Lanthanum Induced Primary Neuronal Apoptosis Through Mitochondrial Dysfunction Modulated by Ca2+ and Bcl-2 Family

As a representative element of lanthanide, lanthanum has been widely used in various fields and eventually entered environment and accumulated in human body. Epidemiological and experimental evidences indicated that lanthanum has neurotoxicity; however, the detailed mechanism is still elusive. Here, we chose primary cerebral cortical neurons as model in vitro to investigate the mechanism underlying the toxic effects of lanthanum chloride (LaCl₃). This study revealed the following findings: (1) LaCl₃ treatment (0.01, 0.1, and 1.0 mM for 24 h) reduced the viability of cortical neurons and elevated apoptotic rate significantly in a dose-dependent manner. (2) LaCl₃ triggered mitochondrial apoptotic pathway in cortical neurons, characterized with collapsed mitochondrial membrane potential, release of cytochrome c into cytosol, and increasing expression of activated caspase-3. (3) LaCl₃ elevated intracellular Ca²⁺ concentration, promoted reactive oxygen species generation, and upregulated pro-apoptotic Bax, whereas it downregulated anti-apoptotic Bcl-2 expression and consequently altered Bax/Bcl-2 ratio, which ultimately lead to neuronal mitochondrial apoptosis. Our results demonstrated that toxicity of lanthanum in cortical neurons perhaps partly attributed to enhanced mitochondrial apoptosis due to mitochondrial dysfunction modulated by Ca²⁺ and Bcl-2 family.     

KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia

Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca²⁺]_i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca²⁺]_i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (K_Ca2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of K_Ca2.2 channels within 3 h after the onset of glutamate exposure. Activation of K_Ca2 channels preserved K_Ca2 expression and significantly reduced pathological increases in [Ca²⁺]_i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for K_Ca2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.     

Kinetics of Apoptosis and Expression of Apoptosis-Related Proteins in Rat CA3 Hippocampus Cells After Experimental Diffuse Brain Injury

The present study examined kinetics of apoptosis and expression of apoptosis-related proteins Bcl-2, Bax, and caspase-3 in the CA3 hippocampus cells after diffuse brain injury (DBI) induced experimentally in rats. Percentage of apoptotic cells and expressions of above proteins were examined by flow cytometry and immunohistochemistry. Substantial neuronal apoptosis was documented in the CA3 hippocampus cells after DBI (22.26 ± 2.97 % at 72 h after DBI vs. 2.92 ± 0.88 % in sham-operated animals). Expression of Bc1-2 decreased, while expression of Bax and caspase-3 increased after DBI, with caspase-3 expression peaking after that of Bax (72 vs. 48 h, respectively). Further, the Bc1-2/Bax expression ratio decreased prior to increase of caspase-3 expression. In conclusion, cell apoptosis and altered expressions of Bcl-2, Bax, and caspase-3 are present in the CA3 region of hippocampus after experimental DBI. Changes in the Bc1-2/Bax expression ratio may facilitate activation of caspase-3 and aggravate neuronal apoptosis after brain injury.     

Molecular mechanisms of apoptosis induction by 2-dodecylcyclobutanone,a radiolytic product of palmitic acid,in human lymphoma U937 cells

The irradiation of fat-containing food forms 2-dodecylcyclobutanone (2-DCB) from palmitic acid (PA). In this study, we investigated whether 2-DCB and PA induce apoptosis in human lymphoma U937 cells. We found that cell viability decreased by 2-DCB and apoptosis was induced by 2-DCB and PA. 2-DCB and PA significantly enhanced the formation of intracellular reactive oxygen species (ROS). Apoptosis induced by 2-DCB and PA was strongly prevented by an antioxidant, N-acetyl-l-cysteine. The treatment with 2-DCB and PA resulted in the loss of mitochondrial membrane potential, and Fas, caspase-8 and caspase-3 activation. Pretreatment with a pan-caspase inhibitor (z-VAD) significantly inhibited apoptosis induced by 2-DCB and PA. Moreover, 2-DCB and PA also induced Bax up-regulation, the reduction in Bcl-2 expression level, Bid cleavage and the release of cytochrome c from the mitochondria to the cytosol. In addition, an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) was observed after the treatment with 2-DCB and PA. Our results indicated that intracellular ROS generation, the modulation of the Fas-mitochondrion-caspase-dependent pathway and the increase in [Ca²⁺]_i involved in apoptosis are induced by 2-DCB and PA in U937 cells.     

