Extracellular ATP-Induced Alterations in Extracellular H+ Fluxes From Cultured Cortical and Hippocampal Astrocytes.

Small alterations in the level of extracellular H+ can profoundly alter neuronal activity throughout the nervous system. In this study, self-referencing H+-selective microelectrodes were used to examine extracellular H+ fluxes from individual astrocytes. Activation of astrocytes cultured from mouse hippocampus and rat cortex with extracellular ATP produced a pronounced increase in extracellular H+ flux. The ATP-elicited increase in H+ flux appeared to be independent of bicarbonate transport, as ATP increased H+ flux regardless of whether the primary extracellular pH buffer was 26 mM bicarbonate or 1 mM HEPES, and persisted when atmospheric levels of CO2 were replaced by oxygen. Adenosine failed to elicit any change in extracellular H+ fluxes, and ATP-mediated increases in H+ flux were inhibited by the P2 inhibitors suramin and PPADS suggesting direct activation of ATP receptors. Extracellular ATP also induced an intracellular rise in calcium in cultured astrocytes, and ATP-induced rises in both calcium and H+ efflux were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin. Replacement of extracellular sodium with choline did not significantly reduce the size of the ATP-induced increases in H+ flux, and the increases in H+ flux were not significantly affected by addition of EIPA, suggesting little involvement of Na+/H+ exchangers in ATP-elicited increases in H+ flux. Given the high sensitivity of voltage-sensitive calcium channels on neurons to small changes in levels of free H+, we hypothesize that the ATP-mediated extrusion of H+ from astrocytes may play a key role in regulating signaling at synapses within the nervous system.

ATP-mediated increase in H+ flux from retinal Müller cells: a role for Na+/H+ exchange.

Small alterations in extracellular H+ can profoundly alter neurotransmitter release by neurons. We examined mechanisms by which extracellular ATP induces an extracellular H+ flux from Müller glial cells, which surround synaptic connections throughout the vertebrate retina. Müller glia were isolated from tiger salamander retinae and H+ fluxes examined using self-referencing H+-selective microelectrodes. Experiments were performed in 1 mM HEPES with no bicarbonate present. Replacement of extracellular sodium by choline decreased H+ efflux induced by 10 µM ATP by 75%. ATP-induced H+ efflux was also reduced by Na+/H+ exchange inhibitors. Amiloride reduced H+ efflux initiated by 10 µM ATP by 60%, while 10 µM cariporide decreased H+ flux by 37%, and 25 µM zoniporide reduced H+ flux by 32%. ATP-induced H+ fluxes were not significantly altered by the K+/H+ pump blockers SCH28080 or TAK438, and replacement of all extracellular chloride with gluconate was without effect on H+ fluxes. Recordings of ATP-induced H+ efflux from cells that were simultaneously whole cell voltage clamped revealed no effect of membrane potential from -70 mV to 0 mV. Restoration of extracellular potassium after cells were bathed in 0 mM potassium produced a transient alteration in ATP-dependent H+ efflux. The transient response to extracellular potassium occurred only when extracellular sodium was present and was abolished by 1 mM ouabain, suggesting that alterations in sodium gradients were mediated by Na+/K+-ATPase activity. Our data indicate that the majority of H+ efflux elicited by extracellular ATP from isolated Müller cells is mediated by Na+/H+ exchange.NEW & NOTEWORTHY Glial cells are known to regulate neuronal activity, but the exact mechanism(s) whereby these "support" cells modulate synaptic transmission remains unclear. Small changes in extracellular levels of acidity are known to be particularly powerful regulators of neurotransmitter release. Here, we show that extracellular ATP, known to be a potent activator of glial cells, induces H+ efflux from retinal Müller (glial) cells and that the bulk of the H+ efflux is mediated by Na+/H+ exchange.

Medium-retaining Petri dish insert to grow and image cultured cells.

