hypoxia-induced generation of nitric oxide free radicals in cerebral cortex of newborn guinea pigs

Neurochemical Research, Vol. 25, No. 12, 2000, pp. 1559–1565

15590364-3190/00/1200–1559$18.00/0 © 2000 Plenum Publishing Corporation

Hypoxia-Induced Generation of Nitric Oxide Free Radicalsin Cerebral Cortex of Newborn Guinea Pigs

Om Prakash Mishra,1,2 Santina Zanelli,1 S. Tsuyoshi Ohnishi,1

and Maria Delivoria-Papadopoulos1

(Accepted August 2, 2000)

Previous studies have shown that brain tissue hypoxia results in increased N-methyl-D-aspartate(NMDA) receptor activation and receptor-mediated increase in intracellular calcium which mayactivate Ca++-dependent nitric oxide synthase (NOS). The present study tested the hypothesis thattissue hypoxia will induce generation of nitric oxide (NO) free radicals in cerebral cortex of new-born guinea pigs. Nitric oxide free radical generation was assayed by electron spin resonance(ESR) spectroscopy. Ten newborn guinea pigs were assigned to either normoxic (FiO2 = 21%,n = 5) or hypoxic (FiO2 = 7%, n = 5) groups. Prior to exposure, animals were injected subcuta-neously with the spin trapping agents diethyldithiocarbamate (DETC, 400 mg/kg), FeSO4.7H2O(40 mg/kg) and sodium citrate (200mg/kg). Pretreated animals were exposed to either 21% or7% oxygen for 60 min. Cortical tissue was obtained, homogenized and the spin adducts extracted.The difference of spectra between 2.047 and 2.027 gauss represents production of NO free rad-ical. In hypoxic animals, there was a difference (16.75 ± 1.70 mm/g dry brain tissue) betweenthe spectra of NO spin adducts identifying a significant increase in NO free radical production.In the normoxic animals, however, there was no difference between the two spectra. We concludethat hypoxia results in Ca2+- dependent NOS mediated increase in NO free radical production inthe cerebral cortex of newborn guinea pigs. Since NO free radicals produce peroxynitrite in pres-ence of superoxide radicals that are abundant in the hypoxic tissue, we speculate that hypoxia-induced generation of NO free radical will lead to nitration of a number of cerebral proteinsincluding the NMDA receptor, a potential mechanism of hypoxia-induced modification of theNMDA receptor resulting in neuronal injury.

KEY WORDS: NO free radicals; NOS; hypoxia; brain; newborn; ESR.

INTRODUCTION

Nitric oxide (NO) is a highly reactive free radicalspecies which is produced by the enzyme nitric oxidesynthase (NOS) (1,2). In the central nervous system,

NO has been proposed to play the role of a neurode-structive as well as a neuroprotective agent (3–7). Inneurons, NO is produced in response to Ca2+-dependentactivation of NOS (8,9). During hypoxia, the increasein intracellular Ca2+ due to activation of the N-methyl-D-aspartate (NMDA) receptor subtype of glutamatereceptor and to susequent release of Ca2+ from inter-cellular stores may activate NOS (10,11). In our previ-ous studies we have demonstrated that cerebral tissuehypoxia results in modification of the recognition, mod-ulatory and ion-channel sites of the NMDA receptorin fetal guinea pig and newborn piglets (12–16). Wehave also demonstrated an increased NMDA receptor

1 Department of Pediatrics, St. Christopher’s Hospital for Children,MCP Hahnemann School of Medicine, MCP Hahnemann Univer-sity, Philadelphia, PA.

2 Address reprint request to: Om Prakash Mishra, Ph.D., Departmentof Pediatrics, Neonatal Research Laboratory, 7th Floor HeritageBuilding, MCP Hahnemann University Hospital, 3300 HenryAvenue, Philadelphia, PA 19129. Tel: 215-842-4960; Fax: 215-843-3505; E-mail: [email protected]

mediated Ca2+ concentration in synaptosomes of hy-poxic guinea pigs (17).

In the present study we have investigated the ef-fect of cerebral tissue hypoxia on generation of NOfree radicals and tested the hypothesis that brain tis-sue hypoxia will result in increased production ofNO free radicals in the cerebral cortex of newbornguinea pigs.

EXPERIMENTAL PROCEDURE

Diethyldithiocarbamate (DETC) was purchased from AldrichChemicals (Milwaukee, WI). All other chemicals including Fe-SO4.TH2O and sodium citrate were purchased from Sigma Chemi-cals (St. Louis, MO). Dunkin Hartely guinea pigs were purchasedfrom Hilltop Laboratory Animals (Scottdale, PA). The experimentalprotocol was approved by the Institutional Animal Care and UseCommittee of MCP Hahnemann University. Animal care was pro-vided by the University Laboratory Animal Resources.