Gastrodin Inhibits Glutamate-Induced Apoptosis of PC12 Cells via Inhibition of CaMKII/ASK-1/p38 MAPK/p53 Signaling Cascade

Previous research demonstrated that glutamate induces neuronal injury partially by increasing intracellular Ca²⁺ concentrations ([Ca²⁺]_i), and inducing oxidative stress, leading to a neurodegenerative disorder. However, the mechanism of glutamate-induced injury remains elusive. Gastrodin, a major active component of the traditional herbal agent Gastrodia elata (GE) Blume, has been recognized as a potential neuroprotective drug. In the current study, a classical injury model based on glutamate-induced cell death of rat pheochromocytoma (PC12) cells was used to investigate the neuroprotective effect of gastrodin, and its potential mechanisms involved. In this paper, the presence of gastrodin inhibits glutamate-induced oxidative stress as measured by the formation of reactive oxygen species (ROS), the level of malondialdehyde (MDA), mitochondrial membrane potential (MMP), and superoxide dismutase (SOD); gastrodin also prevents glutamate-induced [Ca²⁺]_i influx, blocks the activation of the calmodulin-dependent kinase II (CaMKII) and the apoptosis signaling-regulating kinase-1 (ASK-1), inhibits phosphorylation of p38 mitogen-activated kinase (MAPK). Additionally, gastrodin blocked the expression of p53 phosphorylation, caspase-3 and cytochrome C, reduced bax/bcl-2 ratio induced by glutamate in PC12 cells. All these findings indicate that gastrodin protects PC12 cells from the apoptosis induced by glutamate through a new mechanism of the CaMKII/ASK-1/p38 MAPK/p53-signaling pathway.     

Calcium Sequestering Ability of Mitochondria Modulates Influx of Calcium through Glutamate Receptor Channel

The excitotoxicity of glutamate is believed to be mediated by sustained increase in the cytosolic Ca²⁺ concentration. Mitochondria play a vital role in buffering the cytosolic calcium overload in stimulated neurons. Here we have studied the glutamate induced Ca²⁺ signals in cortical brain slices under physiological conditions and the conditions that modify the mitochondrial functions. Exposure of slices to glutamate caused a rapid increase in [Ca²⁺]_i followed by a slow and persistently rising phase. The rapid increase in [Ca²⁺]_i was mainly due to influx of Ca²⁺ through the N-methyl-D-aspartate (NMDA) receptor channels. Glutamate stimulation in the absence of Ca²⁺ in the extracellular medium elicited a small transient rise in [Ca²⁺]_i which can be attributed to the mobilization of Ca²⁺ from IP₃ sensitive endoplasmic reticulum pools consequent to activation of metabotropic glutamate receptors. The glutamate induced Ca²⁺ influx was accompanied by depolarization of the mitochondrial membrane, which was inhibited by ruthenium red, the blocker of mitochondrial Ca²⁺ uniporter. These results imply that mitochondria sequester the Ca²⁺ loaded into the cytosol by glutamate stimulation. Persistent depolarization of mitochondrial membrane observed in presence of extracellular Ca²⁺ caused permeability transition and released the sequestered Ca²⁺ which is manifested as slow rise in [Ca²⁺]_i. Protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) depolarized the mitochondrial membrane and enhanced the glutamate induced [Ca²⁺]_i response. Contrary to this, treatment of slices with mitochondrial inhibitor oligomycin or ruthenium red markedly reduced the [Ca²⁺]_i response. Combined treatment with oligomycin and rotenone further diminished the [Ca²⁺]_i response and also abolished the CCCP mediated rise in [Ca²⁺]_i. However, rotenone alone had no effect on glutamate induced [Ca²⁺]_i response. These changes in glutamate-induced [Ca²⁺]_i response could not be explained on the basis of deficient mitochondrial Ca²⁺ sequestration or ATP dependent Ca²⁺ buffering. The mitochondrial inhibitors reduced the cellular ATP/ADP ratio, however, this would have restrained the ATP dependent Ca²⁺ buffering processes leading to elevation of [Ca²⁺]_i. In contrast our results showed repression of Ca²⁺ signal except in case of CCCP which drastically reduced the ATP/ADP ratio. It was inferred that, under the conditions that hamper the Ca²⁺ sequestering ability of mitochondria, the glutamate induced Ca²⁺ influx could be impeded. To validate this, influx of Mn²⁺ through ionotropic glutamate receptor channel was monitored by measuring the quenching of Fura-2 fluorescence. Treatment of slices with oligomycin and rotenone prior to glutamate exposure conspicuously reduced the rate of glutamate induced fluorescence quenching as compared to untreated slices. Thus our data establish that the functional status of mitochondria can modify the activity of ionotropic glutamate receptor and suggest that blockade of mitochondrial Ca²⁺ sequestration may desensitize the NMDA receptor operated channel.     

