Minocycline prevents glutamate-induced apoptosis of cerebellar granule neurons by differential regulation of p38 and Akt pathways

Minocycline has been shown to have remarkably neuroprotective qualities, but underlying mechanisms remain elusive. We reported here the robust neuroprotection by minocycline against glutamate-induced apoptosis through regulations of p38 and Akt pathways. Pre-treatment of cerebellar granule neurons (CGNs) with minocycline (10-100 microm) elicited a dose-dependent reduction of glutamate excitotoxicity and blocked glutamate-induced nuclear condensation and DNA fragmentations. Using patch-clamping and fluorescence Ca2+ imaging techniques, it was found that minocycline neither blocked NMDA receptors, nor reduced glutamate-caused rises in intracellular Ca2+. Instead, confirmed by immunoblots, minocycline in vivo and in vitro was shown to directly inhibit the activation of p38 caused by glutamate. A p38-specific inhibitor, SB203580, also attenuated glutamate excitotoxicity. Furthermore, the neuroprotective effects of minocycline were blocked by phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 and wortmannin, while pharmacologic inhibition of glycogen synthase kinase 3beta (GSK3beta) attenuated glutamate-induced apoptosis. In addition, immunoblots revealed that minocycline reversed the suppression of phosphorylated Akt and GSK3beta caused by glutamate, as were abolished by PI3-K inhibitors. These results demonstrate that minocycline prevents glutamate-induced apoptosis in CGNs by directly inhibiting p38 activity and maintaining the activation of PI3-K/Akt pathway, which offers a novel modality as to how the drug exerts protective effects.     

Relationships between superoxide levels and delayed calcium deregulation in cultured cerebellar granule cells exposed continuously to glutamate

The relationship is investigated between superoxide levels in single cultured rat cerebellar granule neurons exposed continuously to glutamate in low KCl medium and the deregulation of cytoplasmic Ca2+. Cells that maintain a regulated cytoplasmic-free Ca2+ and mitochondrial polarization in the presence of glutamate show no increase in superoxide levels until the onset of cytoplasmic Ca2+ deregulation. Oxidative stress of mitochondrial origin is readily detectable, as the inhibitors rotenone and antimycin A markedly increase superoxide levels with no effect on cytoplasmic-free Ca2+. The potent cell-permeant superoxide dismutase/catalase mimetic manganese tetrakis (N-ethylpyridinium-2yl) porphyrin, MnTE-PyP, abolishes the deregulation-related increase in superoxide but has no effect on deregulation itself. A combination of catalase with the free radical scavenger 4-hydroxy-TEMPO also fails to reduce deregulation. Following the loss of Ca2+ homeostasis nuclei undergo condensation; this morphological change is not inhibited by MnTE-PyP and cannot account for the increased ethidium fluorescence. Phospholipase A2 inhibitors decrease the deregulation-related increase in superoxide without protecting against deregulation. In conclusion, our study indicates that deregulation is not caused by NMDA receptor-mediated oxidative stress as NMDA receptor activation does not increase superoxide levels until the onset of deregulation. Furthermore, the majority of superoxide is produced in the cytoplasm rather than in mitochondria.     

Characterization of glutamate toxicity in cultured rat cerebellar granule neurons at reduced temperature

We have defined conditions whereby glutamate becomes toxic to isolated cerebellar granule neurons in a physiologic salt solution (pH 7.4). In the presence of a physiologic Mg⁺⁺ concentration, acute glutamate excitotoxicity manifests only when the temperature was reduced from 37°C to 22°C. In contrast to glutamate, N-methyl-D-aspartate (NMDA) was non-toxic at either temperature at concentrations as high as 1 mM. Glycine strongly potentiated both the potency and efficacy of glutamate but revealed only a modest NMDA response. The non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxalinedione (CNQX), potently protected against glutamate challenge, although the contribution of antagonism at strychnine-insensitive glycine sites could not be excluded. To further characterize the non-NMDA receptor contribution to the excitotoxic response, the promiscuity of glutamate interaction with ionotropic receptors was simulated by exposing neurons to NMDA in the presence of non-NMDA receptor agonists. NMDA toxicity was potentiated four- to sevenfold when non-NMDA receptors were coactivated by a subtoxic concentration of AMPA, kainate, or domoate. These results suggest that non-NMDA receptor activation participates in the mechanism of acute glutamate toxicity by producing neuronal depolarization (via sodium influx), which in turn promotes the release of the voltage-dependent magnesium blockade of NMDA receptor ion channels. © 1997 John Wiley & Sons, Inc.     

