Hypoxia-Induced Modification of the InositolTriphosphate Receptor in Neuronal Nuclei ofNewborn Piglets: Role of Nitric Oxide

Om Prakash Mishra, Imaran Qayyum, and Maria Delivoria-PapadopoulosDepartment of Pediatrics, Drexel University College of Medicine, and St. Christopher’s Hospital for Children,Philadelphia, Pennsylvania

Previous studies have shown that hypoxia results in in-creased Ca2� influx in neuronal nuclei and generation ofnitric oxide (NO) free radicals in the cerebral corticaltissue of newborn piglets. The present study tests thehypothesis that hypoxia results in modification of theinositol triphosphate (IP3) receptor characteristics in neu-ronal nuclei and that the hypoxia-induced modification ofthe IP3 receptor is NO mediated. Studies were performedin piglets, 3–5 days old, divided into normoxic (n � 5),hypoxic (n � 5), and NO synthase (NOS) inhibitor N-nitro-L-arginine (NNLA)-treated hypoxic (n � 5) groups. TheNNLA-treated hypoxic group received an infusion ofNNLA (40 mg/kg) over 1 hr prior to hypoxic exposure.The hypoxia was induced by lowering the FiO2 to 0.05–0.07 for 1 hr. Brain tissue hypoxia was documentedbiochemically by determining ATP and phosphocreatine(PCr) levels. Neuronal nuclei were isolated from the ce-rebral cortical tissue, and IP3 receptor binding was per-formed in a medium containing 50 mM HEPES (pH 8.0),2 mM EDTA, 3H-IP3 (7.5–100 nM), and 100 �g nuclearprotein. Nonspecific binding was determined in the pres-ence of 10 �M unlabelled IP3. The IP3 receptor charac-teristics Bmax (number of receptor sites) and Kd (disso-ciation constant) were determined. In normoxic, hypoxic,and NNLA-hypoxic groups, ATP levels were 4.46 � 0.35,1.52 � 0.10 (P � .05 vs. normoxic), and 1.96 �0.33 �moles/g brain, respectively (P � .05 vs. normoxic).PCr levels were 3.75 � 0.35, 0.87 � 0.09 (P � .05 vs.normoxic), and 1.31 � 0.10 �moles/g brain, respectively(P � .05 vs. normoxic). IP3 receptor binding characteris-tics in normoxic nuclear membranes showed that theBmax value was 150.0 � 14.1 pmoles/mg protein com-pared with 239.3 � 13.6 pmoles/mg protein in the hy-poxic group (P � .05). In the NNLA-treated hypoxicgroup, the Bmax value was 159.0 � 42.6 pmoles/mgprotein (P � .05 vs. hypoxic a, P � NS vs. normoxic).Similarly, the Kd was 25.2 � 0.28 nM in the normoxicgroup, 44.6 � 5.4 nM in the hypoxic group (P � .05), and28.1 � 6.4 nM in the NNLA-treated hypoxic group. (P �.05 vs. hypoxic and P � NS vs. normoxic). The resultsshow that hypoxia results in increased Bmax and Kdvalues for the IP3 receptor. Furthermore, the data dem-onstrate that administration of NNLA prior to hypoxiaprevents the hypoxia-induced modification of the IP3

receptor in neuronal nuclei of newborn piglets. BecauseNNLA inhibits NOS and prevents generation of NO, weconclude that the mechanism of hypoxia-induced mod-ification of the neuronal nuclear membrane IP3 receptoris NO mediated. We propose that NO-mediated modifi-cation of the IP3 receptor during hypoxia may lead toincreased intranuclear Ca2�, resulting in altered tran-scription of apoptotic genes and activation of cascadesof hypoxia-induced programmed neuronal death.© 2003 Wiley-Liss, Inc.

