Specific Ouabain Binding to Brain Microvessels and Choroid Plexus

Harik, Sami I.; Doull, Gregory H.; Dick, A. P.

doi:10.1038/jcbfm.1985.20

Cited by 100 publications

(23 citation statements)

References 18 publications

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“…Brain cell membranes were prepared according to the methods of Harik et al (25). Frozen cortex was homogenized in 10 mM Tris-HCl buffer containing 0.3 M sucrose and 0.5 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

Brain Cell Membrane Function during Hypoxia in Hyperglycemic Newborn Piglets

et al. 1995

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To test the hypothesis that acute hyperglycemia reduces changes in cell membrane structure and function during cerebral hypoxia in the newborn, brain cell membrane Na+,K+-A'TPase activity and levels of membrane lipid peroxidation products were measured in four groups of anesthetized, ventilated newborn piglets: normoglycemia/normoxia (control, group 1, n = 12), hyperglycemia/normoxia (group 2, n = 6), untreated hypoxia (group 3, Jl = 10), and hyperglycemialhypoxia (group 4, n = 7). Hyperglycemia (blood glucose concentration 20 mmollL) was induced using the glucose clamp technique. The hyperglycemic glucose clamp was maintained for 90 min before onset of hypoxia and throughout the period of hypoxia. Cerebral tissue hypoxia was induced in groups 3 and 4 by reducing fraction of inspired oxygen for 60 min and was documented by a decrease in the ratio of phosphocreatine to inorganic phosphate as measured using 31P-nuclear magnetic resonance spectroscopy. Blood glucose concentration during hypoxia in hyperglycemic hypoxic animals was 20.7 ± 1.2 mmol/L, compared with 10.3 ± 1.7 mmol/L in untreated hypoxic piglets (p < 0.05). Peak blood lactate concentrations were not significantly different between the two hypoxic groups (8.4 ± 2.8 rnmol/L versus 7.8 ± 1.6 mmollL). In cerebral cortical membranes prepared from the In the brain, an hyp oxic-ischemic insult leads to acc umu latio n of lactate, tissue ene rgy dep letion and aci dosis, and neuro na l necrosis (1-4). Because glucose serves as the major energy so urce for the brain (5, 6), a nu mbe r of stud ies have investigated the role of changes in glucose con centra tion in mo difying the effec ts of ce rebral hypoxia/ischem ia. In adult an imal mo dels, outco me afte r ce reb ral ischemia was improved by mild hypoglycemia, and neuro log ic da mage was more extensive in animals that were hypergl ycemic during or after the insult (2,7,8). Th ese findings have been attributed to the effects of intrace llular lac tic acidosis res ulting from increased anae robic glycolysis in hypergl ycemic anim als (7, 9, 10). untreated animals, cerebral tissue hypoxia caused a 25% reduction in Na+,K+-ATPase activity compared with normoxic controls and an increase in conjugated dienes and fluorescent compounds, markers of lipid peroxidation. In contrast, Na+,K+-ATPase activity and levels of lipid peroxidation products in hyperglycemic hypoxic animals were not significantly different from the values in control normoxic animals. These data suggest that in the newborn piglet model acute hyperglycemia reduces hypoxia-induced brain cell membrane dysfunction. (Pediatr Res 37: 133-139, 1995) Abbrevia tions NMR , nuclear magnetic resonance Fi0 2 , fraction of inspired oxygen PCr, phosphocreatine Pi, inorganic phosphate PCrlPi, ratio of phosphocreatine to inorganic phosphate MAUP, mean arterial blood pressure GSH, reduced glutathione ANOVA, analysis of variance D25W, dextrose 25% wt/vol in water Studies by Myers and coworkers (11 ,12) suggested that hypergl ycemia also increased hypoxic/ische m...

