Water and Electrolyte Contents, Cell pH, and Membrane Potential of Primary Cultures of Astrocytes from DBA, C57, and SW Mice

Some basic properties of primary cultures of astrocytes derived from the cerebral cortex of an audiogenic seizure-sensitive strain of mice, DBA/2J (DBA), were studied with different approaches. The results were compared with those of audiogenic seizure-resistant strains, C57BL/6J (C57) and Swiss Webster (SW). Contents of intracellular water, protein, and DNA of DBA astrocytes were 0.673 +/- 0.019 ml/g cells, 0.082 +/- 0.006 g/g cells, and 0.0072 +/- 0.0005 g/g cells, respectively. These results are not different from those of either C57 or SW astrocytes. Intracellular concentration of K+, Na+, and Cl- ([K+]1, [Na+]1, and [Cl-]1) derived from the flame photometric and from the radioisotope uptake data of DBA astrocytes were 120.4 +/- 8.5, 25.9 +/- 3.2, and 26.8 +/- 1.8 mM/L cell H2O, respectively. [Na+]1 and [Cl-]1 in DBA astrocytes were lower than those in C57 and SW astrocytes. In DBA astrocytes, SITS decreased the cell/medium ratio (C/M) of 36Cl- and increased the C/M of 125I-; ouabain increased the C/M of 22Na+ and decreased the C/M of 125I-; bumetanide decreased the C/M of both 36Cl- and 22Na+; and NaClO4 decreased the C/M of 125I-. Similar results were observed in both C57 and SW astrocytes. Intracellular pH (pHi) as determined with 14C-DMO of astrocytes in HEPES-buffered saline solution averaged 7.04 +/- 0.03 for DBA, 7.01 +/- 0.02 for C57, and 6.97 +/- 0.02 for SW mice when pH of medium was maintained at 7.4. Modification of ion (HCO3-, Cl-, Na+, and K+) concentration and pH of culture medium all changed the pHi of astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH regulation after acid load in primary cultures of mouse astrocytes

Intracellular pH (pHi) recovery in primary cultures of mouse astrocytes after acid-loading was studied with the ion transport inhibitors (amiloride, SITS, acetazolamide, ouabain and bumetanide), and by reducing the concentration of Na+ or Cl- in HCO3- -free HEPES-buffered (HEPES) and in HCO3-/CO2 Hanks' balanced salt solution (HBSS). The pHi of astrocytes exposed to 15 mM NH4Cl decreased abruptly and began to recover slowly after 5 min. Exposure of the cells to NH4Cl for 2 min and reincubation in HEPES HBSS decreased pHi further within 1-2 min after removal of NH4Cl; pHi then recovered toward the control value. Cultures exposed to HCO3-/CO2 HBSS (10 mM/2%) showed changes in pHi in the opposite direction. These responses are unique to astrocytes and differ from those occurring in most other cells. Recovery of pHi after NH4Cl prepulse was markedly inhibited in low-Na+ and in amiloride-containing HEPES HBSS. Ouabain also reduced pHi recovery rate; however, SITS, acetazolamide and bumetanide did not. Therefore, Na(+)-H+ exchange is the major process for pHi recovery from acidification in HCO3- -free solution. In HCO3-/CO2 HBSS pHi recovery was markedly inhibited by SITS and acetazolamide, but not by amiloride, ouabain, or bumetanide. The inhibitory effect of SITS on pHi recovery was enhanced in low-Na+ HBSS. These results indicate that both Na+ and HCO3- are directly related to pHi recovery in HCO3-/CO2 solution after acid-load. Low-Cl HEPES HBSS and low-Cl HCO3-/CO2 HBSS media did not alter pH recovery rate. Thus, pHi recovery after acid-load is not Cl- -dependent, and therefore, does not involve a Na(+)-dependent Cl- -HCO3- exchange process. It appears that mouse astrocytes possess 3 acid-regulating systems: Na(+)-H+ exchange, Na(+)-HCO3- co-transport and Na(+)-independent Cl- -HCO3- exchange.     

Mechanisms of pH recovery from intracellular acid loads in the leech connective glial cell.

