Inhibition of neuronal Ca(2+) influx by gabapentin and pregabalin in the human neocortex.

Gabapentin and pregabalin (S-(+)-3-isobutylgaba) produced concentration-dependent inhibitions of the K(+)-induced [Ca(2+)](i) increase in fura-2-loaded human neocortical synaptosomes (IC(50)=17 microM for both compounds; respective maximal inhibitions of 37 and 35%). The weaker enantiomer of pregabalin, R-(-)-3-isobutylgaba, was inactive. These findings were consistent with the potency of these drugs to inhibit [(3)H]-gabapentin binding to human neocortical membranes. The inhibitory effect of gabapentin on the K(+)-induced [Ca(2+)](i) increase was prevented by the P/Q-type voltage-gated Ca(2+) channel blocker omega-agatoxin IVA. The alpha 2 delta-1, alpha 2 delta-2, and alpha 2 delta-3 subunits of voltage-gated Ca(2+) channels, presumed sites of gabapentin and pregabalin action, were detected with immunoblots of human neocortical synaptosomes. The K(+)-evoked release of [(3)H]-noradrenaline from human neocortical slices was inhibited by gabapentin (maximal inhibition of 31%); this effect was prevented by the AMPA receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide). Gabapentin and pregabalin may bind to the Ca(2+) channel alpha 2 delta subunit to selectively attenuate depolarization-induced Ca(2+) influx of presynaptic P/Q-type Ca(2+) channels; this results in decreased glutamate/aspartate release from excitatory amino acid nerve terminals leading to a reduced activation of AMPA heteroreceptors on noradrenergic nerve terminals.     

Fluoxetine inhibits ATP-induced [Ca(2+)](i) increase in PC12 cells by inhibiting both extracellular Ca(2+) influx and Ca(2+) release from intracellular stores

Fluoxetine, a widely used antidepressant, has additional effects, including the blocking of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells using fura-2-based digital calcium imaging, an assay for [3H]-inositol phosphates (IPs) and whole-cell patch clamping. Treatment with ATP (100 microM) for 2 min induced increases in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Treatment with fluoxetine (100 nM to 30 microM) for 5 min inhibited the ATP-induced [Ca(2+)](i) increases in a concentration-dependent manner (IC(50) = 1.85 microM). Treatment with fluoxetine (1.85 microM) for 5 min significantly inhibited the ATP-induced responses following the removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) stores. Whereas treatment for 10 min with nimodipine (1 microM) significantly inhibited the ATP-induced [Ca(2+)](i) increase, treatment with fluoxetine further inhibited the ATP-induced response. Treatment with fluoxetine significantly inhibited [Ca(2+)](i) increases induced by 50 mM K(+). In addition, treatment with fluoxetine markedly inhibited ATP-induced inward currents in a concentration-dependent manner. However, treatment with fluoxetine did not inhibit ATP-induced [3H]-IPs formation. Therefore, we conclude that fluoxetine inhibits ATP-induced [Ca(2+)](i) increases in PC12 cells by inhibiting both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores without affecting IPs formation.     

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.     

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.     

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.     

The glutathione reductase inhibitor carmustine induces an influx of Ca2+ in PC12 cells

We studied the effects of carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in PC12 cells using fura-2 fluorescence imaging. Carmustine (100 microM) caused a delayed increase in [Ca(2+)](i) that developed within approximately 3 h. This effect was enhanced in cells that were pretreated with an inhibitor of glutathione (GSH) synthesis, buthionine sulfoximine (BSO, 200 microM, 24 h), and was suppressed in cells that were treated with an antioxidant deferoxamine (50 microM). The carmustine-induced increase in [Ca(2+)](i) was absolutely dependent on the presence of extracellular Ca(2+) and could be inhibited by dihydropyridine blockers of L-type voltage-gated Ca(2+) channels (nimodipine or nitrendipine, 10 microM). The increase in [Ca(2+)](i) was also suppressed in Cl(-)-free solution and in the presence of the Cl(-) channel blockers, indanyloxyacetic acid 94 (IAA-94, 100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM). The inhibition was complete when the blockers were applied simultaneously with carmustine and was partial when the blockers were applied after the initial increase in [Ca(2+)](i). We conclude that carmustine induces an influx of extracellular Ca(2+) through L-type Ca(2+) channels and that this effect is mediated by oxidative stress that results from the depletion of GSH following the inhibition by carmustine of glutathione reductase.     

