Effect of hyperglycemia on brain cell membrane function and energy metabolism during hypoxia–ischemia in newborn piglets

The purpose of this study was to test the hypothesis that hyperglycemia ameliorates changes in brain cell membrane function and preserves cerebral high energy phosphates during hypoxia–ischemia in newborn piglets. A total of 42 ventilated piglets were divided into 4 groups, normoglycemic/normoxic(group 1, n=9), hyperglycemic/normoxic(group 2, n=8), normoglycemic/hypoxic–ischemic(group 3, n=13) and hyperglycemic/hypoxic–ischemic(group 4, n=12) group. Cerebral hypoxia–ischemia was induced by occlusion of bilateral common carotid arteries and simultaneous breathing with 8% oxygen for 30 min. Hyperglycemia (blood glucose 350–400 mg/dl) was maintained for 90 min before and throughout hypoxia–ischemia using modified glucose clamp technique. Changes in cytochrome aa3 were continuously monitored using near infrared spectroscopy. Blood and CSF glucose and lactate were monitored. Na⁺, K⁺-ATPase activity, lipid peroxidation products (conjugated dienes), tissue high energy phosphates (ATP and phosphocreatine) levels and brain glucose and lactate levels were determined biochemically in the cerebral cortex. During hypoxia–ischemia, glucose levels in blood and CSF were significantly elevated in hyperglycemic/hypoxic–ischemic group compared with normoglycemic/hypoxic–ischemic group, but lactate levels in blood and CSF were not different between two groups. At the end of hypoxia–ischemia of group 3 and 4, Cyt aa3, Na⁺, K⁺-ATPase activity, ATP and phosphocreatine values in brain were significantly decreased compared with normoxic groups 1 and 2, but were not different between groups 3 and 4. Levels of conjugated dienes and brain lactate were significantly increased in groups 3 and 4 compared with groups 1 and 2, and were significantly elevated in group 4 than in group 3 (0.30±0.11 vs. 0.09±0.02 μmol g⁻¹ protein, 26.4±7.6 vs. 13.1±2.6 mmol kg⁻¹, p<0.05). These findings suggest that hyperglycemia does not reduce the changes in brain cell membrane function and does not preserve cerebral high energy phosphates during hypoxia–ischemia in newborn piglets. We speculate that hyperglycemia may be harmful during hypoxia–ischemia due to increased levels of lipid peroxidation in newborn piglet.     

Blood–brain barrier mechanisms involved in brain calcium and potassium homeostasis

This study examined the potential roles of the plasma membrane Ca²⁺-ATPase (PMCA) at the blood–CSF and blood–brain barriers in brain Ca²⁺ homeostasis and blood–brain barrier Na⁺/K⁺-ATPase subunits in brain K⁺ homeostasis. During dietary-induced hypo- and hypercalcemia (0.59±0.06 and 1.58±0.12 mM [Ca²⁺]) there was no significant change in choroid plexus PMCA (Western Blots) compared to normocalcemic rats (plasma [Ca²⁺]: 1.06±0.11 mM). In contrast, PMCA in cerebral microvessels isolated from hypocalcemic rats was 150% greater than that in controls (p<0.001). Comparison of the α3 subunit of Na⁺/K⁺-ATPase from cerebral microvessels isolated from hypo-, normo- and hyperkalemic rats (2.3±0.1, 3.9±0.1 and 7.2±0.6 mM [K⁺]) showed a 75% reduction in the amount of this isoform during hyperkalemia. None of the other Na⁺/K⁺-ATPase isoforms varied with plasma [K⁺]. These results suggest that both PMCA and the α3 subunit of Na⁺/K⁺-ATPase at the blood–brain barrier play a role in maintaining a constant brain microenvironment during fluctuations in plasma composition.     

Effects of mammalian brain extracts and chlormadinone acetate on neuronal Na, K-ATPase and electrogenic Na, K-pump activity in vitro

