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J Morán

Publications and source records attributed to J Morán.

At least 19 recordsLinked to original sources
PubMed2000

Volume sensitive efflux of taurine in HEK293 cells overexpressing phospholemman.

The role of the phospholemman (PLM) on the efflux of taurine and chloride induced by swelling was studied in HEK293 cells overexpressing stable transfected PLM. PLM, a substrate for protein kinases C and A, is a protein that induces an anion current in Xenopus oocytes and forms taurine-selective channels in lipid bilayers. Taurine contributes as an osmolyte to regulatory volume decrease (RVD) and is highly permeable through PLM channels in bilayers. In PLM-overexpressing cells the process of RVD was more rapid and efficient (75%) than in control cells (44%). Also, [(3)H]taurine and (125)I efflux induced by hyposmolarity were markedly increased (30-100%) in two subclones of cells overexpressing PLM. This increased efflux was sensitive to the Cl channel blockers DDF, NPPB and DIDS. Acute treatment of control cells with isoproterenol and norepinephrine induced a significant potentiation (50-60%) of [(3)H]taurine release induced by hyposmolarity. In PLM-overexpressing cells the potentiation by these drugs was higher (100%). Insulin induced also an increase in [(3)H]taurine release, but only in PLM-overexpressing cells (50%). These results indicate that PLM may play a role in the RVD and that its phosphorylation may have a physiological significance during this process. The mechanisms involved in this process could include the activation of PLM itself as channel or the modulation of other preexisting channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Release of taurine in apoptotic cerebellar granule neurons in culture.

Cell shrinkage is a distinctive feature of apoptotic death, but the mechanisms leading to cell volume loss are unclear at present. Activation of pathways extruding intracellular osmolytes such as K+, Cl- and organic molecules may be part of these mechanisms. This was examined in the present work measuring the release of taurine, gamma-amino-butyric acid (GABA) and glutamate in cerebellar granule neurons cultured in conditions resulting in apoptotic death after 4-7 days in vitro (DIV). The basal release of [3H]taurine from cells started to increase (38%) after 3 DIV and reached a maximal enhancement (250%) at 5 DIV. The increase in taurine efflux closely followed the occurrence of apoptotic death markers such as caspase induction and chromatin condensation. The efflux of glutamate (traced as D-aspartate) and [3H]GABA also increased but notably less than that of taurine (90% and 75%, respectively) at 5 DIV. Taurine release associated with apoptosis was unaffected by 4,4'-diisothiocyanatostilbene 2,2'-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), blockers of the diffusive pathway activated during cell volume regulation in hyposmotic conditions. Taurine efflux was increased in Cl(-)-free (replaced by gluconate) and decreased in Na+-free media. Blockers of the energy-dependent glutamate and taurine carriers, dihydrokainate and guanidinoethane sulfonate, respectively, did not affect the release associated with apoptosis. These results implicate taurine in the mechanism of cell shrinkage during apoptosis.

Animals↗
PubMed2000

Role of heat shock proteins in the effect of NMDA and KCl on cerebellar granule cells survival.

Cerebellar granule cells (CGC) die apoptotically after five days in culture (DIV) at physiological concentrations of potassium (5 mM; K5). When CGC are depolarized (K25) or treated with NMDA (150 microM) cell survival is increased. CGC changed from K25 to K5 die after 24-48 h. It is known that heat shock protein (HSP) may protect from cell death. Here, we found that cells in K5 showed an increase in HSP-70 levels after 3 DIV. Similarly, in cells changed from K25 to K5, HSP-70 levels were increased after 6 h. Neither NMDA nor K25 treatment affected HSP-70 levels from 2-7 DIV. Ethanol or thermal stress induced HSP-70, but cell survival was not affected in K5 medium. These results suggest that HSP, particularly HSP-70, are not involved in the mechanisms by which NMDA and KCl promote cell survival.

Animals↗
PubMed1997

[Multicenter study on the efficacy of advice for the prevention of alcoholism in primary health care].

