Biomedical subjects

J Camarasa

Publications and source records attributed to J Camarasa.

At least 19 recordsLinked to original sources

PubMed2006

Protection against MDMA-induced dopaminergic neurotoxicity in mice by methyllycaconitine: involvement of nicotinic receptors.

Methylenedioxymethamphetamine (MDMA) is a relatively selective dopaminergic neurotoxin in mice. Previous studies demonstrated the participation of alpha-7 nicotinic receptors (nAChR) in the neurotoxic effect of methamphetamine. The aim of this paper was to study the role of this receptor type in the acute effects and neurotoxicity of MDMA in mice. In vivo, methyllycaconitine (MLA), a specific alpha-7 nAChR antagonist, significantly prevented MDMA-induced neurotoxicity at dopaminergic but not at serotonergic level, without affecting MDMA-induced hyperthermia. Glial activation was also fully prevented by MLA. In vitro, MDMA induced intrasynaptosomal reactive oxygen species (ROS) generation, which was calcium-, nitric-oxide synthase-, and protein kinase C-dependent. Also, the increase in ROS was prevented by MLA and alpha-bungarotoxin. Experiments with reserpine point to endogenous dopamine (DA) as the main source of MDMA-induced ROS. MLA also brought the MDMA-induced inhibition of [3H]DA uptake down, from 73% to 11%. We demonstrate that a coordinated activation of alpha-7 nAChR, blockade of DA transporter function and displacement of DA from intracellular stores induced by MDMA produces a neurotoxic effect that can be prevented by MLA, suggesting that alpha-7 nAChR have a key role in the MDMA neurotoxicity in mice; however, the involvement of nicotinic receptors containing the beta2 subunit cannot be conclusively ruled out.

Aconitine↗

PubMed2003

3-Amino thioacridone, a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in neurons.

The mechanisms underlying selective neuronal cell death in kainic acid-mediated neurodegeneration are not fully understood. We have recently demonstrated that in cerebellar granule neurons, kainic acid induces the expression of proteins associated with cell-cycle progression. In the present study we show that 3-amino thioacridone (3-ATA), a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in cerebellar granule neurons. When neurons were pre-treated with 3-ATA 10 microM for 24 h, they were less susceptible to damage induced by kainic acid 500 microM, since the number of dead cells decreased significantly. In flow cytometry studies using propidium iodide staining, 3-ATA also reduced the ratio of apoptotic cells induced by kainic acid. Moreover, 3-ATA decreased the proportion of cells with a condensed nucleus from 55% to 22%. Our data suggest that the cell cycle pathway is involved in the mechanism of apoptosis mediated by kainic acid and that cyclin-dependent kinase 4 plays a prominent role in this process. 3-ATA may to prevent the apoptosis associated with neurodegenerative disorders without the over-activation of excitatory amino acid receptors.

Aminoacridines↗

PubMed2001

C-phycocyanin protects cerebellar granule cells from low potassium/serum deprivation-induced apoptosis.

We tested the potential cytoprotective role of C-phycocyanin in rat cerebellar granule cell cultures. Cell death was induced by potassium and serum (K/S) withdrawal. Cell viability was studied using the neutral red assay and laser scanning cytometry with propidium iodide as fluorochrome. C-phycocyanin (1-3 mg/ml) showed a neuroprotective effect against 24 h of K/S deprivation in cerebellar granule cells. After 4 h K/S deprivation this compound (3 mg/ml) inhibited formation of reactive oxygen species, measured as 2',7'-dichlorofluorescein fluorescence, showing its scavenger capability. Pre-treatment with C-phycocyanin reduced thymidine incorporation into DNA below control values and reduced dramatically apoptotic bodies as visualized by propidium iodide, indicating inhibition of apoptosis induced by K/S deprivation. Flow cytometry studies, using propidium iodide in TritonX100 permeabilized cells, indicated that 24 h K/S deprivation acts as a proliferative signal for cerebellar granule cells, which show an increase in S-phase percentage and cells progressed into the apoptotic pathway. C-phycocyanin protected cerebellar granule cells from the apoptosis induced by deprivation. These results suggest that C-phycocyanin prevents apoptosis in cerebellar granule cells probably through the antioxidant activity. It is proposed that K/S deprivation-induced apoptosis could be due, in part, to an alteration in the cell cycle mediated by an oxidative stress mechanism.

