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Brain cell membrane Na+,K+-ATPase activity following severe hypoxic injury in the newborn piglet
BRAIN RESEARCH ELSEVIER
Brain Research 730 (1996) 52-57
Research report
Brain cell membrane Na+,K+-ATPase activity following severe hypoxic injury in the newborn piglet T e d S.
Rosenkrantz
a,b,* , Joanna Kubin b, O m P. Mishra b, Douglass Smith c Maria Delivoria-Papadopoulos ~
~ Unil,ersity of Connecticut School qf Medicine, Department o[' Pediatrics, Farmington. CT 06030, USA t~ Unil ersiO, Of Pennsyh'ania School of Medicine, Department of Physiology, Philadelphia, PA , USA c UniversiO' Of Pennulvania School c~/'Medicine, Department (~[Neuro.~ttr~et3". Philadelphia, PA , USA
Accepted 2 April 1996
Abstract This study tests the hypothesis that severe brain hypoxia causes decreased Na-,K --ATPase activity, resulting in permanent alterations in the neuronal cell membranes. Seventeen anesthetized piglets (normoxic control (NC), no recovery after hypoxia (Group 1), 6 h normoxic recovery (Group 2), and 48 h normoxic recovery (Group 3)) were studied. Hypoxia was induced by lowering the FiO 2 to maintain P C r / P i ratio at 25% of baseline for 1 h as monitored by 3~P-NMR spectroscopy. PCr/P i returned to 57% of baseline by 6 h and was normal by 48 h. At termination, cortical tissue Na+,K+-ATPase activity was determined. Na +,K+-ATPase activity was measured in cortical membrane preparations by determining the rate of ATP hydrolysis. NC membranes had Na+,K*-ATPasc activity of 58.3 +_ 1.3 IxM Pi/mg protein/h (mean _+ S.E.M.). Na+,K+-ATPase activity was reduced in Groups 1, 2, and 3 (45.8 + 1.3, 47.4 _+ 3.6. 48.7 _+ 2.9 i,tM Pi/mg protein/h) ( P < 0.05 compared to NC). There was no difference in enzyme activity among Groups 1, 2, or 3. The data show that in spite of recovery of neuronal oxidative phosphorylation (PCr/P i) by 48 h, there is a permanent decrease in Na+.K +-ATPase activity in cells that have undergone severe hypoxic injury. The persistent decrease in Na+,K+-ATPase activity indicates ongoing cell injury following severe cerebral hypoxia, and that recovery of oxidative phosphorylation as indicated by PCr/P~ values cannot be used as an index of recovery of cell function. Keywords: Newborn; Brain injury; Hypoxia; Na',K+-ATPase; Outcome; Energy metabolism
1. Introduction There are several mechanisms by which brain hypoxia m a y result in n e u r o n a l d y s f u n c t i o n and death [8,12,19,23,28]. These include adenosine 5'-triphosphate deficiency, increased release of presynaptic glutamate with resultant excessive stimulation of the post-synaptic N M D A receptor followed by opening of the associated ion channel and an increase in intracellular calcium, activation of the arachidonic acid production and the generation of oxygen free radicals. It is the generation of oxygen free radicals and resultant lipid peroxidation that are thought to be
* Corresponding author, at: University of Connecticut Health Center, Department of Pediatrics, MC 2203, Farmingtom CT 06030, USA. Fax: + I (860) 679-1403.
responsible lot modification of membrane-associated receptors and enzymes systems such as Na+,K+-ATPase [11,17,21]. Previous investigations in the newborn piglet model have demonstrated that brain hypoxia results in a 25% reduction in brain cell membrane Na+,K+-ATPase activity immediately after severe brain hypoxia [2]. Normal cellular function following brain hypoxia is dependent upon the normalization of various cellular conditions such cell membrane integrity, oxidative metabolism, and intercellular communications. The goal of the present study was to investigate if there is recovery of N a ~ , K - ATPase activity following severe brain hypoxia in newborn piglets. N a ~ , K + - A T P a s e activity was selected for study as it is an enzyme critical to norntal brain cell function as well as a marker of brain cell membrane integrity.
0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 4 3 0 - I
T,S, Rosenkrantz el a l . / B r a i n Research 730 (1c)96) 52 57
2. Materials and methods
2.1. Study dexi,,,n Three-day-old piglets were divided into four groups: normoxic control ( n = 3), hypoxia without recovery (Group 1) (n = 3), hypoxia followed by 6 h of normoxic recovery (Group 2) (tl = 6 ) , and hypoxia followed by 48 h of normoxic recovery (Group 3) ( , = 5). The control piglets were anesthetized with 4 ~ halothane, sacrificed with removal of the brain which was immediately immersed into liquid nitrogen and stored at - 8 0 ° C until analysis of Na+,K *-ATPase activity could be performed. The remaining three groups of animals underwent induction of anesthesia with 4% halothane and reduced to a dose of 0.4% halothane to maintain sedation. The anterior aspect of the neck was incised after infiltration with 1% lidocaine as a local anesthetic. Polyethylene catheters were placed in the left carotid artery and external ,jugular vein. These catheters were tunnelled under the skin and brought out on the lateral aspec! ol the neck. Following closure of the wound, the piglets were intubated with a 3.5 endotracheal tube (Portex, Kecne. NH), and allowed to breathe spontaneously during mmsport to the NMR facility. The animals were secured in a cradle, mechanically ventilated with oxygen, nitrogen and halothane (0.4-0.6%) and fitted with an NMR coil. Following normoxic baseline NMR measurements, brain hypoxia, defined as a reduction of the PCr/Pl ratio to 25% of normoxic baseline, was maintained for I h [ 1,2,20]. A reduction of the P C r / P i ratio to 25% of baseline for I h is associated with a reduction in the oxidative state as reflected by a quantitative decrease in ~TP and pH,. Previous studies from our laboratory have also shown that this degree of brain hypoxia is associated with brain cell membrane injury [2]. NMR spectra were collected every 5 - 1 0 rain during the period of brain hypoxia. The piglets without recovery (Group 1) were immediately sacrificed at the end of the hour. The brains were removed and preserved as described above. Following I h of hypoxia, the 6 h recovery group (Group 2) was Feoxygenated. provided with an infusion of dextrose (10c~) and water at an infusion rate of 6 - 8 m g / k g / m i n of glucose. Repeat measurements of NMR spectroscopy were made over the 6 h recovery period at 15, 30, 45, 60, 90. 20, 180, 240, 300 and 360 rain of reoxygenation. Animals were sacrificed at the end of the 6-h period. The 48 h recovery group was reoxygenated immediately after 1 h of hypoxia with elinfination of the halothane. They were extubated when respirations became regular and returned to the animal care facility. These animals were reanesthetized and intubated 48 h later and restudied with NMR spectroscopy, The piglets were then sacrificed with re.moval of the brains as with the other groups. All animals subjected to brain hypoxia had continuous monitoring of hearl rate, mean arterial blood pressure,
