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Effect of catecholamines on activity of Na+, K+-ATPase in neonatal piglet brain during posthypoxic reoxygenation
Comparative Biochemistry and Physiology Part A 132 (2002) 139–145
Effect of catecholamines on activity of Naq, Kq-ATPase in neonatal piglet brain during posthypoxic reoxygenation夞 Tatiana Zaitsevaa, Jin Shena, Gregory Schearsb, Jennifer Creeda, David F. Wilsona, Anna Pastuszkoa,* a
Department of Biochemistry & Biophysics, School of Medicine, 264 Anatomy Chemistry Building, University of Pennsylvania, Philadelphia, PA 19104, USA b Department of Anesthesiology & Critical Care, The Children Hospital, Philadelphia, PA 19104, USA Received 10 January 2001; received in revised form 18 May 2001; accepted 24 May 2001
Abstract The present study examined the possible role of dopamine on the response of Naq , Kq -ATPase activity in the striatum of newborn piglets to 1 h of bilateral carotid ligation with hemorrhage and 2 h of recovery. Newborn piglets, 2–4 days of age and with and without prior treatment with a-methyl-p-tyrosine (AMT), an inhibitor of catecholamines synthesis, were used for the study. The oxygen pressure in the microvasculature of the cortex (PcO2) was measured by oxygen dependent quenching of the phosphorescence. In sham-operated animals the PcO2 was 50"3 torr. Following ligation and hemorrhage the PcO2 decreased to 8"0.5 torr. After release of ligation and reperfusion PcO2 increased to 45"4 torr, a value not significantly different from controls, in approximately 30 min. There were no significant differences in PcO2 between AMT treated and untreated animals. In sham-operated animals striatal Naq ,Kq -ATPase was 29.1"3 mmolymg protein per h and decreased by 25% after 2 h of recovery. Depleting the brain of catecholamines prior to ligation and hemorrhage abolished this decrease. It is postulated that the decrease in the level of dopamine in the brain prior to ligation and hemorrhage can be at least partly responsible for the observed decrease in activity of Naq, KqATPase in the striatum of newborn piglets. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Brain; Catecholamines; Hypoxia; Injury; Naq, Kq-ATPase; Piglet; Reperfusion; Striatum
1. Introduction Disturbance of cellular ionic gradients has been implicated as a major mechanism of brain cell death following hypoxicyischemic conditions. Cerebral Naq, Kq-ATPase is an enzyme responsible for maintaining cellular ionic gradients and membrane potential and insufficient Naq, Kq夞 This paper was presented as part of ISOTT2000 held in Nijmegen, The Netherlands, August 20–25, 2000. The Organizer was Dr Berend Oeseburg. *Corresponding author. Tel.: q1-215-898-6382; fax: q1215-573-3787. E-mail address: [email protected] (A. Pastuszko).
ATPase activity results in an intracellular influx of sodium with consequent membrane depolarization. Thus, Naq, Kq-ATPase inhibition would represent an important mechanism of initiating disturbances in brain function and homeostasis. The effect of ischemicyhypoxic conditions on Naq, Kq-ATPase activity has been studied by many authors (for example: Dobrota et al., 1999; Chang et al., 1999a,b; Jamme et al., 1999; Goplerud et al., 1992; Marro et al., 1999; Mishra and Delivoria-Papadopoulos, 1988; Mishra et al., 1989; Razdan et al., 1993; see also review by Lees, 1991). A variety of mechanisms have been proposed for this inhibition, including decrease in
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ATP production, increase in free fatty acid level, and increase in lipid peroxidation (Lees, 1991). The present study describes a possible role of dopamine in loss of Naq, Kq-ATPase activity following ligation with hemorrhage and reperfusion in striatum of newborn piglets. 2. Materials and methods 2.1. Animal preparation Newborn piglets, age 2–4 days, were used for this study. Anesthesia was induced with 4% halothane mixed with 96% oxygen, and 1.5% lidocaine–HCl (Abbott Laboratories) was used as a local anesthetic. The halothane was reduced to 0.6– 0.8% after tracheotomy and tubocurarine–HCl was used to induce respiratory paralysis (Apothecon, Bristol-Myers Squibb, 5 mgykg), and the femoral artery and femoral vein were cannulated. Both carotid arteries were isolated and silk suture was placed loosely around each carotid artery. After the surgery was completed, the halothane was withdrawn entirely and Fentanyl-citrate (ElkinsLinn, Inc., 30 mgykg) was injected intravenously at approximately 1-h intervals throughout the experiments. The animals were paralyzed with tubocurarine and mechanically ventilated with a mixture of oxygen and nitrous oxide (in control conditions with 21–22% oxygen and 78–79% N2O). The head was placed in a Kopf stereotaxic holder and an incision was made along the midline of the scalp. The scalp was removed to expose the skull and a hole approximately 5 mm in diameter was made in the skull over one parietal hemisphere, where the cortical oxygen pressure was measured through the intact dura. In all experiments, the blood pressure, body temperature and heart rate were continuously monitored. Arterial blood samples were taken every 15 min throughout the course of the experiment and the blood pH, PaCO2 and PaO2 were measured using a Model 178 pHyblood gas analyzer (Corning). At the end of each experiment, the anesthetized animals were euthanized by intravenous administration of saturated KCl solution, the brain rapidly removed and the striata dissected out. The tissues were frozen and stored at y60 8C until needed for assays. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee.
