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Research report
Effect of nitric oxide synthase inhibition on high affinity
21
Ca
-ATPase during hypoxia in cerebral cortical neuronal nuclei of
newborn piglets
*
Gopikrishna Gavini , Santina A. Zanelli, Qazi M. Ashraf, Om P. Mishra,
Maria Delivoria-Papadopoulos
Department of Pediatrics /Neonatology, MCP Hahnemann University and St. Christopher’s Hospital for Children, 3300 Henry Avenue, 7th Floor, Room[701, Philadelphia, PA 19129, USA
Accepted 3 October 2000
Abstract
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Previous studies have shown that during hypoxia, neuronal nuclear high affinity Ca -ATPase activity is increased in the cerebral cortex of newborn piglets. The present study tests the hypothesis that pretreatment with N-nitro-L-arginine (NNLA) will prevent the
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hypoxia-induced increase in high affinity Ca -ATPase activity in cortical neuronal nuclear membrane of newborn piglets. We also tested
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the hypothesis that nitration is a mechanism of elevation of the high affinity Ca -ATPase activity during hypoxia. Studies were performed in five normoxic, five hypoxic, and six NNLA-pretreated (40 mg / kg) hypoxic newborn piglets. Cerebral cortical neuronal
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nuclei were isolated and the high affinity Ca -ATPase activity was determined. Further, normoxic samples were aliquoted into two
21
sub-groups for in vitro nitration with 0.5 mM peroxynitrite and subsequent determination of the high affinity Ca -ATPase activity. The activity increased from 309640 nmol Pi / mg protein / h in the normoxic group to 5206108 nmol Pi / mg protein / h in the hypoxic group (P,0.05). In the NNLA-pretreated group, the activity was 442653 nmol Pi / mg protein / h (P,0.05), which is 25% lower than in the hypoxic group. In the nitrated group the enzyme activity increased to 554659 nmol Pi / mg protein / h (P,0.05). Thus
peroxynitrite-21
induced nitration in vitro increased the high affinity Ca -ATPase activity and NNLA administration in vivo partially prevented the
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hypoxia-induced increase in neuronal nuclear high affinity Ca -ATPase activity. We conclude that the hypoxia-induced increase in
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nuclear membrane high affinity Ca -ATPase activity is NO-mediated and that nitration of the enzyme is a mechanism of its modification. 2000 Elsevier Science B.V. All rights reserved.
Theme: Development and regeneration
Topic: Neuronal death
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Keywords: NOS; Hypoxia; Ca -ATPase; Nuclei; Peroxynitrite; Newborn brain
1. Introduction ase (NOS), in a middle cerebral artery occlusion model
reduced the volume of cortical infarct in the mouse, Hypoxic brain injury is associated with the formation of indicating the role of NO in neurotoxicity [36]. Inhibition nitric oxide (NO), a gaseous free radical [2,8]. Although of NOS by NNLA has also been shown to decrease the under normal conditions NO physiologically mediates number of damaged neurons following hypoxia [4]. Fur-cerebral vasodilation [14], recent studies suggest that NO thermore, Numagami et al. [37] showed that NNLA may react with superoxide anion to form peroxynitrite and pretreatment could effectively inhibit free radical gene-cause neurotoxicity [3,12,20]. The administration of N- ration and brain cell membrane damage during hypoxia in nitro-L-arginine (NNLA) an inhibitor of nitric oxide synth- the newborn piglet.
It has been demonstrated that brain tissue hypoxia results in modification of the N-methyl-D-aspartate
*Corresponding author. Tel.: 11-215-842-4960; fax: 1
1-215-843-(NMDA) receptor ion-channel and in increased NMDA
3505.
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E-mail address: [email protected] (G. Gavini). receptor-mediated intracellular Ca influx [16,22,33]. In a
recent study we have shown that cerebral tissue hypoxia Animals were divided into three groups: normoxic, hyp-results in NMDA receptor-mediated increase in intracellu- oxic, and NNLA-pretreated hypoxic. Following 30 min
21 21
lar Ca [44]. Increased intracellular Ca activates sever- post-operative period for stabilization, each animal re-al pathways of free radicre-al generation, including nitric ceived either NNLA (40 mg / kg) or vehicle (pH-adjusted oxide synthase (NOS). Phospholipase A activation, me-2 saline) over a 60-min intravenous infusion. After the diated via NMDA receptor stimulation, produces superox- infusion was complete, hypoxia was then induced by ide anion within the neuron [25]. Thus cerebral hypoxia lowering the FiO to 7% to achieve a PaO of 19 mmHg2 2
concurrently increases the production of superoxide anion for 60 min. The piglets in normoxic group were ventilated and NO, which react to form peroxynitrite. Peroxynitrite to maintain PaO between 80 and 110 mmHg for 60 min.2
induces lipid peroxidation in vitro and is thought to be a At the end of the experiment, the cerebral cortical tissue mediator of direct cell damage [38]. NOS inhibition was removed within 5–10 s, placed in liquid nitrogen, and appears to be neuroprotective, indicating the role of NO in stored at 2808C for biochemical study.
ischemic neuronal damage [6,20,23,28].