NMDA Receptor-Mediated Calcium Influx in Cerebral Cortical Synaptosomes of the Hypoxic Guinea Pig Fetus

Calcium influx via the NMDA receptor has been proposed as a mechanism of hypoxia-induced neuronal injury. The present study tests the hypothesis that the increase of [Ca²⁺]_i observed under hypoxic conditions is the result of an NMDA-mediated Ca²⁺ influx. Changes of [Ca²⁺]_i, measured fluorometrically with Fura-2, were followed after activation of the NMDA receptor with NMDA and glutamate, in the presence of glycine, in cortical synaptosomes prepared from six normoxic and six hypoxic guinea pig fetuses. [Ca²⁺]_i was significantly higher in hypoxic vs normoxic synaptosomes, at baseline and in the presence of glycine as well as following activation of the NMDA receptor. Increase in [Ca²⁺]_i was not observed in a Ca²⁺ free medium and was significantly decreased by MK-801 and thapsigargin. These results demonstrate that hypoxia-induced modifications of the NMDA receptor ion-channel results in increased [Ca²⁺]_i in hypoxic vs normoxic synaptosomes. This increased accumulation may be due to an initial influx of Ca²⁺ via the altered NMDA receptor with subsequent release of Ca²⁺ from intracellular stores. Increase in intracellular calcium may initiate several pathways of free radical generation including cyclooxygenase, lipoxygenase, xanthine oxidase and nitric oxide synthase, and lead to membrane lipid peroxidation resulting in neuronal cell damage.     

Evaluation of Insulin and Ascorbic Acid Effects on Expression of Bcl-2 Family Proteins and Caspase-3 Activity in Hippocampus of STZ-Induced Diabetic Rats

Aims Effects of insulin and ascorbic acid on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of diabetic rats were evaluated in this study. Methods Diabetes was induced in Wistar male rats by streptozotocin (STZ). Six weeks after verification of diabetes, the animals were treated for 2 weeks with insulin or/and ascorbic acid in separate groups. Hippocampi of rats were removed and evaluation of Bcl-2, Bcl-x_L, and Bax proteins expression in frozen hippocampi tissues were done by SDS-PAGE electrophoresis and blotting. The Bcl-2, Bcl-x_L, and Bax proteins bands were visualized after incubation with specific antibodies using enhanced chemiluminescences method. Caspase-3 activity was determined using the caspase-3/CPP32 Fluorometric Assay Kit. Results Diabetic rats showed increase in Bax protein expression and decrease in Bcl-2 and Bcl-x_L proteins expression. The Bax/Bcl-2 and Bax/Bcl-x_L ratios were found higher compared with non-diabetic control group. Treatments with insulin and/or ascorbic acid were resulted in decrease in Bax protein expression and increase in Bcl-2 and Bcl-x_L proteins expression. The Bcl-2/Bax and Bcl-x_L/Bax ratios were found higher in treated groups than untreated diabetic group. Caspase-3 activity level was found higher in diabetic group compared with non-diabetic group. Treatment with insulin and ascorbic acid did downregulated caspase-3 activity. Conclusions Our data provide supportive evidence to demonstrate the antiapoptotic effects of insulin and ascorbic acid on hippocampus of STZ-induced diabetic rats.     

