Evaluation of the osmoregulatory function of taurine in brain cells

Summary Homogenous primary cultures of mouse astrocytes and cortical neurons were used to clarify the role of taurine in ion and osmoregulation in the CNS. This study indicates that both neurons and glial cells have uptake systems for taurine. The cell water content does not change during loading of cells with taurine. Chemical analysis indicates that part of the accumulated taurine is metabolized and that the product(s) are stored in the cells. Extracellular taurine (1 mM) has no effect on K⁺, Na⁺, Cl^-, or Ca²⁺ movements in astrocytes. However, astrocytes loaded to a taurine content which corresponds a concentration of 60 mM (corresponds to normal mouse cortex levels) show a 50% reduction in their K⁺ accumulation by carriers and a 100% increase in Ca²⁺ turnover rates. Movements of Ca²⁺ and K⁺ are involved in neurotransmission. It appears that taurine stored in glial cells, has an important effect on ion homeostasis in the CNS and may act indirectly on neuronal excitability.     

Hyposmolarity-activated fluxes of taurine in astrocytes are mediated by diffusion

In order to obtain information about the mechanism responsible for swelling associated taurine release in astrocytes, the kinetics of taurine uptake in cultured astrocytes from mouse cerebral cortex was studied under isosmotic and hyposmotic (50% osmolarity) conditions. It was found that the V_max for the high affinity component of taurine uptake was unaffected by exposure of the astrocytes to hyposmotic conditions and that the K_m value was somewhat increased. Contrary to V_max, the non-saturable component of the uptake was greatly increased (2.5-fold) after exposure of the cells to hyposmotic media leading to cell swelling. In addition to the kinetic characterization of taurine uptake the actual intracellular taurine content after incubation (15 min) in isosmotic or hyposmotic media with different taurine concentrations (0–100 mM) under Na⁺-free conditions was determined. At taurine concentrations < 30 mM corresponding to the intracellular content in cells not exposed to taurine, exposure to hyposmotic media led to a decrease in the intracellular taurine content. At higher external taurine concentrations (> 30 mM) the intracellular taurine contents were dramatically increased after exposure to hyposmotic conditions. The increase in intracellular taurine seen under hyposmotic conditions at 100 mM external taurine could be significantly reduced by 100 μM DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonate). Altogether these results suggest that a diffusional process rather than the high affinity taurine carrier is involved in the swelling induced increase in astrocytic taurine influx and efflux.     

Ferritin induction protects cortical astrocytes from heme-mediated oxidative injury

Hemin is released from hemoglobin after CNS hemorrhage and may contribute to its cytotoxic effect. In a prior study, we demonstrated that heme oxygenase-1 induction protected murine cortical astrocytes from hemoglobin toxicity. Since heme metabolism releases iron, this observation suggested that these cells are able to effectively sequester and detoxify free iron. In this study, we tested the hypotheses that astrocytes increased ferritin synthesis after exposure to heme-bound iron, and that this induction protected cells from subsequent exposure to toxic concentrations of hemin. Incubation with low micromolar concentrations of hemin, hemoglobin, or ferrous sulfate increased ferritin expression, as detected on immunoblots stained with a polyclonal antibody that was raised against horse spleen ferritin. Time course studies demonstrated an increase in ferritin levels within 2 h. Weak and scattered cellular staining was detected by immunohistochemistry in control, untreated cultures, while diffuse immunoreactivity was observed in cultures exposed to heme-bound iron. An enhanced ferritin band was detected on immunoblots from cultures that were treated with purified apoferritin, consistent with astrocytic ferritin uptake. Immunoreactivity after apoferritin treatment was not altered by concomitant treatment with cycloheximide. Pretreatment with apoferritin protected astrocytes from hemin toxicity in a concentration-dependent fashion between 1 and 4 mg/ml. At the highest concentration, cell death due to a 6-h exposure to 30 microM hemin was decreased by about 85%. A protective effect was also produced by induction of endogenous ferritin with nontoxic concentrations of ferrous sulfate, hemoglobin, or hemin. These results suggest that cortical astrocytes respond to exogenous heme-bound or free iron by rapidly increasing ferritin synthesis. The combined action of heme oxygenase-1 and ferritin may be a primary astrocytic defense against heme-mediated injury.     