53
arterial blood gases and pH, and whole blood glucose and lactate. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. 2.2. +IP-NMR spe~'troscopy Phosphorus nuclear magnetic resonance spectroscopy was utilized to noninvasively measure the relative concentrations of adenosine triphosphate, phosphocreatine (PCr), inorganic phosphate (Pi). and mono- and diphosphoesters. The P C r / P i ratio is proportional to the cellular phosphate potential and was used to reflect neuronal oxidative phosphorylation. Utilizing classic Michaelis-Menten kinetics. intracellular pH (pHi) was calculated bv measuring the chemical shift between PCr and P+ ( p H - 6.77 + log[(8 3.29)/(5.68 - 8)]) [2]. The cerebral magnetic resonance spectroscopy was performed using a 1.9 Tesla magnet with a 12-inch horizontal bore (Otsuka Electronics, Havertown, PA). A single-turn, double tuned circular radiofrequency surface coil (~H frequency = 80.3 mHz, 3Jp frequency = 32.5 mHz) with a diameter of 3 cm wax placed over the vertex of the skulk positioned in the center of the homogeneous magnetic field. Seventy-five FIDS (free induction decays) were collected over 5 min fl)r each spectra. 2.3. Biochemical measuremellts Whole blood glucose and lactate were measured serially using a YSI 2300 dual analyzer (Yellow Springs Instruments Co., Yellow Springs, OH). Arterial blood gases and pH were measured using a Coming 178 Blood Gas Analyzer (Coming Diagnostics Corp., Medfield, MA). 2.4. Brain membrane preparation Brain cell membrane fraction were prepared according to Harik [9]. Frozen cerebral tissue was weighed and a 5% homogenate in 10 mM Tris-HC1 buffer, pH 7.4, containing 0.3 M sucrose and 0.5 mM EDTA wax prepared. The homogenate was centrifuged at 1000 × ~,, fi)r 10 rain, and this initial pellet was discarded while the supernatant was centrifuged at 48 000 × g for 60 min. The resulting pellet was rehomogenized in 10 mM Tris-HC1, containing 0.5 mM EDTA and centrifuged at 48 000 × g liar 60 rain. The final pellet, consisting of the P2 membrane fraction, was resuspended in the same buffet and diluted to a concentration of 1 mg protein/ml. 2.5. Na +, K +-A TPase actiz:itv The activity of Na+,K+-ATPase was determined as previously described [17]. The rate of ATP hydrolysis was
T.S. Rosenkrant: et al. / Brain Research 730 (1996) 52 57
54
eo
Table 2 Cellular energy metabolites and intracellular pH in hypoxic piglets during baseline, hypoxia and recovery periods
50
Group
Baseline
I
P C r / P i 2,0 ± 0 . 2 pH i 7,29_+ 0.07 P C r / P i 2,0 _+0.2 pH i 7.14_+0.05 P C r / P i 2.1 _+0.1 pill 7.25_+0.04
Q
2
~.a~
3 10
Hypoxia
6 h rec.
48 h rec.
0.6 ±0.1 * 6,72_+0.01 0.6 _+0.1 6.88_+0.04 * 0.5 ± 0 . 0 ' 6.90±0,04 "
1.01±0,1 6.89_+0.12 -
2.1 ±0.1 7.20 ± 0.02
Mean + S.E.M. ~ P < 0.001, Baseline vs. Hypoxia.
0 Control
1
2
3
(GROUTS) Fig. 1. ATPase activity for Control and the three groups subjected to brain hypoxia (mean ± S.E.M., * P < 0.05 compared to Control).
carried out in a 1.0 ml reaction mixture containing 100 mM NaC1, 20 mM KCI, 3 mM MgC12, 3 mM Tris-ATP, 50 mM Tris-HC1 buffer (pH 7.40) and 100 ixg membrane protein. The reaction was carried out at 37°C in the presence and absence of 1.0 mM ouabain for 5 rain, a period during which the rate of ATP hydrolysis was linear. The reaction was stopped by the addition of 0.5 ml 12,5% trichloroacetic acid. The samples were kept on ice, centrifuged at 2000 × g for 15 rain and the supernatant was analyzed for liberated inorganic phosphate. Na+,K ~ATPase activity is represented by the ouabain sensitive activity and expressed as IxM Pi/mg protein/h.
2.6. Data analysis Data are expressed as mean + S.E.M. NMR data obtained prior to and during hypoxia were analyzed using the Student's paired t-test. All other intergroup data were analyzed by factorial ANOVA. Data from the 6 h and 48 h recovery groups were analyzed with ANOVA for repeated measures. Na+,K+-ATPase activity [%r the four groups was compared utilizing factorial ANOVA.
in Tables I and 2. The three groups were similar /'or all measurements during the baseline and brain hypoxia periods. There were significant changes in p~O 2, pH, whole blood lactate, P C r / P i and pH i during brain hypoxia. Whole blood glucose was unchanged during the baseline, hypox±a, and 6 h recovery period. Lactate increased significantly during hypoxia and slowly returned to baseline levels after 90 rain of recovery. The mean arterial blood pressure was 60 ± 4 mmHg during the baseline period and unchanged during the period of hypoxia. Following a significant decrease in the P C r / P i ratio during hypoxia, the P C r / P i ratio increased to approximately 50% of the baseline value by 90 rain of reoxygenation and remained at that value for the remainder of the 6 h recovery period (Group 2). At 48 h of recovery the P C r / P i was similar to the pre-hypoxia values (Group 3). Na+,K+-ATPase activity was 58.3 ± 1.3 txM Pi/mg protein/h in normoxic control animals (Fig. 1). There was a significant decrease in Na+,K~-ATPase activity in all three groups who were exposed to brain hypoxia ( P < 0.05). There was no difference in Na-,K~-ATPase activity among the three groups: 45.8 +_ 1.3, 47.4 ± 3.6, and 48.7 _+ 2.9 IxM Pi/mg protein/h (Groups 1, 2, and 3, respectively).
4. Discussion 3. Results
N a ' , K * ATPase is a membrane bound enzyme and its activity depends on the lipid microenvironment of the enzyme. The enzyme is inhibited by hypoxia and lipid
Physiologic, biochemical and NMR data [or the three groups that were subjected to brain hypoxia are presented Table I Physiological and biochemical data during baseline and hypoxia conditions Group 1
p~,O2 p~CO: pH Hematocrit(%) Glucose ( m g / d l ) Lactate(mM/1)
Group 2
Group 3
Baseline
Hypoxia
Baseline
Hypoxia
Baseline
Hypoxia
206 36 7.67 22 85 3.0
33 39 7.13 30 38 15.4
213 38 7.57 31 99 3.1
24 40 7.34 32 63 10.3
202 40 7.58 25 106 2.9
38 43 7.13 31 75 13.5
+_20 ! 2 !0.06 ± 2 ± 27 _+ 0.4
_+ 8 * ± 3 ± 0.08 " ± 3 _+ 11 _+ 3.() ~
_+40 + 3 ± 0.06 +_ 2 _+ 10 ± 0.6
Mean _ S.E.M. * P < 0.002, Baseline vs. Hypoxia: ~ P < 0.001, Baseline vs. Hypoxia.