Key words: nitric oxide; IP3 receptor; hypoxia; brain;nuclear Ca2� influx

The increased intracellular Ca2� is a primary medi-ator of activity-dependent gene transcription under anumber of experimental conditions (Ghosh and Green-berg, 1995; Bito et al., 1997; Hardigham and Bading,1998; Chawla and Bading, 2001). The patterns of neuro-nal impulse and the specific properties of the stimulus-induced calcium transients determine the nature and am-plitude of the genomic response (Fields et al., 1997;Hardigham and Bading, 1998). Several factors, includingthe site of calcium entry, the amplitude, and the spatialproperties of the calcium signals, determine the calcium-regulated gene expression (Lerea et al., 1992; Bading et al.,1993; Larea and McNamara, 1993; Hardigham et al.,1997, 1999; Dolmetsch et al., 2001). Furthermore, theduration of calcium signal also contributes to the specific-ity of the transcription induction. In cells of the immunesystem, only a continuous rise in intracellular Ca2� con-centration, and not a brief spike, induced translocation of

Contract grant sponsor: National Institutes of Health; Contract grant num-bers: HD-20337 and HD-38079.

*Correspondence to: Om Prakash Mishra, PhD, Department of Pediatrics,Room 701, 7th Floor Heritage Building, Medical College of PennsylvaniaHospital, 3300 Henry Avenue, Philadelphia, PA 19129.E-mail: mishra@drexel.edu

Received 7 February 2003; Revised 16 June 2003; Accepted 7 July 2003

Journal of Neuroscience Research 74:333–338 (2003)

© 2003 Wiley-Liss, Inc.

transcription factors NF-ATc (Dolmetsch et al., 1997). Ina recent study, it was demonstrated that gene expression inneurons is also determined by the duration of calciumtransients and that the activity-dependent transcription isregulated by the duration of calcium transients (Chawlaand Bading, 2001).

During hypoxia, the increase in intracellular Ca2� isdue to activation of the N-methyl-D-aspartate (NMDA)receptor subtype of the glutamate receptor and subsequentrelease of Ca2� from intracellular stores. In previous stud-ies, we have demonstrated that cerebral tissue hypoxiaresults in modification of the recognition and modulatorysites of the NMDA receptor in the fetal guinea pig andnewborn piglets (Mishra and Delivoria-Papadopoulos,1992; Hoffman et al., 1994; Fritz et al., 1996). We havealso demonstrated an increased NMDA receptor-mediatedCa2� concentration in synaptosomes and ATP-dependentCa2� influx in neuronal nuclei from hypoxic animals(Zanelli et al., 1999; Mishra and Delivoria-Papadopoulos,2002).

The present study specifically focuses on the effect ofhypoxia on the mechanisms of Ca2� flux in the neuronalnucleus that leads to increased intranuclear Ca2�, resultingin increased hypoxia-induced gene expression. Studieshave shown that the nuclear membrane possesses a high-affinity Ca2�-ATPase and an inositol tetrakisphosphate(IP4) receptor that allow the enrty of Ca2� into thenuclear lumen, from which the inositol triposphate (IP3)receptor, present on the inner nuclear membrane, releasesinto the nucleoplasm. In the present study, we have in-vestigated the effect of hypoxia on characteristics of theIP3 receptor in neuronal nuclear membrane and tested thehypothesis that cerebral hypoxia results in modification ofthe IP3 receptor in nuclear membranes and that thehypoxia-induced modification of the IP3 receptor in nu-clear membrane is nitric oxide (NO)-mediated.

