Glatiramer Acetate modulates ion channels expression and calcium homeostasis in B cell of patients with relapsing-remitting multiple sclerosis.

To investigate the effects of Glatiramer Acetate (GA) on B cells by an integrated computational and experimental approach. GA is an immunomodulatory drug approved for the treatment of multiple sclerosis (MS). GA effect on B cells is yet to be fully elucidated. We compared transcriptional profiles of B cells from treatment-naïve relapsing remitting MS patients, treated or not with GA for 6 hours in vitro, and of B cells before and after six months of GA administration in vivo. Microarrays were analyzed with two different computational approaches, one for functional analysis of pathways (Gene Set Enrichment Analysis) and one for the identification of new drug targets (Mode-of-action by Network Analysis). GA modulates the expression of genes involved in immune response and apoptosis. A differential expression of genes encoding ion channels, mostly regulating Ca2+ homeostasis in endoplasmic reticulum (ER) was also observed. Microfluorimetric analysis confirmed this finding, showing a specific GA effect on ER Ca2+ concentration. Our findings unveils a GA regulatory effect on the immune response by influencing B cell phenotype and function. In particular, our results highlight a new functional role for GA in modulating Ca2+ homeostasis in these cells.

Endoplasmic reticulum refilling and mitochondrial calcium extrusion promoted in neurons by NCX1 and NCX3 in ischemic preconditioning are determinant for neuroprotection.

Ischemic preconditioning (IPC), an important endogenous adaptive mechanism of the CNS, renders the brain more tolerant to lethal cerebral ischemia. The molecular mechanisms responsible for the induction and maintenance of ischemic tolerance in the brain are complex and still remain undefined. Considering the increased expression of the two sodium calcium exchanger (NCX) isoforms, NCX1 and NCX3, during cerebral ischemia and the relevance of nitric oxide (NO) in IPC modulation, we investigated whether the activation of the NO/PI3K/Akt pathway induced by IPC could regulate calcium homeostasis through changes in NCX1 and NCX3 expression and activity, thus contributing to ischemic tolerance. To this aim, we set up an in vitro model of IPC by exposing cortical neurons to a 30-min oxygen and glucose deprivation (OGD) followed by 3-h OGD plus reoxygenation. IPC was able to stimulate NCX activity, as revealed by Fura-2AM single-cell microfluorimetry. This effect was mediated by the NO/PI3K/Akt pathway since it was blocked by the following: (a) the NOS inhibitors L-NAME and 7-Nitroindazole, (b) the IP3K/Akt inhibitors LY294002, wortmannin and the Akt-negative dominant, (c) the NCX1 and NCX3 siRNA. Intriguingly, this IPC-mediated upregulation of NCX1 and NCX3 activity may control calcium level within endoplasimc reticulum (ER) and mitochondria, respectively. In fact, IPC-induced NCX1 upregulation produced an increase in ER calcium refilling since this increase was prevented by siNCX1. Moreover, by increasing NCX3 activity, IPC reduced mitochondrial calcium concentration. Accordingly, the inhibition of NCX by CGP37157 reverted this effect, thus suggesting that IPC-induced NCX3-increased activity may improve mitochondrial function during OGD/reoxygenation. Collectively, these results indicate that IPC-induced neuroprotection may occur through the modulation of calcium homeostasis in ER and mitochondria through NO/PI3K/Akt-mediated NCX1 and NCX3 upregulation.

Silencing or knocking out the Na(+)/Ca(2+) exchanger-3 (NCX3) impairs oligodendrocyte differentiation.

Changes in intracellular [Ca(2+)](i) levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation.

ncx1, ncx2, and ncx3 gene product expression and function in neuronal anoxia and brain ischemia.