Molecular and functional characterization of the human platelet Na(+) /Ca(2+) exchangers

BACKGROUND AND PURPOSE The Na(+) /Ca(2+) exchanger is a bi-directional transporter that plays an important role in maintaining the concentration of cytosolic Ca(2+) ([Ca(2+) ](i) ) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na(+) /Ca(2+) exchangers: K(+) -independent Na(+) /Ca(2+) exchanger (NCX) and K(+) -dependent Na(+) /Ca(2+) exchanger (NCKX). Platelets have previously been shown to express NCKX1. However, initial studies from our laboratory suggest that NCX may also play a role in platelet activation. The objective of this study was to determine if the human platelet expresses functional NCXs. EXPERIMENTAL APPROACH RT-PCR, DNA sequencing and Western blot analysis were utilized to characterize the human platelet Na(+) /Ca(2+) exchangers. Their function during quiescence and collagen-induced activation was determined by measuring [Ca(2+) ](i) with calcium-green/fura-red in response to: changes in the Na(+) and K(+) gradient, NCX pharmacological inhibitors (CBDMB, KB-R7943 and SEA0400) and antibodies specific to extracellular epitopes of the exchangers. KEY RESULTS Human platelets express NCX1.3, NCX3.2 and NCX3.4. The NCXs operate in the Ca(2+) efflux mode in resting platelets and also during their activation with thrombin but not collagen. Collagen-induced increase in [Ca(2+) ](i) was reduced with the pharmacological inhibitors of NCX (CBDMB, KB-R7943 or SEA0400), anti-NCX1 and anti-NCX3. In contrast, anti-NCKX1 enhanced the collagen-induced increase in [Ca(2+) ](i) . CONCLUSIONS AND IMPLICATIONS Human platelets express K(+) -independent Na(+) /Ca(2+) exchangers NCX1.3, NCX3.2 and NCX3.4. During collagen activation, NCX1 and NCX3 transiently reverse to promote Ca(2+) influx, whereas NCKX1 continues to operate in the Ca(2+) efflux mode to reduce [Ca(2+) ](i) .     

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

Summary The modes by which Endothelin-1 (ET) induces Ca²⁺-influx and the relative functional importance of the different sources of Ca²⁺ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca²⁺ level ([Ca²⁺]_i) followed by a tonic contraction in Ca²⁺-containing solution, and produced a transient elevation of [Ca²⁺]_i followed by a small sustained contraction in Ca²⁺-free medium. ET also stimulated ⁴⁵Ca influx into La²⁺-inaccessible fraction significantly. With the same change of [Ca²⁺]_i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca²⁺]_i elevation were not significantly inhibited by 0.1–0.3 M nicardipine which nearly abolished the contraction and [Ca⁺]_i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca²⁺]_i and muscle tension, and vice versa. In Ca²⁺-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca²⁺]_i transient induced by ET. Further, the ET-induced sustained contraction under Ca²⁺-free conditions began to develop after the [Ca²⁺]_i level returned to the baseline. Thus, it seems that the Ca²⁺ released from the ryanodine-sensitive and -insensitive Ca²⁺ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca²⁺-influx through Ca²⁺ channel(s) other than voltage-dependent Ca²⁺ channels in character, and by increasing the sensitivity of the contractile filaments to Ca²⁺ or activating them Ca²⁺-independently.Visiting from Zun Yi Medical College, China Send offprint requests to I. Takayanagi at the above address     

Involvement of Ca(2+)-induced Ca2+ releasing system in interleukin-1beta-associated adenosine release