BACKGROUND: The aim of this study was to analyse and to verify the efficacy of systematic advice for alcoholism prevention, assessing the reduction of the number in risk drinkers. PATIENTS AND METHODS: A multicenter randomized controlled clinical trial was designed, to perform in general practitioner setting, on a sample of risk drinkers (alcohol intake > 280 g weekly, without dependence) sent by random in intervention group (systematic brief advice with support material and a five visit program during a year) and control group (once brief advice and a control in 1 year). The procedure to incorporate in both groups included physical exam, a blood test and the MALT questionnaire. A descriptive and analytic study on included variables was realised, assessing the percentage of drinkers who reduced alcohol intake below risk limit at the end of a year follow up, as well as the reduction intake in each group. RESULTS: Of the 139 included males, 75 were in the intervention group and 64 in the control group. The percentage of patients not excluded by MALT > 10, and/or liver disease, that finished the 1 year follow up, was 46%, being the sample average age of 43 +/- 11.8. Patients included in both groups were initially comparable. At the end of a year follow up there were statistically significant differences in: percentage of risk drinkers who decreased alcohol intake below 280 g weekly (82% intervention group; 47% control group); percentage of reduction in GPT, GGT, triglycerides, systolic blood pressure and the MALT questionnaire. CONCLUSIONS: The efficacy of isolated advice of general practitioner was proved to achieve the alcohol intake reduction below the risk limit accepted in male risk drinkers without alcohol dependence. The systematic follow up during a year significantly improves the results achieved with the isolated advice.

Adult↗
PubMed1997

Regulatory volume decrease and associated osmolyte fluxes in cerebellar granule neurons are calcium independent.

To investigate a possible role for Ca as a transduction signal for regulatory volume decrease (RVD), the effects of external Ca removal, Ca channel blockers (Cd, Co, La, Gd, verapamil, diltiazem, dihydropyridines) and inhibitors of endoplasmic reticulum Ca release (dantrolene, ryanodine, TMB-8) were examined on RVD and on the swelling-activated efflux of two main osmolytes: Cl (traced by 125I) and [3H]taurine. Omission of Ca plus EGTA did not affect RVD or osmolyte release but when BAPTA was the chelator, RVD decreased 20%, 125I fluxes were unaffected and taurine stimulated efflux decreased (20%) while the basal efflux slightly increased (<10%). Verapamil, diltiazem, Co, Cd, La and Gd did not affect RVD or osmolyte fluxes. Nimodipine and nitrendipine (25-50 microM) markedly decreased RVD and osmolyte fluxes (>90%) through a mechanism independent of extracellular Ca. Swelling elicited an increase in cytosolic Ca measured by fura-2, which was notably variable ranging 50-350 nM. However, RVD and osmolyte fluxes were not affected by the blockers of endogenous Ca release dantrolene, ryanodine and TMB-8 or by the permeable Ca chelator BAPTA-AM, even when the cytosolic Ca increase was abolished by the chelator. These results indicate that 1) RVD and osmolyte fluxes are independent of extracellular Ca 2) RVD, osmolyte release and cytosolic Ca raise are only coincident events. Consequently, Ca is unlikely to be a transducing signal for RVD in neurons.

Animals↗
PubMed1997

Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. I. Regulatory volume decrease.

Exposure of NIH/3T3 fibroblasts not expressing P-glycoprotein to 50, 30, 20, and 10% hyposmotic solutions led to cell volume increases of 70, 32, 21, and 12%, respectively. After swelling, NIH/3T3 cells exhibited regulatory volume decrease (RVD), attaining complete volume recovery after 30 min except in 50% hyposmotic solution, in which volume recovery was 76%. RVD was accelerated by gramicidin and inhibited by the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid and by the K channel, blocker quinidine. RVD was reduced 15% by removal of extracellular Ca. The pathway opened by hypotonicity was highly permeable to K and Rb and only partly permeable to other cations. Most anions were able to permeate, with a permeability ranking of nitrate > benzoate = iodide > thiocyanate > chloride > > gluconate. The pathway was permeable to neutral amino acids, with a permeability ranking of glycine > alanine > glutamate > taurine > gamma-aminobutyric acid > glutamine. The pathway was not permeable to basic amino acids. These results show that, despite the absence of P-glycoprotein, NIH/3T3 cells exhibit RVD with properties similar to those expressed in most cell types.

