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Prostate cancer (PCa) is the most commonly diagnosed malignancy in men and the second leading cause of cancer-related death in industrialized countries. Epidemiologic evidence suggests that obesity promotes aggressive PCa. Recently, a family of Free Fatty Acid (FFA) receptors (FFARs) has been identified and reported to affect several crucial biological functions of tumor cells such as proliferation, invasiveness, and apoptosis. Here we report that oleic acid (OA), one of the most prevalent FFA in human plasma, increases proliferation of highly malignant PC3 and DU-145 PCa cells. Furthermore, docetaxel cytotoxic action, the first-line chemotherapeutic agent for the treatment of androgen-independent PCa, was significantly reduced in the presence of OA, when measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, suggesting that this FFA plays also a role in chemoresistance. OA induced intracellular calcium increase, in part due to the store operated calcium entry (SOCE), measured by a calcium imaging technique. Moreover, PI3K/Akt signaling pathway was enhanced, as revealed by increased Akt phosphorylation levels. Intriguingly, attenuating the expression of FFA1/GPR40, a receptor for long chain FFA including OA, prevented the OA-induced effects. Of relevance, we found that FFA1/GPR40 is significantly overexpressed in tissue specimens of PCa, compared to benign prostatic hyperplasia tissues, at both mRNA and protein expression level, analyzed by Real Time RT-PCR and immunofluorescence experiments, respectively. Our data suggest that OA promotes an aggressive phenotype in PCa cells via FFA1/GPR40, calcium and PI3K/Akt signaling. Thus, FFA1/GPR40, might represent a potential useful prognostic biomarker and therapeutic target for the treatment of advanced PCa. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 122, 911-922. Abstract We evaluated whether changes in expression and activity of the three sodium/calcium exchanger isoforms, NCX1, NCX2, and NCX3 occurred in PC12 cells when the extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs) were silenced, pharmacologically blocked, or activated with nerve growth factor (NGF). Several findings suggesting that MAPKs control NCX emerged: (1) A decrease in NCX1 and NCX3 basal expression occurred when JNK or MEK1, the extracellular-signal-regulated kinases 1/2 upstream activator, were pharmacologically blocked, respectively; (2) NGF increased cAMP response element-binding 1 (CREB1) and Specificity Protein 1 (Sp1) binding to ncx1 promoter and CREB1 binding to two different sequences close to ncx2 transcription start site on genomic DNA; (3) An up-regulation of NCX1 and NCX3, abrogated upon either MEK1 or p38 blockade, and a down-regulation of NCX2, abolished upon p38 blockade, occurred upon NGF-induced MAPK activation. The NCX1 up-regulation was abolished upon either CREB1 or Sp1 silencing, whereas NCX2 down-regulation was abrogated only by CREB1 silencing. The NCX3 up-regulation was unaffected by CREB1 or Sp1 silencing and abolished upon proteasomal inhibition; (4) Whole-cell Na+/Ca2+ exchange decreased when MEK1 and JNK were blocked and increased when MAPKs were activated by NGF. Collectively, these results demonstrate a MAPK-dependent regulation of NCX expression and activity which could be relevant in mediating some of the effects of MAPKs in neurons. [ABSTRACT FROM AUTHOR]

In this study, the functional consequences of the pharmacological modulation of the M-current ( IKM) on cytoplasmic Ca2+ intracellular Ca2+concentration ([Ca2+]i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. Kv7.2 immunoreactivity was identified in pre-synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K+]e, the IKM activator retigabine (RT, 10 μM) inhibited [3H]d-aspartate ([3H]d-Asp) release and caused membrane hyperpolarization; both these effects were prevented by the IKM blocker XE-991 (20 μM). The IKM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS-204352 (10 μM) inhibited 20 mM [K+]e-induced synaptosomal [Ca2+]i increases; XE-991 (20 μM) abolished RT-induced inhibition of depolarization-triggered [Ca2+]i transients. The P/Q-type voltage-sensitive Ca2+channel (VSCC) blocker ω-agatoxin IVA prevented RT-induced inhibition of depolarization-induced [Ca2+]i increase and [3H]d-Asp release, whereas the N-type blocker ω-conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca2+]i and the resulting enhancement of [3H]d-Asp release induced by [Ca2+]i mobilization from intracellular stores, or by store-operated Ca2+channel activation. Collectively, the present data reveal that the pharmacological activation of IKM regulates depolarization-induced [3H]d-Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca2+]i occurring through P/Q-type VSCCs. [ABSTRACT FROM AUTHOR]

