Calcium mobilization and protease-activated receptor cleavage after thrombin stimulation in motor neurons

Thrombin, the ultimate enzyme in the blood coagulation cascade, has prominent actions on various cells, including neurons. As in platelets, thrombin increases [Ca²⁺ _i mobilization in neurons, and also retracts neurites. Both these effects are mediated through a G protein-coupled, proteolytically activated receptor for thrombin (PAR-1). Prolonged exposure to thrombin kills neurons via apoptosis, that may also involve PAR-1 activation. Increased [Ca²⁺]ⁱ has been a unifying mechanism proposed for cell death in several neurodegenerative diseases. Thrombin-elevated calcium levels may activate intracellular cascades in neurons leading to cell death. Since thrombin mediates its diverse effects on cells through both heterotrimeric and monomeric G proteins, we also explored what effect altering differential G protein coupling would have on the neuronal response to thrombin. We studied calcium mobilization by thrombin in a model motor neuronal cell line, NSC19, using fluorescence image analysis. Confirming effects in other neuronal types, thrombin caused dramatic increases in [Ca²⁺]_i levels, both transiently and after prolonged exposure, which involved activation and cleavage of the PAR-1 receptor. Using enzyme linked immunosorbent assay (ELISA) and dot-blot analysis, we found that the N-terminal fragment of PAR-1 was released into the medium after exposure to thrombin. We confirmed that PAR-1 protein and mRNA expression occurred in motor neurons. We found that cholera toxin inhibited thrombin-mediated Ca²⁺ influx, pertussis toxin did not significantly alter thrombin action, and lovastatin, a small 21-kDa Ras GTPase (Rho) modulator, showed a tendency to reduce the thrombin effect. These data indicate that thrombin-increased [Ca²⁺]_i, sufficient to trigger cell death in motor neurons, might be approached in vivo by modulating thrombin signaling through PAR-1.     

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.     

Activation of alpha-2 adrenergic receptors stimulates GABA release by astrocytes

Norepinephrine is one of the key neurotransmitters in the hippocampus, but its role in the functioning of the neuroglial networks remains unclear. Here we show that norepinephrine suppresses NH₄Cl-induced oscillations of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in hippocampal neurons. We found that the inhibitory effect of norepinephrine against ammonium-induced [Ca²⁺]_i oscillations is mediated by activation of alpha-2 adrenergic receptors. Furthermore, UK 14,304, an agonist of alpha-2 adrenergic receptors, evokes a biphasic [Ca²⁺]_i elevation in a minor population of astrocytes. This elevation consists of an initial fast, peak-shaped [Ca²⁺]_i rise, mediated by G_iβγ subunit and subsequent PLC-induced mobilization of Ca²⁺ from internal stores, and a plateau phase, mediated by a Ca²⁺ influx from the extracellular medium through store-operated and TRPC3 channels. We show the correlation between the Ca²⁺ response in astrocytes and suppression of [Ca²⁺]_i oscillations in neurons. The inhibitory effect of UK 14,304 is abolished in the presence of gallein, an inhibitor of G_βγ-signaling. In turn, application of the agonist in the presence of the PLC inhibitor decreases the frequency and amplitude of [Ca²⁺]_i oscillations in neurons but does not suppress them. The same effect is observed in the presence of bicuculline, a GABA(A) receptor antagonist. We demonstrate that UK 14,304 application increases the frequency and amplitude of slow outward chloride currents in neurons, indicating the release of GABA by astrocytes. Thus, our findings indicate that the activation of astrocytic alpha-2 adrenergic receptors stimulates GABA release from astrocytes via G_iβγ subunit-associated signaling pathway, contributing to the suppression of neuronal activity.     

The effect of sevoflurane on intracellular calcium concentration from cholinergic cells

