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Due to the neuroprotective role of the Na+/Ca2+exchanger (NCX) isoforms NCX1 and NCX3, we synthesized novel benzodiazepinone derivatives of the unique NCX activator Neurounina-1, named compounds 1–19. The derivatives are characterized by a benzodiazepinonic nucleus linked to five- or six-membered cyclic amines via a methylene, ethylene, or acetyl spacer. The compounds have been screened on NCX1/NCX3 isoform activities by a high-throughput screening approach, and the most promising were characterized by patch-clamp electrophysiology and Fura-2AM video imaging. We identified two novel modulators of NCX: compound 4, inhibiting NCX1 reverse mode, and compound 14, enhancing NCX1 and NCX3 activity. Compound 1displayed neuroprotection in two preclinical models of brain ischemia. The analysis of the conformational and steric features led to the identification of the molecular volume required for selective NCX1 activation for mixed NCX1/NCX3 activation or for NCX1 inhibition, providing the first prototypal model for the design of optimized isoform modulators.

Acid-sensitive ion channels (ASICs) are sodium channels partially permeable to Ca2+ions, listed among putative targets in central nervous system (CNS) diseases in which a pH modification occurs. We targeted novel compounds able to modulate ASIC1 and to reduce the progression of ischemic brain injury. We rationally designed and synthesized several diminazene-inspired diaryl mono- and bis-guanyl hydrazones. A correlation between their predicted docking affinities for the acidic pocket (AcP site) in chicken ASIC1 and their inhibition of homo- and heteromeric hASIC1 channels in HEK-293 cells was found. Their activity on murine ASIC1a currents and their selectivity vs mASIC2a were assessed in engineered CHO-K1 cells, highlighting a limited isoform selectivity. Neuroprotective effects were confirmed in vitro, on primary rat cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation, and in vivo, in ischemic mice. Early lead 3b, showing a good selectivity for hASIC1 in human neurons, was neuroprotective against focal ischemia induced in mice.

While the urotensinergic system plays a role in influencing various pathologies, its potential remains untapped because of the absence of therapeutically effective urotensin II receptor (UTR) modulators. Herein, we developed analogues of human urotensin II (hU-II) peptide in which, along with well-known antagonist-oriented modifications, the Glu1residue was subjected to single-point mutations. The generated library was tested by a calcium mobilization assay and ex vivo experiments, also in competition with selected ligands. Interestingly, many derivatives showed noncompetitive modulation that was rationalized by the lateral allostery concept applied to a G protein-coupled receptor (GPCR) multimeric model. UPG-108showed an unprecedented ability to double the efficacy of hU-II, while UPG-109and UPG-111turned out to be negative allosteric modulators of UTR. Overall, our investigation will serve to explore and highlight the expanding possibilities of modulating the UTR system through N-terminally modified hU-IIanalogues and, furthermore, will aim to elucidate the intricate nature of such a GPCR system.

Synthetic cathinones are commonly abused novel psychoactive substances (NPS) that are consumed alone or in combination with other psychoactive drugs via the oral, intranasal, or intravenous routes. These substances induce physiological and subjective effects typical of psychostimulant drugs, although with a faster onset and shorter duration when compared to amphetamine derivatives. A common consequence of their short half-lives and duration of effects, is repeated administration or bingeing that results in an increase in the number of associated deaths. New synthetic cathinones, now in the fourth generation, have increasing potency and toxicity as compared to those from previous generations. NPS may interfere with the electrical activity of the catecholaminergic neuron, causing damages to neuronal function. The aim of this research is to investigate the effects of 3-methylmethcathinone (3-MMC) on excitability of neuronal cells to identify mechanisms underlying its neurotoxicity.

