2-Aminoethoxydiphenyl borate perturbs hormone-sensitive calcium stores and blocks store-operated calcium influx pathways independent of cytoskeletal disruption in human A549 lung cancer cells

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca2+-signaling pathways, revealing differentially sensitive Ca2+-influx pathways and long-term perturbation of hormone-sensitive Ca2+ stores.     

Mechanisms of Na+ and Ca2+ influx into respiratory neurons during hypoxia

Changes in intracellular Na+ and Ca2+ in inspiratory neurons of neonatal mice were examined by using ion-selective fluorescent indicator dyes SBFI and fura-2, respectively. Both [Na+]i and [Ca2+]i signals showed rhythmic elevations, correlating with the inspiratory motor output. Brief (2-3 min) hypoxia, induced initial potentiation of rhythmic transients followed by their depression. During hypoxia, the basal [Na+]i and [Ca2+]i levels slowly increased, reflecting development of an inward current (Im). By antagonizing specific mechanisms of Na+ and Ca2+ transport we found that increases in [Na+]i, [Ca2+]i and Im due to hypoxia are suppressed by CNQX, nifedipine, riluzole and flufenamic acid, indicating contribution of AMPA/kainate receptors, persistent Na+ channels, L-type Ca2+ channels and Ca2+-sensitive non-selective cationic channels, respectively. The blockers decreased also the amplitude of the inspiratory bursts. Modification of mitochondrial properties with FCCP and cyclosporine A decreased [Ca2+]i elevations due to hypoxia by about 25%. After depletion of internal Ca2+ stores with thapsigargin, the blockade of NMDA receptors, Na+/K+ pump, Na+/H+ and Na+/Ca2+ exchange, the hypoxic response was not changed. We conclude that slow [Na+]i and [Ca2+]i increases in inspiratory neurons during hypoxia are caused by Na+ and Ca2+ entry due to combined activation of persistent Na+ and L-type Ca2+ channels and AMPA/kainate receptors.     

Inhibitory effects of (1R,9S)-β-hydrastine on calcium transport in PC12 cells

(1R,9S)-beta-Hydrastine (BHS), at 100 microM, has been shown to mainly reduce the K+-induced dopamine release and Ca2+ influx by blocking the L-type Ca2+ channel and inhibit the caffeine activated store-operated Ca2+ channels, but not those activated by thapsigargin, in PC12 cells. In this study, the effects of BHS on Ca2+ transport from Ca2+ stores in the absence of external Ca2+ were investigated in PC12 cells. BHS decreased the basal intracellular Ca2+ concentration ([Ca2+]i) in the absence of external Ca2+ in PC12 cells. In the absence of external Ca2+, pretreating PC12 cells with 100 microM BHS reduced the rapid increase in the [Ca2+]i elicited by 20 mM caffeine, but not that by 1 microM thapsigargin. In addition, BHS inhibited the increase in the [Ca2+]i elicited by restoration of 2 mM CaCl2 after the Ca2+ stores had been depleted by 20 mM caffeine, but not those depleted by 1 microM thapsigargin, in the absence of external Ca2+. These results suggested that BHS mainly inhibited Ca2+ leakage from the Ca2+ stores and the caffeine-stimulated release of Ca2+ from the caffeine-sensitive Ca2+ stores in PC12 cells.     

Suppression of Ca2+ influx by unfractionated heparin in non-excitable intact cells via multiple mechanisms

Effect of unfractionated heparin (UFH), described as a cell-impermeant IP3 receptor antagonist, was studied on the capacitive Ca(2+) entry in non-permeabilized, intact cells, measuring the intracellular Ca(2+) levels using fluorescence microplate technique. Ca(2+) influx induced via Ca(2+) mobilization by histamine in Hela cells or evoked by store depletion with thapsigargin in RBL-2H3 cells was dose-dependently suppressed by UFH added either before or after the stimuli. UFH also prevented the spontaneous Ba(2+) entry indicating that the non-capacitive Ca(2+) channels may also be affected. In addition, UFH caused a significant and dose-dependent delay in Ca(2+), and other bivalent cation inflow after treatment of the cells with Triton X-100, but it did not diminish the amount of these cations indicating that UFH did not act simply as a cation chelator, but modulated the capacitive Ca(2+) entry possibly via store operated Ca(2+) channels (SOCCs). Inhibitory activities of UFH and 2-aminoethyl diphenyl borate on the capacitive Ca(2+) influx was found reversible, but the time courses of their actions were dissimilar suggesting distinct modes of action. It was also demonstrated using a fluorescence potentiometric dye that UFH had a considerable hyperpolarizing effect and could alter the changes of membrane potential during Ca(2+) influx after store depletion by thapsigargin. We presume that the hyperpolarizing property of this agent might contribute to the suppression of Ca(2+) influx. We concluded that UFH can negatively modulate SOCCs and also other non-capacitive Ca(2+) channels and these activities might also account for its multiple biological effects.     

