Stimulation of astrocyte Na+/H+ exchange activity in response to in vitro ischemia depends in part on activation of ERK1/2

We recently reported that Na⁺/H⁺ exchanger isoform 1 (NHE1) activity in astrocytes is stimulated and leads to intracellular Na⁺ loading after oxygen and glucose deprivation (OGD). However, the underlying mechanisms for this stimulation of NHE1 activity and its impact on astrocyte function are unknown. In the present study, we investigated the role of the ERK1/2 pathway in NHE1 activation. NHE1 activity was elevated by 75% in NHE1^+/+ astrocytes after 2-h OGD and 1-h reoxygenation (REOX). The OGD/REOX-mediated stimulation of NHE1 was partially blocked by 30 µM PD-98059. Increased expression of phosphorylated ERK1/2 was detected in NHE1^+/+ astrocytes after OGD/REOX. Moreover, stimulation of NHE1 activity disrupted not only Na⁺ but also Ca²⁺ homeostasis via reverse-mode operation of Na⁺/Ca²⁺ exchange. OGD/REOX led to a 103% increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in NHE1^+/+ astrocytes in the presence of thapsigargin. Inhibition of NHE1 activity with the NHE1 inhibitor HOE-642 decreased OGD/REOX-induced elevation of [Ca²⁺]_i by 73%. To further investigate changes of Ca²⁺ signaling, bradykinin-mediated Ca²⁺ release was evaluated. Bradykinin-mediated intracellular Ca²⁺ transient in NHE1^+/+ astrocytes was increased by 84% after OGD/REOX. However, in NHE1^–/– astrocytes or NHE1^+/+ astrocytes treated with HOE-642, the bradykinin-induced Ca²⁺ release was increased by only 34%. Inhibition of the reverse mode of Na⁺/Ca²⁺ exchange abolished OGD/REOX-mediated Ca²⁺ rise. Together, our data suggest that ERK1/2 is involved in activation of NHE1 in astrocytes after in vitro ischemia. NHE1-mediated Na⁺ accumulation subsequently alters Ca²⁺ homeostasis via Na⁺/Ca²⁺ exchange. intracellular pH; cortical astrocytes; sodium/calcium exchange; intracellular sodium ion     

Reversed Actrocytic GLT-1 during Ischemia is Crucial to Excitotoxic Death of Neurons, but Contributes to the Survival of Astrocytes themselves

During ischemia, the operation of astrocytic/neuronal glutamate transporters is reversed and glutamate and Na⁺ are co-transported to the extracellular space. This study aims to investigate whether this reversed operation of glutamate transporters has any functional meanings for astrocytes themselves. Oxygen/glucose deprivation (OGD) of neuron/astrocyte co-cultures resulted in the massive death of neurons, and the cell death was significantly reduced by treatment with either AP5 or DHK. In cultured astrocytes with little GLT-1 expression, OGD produced Na⁺ overload, resulting in the reversal of astrocytic Na⁺/Ca²⁺-exchanger (NCX). The reversed NCX then caused Ca²⁺ overload leading to the damage of astrocytes. In contrast, the OGD-induced Na⁺ overload and astrocytic damage were significantly attenuated in PACAP-treated astrocytes with increased GLT-1 expression, and the attenuation was antagonized by treatment with DHK. These results suggested that the OGD-induced reversal of GLT-1 contributed to the survival of astrocytes themselves by releasing Na⁺ with glutamate via reversed GLT-1.     

