Taurine and β-alanine uptake in primary astrocytes differentiating in culture: Effects of ions

The effects of ions on taurine and -alanine uptake were studied in astrocytes during cellular differentiation in primary cultures. The uptakes were strictly Na⁺-dependent and also inhibited by the omission of K⁺ and in the presence of ouabain suggesting that their transport is fuelled mainly by these cation gradients. Two sodium ions were associated in the transport of one taurine and -alanine molecule across cell membranes. A reduction in Cl^– concentration also markedly inhibited the uptake of both amino acids, indicating that this anion is of importance in the transport processes. The similar ion dependency profiles of taurine and -alanine uptake corroborate the assumption that the uptake of these amino acids in astrocytes is mediated by the same carrier. In Na⁺- and K⁺-free media both taurine and -alanine uptakes were reduced significantly more in 14-day-old or older than in 7-day-old cultures. No significant changes occurred in the coupling ratio between Na⁺ and taurine or -alanine as a function of spontaneous cellular differentiation or upon dBcAMP treatment. These results suggest that the uptake systems of these structurally related amino acids in astrocytes have reached a relatively high degree of functional maturity by two weeks in culture.     

Effects of β-alanine treatment on the taurine and DNA content of the rat heart and retina

Experimental evidence has suggested that the high endogenous levels of taurine found in the rat heart and retina are maintained to a large extent by transport processes out of the blood, rather than by endogenous biosynthesis. When these high levels are depleted, dysfunction ensues. In vitro studies have shown that -alanine is a good antagonist of these transport processes. The current studies were done to evaluate the feasibility of depleting heart and retinal taurine levels in vivo through treatment of adult rats either orally or with injections of -alanine. None of the treatments had significant effects on retinal taurine content; ventricular taurine was reduced in some situations, but the effects were not maintained, nor as large as with another transport antagonist. No functional changes were observed. Oral treatment with -alanine had fewer obvious side effects than injections, but all treated rats had body weights less than age-matched controls.     

Evidence for net uptake of GABA into mouse astrocytes in primary cultures—Its sodium dependence and potassium independence

Content of GABA was measured in cultured, normal astrocytes (from the brain cortex of newborn mice) together with the effect of nonradioactive GABA on the efflux of labeled GABA from cells previously loaded with [¹⁴C]GABA. An increase of external GABA concentration from 0 to 25 M evoked a rise of the GABA content in the cells to a level which was approximately 50 times that of the incubation medium. Neither 200 nor 2000 M nonradioactive GABA had any effect on the rate of release of radioactivity from cells loaded with [¹⁴C]GABA. Both the high tissue/medium ratio and the lack of a GABA-induced enhancement of the release of radioactivity indicate that the previously observed high-affinity uptake of GABA in cultured astrocytes represents a net uptake and not a homoexchange with endogenous GABA. This uptake is sodium dependent but was found to be unaffected in potassium-free media; the quantitative correlation between GABA transport and sodium transport differed from that reported for synaptosomes.     

Similar effects ofβ-alanine and taurine in cholesterol metabolism

β-Alanine, though producing a deficiency of taurine in the tissues, had a similar effect on cholesterol metabolism as taurine. Both caused increased activity of hepatic hydroxymethylglutaryl coenzyme A reductase and increased incorporation of 1, 2 of [¹⁴C]-acetate into liver cholesterol. Both caused increased concentration of biliary cholesterol and bile acids. There was increased activity of lipoprotein lipase in heart, but decreased activity in the adipose tissue in both cases. Release of lipoproteins into circulation was decreased in both cases.     

The level of β-alanine aminotransferase activity in regenerating and differentiating rat liver

β-Alanine aminotransferase from rat liver was purified to electrophoretic homogeneity. The immunological and kinetic properties of this enzyme were similar to those of the enzyme from rat brain. However, the liver enzyme transaminates from β-alanine to 2-oxoglutaric acid, while the brain enzyme transaminates from γ-aminobutyric acid. β-Alanine aminotransferase activity in regenerating rat liver was lower than that in control rat liver. Activity of this enzyme, as well as of other uracil-catabolizing enzymes (Weber, G., Queener, S.F. and Ferdinandus, A. (1970) in Advances in Enzyme Regulation (Weber, G., ed.), Vol. 9, pp. 63–95, Pergamon Press, Oxford), was low in newborn rat liver and increased about 5-fold, reaching the level observed in adult rat liver. β-Alanine and prednisolone induced β-alanine aminotransferase in rat liver.     