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“…Brain cell membranes were prepared according to the methods of Harik et al (25). Frozen cortex was homogenized in 10 mM Tris-HCl buffer containing 0.3 M sucrose and 0.5 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

Brain Cell Membrane Function during Hypoxia in Hyperglycemic Newborn Piglets

et al. 1995

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“…Membranes were prepared as described previously (7). Vesicle formation during this procedure is very limited (13). The Na ϩ ,K ϩ -ATPase activity was determined by the rate of ATP hydrolysis in a 1.0-mL reaction mixture containing NaCl 100 mM, KCl 20 mM, MgCl 2 3 mM, Tris-ATP 3 mM, Tris HCl buffer (pH 7.4) 50 mM, and membrane protein 100 g. The reaction was carried out at 37°C in the presence and absence of 1.0 mM ouabain for 5 min, during which period ATP hydrolysis was linear.…”

Section: Methodsmentioning

confidence: 99%

Effects of Deferoxamine, a Chelator of Free Iron, on Na+,K+-ATPase Activity of Cortical Brain Cell Membrane during Early Reperfusion after Hypoxia-Ischemia in Newborn Lambs

et al. 2000

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Free iron chelation after hypoxia-ischemia can reduce free radical-induced damage to brain cell membranes and preserve electrical brain activity. We investigated whether chelation of free iron with deferoxamine (DFO) preserved cortical cell membrane activity of Na ϩ ,K ϩ -ATPase and electrocortical brain activity (ECBA) of newborn lambs during early reperfusion after severe hypoxia-ischemia. Hypoxia was induced in 16 lambs by decreasing the fraction of inspired oxygen to 0.07 for 30 min, followed by a 5-min period of hypotension (mean arterial blood pressure Ͻ35 mm Hg). ECBA (in microvolts) was measured using a cerebral function monitor. Immediately after hypoxia and additional ischemia, eight lambs received DFO (2.5 mg/kg, i.v.), and seven lambs received a placebo (PLAC). Two lambs underwent sham operation. One hundred eighty minutes after completion of hypoxia and ischemia, the brains were obtained and frozen. Na ϩ ,K ϩ -ATPase activity was measured in the P 2 fraction of cortical tissue. Na ϩ ,K ϩ -ATPase activity was 35.1 Ϯ 7.4, 42.0 Ϯ 7.6, and 40.7 Ϯ 1.4 mol inorganic phosphate/mg protein per hour in PLAC-treated, DFO-treated, and sham-operated lambs, respectively (p Ͻ 0.05: DFO versus PLAC). ECBA was 11.2 Ϯ 6.1, 14.8 Ϯ 4.8, and 17.5Ϯ.0.5 V in PLAC-treated, DFO-treated, and sham-operated lambs, respectively (p ϭ 0.06: DFO versus PLAC). Na Abbreviations DFO, deferoxamine ECBA, electrocortical brain activity PLAC, placebo Q car , carotid blood flow SHAM, sham-operated animals MRS, magnetic resonance spectroscopy P i , inorganic phosphate Hypoxia and ischemia during perinatal asphyxia give rise to an inadequate substrate supply to brain tissue, resulting in damage of neuronal cells. Although recovery of oxygenation and perfusion of the brain is mandatory to prevent further damage, reoxygenation of previously ischemic brain tissue has increasingly been recognized as an important mechanism for additional injury to the neuronal cells and the cerebral microcirculation (1, 2). Production of reactive oxygen species in the early reperfusion phase plays a substantial role in this type of brain cell damage: Reactive oxygen species such as superoxide and hydrogen peroxide can be converted into the highly reactive hydroxyl radical by transition metals, in particular free iron, ultimately leading to lipid peroxidation of the brain cell membrane and cellular damage (3). In recent experimental and clinical studies, we showed that chelation of free iron prevented posthypoxic-ischemic hypoperfusion and metabolic derangements of the brain and preserved ECBA (4, 5). We also found that the free iron chelator DFO effectively lowered free iron in cortical brain tissue (6).Inasmuch as the transmembrane enzyme Na ϩ ,K ϩ -ATPase is very susceptible to free radical-related lipid peroxidation (7, 8), we investigated in the present study whether DFO prevented free radical-induced alterations of the brain cell membrane after global hypoxia and ischemia, simulating severe birth

