Effects of Dibutyryl Cyclic AMP on Na+, K+-ATPase Activity and Intracellular Na+ and K+ in Primary Cultures of Astrocytes from DBA and C57 Mice

Summary: Effects of chronic treatment of dibutyryl cyclic AMP (db-cyclic AMP) on Na⁺, K⁺-ATPase activity in cell homogenates and intracellular N a f and K+ contents [(Na⁺)_i and (K⁺)_i] were studied in primary cultures of astrocytes derived from cerebral cortex of neonatal audiogenic seizure-susceptible DBA and audiogenic seizure-resistant C57 mice. Na⁺, K⁺-ATPase activity in cell homogenates was greater and (Na⁺)_i was less in DBA astrocytes than in C57 astrocytes. There was no difference in (K⁺)_i between astrocytes from DBA and C57 mice. Addition of db-cyclic AMP to the medium from day 14 to day 21 in culture (final concentration 0.25 mM) increased Na⁺, K⁺-ATPase activity in cell homogenates and decreased (Na⁺)_i, but had no significant effect on (K⁺)_i in astrocytes from either DBA or C57 mice. Chronic treatment with db-cyclic AMP altered cell growth. Protein and DNA content of cultured astrocytes from both DBA and C57 mice was decreased. DNA was more affected than protein. Modifying K⁺ and Na⁺ concentration in medium altered Na⁺, K⁺-ATPase activity in cell homogenates as well as (Na⁺)_i and (K⁺)_i in cultured astrocytes of both DBA and C57 mice. Changes in (Na⁺)_i and (K⁺)_i at different K⁺ concentrations in medium paralleled those in Na⁺, K⁺-ATPase activity in cell homogenates. Results indicate that the ability to transport Na⁺ across the cell membrane and the response of Na⁺, K⁺-ATPase to db-cyclic AMP and to the changes in K + in medium of cultured astrocytes from audiogenic seizure-susceptible DBA mice are sufficient.     

Gap junctions equalize intracellular Na+ concentration in astrocytes

Gap junctions between glial cells allow intercellular exchange of ions and small molecules. We have investigated the influence of gap junction coupling on regulation of intracellular Na⁺ concentration ([Na⁺]_i) in cultured rat hippocampal astrocytes, using fluorescence ratio imaging with the Na⁺ indicator dye SBFI (sodium-binding benzofuran isophthalate). The [Na⁺]_i in neighboring astrocytes was very similar (12.0 ± 3.3 mM) and did not fluctuate under resting conditions. During uncoupling of gap junctions with octanol (0.5 mM), baseline [Na⁺]_i was unaltered in 24%, increased in 54%, and decreased in 22% of cells. Qualitatively similar results were obtained with two other uncoupling agents, heptanol and α-glycyrrhetinic acid (AGA). Octanol did not alter the recovery from intracellular Na⁺ load induced by removal of extracellular K⁺, indicating that octanol's effects on baseline [Na⁺]_i were not due to inhibition of Na⁺, K⁺-ATPase activity. Under control conditions, increasing [K⁺]_o from 3 to 8 mM caused similar decreases in [Na⁺]_i in groups of astrocytes, presumably by stimulating Na⁺, K⁺-ATPase. During octanol application, [K⁺]_o-induced [Na⁺]_i decreases were amplified in cells with increased baseline [Na⁺]_i, and reduced in cells with decreased baseline [Na⁺]_i. This suggests that baseline [Na⁺]_i in astrocytes “sets” the responsiveness of Na⁺, K⁺-ATPase to increases in [K⁺]_o. Our results indicate that individual hippocampal astrocytes in culture rapidly develop different levels of baseline [Na⁺]_i when they are isolated from one another by uncoupling agents. In astrocytes, therefore, an apparent function of coupling is the intercellular exchange of Na⁺ ions to equalize baseline [Na⁺]_i, which serves to coordinate physiological responses that depend on the intracellular concentration of this ion. GLIA 20:299–307, 1997. © 1997 Wiley-Liss, Inc.     