Carnosine-synthesis in cultures of rat glial cells is restricted to oligodendrocytes and carnosine uptake to astrocytes

Cultures of glial cells consisting predominantly of oligodendrocytes and astrocytes were prepared to study whether the biosynthesis of carnosine (β-Ala-His) and the cellular uptake of this dipeptide are processes which are associated with a specific cell type. Uptake of the radiolabeled precursor β-alanine was observed in both cultures. Synthesis of radiolabeled carnosine, however, was only observed in oligodendrocyte cultures prepared from rat brain and spinal cord. During oligodendrocyte cultivation we observed a significant increase in the rate of carnosine synthesis which correlates with the differentiation of these cells as revealed by immunostaining with antibodies against oligodendrocyte markers. Carnosine synthesis was not observed in astroglia cell cultures that were depleted of residual O-2A progenitor cells and oligodendrocytes by antibody mediated complement cell killing. Contrary to the synthesis, carnosine was found to be taken up effectively only by astrocytes but not by oligodendrocytes.     

Analysis of growth rate in sucrose-supplemented cultures of Streptococcus mutans.

In the presence of sucrose, Streptococcus mutans grows in large glucan-containing aggregates. Because of reports of linear rather than exponential growth of sucrose-grown cultures, the kinetics of growth of sucrose-grown cultures of S. mutans strain OMZ-176 were compared with those of glucose-grown cultures. Culture turbidity measurements indicated that growth of sucrose cultures was slower, did not follow exponential kinetics, and slowed and stopped at lower absorbance values than did glucose-grown cultures. However, measurements of the rates of accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein using fully equilibrated radioactively labeled precursors of each of these macromolecular species in sucrose and glucose-grown cultures showed that: (i) for glucose cultures the synthesis of each of the three informational molecules occurred at the same exponential rate, which was identical to the rate of turbidity increase; (ii) for sucrose cultures each macromolecular species was synthesized at the same exponential rate and these rates were identical to the rate of increase of turbidity of the glucose-grown culture for periods of up to 7 h. Furthermore, the ratios of DNA to RNA, RNA to protein, and protein to DNA for the sucrose cultures were identical to those for the glucose cultures for up to 10 doublings. From these data it was concluded that in the presence of sucrose S. mutans grows in a balanced fashion at the same exponential rate as it does in glucose. The deviation from an exponential growth model of the absorbance in sucrose cultures was attributed to an optical artifact due to the formation of large glucan-containing aggregates of cells. The addition of dextranase to sucrose cultures resulted in cultures which increased in turbidity at the same exponential rate as glucose-grown cultures, without affecting the rate or extent of macromolecular synthesis.     

86Rubidium release from cultured primary astrocytes: effects of excitatory and inhibitory amino acids

The effects of high K+, glutamate and its analogue, kainate, on K+ release were studied in primary astrocyte cultures prepared from newborn rat brains using 86Rb+ as a tracer for K+. An increase in 86Rb+ release was observed when the extracellular K+ concentration was elevated (10-40 mM). Glutamate and kainate stimulated the release in a dose-dependent manner, 100 microM concentrations being about as equally effective as high K+ (40 mM). Both compounds also caused an increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine, indicating depolarization of the membrane. No significant Na+-dependent uptake of [3H]kainate occurred in the cells, thus excluding the possibility that depolarization was due to electrogenic uptake of amino acid into the cells. GABA and taurine significantly depressed the high K+- and glutamate-induced 86Rb+ release. Taurine itself caused a small increase in 86Rb+ release and the membrane was depolarized, judging from the increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine. No effect of taurine was observed when the Cl- concentration was reduced in the experimental medium. The results suggest that cultured astrocytes respond by membrane depolarization to high external K+ and to glutamate and kainate. The degree of this depolarization can be modified by the inhibitory amino acids GABA, taurine and glycine, the effect of taurine probably being mediated by an increase in Cl- conductance across the cell membrane. The role of functional receptors for amino acid transmitters and the effects observed are discussed.     