Lack of excitatory amino acid-induced effects on calcium fluxes measured with45Ca2+ in rat cerebral cortex synaptosomes

Ca²⁺ uptake was measured in purified rat cerebral cortex synaptosomes (P₃ pellets) using⁴⁵Ca²⁺ as a tracer. Ca²⁺ influx increased in time, and with an increase in external K⁺ concentration and temperature. The net (external K⁺-induced, depolarization-dependent) uptake follows a two-component course. The exponential term, due to the opening of voltage-operated calcium channels (VOC), has a rate constant which increases with an increase in the depolarization level (1.04 versus 0.54 nmol/s/mg protein for 50 mM—versus 15 mM [K⁺]-dependent net influx). The linear term, due to the Na⁺/Ca²⁺ exchange system, has a similar rate constant at all depolarization levels (0.16+/–0.05 and 0.11+/–0.02 nmol/s/mg protein). Excitatory amino acids (glutamate, kainate and n-methyl-d-aspartate-NMDA-) were tested on this preparation at doses ranging between 5×10^–5 M and 5×10^–3M and at multiple incubation times, under resting conditions and under two depolarizing conditions (partial depolarization: 15 mM external K⁺ and maximal depolarization: 50 mM external K⁺). NMDA was also tested in the absence of Mg²⁺. No effect was detectable under any of these experimental conditions. Hypotheses to interpret these data are discussed. Further studies on other preparations are needed in order to directly investigate the presynaptic effects of excitatory amino acids.     

The pituitary adenylate cyclase-activating polypeptide modulates glutamatergic calcium signalling: investigations on rat suprachiasmatic nucleus neurons

Circadian rhythms generated by the hypothalamic suprachiasmatic nucleus (SCN) are synchronized with the external light/dark cycle by photic information transmitted directly from the retina via the retinohypothalamic tract (RHT). The RHT contains the neurotransmitters glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), which code chemically for 'light' or 'darkness' information, respectively. We investigated interactions of PACAP and glutamate by analysing effects on the second messenger calcium in individual SCN neurons using the Fura-2 technique. PACAP did not affect NMDA-mediated calcium increases, but influenced signalling cascades of non-NMDA glutamate receptors, which in turn can regulate NMDA receptors. On the one hand, PACAP amplified/induced glutamate-dependent calcium increases by interacting with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate signalling. This was not related to direct PACAPergic effects on the second messengers cAMP and calcium. On the other hand, PACAP reduced/inhibited calcium increases elicited by glutamate acting on metabotropic receptors. cAMP analogues mimicked this inhibition. Most neurons displaying PACAPergic neuromodulation were immunoreactive for vasoactive intestinal polypeptide, which is a marker for retinorecipient SCN neurons. The observed PACAPergic effects provide a broad range of interactions that allow a fine-tuning of the endogenous clock by the integration of 'light' and 'darkness' information on the level of single SCN neurons.     

Dopamine protects neurons against glutamate-induced excitotoxicity

Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders including stroke, trauma and neurodegenerative disease. Loss of calcium homeostasis is a key mediator of glutamate-induced cell death. The neurotransmitter dopamine (DA) is known to modulate calcium signalling, and here we show that it can do so in response to physiological concentrations of glutamate. Furthermore, DA is able to protect neurons from glutamate-induced cell death at pathological concentrations of glutamate. We demonstrate that DA has a novel role in preventing delayed calcium deregulation in cortical, hippocampal and midbrain neurons. The effect of DA in abolishing glutamate excitotoxicity can be induced by DA receptor agonists, and is abolished by DA receptor antagonists. Our data indicate that the modulation of glutamate excitotoxicity by DA is receptor-mediated. We postulate that DA has a major physiological function as a safety catch to restrict the glutamate-induced calcium signal, and thereby prevent glutamate-induced cell death in the brain.     