Animals↗

PubMed2001

Orphenadrine prevents 3-nitropropionic acid-induced neurotoxicity in vitro and in vivo.

1. Previous studies indicate that 3-nitropropionic acid (3-NPA) neurotoxicity involves the excitotoxic activation of N-methyl-D-aspartate (NMDA) receptors. Thus, we examined the effect of orphenadrine (an anticholinergic drug with NMDA receptor antagonist properties) on 3-NPA neurotoxicity in both cultured rat cerebellar granule cells (CGCs) and in rats. 2. Orphenadrine protected CGCs from 3-NPA-induced mortality, as assessed by both the neutral red viability assay and laser scanning cytometry, using propidium iodide staining. 3. For rats, two indirect markers of neuronal damage were used: the binding of [(3)H]-PK 11195 to the peripheral-type benzodiazepine receptor (PBR), a microglial marker, and expression of the 27 kD heat-shock protein (HSP27), a marker of activated astroglia. Systemic administration of 3-NPA (30 mg kg(-1) per day for 3 days) induced a 170% increase in [(3)H]-PK 11195 binding, and expression of HSP27. 4. Both the increase in [(3)H]-PK 11195 and HSP 27 expression were prevented by previous administration of 30 mg kg(-1) per day of orphenadrine for 3 days. Lower doses (10 and 20 mg kg(-1)) had no protective effect. Orphenadrine also reduced 3-NPA-induced mortality in a dose-dependent manner. 5. We propose that orphenadrine or orphenadrine-like drugs could be used to treat neurodegenerative disorders mediated by overactivation of NMDA receptors.

Animals↗

PubMed2000

Further characterization of an adenosine transport system in the mitochondrial fraction of rat testis.

Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [3H]nitrobenzylthioinosine ([3H]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [3H]adenosine uptake assays. This system (K(m)=2+/-1.3 microM; V(max)=86.2+/-15.5 pmol/mg protein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [3H]Adenosine incorporation was potently inhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC(50)=3 nM) although NBTI inhibited this uptake weakly (IC(50)=72. 7+/-37.1 microM). Dilazep>dipyridamole>/=hexobendine inhibited [3H]adenosine incorporation at low micromolar concentrations. The nucleosides inosine and uridine were weak inhibitors of this system. The adenosine receptor ligands N(6)-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptake only at micromolar concentrations. Neither 5'-(N-ethylcarboxamido)-adenosine (NECA) nor theophylline inhibited adenosine uptake by more than 60% but the mitochodrial benzodiazepine receptor ligands 4'-chloro-diazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl) isoquinoline carboxamide (PK 11195) were able to inhibit it. The lack of inhibition by the blockers of the mitochondrial adenine-nucleotide carrier, atractyloside and alpha, beta-methylene-ATP, indicates that [3H]adenosine uptake occurs via a transporter other than this carrier. All these results support the existence of an equilibrative adenosine transport system, which might mediate the passage of adenosine formed in the mitochondria to the cytoplasm.

Adenosine↗

PubMed2000

Inhibitors of NO-synthase and donors of NO modulate kainic acid-induced damage in the rat hippocampus.

