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

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

Glutamate Regulates the Frequency of Spontaneous Synchronized Ca<Superscript>2+</Superscript> Spikes Through Group II Metabotropic Glutamate Receptor in Cultured Mouse Cortical Networks

1. Synchronized spontaneous intracellular Ca²⁺ spikes in networked neurons are believed to play a major role in the development and plasticity of neural circuits. Glutamate-induced signals through the ionotropic glutamate receptors (iGluRs) are profoundly involved in the generation of synchronized Ca²⁺ spikes.1 2. In this study, we examined the involvement of metabotropic glutamate receptors (mGluRs) in cultured mouse cortical neurons. We pharmacologically revealed that glutamate-induced signals through inclusive mGluRs decreased the frequency of Ca²⁺ spikes. Further experiments indicated that this suppressive effect on the spike frequency was mainly due to the signal through group II mGluR, inactivation of adenylate cyclase-cAMP-PKA signaling pathway. Group I mGluR had little involvement in the spike frequency.3. Taken together, glutamate generates the synchronized Ca²⁺ spikes through iGluRs and modulates simultaneously their frequency through group II mGluR–adenylate cyclase–cAMP–PKA signaling pathway in the present in vitro neural network. These results provide the evidence of the profound role of group II mGluR in the spontaneous and synchronous neural activities.     

H2O2 Mobilizes Ca2+ from Agonist- and Thapsigargin-sensitive and Insensitive Intracellular Stores and Stimulates Glutamate Secretion in Rat Hippocampal Astrocytes

The effect of hydrogen peroxide (H₂O₂) on cytosolic free calcium concentration ([Ca²⁺]_c) as well as its effect on glutamate secretion in rat hippocampal astrocytes have been the aim of the present research. Our results show that 100 μM H₂O₂ induces an increase in [Ca²⁺]_c, that remains at an elevated level while the oxidant is present in the perfusion medium, due to its release from intracellular stores as it was observed in the absence of extracellular Ca²⁺, followed by a significant increase in glutamate secretion. Ca²⁺-mobilization in response to the oxidant could only be reduced by thapsigargin plus FCCP, indicating that the Ca²⁺-mobilizable pool by H₂O₂ includes both endoplasmic reticulum and mitochondria. We conclude that ROS in hippocampal astrocytes might contribute to an elevation of resting [Ca²⁺]_c which, in turn, could lead to a maintained secretion of the excitatory neurotransmitter glutamate, which has been considered a situation potentially leading to neurotoxicity in the hippocampus.     

Infrared neural stimulation induces intracellular Ca2+ release mediated by phospholipase C

The influence of infrared laser pulses on intracellular Ca²⁺ signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo‐4 was used to measure Ca²⁺ in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo‐4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca²⁺ transient with similar dose‐response characteristics in hippocampal neurons and glioblastoma cells (half‐maximal effective energy density EC₅₀ of around 58 J.cm^?2). For both type of cells, the source of the infrared‐induced Ca²⁺ transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP₃‐induced Ca²⁺ release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP₃ cell signaling cascade has many potential applications including the development of optoceutical therapeutics.      

Both Neurons and Astrocytes Exhibited Tetrodotoxin-Resistant Metabotropic Glutamate Receptor-Dependent Spontaneous Slow Ca2+ Oscillations in Striatum

The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson''s disease. The group I mGluRs are known to modulate the intracellular Ca²⁺ signaling. To characterize Ca²⁺ signaling in striatal cells, spontaneous cytoplasmic Ca²⁺ transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca²⁺ transients (referred to as slow Ca²⁺ oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca²⁺ oscillation. Depletion of the intracellular Ca²⁺ store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca²⁺ oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. The slow Ca²⁺ oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca²⁺ oscillation may involve in the neuron-glia interaction in the striatum.     