+ I ' i 2 + 0.05 • + 2 +20 ± 1.2 ~
±19 ± 4 ± 0.06 ± I ± 8 ± 0.8
2 7 ~ 2 ± 0.07 " _£ 2 ±30 ± 1.6
T.S. Rosenkrant: et al. / Brain Research 730 (199(H 52 57
peroxidation in vitro and in vivo [6,14,18,21]. The present study investigated the potential recovery of this enzyme following acute severe brain hypoxia. In our experiments the brain was subjected to severe hypoxia as indicated by a 75% reduction in the P C r / P i ratio as measured by NMR spectroscopy. Following I h of hypoxia, animals were allowed to recover for 0, 6 or 48 h with measurement of enzyme activity at these time points. The results show that the enzyme activity is reduced by hypoxia. As in previous studies from our laboratory, a 25% reduction from the control was observed following acute hypoxia [11,21]. The 25% reduction in enzyme activity remained unchanged up to 48 h of recovery despite of return of the P C r / P i ratio to pre-hypoxia values A decrease in enzyme activity reflects a cascade of events that culminate in lipid peroxidation of the cell membrane and cell death. Brain hypoxia results in degradation of high energy compounds such as ATP, ADP, and AMP. [22,28]. Adenosine is converted to xanthine via purine metabolism. The enzyme xanthine oxidase promotes the conversion of xanthine to uric acid and O 3 . The oxygen radical is available for peroxidation of the lipid rich neuronal membrane. A second mechanism for the generation of free radical and membrane peroxidation is due directly to hypoxia which causes depolarization of the neuronal membrane, leading to an increase in the intracellular sodium concentration. [3] The increased intracellular sodium concentration causes altered sodium and calcium exchange mechanisms, resulting in an increased calcium concentration in the cell. Cell membrane depolarization also induces release of excitatory amino acids such as glutamate which might further increase the inlracellular calcium concentration via the NMDA receptor ion channel [12]. The increased intracellular calcium activates phospholipase A, that results in the release of arachidonic acid and the subsequent generalion {71 oxygen free radicals through the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism !4,21,23]. In addition, NMDA receptor activation and increased intracellular calcium can activate nitric oxide synlhetase which leads to generation of nitric oxide and other flee radicals such as hydroxy radicals [12.20]. Lastly, as previously stated, conversion of xanthine, via the action of xanthine oxidasc, to uric acid plus an oxygen free radical ~s another source of free radicals available for lipid per~xidation [22,28]. The summation of these events leads to increased generation of free radicals which causes increased and ongoing membrane lipid peroxidation, and contributes to cell dysfunction and cell death following hrain hypoxia. Since the activity of the enzyme Na--,K+-ATPase is dependent on the lipid component of the cell membrane being intact, the decreased enzyme activity could be due either to the interaction of the oxygen tree radicals with the lipid component of the membrane, resulting in the alteration of the lipid microenvironment of the enzyme or
.~
the protein molecule itself [13,21J. In addition, the accumulation of lipid peroxidation products could directly interact with enzyme protein causing a decrease in activity. Several recent studies have help to clarify this area. Graham et al. have delineated the mechanism by which the brain cell membrane Na+,K LATPase activity is modified by hypoxia [5]. Hypoxia results in decreased affinity for N a and K + and increased affinity for ATP. The net affect on the enzyme system is decreased dephosphorylation and energy production for ion transmembrane transport. The mediator of injury to brain cell membrane Na~,K+-ATPase activity subjected to hypoxia appears to be the generation of free oxygen radicals and lipid peroxidation. Marro el al. demonstrated that pretreatment of piglets with allopurinol who were then subjected to hypoxia reduced or prevented the hypoxia-induced decrease in brain cell membrane Na~.K+-ATPase activity and decreased concentrations of conjugated dienes and fluorescent compounds compared to non-treated hypoxic piglets [13]. In another study, pretreatment with (,-tocopherol preserved brain cell membrane N a . K - - A T P a s e activity. [24]. These studies demonstrate that the mechanism of hypoxic injury to the brain cell membrane is the result of oxidative damage. We have also demonstrated that brain tissue hypoxia modifies the NMDA receptor in both fetal guinea pigs and newborn piglets [11,15,16]. We have also demonstrated that the blockage of the NMDA receptor prior to brain hypoxia by MgSO 4, prevents the hypoxia-induced alteration in enzyme activity, indicating an impo,'rant role of the NMDA receptor ion channel in hypoxic brain injury [I1]. While direct relationships have not been found between all of these events and the loss of brain cell membrane N a ' , K ~-ATPase activity, it is clear that the lipid peroxidation responsible for the loss of enzyme activity is also responsible for injury to other cell components. Goel et al. have shown changes in the NMDA receptor binding characteristics following lipid peroxidation without changes in receptor number [5]. This would suggest that lipid peroxidation in association with hypoxia will alter the normoxic and as well as hypoxic response of this receptor. The consequences of the receptor modification on hypoxia-associated calcium influx have not be studied. However, Hoffman et al. have shown that pretreatment with MgSO 4 protects the NMDA receptor and also preserves brain cell membrane Na+,K~-ATPase activity [10]. The proposed mechanism is that by blocking the NMDA receptor channel and calcium influx, there is a decrease in oxygen radical production secondary to decreased arachidonic acid metabolism by the lipooxygenase and cyclooxygenase reactions. It is known that the greater the degree of unsaturation in fatty acids, the higher the rate of lipid peroxidation. Therefore the neuronal membrane, which is rich in unsaturated fatty acids is highly susceptible lo lipid peroxidative damage [27]. Our previous studies have shown that the neuronal membrane is much more susceptiMe to hypoxia
56
T.S. Rosenkrantz et al. / Brain Research 730 (1996) 52 57
induced lipid peroxidation and decreased enzyme activity as compared to the glial membrane [21]. Based on these data, one would expect greater injury to neuronal cells compared to glial cells. Indeed, this is consistent with the histopathology in the current study that shows that almost all of the cellular injury is confined to the neurons, which are either dead or abnormal as opposed to the glial cells which are intact. There are three o~ isoforms of Na+,K+ATPase in the neuronal membrane, c~, old, and c~3 [25]. The classification is based on the affinity of the enzyme for its specific inhibitor, ouabain. Isoforms o~ and c~+ are primarily found in the glial membrane, whereas u 3 is predominantly found in the neuronal membrane [26]. Studies from our laboratory have shown that the isoform with the highest affinity, eL3, is highly susceptible to hypoxic injury compared to o~ and c~+ [7]. These enzyme data suggest that most of the decrease in Na+,K+-ATPase activity found in the current study is due to a diminishment in Na+,K ~ATPase derived from the neurons as opposed to the glia. In addition there is a marked increase in the number of high affinity, ouabain-sensitive, c~ isoform sites during the last portion of gestation and that the Bm~~ or number of these sites is significantly decreased by hypoxia. The high concentration of the c~ isoforms on the neurons by the time of birth may help explain the susceptibility of the newborn brain to hypoxia-induced injury and the decrease in enzyme activity found after hypoxia. In summary, the decrease in enzyme activity which was observed immediately alter hypoxia does not recover following reoxygenation. This indicates that the enzyme activity, an index of cell membrane function, is not repaired or restored following recovery from brain hypoxia even though the oxidative phosphorylation, as measured by NMR spectroscopy, is normal by 48 h. The lack of recovery would appear to be due to severe lipid peroxidation of the neuronal membrane and not a failure of oxidative phosphorylation. The data from this study strongly suggest that the measurement of brain membrane function, such as activity of Na+,K + ATPase, appears to be a good predictor of permanent structural neuronal cell injury and death.