Acid-acetone extracts of brain (from beef and guinea pig) and chlormadinone acetate (CMA) were compared with ouabain for their ability to inhibit the electrogenic Na⁺,K⁺-pump and the Na⁺,K⁺-ATPase of neuronal tissues. The membrane potential of neurones in the paravertebral sympathetic ganglion of the bullfrog was recorded in K⁺-free Ringer's solution by means of the sucrose gap technique. The potassium activated hyperpolarization (K_H⁺), induced by the re-introduction of potassium, was used as an index of electrogenic Na⁺,K⁺-pumping. The K_H⁺ was blocked by 1 μM ouabain. Na⁺,K⁺-ATPase activity was measured in microsomal membrane preparations of frog and beef brain using a continuous spectrophotometric assay. Although ouabain consistently inhibited beef brain Na⁺,K⁺-ATPase (IC₅₀ = 2.2 μM), acid-acetone extracts prepared from guinea pig and beef brain produced only partial inhibition. Neither of the extracts significantly reduced the K_H⁺ of the frog ganglion. CMA inhibited Na⁺,K⁺-ATPase prepared from bullfrog brain and spinal cord with slightly greater potency (IC₅₀ = 4.5 μM) than did ouabain (IC₅₀ = 10 μM). In contrast, electrogenic Na⁺,K⁺-pumping (i.e. the K_H⁺) in the frog ganglion was not affected by this steroid. It is concluded that although both the extracts and CMA inhibited Na⁺,K⁺-ATPase, neither can be considered ouabain-like due to their failure to affect the electrogenic Na⁺,K⁺-pump in situ.     

Basic FGF attenuates amyloid β-peptide-induced oxidative stress, mitochondrial dysfunction, and impairment of Na/K-ATPase activity in hippocampal neurons

Basic fibroblast growth factor (bFGF) exhibits trophic activity for many populations of neurons in the brain, and can protect those neurons against excitotoxic, metabolic and oxidative insults. In Alzheimer's disease (AD), amyloid β-peptide (Aβ) fibrils accumulate in plaques which are associated with degenerating neurons. Aβ can be neurotoxic by a mechanism that appears to involve induction of oxidative stress and disruption of calcium homeostasis. Plaques in AD brain contain high levels of bFGF suggesting a possible modulatory role for bFGF in the neurodegenerative process. We now report that bFGF can protect cultured hippocampal neurons against Aβ25-35 toxicity by a mechanism that involves suppression of reactive oxygen species (ROS) accumulation and maintenance of Na⁺/K⁺-ATPase activity. Aβ25-35 induced lipid peroxidation, accumulation of H₂O₂, mitochondrial ROS accumulation, and a decrease in mitochondrial transmembrane potential; each of these effects of Aβ25-35 was abrogated in cultures pre-treated with bFGF. Na⁺/K⁺-ATPase activity was significantly reduced following exposure to Aβ25-35 in control cultures, but not in cultures pre-treated with bFGF. bFGF did not protect neurons from death induced by ouabain (a specific inhibitor of the Na⁺/K⁺-ATPase) or 4-hydroxynonenal (an aldehydic product of lipid peroxidation) consistent with a site of action of bFGF prior to induction of oxidative stress and impairment of ion-motive ATPases. By suppressing accumulation of oxyradicals, bFGF may slow Aβ-induced neurodegenerative cascades.     

Effects of fasting and insulin-induced hypoglycemia on brain cell membrane function and energy metabolism during hypoxia–ischemia in newborn piglets

This study was done to determine the effects of 12 h fasting-induced mild hypoglycemia (blood glucose 60 mg/dl) and insulin-induced moderate hypoglycemia (blood glucose 35 mg/dl) on brain cell membrane function and energy metabolism during hypoxia–ischemia in newborn piglets. Sixty-three ventilated piglets were divided into six groups; normoglycemic control (NC, n=8), fasting-induced mildly hypoglycemic control (FC, n=10), insulin-induced moderately hypoglycemic control (IC, n=10), normoglycemic/hypoxic–ischemic (NH, n=11), fasting-induced mildly hypoglycemic/hypoxic–ischemic (FH, n=12) and insulin-induced moderately hypoglycemic/hypoxic–ischemic (IH, n=12) group. Cerebral hypoxia–ischemia was induced by occlusion of bilateral common carotid arteries and simultaneous breathing with 8% oxygen for 30 min. The brain lactate level was elevated in NH group and this change was attenuated in FH and IH groups. The extent of cerebral lactic acidosis during hypoxic–ischemic insult showed significant positive correlation with blood glucose level (r=0.55, p<0.001). Cerebral Na⁺, K⁺-ATPase activity and concentrations of high-energy phosphate compounds were reduced in NH group and these changes were not ameliorated in FH or IH group. Cortical levels of conjugated dienes, measured as an index of lipid peroxidation of brain cell membrane, were significantly elevated in NH, FH and IH groups compared with NC, FC and IC groups and these increases were more profound in FH and IH with respect to NH. Blood glucose concentration showed significant inverse correlation with levels of conjugated dienes (r=−0.35, p<0.05). These findings suggest that, unlike in adults, mild or moderate hypoglycemia, regardless of methods of induction such as fasting or insulin-induced, during cerebral hypoxia–ischemia is not beneficial and may even be harmful in neonates.     