Bcl-2 acts subsequent to and independent of Ca fluxes to inhibit apoptosis in thapsigargin- and glucocorticoidmtreated mouse lymphoma cells

The mechanism by which Bcl-2 inhibits apoptosis is unknown. One proposal is that Bcl-2 regulates intracellular Ca²⁺ fluxes thought to mediate apoptosis. In the present study, we investigated Bcl-2's mechanism of action by determining the effect of Bcl-2 on intracellular Ca²⁺ fluxes in the WEH17.2 mouse lymphoma cell line, which does not express Bcl-2, and its stable transfectant, which expresses a high level of Bcl-2. Treatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin produced marked alterations in intracellular Ca²⁺ homeostasis in both WEH17.2 and W.Hb12 cells, including elevation of free cytosolic Ca²⁺, endoplasmic reticulum Ca²⁺ pool depletion, capacitative entry of extracellular Ca²⁺, and increased loading of Ca²⁺ into mitochondria. Similar changes in intracellular Ca²⁺ occurred spontaneously in both cell lines following exponential growth. In both situations, W.Hb12 cells maintained optimal viability despite marked alterations in intracellular Ca ²⁺' whereas WEH17.2 cells underwent apoptosis. Treatment with the glucocorticoid hormone, dexamethasone, induced apoptosis in WEH17.2 cells, but not in W.HB12 cells, even though dexamethasone treatment did not alter intracellular Ca²⁺ homeostasis in either cell line. These findings indicate that Bcl-2 acts downstream from intracellular Ca ²⁺ fluxes in a pathway where Ca²⁺-dependent and Ca²⁺-independent death signals converge.     

Localization and regulation of Ca entry and exit pathways in exocrine gland cells

Studies of Ca²⁺ transport pathways in exocrine gland cells have been useful, chiefly because of the polarized nature of the secretory epithelial cells. In pancreatic acinar cells, for example, Ca²⁺ reloading of empty intracellular stores can occur solely via Ca²⁺ entry through the basal part of the plasma membrane. On the other hand, the principal site for intracellular Ca²⁺ release—with the highest concentration of inositol 1,4,5-trisphosphate (IP₃) receptors—is in the apical secretory pole close to the apical plasma membrane. This apical part of the plasma membrane contains the highest density of Ca²⁺ pumps and is therefore the principal site for Ca²⁺ extrusion. On the basis of the known properties of Ca²⁺ entry and exit pathways in exocrine gland cells, the mechanisms controlling Ca²⁺ exit and entry are discussed in relation to recent direct information about Ca²⁺ transport into and out of the endoplasmic reticulum (ER) and the mitochondria in these cells.     

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3-dependent Ca2+ release in mouse brain endothelial cells

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the target cells, which activates the Ca²⁺/Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca²⁺]_i and NO production. The current study assessed whether and how glutamate drives Ca²⁺-dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca²⁺]_i, which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca²⁺ oscillations were triggered by rhythmic endogenous Ca²⁺ mobilization and maintained over time by extracellular Ca²⁺ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca²⁺ release was mediated by InsP₃-sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca²⁺ entry mediated Ca²⁺ entry during ongoing Ca²⁺ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca²⁺ signals. Of note, glutamate induced Ca²⁺-dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca²⁺ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.     

Neurons respond to hyposmotic conditions by an increase in intracellular free calcium

The effect of hyposmotic conditions on the concentration of intracellular free calcium ([Ca²⁺]_i) was studied in cultured cerebellar granule cells and cerebral cortical neurons after loading of the cells with the fluorescent Ca²⁺ chelator Fluo-3. It was found that in both types of neurons exposure to media with a decrease in osmolarity of 20 to 50% of the osmolarity in the isosmotic medium (320 mOsm) led to a dose dependent increase in [Ca²⁺]_i with a time course showing the highest value at the earliest measured time point, i.e. 40 s after exposure to the hyposmotic media and a subsequent decline towards the basal level during the following 320 s. The response in the cortical neurons was larger than in the granule cells but both types of neurons exhibited a similar increase in [Ca²⁺]_i after expoxure to 50 mM K⁺ which was of the same magnitude as the increase in [Ca²⁺]_i observed in the cortical neurons exposed for 40 s to a medium with a 50% reduction in osmolarity. In both types of neurons the blocker of voltage gated Ca²⁺ channels verapamil had no effect on the hyposmolarity induced increase in [Ca²⁺]_i. On the contrary, this increase in [Ca²⁺]_i was dependent upon external calcium and could be inhibited partly or completely by the inorganic blockers of Ca²⁺ channels Mg²⁺ and La³⁺. Dantrolene which prevents release of Ca²⁺ from internal stores had no effect. The results show that exposure of neurons to hyposmotic conditions leading to swelling results in a large increase in free intracellular Ca²⁺ which represents an influx of Ca²⁺ rather than a release of Ca²⁺ from internal, dantrolene sensitive stores.     