Previous studies have shown that during hypoxia,
neuro-21 2.2. Isolation of neuronal nuclei
nal nuclear high affinity Ca -ATPase activity is increased in the cerebral cortex of newborn piglets. The present
Cerebral cortical nuclei were isolated according to study tests the hypothesis that nitration is a mechanism of
21 method described by Giuffrida et al. [18] with some
hypoxia-induced modification of the high affinity Ca
-modifications. Cortical tissue was homogenized by hand in ATPase activity and that NNLA pretreatment of hypoxic
15 volumes of a medium containing 0.32 M sucrose, 10 newborn piglets will prevent the hypoxia-induced increase
21 mM Tris–HCl (pH 6.8), 1 mM MgCl using a dounce-type 2
in high affinity Ca -ATPase activity in cerebral cortical
glass homogenizer in 22 strokes. The homogenate was neuronal nuclei of newborn piglets. To determine the role
21 filtered through nylon mesh cloth and centrifuged at 8503
of NO during hypoxia, the high affinity Ca -ATPase
g for 10 min. The resulting pellet was resuspended in
activity was measured in cerebral cortical neuronal nuclei
homogenizing buffer. The suspension was mixed with a of normoxic, hypoxic and NNLA-pretreated hypoxic
new-medium containing 2.4 M sucrose, 10 mM Tris–HCl (pH born piglets. In addition, to determine the role of nitration
6.8), and 1 mM MgCl to achieve a final concentration of2
as a possible mechanism of modification of the high
21 2.1 M sucrose, which increases the yield of large neuronal
affinity Ca -ATPase activity, neuronal nuclei from
nor-nuclei. The nuclei were then purified by centrifugation at moxic newborn piglets were treated with peroxynitrite in
53 0003g for 60 min. The resulting nuclear pellet was
vitro. Nuclear membrane nitration was confirmed by
resuspended in the homogenization medium and the purity Western blot analysis using an anti-nitrotyrosine antibody
21 of each nuclear preparation was assessed by phase contrast
and the activity of high affinity Ca -ATPase was
sub-microscopy (Olympus). The above method of neuronal sequently measured in nitrated and non-nitrated samples.
nuclei isolation yielded 90–95% pure preparation. This constituted the final nuclear preparation devoid of any microsomal, mitochondrial, or plasma membrane
contami-2. Materials and methods
nation. All procedures were carried out at 48C. 2.1. Animal preparation
2.3. ATP and Phosphocreatine (PCr) determination The experimental protocol was approved by the
Institu-tional Animal Care and Use Committee of MCP Brain tissue concentrations of ATP and PCr were Hahnemann University. Studies were conducted in 16 determined with a coupled enzyme assay using the method anesthetized, ventilated, and instrumented newborn piglets, of Lamprecht et al. [26]. The ATP concentration was 2–5 days of age. Anesthesia was induced with 4% calculated from the increase in absorbance at 340 nm for halothane and lowered to 1% during surgery while allow- the 20-min period following the addition of hexokinase. ing the animals to breathe spontaneously through a mask. Twentyml of ADP and 20 ml creatine kinase (CK) were Lidocaine 2% was injected locally before instrumentation added and readings taken at 5-min intervals from zero time for endotracheal tube insertion and femoral arterial and until a steady-state was restored. PCr concentration was venous catheter insertion. After instrumentation, the use of calculated from the increase in absorbance at 340 nm halothane was discontinued, and anesthesia was main- between 0 and 20 min after the addition of CK.
