxia-induced activation of epidermal growth factor receptor (EGFR) kinase in the cerebral cortex of newborn piglets: the role of nitric oxide.

The present study aims to investigate the mechanism of EGFR kinase activation during hypoxia and tests the hypothesis that hypoxia-induced increased activation of EGFR kinase in the cerebral cortical membrane fraction of newborn piglets is mediated by nitric oxide (NO) derived from neuronal nitric oxide synthase (nNOS). Fifteen newborn piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-treated with nNOS inhibitor (Hx-nNOSi, n = 5). Hypoxia was induced by an FiO2 of 0.07 for 60 min. nNOS inhibitor I (selectivity >2,500 vs. endothelial NOS, eNOS, and >500 vs. inducible NOS, iNOS) was administered (0.4 mg/kg, i. v.) 30 min prior to hypoxia. EGFR kinase tyrosine phosphorylation at Tyr1173, an index of activation of EGFR kinase, was determined by Western blot analysis using an anti-phospho (pTyr(1173))-EGFR kinase antibody. Protein bands were analyzed by imaging densitometry and expressed as absorbance (OD x mm(2)). EGFR kinase activity was determined radiochemically using immunopurified enzyme. EGFR kinase activity was expressed as pmols/mg protein/hr. Density of phosphor (pTyr(1173))-EGFR kinase (OD x mm(2)) was 60.2 +/- 9.8 in Nx, 177.0 +/- 26.9 in Hx (P < 0.05 vs. Nx) and 79.9 +/- 15.7 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Activity of EGFR kinase (pmoles/mg protein/hr) was 4,603 +/- 155 in Nx, 8,493 +/- 427 in Hx (P < 0.05 vs. Nx) and 4,516 +/- 104 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Pretreatment with nNOS inhibitor prevented the hypoxia-induced increased phosphorylation and increased activity of EGFR kinase. We conclude that the mechanism of hypoxia-induced increased activation of EGFR kinase is mediated by nNOS-derived NO.

Mechanism of increased tyrosine (Tyr(99)) phosphorylation of calmodulin during hypoxia in the cerebral cortex of newborn piglets: the role of nNOS-derived nitric oxide.

The present study aims to investigate the mechanism of calmodulin modification during hypoxia and tests the hypothesis that hypoxia-induced increase in Tyr(99) phosphorylation of calmodulin in the cerebral cortex of newborn piglets is mediated by NO derived from nNOS. Fifteen piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, F(i)O(2) of 0.07 for 1 h, n = 5) and hypoxic-pretreated with nNOSi (Hx-nNOSi, n = 5) groups. nNOS inhibitor I (selectivity >2,500 vs. eNOS and >500 vs. iNOS) was administered (0.4 mg/kg, I.V.) 30 min prior to hypoxia. Cortical membranes were isolated and tyrosine phosphorylation (Tyr(99) and total) of calmodulin determined by Western blot using anti-phospho-(pTyr(99))-calmodulin and anti-pTyr antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by densitometry and expressed as absorbance. The pTyr(99) calmodulin (ODxmm(2)) was 78.55 +/- 10.76 in Nx, 165.05 +/- 12.26 in Hx (P < 0.05 vs. Nx) and 96.97 +/- 13.18 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Expression of total tyrosine phosphorylated calmodulin was 69.24 +/- 13.69 in Nx, 156.17 +/- 16.34 in Hx (P < 0.05 vs. Nx) and 74.18 +/- 3.9 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). The data show that administration of nNOS inhibitor prevented the hypoxia-induced increased Tyr(99) phosphorylation of calmodulin. Total tyrosine phosphorylation of calmodulin was similar to Tyr(99) phosphorylation. We conclude that the mechanism of hypoxia-induced modification (Tyr(99) phosphorylation) of calmodulin is mediated by NO derived from nNOS. We speculate that Tyr(99) phosphorylated calmodulin, as compared to non-phosphorylated, binds with a higher affinity at the calmodulin binding site of nNOS leading to increased activation of nNOS and increased generation of NO.

Tyrosine phosphorylation of neuronal nitric oxide synthase (nNOS) during hypoxia in the cerebral cortex of newborn piglets: the role of nitric oxide.