Calcium channels and cation transport ATPases in cardiac hypertrophy induced by aortic constriction in newborn rats

In order to evaluate the contribution of pinocytosis to basal (no agonist) and lanthanide-insensitive store-activated Ca²⁺ inflow in freshly-isolated rat hepatocytes, the uptake of extracellular fluid by pinocytosis was measured at 20°C and used to predict the amount of extracellular Ca²⁺ taken up by pinocytosis. This was compared with the measured rate of Ca²⁺ uptake in the basal state, and with the measured lanthanide-insensitive component of divalent cation uptake stimulated by 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the smooth endoplasmic reticulum (Ca²⁺ + Mg²⁺)ATP-ase. Fluid uptake by pinocytosis was measured using [¹⁴C]sucrose. In hepatocytes incubated at 20°C, DBHQ increased the initial rate of sucrose uptake by about 35%. The data for sucrose uptake were used to calculate the volume of extracellular fluid taken up by pinocytosis which, in turn, was used to predict the amount of extracellular Ca ²⁺ taken up through pinocytosis in the basal and DBHQ-stimulated states. Rates of divalent cation inflow in the basal state were determined at 20°C by measuring the uptake of ⁴⁵Ca²⁺. The degree of stimulation of Ca²⁺ inflow by DBHQ and the lanthanide-insensitive component of DBHQ-stimulated divalent cation inflow were determined by measuring the rate of Mn²⁺-induced quenching of intracellular quin-2 in the absence of an agonist, and in the presence of DBHQ or DBHQ plus Gd³⁺. It was calculated that the process of pinocytosis accounts for at least 15% of Ca²⁺ uptake in the basal (no agonist) state, and for about 10% of DBHQ-stimulated lanthanide-insensitive Ca²⁺ uptake. It is concluded that in isolated hepatocytes (i) the release of Ca²⁺ from intracellular stores stimulates pinocytosis and (ii) the process of pinocytosis can account for a substantial proportion of basal Ca²⁺ inflow and a small proportion of DBHQ-stimulated lanthanide-insensitive Ca²⁺ inflow.Abbreviations RACC receptor-activated Ca²⁺ channel - DBHQ 2,5-di-tert-butylhydroquinone - [Ca²⁺] intracellular free Ca²⁺ concentration     

S100B Secretion in Acute Brain Slices: Modulation by Extracellular Levels of Ca<Superscript>2+</Superscript> and K<Superscript>+</Superscript>

Hippocampal slices have been widely used to investigate electrophysiological and metabolic neuronal parameters, as well as parameters of astroglial activity including protein phosphorylation and glutamate uptake. S100B is an astroglial-derived protein, which extracellularly plays a neurotrophic activity during development and excitotoxic insult. Herein, we characterized S100B secretion in acute hippocampal slices exposed to different concentrations of K⁺ and Ca²⁺ in the extracellular medium. Absence of Ca²⁺ and/or low K⁺ (0.2 mM KCl) caused an increase in S100B secretion, possibly by mobilization of internal stores of Ca²⁺. In contrast, high K⁺ (30 mM KCl) or calcium channel blockers caused a decrease in S100B secretion. This study suggests that exposure of acute hippocampal slices to low- and high-K⁺ could be used as an assay to evaluate astrocyte activity by S100B secretion: positively regulated by low K⁺ (possibly involving mobilization of internal stores of Ca²⁺) and negatively regulated by high-K⁺ (likely secondary to influx of K⁺).     