Expression of estrogen receptor α and β in rat astrocytes in primary culture: effects of hypoxia and glucose deprivation

Estrogen replacement therapy could play a role in the reduction of injury associated with cerebral ischemia in vivo, which could be, at least partially, a consequence of estrogen influence of glutamate buffering by astrocytes during hypoxia/ischemia. Estrogen exerts biological effects through interaction with its two receptors: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), which are both expressed in astrocytes. This study explored effects of hypoxia and glucose deprivation (HGD), alone or followed by 1 h recovery, on ERα and ERβ expression in primary rat astrocyte cultures following 1 h exposure to: a) 5 % CO(2) in air (control group-CG); b) 2 % O(2)/5 % CO(2) in N(2) with glucose deprivation (HGD group-HGDG); or c) the HGDG protocol followed by 1 h CG protocol (recovery group-RG). ERα mRNA expression decreased in HGDG. At the protein level, full-length ERα (67 kDa) and three ERα-immunoreactive protein bands (63, 60 and 52 kDa) were detected. A significant decrease in the 52 kDa band was seen in HGDG, while a significant decrease in expression of the full length ERα was seen in the RG. ERβ mRNA and protein expression (a 54 kDa single band) did not change. The observed decrease in ERα protein may limit estrogen-mediated signalling in astrocytes during hypoxia and recovery.     

High-affinity uptake of γ-aminobutyric acid in cultured glial and neuronal cells

Both glial and neuronal cells maintained in primary culture were found to accumulate [³H]GABA by an efficient high-affinity uptake system (apparentK _m=9 M,V _max=0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1 mM) also strongly inhibited uptake of [³H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, -alanine, and 2,4-diaminobutyrate) inhibited uptake of [³H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine,l-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (K _m=92 M,V _max=0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [³H]GABA against a concentration gradient. ApparentK _m of this uptake was relatively high (819 M), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, none of the nontransformed continuous cell lines of either tumoral (glioma, C₆; neuroblastoma, Ml; MINN) or normal (NN; I₆) origin actively accumulated [³H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.     

GDNF and TGF-β1 promote cell survival in serum-free cultures of primary rat microglia

Recent evidence indicates that glial cell line-derived neurotrophic factor (GDNF) may influence microglial survival, proliferation, and activation, but this has not yet been tested on isolated primary microglia. We compared the effects of individual and combined application of 10 ng/ml GDNF and 1 ng/ml transforming growth factor-beta1 (TGF-beta1) on total cell number, 5-bromo-2'-deoxyuridine (BrdU) incorporation, DNA nick-end labelling (TUNEL staining), and nitrite and lactate dehydrogenase (LDH) secretion in serum-free cultures of primary rat microglia. GDNF as well as TGF-beta1 enhanced the total number of lectin-positive cells and decreased the number of TUNEL-positive nuclei, while no effect on proliferation was observed. Both factors suppressed the secretion of nitrite during the first 4 days of culturing, and GDNF but not TGF-beta1 reduced the secretion of LDH in 2-week-old cultures. These findings suggest that GDNF and TGF-beta1 support survival of primary microglia in vitro.     

δ and κ Opiate receptors in primary astroglial cultures from rat cerebral cortex

The effects of , , and receptor-agonists on forskolin stimulated cyclic adenosine-3, 5-monophosphate (cAMP) formation were examined in astroglial enriched primary cultures from the cerebral cortex of newborn rats. Intracellular cAMP accumulation was quantified by radioimmunoassay. Morphine was used as a -receptor agonist, D-Ala-D-Leu-Enkephalin (DADLE) as a -receptor agonist and dynorphine 1–13 (Dyn) as a -receptor agonist. Basal cAMP levels were unaffected by either the opiate agonists or the antagonists used. In the presence of the cAMP stimulator forskolin, morphine had no significant effect on the cytoplasmic cAMP levels. DADLE caused a dose related inhibition of the forskolin stimulated cAMP accumulation. The effects of this receptor stimulation was blocked with the selective antagonist ICI 174.864. In the presence of Dyn, the forskolin stimulated cAMP accumulation was inhibited in a dose related manner. This receptor stimulation was blocked with the selective antagonist MR 2266. Co-administration of DADLE and Dyn resulted in a non additive inhibition of the forskolin stimulated accumulation of cAMP. These findings indicate that astroglial enriched cultures from the cerebral cortex of rats express and -receptors co-localized ont he same population of cells, and that these receptors are inhibitory coupled to adenylate cyclase.     

Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells,cell suspensions,and primary monolayer cultures

Summary Leakage of lactate dehydrogenase and staining by the vital dye trypan blue were investigated in adult rat hepatocytes at the time of isolation, in suspensions up to 3 h and in primary monolayer cultures up to 3 d. These two parameters of plasma membrane integrity were found to correlate closely in hepatocyte suspensions, but to a lesser degree in monolayer cultures. Functional activity was demonstrated in culture by glucose consumption and lactic acid production. There was a balance of total lactate dehydrogenase (LDH) activity over time for both hepatocyte suspensions and cultures. Loss of LDH activity in the cell fraction was accompanied by a corresponding increase in enzyme activity in the media fraction. Lactate dehydrogenase activity per dye-excluding hepatocyte was calculated to be 9.2±1.5×10⁻⁶ IU assayed at 37°C for 25 preparations of isolated hepatocytes. The results suggest that leakage of cytoplasmic enzyme and vital dye staining are of comparable sensitivity in evaluating hepatocyte preparations. Measurement of LDH leakage offers a less subjective alternative to cell counting procedures and is applicable to both attached and suspended cells. This study was supported in part by Grants HL-11945-11 and 1-RO1-AM 26520-01A1 from the National Institutes of Health, Bethesda, MD.     

Differences in nutrient uptake between the fat body and embryonic primary cultures of silkworm （Bombyx mori）

Nutrition utilization and by-product formation in cultured insect cells has been investigated in several insect cells and has been of great interest to cell culturists and physiologists. In this research the biochemical changes in embryonic and fat body primary cultures of silkworm, Bombyx mori, have been compared. TC-100 medium supplemented with 10% and 20% FBS was used in embryonic and fat body primary cultures, respectively. Medium was renewed every week and the amount of glucose, uric acid, urea, total protein and alkaline phosphatase were measured in the samples from medium of primary cultures using spectrophotometeric methods. All biochemical macromolecules except uric acid showed significant changes. Glucose decreased in embryonic tissues, while in fat body culture its amount increased. Urea accumulation in embryonic culture was higher than in the fat body cultures. Since urea is a by-product, this accumulation could be due to higher utilization of amino acids. Total protein showed considerable changes and was consumed by embryonic culture more than the fat body＇ s. Alkaline phosphatase showed stronger activity in embryonic cells.     

Production and degradation of β-Poly-L-malate in cultures of Physarum polycephalum

β-Poly-L-malate (PMA) is synthesized by plasmodia of Physarum polycephalum during growth and secreted into the culture medium. There it is degraded to L-malate after growth has ceased. Its concentration is highest in cell nuclei, where it probably performs a plasmodium-specific function.     

Mitogenic response of rat lung and tracheal epithelial cells in monolayer primary cultures—Modulation of TNF-α expression

Epithelial cells isolated from rat lung and trachea were grown on monolayers and their response to a number of hormones and growth factors were studied. Maximum proliferative response in serum containing media was observed when insulin, cholera toxin and cortisol were present together. However, these additives when present independently showed a marginal response. The synergism, due to these factors in promoting growth was seen very early in culture (day 4) as shown by thymidine labelling studies, On examining the indices of early mitogenesis, such as the expression ofc-myc, our data suggests that these factors stimulate the expression ofc-myc within 4 h. With respect to expression of TNF-α mRNA, this study suggests a possible modulation of TNF-α expression in response to these mitogens that stimulate proliferation maximally. Whether this expression of TNF-α by these epithelial cells is due to a maximal proliferative stimulus and/or is an early step in the cascade of intracellular signalling events is to be investigated in detail.     

Differential effects of ammonia and β-methylene-dl-aspartate on metabolism of glutamate and related amino acids by astrocytes and neurons in primary culture

The effects of ammonium chloride (3 mM) and -methylene-dl-aspartate (BMA; 5 mM) (an inhibitor of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle (MAS)) on the metabolism of glutamate and related amino acids were studied in primary cultures of astrocytes and neurons. Both ammonia and BMA inhibited¹⁴CO₂ production from [U-¹⁴C]-and [1-¹⁴C]glutamate by astrocytes and neurons and their effects were partially additive. Acute treatment of astrocytes with ammonia (but not BMA) increased astrocytic glutamine. Acute treatment of astrocytes with ammonia or BMA decreased astrocytic glutamate and aspartate (both are key components of the MAS). Acute treatment of neurons with ammonia decreased neuronal aspartate and glutamine and did not apparently affect the efflux of aspartate from neurons. However, acute BMA treatment of neurons led to decreased neuronal glutamate and glutamine and apparently reduced the efflux of aspartate and glutamine from neurons. The data are consistent with the notion that both ammonia and BMA may inhibit the MAS although BMA may also directly inhibit cellular glutamate uptake. Additionally, these results also suggest that ammonia and BMA exert differential effects on astroglial and neuronal glutamate metabolism.This paper is dedicated to Professor E. Kvamme. Dr. Kvamme has conducted numerous pioneering studies on the regulation of the metabolism of glutamine, glutamate and ammonia in nervous and other tissues (see Refs. 1 and 3 for a complete discussion and citation of his many papers). Many important ideas in this exciting field of research have emerged from the work carried out in his laboratory.     

