Salvatore Amoroso

Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions

Reactive oxygen species (ROS) have been implicated in the pathophysiology of many neurologic disorders and brain dysfunction. In the same pathological settings evidence has been provided in favour of a participation of intracellular Ca(2+) concentration altered homeostasis in the chain of events leading to neuronal apoptosis. In the present review literature reports and experimental data on the relationship between caspase activation and alteration of intracellular calcium concentrations in the mechanisms triggering neuronal apoptosis are discussed. The data gathered support the conclusion that during oxidative stress in neuronal cells the production of ROS triggers a mechanism that, through the release of cytochrome c from mitochondria and caspase-3 activation, leads to apoptosis; the concomitant ROS-mediated elevation of intracellular Ca(2+) concentration triggers caspase-2 activation but both events do not seem to be involved in cell death.

Two Sodium/Calcium Exchanger Gene Products, NCX1 and NCX3, Play a Major Role in the Development of Permanent Focal Cerebral Ischemia

Background and Purpose— The Na+/Ca2+ exchanger, by mediating Ca2+ and Na+ fluxes in a bidirectional way across the synaptic plasma membrane, may play a pivotal role in the events leading to anoxic damage. In the brain, there are 3 different genes coding for 3 different proteins: NCX1, NCX2, and NCX3. The aim of this study was to determine whether NCX1, NCX2, and NCX3 might play a differential role in the development of cerebral injury induced by permanent middle cerebral artery occlusion (pMCAO). Methods— By means of Western blotting, NCX1, NCX2, and NCX3 protein expression was evaluated in the ischemic core and in the remaining nonischemic area of the slice at different time intervals starting from ischemia induction. The role of each isoform was also assessed with antisense oligodeoxynucleotides (ODNs) targeted for each isoform. These ODNs were continuously intracerebroventricularly infused with an osmotic minipump (1 &mgr;L/h) for 48 hours, 24 hours before pMCAO. Results— The results showed that after pMCAO all 3 NCX proteins were downregulated in ischemic core; NCX3 decreased in periinfarctual area whereas NCX1 and NCX2 were unchanged. The ODNs for NCX1 and NCX3 gene products were capable of inducing an increase in the ischemic lesion and to worsen neurological scores. Conclusions— The results of this study suggest that in the neuroprotective effect exerted by NCX during ischemic injury, the major role is prevalently exerted by NCX1 and NCX3 gene products.

Evidence for a protective role played by the Na+/Ca2+ exchanger in cerebral ischemia induced by middle cerebral artery occlusion in male rats ☆

In the present paper, the role played by Na+/Ca2+ exchanger (NCX) in focal cerebral ischemia was investigated. To this aim, permanent middle cerebral artery occlusion (pMCAO) was performed in male rats. The effects on the infarct volume of some inhibitors, such as tyrosine-6 glycosylated form of the exchanger inhibitory peptide (GLU-XIP), benzamil derivative (CB-DMB) and diarylaminopropylamine derivative (bepridil), and of the NCX activator, FeCl3, were examined. FeCl3, CB-DMB, bepridil and GLU-XIP, a modified peptide synthesized in our laboratory in order to facilitate its entrance into the cells through the glucose transporter, were intracerebroventricularly (i.c.v.) infused. FeCl3 (10 microg/kg) was able to reduce the extension of brain infarct volume. This effect was counteracted by the concomitant icv administration of CB-DMB (120 microg/kg). All NCX inhibitors, GLU-XIP, CB-DMB and bepridil, caused a worsening of the brain infarct lesion. These results suggest that a stimulation of NCX activity may help neurons and glial cells that are not irreversibly damaged in the penumbral zone to survive, whereas its pharmacological blockade can compromise their survival.