Amyotrophic lateral sclerosis (ALS) is a severe human adult-onset neurodegenerative disease affecting lower and upper motor neurons. In >20% of cases, the familial form of ALS is caused by mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1). Interestingly, administration of wild-type SOD1 to SOD1G93Atransgenic rats ameliorates motor symptoms through an unknown mechanism. Here we investigated whether the neuroprotective effects of SOD1 are due to the Ca2+-dependent activation of such prosurvival signaling pathway and not to its catalytic activity. To this aim, we also examined the mechanism of neuroprotective action of ApoSOD1, the metal-depleted state of SOD1 that lacks dismutase activity, in differentiated motor neuron-like NSC-34 cells and in primary motor neurons exposed to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA). Preincubation of ApoSOD1 and SOD1, but not of human recombinant SOD1G93A, prevented cell death in motor neurons exposed to L-BMAA. Moreover, ApoSOD1 elicited ERK1/2 and Akt phosphorylation in motor neurons through an early increase of intracellular Ca2+concentration ([Ca2+]i). Accordingly, inhibition of ERK1/2 by siMEK1 and PD98059 counteracted ApoSOD1- and SOD1-induced neuroprotection. Similarly, transfection of the dominant-negative form of Akt in NSC-34 motor neurons and treatment with the selective PI3K inhibitor LY294002 prevented ApoSOD1- and SOD1-mediated neuroprotective effects in L-BMAA-treated motor neurons. Furthermore, ApoSOD1 and SOD1 prevented the expression of the two markers of L-BMAA-induced ER stress GRP78 and caspase-12. Collectively, our data indicate that ApoSOD1, which is devoid of any catalytic dismutase activity, exerts a neuroprotective effect through an early activation of Ca2+/Akt/ERK1/2 pro-survival pathway that, in turn, prevents ER stress in a neurotoxic model of ALS.

Activation of CBs on HLMs reduces production of lymph/angiogenic factors; a possible novel strategy to modulate macrophage‐assisted vascular remodeling. Macrophages are pivotal effector cells in immune responses and tissue remodeling by producing a wide spectrum of mediators, including angiogenic and lymphangiogenic factors. Activation of cannabinoid receptor types 1 and 2 has been suggested as a new strategy to modulate angiogenesis in vitro and in vivo. We investigated whether human lung‐resident macrophages express a complete endocannabinoid system by assessing their production of endocannabinoids and expression of cannabinoid receptors. Unstimulated human lung macrophage produce 2‐arachidonoylglycerol, N‐arachidonoyl‐ethanolamine, N‐palmitoyl‐ethanolamine, and N‐oleoyl‐ethanolamine. On LPS stimulation, human lung macrophages selectively synthesize 2‐arachidonoylglycerol in a calcium‐dependent manner. Human lung macrophages express cannabinoid receptor types 1 and 2, and their activation induces ERK1/2 phosphorylation and reactive oxygen species generation. Cannabinoid receptor activation by the specific synthetic agonists ACEA and JWH‐133 (but not the endogenous agonist 2‐arachidonoylglycerol) markedly inhibits LPS‐induced production of vascular endothelial growth factor‐A, vascular endothelial growth factor‐C, and angiopoietins and modestly affects IL‐6 secretion. No significant modulation of TNF‐α or IL‐8/CXCL8 release was observed. The production of vascular endothelial growth factor‐A by human monocyte‐derived macrophages is not modulated by activation of cannabinoid receptor types 1 and 2. Given the prominent role of macrophage‐assisted vascular remodeling in many tumors, we identified the expression of cannabinoid receptors in lung cancer‐associated macrophages. Our results demonstrate that cannabinoid receptor activation selectively inhibits the release of angiogenic and lymphangiogenic factors from human lung macrophage but not from monocyte‐derived macrophages. Activation of cannabinoid receptors on tissue‐resident macrophages might be a novel strategy to modulate macrophage‐assisted vascular remodeling in cancer and chronic inflammation.