Cholesterol oxidation reduces Ca + Mg-ATPase activity, interdigitation, and increases fluidity of brain synaptic plasma membranes

These experiments examined effects of cholesterol oxidation on Ca²⁺+Mg²⁺-ATPase activity, Na⁺+K⁺-ATPase activity, and membrane structure of brain synaptic plasma membranes (SPM). Cholesterol oxidase [E.C.1.1.3.6 fromBrevibacterium sp.] was used to oxidize cholesterol. Two cholesterol pools were identified in synaptosomal membranes based on their accessibility to cholesterol oxidase. A rapidly oxidized cholesterol pool was observed with a¹t₁₂ of 1.19±0.09 min and a second pool with a²t₁₂ of 38.30±4.16 min. Activity of Ca²⁺+Mg²⁺-ATPase was inhibited by low levels of cholesterol oxidation. Ten percent cholesterol oxidation, for example, resulted in approximately 35% percent inhibition of Ca²⁺+Mg²⁺-ATPase activity. After 13% cholesterol oxidation, further inhibition of Ca²⁺+Mg²⁺-ATPase activity was not observed. Activity of Na⁺+K⁺-ATPase was not affected by different levels of cholesterol oxidation (5%–40%). SPM interdigitation was significantly reduced and fluidity was significantly increased by cholesterol oxidation. The relatiobship observed between SPM interdigitation and Ca²⁺+Mg²⁺-ATPase activity was consistent with studies using model membranes [7]. Brain SPM function and structure were altered by relatively low levels of cholesterol oxidation and is a new approach to understanding cholesterol dynamics and neuronal function. The sensitivity of brain SPM to cholesterol oxidation may be important with respect to the proposed association between oxygen free radicals and certain neurodegenerative diseases.     

Energetic demands of the Na+/K+ ATPase in mammalian astrocytes

Cultured astrocytes and cell lines derived therefrom maintain a high energy level ([ATP]/[ADP]) through operation of oxidative phosphorylation and glycolysis. The contribution from the latter to total ATP production is 25–32%. A powerful Na⁺/K⁺ pump maintains potassium, sodium, and calcium gradients out of equilibrium. [Na⁺]_i is about 20 mM, [K⁺]_i is 130 mM and [Ca²⁺]_i is less than 100 nM. Under non-stimulated conditions, the Na⁺/K⁺ ATPase consumes 20% of astrocytic ATP production. Inhibition of the pump by ouabain decreases energy expenditure, raises [creatine phosphate]/[creatine], and leads to a leakage of sodium, potassium, and calcium ions. Decrease in the pump function via a fall in [ATP] also collapses ion gradients; the rate and extent of the fall correlates positively with cellular energy state. Under “normal” conditions (i.e., when ATP production pathways are not inhibited), there appears to be no preferential utilization of energy produced by either glycolysis or oxidative phosphorylation for the support of pump function. GLIA 21:35–45, 1997. © 1997 Wiley-Liss, Inc.     

Ammonium‐evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity

The clearance of extracellular glutamate is mainly mediated by pH‐ and sodium‐dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH₄⁺/NH₃). Here, we examined the effects of NH₄⁺/NH₃ on steady‐state intracellular pH (pH_i) and sodium concentration ([Na⁺]_i) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH₄⁺/NH₃ on glutamate transporter activity by measuring D ‐aspartate‐induced pH_i and [Na⁺]_i transients. In 20–34 days in vitro (DIV) astrocytes, NH₄⁺/NH₃ decreased steady‐state pH_i by 0.19 pH units and increased [Na⁺]_i by 21 mM. D ‐Aspartate‐induced pH_i and [Na⁺]_i transients were reduced by 80–90% in the presence of NH₄⁺/NH₃, indicating a dramatic reduction of glutamate uptake activity. In 9–16 DIV astrocytes, in contrast, pH_i and [Na⁺]_i were minimally affected by NH₄⁺/NH₃, and D ‐aspartate‐induced pH_i and [Na⁺]_i transients were reduced by only 30–40%. Next we determined the contribution of Na⁺, K⁺, Cl^?‐cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20–34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH₄⁺/NH₃‐evoked changes in steady‐state pH_i and [Na⁺]_i and attenuated the reduction of D ‐aspartate‐induced pH_i and [Na⁺]_i transients by NH₄⁺/NH₃ to 30% in 20–34 DIV astrocytes. Our results suggest that NH₄⁺/NH₃ decreases steady‐state pH_i and increases steady‐state [Na⁺]_i in astrocytes by an age‐dependent activation of NKCC. These NH₄⁺/NH₃‐evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE. © 2008 Wiley‐Liss, Inc.     

