Histamine induces intracellular Ca2+ oscillations and nitric oxide release in endothelial cells from brain microvascular circulation

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H₁Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca ²⁺ signals drive histamine-induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca ²⁺ signaling in histamine-induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca ²⁺- and NO-sensitive dyes, Fura-2/AM and DAF-FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca ²⁺ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 μM. The oscillatory Ca ²⁺ response was suppressed by the inhibition of H ₁Rs with pyrilamine, whereas H ₁R was abundantly expressed at the protein level. We further found that histamine-induced intracellular Ca ²⁺ oscillations were initiated by endogenous Ca ²⁺ mobilization through inositol-1,4,5-trisphosphate- and nicotinic acid dinucleotide phosphate-sensitive channels and maintained over time by store-operated Ca ²⁺ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca ²⁺ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca ²⁺ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca ²⁺ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow.     

Glutamate triggers elevation of intracellular Ca2+ concentration in neural precursor cells

Both neurons and glial cells are derived from neuralprecursor cells in the ventricular zone during braindevelopment. The fate of the neural precursor cells isaffected by neurotransmitters such as glutamate. Inthis study, we examined glutamate-triggeredintracellular Ca²⁺ signaling in neural precursorcell lines by the calcium digital imaging method. Whenimmortalized primary-cultured neural precursor cellswere treated with glutamate, a subpopulation of thesecells showed an increase in intracellular Ca²⁺concentration. In an effort to determine the role ofthe glutamate-triggered intracellular Ca²⁺ signalin neural precursor cells, we tried to cultureimmortalized basal ganglial and hippocampal neuralprecursor cell lines in glutamate-free medium. Thehippocampal (MHP-2) cells became adapted to theglutamate-free medium, and when treated with glutamatethe adapted subline (MHP-2-E1) showed an increase inintracellular Ca²⁺ concentration. In contrast,the basal ganglial neural precursor cell lines failedto become adapted to the glutamate-free medium. Theseresults suggest that hippocampal and basal ganglialneural precursor cells differ in their cellularresponse to glutamate as an exogenous stimulus.     

Na+ entry via glutamate transporter activates the reverse Na+/Ca2+ exchange and triggers Ca(i)2+-induced Ca2+ release in rat cerebellar Type-1 astrocytes

We have previously demonstrated that rat cerebellar Type-1 astrocytes express a very active genistein sensitive Na(+)/Ca(2+) exchanger, which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of loads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements of Cai(2+) with Fluo-3 demonstrate that the Cai(2+) signals associated long (> 20 s) periods of reverse operation of the Na(+)/Ca(2+) exchange are amplified by a mechanism compatible with calcium-calcium release, while those associated with short (< 20 s) pulses are not amplified. This was confirmed by pharmacological experiments using ryanodine receptors agonist (4-chloro-m-cresol) and the endoplasmic reticulum ATPase inhibitor (thapsigargin). Confocal microscopy demonstrates a high co-localization of immunofluorescent labeled Na(+)/Ca(2+) exchanger and RyRs. Low (< 50 micromol/L) or high (> 500 micromol/L) concentrations of L-glutamate (L-Glu) or L-aspartate causes a rise in which is completely blocked by the Na(+)/Ca(2+) exchange inhibitors KB-R7943 and SEA0400. The most important novel finding presented in this work is that L-Glu activates the reverse mode of the Na(+)/Ca(2+) exchange by inducing Na(+) entry through the electrogenic Na(+)-Glu-co-transporter and not through the ionophoric L-Glu receptors, as confirmed by pharmacological experiments with specific blockers of the ionophoric L-Glu receptors and the electrogenic Glu transporter.     

Roles of Ca2+ and the Ca2+-sensing receptor (CASR) in the expression of inducible NOS (nitric oxide synthase)-2 and its BH4 (tetrahydrobiopterin)-dependent activation in cytokine-stimulated adult human astrocytes

Since NO production by NOS-2 made by astrocytes activated by proinflammatory cytokines contributes to the killing of neurons in variously damaged human brains, knowing the mechanisms responsible for NOS-2 expression should contribute to developing effective therapeutics. The expression and activation of NOS-2 in normal adult human cerebral cortical astrocytes treated with three proinflammatory cytokines, IL-1beta, TNF-alpha, and IFN-gamma, are driven by two separable mechanisms. NOS-2 expression requires a burst of p38 MAPK activity, while the activation of the resulting enzyme protein requires MEK/ERK-dependent BH4 (tetrahydrobiopterin) synthesis between 24 and 24.5 h after adding the cytokines to the culture medium. Here we show that NOS-2 expression in the activated astrocytes requires that the culture medium contain 1.8 mM Ca2+, but it is unaffected by inhibiting calcium-sensing receptors (CASRs) with NPS 89636. However, NOS-2 activation is inhibited by NPS 89626 during the MEK/ERK-dependent stage between 24 and 24.5 h after adding the cytokines, and this inhibition can be overridden by exogenous BH4. Therefore, NOS-2 expression and the subsequent BH4-dependent NOS-2-activation in human astrocytes need 1.8 mM Ca2+ to be in the culture medium, while NOS-2 activation also needs functional CASRs between 24 and 24.5 h after cytokine addition. These findings raise the possibility that calcilytic drugs prevent NO-induced damage and death of human neurons.     

