Inhibition of Na+/Ca2+ exchange enhances delayed neuronal death elicited by glutamate in cerebellar granule cell cultures

doi:10.1016/0006-8993(91)91141-M

Brain Research

Volume 548, Issues 1–2, 10 May 1991, Pages 322-325

https://doi.org/10.1016/0006-8993(91)91141-M Get rights and content

Abstract

Experiments have been carried out on the primary cerebellar granule cell cultures from 7- to 8-day-old Wistar rats. To study a possible contribution of Na⁺/Ca²⁺ exchange to the toxic effect of glutamate, two amiloride derivatives, 3′,4′-dichlorobenzamil (DCB) and 5-(N-4-chlorobenzyl)-2′,4′-dimethylbenzamil (CBDMB), known to be the potent inhibitors of this exchange system, were used. Addition of DCB or CBDMB (at 30 and 10 μM, respectively) to a 25 μM glutamate solution dramatically enhanced the delayed neuronal death observed during the 4 h after termination of glutamate treatment. Similar but insignificantly smaller effects were obtained when these agents were added to the cultures in the post-glutamate period. Removal of Na⁺ (by substituting for choline chloride) from the external Mg²⁺ - free solution in the post-glutamate period also enhanced a delayed neuronal damage. The data obtained suggest that Na⁺/Ca²⁺ exchanger does not constitute the route for Ca²⁺ entry during the post-glutamate period but, on the contrary, attenuates glutamate neurotoxicity providing Ca²⁺ extrusion from the cells under the conditions of a sustained Ca²⁺ influx.