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.     

Intracellular calcium plays a critical role in the alcohol‐mediated death of cerebellar granule neurons

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol‐induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol‐induced increase in intracellular calcium [Ca²⁺]_i causes the death of CGN. Alcohol induced a dose‐dependent (200–800 mg/dL) neuronal death within 24 h. Ratiometric Ca²⁺ imaging with Fura‐2 revealed that alcohol causes a rapid (1–2 min), dose‐dependent increase in [Ca²⁺]_i, which persisted for the duration of the experiment (5 or 7 min). The alcohol‐induced increase in [Ca²⁺]_i was observed in Ca²⁺‐free media, suggesting intracellular Ca²⁺ release. Pre‐treatment of CGN cultures with an inhibitor (2‐APB) of the inositol‐triphosphate receptor (IP₃R), which regulates Ca²⁺ release from the endoplasmic reticulum (ER), blocked both the alcohol‐induced rise in [Ca²⁺]_i and the neuronal death caused by alcohol. Similarly, pre‐treatment with BAPTA/AM, a Ca²⁺‐chelator, also inhibited the alcohol‐induced surge in [Ca²⁺]_i and prevented neuronal death. In conclusion, alcohol disrupts [Ca²⁺]_i homeostasis in CGN by releasing Ca²⁺ from intracellular stores, resulting in a sustained increase in [Ca²⁺]_i. This sustained increase in [Ca²⁺]_i may be a key determinant in the mechanism underlying alcohol‐induced neuronal death.     

Transient overexpression of TGFBR3 induces apoptosis in human nasopharyngeal carcinoma CNE-2Z cells

NPC (nasopharyngeal carcinoma) is a common malignancy in southern China without defined aetiology. Recent studies have shown that TGFBR3 (transforming growth factor type III receptor, also known as betaglycan), exhibits anticancer activities. This study was to investigate the effects of TGFBR3 on NPC growth and the mechanisms for its actions. Effects of TGFBR3 overexpression on cell viability and apoptosis were measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], AO/EB (acridine orange/ethidium bromide) staining and electron microscopy in human NPC CNE-2Z cells. The expression of apoptosis-related proteins, p-Bad, Bad, XIAP (X-linked inhibitor of apoptosis), AIF (apoptosis-inducing factor), Bax and Bcl-2, was determined by Western blot or immunofluorescence analysis. Caspase 3 activity was measured by caspase 3 activity kit and [Ca²⁺]_i (intracellular Ca²⁺ concentration) was detected by confocal microscopy. Transfection of TGFBR3 containing plasmid DNA at concentrations of 0.5 and 1 μg/ml reduced viability and induced apoptosis in CNE-2Z in concentration- and time-dependent manners. Forced expression of TGFBR3 up-regulated pro-apoptotic Bad and Bax protein, and down-regulated anti-apoptotic p-Bad, Bcl-2 and XIAP protein. Furthermore, transient overexpression of TGFBR3 also enhanced caspase 3 activity, increased [Ca²⁺]_i and facilitated AIF redistribution from the mitochondria to the nucleus in CNE-2Z cells, which is independent of the caspase 3 pathway. These events were associated with TGFBR3-regulated multiple targets involved in CNE-2Z proliferation. Therefore transient overexpression of TGFBR3 may be a novel strategy for NPC prevention and therapy.     

Post-conditioning protects cardiomyocytes from apoptosis via PKCε-interacting with calcium-sensing receptors to inhibit endo(sarco)plasmic reticulum–mitochondria crosstalk