We used double-barrelled, neutral carrier, pH-sensitive microelectrodes to study the mechanisms by which the intracellular pH (pHi) is regulated in the connective glial cells of the medicinal leech. In HEPES-buffered, nominally CO2/HCO3(-)-free solutions the recovery of pHi from intracellular acidosis is Na(+)-dependent and reduced by at least half in the presence of amiloride, suggesting the action of Na+:H+ exchange. The rate of pHi recovery by this mechanism can be increased by raising the extracellular buffering power or by increasing extracellular pH. The presence of CO2/HCO3(-)-greatly increases the rate of pHi recovery from intracellular acidosis. This CO2/HCO3(-)-stimulated recovery is also dependent on external Na+, largely Cl(-)-independent, inhibited by DIDS, and accompanied by membrane hyperpolarization. This is consistent with it being mediated by the electrogenic cotransport of Na+ and HCO3- into the cells. A Cl(-)-dependent component to Na(+)- and HCO3(-)-dependent regulation is most easily explained by the added presence of a Na(+)-dependent exchange of HCO3- and Cl-.     

Sodium-bicarbonate cotransport in retinal Müller (glial) cells of the salamander.

An electrogenic Na+/HCO3- cotransport system was studied in freshly dissociated Müller cells of the salamander retina. Cotransporter currents were recorded from isolated cells using the whole-cell, voltage-clamp technique following the block of K+ conductance with external Ba2+ and internal Cs+. At constant pHo, an outward current was evoked when extracellular HCO3- concentration was raised by pressure ejecting a HCO3(-)-buffered solution onto the surface of cells bathed in nominally HCO3(-)-free solution. The HCO3(-)-evoked outward current was reduced to 4.4% of control by 0.5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate), to 28.8% of control by 2 mM DNDS (4,4'-dinitrostilbene-2,2'-disulfonate), and to 28.4% of control by 2 mM harmaline. Substitution of choline for Na+ in bath and ejection solutions reduced the response to 1.3% of control. Bicarbonate-evoked currents of normal magnitude were recorded when methane sulfonate was substituted for Cl- in bath, ejection, and intracellular solutions. Similarly, an outward current was evoked when extracellular Na+ concentration was raised in the presence of HCO3-. The Na(+)-evoked response was reduced to 16.2% of control by 2 mM DNDS and was abolished by removal of HCO3- from bath and ejection solutions. Taken together, these results (block by stilbenes and harmaline, HCO3- and Na+ dependence, Cl- independence) indicate that salamander Müller cells possess an electrogenic Na+/HCO3- cotransport system. Na+/HCO3- cotransporter sites were localized primarily at the endfoot region of Müller cells. Ejection of HCO3- onto the endfoot evoked outward currents 10 times larger than currents evoked by ejections onto the opposite (distal) end of the cell. The reversal potential of the cotransporter was determined by DNDS block of cotransport current. In the absence of a transmembrane HCO3- gradient, the reversal potential varied systematically as a function of the transmembrane Na+ gradient. The reversal potential was -0.1 mV for a [Na+]o:[Na+]i ratio of 1:1 and -25.2 mV for a Na+ gradient ratio of 7.4:1. Based on these values, the estimated stoichiometry of the cotransporter was 2.80 +/- 0.13:1 (HCO3-:Na+). Possible functions of the glial cell Na+/HCO3- cotransporter, including the regulation of CO2 in the retina and the regulation of cerebral blood flow, are discussed.     

Thrombin stimulation of Cl-/HCO3- exchange in human platelets

The presence of one acidifying Cl-/HCO3- exchange mechanism in human platelets has not been previously reported. This paper demonstrates that this mechanism does function and that it increases its activity after stimulation with thrombin. On resuspension of BCECF-loaded platelets in a chloride-free medium (gluconate replaced) that contains bicarbonate, cytosolic pH (pHi) increased and stabilized after 10 min at an alkaline value. After addition of 50 mM NaCl, pHi fell rapidly reaching steady state in the succeeding 5 min. The stilbene derivative 4-acetamido-4'-isothiocyanato stilbene-2,2' disulfonic acid (SITS) inhibited both, the alkalization in chloride-poor solution and the recovery from the alkaline load after chloride enrichment. The decline in pHi was observed whether chloride was delivered to the solution in the form of LiCl or NaCl, or when the later was applied after blockage of the Na+/H+ exchanger. The recovery in chloride-containing solution was in contrast to the effect of a similar change in osmolarity by addition of 50 mM sodium gluconate that did not produced a significant variation of pHi. Posterior addition of NaCl after 5 min in high gluconate reproduced the pHi fall of the control experiment. Alkali loads produced by 25 mM trimethylamine hydrochloride (TMA) were also counteracted by HCO(3-)-equivalent efflux via Cl-/HCO3- exchange. One of the major observations of the present study is that HCO3- equivalent efflux was twice as high when the platelets were previously stimulated with 0.1 IU of thrombin, but thrombin did not produce significant changes of the pHi recovery rate in a bicarbonate-free solution. The increase of the decline in pHi elicited by preexposure to thrombin was still observed in the presence of an inhibitor of the Na+/H+ exchange or in sodium-free solutions. It is concluded that a Na-independent Cl-/HCO3- exchange mechanism mediates the recovery of pHi from alkalosis in platelets and that thrombin activates this exchanger by a direct regulatory pathway.     