MATERIALS AND METHODSAnimal Experimentation and Induction of Hypoxia

Studies were performed on 2–4-day-old Yorkshire pigletsobtained from the Willow Glenn Farm (Strausburg, PA). Theexperimental animal protocol was approved by the InstitutionalAnimal Care and Use Committee of the MCP HahnemannUniversity. Newborn piglets were randomly assigned to one ofthree groups: normoxic (n � 5), hypoxic (n � 5), or NOsynthase (NOS) inhibitor N-nitro-L-arginine (NNLA)-treatedhypoxic (n � 5). The animals were ventilated for 1 hr undereither normoxic conditions (FiO2 � 0.21) or hypoxic condi-tions; hypoxia was induced by lowering the FiO2 to 0.07 for60 min. The hypoxic-NNLA group received an infusion ofNNLA (40 mg/kg) over 1 hr prior to hypoxia. At the end of theexperimental period, the animal was sacrificed; the cortical tissuewas then removed and placed either in homogenization bufferfor isolation of neuronal nuclei or in liquid nitrogen, and thenstored at –80°C for later biochemical studies.

Isolation of Cerebral Cortical Neuronal Nuclei

Cerebral neuronal nuclei were isolated and purified ac-cording to the methods of Giuffrida et al. (1975) and purified as

described by Austoker et al. (1972). The nuclear pellet wassuspended in the medium (0.32 M sucrose, 10 mM Tris-HClbuffer, pH 6.8, and 1 mM MgCl2) and examined microscopi-cally for purity assessment. The final nuclear preparation wasdevoid of any microsomal, mitochondrial, or plasma membranecontaminants, with a purity for neuronal nuclei of 90%. Proteincontent was determined by the method of Lowry et al. (1951).The nuclear membrane preparation was diluted to a final con-centration of 100 �g protein/100 �l.

Determination of 3H-IP3 Receptor Binding3H-IP3 binding was performed according to Humbert et

al. (1996) at 0°C for 30 min in a medium containing 50 mMHEPES buffer (pH 8.0), 2 mM EDTA, increasing concentra-tions of 3H-IP3 ranging from 7.5 to 100 nM, and 150 �g nuclearmembrane protein. Nonspecific binding was determined in thepresence of 10 �M unlabeled IP3. Bound and free radioligandswere separated by centrifugation at 12,000 rpm, and the super-natant was removed by aspiration. The pellet was subsequentlywashed three times and centrifuged before being suspended in300 �l HEPES buffer containing 0.1 N NaOH. The suspensionwas then transferred to scintillation vials containing 10 ml scin-tillation fluid (EcoLume; ICN, Costa Mesa, CA) and counted inan LKB Rackbeta 1209 Scintillation Counter with an efficiencyof 65% for 3H. Kd and Bmax values were calculated by scatchardplot analysis from saturation experiments.

Determination of ATP and Phosphocreatine

ATP and phosphocreatine (PCr) concentrations were de-termined according to the method of Lamprecht et al. (1974).

Statistical Analysis

Data were analyzed using one-way ANOVA to comparenormoxic, hypoxic, and NNLA-hypoxic groups. A P value ofless than 0.05 was considered statistically significant. All valuesare presented as mean � SD.

RESULTSBrain tissue hypoxia in piglets was documented by

determining the ATP and PCr levels in the cerebral cor-tical tissue. The level of high-energy phosphates decreasedsignificantly in the hypoxic groups, demonstrating thatcerebral tissue hypoxia was achieved. ATP levels were4.46 � 0.35, 1.52 � 0.10, and 1.96 � 0.33 �moles/gbrain in the normoxic, hypoxic, and NNLA-hypoxicgroups, respectively (P � .05, normoxic vs. hypoxic andNNLA-hypoxic groups). PCr levels were 3.75 � 0.0.35(Nx), 0.87 � 0.09 (Hx), and 1.31 � 0.10 �moles/brain(Hx-NNLA; P � .05, Nx vs. Hx and Hx-NNLA). Theresults demonstrate that comparable degrees of hypoxiawere achieved in the hypoxic and NNLA-hypoxicgroups.