Glial contribution to seizure: K activation of (Na, K)-ATPase in bulk isolated glial cells and synaptosomes of epileptogenic cortex

Glial and neuronal (Na⁺, K⁺)-ATPase have dissimilar apparent affinities for K⁺ ions. Glial (Na⁺, K⁺)-ATPase is maximally activated by 20 mM K⁺ while neuronal (Na⁺, K⁺)-ATPase is maximally stimulated by 3–5 mM K⁺. Because this glial property may play an important role in the clearance of [K⁺]₀ during seizures, we investigated the K⁺ activation of (Na⁺, K⁺)-ATPase within bulk isolated glial cells and synaptosomes isolated from epileptogenic brains. The primary focus (F), the homolateral brain area around the focus (PF) and the mirror (M) or secondary focus induced by freezing lesions were studied.Results show that both glial and synaptosomal enzyme activities in the epileptogenic state change in comparison with controls, i.e. sham-operated cats. In F and M., glial enzyme decreased reaction velocities between 3 and 18 mM K⁺. In PF, maximum velocities increased in glial (Na⁺, K⁺)-ATPase. These results indicate that in actively firing epileptogenic tissue (F, M), glial (Na⁺, K⁺)-ATPase decreased rate reactions while the catalytic activity was increased in cortical tissues surrounding the focus. These phenomena appeared early, i.e. 1–3 days after production of the freezing lesion, and was associated with a sharp decrease in absolute levels of enzyme activity.Synaptosomal (Na⁺, K⁺)-ATPase from controls always exhibited a saturation curve at 3–6 mM K⁺. Synaptosomal enzyme activities in the primary (F) lesion increased slightly 24 h after lesion production, then progressively decreased 3 days after lesion production. No significant changes were seen in M and PF.     

Effect of induced hyperglycemia on brain cell membrane function and energy metabolism during the early phase of experimental meningitis in newborn piglets

This study was done to elucidate the mechanism of hypoglycorrhachia and elevated lactate concentrations leading to neuronal dysfunction in neonatal meningitis, and to determine the effects of induced hyperglycemia on these disturbances. Thirty-eight newborn piglets were divided into three groups: 12 in the control group (CG), 12 in the normoglycemic meningitis group (NG), and 14 in the hyperglycemic meningitis group (HG). Meningitis was induced by intracisternal injection of 108 cfu of Escherichia coli. Hyperglycemia (blood glucose 300–400 mg dl⁻¹) was induced and maintained for 60 min before induction of meningitis and throughout the experiment using modified glucose clamp technique. CSF-to-blood glucose ratio decreased significantly in NG. In HG, baseline CSF-to-blood glucose ratio was lower than two other groups, but increased at 1 h after induction of meningitis. CSF lactate concentration was increased progressively in both meningitis groups, and positively correlated with CSF leukocyte numbers (r=0.41, p<0.001) and TNF-α level (r=0.43, p<0.001). Brain glucose concentration was significantly increased in HG and showed inverse correlation with CSF leukocyte numbers (r=−0.59, p<0.01). Brain lactate concentration was not significantly different among three groups and positively correlated with the CSF TNF-α level (r=0.51, p<0.05). Lipid peroxidation products were increased in NG. Na⁺,K⁺-ATPase activity, ATP/PCr concentrations were not different among three groups. Increased intracranial pressure, CSF pleocytosis (214±59 vs. 437±214/mm³, p<0.02) and increased lipid peroxidation products observed in NG were reduced in HG. These results suggest that hypoglycorrhachia and elevated lactate concentration in the CSF during meningitis originates primarily from the increased anaerobic glycolysis in the subarachnoid space, induced by TNF-α and leukocytes. Induced hyperglycemia attenuates the inflammatory responses of meningitis and might be beneficial by providing an increased glucose delivery to meet its increased demand in meningitis.     