Astrocytes fail to regulate intracellular pH at moderately reduced extracellular pH.

Microspectrofluorometry was used to study the regulation of intracellular pH (pHi) in 2'-7'-bis (carboxyethyl-)-5,6-carboxyfluorescein (BCECF)-loaded astrocytes. They rapidly regulated an acid transient induced by an NH4+ prepulse. This back regulation was blocked by removal of Na+, or by addition of amiloride, but was also inhibited when extracellular pH (pHe) was lowered. Furthermore, when cells were exposed to HEPES buffer with reduced or increased pHe, pHi changed in parallel. Thus, although the cells possess an efficient H+ extrusion mechanism they fail to regulate pHi to a normal value unless pHe is held constant. The results challenge the concept of a H+ regulatory site at the internal side of the exchanger regulating pHi to a constant value.     

Increases in [Ca2+]i and changes in intracellular pH during chemical anoxia in mouse neocortical neurons in primary culture.

The effect of chemical anoxia (azide) in the presence of glucose on the free intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) in mouse neocortical neurons was investigated using Fura-2 and BCECF. Anoxia induced a reversible increase in [Ca2+]i which was significantly inhibited in nominally Ca2+-free medium. A change in pHo (8.2 or 6.6), or addition of NMDA and non-NMDA receptor antagonists (D-AP5 and CNQX) in combination, significantly reduced the increase in [Ca2+]i, pointing to a protective effect of extracellular alkalosis or acidosis, and involvement of excitatory amino acids. An initial anoxia-induced acidification was observed under all experimental conditions. In the control situation, this acidification was followed by a recovery/alkalinization of pHi in about 50% of the cells, a few cells showed no recovery, and some showed further acidification. EIPA, an inhibitor of Na+/H+ exchangers, prevented alkalinization, pointing towards anoxia-induced activation of a Na+/H+ exchanger. In a nominally Ca2+-free medium, the initial acidification was followed by a significant alkalinization. At pHo 8.2, the alkalinization was significantly increased, while at pHo 6.2, the initial acidification was followed by further acidification in about 50% of the cells, and by no further change in the remaining cells.     

Subcellular distribution of carbonic anhydrase and Na+, K+- and HCO3--ATPases in brains of DBA and C57 mice

DBA/2J mice are susceptible to audiogenic seizures (ASs) in an age-dependent manner, susceptibility being maximal at 21 days of age and declining thereafter. DBA, as compared with AS-resistant C57BL/6J (C57) mice, had higher carbonic anhydrase (CA) activity in cerebral cortex, brainstem, and cerebellum homogenates at 21 days of age. CA activity was also increased in cytosolic (82%), microsomal (167%), and myelin (68%) subcellular fractions from cerebral cortex, and in cytosolic (51%) and mitochondrial (102%) fractions from brainstem of DBA mice at 21 days of age. In addition, DBA mice had a higher Na+, K+-ATPase activity in myelin from cerebral cortex, and a lower HCO3--ATPase activity in mitochondria from brainstem. The differences in CA activity in the cerebral cortex and in HCO3--ATPase were not present at 110 days of age, when DBA mice are no longer susceptible to ASs. Because CA and HCO3--ATPase are involved in maintaining a proper ionic environment for neuronal function, these data suggest that alterations in activity of these enzymes are related to the age-dependent changes in AS susceptibility in DBA mice.     