The mechanism of action of volatile anesthetics is not completely understood. Calcium release from internal stores may alter signaling pathways that influence neurotransmission. Abnormalities of the regulation of intracellular calcium concentration ([Ca²⁺]_i) from patients with malignant hyperthermia is a hallmark of this syndrome indicating the potential of these agents to interact with proteins involved in Ca²⁺ signaling. In the present study, a cholinergic cell line (SN56) was used to examine whether the release of calcium from intracellular stores occurs in the presence of sevoflurane. Changes in [Ca²⁺]_i were measured using fluo-4, a fluorescent calcium sensitive dye and laser scanning confocal microscopy. Sevoflurane induced an increase on [Ca²⁺]_i from SN56 cells. The sevoflurane-induced increase on [Ca²⁺]_i remained even when the cells were perfused with medium lacking extracellular calcium. However, this effect was abolished by BAPTA-AM, a chelator of intracellular calcium, suggesting the involvement of intracellular Ca²⁺ stores. Using cyclopiazonic acid, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase, we investigated whether the depletion of intracellular Ca²⁺ stores interfered with the effect of sevoflurane. In the presence of this agent, sevoflurane caused a small but not significant rise on [Ca²⁺]_i of the SN56 cells. Dantrolene, an inhibitor of ryanodine-sensitive calcium stores did not modify the sevoflurane increase on [Ca²⁺]_i. Carbachol, a drug that releases Ca²⁺ from the IP₃ pool, abolished the effect of sevoflurane. In addition, xestospongin D, a cell-permeant IP₃ receptor antagonist, decreased significantly the sevoflurane increase on [Ca²⁺]_i. Our data suggest that the sevoflurane-induced increase on [Ca²⁺]_i from SN56 cells occurs through the release of calcium from IP₃-sensitive calcium stores.     

Halothane-induced intracellular calcium release in cholinergic cells

Low concentrations of halothane and isoflurane can release acetylcholine in an extracellular Ca²⁺-independent manner. In the present study, a cholinergic cell line (SN56) was used to examine whether release of calcium from intracellular stores occurs in the presence of halothane. Changes in intracellular calcium concentration ([Ca²⁺]_i) were measured using fluo-3, a fluorescent calcium-sensitive dye and laser scanning confocal microscopy. Halothane, at sub-anesthetic concentrations (14, 28, 40 and 56 μM), increased [Ca²⁺]_i in SN56 cells. This effect remained even when the cells were perfused with medium lacking extracellular calcium, suggesting the involvement of intracellular Ca²⁺ sources. SN56 cells responded to ryanodine by increasing [Ca²⁺]_i and this effect was blocked by dantrolene, an inhibitor of Ca²⁺-release from ryanodine-sensitive stores. The effect of halothane was attenuated after the increase in [Ca²⁺]_i induced by ryanodine and it was suppressed by dantrolene, suggesting the participation of ryanodine-sensitive stores. Using cyclopiazonic acid, a Ca²⁺-ATPase inhibitor, we investigated whether the depletion of intracellular Ca²⁺ stores interfered with the effect of halothane. Cyclopiazonic acid significantly decreased the increase in [Ca²⁺]_i induced by the volatile anesthetic. It is suggested that sub-anesthetic concentrations of halothane may increase [Ca²⁺]_i by releasing Ca²⁺ from intracellular stores in cholinergic cells.     

Depolarization triggers intracellular magnesium surge in cultured dorsal root ganglion neurons

Intracellular magnesium concentration ([Mg²⁺]_i) of cultured dorsal root ganglion (DRG) neurons was measured using the magnesium indicator Mag-Fura-2/AM. [Mg²⁺]_i was 0.48±0.08 mM (mean±SEM, n=23) at rest, and it increased 3-fold by depolarization with a 60-mM K⁺ solution. The [Mg²⁺]_i increase was observed in the absence of extracellular Mg²⁺, but the increase disappeared in the absence of extracellular Ca²⁺. 50 μM cadmium or 100 μM verapamil, a Ca²⁺ channel blocker, also diminished the rise of [Mg²⁺]_i. The additional measurement of an intracellular Ca²⁺ concentration ([Ca²⁺]_i) indicated that the [Mg²⁺]_i rise requires a threshold concentration of [Ca²⁺]_i to be reached; above 60 nM. The present results indicate that depolarization induces a Ca²⁺-influx through voltage dependent Ca channels and this causes the release of Mg²⁺ from intracellular stores into the cytoplasm.     

Secondary Ca overload indicates early neuronal injury which precedes staining with viability indicators

Spinal neurons, lethally challenged with excitatory amino acids (EAAs) or with high-K⁺, underwent a biphasic rise in free intracellular calcium concentration ([Ca²⁺]_i). In contrast to the initial rise in [Ca²⁺]_i which recovered, the secondary, irreversible [Ca²⁺]_i increase was unaffected by antagonists of EAA receptors of Ca²⁺ channels. Also, it correlated highly with cell death, but preceded vital staining with trypan blue and ethidium homodimer, reflecting damaged cellular Ca²⁺ regulation rather than plasma membrane leakiness. Our findings suggest that delayed Ca²⁺ overload is the end-product rather than the cause of Ca²⁺-triggered neurotoxic processes.     