Over the last few years, although extensive studies have focused on the relevant function played by the sodium-calcium exchanger (NCX) during focal ischemia, a thorough understanding of its role still remains a controversial issue. We explored the consequences of the pharmacological inhibition of this antiporter with conventional pharmacological approach, with the synthetic inhibitory peptide, XIP, or with an antisense strategy on the extent of brain damage induced by the permanent occlusion of middle cerebral artery (pMCAO) in rats. Collectively, the results of these studies suggest that ncx1 and ncx3 genes could be play a major role to limit the severity of ischemic damage probably as they act to dampen [Na+]i and [Ca2+]i overload. This mechanism seems to be normally activated in the ischemic brain as we found a selective upregulation of NCX1 and NCX3 mRNA levels in regions of the brain surviving to an ischemic insult. Despite this transcript increase, NCX1, NCX2, and NCX3 proteins undergo an extensive proteolytic degradation in the ipsilateral cerebral hemisphere. All together these results suggest that a rescue program centered on an increase NCX function and expression could halt the progression of the ischemic damage. On the basis of this evidence we directed our attention to the understanding of the transductional and transcriptional pathways responsible for NCX upregulation. To this aim, we are studying whether the brain isoform of Akt, Akt1, which is a downstream effector of neurotrophic factors, such as NGF can, in addition to affecting the other prosurvival cascades, also exert its neuroprotective effect by modulating the expression and activity of ncx1, ncx2, and ncx3 gene products.

Neuronal NOS activation during oxygen and glucose deprivation triggers cerebellar granule cell death in the later reoxygenation phase.

The present study investigated the temporal relationship between neuronal nitric oxide synthase (nNOS) activity and expression and the development of neuronal damage occurring during anoxia and anoxia followed by reoxygenation. For this purpose, cerebellar granule cells were exposed to 2 hr of oxygen and glucose deprivation (OGD) and 24 hr of reoxygenation. To clarify the consequences of nNOS activity inhibition on neuronal survival, cerebellar granule cells were exposed to OGD, both in the absence of extracellular Na(+) ([Na(+)](e)), a condition that by reducing intracellular Ca(2+) ([Ca(2+)](I)) prevents Ca(2+)-dependent nNOS activation, and in the presence of selective and nonselective nNOS inhibitors, such as N(omega)-L-allyl-L-arginine (L-ALA), N(omega)-propyl-L-arginine (NPLA), and L-nitro-arginine-methyl-ester (L-NAME), respectively. The results demonstrated that the removal of [Na(+)](e) hampered the [Ca(2+)](i) increase and decreased expression and activity of nNOS. Similarly, the increase of free radical production present in cerebellar neurons, exposed previously to OGD and OGD/reoxygenation, was abolished completely in the absence of [Na(+)](e). Furthermore, the absence of [Na(+)](e) in cerebellar neurons exposed to 2 hr of OGD led to the improvement of mitochondrial activity and neuronal survival, both after the OGD phase and after 24 hr of reoxygenation. Finally, the exposure of cerebellar neurons to L-ALA (200 nM), and L-NAME (500 microM) was able to effectively reduce NO(*) production and caused an increase in mitochondrial oxidative activity and an improvement of neuronal survival not only during OGD, but also during reoxygenation. Similar results during OGD were obtained also with NPLA (5 nM), another selective nNOS inhibitor. These data suggest that the activation of nNOS is highly accountable for the neuronal damage occurring during the OGD and reoxygenation phases.

Inhibition of depolarization-induced [3H]noradrenaline release from SH-SY5Y human neuroblastoma cells by some second-generation H(1) receptor antagonists through blockade of store-operated Ca(2+) channels (SOCs).

