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H Pasantes-Morales

Publications and source records attributed to H Pasantes-Morales.

At least 19 recordsLinked to original sources
PubMed1998

Ca2+ changes and 86Rb efflux activated by hyposmolarity in cerebellar granule neurons.

Hyposmotic swelling increased 86Rb release in cultured cerebellar granule neurons (1 day in vitro [DIV]) with a magnitude related to the change in osmolarity. 86Rb release was partially blocked by quinidine, Ba2+, and Cs+ but not by TEA, 4-AP, or Gd3+. 86Rb efflux decreased in Cl(-)-depleted cells or cells treated with DDF or DIDS, suggesting an interconnection between Cl- and K+ fluxes. Swelling induced a substantial increase in [Ca2+]i to which both external and internal sources contribute. However, 86Rb efflux was independent of [Ca2+]0, unaffected by depleting the endoplasmic reticulum (ER) by ionomycin or thapsigargin and insensitive to charybdotoxin, iberiotoxin, and apamin. Swelling-activated 86Rb efflux in differentiated granule neurons after 8 DIV, which express Ca2+-sensitive K+ channels, was not different from that in 1 DIV neurons, nor in time course, net release, Ca2+-dependence, or pharmacological sensitivity. We conclude that the swelling-activated K+ efflux in cerebellar granule neurons is not mediated by Ca2+-sensitive large conductance K+ channels (BK) as in many cell types but resembles that in lymphocytes where it is possibly carried by voltage-gated K+ channels.

Animals↗
PubMed1998

Effects of NaCl removal on osmolyte fluxes and regulatory volume decrease in cultured astrocytes.

Cultured cerebellar astrocytes exhibit regulatory volume decrease (RVD) after anisosmotic swelling by extrusion of potassium (K), chloride (Cl), and organic osmolytes. The volume decay curve after swelling in 50% hyposmotic solutions has a first-order rate constant k min(-1) of 0.18 and 70% cell volume recovery is attained after 15 min. Replacing NaCl in the hyposmotic medium by sugars and polyalcohols markedly accelerated RVD increasing k to 0.37-0.39 min(-1) and full recovery in 3-5 min. NaCl removal increased (80%) K efflux (measured as 86Rb), delayed the inactivation of Cl efflux (measured as 125I) and enhanced taurine and D-aspartate release by 25%. These effects appear due to NaCl removal rather than to specific actions of the organic molecules. Replacing Na by other cations or Cl by gluconate increased k to 0.22 min(-1) and 0.26 min(-1), respectively, and 86Rb efflux by 4-23% and 39, respectively. It is concluded that the electrochemical gradient after NaCl removal increasing the K driving force, delaying the equilibrium of Cl and increasing amino acid efflux, accounts for the observed RVD acceleration. Other consequences of NaCl removal possibly modulating the K efflux pathway include changes in the surface charge screening, decreased ion strength, a rise in [Ca]i and a decrease in intracellular Na.

Amino Acids↗
PubMed1998

Effects of taurine depletion on cell migration and NCAM expression in cultures of dissociated mouse cerebellum and N2A cells.

Cultures of dissociated cerebellum from 5- to 6-day-old mice as well as of the N2A neuronal cell line were exposed to guanidino ethane sulfonate (GES, 2-5 mM) to reduce the cellular taurine content. Control cultures were kept in culture medium or medium containing 2-5 mM GES plus 2-5 mM taurine to restore the intracellular taurine content. Taurine depletion led to changes in the expression of certain splice variants of NCAM mRNA such as the AAG and the VASE containing forms, while no differences were seen in the expression of the three forms of NCAM protein. In the N2A cells taurine depletion led to a decreased migration rate of the cells. The results suggest that the reduced migration rate of neurons caused by taurine depletion may be correlated to changes in expression of certain adhesion molecules such as NCAM. Moreover, taurine appears to be involved in regulation of transcription processes.

Alternative Splicing↗
PubMed1998

Osmotic swelling-induced changes in cytosolic calcium do not affect regulatory volume decrease in rat cultured suspended cerebellar astrocytes.