3T3 Cells↗
PubMed1997

Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. II. Chloride and amino acid fluxes.

The osmolyte function of amino acids and Cl in native NIH/3T3 cells not expressing the P-glycoprotein was examined by investigating the free amino acid concentration and the swelling-activated efflux of [3H]taurine, as representative of amino acids, and of 125I, as a tracer for Cl. Taurine and 125I efflux was activated by 20 and 30% hyposmotic solutions. At 50% hyposmotic solutions, the osmolyte pool was essentially depleted. The Cl channel blockers 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid inhibited the release of the two osmolytes by 80-95%. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (400 microM) decreased the efflux of taurine 80% without affecting that of 125I. Linolenic and arachidonic acids (5-20 microM) showed a concentration-dependent inhibitory effect on taurine and 125I fluxes. Omission of Ca decreased osmolyte fluxes by 16%. Verapamil inhibited the osmolyte release only at 500 microM. Nimodipine at 25 and 50 microM decreased the release of [3H]taurine and 125I by approximately 60 and 80%, respectively, but this effect was independent of the presence of extracellular Ca. These results indicate that amino acids and Cl function as osmolytes during regulatory volume decrease in native NIH/ 3T3 cells.

3T3 Cells↗
PubMed1996

Characterization of the activation of glutaminase induced by N-methyl-D-aspartate and potassium in cerebellar granule cells.

Chronic stimulation of cerebellar granule cells with N-methyl-D-aspartate (NMDA) or KCI induces a specific activation of the enzymes directly involved in glutamate neurotransmitter synthesis. Phosphate-activated glutaminase (PAG) activity is enhanced in cultured granule neurons incubated with 150 microM NMDA or 25 mM KCI. Other enzymes are not affected by this treatment like lactate dehydrogenase (LDH) and glutamate dehydrogenase (GLDH), which is also a mitochondrial enzyme but not directly involved in neurotransmitter synthesis. This effect is dependent on protein synthesis and is induced after 12 hr of NMDA or KCI stimulation. Kinetics of PAG activity showed that Km values were unaffected, in contrast to Vmax values that were increased approximately 70% and 215% over control by NMDA and KCI treatment, respectively. For GLDH, we found two isoforms that were affected differentially by the experimental conditions. Western blot analysis clearly evidenced an increase of approximately 120-180% in the amount of PAG in NMDA- and KCI-treated cells, whereas GLDH was not significantly modified. These results demonstrate that the NMDA- and KCI-induced activation of PAG are not due to the modification of the preexisting enzyme, but to an increase in the synthesis of this enzyme. This suggests that NMDA receptor stimulation during critical periods of the cerebellar granule cell development leads to the activation of gene expression involved in the process of cell differentiation.

Animals↗
PubMed1996

Changes of actin cytoskeleton during swelling and regulatory volume decrease in cultured astrocytes.

Swelling of cultured astrocytes exposed to hyposmotic medium modified the organization of the filamentous actin (F-actin) cytoskeleton, making the actin network diffuse in the cell body but concentrated at foci corresponding to the tips of the cell projections retracted by swelling. This change was reversible, and, after 2 h, the actin cytoskeleton tended to recover, and cells regained their flat and stellate shape. Cytochalasins B and D (CB and CD, respectively), which disrupt the actin cytoskeleton, did not affect regulatory volume decrease (RVD) or the swelling-activated efflux of Cl- and inositol, although 10 microM CD increased the basal efflux of taurine. The mercurial p-chloromercuribenzenesulfonate (0.5-1 mM), known to disrupt the membrane cytoskeleton in isosmotic conditions, induced a 46, 50, and 38% release of [3H]taurine, 125I, and [3H]inositol, respectively, causing cell shrinkage and retraction of the cytoskeleton. Coincidently, the swelling-stimulated release of [3H]taurine and 125I was reduced by 60 and 30%, respectively. Results of this study do not exclude the possibility that changes in the actin cytoskeleton elicited by swelling are involved in mechanisms of RVD and only indicate that the disruption caused by cytochalasins is unrelated to that process.

Actins↗
PubMed1995

Inhibition by dihydropyridines of regulatory volume decrease and osmolyte fluxes in cultured astrocytes is unrelated to extracellular calcium.