The intracellular Ca²⁺ concentration ([Ca²⁺]_i) is increased during cardiac ischemia/reperfusion injury (IRI), leading to endo(sarco)plasmic reticulum (ER) stress. Persistent ER stress, such as with the accumulation of [Ca²⁺]_i, results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca²⁺]_i via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP₃) to increase the release of intracellular Ca²⁺ from the ER. This process can be negatively regulated by PKC through the phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that PC prevents cardiomyocyte apoptosis by reducing the [Ca²⁺]_i through an interaction of PKC with CaR to alleviate [Ca²⁺]_ER depletion and [Ca²⁺]_m elevation by the ER-mitochondrial associated membrane (MAM). Cardiomyocytes were post-conditioned after 3 h of ischemia by three cycles of 5 min of reperfusion and 5 min of re-ischemia before 6 h of reperfusion. During PC, PKC_ε translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca²⁺]_i, the [Ca²⁺]_ER was not reduced and [Ca²⁺]_m was not increased in the PC and GdCl₃–PC groups. Furthermore, there was no evident ?ψ_m collapse during PC compared with ischemia/reperfusion (I/R) or PKC inhibitor groups, as evaluated by laser confocal scanning microscopy. The apoptotic rates detected by TUNEL and Hoechst33342 were lower in PC and GdCl₃–PC groups than those in I/R and PKC inhibitor groups. Apoptotic proteins, including m-calpain, BAP31, and caspase-12, were significantly increased in the I/R and PKC inhibitor groups. These results suggested that PKC_ε interacting with CaR protected post-conditioned cardiomyocytes from programmed cell death by inhibiting disruption of the mitochondria by the ER as well as preventing calcium-induced signaling of the apoptotic pathway.     

Bcl-2 Protects Against Apoptosis in Neuronal Cell Line Caused by Thapsigargin-Induced Depletion of Intracellular Calcium Stores

Abstract: The toxicity of thapsigargin, a selective inhibitor of endoplasmic reticular Ca²⁺-ATPase, was investigated in GT1-7 cells, a murine hypothalamic cell line. Treatment of these cells with 50 or 100 nM thapsigargin greatly reduced cell viability at 24 and 48 h. These doses of thapsigargin induced a rapid rise in free cytosolic Ca²⁺ ([Ca²⁺]_i), followed by a sustained increase. Addition of EGTA to chelate extracellular Ca²⁺ diminished somewhat the size of the initial increase of [Ca²⁺]_i caused by thapsigargin, and abolished the sustained increase. The sustained increase could also be abolished by addition of La³⁺ and by SKF 96365, a drug selective for receptor-mediated calcium entry, but not by verapamil or flunarizine. Pretreatment with 50 µM BAPTA/AM, a cytosolic Ca²⁺ chelator, inhibited the peak [Ca²⁺]_i caused by thapsigargin but did not inhibit the sustained elevation of [Ca²⁺]_i. Neither EGTA nor BAPTA/AM inhibited the cell death induced by thapsigargin. The cell death was characterized by DNA fragmentation (“laddering”), nuclear condensation and fragmentation, and was inhibited by protein synthesis inhibitor cycloheximide, all characteristic of apoptotic cell death. Overexpression of the proto-oncogene bcl-2 in GT1-7 cells inhibited significantly DNA fragmentation, nuclear condensation and fragmentation, and cell death induced by thapsigargin. However, Bcl-2 did not alter either basal [Ca²⁺]_i or the elevation of [Ca²⁺]_i induced by thapsigargin. Our results suggest that abnormal Ca²⁺ release from endoplasmic reticulum caused by thapsigargin induces GT1-7 death by apoptosis and that this effect does not depend on Ca²⁺ influx from the extracellular space. Bcl-2 inhibited apoptosis induced by thapsigargin, but the mechanism is unlikely to be inhibition of endoplasmic reticular Ca²⁺ release in GT1-7 neuronal cells.     

Single-Prolonged Stress Induces Endoplasmic Reticulum - Dependent Apoptosis in the Hippocampus in a Rat Model of Post-Traumatic Stress Disorder

Background

Our previous research indicated that apoptosis induced atrophy in the hippocampus of post-traumatic stress disorder (PTSD) rats. Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in the development of several disorder diseases. The aim of this study was to investigate whether endoplasmic reticulum-related pathway is involved in single-prolonged stress (SPS) induces apoptosis in the hippocampus of PTSD rats by examining the expression levels of three important indicators in the ER-related apoptotic pathway: Glucose-regulated protein (GRP) 78, caspase-12 and Ca²⁺/CaM/CaMkinaseIIα (CaMkIIα).