BACKGROUND: Microscope chambers that accept glass coverslips with cultured cells are often used to monitor intracellular Ca2+ concentration ([Ca2+]i) during cell superfusion. Unfortunately, the experimental maneuvers associated with the coverslip installation in these chambers (medium removal and re-application) trigger unintended [Ca2+]i elevations. NEW METHOD: To prevent these [Ca2+]i elevations, a Petri dish insert has been constructed. The insert features a superfusion-optimized well to grow cell cultures. After this insert is removed from the Petri dish, the well retains the medium. This feature allows the inserts to be installed in microscope chambers while keeping the cells submerged at all times. RESULTS: These inserts were used to test the impact of a transient medium removal from the well (an equivalent of a coverslip removal from the medium) on [Ca2+]i in primary murine cortical neurons and astrocytes, and in HEK-293 cells. In all of these models, the medium removal/re-application caused a micromolar [Ca2+]i spike. While in neurons this spike was caused by a Ca2+ influx, in astrocytes and HEK-293 cells, it was caused by a Ca2+ release from intracellular stores. After the spike, a subpopulation of neurons failed to restore low [Ca2+]i; in 24% of the astrocytes, the spike triggered [Ca2+]i oscillations. However, prior to the spike, [Ca2+]i was low and uniform in all these cells. COMPARISON WITH EXISTING METHOD(S): The new method avoids the artificially-induced [Ca2+]i elevations that take place during the handling of glass coverslips with cultured cells. CONCLUSIONS: The new method allows monitoring [Ca2+]i without disturbing the basal [Ca2+]i levels.

Visualizing Metal Content and Intracellular Distribution in Primary Hippocampal Neurons with Synchrotron X-Ray Fluorescence.

Increasing evidence suggests that metal dyshomeostasis plays an important role in human neurodegenerative diseases. Although distinctive metal distributions are described for mature hippocampus and cortex, much less is known about metal levels and intracellular distribution in individual hippocampal neuronal somata. To solve this problem, we conducted quantitative metal analyses utilizing synchrotron radiation X-Ray fluorescence on frozen hydrated primary cultured neurons derived from rat embryonic cortex (CTX) and two regions of the hippocampus: dentate gyrus (DG) and CA1. Comparing average metal contents showed that the most abundant metals were calcium, iron, and zinc, whereas metals such as copper and manganese were less than 10% of zinc. Average metal contents were generally similar when compared across neurons cultured from CTX, DG, and CA1, except for manganese that was larger in CA1. However, each metal showed a characteristic spatial distribution in individual neuronal somata. Zinc was uniformly distributed throughout the cytosol, with no evidence for the existence of previously identified zinc-enriched organelles, zincosomes. Calcium showed a peri-nuclear distribution consistent with accumulation in endoplasmic reticulum and/or mitochondria. Iron showed 2-3 distinct highly concentrated puncta only in peri-nuclear locations. Notwithstanding the small sample size, these analyses demonstrate that primary cultured neurons show characteristic metal signatures. The iron puncta probably represent iron-accumulating organelles, siderosomes. Thus, the metal distributions observed in mature brain structures are likely the result of both intrinsic neuronal factors that control cellular metal content and extrinsic factors related to the synaptic organization, function, and contacts formed and maintained in each region.

Neuronal acid-induced [Zn²âº]i elevations calibrated using the low-affinity ratiometric probe FuraZin-1.