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca²⁺ in brain microsomes after Ca²⁺ loading by the Ca²⁺-ATPase or by the Na⁺/Ca²⁺ exchanger. The results show that in microsomes loaded with Ca²⁺ by the Ca²⁺-ATPase, Ins(1,4,5)P₃ (5 μM) release 21±2% of the total Ca²⁺ accumulated, and that in the microsomes loaded with Ca²⁺ by the Na²⁺/Ca²⁺ exchanger, Ins(1,4,5)P₃ released 28±3% of the total Ca²⁺ accumulated. These results suggest that receptors of Ins(1,4,5)P₃ may be co-localized with the Na²⁺/Ca²⁺ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P₃ receptors in the plasma membrane where the Na²⁺/Ca²⁺ exchanger is normally present, or both. We also found that Ins(1,4,5)P₃ inhibited the Ca²⁺-ATPase by 33.7%, but that it had no significant effect on the Na²⁺/Ca²⁺ exchanger.     

Spatial and dye correlation analysis of intracellular Ca2+ distribution

Intracellular Ca²⁺ is an important regulator of many cellular processes. Besides ion channels and transporters in the plasmalemma, changes in [Ca]_i can be mediated by uptake and release mechanisms of internal organelles. Theoretical and experimental procedures are developed aiming to reveal the distribution of internal Ca²⁺ pools and their role in generating complicated spatial patterns of [Ca]_i gradients. Cultured pyramidal neurons from rat hippocampus were loaded with Ca²⁺-sensitive fluorescent dyes, fura-2 and fluo-3. Cell images were partitioned according to pixel amplitude and highlighted pictures were characterized by their intensity, relative area and connectivity. This approach facilitates the localization of the sites of Ca²⁺ release from internal stores induced by application of different agents. After each trial, neurons were stained with dyes, acridine orange or DiOC₆, which bind preferentially to nucleus and endoplasmic reticulum. A correlation between images confirmed the spatial localization of Ca²⁺ release sites. Application of the partition procedure also gave a clear evidence for the importance of Ca²⁺ influx in the mechanism of [Ca]_i oscillations.     

Protein phosphorylation is a prerequisite for intracellular Ca2+ release and ion channel control by nitric oxide and abscisic acid in guard cells

Recent work has indicated that nitric oxide (NO) and its synthesis are important elements of signal cascades in plant-pathogen defence, and are a prerequisite for drought and abscisic acid (ABA) responses in Arabidopsis thaliana and Vicia faba guard cells. NO regulates inward-rectifying K+ channels and Cl- channels of Vicia guard cells via intracellular Ca2+ release. However, its integration with related signals, including the actions of serine-threonine protein kinases, is less well defined. We report here that the elevation of cytosolic-free [Ca2+] ([Ca2+]i) mediated by NO in guard cells is reversibly inhibited by the broad-range protein kinase antagonists staurosporine and K252A, but not by the tyrosine kinase antagonist genistein. The effects of kinase antagonism translate directly to a loss of NO-sensitivity of the inward-rectifying K+ channels and background (Cl- channel) current, and to a parallel loss in sensitivity of the K+ channels to ABA. These results demonstrate that NO-dependent signals can be modulated through protein phosphorylation upstream of intracellular Ca2+ release, and they implicate a target for protein kinase control in ABA signalling that feeds into NO-dependent Ca2+ release.     

Function of caveolae in Ca2+ entry and Ca2+-dependent signal transduction

The correct spatial and temporal control of Ca²⁺ signaling is essential for such cellular activities as fertilization, secretion, motility, and cell division. There has been a long-standing interest in the role of caveolae in regulating intracellular Ca²⁺ concentration. In this review we provide an updated view of how caveolae may regulate both Ca²⁺ entry into cells and Ca²⁺-dependent signal transduction     

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

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

Suppression of EGF-induced cell proliferation by the blockade of Ca2+ mobilization and capacitative Ca2+ entry in mouse mammary epithelial cells