K(+)- and temperature-evoked taurine efflux from hypothalamic astrocytes

Hypothalamic astrocytes in culture released taurine, a suspected inhibitory amino acid neurotransmitter/neuromodulator/osmoregulator, in response to isoosmotically increasing extracellular K+ in a dose-dependent fashion. In the absence of added Ca2+, basal release levels rose to approach those obtained after exposure to 60 mM K+ in the presence of 2.5 mM Ca2+, and were only partially lowered by the addition of 10 mM Mg2+. Stimulation with K+ (60 mM) did not further increase taurine efflux above the high basal levels seen in the absence of Ca2+. Under standard conditions complete replacement of Na+ with choline Cl had little effect on basal taurine release, but reduced K(+)-evoked (60 mM) efflux by 60%. The temperature dependence of the basal levels of taurine released from hypothalamic astrocytes was similar to that seen for cultured cerebellar astrocytes and neurons over the range 5-50 degrees C. Taurine release increased from 5 to 15 degrees C, remained constant between 15 and 33 degrees C, decreased between 33 and 37 degrees C and increased thereafter. The infection point of increased basal taurine release seen around 37 degrees C (most prominent in astrocytes), may be of physiological significance. Results presented also show that the ion (Na+, Ca2+ and K+) sensitivities of taurine efflux for cultured hypothalamic astrocytes are similar to those previously reported for cultured astrocytes from the cerebellum.     

Release of taurine from cultured cerebellar granule cells and astrocytes: co-release with glutamate

The properties of the release of preloaded [3H]taurine and endogenous taurine were studied with cultured cerebellar granule cells (7-8 days in vitro) and astrocytes (14-15 days in vitro) from the rat. The spontaneous release of taurine from both cell types was slow. The release from both neurons and astrocytes was significantly enhanced by 0.1 mM veratridine, the stimulatory effect being more pronounced in granule cells than in astrocytes. No homo or heteroexchange with extracellularly added taurine or its structural analogues could be detected, suggesting that the efflux is probably not mediated via the membrane transport sites. Kainate stimulated the release more from granule cells than from astrocytes, the effect apparently being mediated by kainate-sensitive receptors. Depolarization of cell membranes by 50 mM K+ induced co-release of endogenous taurine and glutamate from both cell types. Preloaded [3H]taurine was readily released from astrocytes by potassium stimulation. Stimulated release occurred from granule cells if they had been cultured for 4 days with the label but not from the cells preloaded for only 15 min.     

Endogenous glutathione and catalase protect cultured rat astrocytes from the iron-mediated toxicity of hydrogen peroxide

Primary astrocyte cultures from rat brain were exposed to hydrogen peroxide (H2O2) to investigate peroxide toxicity and clearance by astrocytes. After bolus application of H2O2 (100 microM), the peroxide was eliminated from the incubation medium following first-order kinetics with a half-time of approximately 4 min. The rate of peroxide detoxification was significantly slowed by pre-incubating the cells with the glutathione synthesis inhibitor buthionine sulfoximine (BSO), or the catalase inhibitor 3-amino-1,2,4-triazole (3AT), and was retarded further when both treatments were combined. H2O2 application killed a small proportion of cells, as indicated by the levels of the cytosolic enzyme lactate dehydrogenase in the media 1 and 24h later. In contrast, cell viability was strongly compromised when the cells were pre-incubated with 3AT and/or BSO before peroxide application. The iron chelator deferoxamine completely prevented this cell loss. These results demonstrate that chelatable iron is involved in the toxicity of H2O2 and that both the glutathione system and catalase protect astrocytes from this toxicity.     

Noradrenaline- and glutamate-induced taurine release from bulk isolated adult rat astrocytes.