Behavior of extracellular K+ and pH in skate (Raja erinacea) cerebellum

Ion-selective microelectrodes were used to measure extracellular K+ concentrations ([K+]o) and extracellular pH (pHo) in skate cerebellum under resting and stimulated conditions. Consistent with earlier ion analysis of elasmobranch cerebrospinal fluid (CSF), [K+]o was 3.6 +/- 0.1 mM. During parallel fiber activation, [K+]o increased to an upper limit of 12-14 mM with an approximately linear dependence on stimulation frequency (1-20 Hz). Post-stimulus undershoots of 0.1-0.6 mM were seen throughout an animal temperature range of 13-18 degrees C. When stimulation produced spreading depression (SD), [K+]o first increased to about 10 mM, then rose more rapidly to about 30 mM. These observations indicate a K+ ceiling of 10-12 mM in elasmobranchs. This ceiling is the same as that seen in mammals, despite marked differences in CSF composition and osmolality between mammalian and elasmobranch species. Extracellular pH (resting pHo was 7.1-7.3) was also altered during parallel fiber stimulation. An initial alkaline shift and subsequent extracellular acidification were characteristic of the response. These pHo transients were similar to those reported in other preparations, although the alkaline shift was enhanced. This may be attributed to the relatively low buffering capacity of elasmobranch CSF and to summation with a generally smaller acid shift.     

Effect of Milacemide on Audiogenic Seizures and Cortical (Na+, K+)-ATPase of DBA/2J Mice

Milacemide (MLM, CP 1552 S, 2-N-pentylaminoacetamide), a glycinamide derivative, is currently being evaluated clinically for antiepileptic activity. Anticonvulsant properties have been shown in various animal models, but the mechanism of action of MLM is unclear. We studied its activity in audiogenic seizures of DBA/2J mice. MLM was effective in inhibiting the convulsions induced by sound with a biphasic dose-effect relation. The ED50 was 109 mg/kg orally against tonic extension. Higher doses were necessary to abolish clonic convulsion and running response. Because impaired cerebral (Na+, K+)-ATPase activity is supposed to play a role in epileptogenesis, we tested MLM on in vitro cortical enzymatic activity of DBA/2J mice. Basal (Na+, K+)-ATPase activity was unchanged by several concentrations of MLM in normal C57BL/6J and audiogenic DBA/2J mice. K+ activation (from 3 to 18 mM) of (Na+, K+)-ATPase is abolished in DBA/2J mice as compared with C57BL/6J mice, suggesting impaired glial (Na+, K+)-ATPase. In the presence of MLM (from 30 to 1000 mg/L), cortical (Na+, K+)-ATPase of DBA/2J mice is activated by high concentrations of K+, as in C57BL/6J mice. Results suggest that the antiepileptic activity of MLM in audiogenic mice may be secondary to an activation of a deficient glial (Na+, K+)-ATPase.     

Phosphorylation of brain (Na+,K+)-ATPase alpha catalytic subunits in normal and epileptic cerebral cortex: I. The audiogenic mice and the cat with a freeze lesion.

Partially purified (Na+,K+)-ATPase (E.C. 3.6.1.3.) was investigated in the epileptic cortex of audiogenic DBA/2 mice and in the primary and secondary foci of cats with acute or chronic freeze lesions. No differences in specific activities measured at 3 mM K+ were observed between epileptic and control cortex, except an increase of enzymic activities in the primary focus of acutely lesioned cats. The (Na+,K+)-ATPase catalytic subunits were resolved by SDS-gel electrophoresis and their phosphorylation levels were measured in presence of K+ ions and phenytoin. K+ was more effective in inducing maximal dephosphorylation of (Na+,K+)-ATPase in C57/BL, with identical affinity in the two strains. Phenytoin decreased the net phosphorylation level of (Na+,K+)-ATPase by about 50% in C57/BL mice, but only by 20% in DBA/2 mice. Both K+ and phenytoin dephosphorylating influences were decreased in primary and secondary foci of acutely lesioned cats. Those changes were limited to the alpha(-) subunit. In chronic cats, the dephosphorylating step of the (Na+,K+)-ATPase catalytic subunit recovered a normal affinity to K+, but its sensitivity to phenytoin remained decreased. Those differences in K+ and phenytoin influences on brain (Na+,K+)-ATPases between control and epileptic cortex might be responsible for the ictal transformation and seizure spread. In cats, the alteration of the alpha(-) isoform could mainly affect the glial cells.     

