Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by κ-opioids

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca²⁺]_i) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by κ-opioids. [Ca²⁺]_i responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca²⁺]_i increases which were abolished in Ca²⁺-free solution, but unaffected by lanthanum (25 μM) or tetrodotoxin (10⁻⁶ M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd³⁺; 250 μM) and amiloride (100 μM) abolished the [Ca²⁺]_i transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca²⁺]_i transients. The κ-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca²⁺]_i transients but had little effect on capsaicin-induced [Ca²⁺]_i transients. The inhibitory effect of U50,488 was abolished by the κ-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30–100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca²⁺]_i transients in small diameter DRG somas are mediated by influx of Ca²⁺ through a Gd³⁺- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca²⁺ transients are sensitive to κ-opioid agonists.     

Glutamate‐induced calcium increase mediates magnesium release from mitochondria in rat hippocampal neurons

Excess administration of glutamate is known to induce Ca²⁺ overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg²⁺ in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg²⁺ in the excitotoxicity, we simultaneously measured intracellular Ca²⁺ and Mg²⁺, using fluorescent dyes, Fura red, a fluorescent Ca²⁺ probe, and KMG‐104, a highly selective fluorescent Mg²⁺ probe developed by our group, respectively. Administration of 100 μM glutamate supplemented with 10 μM glycine to rat hippocampal neurons induced an increase in intracellular Mg²⁺ concentration ([Mg²⁺]_i). Extracellular Mg²⁺ was not required for this glutamate‐induced increase in [Mg²⁺]_i, and no increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) or [Mg²⁺]_i was observed in neurons in nominally Ca²⁺‐free medium. Application of 5 μM carbonyl cyanide p‐(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in [Ca²⁺]_i and [Mg²⁺]_i. Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg²⁺]_i but did induce an additive increase in [Ca²⁺]_i. Moreover, the glutamate‐induced increase in [Mg²⁺]_i was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg²⁺ efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, prevented this [Mg²⁺]_i increase. These results indicate that glutamate‐induced increases in [Mg²⁺]_i result from the Mg²⁺ release from mitochondria and that Ca²⁺ accumulation in the mitochondria is required for this Mg²⁺ release. © 2010 Wiley‐Liss, Inc.     

Acute action of rotenone on nigral dopaminergic neurons – involvement of reactive oxygen species and disruption of Ca2+ homeostasis

Rotenone is a toxin used to generate animal models of Parkinson’s disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05–1 μm ) effects on SNc neurons in acute rat midbrain slices, using whole‐cell patch‐clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide‐sensitive outward current (94 ± 15 pA) associated with increases in intracellular [Ca²⁺] ([Ca²⁺]_i) (73.8 ± 7.7 nm ) and intracellular [Na⁺] (3.1 ± 0.6 mm ) (all with 1 μm ). The outward current was not affected by a high ATP level (10 mm ) in the patch pipette but was decreased by Trolox. The [Ca²⁺]_i rise was abolished by removing extracellular Ca²⁺, and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N‐(p‐amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine‐123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm ) that, by itself, did not evoke a [Ca²⁺]_i rise resulted in a large (46.6 ± 25.3 nm ) Ca²⁺ response when baseline [Ca²⁺]_i was increased by a ‘priming’ protocol that activated voltage‐gated Ca²⁺ channels. There was also a positive correlation between ‘naturally’ occurring variations in baseline [Ca²⁺]_i and the rotenone‐induced [Ca²⁺]_i rise. This correlation was not seen in non‐dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP‐gated K⁺ channels and TRPM2‐like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone‐induced [Ca²⁺]_i rise by a small increase in baseline [Ca²⁺]_i.     

Selective enhancement by basic fibroblast growth factor of NMDA receptor-mediated increase of intracellular Ca concentration in hippocampal neurons

The short-term effect of bFGF on intracellular Ca²⁺ concentration ([Ca²⁺]_i) of hippocampal neurons was investigated using dissociated cell cultures. Changes in [Ca²⁺]_i were measured by microfluorometrically monitoring the fluorescence intesities from indivudual neurons loaded with fura-2. Perfusion of bFGF (20 ng/ml) alone did not affect the basal level of [Ca²⁺]_i in hippocampal neurons, but clearly enhanced the [Ca²⁺]_i increase induced by NMDA. Quisqualate or KCl-induced [Ca²⁺]_i increase was not influenced by bFGF. These results suggest that bFGF selectively enhances the NMDA receptor-mediated response in hippocampal neurons.     