2.2. Experimental protocol The study was performed on four experimental groups of animals: (1) sham-operated normoxic group; (2) sham-operated normoxic group treated with a-methyl-para-tyrosine; (3) hypoxicyischemic group; (4) hypoxicyischemic group treated with a-methyl-para-tyrosine. Hypoxia–ischemia was induced by ligation of both carotid arteries by pulling the snares snugly around them. Blood, approximately 80 ml, was then withdrawn from the arterial catheter into heparinized syringes over a 12–15-min period to reduce the systemic arterial pressure to approximately 40 torr. After 1 h of hypoxia–ischemia, the carotid ligatures were released and the blood that had been previously withdrawn was reinfused. The animals were then maintained through a 2-h period of recovery. In the a-methyl-para-tyrosine treated groups of animals the injection of a-methyl-para-tyrosine (i.p., 300 mgykg) was given 5 h prior to experiments. The a-methyl-para-tyrosine, an inhibitor of dopamine synthesis, decreased the dopamine content of the striatum by 82"6% (Olano et al., 1995). 2.3. Measurements of oxygen by the oxygen dependent quenching of phosphorescence The cortical oxygen pressure was measured by the oxygen dependent quenching of phosphorescence (Vanderkooi et al., 1987; Wilson et al., 1988; Lo et al., 1996). This is a non-invasive optical method in which an oxygen sensitive phosphor (Oxyphor R2) was injected i.v. at approximately 22 mgykg. The measurements were made with a frequency domain phosphorometer (PMOD 1000, Oxygen Enterprises, Ltd.). The sinusoidally modulated excitation light (524 nm) was carried to the tissue through one branch of a bifurcated light guide and illuminated an approximately 5 mm diameter area. The phosphorescence (690 nm maximum) emitted from the tissue was returned through the second branch of the bifurcated light guide, filtered to remove the excitation light and measured. The resulting signal was amplified, digitized and analyzed with a microcomputer. The phosphorescence lifetime was determined from the phase relationship relative to the excitation light. The instrument was set to measure at a phase shift of 25"2 degrees. With these settings, the calcu-
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Table 1 Effect of ligation and hemorrhage on the physiological parameters of newborn piglets Conditions
MABP (torr)
pHa
PaO2 (torr)
PaCO2 (torr)
Heart rate (beatsymin)
Prior to ligation and hemorrhage End of ligation and hemorrhage Reoxygenation, 1 h Reoxygenation, 2 h
95"9
7.35"0.05
108.7"14.1
41.0"3.4
182"31
41"10*
7.31"0.05
120.2"20.1
19.1"4.6*
193"39
83"14 73"14
7.16"0.11** 7.21"0.07***
120.9"13.8 104.1"17.3
42.9"6.3 40.1"4.9
183"31 210"37
Values are the means"S.D. for 11 animals (six AMT untreated and five AMT treated). *P-0.001; **P-0.005; ***P-0.05 for difference prior to ligation and hemorrhage as determined by one-way analysis of variance with repeated measures by Wilcoxon signedrank test.
lated lifetime is approximately the mean for the phosphor in the tissue microvasculature and the measured oxygen pressures are in good agreement with values obtained using a time domain phosphorometer. 2.4. Determination of activity striatal Naq, KqATPase The striatal membranes were prepared as described by Mishra and Delivoria-Papadopoulos (1988). The rate of ATP hydrolysis was determined in a 1-ml reaction mixture of the following composition: 100 mM NaCl, 20 mM KCl, 3 mM Na-ATP, 3 mM MgCl2 and 50 mM Tris–HCl buffer (pH 7.4), including 50–100 mg membrane protein. In the second reaction mixture KCl was replaced by 1.0 mM ouabain. The ouabain sensitive activity is referred to as the Naq, Kq-ATPase activity and expressed in mmol Pymg protein per h. 2.5. Determination of striatal content of orthotyrosine Striatal tissue (approx. 1 mg proteinyml) was hydrolyzed with 6 N HCl under nitrogen atmosphere for 12 h at 100 8C. The hydrolysates were then dried, redissolved in mobile phase and analyzed for ortho-tyrosine (o-tyr) using a BAS liquid chromatography system with electrochemical detection. The mobile phase consisted of 0.1 M chloroacetic acid, 0.1 M KH2PO4, 1 mM EDTA, 1% methanol, 1 mgyml sodium octyl sulfonate, 10 mM NaCl and was adjusted to a final pH of 3.00.
2.6. Statistical evaluation All values are expressed as means for n experiments"S.E.M. or S.D. Statistical significance was determined using one-way analysis of variance with repeated measures by Wilcoxon signed-rank test. P-0.05 was considered statistically significant. 3. Results 3.1. Effect of ligation and hemorrhage on the physiological parameters of newborn piglets The piglets were subjected to 1-h bilateral ligation and hemorrhage, followed by a recovery period of 2 h. There were no differences between the physiological parameters for untreated and treated with AMT animals. The measured values for "AMT groups of animals of blood pH, arterial CO2, O2 and heart rate are given in Table 1. As can be seen, the heart rate and arterial CO2 did not differ significantly during the course of the experimental protocols. There was, however, a significant decrease in arterial pH, from a control value of 7.35"0.05 to 7.16"0.11 and 7.21"0.07, respectively, at 1 h and 2 h following ligation release and retransfusion. The mean arterial pressure under control conditions was 95"9 torr. During 1-h ligation and hemorrhage, the blood pressure decreased to 41"10 torr (P-0.001). Reinfusion of the removed blood and release of carotid ligation resulted in the blood pressure increasing to 83"14 torr, a value not significantly different from control.
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Fig. 1. Effect of carotid ligation and hemorrhage on cortical oxygen pressure in newborn piglets. The cortical oxygen concentration was measured using the oxygen dependent quenching of phosphorescence. The results are expressed as the mean"S.E.M. for 11 animals (six AMT untreated and five AMT treated). *P-0.05; **P-0.001 for significant difference from baseline values as determined by one-way analysis of variance, followed by the Wilcoxon signed-rank test.