The present study aims to investigate the mechanism of activation of nNOS during hypoxia and tests the hypothesis that the hypoxia-induced increased tyrosine phosphorylation of nNOS in the cerebral cortical membranes of newborn piglets is mediated by nNOS-derived nitric oxide (NO). Fifteen newborn piglets were divided into normoxic (Nx, n=5), hypoxic (Hx, n=5) and hypoxic-pretreated with nNOS inhibitor I (Hx-nNOSi) groups. Hypoxia was induced by an FiO(2) of 0.07 for 60 min. nNOS inhibitor I (selectivity>2500 vs endothelial NOS and >500 vs inducible NOS) was administered (0.4 mg/kg, i.v.) 30 min prior to hypoxia. Cortical membranes were isolated and tyrosine phosphorylation of nNOS determined by Western blot. Membrane protein was immunoprecipitated with nNOS antibody, separated on 12% SDS-PAGE and blotted with anti-phosphotyrosine antibody. Protein bands were detected by enhanced chemiluminescence, analyzed by densitometry and expressed as absorbance (OD x mm(2)). Density (OD x mm(2)) of tyrosine phosphorylated nNOS was 51.66+/-14.11 in Nx, 118.39+/-14.17 in Hx (p<0.05 vs Nx) and 45.56+/-10.34 in Hx-nNOSi (p<0.05 vs Hx, p=NS vs Nx). The results demonstrate that pretreatment with nNOS inhibitor prevents the hypoxia-induced increased tyrosine phosphorylation of nNOS. We conclude that the mechanism of hypoxia-induced increased tyrosine phosphorylation of nNOS is mediated by nNOS-derived NO.

NO-mediated activation of Src kinase during hypoxia in the cerebral cortex of newborn piglets.

The present study aims to investigate the mechanism of Src kinase activation during hypoxia and tests the hypothesis that the hypoxia-induced activation of Src kinase, as determined by Src kinase phosphorylation, in the cerebral cortical membranes of newborn piglets is mediated by NO derived from neuronal nitric oxide synthase (nNOS). Fifteen piglets were divided into normoxic (Nx, n=5), hypoxic (Hx, n=5) and hypoxic-treated with nNOS inhibitor I (Hx-nNOSi) groups. Hypoxia was induced by decreasing FiO(2) to 0.06 for 1h. nNOS inhibitor I (selectivity >2500 vs eNOS and >500 vs iNOS) was administered (0.4 mg/kg, i.v.) 30 min prior to hypoxia. Cortical membranes were isolated and phosphorylation of Src kinase was determined by Western blot analysis. Src kinase activity was determined by radioactive assay using immunopurified enzyme. Membrane proteins were separated by 12% SDS-PAGE and probed with anti-phospho (pTyr(418))-Src kinase antibody. Protein bands were detected, analyzed by densitometry and expressed as absorbance (ODxmm(2)). Density (ODxmm(2)) of phosphorylated Src kinase was 111.7+/-21.1 in Nx, 234.5+/-23.8 in Hx (p<0.05 vs Nx) and 104.7+/-18.1 in Hx-nNOSi (p<0.05 vs Hx, p=NS vs Nx). Src kinase activity (pmol/mgprotein/ h) was 2472+/-75 in Nx, 4556+/-358 in Hx (p<0.05 vs Nx) and 2259+/-207 in Hx-nNOSi (p<0.05 vs Hx, p=NS vs Nx). The data show that pretreatment with nNOS inhibitor prevents the hypoxia-induced increase in tyrosine phosphorylation and the activity of Src kinase. We conclude that the mechanism of hypoxia-induced increased activation of Src kinase is mediated by nNOS derived NO. We propose that NO mediated inhibition of protein tyrosine phosphatases SH-PTP-1 and SH-PTP-2 leads to increased tyrosine phosphorylation and activation of Src kinase in the cerebral cortex of newborn piglets.

Effect of hyperoxia on serine phosphorylation of apoptotic proteins in mitochondrial membranes of the cerebral cortex of newborn piglets.