The experiments were carried out on primary cultures of murine cortical neurons from cryopreserved preparations obtained from embryonic-day-16 fetuses. To calibrate acid-induced intracelluar [Zn(2+) ] ([Zn(2+) ]i ) elevations, a low affinity (Kd = 39 µM at pH 6.1) ratiometric Zn(2+) probe, FuraZin-1, was used. A pHi drop from 7.2 to 6.1 caused [Zn(2+) ]i elevations reaching 2 µM; when the thiol-reactive agent N-ethylmaleimide (NEM) was subsequently applied, [Zn(2+) ]i increased further to 5.6 µM; analogous acid- and NEM-induced [Zn(2+) ]i elevations could also be detected but not calibrated, using the high affinity Zn(2+) probe FluoZin-3. The data indicate that NEM causes Zn(2+) release from ligands that chelate Zn(2+) at pH 6.1. ATP could also chelate Zn(2+) at pH 6.1 because its pKa is about 6.8. Therefore, it was tested whether an ATP depletion affects the acid-induced [Zn(2+) ]i elevations. The ATP depletion was induced by inhibiting mitochondrial and glycolytic ATP production. Interestingly, an almost complete ATP depletion (confirmed using a luciferin/luciferase assay) failed to affect the acid-induced [Zn(2+) ]i increases. These data suggest that the total amount of Zn(2+) accumulated in intracellular ATP-dependent stores (Zn(2+) -ATP complexes and organelles that accumulate Zn(2+) in an ATP-dependent manner) is negligible compared to the amount of Zn(2+) accumulated in the acid-sensitive intracellular ligands. In vitro, upon acidification, Zn(2+) -cysteine complexes release Zn(2+) and ATP chelates the released Zn(2+) . However, in vivo (cultured neurons), an ATP depletion failed to enhance acid-induced [Zn(2+) ]i elevations. These [Zn(2+) ]i elevations were calibrated using a low affinity ratiometric probe FuraZin-1; they reached 2 µM levels and increased to 5 µM when a thiol-reactive agent, N-ethylmaleimide, compromised Zn(2+) binding by cysteines.

Proton-dependent zinc release from intracellular ligands.

In cultured cortical and hippocampal neurons when intracellular pH drops from 6.6 to 6.1, yet unclear intracellular stores release micromolar amounts of Zn(2+) into the cytosol. Mitochondria, acidic organelles, and/or intracellular ligands could release this Zn(2+) . Although exposure to the protonophore FCCP precludes reloading of the mitochondria and acidic organelles with Zn(2+) , FCCP failed to compromise the ability of the intracellular stores to repeatedly release Zn(2+) . Therefore, Zn(2+) -releasing stores were not mitochondria or acidic organelles but rather intracellular Zn(2+) ligands. To test which ligands might be involved, the rate of acid-induced Zn(2+) release from complexes with cysteine, glutathione, histidine, aspartate, glutamate, glycine, and carnosine was investigated; [Zn(2+) ] was monitored in vitro using the ratiometric Zn(2+) -sensitive fluorescent probe FuraZin-1. Carnosine failed to chelate Zn(2+) but did chelate Cu(2+) ; the remaining ligands chelated Zn(2+) and upon acidification were releasing it into the medium. However, when pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes rapidly accelerated the rate of Zn(2+) release. The zinc-cysteine complexes also released Zn(2+) when a histidine-modifying agent, diethylpyrocarbonate, was applied at pH 7.2. Since the cytosolic zinc-cysteine complexes can contain micromolar amounts of Zn(2+) , these complexes may represent the stores responsible for an acid-induced intracellular Zn(2+) release. This study aimed at identifying intracellular stores which release Zn(2+) when pHi drops from 6.6 to 6.1. It was found that these stores are not mitochondria or acidic organelles, but rather intracellular Zn(2+) ligands. When the pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes showed a rapid acceleration in the rate of Zn(2+) release. Therefore, the stores responsible for an acid-induced intracellular Zn(2+) release in neurons may be the cytosolic zinc-cysteine complexes.

Cytosolic acidification and intracellular zinc release in hippocampal neurons.

