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H Pasantes-Morales

Publications and source records attributed to H Pasantes-Morales.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1995

Volume-associated osmolyte fluxes in cell lines with or without the anion exchanger.

To investigate the involvement of a red cell-type anion exchanger in the volume-sensitive amino acid release, the hyposmolarity-evoked release of D-[3H]aspartate and [3H]taurine was examined in three cell lines: 1) wild-type Chinese hamster ovary (CHO-K1) cells, expressing an anion exchanger activity (Cl-/SO4(2-)) functionally similar to the erythroid band 3; 2) a mutant CHO cell type (CHO 605) lacking this anion exchanger activity; and 3) 293 cells in which the Cl-/HCO3(-) anion exchanger is absent. All cell types accumulated D-[3H]aspartate and [3H]taurine under isosmotic conditions, and, similarly, in the three cell lines, cell swelling evoked by hyposmolarity induced a rapid and transient increase in the amino acid efflux. Blockers of the anion exchanger and/or Cl- channels [niflumic acid, dipyridamole, diphenylamine-2-carboxylate,5-nitro-2-(3-phenylpropylamino)-benzoi c acid, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid] were potent inhibitors of amino acid efflux in the three cell lines. 125I- efflux, used as a marker for Cl- fluxes, was also markedly increased in response to cell swelling in all cell lines, and this efflux was inhibited by the anion exchanger/Cl- channel blockers. These results do not support a role for an anion exchanger in the hyposmolarity-induced amino acid efflux and suggest that amino acids and Cl- may be transported by the same or a similar mechanism, presumably an anion channel-like structure.

Animals↗
PubMed1995

Taurine content in breast milk of Mexican women from urban and rural areas.

The concentration of the most abundant free amino acids in breast milk, taurine, glutamic acid, glycine + threonine, alanine and glycine was measured in breast milk samples obtained from Mexican women from an urban population (38 samples) and from a rural population (106 samples). Free amino acid levels including taurine in the urban group were essentially similar to those reported in samples from American and Canadian women (taurine concentration: 332-357 nmol/ml) but significantly lower content of taurine (237-259 nmol/ml) was found in the rural group. The only other significant difference between the two groups was a 32% higher concentration of alanine in the rural group as compared to the urban group. The observed differences may result from a lower dietary intake of taurine containing food in the rural group as this group reported a restricted consumption of meat, the most abundant dietary source of taurine. Because taurine synthesis is low in primates including human, a decreased external supply of taurine may explain the observed reduction in the taurine content of milk. The increase in alanine may represent a compensatory mechanism for the decrease in taurine.

Amino Acids↗
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↗
PubMed1994

Impaired cell volume regulation in taurine deficient cultured astrocytes.

Taurine concentration was reduced by 40 and 65%, respectively in rat cerebellar astrocytes grown in a chemically defined medium or in culture medium containing a blocker of taurine transport (GES). Cell volume in these taurine deficient cells was 10%-16% higher than in controls. When challenged by hyposmotic conditions, astrocytes release taurine and this efflux contributes to the volume regulatory decrease observed in these cells. Taurine deficient astrocytes showed a less efficient volume recovery as compared to controls with normal taurine levels. Exposed to 50% hyposmotic medium, astrocytes with normal taurine concentration recovered 60% of their original volume whereas taurine deficient cells recovered only 30-35%. Similarly, in 30% hyposmotic medium, taurine deficient astrocytes recovered only 40% as compared to 75% in controls. No compensatory increases in the efflux of other osmolytes (free amino acids or potassium) were observed during regulatory volume decrease in taurine deficient astrocytes.

Amino Acids↗
PubMed1994

Regulatory volume decrease in cultured astrocytes. I. Potassium- and chloride-activated permeability.

Regulatory volume decrease (RVD) in detached cerebellar astrocytes in culture after acute exposure to hyposmolarity was characterized in this and the accompanying paper [H. Pasantes-Morales, R. A. Murray, R. Sanches-Olea, and J. Moran. Am. J. Physiol. 266 (Cell Physiol. 35): C172-C178, 1994]. RVD was independent of extracellular calcium, was accelerated at pH 8-9 and retarded at pH 6, and was reduced at temperatures < 18 degrees C. The cationic pathway activated by hyposmolarity was specific for K+ and Rb+, since RVD was abolished and secondary swelling occurred when these ions replaced Na+. However, Li+, choline, tris(hydroxymethyl)aminomethane, and glucosamine, all as Cl- salts, did not affect RVD. The anion pathway was unselective, since RVD was inhibited when NaCl was replaced by anion K+ salts with a permeability rank of SCN- = I- > NO3- > Cl- > benzoate > acetate >> SO3- > gluconate. RVD was unaffected by bumetanide (50 microM) and weakly inhibited by furosemide (2 mM). Quinidine but not other K+ channel blockers inhibited RVD, and its effect was reversed by gramicidin. RVD was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and dipyridamole but not by diphenylamine-2-carboxylate or anthracene-9-carboxylate. These results suggest that diffusion possibly via channels rather than cotransporters is involved in the swelling-activated K+ and Cl- fluxes. Gramicidin did not change astrocyte volume in isosmotic conditions, but greatly accelerated RVD, suggesting that low Cl- permeability in isosmotic conditions markedly increases by swelling, thus making K+ permeability the rate-limiting step for RVD.

Animals↗
PubMed1994

Regulatory volume decrease in cultured astrocytes. II. Permeability pathway to amino acids and polyols.

The permeability of the hyposmolarity-activated pathway to amino acids and polyols in cultured astrocytes was examined following the change in rate and direction of regulatory volume decrease (RVD) when the extracellular concentration of the osmolytes was increased to reverse their intracellular-extracellular concentration gradient. Activation of the pathway by swelling would allow those permeable osmolytes to enter the cell and inhibit RVD. The pathway was found to be permeable to neutral amino acids, with beta-amino acids (beta-alanine = taurine > gamma-aminobutyric acid) more permeable than alpha-amino acids. Glycine, alanine, threonine, phenylalanine, and asparagine, but not glutamine, were permeable through this pathway. Aspartate was more permeable than glutamate, and K+ and not Na+ must be the accompanying cation. Basic amino acids were excluded. The dimension of the amino acid pore activated by hyposmolarity seems to be at the limit of glutamate-glutamine size. Influx rather than efflux of amino acids was observed when extracellular concentration was greater than intracellular concentration, with differences in the amount accumulated by cells correlating with their efficiency as RVD blockers. Influx of taurine (as representative of permeable amino acids) was inhibited by the Cl- channel blockers/exchangers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (40%) and dipyridamole (85%) , and it is suggested that amino acids permeate through an anion channel. Sorbitol and mannitol, but not inositol, exhibited a small inhibitory effect on the later phase of RVD, whereas inositol slightly accelerated RVD.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Inhibition of volume regulation and efflux of osmoregulatory amino acids by blockers of Cl- transport in cultured astrocytes.

Regulatory volume decrease (RVD) in astrocytes was inhibited by the Cl- exchanger blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), dipyridamole and niflumic acid but not by the Cl- channel inhibitors diphenylamine-2-carboxylate (DPC) and anthracene-9-carboxylate (9-AC). The volume activated efflux of [3H]taurine and [3H]D-aspartate (as marker for glutamate) was similarly affected by these compounds. However, neither RVD nor osmolyte fluxes were significantly reduced by removal of external Cl-, suggesting that an anion exchanger activity is not required for the volume regulatory process. Alternatively, these results suggest that the anion exchanger molecule may function as an unidirectional Cl- channel possibly permeable also to amino acids.

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