3.2. Effect of carotid artery ligation on cortical oxygen pressure in newborn piglets Cortical oxygen pressure was measured every 5 min during the experimental protocol, including the period after release of carotid artery ligation and reperfusion. Bilateral ligation of carotid artery did not induce a significant change in cortical oxygen pressure compared with control. However, as can be seen in Fig. 1, the cortical oxygen pressure decreased rapidly after ligation and hemorrhage. During the 1 h of ligation and hemorrhage the cortical oxygen pressure decreased from 50"3
torr (control) to 8"0.5 torr (P-0.001). Following reperfusion and release of ligation cortical oxygen pressure increased to 45"4 torr. There were no differences in PcO2 between qAMT-treated animals. 3.3. Effect of ligation and hemorrhage on the activity of Naq, Kq-ATPase measured in striatal homogenates The effect of ligation and hemorrhage followed by 2 h of perfusion on the activity of Naq, KqATPase activity is shown in Table 2. In sham-
Table 2 Effects of ligationyhemorrhage and catecholamine depletion on activity of Naq, Kq-ATPase in striatum of newborn piglets Experimental conditions
Sham operated Ligationyhemorrhageq1 h recovery Ligationyhemorrhageq2 h recovery
Activity of Naq, Kq-ATPase (mmolymg protein per h) AMT untreated
AMT treated
29.1"3.0 33.9"6.5 22.0"2.7*
28.3"4.5 29.7"3.1 27.4"5.0
Values are the means"S.D. for six animals in each AMT untreated group and for five animals in each AMT treated group. *P0.05 for difference from the sham-operated group as determined by one-way analysis of variance with repeated measures by Wilcoxon signed-rank test.
T. Zaitseva et al. / Comparative Biochemistry and Physiology Part A 132 (2002) 139–145 Table 3 Effects of ligation and hemorrhage on the level of o-tyrosine in striatum of newborn piglets Experimental conditions
o-Tyrosine level (nmolymg protein)
Sham operated Ligationyhemorrhageq1 h recovery Ligationyhemorrhageq2 h recovery
0.59"0.23 2.92"0.49a 5.56"0.64*
Values are the means"S.D. for six animals in each experimental group. *P-0.005 for difference from sham-operated animals as determined by one-way analysis of variance with repeated measures by Wilcoxon signed-rank test. a Data from Shen et al. (2000) in press.
operated animals (control, no AMT) the activity of striatal Naq, Kq-ATPase was 29.1"3 mmoly mg protein per h. At 1 h following a release of carotid ligation and a reperfusion of whole removed blood, the activity of enzyme was not significantly different from controls. After 2 h of recovery, however, the activity of Naq, Kq-ATPase was 22"2.7 mmolymg protein per h, a value statistically significant lower than controls (P0.05). This decrease was completely abolished by depleting the brain of catecholamines prior to hypoxiayischemia. 3.4. Effect of ligation and hemorrhage on striatal level of ortho-tyrosine The level of o-tyr in striatal tissue of shamoperated animals was 0.59"0.23 nmolyg striatal tissue. The level of o-tyr increased after 2 h of recovery from ligation and hemorrhage to 5.56"0.64 nmolymg (P-0.001) (Table 3). The o-tyr could not be measured in striatum of piglets depleted of catecholamines prior to ligation and hemorrhage due to AMT interference with the assay. 4. Discussion The present study demonstrated that following a period of 1-h ligation and hemorrhage and 2 h of recovery the activity of Naq, Kq-ATPase is significantly suppressed in striatum of newborn piglets. Inhibition of activity of enzyme was not observed when the brain had been depleted of catecholamines prior to ischemiayhypoxia. Therefore, it is reasonable to suggest that catecholamines, particularly dopamine, were involved in the inhibitory process.
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Several studies from our laboratory have shown that the dopaminergic system in newborn brain is very sensitive to hypoxia and that extracellular levels of dopamine increase with even small decreases in oxygen pressure (Pastuszko et al., 1992, 1996; Huang et al., 1994, 1995; Yonetani et al., 1994). This increase in extracellular dopamine during hypoxicyischemic insult can affect the activity of Naq, Kq-ATPase in different ways. Bertorello et al. (1990) showed modulation of this enzyme activity via the D1 and D2 receptors. Similarly, Nishi et al. (1999a) reported that D1 agonists and D2 agonists inhibit Naq, Kq-ATPase activity in dissociated cells from the mouse neostriatum and that this effect is abolished in cells from mice deficient in DARPP-32. They also reported that in rat neostriatal neurons, treatment with dopamine results in inhibition of specific alpha3 andyor alpha2 isoforms of enzyme, but that this is not mediated through direct phosphorylation of the enzyme (Nishi et al., 1999b). Another proposed mechanism for the effect of dopamine on activity of Naq, Kq-ATPase is through an increase in the production of free radicals. Many studies already established the inhibitory effects of free radicals and lipid peroxidation on activity of Naq, Kq-ATPase (see Section 1). Oxidation of the excess dopamine released during hypoxiayischemia by molecular oxygen, as may occur during reoxygenation, can result in the formation of two superoxide anion radicals (Graham et al., 1978; Graham, 1984; Tse et al., 1976). Enzymatic oxidation of dopamine by monoamine oxidase also results in formation of hydrogen peroxide, a hydroxyl radical ‘precursor’. It has been proposed that a major factor in the CNS oxygen toxicity involves excess H2O2 production by monoamine oxidase during hyperoxia (Matsui and Kumagae, 1991; Zhang and Piantadosi, 1991). In the present study, a statistically significant decrease in enzyme activity was observed after 2 h of reoxygenation. At this point, the level of ortho-tyrosine (o-tyr), produced by hydroxyl radical attack on phenylalanine was 10 times higher than in control tissue. We have also observed (unpublished) that maximal increase of the level 2,3-dihydroxybenzoic acid, a marker of hydroxyl radical production, occurred at approximately 2 h reoxygenation following ligation and hemorrhage. These results are similar to those obtained after hypoxic insult induced by decreasing the fraction of inspired oxygen (FiO2) from 21 to 6% and