Homeostatic regulation mechanism of spontaneous firing determines glutamate responsiveness in the midbrain dopamine neurons

Autonomous tonic firing of the midbrain dopamine neuron is essential for maintenance of ambient dopamine level in the brain, in which intracellular Ca²⁺ concentration ([Ca²⁺]c) plays a complex but pivotal role. However, little is known about Ca²⁺ signals by which dopamine neurons maintain an optimum spontaneous firing rate. In the midbrain dopamine neurons, we here show that spontaneous firing evoked [Ca²⁺]c changes in a phasic manner in the dendritic region but a tonic manner in the soma. Tonic levels of somatic [Ca²⁺]c strictly tallied with spontaneous firing rates. However, manipulatory raising or lowering of [Ca²⁺]c with caged compounds from the resting firing state proportionally suppressed or raised spontaneous firing rate, respectively, suggesting presence of the homeostatic regulation mechanism for spontaneous firing rate via tonic [Ca²⁺]c changes of the soma. More importantly, abolition of this homeostatic regulation mechanism significantly exaggerated the responses of tonic firings and high-frequency phasic discharges to glutamate. Therefore, we conclude that this Ca²⁺-dependent homeostatic regulation mechanism is responsible for not only maintaining optimum rate of spontaneous firing, but also proper responses to glutamate. Perturbation of this mechanism could cause dopamine neurons to be more vulnerable to glutamate and Ca²⁺ toxicities.     

Functional Characteristics of ES Cell–derived Cardiac Precursor Cells Identified by Tissue-specific Expression of the Green Fluorescent Protein

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions.In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac α-actin promoter. First, fluorescent areas in ES cell–derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca²⁺ homeostasis plays a key role in cardiac function, we investigated how Ca²⁺ channels and Ca²⁺ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca²⁺ imaging experiments proved the functional expression of the L-type Ca²⁺ current (I_Ca) starting from day 7 of EB development. On day 7, using 10 mM Ca²⁺ as charge carrier, I_Ca was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of I_Ca proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, I_Ca was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP₃-sensitive intracellular Ca²⁺ stores and Ca²⁺-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca²⁺ stores, detected by caffeine-induced Ca²⁺ release, appeared in most GFP-positive cells 1–2 d after I_Ca. Coexpression of both I_Ca and ryanodine-sensitive Ca²⁺ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs.Thus, the functional expression of voltage-dependent L-type Ca²⁺ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP₃ receptors and sarcoplasmic Ca²⁺-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.     

Participation of the microtubular-microfilamentous system on intracellular Ca transport and acid secretion in dispersed parietal cells

Biphasic responses of amino[¹⁴C]pyrine accumulation and oxygen consumption were registered by gastrin stimulation in dispersed parietal cells from guinea pig gastric mucosa, and this was mimicked with the calcium ionophore A23187. The characteristics of these phases (first phase and second phase) were distinguished by the differences in the requirements of extracellular Ca²⁺. The first phase evoked by gastrin or ionophore A23187 was independent of extracellular Ca²⁺, whereas the second phase was not. In the first phase, fluorescence of a cytosolic Ca²⁺ indicator (quin2-AM) increased with the stimulation of ionophore A23187 and carbamylcholine chloride in the presence of extracellular Ca²⁺. In addition, an increase in cytosolic Ca²⁺ induced by ionophore A23187, but not by carbamylcholine chloride was also observed in the absence of extracellular Ca²⁺, suggesting that Ca²⁺ pool(s) in parietal cells might be present in the intracellular organelle. Cytochalasin B and colchicine, but not oligomycin, could eliminate this cytosolic Ca²⁺ increase induced by A23187 in a Ca²⁺-free medium. On the other hand, in a Ca²⁺-free medium, addition of ATP after pretreatment with digitonin could diminish the cytosolic Ca²⁺ increase brought about by A23187. This was also observed with oligomycin-treated cells, but not with cytochalasin B-treated cells. Similarly, subcellular fractionation of a parietal cell which had been pretreated with cytochalasin B or colchicine in an intact cell system reduced the rate of ATP-dependent Ca²⁺ uptake. These observations indicate that intracellular Ca²⁺ transport in dispersed parietal cells may be regulated by the microtubular-microfilamentous system. In conclusion, this study demonstrates the possibility of the existence of intracellular Ca²⁺ transport mediated by gastrin or ionophore A23187 and regulated by the microtubular-microfilamentous system in parietal cells.     

