Effects of Acute and Chronic Phenytoin on the Electrolyte Content and the Activities of Na+, K+-, Ca2+, Mg2+ -, and HCO3--ATPases and Carbonic Anhydrase of Neonatal and Adult Rat Cerebral Cortex

Various parameters of anion and cation transport were measured in the cerebral cortex of neonatal (3-day-old) and adult rats following acute and chronic treatment with phenytoin (PHT). Acutely, PHT significantly inhibited the enzyme Na+, K+-ATPase in both neonatal and adult rats. This effect was accompanied by a significant increase in cerebral cortical Na+ content and a decrease in K+ content only in neonatal animals. Chronic treatment (two and four times a day for 7 days) of adult rats with PHT significantly reduced Na+ content without affecting whole homogenate Na+, K+-ATPase activity. The activity of this enzyme was markedly increased in the myelin- (glial product) and slightly decreased in the synaptosomal- (neuronal) fractions following chronic (four times a day for 7 days) PHT treatment. These results suggest that PHT differentially affects the two forms (neuronal and glial) of the enzyme Na+, K+-ATPase. The possible relevance of this hypothesis in relationship to the anticonvulsant and excitatory properties of PHT is discussed. Chronic (two and four times a day for 7 days) PHT treatment increased both DNA content and activity of the glial marker enzyme carbonic anhydrase. Activity of the mitochondrial enzyme HCO3- -ATPase was also increased following chronic PHT treatment. These two enzymes are intimately involved in the regulation of HCO3- -Cl- transport across glial cell and mitochondrial membranes, and these results suggest that PHT is able to affect beneficially glial regulatory processes. The ability to enhance glial regulation of anions and cations in extracellular fluid provides new and important insights into the mechanism of the anticonvulsant action of PHT.     

Electrophysiological characterization of a nicotinic acetylcholine receptor on leech neuropile glial cells

Ion-selective double-barrelled microelectrodes were used to measure the activities of intracellular K+, Na+, Cl-, and H+ (aiK, aiNa, aiCl, pHi) and membrane potential (Em) in neuropile glial cells as well as extracellular K+ activity (aeK) in the neuropile of the leech, Hirudo medicinalis, during bath application of carbachol. As measured with conventional single-barrelled microelectrodes, acetylcholine (ACh), nicotine, carbachol, tetramethylammonium (TMA), and choline elicited concentration-dependent (10(-6)-5 X 10(-3) M) transient membrane depolarizations of up to 60 mV amplitude whereas muscarine (10(-6)-10(-3) M) did not affect Em. alpha-Bungarotoxin (10(-7) M), decamethonium (10(-5) M), d-tubocurarine (5 X 10(-5) M), and strychnine (5 X 10(-5) M) blocked the carbachol depolarization by about 90%. Atropine (5 X 10(-5) M) blocked the response by about 75%, whereas hexamethonium was only effective at millimolar concentrations. Average baseline levels of aeK in the neuropile and of aiK, aiNa, and aiCl in the neuropile glial cells were about 3, 70, 10, and 7 mM, respectively. During the carbachol depolarization aeK and aiNa transiently increased, whereas aiK decreased. In contrast, a rise of aiK and a fall of aiNa were observed during glial depolarizations in solutions with elevated K+ concentration. aiCl increased during both the carbachol- and the K+-induced depolarization. During carbachol, pHi transiently fell by about 0.2 units from its average baseline level of 6.9, whereas an alkalinization of small amplitude was observed in high-K+ solutions. Bath-applied choline, TMA, and decamethonium rapidly accumulated in the neuropile glial cells as intracellularly monitored with double-barrelled microelectrodes filled with Corning K+ exchanger resin, which is highly selective for these agents. The results suggest that leech neuropile glial cells have a nicotinic ACh receptor coupled to a cation channel. It is hypothesized that this channel might also be permeable to choline, TMA, and decamethonium.     

Arachidonate activates muscle electrogenic sodium pump and brain microsome Na+,K+-ATPase under suboptimal conditions

Arachidonate 5 x 10(-5) mol.l-1 increased the rate of hyperpolarization induced in Na+-loaded mouse diaphragm fibers by 5 mmol.l-1 K+. When applied to Na+-loaded muscles without potassium, arachidonate 1 x 10(-6) and 5 x 10(-5) mol.l-1 induced a ouabain-sensitive hyperpolarization of the muscle fibers. The activity of rat brain microsomal Na+,K+-ATPase was stimulated by 1 x 10(-7)-5 x 10(-6) mol.l-1 arachidonate in reaction media with reduced amounts of ATP or K+ and after short-lasting sonication of the samples. It was concluded that, under particular conditions, arachidonate might serve as a Na+,K+-ATPase activator or inhibitor regulating its ion transport and electrogenicity.     