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.     

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.     

Characteristics of thrombin-induced calcium signals in rat astrocytes

The protease thrombin seems to play a central role in events following neural injury, whereby the enzyme can act, in concert with other molecules as a hormone or as a growth factor. In cells derived from the nervous system, thrombin induces changes in morphology and proliferation. The signalling mechanisms involved in these thrombin-activated processes are still unclear. In the present study we investigated Ca²⁺ signals in fura-2 loaded rat astrocytes in primary culture. Brief stimulation of astrocytes with thrombin induced a dose-dependent transient elevation of [Ca²⁺]_i, best fitted by a double-sigmoidal curve giving two EC₅₀ values of 3 pM and 150 pM. Continuous superfusion of cells with thrombin induced Ca²⁺ responses with three different types of kinetics. In 48% of the cells tested a single transient rise superimposed with fast fluctuations of [Ca²⁺]_i was seen. The following complex long-term changes of [Ca²⁺]_i, dependent on the presence of the agonist thrombin, were observed: i) a biphasic [Ca²⁺]_i elevation, characterized by an initial peak followed by a sustained plateau phase (in 43% of the cells) and ii) oscillations of [Ca²⁺]_i (in 9% of the cells). The observed Ca²⁺ responses were inhibited by the phospholipase C (PLC) inhibitor U-73122 and the thrombin inhibitor protease nexin-1/glia-derived nexin. The synthetic thrombin receptor activating peptide could mimic the thrombin-induced changes of [Ca²⁺]_i. In astrocytes in Ca²⁺-free medium, thrombin induced a sharp single transient Ca²⁺ rise, without superimposed fluctuations. After depletion of intracellular Ca²⁺ stores with thapsigargin the Ca²⁺ response to thrombin was diminished or completely suppressed indicating that thrombin induces the release of Ca²⁺ from intracellular stores. During long-term Ca²⁺ responses, omission of extracellular Ca²⁺ resulted in a reversible interruption of the signal. In conclusion our results demonstrate that thrombin by activation of its plasma membrane receptor induces through activation of PLC different types of Ca²⁺ responses. The complex Ca²⁺ signals are generated by an interplay of InsP₃-mediated Ca²⁺ release from intracellular stores and Ca²⁺ entry across the plasma membrane. GLIA 21:361–369, 1997. © 1997 Wiley-Liss, Inc.     

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.     

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.     

Calcium oscillations in a subpopulation of S-antigen-immunoreactive pinealocytes of the rainbow trout (Oncorhynchus mykiss)

By means of the fura-2 technique and image analysis the intracellular concentration of free calcium ions [Ca²⁺]_i was examined in isolated rainbow trout pinealocytes identified by S-antigen immunocytochemistry. Approximately 30% of the pinealocytes exhibited spontaneous [Ca²⁺]_i oscillations whose frequency differed from cell to cell. Neither illumination with bright light nor dark adaptation of the cells had an apparent effect on the oscillations. Removal of extracellular Ca²⁺ or application of 10 μM nifedipine caused a reversible breakdown of the [Ca²⁺]_i oscillations. Application of 60 mM KCl elevated [Ca²⁺]_i in 90% of the oscillating and 50% of the non-oscillating pinealocytes. The effect of KCl was blocked by 50 μM nifedipine. These results suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca²⁺]_i in trout pinealocytes. Experiments with thapsigargin (2 μM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role for regulation of [Ca²⁺]_i remains elusive. Treatment with norepinephrine (100 pM–50 μM), previously shown to induce calcium release from intracellular calcium stores in rat pinealocytes, had no apparent effect on [Ca²⁺]_i in any trout pinealocyte. This finding conforms to the concept that noradrenergic mechanisms are not involved in signal transduction in the directly light-sensitive pineal organ of anamniotic vertebrates.     