Effects of amyloid-β peptides on voltage-gated L-type Ca(V)1.2 and Ca(V)1.3 Ca(2+) channels

Overload of intracellular Ca²⁺ has been implicated in the pathogenesis of neuronal disorders, such as Alzheimer’s disease. Various mechanisms produce abnormalities in intracellular Ca²⁺ homeostasis systems. L-type Ca²⁺ channels have been known to be closely involved in the mechanisms underlying the neurodegenerative properties of amyloid-β (Aβ) peptides. However, most studies of L-type Ca²⁺ channels in Aβ-related mechanisms have been limited to Ca_V1.2, and surprisingly little is known about the involvement of Ca_V1.3 in Aβ-induced neuronal toxicity. In the present study, we examined the expression patterns of Ca_V1.3 after Aβ_25–35 exposure for 24 h and compared them with the expression patterns of Ca_V1.2. The expression levels of Ca_V1.3 were not significantly changed by Aβ_25–35 at both the mRNA levels and the total protein level in cultured hippocampal neurons. However, surface protein levels of Ca_V1.3 were significantly increased by Aβ_25–35, but not by Aβ_35–25. We next found that acute treatment with Aβ_25–35 increased Ca_V1.3 channel activities in HEK293 cells using whole-cell patch-clamp recordings. Furthermore, using GTP pulldown and co-immunoprecipitation assays in HEK293 cell lysates, we found that amyloid precursor protein interacts with β₃ subunits of Ca²⁺ channels instead of Ca_V1.2 or Ca_V1.3 α₁ subunits. These results show that Aβ_25–35 chronically or acutely upregulates Ca_V1.3 in the rat hippocampal and human kidney cells (HEK293). This suggests that Ca_V1.3 has a potential role along with Ca_V1.2 in the pathogenesis of Alzheimer’s disease.     

High Concentrations of Tricyclic Antidepressants Increase Intracellular Ca2+ in Cultured Neural Cells

We examined the effect of tricyclic antidepressants on intracellular Ca²⁺ signalling in cultured cells of neuronal and glial origin. High concentrations of amitriptyline and desipramine increased the intracellular Ca²⁺ in PC-12 and U-87 MG cells. In PC-12 cells amitriptyline induced a biphasic rise in intracellular Ca²⁺. A rapid and transient increase due to release of Ca²⁺ from intracellular pools was followed by sustained elevation of [Ca²⁺]_i due to influx from the extracellular medium. Desipramine evoked the Ca²⁺ release from intracellular pools but the influx of Ca²⁺ was not elicited. In U-87 MG cells both the drugs induced Ca²⁺ release from intracellular pools, however amitriptyline also induced a transient influx of Ca²⁺. To delineate the mechanisms involved in mobilization of Ca²⁺ by the drugs pharmacological agents that inhibit IP₃ formation in cells and Ca²⁺ channel blockers were used and changes in [Ca²⁺]_i and membrane potential were monitored. The results show that both the drugs release Ca²⁺ from IP₃ sensitive pools by activation of phospholipase C and amitriptyline in addition activates a non specific cation channel in the plasma membrane of cells. Paradoxically at relatively lower concentrations (< 50 M) amitriptyline and desipramine inhibited the Ca²⁺ signal induced by adenosine triphosphate in both the cell types. Our data demonstrate that tricyclic antidepressants at different doses may have inhibitory or stimulatory effects on cellular Ca²⁺ signalling.     

Glutamate receptor agonists evoked Ca2+-dependent and Ca2+-independent release of [3H]d-Aspartate from cultured chick retina cells

We studied the release of [³H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca²⁺-dependent and Ca²⁺-independent release of [³H]d-aspartate. In Ca²⁺-free (no added Ca²⁺) Na⁺ medium, the agonists of the glutamate receptors induced the release of [³H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl₂ the release of [³H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca²⁺-free medium. NMDA was the most potent agonist in stimulating the Ca²⁺-dependent release of [³H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca²⁺-dependent release of [³H]d-aspartate evoked by kainate was dependent on the influx of Ca²⁺ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca²⁺ channels (VSCC). The exocytotic release of [³H]d-aspartate evoked by AMPA relied exclusively on Ca²⁺ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca²⁺ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca²⁺ entry modulating vesicular release may be selectively recruited.     

Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture

We have addressed the possibility that Ca²⁺, Mg²⁺ and K⁺ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca²⁺, Mg²⁺ or K⁺ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their respective intracellular levels. From several lines of evidence, the deprivation of K⁺ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells. Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes. Removal of Mg²⁺ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death. The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features. These results highlighted the importance of utilizing several assays for the determination of apoptosis. The absence of Ca²⁺ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death. Hybridoma cells overexpressing the apoptotic suppresser gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg²⁺ ion deprivation and apoptosis inducing effects of Ca²⁺ ion deprivation. However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K⁺ free medium. The inclusion of bcl-2 activity in the mechanisms of Ca²⁺ Mg²⁺ or K⁺ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.     

Glutamate agonists and [3H]GABA release from rat hippocampal slices: Involvement of metabotropic glutamate receptors in the quisqualate-evoked release

The effects of glutamate agonists and their selective antagonists on the Ca²⁺-dependent and independent releases of [³H]GABA from rat coronal hippocampal slices were studied in a superfusion system. The Ca²⁺-dependent release evoked by glutamate, kainate and N-methyl-D-aspartate (NMDA) gradually declined with time despite the continuous presence of the agonists. Quisqualate (QA) caused a sustained release which exhibited no tendency to decline within the 20-min period of stimulation. This release was enhanced in Ca²⁺-free medium. The release evoked by QA in Ca²⁺-containing medium was significantly inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing that QA activates NMDA receptors directly or indirectly through (RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The inhibition of MK-801 was slightly diminished and that of CNQX totally abolished in Ca²⁺-free medium. Verapamil inhibited the QA-activated release in both Ca²⁺-containing and Ca²⁺-free media. The effect of QA but not that of AMPA was blocked in Ca²⁺-free medium by L(+)-2-amino-3-phosphonopropionate (L-AP3), a selective antagonist of the metabotropic glutamate receptor. It is suggested that the sustained release of GABA is also mediated partly by activation of metabotropic receptors and mobilization of Ca²⁺ from intracellular stores.     

Depletion and refilling of intracellular calcium pools in bovine chromaffin cells

To investigate Ca²⁺ uptake by Ca²⁺-depleted bovine chromaffin cells we depleted these cells of Ca²⁺ by incubating them in Ca²⁺-free buffer, then measured changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺ ₁)⁴⁵Ca²⁺ uptake, and Mn²⁺ uptake in response to added Ca²⁺ or MN²⁺. In depleted cells, the increase in [Ca²⁺]_i after Ca²⁺ addition, and the Mn²⁺ and⁴⁵Ca²⁺ uptakes were higher than in control cells, and were inhibited by verapamil. The size of the intracellular Ca²⁺ pools in depleted cells increased after Ca²⁺ addition. The times for [Ca²⁺]_i rise and Mn²⁺ entry to reach plateau levels were much shorter than the time for refilling of intracellular Ca²⁺ stores. In Ca²⁺-depleted cells and cells which had been loaded with BAPTA,⁴⁵Ca²⁺ uptake was much higher than in control cells. These results suggest that extracellular Ca²⁺ enters the cytoplasm first before refilling the intracellular stores. The rate of Mn²⁺ influx depended on the level of filling of the Ca²⁺ stores, suggesting that some signalling takes place between the intracellular stores and Ca²⁺ entry pathways through the plasma membrane.Abbreviations used BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid - BAPTA/AM acetoxymethyl ester of BAPTA - [Ca²⁺]_i cytosolic Ca²⁺ concentration - IP₃ inositol 1,4,5-trisphosphate - tBHQ 2,5-di-(t-butyl)-1,4-benzohydroquinone This work was included in a thesis submitted by A.-L. Sui to the Department of Biochemistry, National Yang-Ming Medical College, in partial fulfillment of the requirements for the degree of Doctor of Philosophy