The effects of nitric oxide synthase (NOS) inhibitors, N(omega)-nitro-L-arginine and 7-nitroindazole, and the NOS substrate L-arginine on kainic acid (KA)-induced microglial reactivity and stress response were studied in the hippocampus 7 and 1 days after KA, respectively. Density of peripheral-type benzodiazepine receptors was measured as an index of microglial reactivity. Histological damage in hippocampus was evaluated at 7 days by neuronal counting. KA increased the maximal number of binding sites (B(max)) versus controls. Administration of either 7-nitroindazole (25 mg/kg) or N(omega)-nitro-L-arginine (20 and 50 mg/kg) 24 hr before KA, further increased B(max). This later effect was abolished by L-arginine (1 g/kg), which given 24 hr before KA decreased B(max) to control values. Also, KA-induced HSP72 stress response was attenuated by pre-treatment with L-arginine. Histological evaluation showed reduced cell numbers in the pyramidal cell layer of the hippocampus in groups receiving KA, either alone or in combination with 7-nitroindazole. Administration of L-arginine before KA attenuated neuronal loss in CA3 but not CA1. A clear protective effect was observed, however, in CA1 and CA3, in rats receiving both L-arginine plus 7-nitroindazole before KA. The results show that the combination of a NO substrate with a NOS inhibitor reduces the neurotoxic effects of KA in the rat hippocampus. This study suggests that extremely fine regulation of NO levels in the different neural cell types can modulate excitotoxicity.

Animals↗

PubMed2000

MPP(+) injection into rat substantia nigra causes secondary glial activation but not cell death in the ipsilateral striatum.

Injection of MPP(+) into the substantia nigra causes extensive necrosis and anterograde degeneration of pars compacta dopaminergic neurons. We studied secondary effects in the ipsilateral striatum by examining dopaminergic terminals, signs of neuronal damage, and glial reactivity at 1, 2, 3, and 7 days after injection of MPP(+) into the substantia nigra. Dopaminergic terminals and uptake sites were evaluated with [(3)H]GBR-12935 binding and tyrosine hydroxylase immunoreactivity. Glial reaction was examined with markers of astrocytes and microglia. Stereology was used to evaluate any changes in neuronal density. Tyrosine hydroxylase immunoreactivity and [(3)H]GBR-12935 binding markedly decreased (74%) from days 2 to 7. Loss of dopaminergic terminals in the ipsilateral striatum was accompanied by an intense astroglial and, to a lesser extent, microglial reaction. However, no signs of cell damage, neuronal loss, or disruption of the blood-brain barrier were found in the striatum. Resident astroglial and microglial cells showed a morphological shift and notable changes in protein expression typical of glial reactivity, yet the presence of macrophage-like cells was not detected. This study shows that injection of MPP(+) in the substantia nigra causes a secondary reaction within the ipsilateral striatum involving the transformation of quiescent glia to reactive glia. It is suggested that stimuli derived from damaged dopaminergic terminals within the striatum are able to activate resident glia and that this glial transformation may promote repair and regeneration.

1-Methyl-4-phenylpyridinium↗

PubMed2000

Chronic urticaria: diagnostic recommendations.

A group of European recognized dermatologists - the European Working Group on the Diagnosis of Chronic Urticaria - met on 12 March 1999 to discuss best practice for assessing and diagnosing patients with chronic urticaria. These are their recommendations. The recommendations will be valuable for the majority of cases, but are not exhaustive and may not include every possible precipitating factor.

Chronic Disease↗

PubMed1999

Effects of U-83836E on glutamate-induced neurotoxicity in dissociated rat cerebellar granule cells.

The effects of the lazaroid compound U-83836E on the glutamate-induced production of reactive oxygen species (ROS) were studied in dissociated rat cerebellar granule cells by flow cytometry. U-83836E completely inhibited ROS production with an estimated IC50 value of 21.7 +/- 9.1 nM. However, U-83836E did not inhibit the glutamate-evoked decrease in mitochondrial membrane potential (MMP). Nevertheless, U-83836E (10 nM to 10 microM) prevented cell death induced by 10 mM of glutamate. At concentrations above 10 microM, U-83836E by itself showed slight cytotoxicity, which was significant at a 100 microM concentration. U-83836E (25 to 200 microM) also increased the cytosolic calcium levels in a concentration-dependent manner. Our results indicate that the cytotoxic effects found at micromolar concentrations of U-83836E could be explained by an increase in [Ca2+]i. Finally, since U-83836E did not prevent the MMP decrease evoked by glutamate, it is suggested that antioxidant pharmacotherapy would not be sufficient to block the neurotoxic effects of glutamate.