In vitro ischemia-induced intracellular Ca2+ elevation in cerebellar slices: a comparative study with the values found in hippocampal slices

Changes in levels of intracellular calcium ion ([Ca²⁺]_i) induced by in vitro ischemic conditions in gerbil cerebellar and hippocampal slices were investigated using a calcium imaging system and electron microscopy. When the cerebellar slice was perfused with a glucose-free physiological medium equilibrated with a 95% N₂/5% CO₂ gas mixture (in vitro ischemic medium), a large [Ca²⁺]_i elevation was region-specifically induced in the molecular laver of the cerebellar cortex (a dendritic field of Purkinje cells). When the hippocampal slice was perfused with in vitro ischemic medium, a large [Ca²⁺]_i elevation was region-specifically induced in CA1 field of the hippocampal slices. Electron microscopic examinations showed that the large [Ca²⁺]_i elevations occurred in Purkinje cells and CA1 pyramidal neurons. To isolate Ca²⁺ release from intracellular Ca²⁺ store sites, the slices were perfused with Ca²⁺-free in vitro ischemic medium. the increases in [Ca²⁺]_i in both cerebellar and hippocampal slices were significantly lower than those observed in the slices perfused with the Ca²⁺-containing in vitro ischemic medium. However, the suppression of the [Ca²⁺]_i-elevation in the molecular layer of the cerebellar slices was smaller than that in the CA1 field of the hippocampal slices. These results reinforce the hypothesis that calcium plays a pivotal role in the development of ischemia-induced neuronal death, and suggest that Ca²⁺ release from intracellular Ca²⁺ store sites may play an important role in the ischemia-induced [Ca²⁺]_i elevation in Purkinje cells.     

U73122 inhibits phospholipase C-dependent calcium mobilization in neuronal cells

The aminosteroid U73122 inhibited phospholipase C (PLC)-mediated intracellular Ca²⁺ release in differentiated and undifferentiated NG108-15 cells, as well as rat dorsal root ganglion (DRG) neurons grown in primary culture. 1 μM U73122 blocked bradykinin (BK)-induced increases in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) measured in single cells with indo-1-based dual emission microfluorimetry. A close structural analog, U73343, was without effect. The effects of U73122 were time and concentration-dependent. 1 μM drug produced half maximal inhibition in approximately 3 min. The IC₅₀ for a 20-min exposure was approximately 200 nM. The effects of the compound were irreversible for the duration of experiments as long as 1 h. Treatment with 1 μM U73122, but not U73343 produced a small but significant increase in [Ca²⁺]_i which resulted from Ca²⁺ release from an intracellular store. It is not clear whether this [Ca²⁺]_i increase resulted from inhibition of PLC or an action on the store directly. In differentiated NG108-15 cells U73122 blocked completely depolarization-induced Ca²⁺ influx. In contrast, in DRG neurons U73122 inhibited only slightly voltage-sensitive Ca²⁺ channels. Thus, we caution that U73122 may not be selective at concentrations required for maximal block of PLC and that the selectivity of U73122 is dependent on cell type. Overall, our results are consistent with U73122 inhibiting PLC in neuronal cells and indicate that under the appropriate conditions, this compound is a useful tool for studying inositol 1,4,5-trisphosphate (IP₃)-mediated Ca²⁺ mobilization.     

Different stimulatory opioid effects on intracellular Ca in SH-SY5Y cells

Present study revealed the stimulatory effects of δ opioid receptor on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in SH-SY5Y cells. Fura-2 based single cell fluorescence ratio (F345/F380) was used to monitor the fluctuation of [Ca²⁺]_i. Application of the selective delta-opioid receptor agonist alone, [D-Pen^2,5]-enkephalin (DPDPE), hardly had any effects on cells cultivated for 3–10 days. However, after the cells had been pre-stimulated with cholinoceptor agonist, carbachol, variable calcium elevation was found in 59% of the cultures. The response was naltridole-reversible and dose-dependent, and was abolished completely by thapsigargin (TG) treatment but not by administration of CdCl₂ or 0-Ca²⁺ bath solutions. DPDPE-mediated [Ca²⁺]_i elevation was abolished by pertussis toxin (PTX) pretreatment but not cholera toxin (CTX), indicating coupling via G proteins of G_i/G_o subfamily. In 17.5% of the responding cells, biphase response was found which may be due to both the stimulatory and the inhibitory effects of opioid. On the other hand, in acutely dissociated cells, DPPDE alone induced [Ca²⁺]_i increase in 50% of the cultures. The probability and the amplitude of the elevation were decreased considerably by application of nifedipine or 0-Ca²⁺ bath solution and was little affected by application of TG. DPDPE activated [Ca²⁺]_i increase via a PTX-insensitive and CTX-sensitive pathway suggesting coupling through G_s subunit. All these indicated the opioid modulated the intracellular Ca²⁺ regulation system through different pathways. SH-SY5Y cell line might be a suitable model for the investigation of the complex mechanism which underlies opioid function.     

