Neuron-derived factors negatively modulate ryanodine receptor-mediated calcium release in cultured mouse astrocytes

Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) produced by ryanodine receptor (RyR) agonist, caffeine (caf), and ionotropic agonists: N-methyl-d-aspartate (NMDA) receptor (NMDAR) agonist, NMDA and P2X7 receptor (P2X7R) agonist, 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP) were measured in cultured mouse cortical astrocytes loaded with the fluorescent calcium indicator Fluo3-AM in a confocal laser scanning microscope. In mouse astrocytes cultured in standard medium (SM), treatment with caf increased [Ca²⁺]_i, with a peak response occurring about 10 min after stimulus application. Peak responses to NMDA or BzATP were observed about <1 min and 4.5 min post stimulus, respectively. Co-treatment with NMDA or BzATP did not alter the peak response to caf in astrocytes cultured in SM, the absence of the effects being most likely due to asynchrony between the response to caf, NMDA and BzATP. Incubation of astrocytes with neuron-condition medium (NCM) for 24 h totally abolished the caf-evoked [Ca²⁺]_i increase. In NCM-treated astrocytes, peak of [Ca²⁺]_i rise evoked by NMDA was delayed to about 3.5 min, and that induced by BzATP occurred about three minutes earlier than in SM. The results show that neurons secrete factors that negatively modulate RyR-mediated Ca²⁺-induced Ca²⁺ release (CICR) in astrocytes and alter the time course of Ca²⁺ responses to ionotropic stimuli.     

Separate Intramolecular Targets for Protein Kinase A Control N-Methyl-d-aspartate Receptor Gating and Ca2+ Permeability

Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca²⁺ flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca²⁺ permeability (P_Ca/P_Na) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca²⁺ conductance, because neither Na⁺ conductance nor Ca²⁺-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca²⁺ permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca²⁺ permeability.     

Identification of new functions of Ca2+ release from intracellular stores in central nervous system

Ca²⁺ release from intracellular stores regulates muscle contraction and a vast array of cell functions, but its role in the central nervous system (CNS) has not been completely elucidated. A new method of blocking IP₃ signaling by artificially expressing IP₃ 5-phosphatase has been used to clarify the functions of intracellular Ca²⁺ mobilization in CNS. Here I review two of such functions: the activity-dependent synaptic maintenance mechanism and the regulation of neuronal growth by spontaneous Ca²⁺ oscillations in astrocytes. These findings add new bases for better understanding CNS functions and suggest the presence of as yet unidentified neuronal and glial functions that are regulated by Ca²⁺ store-dependent Ca²⁺ signaling.     

Selective Inhibition by Ethanol of Mitochondrial Calcium Influx Mediated by Uncoupling Protein-2 in Relation to N-Methyl-D-Aspartate Cytotoxicity in Cultured Neurons

Background

We have shown the involvement of mitochondrial uncoupling protein-2 (UCP2) in the cytotoxicity by N-methyl-D-aspartate receptor (NMDAR) through a mechanism relevant to the increased mitochondrial Ca²⁺ levels in HEK293 cells with acquired NMDAR channels. Here, we evaluated pharmacological profiles of ethanol on the NMDA-induced increase in mitochondrial Ca²⁺ levels in cultured murine neocortical neurons.

Methodology/Principal Findings

In neurons exposed to glutamate or NMDA, a significant increase was seen in mitochondrial Ca²⁺ levels determined by Rhod-2 at concentrations of 0.1 to 100 µM. Further addition of 250 mM ethanol significantly inhibited the increase by glutamate and NMDA in Rhod-2 fluorescence, while similarly potent inhibition of the NMDA-induced increase was seen after exposure to ethanol at 50 to 250 mM in cultured neurons. Lentiviral overexpression of UCP2 significantly accelerated the increase by NMDA in Rhod-2 fluorescence in neurons, without affecting Fluo-3 fluorescence for intracellular Ca²⁺ levels. In neurons overexpressing UCP2, exposure to ethanol resulted in significantly more effective inhibition of the NMDA-induced increase in mitochondrial free Ca²⁺ levels than in those without UCP2 overexpression, despite a similarly efficient increase in intracellular Ca²⁺ levels irrespective of UCP2 overexpression. Overexpression of UCP2 significantly increased the number of dead cells in a manner prevented by ethanol in neurons exposed to glutamate. In HEK293 cells with NMDAR containing GluN2B subunit, more efficient inhibition was similarly induced by ethanol at 50 and 250 mM on the NMDA-induced increase in mitochondrial Ca²⁺ levels than in those with GluN2A subunit. Decreased protein levels of GluN2B, but not GluN2A, subunit were seen in immunoprecipitates with UCP2 from neurons with brief exposure to ethanol at concentrations over 50 mM.