Interleukin-1beta (IL-1beta) plays an important role in neuroprotective and neurodegenerative events in the central nervous system. To clarify the mechanism of controversial actions of IL-1beta, we determined the effect of IL-1beta, as well as the interaction between IL-1beta and Ca(2+)-induced Ca2+ releasing system (CICR), on adenosine releases in mice hippocampus using mini-slices method. Basal and K(+)-stimulated adenosine releases were regulated by two types of CICRs, including inositol-1,4,5-trisphosphate (IP3) receptor and ryanodine receptor. Lower concentration of IL-1beta increased both adenosine releases, whereas higher concentration did not affect their releases. The stimulatory effect of IL-1beta on basal adenosine release was reduced by removal of extracellular Ca2+ and IP3 receptor inhibitor, while the stimulatory effect of IL-1beta on K(+)-stimulated adenosine release was reduced by ryanodine receptor inhibitor. These results suggest that the potent effect of IL-1beta upon adenosine release might contribute to the neuroprotective action of IL-1beta, whereas IL-1beta-induced neurodegeneration might be due to the overload response of Ca2+ mobilization and the inactivation of adenosine exocytosis.     

Effects of presynaptic modulators on Ca2+-induced noradrenaline relase from cardiac sympathetic nerves

Summary In order to elucidate the mode of action of the Ca²⁺-antagonistic inhibitor nifedipine, its effect on Ca²⁺-mediated action potentials and transmembrane slow inward current in papillary muscles of guinea pigs and cats was studied.Nifedipine (0.5 mg/l1.4×10^–6M) depressed upstroke velocity and overshoot of the Ca²⁺-mediated action potential and reduced the transmembrane slow inward current by about 50%, but the kinetics of inactivation and recovery from inactivation were not affected. The decrease of upstroke velocity was accompanied by a proportional diminution of isometric contractile force. This indicates that nifedipine exerts its Ca²⁺-antagonistic effect on excitation-contraction coupling in mammalian ventricular myocardium by inhibition of the transmembrane Ca²⁺ inward current. The inhibitory action of nifedipine on contractile tension development could be neutralized by an augmentation of the extracellular Ca²⁺ concentration from 2 mM to 4 mM or by -receptor stimulation (isoproterenol) that promotes the transmembrane Ca²⁺-rich medium or under the influence of isoproterenol the upstroke velocity of the Ca²⁺-mediated action potentials rose even above the initial values which were measured prior to the nifedipine administration.     

Dantrolene: effects on abnormal intracellular Ca(2+) handling and inotropy in postinfarcted rat myocardium

The present study was designed to investigate the effects of dantrolene on intracellular Ca(2+) ([Ca(2+)](i)) handling and inotropy in rat infarcted myocardium. Dantrolene-treated rats with myocardial infarction were placed into two different dosage groups. The infarcted control group received placebo only. Isometric contractility and intracellular Ca(2+) transients were recorded simultaneously in isolated papillary muscles. Diastolic [Ca(2+)](i) was significantly lower in muscle preparations from infarcted rats receiving dantrolene compared to the placebo control group. Additionally, treatment with dantrolene in infarcted rats significantly improved the inotropic response to 10(-4) M isoproterenol. The protein levels of the sarcoplasmic reticulum Ca(2+) ATPase were increased in infarcted rat hearts with dantrolene treatment. We conclude that dantrolene improved the inotropic response to beta-adrenoceptor stimulation in rat postinfarcted myocardium, which is related to improved intracellular Ca(2+) handling, and lowered diastolic Ca(2+) concentration.     

Capsaicin sensitizes TRAIL-induced apoptosis through Sp1-mediated DR5 up-regulation: involvement of Ca(2+) influx

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various malignant cells, several cancers including human hepatocellular carcinoma (HCC) exhibit potent resistance to TRAIL-induced cell death. The aim of this study is to evaluate the anti-cancer potential of capsaicin in TRAIL-induced cancer cell death. As indicated by assays that measure phosphatidylserine exposure, mitochondrial activity and activation of caspases, capsaicin potentiated TRAIL-resistant cells to lead to cell death. In addition, we found that capsaicin induces the cell surface expression of TRAIL receptor DR5, but not DR4 through the activation Sp1 on its promoter region. Furthermore, we investigated that capsaicin-induced DR5 expression and apoptosis are inhibited by calcium chelator or inhibitors for calmodulin-dependent protein kinase. Taken together, our data suggest that capsaicin sensitizes TRAIL-mediated HCC cell apoptosis by DR5 up-regulation via calcium influx-dependent Sp1 activation.     