NOX activation in reactive astrocytes regulates astrocytic LCN2 expression and neurodegeneration

Reactive astrocytes (RA) secrete lipocalin-2 (LCN2) glycoprotein that regulates diverse cellular processes including cell death/survival, inflammation, iron delivery and cell differentiation. Elevated levels of LCN2 are considered as a biomarker of brain injury, however, the underlying regulatory mechanisms of its expression and release are not well understood. In this study, we investigated the role of astrocytic Na⁺/H⁺ exchanger 1 (NHE1) in regulating reactive astrocyte LCN2 secretion and neurodegeneration after stroke. Astrocyte specific deletion of Nhe1 in Gfap-Cre^ER+/−;Nhe1^f/f mice reduced astrogliosis and astrocytic LCN2 and GFAP expression, which was associated with reduced loss of NeuN⁺ and GRP78⁺ neurons in stroke brains. In vitro ischemia in astrocyte cultures triggered a significant increase of secreted LCN2 in astrocytic exosomes, which caused neuronal cell death and neurodegeneration. Inhibition of NHE1 activity during in vitro ischemia with its potent inhibitor HOE642 significantly reduced astrocytic LCN2⁺ exosome secretion. In elucidating the cellular mechanisms, we found that stroke triggered activation of NADPH oxidase (NOX)-NF-κB signaling and ROS-mediated LCN2 expression. Inhibition of astrocytic NHE1 activity attenuated NOX signaling and LCN2-mediated neuronal apoptosis and neurite degeneration. Our findings demonstrate for the first time that RA use NOX signaling to stimulate LCN2 expression and secretion. Blocking astrocytic NHE1 activity is beneficial to reduce LCN2-mediated neurotoxicity after stroke.Subject terms: Cell death in the nervous system, Astrocyte     

Role of Na+-K+-Cl- cotransport and Na+/Ca2+ exchange in mitochondrial dysfunction in astrocytes following in vitro ischemia

Na⁺-K⁺-Cl^– cotransporter isoform 1 (NKCC1) and reverse mode operation of the Na⁺/Ca²⁺ exchanger (NCX) contribute to intracellular Na⁺ and Ca²⁺ overload in astrocytes following oxygen-glucose deprivation (OGD) and reoxygenation (REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca²⁺ (Ca_m²⁺) overload and dysfunction. OGD/REOX caused a doubling of mitochondrial-releasable Ca²⁺ (P < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial-releasable Ca²⁺ was reduced by 42% (P < 0.05). Genetic ablation of NKCC1 also reduced Ca_m²⁺ accumulation. Moreover, OGD/REOX in NKCC1^+/+ astrocytes caused dissipation of the mitochondrial membrane potential (_m) to 42 ± 3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of _m was attenuated significantly (66 ± 10% of controls, P < 0.05). Cells were also subjected to severe in vitro hypoxia by superfusion with a hypoxic, acidic, ion-shifted Ringer buffer (HAIR). HAIR/REOX triggered a secondary, sustained rise in intracellular Ca²⁺ that was attenuated by reversal NCX inhibitor KB-R7943. The hypoxia-mediated increase in Ca_m²⁺ was accompanied by loss of _m and cytochrome c release in NKCC1^+/+ astrocytes. Bumetanide or genetic ablation of NKCC1 attenuated mitochondrial dysfunction and astrocyte death following ischemia. Our study suggests that NKCC1 acting in concert with NCX causes a perturbation of Ca_m²⁺ homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia. intracellular calcium ion; mitochondrial membrane potential; sodium ion influx; bumetanide; cytochrome c; glial cell death     

ERK1/2-p90RSK-mediated phosphorylation of Na+/H+ exchanger isoform 1. A role in ischemic neuronal death

The function and regulation of Na(+)/H(+) exchanger isoform 1 (NHE1) following cerebral ischemia are not well understood. In this study, we demonstrate that extracellular signal-related kinases (ERK1/2) play a role in stimulation of neuronal NHE1 following in vitro ischemia. NHE1 activity was significantly increased during 10-60 min reoxygenation (REOX) after 2-h oxygen and glucose deprivation (OGD). OGD/REOX not only increased the V(max) for NHE1 but also shifted the K(m) toward decreased [H(+)](i). These changes in NHE1 kinetics were absent when MAPK/ERK kinase (MEK) was inhibited by the MEK inhibitor U0126. There were no changes in the levels of phosphorylated ERK1/2 (p-ERK1/2) after 2 h OGD. The p-ERK1/2 level was significantly increased during 10-60 min REOX, which was accompanied by nuclear translocation. U0126 abolished REOX-induced elevation and translocation of p-ERK1/2. We further examined the ERK/90-kDa ribosomal S6 kinase (p90(RSK)) signaling pathways. At 10 min REOX, phosphorylated NHE1 was increased with a concurrent elevation of phosphorylation of p90(RSK), a known NHE1 kinase. Inhibition of MEK activity with U0126 abolished phosphorylation of both NHE1 and p90(RSK). Moreover, neuroprotection was observed with U0126 or genetic ablation or pharmacological inhibition of NHE1 following OGD/REOX. Taken together, these results suggest that activation of ERK1/2-p90(RSK) pathways following in vitro ischemia phosphorylates NHE1 and increases its activity, which subsequently contributes to neuronal damage.     