Pharmacological characterization of serotonin receptors involved in the control of prolactin secretion

The present study was undertaken to characterize the type of serotonin (5-HT) receptors involved in the control of prolactin (PRL) secretion in male rats. d-Fenfluramine (10 mg/kg i.p.), a potent 5-HT releaser and quipazine, (20 mg/kg i.p.) a 5-HT agonist, caused a marked increase in serum PRL levels. Ritanserin (200 micrograms/kg i.p.), a specific antagonist of 5-HT2 receptors, administered 1 h before the administration of d-fenfluramine or quipazine, completely prevented the PRL-releasing effect of these drugs. Furthermore, the administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) (1.5, 3 and 6 mg/kg i.p.), a compound considered to be a prototypical 5-HT1A agonist, failed to induce any change in serum PRL levels. The same lack of effect on PRL secretion was observed after the administration of 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridin-4-yl)-1-H-indole (RU 24969) (1, 3 and 10 mg/kg i.p.), a compound which has been shown to possess a higher selectivity for 5-HT1B receptor subtypes than for 5-HT1A subtypes. These results suggest that 5-HT receptors involved in the control of PRL secretion are of the 5-HT2 type.

Sodium Nitroprusside Prevents Chemical Hypoxia-Induced Cell Death Through Iron Ions Stimulating the Activity of the Na+-Ca2+ Exchanger in C6 Glioma Cells

Abstract: 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 hypoxiaevoked 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.

Role of the mitochondrial sodium/calcium exchanger in neuronal physiology and in the pathogenesis of neurological diseases.

In neurons, as in other excitable cells, mitochondria extrude Ca(2+) ions from their matrix in exchange with cytosolic Na(+) ions. This exchange is mediated by a specific transporter located in the inner mitochondrial membrane, the mitochondrial Na(+)/Ca(2+) exchanger (NCX(mito)). The stoichiometry of NCX(mito)-operated Na(+)/Ca(2+) exchange has been the subject of a long controversy, but evidence of an electrogenic 3 Na(+)/1 Ca(2+) exchange is increasing. Although the molecular identity of NCX(mito) is still undetermined, data obtained in our laboratory suggest that besides the long-sought and as yet unfound mitochondrial-specific NCX, the three isoforms of plasmamembrane NCX can contribute to NCX(mito) in neurons and astrocytes. NCX(mito) has a role in controlling neuronal Ca(2+) homeostasis and neuronal bioenergetics. Indeed, by cycling the Ca(2+) ions captured by mitochondria back to the cytosol, NCX(mito) determines a shoulder in neuronal [Ca(2+)](c) responses to neurotransmitters and depolarizing stimuli which may then outlast stimulus duration. This persistent NCX(mito)-dependent Ca(2+) release has a role in post-tetanic potentiation, a form of short-term synaptic plasticity. By controlling [Ca(2+)](m) NCX(mito) regulates the activity of the Ca(2+)-sensitive enzymes pyruvate-, alpha-ketoglutarate- and isocitrate-dehydrogenases and affects the activity of the respiratory chain. Convincing experimental evidence suggests that supraphysiological activation of NCX(mito) contributes to neuronal cell death in the ischemic brain and, in epileptic neurons coping with seizure-induced ion overload, reduces the ability to reestablish normal ionic homeostasis. These data suggest that NCX(mito) could represent an important target for the development of new neurological drugs.

Differential vulnerability of cortical and cerebellar neurons in primary culture to oxygen glucose deprivation followed by reoxygenation

The effects of glucose and O2 deprivation (OGD) on the survival of cortical and cerebellar neurons were examined to characterize the biochemical mechanisms involved in OGD and OGD followed by reoxygenation. To this aim, neurons were kept for different time periods in a hypoxic chamber with a controlled atmosphere of 95% N2 and 5% CO2 in a glucose‐free medium. After OGD, reoxygenation was achieved by exposing the cells to normal O2 and glucose levels. Neither MTT, an index of mitochondrial oxidative phosphorylation, nor malondialdehyde (MDA) production, a parameter measuring lipid peroxidation, were affected by 1 hr of OGD in cortical neurons. When OGD was followed by 24 hr of reoxygenation, MTT levels were reduced by 40% and MDA was significantly increased, whereas cellular ATP content did not change. Cerebellar granule cells, on the other hand, did not show any reduction of mitochondrial activity after exposure to 1 hr OGD or to 1 hr OGD plus 24 hr of reoxygenation. When OGD was prolonged for 2 hr, a significant reduction of the mitochondrial activity and of cellular ATP content occurred, coupled to a significant MDA increase in cerebellar granule cells, whereas in cortical neurons a reduction of MTT levels after 2 hr OGD was not accompanied by a decrease of cellular ATP content nor by an increase of MDA production. Moreover, 24 hr of reoxygenation further reinforced lipid peroxidation, LDH release, propidium iodide positive neurons and the reduction of ATP content in cerebellar granule cells. The results of the present study collectively show that cortical and cerebellar neurons display different levels of vulnerability to reoxygenation followed by OGD. Furthermore, the impairment of mitochondrial activity and the consequent overproduction of free radicals in neurons were observed for the first time occurring not only during the reoxygenation phase, but already beginning during the OGD phase. J. Neurosci. Res. 63:20–26, 2001.

