TRPC5 channel is the mediator of neurotrophin-3 in regulating dendritic growth via CaMKIIα in rat hippocampal neurons

Neurotrophin-3 (NT-3) plays numerous important roles in the CNS and the elevation of intracellular Ca(2+) ([Ca(2+)](i)) is critical for these functions of NT-3. However, the mechanism by which NT-3 induces [Ca(2+)](i) elevation remains largely unknown. Here, we found that transient receptor potential canonical (TRPC) 5 protein and TrkC, the NT-3 receptor, exhibited a similar temporal expression in rat hippocampus and cellular colocalization in hippocampal neurons. Stimulation of the neurons by NT-3 induced a nonselective cation conductance and PLCγ-dependent [Ca(2+)](i) elevation, which were both blocked when TRPC5, but not TRPC6 channels, were inhibited. Moreover, the Ca(2+) influx through TRPC5 induced by NT-3 inhibited the neuronal dendritic growth through activation of calmodulin-dependent kinase (CaMK) IIα. In contrast, the Ca(2+) influx through TRPC6 induced by NT-4 promoted the dendritic growth. Thus, TRPC5 acts as a novel and specific mediator for NT-3 to regulate dendrite development through CaMKIIα.     

Interferon-gamma acutely induces calcium influx in human microglia

The acute actions of the cytokine, interferon-gamma (IFN-gamma), on intracellular calcium [Ca(2+)](i) levels in human microglia were investigated. In the presence of a calcium-containing physiological solution (Ca(2+)-PSS), IFN-gamma caused a progressive increase in [Ca(2+)](i) to a plateau level with a mean rate of increase of 0.81 +/- 0.17 nM/s and mean amplitude of 102 +/- 12 nM (n = 67 cells). Washout of the cytokine did not alter the plateau established with IFN-gamma in Ca(2+)-PSS; however, introduction of a Ca(2+)-free PSS diminished [Ca(2+)](i) to baseline levels. The decrease in [Ca(2+)](i) with Ca(2+)-free PSS would indicate that the response to IFN-gamma was mediated by an influx pathway. This result was confirmed in separate experiments showing the lack of an induced change in [Ca(2+)](i) with IFN-gamma applied in Ca(2+)-free PSS. The increase in [Ca(2+)](i) induced in Ca(2+)-PSS was reduced to near baseline levels when the external solution contained low Cl(-) in the maintained presence of IFN-gamma suggesting that cellular depolarization inhibited the cytokine mediated entry pathway. The compound SKF96365, which blocks store operated influx of Ca(2+) in human microglia, was ineffective in altering the increase in [Ca(2+)](i), however, La(3+) completely inhibited the Ca(2+) response induced by IFN-gamma. Whole-cell patch clamp studies showed no effect of IFN-gamma to alter outward currents and inward rectifier K(+) currents. The influx of Ca(2+) may serve a signaling role in microglia linking IFN-gamma to functional responses of the cells to infiltrating T lymphocytes into the central nervous system (CNS) during inflammatory processes.     

Recovery of deficient cholinergic calcium signaling by adenosine in cultured rat cortical astrocytes

The regulation of the cholinergic calcium signaling in astroglial cells is thought to play a crucial role in the pathogenesis of Alzheimer's disease. We investigated the action of the cell modulator adenosine on acetylcholine (Ach)-mediated intracellular calcium ([Ca(2+)](i)) transients in cultured rat cortical astrocytes using the Ca(2+) imaging technique. The stable adenosine analog 2-chloroadenosine (2ClA) potentiated the [Ca(2+)](i) rise induced by activation of muscarinic Ach receptors by shifting approximately 30-fold the half-effective Ach concentration. This 2ClA effect was maintained upon removal of extracellular Ca(2+), indicating that Ach-induced [Ca(2+)](i) elevation was due mainly to Ca(2+) mobilization from intracellular stores. Pharmacological studies demonstrated that the 2ClA action was mediated by A1 receptors. Incubation with pertussis toxin abrogated the 2ClA effect but left unchanged the [Ca(2+)](i) rise produced by Ach alone. The [Ca(2+)](i) response elicited by Ach alone was abolished upon blockade of muscarinic receptor subtypes that stimulate phospholipase C, whereas the [Ca(2+)](i) elevation generated by the combined action of subthreshold Ach and 2ClA was not affected. Collectively, these results suggest that the impaired cholinergic signaling, the cardinal symptom of Alzheimer's disease, can be reinforced at the second messenger level by an alternative intracellular Ca(2+) mobilizing path, which can be brought into play by the concomitant activation of A1 purinoceptors and muscarinic receptors negatively coupled to adenylyl cyclase.     