The role of intracellular Ca2+ stores and capacitative Ca2+ entry on EGF-induced cell proliferation was investigated in mouse mammary epithelial cells. We have previously demonstrated that EGF enhances Ca2+ mobilization (release of Ca2+ from intracellular Ca2+ stores) and capacitative Ca2+ entry correlated with cell proliferation in mouse mammary epithelial cells. To confirm their role on EGF-induced cell cycle progression, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), a reversible inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and SK&F 96365, a blocker of capacitative Ca2+ entry, on mitotic activity induced by EGF. Mitotic activity was examined using an antibody to PCNA for immunocytochemistry. SK&F 96365 inhibited capacitative Ca2+ entry in a dose-dependent manner (I50: 1-5 microM). SK&F 96365 also inhibited EGF-induced cell proliferation in the same range of concentration (I50: 1-5 microM). DBHQ suppressed [Ca2+]i response to UTP and thus depleted completely Ca2+ stores at 5 microM. DBHQ also inhibited EGF-induced cell proliferation at an I50 value of approximately 10 microM. The removal of these inhibitors from the culture medium increased the reduced mitotic activity reversibly. Using a fluorescent assay of DNA binding of ethidium bromide, no dead cells were detected in any of the cultures. These results indicate that the inhibitory effects of SK&F 96365 and DBHQ on cell proliferation were due to the inhibition of capacitative Ca2+ entry and Ca2+ mobilization suggesting the importance of capacitative Ca2+ entry and Ca2+ mobilization in the control of EGF-induced cell cycle progression in mouse mammary epithelial cells.     

Cytotoxicity of IFN-gamma and TNF-alpha for vascular endothelial cell is mediated by nitric oxide

Endothelial cell injury is a critical event in tissue damage accompanying inflammation, in which both inflammatory cytokines and reactive oxygen species may play pivotal roles, although the exact mechanism has not yet been clarified. We found that combined stimulation with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) induced both cytotoxicity to murine vascular endothelial cell line F-2 and an increase in nitric oxide (NO). Therefore, in the present study, the implication of NO in cytotoxicity was examined. A potent iNOS-specific inhibitor ONO-1714 completely blocked both cytokine-induced cytotoxicity and NO production. NO scavengers such as carboxy-PTIO and hemoglobin blocked cytotoxicity. Moreover, exogenous NO from NOC 18 also caused cytotoxicity. These results together demonstrated that cytotoxicity of IFN-gamma and TNF-alpha for endothelial cell F-2 was mediated by NO, suggesting a pathogenic role of cytokine-induced NO production in endothelial damage under inflammatory conditions.     

Spontaneous Ca^2＋ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca^2＋ pools

INTRODUCTION In vascular smooth muscle, as in other types of muscle,an increase in intracellular Ca2 is the immediate triggerfor contraction, which ultimately determines vascular toneand peripheral resistance. In the past 12 years, investiga-tors have …     

Release of nitric oxide and expression of constitutive nitric oxide synthase of human endothelial cells: Enhancement by a 14-membered ring macrolide

A 14-membered ring macrolide, erythromycin, acts not only as an antibacterial but also as an anti-inflammatory agent. We have previously reported that erythromycin modulates neutrophil functions and ameliorates neutrophil-induced endothelial cell damage through the action of cyclic AMP-dependent protein kinase (PKA) and nitric oxide (NO). We investigated the effect of erythromycin on human endothelial cell functions. Erythromycin enhanced intracellular calcium ion concentration ([Ca²⁺]_i) of endothelial cells and NO release from endothelial cells. The enhancement of NO release from endothelial cells by erythromycin was abolished by addition of EGTA in the medium and was partially reduced by addition of H-89, an inhibitor of PKA. These results suggest that erythromycin enhances NO release from endothelial cells through the action of PKA and [Ca²⁺]_i. In addition, constitutive NO synthase (cNOS) protein expression of endothelial cells was dose-dependently enhanced by treatment with erythromycin, which might also contribute to the enhancement of NO release from endothelial cells by erythromycin. The effect of erythromycin as an anti-inflammatory agent might be partially mediated through the enhancement of NO release from endothelial cells and the drug might be a useful tool for the investigation of cNOS of endothelial cells.     

Melatonin protects rat cerebellar granule cells against electromagnetic field‐induced increases in Na+ currents through intracellular Ca2+ release

Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low‐frequency electromagnetic field (ELF‐EMF)‐induced Na_v activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF‐EMF for 60 min. significantly increased the Na_v current (I_Na) densities by 62.5%. MT (5 μM) inhibited the ELF‐EMF‐induced I_Na increase. This inhibitory effect of MT is mimicked by an MT₂ receptor agonist and was eliminated by an MT₂ receptor antagonist. The Na_v channel steady‐state activation curve was significantly shifted towards hyperpolarization by ELF‐EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF‐EMF. ELF‐EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK‐7 did not reduce the ELF‐EMF‐induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF‐EMF‐induced Na_v activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca²⁺ level, but it significantly elevated the intracellular Ca²⁺ level evoked by the high K⁺ stimulation in cerebellar GC that were either exposed or not exposed to ELF‐EMF. In the presence of ruthenium red, a ryanodine‐sensitive receptor blocker, the MT‐induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal I_Na that result from ELF‐EMF exposure through Ca²⁺ influx‐induced Ca²⁺ release.     

