A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics

The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.     

Ouabain enhances basal and stimulus-induced cytoplasmic calcium concentrations in platelets

Incubation of platelet-rich plasma (PRP) with ouabain, an inhibitor of sodium/potassium ATPase (Na+/K+ ATPase), induced a significant rise in basal platelet intracellular calcium concentration [( Ca2+]i) when measured using fura 2. Ouabain induced an enhanced aggregation response to low doses of collagen in both PRP and washed platelets loaded with aequorin. In aequorin loaded platelets this enhanced aggregation response was associated with an enhanced rise in [Ca2+]i such that the relationship between [Ca2+]i and aggregation was unchanged. As inhibition of plasma membrane Na+/K+ ATPase would lead to a raised intracellular sodium ion concentration [( Na+]i) the results suggest that in the platelet, [Na+]i can modulate [Ca2+]i and hence influence the response of platelets to stimuli such as collagen.     

Studies on pH Regulatory Mechanisms in Cultured Astrocytes of DBA and C57 Mice

pH regulatory mechanisms in primary cultures of astrocytes from the cerebral cortex of neonatal audiogenic-seizure-susceptible DBA/2J (DBA) and genetically controlled C57BL/6J (C57) mice were studied with [14C]dimethyloxazolidine-2-4-dione (DMO) and [3H]-methyl-D-glucose (MDG). Effects of changing the concentration of Na+, K+, HCO3- or Cl- in medium, and/or of different transport blockers and metabolite inhibitor on intracellular pH (pHi) of cultured astrocytes were also studied. In nominal HCO3(-)-free HEPES-buffered Hanks' balanced salt solution (HEPES HBSS), when the pH of medium (pHo) was maintained at 7.4, the steady-state pHi of cultured astrocytes from DBA mice was 6.98 +/- 0.03, and that from C57 mice was 7.01 +/- 0.03. When the cells were incubated in HBSS containing 25 mM HCO3- and equilibrated with 5% CO2 (HCO3- HBSS, pHo = 7.4), pHi of both DBA and C57 astrocytes was approximately 0.1-0.15 pH units higher than that in HEPES HBSS. Reducing the pH or the Na+ concentration in media (pHo, [Na+]o) of either HEPES HBSS or HCO3- HBSS, pHi of both DBA and C57 astrocytes decreased markedly (0.25-0.45 pH units lower than the controls). The decrease in pHi was greater in HEPES HBSS than in HCO3- HBSS. Reducing the Cl- concentration ([Cl-]o) in either HEPES or HCO3- HBSS, pHi of astrocytes increased by 0.05-0.1 pH units. Increasing the K+ concentration ([K+]o) of or adding Ba2+ to the media increased the pHi of both DBA and C57 astrocytes accordingly. SITS, an anion transport inhibitor, decreased the pHi of both DBA and C57 astrocytes in HCO3- HBSS but not in HEPES HBSS. It enhanced the response of pHi to reduction in pHo. Amiloride, a Na(+)-H+ exchange inhibitor, decreased the pHi of both DBA and C57 astrocytes more in HEPES HBSS than in HCO3- HBSS. It enhanced the response of pHi to reduction in pHo and [Na+]o. Ouabain, an Na+,K(+)-ATPase inhibitor, decreased the pHi of cultured astrocytes in HEPES HBSS, but not in HCO3- HBSS. It also enhanced the response of pHi to changing pHo and [Na+]o in HEPES HBSS. Acetazolamide, a carbonic anhydrase inhibitor, decreased the pHi of astrocytes in both HEPES and HCO3- HBSS. Both bumetanide, an Na+,K+/Cl- cotransport blocker, and KCN, a metabolic inhibitor, produced no significant effect on the steady-state pHi or the response of pHi to changing ionic concentration in media in both DBA and C57 astrocytes.     

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.     

Effects of Na+ and Ca2+ gradients on intracellular free Ca2+ in voltage-clamped Aplysia neurons