Functional role of calcium signals for microglial function

In this review we summarize mechanisms of Ca(2+) signaling in microglial cells and the impact of Ca(2+) signaling and Ca(2+) levels on microglial function. So far, Ca(2+) signaling has been only characterized in cultured microglia and thus these data refer rather to activated microglia as observed in pathology when compared with the resting form found under physiological conditions. Purinergic receptors are the most prominently expressed ligand-gated Ca(2+)-permeable channels in microglia and control several microglial functions such as cytokine release in a Ca(2+)-dependent fashion. A large variety of metabotropic receptors are linked to Ca(2+) release from intracellular stores. Depletion of these intracellular stores triggers a capacitative Ca(2+) entry. While microglia are already in an activated state in culture, they can be further activated, for example, by exposure to bacterial endotoxin. This activation leads to a chronic increase of [Ca(2+)](i) and this Ca(2+) increase is a prerequisite for the release of nitric oxide and cytokines. Moreover, several factors (TNFalpha, IL-1beta, and IFN-gamma) regulate resting [Ca(2+)](i) levels.     

Coupling of AMPA receptors with the Na(+)/Ca(2+) exchanger in cultured rat astrocytes

Astrocytes exhibit three transmembrane Ca(2+) influx pathways: voltage-gated Ca(2+) channels (VGCCs), the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na(+)/Ca(2+) exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca(2+) concentration ([Ca(2+)](i)); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca(2+)](i) is not known. We demonstrate here that 100 microM AMPA in the presence of 100 microM cyclothiazide (CTZ) causes an increase in [Ca(2+)](i) in cultured cerebral astrocytes that requires transmembrane Ca(2+) influx. This increase of [Ca(2+)](i) is blocked by 100 microM benzamil or 0.5 microM U-73122, which inhibit reverse-mode operation of the Na(+)/Ca(2+) exchanger by independent mechanisms. This response does not require Ca(2+) influx through VGCCs, nor does it depend upon a significant Ca(2+) influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca(2+) stores, although depletion of these Ca(2+) stores does not decrease the peak [Ca(2+)](i) response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca(2+)](i) to increase through the reverse mode operation of the Na(+)/Ca(2+) exchanger with an associated release of Ca(2+) from intracellular stores. This proposed mechanism requires neither Ca(2+)-permeant AMPARs nor the activation of VGCCs to be effective.     

Hypoxic enhancement of evoked noradrenaline release from the human neuroblastoma SH-SY5Y

The effects of chronic hypoxia (2.5% O(2), 24 h) on [3H]noradrenaline ([3H]NA) release evoked from human neuroblastoma SH-SY5Y cells by depolarisation and by activation of muscarinic receptors was investigated. Depolarization of cells with 100 mM K(+) evoked [3H]NA release, and chronic hypoxia enhanced this release significantly. In fluorimetric studies, the K(+)-evoked rises of [Ca(2+)](i) observed in response to 100 mM K(+) were also significantly enhanced. Muscarine-evoked [3H]NA release was also dramatically enhanced by chronic hypoxia. However, muscarine-induced release of Ca(2+) from intracellular stores and subsequent capacitative Ca(2+) entry was unaffected. The protein kinase C inhibitors GF 109 203X and RO-31-8220 did not prevent the enhancement of muscarine-evoked release caused by chronic hypoxia. These findings indicate that chronic hypoxia increases release of [3H]NA from human neuroblastoma SH-SY5Y cells. Enhancement of K(+)-evoked release was attributable to an enhancement of depolarisation-mediated Ca(2+) influx. In contrast, the larger enhancement of muscarine-evoked [3H]NA release was not due to greater release of Ca(2+) from internal stores, nor due to enhanced Ca(2+) influx. Furthermore, it was not attributable to activation of protein kinase C. These findings suggest that enhancement of sympathetic output, known to occur following prolonged hypoxia, may be mediated in part by enhancement of exocytosis.     