Orai1-STIM1 formed store-operated Ca2+ channels (SOCs) as the molecular components needed for Pb2+ entry in living cells

Heavy metal lead (Pb2+) is a pollutant and causes severe toxicity when present in human tissues especially the nervous system. Recent reviews have suggested that Pb2+ can target Ca2+-related proteins within neurons and that Ca2+ channels might be a candidate for Pb2+ entry. This study's main aim was to identify the functional entry pathway of Pb2+ into living cells. We firstly characterized the endogenous expression of Orai1 and STIM1 mRNA together with the level of thapsigargin (TG) stimulated capacitative Ca2+ entry in PC12 and HeLa cells; this was done by RT-PCR and time-lapse Ca2+ imaging microscopy, respectively. Our data supported Orai1 and STIM1 as contributing to store-operated Ca2+ channel (SOC) basal activity. Secondly, using the indo-1 quenching method with the SOC blocker 2-APB, we observed that Pb2+ was able to enter cells directly through unactivated SOCs without TG pretreatment. Thirdly, we further demonstrated that co-expression of Orai1 and STIM1 differentially enhanced SOC functional activity (4-fold with PC12 and 5-fold with HeLa cells) and Pb2+ entry (5- to 7-fold with PC12 and 2-fold with HeLa cells). Furthermore, after a 1 h of Pb2+ exposure, the depolarization- and histamine-induced Ca2+ responses were significantly decreased in both PC12 and HeLa cells in a dose-dependent manner. This result indicated that the decreased Ca2+ responses were, in part, due to Pb2+ entry. In summary, our results suggest that SOCs are responsible for Pb2+ permeation and that the Orai1-STIM1 protein complex formed by functional SOCs is one of the molecular components involved in Pb2+ entry.     

ML-9, a myosin light chain kinase inhibitor, reduces intracellular Ca2+ concentration in guinea pig trachealis

We investigated the effects of ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], a myosin light chain kinase (MLCK) inhibitor, on intracellular Ca2+ concentration ([Ca2+]i), contraction induced by high K+ and an agonist, and capacitative Ca2+ entry in fura-2-loaded guinea pig tracheal smooth muscle. ML-9 inhibited both the increase in [Ca2+]i and the contraction induced by 60 mM K+, 1 microM methacholine or 1 microM thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. However, another MLCK inhibitor, wortmannin (3 microM), inhibited the contraction elicited by these stimuli without affecting [Ca2+]i. Under the condition that the thapsigargin-induced contraction was fully suppressed by 3 microM wortmannin, 30 microM ML-9 caused a further decrease in [Ca2+]i. The inhibitory effects of ML-9 on [Ca2+]i and the contraction elicited by methacholine were similar to those of SKF-96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), a Ca2+ channel blocker. These results indicate that ML-9 acts as a potent inhibitor of Ca2+-permeable channels independently of MLCK inhibition in tracheal smooth muscle.     

Effects of phosphoinositide 3-kinase on endothelin-1-induced activation of voltage-independent Ca2+ channels and vasoconstriction