Contribution of Na+,K(+)-ATPase to focal epilepsy: a brief review.

The authors review some of their experimental data on the contribution of Na(+)- and K(+)-dependent adenosine triphosphatase (Na+,K(+)-ATPase) to focal epilepsy. It has been previously demonstrated that high extracellular K+ concentration increases glial Na+,K(+)-ATPase specific activities in normal conditions while this was not observed in neuronal preparations. At this time, it was hypothesized that this molecular mechanism could play a role in removing K+ released in the extracellular space during neuronal firing. These results have therefore been investigated in acute and chronic epileptogenic lesions of cats with freeze lesion. It was demonstrated that within the primary (F) and the secondary or 'mirror' (M) focus the K+ activation of the glial Na+,K(+)-ATPase dramatically decreased compared to both control animals (C) and the perifocal (PF) non epileptogenic area. Similar results were observed in man when using specimens of anterolateral temporal neocortex obtained during temporal lobectomies in patients with intractable temporal lobe epilepsy, compared with postmortem human specimens or control brain tissues. The modifications of the level of phosphorylation of partially purified Na+,K(+)-ATPase was also investigated in the epileptic cortex in these two experimental conditions. The catalytic subunits were resolved by sodium dodecylsulfate (SDS) gel electrophoresis and their phosphorylation levels were measured in the presence of various concentrations of K+ ions which dephosphorylate the catalytic subunit. K(+)-induced dephosphorylation was decreased in primary and secondary foci of acutely lesioned cats. Those alterations, due to a decreased affinity for K+, were limited to the alpha (-) subunit. In cats with chronic lesions, the dephosphorylating step of the Na+,K+-ATPase catalytic subunit recovered to normal affinity for K+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of morphine on Na+,K(+)-ATPase from homogenate of synaptosomes and of synaptic membrane of rat cerebral cortex

Effects of morphine on noradrenaline release from rat cerebrocortical synaptosomes and on the Na+,K(+)-ATPase activity in homogenates of synaptosomes and of synaptic membranes were examined. Both morphine (10(-3)-10(-5) M) and methionine-enkephalin (M-Enk; 10(-5) M) inhibited the enhanced [3H]noradrenaline [( 3H]NA) release evoked by high concentrations of K+ from synaptosomes and these inhibitory actions were antagonized by naloxone (10(-4), 10(-5) M). Morphine (10(-3)-10(-5) M) and M-Enk (10(-5) M) stimulated the Na+,K(+)-ATPase activity in homogenates of synaptosomes but not of synaptic membranes in the incubation medium containing 2.2 X 10(-6)-4.7 X 10(-7) M free Ca2+ and these stimulatory effects were antagonized by naloxone. In homogenates of synaptic membranes, the same concentrations of morphine and M-Enk stimulated the Na+,K(+)-ATPase activity suppressed by FeCl2 (5 X 10(-7) M) but not by CuCl2 nor ZnCl2, and these stimulatory effects were antagonized by naloxone. Significant levels of Fe2+ were liberated from synaptosomes during the preparation of synaptic membrane using distilled water. These results suggest that both morphine and M-Enk stimulate the suppressed Na+,K(+)-ATPase activity by interacting with Fe2+ at opioid receptor sites, and they may play a role in the suppression of membrane depolarization and/or the release of NA through their stimulatory action on the Na+,K(+)-ATPase activity probably suppressed by Fe2+ in the rat cerebral cortex.     

Glutamate and kainate increase intracellular sodium activity in leech neuropile glial cells

Na+-selective, double-barrelled microelectrodes were used to measure intracellular Na+ activity (aiNa) and membrane potential (Em) in neuropile glial cells of isolated segmental ganglia in the leech Hirudo medicinalis. Bath application of glutamate (10(-3) M) resulted in membrane depolarizations of about 5 mV and a concomitant increase of aiNa by between 2 and 10 mM. Kainate (10(-4) M) elicited depolarizations of up to 40 mV amplitude followed by a prominent after hyperpolarization. During kainate, aiNa increased by 7 to 25 mM. In contrast to glutamate, an initial decrease of aiNa was detected during the action of kainate. N-methyl-D-aspartate (NMDA, 10(-5)-10(-3) M) had no effect of Em and aiNa. The results indicate that leech glial cells have a kainate-preferring non-NMDA glutamate receptor.     

Separate neuronal and glial Na+,K+-ATPase isoforms regulate glucose utilization in response to membrane depolarization and elevated extracellular potassium.