Activation of mouse microglial cells affects P2 receptor signaling

Microglial cells are the immunocompetent cells of the CNS, which are known to exist in several activation states. Here we investigated the impact of microglial activation on the P2 receptor-mediated intracellular calcium ([Ca(2+)](i)) signaling by means of fluo-3 based Ca(2+)-imaging. Cultured mouse microglial cells were treated with either astrocyte-conditioned medium to induce a ramified morphology or LPS to shift the cells toward the fully activated stage. The extracellular application of ATP (100 microM) induced a [Ca(2+)](i) elevation in 85% of both untreated and ramified microglial cells, whereas only 50% of the LPS-activated cells responded to the stimulus. To characterise the pharmacological profile of microglial P2 receptors we investigated the effects of various P2 agonists on [Ca(2+)](i) in cultured microglial cells. Untreated and ramified microglial cells demonstrated a very similar sensitivity to the different P2 agonists. In contrast, in LPS-activated microglia, a sharp decrease of responses to P2 agonist stimulation was seen. This indicates that microglial activation influences the capability of microglial cells to generate [Ca(2+)](i) signals upon P2 receptor activation.     

The chemokine CCL2 modulates Ca2+ dynamics and electrophysiological properties of cultured cerebellar Purkinje neurons

The chemokine CCL2 is produced at high levels in the central nervous system (CNS) during infection, injury, neuroinflammation and other pathological conditions. Cells of the CNS including neurons and glia express receptors for CCL2 and these receptors may contribute to a signaling system through which pathologic conditions in the CNS are communicated. However, our understanding of the consequences of activation of chemokine signaling in the CNS is limited, especially for neurons. In many cell types, chemokine signaling alters intracellular Ca(2+) dynamics. Therefore, we investigated the potential involvement of this mechanism in neuronal signaling activated by CCL2. In addition, we examined the effects of CCL2 on neuronal excitability. The studies focused on the rat cerebellar Purkinje neuron, an identified CNS neuronal type reported to express both CCL2 and its receptor, CCR2. Immunohistochemical studies of Purkinje neurons in situ confirmed that they express CCR2 and CCL2. The effect of exogenous application on Purkinje neurons was studied in a cerebellar culture preparation. CCL2 was tested by micropressure or bath application, at high concentrations (13-100 nm) to simulate conditions during a pathologic state. Results show that Purkinje neurons express receptors for CCL2 and that activation of these receptors alters several neuronal properties. CCL2 increased resting Ca(2+) levels, enhanced the Ca(2+) response evoked by activation of metabotropic glutamate receptor 1 and depressed action potential generation in the cultured Purkinje neurons. Passive membrane properties were unaltered. These modulatory effects of CCL2 on neuronal properties are likely to contribute to the altered CNS function associated with CNS disease and injury.     

Inhibition of depolarisation-evoked [(3)H]noradrenaline release from SH-SYFY human neuroblastoma cells by muscarinic (M1) receptors is not mediated by changes in [Ca(2+)

The aim of this study was to obtain further understanding of the mechanism by which activation of muscarinic M(1) receptors inhibits K(+)-evoked noradrenaline (NA) release in the human neuroblastoma SH-SY5Y. Previous studies have found that muscarinic M(1) and M(3) receptors couple to the activation of phospholipase C in SH-SY5Y cells leading to an increase in (a) intracellular calcium ([Ca(2+)](i)) and (b) activation of protein kinase C (PKC). This study used specific inhibitors of PKC and conditions which deplete Ca(2+)(i) stores to examine the role of protein kinase C and changes in [Ca(2+)](i) in mediating the inhibition of K(+)-evoked NA release by muscarine. Our data show that pretreatment of SH-SY5Y cell layers with bisindolylmaleimide I (BIM-I) (i) failed to reverse inhibition of K(+)-evoked NA release by muscarine but (ii) did overcome the attenuation of muscarine inhibition following pretreatment with TPA. Furthermore pretreating cell layers with Ca(2+)-free Hepes buffered saline in the presence of thapsigargin, conditions which prevented muscarine induced increases in [Ca(2+)](i), failed to prevent inhibition of K(+)-evoked NA release by muscarine. The effect of muscarine on K(+)-evoked uptake of Ca(2+)(e) was examined in SH-SY5Y cells loaded with Fura-2. Muscarine inhibited Ca(2+)(e)-uptake by decreasing the rate at which Ca(2+) entered SH-SY5Y cells via voltage sensitive Ca(2+)-channels. Thus this study shows that muscarine inhibits depolarisation-evoked NA release by a mechanism which is not dependent on activation of PKC or release of Ca(2+) from internal stores.     