Amyloid β-peptides inhibit Na/K-ATPase: tissue slices versus primary cultures

Aβ 1-40 (20 μM) has been reported to selectively inhibit Na⁺/K⁺-ATPase activity in rat primary hippocampal cultures after 2–6 days of exposure [10]. We expanded these studies to include Aβ’s effects on Na⁺/K⁺-ATPase activity in rat primary cortical cultures and hippocampal slices, and we correlated these effects with estimates of cell survival in rat brain primary cultures. Using optimized assay conditions, a 5-day exposure to 50 μM Aβ 25-35, 20 μM Aβ 1-40, and 20 μM Aβ 1-42 decreased Na⁺/K⁺-ATPase activity in rat primary cortical cultures 66%, 60%, and 22%, respectively. Aβ 25-35 (50 μM) at 24 h was the only condition that caused inhibition of Na⁺/K⁺-ATPase activity in the absence of cell death, defined as an extracellular shift in the localization of the cytoplasmic enzyme lactate dehydrogenase (LDH). We also found that hippocampal slices were sensitive to Aβ, exhibiting a 40–60% reduction in membrane Na⁺/K⁺-ATPase activity when exposed to 1–30 nM of Aβ 1-40 for 60 min. This inhibition was not readily reversible, as it withstood homogenization and repeated dilution and centrifugation. Additionally, this inhibition occurred only after amyloid incubation with intact hippocampal slices, not with disrupted membranes. The inhibition of Na⁺/K⁺-ATPase in brain slices by physiological, low nM concentrations of Aβ 1-40 is consistent with effects on neurotransmitter release and intrasynaptosomal calcium responses [4 and 7].     

Alterations of cerebromicrovascular Na, K-ATPase activity due to fatty acids and acute hypertension

Acute hypertension, induced in rats by intravenous injection of angiotensin II, previously has been shown to increase cerebrovascular permeability to macromolecules. The purpose of this study was to examine the effect of acute hypertension on Na⁺, K⁺-ATPase, the enzyme responsible for controlling ionic permeability of the cerebromicrovascular endothelium. The K⁺-dependent p-nitrophenylphosphatase activity of the cerebromicrovascular Na⁺, K⁺-ATPase was determined using microvessels prepared from hypertensive and normotensive rats. When compared to controls, a 70% decrease (P < 0.02) in the maximum rate (V_max) of the Na⁺, K⁺-ATPase from hypertensive rats was evident with no change in the Michaelis constant (K_M). In contrast, γ-glutamyltranspeptidase, a marker enzyme for cerebral endothelic cells, was not significantly affected. Sodium arachidonate (1–100 μM) inhibited the phosphatase activity of the Na⁺, K⁺-ATPase in microvessels isolated from both normotensive and hypertensive rats in a dose-dependent manner. Furthermore, poly-unsaturated fatty acids (sodium linoleate and arachidonate) evoked the greatest inhibition of the enzyme, while sodium oleate and sodium palmitate inhibited the Na⁺, K⁺-ATPase to lesser extents. This regulation of enzyme activity by fatty acids was comparable in control and hypertensive groups. In summary, the data indicate that the cerebromicrovascular Na⁺, K⁺-ATPase was altered as a consequence of acute hypertension and that poly-unsaturated fatty acids can modulate this enzyme in microvessels derived from hypertensive or control rats     

Effect of K ions on kinetic properties of the (Na, K)-ATPase (EC 3.6.1.3) of bulk isolated glial cells, perikarya and synaptosomes from rabbit brain cortex

Progress curves of the enzymatic reactions show that ATPases of bulk isolated glial cells, perikarya and synaptosomes exhibit hysteretic change. Initial velocities of enzyme activities were therefore obtained according to the equation valid for the hysteretic model.The (Na⁺, K⁺)-ATPase activities of the same brain fractions were measured before or after NaI treatment. Only glial and synaptosomal enzyme could be adequately extracted by using this procedure. Attempts to purify the (Na⁺, K⁺)-ATPase from brain perikarya by NaI extraction were unsuccessful.In order to determine the effect of the K⁺ ions on enzymic physiological efficiency (phys. eff.; i.e., the ratio V_max/K_mapp) the variation of (Na⁺, K⁺)-ATPase activities from each brain fraction was measured as a function of Mg·ATP²⁻ concentration in the presence of 5 and 20 mM K⁺ ions. High K⁺ ion concentrations (20mM) increased the physiological efficiency of glial enzyme and decreased the same kinetic parameter in neuronal (perikaryal as well as synaptosomal) enzyme preparations.Results are discussed in relation to a possible distribution of distinct enzyme in different brain cell populations as well as a possible role of glial cells in an active regulation of K⁺ ion extracellular fluid in the CNS.     