Conclusions/Significance

Ethanol could inhibit the interaction between UCP2 and NMDAR channels to prevent the mitochondrial Ca²⁺ incorporation and cell death after NMDAR activation in neurons.     

Ca2+-Dependent Inactivation of GluN2A and GluN2B NMDA Receptors Occurs by a Common Kinetic Mechanism

N-Methyl-d-aspartate (NMDA) receptors are Ca²⁺-permeable channels gated by glutamate and glycine that are essential for central excitatory transmission. Ca²⁺-dependent inactivation (CDI) is a regulatory feedback mechanism that reduces GluN2A-type NMDA receptor responses in an activity-dependent manner. Although CDI is mediated by calmodulin binding to the constitutive GluN1 subunit, prior studies suggest that GluN2B-type receptors are insensitive to CDI. We examined the mechanism of CDI subtype dependence using electrophysiological recordings of recombinant NMDA receptors expressed in HEK-293 cells. In physiological external Ca²⁺, we observed robust CDI of whole-cell GluN2A currents (0.42 ± 0.05) but no CDI in GluN2B currents (0.08 ± 0.07). In contrast, when Ca²⁺ was supplied intracellularly, robust CDI occurred for both GluN2A and GluN2B currents (0.75 ± 0.03 and 0.67 ± 0.02, respectively). To examine how the source of Ca²⁺ affects CDI, we recorded one-channel Na⁺ currents to quantify the receptor gating mechanism while simultaneously monitoring ionomycin-induced intracellular Ca²⁺ elevations with fluorometry. We found that CDI of both GluN2A and GluN2B receptors reflects receptor accumulation in long-lived closed (desensitized) states, suggesting that the observed subtype-dependent differences in macroscopic CDI reflect intrinsic differences in equilibrium open probabilities (P_o). We tested this hypothesis by measuring substantial macroscopic CDI, in physiologic conditions, for high P_o GluN2B receptors (GluN1^A652Y/GluN2B). Together, these results show that Ca²⁺ flux produces activity-dependent inactivation for both GluN2A and GluN2B receptors and that the extent of CDI varies with channel P_o. These results are consistent with CDI as an autoinhibitory feedback mechanism against excessive Ca²⁺ load during high P_o activation.     

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca²⁺ concentration fluctuations ([Ca²⁺]_i) and in several settings respond to neuronal excitation with enhanced [Ca²⁺]_i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca²⁺]_i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca²⁺]_i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca²⁺]_i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca²⁺]_i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca²⁺]_i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca²⁺]_i signals in the striatal microcircuitry.     

Inositol 1,4,5‐trisphosphate receptor 1 degradation in mouse eggs and impact on [Ca2+]i oscillations

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca²⁺]_i), which is almost entirely mediated by inositol 1,4,5‐trisphosphate receptor 1 (IP₃R1). In mammalian eggs, fertilization‐induced [Ca²⁺]_i responses exhibit a periodic pattern that are called [Ca²⁺]_i oscillations. These [Ca²⁺]_i oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP₃R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP₃R1 degradation and examined the impact of the IP₃R1 levels on the pattern of [Ca²⁺]_i oscillations. Using microinjection of IP₃ and of its analogs and conditions that prevent the development of [Ca²⁺]_i oscillations, we show that IP₃R1 degradation requires uniform and persistently elevated levels of IP₃. We also established that progressive degradation of the IP₃R1 results in [Ca²⁺]_i oscillations with diminished periodicity while a near complete depletion of IP₃R1s precludes the initiation of [Ca²⁺]_i oscillations. These results provide insights into the mechanism involved in the generation of [Ca²⁺]_i oscillations in mouse eggs. J. Cell. Physiol. 222:238–247, 2010. © 2009 Wiley‐Liss, Inc.     