Ethanol and nerve growth factor effects on calcium homeostasis in cultured embryonic rat medial septal neurons before and during depolarization

Ethanol and nerve growth factor (NGF) affect the survival of cholinergic neurons in the rat medial septum. To investigate whether calcium (Ca²⁺) homeostasis in these neurons is affected by ethanol or NGF treatment, changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) were studied in embryonic (E21) cultured medial septal neurons before stimulation (basal) and during stimulation with high potassium (K⁺). Changes in [Ca²⁺]; across time were measured in cultures of neurons treated without ethanol or with 100, 2110, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (-NGF). Changes in [Ca²⁺]_i were analyzed from fluorescence images, using indo-1. The effect of ethanol or NGF treatment was to reduce the rise in basal [Ca²⁺]_i. The combination of ethanol and NGF treatment in +NGF neurons led to increases in basal [Ca²⁺]_i with the greatest increase in basal [Ca²⁺]_i occurring with 200 mg% ethanol. The effect of ethanol or NGF was to increase [Ca²⁺]_i; during stimulation with high K⁺. The greatest increases in [Ca⁺]_i occurred with 100 and 800 mg% ethanol. Together, ethanol and NGF treatment in +NGF-treated neurons led to significantly greater increases or decreases in K⁺ stimulated changes in [Ca²⁺]_i compared to similarly treated -NGF neurons. We conclude that in medial septal neurons (before and during depolarization) changes in Ca²⁺ homeostasis occur in the presence of ethanol or NGF. The changes in [Ca²⁺]_i, following ethanol treatment are greater when NGF is present.     

Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin

Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca²⁺]_i) that was communicated to surrounding cells. Following propagation of the Ca²⁺ wave, many cells showed asynchronous oscillations in [Ca²⁺]_i. Dantrolene sodium (10 μM) inhibited the increase in [Ca²⁺]_i associated with this Ca²⁺ wave by 60-80%, and prevented subsequent Ca²⁺ oscillations. Despite the markedly decreased magnitude of the increase in [Ca²⁺]_i, the rate of propagation and the extent of communication of the Ca²⁺ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 μM) induced an initial increase in [Ca²⁺]_i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca²⁺]_i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of a Ca²⁺ wave from the stimulated cell to adjacent cells. Glutamate (50 μM) induced an initial increase in [Ca²⁺]_i in most cells that was followed by sustained oscillations in [Ca²⁺]_i in some cells. Dantrolene (10 μM) inhibited this initial [Ca²⁺]_i increase caused by glutamate by 65-90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 μm) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca²⁺ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca²⁺ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP₃) mediates the propagation of Ca²⁺ waves whereas Ca²⁺ -induced Ca²⁺ release amplifies Ca²⁺ waves and generates subsequent Ca²⁺ oscillations.     

Divalent Cation Entry in Cultured Rat Cerebellar Granule Cells Measured Using Mn2+ Quench of Fura 2 Fluorescence

In this study the rate of Mn²⁺ quench of fura-2 fluorescence evoked by glutamatergic and cholinergic agonists, depolarization and Ca²⁺ store modulators was measured in cultured cerebellar granule cells, in order to study their effects on Ca²⁺ entry in isolation from effects on Ca²⁺ store release. The rate of fluorescence quench by 0.1 mM Mn²⁺ was markedly increased by 25 mM K⁺- evoked depolarization or by 200 μM N-methyl-D-aspartate (NMDA), with a significantly greater increase occurring during the rapid-onset peak phase compared to the plateau phase of the K⁺- or NMDA-evoked [Ca²⁺]_i response. The stimulatory effect of NMDA on Mn²⁺ quench was abolished by dizocilpine (10 μM), but nitrendipine (2 μM), while decreasing the rate of basal quench, did not affect NMDA-stimulated Mn²⁺ entry. This suggests that nitrendipine may not act on NMDA channels in granule cells, at least under these conditions, and that voltage-operated Ca²⁺ channels are involved in control quench whereas the NMDA-evoked quench is dependent on entry through the receptor channel. The t_1/2 of quench was unaffected by α-amino-hydroxyisoxazole propionic acid (200 μM) and carbamyl choline (1 mM). Neither thapsigargin (10 μM) nor dantrolene (30 μM) significantly affected the rate of quench under control or NMDA- or K⁺-stimulated conditions, which confirms that the previously reported inhibitory effects on [Ca²⁺]_i elevations evoked by these agents are due to actions on Ca²⁺ stores. However, thapsigargin elevated [Ca²⁺Ii in the presence of normal [Ca²⁺]_i, but not in nominally Ca²⁺-free medium, indicating that it evokes Ca²⁺ entry in cerebellar granule cells, probably subsequent to store depletion, which appears to be either too small to be detected by Mn²⁺ quench or to occur via Mn²⁺-impermeant channels.     