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Over the past several decades, an overwhelming body of research has greatly expanded our understanding of the mechanisms underlying excitotoxicity in brain ischemia as well as in many chronic neurodegenerative diseases. The identification of an array of molecular targets has opened avenues for neuroprotective strategies and, consequently, has sparked considerable interest for their attractive therapeutic means as pharmacological options. The purpose of this work is to provide a general overview of neuronal excitotoxicity and the inevitable downstream consequences of Ca²⁺ overload. We also discuss the contribution of Ca²⁺ transporters in excitotoxicity. This article is part of a Special Issue entitled “Calcium Pumps and Exchangers in Neuronal Injury and Neurodegeneration”.
Effects of preconditioning with normobaric hyperoxia on Na+/Ca2+ exchanger in the rat brain
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Citation Excerpt :
Thus, NCX plays a relevant role in the maintenance of the intracellular balance of these two ions. A great number of conflicting reports on the effects of NCX modulation on cell damage, induced by anoxic conditions, have been published (Andreeva et al., 1991; Amoroso et al., 1997, 2000; Masada et al., 2001; Takahashi et al., 2003). The results of studies provided evidence in vivo regarding the ability of NCX activation to reduce the extent of brain infarct volume after permanent middle cerebral artery occlusion (MCAO) and its selective pharmacological blockade produced a worsening of the brain lesion, thus suggesting a protective role played by the antiporter during the events leading to brain ischemia (Pignataro et al., 2004).
Recent studies suggest that normobaric hyperoxia (HO) reduces hypoxia-reoxygenation injury in the rat brain. We have attempted to determine the effect of HO on Na⁺–Ca²⁺ exchangers (NCX) in the rat stroke model.
Rats were divided into two experimental groups. The first group was exposed to 95% inspired HO for 4 h/day for 6 consecutive days (HO). The second group acted as the control, and was exposed to 21% oxygen in the same chamber. Each main group was subdivided to middle cerebral artery occlusion (MCAO-operated) and intact (without any surgery) subgroups. After 48 h from pretreatment, MCAO-operated subgroups were subjected to 60 min of right MCAO. After 24 h reperfusion, neurologic deficit score (NDS) and infarct volume were measured in MCAO-operated subgroups. The NCXs expression levels of the core, penumbra and subcortical regions were assessed in sham-operated and intact subgroups.
Preconditioning with HO decreased NDS and infarct volume, and increased the expression of NCX1, NCX2 and NCX3 in the penumbra, NCX2, NCX3 in the core and NCX1 and NCX3 in the subcortex.
Although further studies are needed to clarify the mechanisms of ischemic tolerance, HO partly is associated with the expression of NCX1, 2, 3 consistent with an active role in the genesis of ischemic protection.
Genomic analysis of [d-Ala2, d-Leu5] enkephalin preconditioning in cortical neuron and glial cell injury after oxygen deprivation
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Citation Excerpt :
The extent of excitotoxic cell death correlates with the total amount of Ca2 + uptake and is independent of the route of entry (Hartley et al., 1993; Lu et al., 1996). Ca2 + overload results from the instability of cellular Ca2 + homeostasis, such as extrusion of Ca2 + across the plasma membrane by the Na+/Ca2 + antiporter (Andreeva et al., 1991; Mattson et al., 1989; White and Reynolds, 1997) and Ca2 +ATPase (Carafoli, 1991), or Ca2 + sequestration by the endoplasmic reticulum and mitochondria (Gunter et al., 1994) to remove the large influx of Ca2 +. Consequently, a variety of Ca2 +-dependent hydrolytic enzymes, including lipases and proteases, has been suggested to be involved in excitotoxic neuronal damage.
[d-Ala², d-Leu⁵] enkephalin (DADLE) is a synthetic δ-opioid agonist that induces hibernation and promotes survival of neurons and glial cells in the central nervous system. Several mechanisms for the attenuation of hypoxic injury have been suggested, including control of intracellular signaling pathways via the δ-opioid receptor (DOR). However, the cellular and molecular mechanisms of DADLE in hypoxic injury are largely unknown. To investigate neuronal injury after oxygen-deprivation (OD) and DOR stimulation by DADLE, we used a lactate dehydrogenase assay, MTT assay, and immunofluorescence live/dead staining. And we used cDNA microarrays to investigate the influence of DADLE exposure on transcription after OD in rat cortical glial and neuronal co-culture. DADLE reduced neuronal injury after 24 h of OD. Preconditioning with DADLE before 24 h hypoxia exposure also altered gene expression in comparison with 24 h OD without pretreatment. After DADLE exposure and hypoxia, 1917 of 39,511 genes (4.9%) were significantly induced or repressed at least 2.5-fold. Assigning differentially expressed ESTs (expressed sequence tags) to molecular functional groups revealed that DADLE affected many pathways including apoptosis, intracellular ion homeostasis, molecular chaperones, and glucose metabolism. We observed a coordinated change in expression of many genes (increased expression of potentially protective genes and decreased expression of potentially harmful genes) after DADLE exposure. A comprehensive list of regulated genes should prove valuable in advancing our understanding of the neuroprotective mechanisms of DADLE under OD.
Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity
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The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca²⁺]_i) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca²⁺]_i). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca²⁺]_i increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca²⁺]_i. Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca²⁺]_i, demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca²⁺]_i and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca²⁺]_i and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival.
Calcium clearance and its energy requirements in cerebellar neurons
2010, Cell Calcium
Quick cytosolic calcium clearance is essential for the effective modulation of various cellular functions. An excess of cytosolic calcium after influx is largely removed via ATP-dependent mechanisms located in the plasma membrane and the endoplasmic reticulum. Therefore, calcium clearance depends critically on the adequate supply of ATP, which may come from either glycolysis or mitochondria, or both. However, it presently remains unknown the degree to which individual ATP generating pathways – glycolysis and mitochondria power ATP-dependent calcium as well as other vital ion clearance mechanisms in neurons. In this study, we explored the relationship between the energy generating pathways and ion clearance mechanisms in neurons by characterizing the effects of glycolytic and mitochondrial inhibitors of ATP synthesis on calcium clearance kinetics in the soma, dendrites and spines. Stimulation of cultured cerebellar granule cells by brief pulses of 60 mM potassium ACSF, and electrical stimulation of purkinje cells in acutely prepared slices led to a transient calcium influx, whose clearance was largely mediated by the plasma membrane Ca²⁺-ATPase pump. Inhibition of glycolysis by deoxyglucose or iodoacetic acid resulted in a marked slowing in calcium clearance in the soma, dendrites, and spines with the spines affected the most. However, inhibition of the mitochondrial citric acid cycle with fluoroacetate and arsenite, or mitochondrial ATP synthase with oligomycin did not produce any immediate effects on calcium clearance kinetics in any of those neuronal regions. Although cytosolic calcium clearance was not affected by the inhibition of mitochondria, the magnitude of the calcium clearance delay induced by glycolytic inhibitors in different neuronal compartments was related to their mitochondrial density. Conversely, the endoplasmic reticulum Ca²⁺-ATPase pump activity is fuelled by both glycolytic and mitochondrial ATP, as evidenced by a minimal change in the endoplasmic reticulum calcium contents in cells treated with either deoxyglucose supplemented with lactate or arsenite. Taken together, these data suggest that calcium clearance in cerebellar granule and purkinje cells relies on the plasma membrane Ca²⁺-ATPase, and is powered by glycolysis.
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There is uncertainty as to whether the plasma membrane Na⁺/Ca²⁺exchanger (NCX) has a neuroprotective or neurodamaging role following cerebral ischemia. To address this issue we compared hippocampal neuronal injury in NCX3 knockout mice (Ncx3^-/-) and wild-type mice (Ncx3^+/+) following global cerebral ischemia. Using a bilateral common carotid artery occlusion (BCCAO) model of global ischemia we subjected NCX3 knockout and wild-type mice to 17 and 15 minutes of ischemia. Following the 17 minute period of ischemia, wild-type mice exhibited ≈ 80% CA1 neuronal loss and ≈ 40% CA2 neuronal loss. In contrast, NCX3 knockout mice displayed > 95% CA1 neuronal loss and ≈ 95% CA2 neuronal loss. Following the 15 minute period of ischemia, wild-type mice did not exhibit any significant hippocampal neuronal loss. In contrast, NCX3 knockout mice displayed ≈ 45% CA1 neuronal loss and ≈ 25% CA2 neuronal loss. The results clearly demonstrate that mice deficient in the NCX3 protein are more susceptible to global cerebral ischemia than wild-type mice. Our findings suggest NCX3 has a positive role in maintaining neuronal intracellular calcium homeostasis following ischemia, and that when exchanger function is compromised neurons are more susceptible to calcium deregulation and cell death.