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reoxygenation (Olano et al., 1995). In the latter case as well, the increase in the level of 2,3 dihydroxybenzoic acid was abolished by depletion of catecholamines from the brain, suggesting that oxidation of striatal dopamine during the period of posthypoxic reoxygenation is at least partly responsible for the increase (Olano et al., 1995). We conclude that dopamine dependent increase of free radicals during postischemic reoxygenation can be at least partly responsible for the decrease in activity of Naq, Kq-ATPase in striatum of newborn piglets. Acknowledgments Supported in part by grant NS-31465 and HL58669. References Bertorello, A.M., Hopfield, J.F., Aperia, A., Greengard, P., 1990. Inhibition by dopamine of Naq,Kq-ATPase activity in neostriatal neurons through D1 and D2 dopamine receptor synergism. Nature 347, 386–388. Chang, Y.S., Park, W.S., Ko, S.Y., Kang, M.J., Lee, M., Choi, J., 1999a. Effects of fasting and insulin-induced hypoglycemia on brain cell membrane function and energy metabolism during hypoxia–ischemia in newborn piglets. Brain Res. 844, 135–142. Chang, Y.S., Park, W.S., Lee, M., Kim, K.S., Shin, S.M., Choi, J., 1999b. Near infrared spectroscopic monitoring of secondary cerebral energy failure after transient global hypoxiaischemia in the newborn piglet. Neurol. Res. 21, 216–224. Dobrota, D., Matejoviciva, M., Kurella, E.G., Boldrev, A.A., 1999. NayK-ATPase under oxidative stress: molecular mechanisms of injury. Cell. Mol. Neurobiol. 19, 141–149. Goplerud, J.M., Mishra, O.P., Delivoria-Papadopoulos, M., 1992. Brain cell membrane dysfunction following acute asphyxia in newborn piglets. Biol. Neonate 61, 33–41. Graham, D.G., Tiffany, S.M., Bell, W.R., Gutknecht, W.F., 1978. Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine and related compounds toward C 1300 Neuroblastoma cells in vitro. Mol. Pharmacol. 14, 644–653. Graham, D.T., 1984. Catecholamine toxicity: a proposal for the molecular pathogenesis of manganese neurotoxicity and Parkinson’s disease. Neurotoxicology 5, 83–96. Huang, Ch.-Ch., Lajevardi, N.S., Tammela, O., Pastuszko, A., Delivoria-Papadopoulos, M., Wilson, D.F., 1994. Relationship of extracellular dopamine in striatum of newborn piglets to cortical oxygen pressure. Neurochem. Res. 19, 640–655. Huang, Ch-Ch., Yonetani, M., Lajevardi, N., Delivoria-Papadopoulos, M., Pastuszko, A., Wilson, D.F., 1995. Comparison of post-asphyxial resuscitation with 100% and 21% oxygen on striatal dopamine metabolism in newborn piglets. J. Neurochem. 64, 292–298. Jamme, I., Barbey, O., Trouve, P., et al., 1999. Focal cerebral ischemia induces a decrease in activity and a shift in ouabain
affinity of Naq,Kq-ATPase isoforms without modifications in mRNA and protein expression. Brain Res. 819, 132–142. Lees, G.J., 1991. Inhibition of sodium-potassium-ATPase: a potentially ubiquitous mechanism contributing to central nervous system neuropathology. Brain Res. Rev. 16, 283–300. Lo, L-W., Koch, C.J., Wilson, D.F., 1996. Calibration of oxygen dependent quenching of the phosphorescence of Pdmeso-tetra (4-carboxyphenyl) porphine: a phosphor with general application for measuring oxygen concentration in biological systems. Anal. Biochem. 236, 153–160. Marro, P.J., Anderson, C.B., Mishra, O.P., Delivoria-Papadopoulos, M., 1999. Effect of allopurinol on hypoxia induced modification of the NMDA receptor in newborn piglets. Neurochem. Res. 24, 1301–1306. Matsui, Y., Kumagae, Y., 1991. Monoamine oxidase inhibitors prevents striatal neuronal necrosis induced by transient forebrain ischemia. Neurosci. Lett. 126, 175–178. Mishra, O.P., Delivoria-Papadopoulos, M., 1988. Naq,KqATPase in developing fetal guinea pig brain and the effect of maternal hypoxia. Neurochem. Res. 13, 765–770. Mishra, O.P., Delivoria-Papadopoulos, M., Cahillane, G., Wagerle, L.G., 1989. Lipid peroxidation as the mechanism of modification of the affinity of the Naq,Kq-ATPase active sites for ATP, K, Na and strophanthidin in vitro. Neurochem. Res. 14, 845–851. Nishi, A., Fisone, G., Snyder, G.L., et al., 1999a. Requirement for DARPP-32 in mediating effect of dopamine D2 receptor activation. Eur. J. Neurosci. 11, 2589–2592. Nishi, A., Fisone, G., Snyder, G.L., et al., 1999b. Regulation of Naq,Kq-ATPase isoforms in rat neostriatum by dopamine and protein kinase C. J. Neurochem. 73, 1492–1501. Olano, M., Song, D., Murphy, S., Wilson, D.F., Pastuszko, A., 1995. Relationships of dopamine, cortical oxygen pressure and hydroxyl radicals in brain of newborn piglets during hypoxia and posthypoxic recovery. J. Neurochem. 65, 1205–1212. Pastuszko, A., Lajevardi, N.S., Chen, J., Tammela, O., Wilson, D.F., Delivoria-Papadopoulos, M., 1992. The effects of graded levels of tissue oxygen pressure on dopamine metabolism in the striatum of newborn piglets. J. Neurochem. 60, 161–166. Pastuszko, A., Song, D., Olano, M., Huang, Ch-Ch., Wilson, D.F., 1996. Response of cortical oxygen pressure and striatal extracellular dopamine in the brain of newborn and adult animals to hypoxia. In: Ince, C., Kesecioglu, J., Telci, L., Akpir, K. (Eds.), Oxygen Transport to Tissue XVII. Advances in Experimental Medicine and Biology, Vol. 388. Plenum Press, New York pp. 415–421. Razdan, B., Marro, P.J., Tammela, O., Goel, R., Mishra, O.P., Delivoria-Papadopoulos, M., 1993. Selective sensitivity of synaptosomal membrane function to cerebral cortical hypoxia in newborn piglets. Brain Res. 600, 308–314. Shen, J., Creed, J., Zaitseva, T., Schears, G., Wilson, D.F., Pastuszko, A. 2000. Changes in cortical oxygen pressure and o-tyrosine in response to bilateral carotid ligation and hemorrhage in newborn piglets, Oxygen Transport to Tissue XXII, Adv. Exp. Med. Biol. in press. Tse, D.C.S., McCreery, R.L., Adams, R.N., 1976. Potential oxidative pathways of brain catecholamines. J. Med. Chem. 19, 37–40.