Coupling of AMPA receptors with the Na/Ca exchanger in cultured rat astrocytes

Astrocytes exhibit three transmembrane Ca²⁺ influx pathways: voltage-gated Ca²⁺ channels (VGCCs), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na⁺/Ca²⁺ exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca²⁺ concentration ([Ca²⁺]_i); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca²⁺]_i is not known. We demonstrate here that 100 μM AMPA in the presence of 100 μM cyclothiazide (CTZ) causes an increase in [Ca²⁺]_i in cultured cerebral astrocytes that requires transmembrane Ca²⁺ influx. This increase of [Ca²⁺]_i is blocked by 100 μM benzamil or 0.5 μM U-73122, which inhibit reverse-mode operation of the Na⁺/Ca²⁺ exchanger by independent mechanisms. This response does not require Ca²⁺ influx through VGCCs, nor does it depend upon a significant Ca²⁺ influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca²⁺ stores, although depletion of these Ca²⁺ stores does not decrease the peak [Ca²⁺]_i response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca²⁺]_i to increase through the reverse mode operation of the Na⁺/Ca²⁺ exchanger with an associated release of Ca²⁺ from intracellular stores. This proposed mechanism requires neither Ca²⁺-permeant AMPARs nor the activation of VGCCs to be effective.     

Effect of dantrolene on KCl- or NMDA-induced intracellular Ca2+ changes and spontaneous Ca2+ oscillation in cultured rat frontal cortical neurons

Summary Dantrolene has been known to affect intracellular Ca²⁺ concentration ([Ca²⁺]_i) by inhibiting Ca²⁺ release from intracellular stores in cultured neurons. We were interested in examining this property of dantrolene in influencing the [Ca²⁺]_i affected by the NMDA receptor ligands, KCl, L-type Ca²⁺ channel blocker nifedipine, and two other intracellular Ca²⁺-mobilizing agents caffeine and bradykinin. Effect of dantrolene on the spontaneous oscillation of [Ca²⁺]_i was also examined. Dantrolene in M concentrations dose-dependently inhibited the increase in [Ca²⁺]_i elicited by NMDA and KCl. AP-5, MK-801 (NMDA antagonists), and nifedipine respectively reduced the NMDA and KCl-induced increase in [Ca²⁺]_i. Dantrolene, added to the buffer solution together with the antagonists or nifedipine, caused a further reduction in [Ca²⁺]_i to a degree similar to that seen with dantrolene alone inhibiting the increase in [Ca₂₊]_i caused by NMDA or KCl. At 30 M, dantrolene partially inhibited caffeine-induced increase in [Ca²⁺]_i whereas it has no effect on the bradykinin-induced change in [Ca²⁺]_i. The spontaneous oscillation of [Ca²⁺]_i in frontal cortical neurons was reduced both in amplitude and in base line concentration in the presence of 10 M dantrolene. Our results indicate that dantrolene's mobilizing effects on intracellular Ca²⁺ stores operate independently from the influxed Ca²⁺ and that a component of the apparent increase in [Ca²⁺]_i elicited by NMDA or KCl represents a dantrolene-sensitive Ca₂₊ release from intracellular stores. Results also suggest that dantrolene does not affect the IP₃-gated release of intracellular Ca²⁺ and that the spontaneous Ca²⁺ oscillation is, at least partially, under the control of Ca²⁺ mobilization from internal stores.Abbreviations AP-5 (±)-2-amino-5-phosphonopentanoic acid - AMPA amino-3-hydroxy-5-methyl-isoxazole-4-propionate - BSS balanced salt solution - CNS central nervous system - CICR Ca²⁺-induced Ca²⁺ release - DCKA 5,7-dichlorokynurenate - DNasel deoxyribonuclease I - DMEM Dulbecco's Modified Eagle's Medium - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N,N,-tetraacetic acid - FCS fetal calf serum - fura-2-AM 1-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy-2-ethane-N,N,N,N-te-traacetic acid, pentaacetoxymethyl ester - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - [Ca ²⁺] _i intracellular free Ca²⁺ concentration - LTP long-term potantiation - MK-801 (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate     

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.