Previous studies have shown that hyperoxia results in cerebral cortical neuronal apoptosis. Studies have also shown that phosphorylation of anti-apoptotic proteins Bcl-2 and Bcl-xl results in loss of their anti-apoptotic potential leading to alteration in mitochondrial membrane permeability and the release of apoptogenic proteins in the neuronal cell of the newborn piglets. The present study tests the hypothesis that cerebral hyperoxia will result in increased serine phosphorylation of apoptotic proteins Bcl-2, Bcl-xl, Bax, and Bad in the mitochondrial membranes of the cerebral cortex of newborn piglets. Twelve newborn piglets were divided into normoxic (Nx, n = 6) exposed to an FiO(2) of 0.21 for 1 h and hyperoxic (Hyx, n = 6) exposed to FiO(2) of 1.0 for 1 h. In the Hyx group, PaO(2) was maintained above 400 mmHg while the Nx group was kept at 80-100 mmHg. Cerebral cortical tissue was harvested and mitochondrial fractions were isolated. Mitochondrial membrane proteins were separated using 12% SDS-PAGE, and probed with anti-serine phosphorylated Bcl-2, Bcl-xl, Bax, and Bad antibodies. Protein bands were detected, analyzed by imaging densitometry and density expressed as absorbance (OD x mm(2)). Phosphorylated Bcl-2 (p-Bcl-2) protein density (OD x mm(2)) was 81.81 +/- 9.24 in Nx and 158.34 +/- 10.66 in Hyx (P < 0.05). Phosphorylated Bcl-xl (p-Bcl-xl) protein density was 52.98 +/- 3.59 in Nx and 99.62 +/- 18.22 in Hyx (P < 0.05). Phosphorylated Bax (p-Bax) protein was 161.13 +/- 6.27 in Nx and 174.21 +/- 15.95 in Hyx (P = NS). Phosphorylated Bad (p-Bad) protein was 166.24 +/- 9.47 in Nx 155.38 +/- 12.32 in Hyx (P = NS). The data show that there is a significant increase in serine phosphorylation of Bcl-2 and Bcl-xl proteins while phosphorylation of Bad and Bax proteins were not altered during hyperoxia in the mitochondrial fraction of the cerebral cortex of newborn piglets. We conclude that hyperoxia results in differential post-translational modification of anti-apoptotic proteins Bcl-2 and Bcl-xl as compared to pro-apoptotic proteins Bax and Bad in mitochondria. We speculate that phosphorylation of Bcl-2 and Bcl-xl will result in loss of their anti-apoptotic potential by preventing their dimerization with Bax leading to activation of the caspase cascade of neuronal death.

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.

Nuclear mechanisms of hypoxic cerebral injury in the newborn: the role of caspases.

Our laboratory has been actively engaged in investigating mechanisms of activation of initiator caspase-9 during hypoxia in the developing newborn and fetal brains. The present review has been organized as follows: (a) the effect of hypoxia on the expression and activation of caspase-3, -8, and -9 in the newborn brain; (b) the role of nitric oxide in caspase-9, and caspase-3 activation during hypoxia in the newborn brain; (c) the role of nuclear Ca(2+)-influx in caspase-9 and caspase-3 activation during hypoxia in the newborn brain; (d) the effect of caspase-9 inhibition during hypoxia on preventing downstream events including caspase-3 activation. The results of our research investigations presented in (b), (c), and (d) elucidate mechanisms of caspase activation during hypoxia in the newborn brain. These studies provide the fundamental framework for developing neuroprotective strategies in the hypoxic newborn.

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.