Previous studies have demonstrated that the knockdown or knockout of the three Na+/Ca2+exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, worsens ischemic brain damage. This suggests that the activation of these antiporters exerts a neuroprotective action against stroke damage. However, drugs able to increase the activity of NCXs are not yet available. We have here succeeded in synthesizing a new compound, named neurounina-1 (7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one), provided with an high lipophilicity index and able to increase NCX activity. Ca2+radiotracer, Fura-2 microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC50in the picomolar to low nanomolar range, whereas it did not affect NCX3 activity. Furthermore, by using chimera strategy and site-directed mutagenesis, three specific molecular determinants of NCX1 responsible for neurounina-1 activity were identified in the α-repeats. Interestingly, NCX3 became responsive to neurounina-1 when both α-repeats were replaced with the corresponding regions of NCX1. In vitro studies showed that 10 nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced γ-aminobutyric acid (GABA) release, enhanced GABAAcurrents, and inhibited both glutamate release and N-methyl-d-aspartate receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered at doses of 0.003 to 30 μg/kg i.p., it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3 to 5 hours after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities.

Chronic exposure to polychlorinated biphenyls (PCBs), a class of ubiquitous environmental toxicants, causes neurocognitive anomalies. The transcription factor repressor element 1-silencing transcription factor (REST) plays a critical role in neuronal phenotype elaboration in both neural progenitor cells and non-neuronal cells. Here, we investigated the possible relationship between PCBs and REST in neuroblastoma SH-SY5Y cells. In these cells, chronic exposure to the PCB mixture Aroclor 1254 (A-1254; 5-30 μg/ml) caused dose-dependent cell death via the induction of calpain but not caspase-3. Intriguingly, this effect was prevented by the calpain inhibitor calpeptin. Furthermore, A-1254 enhanced REST mRNA and protein expression levels after both 24 and 48 h. REST down-regulation by small interfering RNA prevented A-1254-induced cell death. In addition, A-1254 enhanced the binding of REST to the synapsin 1 gene promoter, and synapsin 1 knockdown potentiated A-1254-induced cell death. A-1254 (10 μg/ml) also increased the expression of the two REST cofactors, the REST corepressor and the mammalian SIN3 homolog A transcription regulator. Moreover, the PCB mixture decreased acetylation of the histone proteins H3 and H4. It is noteworthy that the histone deacetylase inhibitor trichostatin A prevented such decreases and reduced the A-1254-induced neurotoxic effect. Collectively, these results suggest that A-1254 exerts its toxic effect via REST by down-regulating synapsin 1 and decreasing H3 and H4 acetylation.

In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and NCX3 exchangers was investigated in baby hamster kidney cells singly transfected with each of these isoforms by single-cell Fura-2-microfluorometry and patch clamp. Furthermore, the molecular determinants of NO on each isoform were identified by deletion, site-directed mutagenesis, and chimera strategies. Our data revealed four main findings. First, the NO-donor S-nitroso-N-acetylpenicillamine (SNAP; 10 nM) and the NO-precursor L-arginine (10 mM) were both able to increase NCX1 activity in a cGMP-independent way. Moreover, within the amino acid sequence 723 to 734 of the f-loop, Cys730 resulted as the target of NO on NCX1. Second, SNAP and L-arginine were able to increase NCX2 activity, but this effect was prevented by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In addition, the membrane-permeable 8-bromoguanosine-cGMP alone was able to mimic the stimulatory effect of the gaseous mediator, suggesting the involvement of a cGMP-dependent mechanism. Within the amino acid sequence 699 to 744 of the f-loop, Ser713 was the NO molecular determinant on the NCX2 protein; Third, NCX3 activity was instead down-regulated by NO in a cGMP-independent manner. This NO-inhibitory action was exerted at the level of Cys156 in the α1-region outside the f-loop. Finally, the activity of the two NCX3 chimeras—obtained by the replacement of the NO-insensitive NCX3 region with the homologous NO-sensitive segments of NCX1 or NCX2—was potentiated by SNAP. Together, the present data demonstrate that NO differently regulates the activity of the three gene products NCX1, NCX2, and NCX3 by modulating specific molecular determinants.