Testing competing path models linking the biochemical variables in red blood cells from Li+-treated bipolar patients

Objectives: Red blood cells (RBCs) from Li⁺‐treated bipolar patients have shown abnormalities in intracellular Li⁺ concentration ([Li⁺]_i), Na⁺/Li⁺ exchange rates, and membrane phospholipid levels. Based on Li⁺‐loaded RBC studies, we hypothesized that Li⁺‐treated bipolar patients also have varied intracellular free Mg²⁺ concentrations ([Mg²⁺]_f) as compared with normotensive patients. We addressed how these experimentally determined values are intercorrelated. Assuming that Li⁺ treatment alters these biochemical parameters, we provide hypothetical pathways based upon structural equation modeling statistics. Methods: In RBCs from 30 Li⁺‐treated bipolar patients, we determined [Li⁺]_i, serum [Li⁺] ([Li⁺]_e), Na⁺/Li⁺ exchange parameters, membrane phospholipid levels, [Mg²⁺]_f, and Li⁺ membrane binding affinities. Comprehensive statistical analyses assessed correlations among the biochemical data. We used path analysis statistics to propose potential pathways in which the data were correlated. Results: We found significant correlations within the three Na⁺/Li⁺ exchange parameters and percentage composition of the membrane phospholipids. Additional correlations existed between [Mg²⁺]_f and V_std, K_m, or phospholipid composition, between [Li⁺]_i and percentage of phosphatidylcholine, and between percentage of phosphatidylserine and K_m. Based on these findings, we hypothesized and statistically determined the most probable pathway through which these parameters were intercorrelated. Conclusions: Significant correlations existed between the biochemical parameters that describe the cell membrane abnormality and the Li⁺/Mg²⁺ competition hypotheses. Using path analysis statistics, we identified a biochemical pathway by which Li⁺ may assert its cellular effects. This study serves as an illustrative example how path analysis is a valuable tool in determining the direction of a certain biochemical pathway.     

Functional activity of Na+, K+-pump in normal and pathological tissues

Na⁺,K⁺-ATPase, supporting the ionic homeostasis of the cell, is under control of Na⁺, K⁺, Mg²⁺, and ATP. The regulating effect of Mg²⁺ is rather unclear, whereas the Na⁺/K⁺ ratio in the cytoplasm is a potent regulatory factor, especially for osmotic balance in excitable cells. We have demonstrated two possibilities for regulation of ion pumping activity: First, via the number of Na⁺,K⁺-ATPase molecules under operation, and second, via changes in the turnover rate of the active molecules. In the presence of low ATP concentration, which is typical for cells with membrane damage (ischemic cardiac myocytes, tumor cells, fatigued muscles) Na⁺,K⁺-ATPase is transformed to a regime of the decreased efficiency. Radiation inactivation study demonstrates the weakening of the interprotein interactions in the enzyme complexes during ATP deficiency. Thus, measurements of ATPase activity of the purified enzyme under optimal conditions in vitro may be useless for the discrimination of pathological from normal tissues. In such a case, the estimation of lipid composition and microviscosity of the membranes under study could be important. This review briefly discusses several basic mechanisms of the regulation of Na⁺,K⁺-ATPase—an integral protein of the outer cell membranes.     