Differential activation of subtype purinergic receptors modulates Ca(2+) mobilization and COX-2 in human microglia

We have studied modulation of purinergic receptors (P(2Y) and P(2X) subtypes) on changes in intracellular Ca(2+) [Ca(2+)](i) and expression and production of COX-2 in human microglia. Measurements using Ca(2+)-sensitive spectrofluorometry showed adenosine triphosphate (ATP) to cause rapid transient increases in [Ca(2+)](i). Application of ATP plus the P(2X) antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), or treatment with adenosine diphosphate-beta-S (ADP-beta-S), a selective P(2Y) agonist, led to a considerable prolongation in [Ca(2+)](i) responses compared with ATP. The prolonged time courses were consistent with sustained activation of store-operated channels (SOC) since SKF96365, an inhibitor of SOC, blocked this component of the response. RT-PCR data showed that microglia expressed no COX-2 either constitutively or following treatment of cells with ATP (100 microM for 8 h). However, treatment using ATP plus PPADS or with ADP-beta-S led to marked expression of COX-2. The enhanced COX-2 with ATP plus PPADS treatment was absent in the presence of SKF96365 or using Ca(2+)-free solution. Immunocytochemistry, using a specific anti-COX-2 antibody, also revealed a pattern of purinergic modulation whereby lack of P(2X) activation enhanced the production of COX-2 protein. These results suggest that modulation of subtypes of purinergic receptors regulates COX-2 in human microglia with a link involving SOC-mediated influx of Ca(2+).     

PKC and PKA, but not PKG mediate LPS-induced CGRP release and [Ca(2+)](i) elevation in DRG neurons of neonatal rats.

Calcitonin gene-related peptide (CGRP), is produced in dorsal root ganglia (DRG) neurons and released from primary afferent neurons to mediate hemodynamic effects and neurogenic inflammation. In this work, we determined whether lipopolysaccharide (LPS), an inflammatory stimulator, could trigger CGRP release from cultured DRG neurons and if so, which cellular signaling pathway was involved in this response. Cytoplasmic concentration of calcium ([Ca(2+)](i)) plays a key role in neurotransmitter release, therefore [Ca(2+)](i) was also determined in cultured DRG cells using fluo-3/AM. The results showed that LPS (0.1-10 microg/ml) evoked CGRP release in a time- and concentration-dependent manner from DRG neurons. LPS also increased [Ca(2+)](i) in a concentration-dependent manner. The protein kinase C (PKC) inhibitors, calphostin C 0.5 microM or RO-31-8220 0.1 microM, and the cAMP-dependent protein kinase (PKA) specific inhibitor RP-CAMPS 30 microM or nonspecific inhibitor H8 1 microM inhibited 1 microg/ml LPS-evoked CGRP release and [Ca(2+)](i) increase from DRG neurons. The cGMP-dependent protein kinase (PKG) inhibitor Rp-8-pCPT-cGMPS 30 microM did not block the LPS response. These data suggest that LPS may stimulate CGRP release and [Ca(2+)](i) elevation through PKC and PKA, but not PKG signaling pathway in DRG neurons of neonatal rats.     