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in rabbit basilar artery (BA) vascular smooth muscle cells (VSMCs). In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on ET-1-induced activation of these channels and BA contraction by using PI3K inhibitors, wortmannin and LY 249002. To determine which Ca(2+) channels are activated via PI3K, monitoring of intracellular Ca(2+) concentration was performed. Role of PI3K in ET-1-induced vasoconstriction was examined by tension study using rabbit BA rings. Only NSCC-1 was activated by ET-1 in wortmannin- or LY 294002-pretreated VSMCs. In contrast, addition of these drugs after ET-1 stimulation did not suppress Ca(2+) influx. Wortmannin inhibited the ET-1-induced contraction of rabbit BA rings that depends on the Ca(2+) influx through NSCC-2 and SOCC. The IC(50) values of wortmannin for the ET-1-induced Ca(2+) influx and vasoconstriction were similar to those for the ET-1-induced PI3K activation. These results indicate that (1) NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade; (2) PI3K is required for the activation of the Ca(2+) entry, but not for its maintenance; and (3) PI3K is involved in the ET-1-induced contraction of rabbit BA rings that depends on the extracellular Ca(2+) influx through SOCC and NSCC-2.     

Characterization of beta-leptinotarsin-h and the effects of calcium flux antagonists on its activity.

beta-Leptinotarsin-h, purified from the hemolymph of the beetle Leptinotarsa haldemani, is a potent ( approximately 1 nM) neuroactive protein that rapidly (few seconds) stimulates Ca(2+) influx and neurotransmitter release. Our goals were to further characterize beta-leptinotarsin-h and to test the hypothesis that it stimulates Ca(2+) influx through presynaptic Ca(2+) channels. Analysis of partial amino acid sequences revealed that beta-leptinotarsin-h is a unique protein with significant similarity to only one other protein, the juvenile hormone esterase of Leptinotarsa decemlineata, commonly known as the Colorado potato beetle. We have examined the effect of beta-leptinotarsin-h on Ca(2+) current, Ca(2+) uptake, Ca(2+) levels, and neurotransmitter release in synaptosomes, cell lines, and neuronal systems. We found that its preferred site of action appears to be mammalian presynaptic nerve terminals. We tested antagonists of Ca(2+) flux for their effects on beta-leptinotarsin-h-stimulated Ca(2+) uptake in rat brain synaptosomes. The non-selective Ca(2+) channel blockers flunarizine, Ni(2+), ruthenium red, high-concentration thapsigargin, and SKF 96365 inhibited beta-leptinotarsin-h's activity, but none of the tested selective blockers of voltage-operated Ca(2+) channels (omega-agatoxin IVA, omega-conotoxin GVIA, omega-conotoxin MVIIC, nicardipine, nifedipine, SNX-482) was inhibitory. Selective inhibitors of ligand-operated, store-operated, and transduction-operated channels were also not inhibitory. beta-Leptinotarsin-h did not stimulate Na(+) uptake, ruling out Na(+) channels and many non-selective cation channels as targets. We conclude that beta-leptinotarsin-h stimulated Ca(2+) uptake through presynaptic Ca(2+) channels; which channel is yet to be determined. beta-Leptinotarsin-h may prove to be a useful tool with which to investigate calcium channels and calcium flux.     

Inhibition of store-operated calcium entry-mediated superoxide generation by histamine trifluoromethyltoluide independent of histamine receptors

Store-operated calcium entry (SOCE) plays an important role in shaping the Ca(2+) response of various tissues and cell types. In this report, we show that thapsigargin (TG)-induced SOCE was inhibited by the histamine receptor agonist, histamine-trifluoromethyltoluide (HTMT), in U937 and HL-60 human promyelocytes. Preincubation of HTMT resulted in a significant inhibition of subsequent TG-induced Ca(2+) elevation without affecting Ca(2+) release from intracellular stores. HTMT also inhibited TG-induced Ca(2+) current and Ba(2+)/Mn(2+) influx in a concentration-dependent manner. In contrast with HTMT, other H1 histamine receptor agonists, histamine, 2-methylhistamine and 2-thiazolylethylamine, did not affect TG-induced SOCE. In addition, HTMT also attenuated TG-induced cytosolic superoxide generation. Taken together, our data clearly suggest that the anti-inflammatory effect of HTMT may occur through direct inhibition of SOCE.     