Endoneurial ATPase activity in tangier disease and other peripheral neuropathies

Endoneurial sodium, potassium adenosine triphosphatase (Na⁺, K⁺-ATPase) and Mg²⁺-ATPase activities were determined in routine sural nerve biopsies from patients being evaluated for peripheral neuropathy. A significant reduction of endoneurial Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities was shown in six sural nerve biopsies from patients with Tangier disease complicated by mononeuopathy multiplex or progressive axonal neuropathy. Peripheral nerve ATPase activities did not correlate with myelinated or unmyelinated nerve fiber densities in these biopsies. Other peripheral neuronal disorders with reduced endoneurial Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities included severe vasculitic neuropathy, diabetic neuropathy, tomaculous neuropathy, and motoneuron disease. Such reduced levels of ATPase activity in peripheral nerve may relate to altered endoneurial lipid metabolism and impaired axoplasmic flow.     

Erythrocyte membrane abnormalities in human myotonic dystrophy

Membrane-bound enzyme activities and cardiac glycoside binding were determined in red blood cell membrane preparations from patients with myotonic dystrophy and in age matched controls. Na⁺-K⁺-activated ATPase activity was signficantly increased in myotonic patients. [³H]Ouabain binding to erythrocyte membranes was also significantly increased in myotonic dystrophy patients. The Mg²⁺-ATPase (ouabain-insensitive) was, however, unchanged. The K⁺-stimulated paranitrophenyl phosphatase (KPNPPase) activity was markedly enhanced in myotonic patients as compared to controls. The kinetic analysis showed a marked change in V_max of Na⁺-K⁺ ATPase with respect to the activation by Na⁺, K⁺ and ATP. However, the K_m values were the same in control as well as in myotonic groups. The increased erythrocyte membrane Na⁺-K⁺-ATPase activity, KPNPPase and [³H]ouabain binding in myotonic patients supports the hypothesis that generalized membrane abnormality may be involved in pathogenesis of the human myotonic dystrophy.     

Gray and white matter astrocytes differ in basal metabolism but respond similarly to neuronal activity

Astrocytes are a heterogeneous population of glial cells in the brain, which adapt their properties to the requirements of the local environment. Two major groups of astrocytes are protoplasmic astrocytes residing in gray matter as well as fibrous astrocytes of white matter. Here, we compared the energy metabolism of astrocytes in the cortex and corpus callosum as representative gray matter and white matter regions, in acute brain slices taking advantage of genetically encoded fluorescent nanosensors for the NADH/NAD⁺ redox ratio and for ATP. Astrocytes of the corpus callosum presented a more reduced basal NADH/NAD⁺ redox ratio, and a lower cytosolic concentration of ATP compared to cortical astrocytes. In cortical astrocytes, the neurotransmitter glutamate and increased extracellular concentrations of K⁺, typical correlates of neuronal activity, induced a more reduced NADH/NAD⁺ redox ratio. While application of glutamate decreased [ATP], K⁺ as well as the combination of glutamate and K⁺ resulted in an increase of ATP levels. Strikingly, a very similar regulation of metabolism by K⁺ and glutamate was observed in astrocytes in the corpus callosum. Finally, strong intrinsic neuronal activity provoked by application of bicuculline and withdrawal of Mg²⁺ caused a shift of the NADH/NAD⁺ redox ratio to a more reduced state as well as a slight reduction of [ATP] in gray and white matter astrocytes. In summary, the metabolism of astrocytes in cortex and corpus callosum shows distinct basal properties, but qualitatively similar responses to neuronal activity, probably reflecting the different environment and requirements of these brain regions.     