Immunohistochemical localization of Na, K-ATPase in the choroid plexus

To determine if canine and rat choroid plexus Na⁺, K⁺-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified canine kidney medulla Na⁺, K⁺-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified canine kidney Na⁺, K⁺-ATPase and canine kidney microsomes were treated with antiserum followed by [¹²⁵I]protein A and autoradiography, the canine microsomes and purified Na⁺, K⁺-ATPase showed a prominent radioactive band coincident with the α-, β- and γ-subunits of the purified canine kidney enzyme.When the rabbit immunoglobulin that was purified from the Na⁺, K⁺-ATPase antiserum through DEAE-cellulose ion exchange chromatography was used for immunoperoxidase staining of the choroid plexus fixed with Bouin's fixative, intense immunoreactive staining was present on the epithelial cells of both choroid plexuses but was not found in the tissue around the vessel. The staining was especially confined to apical surfaces of the epithelial cells. The same procedure was performed in the canine kidney, and immunostaining was obtained in the tubules where Baskin and Stahl described the enzyme localization. No staining was seen with pre-immune serum of the normal rabbit. We concluded that both the canine and rat choroid plexus are rich in Na⁺, K⁺-ATPase, which plays an important role in cerebrospinal fluid (CSF) secretion.     

Stimulation by noradrenaline of Na+K+ ATPase in different fractions of rat brain cortex

Summary Distribution of Na⁺K⁺-ATPase (EC. 3.6.1.3) and its susceptibility to noradrenaline (NA) were studied on electronmicroscopically characterized subcellular fractions of rat brain cortex. The highest specific activity of Na⁺K⁺-ATPase was present in synaptosomal fraction disrupted in distilled water (29.52±5.53moles phosphate/mg protein/hour). In the presence of 1 mM EGTA significantly higher specific activity was determined in all fractions studied, except homogenate and synaptosome. The effect of NA was studied in concentration range from 10^–6-10^–3M. 10^–4M of NA produced the highest activation of the enzyme in different fractions. Also in the presence of EGTA NA was able to increase the enzyme activity. The effect of NA was much more marked on disrupted synaptosomal fraction. No qualitative differences have been found between the Na⁺K⁺-ATPase activities exhibited by the synaptosomal fraction and by other subcellular fractions with respect to susceptibility to NA. Therefore, it seems very probable that NA might modulate neurochemical transmission not only via an effect on nerve terminals but also via stimulating other part of the neuron.     

Hydrogen ion buffering during complete brain ischemia

As a first step to quantify [H⁺] changes in brain ischemia we used H⁺-selective microelectrodes and enzyme fluorometric techniques to describe the relationship between interstitial [H⁺] ([H⁺]₀) and peak tissue lactate after cardiac arrest. We found a step function relationship between [H⁺]₀ and tissue lactate rather than the linear titration expected in a homogeneous protein solution. Within a blood glucose range from 3–7 mM, brain lactate rose from 8–13 mmol/kg along with a rise in [H⁺]₀ of99 ± 6nM(0.44 ± 0.02pH At higher blood glucose levels (17–80 mM), brain lactate accumulated to levels of 16–31 mmol/kg; concurrently [H⁺]₀ rose by608 ± 16nM(1.07 ± 0.02pH). The unchanging level of [H⁺]₀ between 8–13 and 16–31 mmol/kg lactate implies that [H⁺]₀ is at a steady-state, but not equilibrium with respect to [H⁺] in other brain compartments. We propose that ion-transport characteristics of astroglia account for the observed relationship of [H⁺]₀ to tissue lactate during complete ischemia and suggest that brain infarction develops after plasma membranes in brain cells can no longer transport ions to regulate [H⁺].     