Inhibition of T-type Ca(2+) currents in mouse spermatogenic cells by gossypol, an antifertility compound

Gossypol, a male antifertility compound isolated from cotton, has been proved to inhibit capacitation and the acrosome reaction in human and mammalian sperm. Here, by using whole-cell recording, we observed the effects of gossypol on Ca(2+) and Cl(-) currents in mouse spermatogenic cells obtained by mechanical dissociation. The results showed that gossypol concentration-dependently and irreversibly inhibited T-type Ca(2+) currents in the cells. When the concentration of gossypol was > or =5 microM, the currents were blocked completely. The time to current block was progressively shortened as the gossypol concentration was increased from 5 to 80 microM. Moreover, the drug increased the time constant of inactivation in a concentration-dependent manner, while it did not affect the activation of the current. The inhibitory effect on the T-type Ca(2+) current did not correlate with signaling mediated by G proteins and tyrosine phosphorylation. No obvious effect of gossypol on Cl(-) currents was observed. These data suggest that the gossypol-induced inhibition of T-type Ca(2+) currents could be responsible for the antifertility activity of the compound, indicating a possibility to use gossypol as a local contraceptive drug.     

Group I metabotropic glutamate receptor-induced Ca(2+)-gradients in rat superficial spinal dorsal horn neurons

Here, we investigated changes in the free cytosolic Ca(2+) concentration ([Ca(2+)](i)), induced by the pharmacological activation of metabotropic glutamate receptors (mGluRs), in nociceptive neurons of the superficial spinal dorsal horn. Microfluorometric Ca(2+) measurements with fura-2 in a lumbar spinal cord slice preparation from young rats were used. Bath application of the specific group I mGluR agonist (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) resulted in a distinct increase of [Ca(2+)](i) in most of the neurons in superficial dorsal horn. In contrast, activation of groups II or III mGluRs by DCG-IV or l-AP4, respectively, failed to evoke any significant change in [Ca(2+)](i). The effect of (S)-3,5-DHPG was mediated by both group I subtypes mGluR1 and mGluR5, since combined pre-treatment with the subtype antagonists (S)-4-CPG and MPEP was necessary to abolish the [Ca(2+)](i) increase. Depleting intracellular Ca(2+) stores with CPA or inhibiting IP(3)-receptors with 2-APB, respectively, reduced the (S)-3,5-DHPG-evoked [Ca(2+)](i) increase significantly. Inhibition of voltage-dependent L-type Ca(2+) channels (VDCCs) by verapamil or nicardipine reduced the (S)-3,5-DHPG-induced [Ca(2+)](i) rise likewise. Thus, in rat spinal cord, (S)-3,5-DHPG enhances Ca(2+) signalling in superficial dorsal horn neurons, mediated by the release of Ca(2+) from IP(3)-sensitive intracellular stores and by an influx through L-type VDCCs. This may be relevant to the processing of nociceptive information in the spinal cord.     

Activation by high potassium of a novel voltage-operated Ca2+ channel in rat spleen