On the special role of NCX in astrocytes: Translating Na+-transients into intracellular Ca2+ signals

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na⁺, Ca²⁺) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca²⁺ at the expense of the Na⁺ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca²⁺ extrusion and thus to the recovery from intracellular Ca²⁺ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na⁺ to 1 Ca²⁺, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na⁺ concentration or minor depolarisations switch it into the “reverse mode” (Ca²⁺ import/Na⁺ export). Notably, transient Na⁺ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na⁺-permeable ion channels in response to neuronal activity. Activity-related Na⁺ transients result in NCX reversal, which mediates Ca²⁺ influx from the extracellular space, thereby generating astrocyte Ca²⁺ signalling independent from InsP₃-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca²⁺ overload, while dampening Na⁺ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na⁺-driven, reverse NCX-mediated astrocyte Ca²⁺ signals are involved neurone-glia interaction. Na⁺ transients, translated by the NCX into Ca²⁺ elevations, thereby emerge as a new signalling pathway in astrocytes.     

Amyloid-β and Alzheimer's disease type pathology differentially affects the calcium signalling toolkit in astrocytes from different brain regions

The entorhinal–hippocampal circuit is severely affected in Alzheimer''s disease (AD). Here, we demonstrate that amyloid-β (Aβ) differentially affects primary cultured astrocytes derived from the entorhinal cortex (EC) and from the hippocampus from non-transgenic controls and 3xTg-AD transgenic mice. Exposure to 100 nM of Aβ resulted in increased expression of the metabotropic glutamate receptor type 5 (mGluR5) and its downstream InsP₃ receptor type 1 (InsP₃R1) in hippocampal but not in EC astrocytes. Amplitudes of Ca²⁺ responses to an mGluR5 agonist, DHPG, and to ATP, another metabotropic agonist coupled to InsP₃Rs, were significantly increased in Aβ-treated hippocampal but not in EC astrocytes. Previously we demonstrated that senile plaque formation in 3xTg-AD mice triggers astrogliosis in hippocampal but not in EC astrocytes. The different sensitivities of the Ca²⁺ signalling toolkit of EC versus hippocampal astrocytes to Aβ may account for the lack of astrogliosis in the EC, which in turn can explain the higher vulnerability of this region to AD.     