Animals↗

PubMed1999

In vitro and in vivo protective effect of orphenadrine on glutamate neurotoxicity.

The anticholinergic drug orphenadrine is used in the treatment of Parkinson's disease. In this study we evaluate the neuroprotective effects of orphenadrine on excitotoxicity in vivo and in vitro. Orphenadrine prevented the mitochondrial and the cytoplasmic membrane potential decrease evoked by NMDA (100 microM) in rat dissociated cerebellar granule cells showing an IC50 value of 11.6 +/- 4.7 microM (mean +/- SEM, n = 5) and 13.5 +/- 2.3 microM (n = 3), respectively. Orphenadrine was able to protect cerebellar granule cell cultures from glutamate-induced neurotoxicity. Kainic acid (KA, 10 mg/kg)-induced excitotoxicity was evaluated in vivo using the microglial marker peripheral-type benzodiazepine receptor (PBR) and heat shock protein 72 (HSP72) expression in the hippocampus. The Bmax of PBR for control tissues was 589.1 +/- 40.0 fmol/mg protein (n = 4), increasing to 1692.5 +/- 51.6 fmol/mg protein (n = 5) after the KA treatment. Pretreatment with orphenadrine (10 mg/kg) blocked the KA-induced increase in PBR density. As expected, KA-administration induced the expression of HSP72 that was blocked in the orphenadrine + KA-treated rats. We demonstrate that orphenadrine, interacting at the NMDA receptor, is able to prevent the neurotoxicity mediated by activation at glutamate ionotropic receptors.

Animals↗

PubMed1999

Evaluation of free radical production, mitochondrial membrane potential and cytoplasmic calcium in mammalian neurons by flow cytometry.

The overexcitation of glutamate receptors is believed to be the cause of several neurodegenerative disorders. The determination of calcium fluxes, mitochondrial membrane potential (MMP) variations or the production of reactive oxygen species (ROS) in mammalian cells are usually measured during the development of potentially useful drugs that might interfere in the events induced by glutamate receptor activation. By using flow cytometry with dissociated cerebellar granule cells, we have developed a rapid and economical method to measure changes in biochemical parameters that are involved in neuronal cell death. The formation of intracellular ROS is measured using 2',7'-dichlorofluorescin diacetate (DCFH-DA). The mitochondrial membrane potential is assessed by the retention of rhodamine 123 (Rh123), a specific fluorescent cationic dye that is readily sequestered by active mitochondria, depending on their transmembrane potential. Finally, intracellular calcium increases are detected by using the calcium-selective indicator Indo-1. Cell viability is also assessed by using propidium iodide (PI) which stains DNA strands of permeabilized cells. This method might be useful for the screening of new drugs with potential neuroprotective activity, with improved cost/effectiveness ratio compared to other techniques.

Animals↗

PubMed1999

Assessment of the adrenergic effects of orphenadrine in rat vas deferens.

The peripheral adrenergic effects of orphenadrine, an antiparkinsonian drug, have been evaluated in the rat vas deferens to investigate whether these properties are the same as those of other phencyclidine ligands. In the low micromolar range, orphenadrine enhanced electrically-evoked and exogenous noradrenaline contractile responses in the epididymal portion of rat vas deferens. It also induced spontaneous activity that was inhibited by prazosin (1 microM) but not by atropine (20 nM). It inhibited accumulation of [3H]noradrenaline in rat vas deferens (IC50 = 14.2+/-2.3 microM). Orphenadrine competitively inhibited [3H]nisoxetine binding in rat vas deferens membranes (Ki = 1.05+/-0.20 microM). It can be concluded that orphenadrine, at low micromolar concentrations, interacts with the noradrenaline reuptake system inhibiting its functionality and thus potentiating the effect of noradrenaline.

Adrenergic Agents↗

PubMed1998

Microgliosis and down-regulation of adenosine transporter induced by methamphetamine in rats.