The 1,4-dihydropyridines (DHP), nimodipine (NMD) and nitrendipine (NTD) were potent blockers of regulatory volume decrease (RVD) and the volume-associated release of [3H]taurine and chloride (measured as 125I) in 2-weeks cultured rat cerebellar astrocytes. The IC50 were 30 microM and 29 microM for taurine efflux and 26 and 27 microM for C1 efflux for NMD and NTD, respectively. Inhibition by DHP was independent of extracellular Ca, as the effect was the same in media with 1 mM Ca or without Ca and 0.5 mM EGTA. DHP did not affect the basal (isosmotic) release of [3H]taurine or 125I inhibition by DHP (measured only on [3H]taurine efflux) was the same in 3-4 weeks cultured cerebellar astrocytes, 2-4 weeks cultured cortical astrocytes and 2-weeks cultured cerebellar astrocytes treated with dibutyril cAMP. Diltiazem (50 microM) and verapamil (100 microM) failed to inhibit RVD or osmolyte efflux.

Animals↗
PubMed1995

Selective neurotoxicity of ruthenium red in primary cultures.

The inorganic dye ruthenium red (RuR) has been shown to be neurotoxic in vivo when injected intracerebrally. In this work the toxicity of RuR was compared in primary cultures of rat cortical neurons, cerebellar granule neurons and cerebellar astroglia. Microscopic examination of the cultures revealed that RuR penetrates the somata of both types of neurons used and produces vacuolization and loss and fragmentation of neurites. In contrast, no RuR was seen inside cultured astrocytes and no morphological signs of damage were observed in these cells. RuR toxicity was also assessed by immunocytochemistry of alpha-tubulin and by biochemical measurement of the reduction of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by the cultured cells. The morphological alterations in the neurons were closely correlated with loss of tubulin immunoreactivity and particularly with a notable decrement in the ability to reduce MTT. Using the latter parameter, it was found that neuronal damage was independent of the age of the cultures, augmented progressively with time of incubation with RuR, from 8 to 24 h, and showed a clear dose-response curve from 20 to 100 microM RuR. Astrocytes showed only a slight decrease in MTT reduction after 24 h of incubation with 100 microM RuR. It is concluded that RuR seems to be toxic for neurons but not for astroglia, and that this selectivity is probably related to the ability of the neurons to internalize the dye. The possible mechanisms of RuR penetration and neuronal damage are discussed.

Animals↗
PubMed1995

Taurine deficiency in dissociated mouse cerebellar cultures affects neuronal migration.

The role of taurine in the process of neuronal migration was studied in a microwell cell culture system. Immunocytochemical analysis of the cellular composition of this culture system revealed the presence of the astrocytic marker GFAP in some structures such as the aggregates of neuronal bodies and in those cables used for migration, resembling what is described in vivo. The neuronal marker gamma-enolase stained practically all structures, including the aggregates and all cables. The intracellular taurine concentration was reduced by 60% in mouse cerebellar granule cells treated with a blocker of taurine transport, guanidinoethane sulfonate (GES). Under these conditions cell migration was markedly reduced to approximately 50% of that in untreated cultures. Both, taurine depletion and impairment of cell migration induced by GES were prevented by adding taurine to the culture medium. Taurine deficiency similarly affected different morphological parameters such as the number of cables suitable for neuronal migration as well as the number of migrating neurons. The number of aggregates of neuronal bodies was significantly increased, by about 30%, as a consequence of the reduced migration. Taurine alone did not exert any effect on the parameters evaluated. GES treatment of granule cells did not affect mitochondrial metabolism or K(+)-stimulated Ca(2+)-dependent [3H]-D-aspartate release. This suggests that the described effects of taurine deficiency were not due to an alteration of neuronal viability and that the action of GES was not simply due to unspecific and deleterious effects. These results are in agreement with those obtained in in vivo studies. This approach represents a useful model to investigate the role played by taurine in the process of neuronal migration.

Amino Acids↗
PubMed1995

Inhibition by polyunsaturated fatty acids of cell volume regulation and osmolyte fluxes in astrocytes.