T. Zaitseva et al. / Comparative Biochemistry and Physiology Part A 132 (2002) 139–145 Vanderkooi, J.M., Maniara, G., Green, T.J., Wilson, D.F., 1987. An optical method for measurement of dioxygen concentration based on quenching of phosphorescence. J. Biol. Chem. 262, 5476–5482. Wilson, D.F., Rumsey, W.L., Green, T.J., Vanderkooi, J.M.., 1988. The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen. J. Biol. Chem. 263, 2712–2718.
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Yonetani, M., Huang, Ch.-Ch., Lajevardi, N., Pastuszko, A., Delivoria-Papadopoulos, M., Wilson, D.F., 1994. Effect of hemorrhagic hypotension on extracellular level of dopamine, cortical oxygen pressure and blood flow in brain of newborn piglets. Neurosci. Lett. 180, 247–252. Zhang, J., Piantadosi, C.A., 1991. Preventation of H2O2 generation by monoamine oxidase protects against CNS O2 toxicity. J. Appl. Physiol. 71, 1056–1061.
Effect of catecholamines on activity of Naq, Kq-ATPase in neonatal piglet brain during posthypoxic reoxygenation夞 Tatiana Zaitsevaa, Jin Shena, Gregory Schearsb, Jennifer Creeda, David F. Wilsona, Anna Pastuszkoa,* a
Department of Biochemistry & Biophysics, School of Medicine, 264 Anatomy Chemistry Building, University of Pennsylvania, Philadelphia, PA 19104, USA b Department of Anesthesiology & Critical Care, The Children Hospital, Philadelphia, PA 19104, USA Received 10 January 2001; received in revised form 18 May 2001; accepted 24 May 2001
Abstract The present study examined the possible role of dopamine on the response of Naq , Kq -ATPase activity in the striatum of newborn piglets to 1 h of bilateral carotid ligation with hemorrhage and 2 h of recovery. Newborn piglets, 2–4 days of age and with and without prior treatment with a-methyl-p-tyrosine (AMT), an inhibitor of catecholamines synthesis, were used for the study. The oxygen pressure in the microvasculature of the cortex (PcO2) was measured by oxygen dependent quenching of the phosphorescence. In sham-operated animals the PcO2 was 50"3 torr. Following ligation and hemorrhage the PcO2 decreased to 8"0.5 torr. After release of ligation and reperfusion PcO2 increased to 45"4 torr, a value not significantly different from controls, in approximately 30 min. There were no significant differences in PcO2 between AMT treated and untreated animals. In sham-operated animals striatal Naq ,Kq -ATPase was 29.1"3 mmolymg protein per h and decreased by 25% after 2 h of recovery. Depleting the brain of catecholamines prior to ligation and hemorrhage abolished this decrease. It is postulated that the decrease in the level of dopamine in the brain prior to ligation and hemorrhage can be at least partly responsible for the observed decrease in activity of Naq, KqATPase in the striatum of newborn piglets. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Brain; Catecholamines; Hypoxia; Injury; Naq, Kq-ATPase; Piglet; Reperfusion; Striatum
1. Introduction Disturbance of cellular ionic gradients has been implicated as a major mechanism of brain cell death following hypoxicyischemic conditions. Cerebral Naq, Kq-ATPase is an enzyme responsible for maintaining cellular ionic gradients and membrane potential and insufficient Naq, Kq夞 This paper was presented as part of ISOTT2000 held in Nijmegen, The Netherlands, August 20–25, 2000. The Organizer was Dr Berend Oeseburg. *Corresponding author. Tel.: q1-215-898-6382; fax: q1215-573-3787. E-mail address: [email protected] (A. Pastuszko).