Pharmacological evidence that the activation of the Na+‐Ca2+ exchanger protects C6 glioma cells during chemical hypoxia

In C6 glioma cells exposed to chemical hypoxia a massive release of lactate dehydrogenase (LDH) occurred at 3 and 6 h, coupled with an increased number of propidium‐iodide positive dead cells. Extracellular Na+ removal, which activates the Na+‐Ca2+ exchanger as a Na+ efflux pathway and prevents Na+ entrance, significantly reduced LDH release and the number of propidium iodide positive C6 cells. During chemical hypoxia, in the presence of extracellular Na+ ions, a progressive increase of [Ca2+]i occurred; in the absence of extracellular Na+ ions [Ca2+]i was enhanced to a greater extent. The blockade of the Na+‐Ca2+ exchanger by the amiloride derivative 5‐(N‐4‐chlorobenzyl)‐2′,4′‐dimethylbenzamil (CB‐DMB), lanthanum (La3+) and the Ca2+ chelator EGTA, completely reverted the protective effect exerted by the removal of Na+ ions on C6 glioma cells exposed to chemical hypoxia. The inhibition of the Na+‐Ca2+ antiporter enhanced chemical hypoxia‐induced LDH release when C6 glioma cells were incubated in the presence of physiological concentrations of extracellular Na+ ions (145 mM), suggesting that the blockade of the Na+‐Ca2+ antiporter during chemical hypoxia can lead to increased cell damage. Collectively, these results suggest that activation of the Na+‐Ca2+ exchanger protects C6 glioma cells exposed to chemical hypoxia, whereas its pharmacological blockade can exacerbate cellular injury.

Involvement of PI3′-K, mitogen-activated protein kinase and protein kinase B in the up-regulation of the expression of nNOSα and nNOSβ splicing variants induced by PRL-receptor activation in GH3 cells

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 NO•. In conclusion, PRL receptor activation up‐regulated the expression of both nNOSα and nNOSβ proteins via a PTK, PI3′‐K, MAPK and PKB signalling transduction components. This action may represent the molecular mechanism by which PRL exerts the ‘short‐loop’ feedback on its own secretion.

Prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 increases glutamate uptake through overexpression of GLT1 and EAAC1 glutamate transporter subtypes in rat frontal cerebral cortex

Prenatal exposure to the CB1 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone) mesylate (WIN) at a daily dose of 0.5 mg/kg, and Delta9-tetrahydrocannabinol (Delta9-THC) at a daily dose of 5 mg/kg, reduced dialysate glutamate levels in frontal cerebral cortex of adolescent offspring (40-day-old) with respect to those born from vehicle-treated mothers. WIN treatment induced a statistically significant enhancement of Vmaxl-[3H]glutamate uptake, whereas it did not modify glutamate Km, in frontal cerebral cortex synaptosomes of adolescent rats. Western blotting analysis, performed either in membrane proteins derived from homogenates and in proteins extracted from synaptosomes of frontal cerebral cortex, revealed that prenatal WIN exposure enhanced the expression of glutamate transporter 1 (GLT1) and excitatory amino acid carrier 1 (EAAC1). Moreover, immunocytochemical analyses of frontal cortex area revealed a more intense GLT1 and EAAC1 immunoreactivity (ir) distribution in the WIN-treated group. Collectively these results show that prenatal exposure to the cannabinoid CB1 receptor agonist WIN increases expression and functional activity of GLT1 and EAAC1 glutamate transporters (GluTs) associated to a decrease of cortical glutamate outflow, in adolescent rats. These findings may contribute to explain the mechanism underlying the cognitive impairment observed in the offspring of mothers who used marijuana during pregnancy.