In the present study, the effect of the blockade of membrane calcium channels activated by intracellular Ca(2+) store depletion on basal and depolarization-induced [3H]norepinephrine ([3H]NE) release from SH-SY5Y human neuroblastoma cells was examined. The second-generation H(1) receptor blockers astemizole, terfenadine, and loratadine, as well as the first-generation compound hydroxyzine, inhibited [3H]NE release induced by high extracellular K(+) concentration ([K(+)](e)) depolarization in a concentration-dependent manner (the IC(50)s were 2.3, 1.7, 4.8, and 9.4 microM, respectively). In contrast, the more hydrophilic second-generation H(1) receptor blocker cetirizine was completely ineffective (0.1-30 microM). The inhibition of high [K(+)](e)-induced [3H]NE release by H(1) receptor blockers seems to be related to their ability to inhibit Ca(2+) channels activated by Ca(i)(2+) store depletion (SOCs). In fact, astemizole, terfenadine, loratadine, and hydroxyzine, but not cetirizine, displayed a dose-dependent inhibitory action on the increase in intracellular Ca(2+) concentrations ([Ca(2+)](i)) obtained with extracellular Ca(2+) reintroduction after Ca(i)(2+) store depletion with thapsigargin (1 microM), an inhibitor of the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pump. The rank order of potency for SOC inhibition by these compounds closely correlated with their inhibitory properties on depolarization-induced [3H]NE release from SH-SY5Y human neuroblastoma cells. Nimodipine (1 microM) plus omega-conotoxin (100 nM) did not interfere with the present model for SOC activation. In addition, the inhibition of depolarization-induced [3H]NE release does not seem to be attributable to the blockade of the K(+) currents carried by the K(+) channels encoded by the human Ether-a-Gogo Related Gene (I(HERG)) by these antihistamines. In fact, whole-cell voltage-clamp experiments revealed that the IC(50) for astemizole-induced hERG blockade is about 300-fold lower than that for the inhibition of high K(+)-induced [3H]NE release. Furthermore, current-clamp experiments in SH-SY5Y cells showed that concentrations of astemizole (3 microM) which were effective in preventing depolarization-induced [3H]NE release were unable to interfere with the cell membrane potential under depolarizing conditions (100 mM [K(+)](e)), suggesting that hERG K(+) channels do not contribute to membrane potential control during exposure to elevated [K(+)](e). Collectively, the results of the present study suggest that, in SH-SY5Y human neuroblastoma cells, the inhibition of SOCs by some second-generation antihistamines can prevent depolarization-induced neurotransmitter release.

Histamine induces exocytosis and IL-6 production from human lung macrophages through interaction with H1 receptors.

Increasing evidence suggests that a continuous release of histamine from mast cells occurs in the airways of asthmatic patients and that histamine may modulate functions of other inflammatory cells such as macrophages. In the present study histamine (10(-9)-10(-6) M) increased in a concentration-dependent fashion the basal release of beta-glucuronidase (EC(50) = 8.2 +/- 3.5 x 10(-9) M) and IL-6 (EC(50) = 9.3 +/- 2.9 x 10(-8) M) from human lung macrophages. Enhancement of beta-glucuronidase release induced by histamine was evident after 30 min and peaked at 90 min, whereas that of IL-6 required 2-6 h of incubation. These effects were reproduced by the H(1) agonist (6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptane carboxamide but not by the H(2) agonist dimaprit. Furthermore, histamine induced a concentration-dependent increase of intracellular Ca(2+) concentrations ([Ca(2+)](i)) that followed three types of response, one characterized by a rapid increase, a second in which [Ca(2+)](i) displays a slow but progressive increase, and a third characterized by an oscillatory pattern. Histamine-induced beta-glucuronidase and IL-6 release and [Ca(2+)](i) elevation were inhibited by the selective H(1) antagonist fexofenadine (10(-7)-10(-4) M), but not by the H(2) antagonist ranitidine. Inhibition of histamine-induced beta-glucuronidase and IL-6 release by fexofenadine was concentration dependent and displayed the characteristics of a competitive antagonism (K(d) = 89 nM). These data demonstrate that histamine induces exocytosis and IL-6 production from human macrophages by activating H(1) receptor and by increasing [Ca(2+)](i) and they suggest that histamine may play a relevant role in the long-term sustainment of allergic inflammation in the airways.

Pharmacological blockade of ERG K(+) channels and Ca(2+) influx through store-operated channels exerts opposite effects on intracellular Ca(2+) oscillations in pituitary GH(3) cells.