ATPase activity results in an intracellular influx of sodium with consequent membrane depolarization. Thus, Naq, Kq-ATPase inhibition would represent an important mechanism of initiating disturbances in brain function and homeostasis. The effect of ischemicyhypoxic conditions on Naq, Kq-ATPase activity has been studied by many authors (for example: Dobrota et al., 1999; Chang et al., 1999a,b; Jamme et al., 1999; Goplerud et al., 1992; Marro et al., 1999; Mishra and Delivoria-Papadopoulos, 1988; Mishra et al., 1989; Razdan et al., 1993; see also review by Lees, 1991). A variety of mechanisms have been proposed for this inhibition, including decrease in
1095-6433/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 1 . 0 0 5 4 0 - 2
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ATP production, increase in free fatty acid level, and increase in lipid peroxidation (Lees, 1991). The present study describes a possible role of dopamine in loss of Naq, Kq-ATPase activity following ligation with hemorrhage and reperfusion in striatum of newborn piglets. 2. Materials and methods 2.1. Animal preparation Newborn piglets, age 2–4 days, were used for this study. Anesthesia was induced with 4% halothane mixed with 96% oxygen, and 1.5% lidocaine–HCl (Abbott Laboratories) was used as a local anesthetic. The halothane was reduced to 0.6– 0.8% after tracheotomy and tubocurarine–HCl was used to induce respiratory paralysis (Apothecon, Bristol-Myers Squibb, 5 mgykg), and the femoral artery and femoral vein were cannulated. Both carotid arteries were isolated and silk suture was placed loosely around each carotid artery. After the surgery was completed, the halothane was withdrawn entirely and Fentanyl-citrate (ElkinsLinn, Inc., 30 mgykg) was injected intravenously at approximately 1-h intervals throughout the experiments. The animals were paralyzed with tubocurarine and mechanically ventilated with a mixture of oxygen and nitrous oxide (in control conditions with 21–22% oxygen and 78–79% N2O). The head was placed in a Kopf stereotaxic holder and an incision was made along the midline of the scalp. The scalp was removed to expose the skull and a hole approximately 5 mm in diameter was made in the skull over one parietal hemisphere, where the cortical oxygen pressure was measured through the intact dura. In all experiments, the blood pressure, body temperature and heart rate were continuously monitored. Arterial blood samples were taken every 15 min throughout the course of the experiment and the blood pH, PaCO2 and PaO2 were measured using a Model 178 pHyblood gas analyzer (Corning). At the end of each experiment, the anesthetized animals were euthanized by intravenous administration of saturated KCl solution, the brain rapidly removed and the striata dissected out. The tissues were frozen and stored at y60 8C until needed for assays. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee.
2.2. Experimental protocol The study was performed on four experimental groups of animals: (1) sham-operated normoxic group; (2) sham-operated normoxic group treated with a-methyl-para-tyrosine; (3) hypoxicyischemic group; (4) hypoxicyischemic group treated with a-methyl-para-tyrosine. Hypoxia–ischemia was induced by ligation of both carotid arteries by pulling the snares snugly around them. Blood, approximately 80 ml, was then withdrawn from the arterial catheter into heparinized syringes over a 12–15-min period to reduce the systemic arterial pressure to approximately 40 torr. After 1 h of hypoxia–ischemia, the carotid ligatures were released and the blood that had been previously withdrawn was reinfused. The animals were then maintained through a 2-h period of recovery. In the a-methyl-para-tyrosine treated groups of animals the injection of a-methyl-para-tyrosine (i.p., 300 mgykg) was given 5 h prior to experiments. The a-methyl-para-tyrosine, an inhibitor of dopamine synthesis, decreased the dopamine content of the striatum by 82"6% (Olano et al., 1995). 2.3. Measurements of oxygen by the oxygen dependent quenching of phosphorescence The cortical oxygen pressure was measured by the oxygen dependent quenching of phosphorescence (Vanderkooi et al., 1987; Wilson et al., 1988; Lo et al., 1996). This is a non-invasive optical method in which an oxygen sensitive phosphor (Oxyphor R2) was injected i.v. at approximately 22 mgykg. The measurements were made with a frequency domain phosphorometer (PMOD 1000, Oxygen Enterprises, Ltd.). The sinusoidally modulated excitation light (524 nm) was carried to the tissue through one branch of a bifurcated light guide and illuminated an approximately 5 mm diameter area. The phosphorescence (690 nm maximum) emitted from the tissue was returned through the second branch of the bifurcated light guide, filtered to remove the excitation light and measured. The resulting signal was amplified, digitized and analyzed with a microcomputer. The phosphorescence lifetime was determined from the phase relationship relative to the excitation light. The instrument was set to measure at a phase shift of 25"2 degrees. With these settings, the calcu-
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Table 1 Effect of ligation and hemorrhage on the physiological parameters of newborn piglets Conditions
MABP (torr)
pHa
PaO2 (torr)
PaCO2 (torr)
Heart rate (beatsymin)
Prior to ligation and hemorrhage End of ligation and hemorrhage Reoxygenation, 1 h Reoxygenation, 2 h
95"9
7.35"0.05
108.7"14.1
41.0"3.4
182"31
41"10*
7.31"0.05
120.2"20.1
19.1"4.6*
193"39
83"14 73"14
7.16"0.11** 7.21"0.07***
120.9"13.8 104.1"17.3
42.9"6.3 40.1"4.9
183"31 210"37
Values are the means"S.D. for 11 animals (six AMT untreated and five AMT treated). *P-0.001; **P-0.005; ***P-0.05 for difference prior to ligation and hemorrhage as determined by one-way analysis of variance with repeated measures by Wilcoxon signedrank test.
lated lifetime is approximately the mean for the phosphor in the tissue microvasculature and the measured oxygen pressures are in good agreement with values obtained using a time domain phosphorometer. 2.4. Determination of activity striatal Naq, KqATPase The striatal membranes were prepared as described by Mishra and Delivoria-Papadopoulos (1988). The rate of ATP hydrolysis was determined in a 1-ml reaction mixture of the following composition: 100 mM NaCl, 20 mM KCl, 3 mM Na-ATP, 3 mM MgCl2 and 50 mM Tris–HCl buffer (pH 7.4), including 50–100 mg membrane protein. In the second reaction mixture KCl was replaced by 1.0 mM ouabain. The ouabain sensitive activity is referred to as the Naq, Kq-ATPase activity and expressed in mmol Pymg protein per h. 2.5. Determination of striatal content of orthotyrosine Striatal tissue (approx. 1 mg proteinyml) was hydrolyzed with 6 N HCl under nitrogen atmosphere for 12 h at 100 8C. The hydrolysates were then dried, redissolved in mobile phase and analyzed for ortho-tyrosine (o-tyr) using a BAS liquid chromatography system with electrochemical detection. The mobile phase consisted of 0.1 M chloroacetic acid, 0.1 M KH2PO4, 1 mM EDTA, 1% methanol, 1 mgyml sodium octyl sulfonate, 10 mM NaCl and was adjusted to a final pH of 3.00.