Selected neurons of the abdominal ganglion of Aplysia californica were voltage-clamped and intracellular free Ca [( Ca2+]i) and Na [( Na+]i) concentrations were monitored with ion selective microelectrodes. Reducing [Na+]o from 500 mM (normal seawater, NSW) to 5 mM resulted in a decrease of the potential measured by the Ca electrode (VCa). Increasing [Ca2+]o from 10 to 50 mM increased [Ca2+]i two-fold, keeping [Ca2+]o at 50 mM and decreasing [Na+]o to 5 mM still led to a decrease in VCa. With 100 mM [Ca2+]o, which also increased [Ca2+]i, decreasing [Na+]o increased VCa in two of the eight cells tested. This indicates that in normal or moderately high resting [Ca2+]i, Ca2+ extrusion by Na/Ca exchange (forward mode) is not essential for [Ca2+]i buffering. [Na+]i was 12.9 +/- 3.6 mM (S.E.M., n = 7) in NSW; reducing [Na+]o to 5 mM decreased [Na+]i to 2.0 +/- 1.1 mM (S.E.M.). Keeping [Na+]o at 5 mM and increasing [Ca2+]o from 10 to 20 mM further decreased [Na+]i to about 1.0 mM, evidence of Na/Ca exchange operating in the reverse mode. Attempts to increase [Ca2+]i by bath application of the Ca ionophores A23187, X537A, ionomycin or ETH 1001 resulted in no measurable change of the resting [Ca2+]i. Application of Ouabain caused an apparent increase in [Ca2+]i in two of the six cells tested. In cells injected with the metallochromic indicator arsenazo III (AIII), the rate of the falling phase of the AIII absorbance increase, following a voltage-clamp pulse, was significantly slower in 5 mM [Na+]o. This indicates that in its forward mode Na-Ca exchange is active in clearing large submembrane increases in [Ca2+]i.     

Hypoxic and hypercapnic responses of [Ca]0 and [K]0 in the cat carotid body in vitro

Measurements of extracellular Ca2+ and K+ activities [( Ca2+]o, [K+]o) in the superfused cat carotid body in vitro with triple-barrelled ion-selective electrodes have shown that hypoxia induced a decrease in [Ca2+]o of 0.035 +/- 0.17 mM (mean +/- S.D.; n = 17) and a biphasic change in [K+]o which consisted of an increase of 2.3 +/- 1.8 mM followed by an undershoot of -0.52 +/- 0.34 mM (mean +/- S.D.; n = 17). Hypercapnia induced a monophasic upward deflection increase of both [Ca2+]o and [K+]o of about 0.037 +/- 0.013 mM and 0.33 +/- 0.15 mM, respectively (n = 17). During hypoxia, lowering [Ca2+] in the medium to 0.1 mM resulted in a reversed [Ca2+]o response, attenuated [K+]o increase and absence of chemosensory nerve discharges. TTX generally did not affect the hypoxic and hypercapnic induced ionic changes, although the [K+]o undershoot was reduced by 30%. Co2+ competitively blocked the changes in [Ca2+]o and the increase in the sensory nerve discharge elicited by hypoxia and, not competitively, the changes of [K+]o. The ionic changes to hypercapnia were less affected by Co2+. Ouabain inhibited the [K+]o undershoot induced by hypoxia, as did the removal of Na+ from medium. It is concluded that changes in extracellular free Ca2+ and K+ ions concentration induced by hypoxia and hypercapnia represent ionic fluxes related to the transduction process of carotid body cells (glomus and/or sustentacular).     

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)     

Activity-related changes in intracellular pH in rat thalamic relay neurons

Activity-related shifts in intracellular pH (pHi) can exert potent neuromodulatory actions. Different states of neuronal activity of thalamocortical neurons were found to differentially modulate pHi. Tonic activity evoked by injection of depolarizing current led to a reversible rise in [H+]i which was nearly abolished in the presence of TTX. Block of voltage-gated calcium channels with I mM Ni2+ reduced the [H+]i transients related to tonic activity. Rhythmic activation of burst discharges caused changes of [H+]i which were decreased by TTX, whereas I mM Ni2+ almost abolished the [H+]i transients. The present results show that different forms of neuronal activity can lead to intracellular acidification caused by different mechanisms, i.e. Na+ and Ca2+ influx through sodium and Ca2+ channels, respectively, and the subsequent activation of a Ca2+/H+ pump. The resulting acidosis is suggested to reduce further Ca2+ influx and prevent excessive neuronal excitation.     

Control of cation transport in cultured glial cells by external Ca++: a possible signal in glial-neuronal interaction

In cultured glial cells from chick embryonic brain, both influx and efflux of 42K+ and 22Na+ are dependent on the external Ca++ and concentration ([Ca++]0) between 2 and 0.1 mM although intracellular concentrations of K+ ([K+]i) and Na+ ([Na+]i) do not change. Only a reduction of [Ca++]0 below 0.1 mM results in both a decrease of [K+]i and an increase of [Na+]i. Ouabain significantly decreases the [Ca++]0 sensitivity of uphill cation movements (K+ influx and Na+ efflux), while the [Ca++]0 sensitivity of downhill cation movements (K+ efflux and Na+ influx) is almost not affected by the presence of ouabain. Additionally, a decrease in [Ca++]0 triggers an increase in intracellular concentration of adenosine 3':5'-cyclic monophosphoric acid (cAMP). These findings suggest that changes of [Ca++]0, which take place in vivo in the microenvironment of the glia after neuronal firing, represent a signal in the glial-neuronal interaction controlling cation transport and that this control is achieved by a co-operation between the cAMP-generating and the cation transport system.     