Representative plots of IP3 binding in neuronal nu-clei of normoxic, hypoxic, and NNLA-treated hypoxicgroups are shown in Figure 1. The results from all theanimals in the characteristics of IP3 receptor binding(Bmax � number of receptor sites and Kd � the dissoci-ation constant values) in neuronal nuclei of normoxic,hypoxic, and NNLA-hypoxic newborn piglets are shown

334 Mishra et al.

in Figures 2 and 3. Bmax was 150 � 14.1 (Nx), 239 �13.6 (Hx), and 159.0 � 42.6 (Hx-NNLA) pmoles/mgprotein (P � .05, Nx vs. Hx; P � NS, Nx vs. Hx-NNLA). Similarly, the Kd was 25.2 � 0.28 (Nx), 44.6 �5.4 (Hx), and 28.1 � 6.4 (Hx-NNLA) nM (P � .05, Nxvs. Hx; P � NS, Nx vs. Hx-NNLA). The results showthat hypoxia resulted in increased numbers and decreasedaffinity of the IP3 receptor sites, demonstrating a hypoxia-induced modification of the IP3 receptor in neuronalnuclear membranes. Furthermore, the data demonstratethat the administration of NNLA prevented the hypoxia-induced modification of the IP3 receptor (Figs. 2, 3).

DISCUSSIONSeveral critical nuclear functions, including regula-

tion of transcription factors, cell cycle, gene transcription,DNA replication, and nuclear envelope breakdown, arecontrolled by intranuclear Ca2� (Mishra and Delivoria-Papadopoulos, 1999). Furthermore, nuclear Ca2� signalspotentially control a number of events leading to hypoxia-induced programmed cell death. Nuclear and cytosolicCa2� signals are differentially regulated, and the extranu-clear Ca2� concentration determines the mode of Ca2�

entry into the nucleus.

Fig. 1. Effect of nitric oxide synthase inhibition on IP3 receptor inneuronal nuclei of newborn piglets. Representative Scatchard plots forIP3 binding in neuronal nuclei of normoxic (A), hypoxic (B), andNNLA-pretreated hypoxic (C) groups. The data from all the animalsare shown as mean � SD in Figures 2 and 3.

Fig. 2. Effect of nitric oxide synthase inhibition on IP3 receptor inneuronal nuclei of newborn piglets. Experiments were performed onfive normoxic, five hypoxic, and five NNLA-treated hypoxic newbornpiglets. The Bmax (number of receptor sites) is expressed as pmoles/mgprotein and is shown on the Y axis.

Fig. 3. Effect of nitric oxide synthase inhibition on IP3 receptor inneuronal nuclei of newborn piglets. Experiments were performed onfive normoxic, five hypoxic, and five NNLA-treated hypoxic newbornpiglets. The Kd (dissociation constant) is expressed as nM and is shownon the Y axis.

NO-Mediated Modification of IP3 Receptor 335

An increase in intracellular Ca2� is a critical event inhypoxic-ischemic neuronal excitotoxicity. The Ca2�-dependent neuronal damage is due to NMDA receptor-mediated excitotoxicity that is initiated as a result of hyp-oxia. During hypoxia, an increase in intracellular Ca2� isa result of the NMDA receptor ion channel-mediatedCa2� influx as well as the release of Ca2� from intracel-lular stores, such as mitochondria and the endoplasmicreticulum (Frandsen and Schousboe, 1991). The presentstudy specifically investigated the effect of hypoxia oncharacteristics of the IP3 receptor in neuronal nuclearmembrane and tested the hypothesis that cerebral hypoxiaresults in modification of the IP3 receptor in neuronalnuclear membranes and that the hypoxia-induced modi-fication of the IP3 receptor in nuclear membrane is NOmediated.

The results of the present study show that cerebralhypoxia results in increased Bmax (number of IP3 receptorsites) and increased Kd (dissociation constant). Furhter-more, the results of the present study demonstrate thathypoxia-induced modification of the IP3 receptor thatresults in increased Bmax and increased Kd of the IP3receptor site is prevented by prior administration ofNNLA, an NOS inhibitor, demonstrating that thehypoxia-induced modification of the IP3 receptor in neu-ronal nuclear membrane is NO mediated.