Hyposmotic swelling-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and their influence on regulatory volume decrease (RVD) were examined in rat cultured suspended cerebellar astrocytes. Hyposmotic media (50 or 30%) evoked an immediate rise in [Ca2+]i from 117 nM to a mean peak increase of 386 (50%) and 220 nM (30%), followed by a maintained plateau phase. Ca2+ influx through the plasmalemma as well as release from internal stores contributed to this osmosensitive [Ca2+]i elevation. Omission of external Ca2+ or addition of Cd2+, Mn2+, or Gd3+ did not reduce RVD, although it was decreased by La3+ (0.1-1 mM). Verapamil did not affect either the swelling-evoked [Ca2+]i or RVD. Maneuvers that deplete endoplasmic reticulum (ER) Ca2+ stores, such as treatment (in Ca2+-free medium) with 0.2 microM thapsigargin (Tg), 10 microM 2,5-di-tert-butylhydroquinone, 1 microM ionomycin, or 100 microM ATP abolished the increase in [Ca2+]i but did not affect RVD. However, prolonged exposure to 1 microM Tg blocked RVD regardless of ER Ca2+ content or cytosolic Ca2+ levels. Ryanodine (up to 100 microM) and caffeine (10 mM) did not modify [Ca2+]i or RVD. BAPTA-acetoxymethyl ester (20 microM) abolished [Ca2+]i elevation without affecting RVD, but at higher concentrations BAPTA prevented cell swelling and blocked RVD. We conclude that the osmosensitive [Ca2+]i rise occurs as a consequence of increased Ca2+ permeability of plasma and organelle membranes, but it appears not relevant as a transduction signal for RVD in rat cultured cerebellar astrocytes.

Animals↗
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

Characterization of microwell cultures of dissociated brain tissue for studies of cell-cell interactions.

Microwell cultures of dissociated tissue from prenatal rat hippocampus and cerebral cortex as well as from early postnatal cerebellum were used for quantification of neuronal aggregation, process extension, and fasciculation. It was shown that the cells in culture from these different brain regions developed differently with regard to both architecture and rate of differentiation. The effect of a polyclonal antibody against the neural cell adhesion molecule (NCAM), the excitatory amino acid receptor agonist N-methyl-D-aspartate (NMDA), and the neurotoxin acrylamide on aggregation and fiber formation was investigated. Exposure to the NCAM antibody led to formation of fewer but larger aggregates and stimulated the morphological development of the cultures. Acrylamide affected aggregate formation, leading to smaller but more numerous aggregates, and it inhibited process extension and fasciculation. Treatment with NMDA affected process formation and led to formation of more numerous but smaller aggregates. Some of these effects were strongly tissue-dependent. Thus, large differences were seen regarding the effect of the NCAM antibody on aggregation and process extension in cultures from the different brain areas. The culture systems appear to represent convenient and reliable screening tools to study the influence of putative morphoregulatory substances on cell-cell interactions during early neuronal development.

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

Cl channel blockers inhibit the volume-activated efflux of Cl and taurine in cultured neurons.

The effects of the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 1,9-dideoxyforskolin (DDF), dipyridamole, and niflumic acid and of the polyunsaturated fatty acids arachidonic, linolenic, and linoleic acids on regulatory volume decrease (RVD) and associated 125I and [3H]taurine fluxes in cultured rat cerebellar granule neurons were examined. Dose-response curves of NPPB, DDF, and dipyridamole showed 20-100% inhibition of RVD and osmolyte fluxes. Niflumic acid was less potent, requiring 150-600 microM to show effects of this magnitude. The polyunsaturated fatty acids (5-20 microM) inhibited 80-90% RVD and osmolyte fluxes, with arachidonic acid exhibiting the most potent effect. The volume-associated taurine efflux was somewhat higher in younger neurons, but the pharmacological sensitivity was essentially the same in immature and mature cells. The effects of all tested drugs on 125I and [3H]taurine fluxes were remarkably similar, indicating a close pharmacological sensitivity of the transport mechanism for the two osmolytes. This is in line with the suggestion of a common pathway for the volume-associated release of Cl and amino acids functioning as osmolytes.

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

Inhibition by Cl- channel blockers of the volume-activated, diffusional mechanism of inositol transport in primary astrocytes in culture.

[3H]Inositol accumulated by rat brain cultured astrocytes is released when cells swell by exposure to solutions of decreased osmolarity. Activation of inositol efflux was proportional to reductions in osmolarity from 30%-70%. This volume-activated inositol efflux pathway was increased (27%) in Na(+)-free medium and decreased (22%) in Cl(-)-free medium. It was independent of extracellular Ca2+ and was reduced (30%) in the presence of the intracellular chelator [1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester] (BAPTA-AM). The inositol efflux pathway was markedly inhibited by Cl- channel blockers, which at maximal inhibitory concentrations decreased inositol efflux by 70%-83%. The potency range of the drugs was: 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > 1-9, dideoxyforskolin > 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) > niflumic acid. Inositol efflux was strongly inhibited by the SH blocker N-ethyl maleimide (NEM), which at 100 microM abolished inositol release. Inositol efflux can be reversed by increasing its extracellular concentration, suggesting that the efflux is mediated by a diffusional pathway whose direction is given by the concentration gradient. The inhibition of volume-associated fluxes of inositol by Cl- channel blockers supports the suggestion of an anion channel as the common pathway for inorganic and organic osmolytes in cultured astrocytes.

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