Regulation of BK(Ca) channels expressed in human embryonic kidney 293 cells by epoxyeicosatrienoic acid

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 monooxygenase, which are released from endothelial cells and dilate arteries. Dilation seems to be caused by activation of large-conductance Ca2+ activated K+ channels (BK(Ca)) leading to membrane hyperpolarization. Previous studies suggest that EETs activate BK(Ca) channels via ADP-ribosylation of the G protein Galphas with a subsequent membrane-delimited action on the channel [Circ Res 78:415-423, 1996; 80:877-884, 1997; 85:349-356, 1999]. The present study examined whether this pathway is present in human embryonic kidney (HEK) 293 cells when the BK(Ca) alpha-subunit (cslo-alpha) is expressed without the beta-subunit. 11,12-EET increased outward K+ current in whole-cell recordings of HEK293 cells. In cell-attached patches, 11,12-EET also increased the activity of cslo-alpha channels without affecting unitary conductance. This action was mimicked by cholera toxin. The ADP-ribosyltransferase inhibitors 3-aminobenzamide and m-iodobenxylguanidine blocked the stimulatory effect of 11,12-EET. In inside-out patches 11,12-EET was without effect on channel activity unless GTP was included in the bathing solution. GTP and GTPgammaS alone also activated cslo-alpha channels. Dialysis of cells with anti-Galphas antibody completely blocked the activation of cslo-alpha channels by 11,12-EET, whereas anti-Galphai/o and anti-Gbetagamma antibodies were without effect. The protein kinase A inhibitor KT5720 and the adenylate cyclase inhibitor SQ22536 did not reduce the stimulatory effect of 11,12-EET on cslo-alpha channels in cell-attached patches. These data suggest that EET leads to Galphas-dependent activation of the cslo-alpha subunits expressed in HEK293 cells and that the cslo-beta subunit is not required.     

Azaspiracid-4 inhibits Ca2+ entry by stored operated channels in human T lymphocytes

Azaspiracids (AZs) are a new group of phycotoxins discovered in the Ireland coast that includes the isolated analogues: AZ-1, AZ-2, AZ-3, AZ-4 and AZ-5 and the recently described AZ-6-11. Azaspiracid toxic episodes show gastrointestinal illness, but neurotoxic symptoms are also observed in mouse bioassay. Despite their great importance in human health, so far its mechanism of action is largely unknown. In this report, we present the first data about the effect of AZ-4 on cytosolic calcium concentration [Ca2+]i in freshly human lymphocytes. Cytosolic Ca2+ variations were determined by fluorescence digital imaging microscopy using Fura2 acetoxymethyl ester (Fura2-AM). AZ-4 did not modify cytosolic Ca2+ in resting cells. However, the toxin dose-dependent inhibited the increase in cytosolic Ca2+ levels induced by thapsigargin (Tg). AZ-4 decreased Ca2+-influx induced by Tg but did not affect the Ca2+-release from internal stores induced by this drug. The effects of AZ-4 on Ca2+-influx induced by Tg were reversible and not regulated by adenosine 3',5'-cyclic monophosphate (cAMP) pathway. When AZ-4 was added before, after or together with nickel, an unspecific blocker of Ca2+ channels, the effects were indistinguishable and additive. AZ-4 also inhibited maitotoxin (MTX)-stimulated Ca2+-influx by 5-10%. Thus, AZ-4 appeared to be a novel inhibitor of plasma membrane Ca2+ channels, affecting at least to store operated channels, showing an effect clearly different from other azaspiracid analogues.     

Potentiation of Ca2+ signaling in endothelial cells by 11,12-epoxyeicosatrienoic acid

Incubation of endothelium with an increased epoxyeicosatrienoic acid (EET) concentration specifically augments the endothelium-dependent relaxation ascribed to endothelium-derived hyperpolarizing factor in porcine coronary arteries (Weintraub et al., Circ Res 1997;81:258-267). Experiments were designed to test whether such sustained increased levels of EETs in the environment of endothelial cells alters Ca2+ signaling. Changes in cytosolic Ca2+ were monitored in cultured porcine aortic endothelial cells (PAECs) and in the human endothelial EA.hy926 cell line after incubation (or not) with 5 microM 11,12-epoxyeicosatrienoic acid (EET). Although the mobilization of intracellular Ca2+ induced by 2 microM thapsigargin was not affected significantly, EET treatment augmented the capacitative Ca2+ entry evoked by the Ca(2+)-ATPase) inhibitor in both cell types. Similar observations were obtained by using histamine as a stimulant in EA.hy926 cells. As assessed in PAECs, 2 micrograms/ml triacsin C, a known inhibitor of the incorporation of EETs into phospholipids, did not significantly affect the potentiating action of EETs on Ca2+ signaling in response to thapsigargin. However, in solvent-control cells, triacsin C significantly reduced both the mobilization of Ca2+ from intracellular stores and the capacitative Ca2+ entry provoked by thapsigargin. Thus the EET-potentiating effect overcomes the inhibitory action of triacsin C on Ca2+ signaling in endothelial cells. Taken together, these results demonstrate that sustained increases in EETs may amplify Ca2+ signaling. However, contrary to the EET-induced augmentation of endothelium-dependent relaxation in the porcine coronary artery, resistance of this novel action of EETs to triacsin C suggests that the mechanism involved does not depend on incorporation into phospholipids.     