The effect of N-arachidonoyldopamine on the dynamics of the intracellular calcium concentration in hippocampal neurons in the model of postischemic epileptogenesis in vitro

We studied the dynamics of the intracellular concentration of calcium ions ([Ca²⁺] _i ) and the influence of the endogenous cannabinoid N-arachidonoyldopamine (N-ADA) on disturbances of calcium homeostasis in cultured hippocampal neurons in the model of postischemic epileptogenesis (PE) in vitro, in accordance with a previously published method. It was found that 24 h after treatment with 20 μM glutamate, its application at a concentration of 50 μM results in a persistent increase in [Ca²⁺] _i whereas in neurons that were not previously subjected to glutamate treatment an increase in [Ca²⁺] _i after the application of 50 μM glutamate was reversible. The presence of N-ADA (5 μM) in the incubation medium both simultaneously with 20 μM glutamate exposure and for 24 h after it promoted recovery of the [Ca²⁺] _i level to the initial level. The results indicate that application of N-ADA promotes normalization of neuronal calcium homeostasis in a PE model in vitro.     

Spatiotemporal Distribution of Ca2+ Following Axotomy and Throughout the Recovery Process of Cultured Aplysia Neurons

This study investigates the alterations in the spatiotemporal distribution pattern of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i) during axotomy and throughout the recovery process of cultured Aplysia neurons, and correlates these alterations with changes in the neurons input resistance and trans-membrane potential. For the experiments, the axons were transected while imaging the changes in [Ca²⁺]_i with fura-2, and monitoring the neurons’resting potential and input resistance (R_i) with an intracellular microelectrode inserted into the cell body. The alterations in the spatiotemporal distribution pattern of [Ca²⁺]_i were essentially the same in the proximal and the distal segments, and occurred in two distinct steps: concomitantly with the rupturing of the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, [Ca²⁺]_i increased from resting levels of 0.05 – 0.1 μM to 1 – 1.5 μM along the entire axon. This is followed by a slower process in which a [Ca²⁺]_i front propagates at a rate of 11 – 16 μm/s from the point of transection towards the intact ends, elevating [Ca²⁺]_i to 3 – 18 μM. Following the resealing of the cut end 0.5 – 2 min post-axotomy, [Ca²⁺]_i recovers in a typical pattern of a retreating front, travelling from the intact ends towards the cut regions. The [Ca²⁺]_i recovers to the control level 7 – 10 min post-axotomy. In Ca²⁺-free artificial sea water (2.5 mM EGTA) axotomy does not lead to increased [Ca²⁺]_i and a membrane seal is not formed over the cut end. Upon reperfusion with normal artificial sea water, [Ca²⁺]_i is elevated at the tip of the cut axon and a membrane seal is formed. This experiment, together with the observations that injections of Ca²⁺, Mg²⁺ and Na⁺ into intact axons do not induce the release of Ca²⁺ from intracellular stores, indicates that Ca²⁺ influx through voltage gated Ca²⁺ channels and through the cut end are the primary sources of [Ca²⁺]_i following axotomy. However, examination of the spatiotemporal distribution pattern of [Ca²⁺]_i following axotomy and during the recovery process indicates that diffusion is not the dominating process in shaping the [Ca²⁺]_i gradients. Other Ca²⁺ regulatory mechanisms seem to be very effective in limiting these gradients, thus enabling the neuron to survive the injury.     