The influence of noradrenaline and various agonists of glutamatergic receptors on preloaded [3H]taurine release from bulk isolated adult rat brain astrocytes was investigated by a superfusion technique. In the presence of 1 mM noradrenaline a stimulation of taurine release, resembling that observed in astroglial cultures, was preceded by an inhibition of the efflux, thus demonstrating different dynamics of noradrenaline-evoked taurine release from that observed with beta-agonists on cultured astroglia. Application of 1 mM glutamate and kainate produced stimulation of the release, while 1 mM N-methyl-D-aspartate (NMDA) and 1 mM NMDA together with 65 mM K+ had no effect on the [3H]taurine release. These data suggest the presence of kainate-sensitive and the absence of NMDA-sensitive glutamate receptors on bulk isolated astrocytes, which is consistent with previous observations on astrocytes in culture.     

Changes in ganglioside composition and morphological features during the development of cultured astrocytes from rat brain

Changes in ganglioside content over a period of days were examined in astrocytes obtained via cell passage from rat cerebral cortex. Thin-layer chromatography revealed that, in the astrocytes, ganglioside GM1 was absent, the predominant ganglioside being GM3. Also, an increased GD3 content in long-term astrocyte cultures was detected. The morphological features of astrocytes were also studied using immunoperoxidase staining. Astroglial features were characterized by high levels of glial fibrillary acidic protein (GFAP) and vimentin, which are the major intermediate-filament proteins present in astrocytes at an early culture stage. In long-term-cultured (greater than 7 months) astrocytes, vimentin and GFAP were increased in process-bearing cells. Ganglioside GD3 recognized by R24 monoclonal antibody was also expressed in these cells. These results suggest that the increase of ganglioside GD3 in long-term-cultured astrocytes may be related to the appearance of multistellate cells showing strong reactivity against GFAP and vimentin during development over a specified period in culture.     

Aspartate, glutamate and gamma-aminobutyric acid depolarize cultured astrocytes

Cultures of differentiated, glial fibrillary acidic protein-positive astrocytes from early postnatal rat cerebral hemispheres respond with depolarization of 2-36 mV to glutamate, gamma-aminobutyric acid (GABA) and aspartate but not to glycine or taurine. While GABA resulted in a transient depolarization, the effect of glutamate and aspartate persisted during the application. Since neurons were not present in these cultures a contribution of transmitter-mediated K+ release from adjacent neurons could be excluded. The depolarization triggered by these neurotransmitters is therefore an intrinsic reaction of astrocytes.     

Hydrocortisone enhances total IgE levels--but not the synthesis of allergen-specific IgE--in a monocyte-dependent manner.

Recently, hydrocortisone (HC), when combined with human IL-4, has been reported to increase IgE levels in supernatants (SN) of in vitro cultured leucocytes. In this study we investigated the influence of HC on allergen-specific IgE synthesis. Moreover, we examined the relevance of different cell types in this respect. Peripheral blood mononuclear cells (PBMC), T-cell depleted PBMC, CD14-depleted PBMC and highly purified B cells from 10 allergic (birch pollen and/or grass pollen) patients and five non-allergic individuals were investigated. The cells were incubated with HC and/or recombinant human IL-4 (rIL-4) for 8 days. A considerable increase of total IgE was observed in HC/rIL-4-stimulated cultures compared with rIL-4 alone, HC alone or non-stimulated cultures. We demonstrate that this effect depends on the presence of monocytes in in vitro cultures. These results were seen in every experiment, irrespective of healthy or atopic state of the blood donor. The increase of IgE could not be attributed to a rise of birch pollen-and/or grass pollen-specific IgE in patients allergic to these allergens, as shown by IgE-immunoblot. Radio-allergosorbent test (RAST) investigations of HC/rIL-4-stimulated cells cultures from allergic and non-allergic patients confirmed that HC/rIL-4-induced elevated IgE production was also not due to increased production of IgE, specific for important aero-allergens (pollens, house dust mite or animal dander). Therefore we conclude that newly synthesized IgE is not specific for allergens, but that sequential isotype switching in human B cells leads to increased polyclonal IgE production.     