Taurine and neural cell damage

Summary. The inhibitory amino acid taurine is an osmoregulator and neuromodulator, also exerting neuroprotective actions in neural tissue. We review now the involvement of taurine in neuron-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress, and the presence of free radicals, metabolic poisons and an excess of ammonia. The brain concentration of taurine is increased in several models of ischemic injury in vivo. Cell-damaging conditions which perturb the oxidative metabolism needed for active transport across cell membranes generally reduce taurine uptake in vitro, immature brain tissue being more tolerant to the lack of oxygen. In ischemia nonsaturable diffusion increases considerably. Both basal and K⁺-stimulated release of taurine in the hippocampus in vitro is markedly enhanced under cell-damaging conditions, ischemia, free radicals and metabolic poisons being the most potent. Hypoxia, hypoglycemia, ischemia, free radicals and oxidative stress also increase the initial basal release of taurine in cerebellar granule neurons, while the release is only moderately enhanced in hypoxia and ischemia in cerebral cortical astrocytes. The taurine release induced by ischemia is for the most part Ca²⁺-independent, a Ca²⁺-dependent mechanism being discernible only in hippocampal slices from developing mice. Moreover, a considerable portion of hippocampal taurine release in ischemia is mediated by the reversal of Na⁺-dependent transporters. The enhanced release in adults may comprise a swelling-induced component through Cl⁻ channels, which is not discernible in developing mice. Excitotoxic concentrations of glutamate also potentiate taurine release in mouse hippocampal slices. The ability of ionotropic glutamate receptor agonists to evoke taurine release varies under different cell-damaging conditions, the N-methyl-D-aspartate-evoked release being clearly receptor-mediated in ischemia. Neurotoxic ammonia has been shown to provoke taurine release from different brain preparations, indicating that the ammonia-induced release may modify neuronal excitability in hyperammonic conditions. Taurine released simultaneously with an excess of excitatory amino acids in the hippocampus under ischemic and other neuron-damaging conditions may constitute an important protective mechanism against excitotoxicity, counteracting the harmful effects which lead to neuronal death. The release of taurine may prevent excitation from reaching neurotoxic levels. Received January 25, 2000/Accepted January 31, 2000     

Mechanisms of D-Aspartate Release Under Ischemic Conditions in Mouse Hippocampal Slices

The release of preloaded D-[³H]aspartate, an unmetabolizable analogue of L-glutamate, was studied in superfused hippocampal slices from 7-day-old and 3-month-old (adult) mice under various cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and the presence of free radicals and metabolic poisons. The release was generally markedly enhanced in most of the above conditions, the responses being greater in adults than in developing mice. The presence of dinitrophenol had the most pronounced effect at both ages, followed by NaCN- and free-radical-containing media and ischemia. Hypoxia did not affect release in the immature hippocampus. Under most conditions K⁺ stimulation (50 mM) was still able markedly to enhance D-aspartate release. This potentiation under cell-damaging conditions in both adult and developing hippocampus signifies that increased L-glutamate release contributes to excitotoxicity and subsequent cell death. The mechanisms of ischemia-induced release of D-aspartate were analyzed in the adult hippocampus using ion channel inhibitors and modified superfusion media. The induced release proved to be partly Ca²⁺-dependent and partly Ca²⁺-independent. The results obtained with Na⁺ omission and homo- and heteroexchange with D-aspartate and L-glutamate demonstrated that a part of the release in normoxia and ischemia is mediated by the reversal of Na⁺-dependent glutamate transporters. The Na⁺ channel blockers amiloride and riluzole reduced the ischemia-induced release, also indicating the involvement of Na⁺ channels. In addition to this, the enhanced release of D-aspartate may comprise a swelling-induced component through chloride channels.     

Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes

Astroglial excitability operates through increases in Ca²⁺_cyt (cytosolic Ca²⁺), which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na⁺_cyt (cytosolic Na⁺) control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca²⁺-ATPase), NCX (plasma membrane Na⁺/Ca²⁺ exchanger) and NKA (Na⁺/K⁺-ATPase) in complex and coordinated regulation of Ca²⁺_cyt and Na⁺_cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na⁺ and Ca²⁺ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca²⁺ and Na⁺ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca²⁺ and Na⁺ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca²⁺-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca²⁺-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca²⁺ release from the ER (endoplasmic reticulum).     

Extracellular calcium depletion transiently elevates oxygen consumption in neurosecretory PC12 cells through activation of mitochondrial Na/Ca exchange