Acknowledgements This work was supported by NIH Grant HD-20337. It was presented at the 1994 Annual Meeting of the Society for Pediatric Research in Seattle, WA, USA.
References [1] Delivoria-Papadopoulos, M. and Chance, B.. In E.M. Scarpelli (Ed.), Pulmonary Physiology: Fetus, Newborn. Child and Adolescent, Lea and Berger, Philadelphia, 1989, pp. 365-370. [2] DiGiacomo+ J.E., Pane, C.R., Gwiazdowski, S., Mishra, O.P. and
Delivoria-Papadopoulos, M., Effect of graded hypoxia on brain cell membrane injury in newborn piglets, Biol. Neonate+ 61 (1992) 25-32. [3] Erecinska, M. and Silver, I.A., ATP and brain function, ./. Cereh. Blood Flow Metab.. 9 (1989) 2-19. [4] Espinoza, M.I. and Parer, J.T., Mechanism of asphyxial brain datnage and possible pharmacological interventions in the ti:tus, Am. ,I. Obstet. Gylwcol., 164 (1991) 1582-1591. [5] Goel, R.. Mishra, O.P., Razdan, B. and Delivoria-Papadopoulos, M., Modification of the NMDA receptor by in vitro lipid peroxidation in fetal guinea pig brain, Nemwsei. Lett.. 151 (1993) 219-223. [6] Graham, E.. Mishra, O.P. and Delivoria-Papadopoulos, M., Brain cell membrane Na+,K+-ATPase modification following hypoxia m the guinea pig t~tus, Neurosci. Lett.. 153 (1993) 93--97. [7] Graham+ E., Mishra, O.P. and Delivoria-Papadopoulos, M., Effect ot in utero hypoxia on the ouabainstrphanthidin binding site of the fetal guinea pig brain cell membrane Na*+K+-ATPase, Neuro~ei. Letl., 185 (1995) 159-162. [8] Haddad, 0.(3. and Jiang, C., O e deprivation in the central nervous system: On mechanisms of neuronal response, differential sensitivity and injury. Prof. Neurobiol., 40 (1993) 277--318. [9] Harik, S.l., Doul, G.H. and Dick, A.P.K.. Specific ouabain binding to brain rnicrovessels and choroid plexus. ,l. Cereh. Blood Flow Metab.. 5 (1985) 156-160. [10] Hoffman, D,J., Marro, P.J., McGowan, J.E.. Mishra, O.P. and Delivoria-Papadopoulos, M., Protective effect of MgSO 4 infusion on NMDA receptor binding characteristics during cerebral cortical hypoxia in the newborn piglet, Brain Res., 644 (1994) 144 149. [11] Hoffman, D.J., McGowan, J.E., Marro. P.J., Mishra. O.P. and Delivoria-Papadopoulos. M., Hypoxia-induced modification of the N-methyl-l)-aspartatc receptor in the brain of the newborn piglet. Neurosci. l+ett.. 167 (1994) 156-160. [12] Lipton, S.A. and Rosenberg+ P.A., Excitatou anfino acids as a final common pathwa5 for neurologic disorders, N. Engl. ,I. Med., 330 (1994) 613 622. [13] Marro, P.J., McGowan. J.E., Razdan, B., Mishra, O.P. and Deliw+ ria-Papadopoulos. M., Effect of allopurinol on uric acid levels and brain cell membrane Na+.K~-ATPase activity during hyoxia in newborn piglets, Brain Re,s'.. 650 (1994) 9 15, [14] Mishra, O.P, and Deliw~ria-Papadopoulos, M.. Lipid pcroxidation in developing lietal guinea pig brain during normoxia and hypoxia, Det,. Brain Res.. 45 (1989) 129-135. [15] Mishra, O.P. and Delivoria-Papadopoulos, M., NMDA receptor modification of the fetal guinea pig brain during hypoxia, Neuroehem, Res., 17 (1992) 1211 - 1216. [16] Mishra, O.P. and Delivoria-Papadopoulos, M.. Modification of modulatory sites of NMDA receptor in the fetal guinea pig brain during development, Neuroehem, Res.. 17 (1992) 1223-1228. [17] Mishra. O.P., Delivoria-Papadopoulos+ M., Cahillane. G+ and Wagerle, L.C., Lipid peroxidation as the mechanism of modification of the affinity of the Na +,K ~-ATPase active sites for ATP+ K + , Na + and strophanthidin in vitro, Neurochem. Re,s, 14(1989) 845 851. [18] Mishra, O.P. and Deliw)ria-Papadopoulos, M., Na+,K + ATPase in developing fetal guinea pig brain and the effect of maternal hypoxia, Neurochem. Res., 13 (1988) 765-770. [19] Monaghan, D.T,, Bridges. R.J. and Cotman. C.W., The excitator~ amino acid receptors: Their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu+ Rel. Pharm~u'of 7)~.vieol., 29 (1989)365-402. [20] Montague, PR., Gancayco, C.D., Winn, M.J., Marchasc, R.B. and Friedlander, M.J., Role of NO production in NMDA receptor-mediated neurotransmitter release in cerebral cortex, Seiem'e. 263 (1994) 973-977. [21] Razdan, B., Marro, P.J., Tammela, O.+ Goel. R., Mishra. O.P. and Delivoria-Papadopoulos, M.+ Selective sensitivity of syaptosomal membrane function to cerebral cortical hypoxia in nexvborn piglets. Brain Res.. 600(1993) 308 314.
T.S. Rosenkrant= et al. / Brain Research Z~O (1996! 52 57
[22] Roy, R.S. and McCord, J.M., Superoxide and ischemia: conversion of xanthine dehydrogenase to xanthine oxidase. In R. Greenald and G. Ccohen (Eds.), O D'-radicals and their Scavenger ~vstems. Vof 2. Celhdar a , d MolecMar A.slWCts. Elsevier Science, New York, 1983, pp. 145 153. [23] Sanleliu, C.. Hunl, A. and Patel, A., Exposure to N-methyl-Daspartate increases release of arachidonic acid in primary cultures of rat hippocampal neurons and nol in astrocytes. Brain Re.~., 526 (1990) 241 248. [24] Shin, S.M.. Razdan, B.. Mi>hra, O.P.. Johnson, L. and DelivoriaPapadopoulos. M., Prnlective effect of a-tocopherol on brain cell nlenlbranc funclion during cerebral cortical hypoxia in newborn piglets. Blain Rev.. 553 (1994) 45-50.
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[25] Sweadner. K.J.. lsozymes of the N a ' . K - A'PPase. Biochim. Biophy.s. ,4eta. 988 (1989) 185-220. [26] Sweadner, K.J. and Gilkeson, R., Enzymatic properties of separated ioszymes of the Na +.K ~-ATPase: substrate affinities, kinetic coperativity, and ion transport stoichiometrv..L Biol. Chem.. 260 (1985) 9016-9022, [27] Sun, G.Y. and Sun, A.Y., Synaptosomal plasma membranes: acyl group composition of phosphoglycerides and N a , K ' ATPasc aciiv iD during fatty acid deficiency, J. Neuroctu'm., 22 (1~,~74) 15--IK [28] Vannucci. R.C., Experimental biology of cerebral hypoxia-ischemia: Relation to perinatal brain damagc, Pediarr Rc.~., 27 (1990)1117 326.