Effect of magnesium on calcium influx activated by glutamate and its agonists in cultured cerebellar granule cells

The effects of Mg²⁺ on the glutamate-, kainate-, N-methyl-d-aspartate- and quisqualate-induced influx of⁴⁵Ca²⁺ were studied in cultured cerebellar granule cells. The N-methyl-d-aspartate- and quisqualate-evoked influx was totally and the kainate- and glutamate-evoked influx partially blocked in 1.3 mM extracellular Mg²⁺. The increase in influx induced by kainate, quisqualate and glutamate was maximal at 0.1 mM Mg²⁺, whereas N-methyl-d-aspartate was most effective in totally Mg²⁺-free media.d-2-Amino-5-phosphonovalerate blocked partially and phencyclidine completely the enhancement of Ca²⁺ influx by 1 mM quisqualate in 0.1-mM Mg²⁺ medium. The effect of 10 M quisqualate was also significantly inhibited by antagonists specific for different glutamate receptor subtypes, including N-methyl-d-aspartate, (RS)-amino-3-hydroxy-5-methyl-4-isozazolepropionate and metabotropic recptors. This evidences a heterogeneous action of quisqualate, mediated by different glutamate receptor subtypes in 0.1 mM Mg²⁺ medium. The efficacy of quisqualate in inducing influx of Ca⁺ and the selectivity of antagonists for different receptors are also modified by extracellular Mg²⁺.     

'Mild Uncoupling' does not decrease mitochondrial superoxide levels in cultured cerebellar granule neurons but decreases spare respiratory capacity and increases toxicity to glutamate and oxidative stress

Cultured rat cerebellar granule neurons were incubated with low nanomolar concentrations of the protonophore carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) to test the hypothesis that 'mild uncoupling' could be neuroprotective by decreasing oxidative stress. To quantify the uncoupling, respiration and mitochondrial membrane potential (Deltapsi(m)) were determined in parallel as a function of FCCP concentration. Deltapsi(m) dropped by less than 10 mV before respiratory control was lost. Conditions for the valid estimation of matrix superoxide levels were determined from the rate of oxidation of the matrix-targeted fluorescent probe MitoSOX. No significant change in the level of matrix superoxide could be detected on addition of FCCP while respiratory control was retained, although cytoplasmic superoxide levels measured by dihydroethidium oxidation increased. 'Mild uncoupling' by 30 nmol/L FCCP did not alleviate neuronal dysregulation induced by glutathione depletion and significantly enhanced that due to menadione-induced oxidative stress. Low protonophore concentrations enhanced N-methyl-d-aspartate receptor-induced delayed calcium deregulation consistent with a decrease in the spare respiratory capacity available to match the bioenergetic demand of chronic receptor activation. It is concluded that the 'mild uncoupling' hypothesis is not supported by this model.     

Contributing mechanisms for cysteine excitotoxicity in cultured cerebellar granule cells

Several possible mechanisms for cysteine toxicity on rat cerebellar granule cells were studied and compared with the excitotoxic effect of glutamate. It was shown that the excitotoxic potency of both cysteine and glutamate increased in the presence of elevated concentrations, of bicarbonate or increased pH. Pharmacological studies showed that the cysteine toxicity was specifically coupled to the NMDA receptor, whereas the glutamate toxicity was mediated to a smaller extent also by non-NMDA receptors. Treatment of cerebellar granule cells with cysteine led to an increased extracellular level of glutamate. In addition, cysteine sensitized NMDA receptors by reducing disulfide bonds in the receptor to sulfhydryl groups. A mechanism for cysteine excitotoxicity may therefore be formation of cysteine-sensitized NMDA receptors that are stimulated either by cysteine and/or by endogenous glutamate. This mechanism may also be important for the effects observed during regulated physiological release of cysteine.     