Experimental Protocol.Experiments were conducted on 10newborn guinea pigs (1–2 days old) divided into normoxic (n = 5)and hypoxic (n = 5) groups. Prior to exposure, animals were injectedsubcutaneously with the spin trapping agents diethyldithiocarba-mate (DETC, 400 mg/kg), FeSO4.7H2O (40 mg/kg) and sodiumcitrate (200 mg/kg) at separate locations of the lower abdomen. Pre-treated animals were exposed to either 21% or 7% oxygen for 60 min.Following the exposure, the animals were anesthetized (nembutal50 mg/kg, i.p.) and the cerebral cortical tissue was obtained. Thecortical tissue was transferred into a 3 ml plastic syringe mountedwith a 12 inch long infusion tubing and extruded into a quartz capi-lary ESR tubes (4 mm outer diameter, Wilmad Glass Co, Buena, NJ)and frozen in liquid nitrogen for the analysis of spin adduct by elec-tron spin resonance (ESR) spectroscopy.

Determination of Nitric Oxide Free Radicals.Nitric oxide freeradicals were determined as described by Tominaga et.al. (18). ESRspectra were obtained using Varian E-109 spectrometer with spec-tral measurements conditions as follows: 20 mV micro-wave power,0.2 mT modulation, 9.1 GHz microwave frequency, 100KHz modu-lation frequency, 10 mt. scan range, 0.25 sec scan time constant and4 min scan time. The temperature of ESR measurement was −150°C.Two scans were taken and the signals averaged. The height of dif-ference of spectra between 2.047 and 2.027 guass represents the tis-sue production of nitric oxide free radicals.

Determination of ATP and Phosphocreatine (PCr).The level ofcerebral tissue hypoxia was documented by determining the con-centrations of high energy phosphates. The cerebral tissue concen-trations of ATP and PCr were determined using a coupled enzymeassay by the method of Lamprecht et. al. (19). The frozen tissue wasground in perchloric acid under liquid nitrogen. The sample was al-lowed to thaw on ice and then centrifuged at 12,000 g for 5 min.Aliquots of the supernatant were neutralized and then centrifuged at2,000 g for 5 min. ATP and PCr concentrations were determined ina medium containing 1 ml buffer (50 mM triethanolamine, 5 mMMgCl2, 1 mM EDTA, 2mM glucose), 400µl aliquot of the 2,000g su-pernatant and 20 µl NADP. Ten µl of glucose-6-phosphate dehy-drogenase were added to initiate the reaction, which was followedspectrophotometrically at 340 nm. Ten µl of hexokinase were ad-ded and readings were taken at 0,5, 10, 15 and 20 min until a steadystate was achieved. The ATP concentration was calculated from the

increase in absorbance at 340 nm for 20 min after the addition ofhexokinase. Twenty µl of ADP and 20µl of creatine kinase (CK)were added and readings taken at 5 min intervals from zero timeuntil a steady-state was restored. PCr concentration was calculatedfrom the increase in absorbance at 340 nm between 0 - 20 min afterthe addition of CK.

Statistical Analysis.Statistical analysis of the biochemicalmeasurements and the free radical data were performed using a two-tailed Students’s t-test. A p value less than 0.05 was considered sig-nificant. All values are mean ± SD.

RESULTS

Brain tissue hypoxia in the guinea pigs was docu-mented by determining the levels of high energyphosphates (Table 1). The levels of ATP and phospho-creatine in the normoxic group were 4.24 ± 0.55 µmol/gbrain and 4.03 ± 0.67 µmol/g brain, respectively. In thehypoxic group the levels of ATP and phosphocreatinedecreased to 1.22 ± 0.31 µmol/ g brain (p < 0.001) and1.03 ± 0.34 µmol/g brain (p < 0.001), respectively.There were 71.3% and 74.5% decreases in the levels ofATP and phosphocreatine in the hypoxic group, docu-menting cerebral tissue hypoxia in these animals.

Fig.1 shows a representative electron spin reso-nance (ESR) spectrum of spin adducts in cerebralcortex of normoxic newborn guinea pigs. In Fig. 2, arepresentative ESR spectra of the spin adduct NO-Fe-DETC in cerebral cortex of hypoxic guinea pigs isshown. An arrow shows the presence of NO-Fe-DETCadduct which is not present in the normoxic animals asseen in Fig. 1. The spectral data of hypoxic animalswere subtracted from the normoxic and differences inspectra were obtained. Fig. 3 shows representativespectral data from normoxic and hypoxic, and theirdifference spectrum. The height of the difference spec-tra between the 2.047 and 2.027 gauss represents theproduction of NO free radicals as described by Tomi-naga et al. Fig. 3a shows a spin adduct spectrum fromthe hypoxic group and Fig. 3b shows a spectrum from