Dihydropyridines as inhibitors of capacitative calcium entry in leukemic HL-60 cells

A series of 1,4-dihydropyridines (DHPs) were investigated as inhibitors of capacitative calcium influx through store-operated calcium (SOC) channels. Such channels activate after ATP-elicited release of inositol trisphosphate (IP(3))-sensitive calcium stores in leukemia HL-60 cells. The most potent DHPs were those containing a 4-phenyl group with an electron-withdrawing substituent, such as m- or p-nitro- or m-trifluoromethyl (IC(50) values: 3-6 microM). Benzyl esters, corresponding to the usual ethyl/methyl esters of the DHPs developed as L-type calcium channel blockers, retained potency at SOC channels, as did N-substituted DHPs. N-Methylation reduced by orders of magnitude the potency at L-type channels resulting in DHPs nearly equipotent at SOC and L-type channels. DHPs with N-ethyl, N-allyl, and N-propargyl groups also had similar potencies at SOC and L-type channels. Replacement of the usual 6-methyl group of DHPs with larger groups, such as cyclobutyl or phenyl, eliminated activity at the SOC channels; such DHPs instead elicited formation of inositol phosphates and release of IP(3)-sensitive calcium stores. Other DHPs also caused a release of calcium stores, but usually at significantly higher concentrations than those required for the inhibition of capacitative calcium influx. Certain DHPs appeared to cause an incomplete blockade of SOC channel-dependent elevations of calcium, suggesting the presence of more than one class of such channels in HL-60 cells. N-Methylnitrendipine (IC(50) 2.6 microM, MRS 1844) and N-propargylnifrendipine (IC(50) 1.7 microM, MRS 1845) represent possible lead compounds for the development of selective SOC channel inhibitors.     

Mechanisms of AM404-induced [Ca(2+)](i) rise and death in human osteosarcoma cells

The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca2+ levels ([Ca2+]i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations > or = 5 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 60 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. AM404 induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was sensitive to La3+, Ni2+, nifedipine and verapamil. In Ca2+-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), AM404-induced [Ca2+]i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca2+]i rise. At concentrations between 10 and 200 microM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 microM AM404 was partly reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via L-type Ca2+ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.     

Ceramide 1-(2-cyanoethyl) phosphate enhances store-operated Ca2+ entry in thyroid FRTL-5 cells

Sphingolipid derivatives cause diverse effects towards the regulation of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in a multitude of nonexcitable cells. In the present investigation, the effect of C-8 ceramide-1-(2-cyanoethyl) phosphate (C1CP) on store-operated Ca(2+) (SOC) entry was investigated. C1CP evoked a modest increase in [Ca(2+)](i). The increase was inhibited by the SOC channel antagonist 1-(beta-[3-(4methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SKF96365) but not by overnight pretreatment of the cells with pertussis toxin. C1CP did not invoke the production of inositol phosphates. When cells were stimulated with both C1CP and thapsigargin, the thapsigargin-invoked increase in [Ca(2+)](i) was enhanced in comparison to control cells. When Ca(2+) was added to cells treated with both C1CP and thapsigargin in a Ca(2+)-free buffer, the increase in [Ca(2+)](i) was enhanced in comparison to control cells. In patch-clamp experiments, C1CP hyperpolarized the membrane potential (E(m)) of the cells and attenuated the thapsigargin-invoked depolarization of the E(m). The effects of C1CP came, in part, as a result of a decreased conductance of the cell membrane towards Cl(-) ions, as C1CP in a Cl(-)-free solution also enhanced Ca(2+) entry. Barium 2-cyanoethylphosphate (Ba2Cy), which also contains the 2-cyanoethyl group, did not modulate thapsigargin-invoked changes in [Ca(2+)](i) nor did it modulate the E(m). In conclusion, C1CP enhances SOC entry, in part, via hyperpolarization of the E(m) and attenuation of the thapsigargin-invoked membrane depolarization, thus increasing the electrochemical gradient for Ca(2+) ions. Hence, C1CP may be a useful reagent for investigating the cellular effects of ceramide derivatives.     