Mitochondrial free radical production induced by glucose deprivation in cerebellar granule neurons

Using a fluorescent probe for superoxide, hydroethidine, we have demonstrated that glucose deprivation (GD) activates production of reactive oxygen species (ROS) in cultured cerebellar granule neurons. ROS production was insensitive to the blockade of ionotropic glutamate channels by MK-801 (10 microM) and NBQX (10 microM). Inhibitors of mitochondrial electron transport, i.e. rotenone (complex I), antimycin A (complex III), or sodium azide (complex IV), an inhibitor of mitochondrial ATP synthase--oligomycin, an uncoupler of oxidative phosphorylation--CCCP, a chelator of intracellular Ca2+--BAPTA, an inhibitor of electrogenic mitochondrial Ca2+ transport--ruthenium red, as well as pyruvate significantly decreased neuronal ROS production induced by GD. GD was accompanied by a progressive decrease in the mitochondrial membrane potential and an increase in free cytosolic calcium ions, [Ca2+](i). Pyruvate, BAPTA, and ruthenium red lowered the GD-induced calcium overload, while pyruvate and ruthenium red also prevented mitochondrial membrane potential changes induced by GD. We conclude that GD-induced ROS production in neurons is related to potential-dependent mitochondrial Ca2+ overload. GD-induced mitochondrial Ca2+ overload in neurons in combination with depletion of energy substrates may result in the decrease of the membrane potential in these organelles.     

Cannabinoid receptor agonists inhibit depolarization-induced calcium influx in cerebellar granule neurons.

Neuronal cannabinoid receptors (CB(1)) are coupled to inhibition of voltage-sensitive Ca(2+) channels (VSCCs) in several cell types. The purpose of these studies was to characterize the interaction between endogenous CB(1) receptors and VSCCs in cerebellar granule neurons (CGN). Ca(2+) transients were evoked by KCl-induced depolarization and imaged using fura-2. The CB(1) receptor agonists CP55940, Win 55212-2 and N-arachidonylethanolamine (anandamide) produced concentration-related decreases in peak amplitude of the Ca(2+) response and total Ca(2+) influx. Pre-treatment of CGN with pertussis toxin abolished agonist-mediated inhibition. The inhibitory effect of Win 55212-2 on Ca(2+) influx was additive with inhibition produced by omega-agatoxin IVA and nifedipine but not with omega-conotoxin GVIA, indicating that N-type VSCCs are the primary effector. Paradoxically, the CB(1) receptor antagonist, SR141716, also inhibited KCl-induced Ca(2+) influx into CGN in a concentration-related manner. SR141716 inhibition was pertussis toxin-insensitive and was not additive with the inhibition produced by Win 55212-2. Confocal imaging of CGN in primary culture demonstrate a high density of CB(1) receptor expression on CGN plasma membranes, including the neuritic processes. These data demonstrate that the CB(1) receptor is highly expressed by CGN and agonists serve as potent and efficacious inhibitory modulators of Ca(2+) influx through N-type VSCC.     

Mitochondrial permeability transition and calcium dynamics in striatal neurons upon intense NMDA receptor activation

Deregulation of the intracellular Ca2+ homeostasis by NMDA receptor activation leads to neuronal cell death. Induction of the mitochondrial permeability transition pore (MPT) by Ca2+ is a critical event in mediating cell death. In this study, we used fluorescent Ca2+ indicators to investigate the effect of high concentrations of NMDA on cytosolic and mitochondrial Ca2+ concentrations ([Ca2+]c and [Ca2+]m, respectively) in cultured striatal neurons. Exposure to NMDA resulted in an immediate, sustained increase in [Ca2+]c followed by a secondary increase in [Ca2+]c. This second increase of [Ca2+]c was prevented by pretreatment with N-methyl-valine-4-cyclosporin (NMV-Cys). Exposure of neurons to NMDA also resulted in an increase in [Ca2+]m that was followed by a precipitous decrease in the rhod-2 signal. This decrease followed the time frame of the secondary increase in [Ca2+]c. Preincubation of the neurons with NMV-Cys prevented the decrease in rhod-2 fluorescence. These dynamic changes in the rhod-2 signal and [Ca2+]m in response to NMDA were confirmed by using confocal microscopy. The presented results indicate that MPT can be detected in living neurons using fluorescent Ca2+ indicators, which would allow the study of the physiological role of MPT in cell death.     