Simultaneous determination of leu-enkephalin localization and [3H]γ-aminobutyric acid uptake in rat striatal cell cultures

The purpose of this study was to investigate the coexistence of gamma-aminobutyric acid (GABA) and leu-enkephalin in single neurons from the corpus striatum. Monolayer cell cultures, started from newborn rat corpus striatum, were grown in serum-free medium and examined using GABA autoradiography and leu-enkephalin immunocytochemistry in a double-label protocol. Examples of cells were found which were positive for one or the other neurotransmitter or for neither transmitter, but not for both. Furthermore, cells which appear similar by morphological criteria alone differed in transmitter specificity. We conclude that the two transmitters tested are not localized within single cells and that morphology alone is inadequate to identify functional cell classes in this area.     

Glial α1-receptors probably inhibit the high-affinity uptake of noradrenaline into astrocytes in the rat brain in vivo

The effect of ₂-receptor blockage on the extraneuronal turnover of noradrenaline (NA) has been studied in the intact rat brain. Tropolone and yohimbine, along with reserpine or desmethylimipramine, were given 30 min after intracerebroventricular injection of [7-³H]NA, i.e. after the tracer had been stored or inactivated. Tropolone given alone did not change the fractions of³H-activity recovered as [³H]NA from hypothalamus, septum, striatum and pons-medulla, but in the presence of yohimbine improved the [³H]NA recovery in all areas except pons-medulla. The maximum effect was seen in the hypothalamus of reserpine-treated rats. Since the ₂-autoreceptors were blocked, the increased [³H]NA recovery does not reflect a down-regulated neuronal NA turnover. Instead it seems to show that a fraction greater than normal of neuronally released NA had been taken up into astrocytes and remained unmetabolized if catechol-O-methyltransferase was inactive. It is assumed that yohimbine enabled the protective tropolone effect by blocking astrocytic ₂-receptors that otherwise, either by itself or by antagonizing -receptor-induced hyperpolarization or cAMP formation, had impaired parameters that stimulate the high-affinity NA Uptake₁ of astrocytes (e.g. membrane potential, Na⁺, K⁺-ATPase) or control the gap junction permeability in the glial syncytium.     

Leptin inhibits amyloid β-protein degradation through decrease of neprilysin expression in primary cultured astrocytes

Pathogenesis of Alzheimer’s disease (AD) is characterized by accumulation of extracellular deposits of amyloid β-protein (Aβ) in the brain. The steady state level of Aβ in the brain is determined by the balance between its production and removal; the latter occurring through egress across blood and CSF barriers as well as Aβ degradation. The major Aβ-degrading enzymes in the brain are neprilysin (NEP) and insulin-degrading enzyme (IDE), which may promote Aβ deposition in patients with sporadic late-onset AD. Epidemiological studies have suggested an inverse relationship between the adipocytokine leptin levels and the onset of AD. However, the mechanisms underlying the relationship remain uncertain. We investigated whether leptin is associated with Aβ degradation by inducing NEP and IDE expression within primary cultured astrocytes. Leptin significantly decreased the expression of NEP but not IDE in a concentration- and time-dependent manner through the activation of extracellular signal-regulated kinase (ERK) in cultured rat astrocytes. Furthermore, leptin inhibited the degradation of exogenous Aβ in primary cultured astrocytes. These results suggest that leptin suppresses Aβ degradation by NEP through activation of ERK.     

Simultaneous determination of F-β-alanine and β-alanine in plasma and urine with dual-column reversed-phase high-performance liquid chromatography

F-β-Alanine and β-alanine were detected in plasma and urine samples with fluorescence detection of orthophthaldialdehyde derivatives of F-β-alanine and β-alanine after separation with dual-column reversed-phase HPLC. The detection limits of F-β-alanine and β-alanine in the HPLC system were approximately 0.3 and 0.7 pmol, respectively. The procedure proved to be very reproducible with intra-assay RSDs and inter-assay RSDs being less than 8%. The usefulness of the method was demonstrated by the analysis of the F-β-alanine and β-alanine concentrations in plasma and urine samples from tumor patients treated with S-1 (Tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate in a molar ratio of 1:0.4:1).