Fluctuating extracellular Ca²⁺ regulates many aspects of neuronal (patho)physiology including cell metabolism and respiration. Using fluorescence-based intracellular oxygen sensing technique, we demonstrate that depletion of extracellular Ca²⁺ from 1.8 to ≤ 0.6 mM by chelation with EGTA induces a marked spike in O₂ consumption in differentiated PC12 cells. This respiratory response is associated with the reduction in cytosolic and mitochondrial Ca²⁺, minor depolarization on the mitochondrial membrane, moderate depolarization of plasma membrane, and no changes in NAD(P)H and ATP. The response is linked to the influx of extracellular Na⁺ and the subsequent activation of mitochondrial Na⁺/Ca²⁺ and Na⁺/H⁺ exchange. The mitochondrial Na⁺/Ca²⁺ exchanger (_mNCX) activated by Na⁺ influx reduces Ca²⁺ and increases Na⁺ levels in the mitochondrial matrix. The excess of Na⁺ activates the mitochondrial Na⁺/H⁺ exchanger (NHE) increasing the outward pumping of protons, electron transport and O₂ consumption. Reduction in extracellular Na⁺ and inhibition of Na⁺ influx through the receptor operated calcium channels and plasmalemmal NHE reduce the respiratory response. Inhibition of the _mNCX, L-type voltage gated Ca²⁺ channels or the release of Ca²⁺ from the endoplasmic reticulum also reduces the respiratory spike, indicating that unimpaired intercompartmental Ca²⁺ exchange is critical for response development.     

Protective effects of Lycium barbarum polysaccharide on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion

This study investigated the protective effects of Lycium barbarum polysaccharide (LBP) on alleviating injury from oxygen-glucose deprivation/reperfusion (OGD/RP) in primary cultured rat hippocampal neurons. Cultured hippocampal neurons were exposed to oxygen-glucose deprivation (OGD) for 2?h followed by a 24?h re-oxygenation. The MTT assay and the lactate dehydrogenase (LDH) release were used to determine the neuron viability. Superoxide dismutase (SOD), Glutathione peroxidase (GSH-PX), malondialdehyde (MDA) were determined by spectrophotometry using commercial kits. Mitochondrial membrane potential (MMP) and the intracellular free calcium concentration ([Ca²⁺]_i) in hippocampal neurons were measured using the confocal laser scanning microscope (CLSM). Treatment with LBP (10–40?mg/l) significantly attenuated neuronal damage and inhibited LDH release in a dose-dependent manner. Furthermore, LBP enhanced activities of SOD and GSH-PX but it decreased their MDA content, inhibited [Ca²⁺]_i elevation and decrease of MMP in ischemia–reperfusion treated hippocampal neurons. These findings suggested that LBP may be a potential neuroprotective agent for cerebral?ischemia–reperfusion injury.     

Characterization of endogenous amino acid efflux from hippocampal slices during chemically-induced ischemia

Using sodium (NaN₃)-induced anoxia plus aglycaemia as a model of chemically-induced ischemia, we have characterized the endogenous release of excitatory and inhibitory amino acids from superfused hippocampal slices. Chemical ischemia produced an azide (1–30 mM) dose-dependent increase in the efflux of glutamate, aspartate and GABA. These increases were attenuated to varying degrees by removal of Ca²⁺, or the addition of the voltage dependent Na⁺-channel blocker tetrodotoxin (TTX), the selective Ca²⁺ channel blockers conotoxin MVIIA, MVIIC, and nifedipine, the NMDA antagonist MK801, the AMPA antagonist GYKI-52466. Similarly, addition of the GLT-1 glutamate transport inhibitor dihydrokainate (DHK) and the anti-estrogen/anion channel blocker tamoxifen also attenuated the efflux of glutamate and GABA. It would therefore appear that the increases in amino acid efflux induced by chemical ischemia originates from both the neuronal pool, via conventional exocytotic release, and glial sources via reversal of the GLT-1 transporter and anion channel regulated cell swelling.     

Distinct Subunit-specific α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Trafficking Mechanisms in Cultured Cortical and Hippocampal Neurons in Response to Oxygen and Glucose Deprivation