Chronic administration of methamphetamine to rats induces neurotoxicity characterized by a loss of striatal dopaminergic terminals and reactive gliosis. Subcutaneous administration of methamphetamine in a scheduled procedure of four doses (10 mg/kg) at 2 h interval also induces a significant increase in the peripheral-type benzodiazepine receptor (PBR) density. This increase is maximum (76%) at 72 h post-treatment in the striatum and disappears at 7 days, suggesting that microglia may have a predominant role in necrosis-phagocytosis of neuronal debris rather than acting in a restorative manner. Microgliosis is not restricted to the striatum since it is also evident in cerebellum (75.4% of PBR increase) and hippocampus (37.2% of PBR increase). In the areas with high density of adenosine transporter, the microgliosis phenomenon correlates well with a decrease of this nucleoside transporter (about 39%). Although the microgliosis and the decrease in adenosine transporter could be parallel and not related events, we can speculate that when microglia are activated, a down-regulation of adenosine transporter occurs, playing a role in tissue homeostasis. With the same dosing schedule, methamphetamine induces HSP72 expression in both cytoplasmic and nuclear fractions of the striatum, cerebellum and hippocampus. This expression is also evident in the cerebral cortex, where adenosine transporter population did not show any variation.

Adenosine↗

PubMed1998

Flow cytometric study of mitochondrial dysfunction after AMPA receptor activation.

The effect of AMPA-receptor stimulation on MMP and on the concentration of intracellular calcium ([Ca2+]i) was studied in dissociated CGC from rat pups, by flow cytometry. In the presence of cyclothiazide, AMPA induced a sodium-independent decrease in MMP up to 30.7+/-2.5%. This effect was antagonized by CNQX and NBQX. Mepacrine and dibucaine reversed the effect of AMPA on MMP, suggesting that it is mediated by a release of arachidonic acid. AMPA alone induced a slight (about 7%) increase in [Ca2+]i. In the presence of cyclothiazide, AMPA induced a concentration-dependent [Ca2+]i increase up to 29.10+/-2.10% that was not reversed by flunarizine. This increase was similar to that observed in a Na+-free medium, and was antagonized by CNQX and NBQX, but not by MK-801. Mitochondria play a key role in the modulation of [Ca2+]i since a significant [Ca2+]i increase was found in the presence of FCCP. On the other hand, the dantrolene-sensitive calcium pools do not participate in the [Ca2+]i increase induced by stimulation of AMPA receptors. It is concluded that when AMPA-receptor desensitization is blocked, a decrease in MMP and an increase in [Ca2+]i occurs, which could be additional events to potentiate neuronal cell death induced by glutamate.

Animals↗

PubMed1998

U-83836E prevents kainic acid-induced neuronal damage.

The effect of kainic acid (KA) on mitochondrial membrane potential (MMP) and reactive-oxygen species (ROS) production was studied in dissociated cerebellar granule cells from rat pups. KA induced a maximum increase of 361%+/-35% in ROS production. The lazaroid compound U-83836E (at concentrations ranging from 10(-9) to 5x10(-6) M) completely inhibited this increase, with an IC50 value of 3.02+/-1.08x10(-7) M. KA also decreased the mitochondrial membrane potential (MMP), with a maximum decrease of about 30%. Absence of Na+ in the incubation medium did not significantly alter the effect of KA on MMP. As expected, the AMPA/kainate receptor antagonist NBQX inhibited the effects of KA on MMP with an IC50 value of 1.1+/-0.8 microM. However, the lazaroid U-83836E, indomethacin, nor-dihydroguaiaretic acid and L-nitroarginine all failed to inhibit the KA-induced decrease in the MMP. Finally, to assess the neuroprotective effect of U-83836E on KA-induced neurotoxicity in vivo, the increase in the peripheral-type benzodiazepine receptor density in rat hippocampus was measured. Treatment with KA increased the Bmax to 1341+/-192 fmol mg(-1). When U-83836E was coadministered with KA, the Bmax was reduced to 765+/-122 fmol mg(-1), which was not significantly different from the Bmax obtained from untreated rats (Bmax: 518+/-33 fmol mg(-1)). We conclude that treatment with the lazaroid U-83836E might be a suitable therapeutic strategy in neurodegenerative disorders.