In previous studies, we have observed that NO do-nors increase neuronal nuclear Ca2� influx (Mishra andDelivoria-Papadopoulos, 2002). We have also shown thathypoxia results in generation of NO free radicals (Mishraet al., 2000). Furthermore, we have observed that IP3-dependent Ca2� influx is increased in neuronal nuclei ofhypoxic animals compared with normoxic animals andthat the increase in nuclear Ca2� influx by IP3 increaseswith increasing cerebral tissue hypoxia.

We propose that NO-mediated modification of thenuclear membrane IP3 receptor is a potential mechanismof increased intranuclear Ca2� during hypoxia that leadsto activation of Ca2�-dependent nuclear mechanisms andactivates cascades of hypoxia-induced programmed celldeath. In recent studies, we have demonstrated that ad-ministration of the NOS inhibitor NNLA prevents thehypoxia-induced increase in CaM kinase IV activity, in-crease in cyclic AMP response element-binding protein(CREB) phosphorylation, increase in expression of theproapoptotic protein Bax, and increase in fragmentation ofnuclear DNA (Zubrow et al., 2002a,b; Mishra et al.,2002).

Studies have demonstrated a calcium gradient be-tween the nucleus and the cytosol, indicating that calciummovement in and out of the nucleus is a regulated process(Al-Mohanna et al., 1994). The nuclear envelope consistsof an inner and an outer membrane interrupted by nuclearpores. The outer nuclear membrane has been shown tocontain IP4 receptor as well as the high-affinity Ca2�-ATPase. The IP3 receptor is located on the inner mem-brane. Thus IP4 and IP3 receptors can mediate the Ca2�

influx into the nucleus (Humbert et al., 1996). In the

present study, we have focused on determining thehypoxia-induced modification of the IP3 receptor charac-teristics in nuclei from normoxic and hypoxic animals.The increase in density and decrease in affinity of the IP3receptor observed in nuclei from the hypoxic group mightpotentially result in a hypoxia-induced increase in intranu-clear Ca2�. We have observed that hypoxia results inincreased Ca2� influx in neuronal nuclei (Mishra andDelivoria-Papadopoulos, 2002). In the present study, wehave shown that administration of NNLA 1 hr prior tohypoxia prevents the hypoxia-induced increase in IP3receptor sites in the neuronal nuclei of newborn piglets.These results demonstrate that hypoxia-induced alterationof the IP3 receptor is NO mediated. The data suggest thathypoxia-induced, NO-mediated modification of nuclearmembrane results in alteration of the IP3 receptors thatmay lead to increased nuclear Ca2� influx. We have alsoshown that the presence of NO increases Ca2� influx inneuronal nuclei (Mishra and Delivoria-Papadopoulos,2002).

Furthermore, pretreatment of animals with NNLAdecreased the hypoxia-induced increase in IP3 receptordensity, indicating an NO-mediated modification of neu-ronal nuclear membrane during hypoxia. We have alsoobserved that hypoxia results in peroxidation of neuronalnuclear membranes as well as increased nuclear Ca2�

influx (Maulik et al., 2001, 2002). The results of this studyindicate that NOS inhibitor may prevent the hypoxia-induced, free radical-mediated nuclear membrane peroxi-dation that results in modification of the IP3 receptor.

NO and superoxide radicals are concurrently pro-duced during hypoxia. NO produces peroxynitrite uponcombining with superoxide. The reaction between NOand superoxide is several orders of magnitude faster thanthe reaction between superoxide and superoxide dis-mutase. Therefore, production of peroxynitrite is favoredover dismutation of superoxide. Thus NO produced dur-ing hypoxia may result in nitrosylation as well asperoxynitrite-mediated nitration of a number of proteins,including the IP3 receptor in the nuclear membrane.