The sodium–calcium exchanger NCX1 represents a key mediator for maintaining Naiand Ca2iin anoxic conditions. To date, no information is available on NCX1 protein expression and activity in microglial cells under ischemic conditions.

With the help of single-cell microflorimetry, (45)Ca(2+) radiotracer fluxes, and patch-clamp in whole-cell configuration, we examined the effect of the amiloride derivative 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) on the activity of the three isoforms of the Na(+)/Ca(2+) exchanger (NCX) and on several other membrane currents including voltage- and pH-sensitive ones. This amiloride analog suppressed the bidirectional activity of all NCX isoforms in a concentration-dependent manner. The IC(50) values of CB-DMB were in the nanomolar range for the outward and the inward components of the bidirectional NCX1, NCX2, and NCX3 activity. Deletion mutagenesis showed that CB-DMB inhibited NCX activity mainly at level of the f-loop but not through the interaction with Gly833 located at the level of the alpha(2) repeat. On the other hand, CB-DMB suppressed in the micromolar range the other plasma membrane currents encoded by voltage-dependent Ca(2+) channels, tetrodotoxin-sensitive Na(+) channels, and pH-sensitive ASIC1a. Collectively, the data of the present study showed that CB-DMB, when used in the nanomolar range, is one of the most potent compounds that can block the activity of the three NCX isoforms when they work both in the forward and in the reverse modes of operation without interfering with other ionic channels.

The 3 gene products of the Na/Ca2exchanger (NCX), viz, NCX1, NCX2, and NCX3, may play a pivotal role in the pathophysiology of brain ischemia. The aim of this study was to investigate the transductional and posttranslational mechanisms involved in the expression of these isoforms during oxygen and glucose deprivation and their role in endoplasmic reticulum Ca2refilling in cortical neurons.

The proteins NCX1, NCX2, and NCX3 expressed on the plasma membrane of neurons play a crucial role in ionic regulation because they are the major bidirectional system promoting the efflux and influx of Na+and Ca2+ions. Here, we demonstrate that NCX1 and NCX3 proteins are novel additional targets for the survival action of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Indeed, the doxycycline-dependent overexpression of constitutively active Akt1 in tetracycline (Tet)-Off PC-12 positive mutants and the exposure of Tet-Off PC-12 wild type to nerve growth factor induced an up-regulation of NCX1 and NCX3 proteins. NCX1 up-regulation induced by Akt1 activation occurred at the transcriptional level because NCX1 mRNA increased, and it was counteracted by cAMP response element-binding protein 1 inhibition through small interfering RNA strategy. In contrast, Akt1-induced NCX3 up-regulation recognized a post-transcriptional mechanism occurring at the proteasome level because 1) NCX3 transcript did not increase and 2) the proteasome inhibitor N-benzyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) did not further enhance NCX3 protein levels in Akt1 active mutants as it would be expected if the ubiquitin-proteasome complex was not already blocked by Akt1 pathway. As expected, in PC-12 Tet-Off wild-type cells MG-132 enhanced NCX3 protein levels. This up-regulation produced an increased activity of NCX function. Furthermore, NCX1 and NCX3 up-regulation contributed to the survival action of Akt1 during chemical hypoxia because both the silencing of NCX1 or NCX3 and the pharmacological paninhibition of NCX isoforms reduced the prosurvival property of Akt1. Together, these results indicated that NCX1 and NCX3 represent novel additional molecular targets for the prosurvival action of PI3-K/Akt pathway.