The lysosome or lysosome-related organelle may serve as a Ca2+ store in the boutons of hippocampal pyramidal cells

Boutons are specialised presynaptic compartments that lie along the axons of central neurons. Release of neurotransmitter from boutons is tightly regulated by the level of intracellular calcium [Ca2+]i. A rise in Ca2+ level may be generated in several ways; entry of extracellular Ca2+ via voltage gated calcium channels (VGCCs), entry via ligand-operated channels (LOCs) or the release of Ca2+ from intracellular Ca2+ stores. The role of Ca2+ stores in boutons remains poorly understood, despite recent work indicating that the release of Ca2+ from the endoplasmic reticulum (ER) may contribute to transmitter release. In this study we assess whether the lysosome or a closely related organelle functions as a Ca2+ store in the boutons of hippocampal pyramidal neurones. Lysosomes are small acidic organelles more commonly known for their role in degrading redundant cellular constituents. Using a fluorescent lysosomal marker, we show that lysosomes are located in the axons of hippocampal CA3 neurones. Selective pharmacological lysis of the lysosomes with glycyl-phenylalanine 2-naphthylamide (GPN) generates rapid, highly focal Ca2+ transients within the axon and increases the frequency of spontaneous miniature excitatory post-synaptic currents (mEPSCs), revealing that the organelle contains Ca2+ at a concentration sufficient to evoke transmitter release. Confocal laser scanning microscopy, combined with electrophysiology is used to monitor the action potential evoked increases in [Ca2+]i in boutons. We show that disruption of lysosomes compromises action potential evoked [Ca2+]i but this effect is occluded if the ER is discharged. Conversely, disruption of the lysosome does not appear to impact on the capacity of the ER to release Ca2+. These results suggest that the lysosome may serve as a Ca2+ store within hippocampal boutons, with a Ca2+ signalling role that is unique from that of the ER.     

Cannabidiol-induced intracellular Ca2+ elevations in hippocampal cells

The phytocannabinoid cannabidiol (CBD) is at the forefront of therapeutic cannabinoid research due to its non-psychotropic properties. Research supports its use in a variety of disorders, yet the cellular mechanisms of its action remain unclear. In this study, the effect of CBD upon Ca2+ homeostasis in hippocampal cells was characterised. CBD (1 microM) elevated intracellular Ca2+ ([Ca2+]i) by approximately +45% of basal Ca2+ levels in both glia (77% responders) and neurones (51% responders). Responses to CBD were reduced in high excitability HEPES buffered solution (HBS), but not affected in low excitability/low Ca2+ HBS. CBD responses were also significantly reduced (by 50%) by the universal Ca2+ channel blocker cadmium (50 microM) and the L-type specific Ca2+ channel blocker nifedipine (20 microM). Interestingly, intracellular store depletion with thapsigargin (2 microM) had the most dramatic effect on CBD responses, leading on average to a full block of the response. Elevated CBD-induced [Ca2+]i responses (>+100%) were observed in the presence of the CB1 receptor antagonist, AM281 (1 microM), and the vanilloid receptor antagonist, capsazepine (CPZ, 1 microM). Overall, our data suggest that CBD modulates hippocampal [Ca2+]i homeostasis via intracellular Ca2+ stores and L-type VGCC-mediated Ca2+ entry, with tonic cannabinoid and vanilloid receptor signalling being negatively coupled to this pathway.     