NO is a critical mediator of neuronal injury, asevidenced by the administration of pharmacological in-hibitors of NOS that reduce neuronal injury from focalischemia, NMDA-dependent excitotoxicity, and cerebralhypoxia (Huang et al., 1994; Yun et al., 1997; Numagamiet al., 1997). Mice deficient in neuronal NOS gene exhibitsignificant protection against cerebral ischemia andNMDA-mediated neurotoxicity (Huang et al., 1994; Yunet al., 1997). Furthermore, cerebral hypoxia results in thegeneration of NO free radicals (Mishra et al., 2000). Inaddition, administration of NOS inhibitor prevents thehypoxia-induced generation of free radicals, nitration ofthe NMDA receptor subunits, CaM kinase IV activation,increased phosphorylation of cAMP at Ser133, increasedexpression expression of the apoptotic protein Bax, andfragmentation of nuclear DNA (Numagami et al., 1997;Zanelli et al., 2002; Zubrow et al., 2002a,b; Mishra et al.,2002).

336 Mishra et al.

In view of these observations, NO can play a centralrole in hypoxia-induced neuronal death by the necrotic aswell as the apoptotic or programmed cell death mecha-nisms. First, the NO-induced increase in NMDAreceptor-mediated intracellular Ca2� potentially initiates anumber of reactions leading to increased free radical gen-eration via a number of enzymatic pathways, such as Ca2�

activation of phospholipase A2, causing release of arachi-donic acid, which then can be metabolized by cyclooxy-genase and lipoxygenase, conversion of xanthine dehydro-genase to xanthine oxidase by Ca2�-dependent activationof proteases, and activation of NOS by Ca2� to generateNO further, leading to formation of peroxynitrite andoxygen free radical species. The increased free radicalsgenerated result in increased peroxidation of cellular andsubcellular membranes, leading to necrotic cell death.Second, the increased intracellular Ca2� may lead to in-creased intranuclear Ca2� by mechanisms of Ca2� influx,such as the IP3 receptors and the nuclear membrane high-affinity Ca2� ATPase. Furthermore, we have demon-strated that NO increases nuclear Ca2� influx. In light ofthese observations, NO is capable of increasing intranu-clear Ca2� by more than one mechanism in vivo. In-creased intranuclear Ca2� may activate Ca2�-dependentendonucleases, leading to DNA fragmentation. In addi-tion, increased intranuclear Ca2� can activate CaM kinaseIV in the nucleus, leading to increased phosphorylation ofCREB, resulting in increased transcription of apoptoticgenes such as Bax and initiating the early events of DNAfragmentation and programmed cell death. Thus a centralrole for NO is proposed in regulating neuronal functionand specifically in hypoxia-induced neuronal death, byaltering the nuclear membrane mechanisms of Ca2� influxresulting in increased nuclear Ca2�.

In summary, these results show that hypoxia resultsin modification of the IP3 receptor (increase in the num-ber and increase in the affinity) in neuronal nuclei ofnewborn piglets. Furthermore, the data demonstrate thatthe administration of the NOS inhibitor NNLA preventshypoxia-induced modification of the IP3 receptor. Weconclude that cerebral hypoxia results in modification ofthe IP3 receptor in neuronal nuclei of newborn piglets andthat the hypoxia-induced modification of the nuclearmembrane IP3 receptor is NO mediated. We propose thatNO-mediated modification (nitrosylation/nitration) ofIP3 receptors of the neuronal membrane during hypoxia isa potential mechanism of increased nuclear Ca2� that leadsto hypoxic neuronal injury.

ACKNOWLEDGMENTThe authors thank Mrs. Joanna Kubin for her expert

technical assistance.

REFERENCESAl-Mohanna FA, Caddy KWT, Boisover SR. 1994. The nucleus is insu-

lated from large cytosolic calcium ion changes. Nature 367:745–750.Austoker J, Cox D, Mathias P. 1972. Fractionation of nuclei from brain by

zonal centrifugation and a study of ribonucleic acid polymerase activity inthe various classes of nuclei. Biochem J 129:1139–1155.