1. High potassium produced a concentration-dependent contraction in rat isolated spleen.
2. The high potassium-induced contraction of rat spleen was abolished in Ca²⁺-free Krebs solution containing 1 mM EGTA, and the subsequent addition of 3 mM Ca²⁺ restored the high potassium-induced contraction to the control level.
3. Nifedipine, verapamil, diltiazem, Cd²⁺, Ni²⁺, Co²⁺, R-(+)-Bay K 8644 and pimozide inhibited and relaxed high potassium-induced contraction of rat spleen with IC₅₀ and EC₅₀ values much higher than those values in rat aorta.
4. In addition, high potassium-stimulated contraction of rat spleen was insensitive to ω-conotoxin GVIA, ω-conotoxin MVIIC and ω-agatoxin IVA.
5. The high potassium-induced contraction of rat spleen was also unaffected by tetrodotoxin (TTX), prazosin, chloroethylclonidine (CEC), yohimbine, propranolol, atropine, diphenhydramine, cimetidine, ketanserin, 3-tropanyl-indole-3-carboxylate, saralasin, indomethacin, nordihydroguaiaretic acid, GR32191B, domperidone, naloxone, chlorpromazine, suramin, (±)-2-amino-5-phosphonopentanoic acid, 6,7-dinitroquinoxaline-2,3-dione (DNQX), L-659,877, L-703,606, lorglumide, PD 135,158 N-methyl-D-glucamine, benextramine, amiloride, dantrolene, TMB-8, econazole, staurosporine and neomycin.
6. Forskolin and sodium nitroprusside relaxed high potassium-induced contraction of rat spleen with EC₅₀ values of 0.55±0.04 and 20.0±2.7 μM, respectively.
7. It is concluded that high potassium may activate a novel, pharmacologically uncharacterized voltage-operated Ca²⁺ channel in rat spleen.

     

Ca2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes

BACKGROUND AND PURPOSE

The Ca²⁺ paradox is an important phenomenon associated with Ca²⁺ overload-mediated cellular injury in myocardium. The present study was undertaken to elucidate molecular and cellular mechanisms for the development of the Ca²⁺ paradox.

EXPERIMENTAL APPROACH

Fluorescence imaging was performed on fluo-3 loaded quiescent mouse ventricular myocytes using confocal laser scanning microscope.

KEY RESULTS

The Ca²⁺ paradox was readily evoked by restoration of the extracellular Ca²⁺ following 10–20 min of nominally Ca²⁺-free superfusion. The Ca²⁺ paradox was significantly reduced by blockers of transient receptor potential canonical (TRPC) channels (2-aminoethoxydiphenyl borate, Gd³⁺, La³⁺) and anti-TRPC1 antibody. The sarcoplasmic reticulum (SR) Ca²⁺ content, assessed by caffeine application, gradually declined during Ca²⁺-free superfusion, which was further accelerated by metabolic inhibition. Block of SR Ca²⁺ leak by tetracaine prevented Ca²⁺ paradox. The Na⁺/Ca²⁺ exchange (NCX) blocker KB-R7943 significantly inhibited Ca²⁺ paradox when applied throughout superfusion period, but had little effect when added for a period of 3 min before and during Ca²⁺ restoration. The SR Ca²⁺ content was better preserved during Ca²⁺ depletion by KB-R7943. Immunocytochemistry confirmed the expression of TRPC1, in addition to TRPC3 and TRPC4, in mouse ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

These results provide evidence that (i) the Ca²⁺ paradox is primarily mediated by Ca²⁺ entry through TRPC (probably TRPC1) channels that are presumably activated by SR Ca²⁺ depletion; and (ii) reverse mode NCX contributes little to the Ca²⁺ paradox, whereas inhibition of NCX during Ca²⁺ depletion improves SR Ca²⁺ loading, and is associated with reduced incidence of Ca²⁺ paradox in mouse ventricular myocytes.     

The TRPC channel blocker SKF 96365 inhibits glioblastoma cell growth by enhancing reverse mode of the Na+/Ca2+ exchanger and increasing intracellular Ca2+

BACKGROUND AND PURPOSE

SKF 96365 is well known for its suppressing effect on human glioblastoma growth by inhibiting pre-activated transient receptor potential canonical (TRPC) channels and Ca²⁺ influx. The effect of SKF 96363 on glioblastoma cells, however, may be multifaceted and this possibility has been largely ignored.

EXPERIMENTAL APPROACH

The effects of SKF 96365 on cell cycle and cell viability of cultured human glioblastoma cells were characterized. Western blot, Ca²⁺ imaging and patch clamp recordings were used to delineate cell death mechanisms. siRNA gene knockdown provided additional evidence.