Changes in mitochondrial NAD(P)H and glutamate-induced delayed calcium deregulation in cultured rat cerebellar granule neurons

Changes in cytosolic [Ca²⁺]_i, mitochondrial potential (ΔVm), and mitochondrial NAD(P)H autofluorescence were compared in experiments on cultured cerebellar granule cells co-loaded with Ca²⁺ indicator Fluo-3FF or mitochondrial potential-sensitive probe Rh123. In the majority of neurons (94% of cells, n = 205, 28 experiments) the delayed Ca²⁺ deregulation (DCD) induced by Glu (100 μM) was preceded by more or less prolonged decrease in NAD(P)H, which in 57% of cells underwent a further (secondary) reduction during DCD development. To clarify the origin of these changes in NAD(P)H production during DCD we examined the effects of the protonophore FCCP on NAD(P)H increase induced by the electron chain blocker CN (3 mM) application. The data suggest that a pronounced lowering of mitochondrial pH during DCD contributed to the mechanism of Glu-induced suppression of NAD(P)H production.     

Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu>quisqualate (quis)=ibotenate (ibo)>(1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg²⁺). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1_S,3_R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg²⁺, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg²⁺-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms). Thus, in cerebellar granule cells EAAs elicit PPI hydrolysis by acting at two distinct receptor types: (i) metabotropic Glu receptors (mGluR), with pharmacological characteristics suggesting the expression of a unique mGluR receptor that shows certain similarities to those observed for the mGluR1 subtype (Aramori and Nakanishi, 1992) and (ii) NMDA receptors. The physiological agonist, Glu, is able to stimulate both receptor classes.Abbreviations ACPD (1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - AP₃ L-2-amino-3-phosphono-propionate - AP₅ D-2-amino-5-phosphonopentenoate - BMAA -methyl-amino-L-alanine - DIV days in vitro - DNOX 6,7-dinitroouinoxoline-2,3-dione - EAA excitatory amino acids - Glu glutamate - InsP inositol monophosphate - mGluR metabotropic glutamate receptors - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohept-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate - PPI polyphosphoinositide - quis quisqualate     

Topiramate modulation of kainate-induced calcium currents is inversely related to channel phosphorylation level

Topiramate (TPM) is a structurally novel broad-spectrum anticonvulsant known to modulate the activity of several ligand- and voltage-gated ion channels in neurons. These include an inhibitory effect on the AMPA and kainate subtypes of glutamate receptors, mixed modulatory effects (usually positive) on some types of GABAA receptors, negative modulatory effects on some types of voltage-gated Na+ and Ca2+ channels, and a positive modulatory effect on at least one type of K+ channel. The nature of these effects at the molecular level has not been established, but two previous studies have implicated the phosphorylation state of these receptor/channel complexes as an influencing factor in the activity of TPM. Here, we report that the ability of TPM to inhibit a kainate-induced accumulation of free Ca2+ in cultured neurons from rat cerebral cortex is inversely related to the level of cAMP-dependent protein kinase (cAPK) mediated phosphorylation of kainate-activated receptors/channels. Specifically, when cell cultures were pre-treated with forskolin or dibutyryl cAMP, indirect activators of cAPK, the activity of TPM was abolished, whereas when the cells were pre-treated with H89, an inhibitor of cAPK, the relative activity of TPM was enhanced. The results of this study support the hypothesis that TPM binds to phosphorylation sites on AMPA and kainate receptors, but only in the dephosphorylated state and thereby exerts an allosteric modulatory effect on channel conductance.     

Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.     