Animals↗

PubMed1998

Characterization of [3H]nisoxetine binding in rat vas deferens membranes: modulation by sigma and PCP ligands.

Sigma (sigma) and phencyclidine (PCP) receptor ligands, apart from their main effects on sigma receptors and NMDA receptor-mediated neurotransmission, have been found to interact with catecholamine systems in several central and peripheral tissues. In the present study the binding profile of [3H]nisoxetine ([3H]NIS), a selective marker of the noradrenaline transporter, has been characterized in rat vas deferens membranes to further study its modulation by a number of characteristic sigma and PCP ligands. The binding of [3H]NIS was found to be of high affinity (Kd = 1.63 +/- 0.36 nM), saturable, sodium-dependent and to a single population of binding sites (nH = 1.003 +/- 0.017). The maximal binding capacity was 1,625 +/- 500 fmol/mg of protein. Kinetic experiments gave a k(+1) of 3.9 x 10(7) min(-1)M(-1) and a k(-1) of 0.005 min(-1). The [3H]NIS binding was totally inhibited, with IC50 values in the micromolar range, by all the sigma and PCP ligands tested, with the following order of potency: haloperidol > dextromethorphan > dizocilpine > dextrorphan > (+)-3-PPP > PCP > tenocyclidine. This order correlates well with that described in other tissues using [3H]desmethylimipramine. The inhibition by all these compounds, except that of (+)-3-PPP, was competitive. These results suggest that sigma and PCP ligands bind, at low micromolar concentrations, to a site in the noradrenaline transporter that is labelled by [3H]NIS.

Animals↗

PubMed1998

Reactive oxygen production by glutamate agonists in dissociated cerebellar cells: a flow cytometric study.

1. The effect of glutamate, N-methyl-D-aspartate (NMDA) and kainate on radical oxygen species (ROS) production and calcium influx was studied in dissociated cerebellar granule cells with the use of flow cytometry. 2. Glutamate and NMDA induced an intracellular ROS increase by an activation of NMDA receptors. 3. (+)MK-801 inhibited the effect on ROS production of both agonists (IC50 values of 1.52 +/- 0.05 and 0.71 +/- 0.02 microM, respectively). 4. (+)MK-801 inhibited the intracellular calcium increase induced by glutamate and NMDA, whereas 6-cyano-7-nitroquinoxaline-2,3-dione inhibited that induced by kainate. 5. NG-Nitro-L-arginine, but not nitrendipine, inhibited the ROS production induced by glutamate agonists. Consequently, nitric oxide synthase might play an important role in the neurotoxic process induced by excitatory amino acids.

Animals↗

PubMed1998

Effect of PCP and sigma ligands on both noradrenaline- and electrically-induced contractions and on [3H]-noradrenaline uptake in rat vas deferens.

1. Electrically induced contractions of the epididymal portion of rat vas deferens were potentiated in concentration-dependent manner (0.1-30 microM) by different sigma and PCP receptor ligands (PCP, TCP, (+)-MK-801, dextromethorphan and (+)-3-PPP); dextrorphan did it in a minor extent. 2. Sigma and PCP receptor ligands also potentiated the effect of noradrenaline, inducing a reduction of the noradrenaline EC50 value in the rat vas deferens. The rank order of potencies was: PCP > TCP > (+)-3-PPP > (+)-MK-801 > dextrorphan > > > dextrometorphan. 3. In contrast, haloperidol (1 microM), a sigma receptor ligand, inhibited both the neurogenic and noradrenaline-induced responses in this tissue. 4. The effect of PCP and sigma receptor ligands on noradrenaline uptake was evaluated. All compounds tested, including haloperidol, inhibited the tritiated noradrenaline incorporation to the tissue. IC50 values were in the micromolar range, between 1.09 microM for dextrophan and 18 microM for dextrometorphan. 5. It is concluded that a direct interaction with the noradrenaline uptake system is involved in the potentiating effect of some sigma and PCP receptor ligands in the epididymal portion of rat vas deferens.

Adrenergic alpha-Agonists↗