Bading H, Ginty DD, Greenberg ME. 1993. Regulation of gene expressionin hippocampal neurons by distinct calcium signaling pathways. Science260:181–186.

Bito H, Deisseroth K, Tsien RW. 1997. Ca2�-dependent regulation inneuronal gene expression. Curr Opin Neurobiol 7:419–429.

Chawla S, Bading H. 2001. CREB/CBP and SRE-interacting transcrip-tional regulators are fast on-off switches: duration of calcium transientsspecifies the magnitude of transcriptional responses. J Neurochem 79:849–858.

Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI. 1997. Differentialactivation of transcrition factors induced Ca2� response amplitude andduration. Nature 386:855–858.

Dolmetsch RE, Pajvani U, Fife K, Spotts JM, Greenberg ME. 2001.Signaling to the nucleus by an L-type calcium channel-calmodulin com-plex through the MAP kinase pathway. Science 294:333–339.

Fields RD, Esthete F, Stevens B, Itoh K. 1997. Action potential-dependentregulation of gene expression: temporal specificity in calcium cyclicAMP-responsice element binding proteins, and mitogen-activated pro-tein kinase signaling. J Neurosci 17:7252–7266.

Frandsen A, Schousboe A. 1991. Dantrolene prevents cytotoxicity andCa2� release from intracellular stores in cultured cerebral cortical neurons.J Neurchem 56:1075–1078.

Fritz KI, Groenendaal F, McGowan JE, Mishra OP, Delivoria-Papadopoulos M. 1996. Effects of 3-(2-carboxy-piperzine-4-yl)-propy-1-phosphonic acid (CPP) on NMDA receptor binding characteristics andbrain cell membrane function during cerebral hypoxia in newborn piglets.Brain Res 729:66–74.

Ghosh A, Greenberg ME. 1995. Calcium signaling in neurons: molecularmechanisms and cellular consequences. Science 268:239–247.

Giuffrida AM, Cox D, Mathias AP. 1975. RNA polymerase activity in-various classes of nuclei from different regions of rat brain during postnataldevelopment. J Neurochem 24:749–755.

Hardigham GE, Bading H. 1998. Nuclear calcium: a key regulator of geneexpression. Biometals 11:345–358.

Hardigham GE, Chawla S, Johnson CM, Bading H. 1997. Distinct func-tions of nuclear and cytoplasmic calcium in the control of gene expres-sion. Nature 385:260–265.

Hardigham GE, Chawla S, Cruzalegui FH, Bading H. 1999. Control ofrecruitment and transcription activating function of CBP determinedgene regulation by NMDA receptor and L-type calcium channels. Neu-ron 22:789–798.

Hoffman DJ, DiGiacomo JE, Marro PJ, Mishra OP, Delivoria-Papadopoulos M. 1994. Hypoxia-induced modification of the N-methyl-D-aspartate (NMDA) receptor in the brain of newborn piglets. NeurosciLett 167:156–160.

Huang Z, Huang PL, Panathian N, Dalkara T, Fishman MC, MoskowitzMA. 1994. Effects of cerebral ischemiain mice deficient in neuronal nitricoxide synthase. Science 265:1183–1885.

Humbert J-P, Matter N, Artault J-C, Koppler P, Malviya AN. 1996.Inositol 1,4,5-triphosphate receptor is located to the inner nuclear mem-brane vindicating regulation of nuclear calcium signaling by inositol1,4,5-triphosphate. J Biol Chem 271:478–485.

Lamprechet W, Stein P, Heinz F, Weissner H. 1974. Creatine phosphate.In: Bergmeyer HU, editor. Methods of enzymatic analysis, vol 4. NewYork: Academic Press. p 1777–1781.