Identification of Bax-voltage-dependent anion channel 1 complexes in digitonin-solubilized cerebellar granule neurons

Mitochondrial outer membrane Bax oligomers are critical for cytochrome c release, but the role of resident mitochondrial proteins in this process remains unclear. Membrane-associated Bax has primarily been studied using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) as the solubilizing agent, as it does not induce conformational artifacts, although recent evidence indicates it may have other artifactual effects. The objective of this study was to investigate digitonin as an alternative detergent to assess Bax oligomeric state, and possible interaction with voltage-dependent anion channel (VDAC)1 in cerebellar granule neurons. VDAC1 co-immunoprecipitated with Bax in digitonin extracts from healthy and apoptotic neurons. Two-dimensional blue native-SDS-PAGE revealed five Bax and VDAC1 oligomers having similar masses from 120 to 500 kDa. The levels of two VDAC1 oligomers in Bax 1D1 immunodepleted extracts negatively correlated with levels of co-precipitated VDAC1, indicating the co-precipitated VDAC1 was derived from these oligomers. Immunodepletion with the 6A7 antibody modestly reduced the levels of Bax oligomers from apoptotic but not healthy neurons. A sixth 170 kDa oligomer containing exclusively 6A7 Bax and no VDAC1 was identified after apoptosis induction. CHAPS failed to solubilize VDAC1, and additionally yielded no distinct oligomers. We conclude that digitonin is a potentially useful detergent preserving Bax-VDAC1 interactions that may be disrupted with CHAPS.     

Redistribution of subcellular calcium in rat liver on administration of vanadate

Summary Mitochondria isolated from the livers of rats administered with sodium meta-, ortho-, or polyvanadate, but not vanadyl sulphate, exhibited enhanced Ca²⁺ — stimulated respiration and uptake of calcium. These effects were shown also by mitochondria isolated from livers perfused with polyvanadate. The concentration of acid-soluble calcium decreased significantly in the mitochondrial fraction on vanadate treatment, while that in the cytosol showed a corresponding increase. Phenoxybenzamine, an antagonist to a-adrenergic receptors, effectively inhibited vanadate-induced Ca²⁺ mobilization, but surgical sympathectomy was without effect. This is the first demonstration of vanadate mimicking -adrenergic agonists in vivo.     

Pharmacologically induced calcium oscillations protect neurons from increases in cytosolic calcium after trauma

Increases in cytosolic calcium ([Ca(2+)](i)) following mechanical injury are often considered a major contributing factor to the cellular sequelae in traumatic brain injury (TBI). However, very little is known on how developmental changes may affect the calcium signaling in mechanically injured neurons. One key feature in the developing brain that may directly impact its sensitivity to stretch is the reduced inhibition which results in spontaneous [Ca(2+)](i) oscillations. In this study, we examined the mechanism of stretch-induced [Ca(2+)](i) transients in 18-days in vitro (DIV) neurons exhibiting bicuculline-induced [Ca(2+)](i) oscillations. We used an in vitro model of mechanical trauma to apply a defined uniaxial strain to cultured cortical neurons and used increases in [Ca(2+)](i) as a measure of the neuronal response to the stretch insult. We found that stretch-induced increases in [Ca(2+)](i) in 18-DIV neurons were inhibited by pretreatment with either the NMDA receptor antagonist, APV [D(-)-2-Amino-5-phosphonopentanoic acid], or by depolymerizing the actin cytoskeleton prior to stretch. Blocking synaptic NMDA receptors prior to stretch significantly attenuated most of the [Ca(2+)](i) transient. In comparison, cultures with pharmacologically induced [Ca(2+)](i) oscillations showed a substantially reduced [Ca(2+)](i) peak after stretch. We provide evidence showing that a contributing factor to this mechanical desensitization from induced [Ca(2+)](i) oscillations is the PKC-mediated uncoupling of NMDA receptors (NMDARs) from spectrin, an actin-associated protein, thereby rendering neurons insensitive to stretch. These results provide novel insights into how the [Ca(2+)](i) response to stretch is initiated, and how reduced inhibition - a feature of the developing brain - may affect the sensitivity of the immature brain to trauma.