The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, it is not clear whether SOD1 secretion is only constitutive or can be regulated in an activity-dependent fashion. Using rat pituitary GH3 cells that express voltage-dependent calcium channels and are subjected to Ca2+ oscillations, we found that treatment with high K+-induced SOD1 release that was significantly higher than the constitutive secretion. Evoked SOD1 release was correlated with depolarization-dependent calcium influx and was virtually abolished by removal of extracellular calcium with EGTA or by pre-incubation of GH3 cells with Botulinum toxin A that cleaves the SNARE protein SNAP-25. Immunofluorescence experiments performed in GH3 cells and rat brain synaptosomes showed that K+-depolarization induced a marked depletion of intracellular SOD1 immunoreactivity, an effect that was again abolished in the absence of extracellular calcium or after treatment with Botulinum toxin A. Subcellular fractionation analysis showed that SOD1 was present in large dense core vesicles. These data clearly show that, in addition to the constitutive SOD1 secretion, depolarization induces an additional rapid calcium-dependent SOD1 release in GH3 cells and in rat brain synaptosomes. This likely occurs through exocytosis from SOD1-containing vesicles operated by the SNARE complex. [ABSTRACT FROM AUTHOR]

Chemical hypoxia produces depletion of ATP, intracellular Ca2+ overload, and cell death. The role of Na+/Ca2+ exchanger (NCX), the major plasma membrane Ca2+ extruding system, has been explored in chemical hypoxia using BHK cells stably transfected with the three mammalian NCX isoforms: NCX1, NCX2, and NCX3. Here we report that the three isoforms show similar activity evaluated as [Ca2+]i increase evoked by Na+-free medium exposure in Fura-2-loaded single cells and NCX3 transfected cells are less vulnerable to chemical hypoxia compared to NCX1- and NCX2-transfected cells, suggesting that NCX3 could play a more relevant protective role during chemical hypoxia. [ABSTRACT FROM AUTHOR]

Increased activity of plasma membrane K+ channels, leading to decreased cytoplasmic K+ concentrations, occurs during neuronal cell death. In the present study, we showed that the neurotoxic β-amyloid peptide Aβ25−35 caused a dose-dependent (0.1–10 µ m) and time-dependent (> 12 h) enhancement of both inactivating and non-inactivating components of voltage-dependent K+ (VGK) currents in nerve growth factor-differentiated rat phaeochromocytoma (PC-12) cells and primary rat hippocampal neurones. Similar effects were exerted by Aβ1−42, but not by the non-neurotoxic Aβ35−25 peptide. Aβ25−35 and Aβ1−42 caused an early (15–20 min) increase in intracellular Ca2+ concentration. This led to an increased production of reactive oxygen species (ROS), which peaked at 3 h and lasted for 24 h; ROS production seemed to trigger the VGK current increase as vitamin E (50 µ m) blocked both the Aβ25−35- and Aβ1−42-induced ROS increase and VGK current enhancement. Inhibition of protein synthesis (cycloheximide, 1 µg/mL) and transcription (actinomycin D, 50 ng/mL) blocked Aβ25−35-induced VGK current enhancement, suggesting that this potentiation is mediated by transcriptional activation induced by ROS. Interestingly, the specific nuclear factor-κB inhibitor SN-50 (5 µ m), but not its inactive analogue SN-50M (5 µ m), fully counteracted Aβ1−42- or Aβ25−35-induced enhancement of VGK currents, providing evidence for a role of this family of transcription factors in regulating neuronal K+ channel function during exposure to Aβ. [ABSTRACT FROM AUTHOR]

Abstract It is well known that GH-PRL secreting GH3 cells express constitutive neuronal nitric oxide synthase (nNOS) and produce nitric oxide (NO• ). In addition, these cells possess plasma membrane prolactin (PRL) receptors which can be responsible for an autocrine ‘short-loop’ feedback. The aim of the present study was to investigate whether the activation of PRL receptors modulates the expression of the different spliced forms of nNOS gene, and the transductional mechanisms involved in this action. In GH3 cells, both exon 2-containing nNOSα and exon 2-lacking nNOSβ were time-dependently expressed, whereas the other two isoforms eNOS and iNOS were not. The antibodies directed against the residues 53–68 of the external domain common to both the long and short form of rat PRL receptors, and the selective D2 agonist cabergoline (1 nm) reduced both basal and exogenous PRL-induced expressions of nNOSα and nNOSβ, but to a greater extent for the β splicing form. In line with these results, oPRL (1 and 10 μm) added to the incubation medium increased to a greater extent the expression of nNOSβ form than of the nNOSα. The receptor and non-receptor protein tyrosine kinase (PTK) inhibitors, genistein (10 μm), the Src -specific tyrosine kinase inhibitor PP2 (100 μm), the MAPK inhibitor PD 098059 (50 nm) and the two PI3′-K inhibitors, wortmannin (300 nm) and LY-294002 (25 μm) prevented both basal and exogenous PRL-induced expression of nNOSα and nNOSβ isoforms. In addition, exogenous PRL induced a phosphorylation of protein kinase B (PKB) (Akt) that was prevented both by the two MAPK inhibitors PD 098059 and U 0126, and by the PI3′-K inhibitors wortmannin and LY-294002. Up-regulation of the expression of the two splicing forms of nNOS elicited by PRL-receptor activation was mirrored by the increased synthesis of... [ABSTRACT FROM AUTHOR]