Mechanisms of calcium signaling in smooth muscle cells explored with fluorescence confocal imaging

A rise in the intracellular concentration of ionized calcium ([Ca²⁺]_i) is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques, with special accent on fluorescence confocal microscopy, and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for studying the relationship between the structural organization of living smooth muscle cells (SMCs) and features of calcium signaling at the subcellular level. Applying fluorescent confocal imaging, patch-clamp recording, immunostaining, and flash photolysis techniques to freshly isolated SMCs, we have demonstrated that: (i) Ca²⁺ sparks are mediated by spontaneous clustered opening of ryanodine receptors (RyRs) and occur at the highest rate at preferred sites (frequent discharge sites, FDSs), the number of which depends on SMC type; (ii) FDSs are associated with sub-plasmalemmal sarcoplasmic reticulum (SR) elements, but not with polarized mitochondria; (iii) Ca²⁺ spark frequency increases with membrane depolarization in voltage-clamped SMCs or following neurotransmitter application to SMCs, in which the membrane potential was not controlled, leading to spark summation and resulting in a cell-wide increase in [Ca²⁺]_i and myocyte contraction; (iv) cross-talk between RyRs and inositol trisphosphate receptors (IP₃Rs) is an important determinant of the [Ca²⁺]_i dynamics and recruits neighboring Ca²⁺-release sites to generate [Ca²⁺]_i waves; (v) [Ca²⁺]_i waves induced by depolarization of the plasma membrane or by noradrenaline or caffeine, but not by carbachol (CCh), originate at FDSs; (vi) Ca²⁺-dependent K⁺ and Cl^- channels sense the local changes in [Ca²⁺]_i during a Ca²⁺ spark and thereby may couple changes in [Ca²⁺]_i within a microdomain to changes in the membrane potential, thus affecting the cell excitability; (vii) the muscarinic cation current (mI _cat) does not mirror changes in [Ca²⁺]_i, thus reflecting the complexity of [Ca²⁺]_i — muscarinic cationic channel coupling; (viii) RyR-mediated Ca²⁺ release, either spontaneous or caffeine-induced, does not augment mI _cat; (ix) intracellular flash release of Ca²⁺ is less effective in augmentation of mI _cat than flash release of IP₃, suggesting that IP₃ may sensitize muscarinic cationic channels to Ca²⁺; (x) intracellular flash release of IP₃ fails to augment mI _cat in SMCs, in which [Ca²⁺]_i was strongly buffered, suggesting that IP₃ exerts no direct effect on muscarinic cationic channel gating, and that these channels sense an increase in [Ca²⁺]_i rather than depletion of the IP₃-dependent Ca²⁺ store; and (xi) predominant expression of IP₃R type 1 in the peripheral SR provides a structural basis for a tight functional coupling between IP₃R-mediated Ca²⁺ release and muscarinic cationic channel opening.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 455–465, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

Oxytocin induced a biphasic increase in the intracellular Ca2+ concentration of porcine myometrial cells: Participation of a pertussis toxin—insensitive G-protein,inositol 1,4,5-trisphosphate—sensitive Ca2+ pool,and Ca2+ channels

This study investigated the underlying mechanisms of oxytocin (OT)-induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in acutely dispersed myometrial cells from prepartum sows. A dosedependent increase in [Ca²⁺]_i was induced by OT (0.1 nM to 1 μM) in the presence and absence of extracellular Ca²⁺ ([Ca²⁺]_e). [Ca²⁺]_i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca²⁺]_e. However, in the absence of [Ca²⁺]_e, the [Ca²⁺]_i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca²⁺ ionophore. Administration of OT (1 μM) for 15 sec increased inositol 1,4,5-trisphosphate (IP₃) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 μg/ml) for 2 hr failed to alter the OT-induced increase in [Ca²⁺]_i and IP₃ formation. U-73122 (30 nM to 3 μM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca²⁺]_i by OT dose dependently. U-73122 (3 μM) also abolished the OT-induced IP₃ formation. Thapsigargin (2 μM), an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, did not increase [Ca²⁺]_i. However, it did time-dependently inhibit the OT-induced increase in [Ca²⁺]_i. Nimodipine (1 μM), a Voltage-dependent Ca²⁺ channel (VDCC) blocker, inhibited the OT-induced plateau by 26%. La³⁺ (1 μM), a nonspecific Ca²⁺ channel blocker, abrogated the OT-induced plateau. In whole-cell patch-clamp studies used to evaluate VDCC activities, OT (0.1 μM) increased Ca²⁺ Current (I_ca) by 40% with no apparent changes in the current-voltage relationship. The OT-induced increase in I_ca reached the maximum in 5 min, and the increase was abolished by nimodipine (1 μM). These results suggested that (1) activation of OT receptors in porcine myometrium evokes a cascade in the PTX-insensitive G-protein–PLC-IP₃ signal transduction, resulting in an increase in [Ca²⁺]_i; (2) the OT-induced increase in [Ca²⁺]_i is characterized by a biphasic pattern, in which the spike is predominately contributed by the intracellular Ca²⁺ release from the IP₃-sensitive pool, and to a lesser extent by Ca²⁺ influx, whereas the plateau is from increased Ca²⁺ influx; and (3) the influx is via VDCC and receptor-operated Ca²⁺ channels. © 1995 Wiley-Liss, Inc.     

