Chloride dependence of the K+-stimulated release of taurine from synaptosomes

Exposure of a crude synaptosomal fraction to K⁺ concentrations ranging from 25 to 100 mM evokes the release of [³H]taurine and [³H]GABA. These high concentrations of K⁺ induce, besides depolarization, a marked synaptosomal swelling, which is prevented by replacing chloride in the solutions with the largely impermeant anion gluconate. The depolarizing effect of K⁺ is unaffected by omission of chloride. The K⁺-evoked release of taurine seems related to K⁺-induced changes in synaptosomal volume rather than to a depolarizing effect, since it is totally calcium-independent but is abolished by reducing chloride and by making solutions hypertonic with mannitol. The release of [³H]GABA, in contrast is unaffected in chloride-free or hypertonic solutions.     

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.     

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 M 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.     

Taurine release associated to volume regulation in rabbit lymphocytes.

Rabbit lymphocytes exposed to hyposmotic media first swell and then recover their initial volume within 6 min. During volume recovery, free amino acids (FAA) decrease from 451.1 to 208 nmoles/mg protein. Taurine was the dominating FAA, accounting for 70% of the FAA pool. The time course of 3H-taurine release induced by hyposmolarity followed that of volume recovery. Efflux of 3H-taurine in an 8 min period was 17.8% (of total labeled taurine accumulated during loading) in an isosmotic medium. Reducing osmolarity to 0.87, 0.75, 0.62, and 0.5 increased this release to 24.8%, 38.1%, 56.4% and 70.9%, respectively. The volume-sensitive release of 3H-taurine was unaffected by omission of external Na+ or Ca++ and was reduced by 23% in the absence of Cl-. It was unaffected by agents disrupting the cytoskeleton or by tetraethylammonium, barium, quinidine, and gadolinium, but was 26% reduced by DIDS. Taurine release was inhibited at 4 degrees C, but was unchanged at 15 degrees C or 25 degrees C. An involvement of FAA, particularly taurine, in lymphocyte volume regulation is suggested.     

Seizure susceptibility in the developing mouse and its relationship to glutamate decarboxylase and pyridoxal phosphate in brain.

The relationship between the susceptibility to convulsions, the content of pyridoxal 5'-phosphate and the activity of pyridoxal kinase (EC 2.7.1.35) and glutamate decarboxylase (EC 4.1.1.15) in brain, was studied in the developing mouse. Seizures were induced by pyridoxal phosphate-gamma-glutamyl hydrazone (PLPGH), a drug previously reported to reduce the levels of pyridoxal 5'-phosphate and as a consequence to inhibit the activity of glutamate decarboxylase in brain of adult mice. It was found that the seizure pattern, as well as the time of appearance of convulsions, differed between 2- and 5-day old mice and 10-day old or older mice, indicating a progressive increase in seizure susceptibility during development. In brain, pyridoxal kinase activity and pyridoxal 5'-phosphate levels were decreased by the administration of PLPGH at all ages studied, whereas glutamate decarboxylase activity was inhibited less than 25% in 2- and 5-day old mice, and about 50% thereafter. Parallelly, the activation of glutamate decarboxylase by pyridoxal 5'-phosphate added in vitro to control homogenates was less in 2- and 5-day old mice than in older animals. It is concluded that the increase in the susceptibility to seizures induced by PLPGH during development is probably related to the increase observed in the sensitivity of glutamate decarboxylase in vivo to a decrease of pyridoxal 5'-phosphate levels. The correlation between pyridoxal 5'-phosphate, glutamate decarboxylase, and seizure susceptibility seems to be established at about 10 days of age.     

Isovolumetric regulation mechanisms in cultured cerebellar granule neurons

Cultured cerebellar granule neurons exposed to gradual reductions in osmolarity (-1.8 mOsm/min) maintained constant volume up to -50% external osmolarity (pi(o)), showing the occurrence of isovolumetric regulation (IVR). Amino acids, Cl-, and K+ contributed at different phases of IVR, with early efflux threshold for [3H]taurine, D-[3H]aspartate (as marker for glutamate) of pi(o) -2% and -19%, respectively, and more delayed thresholds of -30% for [3H]glycine and -25% and -29%, respectively, for Cl- (125I) and K+ (86Rb). Taurine seems preferentially involved in IVR, showing the lowest threshold, the highest efflux rate (five-fold over other amino acids) and the largest cell content decrease. Taurine and Cl- efflux were abolished by niflumic acid and 86Rb by 15 mM Ba2+. Niflumic acid essentially prevented IVR in all ranges of pi(o). Cl--free medium impaired IVR when pi(o) decreased to -24% and Ba2+ blocked it only at a late phase of -30% pi(o). These results indicate that in cerebellar granule neurons: (i) IVR is an active process of volume regulation accomplished by efflux of intracellular osmolytes; (ii) the volume regulation operating at small changes of pi(o) is fully accounted for by mechanisms sensitive to niflumic acid, with contributions of both Cl- and amino acids, particularly taurine; (iii) Cl- contribution to IVR is delayed with respect to other niflumic acid-sensitive osmolyte fluxes (osmolarity threshold of -25% pi(o)); and (iv), K+ fluxes do not contribute to IVR until a late phase (< -30% pi(o)).     