Calcium transport in and out of brain nerve endings in vitro—the role of synaptosomal plasma membrane Ca-ATPase in Ca-extrusion

The effects of cellular cations and ATP on calcium transport in and out of the nerve endings (synaptosomes) of mice brain were studied. The synaptosomes accumulated⁴⁵Ca time-dependently in the absence of ATP or other additions for at least 10 min. When ATP was present, the overall⁴⁵Ca accumulation was decreased and was maximal at about 4 min, after which it started to decline. Studies on the effects of cations with or without ATP at 4 min revealed selective activities for different cations. Mg²⁺ inhibited⁴⁵Ca accumulation in the absence of ATP but increased⁴⁵Ca accumulation when ATP was present. Similarly, ATP increased⁴⁵Ca accumulation only when Mg²⁺ was present. Na⁺, on the other hand, inhibited⁴⁵Ca accumulation both in the presence and absence of ATP and/or Mg²⁺. K⁺ increased⁴⁵Ca accumulation in the presence of ATP with or without Mg²⁺; however, K⁺-stimulation was not noted in the presence of 100 mM Na⁺, and in fact, K⁺ became inhibitory. The ATP-stimulated⁴⁵Ca accumulation in the presence of Mg²⁺ peaked within 4–6 min and then declined, suggesting release of⁴⁵Ca. Compatible with this notion, in⁴⁵Ca-loaded synaptosomes, ATP evoked⁴⁵Ca release which was accompanied by the appearance of P_i in the medium. Although ATP-activated⁴⁵Ca-release can occur in the presence of Mg²⁺, Mg²⁺ is not required and, infact, is inhibitory. Rapid release of⁴⁵Ca was also noted when⁴⁵Ca-loaded synaptosomes were incubated in the presence of Na⁺ without ATP. It is concluded that Mg²⁺, Na⁺,⁺K and ATP each has a specific role in regulating Ca²⁺ permeability of the plasma membrane, calcium binding and calcium extrusion.     

Sodium-dependent glutamate uptake as an activator of oxidative metabolism in primary astrocyte cultures from newborn rat

In the present report we describe the effect of glutamate on respiratory activity in primary cultures of astrocytes, derived from cerebral cortex of newborn rat. Glutamate (100 μM) caused an increased oxygen consumption. This effect could not be inhibited by antagonists to the NMDA or AMPA/kainate receptors. Neither trans-ACPD (an agonist to the metabotropic glutamate receptor) nor the Krebs cycle intermediate α-ketoglutarate had any effect on the respiratory rate. An uncontrolled influx of Na⁺, caused by gramicidin, could mimic the glutamate effect on respiratory activity. In addition, the glutamate effect was abolished by addition of ouabain or replacement of Na⁺ by Li⁺ in the perfusion buffer. We conclude that the co-transport of Na⁺, in the Na⁺ -dependent high-affinity glutamate uptake system, mediated the glutamate-induced increase in oxygen consumption through an increased activity of Na⁺/K⁺-ATPases. © 1995 Wiley-Liss, Inc.     

Effect of intermittent mild hypoxia and drug treatment on synaptosomal nonmitochondrial ATPase activities