Brain ischemia occurs when the blood supply to the brain is interrupted, leading to oxygen and glucose deprivation (OGD). This triggers a cascade of events causing a synaptic accumulation of glutamate. Excessive activation of glutamate receptors results in excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts. The mechanisms that underlie this difference are unclear. Cultured hippocampal neurons respond to OGD with a rapid internalization of AMPA receptor (AMPAR) subunit GluA2, resulting in a switch from GluA2-containing Ca²⁺-impermeable receptors to GluA2-lacking Ca²⁺-permeable subtypes (CP-AMPARs). GluA2 internalization is a critical component of OGD-induced cell death in hippocampal neurons. It is unknown how AMPAR trafficking is affected in cortical neurons following OGD. Here, we show that cultured cortical neurons are resistant to an OGD insult that causes cell death in hippocampal neurons. GluA1 is inserted at the plasma membrane in both cortical and hippocampal neurons in response to OGD. In contrast, OGD causes a rapid endocytosis of GluA2 in hippocampal neurons, which is absent in cortical neurons. These data demonstrate that populations of neurons with different vulnerabilities to OGD recruit distinct cell biological mechanisms in response to insult, and that a crucial aspect of the mechanism leading to OGD-induced cell death is absent in cortical neurons. This strongly suggests that the absence of OGD-induced GluA2 trafficking contributes to the relatively low vulnerability of cortical neurons to ischemia.     

Astrocyte dystrophy in ageing brain parallels impaired synaptic plasticity

Little is known about age‐dependent changes in structure and function of astrocytes and of the impact of these on the cognitive decline in the senescent brain. The prevalent view on the age‐dependent increase in reactive astrogliosis and astrocytic hypertrophy requires scrutiny and detailed analysis. Using two‐photon microscopy in conjunction with 3D reconstruction, Sholl and volume fraction analysis, we demonstrate a significant reduction in the number and the length of astrocytic processes, in astrocytic territorial domains and in astrocyte‐to‐astrocyte coupling in the aged brain. Probing physiology of astrocytes with patch clamp, and Ca²⁺ imaging revealed deficits in K⁺ and glutamate clearance and spatiotemporal reorganisation of Ca²⁺ events in old astrocytes. These changes paralleled impaired synaptic long‐term potentiation (LTP) in hippocampal CA1 in old mice. Our findings may explain the astroglial mechanisms of age‐dependent decline in learning and memory.     

A Model of Na/H Exchanger and Its Central Role in Regulation of pH and Na in Cardiac Myocytes

A new kinetic model of the Na⁺/H⁺ exchanger (NHE) was developed by fitting a variety of major experimental findings, such as ion-dependencies, forward/reverse mode, and the turnover rate. The role of NHE in ion homeostasis was examined by implementing the NHE model in a minimum cell model including intracellular pH buffer, Na⁺/K⁺ pump, background H⁺, and Na⁺ fluxes. This minimum cell model was validated by reconstructing recovery of pH_i from acidification, accompanying transient increase in [Na⁺]_i due to NHE activity. Based on this cell model, steady-state relationships among pH_i, [Na⁺]_I, and [Ca²⁺]_i were quantitatively determined, and thereby the critical level of acidosis for cell survival was predicted. The acidification reported during partial blockade of the Na⁺/K⁺ pump was not attributed to a dissipation of the Na⁺ gradient across the membrane, but to an increase in indirect H⁺ production. This NHE model, though not adapted to the dimeric behavioral aspects of NHE, can provide a strong clue to quantitative prediction of degree of acidification and accompanying disturbance of ion homeostasis under various pathophysiological conditions.     

Mitochondrial handling of excess Ca2+ is substrate-dependent with implications for reactive oxygen speciesgeneration

The mitochondrial electron transport chain is the major source of reactive oxygen species (ROS) during cardiac ischemia. Several mechanisms modulate ROS production; one is mitochondrial Ca²⁺ uptake. Here we sought to elucidate the effects of extramitochondrial Ca²⁺ (e[Ca²⁺]) on ROS production (measured as H₂O₂ release) from complexes I and III. Mitochondria isolated from guinea pig hearts were preincubated with increasing concentrations of CaCl₂ and then energized with the complex I substrate Na⁺ pyruvate or the complex II substrate Na⁺ succinate. Mitochondrial H₂O₂ release rates were assessed after giving either rotenone or antimycin A to inhibit complex I or III, respectively. After pyruvate, mitochondria maintained a fully polarized membrane potential (ΔΨ; assessed using rhodamine 123) and were able to generate NADH (assessed using autofluorescence) even with excess e[Ca²⁺] (assessed using CaGreen-5N), whereas they remained partially depolarized and did not generate NADH after succinate. This partial ΔΨ depolarization with succinate was accompanied by a large release in H₂O₂ (assessed using Amplex red/horseradish peroxidase) with later addition of antimycin A. In the presence of excess e[Ca²⁺], adding cyclosporin A to inhibit mitochondrial permeability transition pore opening restored ΔΨ and significantly decreased antimycin A-induced H₂O₂ release. Succinate accumulates during ischemia to become the major substrate utilized by cardiac mitochondria. The inability of mitochondria to maintain a fully polarized ΔΨ under excess e[Ca²⁺] when succinate, but not pyruvate, is the substrate may indicate a permeabilization of the mitochondrial membrane, which enhances H₂O₂ emission from complex III during ischemia.     