In the present study, the effects on intracellular calcium concentration ([Ca(2+)](i)) oscillations of the blockade of ether-a-go-go-related gene (ERG) K(+) channels and of Ca(2+) influx through store-operated channels (SOC) activated by [Ca(2+)](i) store depletion have been studied in GH(3) cells by means of a combination of single-cell fura-2 microfluorimetry and whole-cell mode of the patch-clamp technique. Nanomolar concentrations (1-30 nM) of the piperidinic second-generation antihistamines terfenadine and astemizole and of the class III antiarrhythmic methanesulfonanilide dofetilide, by blocking ERG K(+) channels, increased the frequency and the amplitude of [Ca(2+)](i) oscillations in resting oscillating GH(3) cells. These compounds also induced the appearance of an oscillatory pattern of [Ca(2+)](i) in a subpopulation of nonoscillating GH(3) cells. The effects of ERG K(+) channel blockade on [Ca(2+)](i) oscillations appeared to be due to the activation of L-type Ca(2+) channels, because they were prevented by 300 nM nimodipine. By contrast, the piperazinic second-generation antihistamine cetirizine (0.01-30 microM), which served as a negative control, failed to affect ERG K(+) channels and did not interfere with [Ca(2+)](i) oscillations in GH(3) cells. Interestingly, micromolar concentrations of terfenadine and astemizole (0.3-30 microM), but not of dofetilide (10-100 microM), produced an inhibition of the spontaneous oscillatory pattern of [Ca(2+)](i) changes. This effect was possibly related to an inhibition of SOC, because these compounds inhibited the increase of [Ca(2+)](i) achieved by extracellular calcium reintroduction after intracellular calcium store depletion with the sarcoplasmic or endoplasmic reticulum calcium ATPase pump inhibitor thapsigargin (10 microM) in an extracellular calcium-free medium. The same inhibitory effect on [Ca(2+)](i) oscillations and SOC was observed with the first-generation antihistamine hydroxyzine (1-30 microM), the more hydrophobic metabolic precursor of cetirizine. Collectively, the results of the present study obtained with compounds that interfere in a different concentration range with ERG K(+) channels or SOC suggest that 1) ERG K(+) channels play a relevant role in controlling the oscillatory pattern of [Ca(2+)](i) in resting GH(3) cells and 2) the inhibition of SOC might induce an opposite effect, i.e., an inhibition of [Ca(2+)](i) oscillations.

Sodium nitroprusside prevents chemical hypoxia-induced cell death through iron ions stimulating the activity of the Na+-Ca2+ exchanger in C6 glioma cells.

In C6 glioma cells exposed to chemical hypoxia, an increase of extracellular lactate dehydrogenase (LDH) activity, cell death, and intracellular Ca2+ concentration ([Ca2+]i) occurred. Sodium nitroprusside (SNP), a nitric oxide donor and an iron-containing molecule, reduced chemical hypoxia-induced LDH release and cell death. These effects were counteracted by bepridil and by 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), two specific inhibitors of the Na+-Ca2+ exchanger. SNP also increased the activity of the Na+-Ca2+ exchanger as a Na+ efflux pathway, stimulated by Na+-free conditions and evaluated by monitoring [Ca2+]i in single cells. In addition, SNP produced a further increase of chemical hypoxia-elicited [Ca2+]i elevation, and this effect was blocked by bepridil. Chemical hypoxia-evoked cell death and LDH release were counteracted by the ferricyanide moiety of the SNP molecule, K3Fe(CN)6, and by ferric chloride (FeCl3), and this effect was counteracted by CB-DMB. In addition, the iron ion chelator deferoxamine reversed the protective effect exerted by SNP on cell injury. Collectively, these findings suggest that the protective effect of SNP on C6 glioma cells exposed to chemical hypoxia is due to the activation of the Na+-Ca2+ exchanger operating as a Na+ efflux-Ca2+ influx pathway induced by iron present in the SNP molecule.

Effects of manidipine and nitrendipine enantiomers on the plateau phase of K+-induced intracellular Ca2+ increase in GH3 cells.

The aim of the present study was to investigate whether the chirality and type of substitution at position 3 of the dihydropyridine ring influences the pattern of voltage-gated Ca2+ channel blockade. For this purpose, the effect of R- and S-enantiomers of manidipine and nitrendipine, separated by chiral High-Pressure-Liquid-Chromatography columns, were investigated by fura-2 microfluorimetry during the plateau phase of the intracellular Ca2+ ([Ca2+]i) increase induced by 55 mM K+ and by patch-clamp recording of Ca2+ channel activity in GH3 cells. R- and S-enantiomers of both nitrendipine and manidipine produced a [Ca2+]i decay of the K+-induced plateau phase that followed a biexponential pattern with a 'fast' and a 'slow' phase. The S-configuration of both nitrendipine and manidipine produced a larger [Ca2+]i decrease during the 'fast phase', and a faster and smaller [Ca2+]i decrease in the 'slow phase' than did the R-enantiomers. The S- and R-enantiomers of manidipine, which possess a longer and more lipophilic side chain at position 3 of the dihydropyridine ring, induced a slower [Ca2+]i decrease than that observed with the respective nitrendipine enantiomers. Accordingly, patch-clamp experiments revealed that the S-enantiomers of both dihydropyridines displayed a faster onset of action and produced a greater blockade than the R-enantiomers. These results suggest that the enantiomeric configuration and a small side chain at position 3 of the dihydropyridine ring are factors in the chemical structure which influence the pattern of blockade of voltage-sensitive Ca2+ channels.