Effect of hypoxia on the expression of procaspase-9 and procaspase-3 in neuronal nuclear, mitochondrial and cytosolic fractions of 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, in the cytosolic fractions of the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypoxia results in increased expression of procaspase-9 and procaspase-3 in neuronal nuclear, mitochondrial and cytosolic fractions of the cerebral cortex of newborn piglets. To test this hypothesis, expression of procaspase-9 and procaspase-3 was determined in 10 newborn piglets divided into two groups: normoxic (Nx, n=5) and hypoxic (Hx, 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. Neuronal nuclear, mitochondrial and cytosolic fractions were isolated and the expression of procaspase-9 and procaspase-3 was determined by immunoblotting using specific anti-procaspase-9 and anti-procaspase-3 antibodies. ATP levels (micromol/g brain) were 4.34+/-0.36 in the Nx and 1.43+/-0.28 in the Hx (p<0.001 vs. Nx) groups. PCr levels (micromol/g brain) were 3.75+/-0.27 in the Nx and 0.69+/-0.26 in the Hx (p<0.001 vs. Nx) group. Cytosolic procaspase-9 density (ODxmm(2)) was 88.82+/-17.55 in the Nx and 215.54+/-22.77 in the Hx (p<0.001 vs. Nx). Mitochondrial procaspase-9 density (ODxmm(2)) was 104.67+/-12.75 in the Nx and 183.44+/-16.69 in the Hx (p<0.001 vs. Nx). Nuclear procaspase-9 density (ODxmm(2)) was 135.56+/-15.36 in the Nx and 190.66+/-29.35 in the Hx (p<0.001 vs. Nx). Cytosolic procaspase-3 density (ODxmm(2)) was 23.72+/-3.71 in the Nx and 92.44+/-8.46 in the Hx (p<0.001 vs. Nx). Mitochondrial procaspase-3 density (ODxmm(2)) was 22.12+/-2.97 in the Nx and 51.22+/-10.67 in the Hx (p<0.001 vs. Nx). Nuclear procaspase-3 density (ODxmm(2)) was 53.80+/-7.18 in the Nx and 84.67+/-5.63 in the Hx (p<0.001 vs. Nx). We conclude that procaspase-9 and procaspase-3 proteins increased in all cell compartments including cytosolic, mitochondrial and nuclear during hypoxia, indicating increased expression of procaspase-9 during hypoxia. We propose that following increased expression of procaspase-9 and procaspase-3, these molecules traffic among the various cell compartments and become available for their activation resulting in increased caspase-9 and caspase-3 activity.

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.

Mechanism of DNA fragmentation during hypoxia in the cerebral cortex of newborn piglets.

We have previously shown that hypoxia results in increased activity of caspase-9, caspase-3 and fragmentation of nuclear DNA in the cerebral cortex of newborn piglets. The present study tested the hypothesis that mechanism of DNA fragmentation during hypoxia in the cerebral cortex of newborn piglets is mediated by caspase-9-dependent caspase-3 activation. Newborn piglets were randomly assigned to normoxic, hypoxic, and hypoxic pretreated with a highly selective caspase-9 inhibitor, Z-LEHD-FMK groups. The data showed that cerebral tissue hypoxia results in increased expression of caspase-activated DNase (CAD) protein in the nucleus and fragmentation of nuclear DNA. A pretreatment with Z-LEHD-FMK attenuated the expression of CAD protein in the nucleus and the fragmentation of nuclear DNA. Based on these results, we conclude that the mechanism by which the nuclear DNA was fragmented is mediated by caspase-9-dependent caspase-3 activation and the consequence of caspase-activated DNase activation in the cerebral cortex of newborn piglets.

Effect of hypoxia on expression of apoptotic proteins in nuclear, mitochondrial and cytosolic fractions of the cerebral cortex of newborn piglets: the role of nuclear Ca++ -influx.

We have shown that hypoxia results in increased influx of nuclear Ca++ and increased expression of nuclear apoptotic proteins. The present study tests the hypothesis that hypoxia alters the distribution of pro-apoptotic proteins Bad and Bax, and the anti-apoptotic proteins Bcl-xl, and Bcl-2 in the nuclear, mitochondrial and cytosolic compartments of the cerebral cortex of newborn piglets and the administration of Clonidine, an inhibitor of high affinity nuclear Ca++ -ATPase, will prevent the hypoxia-induced increase in apoptotic proteins' expression. Studies were conducted in 19 newborn piglets, 6 normoxic (Nx), 7 hypoxic and 6 Clonidine-treated hypoxic (Hx-Clo). Tissue hypoxia was documented biochemically by measuring cerebral tissue ATP and phosphocreatine (PCr) levels. Bax and Bad protein expression increased in all the three compartments during hypoxia, while there was no significant change in the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. In Clonidine pretreated hypoxic group, the hypoxia-induced increased expression of pro-apoptotic proteins Bad and Bax was prevented in all the three fractions. We conclude that hypoxia results in increased expression of pro-apoptotic proteins in nuclear, mitochondrial and cytosolic compartments and that the increased expression of pro-apoptotic proteins during hypoxia is nuclear Ca++ -influx-dependent. We propose that during hypoxia the increased ratio of (pro-apoptotic Bad and Bax/anti-apoptotic Bcl-xl and Bcl-2) in all the three compartments, will lead to altered mitochondrial and nuclear membrane permeability as well as caspase-9 activation in the cytosolic compartment.