Complex rectification of Müller cell Kir currents

Although Kir4.1 channels are the major inwardly rectifying channels in glial cells and are widely accepted to support K+- and glutamate-uptake in the nervous system, the properties of Kir4.1 channels during vital changes of K+ and polyamines remain poorly understood. Therefore, the present study examined the voltage-dependence of K+ conductance with varying physiological and pathophysiological external [K+] and intrapipette spermine ([SP]) concentrations in Müller glial cells and in tsA201 cells expressing recombinant Kir4.1 channels. Two different types of [SP] block were characterized: "fast" and "slow." Fast block was steeply voltage-dependent, with only a low sensitivity to spermine and strong dependence on extracellular potassium concentration, [K+]o. Slow block had a strong voltage sensitivity that begins closer to resting membrane potential and was essentially [K+]o-independent, but with a higher spermine- and [K+]i-sensitivity. Using a modified Woodhull model and fitting i/V curves from whole cell recordings, we have calculated free [SP](in) in Müller glial cells as 0.81 +/- 0.24 mM. This is much higher than has been estimated previously in neurons. Biphasic block properties underlie a significantly varying extent of rectification with [K+] and [SP]. While confirming similar properties of glial Kir and recombinant Kir4.1, the results also suggest mechanisms underlying K+ buffering in glial cells: When [K+]o is rapidly increased, as would occur during neuronal excitation, "fast block" would be relieved, promoting potassium influx to glial cells. Increase in [K+]in would then lead to relief of "slow block," further promoting K+-influx.     

Intracellular Na+ activity in cultured mouse oligodendrocytes

Na(+)-selective double-barrelled microelectrodes were used to measure the intracellular Na+ activity (aiNa) and membrane potential (Em) in oligodendrocytes from cultures of embryonic mouse spinal cord. In Na(+)-free solutions aiNa rapidly fell from its baseline of about 15 mM to values below 1 mM. Elevation of the K+ concentration in the bath ([K+]o) from 5.4 to 15 or 50 mM elicited an aiNa decrease of 4.7 or 9.0 mM, respectively. Ouabain blocked the aiNa decrease in response to 50 mM K+ by 37%. Bath application of 1 mM glutamate resulted in a membrane depolarization of 4.5 mV and a concomitant rise of aiNa by 8.6 mM. aiNa increased by approximately 11 mM after washout of a solution containing 20 mM NH4+. This aiNa increase was not blocked by amiloride, excluding a major contribution of a Na+/H+ antiporter. We conclude that, in cultured oligodendrocytes, transmembraneous Na+ movements are involved in pH regulation, glutamate causes an influx of Na+, and that the Na+/K+ pump and passive KCl uptake contribute to K+ accumulation.     

Early ionic and membrane potential changes caused by the pesticide rotenone in striatal cholinergic interneurons

Mitochondrial metabolism impairment has been implicated in the pathogenesis of several neurodegenerative disorders. In the present work, we combined electrophysiological recordings and microfluorometric measurements from cholinergic interneurons obtained from a rat neostriatal slice preparation. Acute application of the mitochondrial complex I inhibitor rotenone produced an early membrane hyperpolarization coupled to a fall in input resistance, followed by a late depolarizing response. Current-voltage relationship showed a reversal potential of -80 +/- 3 mV, suggesting the involvement of a potassium (K+) current. Simultaneous measurement of intracellular sodium [Na+]i or calcium [Ca2+]i concentrations revealed a striking correlation between [Na+]i elevation and the early membrane hyperpolarization, whereas a significant [Ca2+]i rise matched the depolarizing phase. Interestingly, ion and membrane potential changes were mimicked by ouabain, inhibitor of the Na+-K+ATPase, and were insensitive to tetrodotoxin (TTX) or to a combination of glutamate receptor antagonists. The rotenone effects were partially reduced by blockers of ATP-sensitive K+ channels, glibenclamide and tolbutamide, and largely attenuated by a low Na+-containing solution. Morphological analysis of the rotenone effects on striatal slices showed a significant decrease in the number of choline acetyltransferase (ChAT) immunoreactive cells. These results suggest that rotenone rapidly disrupts the ATP content, leading to a decreased Na+-K+ATPase function and, therefore, to [Na+]i overload. In turn, the hyperpolarizing response might be generated both by the opening of ATP-sensitive K+ channels and by Na+-activated K+ conductances. The increase in [Ca2+]i occurs lately and does not seem to influence the early events.     