Action potential-like responses in B 104 cells with low Na channel densities

Action potential generation and Na⁺ currents were studied in B104 neuroblastoma cells in vitro using the whole-cell patch-clamp method in voltage-clamp and current-clamp mode. Action potential-like responses were elicited in 38 of 42 cells, with a threshold close to −55 mV for depolarizing stimuli, and −56 mV for anode-break stimuli. Response amplitudes were larger when cells were held at more negative prepulse potentials, and were well fit by a Boltzmann distribution with a midpoint of approx. −75 mV, close to theV_1/2 for Na⁺ current steady-state inactivation in these cells. Cells displaying action potential-like responses exhibited a peak Na⁺ current density of 133 ± 0.14 pA/pF (range, 10.2–296.2 pA/pF) and a lowg_K: g_Na ratio (0.0067 ± 0.0023). Exposure to 0.1 mM Cd²⁺ did not block the generation of action potential-like responses in B104 cells, while 1 μM TTX abolished the responses. We conclude that low densities of Na⁺ channels ( < 3/μm² and < 1/μm² in some cells) can support the generation of action potential-like responses in B104 cells if they are held at hyperpolarized levels to remove inactivation. The low leak and K⁺ conductance of these cells may contribute to their ability to generate action potential-like responses under these circumstances.     

Effect of allopurinol on NMDA receptor modification following recurrent asphyxia in newborn piglets

The present study tests the hypothesis that repeated episodes of asphyxia will lead to alterations in the characteristics of the N-methyl-d-aspartate (NMDA) receptor in the brain cell membrane of newborn piglets and that pre-treatment with allopurinol, a xanthine oxidase inhibitor, will prevent these modifications. Eighteen newborn piglets were studied. Six untreated and six allopurinol treated animals were subjected to eight asphyxial episodes and compared to six normoxic, normocapneic controls. Brain cell membrane Na⁺,K⁺-ATPase activity was determined to assess membrane function. Na⁺,K⁺-ATPase activity was decreased from control following asphyxia in both the untreated and treated animals (47.7±3.2 vs. 43.0±2.2 and 41.0±5.3 μmol Pi/mg protein/h, p<0.05, respectively). ³H-MK-801 binding studies were performed to measure NMDA receptor binding characteristics. The receptor density (B_max) in the untreated asphyxia group was decreased compared to control animals (0.80±0.11 vs. 1.13±0.33, p<0.05); furthermore, the dissociation constant (K_d) was also decreased (3.8±0.7 vs. 9.2±2.2, p<0.05), indicating an increase in receptor affinity. In contrast, B_max in the allopurinol treated asphyxia group was similar to control (1.06±0.37); and K_d was higher (lower affinity) than in the untreated group (6.5±1.4, p<0.05). The data indicate that recurrent asphyxial episodes lead to alterations in NMDA receptor characteristics; and that despite cell membrane dysfunction as seen by a decrease in Na⁺,K⁺-ATPase activity, allopurinol prevents modification of NMDA receptor–ion channel binding characteristics induced by repeated episodes of asphyxia.     

Brain (Na, K)-ATPase and nonadrenergic function: recovery of enzyme activity after norepinephrine depletion

We examined the time course of K⁺-p-nitrophenylphosphatase and ouabain binding associated with cerebral cortex (Na⁺,K⁺) -AT-Pase after depletion of norepinephrine. Norepinephrine depletion by the norepinephrine-selective neurotoxin DSP4 initially reduced the indices of (Na⁺,K⁺)-ATPase, with a significant correlation between ouabain binding and tissue norepinephrine levels 16 h after DSP4. Tissue norepinephrine content and DMI binding rapidly declined after DSP4 and remained essentially unchanged for at least 8 weeks. By contrast, the indices of (Na⁺,K⁺)-ATPase remained low for about two weeks but then gradually increased, returning to baseline levels by 8 weeks after DSP4. These data indicate that, while usually regulated in part by exposure to norepinephrine, brain (Na⁺,K⁺)-ATPase undergoes adaptation to prolonged noradrenergic depletion.     

The amiloride-sensitive Na/H exchange antiporter and control of intracellular pH in hippocampal brain slices

The intracellular pH, 7.54 ± 0.03 (mean ± S.D., n = 15), determined with the Neutral red method, of the hippocampal brain slice preparation under baseline incubation conditions is considerably more alkaline than the bath buffer pH. Neutralization by amiloride suggests that the alkalinity was due to Na⁺/H⁺ exchange antiporter activation. To characterize the brain Na⁺/H⁺ exchange antiporter we compared the inhibitory effects of MIA, amiloride and other 5-N substituted analogues on proton extrusion after acid loading by transient exposure to ammonium chloride in the isolated hippocampal brain slice preparation. The potencies of amiloride compounds on the initial recovery rate of intracellular pH after acid-loading were DMA > MIA > HMA = MHA ≥ IPA-HCl > IPA > MNPA = Amil > Benzamil. The greater potency of the 5-N substituted analogs of amiloride over amiloride and benzamil strongly suggest that Na⁺/H⁺ exchange antiporter is the mechanism responsible for alkalinization in the isolated hippocampal brain slice in vitro.     