In neurons exposed to glutamate, Ca²âº influx triggers intracellular Zn²âº release via an as yet unclear mechanism. As glutamate induces a Ca²âº-dependent cytosolic acidification, the present work tested the relationships among intracellular Ca²âº concentration ([Ca²âº](i)), intracellular pH (pH(i) ), and [Zn²âº](i). Cultured hippocampal neurons were exposed to glutamate and glycine (Glu/Gly), while [Zn²âº](i), [Ca²âº](i) and pH(i) were monitored using FluoZin-3, Fura2-FF, and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Glu/Gly applications decreased pH(i) to 6.1 and induced intracellular Zn²âº release in a Ca²âº-dependent manner, as expected. The pH(i) drop reduced the affinity of FluoZin-3 and Fura-2-FF for Zn²âº. The rate of Glu/Gly-induced [Zn²âº](i) increase was not correlated with the rate of [Ca²âº](i) increase. Instead, the extent of [Zn²âº](i) elevations corresponded well to the rate of pH(i) drop. Namely, [Zn²âº](i) increased more in more highly acidified neurons. Inhibiting the mechanisms responsible for the Ca²âº-dependent pH(i) drop (plasmalemmal Ca²âº pump and mitochondria) counteracted the Glu/Gly-induced intracellular Zn²âº release. Alkaline pH (8.5) suppressed Glu/Gly-induced intracellular Zn²âº release whereas acidic pH (6.0) enhanced it. A pH(i) drop to 6.0 (without any Ca²âº influx or glutamate receptor activation) led to intracellular Zn²âº release; the released Zn²âº (free Zn²âº plus Zn²âº) bound to Fura-2FF and FluoZin-3) reached 1 µM.

Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate--the role of pH and sodium.

Although Zn(2+) homeostasis in neurons is tightly regulated and its destabilization has been linked to a number of pathologies including Alzheimer's disease and ischemic neuronal death, the primary mechanisms affecting intracellular Zn(2+) concentration ([Zn(2+) ](i)) in neurons exposed to excitotoxic stimuli remain poorly understood. The present work addressed these mechanisms in cultured hippocampal neurons exposed to glutamate and glycine (Glu/Gly). [Zn(2+)](i) and intracellular Ca(2+) concentration were monitored simultaneously using FluoZin-3 and Fura-2FF, and intracellular pH (pH(i)) was studied in parallel experiments using 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Glu/Gly applications under Na(+)-free conditions (Na(+) substituted with N-methyl-D-glucamine(+)) caused Ca(2+) influx, pH(i) drop, and Zn(2+) release from intracellular stores. Experimental maneuvers resulting in a pH(i) increase during Glu/Gly applications, such as stimulation of Na(+) -dependent pathways of H(+) efflux, forcing H(+) efflux via gramicidin-formed channels, or increasing extracellular pH counteracted [Zn(2+)](i) elevations. In the absence of Na(+), the rate of [Zn(2+)](i) decrease could be correlated with the rate of pH(i) increase. In the presence of Na(+), the rate of [Zn(2+) ](i) decrease was about twice as fast as expected from the rate of pH(i) elevation. The data suggest that Glu/Gly-induced cytosolic acidification promotes [Zn(2+) ](i) elevations and that Na(+) counteracts the latter by promoting pH(i)-dependent and pH(i)-independent mechanisms of cytosolic Zn(2+) clearance.

Contribution of Na(+)/Ca(2+) exchange to excessive Ca(2+) loading in dendrites and somata of CA1 neurons in acute slice.