Synaptosomal nonmitochondrial ATPases linked to the energy-utilizing systems were evaluated in cerebral cortex from normoxic rats and rats submitted to mild intermittent normobaric hypoxia [12 hr daily exposure to N₂:O₂ (90:10) mixture for 4 weeks]. The activities of Na⁺, K⁺-ATPase; high- and low-affinity Ca²⁺-ATPase; basal Mg²⁺-ATPase; and Ca²⁺, Mg²⁺-ATPase were assayed in synaptosomes and synaptosomal subfractions, namely, synaptosomal plasma membranes and synaptic vesicles. The evaluations were performed either in normoxic rats or in hypoxic rats submitted to 4-week treatment with saline (controls) or a vasodilator agent (papaverine), an energy-metabolism interfering agent (theniloxazine), a calcium blocker (nicardipine), and a lipidmetabolism interfering agent (phosphatidylcholine) in order to define the plasticity and the selective changes in individual ATPases. In synaptosomes from rat cerebral cortex, the enzyme adaptation to the daily mild intermittent hypoxia for 4 weeks was characterized by an increase in the activity of Mg²⁺-ATPase, concomitant with a decrease in the activities of Na⁺, K⁺-ATPase, high-affinity Ca²⁺-ATPase, and Ca²⁺, Mg²⁺-ATPase. In hypoxic rats the enzyme adaptation to the 4-week treatment with phosphatidylcholine was characterized by an increase in Ca²⁺, Mg²⁺-ATPase activity and a decrease in Mg²⁺-ATPase activity. The action involves the enzymatic form located in the synaptic plasma membranes. In hypoxic rats the adaptation to the 4 week treatment with nicardipine was characterized by an increase in high-affinity Ca²⁺-ATPase activity, while the 4-week-treatment with theniloxzine induced an increase in Na⁺, K²⁺-ATPase activity. The actions of both nicardipine and theniloxazine were related to the enzymatic forms located in the synaptic plasma membranes. The effects on the biophase induced by the sequential cycles of hypoxia/normoxia and the treatment with the various agents tested should also be related to the changes induced in the activity of some synaptosomal ATPases. © 1993 Wiley-Liss, Inc.     

AMPA/kainate receptor activation blocks K+ currents via internal Na+ increase in mouse cultured stellate astrocytes

Cultured mouse cortical astrocytes of the stellate type were studied by using the patch-clamp technique in whole-cell configuration. The astrocytes express at least two types of outwardly rectifying K⁺ channels which mediate a transient and a sustained current. Activation of AMPA receptors by kainate leads to a substantial blockade of both types of K⁺ currents. The blockade is absent when Na⁺ is withdrawn from the external medium, suggesting that it is caused by constant Na⁺ influx through AMPA receptors. The presence of high Na⁺ solutions in the pipette induces a blockade of both K⁺ currents which is very similar to the blockade induced by kainate, supporting thus the view that the mechanism of the blockade of K⁺ channels by kainate involves Na⁺ increases in the submembrane area. The blockade occurs between 20 and 40 mM [Na⁺]_i, which is within the physiological range of [Na⁺]_i in astrocytes. The data may suggest that the blockade of K⁺ channels by high [Na⁺]_i conditions could provide a mechanism to prevent K⁺ leakage from the astrocytes into the extracellular space during periods of intense neuronal activity. GLIA 20:38-50, 1997. © 1997 Wiley-Liss, Inc.     

Familial hemiplegic migraine

Familial hemiplegic migraine (FHM) is a rare and genetically heterogeneous autosomal dominant subtype of migraine with aura. Mutations in the genes CACNA1A and SCNA1A, encoding the pore-forming α₁ subunits of the neuronal voltage-gated Ca²⁺ channels Ca_v2.1 and Na⁺ channels Na_v1.1, are responsible for FHM1 and FHM3, respectively, whereas mutations in ATP1A2, encoding the α₂ subunit of the Na⁺, K⁺ adenosinetriphosphatase (ATPase), are responsible for FHM2. This review discusses the functional studies of two FHM1 knockin mice and of several FHM mutants in heterologous expression systems (12 FHM1, 8 FHM2, and 1 FHM3). These studies show the following: (1) FHM1 mutations produce gain-of-function of the Ca_v2.1 channel and, as a consequence, increased Ca_v2.1-dependent neurotransmitter release from cortical neurons and facilitation of in vivo induction and propagation of cortical spreading depression (CSD: the phenomenon underlying migraine aura); (2) FHM2 mutations produce loss-of-function of the α₂ Na⁺,K⁺-ATPase; and (3) the FHM3 mutation accelerates recovery from fast inactivation of Na_v1.5 (and presumably Na_v1.1) channels. These findings are consistent with the hypothesis that FHM mutations share the ability of rendering the brain more susceptible to CSD by causing either excessive synaptic glutamate release (FHM1) or decreased removal of K⁺ and glutamate from the synaptic cleft (FHM2) or excessive extracellular K⁺ (FHM3). The FHM data support a key role of CSD in migraine pathogenesis and point to cortical hyperexcitability as the basis for vulnerability to CSD and to migraine attacks. Hence, they support novel therapeutic strategics that consider CSD and cortical hyperexcitability as key targets for preventive migraine treatment.     