Studies on maitotoxin-induced intracellular Ca(2+) elevation in chinese hamster ovary cells stably transfected with cDNAs encoding for L-type Ca(2+) channel subunits.

The aim of the present study was to characterize the role played by different L-type Ca(2+) channel subunits in [Ca(2+)](i) increase induced by maitotoxin (MTX). In the presence of 5 mM extracellular K(+), MTX (0.01-0.5 ng/ml) induced a significant concentration-dependent increase in Fura-2-monitored [Ca(2+)](i) in single Chinese hamster ovary (CHO) cells expressing the alpha(1c) (CHOCalpha9 cells) or the alpha(1c)beta(3)alpha(2)delta (CHOCalpha9beta3alpha2/delta4 cells) subunits of voltage-gated Ca(2+) channels (VGCCs), whereas the effect was much reduced in wild-type CHO cells lacking VGCCs. In addition, MTX effect on CHOCalpha9, CHOCalpha9beta3alpha2/delta4, and GH(3) cells (0.01-0.1 ng/ml) was inhibited by the selective L-type Ca(2+) channel entry-blocker nimodipine (10 microM); a nimodipine-insensitive component was still present, particularly at high (>1 ng/ml) toxin concentrations. In CHOCalpha9beta3alpha2/delta4 cells, depolarizing concentrations of extracellular K(+) (55 mM) reinforced the [Ca(2+)](i) increase induced by MTX (0.1 ng/ml), and this effect was prevented by nimodipine (10 microM). Finally, patch-clamp experiments in CHOCalpha9beta3alpha2/delta4 cells showed that low MTX concentrations (0.03 ng/ml) induced the occurrence of an inward current at -60 mV, which was completely prevented by Cd(2+) (100 microM) and by nimodipine (10 microM), whereas the same dihydropyridine concentration (10 microM) failed to prevent the electrophysiological effects of a higher toxin concentration (3 ng/ml). In conclusion, the results of the present study showed that MTX-induced [Ca(2+)](i) elevation involves two components: 1) an action on L-type VGCCs at the pore-forming alpha(1c) subunit level, which is responsible for the greatest rise of [Ca(2+)](i); and 2) a VGCC-independent mechanism that is present both in excitable and in nonexcitable cells and is responsible for a lower elevation of [Ca(2+)](i).

Involvement of phosphodiesterase-cGMP-PKG pathway in intracellular Ca2+ oscillations in pituitary GH3 cells.

The present study investigates the potential role of the Ca2+-calmodulin-dependent type I phosphodiesterase (PDE)-cGMP-protein kinase G (PKG) pathway in spontaneous [Ca2+]i oscillations in GH3 cells using fura-2 single cell videoimaging. Vinpocetine (2.5-50 microM), a selective inhibitor of type I PDE, induced a concentration-dependent inhibition of spontaneous [Ca2+]i oscillations in these pituitary cells, and at the same time produced an increase of the intracellular cGMP content. The cell permeable cGMP analog N2,2'-O-dibutyryl-cGMP (dB-cGMP) (1 mM) caused a progressive reduction of the frequency and the amplitude of spontaneous [Ca2+]i oscillations when added to the medium. KT5823 (400 nM), a selective inhibitor of cGMP-dependent protein kinase (PKG), produced an increase of baseline [Ca2+]i and the disappearance of spontaneous [Ca2+]i oscillations. When KT5823 was added before vinpocetine, the PKG inhibitor counteracted the [Ca2+]i lowering effect of the cGMP catabolism inhibitor. Finally, the removal of extracellular Ca2+ or the blockade of L-type voltage-sensitive calcium channels (VSCC) by nimodipine produced a decrease of cytosolic cGMP levels. Collectively, the results of the present study suggest that spontaneous [Ca2+]i oscillations in GH3 cells may be regulated by the activity of type I PDE-cGMP-PKG pathway.