Multiple Ca(2+) entry routes have been implicated in excitotoxic Ca(2+) loading in neurons and reverse-operation of sodium-calcium exchangers (NCX) has been shown to contribute under conditions where intracellular Na(+) levels are enhanced. We have investigated effects of KB-R7943, an inhibitor of reverse-operation NCX activity, on Ca(2+) elevations in single CA1 neurons in acute hippocampal slices. KB-R7943 had no significant effect on input resistance, action potential waveform, or action potential frequency adaptation, but reduced L-type Ca(2+) entry in somata. Nimodipine was therefore included in subsequent experiments to prevent complication from effects of L-type influx on evaluation of NCX activity. NMDA produced transient primary Ca(2+) increases, followed by propagating secondary Ca(2+) increases that initiated in apical dendrites. KB-R7943 had no significant effect on primary or secondary Ca(2+) increases generated by NMDA. The Na(+)/K(+) ATPase inhibitor ouabain (30 microM) produced degenerative Ca(2+) overload that was initiated in basal dendrites. KB-R7943 significantly reduced initial Ca(2+) increases and delayed the propagation of degenerative Ca(2+) loads triggered by ouabain, raising the possibility that excessive intracellular Na(+) loading can trigger reverse-operation NCX activity. A combination of NMDA and ouabain produced more rapid Ca(2+) overload, that was contributed to by NCX activity. These results suggest that degenerative Ca(2+) signaling can be triggered by NMDA in dendrites, before intracellular Na(+) levels become sufficient to reverse NCX activity. However, since Na(+)/K(+) ATPase inhibition does appear to produce significant reverse-operation NCX activity, this additional Ca(2+) influx pathway may operate in ATP-deprived CA1 neurons and play a role in ischemic neurodegeneration.

AMPA-mediated excitotoxicity in oligodendrocytes: role for Na(+)-K(+)-Cl(-) co-transport and reversal of Na(+)/Ca(2+) exchanger.

We investigated the role of Na(+)-K(+)-Cl(-) co-transporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchanger (NCX(rev)) in glutamate-mediated excitotoxicity in oligodendrocytes obtained from rat spinal cords (postnatal day 6-8). An immunocytochemical characterization showed that these cultures express NKCC1 and Na(+)/Ca(2+) exchanger isoforms 1, 2, and 3 (NCX1, NCX2, NCX3). Exposing the cultures to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) plus cyclothiazide (CTZ) led to a transient rise in intracellular (), which was followed by a sustained overload, NKCC1 phosphorylation, and a NKCC1-mediated Na(+) influx. In the presence of a specific AMPA receptor inhibitor 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), the AMPA/CTZ failed to elicit any changes in . The AMPA/CTZ-induced sustained rise led to mitochondrial Ca(2+) accumulation, release of cytochrome c from mitochondria, and cell death. The AMPA/CTZ-elicited increase, mitochondrial damage, and cell death were significantly reduced by inhibiting NKCC1 or NCX(rev). These data suggest that in cultured oligodendrocytes, activation of AMPA receptors leads to NKCC1 phosphorylation that enhances NKCC1-mediated Na(+) influx. The latter triggers NCX(rev) and NCX(rev)-mediated overload and compromises mitochondrial function and cellular viability.

NCX and NCKX operation in ischemic neurons.

Within the first 2 min of global brain ischemia, extracellular [K+] ([K+]o) increases above 60 mM and [Na+](o) drops to about 50 mM, indicating a massive K+ efflux and Na+ influx, a phenomenon known as anoxic depolarization (AD). Similar ionic shifts take place during repetitive peri-infarct depolarizations (PID) in the area penumbra in focal brain ischemia. The size of ischemic infarct is determined by the duration of AD and PID. However, the mechanism of cytosolic [Ca2+] ([Ca2+]c) elevation during AD or PID is poorly understood. Our data show that the exposure of cultured rat hippocampal CA1 neurons to AD-like conditions promptly elevates [Ca2+]c to about 30 microM. These high [Ca2+]c elevations depend on external Ca2+ and can be prevented by removing Na+ or by simultaneously inhibiting NMDA and AMPA/kainate receptors. These data indicate that [Ca2+]c elevations during AD result from Na+ influx via either NMDA or AMPA/kainate channels. The mechanism of the Na-dependent [Ca2+]c elevations may involve a reversal of plasmalemmal Na+/Ca2+ (NCX) and/or Na+/Ca2+ + K+ (NCKX) exchangers. KB-R7943, an NCX inhibitor, suppresses a fraction of the Na-dependent Ca2+ influx during AD. Therefore, Ca2+ influx via NCX and a KB-R7943-resistant pathway (possibly NCKX) is involved. Inhibition of the Na-dependent Ca2+ influx is likely to decrease ischemic brain damage. No drugs are known that are able to inhibit the KB-R7943-resistant component of Na-dependent Ca2+ influx during AD. The present data encourage development of such agents as potential therapeutic means to limit ischemic brain damage after stroke or heart attack.