Volume-sensitive release of taurine from cultured astrocytes: properties and mechanism

Release of taurine in response to cell swelling induced by hyposmolarity was observed in cultured astrocytes. Efflux of 3H-taurine increased by 30% and 70% upon reductions in osmolarity of only 5% and 10%. Reductions in osmolarity of 20%, 30%, and 50% stimulated basal taurine release by 300%, 500%, and 1,500%, respectively. The properties of this volume-sensitive release of taurine were examined to investigate: 1) its association with K+ and Cl- fluxes, currently activated during volume regulation: 2) its relationship with Ca2(+)-dependent reactions; and 3) the mechanism of the taurine efflux process. Taurine release was unaffected by removal of Na+, Ca2+, or Cl-, by pimozide and trifluoperazine, or by agents disrupting the cytoskeleton. The K+ channel inhibitors barium, quinidine, tetraethylammonium, and gadolinium had no effect. Taurine release was reduced by furosemide, a blocker of K+/Cl- cotransport, but not by the more specific inhibitor, bumetanide. It was markedly reduced by the inhibitors of Cl- channels DIDS, SITS, and anthracene-9-carboxylate. Taurine efflux was pH-dependent, being reduced at low pH values. It was decreased at 4 degrees C but not at 14 degrees C or 20 degrees C. These results suggest that the volume-sensitive release of taurine is independent of K+ fluxes but may be associated with Cl- conductances. It also seems unrelated to Ca2(+)-dependent transduction mechanisms. The Na(+)-dependent taurine carrier apparently is not involved in the swelling-induced release process.     

The effects of temperature and inhibitors on HCO3-stimulated swelling and ion uptake of monkey cerebral cortex.

In the presence of high concentrations of K+, additions of HCO3- as low as 0.35 mM caused a 23% increase in swelling, and concomitant increases in the chloride content of incubating monkey cerebrocortical slices. The uptake of chloride was accompanied by increased uptake of sodium and was highly temperature dependent, showing a marked activation at approximately 30 degrees C. A similar temperature activation was also found for a Mg2+-dependent, HCO3-stimulated ATPase activity in monkey cerebral cortex, consistent with a possible role for this enzyme in the K+ and HCO3-dependent swelling process and its associated ion movements. K+-dependent, HCO3-stimulated cerebrocortical tissue swelling with uptake of Na+ and Cl- was inhibited by acetazolamide indicating that carbonic anhydrase was also involved. The addition of ouabain also inhibited swelling and K+ and Cl- uptake at low concentrations, but led to increased swelling at higher concentrations ( greater than 10 mum). A similar biphasic effect on swelling was also seen following addition of ethacrynic acid.     

Sodium-bicarbonate cotransport in retinal astrocytes and Müller cells of the rat.

Sodium-bicarbonate cotransport in retinal glial cells was studied in the everted eyecup preparation of the rat. Intracellular pH was monitored with the indicator dye BCPCF and fluorescence confocal microscopy. Raising the K+ concentration from 3 to 12 mM in HCO3- -buffered perfusate evoked an intracellular alkalinization in both astrocytes and Müller cells. The alkalinization developed more rapidly and was larger in astrocytes. The K+ -induced alkalinization was HCO3- -dependent; it was reduced by 33% in astrocytes and 71% in Müller cells when HCO3- was removed from the perfusate. The alkalinization was effectively blocked by addition of 0.5 mM 4,4"-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). Removal of Na+ from the perfusate evoked a rapid acidification in both types of glial cells. The results indicate that astrocytes and Müller cells in situ in the rat retina possess an electrogenic Na+/HCO3- cotransporter.     

Na(+)- and Ca(2+)-dependent pH regulation in unstimulated human platelets.

The influence of transmembrane Na+ and Ca2+ gradients on cytosolic pH (pHi) and free Ca2+ concentration ([Ca2+]i) have been examined in unstimulated human platelets with the aid of BCECF and Fura-2 fluorescent dyes. The removal of external Na+ (Na+o) acidified the cytosol in a pHo-dependent manner which was insensitive to EIPA and DIDS, the inhibitors of the Na+/H+ exchanger and bicarbonate transporters. Na+o removal also increased [Ca2+]i by 17 +/- 5%, but the amplitude of the concomitant acidification was independent on Ca2+ influx or cytosolic Ca2+ concentration. In contrast, in the presence of 145mM Na+o, a rise in external Ca2+ concentration from 1 to 2mM increased [Ca2+]i by 38 +/- 11% and acidified the cytosol by 0.16 +/- 0.04 pH units. These results indicated that, in resting human platelets, the transmembrane Na+ gradient is a major determinant of pHi. Two Na(+)-dependent processes have been found: one is triggered by an external acidification and the other activated by a rise in Ca2+ influx or cytosolic concentration.     