Brain Research 730 (1996) 52-57
Research report
Brain cell membrane Na+,K+-ATPase activity following severe hypoxic injury in the newborn piglet T e d S.
Rosenkrantz
a,b,* , Joanna Kubin b, O m P. Mishra b, Douglass Smith c Maria Delivoria-Papadopoulos ~
~ Unil,ersity of Connecticut School qf Medicine, Department o[' Pediatrics, Farmington. CT 06030, USA t~ Unil ersiO, Of Pennsyh'ania School of Medicine, Department of Physiology, Philadelphia, PA , USA c UniversiO' Of Pennulvania School c~/'Medicine, Department (~[Neuro.~ttr~et3". Philadelphia, PA , USA
Accepted 2 April 1996
Abstract This study tests the hypothesis that severe brain hypoxia causes decreased Na-,K --ATPase activity, resulting in permanent alterations in the neuronal cell membranes. Seventeen anesthetized piglets (normoxic control (NC), no recovery after hypoxia (Group 1), 6 h normoxic recovery (Group 2), and 48 h normoxic recovery (Group 3)) were studied. Hypoxia was induced by lowering the FiO 2 to maintain P C r / P i ratio at 25% of baseline for 1 h as monitored by 3~P-NMR spectroscopy. PCr/P i returned to 57% of baseline by 6 h and was normal by 48 h. At termination, cortical tissue Na+,K+-ATPase activity was determined. Na +,K+-ATPase activity was measured in cortical membrane preparations by determining the rate of ATP hydrolysis. NC membranes had Na+,K*-ATPasc activity of 58.3 +_ 1.3 IxM Pi/mg protein/h (mean _+ S.E.M.). Na+,K+-ATPase activity was reduced in Groups 1, 2, and 3 (45.8 + 1.3, 47.4 _+ 3.6. 48.7 _+ 2.9 i,tM Pi/mg protein/h) ( P < 0.05 compared to NC). There was no difference in enzyme activity among Groups 1, 2, or 3. The data show that in spite of recovery of neuronal oxidative phosphorylation (PCr/P i) by 48 h, there is a permanent decrease in Na+.K +-ATPase activity in cells that have undergone severe hypoxic injury. The persistent decrease in Na+,K+-ATPase activity indicates ongoing cell injury following severe cerebral hypoxia, and that recovery of oxidative phosphorylation as indicated by PCr/P~ values cannot be used as an index of recovery of cell function. Keywords: Newborn; Brain injury; Hypoxia; Na',K+-ATPase; Outcome; Energy metabolism
1. Introduction There are several mechanisms by which brain hypoxia m a y result in n e u r o n a l d y s f u n c t i o n and death [8,12,19,23,28]. These include adenosine 5'-triphosphate deficiency, increased release of presynaptic glutamate with resultant excessive stimulation of the post-synaptic N M D A receptor followed by opening of the associated ion channel and an increase in intracellular calcium, activation of the arachidonic acid production and the generation of oxygen free radicals. It is the generation of oxygen free radicals and resultant lipid peroxidation that are thought to be
* Corresponding author, at: University of Connecticut Health Center, Department of Pediatrics, MC 2203, Farmingtom CT 06030, USA. Fax: + I (860) 679-1403.
responsible lot modification of membrane-associated receptors and enzymes systems such as Na+,K+-ATPase [11,17,21]. Previous investigations in the newborn piglet model have demonstrated that brain hypoxia results in a 25% reduction in brain cell membrane Na+,K+-ATPase activity immediately after severe brain hypoxia [2]. Normal cellular function following brain hypoxia is dependent upon the normalization of various cellular conditions such cell membrane integrity, oxidative metabolism, and intercellular communications. The goal of the present study was to investigate if there is recovery of N a ~ , K - ATPase activity following severe brain hypoxia in newborn piglets. N a ~ , K + - A T P a s e activity was selected for study as it is an enzyme critical to norntal brain cell function as well as a marker of brain cell membrane integrity.
0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 4 3 0 - I
T,S, Rosenkrantz el a l . / B r a i n Research 730 (1c)96) 52 57
2. Materials and methods
2.1. Study dexi,,,n Three-day-old piglets were divided into four groups: normoxic control ( n = 3), hypoxia without recovery (Group 1) (n = 3), hypoxia followed by 6 h of normoxic recovery (Group 2) (tl = 6 ) , and hypoxia followed by 48 h of normoxic recovery (Group 3) ( , = 5). The control piglets were anesthetized with 4 ~ halothane, sacrificed with removal of the brain which was immediately immersed into liquid nitrogen and stored at - 8 0 ° C until analysis of Na+,K *-ATPase activity could be performed. The remaining three groups of animals underwent induction of anesthesia with 4% halothane and reduced to a dose of 0.4% halothane to maintain sedation. The anterior aspect of the neck was incised after infiltration with 1% lidocaine as a local anesthetic. Polyethylene catheters were placed in the left carotid artery and external ,jugular vein. These catheters were tunnelled under the skin and brought out on the lateral aspec! ol the neck. Following closure of the wound, the piglets were intubated with a 3.5 endotracheal tube (Portex, Kecne. NH), and allowed to breathe spontaneously during mmsport to the NMR facility. The animals were secured in a cradle, mechanically ventilated with oxygen, nitrogen and halothane (0.4-0.6%) and fitted with an NMR coil. Following normoxic baseline NMR measurements, brain hypoxia, defined as a reduction of the PCr/Pl ratio to 25% of normoxic baseline, was maintained for I h [ 1,2,20]. A reduction of the P C r / P i ratio to 25% of baseline for I h is associated with a reduction in the oxidative state as reflected by a quantitative decrease in ~TP and pH,. Previous studies from our laboratory have also shown that this degree of brain hypoxia is associated with brain cell membrane injury [2]. NMR spectra were collected every 5 - 1 0 rain during the period of brain hypoxia. The piglets without recovery (Group 1) were immediately sacrificed at the end of the hour. The brains were removed and preserved as described above. Following I h of hypoxia, the 6 h recovery group (Group 2) was Feoxygenated. provided with an infusion of dextrose (10c~) and water at an infusion rate of 6 - 8 m g / k g / m i n of glucose. Repeat measurements of NMR spectroscopy were made over the 6 h recovery period at 15, 30, 45, 60, 90. 20, 180, 240, 300 and 360 rain of reoxygenation. Animals were sacrificed at the end of the 6-h period. The 48 h recovery group was reoxygenated immediately after 1 h of hypoxia with elinfination of the halothane. They were extubated when respirations became regular and returned to the animal care facility. These animals were reanesthetized and intubated 48 h later and restudied with NMR spectroscopy, The piglets were then sacrificed with re.moval of the brains as with the other groups. All animals subjected to brain hypoxia had continuous monitoring of hearl rate, mean arterial blood pressure,