Effect of Magnesium tanshinoate B on the production of nitric oxide in endothelial cells

Nitric oxide (NO) is a potent vasodilator which plays an important role in regulating vascular tones. Danshen, a Chinese herbal medicine has been widely used for the treatment of cardiovascular diseases. The objective of this study was to investigate the effect of magnesium tanshinoate B (MTB), a compound purified from Danshen, on the production of NO in human endothelial cell line (ECV304). After cells were incubated with MTB (1-10 µM) for 1 or 4 h, amounts of NO metabolites released by cells were quantified and cellular NOS activities were determined following the conversion of [³H]arginine to [³H]citrulline. The NOS protein expression was determined by Western immunoblotting analysis. MTB (1-10 µM) stimulated the release of NO and its metabolites from endothelial cells. Following MTB treatment, the cellular NOS activities were significantly enhanced with a concomitant increase in the levels of constitutive NOS (cNOS) protein mass (110-178%). Selective activation of cNOS by MTB may be employed therapeutically in modulating NO production in endothelial cells.     

PLC-dependent intracellular Ca2+ release was associated with C6-ceramide-induced inhibition of Na+ current in rat granule cells

In this report, the effects of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)), two types of main K(+) current [outward rectifier delayed K(+) current (I(K)) and outward transient K(+) current (I(A))], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01-100 microM, ceramide produced a dose-dependent and reversible inhibition of I(Na) without alteration of the steady-state activation and inactivation properties. Treatment with C(2)-ceramide caused a similar inhibitory effect on I(Na). However, dihydro-C(6)-ceramide failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent, 1,2-bis (2-aminophenoxy) ethane-N, N, N9, N9-tetraacetic acid, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with ryanodine receptor blocker induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide. Intracellular application of GTPgammaS also induced a gradual decrease in I(Na) amplitude, while GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Furthermore, the C(6)-ceramide effect on I(Na) was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C(6)-ceramide decreased cell viability and blocking I(Na) by tetrodotoxin did not mimic the effect of C(6)-ceramide, and inhibiting intracellular Ca(2+) release by dantrolene could not decrease the C(6)-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca(2+) release through the ryanodine-sensitive Ca(2+) receptor may be responsible for the C(6)-ceramide-induced inhibition of I(Na), which does not seem to be associated with C(6)-ceramide-induced granule neuron death.     

Calcium signaling in non-excitable cells: Ca2+ release and influx are independent events linked to two plasma membrane Ca2+ entry channels

The regulatory mechanism of Ca2+ influx into the cytosol from the extracellular space in non-excitable cells is not clear. The "capacitative calcium entry" (CCE) hypothesis suggested that Ca2+ influx is triggered by the IP(3)-mediated emptying of the intracellular Ca2+ stores. However, there is no clear evidence for CCE and its mechanism remains elusive. In the present work, we have provided the reported evidences to show that inhibition of IP(3)-dependent Ca2+ release does not affect Ca2+ influx, and the experimental protocols used to demonstrate CCE can stimulate Ca2+ influx by means other than emptying of the Ca2+ stores. In addition, we have presented the reports showing that IP(3)-mediated Ca2+ release is linked to a Ca2+ entry from the extracellular space, which does not increase cytosolic [Ca2+] prior to Ca2+ release. Based on these and other reports, we have provided a model of Ca2+ signaling in non-excitable cells, in which IP(3)-mediated emptying of the intracellular Ca2+ store triggers entry of Ca2+ directly into the store, through a plasma membrane TRPC channel. Thus, emptying and direct refilling of the Ca2+ stores are repeated in the presence of IP(3), giving rise to the transient phase of oscillatory Ca2+ release. Direct Ca2+ entry into the store is regulated by its filling status in a negative and positive manner through a Ca2+ -binding protein and Stim1/Orai complex, respectively. The sustained phase of Ca2+ influx is triggered by diacylglycerol (DAG) through the activation of another TRPC channel, independent of Ca2+ release. The plasma membrane IP(3) receptor (IP(3)R) plays an essential role in Ca2+ influx, by interacting with the DAG-activated TRPC, without the requirement of binding to IP(3).