Vasopressin-induced taurine efflux from rat pituicytes: a potential negative feedback for hormone secretion

Previous work on the whole neurohypophysis has shown that hypotonic conditions increase release of taurine from neurohypophysial astrocytes (pituicytes). The present work confirms that taurine is present in cultured pituicytes, and that its specific release increases in response to a hypotonic shock. We next show that vasopressin (VP) and oxytocin (OT) also specifically release taurine from pituicytes. With an EC₅₀ of ∼2 n m , VP is much more potent than OT, and the effects of both hormones are blocked by SR 49059, a V_1a receptor antagonist. This pharmacological profile matches the one for VP- and OT-evoked calcium signals in pituicytes, consistent with the fact that VP-induced taurine efflux is blocked by BAPTA-AM. However, BAPTA-AM also blocks the taurine efflux induced by a 270 mosmol l⁻¹ challenge, which per se does not evoke any calcium signal, suggesting a permissive role for calcium in this case. Nevertheless, the fact that structurally unrelated calcium-mobilizing agents and ionomycin are able to induce taurine efflux suggests that calcium may also play a signalling role in this event. It is widely accepted that in hypotonic conditions taurine exits cells through anionic channels. Antagonism by the chloride channel inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) suggests the same pathway for VP-induced taurine efflux, which is also blocked in hypertonic conditions (330 mosmol l⁻¹). Moreover, it is likely that the osmosensitivity of the taurine channel is up-regulated by calcium. These results, together with our in situ experiments showing stimulation of taurine release by endogenous VP, strengthen the concept of a glial control of neurohormone output.     

Taurine and hypotaurine transport by a single system in cultured neuroblastoma cells

The kinetics of mutual inhibition of taurine and hypotaurine uptake were studied using neuroblastoma C1300 cells as neuronal model. Hypotaurine and GABA inhibited taurine uptake competitively, increasing the apparent K_m. High-affinity uptake of hypotaurine was completely abolished and the low-affinity component competitively inhibited by taurine. GABA affected noncompetitively low-affinity hypotaurine uptake, whereas the effect on high-affinity uptake was competitive, with an increase in the apparent K_m. All structural analogues tested inhibited taurine and hypotaurine uptakes similarly. The most potent inhibitors were β-alanine and 2-guanidinoethanesulphonic acid. The mutual inhibition and similar specificity profiles of taurine and hypotaurine uptakes showed that these amino acids employ a single transport system in neuroblastoma cells. Competitive inhibition by GABA of the high-affinity uptake of taurine and hypotaurine further suggests that also GABA uses the same carrier system.     

The relationship between type-1 astrocytes, Schwann cells and oligodendrocytes following transplantation of glial cell cultures into demyelinating lesions in the adult rat spinal cord

Summary Remyelination of ethidium bromide induced areas of demyelination in the adult rat spinal cord is normally carried out by Schwann cells. When CNS cultures containing large numbers of oligodendrocytes, oligodendrocyte precursors and type-1 astrocytes were injected into such lesions 3 days after the injection of ethidium bromide, remyelination was carried out by oligodendrocytes. When cultures deficient in type-1 astrocytes, prepared by shaking off and subculturing top-dwelling cells, were used there was only a modest increase in the extent of oligodendrocyte remyelination over that seen in uninjected lesions; the majority of axons being remyelinated by Schwann cells. To prove that these Schwann cells were mainly locally derived, shaken cultures were injected into lesions prepared in areas of the spinal cord locally X-irradiated with 40 Grays to inhibit host repair. In these animals the extent of oligodendrocyte remyelination achieved was similar to that seen when unshaken cultures (rich in type-1 astrocytes) were injected into lesions made in non-irradiated tissue. These results indicate that type-1 astrocytes control Schwann cell remyelination of CNS axons.     