Glial depolarization evokes a larger potassium accumulation around oligodendrocytes than around astrocytes in gray matter of rat spinal cord slices.

The cell membrane of astrocytes and oligodendrocytes is almost exclusively permeable for K+. Depolarizing and hyperpolarizing voltage steps produce in oligodendrocytes, but not in astrocytes, decaying passive currents followed by large tail currents (Itail) after the offset of a voltage jump. The aim of the present study was to characterize the properties of Itail in astrocytes, oligodendrocytes, and their respective precursors in the gray matter of spinal cord slices. Studies were carried out on 5- to 11-day-old rats, using the whole-cell patch clamp technique. The reversal potential (Vrev) of Itail evoked by membrane depolarization was significantly more positive in oligodendrocytes (-31.7+/-2.58 mV, n = 53) than in astrocytes (-57.9+/-2.43 mV, n = 21), oligodendrocyte precursors (-41.2+/-3.44 mV, n = 36), or astrocyte precursors (-52.1+/-1.32 mV, n = 43). Analysis of the Itail (using a variable amplitude and duration of the de- and hyperpolarizing prepulses as well as an analysis of the time constant of the membrane currents during voltage steps) showed that the Itail in oligodendrocytes arise from a larger shift of K+ across their membrane than in other cell types. As calculated from the Nernst equation, changes in Vrev revealed significantly larger accumulation of the extracellular K+ concentration ([K+]e) around oligodendrocytes than around astrocytes. The application of 50 mM K+ or hypotonic solution, used to study the effect of cell swelling on the changes in [K+]e evoked by a depolarizing prepulse, produced in astrocytes an increase in [K+]e of 201% and 239%, respectively. In oligodendrocytes, such increases (22% and 29%) were not found. We conclude that K+ tail currents, evoked by a larger accumulation of K+ in the vicinity of the oligodendrocyte membrane, could result from a smaller extracellular space (ECS) volume around oligodendrocytes than around astrocytes. Thus, in addition to the clearance of K+ from the ECS performed by astrocytes, the presence of the K+ tail currents in oligodendrocytes indicates that they might also contribute to efficient K+ homeostasis.     

Astrocytic modulation of potassium under seizures

The contribution of an impaired astrocytic K^+ regulation system to epileptic neuronal hyperexcitability has been increasingly recognized in the last decade.A defective K^+ regulation leads to an elevated extracellular K^+ concentration([K^+]o).When[K^+]o reaches peaks of 10-12 mM,it is strongly associated with seizure initiation during hypersynchronous neuronal activities.On the other hand,reactive astrocytes during a seizure attack restrict influx of K^+ across the membrane both passively and actively.In addition to decreased K^+ buffering,aberrant Ca^2+ signaling and declined glutamate transport have also been observed in astrogliosis in epileptic specimens,precipitating an increased neuronal discharge and induction of seizures.This review aims to provide an overview of experimental findings that implicated astrocytic modulation of extracellular K^+ in the mechanism of epileptogenesis.     

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.     

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.     

Role of Na+-H+ and Na+-Ca2+ exchange in hypoxia-related acute astrocyte death

Cultured astrocytes do not succumb to hypoxia/zero glucose for up to 24 h, yet astrocyte death following injury can occur within 1 h. It was previously demonstrated that astrocyte loss can occur quickly when the gaseous and interstitial ionic changes of transient brain ischemia are simulated: After a 20-40-min exposure to hypoxic, acidic, ion-shifted Ringer (HAIR), most cells died within 30 min after return to normal saline (i.e., "reperfusion"). Astrocyte death required external Ca2+ and was blocked by KB-R7943, an inhibitor of reversed Na+-Ca2+ exchange, suggesting that injury was triggered by a rise in [Ca2+]i. In the present study, we confirmed the elevation of [Ca2+]i during reperfusion and studied the role of Na+-Ca2+ and Na+-H+ exchange in this process. Upon reperfusion, elevation of [Ca2+]i was detectable by Fura-2 and was blocked by KB-R7943. The low-affinity Ca2+ indicator Fura-FF indicated a mean [Ca2+]i rise to 4.8+/-0.4 microM. Loading astrocytes with Fura-2 provided significant protection from injury, presumably due to the high affinity of the dye for Ca2+. Injury was prevented by the Na+-H+ exchange inhibitors ethyl isopropyl amiloride or HOE-694, and the rise of [Ca2+]i at the onset of reperfusion was blocked by HOE-694. Acidic reperfusion media was also protective. These data are consistent with Na+ loading via Na+-H+ exchange, fostering reversal of Na+-Ca2+ exchange and cytotoxic elevation of [Ca2+]i. The results indicate that mechanisms involved in pH regulation may play a role in the fate of astrocytes following acute CNS injuries.     