Adenosine triphosphate induces proliferation of human neural stem cells: Role of calcium and p70 ribosomal protein S6 kinase

Human neural stem cells (NSCs) grown in culture responded to extracellularly applied adenosine triphosphate (ATP), and the rate of proliferation increased as shown by immunocytochemical and RT-PCR analysis. Activation of P2 purinoceptors by ATP is coupled to the release of intracellular calcium ([Ca(2+)](i)) from thapsigargin-sensitive intracellular stores. ATP-induced proliferation was blocked by thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. Neither EGTA, a calcium chelator, nor caffeine had any effect on ATP-induced [Ca(2+)](i) increases. Multiblot kinase analysis, by which activation of 24 different kinases could be determined, showed that application of ATP to NSCs predominantly activated p70 ribosomal protein S6 kinase (p70 S6 kinase). As well, rapamycin, a p70 S6 kinase inhibitor, blocked the ATP-mediated proliferative response in NSCs. After outlining a role for p70 S6 kinase in ATP-mediated NSC proliferation, we examined the possibility that phosphatidylinositol 3-kinase (PI3-kinase) acts upstream of p70 S6 kinase. The application of wortmannin, a PI3-kinase inhibitor, decreased both ATP-mediated p70 S6 kinase activation and NSC proliferation. From these results, we conclude that ATP application to NSCs induces release of Ca(2+) from intracellular Ca(2+) stores and that this increase in intracellular Ca(2+) in turn promotes NSC proliferation. The increase in NSC proliferation observed following ATP application can also be mediated by PI3-kinase-dependent p70 S6 kinase activation.     

Characterization of neurotransmitters and dopamine attenuation of inducible nitric oxide synthase in glioma cells

Autoreactive T lymphocytes are considered to play a crucial role in orchestrating a chronic inflammation in the central nervous system (CNS) of multiple sclerosis (MS) patients and in the joints of rheumatoid arthritis (RA) patients. However, it has been suggested that the majority of T cells in the immune infiltrate are nonspecifically recruited into the CNS and into the inflamed joint. In addition, several lines of evidence suggest an important role for interferon-gamma (IFN-gamma) in the pathogenesis of MS and RA. We have studied whether peripheral blood T cells from patients with autoimmune diseases are more susceptible to activation in the presence of IFN-gamma. The results indicate that IFN-gamma mediates a sustained elevated [Ca(2+)](i) in T cells of (active) MS and RA patients as compared to healthy controls and patients with common viral infections. No [Ca(2+)](i) increase was observed in Ca(2+)-free medium, excluding an effect of IFN-gamma on Ca(2+)-release from intracellular stores. Although the IFN-gamma-activated Ca(2+)-influx is insufficient to induce T cell proliferation in vitro, our data indicate a significantly augmented proliferation in response to suboptimal doses of PHA in the presence of IFN-gamma. This study suggests that the IFN-gamma-induced Ca(2+)-influx can act as a complementary mechanism in the activation of blood T lymphocytes from MS and RA patients.     

Pharmacological characterization of P2Y receptor subtypes on isolated tiger salamander Müller cells

Müller cells express a variety of neurotransmitter receptors that permit them to "sense" the extracellular environment within the retina. We have used a battery of agonists and antagonists to characterize the purinergic receptor subtypes expressed on isolated tiger salamander Müller cells. Changes in intracellular calcium ion concentration ([Ca(2+)](i)) in Müller cells were measured using the Ca(2+) indicator dye Fura-2 and digital imaging microscopy. ATP, 2-methylthio-ATP, 2-methylthio-ADP, ADP, UTP, UDP, deoxyATP, and 3'-O-(4-benzoyl)benzoyl ATP evoked increases in [Ca(2+)](i) in both the presence and absence of extracellular Ca(2+). Therefore, the increases we observed were likely due to intracellular Ca(2+) release mediated by G-protein-coupled P2Y receptor activation, rather than Ca(2+) influx via P2X receptor channels. The P2Y(1) receptor agonists 2-methylthio-ATP, 2-methylthio-ADP, and ADP evoked increases in [Ca(2+)](i) that were inhibited by the P2Y(1) receptor antagonists adenosine 3'-phosphate 5'-phosphosulfate and 2'-deoxy-N(6)-methyleneadenosine-3',5'-bisphosphate. Responses to ADP were not completely inhibited by the P2Y(1) receptor antagonists. The residual response to ADP could be mediated by P2Y(13) receptors. UTP evoked an increase in [Ca(2+)](i) that was partially inhibited by suramin, suggesting that Müller cells express P2Y(2) and P2Y(4) receptors. The P2Y(6) receptor agonist UDP, and the P2Y(11) receptor agonists deoxyATP, and 3'-O-(4-benzoyl)benzoyl ATP, evoked increases in [Ca(2+)](i) in Müller cells. We conclude that isolated tiger salamander Müller cells express P2Y(1), P2Y(2), P2Y(6), P2Y(11), and possibly P2Y(4) and P2Y(13) receptors. Therefore, the physiological release of ATP, ADP, UTP, and UDP and/or their accumulation in the retina under pathological conditions could stimulate increases in [Ca(2+)](i) in Müller cells.     