The polyunsaturated fatty acids, arachidonic, linoleic, and linolenic acids, were potent blockers of regulatory volume decrease (RVD) and of the swelling-activated efflux of [3H]taurine, D-[3H]aspartate, [3H]inositol, and 125I (used as marker of Cl) from rat cerebellar astrocytes in culture. The monounsaturated oleic and ricinoleic acids and saturated fatty acids were ineffective. The amino acid and 125I fluxes were similarly inhibited by fatty acids, whereas inositol release was less sensitive. Polyunsaturated fatty acids appear to directly affect RVD in trypsinized astrocytes as the inhibition was immediate and fully reversible. Blockers of the arachidonic acid metabolic pathways, indomethacin (cyclooxygenase), esculetin (lipoxygenases), and metyrapone (P-450 monooxygenases), did not prevent the effect of arachidonic acid, suggesting that further metabolism is not required for displaying the effects of arachidonic acid on RVD and osmolyte fluxes. Some blockers of arachidonic acid metabolic pathways, such as nordihydroguaiaretic acid (lipoxygenases) and naphthoflavone (P-450 monooxygenases), also exhibited marked inhibitory effects on RVD and on osmolyte fluxes. The predominant arachidonic acid metabolite in astrocytes, 12-hydroxyeicosatetraenoic acid, did not affect RVD or osmolyte fluxes. These results suggest that arachidonic acid and other polyunsaturated fatty acids directly inhibit the permeability pathways correcting cell volume after swelling in cultured astrocytes.

Animals↗
PubMed1994

Sulfhydryl groups essential for the volume-sensitive release of taurine from astrocytes.

The volume-sensitive [3H]taurine efflux from cultured astrocytes was found to be strongly inhibited by the sulfhydryl group-modifying reagent N-ethylmaleimide (NEM). This maleimide inhibited taurine efflux evoked by 50% hyposmotic solutions with an IC50 of 54 microM. The inhibition by NEM followed pseudo-first order reaction kinetics. A double log plot of the pseudo-first order constant against NEM concentration gave a linear relationship with a slope of 1.2. The data are consistent with a simple bimolecular reaction mechanism in which one molecule of NEM reacts with one sulfhydryl group per transport unit. The membrane-impermeant maleimide derivative 5-eosin maleimide did not affect the volume-stimulated taurine efflux. The sulfhydryl-modifying mercurial reagents mersalyl and p-chloro mercuribenzenesulfonate (0.5-1 mM) increased [3H]taurine efflux under isosmotic conditions and concomitantly decreased the hyposmolarity-evoked efflux. The results demonstrate an essential requirement for sulfhydryl groups for the volume-sensitive taurine efflux.

Animals↗
PubMed1994

Properties of osmolyte fluxes activated during regulatory volume decrease in cultured cerebellar granule neurons.

Efflux pathways for amino acids, K, and Cl activated during regulatory volume decrease (RVD) were characterized in cultured cerebellar granule neurons exposed to hyposmotic conditions. Results of this study favor diffusion pores (presumably channels) over energy-dependent transporters as the mechanisms responsible for the efflux of these osmolytes. The selectivity of osmolyte pathways activated by RVD was assessed by increasing the extracellular concentrations of cations, anions, and amino acids to such an extent that upon opening of the pathway, a permeable compound will enter the cell and block RVD by reducing the efflux of water carried by the exit of intracellular osmolytes. The cationic pathway was found selective for K (and Rb), whereas the anionic pathway was rather unselective being permeable to Cl, nitrate, iodine, benzoate, thiocyanate, and sulfate but impermeable to gluconate. Glutamate and aspartate as K but not as Na salts were permeable through the anion channel. RVD was slightly inhibited by quinidine but otherwise was insensitive to known K channel blockers. RVD was inhibited by 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS), niflumic acid, and dipyridamole. Gramicidin did not affect cell volume in isosmotic conditions but greatly accelerated RVD, suggesting that cell permeability to Cl is low in isosmotic conditions but increases markedly during RVD making K permeability the rate limit of the process. The permeability pathway for amino acids activated during RVD as permeable to short chain alpha- and beta-amino acids, but excluded glutamine and basic amino acids.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