1560 Mishra, Zanelli, Ohnishi, and Delivoria-Papadopoulos

Table I. Tissue Levels of High Energy Phosphates in CerebralCortex of Newborn Guinea Pigs

ATP PCrStudy (µmol/g wet (µmol/g wet Groups brain tissue) brain tissue)

Normoxic (n = 4) 4.24 ± 0.55 4.03 ± 0.67Hypoxic (n = 4) 1.22 ± 0.31* 1.03 ± 0.34*

* p < 0.001 vs NormoxicMean ± SD

the normoxic group. The difference spectrum is shownin Fig. 3c. It is very clear in figure 3a that there is adifference between the spectra at g = 2.047 and g =2.027, indicating the production of NO free radicals.In contrast, in Fig. 3b, there is no difference betweenthe spectra at g = 2.047 and g = 2.027. Both are at thesame level at these values indicating the absence ofNO dependent spin adduct. The difference spectrashown in Fig. 3c clearly shows the typical NO-Fe-DETC signal with a peak at g = 2.047 and a trough atg = 2.027. These results demonstrate hypoxia-inducedgeneration of NO free radicals. Data from all the hy-poxic animals (Table II) showed an increase in spectradue to NO spin adducts in cerebral cortical tissue(16.75 ± 1.70 mm/ g dry brain tissue) identifying a sig-nificant increase in NO free radical production duringhypoxia.

DISCUSSION

In mammalian systems, three isoforms of NOShave been identified. NOS I (nNOS) is present in neu-

rons and is a constitutively expressed enzyme whoseactivity is regulated by Ca++ and calmodulin (20). NOSII (iNOS) is an inducible enzyme whose activity is in-dependent of Ca++. NOS III (eNOS) is constitutivelyexpressed in endothelial cells and is also regulated byCa++ and calmodulin. Increasing evidence suggest thatNOS expression can be regulated by various physio-logical and pathological conditions. nNOS mRNA up-regulation represents a general response of neuronalcells to stress conditions including hypoxia(21–23) andischemia (24–25). The present study demonstrates thehypoxia-induced generation of NO radicals in cerebralcortex of newborn guinea pigs.

The results demonstrate that there is an increasedgeneration of NO free radicals in the cerebral cortexof hypoxic newborn guinea pigs. The height of the dif-ference of spectra between g = 2.047 and 2.027 repre-sents the production of NO free radicals. In hypoxicanimals, there is a substantial difference between spec-

Nitric Oxide Free Radical Generation during Hypoxia 1561

Fig. 1. A representative electron spin resonance (EPR) spectra ofspin adducts in cerebral cortex of normoxic newborn guinea pigs.

Fig. 2. A representative electron spin resonance (EPR) spectra ofNO-Fe-DETC in cerebral cortex of hypoxic newborn guinea pigs.

Fig. 3. A representative difference spectra of NO-spin adducts.Difference between the spectra at g = 2.047 and g = 2.027 indicatesNO production. Straight lines have been drawn in a and b to show thedifference at these values. In a, there is a NO-spin adduct signal asindicated by the difference. In b, there is no difference indicatingabsence of NO-spin adduct signal. c shows the subtraction ofspectrum for normoxic from hypoxic (a–b), demonstrating thegeneration of NO-spin adduct (at g = 2.047) in the hypoxic group.

tra at g = 2.47 and 2.027. In normoxic animals, there isno difference between these two points. These resultsdemonstrate and identify an NO free radical signal in hy-poxic tissue. Spectral data from all the hypoxic animalsshowed a significant increase in difference of spectrafor NO spin adducts 16.75 ± 1.70 mm/g dry cortical tis-sue) as compared to absence of NO-spin adducts in thecortical tissue from normoxic animals.

There are a number of potential mechanisms forfree radical generation during hypoxia (26). Under hy-poxic condition, an increased accumulation of intra-cellular Ca2+ due to excessive activation of the NMDAreceptor and the non-NMDA receptor is a crucial stepin hypoxia-induced excitotoxicity (27,28). Increasedintracellular Ca2+ can activate a number of biochemi-cal pathways that can lead to free radical generationand cell death including: (1) activation of phospholi-pase A2 leading to increased generation of oxygen freeradicals from cyclooxygenase and lipoxygenase path-ways, (2) activation of NOS pathway leading to gen-eration of nitric oxide free radicals, peroxynitrite andoxygen derived free radicals, (3) activation of pro-teases leading to conversion of xanthine dehydroge-nase to xanthine oxidase resulting in increased freeradical generation, (4) activation of phospholipase Cleading to inositol triphosphate (IP3) formation and re-lease of Ca2+ from intracellular stores. In addition, freeradical generation can further trigger the release of ad-ditional excitatory amino acid neurotransmitters aswell as influence the activation of the NMDA receptorion-channel activity through the redox sites.