Critical role of sodium in cytosolic [Ca2+] elevations in cultured hippocampal CA1 neurons during anoxic depolarization.

Although the extent of ischemic brain damage is directly proportional to the duration of anoxic depolarization (AD), the mechanism of cytosolic [Ca(2+)] ([Ca(2+)](c)) elevation during AD is poorly understood. To address the mechanism in this study, [Ca(2+)](c) was monitored in cultured rat hippocampal CA1 neurons loaded with a Ca-sensitive dye, fura-2FF, and exposed to an AD-simulating medium containing (in mmol/L): K(+) 65, Na(+) 50, Ca(2+) 0.13, glutamate 0.1, and pH reduced to 6.6. Application of this medium promptly elevated [Ca(2+)](c) to about 30 micromol/L, but only if oxygen was removed, the respiratory chain was inhibited, or if the mitochondria were uncoupled. These high [Ca(2+)](c) elevations depended on external Ca(2+) and could not be prevented by inhibiting NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors, or gadolinium-sensitive channels. However, they could be prevented by removing external Na(+) or simultaneously inhibiting NMDA and AMPA/kainate receptors; 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), an inhibitor of plasmalemmal Na(+)/Ca(2+) exchanger, partly suppressed them. The data indicate that the [Ca(2+)](c) elevations to 30 micromol/L during AD result from Na(+) influx. Activation of either NMDA or AMPA/kainate channels provides adequate Na(+) influx to induce these [Ca(2+)](c) elevations, which are mediated by KB-R7943-sensitive and KB-R7943-resistant mechanisms.

Unconventional neuroprotection against Ca2+ -dependent insults by metalloporphyrin catalytic antioxidants.

We evaluated whether both inert and catalytically active metalloporphyrin antioxidants, meso-substituted with either phenyl-based or N-alkylpyridinium-based groups, suppress Ca(2+)-dependent neurotoxicity in cell culture models of relevance to cerebral ischemia. Representatives from both metalloporphyrin classes, regardless of antioxidant strength, protected cultured cortical neurons or PC-12 cultures against the Ca(2+) ionophores ionomycin or A23187, by suppressing neurotoxic Ca(2+) influx. Some metalloporphyrins suppressed excitotoxic Ca(2+) influx indirectly induced by the Ca(2+) ionophores in cortical neurons. Metalloporphyrins did not quench intracellular fluorescence, suggesting localization to the plasma membrane interface and/or interference with Ca(2+) ionophores. Metalloporphyrins suppressed ionomycin-induced Mn(2+) influx, but did not protect cortical neurons against pyrithione, a Zn(2+) ionophore. In other Ca(2+)-dependent paradigms, Ca(2+) influx via plasma membrane depolarization, but not through reversal of plasmalemmal Na(+)/Ca(2+) exchangers, was modestly suppressed by Mn(III)meso-tetrakis(4-benzoic acid)porphyrin (Mn(III)TBAP) or by an inert analog, Zn(II)TBAP. Mn(III)TBAP and Zn(II)TBAP potently protected cortical neurons against long-duration oxygen-glucose deprivation (OGD), performed in the presence of antagonists of NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and L-type voltage-gated Ca(2+) channels, raising the possibility of an unconventional mode of blockade of transient receptor protein melastatin 7 channels by a metalloTBAP family of metalloporphyrins. The present study extends the range of Ca(2+)-dependent insults for which metalloporphyrins demonstrate unconventional neuroprotection. MetalloTBAPs appear capable of targeting an OGD temporal continuum.

Importance of K+-dependent Na+/Ca2+-exchanger 2, NCKX2, in motor learning and memory.