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“…All procedures were carried out at 0-40C unless stated otherwise. Purity of the microvessel preparations was assessed by morphological and biochemical criteria (9)(10)(11). These microvessels consist mostly of capillaries less than 15 ,um in diameter, with small arterioles and venules contributing 10-15% of the preparation.…”

Section: Methodsmentioning

confidence: 99%

“…The cerebral cortical mantles and cerebella of two dogs were pooled for each microvessel preparation to obtain sufficient microvessels. Preparations enriched with brain microvessels were obtained from these two regions by bulk isolation as described previously (9). All procedures were carried out at 0-40C unless stated otherwise.…”

Section: Methodsmentioning

confidence: 99%

Angiotensin II receptor binding sites in brain microvessels.

Speth¹,

Harik²

1985

Proc. Natl. Acad. Sci. U.S.A.

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We assessed the specific binding of 125I-labeled angiotensin II to particulate fractions of the cerebral cortex and cerebellum and to microvessels obtained by bulk isolation from these two brain regions in the dog. MATERIALS AND METHODSAdult male mongrel dogs were killed by lethal doses of pentobarbital and their cerebral cortical mantles and cerebella were quickly removed and dissected free of meninges and choroid plexus. The cerebral cortical mantles and cerebella of two dogs were pooled for each microvessel preparation to obtain sufficient microvessels. Preparations enriched with brain microvessels were obtained from these two regions by bulk isolation as described previously (9). All procedures were carried out at 0-40C unless stated otherwise. Purity of the microvessel preparations was assessed by morphological and biochemical criteria (9-11). These microvessels consist mostly of capillaries less than 15 ,um in diameter, with small arterioles and venules contributing 10-15% of the preparation.The microvessels were washed two times by dispersion and centrifugation in cold 0.9% NaCl. Particulate fractions of microvessels, and of samples of the cerebral cortex and cerebellum [obtained prior to microvessel isolation ("total") and during the process of separation ("float")], were prepared by homogenization in 50 mM sodium phosphate buffer, pH 7.1/150 mM NaCl/10 mM MgCl2/1 mM EGTA/1 mM dithiothreitol (incubation buffer) in a Brinkmann Polytron. Homogenates were centrifuged at 48,000 x g for 20 min and the pellets were resuspended in incubation buffer to a concentration of about 2 mg of tissue protein per ml.125I-Ang II binding to microvessel and brain membrane fractions was performed as detailed by Speth and Husain (12). Briefly, 0.1-0.15 ml of tissue suspension was incubated with 125I-Ang II and unlabeled Ang II and/or one of its analogues in glass tubes for 1 hr at 23TC. Nonspecific binding was determined in the presence of 1 ,uM unlabeled native Ang II. Specific 125I-Ang II binding was calculated by subtracting nonspecific binding from total binding, measured in the absence of unlabeled Ang II. In all binding assays, addition of tissue suspension to the incubation mixture followed the addition of 125I-Ang II and unlabeled Ang II or one of its analogues, and it was the initiating step for the binding process. Bound 125I-Ang II was separated from free 125I-Ang II by filtration through Whatman GF/B glass fiber filters using a cell harvester (M24R, Brandel). The filters were assayed for their 125I content by -ray scintillation counting.Ang II was radioiodinated in our laboratory as described previously (12).

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Specific Ouabain Binding to Brain Microvessels and Choroid Plexus

Cited by 100 publications

References 18 publications

Brain Cell Membrane Function during Hypoxia in Hyperglycemic Newborn Piglets

Brain Cell Membrane Function during Hypoxia in Hyperglycemic Newborn Piglets

Effects of Deferoxamine, a Chelator of Free Iron, on Na+,K+-ATPase Activity of Cortical Brain Cell Membrane during Early Reperfusion after Hypoxia-Ischemia in Newborn Lambs

Angiotensin II receptor binding sites in brain microvessels.

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