tained with nitrous oxide (79%), oxygen (21%) and fentanyl (50mg / kg), as needed. Tubocurarine (0.3 mg / kg)
was administered after connecting the piglet to ventilator. 2.4. In vitro treatment with peroxynitrite Arterial blood gases, pH, heart rate, and blood pressure
Table 1
Al-Medhi [24]. The concentration of the stock solution of
Cerebral tissue ATP and phosphocreatine levels in normoxic, hypoxic and
peroxynitrite was determined before each experiment by a
NNLA pretreated hypoxic newborn piglets
the absorbance at 302 nm in 1.2 M sodium hydroxide. The
Groups ATP Phosphocreatine
reaction was performed in 100 mM sodium phosphate
(mmol / g brain) (mmol / g brain)
buffer containing 100 mM DTPA and 25 mM sodium
Normoxia1saline 4.2760.08 3.4060.99
bicarbonate (pH 7.4). The peroxynitrite solution used for
Hypoxia1saline 1.2060.39* 0.7160.19*
the study may contain other compounds such as nitrite,
Hypoxia1NNLA 0.9860.21* 0.4760.06*
nitrate, and hydrogen peroxide and therefore to account for
a
Mean6S.D. * P,0.001 vs. normoxia.
their potential effects, peroxynitrite was allowed to de-compose for 5 min in the phosphate buffer before the
addition of the nuclear membrane protein. This reverse 3. Results
order reaction was used as control. Nuclear membrane
protein nitration was confirmed by Western blot analysis Tissue hypoxia was documented by determining the using monoclonal anti-nitrotyrosine antibody. levels of ATP and PCr in the cerebral cortex of newborn piglets. Table 1 shows the ATP and PCr values for the normoxic, hypoxic, and NNLA-pretreated hypoxic groups. 2.5. Western blot analysis In the hypoxic group, there was a 72 and 79% decrease in the ATP and PCr values, respectively (P,0.001 vs. Nuclear membranes were separated on 8% SDS–PAGE. normoxic group). Similarly, the ATP and PCr values Proteins were transferred overnight onto nitrocellulose decreased by 77 and 86%, respectively, in the NNLA-membranes. The nitrocellulose membranes were blocked pretreated hypoxic group (P,0.001 vs. normoxic group). with 10% milk in PBS at 48C with constant agitation for There was no statistical difference between the hypoxia 4–6 h and then incubated with monoclonal anti-nitro- and NNLA-pretreated hypoxic group.
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tyrosine antibody (Upstate Biotech., Lake Placid, MA) in High affinity Ca -ATPase activity was determined in 3% milk in PBS at 48C, at the concentration of 1:1000. neuronal nuclei of normoxic, hypoxic, and NNLA-pre-The membranes were incubated with horseradish peroxi- treated hypoxic newborn piglets (Fig. 1). The data show
21
dase-conjugated secondary antibody (Rockland, Gil- that the high affinity Ca -ATPase activity increased bertsville, PA) for 1.5 h. Immune complexes were detected following hypoxia: from 309633 nmol Pi / mg protein / h in using an enhanced chemiluminescence (ECL) method the normoxic group to 5206108 nmol Pi / mg protein / h in (Amersham Pharmacia Biotech., Buckinghamshire, UK). the hypoxic group, a 68% increase (P,0.05 vs. normoxic group). In the NNLA-pretreated hypoxic group, the high
21
affinity Ca -ATPase activity increased to 442653 nmol
21
2.6. Determination of high-affinity Ca -ATPase activity Pi / mg protein / h (P,0.05 vs. normoxic group), represent-ing a 43% increase in activity as compared to a 68%
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The activity of high affinity Ca -ATPase was de- increase in activity in the untreated hypoxic group (P,
termined in a 1-ml assay medium containing 20 mM 0.05 vs. hypoxic group). HEPES, 100 mM KCl, 250 mM MgCl , 1002 mM EGTA,
95 mM CaCl , 1 mM ouabain, 1 mM ATP, and 1502 mg nuclear membrane protein. The reaction was carried out at 378C for 30 min, a period during which the rate of reaction was linear. The reaction was stopped by the addition of 0.5 ml 12.5% tricholoroacetic acid. The samples were then centrifuged and the supernatant was analyzed for inorganic phosphate. The activity of the enzyme was expressed as nmol Pi / mg protein / h. Exposure of the nuclear membrane to the reverse order reaction did not affect the enzyme activity.