Plasma membrane Na+/Ca2+-exchangers play a predominant role in Ca2+ extrusion in brain. Neurons express several different Na+/Ca2+-exchangers belonging to both the K+-independent NCX family and the K+-dependent NCKX family. The unique contributions of each of these proteins to neuronal Ca2+ homeostasis and/or physiology remain largely unexplored. To address this question, we generated mice in which the gene encoding the abundant neuronal K+ -dependent Na+/Ca2+-exchanger protein, NCKX2, was knocked out. Analysis of these animals revealed a significant reduction in Ca2+ flux in cortical neurons, a profound loss of long term potentiation and an increase in long term depression at hippocampal Schaffer/CA1 synapses, and clear deficits in specific tests of motor learning and spatial working memory. Surprisingly, there was no obvious loss of photoreceptor function in cones, where expression of the NCKX2 protein had been reported previously. These data emphasize the critical and non-redundant role of NCKX2 in the local control of neuronal [Ca2+] that is essential for the development of synaptic plasticity associated with learning and memory.

High activity of K+-dependent plasmalemmal Na+/Ca2+ exchangers in hippocampal CA1 neurons.

Ca(2+) influx via reversed K(+)-dependent (NCKX) and/or K(+)-independent (NCX) plasmalemmal Na(+)/Ca(2+) exchangers may play a role in neuronal death following global brain ischemia to which CA1 neurons are particularly vulnerable. Therefore, this work tested whether the rates of Ca(2+) influx via reversed NCKX or NCX in cultured rat CA1 neurons differ from those in forebrain neurons (FNs) or cerebellar granule cells (CGCs). The NCKX-mediated Ca(2+) influx was several times more rapid in CA1 neurons than in FNs or CGCs and was not affected by Na(+)/Ca(2+) exchange inhibitors, KB-R7943 or bepridil. NCKX reversal inhibitors are not yet available. Their development would greatly facilitate further testing the role of NCKX in ischemic death of CA1 neurons.

Differential contribution of plasmalemmal Na/Ca exchange isoforms to sodium-dependent calcium influx and NMDA excitotoxicity in depolarized neurons.

Inhibition of Na(+),K(+)-ATPase during NMDA applications greatly increased NMDA-induced excitotoxicity in primary cultures of forebrain neurons (FNs), but not in cerebellar granule cells (CGCs). Because Na(+),K(+)-ATPase inhibition promotes reversal of plasmalemmal Na(+)/Ca(2+) exchangers, we compared the activities of reversed K(+)-independent (NCX) and K(+)-dependent (NCKX) Na(+)/Ca(2+) exchangers in these cultures. To this end, we measured gramicidin-induced and Na(+)-dependent elevation in cytosolic [Ca(2+)] ([Ca(2+)](c)) that represents Ca(2+) influx via reversed NCX and NCKX; NCX activity was dissected out by removing external K(+). The [Ca(2+)](c) elevations mediated by NCX alone, and NCX plus NCKX combined, were 17 and 6 times more rapid in FNs than in CGCs, respectively. Northern blot analysis showed that FNs preferentially express NCX1 whereas CGCs expressed NCX3. Differences in expression of other isoforms (NCX2, NCKX2, NCKX3 and NCKX4) were less pronounced. We tested whether the NCX or NCKX family of exchangers contributes most to the toxic NMDA-induced Ca(2+) influx in depolarized neurons. We found that in FNs, inhibition of NCX alone was sufficient to significantly limit NMDA excitotoxicity, whereas in CGCs, inhibition of both NCX and NCKX was required. The data suggest that the high activity of NCX isoforms expressed in FNs, possibly NCX1, sensitizes these neurons to NMDA excitotoxicity.

Inhibition of plasmalemmal Na(+)/Ca(2+) exchange by mitochondrial Na(+)/Ca(2+) exchange inhibitor 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157) in cerebellar granule cells.