Intracellular pH shifts capable of uncoupling cultured oligodendrocytes are seen only in low HCO3- solution

Electrical coupling between cultured mouse oligodendrocytes was transiently blocked when pHi was decreased below about 6.5 using the NH4+ prepulse method. This uncoupling could, however, only be achieved if the dominant pHi regulating mechanism in these cells, the Na+/HCO3- cotransporter, was blocked by lowering bath [HCO3-]. Under this condition, an NH4+ prepulse caused pHi to decrease toward the passive distribution for H+ (i.e., about pH 6.2). In the presence of normal bath [HCO3-] an NH4+ prepulse did not decrease pHi below 6.5 even when the second pHi regulating mechanism, the Na+/H+ exchanger, was blocked by amiloride, and consequently oligodendrocytes could not be uncoupled. Increasing CO2, which uncouples glial cells in situ (Connors et al: J. Neurosci. 4:1324-1330, 1984), did not uncouple cultured oligodendrocytes in the presence of normal bath [HCO3-], but did cause uncoupling in low [HCO3-] solution. These results indicate that electrical coupling between cultured oligodendrocytes is sensitive to pHi; in normal bath [HCO3-], however, the pHi regulation of these cells is so effective that standard techniques for intracellular acidification are unable to lower pHi to levels which cause the closure of oligodendrocyte gap junctions.     

Studies on uptake of gamma-aminobutyric acid by mouse brain particles; toward the development of a model

Several substances were studied for their effect on enhancement and/or inhibition of uptake of GABA into a mouse brain microsomal fraction (P3) at pH 7.3 in the presence and absence of buffer. These were diverse: Na+, K+, NH4+, Hg2+, Cl-, and HCO3-; beta-guanidinopropionic and L-2,3-diaminopropionic acids and 1,2-diaminoethane; pyridine and several methylated pyridines; chlorpromazine and ketamine; and melittin. Kinetic experiments tested these substances for competition with GABA and Na+. Assuming the GABA transporter to consist of a GABA recognition entity and a Na+- and Cl-dependent protein required for its activity, a minimal provisional model for the GABA uptake process is proposed that is consistent with all current data and with relevant observations in the literature. It accounts for the activational effects of proton removal on GABA uptake, the stoichiometry of 2 Na+ and 1 Cl- associated with uptake of one GABA molecule, and the types of inhibition of uptake shown by the substances listed above. Factors are considered that may be necessary to maintain the transporter in a GABA-receptive configuration and that allow it the freedom of movement to undergo the structural variations necessary for the transport process to take place at rates that may be regulated by environmental factors.     

Brain cortical (Na+ K+)-ATPase in epilepsy. A biochemical study in animals and humans

(Na+, K+)-ATPase (E.C.3.6.1.3) was partially purified from the cerebral cortex of audiogenic DBA/2 mice, from the primary and secondary epileptogenic foci of cats with a freeze lesion and from normal and epileptic human cortices. No differences in the specific activities of the microsomal enzyme were observed between normal and epileptic cortex. The influence of K+ ions and phenytoin, a potent antiepileptic drug, was then studied on the phosphorylation level of (Na+, K+)-ATPase alpha(+) (neuronal) and alpha(-) (non-neuronal) catalytic subunits resolved by SDS-gel electrophoresis. In normal cortex, the apparent affinity of the non-neuronal enzyme to K+ ions was reduced compared to the affinity of the neuronal enzyme. Phenytoin decreased the phosphorylation level of (Na+, K+)-ATPase purified from non-epileptogenic cortex of control C57/BL mice, cats and human patients. In fact, the drug induced the dephosphorylation of the (Na+, K+)-ATPase catalytic subunits, mainly of its alpha(-), non-neuronal subtype. In the cortex of audiogenic DBA/2 mice, K+ ions induced the dephosphorylation of (Na+, K+)-ATPase, with the same affinity as in control C57/BL mice. The dephosphorylating influence of phenytoin was however much decreased. In the primary and secondary foci of lesioned cats, both K+ and phenytoin dephosphorylating influences were decreased. Those changes were especially valid for the alpha(-), non-neuronal subunit. In human epileptic cortex, the (Na+, K+)-ATPase catalytic subunit had a decreased affinity to K+, as well as it lost its sensitivity to phenytoin dephosphorylation. Those results confirm the existence of two molecular forms of (Na+, K+)-ATPase in animal and human brain cortex. Those two forms, the neuronal and the non-neuronal or glial (Na+, K+)-ATPases, differ at least by their K+ regulation and their phenytoin sensitivity. Phenytoin studies also suggest that the drug stimulates the cortical (Na+, K+)-ATPase, mainly its glial form, providing central nervous system with an enhanced ability to regulate extracellular K+. In epileptic cortex, (Na+, K+)-ATPase and especially its glial form is altered in its K+ regulation and phenytoin sensitivity. That deficiency of glial (Na+, K+)-ATPase in focal epileptogenic cortex could be responsible for ictal transformation and seizure spread (Acta neurol. belg., 1988, 88, 257-280).     