53
arterial blood gases and pH, and whole blood glucose and lactate. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. 2.2. +IP-NMR spe~'troscopy Phosphorus nuclear magnetic resonance spectroscopy was utilized to noninvasively measure the relative concentrations of adenosine triphosphate, phosphocreatine (PCr), inorganic phosphate (Pi). and mono- and diphosphoesters. The P C r / P i ratio is proportional to the cellular phosphate potential and was used to reflect neuronal oxidative phosphorylation. Utilizing classic Michaelis-Menten kinetics. intracellular pH (pHi) was calculated bv measuring the chemical shift between PCr and P+ ( p H - 6.77 + log[(8 3.29)/(5.68 - 8)]) [2]. The cerebral magnetic resonance spectroscopy was performed using a 1.9 Tesla magnet with a 12-inch horizontal bore (Otsuka Electronics, Havertown, PA). A single-turn, double tuned circular radiofrequency surface coil (~H frequency = 80.3 mHz, 3Jp frequency = 32.5 mHz) with a diameter of 3 cm wax placed over the vertex of the skulk positioned in the center of the homogeneous magnetic field. Seventy-five FIDS (free induction decays) were collected over 5 min fl)r each spectra. 2.3. Biochemical measuremellts Whole blood glucose and lactate were measured serially using a YSI 2300 dual analyzer (Yellow Springs Instruments Co., Yellow Springs, OH). Arterial blood gases and pH were measured using a Coming 178 Blood Gas Analyzer (Coming Diagnostics Corp., Medfield, MA). 2.4. Brain membrane preparation Brain cell membrane fraction were prepared according to Harik [9]. Frozen cerebral tissue was weighed and a 5% homogenate in 10 mM Tris-HC1 buffer, pH 7.4, containing 0.3 M sucrose and 0.5 mM EDTA wax prepared. The homogenate was centrifuged at 1000 × ~,, fi)r 10 rain, and this initial pellet was discarded while the supernatant was centrifuged at 48 000 × g for 60 min. The resulting pellet was rehomogenized in 10 mM Tris-HC1, containing 0.5 mM EDTA and centrifuged at 48 000 × g liar 60 rain. The final pellet, consisting of the P2 membrane fraction, was resuspended in the same buffet and diluted to a concentration of 1 mg protein/ml. 2.5. Na +, K +-A TPase actiz:itv The activity of Na+,K+-ATPase was determined as previously described [17]. The rate of ATP hydrolysis was
T.S. Rosenkrant: et al. / Brain Research 730 (1996) 52 57
54
eo
Table 2 Cellular energy metabolites and intracellular pH in hypoxic piglets during baseline, hypoxia and recovery periods
50
Group
Baseline
I
P C r / P i 2,0 ± 0 . 2 pH i 7,29_+ 0.07 P C r / P i 2,0 _+0.2 pH i 7.14_+0.05 P C r / P i 2.1 _+0.1 pill 7.25_+0.04
Q
2
~.a~
3 10
Hypoxia
6 h rec.
48 h rec.
0.6 ±0.1 * 6,72_+0.01 0.6 _+0.1 6.88_+0.04 * 0.5 ± 0 . 0 ' 6.90±0,04 "
1.01±0,1 6.89_+0.12 -
2.1 ±0.1 7.20 ± 0.02
Mean + S.E.M. ~ P < 0.001, Baseline vs. Hypoxia.
0 Control
1
2
3
(GROUTS) Fig. 1. ATPase activity for Control and the three groups subjected to brain hypoxia (mean ± S.E.M., * P < 0.05 compared to Control).
carried out in a 1.0 ml reaction mixture containing 100 mM NaC1, 20 mM KCI, 3 mM MgC12, 3 mM Tris-ATP, 50 mM Tris-HC1 buffer (pH 7.40) and 100 ixg membrane protein. The reaction was carried out at 37°C in the presence and absence of 1.0 mM ouabain for 5 rain, a period during which the rate of ATP hydrolysis was linear. The reaction was stopped by the addition of 0.5 ml 12,5% trichloroacetic acid. The samples were kept on ice, centrifuged at 2000 × g for 15 rain and the supernatant was analyzed for liberated inorganic phosphate. Na+,K ~ATPase activity is represented by the ouabain sensitive activity and expressed as IxM Pi/mg protein/h.
2.6. Data analysis Data are expressed as mean + S.E.M. NMR data obtained prior to and during hypoxia were analyzed using the Student's paired t-test. All other intergroup data were analyzed by factorial ANOVA. Data from the 6 h and 48 h recovery groups were analyzed with ANOVA for repeated measures. Na+,K+-ATPase activity [%r the four groups was compared utilizing factorial ANOVA.
in Tables I and 2. The three groups were similar /'or all measurements during the baseline and brain hypoxia periods. There were significant changes in p~O 2, pH, whole blood lactate, P C r / P i and pH i during brain hypoxia. Whole blood glucose was unchanged during the baseline, hypox±a, and 6 h recovery period. Lactate increased significantly during hypoxia and slowly returned to baseline levels after 90 rain of recovery. The mean arterial blood pressure was 60 ± 4 mmHg during the baseline period and unchanged during the period of hypoxia. Following a significant decrease in the P C r / P i ratio during hypoxia, the P C r / P i ratio increased to approximately 50% of the baseline value by 90 rain of reoxygenation and remained at that value for the remainder of the 6 h recovery period (Group 2). At 48 h of recovery the P C r / P i was similar to the pre-hypoxia values (Group 3). Na+,K+-ATPase activity was 58.3 ± 1.3 txM Pi/mg protein/h in normoxic control animals (Fig. 1). There was a significant decrease in Na+,K~-ATPase activity in all three groups who were exposed to brain hypoxia ( P < 0.05). There was no difference in Na-,K~-ATPase activity among the three groups: 45.8 +_ 1.3, 47.4 ± 3.6, and 48.7 _+ 2.9 IxM Pi/mg protein/h (Groups 1, 2, and 3, respectively).
4. Discussion 3. Results
N a ' , K * ATPase is a membrane bound enzyme and its activity depends on the lipid microenvironment of the enzyme. The enzyme is inhibited by hypoxia and lipid
Physiologic, biochemical and NMR data [or the three groups that were subjected to brain hypoxia are presented Table I Physiological and biochemical data during baseline and hypoxia conditions Group 1
p~,O2 p~CO: pH Hematocrit(%) Glucose ( m g / d l ) Lactate(mM/1)
Group 2
Group 3
Baseline
Hypoxia
Baseline
Hypoxia
Baseline
Hypoxia
206 36 7.67 22 85 3.0
33 39 7.13 30 38 15.4
213 38 7.57 31 99 3.1
24 40 7.34 32 63 10.3
202 40 7.58 25 106 2.9
38 43 7.13 31 75 13.5
+_20 ! 2 !0.06 ± 2 ± 27 _+ 0.4
_+ 8 * ± 3 ± 0.08 " ± 3 _+ 11 _+ 3.() ~
_+40 + 3 ± 0.06 +_ 2 _+ 10 ± 0.6
Mean _ S.E.M. * P < 0.002, Baseline vs. Hypoxia: ~ P < 0.001, Baseline vs. Hypoxia.