Increased monokines in cytomegalovirus infected myelomonocytic cell cultures

In an investigation of the role of monokines in CMV associated immunosuppression we document modulation of both TNF and IL-1 beta in CMV infected THP-1 cells. CMV infected cultures released almost two-fold more IL-1 beta protein and contained significantly higher IL-1 beta mRNA levels than uninfected cultures for 72-96 h after induction. In both CMV infected and uninfected cultures, significant amounts of IL-1 beta protein were not detected until 24 h post induction, while maximum amounts of TNF were detected in culture supernatants by 3 h post induction, suggesting that TNF may play a role in IL-1 beta induction. TNF levels subsequently declined but in infected cultures remained over 2.5-fold higher than controls through 96 h. The CMV alteration in the kinetics and extent of IL-1 beta release must be indirectly mediated by CMV since only 1% of THP-1 cells were infected. Most infected cells expressed CMV immediate early proteins but did not overexpress IL-1. We speculate that CMV infected cells release excess TNF or other stimulatory factors which increase IL-1 beta synthesis. Since IL-1 beta is increased, the decreased IL-1 'activity' described by others as an explanation in part for the immunosuppressive effects of infection may actually reflect alterations of IL-1 inhibitor levels during CMV infection.     

Characterization of normal human brain cultures. Evidence for the outgrowth of leptomeningeal cells

To establish the histogenetic identity of the predominant cell type in monolayer cultures of normal human adult brain, eight brain specimens were placed into culture and characterized according to cell kinetics, karyotype, antigenic expression, and ultrastructural features. The protein profiles of both the cell layer and the medium were analyzed in selected cultures using sodium dodecyl sulfate polyacrylamide gel electrophoresis and diethylaminoethyl cellulose chromatography. All cultures displayed a limited life span in vitro; marked contact inhibition at confluence; a normal karyotype; an intracytoplasmic and extracellular glycoprotein profile consisting of fibronectin, procollagen type III, laminin, and collagen type IV; specialized intercellular junctions; and interstitial collagen chain synthesis. All of these features were identified in our previous study of human leptomeningeal cultures. The results of immunocytochemical staining for glial fibrillary acidic protein were negative in all cultures of normal human brain, except in early passages in two cultures, which lost the glial cell marker during subsequent passages; immunostains for vimentin were positive in all cells in all cultures. These results support the hypothesis that, in this study, cultures derived from normal human brain are not of glial origin. Our findings also suggest that glial cells are less well-suited to monolayer growth under our culture conditions than are other cell types in enzyme-dissociated brain tissue placed in culture, especially leptomeningeal cells. The identification of leptomeningeal cells as the predominant cell type in normal human brain cultures may prove useful in attempts to foster the growth of human glial cells by culturing brain samples under conditions that prohibit the growth of leptomeningeal cells. Under such conditions, astrocytes, oligodendroglia, and ependymal cells could be isolated with greater ease and cultured separately. These purified cultures of different glial cell types would then provide a more relevant in vitro model for studying human neurological diseases.     

Pentylenetetrazol seizure threshold and extracellular levels of cortical amino acids in taurine-deficient kittens

Directly after weaning, kittens were raised on a semisynthetic diet supplemented with 0.4% taurine or devoid of this amino acid. Eight to twelve weeks later the blood plasma concentration of taurine was decreased by 98-99% in kittens fed a taurine-free regimen. Parietal cortex dialysis, performed in anaesthetized kittens, revealed a selective, but less marked, reduction of extracellular taurine. When kainic acid was included in the dialysis buffer, taurine was doubled in taurine-supplemented kittens, but it was only slightly affected in deficient animals. The animals were also used for determination of the threshold of pentylenetetrazol-induced epilepsy 3 days after the dialysis experiment. This was not significantly different between the groups. The present work shows that taurine deficiency in its own right does not elevate interstitial glutamate, an effect previously observed in the 2-guanidino-ethane sulphonic acid model for taurine deficiency. The results further suggest that taurine is better retained in neural cells in the taurine-deficient state. Moreover, the findings argue against a role for endogenous taurine in the control of epileptiform discharge initiation and/or spread.