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Abstract

References (17)

Glutamate neurotoxicity and diseases of the nervous system

Neuron

Cultured sympathetic neurons: effects of cell-derived and synthetic substrata on survival and development

Dev. Biol.

Cell aggregation: properties of specific cell ligands and their role in the formation of multicelular system

Dev. Biol.

Glutamate receptor activation in cultured cerebellar granule cells increases cytosolic free Ca²⁺ by mobilization of cellular Ca²⁺ and activation of Ca²⁺ influx

Exp. Brain Res.

Dissociated nervous cell cutlures from rat cerebellum. Morphological characterization

Int. J. Neurosci.

Ionic dependence of glutamate neurotoxicity

J. Neurosci.

Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists

J. Neurosci.

Glutamate neurotoxicity in cortical cell culture

J. Neurosci.

Cited by (123)

Beyond the critical point: An overview of excitotoxicity, calcium overload and the downstream consequences

Effects of preconditioning with normobaric hyperoxia on Na<sup>+</sup>/Ca<sup>2+</sup> exchanger in the rat brain

Genomic analysis of [d-Ala<sup>2</sup>, d-Leu<sup>5</sup>] enkephalin preconditioning in cortical neuron and glial cell injury after oxygen deprivation

Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity

Calcium clearance and its energy requirements in cerebellar neurons

NCX3 knockout mice exhibit increased hippocampal CA1 and CA2 neuronal damage compared to wild-type mice following global cerebral ischemia

Inhibition of Na+/Ca2+ exchange enhances delayed neuronal death elicited by glutamate in cerebellar granule cell cultures

Abstract

Neuron

Dev. Biol.

Dev. Biol.

Glutamate receptor activation in cultured cerebellar granule cells increases cytosolic free Ca2+ by mobilization of cellular Ca2+ and activation of Ca2+ influx

Exp. Brain Res.

Dissociated nervous cell cutlures from rat cerebellum. Morphological characterization

Int. J. Neurosci.

Ionic dependence of glutamate neurotoxicity

J. Neurosci.

Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists

J. Neurosci.

Glutamate neurotoxicity in cortical cell culture

J. Neurosci.

Inhibition of Na⁺/Ca²⁺ exchange enhances delayed neuronal death elicited by glutamate in cerebellar granule cell cultures

Glutamate receptor activation in cultured cerebellar granule cells increases cytosolic free Ca²⁺ by mobilization of cellular Ca²⁺ and activation of Ca²⁺ influx