Non-uniform expression of α subunit isoforms of the Na/K pump in rat dorsal root ganglia neurons

Tissue sections and antibodies selectively recognizing isoforms of the α subunit of the Na⁺/K⁺ pump were used to determine the expression of α₁, α₂ and α₃ pump isoforms in the plasma membrane of adult rat dorsal root ganglia (DRG) neurons. There was no detectable membrane signal from DRG neurons that were probed with antibodies to the α₂ isoform of the Na⁺/K⁺ pump. The α₁ isoform of the Na⁺/K⁺ pump was found in most (77±4%) studied DRG neurons, regardless of cell size. Only 16±7% of the neurons expressed a detectable level of the α₃ Na⁺/K⁺ pump and all were apparently from a subpopulation of large DRG neurons. Comparison of cell size distributions and a study of neurons identified in serial sections suggested that of the α₃ positive DRG neurons about 75% coexpressed the α₁ isoform of the Na⁺/K⁺ pump. These data show that the expression of the protein of the α subunit isoforms of the Na⁺/K⁺ pump is not uniform throughout the population of DRG neurons and that α₁ is the predominant isoform in the plasma membrane of these neurons.     

Nω-nitro-L-arginine methyl ester (L-NAME) attenuates the acute inflammatory responses and brain injury during the early phase of experimental Escherichia coli meningitis in the newborn piglet

Abstract

We evaluated the anti-inflammatory and neuroprotective effect of nonselective NOS inhibitor, N^ω-nitro-Larginine methyl ester (L-NAME), in experimental bacterial meningitis in the newborn piglet. Meningitis was induced by intracisternal injection of 10⁸ colony forming units of Escherichia coli. L-NAME 10 mg kg^-1 was given intravenously 30 min before induction of meningitis. L-NAME significantly attenuated the increase in intracranial pressure and decrease in cerebrospinal fluid glucose concentration observed in the meningitis group. Systemic and cerebral perfusion pressure were even higher compared to the control and meningitis groups. However, the meningitis-induced increase in tumor necrosis factor- α level, leukocyte numbers and lactate level in the cerebrospinal fluid was not significantly attenuated with L-NAME administration. Reduced cerebral cortical cell membrane Na⁺ ,K⁺-ATPase activity and increased lipid peroxidation products, indicative of meningitis-induced brain cell membrane dysfunction, were significantly improved with L-NAME treatment. Decreased brain glucose and ATP levels were also significantly improved with L-NAME treatment. These findings suggest that L-NAME was effective in attenuating the acute inflammatory responses and brain injury in neonatal bacterial meningitis. [Neurol Res 2001; 23: 862-868]     

Effects of lithium therapy on Na+-K+-ATPase activity and lipid peroxidation in bipolar disorder

Objectives

Oxidative stress induced lipid peroxidation along with a reduced Na⁺–K⁺-ATPase activity has been implicated in the pathophysiology of bipolar disorders (BPD). Although, lithium therapy results in significant improvement in the symptoms of the disease, studies regarding its effect on the altered sodium pump activity and lipid peroxidation status have come out with conflicting results. The present study was undertaken to evaluate the status of lipid peroxidation and analyze the role of lithium and Na⁺–K⁺-ATPase activity in its regulation in BPD patients in our region.

Method

We measured RBC membrane Na⁺–K⁺-ATPase activity and serum thiobarbituric acid reacting substances (TBARS) level in 73 BPD patients and serum lithium, in addition, in 48 patients receiving lithium therapy among them.

Results

Na⁺–K⁺-ATPase activity and serum TBARS level were significantly decreased and increased respectively in all BPD patients compared to age and sex matched healthy controls. Same trend was observed in the BPD patients stabilized on lithium therapy compared to the lithium naive ones. Although, the enzyme activity showed a reciprocal relationship with TBARS in all patients of BPD, a significant positive correlation and dependence of the enzyme activity was evident with serum lithium level only in the lithium stabilized BPD group.

Conclusions

BPD patients showed significantly compromised Na⁺–K⁺-ATPase activity and increased lipid peroxidation. Lithium induced improvement in the enzyme activity was associated with significant reduction in lipid peroxidation. Enhancement of the Na⁺–K⁺-ATPase activity by optimum dosage of lithium may be a potential contributing factor for reducing oxidative stress in BPD patients.