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK_Ca channel regulation by IP₃ and IP₃Rs in rat and mouse cerebral artery smooth muscle cells. IP₃ activated BK_Ca channels both in intact cells and in excised inside-out membrane patches. IP₃ caused concentration-dependent BK_Ca channel activation with an apparent dissociation constant (K_d) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca²⁺ concentration ([Ca²⁺]_i; 10 µM). IP₃ also caused a leftward-shift in BK_Ca channel apparent Ca²⁺ sensitivity and reduced the K_d for free [Ca²⁺]_i from ∼20 to 12 µM, but did not alter the slope or maximal P_o. BAPTA, a fast Ca²⁺ buffer, or an elevation in extracellular Ca²⁺ concentration did not alter IP₃-induced BK_Ca channel activation. Heparin, an IP₃R inhibitor, and a monoclonal type 1 IP₃R (IP₃R1) antibody blocked IP₃-induced BK_Ca channel activation. Adenophostin A, an IP₃R agonist, also activated BK_Ca channels. IP₃ activated BK_Ca channels in inside-out patches from wild-type (IP₃R1^+/+) mouse arterial smooth muscle cells, but had no effect on BK_Ca channels of IP₃R1-deficient (IP₃R1^−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP₃R1 is located in close spatial proximity to BK_Ca α subunits. The IP₃R1 monoclonal antibody coimmunoprecipitated IP₃R1 and BK_Ca channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP₃R1 activation elevates BK_Ca channel apparent Ca²⁺ sensitivity through local molecular coupling in arterial smooth muscle cells.     

Effects of chronic treatment with fluoxetine on receptor-stimulated increase of [Ca2+]i in astrocytes mimic those of acute inhibition of TRPC1 channel activity

Primary cultures of mouse astrocytes were used to investigate effects by chronic treatment (3-21 days) with fluoxetine (0.5-10 μM) on capacitative Ca²⁺ influx after treatment with the SERCA inhibitor thapsigargin and on receptor agonist-induced increases in free cytosolic Ca²⁺ concentration [Ca²⁺]_i, determined with Fura-2. The agonists were the 5-HT_2B agonist fluoxetine, the α₂-adrenergic agonist dexmedetomidine, and ryanodine receptor (RyR) and IP₃ receptor (IP₃R) agonists. In untreated sister cultures each agonist distinctly increased [Ca²⁺]_i, but in cultures treated for sufficient length of time or with sufficiently high doses of fluoxetine, acute administration of fluoxetine, dexmedetomidine, or RyR or IP₃R agonists elicited reduced, in some cases abolished, effects. Capacitative Ca²⁺ entry, meditated by TRPC1 channels, was sufficiently inhibited to cause a depletion of Ca²⁺ stores, which could explain the reduced agonist effects. All effects of chronic fluoxetine administration could be replicated by TRPC1 channel antibody or siRNA. Since increases in astrocytic [Ca²⁺]_i regulate release of gliotransmitters, these effects may have profound effects on brain function. They may be important for therapeutic effects of all 5 conventional ‘serotonin-specific reuptake inhibitors’ (SSRIs), which at concentrations used therapeutically (∼1 μM) share other of fluoxetine's chronic effects (Zhang et al., Neuron Glia Biol. 16 (2010) 1-13).     

Redox modulation of calcium entry and release of intracellular calcium by thimerosal in GH4C1 pituitary cells