2.6. Statistical evaluation All values are expressed as means for n experiments"S.E.M. or S.D. Statistical significance was determined using one-way analysis of variance with repeated measures by Wilcoxon signed-rank test. P-0.05 was considered statistically significant. 3. Results 3.1. Effect of ligation and hemorrhage on the physiological parameters of newborn piglets The piglets were subjected to 1-h bilateral ligation and hemorrhage, followed by a recovery period of 2 h. There were no differences between the physiological parameters for untreated and treated with AMT animals. The measured values for "AMT groups of animals of blood pH, arterial CO2, O2 and heart rate are given in Table 1. As can be seen, the heart rate and arterial CO2 did not differ significantly during the course of the experimental protocols. There was, however, a significant decrease in arterial pH, from a control value of 7.35"0.05 to 7.16"0.11 and 7.21"0.07, respectively, at 1 h and 2 h following ligation release and retransfusion. The mean arterial pressure under control conditions was 95"9 torr. During 1-h ligation and hemorrhage, the blood pressure decreased to 41"10 torr (P-0.001). Reinfusion of the removed blood and release of carotid ligation resulted in the blood pressure increasing to 83"14 torr, a value not significantly different from control.
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Fig. 1. Effect of carotid ligation and hemorrhage on cortical oxygen pressure in newborn piglets. The cortical oxygen concentration was measured using the oxygen dependent quenching of phosphorescence. The results are expressed as the mean"S.E.M. for 11 animals (six AMT untreated and five AMT treated). *P-0.05; **P-0.001 for significant difference from baseline values as determined by one-way analysis of variance, followed by the Wilcoxon signed-rank test.
3.2. Effect of carotid artery ligation on cortical oxygen pressure in newborn piglets Cortical oxygen pressure was measured every 5 min during the experimental protocol, including the period after release of carotid artery ligation and reperfusion. Bilateral ligation of carotid artery did not induce a significant change in cortical oxygen pressure compared with control. However, as can be seen in Fig. 1, the cortical oxygen pressure decreased rapidly after ligation and hemorrhage. During the 1 h of ligation and hemorrhage the cortical oxygen pressure decreased from 50"3
torr (control) to 8"0.5 torr (P-0.001). Following reperfusion and release of ligation cortical oxygen pressure increased to 45"4 torr. There were no differences in PcO2 between qAMT-treated animals. 3.3. Effect of ligation and hemorrhage on the activity of Naq, Kq-ATPase measured in striatal homogenates The effect of ligation and hemorrhage followed by 2 h of perfusion on the activity of Naq, KqATPase activity is shown in Table 2. In sham-
Table 2 Effects of ligationyhemorrhage and catecholamine depletion on activity of Naq, Kq-ATPase in striatum of newborn piglets Experimental conditions
Sham operated Ligationyhemorrhageq1 h recovery Ligationyhemorrhageq2 h recovery
Activity of Naq, Kq-ATPase (mmolymg protein per h) AMT untreated
AMT treated
29.1"3.0 33.9"6.5 22.0"2.7*
28.3"4.5 29.7"3.1 27.4"5.0
Values are the means"S.D. for six animals in each AMT untreated group and for five animals in each AMT treated group. *P0.05 for difference from the sham-operated group as determined by one-way analysis of variance with repeated measures by Wilcoxon signed-rank test.
T. Zaitseva et al. / Comparative Biochemistry and Physiology Part A 132 (2002) 139–145 Table 3 Effects of ligation and hemorrhage on the level of o-tyrosine in striatum of newborn piglets Experimental conditions
o-Tyrosine level (nmolymg protein)
Sham operated Ligationyhemorrhageq1 h recovery Ligationyhemorrhageq2 h recovery
0.59"0.23 2.92"0.49a 5.56"0.64*
Values are the means"S.D. for six animals in each experimental group. *P-0.005 for difference from sham-operated animals as determined by one-way analysis of variance with repeated measures by Wilcoxon signed-rank test. a Data from Shen et al. (2000) in press.
operated animals (control, no AMT) the activity of striatal Naq, Kq-ATPase was 29.1"3 mmoly mg protein per h. At 1 h following a release of carotid ligation and a reperfusion of whole removed blood, the activity of enzyme was not significantly different from controls. After 2 h of recovery, however, the activity of Naq, Kq-ATPase was 22"2.7 mmolymg protein per h, a value statistically significant lower than controls (P0.05). This decrease was completely abolished by depleting the brain of catecholamines prior to hypoxiayischemia. 3.4. Effect of ligation and hemorrhage on striatal level of ortho-tyrosine The level of o-tyr in striatal tissue of shamoperated animals was 0.59"0.23 nmolyg striatal tissue. The level of o-tyr increased after 2 h of recovery from ligation and hemorrhage to 5.56"0.64 nmolymg (P-0.001) (Table 3). The o-tyr could not be measured in striatum of piglets depleted of catecholamines prior to ligation and hemorrhage due to AMT interference with the assay. 4. Discussion The present study demonstrated that following a period of 1-h ligation and hemorrhage and 2 h of recovery the activity of Naq, Kq-ATPase is significantly suppressed in striatum of newborn piglets. Inhibition of activity of enzyme was not observed when the brain had been depleted of catecholamines prior to ischemiayhypoxia. Therefore, it is reasonable to suggest that catecholamines, particularly dopamine, were involved in the inhibitory process.