+ I ' i 2 + 0.05 • + 2 +20 ± 1.2 ~
±19 ± 4 ± 0.06 ± I ± 8 ± 0.8
2 7 ~ 2 ± 0.07 " _£ 2 ±30 ± 1.6
T.S. Rosenkrant: et al. / Brain Research 730 (199(H 52 57
peroxidation in vitro and in vivo [6,14,18,21]. The present study investigated the potential recovery of this enzyme following acute severe brain hypoxia. In our experiments the brain was subjected to severe hypoxia as indicated by a 75% reduction in the P C r / P i ratio as measured by NMR spectroscopy. Following I h of hypoxia, animals were allowed to recover for 0, 6 or 48 h with measurement of enzyme activity at these time points. The results show that the enzyme activity is reduced by hypoxia. As in previous studies from our laboratory, a 25% reduction from the control was observed following acute hypoxia [11,21]. The 25% reduction in enzyme activity remained unchanged up to 48 h of recovery despite of return of the P C r / P i ratio to pre-hypoxia values A decrease in enzyme activity reflects a cascade of events that culminate in lipid peroxidation of the cell membrane and cell death. Brain hypoxia results in degradation of high energy compounds such as ATP, ADP, and AMP. [22,28]. Adenosine is converted to xanthine via purine metabolism. The enzyme xanthine oxidase promotes the conversion of xanthine to uric acid and O 3 . The oxygen radical is available for peroxidation of the lipid rich neuronal membrane. A second mechanism for the generation of free radical and membrane peroxidation is due directly to hypoxia which causes depolarization of the neuronal membrane, leading to an increase in the intracellular sodium concentration. [3] The increased intracellular sodium concentration causes altered sodium and calcium exchange mechanisms, resulting in an increased calcium concentration in the cell. Cell membrane depolarization also induces release of excitatory amino acids such as glutamate which might further increase the inlracellular calcium concentration via the NMDA receptor ion channel [12]. The increased intracellular calcium activates phospholipase A, that results in the release of arachidonic acid and the subsequent generalion {71 oxygen free radicals through the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism !4,21,23]. In addition, NMDA receptor activation and increased intracellular calcium can activate nitric oxide synlhetase which leads to generation of nitric oxide and other flee radicals such as hydroxy radicals [12.20]. Lastly, as previously stated, conversion of xanthine, via the action of xanthine oxidasc, to uric acid plus an oxygen free radical ~s another source of free radicals available for lipid per~xidation [22,28]. The summation of these events leads to increased generation of free radicals which causes increased and ongoing membrane lipid peroxidation, and contributes to cell dysfunction and cell death following hrain hypoxia. Since the activity of the enzyme Na--,K+-ATPase is dependent on the lipid component of the cell membrane being intact, the decreased enzyme activity could be due either to the interaction of the oxygen tree radicals with the lipid component of the membrane, resulting in the alteration of the lipid microenvironment of the enzyme or
.~
the protein molecule itself [13,21J. In addition, the accumulation of lipid peroxidation products could directly interact with enzyme protein causing a decrease in activity. Several recent studies have help to clarify this area. Graham et al. have delineated the mechanism by which the brain cell membrane Na+,K LATPase activity is modified by hypoxia [5]. Hypoxia results in decreased affinity for N a and K + and increased affinity for ATP. The net affect on the enzyme system is decreased dephosphorylation and energy production for ion transmembrane transport. The mediator of injury to brain cell membrane Na~,K+-ATPase activity subjected to hypoxia appears to be the generation of free oxygen radicals and lipid peroxidation. Marro el al. demonstrated that pretreatment of piglets with allopurinol who were then subjected to hypoxia reduced or prevented the hypoxia-induced decrease in brain cell membrane Na~.K+-ATPase activity and decreased concentrations of conjugated dienes and fluorescent compounds compared to non-treated hypoxic piglets [13]. In another study, pretreatment with (,-tocopherol preserved brain cell membrane N a . K - - A T P a s e activity. [24]. These studies demonstrate that the mechanism of hypoxic injury to the brain cell membrane is the result of oxidative damage. We have also demonstrated that brain tissue hypoxia modifies the NMDA receptor in both fetal guinea pigs and newborn piglets [11,15,16]. We have also demonstrated that the blockage of the NMDA receptor prior to brain hypoxia by MgSO 4, prevents the hypoxia-induced alteration in enzyme activity, indicating an impo,'rant role of the NMDA receptor ion channel in hypoxic brain injury [I1]. While direct relationships have not been found between all of these events and the loss of brain cell membrane N a ' , K ~-ATPase activity, it is clear that the lipid peroxidation responsible for the loss of enzyme activity is also responsible for injury to other cell components. Goel et al. have shown changes in the NMDA receptor binding characteristics following lipid peroxidation without changes in receptor number [5]. This would suggest that lipid peroxidation in association with hypoxia will alter the normoxic and as well as hypoxic response of this receptor. The consequences of the receptor modification on hypoxia-associated calcium influx have not be studied. However, Hoffman et al. have shown that pretreatment with MgSO 4 protects the NMDA receptor and also preserves brain cell membrane Na+,K~-ATPase activity [10]. The proposed mechanism is that by blocking the NMDA receptor channel and calcium influx, there is a decrease in oxygen radical production secondary to decreased arachidonic acid metabolism by the lipooxygenase and cyclooxygenase reactions. It is known that the greater the degree of unsaturation in fatty acids, the higher the rate of lipid peroxidation. Therefore the neuronal membrane, which is rich in unsaturated fatty acids is highly susceptible lo lipid peroxidative damage [27]. Our previous studies have shown that the neuronal membrane is much more susceptiMe to hypoxia
56
T.S. Rosenkrantz et al. / Brain Research 730 (1996) 52 57
induced lipid peroxidation and decreased enzyme activity as compared to the glial membrane [21]. Based on these data, one would expect greater injury to neuronal cells compared to glial cells. Indeed, this is consistent with the histopathology in the current study that shows that almost all of the cellular injury is confined to the neurons, which are either dead or abnormal as opposed to the glial cells which are intact. There are three o~ isoforms of Na+,K+ATPase in the neuronal membrane, c~, old, and c~3 [25]. The classification is based on the affinity of the enzyme for its specific inhibitor, ouabain. Isoforms o~ and c~+ are primarily found in the glial membrane, whereas u 3 is predominantly found in the neuronal membrane [26]. Studies from our laboratory have shown that the isoform with the highest affinity, eL3, is highly susceptible to hypoxic injury compared to o~ and c~+ [7]. These enzyme data suggest that most of the decrease in Na+,K+-ATPase activity found in the current study is due to a diminishment in Na+,K ~ATPase derived from the neurons as opposed to the glia. In addition there is a marked increase in the number of high affinity, ouabain-sensitive, c~ isoform sites during the last portion of gestation and that the Bm~~ or number of these sites is significantly decreased by hypoxia. The high concentration of the c~ isoforms on the neurons by the time of birth may help explain the susceptibility of the newborn brain to hypoxia-induced injury and the decrease in enzyme activity found after hypoxia. In summary, the decrease in enzyme activity which was observed immediately alter hypoxia does not recover following reoxygenation. This indicates that the enzyme activity, an index of cell membrane function, is not repaired or restored following recovery from brain hypoxia even though the oxidative phosphorylation, as measured by NMR spectroscopy, is normal by 48 h. The lack of recovery would appear to be due to severe lipid peroxidation of the neuronal membrane and not a failure of oxidative phosphorylation. The data from this study strongly suggest that the measurement of brain membrane function, such as activity of Na+,K + ATPase, appears to be a good predictor of permanent structural neuronal cell injury and death.