Effect of hypoxia on protein tyrosine phosphatase activity and expression of protein tyrosine phosphatases PTP-1B, PTP-SH1 and PTP-SH2 in the cerebral cortex of guinea pig fetus.

The present study tested the hypothesis that the hypoxia in utero results in decreased protein tyrosine phosphatase (PTP) activity in cytosolic and membrane fractions and increased expression of PTPs (PTP-1B, PTP-SH1 and PTP-SH2) in the cytosol and the membrane fraction of the cerebral cortex of guinea pig fetus. In addition, we hypothesize that the increased expression is mediated by nitric oxide (NO). To test this hypothesis, PTP activity in cytosol and cell membrane, and expression in the cytosol and membrane fraction were measured in the cerebral cortex of normoxic, hypoxic and L-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), pretreated hypoxic (L-NAME+Hx) guinea pig fetuses. PTP activity in the cytosolic and membrane fractions was significantly lower in the Hx group as compared to the Nx group. The density of cytosolic PTP-1B, cytosolic PTP-SH1 and PTP-SH2 was increased in the Hx group and this increase was prevented in the L-NAME+Hx group. The data show that pretreatment with L-NAME, an inhibitor of NOS, prevents the hypoxia-induced increased expression of PTP-1B, PTP-SH1 and PTP-SH2 in the membrane and cytosolic fractions of the cerebral cortex of the guinea pig fetus. We conclude that the decrease in PTP activity during hypoxia is not due to protein modification of PTP and due to alteration in PTP expression.

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.

Mechanism of caspase-3 activation during hypoxia in the cerebral cortex of newborn piglets.

We have previously shown that the activity and the expression of caspase-9 and caspase-3 were increased during hypoxia in the cerebral cortex of newborn piglets. The present study was conducted to test the hypothesis that the hypoxia-induced activation of caspase-3 in the cerebral cortex of newborn piglets is mediated by caspase-9. Twenty-two newborn piglets were randomly assigned to four groups: normoxic (Nx), normoxic pretreated with a selective caspase-9 inhibitor, Z-Leu-Glu(OMe)-His-Asp(OMe)-Fluoromethyl ketone (Z-LEHD-FMK) (Nx+LEHD), hypoxic (Hx), and hypoxic pretreated with Z-LEHD-FMK (Hx+LEHD). Cerebral tissue hypoxia was confirmed biochemically by measuring ATP and phosphocreatine. Caspase-9 and -3 activities were determined spectrofluorometrically. The expression of caspase-9 and -3 proteins was measured by Western blot analysis using active enzyme specific antibodies. Cytosolic caspase-9 activity (nmol/mg protein/h) was 3.70+/-0.40 in Nx, 3.56+/-0.31 in Nx+LEHD (p=NS versus Nx), 4.99+/-0.64 in Hx (p<0.05 versus Nx), and 3.73+/-0.80 in Hx+LEHD (p<0.05 versus Hx, p=NS versus Nx). Cytosolic caspase-3 activity (nmol/mg protein/h) was 7.80+/-1.17 in Nx, 8.15+/-0.87 in Nx+LEHD (p=NS versus Nx), 13.07+/-0.78 in Hx (p<0.05 versus Nx), and 10.05+/-2.09 in Hx+LEHD (p<0.05 versus Hx) The density (ODxmm(2)) of active caspase-9 protein was 18.52+/-1.89 in Nx, 20.53+/-1.12 in Nx+LEHD (p=NS versus Nx), 32.36+/-5.03 in Hx (p<0.05 versus Nx), and 19.94+/-3.59 in Hx+LEHD (p<0.05 versus Hx, p=NS versus Nx). The density (ODxmm(2)) of active caspase-3 protein was 55.87+/-8.73 in Nx, 55.69+/-8.18 in Nx+LEHD (p=NS versus Nx), 94.10+/-12.05 in Hx (p<0.05 versus Nx), and 56.12+/-14.56 in Hx+LEHD (p<0.05 versus Hx, p=NS versus Nx). These data show that administration of a selective caspase-9 inhibitor, Z-LEHD-FMK, prior to hypoxia prevents the hypoxia-induced increase in caspase-3 activity and the expression of active caspase-3 protein. We conclude that the hypoxia-induced activation of caspase-3 during hypoxia in the cerebral cortex of newborn piglets is mediated by caspase-9.