Volume changes and whole cell membrane currents activated during gradual osmolarity decrease in C6 glioma cells: contribution of two types of K+ channels

Volume changes and whole cell ionic currents activated by gradual osmolarity reductions (GOR) of 1.8 mosM/min were characterized in C6 glioma cells. Cells swell less in GOR than after sudden osmolarity reductions (SOR), the extent of swelling being partly Ca²⁺ dependent. In nominally Ca²⁺-free conditions, GOR activated predominantly whole cell outward currents. Cells depolarized from the initial –79 mV to a steady state of –54 mV reached at 18% osmolarity reduction [hyposmolarity of –18% (H-18%)]. Recordings of Cl^– and K⁺ currents showed activation at H-3% of an outwardly rectifying Cl^– current, with conductance of 1.6 nS, sensitive to niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, followed at H-18% by an outwardly rectifying K⁺ current with conductance of 4.1 nS, inhibited by clofilium but insensitive to the typical K⁺ channel blockers. With 200 nM Ca²⁺ in the patch pipette, whole cell currents activated at H-3% and at H-13% cells depolarized from –77 to –63 mV. A K⁺ current activated at H-1%, showing a rapid increase in conductance, suppressed by charybdotoxin and insensitive to clofilium. These results show the operation of two different K⁺ channels in response to GOR in the same cell type, activated by Ca²⁺ and osmolarity and with different osmolarity activation thresholds. Taurine and glutamate efflux, monitored by labeled tracers, showed delayed osmolarity thresholds of H-39 and H-33%, respectively. This observation clearly separates the Cl^– and amino acid osmosensitive pathways. The delayed amino acid efflux may contribute to counteract swelling at more stringent osmolarity reductions. volume regulation; taurine; hyposmolarity; isovolumetric regulation; regulatory volume decrease     

Osmosensitive Release of Neurotransmitter Amino Acids: Relevance and Mechanisms

Hyposmolarity activates amino acid efflux as part of the corrective volume process in a variety of cells. This review discusses the mechanism of amino acid release in brain cells preparations. Results present evidence of substantial differences between the efflux of taurine and that of GABA and glutamate, which besides a possible role as osmolytes, have a main function as synaptic transmitters. The differences found concern the efflux time course, the sensitivity to Cl^– channel blockers, the modulation by tyrosine kinases, the influence of PKC and the effect of cytoskeleton disruptive agents. While taurine efflux features fit well with the mechanisms so far described in most cell types, the efflux of GABA and glutamate does not. Alternate mechanisms for the release of these two amino acids are discussed, including a PKC-modulated, actin-dependent exocytosis.     

Influence of protein kinases on the osmosensitive release of taurine from cerebellar granule neurons

The role of phosphorylation events on the activation and modulation of the osmosensitive (3)H-taurine release (OTR) was examined in cultured cerebellar granule neurons (CGN) stimulated with 30% hyposmotic solutions. OTR was not decreased when [Ca(2+)](i) rise evoked by hyposmolarity was prevented by EGTA-AM (50 microM) or depleted by treatment with 1 microM ionomycin in Ca(2+)-free medium. Accordingly, OTR was not inhibited by Ca(2+)-dependent signaling events. The calmodulin (CAM) blocker W-7 (50 microM) potentiated OTR while the Ca(2+)/CAM kinase blocker KN-93 (10 microM) was without effect. Blockade of PKC by H-7, H-8 (50 microM) and G?6976 (1 microM), as well as activation by phorbol myristate acetate (PMA) (100 nM) did not influence OTR, but chronic treatment to down regulate PKC decreased it by 30%. Forskolin (20 microM) and 8-BrcAMP (10 microM) did not change OTR. Protein tyrosine phosphorylation seems to be of crucial importance in the activation and modulation of OTR, as it was markedly inhibited (90%) by tyrphostine A23 (50 microM) and potentiated by the tyrosine phosphatase inhibitor ortho-vanadate (100 microM). The PI3 kinase blocker wortmannin 100 nM essentially abolished OTR but LY294002 (10-100 microM) was without effect. This difference may be accounted for PI3K isoforms in neurons with different sensitivity to the blockers. Alternatively, the effect of wortmannin may be exerted not in PI3 kinase but instead on phospholipases, which are also sensitive to this blocker. The hyposmotic stimulus induced activation of Erk1/Erk2, but blockade of this effect by PD 98059 (50 microM) only marginally decreased OTR suggesting that the Erk1/Erk2 is an epiphenomenon, not directly involved in OTR activation.