Cytosolic Ca2+ under high glucose with suppressed Na+/K+ pump activity in rat sensory neurons

Cytosolic Ca2+ concentration ([Ca2+]i) was measured in isolated rat dorsal root ganglion (DRG) neurons using the fluorescent Ca2+ indicator fura-2. Exposure to high (50 mM) extracellular K+ evoked a robust increase in [Ca2+]i, which was almost totally abolished by concomitant presence of nisoldipine (10 microM) and omega-conotoxin GVIA (10 microM). Whereas either high (30 mM) D-glucose alone or ouabain (100 microM) alone did not appreciably affect the high K+-induced [Ca2+]i elevation, neurons pretreated with high D-glucose together with ouabain exhibited a significantly larger [Ca2+]i response to high K+ stimulation, which was almost completely inhibited by nisoldipine and omega-conotoxin GVIA. These results suggest that a combination of high glucose and suppressed Na+/K+ pump activity potentiates the [Ca2+]i elevation stimulated by activation of the voltage-gated Ca2+ channels in rat DRG neurons.     

Regulation of intracellular free calcium in normal and dystrophic mouse cerebellar neurons.

We measured free intracellular calcium ([Ca2+]i) in cultured cerebellar granule cells from normal and mdx mice. Resting levels of ([Ca2+]i) were 24% higher in the dystrophic neurons (normal: 61.2 +/- 1.5 nM calcium, n = 104; dystrophic: 76.1 +/- 2.4 nM calcium, n = 136, P less than 0.01). Dystrophic neurons showed a significantly greater increase in ([Ca2+]i) in the presence of elevated (18 mM) extracellular calcium levels. Resting sodium levels ([Na+]i), however, were found to be similar in normal and dystrophic granule neurons. In addition, sodium influx rates after ouabain inhibition of the Na+/K+ ATPase were also identical. Therefore, the increased permeability of granule neurons was specific to calcium, and did not result from a non-selective cation-permeable conductance. Unlike granule cells, astrocytes do not express dystrophin. Glial cells from normal and dystrophic mice showed no difference in their resting free calcium levels or their response to a high calcium load. Thus, cerebellar granule neurons from mdx mice show a calcium-specific regulatory defect similar to that found in dystrophic muscle fibers, while cerebellar glial cells, which do not normally express dystrophin, have no such defect.     

Astrocyte membrane responses and potassium accumulation during neuronal activity

Older studies suggest that astrocytes act as potassium electrodes and depolarize with the potassium efflux accompanying neuronal activity. Newer studies suggest that astrocytes depolarize in response to neuronal glutamate release and the activity of electrogenic glial glutamate transporters, thus casting doubt on the fidelity with which astrocytes might sense extracellular potassium rises. Any K(+)-induced astrocyte depolarization might reflect a spatial buffering effect of astrocytes during neuronal activity. For these reasons, we studied stimulus-evoked currents in hippocampal CA1 astrocytes. Hippocampal astrocytes exhibited stimulus-evoked transient glutamate transporter currents and slower Ba(2+)-sensitive inward rectifier potassium (K(ir)) currents. In whole-cell astrocyte recordings, Ba(2+) blocked a very weakly rectifying component of the astrocyte membrane conductance. The slow stimulus-elicited current, like measurements from K(+)-sensitive electrodes under the same conditions, predicted small bulk [K(+)](o) increases (<0.5 mM) following the termination of short-stimulus trains. These currents indicate the potential for astrocyte spatial K(+) buffering. However, Ba(2+) did not significantly affect resting [K(+)](o) or the [K(+)](o) rises detected by the K(+)-sensitive electrode. To test whether local K(+) rises may be significantly higher than those detected by glial recordings or by K(+) electrodes, we assayed EPSCs and fiber volleys, two measures very sensitive to K(+) increases. We found that Ba(2+) had little effect on neuronal axonal or synaptic function during short-stimulus trains, indicating that K(ir)s do not influence local [K(+)](o) rises enough, under these conditions to affect synaptic transmission. In conclusion, our results indicate that hippocampal astrocytes are faithful sensors of [K(+)](o) rises, but we find little evidence for physiologically relevant spatial K(+) buffering during brief bursts of presynaptic activity.