2.7. Statistical analysis
Statistical analysis was performed using one-way
analy-sis of variance (ANOVA) to compare the three groups and 21
Fig. 1. High affinity Ca -ATPase activity in neuronal nuclei of
normox-Tukey test was used for pairwise multiple comparisons. A ic, hypoxic and NNLA-pretreated hypoxic piglets. The enzyme activity
P value ,0.05 was considered significant. All values are was determined as described in the Methods section. The values are
newborns [29,41]. Although the consequences of antepar-tum or perinatal hypoxia can be observed clinically in newborn infants, the specific acute pathologic processes preceding the onset of irreversible cerebral injury are not well understood. Studies suggest that it is a complex combination of processes including increased release of excitatory neurotransmitters, activation of excitatory amino acid neurotransmitter receptors, increased intracellular
21
Ca , production of oxygen free radicals, changes in cellular ion-fluxes, and alteration of cell membrane struc-ture and function as well as changes in the expression of pro- and anti-apoptotic genes that contribute to cerebral function and cell death. The understanding of the basic cellular mechanisms which characterize the response of the immature brain to hypoxia will enable the development of novel and specific strategies for preventing or attenuating
Fig. 2. Western blot analysis showing evidence of nitration of neuronal
the deleterious effects of hypoxia in the human fetus and
nuclear membrane of newborn piglets following peroxynitrite treatment
newborn. The present study examined the effect of
inhibi-(0.5 mM) in vitro. Lanes 1, 3 and 5 are for nitrated samples and 2, 4, and 21
tion of nitric oxide synthase on high affinity Ca ATPase
6 are for non-nitrated samples.
in hypoxic cerebral cortical nuclei of newborn piglets and also tested protein modification via nitration as a mecha-The effect of in vitro nitration on the activity of the high 21
nism of alteration of high-affinity Ca -ATPase activity.
21
affinity Ca -ATPase was determined. Nitration of the
Our data demonstrate a significant increase in the neuronal nuclear membranes was confirmed by Western 21
nuclear high affinity Ca -ATPase activity in the hypoxic blot (Fig. 2). The data shows an increase in protein
group. However, this increase was 25% lower in the nitration in the peroxynitrite treated nuclear membranes as
NNLA-pretreated hypoxic group, suggesting a partial compared to untreated nuclear membranes. The results for
protection by NNLA. Furthermore, the results show a all samples in each groups are presented in Fig. 3. The 21
significant increase in the high-affinity Ca -ATPase
21
high affinity Ca -ATPase activity increased from 309633
activity following in vitro nitration, indicating that nitra-nmol Pi / mg protein / h in non-nitrated samples to 554659 21
tion is a mechanism of high affinity Ca -ATPase activity nmol Pi / mg protein / h in nitrated samples, a 79% increase
modification. These results indicate the role of NO in the (P,0.05 vs. non-nitrated normoxic samples). 21
regulation of the high affinity Ca -ATPase activity. The exact molecular mechanism of hypoxic nuclear damage is not clear. An appealing hypothesis is that
4. Discussion
generation of NO during hypoxia leads to the formation of peroxynitrite that decomposes rapidly to form nitrogen In the newborn, cerebral hypoxia occurs due to
antepar-dioxide and hydroxyl radicals [2,3,38]. It has also been tum or perinatal hypoxia / asphyxia with an incidence
shown that peroxynitrite can cause nitration of proteins in ranging from 1 to 5% of all live births and 20% of preterm
vitro. In the present study, we demonstrate that in vitro nitration of cerebral cortical nuclear membranes of new-born piglets resulted in increased activity of the high
21
affinity Ca -ATPase.
Following ischemia, NO produced from neuronal NOS has toxic effects, but NO produced from endothelial NOS has protective effects in the brain [10,11,21,39,42]. Re-cently, the effects of inhibition of NOS during ischemia or hypoxia–ischemia have been studied. Both neuroprotection [7,9,19,20,32,35,40] as well as absence of protection [5,13,31,36,43] were shown in hypoxic–ischemic, and ischemic animal models as well as cell culture models. These disparities could be attributed to differences in experimental models, specificities of NOS inhibitors, dos-ages and routes of administration. The data presented here
21 demonstrate the role of nitration in modification of high Fig. 3. High affinity Ca -ATPase activity in non-nitrated and nitrated 21
affinity Ca -ATPase activity in neuronal nuclei. In
addi-neuronal nuclear membranes. The values are expressed as mean6S.D.
21
hypoxia-induced modification of neuronal nuclear mem- increase in nuclear membrane high affinity Ca -ATPase
21
brane high affinity Ca -ATPase. activity is NO-mediated and that nitration of the enzyme is There are a number of potential mechanisms of free a mechanism of its modification during hypoxia.
radical generation during hypoxia. Increased accumulation
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of intracellular Ca due to excessive activation of NMDA
and non-NMDA receptors is critical in hypoxia-induced Acknowledgements
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excitotoxicity [15,27,34]. Increase in intracellular Ca
can initiate a number of biochemical events that lead to This study was supported by NIH grants HD-20337 and free radical generation and cell death including (1) activa- HD-38079.
tion of phospholipase A , leading to increased generation2
of oxygen free radicals from cyclooxygenase and
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