Calcium Signalling in Mouse Bergmann Glial Cells Mediated by α1-adrenoreceptors and H1 Histamine - Receptors

The presence of adrenergic and histaminergic receptors in Bergmann glial cells from cerebellar slices from mice aged 20–25 days was determined using fura-2 Ca²⁺ microfluorimetry. To measure the cytoplasmic concentration of Ca²⁺ ([Ca²⁺]_i), either individual cells were loaded with the Ca²⁺-sensitive probe fura-2 using the whole-cell patch-clamp technique or slices were incubated with a membrane-permeable form of the dye (fura-2/AM) and the microfluorimetric system was focused on individual cells. The monoamines adrenalin and noradrenalin (0.1-10 μM) and histamine (10-100 μM) triggered a transient increase in [Ca²⁺]_i. The involvement of the α₁-adrenoreceptor was inferred from the observations that monoamine-triggered [Ca²⁺]_i responses were blocked by the selective α¹-adreno-antagonist prazosin and were mimicked by the α¹-adreno-agonist phenylephrine. The monoamine-induced [Ca²⁺]_i signals were not affected by β- and α₂-adrenoreceptor antagonists (propranolol and yohimbine), and were not mimicked by β- and α₂-adrenoreceptor agonists (isoproterenol and clonidine). Histamine-induced [Ca²⁺]_i responses demonstrated specific sensitivity to only H₁ histamine receptor modulators. [Ca²⁺]_i responses to monoamines and histamine did not require the presence of extracellular Ca²⁺ and they were blocked by preincubation of slices with thapsigargin (500 nM), indicating that the [Ca²⁺]_i increase is due to release from intracellular pools. No [Ca²⁺]_i responses were recorded after application of aspartate, bradykinin, dopamine, GABA, glycine, oxytocin, serotonin, somatostatin, substance P, taurine or vasopressin. We conclude that cerebellar Bergmann glial cells are endowed with α₁ -adrenoreceptors and H₁ histamine receptors which induce the generation of intracellular [Ca²⁺]_i signals via activation of Ca²⁺ release from inositol-l,4,5-trisphosphate-sensitive intracellular stores.     

Taxol stabilizes [Ca]i and protects hippocampal neurons against excitotoxicity

Elevation of intracellular calcium levels [Ca²⁺]_i induces microtubule depolymerization, a process which plays roles in regulation of cell motility and axonal transport. However, excessive Ca²⁺ influx, as occurs in neurons subjected to excitotoxic conditions, can kill neurons. We now provide evidence that the polymerization state of microtubules influences neuronal [Ca²⁺]_i homeostasis and vulnerability to excitotoxicity. The microtubule-stabilizing agent taxol significantly attenuated glutamate neurotoxicity in cultured rat hippocampal neurons. Experiments in which [Ca²⁺]_i was monitored using the Ca²⁺ indicator dye fura-2 showed that the elevation of [Ca²⁺]_i induced by glutamate was significantly attenuated in neurons pretreated with taxol. Experiments using selective glutamate receptor agonists suggested that taxol suppressed Ca²⁺ influx through α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, but not through N-methyl-D-aspartate (NMDA) receptors. Taxol attenuated the neurotoxicity of the microtubule-depolymerizing agent colchicine; colchicine neurotoxicity was, in part, dependent on Ca²⁺ influx. These findings suggest that microtobules play a role in the mechanism of excitotoxicity and suggest that taxol and related compounds may be useful as antiexcitotoxic agents.     

PACAP Increases the Cytosolic Ca2−/ Concentration and Stimulates Somatodendritic Vasopresson Release in Rat Supraoptic Neurons

Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity and its receptor mRNA have been reported in the supraoptic and the paraventricular nucleus (SON and PVN, respectively) and PACAP has been implicated in the regulation of magnocellular neurosecretory cell function. To examine the site and the mechanism of the action of PACAP in the neurosecretory cells, we measured AVP release from SON slice preparations and the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) from single dissociated SON neurons. PACAP at concentrations from 10^?12 to 10^?7 M increased [Ca²⁺]_i in dissociated SON neurons in a dose-dependent manner. The patterns of the PACAP-induced [Ca²⁺]_i increase were either sustained increase or cytosolic Ca²⁺ oscillations. PACAP (10^?7 M) increased [Ca²⁺]_i in 27 of 27 neurons and glutamate (10^?4 M) increased [Ca²⁺]_i in 19 of 19 SON neurons examined, whereas angiotensin II (10^?7 M) increased [Ca²⁺]_i in only 15 of 60 SON neurons examined. PACAP at lower concentrations (10^?10 to 10^?8 M) increased [Ca²⁺]_i in 70–80% of neurons examined. Although the onset and recovery of the PACAP-induced [Ca²⁺]_i increase were slower than those observed with glutamate, the spatial distribution of the [Ca²⁺]_i increases in response to the two ligands were similar: [Ca²⁺]_i increase at the proximal dendrites was larger and faster and that at the center of the soma was smaller and slower. The PACAP-induced [Ca²⁺]_i responseswere abolished by extracellular Ca²⁺ removal, the l -type Ca²⁺-channel blocker, nicardipine, or by replacement of extracellular Na⁺ with N-methyl d-glucamine, and were partially inhibited by the Na⁺-channel blocker, tetrodotoxin. The N-type Ca²⁺-channel blocker, ω-conotoxin GVIA did not significantly inhibit the PACAP-induced [Ca²⁺]_i responses. Furthermore, PACAP (10^?7 M) as well as glutamate (10^?4 M) increased AVP release from SON slice preparations, and extracellular Ca²⁺ removal or nicardipine inhibited the AVP release in response to PACAP. These results indicate that PACAP enhances Ca²⁺ entry via voltage-gated Ca²⁺ channels and increases [Ca²⁺]_i, which, in turn, stimulates somatodendritic vasopressin release by directly activating PACAP receptors on SON neurons. The results also suggest that PACAP in the SON may play a pivotal role in the control of the neurohypophyseal function at the level of the soma or the dendrites.     

Cultured postnatal rat septohippocampal neurons change intracellular calcium in response to ethanol and nerve growth factor

Ethanol exposure affects cellular mechanisms involved in the regulation of calcium (Ca²⁺) homeostasis. Neurotrophins, such as nerve growth factor (NGF), stabilize intracellular Ca²⁺([Ca²⁺]_i) during a variety of neurotoxic insults. In this study, changes in [Ca²⁺]_i during treatment with ethanol and NGF were measured at the cell body of neurons using the Ca²⁺ indicator indo-1. Cultured postnatal day-of-birth (P0) septohippocampal (SH) neurons that were labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI), increased [Ca²⁺]_i in response to ethanol. This response was dose-related. P0 SH neurons treated with NGF had lower [Ca²⁺]_i than neurons withdrawn from NGF, implying that NGF may modulate Ca²⁺ homeostasis in these neurons. NGF also prevented the dose-related increase in [Ca²⁺]_i in ethanol-treated SH neurons. The SH neurons increased [Ca²⁺]_i when they were stimulated with 30 mM potassium chloride (KCl). Ethanol inhibited the potassium-stimulated change in [Ca²⁺]_i but the combination of ethanol and NGF caused [Ca²⁺]_i to increase with 100 mg% and 400 mg% ethanol and to decrease to a lower level with 200 mg% ethanol. These data were compared to data from previously published similar aged medial septal (MS) neurons (B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Clin. Exp. Res. 20 (1996) 1385–1394) and with embryonic gestational day 21 (E21) SH neurons (B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Brain Res. 729 (1996) 176–189). Differences in [Ca²⁺]_i responses were observed in ethanol and NGF-treated postnatal SH neurons compared with P0 MS neurons and E21 SH neurons. Of these differences, most occurred during the combined treatment with ethanol and NGF compared with either treatment alone.     

Cytotoxic actions and effects on intracellular Ca2+ and cGMP concentrations of sulphur-containing excitatory amino acids in cultured cerebral cortical neurons