Methods

Wistar rats were sacrificed at 1, 4 and 7 days after SPS. SPS is a reliable animal model of PTSD. The apoptotic cells in the hippocampus were assessed by TUNEL method and transmission electron microscopy (TEM). Free intracellular Ca²⁺ concentration was measured. GRP78 expression was examined by immunohistochemistry, western blotting and RT-PCR. mRNA of caspase-12 and CaM/CaMkIIα were determined by RT-PCR.

Results

Our results showed that apoptotic cells were increased in the SPS rats. TEM analysis revealed characteristic morphological changes of apoptosis in these cells. We observed that GRP78 was significantly up-regulated during early PTSD, and then recovered at 7 days after SPS. By RT-PCR, we observed that the change in caspase-12 expression level was similar to that in GRP78. Moreover, the free intracellular Ca²⁺ concentration was significantly higher at 1 day after SPS and decreased in 7 days. CaM expression increased significantly, while CaMKIIα expression decreased significantly in the hippocampus at 1 day after SPS.

Conclusion

SPS induced change in the expression levels of GRP78, caspase-12 and Ca²⁺/CaM/CaMkIIα in the hippocampus of PTSD rats indicated that the endoplasmic reticulum pathway may be involved in PTSD-induced apoptosis.     

Ca2+-Permeable Acid-sensing Ion Channels and Ischemic Brain Injury

Acidosis is a common feature of brain in acute neurological injury, particularly in ischemia where low pH has been assumed to play an important role in the pathological process. However, the cellular and molecular mechanisms underlying acidosis-induced injury remain unclear. Recent studies have demonstrated that activation of Ca²⁺-permeable acid-sensing ion channels (ASIC1a) is largely responsible for acidosis-mediated, glutamate receptor-independent, neuronal injury. In cultured mouse cortical neurons, lowering extracellular pH to the level commonly seen in ischemic brain activates amiloride-sensitive ASIC currents. In the majority of these neurons, ASICs are permeable to Ca²⁺, and an activation of these channels induces increases in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i). Activation of ASICs with resultant [Ca²⁺]_i loading induces time-dependent neuronal injury occurring in the presence of the blockers for voltage-gated Ca²⁺ channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASICs, and by reducing [Ca²⁺]_o. In focal ischemia, intracerebroventricular administration of ASIC1a blockers, or knockout of the ASIC1a gene protects brain from injury and does so more potently than glutamate antagonism. Furthermore, pharmacological blockade of ASICs has up to a 5 h therapeutic time window, far beyond that of glutamate antagonists. Thus, targeting the Ca²⁺-permeable acid-sensing ion channels may prove to be a novel neuroprotective strategy for stroke patients.     

Role of STIM1 in Regulation of Store-Operated Ca<Superscript>2+</Superscript> Influx in Pheochromocytoma Cells

Changes in the local environment such as pH (acidosis/alkalosis), temperature (hypothermia/hyperthermia), and agonist (glutamate) can adversely affect neuronal function, and are important factors in clinical situations such as anesthesia and intensive care. Regulation of intracellular Ca²⁺ ([Ca²⁺]_i) is key to neuronal function. Stromal interaction molecule (STIM1) has been recently recognized to trigger store-operated Ca²⁺ entry (SOCE), an important component of [Ca²⁺]_i regulation. Using differentiated, fura-2 loaded rat pheochromocytoma (PC12) cells transfected with small interference RNA for STIM1 (or vehicle), we examined the role of STIM1 in SOCE sensitivity to temperature, pH, and glutamate. SOCE was triggered following endoplasmic reticulum depletion. Cells were washed and exposed to altered pH (6.0–8.0), altered temperature (34–40°C), or to glutamate. In non-transfected cells, SOCE was inhibited by acidosis or hypothermia, but increased with alkalosis and hyperthermia. Increasing glutamate concentrations progressively stimulated SOCE. STIM1 siRNA decreased SOCE at normal temperature and pH, and substantially decreased sensitivity to acidosis and hypothermia, eliminating the concentration-dependence to glutamate. Sensitivity of SOCE to these environmental parameters was less altered by decreased extracellular Ca²⁺ alone (with STIM1 intact). We conclude that STIM1 mediates exquisite susceptibility of SOCE to pH, temperature, and glutamate: factors that can adversely affect neuronal function under pathological conditions.     