In addition to the Ca2+-mediated, there are otherpotential mechanisms of free radical generation duringhypoxia such as: (1) reduction of electron transportchain components including ubiquinone (a componentthat undergoes auto-oxidation to produce free radicals)(29,30), (2) increased release of iron from ferritin underthe condition of decreased cellular high energy com-pounds, and (3) increased degradation of ATP duringhypoxia increases the substrate for the xanthine oxidasereaction leading to increased free radical generation.

The excitatory amino acid glutamate contributesto brain injury during hypoxia (31,32). The excitotoxiceffect of glutamate in the CNS is mediated throughits interaction with specific cell membrane receptors,of which the NMDA, kainate, and the alpha-amino-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)subtypes are the best characterized (33). The protectiveeffect of specific glutamate receptor antagonists sup-port the role of NMDA receptor activation in hypoxic/ischemic cerebral injury (34–40). The NMDA receptoris the predominant mediator of excitotoxicity in the im-mature as compared to the adult brain (41).

NO produced in response to glutaminergic activa-tion (9) has been suggested to be involved in excita-tory amino acid-induced neuronal death (9,42). Theobservation that activation of the NMDA receptor gene-rates NO in Ca2+-dependent manner raised the possi-bility that NO, a well known cytotoxin, participates inneuronal excitotoxicity. This hypothesis was furtherstrengthened by the demonstration that inhibition ofNOS attenuates NMDA-dependent neurotoxicity inneuronal culture as well as reduces brain damage pro-duced by the middle cerebral arteries occlusion model(43–45).

In fact, there is a special structural and functionallink between the NMDA receptor and nNOS. In neuronsof the central nervous system, nNOS is co-localizedwith NMDA receptors (46,47). In addition, nNOS isactivated by Ca2+-influx through the NMDA receptorion-channel, however, nNOS is not efficiently stimu-lated by activation of non-NMDA receptor that alsoinduced Ca2+-influx (48–50). In synaptic plasma mem-branes, nNOS immunoreactivity is associated with theNMDA receptor. The synaptic localization of nNOS inthe brain may be mediated by postsynaptic densityprotein, PSD-95. It was recently demonstrated thatnNOS, PSD-95 and NMDA receptor subunit NR2Bcoimmunoprecipitate and that PSD-95 is sufficient toassemble a tight ternary complex with nNOS and theNR2B subunit of the NMDA receptor (52). The for-mation of this complex is mediated by PDZ domainsof PSD-95, which binds to the -COOH terminus of theNR2B subunit of NMDA receptor and PSD-95 recruitsnNOS by recognizing an internal motif adjacent to theconsensus nNOS PDZ domain (52). In summary, theresults of these studies indicate that NO production inthe brain is preferentially activated by Ca2+-influxthrough the NMDA receptor ion-channel and that thereis a specific structural and functional link between theNMDA receptor and the nNOS. It has also been re-cently demonstrated that there is a specific couplingof the NMDA receptor activation to NO mediated


Table II. Nitric Oxide Free Radical Generation in Cerebral Cortexof Newborn Guinea Pigs

NO Free Radical Spin-Adducts Study Groups (mm/g dry brain tissue)

Normoxic (n = 5) 0 ± 0 (reference)Hypoxic (n = 5) 16.75 ± 1.70*

* p < 0.001 vs normoxicMean ± SD

neurotoxicity by PSD-95 protein in cultured corticalneurons (53).

Activation of NOS generates NO, which leads tothe formation of peroxynitrite. Peroxynitrite, a potentoxidant, is formed by the rapid reaction of NO withsuperoxide anion radicals. This reaction mechanismpredominates (by two orders of magnitude) over thescavenging of superoxide by superoxide dismutase(54,55). Most cytotoxic effects of high levels of NOare mediated by peroxynitrite (55–57). Peroxynitriteadds a nitro group to the 3-position adjacent to the hy-droxyl group of tyrosine to produces stable product3-nitrotyrosine, an index of peroxynitrite-mediated ni-tration (58,59). Nitration of tyrosine residues has beenproposed as a major mechanism of modification ofproteins. Nitrotyrosine formation has been demonstratedin atherosclerotic lesions (60) and in a number of neu-rodegenerative diseases (61–68).

In light of these observations, our demonstration ofhypoxia-induced generation of nitric oxide free radicalsin cerebral cortex of newborn guinea pigs provides thecritical link in cascades of NMDA receptor-mediatedneurotoxicity during hypoxia.

ACKNOWLEDGMENTS

The present study was supported by grants from the NationalInstitutes of Health, NIH-HD-38079 and HD-20337.

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hypoxia-induced generation of nitric oxide free radicals in cerebral cortex of newborn guinea pigs

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