In the present work we have investigated the actions of the oxidizing sulfhydryl reagent thimerosal on different mechanisms which regulate intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in GH₄C₁ pituitary cells. In intact Fura-2 loaded cells, low concentrations of thimerosal potentiated the spike phase of the TRH-induced (thyrotropin-releasing hormone) rise in [Ca²⁺]_i, whereas high thimerosal concentrations inhibited it. The effect of thimerosal on the plateau phase was always inhibitory.The effect of thimerosal on the IP₃-induced calcium release (IICR) was studied in permeabilized cells using the Ca²⁺ indicator Fluo-3. A low concentration of thimerosal (10 μM) stimulated IICR: the Ca²⁺ release induced by 300 nM inositol-1,4,5-trisphosphate (IP₃) was enhanced in cells treated with thimerosal for 1 or 6 min (67 ± 11 nM and 34 ± 5 nM, respectively) as compared to control cells (17 ± 2 nM). On the other hand, a high concentration of thimerosal (100 μ inhibited IICR: when IP₃ (10 μM) was added after a 5 min preincubation with thimerosal, the IP₃-induced rise in [Ca²⁺]_i (46 ± 14 nM) was 57% smaller as compared with that seen in control cells (106 ± 10 nM).The effect of thimerosal on the voltage-operated Ca ²⁺ channels (VOCCs) was studied by depolarizing intact Fura-2 loaded cells by addition of 20 mM K⁺ to the cuvette. The depolarization-evoked increase in [Ca²⁺]_i was inhibited in a dose-dependent manner by thimerosal. Direct evidence for an inhibitory effect of thimerosal on VOCCs was obtained by using the whole-cell configuration of the patch-clamp technique: thimerosal (100 μM) potently inhibited the Ba²⁺ currents through VOCCs.In addition, our results indicated that thimerosal inhibited the caffeine-induced increase in [Ca²⁺]_i, and activated a capacitative Ca²⁺ entry pathway. The actions of thimerosal were apparently due to its oxidizing activity because the effects were mostly reversed by the thiol-reducing agent dithiothreitol (DTT).We conclude that, in GH₄C₁ pituitary cells, the mobilization of intracellular calcium and the different Ca²⁺ entry pathways are sensitive to redox modulation.     

Development of a functional assay for Ca2+ release activity of IP3R and expression of an IP3R gene fragment in the baculovirus-insect cell system

Receptor-stimulated phosphoinositide (PI) hydrolysis is an important and ubiquitous mechanism of intracellular signaling. Inositol 1,4,5-trisphosphate (IP₃), generated by phosphoinositide (PI) hydrolysis, binds to and gates an intracellular Ca²⁺ channel, the IP₃ receptor (IP₃R), which is therefore a central component of this signaling cascade. Here we describe the development of a baculovirus (BV)/Sf(S. frugiperda) cell system that can be used to look at IP₃R function. Agonist-evoked changes in intracellular Ca²⁺ levels [Ca²⁺]_i were measured (using Fura2) in Sf cells expressing the gene encoding the muscarinic acetylcholine receptor (vmlAchR). Furthermore, we have constructed a recombinant BV (vlP₃R), with the core of the IP₃R ligand-binding domain from the Drosophila IP₃R, under the polyhedrin promoter. The recombinant protein from such a virus was expected to act as a large ligand sink for IP3, generated by stimulation of vmlAchR. Cells coinfected with recombinant BV carrying the potential dominant-negative vIP₃R construct and vmlAchR have been used to assay the modulation of IP₃R-mediated Ca²⁺ release, by the ligand sink.     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

Modulation of intracellular calcium concentrations and T cell activation by prickly pear polyphenols

Opuntia ficus indica(prickly pear) polyphenolic compounds (OFPC) triggered an increase in [Ca²⁺]_i in human Jurkat T-cell lines. Furthermore, OFPC-induced rise in [Ca²⁺]_i was significantly curtailed in calcium-free buffer (0% Ca²⁺) as compared to that in 100% Ca²⁺ medium. Preincubation of cells with tyrphostin A9, an inhibitor of Ca²⁺ release-activated Ca²⁺(CRAC) channels, significantly diminished the OFPC-induced sustained response on the increases in [Ca²⁺]_i. Lanthanum and nifedipine, the respective inhibitors of voltage-dependent and L-type calcium channels, failed to curtail significantly the OFPC-induced calcium response. As OFPC still stimulated increases in [Ca²⁺]_i in 0% Ca²⁺ medium, the role of intracellular calcium was investigated. Hence, addition of thapsigargin (TG), an inhibitor of Ca²⁺-ATPase of the endoplasmic reticulum (ER), during the OFPC-induced peak response exerted an additive effect, indicating that the mechanism of action of these two agents are different. Furthermore, U73122, an inhibitor of IP₃ production, completely abolished increases in [Ca²⁺]_i, induced by OFPC, suggesting that these polyphenols induce the production of IP₃ that recruits calcium from ER pool. Polyphenolic compounds do act extracellularly as addition of fatty acid-free bovine serum albumin (BSA) significantly diminished the rise in [Ca²⁺]_i evoked by the formers. OFPC also induced plasma membrane hyperpolarisation which was reversed by addition of BSA. OFPC were found to curtail the expression of IL-2 mRNA and T-cell blastogenesis. Together these results suggest that OFPC induce increases in [Ca²⁺]_i via ER pool and opening of CRAC channels, and exert immunosuppressive effects in Jurkat T-cells.     

Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells

Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca²⁺ ([Ca²⁺]_i) in C₆ glioma cells with an EC₅₀ of 60±4 and 10±5 M, respectively. The threshold concentration of ATP (3 M) for increasing [Ca²⁺]_i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca²⁺ and Na⁺ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca²⁺ channels (Co²⁺, Mn²⁺, La³⁺, or Cd²⁺). In Ca²⁺-free medium, ATP caused only a transient increase in [Ca²⁺]_i as opposed to a sustained [Ca²⁺]_i increase in normal medium. The ATP-induced elevation of [Ca²⁺]_i was resistant to Na⁺ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attentuated by La³⁺. The differences in the characteristics of ATP-caused P₁ hydrolysis and [Ca²⁺]_i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca²⁺ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P₁-purinergic receptors, the C₆ glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na⁺]_o dependency for ATP-induced PI turnover require further investigation.Abbreviations PI phosphoinositide - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - PDBu phorbol 12, 13-dibutyrate - PSS physiological saline solution - IP inositol phosphates - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - IP₄ inositol (1,3,4,5) tetrakisphosphate - PLC phospholipase C     

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.     

Potentiation of Carbachol-Induced Ca2+ Release by Peroxynitrite in Human Neuroblastoma SH-SY5Y Cells

We have investigated the effect of 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, on carbachol-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human neuroblastoma SH-SY5Y cells by means of single cell imaging of [Ca²⁺]_i. SIN-1 potentiated carbachol-induced [Ca²⁺]_i rise regardless of external Ca²⁺, and the potentiation was completely inhibited by superoxide dismutase, indicating that peroxynitrite may enhance Ca²⁺ release from intracellular stores. On the other hand, SIN-1 reduced carbachol-induced inositol 1,4,5-trisphosphate (IP₃) formation. Genistein, a tyrosine kinase inhibitor, potentiated carbachol-induced rise of [Ca²⁺]_i regardless of external Ca²⁺. These results suggest that peroxynitrite may potentiate the release of Ca²⁺ from intracellular stores through the perturbation of regulation in tyrosine phosphorylation-dephosphorylation system.     

A2A R mediated modulation in IP3 levels altering the [Ca2+]i through cAMP-dependent PKA signalling pathway

Stimulation of A_2A receptors (A_2A R) coupled to G_s/olf protein activates Adenylyl cyclase (AC) leading to the release of cAMP which activates the cAMP-dependent PKA phosphorylation. The possible role of A_2A R in the modulation of free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) involving IP₃, cAMP and PKA was investigated in HEK 293-A_2A R. The levels of IP₃ and cAMP were observed by enzyme immunoassay detection method and [Ca²⁺]_i using Fluo-4 AM. Moreover, cAMP-dependent PKA was determined using the PKA Colorimetric Activity Kit. We observed that the cells pre-treated with A_2A R agonist NECA showed increased levels of cAMP, PKA, IP₃ and [Ca²⁺]_i levels. However, the reverse effect was observed with A_2A R antagonists (ZM241385 and caffeine). Blocking the G_αq/PLC/DAG/IP₃ pathway with neomycin, a PLC inhibitor did not affect the modulation of IP₃ and [Ca²⁺]_i levels in HEK 293-A_2A R cells. To investigate the G_αi/AC/cAMP/PKA, HEK 293-A_2A R cells pre-treated with pertussis toxin followed by forskolin in the presence of A_2A R agonist (NECA) showed no effect on cAMP levels. Further, G_αs/AC/cAMP/PKA pathway was investigated to elucidate the role of cAMP-dependent PKA in IP₃ mediated [Ca²⁺]_i modulation. In the HEK 293-A_2A R cells pre-treated with PKA inhibitor KT5720 and treated with NECA led to inhibit the IP₃ and [Ca²⁺]_i levels. The study distinctly demonstrated that A_2A R modulates IP₃ levels to release the [Ca²⁺]_i via cAMP-dependent PKA. The role of A_2A R mediated G_αs pathway inducing IP₃ mediated [Ca²⁺]_i release may open new avenues in the therapy of neurodegenerative disorder.