Acute Restraint Stress Enhances Calcium Mobilization and Glutamate Exocytosis in Cerebrocortical Synaptosomes from Mice

Acute stress is known to enhance the memory of events that are potentially threatening to the organisms. Glutamate, the most abundant excitatory neurotransmitter in the mammalian central nervous system, plays a critical role in learning and memory formation and calcium (Ca²⁺) plays an essential role in transmitter release from nerve terminals (synaptosomes). In the present study, we investigated the effects of acute restraint stress on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in cerebrocortical synaptosomes from mice. Acute restraint stress caused a significant increase in resting [Ca²⁺]_i and significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. It also brought about a significant increase in spontaneous (unstimulated) glutamate release and significantly enhanced K⁺- and 4-AP-induced Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) completely suppressed the enhancements of [Ca²⁺]_i and Ca²⁺-dependent glutamate release in acute restraint-stressed mice. These results indicate that acute restraint stress enhances K⁺- or 4-AP-induced glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through P- and N-type Ca²⁺ channels.     

The Difference in the Effect of Glutamate and NO Synthase Inhibitor on Free Calcium Concentration and Na+,K+-ATPase Activity in Synaptosomes from Various Brain Regions

The significant increase of free calcium concentration ([Ca²⁺]_i) was found in rat cerebral cortex synaptosomes and hippocampal crude synaptosomal fraction after their exposure to glutamate. But no change of [Ca²⁺]_i was revealed in cerebellar synaptosomes, the slight increase of [Ca²⁺]_i in striatal synaptosomes was not significant. The presence of N^g-nitro-L-arginine methyl ester (L-NAME) in the incubation medium practically prevented the increase of [Ca²⁺]_i initiated by glutamate in cerebral cortex synaptosomes, but not in hippocampal ones. The significant diminution of [Ca²⁺]_i in the presence of this inhibitor was shown in striatal synaptosomes exposed to glutamate. Na⁺,K⁺-ATPase activity is significantly lower in cerebral cortex, striatal and hippocampal synaptosomes exposed to glutamate. L-NAME prevented the inactivation of this enzyme by glutamate. In cerebellar synaptosomes the tendency to the decrease of enzymatic activity in the presence of L-NAME was on the contrary noticed. Thus, the data obtained provide evidence of the protective effect of NO synthase inhibitor in brain cortex and striatal synaptosomes, but not in cerebellar synaptosomes. Synaptosomes appear to be an adequate model to study the regional differences in the mechanism of toxic effect of excitatory amino acids.     

Simultaneous single neuron recording of O2 consumption, [Ca2+]i and mitochondrial membrane potential in glutamate toxicity

In order to determine the sequence of cellular processes in glutamate toxicity, we simultaneously recorded O₂ consumption, cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and mitochondrial membrane potential (mΔψ) in single cortical neurons. Oxygen consumption was measured using an amperometric self‐referencing platinum electrode adjacent to neurons in which [Ca²⁺]_i and mΔψ were monitored with Fluo‐4 and TMRE⁺, respectively, using a spinning disk laser confocal microscope. Excitotoxic doses of glutamate caused an elevation of [Ca²⁺]_i followed seconds afterwards by an increase in O₂ consumption which reached a maximum level within 1–5 min. A modest increase in mΔψ occurred during this time period, and then, shortly before maximal O₂ consumption was reached, the mΔψ, as indicated by TMRE⁺ fluorescence, dissipated. Maximal O₂ consumption lasted up to 5 min and then declined together with mΔψ and ATP levels, while [Ca²⁺]_i further increased. mΔψ and [Ca²⁺]_i returned to baseline levels when neurons were treated with an NMDA receptor antagonist shortly after the [Ca²⁺]_i increased. Our unprecedented spatial and time resolution revealed that this sequence of events is identical in all neurons, albeit with considerable variability in magnitude and kinetics of changes in O₂ consumption, [Ca²⁺]_i, and mΔψ. The data obtained using this new method are consistent with a model where Ca²⁺ influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation.