Adenosine Receptors Modulate [Ca2+]i in Hippocampal Astrocytes In Situ

Abstract: Cultured astroglia express both adenosine and ATP purinergic receptors that are coupled to increases in intracellular calcium concentration ([Ca²⁺]_i). Currently, there is little evidence that such purinergic receptors exist on astrocytes in vivo. To address this issue, calcium-sensitive fluorescent dyes were used in conjunction with confocal microscopy and immunocytochemistry to examine the responsiveness of astrocytes in acutely isolated hippocampal slices to purinergic neuroligands. Both ATP and adenosine induced dynamic increases in astrocytic [Ca²⁺]_i that were blocked by the adenosine receptor antagonist 8-( p -sulfophenyl)theophylline. The responses to adenosine were not blocked by tetrodotoxin, 8-cyclopentyltheophylline, 8-(3-chlorostyryl)caffeine, dipyridamole, or removal of extracellular calcium. The P_2Y-selective agonist 2-methylthioadenosine triphosphate was unable to induce increases in astrocytic [Ca²⁺]_i, whereas the P₂ agonist adenosine 5'- O -(2-thiodiphosphate) induced astrocytic responses in a low percentage of astrocytes. These results indicate that the majority of hippocampal astrocytes in situ contain P₁ purinergic receptors coupled to increases in [Ca²⁺]_i, whereas a small minority appear to contain P₂ purinergic receptors. Furthermore, individual hippocampal astrocytes responded to adenosine, glutamate, and depolarization with increases in [Ca²⁺]_i. The existence of both purinergic and glutamatergic receptors on individual astrocytes in situ suggests that astrocytes in vivo are able to integrate information derived from glutamate and adenosine receptor stimulation.     

Net Glutamate Release from Astrocytes Is Not Induced by Extracellular Potassium Concentrations Attainable in Brain

Abstract: Elevated extracellular potassium concentration ([K⁺]_e) has been shown to induce reversal of glial Na⁺-dependent glutamate uptake in whole-cell patch clamp preparations. It is uncertain, however, whether elevated [K⁺]_e similarly induces a net glutamate efflux from intact cells with a physiological intracellular milieu. To answer this question, astrocyte cultures prepared from rat and mouse cortices were incubated in medium with elevated [K⁺]_e (by equimolar substitution of K⁺ for Na⁺), and glutamate accumulation was measured by HPLC. With [K⁺]_e elevations to 60 m M , medium glutamate concentrations did not increase during incubation periods of 5–120 min. By contrast, 45 min of combined inhibition of glycolytic and oxidative ATP production increased medium glutamate concentrations 50–100-fold. Similar results were obtained in both rat and mouse cultures. Studies were also performed using astrocytes loaded with the nonmetabolized glutamate tracer d -aspartate, and parallel results were obtained; no increase in medium d -aspartate content resulted from [K⁺]_e elevation up to 90 m M , whereas a large increase occurred during inhibition of energy metabolism. These results suggest that a net efflux of glutamate from intact astrocytes is not induced by any [K⁺]_e attainable in brain.     

Mitochondria, Calcium Regulation, and Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells

Abstract: Exposure of cultured cerebellar granule cells to 100 µ M glutamate plus glycine in the absence of Mg²⁺ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca²⁺ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca²⁺ concentration), and extensive cell death. Glutamate-evoked Ca²⁺ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca²⁺ transport, allows glutamate-exposed cells to maintain a high ATP/ADP ratio while accumulating little ⁴⁵Ca²⁺ and maintaining a low bulk cytoplasmic free Ca²⁺ concentration determined by fura-2. It is concluded that mitochondrial Ca²⁺ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca²⁺ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca²⁺ accumulation in a microdomain accessible to the mitochondria.     

Tachykinins Potentiate N-Methyl-D-Aspartate Responses in Acutely Isolated Neurons from the Dorsal Horn

Abstract: Substance P and neurokinin A both potentiated N -methyl- d -aspartate (NMDA)-induced currents recorded in acutely isolated neurons from the dorsal horn of the rat. To elucidate the mechanism underlying this phenomenon, we measured the effects of tachykinins and glutamate receptor agonists on [Ca²⁺]_i in these cells. Substance P, but not neurokinin A, increased [Ca²⁺]_i in a subpopulation of neurons. The increase in [Ca²⁺]_i was found to be due to Ca²⁺ influx through voltage-sensitive Ca²⁺ channels. Substance P and neurokinin A also potentiated the increase in [Ca²⁺]_i produced by NMDA, but not by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, or 50 m M K⁺. Phorbol esters enhanced the effects of NMDA and staurosporine inhibited the potentiation of NMDA effects by tachykinins. It is concluded that activation of protein kinase C may mediate the enhancement of NMDA effects by tachykinins in these cells. However, the effects of tachykinins on [Ca²⁺]_i can be dissociated from their effects on NMDA receptors.     

The Metabotropic Glutamate Receptor mGluR5 Induces Calcium Oscillations in Cultured Astrocytes via Protein Kinase C Phosphorylation

Abstract: The metabotropic glutamate receptor mGluR5, but not the closely related mGluR1, is expressed in cultured astrocytes, and this expression is up-regulated by specific growth factors. We investigated the capability and underlying mechanisms of mGluR5 to induce oscillatory responses of intracellular calcium concentration ([Ca²⁺]_i) in cultured rat astrocytes. Single-cell [Ca²⁺]_i recordings indicated that an mGluR-selective agonist, (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate (1 S ,3 R -ACPD), elicits [Ca²⁺]_i oscillations in good agreement with the growth factor-induced up-regulation of mGluR5 in cultured astrocytes. A protein kinase C (PKC) inhibitor, bisindolylmaleimide I, converted a 1 S ,3 R -ACPD-mediated oscillatory response into a nonoscillatory response. In addition, the PKC activator phorbol 12-myristate 13-acetate completely abolished the [Ca²⁺]_i increase. These and other pharmacological properties of 1 S ,3 R -ACPD-induced [Ca²⁺]_i oscillations correlate well with those of the cloned mGluR5 characterized in heterologous expression systems. Furthermore, the potential involvement of protein phosphatases in [Ca²⁺]_i oscillations is suggested. The present study demonstrates that mGluR5 is capable of inducing [Ca²⁺]_i oscillations in cultured astrocytes and that phosphorylation/dephosphorylation of mGluR5 is critical in [Ca²⁺]_i oscillations, analogous to the cloned mGluR5 expressed in heterologous cell lines.     

Responses of Bergmann Glia and Granule Neurons In Situ to N-Methyl-d-Aspartate, Norepinephrine, and High Potassium

Abstract: To gain insight into neuronal-glial signaling in brain, cerebellar Bergmann glia and granule neurons were studied in acutely isolated slices with the aid of laser scanning confocal microscopy. Both Bergmann glia and granule neurons responded to N -methyl- d -aspartate (NMDA) with a rise in [Ca²⁺]_i. However, the glial NMDA response was frequently inhibited by tetrodotoxin, suggesting that the response depended on neuronal action potentials, rather than on direct activation of NMDA receptors on the Bergmann glia. Further experiments demonstrated that the NMDA response in Bergmann glia was not inhibited by a combination of non-NMDA glutamate receptor blockers 6-cyano-7-nitroquinoxaline-2,3-dione and α-methyl-4-carboxyphenylglycine. Bergmann glia also responded to norepinephrine and high K⁺, and the responses were not inhibited by tetrodotoxin. The glial norepinephrine response was blocked by phentolamine but not by the removal of external Ca²⁺, indicating a direct activation of α₁-adrenergic receptors that mediated release of Ca²⁺ from intracellular stores. The KCI-induced response in both neurons and glia was dependent on external Ca²⁺ and was blocked by verapamil or nifedipine. In summary, our data indicate that Bergmann glia in situ recognize a signal(s) released from neurons during neuronal activity.     

Mechanism Underlying the ATP-Induced Increase in the Cytosolic Ca2+ Concentration in Chick Ciliary Ganglion Neurons

Abstract: We examined the mechanism underlying the ATP-induced increase in the cytosolic Ca²⁺ concentration ([Ca]_in) in acutely isolated chick ciliary ganglion neurons, using fura-2 microfluorometry. The ATP-induced increase in [Ca]_in was dependent on external Ca²⁺, was blocked in a dose-dependent manner by reactive blue 2, and was substantially inhibited by both L- and N-type Ca²⁺ channel blockers. ATP was effective in increasing [Ca]_in in the presence of a desensitizing concentration of nicotine (100 µ M ), and simultaneous addition of maximal doses of ATP and nicotine caused an additive increase in [Ca]_in, suggesting that ATP acts on a site distinct from nicotinic acetylcholine receptors. ATP also increased the cytosolic Na⁺ concentration as determined by sodium-binding benzofuran isophthalate microfluorometry. These results suggest that ATP increases Na⁺ influx through P₂ purinoceptor-associated channels resulting in membrane depolarization, which in turn increases Ca²⁺ influx through voltage-dependent Ca²⁺ channels. However, ATP still caused a small increase in [Ca]_in under Na⁺-free conditions, and this [Ca]_in increase was little affected by Ca²⁺ channel blockers. ATP also increased Mn²⁺ influx under Na⁺-free conditions, as indicated by quenching of fura-2 fluorescence. These results suggest that nonselective cationic channels activated by ATP are permeable not only to Ca²⁺ but also to Mn²⁺, in addition to monovalent cations.     

Enteric Glia Exhibit P2U Receptors that Increase Cytosolic Calcium by a Phospholipase C-Dependent Mechanism

Abstract: Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 m M ), glutamate (100 µ M ), norepinephrine (10 µ M ), and substance P (1 µ M ) did not increase the intracellular calcium concentration ([Ca²⁺]_i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 µ M ), bradykinin (11%; 10 µ M ), and histamine (31%; 100 µ M ), whereas 100% of glia responded to ATP (100 µ M ). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca²⁺]_i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca²⁺]_i evoked, ATP = UTP > ADP > β,γ-methyleneadenosine 5'-triphosphate ≫ 2-methylthioadenosine 5'-triphosphate = α,β-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P_2U receptor. Depletion of d - myo -inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 µ M ) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 µ M ), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 n M ), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca²⁺]_i. Our data suggest that glial responses to ATP are mediated by a P_2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.     

Characterization of the Exocytotic Release of Glutamate from Guinea-Pig Cerebral Cortical Synaptosomes

Abstract: A continuous enzyme-linked fluorometric assay was used for determining the characteristics for glutamate exocytosis from guinea-pig cerebrocortical synaptosomes. Ca²⁺-dependent release can be induced not only by K⁺, but also by the Na⁺ channel activator veratridine and the Ca²⁺ ionophore ionomycin. K⁺-induced release can be inhibited by the Ca²⁺ channel inhibitor verapamil. Sr²⁺ and Ba²⁺ substitute for Ca²⁺ in promoting K⁺-induced release. Agents that would be predicted to transform the transvesicular pH gradient into a membrane potential are without effect on glutamate release. However, the protonophore carbonylcy-anide p -trifluoromethoxyphenylhydrazone causes a time-dependent loss of exocytosis that is oligomycin insensitive and may be due to depletion of vesicular glutamate. The Ca²⁺-independent release of glutamate from the cytosol on depolarization is unchanged or promoted by metabolic inhibitors that lower the ATP/ADP ratio. In contrast, Ca²⁺-dependent release is ATP dependent and is blocked by the combined inhibition of oxidative phosphorylation and glycolysis.     

Glutamate Receptor-Mediated Calcium Surges in Neurons Derived from P19 Cells

Abstract: Retinoic acid-treated murine P19 embryonal carcinoma cells differentiate into cells with neuronal morphology that display typical neuronal markers. In this study, the presence of glutamate receptors linked to Ca²⁺-signaling mechanisms on these neurons was demonstrated by testing the effects of glutamate agonists and antagonists on the intracellular calcium ion concentration ([Ca²⁺]_i). Glutamate (1 m M ) induced either sustained or transient increases in [Ca²⁺]_i. The sustained glutamate-induced increase in [Ca²⁺]_i was mimicked by NMDA (40 µ M ). The NMDA-triggered [Ca²⁺]_i response was abolished by incubating the cells in Ca²⁺-free medium or by pretreating them with Mg²⁺ (2 m M ) or MK-801 (0.1 µ M ). These responses were unaffected by the non-NMDA antagonist CNQX (10 µ M ), but they required glycine (3–30 µ M ). Kainate (40 µ M ) and AMPA (40 µ M ) did not affect [Ca²⁺]_i. Without external Ca²⁺, glutamate triggered transient, sometimes oscillating, increases in [Ca²⁺]_i. These responses were mimicked by the metabotropic agonist trans -(1 S ,3 R )-1-amino-1,3-cyclopentanedicarboxylic acid (300 µ M ). These results suggest that neurons derived from P19 embryonal carcinoma cells have NMDA and metabotropic, but not AMPA/kainate receptors, which are linked to Ca²⁺-signaling mechanisms. These cells could provide a consistent and reproducible model with which to study neuronal differentiation, neurotoxicity, and glutamate receptor-signaling mechanisms.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

Mechanism of calcium-dependent salt tolerance in cells of Nitellopsis obtusa: role of intracellular adenine nucleotides

Abstract. The tonoplasts of internodal cells of Nitellopsis were removed by perfusing the vacuoles with media containing a Ca² chelator, EGTA. Treatment of tonoplast-free cells with 100 mol m³ NaCl induces a large membrane depolarization, a drastic decrease in the membrane resistance and an increase in Na⁺ influx. These events are identical to those that occur in intact cells subjected to high NaCl. These responses to NaCl are prevented if 10 mol m³ Ca²⁺ is supplied together with 100 mol m³ NaCl. The protective effect of Ca²⁺ is evident only when the intracellular ATP concentration exceeds 0.1 mol m³ and does not occur full when the intracellular ATP is removed. AMP at concentrations greater than 0.5 mol m³ or 0.25 mol m³ AMPPNP can replace ATP. It is concluded that ATP does not act as an energy source nor as a substrate for protein phosphorylation. ATP seems to exert its effects as a coeffector with Ca²⁺ in regulating the Na⁺ permeability of the plasma membrane.     

Calcium and plant organelles

Abstract. The role of intracellular organelles in the regulation of cytosolic Ca²⁺ levels and whether changes in these levels affect organelle metabolism is considered. We have assessed the biochemical properties of the Ca²⁺ transporting systems in mitochondrial, chloroplast and microsomal fractions. It is proposed that although all of these organelles can transport Ca²⁺ to varying extents it would appear that in some tissues at least mitochondria do not play a significant role in the maintenance of cytosolic Ca²⁺. The most important Ca²⁺ transporting systems are probably the ATP dependent Ca²⁺ extrusion across the plasma membrane and Ca²⁺ uptake by endoplasmic reticulum, as well as light driven Ca²⁺ uptake by chloroplasts. Changes in cytoplasmic [Ca²⁺] do appear to regulate the activity of NAD kinase in chloroplasts, the mitochondrial external NADH dehydrogenase and intra-mitochondrial glutamate dehydrogenase, all of which play a key role in plant cell metabolism. Since some of these enzymes are affected by primary stimuli such as light or hormones, it is concluded that Ca²⁺ may act as a second messenger mediating some of the primary responses.     

Comparison of Type 2 Inositol 1,4,5-Trisphosphate Receptor Distribution and Subcellular Ca2+ Release Sites that Support Ca2+ Waves in Cultured Astrocytes

Abstract: We have examined the mechanisms that underlie Ca²⁺ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca²⁺ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca²⁺ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca²⁺ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP₃R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP₃Rs with Ca²⁺ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP₃Rs (InsP₃R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca²⁺ responses and InsP₃R2 immunofluorescence were compared in the same cell, domains of elevated Ca²⁺ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP₃R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca²⁺ release sites that support regenerative wave propagation include increased levels of InsP₃R2 expression.     