The “protected” glucose transport through the astrocytic endoplasmic reticulum is too slow to serve as a quantitatively-important highway for nutrient delivery

Glycogen levels in resting brain and its utilization rates during brain activation are high, but the functions fulfilled by glycogenolysis in living brain are poorly understood. Studies in cultured astrocytes have identified glycogen as the preferred fuel to provide ATP for Na⁺,K⁺-ATPase for the uptake of extracellular K⁺ and for Ca²⁺-ATPase to pump Ca²⁺ into the endoplasmic reticulum. Studies in astrocyte–neuron co-cultures led to the suggestion that glycogen-derived lactate is shuttled to neurons as oxidative fuel to support glutamatergic neurotransmission. Furthermore, both knockout of brain glycogen synthase and inhibition of glycogenolysis prior to a memory-evoking event impair memory consolidation, and shuttling of glycogen-derived lactate as neuronal fuel was postulated to be required for memory. However, lactate shuttling has not been measured in any of these studies, and procedures to inhibit glycogenolysis and neuronal lactate uptake are not specific. Testable alternative mechanisms to explain the observed findings are proposed: (i) disruption of K⁺ and Ca²⁺ homeostasis, (ii) release of gliotransmitters, (iii) imposition of an energy crisis on astrocytes and neurons by inhibition of mitochondrial pyruvate transport by compounds used to block neuronal monocarboxylic acid transporters, and (iv) inhibition of astrocytic filopodial movements that secondarily interfere with glutamate and K⁺ uptake from the synaptic cleft. Evidence that most pyruvate/lactate derived from glycogen is not oxidized and does not accumulate suggests predominant glycolytic metabolism of glycogen to support astrocytic energy demands. Sparing of blood-borne glucose for use by neurons is a reasonable explanation for the requirement for glycogenolysis in neurotransmission and memory processing.     

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.     

Single-channel Activity in Cultured Cortical Neurons of the Rat in the Presence of a Toxic Dose of Glutamate

Rat cortical neurons grown in cell culture were exposed to 500 μM glutamate for 5 min during continuous current recording from cell-attached patches. The Ca²⁺-dependence and ion selectivity of the membrane channels activated during and after glutamate application were studied in inside-out patches. Glutamate blocked spontaneous action potential firing. In 77% of the experiments glutamate activated several types of ion channels indirectly, i.e. via a change of cytoplasmic factors. Channel activity did not disappear after removing glutamate from the bath. A K⁺ channel requiring intracellular calcium ([Ca²⁺]_i) was activated in 44% of the experiments (conductance for inward currents in cell-attached patches 118 ± 6 pS;‘BK channel'). Another Ca²⁺-dependent channel permeable for Cl^- (conductance for outward currents in cell-attached patches 72±17 pS), acetate and methanesulphonate appeared in 26% of the patches. Other K⁺ channels of smaller conductance were infrequently observed. During and after glutamate application the activity of the BK channel showed an initial increase followed by a transient decay and a second rise to a plateau, probably reflecting a similar time course of changes in [Ca²⁺]_i. Both phases of increasing channel activity required the presence of extracellular Ca²⁺ suggesting that [Ca²⁺]_i was mainly increased by Ca²⁺ influx. The N-methyl-d -aspartate (NMDA) antagonists dizocilpine (MK-801, 10 μM) and dl -2-amino-5-phosphonovaleric acid (AP5; 100 μM), added within 5 min after glutamate application, stopped BK channel activity and restored the spontaneous action potential firing. We conclude that the influx of Ca²⁺ through NMDA receptor channels causes a strong activation of Ca²⁺-dependent K⁺ channels, which is likely to result in pronounced loss of intracellular K⁺. NMDA receptor channels seem to remain active for a long time (>10 min) after the end of glutamate application.     