2-Aminoethoxydiphenyl borate (2-APB) stimulates a conformationally coupled calcium release pathway in the NG115-401L neuronal cell line

We report in this study a 2-aminoethoxydiphenyl borate (2-APB) activated Ca2+ pathway in NG115-401L (401L) neuronal cells bearing resemblance to hormonal and ryanodine receptor activated pathways. We observed that 2-APB, in contrast to much earlier work, did not inhibit store operated Ca2+ channel (SOC) function, but rather induced potent Ca2+ discharge responses that robustly activated SOC-mediated Ca2+ influx. Further, these studies intriguingly revealed that the 2-APB-induced Ca2+ release pathway likely couples conformationally to targets in the plasma membrane, as membrane permeabilization or actin perturbation abolished the ability of the compound to stimulate Ca2+ signals. These findings suggest that conformationally sensitive complexes form between endoplasmic reticulum and plasma membrane components that not only regulate Ca2+ influx, previously proposed as the conformational coupling hypothesis, but are also required to promote Ca2+ release from intracellular stores. These observations further characterize the 401L neuronal cell line as having unique characteristics that may prove useful in gaining insight into the nature of the coupling mechanism linking Ca2+ release to Ca2+ influx.     

Mechanism of apoptosis induced by diazoxide,a K<Superscript>+</Superscript> channel opener,in HepG2 Human hepatoma cells

The effect of diazoxide, a K+ channel opener, on apoptotic cell death was investigated in HepG2 human hepatoblastoma cells. Diazoxide induced apoptosis in a dose-dependent manner and this was evaluated by flow cytometric assays of annexin-V binding and hypodiploid nuclei stained with propidium iodide. Diazoxide did not alter intracellular K+ concentration, and various inhibitors of K+ channels had no influence on the diazoxide-induced apoptosis; this implies that K+ channels activated by diazoxide may be absent in the HepG2 cells. However, diazoxide induced a rapid and sustained increase in intracellular Ca(2+) concentration, and this was completely inhibited by the extracellular Ca(2+) chelation with EGTA, but not by blockers of intracellular Ca(2+) release (dantrolene and TMB-8). This result indicated that the diazoxide-induced increase of intracellular Ca(2+) might be due to the activation of a Ca(2+) influx pathway. Diazoxide-induced Ca(2+) influx was not significantly inhibited by either voltage-operative Ca(2+) channel blockers (nifedipine or verapamil), or by inhibitors of Na+, Ca(2+)-exchanger (bepridil and benzamil), but it was inhibited by flufenamic acid (FA), a Ca(2+)-permeable nonselective cation channel blocker. A quantitative analysis of apoptosis by flow cytometry revealed that a treatment with either FA or BAPTA, an intracellular Ca(2+) chelator, significantly inhibited the diazoxide-induced apoptosis. Taken together, these results suggest that the observed diazoxide-induced apoptosis in the HepG2 cells may result from a Ca(2+) influx through the activation of Ca(2+)-permeable non-selective cation channels. These results are very significant, and they lead us to further suggest that diazoxide may be valuable for the therapeutic intervention of human hepatomas.     

Inhibition of store-operated Ca(2+) channels prevent ethanol-induced intracellular Ca(2+) increase and cell injury in a human hepatoma cell line

Elevated intracellular Ca²⁺ content is implicated in ethanol-induced hepatocyte apoptosis and necrosis. Extracellular Ca²⁺ influx has been suggested to play a role in this process. However, the exact Ca²⁺-permeable channel involved in the plasma membrane is still unclear. This study investigated the role of store-operated calcium entry (SOCE) in ethanol-induced cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) increase and hepatotoxicity. Ethanol (25-800 mM) dose-dependently increased [Ca²⁺]_i content and hepatocyte damage in HepG2 cells. 2-aminoethoxydiphenyl borate (2-APB), the proved efficient antagonist of SOCs, dose-dependently suppressed the ethanol (200 nM)-increased [Ca²⁺]_i content and protected against ethanol-induced viability loss and transaminase leakage. Exposure to 200 mM ethanol for 24 h significantly upregulated the mRNA and protein expression of calcium release-activated calcium channel protein 1 (CRACM1, Orai1) and stromal interaction molecule 1 (STIM1), the two main molecular constituents of SOCs, which was sustained for at least 72 h. In addition, small interfering RNA knockdown of STIM1 attenuated the ethanol-increased [Ca²⁺]_i content and hepatotoxicity. Taken together, these data indicate that the Ca²⁺ channel of SOCE may be involved in the pathogenesis of ethanol-induced intracellular Ca²⁺ elevation and consequent hepatocyte damage.