Effects of Calcium Channel Antagonists on Calcium Entry and Glutamate Release from Cultured Rat Cerebellar Granule Cells

Abstract: Using a range of Ca²⁺ channel blockers we have investigated the Ca²⁺ channel subtypes that mediate the depolarisation-induced elevation of the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release from cultured rat cerebellar granule cells. ω-Conotoxin-GVIA had little effect on either the transient or plateau phase of the depolarisation-induced [Ca²⁺]_i rise or on glutamate release, ruling out a significant role for N-type Ca²⁺ channels. Nifedipine substantially inhibited the initial transient rise in [Ca²⁺]_i and the plateau phase of the [Ca²⁺]_i rise and glutamate release, suggesting the involvement of L-type Ca²⁺ channels. Both ω-agatoxin and ω-conotoxin-MVIIC also inhibited the transient rise in [Ca²⁺]_i and glutamate release but not the plateau phase of the [Ca²⁺]_i rise. The inhibitions by nifedipine were not increased by coaddition of ω-conotoxin-MVIIC, suggesting overlapping sensitivity to these channel blockers. These data show that glutamate release from granule cells in response to depolarisation with a high KCI level involves Ca²⁺ currents that are sensitive to nifedipine, ω-agatoxin-IVA, and also ω-conotoxin-MVIIC. The overlapping sensitivity of the channels to these toxins prevents attribution of any of the phases of the [Ca²⁺]_i rise or glutamate release to distinct P-, Q-, or O-type Ca²⁺ currents.     

P2Y1 receptor-mediated glutamate release from cultured dorsal spinal cord astrocytes

P2 receptors have been implicated in the release of neurotransmitter and proinflammatory cytokines by the response to neuroexcitatory substances in astrocytes. In the present study, we examined the mechanisms of ADP and adenosine 5'-O-2-thiodiphosphate (ADPbetaS, ADP analogue) on glutamate release from cultured dorsal spinal cord astrocytes by using confocal laser scanning microscopy and HPLC. Immunofluorescence activity showed that P2Y₁ receptor protein is expressed in cultured astrocytes. ADP and ADPbetaS-induced [Ca²⁺]_i increase and glutamate release are mediated by P2Y₁ receptor. Ca²⁺ release from IP₃-sensitive calcium stores and protein kinase C (PKC) activation is important for glutamate release from astrocytes. Furthermore, P2Y₁ receptor-evoked glutamate release is regulated by volume-sensitive Cl⁻ channels and anion co-transporter, which open up the possibility that P2Y₁ receptor activation causes the increase of cell volume. Release of glutamate by ADPbetaS was abolished by 5-nitro-2 (3-phenyl propy lamino)–benzoate plus furosemide but was unaffected by botulinum toxin A. These observations indicate that P2Y₁ receptor-evoked glutamate may be mediated via volume-sensitive Cl⁻ channel but not via exocytosis of glutamate containing vesicles. We speculate that P2Y₁ receptors-evoked glutamate efflux, occurring under pathological condition, may modulate the activity of synapses in spinal cord.     

Vesicular release of glutamate mediates bidirectional signaling between astrocytes and neurons

The major excitatory neurotransmitter in the CNS, glutamate, can be released exocytotically by neurons and astrocytes. Glutamate released from neurons can affect adjacent astrocytes by changing their intracellular Ca²⁺ dynamics and, vice versa , glutamate released from astrocytes can cause a variety of responses in neurons such as: an elevation of [Ca²⁺]_i, a slow inward current, an increase of excitability, modulation of synaptic transmission, synchronization of synaptic events, or some combination of these. This astrocyte-neuron signaling pathway might be a widespread phenomenon throughout the brain with astrocytes possessing the means to be active participants in many functions of the CNS. Thus, it appears that the vesicular release of glutamate can serve as a common denominator for two of the major cellular components of the CNS, astrocytes and neurons, in brain function.     

Involvement of Calcineurin in Ca2+ Paradox-Like Injury of Cultured Rat Astrocytes

Abstract: The Ca²⁺/calmodulin-dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca²⁺-containing medium after exposure to Ca²⁺-free medium caused Ca²⁺ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca²⁺-mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca²⁺ challenge-induced injury in a dose-dependent manner (10⁻¹⁰–10⁻⁸ M ). Cyclosporin A at 1 µ M mimicked the effect of FK506. Rapamycin (1 µ M ) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 n M ), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca²⁺ challenge-induced increase in cytosolic Ca²⁺ level nor Na⁺-Ca²⁺ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca²⁺ level. Immunochemical studies showed that both calcineurin A (probably the Aβ2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.