Acknowledgements This work was supported by NIH Grant HD-20337. It was presented at the 1994 Annual Meeting of the Society for Pediatric Research in Seattle, WA, USA.
References [1] Delivoria-Papadopoulos, M. and Chance, B.. In E.M. Scarpelli (Ed.), Pulmonary Physiology: Fetus, Newborn. Child and Adolescent, Lea and Berger, Philadelphia, 1989, pp. 365-370. [2] DiGiacomo+ J.E., Pane, C.R., Gwiazdowski, S., Mishra, O.P. and
Delivoria-Papadopoulos, M., Effect of graded hypoxia on brain cell membrane injury in newborn piglets, Biol. Neonate+ 61 (1992) 25-32. [3] Erecinska, M. and Silver, I.A., ATP and brain function, ./. Cereh. Blood Flow Metab.. 9 (1989) 2-19. [4] Espinoza, M.I. and Parer, J.T., Mechanism of asphyxial brain datnage and possible pharmacological interventions in the ti:tus, Am. ,I. Obstet. Gylwcol., 164 (1991) 1582-1591. [5] Goel, R.. Mishra, O.P., Razdan, B. and Delivoria-Papadopoulos, M., Modification of the NMDA receptor by in vitro lipid peroxidation in fetal guinea pig brain, Nemwsei. Lett.. 151 (1993) 219-223. [6] Graham, E.. Mishra, O.P. and Delivoria-Papadopoulos, M., Brain cell membrane Na+,K+-ATPase modification following hypoxia m the guinea pig t~tus, Neurosci. Lett.. 153 (1993) 93--97. [7] Graham+ E., Mishra, O.P. and Delivoria-Papadopoulos, M., Effect ot in utero hypoxia on the ouabainstrphanthidin binding site of the fetal guinea pig brain cell membrane Na*+K+-ATPase, Neuro~ei. Letl., 185 (1995) 159-162. [8] Haddad, 0.(3. and Jiang, C., O e deprivation in the central nervous system: On mechanisms of neuronal response, differential sensitivity and injury. Prof. Neurobiol., 40 (1993) 277--318. [9] Harik, S.l., Doul, G.H. and Dick, A.P.K.. Specific ouabain binding to brain rnicrovessels and choroid plexus. ,l. Cereh. Blood Flow Metab.. 5 (1985) 156-160. [10] Hoffman, D,J., Marro, P.J., McGowan, J.E.. Mishra, O.P. and Delivoria-Papadopoulos, M., Protective effect of MgSO 4 infusion on NMDA receptor binding characteristics during cerebral cortical hypoxia in the newborn piglet, Brain Res., 644 (1994) 144 149. [11] Hoffman, D.J., McGowan, J.E., Marro. P.J., Mishra. O.P. and Delivoria-Papadopoulos. M., Hypoxia-induced modification of the N-methyl-l)-aspartatc receptor in the brain of the newborn piglet. Neurosci. l+ett.. 167 (1994) 156-160. [12] Lipton, S.A. and Rosenberg+ P.A., Excitatou anfino acids as a final common pathwa5 for neurologic disorders, N. Engl. ,I. Med., 330 (1994) 613 622. [13] Marro, P.J., McGowan. J.E., Razdan, B., Mishra, O.P. and Deliw+ ria-Papadopoulos. M., Effect of allopurinol on uric acid levels and brain cell membrane Na+.K~-ATPase activity during hyoxia in newborn piglets, Brain Re,s'.. 650 (1994) 9 15, [14] Mishra, O.P, and Deliw~ria-Papadopoulos, M.. Lipid pcroxidation in developing lietal guinea pig brain during normoxia and hypoxia, Det,. Brain Res.. 45 (1989) 129-135. [15] Mishra, O.P. and Delivoria-Papadopoulos, M., NMDA receptor modification of the fetal guinea pig brain during hypoxia, Neuroehem, Res., 17 (1992) 1211 - 1216. [16] Mishra, O.P. and Delivoria-Papadopoulos, M.. Modification of modulatory sites of NMDA receptor in the fetal guinea pig brain during development, Neuroehem, Res.. 17 (1992) 1223-1228. [17] Mishra. O.P., Delivoria-Papadopoulos+ M., Cahillane. G+ and Wagerle, L.C., Lipid peroxidation as the mechanism of modification of the affinity of the Na +,K ~-ATPase active sites for ATP+ K + , Na + and strophanthidin in vitro, Neurochem. Re,s, 14(1989) 845 851. [18] Mishra, O.P. and Deliw)ria-Papadopoulos, M., Na+,K + ATPase in developing fetal guinea pig brain and the effect of maternal hypoxia, Neurochem. Res., 13 (1988) 765-770. [19] Monaghan, D.T,, Bridges. R.J. and Cotman. C.W., The excitator~ amino acid receptors: Their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu+ Rel. Pharm~u'of 7)~.vieol., 29 (1989)365-402. [20] Montague, PR., Gancayco, C.D., Winn, M.J., Marchasc, R.B. and Friedlander, M.J., Role of NO production in NMDA receptor-mediated neurotransmitter release in cerebral cortex, Seiem'e. 263 (1994) 973-977. [21] Razdan, B., Marro, P.J., Tammela, O.+ Goel. R., Mishra. O.P. and Delivoria-Papadopoulos, M.+ Selective sensitivity of syaptosomal membrane function to cerebral cortical hypoxia in nexvborn piglets. Brain Res.. 600(1993) 308 314.
T.S. Rosenkrant= et al. / Brain Research Z~O (1996! 52 57
[22] Roy, R.S. and McCord, J.M., Superoxide and ischemia: conversion of xanthine dehydrogenase to xanthine oxidase. In R. Greenald and G. Ccohen (Eds.), O D'-radicals and their Scavenger ~vstems. Vof 2. Celhdar a , d MolecMar A.slWCts. Elsevier Science, New York, 1983, pp. 145 153. [23] Sanleliu, C.. Hunl, A. and Patel, A., Exposure to N-methyl-Daspartate increases release of arachidonic acid in primary cultures of rat hippocampal neurons and nol in astrocytes. Brain Re.~., 526 (1990) 241 248. [24] Shin, S.M.. Razdan, B.. Mi>hra, O.P.. Johnson, L. and DelivoriaPapadopoulos. M., Prnlective effect of a-tocopherol on brain cell nlenlbranc funclion during cerebral cortical hypoxia in newborn piglets. Blain Rev.. 553 (1994) 45-50.
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[25] Sweadner. K.J.. lsozymes of the N a ' . K - A'PPase. Biochim. Biophy.s. ,4eta. 988 (1989) 185-220. [26] Sweadner, K.J. and Gilkeson, R., Enzymatic properties of separated ioszymes of the Na +.K ~-ATPase: substrate affinities, kinetic coperativity, and ion transport stoichiometrv..L Biol. Chem.. 260 (1985) 9016-9022, [27] Sun, G.Y. and Sun, A.Y., Synaptosomal plasma membranes: acyl group composition of phosphoglycerides and N a , K ' ATPasc aciiv iD during fatty acid deficiency, J. Neuroctu'm., 22 (1~,~74) 15--IK [28] Vannucci. R.C., Experimental biology of cerebral hypoxia-ischemia: Relation to perinatal brain damagc, Pediarr Rc.~., 27 (1990)1117 326.