Effects of the sulphur-containing acidic amino acids (SAAs) cysteic acid (CA), homocysteic acid (HCA), cysteine sulphinic acid (CSA), homocysteine sulphinic acid (HCSA), and S-sulphocysteine (SC) on intracellular concentrations of Ca²⁺ ([Ca²⁺]_i) and cGMP ([cGMP]_i) as well as their cytotoxic actions were investigated in cultured cerebral cortical neurons. The glutamate receptor subtype selective antagonists APV (D-(?)-2-amino-5-phosphonopentanoate) acting on N-methyl-D-aspartate (NMDA) receptors and DNQX (6,7-dinitroquinoxaline-2,3-dione) acting on non-NMDA receptors were employed to obtain information about the involvement of glutamate receptor subtypes in these actions of the SAAs. It was found that all SAAs exerted a cytotoxic action on the neurons. The ED₅₀ values for CSA, CA, HCSA, and HCA were around 30 to 50 μM and that for SC was about 150 μM. The glutamate transport blocker L-aspartate-β-hydroxamate increased the efficacy of CSA and CA but had no effect on the cytotoxic actions of the remaining SAAs. In case of CA, HCA, and SC the cytotoxicity could be prevented by APV alone and for HCSA, DNQX could block the toxic action. DNQX reduced the toxicity of HCA somewhat but the presence of APV was required for complete protection. CSA toxicity could only be blocked by the combination of APV and DNQX. All SAAs induced an increase in [cGMP]_i and [Ca²⁺]_i and with regard to [Ca²⁺]_i SC was the most potent and CA the least potent SAA. The effect of all SAAs on [cGMP]_i could be blocked by APV alone whereas DNQX had no effect except in the case of HCSA where the response was blocked completely and HCA where the response was inhibited by 75%. The SAA-induced increase in [Ca²⁺]_i could in all cases be significantly reduced by 0.6 mM Mg²⁺ and in the presence of Mg²⁺, APV dose dependently blocked the remaining SAA induced increase in [Ca²⁺]_i completely. Under these conditions DNQX was also found to block the SAA-induced increase in [Ca²⁺]_i dose dependently. In the absence of Mg²⁺, DNQX (25 μM) inhibited the response of the SAAs only by 65–75%. Under these conditions all SAA responses except that to SC could be fully antagonized by 300 μM APV. The SC-induced increase in [Ca²⁺]_i was inhibited by 60% by APV. The results show that no simple correlation exists between SAA-induced cytotoxicity and their ability to increase intracellular levels of Ca²⁺ and cGMP. However, when both NMDA and non-NMDA receptors were antagonized no toxicity or changes in calcium or cGMP were observed. © 1993 Wiley-Liss, Inc.     

Ionotropic and Metabotropic Glutamate Receptor Agonist-Induced [Ca2+]i Increase in Isolated Rat Supraoptic Neurons

In the present study, the effects of glutamate and of agonists for ionotropic and metabotropic glutamate receptors on intracellular Ca²⁺ concentration ([Ca²⁺]_i) were investigated in neurons of the rat supraoptic nucleus (SON). We used the intracellular Ca²⁺ imaging technique with fura-2, in single magnocellular neurons dissociated from the SON of rats. Glutamate (10^?6?10^?4 M) evoked a dose-dependent increase in [Ca²⁺]_i. The glutamate agonists exerted similar effects, although with some differences in the characteristics of their responses. The [Ca²⁺]_i response to NMDA was smaller than those of glutamate or the non-NMDA receptor agonists, AMPA and kainate, but was significantly enhanced by the removal of extracellular Mg²⁺. Glutamate, as well as quisqualate, an agonist for both ionotropic and metabotropic glutamate receptors, evoked a [Ca²⁺]_i increase in a Ca²⁺-free condition, suggesting Ca²⁺ release from intracellular Ca²⁺ stores. This was further evidenced by [Ca²⁺]_i increases in response to a more selective metabotropic glutamate receptor agonist, t-ACPD, in the absence of extracellular Ca²⁺. Furthermore, the quisqualate-induced Ca²⁺ release was abolished by the selective metabotropic glutamate receptor antagonist, (S)-4-carboxyphenylglycine. The results suggest that metabotropic glutamate receptors as well as non-NMDA and NMDA receptors are present in the SON neurons, and that activation of the first leads to Ca²⁺ release from intracellular Ca²⁺ stores and the activation of the latter two types induces Ca²⁺ entry. These dual mechanisms of Ca²⁺ signalling may play a role in the regulation of SON neurosecretory cells by glutamate.     

GABAB receptors increase intracellular calcium concentrations in chromaffin cells through two different pathways: Their role in catecholamine secretion