ATPase and carbonic anhydrase activities of bulk-isolated neuron, glia and synaptosome fractions from rat brain

The (Na⁺ + K⁺) ATPase, carbonic anhydrase and HCO₃⁻-stimulated ATPase activities of bulk-isolated neuronal perikarya, glial and synaptosome fractions from 20–26-day-old rats were studied. The effects of varying K⁺, Na⁺ and ATP concentrations were investigated to determine limits on how these enzymes might respond to changes in the levels of these substances in vivo.The (Na⁺ + K⁺) ATPase activity of all three fractions had a similar high affinity for K⁺, withK_m values in the range of0.7–1.7mM. TheK_m for Na⁺ was around 10-fold higher, in the range of10–15mM.K_m values for ATP were also not markedly different between the different fractions, being1.2mM for the neurons and1.8mM for the glia and synaptosome fractions, respectively. The V_max at infinite [ATP] in the presence of10mM K⁺ was 2.9-fold higher for the glia as compared to the neuron fraction and 1.5-fold higher than the synaptosome fraction. At infinite K⁺ in the presence of3.3mM ATP the corresponding figures were 3.0 and 2.1. Arrhenius plots of (Na⁺ + K⁺) ATPase activity were different for the neuron and glia fractions as compared to the synaptosome fraction,, suggesting subtle differences in themembrane environment and/or the enzyme molecule itself.The HCO₃⁻-stimulated ATPase activity was only 14% higher in the glial fraction compared to the neurons and was variably stimulated by added K⁺ at concentrations< 10mM. The glial-enriched cell fraction had a 2-fold higher specific carbonic anhydrase activity than the neuron fraction, but the combined activity of these fractions only represented< 2% of the total brain activity. Total brain carbonic anhydrase activity was not stimulated by added K⁺ in the range of5–50mM K⁺.These data indicate that none of the enzymes studied are likely to directly respond to extracellular K⁺ levels in excess of10mM, and therefore would only be secondarily involved in cerebrocortical swelling caused by increasing K⁺ concentrations20mM. The relation of these findings to previous work on increased (Na⁺ + K⁺) ATPase and carbonic anhydrase activity in glial cells, the role of glial cells in passive or mediated transport processes related to increased levels of extracellular K⁺ and the suitability of bulk-isolated glia and neuron-enriched fractions as experimental models are discussed.     

Transport mechanism of glutamate by hypotonic-treated glial plasmalemmal vesicles from rat hippocampus

Recently, we isolated a novel subcellular fraction of glial plasmalemmal vesicles (GPV), which showed a higher activity of Na⁺-dependent glutamate transport than synaptosomes (Nakamura et al., 1993). In order to study kinetically the glutamate transport mechanism, we measured the reaction under various ionic conditions both inside and outside the vesicles. The vesicles treated hypotonically and preloaded with KCl could take up glutamate in the presence of external Na⁺. The level of glutamate uptake was dependent on external concentrations of NaCl ([NaCl]_o) and competitively inhibited by [KCl]_o. However, it was dependent on [KCl]_i, and competitively inhibited by [NaCl]_i. The activation and inhibition constants of K⁺ were about 30 mM inside and 20 mM outside, respectively, whereas those of Na⁺ were 140 mM outside and 4 mM inside, respectively. These results suggest that the transport carrier molecules work asymmetrically to the membranes. Nigericin and monensin, acidic ionophores for K⁺ and Na⁺, respectively, inhibited the glutamate uptake. On the other hand, valinomycin, a neutral ionophore for K⁺, elevated the uptake level, suggesting that the inside-negative membrane potential induced by K⁺ diffusion enhances the uptake activity. We conclude that glutamate transport by glial cells requires both external Na⁺ and internal K⁺ and is regulated by the membrane potential.