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Several studies from our laboratory have shown that the dopaminergic system in newborn brain is very sensitive to hypoxia and that extracellular levels of dopamine increase with even small decreases in oxygen pressure (Pastuszko et al., 1992, 1996; Huang et al., 1994, 1995; Yonetani et al., 1994). This increase in extracellular dopamine during hypoxicyischemic insult can affect the activity of Naq, Kq-ATPase in different ways. Bertorello et al. (1990) showed modulation of this enzyme activity via the D1 and D2 receptors. Similarly, Nishi et al. (1999a) reported that D1 agonists and D2 agonists inhibit Naq, Kq-ATPase activity in dissociated cells from the mouse neostriatum and that this effect is abolished in cells from mice deficient in DARPP-32. They also reported that in rat neostriatal neurons, treatment with dopamine results in inhibition of specific alpha3 andyor alpha2 isoforms of enzyme, but that this is not mediated through direct phosphorylation of the enzyme (Nishi et al., 1999b). Another proposed mechanism for the effect of dopamine on activity of Naq, Kq-ATPase is through an increase in the production of free radicals. Many studies already established the inhibitory effects of free radicals and lipid peroxidation on activity of Naq, Kq-ATPase (see Section 1). Oxidation of the excess dopamine released during hypoxiayischemia by molecular oxygen, as may occur during reoxygenation, can result in the formation of two superoxide anion radicals (Graham et al., 1978; Graham, 1984; Tse et al., 1976). Enzymatic oxidation of dopamine by monoamine oxidase also results in formation of hydrogen peroxide, a hydroxyl radical ‘precursor’. It has been proposed that a major factor in the CNS oxygen toxicity involves excess H2O2 production by monoamine oxidase during hyperoxia (Matsui and Kumagae, 1991; Zhang and Piantadosi, 1991). In the present study, a statistically significant decrease in enzyme activity was observed after 2 h of reoxygenation. At this point, the level of ortho-tyrosine (o-tyr), produced by hydroxyl radical attack on phenylalanine was 10 times higher than in control tissue. We have also observed (unpublished) that maximal increase of the level 2,3-dihydroxybenzoic acid, a marker of hydroxyl radical production, occurred at approximately 2 h reoxygenation following ligation and hemorrhage. These results are similar to those obtained after hypoxic insult induced by decreasing the fraction of inspired oxygen (FiO2) from 21 to 6% and
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reoxygenation (Olano et al., 1995). In the latter case as well, the increase in the level of 2,3 dihydroxybenzoic acid was abolished by depletion of catecholamines from the brain, suggesting that oxidation of striatal dopamine during the period of posthypoxic reoxygenation is at least partly responsible for the increase (Olano et al., 1995). We conclude that dopamine dependent increase of free radicals during postischemic reoxygenation can be at least partly responsible for the decrease in activity of Naq, Kq-ATPase in striatum of newborn piglets. Acknowledgments Supported in part by grant NS-31465 and HL58669. References Bertorello, A.M., Hopfield, J.F., Aperia, A., Greengard, P., 1990. Inhibition by dopamine of Naq,Kq-ATPase activity in neostriatal neurons through D1 and D2 dopamine receptor synergism. Nature 347, 386–388. Chang, Y.S., Park, W.S., Ko, S.Y., Kang, M.J., Lee, M., Choi, J., 1999a. Effects of fasting and insulin-induced hypoglycemia on brain cell membrane function and energy metabolism during hypoxia–ischemia in newborn piglets. Brain Res. 844, 135–142. Chang, Y.S., Park, W.S., Lee, M., Kim, K.S., Shin, S.M., Choi, J., 1999b. Near infrared spectroscopic monitoring of secondary cerebral energy failure after transient global hypoxiaischemia in the newborn piglet. Neurol. Res. 21, 216–224. Dobrota, D., Matejoviciva, M., Kurella, E.G., Boldrev, A.A., 1999. NayK-ATPase under oxidative stress: molecular mechanisms of injury. Cell. Mol. Neurobiol. 19, 141–149. Goplerud, J.M., Mishra, O.P., Delivoria-Papadopoulos, M., 1992. Brain cell membrane dysfunction following acute asphyxia in newborn piglets. Biol. Neonate 61, 33–41. Graham, D.G., Tiffany, S.M., Bell, W.R., Gutknecht, W.F., 1978. Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine and related compounds toward C 1300 Neuroblastoma cells in vitro. Mol. Pharmacol. 14, 644–653. Graham, D.T., 1984. Catecholamine toxicity: a proposal for the molecular pathogenesis of manganese neurotoxicity and Parkinson’s disease. Neurotoxicology 5, 83–96. Huang, Ch.-Ch., Lajevardi, N.S., Tammela, O., Pastuszko, A., Delivoria-Papadopoulos, M., Wilson, D.F., 1994. Relationship of extracellular dopamine in striatum of newborn piglets to cortical oxygen pressure. Neurochem. Res. 19, 640–655. Huang, Ch-Ch., Yonetani, M., Lajevardi, N., Delivoria-Papadopoulos, M., Pastuszko, A., Wilson, D.F., 1995. Comparison of post-asphyxial resuscitation with 100% and 21% oxygen on striatal dopamine metabolism in newborn piglets. J. Neurochem. 64, 292–298. Jamme, I., Barbey, O., Trouve, P., et al., 1999. Focal cerebral ischemia induces a decrease in activity and a shift in ouabain
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