The activation of GABA_B receptors of adrenal chomaffin cells produces an increase of [Ca²⁺]_i measured by fura-2 AM techniques. GABA_B agonists 3-aminopropylphosphinic acid or (-)baclofen, at concentrations of 0.5 mM, increased basal Ca²⁺, values 332 ± 60.9 and 306 ± 40.5 nM, respectively, in cells suspended in a 2.5 mM Ca²⁺ buffer. The GABA_B-induced increase of [Ca²⁺]_i seemed to have two different components. The first was due to an entry from the extracellular medium mainly through L-type voltage-dependent Ca²⁺ channels as the dihydropiridine nifedipine 50 μM was able to decrease it more than 60%, while ω-conotoxin, which blocks N-type channels, did not produce any change in the GABA_B-evoked Ca²⁺ increment. The second component was due to a release of Ca²⁺ from intracellular pools and was about one-third of the total GABAB-induced increase of [Ca²⁺]_i. GABA_B receptors stimulated inositol 1,4,5-trisphosphate-sensitive and not the caffeine-sensitive Ca²⁺ store. In a low Ca²⁺ buffer after treatment with 2 μM angiotensin II, neither 0.5 mM 3-APPA nor baclofen were able to produce an additional increase of [Ca²⁺]_i, whereas 4 mM caffeine had no effect on GABA_B response. This intracellular Ca²⁺ mobilization could be due to inositol 1,4,5-trisphosphate accumulation produced by the activation of GABA_B receptors. In fact, the specific agonists after 10 minutes incubation produced a dosedependent increase of inositol 1,4,5-trisphosphate. The maximal effect was obtained at 100 μM baclofen and 3-APPA, and it was 3.63 ± 0.75 and 3.2 ± 1.5 times the basal levels (7.3 ± 0.3 pmol/10⁶ cells), respectively. In the absence of extracellular Ca²⁺, GABA_B-evoked catecholamine secretion and cyclic AMP formation were reduced more than 70%, suggesting an important role of extracellular Ca²⁺ in GABA_B mechanisms in adrenal chromaffin cells. © 1995 Wiley-Liss, Inc.     

Regulation of Intracellular Ca2+ in Response to Muscarinic and Glutamate Receptor Agonists During the Differentiation of NTERA2 Human Embryonal Carcinoma Cells into Neurons

Single cell microfluorimetry was used to study intracellular calcium ion signals ([Ca²⁺]_i) evoked by acetylcholine (ACh), glutamate receptor agonists and by KCI-induced membrane depolarization, during neuronal differentiation of the human embryonal carcinoma (EC) cell line, NTERA2. In undifferentiated NTERA2 EC cells, [Ca²⁺]_i) was elevated in response to ACh, but not to the glutamate receptor agonists NMDA, kainate or AMPA. The ACh-induced rise in [Ca²⁺]_i) was dependent upon both Ca²⁺ influx and Ca²⁺ mobilization from cytoplasmic calcium stores. Three other human EC cell lines responded similarly to ACh but not to glutamate or KCI-induced depolarization. In neurons derived from NTERA2 cells by retinoic acid induction, [Ca²⁺]_i) signals were evoked by ACh, NMDA, kainate and by an elevation of the extracellular KCI concentration. As in undifferentiated EC cells, the ACh-mediated increases in [Ca²⁺li were governed by both Ca²⁺ influx and Ca²⁺ mobilization. In contrast, the effects of NMDA, kainate and KCI did not involve intracellular Ca²⁺ mobilization. The appearance of glutamate and KCI responsiveness was not detected in non-neuronal differentiated derivatives of NTERA2 cells. Using a number of pharmacologically defined muscarinic receptor antagonists we found that NTERA2 EC cells express M₁, M₃, M₄ and possibly M₅ receptor subtypes linked to changes in [Ca²⁺]_i), whilst only M₃ and M₅ are present in NTERA2-derived neurons. The results were supported by PCR analysis of the muscarinic mRNA species expressed in the cells. The data demonstrate that differentiation of NTERA2 EC cells into neurons involves the induction of functional glutamate receptors coupled to rises in [Ca²⁺]_i), and changes in the expression of muscarinic ACh receptor subtypes.     

Possible involvement of transient receptor potential ankyrin 1 in Ca2+ signaling via T‐type Ca2+ channel in mouse sensory neurons

T‐type Ca²⁺ channels and TRPA1 are expressed in sensory neurons and both are associated with pain transmission, but their functional interaction is unclear. Here we demonstrate that pharmacological evidence of the functional relation between T‐type Ca²⁺ channels and TRPA1 in mouse sensory neurons. Low concentration of KCl at 15 mM (15K) evoked increases of intracellular Ca²⁺ concentration ([Ca²⁺]_i), which were suppressed by selective T‐type Ca²⁺ channel blockers. RT‐PCR showed that mouse sensory neurons expressed all subtypes of T‐type Ca²⁺ channel. The magnitude of 15K‐induced [Ca²⁺]_i increase was significantly larger in neurons sensitive to allylisothiocyanate (AITC, a TRPA1 agonist) than in those insensitive to it, and in TRPA1^?/? mouse sensory neurons. TRPA1 blockers diminished the [Ca²⁺]_i responses to 15K in neurons sensitive to AITC, but failed to inhibit 40 mM KCl‐induced [Ca²⁺]_i increases even in AITC‐sensitive neurons. TRPV1 blockers did not inhibit the 15K‐induced [Ca²⁺]_i increase regardless of the sensitivity to capsaicin. [Ca²⁺]_i responses to TRPA1 agonist were enhanced by co‐application with 15K. These pharmacological data suggest the possibility of functional interaction between T‐type Ca²⁺ channels and TRPA1 in sensory neurons. Since TRPA1 channel is activated by intracellular Ca²⁺, we hypothesize that Ca²⁺ entered via T‐type Ca²⁺ channel activation may further stimulate TRPA1, resulting in an enhancement of nociceptive signaling. Thus, T‐type Ca²⁺ channel may be a potential target for TRPA1‐related pain.     