Lerea LS, McNamara JO. 1993. Ionotropic glutamate receptor subtypesactivate c-fos transcription by distinct calcium requiring intracellularsignaling pathways. Neuron 10:31–41.

Lerea LS, Butler LS, McNamar JO. 1992. NMDA and non-NMDAreceptor-mediated increase of c-fos mRNA in dentate gyrus neuronsinvolved calcium influx via different routes. J Neurosci 12:2973–2981.

Lowry OH, Rosenbrough NJ, Farr AL, Randal RJ. 1951. Protein mea-surement with the Folin phenol reagent. J Biol Chem 193:265–275.

NO-Mediated Modification of IP3 Receptor 337

Maulik D, Qayyum I, Powell SR, Karanza M, Mishra OP, Delivoria-Papadopoulos M. 2001. Post hypoxic magnesium decreases nuclear oxi-dative damage in fetal guinea pig brain. Brain Res 890:130–136.

Maulik D, Ashraf QM, Mishra OP, Delivoria-Papadopoulos M. 2002.Effect of hypoxia on calcium influx and calcium/calmodulin-dependentkinase activity in neuronal nuclei of the guinea pig fetus during develop-ment. Am J Obstet Gynecol 188:658–662.

Mishra OP, Delivoria-Papadopoulos M. 1992. NMDA receptor modifica-tion of the fetal guina pig brain during hypoxia. Neurochem Res 17:1211–1216.

Mishra OP, Delivoria-Papadopoulos M. 1999. Cellular mechanisms ofhypoxic injury in the developing brain. Brain Res Bull 48:233–238.

Mishra OP, Delivoria-Papadopoulos M. 2002. Nitric oxide mediated Ca2�

influx in neuronal nuclei and cortical synaptosomes of normoxic andhypoxic newborn piglets. Neurosci Lett 318:93–97.

Mishra OP, Zanelli SA, Ohnishi ST, Delivoria-Papadopoulos M. 2000.Hypoxia-induced generation of nitric oxide free radicals in cerebral cortexof newborn guinea pigs. Neurochem Res 25:1559–1565.

Mishra OP, Ashraf QM, Delivoria-Papadopoulos M. 2002. Phosphoryla-tion of cAMP response element binding (CREB) protein during hypoxiain cerebral cortex of newborn piglets and the effect of nitric oxidesynthase inhibition. Neuroscience 115:985–991.

Numagami Y, Zubrow AB, Mishra OP, Delivoria-Papadopoulos M. 1997.Lipid free radical generation and brain cell membrane alteration followingnitric oxide synthase inhibition during cerebral hypoxia in the newbornpiglet. J Neurochem 69:1542–1547.

Yun H-Y, Dawson VL, Dawson TM. 1997. Nitric oxide in health anddiseases of the nervous system. Mol Psychiatr 2:300–310.

Zanelli SA, Numagami Y, McGowan JE, Mishra OP, Delivoria-Papadopoulos M. 1999. NMDA receptor mediated calcium influx incerebral cortical synaptosomes of the hypoxic guinea pig fetus. Neuro-chem Res 24:437–446.

Zanelli SA, Ashraf QM, Mishra OP. 2002. Nitration is a mechanism ofregulation of the NMDA receptor function during hypoxia. Neuro-science 112:869–877.

Zubrow AB, Delivoria-Papadopoulos M, Ashraf QM, Fritz KI, Mishra OP.2002a. Nitric oxide-mediated Ca2�/calmodulin-dependent protein ki-nase IV activity during hypoxia in neuronal nuclei from newborn piglets.Neurosci Lett 335:5–8.

Zubrow AB, Delivoria-Papadopoulos M, Ashraf QM, Ballesteros JR, FritzKI, Mishra OP. 2002b. Nitric oxide-mediated expression of Bax proteinand DNA fragmentation during hypoxia in neuronal nuclei from new-born piglets. Brain Res 954:60–67.

338 Mishra et al.