Nitric oxide (NO) is critical for the normal physiological regulation of the nervous system and other tissues. l-Arginine, but not d-arginine, is the natural substrate for nitric oxide synthase (NOS), for it is enzymatically converted to NO and l-citrulline. However, recent evidence suggests that d-arginine can also produce NO and NO-derivatives via a different pathway. The aim of the present paper was to raise NO levels in the cells by increasing the cell permeation of its precursors. To this aim, two galactosyl prodrugs, l-arginine-d-galactos-6‘-yl ester (l-ArgGal) and d-arginine-d-galactos-6‘-yl ester (d-ArgGal) were synthesized. Remarkably, using the HPLC-ESI/MS technique, we found that l-ArgGal and d-ArgGal prodrugs both increased the concentration levels of l- and d-arginine and their derivatives in pituitary GH3cells. Furthermore, we found that d-ArgGal (1) penetrated cell membranes more rapidly than its precursor d-arginine, (2) released arginine more slowly and in greater amounts than l-ArgGal, and (3) produced much higher levels of DAF-2 monitored NO and nitrite than did l-ArgGal under the same experimental conditions. In conclusion, these results indicate that an increase in the cell permeation of l- and d-arginine by l-ArgGal and d-ArgGal can lead to an increase in NO levels.

The present study explores the hypothesis that the new anti-epileptic drug levetiracetam (LEV) could interfere with the inositol 1,4,5-trisphosphate (IP(3))-dependent release of intracellular Ca(2+) initiated by G(q)-coupled receptor activation, a process that plays a role in triggering and maintaining seizures. We assessed the effect of LEV on the amplitude of [Ca(2+)](i) response to bradykinin (BK) and ATP in single Fura-2/acetoxymethyl ester-loaded PC12 rat pheochromocytoma cells, which express very high levels of LEV binding sites. LEV dose-dependently reduced the [Ca(2+)](i) increase, elicited either by 1 microM BK or by 100 microM ATP (IC(50), 0.39 +/- 0.01 microM for BK and 0.20 +/- 0.01 microM for ATP; Hill coefficients, 1.33 +/- 0.04 for BK and 1.38 +/- 0.06 for ATP). Interestingly, although the discharge of ryanodine stores by a process of calcium-induced calcium release also took place as part of the [Ca(2+)](i) response to BK, LEV inhibitory effect was mainly exerted on the IP(3)-dependent stores. In fact, the drug was still effective after the pharmacological blockade of ryanodine receptors. Furthermore, LEV did not affect Ca(2+) stored in the intracellular deposits since it did not reduce the amplitude of [Ca(2+)](i) response either to thapsigargin or to ionomycin. In conclusion, LEV inhibits Ca(2+) release from the IP(3)-sensitive stores without reducing Ca(2+) storage into these deposits. Because of the relevant implications of IP(3)-dependent Ca(2+) release in neuron excitability and epileptogenesis, this novel effect of LEV could provide a useful insight into the mechanisms underlying its antiepileptic properties.

An ever increasing number of reports shows the involvement of free radicals in the functional and structural changes occurring in the brain as a part of the "normal" aging process. Given that plasma membrane and intracellular ion channels play a critical role in maintaining intracellular ion homeostasis, which is crucial for neuronal cell survival, in the present review we have attempted to elaborate on the idea that functional changes in ion channel activity induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) might occur during the aging process. To this aim, we have reviewed the available literature and the data obtained in our laboratory on the ability of ROS and RNS to modify the activity of several plasma membrane and intracellular ion channels and transporters, in an attempt to correlate such changes with those occurring with the aging process. Particular emphasis is given to voltage-gated Na, Ca, and K channels, although second messenger-activated channels like Ca- and ATP-dependent K channels, and intracellular channels controlling intracellular Ca storage and release will also be discussed. On the basis of the available data it is not yet possible to establish a strict correlation between the changes in neuronal electrophysiological properties induced by oxidative modification at the level of ion channels and the neurodegenerative process accompanying brain aging. However, an increasing amount of information suggests that the modulatory effects exerted by ROS and RNS on ion channel proteins might have a relevant role for neuronal cell survival or death. Obviously, more work is needed to establish the possible involvement of ion channels and of their modulation by ROS and RNS as important mechanisms for the aging process. Only when a more complete molecular picture of the aging process will be available, it will be possible to test the fascinating hypothesis that aging might be pharmacologically delayed by modulating ROS and RNS action on ion channels or the biochemical pathways involved in their modulation.