Roberto Sánchez-Olea

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

Abstract[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 μM 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.

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.

Neurons respond to hyposmotic conditions by an increase in intracellular free calcium

The effect of hyposmotic conditions on the concentration of intracellular free calcium ([Ca2+]i) was studied in cultured cerebellar granule cells and cerebral cortical neurons after loading of the cells with the fluorescent Ca2+ chelator Fluo-3. It was found that in both types of neurons exposure to media with a decrease in osmolarity of 20 to 50% of the osmolarity in the isosmotic medium (320 mOsm) led to a dose dependent increase in [Ca2+]i with a time course showing the highest value at the earliest measured time point, i.e. 40 s after exposure to the hyposmotic media and a subsequent decline towards the basal level during the following 320 s. The response in the cortical neurons was larger than in the granule cells but both types of neurons exhibited a similar increase in [Ca2+]i after expoxure to 50 mM K+ which was of the same magnitude as the increase in [Ca2+]i observed in the cortical neurons exposed for 40 s to a medium with a 50% reduction in osmolarity. In both types of neurons the blocker of voltage gated Ca2+ channels verapamil had no effect on the hyposmolarity induced increase in [Ca2+]i. On the contrary, this increase in [Ca2+]i was dependent upon external calcium and could be inhibited partly or completely by the inorganic blockers of Ca2+ channels Mg2+ and La3+. Dantrolene which prevents release of Ca2+ from internal stores had no effect. The results show that exposure of neurons to hyposmotic conditions leading to swelling results in a large increase in free intracellular Ca2+ which represents an influx of Ca2+ rather than a release of Ca2+ from internal, dantrolene sensitive stores.

Volume Regulation in Cultured Neurons: Pivotal Role of Taurine

The ability of cells to adapt by active processes to changes in external osmolarity has been known for a long time (3). Two new concepts have emerged in recent years: first, that the adaptive response is present in cells which grow and live in media of strictly controlled osmolarity, and secondly, that a number of organic molecules are importantly involved in volume regulation in vertebrates (5,8).

Volume regulatory fluxes in glial and renal cells.

A variety of vertebrate cells have the ability to adjust to changes in cell volume by activating transmembrane fluxes of osmotically active solutes in the necessary direction to correct the deviations in cell volume (Macnight, 1988; Hoffmann and Simonsen, 1989). The osmotically active compounds in animal cells fall into two main groups: inorganic ions, Na+, K+ and Cl-and a large variety organic molecules, including polyols, methylamines and amino acids. Among these later, taurine appears to be predominantly involved in volume regulatory processes (Pasantes-Morales and Martin del Rio, 1990).

Hyperosmolarity and taurine content, uptake and release in astrocytes.

There is increasing evidence suggesting an association of free amino acids with the mechanisms of cell volume regulation in the nervous system1. A response of tissue cells to hyposmolarity by massive release of taurine and other free amino acids has been documented in a wide variety of prepartions that includes cultured cells and intact brain2,3,4. There is also an indication that as part of an adaptative response of the brain to hyperosmolarity there is an increase in the intracellular concentration of amino acids, including taurine. Thurston et a1.5 showed that in chronic hypernatremic dehydrated mice a decrease in the water content of the brain is followed by a marked increment in the concentration of free amino acids, particularly taurine. Similarly, in chronically dehydrated Brattelboro rats, taurine concentration in the brain is markedly enhanced6. Recently a study by Olson and Goldfinger7 demonstrated an increase of taurine content in cultured astrocytes grown in hyperosmotic solutions. The mechanism by which these increases in taurine content occur is still unknown. In this work we explored the possible mechanisms responsible for the increase of taurine levels induced by hyperosmolarity in cerebellar astrocytes.

Taurine and Volume Regulation in Isolated Nerve Endings

Taurine is released from nerve endings isolated from different brain regions by a number of depolarizing agents and conditions, including high concentrations of potassium, ouabain, veratridine and electrical stimulation (Rev. in Huxtable, 1989). This response has been considered as evidence supporting a role for taurine as a neurotransmitter. However, the features of this release do not fit well with those currently exhibited by synaptic transmitters. It has been consistently observed that taurine release from synaptosomes is essentially calcium independent (Huxtable, 1989) in opposition to the calcium requirement for exocytotic release of neurotransmitters. In contrast, a predominant chloride dependent component of the potassium stimulated efflux of taurine is observed and evidence has been provided suggesting that this release is associated with synaptosomal swelling (Sanchez-Olea and Pasantes-Morales, 1990). This observation together with the above mentioned properties of taurine release raised the question of whether a component of the taurine efflux elicited by depolarizing conditions is a response to swelling and reflects an involvement of taurine in mechanisms of volume regulation in the nerve endings.