Anoxia-induced changes in extracellular K+ and pH in mammalian central white matter.

In gray matter (GM), anoxia induces prominent extracellular ionic changes that are important in understanding the pathophysiology of this insult. White matter (WM) is also injured by anoxia but the accompanying changes in extracellular ions have not been studied. To provide such information, the time course and magnitude of anoxia-induced changes in extracellular K+ concentration ([K+]o) and extracellular pH (pHo) were measured in the isolated rat optic nerve, a representative central WM tract, using ion-selective microelectrodes. Anoxia produced less extreme changes in [K+]o and pHo in WM than are known to occur in GM; in WM during anoxia, the average maximum [K+]o was 14 +/- 2.9 mM (bath [K+]o = 3 mM) and the average maximum acid shift was 0.31 +/- 0.07 pH unit. The extracellular space volume rapidly decreased by approximately 20% during anoxia. Excitability of the rat optic nerve, monitored as the amplitude of the supramaximal compound action potential, was lost in close temporal association with the increase in [K+]o. Increasing the bath glucose concentration from 10 to 20 mM resulted in a much larger acid shift during anoxia (0.58 +/- 0.08 pH unit) and a smaller average increase in [K]o (9.2 +/- 2.6 mM). The increased extracellular glucose concentration presumably provided more substrate for anaerobic metabolism, resulting in more extracellular lactate accumulation (although not directly measured) and a greater acid shift. Enhanced anaerobic metabolism during anoxia would provide energy for operation of ion pumps, including the sodium pump, that would result in smaller changes in [K+]o. These effects were probably responsible for the observation that the optic nerve showed significantly less damage after 60 min of anoxia in the presence of 20 mM glucose compared to 10 mM glucose. Under normoxic conditions, increasing bath K+ concentration to 30 mM (i.e., well beyond the level shown to occur with anoxia) for 60 min caused abrupt loss of excitability during the period of application but minimal change in the amplitude of the compound action potential following the period of exposure. The anoxia-induced increase in [K+]o, therefore, was not itself directly responsible for irreversible loss of optic nerve function. These observations indicate that major qualitative differences exist between mammalian GM and WM with regard to anoxia-induced extracellular ionic changes.     

Cell swelling following recovery from acidification in C6 glioma cells: an in vitro model of postischemic brain edema

Two consequences of cerebral ischemia are cell acidification and cytotoxic edema. To test the possibility that Na+/H+ exchange mediates acid-induced edema, we measured cytoplasmic pH (pHi) and cell volume changes in C6 glioma cells that were artificially acid-loaded using weak electrolytes. pHi was monitored fluorimetrically with 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein. Upon acidification with sodium propionate, pHi dropped to 6.74 +/- 0.05 (n = 25), and then recovered to levels near the physiological value of 7.23 +/- 0.02 (n = 13). Cell volume, measured by electronic sizing, increased concomitantly by approximately 50% in sodium propionate solution. Both pHi recovery and cell swelling were Na+-dependent, amiloride-sensitive, and inhibited at pHo less than 6.0. These results demonstrate that in vitro: (1) intracellular acidification can lead to cell swelling, and (2) pHi recovery and the concomitant cell swelling are likely mediated by Na+/H+ exchange. These mechanisms may be related to postischemic cytotoxic glial edema.