Abstract: In C6 glioma cells exposed to chemical hypoxia, an increase of extracellular lactate dehydrogenase (LDH) activity, cell death, and intracellular Ca[sup 2+] concentration ([Ca[sup 2+]][sub 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[sup +]-Ca[sup 2+] exchanger. SNP also increased the activity of the Na[sup +]-Ca[sup 2+] exchanger as a Na[sup +] efflux pathway, stimulated by Na[sup +]-free conditions and evaluated by monitoring [Ca[sup 2+]][sub i] in single cells. In addition, SNP produced a further increase of chemical hypoxia-elicited [Ca[sup 2+]][sub 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, K[sub 3]Fe(CN)[sub 6], and by ferric chloride (FeCl[sub 3]), 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[sup +]-Ca[sup 2+] exchanger operating as a Na[sup +] efflux--Ca[sup 2+] influx pathway induced by iron present in the SNP molecule. Key Words: Na[sup +]-Ca[sup 2+] exchanger--Sodium nitroprusside--Iron--Chemical hypoxia--C6 glioma--Cell survival. J... [ABSTRACT FROM AUTHOR]

Glutamate signaling may orchestrate oligodendrocyte precursor cell (OPC) development and myelin regeneration through the activation of glutamate receptors at OPC‐neuron synapses. D‐Aspartate is a D‐amino acid exerting modulatory actions at glutamatergic synapses. Chronic administration of D‐Aspartate has been proposed as therapeutic treatment in diseases related to myelin dysfunction and NMDA receptors hypofunction, including schizophrenia and cognitive deficits. Here, we show, by using an in vivo remyelination model, that administration of D‐Aspartate during remyelination improved motor coordination, accelerated myelin recovery, and significantly increased the number of small‐diameter myelinated axons. Chronically administered during demyelination, D‐Aspartate also attenuated myelin loss and inflammation. Interestingly, D‐Aspartate exposure stimulated OPC maturation and accelerated developmental myelination in organotypic cerebellar slices. D‐Aspartate promoting effects on OPC maturation involved the activation of glutamate transporters, AMPA and NMDA receptors, and the Na+/Ca2+ exchanger NCX3. While blocking NMDA or NCX3 significantly prevented D‐Aspartate‐induced [Ca2+]i oscillations, blocking AMPA and glutamate transporters prevented both the initial and oscillatory [Ca2+]i response as well as D‐Aspartate‐induced inward currents in OPC. Our findings reveal that D‐Aspartate treatment may represent a novel strategy for promoting myelin recovery. Synopsis: Glutamate signaling is critical for oligodendrocyte precursor cell (OPC) repair responses. D‐Aspartate exerts modulatory actions at glutamatergic synapses. D‐Aspartate treatment is here shown to stimulate oligodendrocyte development and benefits demyelination and remyelination processes in vivo. D‐Aspartate exposure promoted OPC maturation and stimulated developmental myelination in organotypic cerebellar slices.D‐Aspartate treatment attenuated demyelination and accelerated remyelination in the cuprizone mouse model of myelin damage and repair.D‐Aspartate boosting effects on OPC differentiation involved an orchestrated stimulation of calcium signalling pathways that are consequent to a cooperative activation of glutamate transporters and AMPA receptors, which then leads to a secondary NMDA receptor and NCX3 exchanger effects. Glutamate signaling is critical for oligodendrocyte precursor cell (OPC) repair responses. D‐Aspartate exerts modulatory actions at glutamatergic synapses. D‐Aspartate treatment is here shown to stimulate oligodendrocyte development and benefits demyelination and remyelination processes in vivo. [ABSTRACT FROM AUTHOR]

Glutamate signaling may orchestrate oligodendrocyte precursor cell (OPC) development and myelin regeneration through the activation of glutamate receptors at OPC-neuron synapses. D-Aspartate is a D-amino acid exerting modulatory actions at glutamatergic synapses. Chronic administration of D-Aspartate has been proposed as therapeutic treatment in diseases related to myelin dysfunction and NMDA receptors hypofunction, including schizophrenia and cognitive deficits. Here, we show, by using an in vivo remyelination model, that administration of D-Aspartate during remyelination improved motor coordination, accelerated myelin recovery, and significantly increased the number of small-diameter myelinated axons. Chronically administered during demyelination, D-Aspartate also attenuated myelin loss and inflammation. Interestingly, D-Aspartate exposure stimulated OPC maturation and accelerated developmental myelination in organotypic cerebellar slices. D-Aspartate promoting effects on OPC maturation involved the activation of glutamate transporters, AMPA and NMDA receptors, and the Na + /Ca 2+ exchanger NCX3. While blocking NMDA or NCX3 significantly prevented D-Aspartate-induced [Ca 2+ ] i oscillations, blocking AMPA and glutamate transporters prevented both the initial and oscillatory [Ca 2+ ] i response as well as D-Aspartate-induced inward currents in OPC Our findings reveal that D-Aspartate treatment may represent a novel strategy for promoting myelin recovery., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)