Differential expression of apoptotic proteins following hypoxia-induced CREB phosphorylation in the cerebral cortex of newborn piglets.

The present study investigates the correlation between the hypoxia-induced phosphorylation of cyclic AMP response element binding protein and the expression of apoptotic proteins (proapoptotic proteins Bax and Bad and antiapoptotic proteins Bcl-2 and Bcl-xl) during hypoxia in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx) and hypoxic (Hx, FiO(2)=0.06 for 1 h) groups. Cerebral tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Ser(133) phosphorylation of cyclic AMP response element binding (CREB) protein was determined by Western blot analysis using a specific anti-phosphorylated Ser(133)-CREB protein antibody. The expression of apoptotic proteins was determined by using specific anti-Bax, anti-Bad, anti-Bcl-2 and anti-Bcl-xl antibodies. ATP and PCr values (mumoles/g brain) in Hx were significantly different from Nx (ATP: 4.40 +/- 0.39 in Nx vs. 1.19 +/- 0.44 in Hx, P<0.05 vs. Nx; PCr: 3.60 +/- 0.40 in Nx vs. 0.70 +/- 0.31 in Hx, P<0.05 vs. Nx). Ser(133) phosphorylated CREB protein (OD x mm(2)) was 74.55 +/- 4.75 in Nx and 127.13 +/- 19.36 in Hx (P<0.05 vs. Nx). The expression of proapoptotic proteins Bax and Bad increased and strongly correlated with the increase in CREB protein phosphorylation (correlation coefficient r=0.82 and r=0.85, respectively). The expression of antiapoptotic proteins Bcl-2 and Bcl-xl did not show correlation with CREB protein phosphorylation. We conclude that cerebral hypoxia results in differential regulation of CREB protein-mediated expression of proapoptotic and antiapoptotic proteins in the cerebral cortex of newborn piglets. We propose that the increased expression of proapoptotic vs antiapoptotic genes will lead to an increased potential for apoptotic programmed cell death in the Hx newborn brain.

Effects of severe hypocapnia on expression of bax and bcl-2 proteins, DNA fragmentation, and membrane peroxidation products in cerebral cortical mitochondria of newborn piglets.

BACKGROUND: Hypocapnia occurs in the newborn infant inadvertently or as a therapeutic modality and may result in neuronal and mitochondrial alterations in the newborn brain. Since mitochondria regulate apoptosis, these alterations may initiate a cascade of reactions that lead to apoptotic cell death. OBJECTIVES: This study tests the hypothesis that hypocapnia results in increased expression of the pro-apoptotic protein Bax, fragmentation of DNA and membrane lipid peroxidation in cerebral cortical mitochondria (mt) of newborn piglets. METHODS: Studies were performed in three groups of anesthetized normoxic newborn piglets: hypocapnic (H, n = 5), ventilated at a PaCO(2) of 11-15 mm Hg; normocapnic (N, n = 5), ventilated at a PaCO(2) of 40 mm Hg; and corrected normocapnic (CN, n = 4), ventilated as H with CO(2) added to maintain normocapnia. Tissue ATP and phosphocreatine levels were determined. Mitochondrial membrane proteins were separated, transblotted and probed with antibodies to Bax and Bcl-2. Bands were detected by enhanced chemiluminescence and analyzed by imaging densitometry. mtDNA was isolated. Cell and mitochondrial membrane lipid peroxidation products were measured spectrofluorometrically. RESULTS: ATP and PCr concentrations were similar in the 3 groups. The ratio of Bax/Bcl-2 increased significantly in H compared to N and CN. mtDNA fragmentation was also significantly greater in H compared to N or CN. Membrane lipid peroxidation was higher in H than in N or CN; and in CN compared to N. CONCLUSIONS: The data demonstrate that severe hypocapnia results in increased Bax expression, DNA fragmentation, and membrane lipid peroxidation in mitochondria of cerebral cortical neurons of newborn piglets, and may result in apoptotic 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.