Developmental Loss of GABA- and Glycine-induced Depolarization and Ca2+ Transients in Embryonic Rat Dorsal Horn Neurons in Culture

More than 90% of dorsal horn neurons from embryonic day 15–16 rats responded to the inhibitory amino acids GABA and glycine by a transient elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) when maintained in culture for <1 week. This [Ca²⁺]_i response has previously been shown to be due to depolarization and subsequent Ca²⁺ entry through voltage-gated Ca²⁺ channels following activation of bicuculline-sensitive GABA_A receptors and strychnine-sensitive glycine receptors. Both the number of cells responding to GABA and glycine and the amplitude of the [Ca²⁺]_i response diminished over time in culture. By 30 days in culture, none of the cells responded to GABA, muscimol or glycine by elevation of [Ca²⁺]_i. The loss of the [Ca²⁺]_i response was not due to a change in the abundance or the properties of voltage-gated Ca²⁺ channels, since over the same period of time dorsal horn neurons showed a large increase in the amplitude of the [Ca²⁺]_i transient in response to 30 mM K⁺. Nor was the loss of the [Ca²⁺]_i response due to a loss of GABA and glycine receptors. Instead, the decrease in the [Ca²⁺]_i response over time paralleled a similar change in the electrophysiological responses. More than 90% of the neurons tested were depolarized in response to inhibitory amino acids during the first week in culture. After 30 days, all neurons tested responded to GABA and glycine with a hyperpolarization. These observations add support to the suggestion that GABA and glycine may excite dorsal horn neurons earlyin development and play a role in postmitotic differentiation.     

AMPA receptor contribution to methylmercury-mediated alteration of intracellular Ca2+ concentration in human induced pluripotent stem cell motor neurons

α motor neurons (MNs) are a target of the environmental neurotoxicant methylmercury (MeHg), accumulating MeHg and subsequently degenerating. In mouse spinal cord MN cultures, MeHg increased intracellular Ca²⁺ [Ca²⁺]_i; the AMPA receptor (AMPAR) antagonist CNQX delayed the increase in [Ca²⁺]_i, implicating the role of AMPARs in this response. Here we used human induced pluripotent stem cell-derived MNs (hiPSC-MNs), to characterize the role of MN AMPARs in MeHg neurotoxicity. Acute exposure to MeHg (0.1, 0.2, 0.5, 1 and 1.5 μM), fura-2 microfluorimetry, and a standard cytotoxicity assay, were used to examine MN regulation of [Ca²⁺]_i, and cytotoxicity, respectively. Contribution of Ca²⁺-permeable and impermeable AMPARs was compared using either CNQX, or the Ca²⁺-permeable AMPAR antagonist N-acetyl spermine (NAS). MeHg-induced cytotoxicity was evaluated following a 24 h delay subsequent to 1 h exposure of hiPSC-MNs. MeHg caused a characteristic biphasic increase in [Ca²⁺]_i, the onset of which was concentration-dependent; higher MeHg concentrations hastened onset of both phases. CNQX significantly delayed MeHg’s effect on onset time of both phases. In contrast, NAS significantly delayed only the 2nd phase increase in fura-2 fluorescence. Exposure to MeHg for 1 h followed by a 24 h recovery period caused a concentration-dependent incidence of cell death. These results demonstrate for the first time that hiPSC-derived MNs are highly sensitive to effects of MeHg on [Ca²⁺]_i, and cytotoxicity, and that both Ca²⁺-permeable and impermeable AMPARs contribute the elevations in [Ca²⁺]_i.