Swelling-activated calcium signalling in cultured mouse primary sensory neurons

The effects of hypo-osmotic membrane stretch on intracellular calcium concentration ([Ca(2+)](i)), cell volume and cellular excitability were investigated in cultured mouse primary sensory trigeminal neurons. Hypotonic solutions (15--45%) led to rapid cell swelling in all neurons. Swelling was accompanied by dose-dependent elevations in [Ca(2+)](i) in a large fraction of neurons. Responses could be classified into three categories. (i) In 57% of the neurons [Ca(2+)](i) responses had a slow rise time and were generally of small amplitude. (ii) In 21% of the neurons, responses had a faster rise and were larger in amplitude. (iii) The remaining cells (22%) did not show [Ca(2+)](i) responses to hypo-osmotic stretch. Slow and fast [Ca(2+)](i) changes were observed in trigeminal neurons of different sizes with variable responses to capsaicin (0.5 microM). The swelling-induced [Ca(2+)](i) responses were not abolished after depletion of intracellular Ca2+ stores with cyclopiazonic acid or preincubation in thapsigargin, but were suppressed in the absence of external Ca(2+). They were strongly attenuated by extracellular nickel and gadolinium. Hypotonic stimulation led to a decrease in input resistance and to membrane potential depolarization. Under voltage-clamp, the [Ca(2+)](i) elevation produced by hypotonic stimulation was accompanied by the development of an inward current and a conductance increase. The time course and amplitude of the [Ca(2+)](i) response to hypo-osmotic stimulation showed a close correlation with electrophysiological properties of the neurons. Fast [Ca(2+)](i) responses were characteristic of trigeminal neurons with short duration action potentials and marked inward rectification. These findings suggest that hypo-osmotic stimulation activates several Ca(2+)-influx pathways, including Gd(3+)-sensitive stretch-activated ion channels, in a large fraction of trigeminal ganglion neurons. Opening of voltage-gated Ca(2+) channels also contributes to the response. The pattern and rate of Ca(2+) influx may be correlated with functional subtypes of sensory neurons.     

Growth factors upregulate astrocyte [Ca2+]i oscillation by increasing SERCA2b expression

Previously, we reported upregulation of astrocyte [Ca(2+)](i) oscillation by growth factors (i.e., conversion of glutamate-induced sustained [Ca(2+)](i) increase in astrocytes cultured in a defined medium to [Ca(2+)](i) oscillation by EGF and bFGF treatment over 48 h) (Morita et al., (2003) J Neurosci 23:10944-10952). As our previous study also showed that these growth factors increase intracellular Ca(2+) stores, this study was performed to investigate the mechanism underlying loading of intracellular Ca(2+) stores in astrocytes, especially sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA), as a candidate mechanism by which growth factors upregulate [Ca(2+)](i) oscillation. The results indicated that the growth factors upregulated a SERCA inhibitor-sensitive component of [Ca(2+)](i) clearance, and increased expression of the SERCA subtype, SERCA2b. Furthermore, treating the growth factor-treated astrocytes with a low concentration of SERCA inhibitor to partially inhibit SERCA reduced the level of intracellular Ca(2+) storage and reversed glutamate-induced [Ca(2+)](i) oscillations to sustained [Ca(2+)](i) increases. Thus, the upregulation of [Ca(2+)](i) oscillations was attributed to the upregulation of SERCA activities. These results indicated that these growth factors regulate the pattern of glutamate-induced astrocyte [Ca(2+)](i) increases via SERCA2b expression.     