The role of cell type-specific Na+,K+-ATPase isozymes in function-related glucose metabolism was studied using differentiated rat brain cell aggregate cultures. In mixed neuron-glia cultures, glucose utilization, determined by measuring the rate of radiolabeled 2-deoxyglucose accumulation, was markedly stimulated by the voltage-dependent sodium channel agonist veratridine (0.75 micromol/L), as well as by glutamate (100 micromol/L) and the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) (10 micromol/L). Significant stimulation also was elicited by elevated extracellular potassium (12 mmol/L KCl), which was even more pronounced at 30 mmol/L KCl. In neuron-enriched cultures, a similar stimulation of glucose utilization was obtained with veratridine, specific ionotropic glutamate receptor agonists, and 30 mmol/L but not 12 mmol/L KCl. The effects of veratridine, glutamate, and NMDA were blocked by specific antagonists (tetrodotoxin, CNQX, or MK801, respectively). Low concentrations of ouabain (10(-6) mol/L) prevented stimulation by the depolarizing agents but reduced only partially the response to 12 mmol/L KCl. Together with previous data showing cell type-specific expression of Na+,K+-ATPase subunit isoforms in these cultures, the current results support the view that distinct isoforms of Na+,K+-ATPase regulate glucose utilization in neurons in response to membrane depolarization, and in glial cells in response to elevated extracellular potassium.     

The Na-K pump in neuropile glial cells of the medicinal leech

The membrane potential of neuropile glial (NG) cells in the central nervous system of the medicinal leech and the K+ concentration in extracellular spaces (ECS) of the neuropile were measured under various experimental conditions to determine properties of a glial Na+-K+ pump. The ganglia were exposed to K+-free saline thereby loading the NG cells with intracellular Na+. Their membranes hyperpolarized transiently when the K+-free solution was replaced by a bathing medium with normal (= 4 mM) K+ concentration. The hyperpolarization increased in amplitude with time of exposure to K+ -free solution and could be abolished by ouabain or by replacing Na+ with Li+. The transient membrane hyperpolarization could not be attributed to K+ depletion in the ECS of the neuropile or to changes in membrane input conductance. In a (bathing) medium containing 5 X 10(-4) M ouabain, the K+ concentration in the ECS increased transiently, and the NG cell membrane depolarized rapidly. This short-term depolarization (duration 2-3 min) was followed by a second long-term depolarization (duration 15 min) of the NG cell membrane, which reached a steady-state 20 min after ouabain application. In a bathing medium with elevated external K+ concentrations, the amplitude of the membrane depolarization was enhanced by ouabain. This depolarizing ouabain effect was a result of K+ accumulation in the ECS. We conclude that the Na+-K+ pump does not contribute directly to the resting membrane potential of NG cells and is not directly involved in K+ homeostasis at the cellular level.     

The effect of vanadate on Na, K-ATPase activity of mouse cerebral cortex during bicuculline-induced seizures

The effect of bicuculline-induced seizures on Na+,K+-ATPase activity of mouse cerebral cortex homogenates, using two different procedures of sample preparation (freezing in situ or decapitation of animals without freezing) is described. Regardless of tissue treatment Na+,K+-ATPase activities during bicuculline-induced seizures did not differ significantly from the appropriate controls when vanadate-free ATP was used as substrate. The response of Na+,K+-ATPase to K+ activation was also similar; the increase in potassium concentration from 2 to 20 mM caused a 33.0 and 32.3% increase of enzyme activity in cortical homogenates from control and convulsing mice, respectively. Vanadate added to the assay medium inhibited Na+,K+-ATPase activity in a dose-dependent manner; with both types of tissue treatment there was, however, a tendency towards lesser inhibition of the enzyme from convulsing mice and at 1 X 10(-7) M vanadate this difference, though slight, was statistically significant: -22.59 vs -27.55% (freezing) and -28.73 vs -38.42% (decapitation) for seizures vs controls, respectively. The reduced sensitivity of Na+,K+-ATPase towards vanadate inhibition in cortical homogenates prepared from mice with convulsions suggests that vanadate might play a role in the modulation of enzyme activity during seizures in vivo.     

Spatial learning transiently disturbed by intraventricular administration of ouabain

The presence of sodium-potassium-adenosine triphosphatase (Na+,K+-ATPase) on the surface of arachnoid cells indicates that active transport of electrolytes and water occurs there. Previously, we accidentally found that intraventricular administration of TGF-beta1 impaired rat spatial learning. Levels of Na+,K+ -ATPase were decreased in arachnoid cells with fibrosis. To characterize the role of the Na+,K+ -ATPase, Wistar rats were intraventricularly administered a total of 200 microl of ouabain, at concentrations of 10(-5), 10(-4) and 10(-3) M, for one week with an osmotic pump, and were examined with a Morris water maze. Latency for reaching the platform did not significantly differ between ouabain-administered rats and controls. Spatial learning was impaired in a dose-dependent manner. Na+,K+ -ATPase activity of arachnoid cells ceased during ouabain administration, and recovered completely three weeks after the end of ouabain administration. The present results suggest that the Na+,K+ -ATPase on the surface of arachnoid cells contributes to maintenance of rat spatial learning.