In the heart, 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157) inhibits mitochondrial but not sarcolemmal Na(+)/Ca(2+) exchange. Therefore, CGP-37157 is often used as an experimental tool to study the role of mitochondrial Na(+)/Ca(2+) exchange in Ca(2+) homeostasis in various cells, including neurons. However, neurons express several K(+)-dependent (NCKX) and/or K(+)-independent (NCX) isoforms of plasmalemmal Na(+)/Ca(2+) exchange not expressed in the sarcolemma. Because it has never been determined whether CGP-37157 inhibits plasmalemmal NCKX and/or NCX isoforms in neurons, we tested this possibility. As an index of NCKX and/or NCX activity, we studied Na-dependent and gramicidin-induced 45Ca(2+) accumulation in the presence and absence of K(+), respectively. In primary cultures of cerebellar granule cells, CGP-37157 with IC(50) of 13 microM inhibited over 70% of plasmalemmal NCX activity (P<0.01) but not NCKX activity. Our data suggest that the effects of CGP-37157 on neuronal Ca(2+) homeostasis include inhibition of certain plasmalemmal NCX isoform(s). Because cerebellar granule cells robustly express NCX3 transcripts, which are not expressed in the heart, it appears that this isoform may be CGP-37157 sensitive.

In depolarized and glucose-deprived neurons, Na+ influx reverses plasmalemmal K+-dependent and K+-independent Na+/Ca2+ exchangers and contributes to NMDA excitotoxicity.

Cerebellar granule cells (CGCs) express K+-dependent (NCKX) and K+-independent (NCX) plasmalemmal Na+/Ca2+ exchangers which, under plasma membrane-depolarizing conditions and high cytosolic [Na+], may reverse and mediate potentially toxic Ca2+ influx. To examine this possibility, we inhibited NCX or NCKX with KB-R7943 or K+-free medium, respectively, and studied how gramicidin affects cytosolic [Ca2+] and 45Ca2+ accumulation. Gramicidin forms pores permeable to alkali cations but not Ca2+. Therefore, gramicidin-induced Ca2+ influx is indirect; it results from fluxes of monovalent cations. In the presence of Na+, but not Li+ or Cs+, gramicidin induced Ca2+ influx that was inhibited by simultaneous application of KB-R7943 and K+-free medium. The data indicate that gramicidin-induced Na+ influx reverses NCX and NCKX. To test the role of NCX and/or NCKX in excitotoxicity, we studied how NMDA affects the viability of glucose-deprived and depolarized CGCs. To assure depolarization of the plasma membrane, we inhibited Na+,K+-ATPase with ouabain. Although inhibition of NCX or NCKX reversal failed to significantly limit 45Ca2+ accumulation and excitotoxicity, simultaneously inhibiting NCX and NCKX reversal was neuroprotective and significantly decreased NMDA-induced 45Ca2+ accumulation. Our data suggest that NMDA-induced Na+ influx reverses NCX and NCKX and leads to the death of depolarized and glucose-deprived neurons.

Preferential expression of plasmalemmal K-dependent Na+/Ca2+ exchangers in neurons versus astrocytes.

Numerous isoforms of plasmalemmal K-dependent (NCKX) and K-independent (NCX) Na+/Ca2+ exchangers are expressed in the brain. The physiological functions of each isoform are presently unknown. Therefore, in this study, we compared expression of NCKX and NCX transcripts between primary cultures of cerebellar granule cells, and astrocytes. Northern blot analysis showed that granule cells expressed NCKX2, NCKX3, NCKX4 and NCX3, whereas astrocytes expressed primarily NCX1. Consistent with this molecular characterization, a significant fraction of 45Ca2+ accumulation in Na-loaded granule cells, but not in astrocytes, depended on external K+. This is the first demonstration of native NCKX activity in neurons derived from the central nervous system. Our data suggest that NCKX isoform expression may correspond to the unique Ca2+ homeostasis requirements of neurons.