Astrocytes refill intracellular Ca2+ stores in the absence of cytoplasmic [Ca2+] elevation: a functional rather than a structural ability

Capacitative Ca(2+) entry (CCE) is a phenomenon triggered by depletion of Ca(2+) content in intracellular stores (ICS). Data about this phenomenon in astrocytes are limited. We analyzed CCE in astrocytes by means of fura-2 based digital imaging. We found that in astrocytes CCE is not associated with an increase of cytosolic Ca(2+) concentration ([Ca(2+)](i)), although ICS are efficiently refilled. We used Mn(2+), thapsigargin and prolonged ATP exposure to show that CCE is not associated with cytosolic diffusion of Ca(2+) entering astrocytes. Our data suggest that the ion is being quickly sequestered in the ICS by the smooth endoplasmic reticulum Ca(2+)-ATP-ase (SERCA). Several experiments were carried out with the goal of failing the efficient uptake in the endoplasmic reticulum (ER). In fact, inhibition of SERCA activity, increased extracellular [Ca(2+)](i) or pharmacologic potentiation of CCE all caused [Ca(2+)](i) elevation during CCE, suggesting that the control of this phenomenon could have physiologic and pathological relevance. The molecular components involved in CCE have been proposed to be organized in a multi-molecular complex tethered by cytoskeleton components and arranged via a secretion coupling model. We show here that the efficient routing of Ca(2+) into the ICS in astrocytes is not affected by disruption of cytoskeleton organization or Golgi's function, but it is instead linked to the high efficiency of SERCA. We conclude that depleted ICS in astrocytes are efficiently refilled by CCE activation, although Ca(2+) influx is not accompanied by elevation of [Ca(2+)](i). This ability seems to be functional rather than structural in nature.     

Ca2+ mobilization mediated by transient receptor potential canonical 3 is associated with thrombin-induced morphological changes in 1321N1 human astrocytoma cells

Activated astrocytes show various patterns of Ca(2+) mobilization under pathological conditions. In the present study we revealed a novel function of astrocytic Ca(2+) dynamics through investigation of thrombin-induced unique Ca(2+) entry. Using 1321N1 human astrocytoma cells, which have been shown to be a good model for detecting morphological dynamics, we observed rapid retraction of bipolar protrusions that were reversibly evoked by 0.03-3 U/mL thrombin. Morphological changes were predominantly dependent on a specific thrombin receptor subtype, proteinase-activated receptor 1 (PAR-1). In parallel, Fura-2 imaging of intracellular Ca(2+) concentration ([Ca(2+)](i)) showed that thrombin induced heterogeneous Ca(2+) responses with asynchronous repetitive peaks. These oscillations were found to be a result of repetitive Ca(2+) release from intracellular stores, followed by refilling of Ca(2+) from the extracellular region without a direct [Ca(2+)](i) increase. Pharmacological manipulation with BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate indicated that Ca(2+) mobilization was involved in thrombin-induced morphological changes. We further addressed the role of Ca(2+) entry using small interfering RNA (siRNA) for transient receptor potential canonical 3 (TRPC3). As a result, both thrombin-induced morphological changes and oscillatory Ca(2+) responses were significantly attenuated in siRNA-transfected cells. Inhibition of TRPC3 with pyrazole-3 also provided support for the contribution of Ca(2+) influx. Moreover, TRPC3-mediated Ca(2+) dynamics regulated thrombin-induced phosphorylation of myosin light chain 2. These results suggest a novel function of astrocytic Ca(2+) dynamics, including Ca(2+) entry, in the pathophysiological effects of PAR-1-mediated astrocytic activation. TRPC3 forms a functional Ca(2+) channel and might modulate astrocytic activation in response to brain hemorrhaging.     

Different stimulatory opioid effects on intracellular Ca(2+) in SH-SY5Y cells

Present study revealed the stimulatory effects of delta opioid receptor on intracellular Ca(2+) concentration ([Ca(2+)](i)) in SH-SY5Y cells. Fura-2 based single cell fluorescence ratio (F345/F380) was used to monitor the fluctuation of [Ca(2+)](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 CdCl(2) or 0-Ca(2+) bath solutions. DPDPE-mediated [Ca(2+)](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 [Ca(2+)](i) increase in 50% of the cultures. The probability and the amplitude of the elevation were decreased considerably by application of nifedipine or 0-Ca(2+) bath solution and was little affected by application of TG. DPDPE activated [Ca(2+)](i) increase via a PTX-insensitive and CTX-sensitive pathway suggesting coupling through G(s) subunit. All these indicated the opioid modulated the intracellular Ca(2+) 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.