Astrocytic gap junctional networks suppress cellular damage in an in vitro model of ischemia

Astrocytes play pivotal roles in both the physiology and the pathophysiology of the brain. They communicate with each other via extracellular messengers as well as through gap junctions, which may exacerbate or protect against pathological processes in the brain. However, their roles during the acute phase of ischemia and the underlying cellular mechanisms remain largely unknown. To address this issue, we imaged changes in the intracellular calcium concentration ([Ca²⁺]_i) in astrocytes in mouse cortical slices under oxygen/glucose deprivation (OGD) condition using two-photon microscopy. Under OGD, astrocytes showed [Ca²⁺]_i oscillations followed by larger and sustained [Ca²⁺]_i increases. While the pharmacological blockades of astrocytic receptors for glutamate and ATP had no effect, the inhibitions of gap junctional intercellular coupling between astrocytes significantly advanced the onset of the sustained [Ca²⁺]_i increase after OGD exposure. Interestingly, the simultaneous recording of the neuronal membrane potential revealed that the onset of the sustained [Ca²⁺]_i increase in astrocytes was synchronized with the appearance of neuronal anoxic depolarization. Furthermore, the blockade of gap junctional coupling resulted in a concurrent faster appearance of neuronal depolarizations, which remain synchronized with the sustained [Ca²⁺]_i increase in astrocytes. These results indicate that astrocytes delay the appearance of the pathological responses of astrocytes and neurons through their gap junction-mediated intercellular network under OGD. Thus, astrocytic gap junctional networks provide protection against tissue damage during the acute phase of ischemia.     

Overexpression of the NHE1 isoform of the Na+/H+ exchanger causes elevated apoptosis in isolated cardiomyocytes after hypoxia/reoxygenation challenge

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH in the myocardium and other tissues. NHE1 is an important mediator of myocardial damage that occurs after ischemia–reperfusion injury. It has also been implicated in apoptotic damage in many tissues and its expression and activity are elevated in disease states in the myocardium. In this study, we examined the effect of additional exogenous NHE1 expression on isolated cardiomyocytes susceptibility to ischemia/reperfusion damage. Exogenous NHE1 elevated Na⁺/H⁺ exchanger expression and activity when introduced into isolated cardiomyocytes through an adenoviral system. Isolated cardiomyocytes were subjected to simulated ischemia and reperfusion after infection with either control or NHE1-containing adenovirus. Cells were placed into an anaerobic chamber and effects of NHE1 expression after hypoxia/reoxygenation were examined. Hypoxia/reoxygenation increased caspase-3-like activity in controls, and the effect was greatly magnified in cells expressing NHE1 protein. It also elevated the percentage of apoptotic cardiomyocytes, which was also aggravated by expression of NHE1 protein. Hypoxia/reoxygenation also increased phospho-ERK levels. Elevated NHE1 expression was coincidental with increased expression of the ER stress protein, protein disulfide isomerase (PDI) and calreticulin (CRT). Our results demonstrate that increased NHE1 protein expression makes cells more susceptible to damage induced by hypoxia/reoxygenation in isolated cardiomyocytes. They suggest that elevated NHE1 in cardiovascular disease could predispose the human myocardium to enhanced apoptotic damage.