KEY RESULTS

SKF 96365 repressed glioblastoma cell growth via increasing intracellular Ca²⁺ ([Ca²⁺]_i) irrespective of whether TRPC channels were blocked or not. The effect of SKF 96365 primarily resulted from enhanced reverse operation of the Na⁺/Ca²⁺ exchanger (NCX) with an EC₅₀ of 9.79 μM. SKF 96365 arrested the glioblastoma cells in the S and G₂ phases and activated p38-MAPK and JNK, which were all prevented by the Ca²⁺ chelator BAPTA-AM or EGTA. The expression of NCX in glioblastoma cells was significantly higher than in normal human astrocytes. Knockdown of the NCX1 isoforms diminished the effect of SKF 96365 on glioblastoma cells.

CONCLUSIONS AND IMPLICATIONS

At the same concentration, SKF 96365 blocks TRPC channels and enhances the reverse mode of the NCX causing [Ca²⁺]_i accumulation and cytotoxicity. This finding suggests an alternative pharmacological mechanism of SKF 96365. It also indicates that modulation of the NCX is an effective method to disrupt Ca²⁺ homeostasis and suppress human glioblastoma cells.     

Effects of flunarizine on spontaneous synaptic currents in rat neocortex

Flunarizine, a non-selective blocker of voltage-dependent Ca²⁺ and Na⁺ channels, is clinically effective against several neurological disorders, including epilepsy, migraine, and alternating hemiplegia of childhood. We examined the effects of flunarizine on spontaneous post-synaptic currents in acute brain slices maintained in vitro using patch-clamp electrophysiology. Flunarizine significantly attenuated the amplitude of spontaneous currents in pyramidal neurons from juvenile rat neocortex. Flunarizine had no effect on miniature spontaneous events recorded in the presence of tetrodotoxin, a blocker of voltage-dependent sodium channels. In high (9 mM) extracellular potassium, flunarizine reduced the amplitude and frequency of the spontaneous currents. Additionally, dimethyl sulfoxide, the solvent used in our experiments, reduced the amplitude of spontaneous currents, but only in high extracellular potassium. Our data suggest that the clinical activity of flunarizine may in part be a consequence of reducing spontaneous synaptic currents in the neocortex, especially under conditions of heightened neuronal activity.     

Protective effects of Ca2+ handling drugs against abnormal Ca2+ homeostasis and cell damage in myopathic skeletal muscle cells

Deficiency of delta-sarcoglycan (delta-SG), a component of the dystrophin-glycoprotein complex (DGC), causes skeletal muscular dystrophy and cardiomyopathy in BIO14.6 hamsters. Here, we studied the involvement of abnormal Ca2+ homeostasis in muscle degeneration and the protective effect of drugs against Ca2+ handling proteins in vivo as well as in vitro. First, we characterized the properties of cultured myotubes from muscles of normal and BIO14.6 hamsters (30-60 days old). While there were no apparent differences in the levels of expression of various Ca2+ handling proteins (L-type Ca2+ channel, ryanodine receptor, SR-Ca2+ ATPase, and Na+/Ca2+ exchanger), muscle-specific proteins (contractile actin and acetylcholine receptor), or DGC member proteins except SGs, BIO14.6 myotubes showed a high degree of susceptibility to mechanical stressors, such as cyclic stretching and hypo-osmotic stress as compared to normal myotubes, as evidenced by marked increases in creatine phosphokinase (CK) release and bleb formation. BIO14.6 myotubes showed abnormal Ca2+ homeostasis characterized by elevated cytosolic Ca2+ concentration, frequent Ca2+ oscillation, and increased 45Ca2+ uptake. These abnormal Ca2+ events and CK release were significantly prevented by Ca2+ handling drugs, tranilast, diltiazem, and FK506. The calpain inhibitor E64 prevented CK release, but not 45Ca2+ uptake. Some of these drugs (tranilast, diltiazem, and FK506) also exerted a significant protective effect for muscle degeneration in BIO14.6 hamsters and mdx mice in vivo. These observations suggest that elevated Ca2+ entry through sarcolemmal Ca2+ channels predominantly contributes to muscle degeneration and that the drugs tested here may have novel therapeutic potential against muscular dystrophy.