Elevation of glutathione levels by ammonium ions in primary cultures of rat astrocytes.

It is well established that ammonia is detoxified in the brain to form glutamine and that astrocytes play a major role in this process. The synthesis of glutamine requires glutamate and ATP. Since glutamate and ATP are also required for the synthesis of glutathione (GSH), we examined the effect of pathophysiological concentrations of ammonia on levels of GSH in primary cultures of astrocytes. GSH content in the medium increased in a dose- and time-dependent manner in the presence of ammonia. After an initial decrease, cellular GSH content increased in a similar manner. The levels of glutathione disulfide (GSSG) were also increased. A linear relationship was observed between ammonia concentration and the increase in GSH levels. An increase in the efflux of GSH from cells into medium was also observed under these conditions. Buthionine sulfoximine and acivicin, but not methionine sulfoximine, blocked the ammonia induced increase in GSH levels. No, or minor, changes in the activities of enzymes (gamma-glutamyl transpeptidase, GSH reductase and GSH-peroxidase) that might influence GSH levels were identified and thus could not account for the ammonia induced increase in GSH levels in astrocytes. These findings indicate that pathophysiological concentrations of ammonium ions result in increased astroglial levels of GSH which may affect the metabolism and function of astrocytes.

Characterization of cystine uptake in cultured astrocytes.

Glutathione is involved in the maintenance of the structural and functional integrity of membrane proteins, in protection against free radicals and oxidative stress, and in the detoxification of xenobiotics. The cellular uptake of cystine is the rate limiting step in the biosynthesis of glutathione. The precise mechanism for such uptake is not clear as some reports indicate that the uptake occurs through a glutamate-cystine antiporter (system X(c)(-)), whereas, others suggest that it is taken up by the glutamate transporter (system X(AG)). Our studies in cultured astrocytes derived from neonatal rats showed that glutamate, D- and L-aspartate inhibited cystine uptake; that factors that increased intracellular glutamate levels, which would have enhanced the activity of the antiporter, did not stimulate cystine uptake; that the uptake was sodium dependent and partially chloride dependent; that the b(o,+) and ASC systems, which have been shown to carry cystine in some cells, did not mediate cystine uptake in astrocytes; that glutamate uptake blockers such as L-aspartate-beta-hydroxamate (AbetaH) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC), as well as cystine uptake inhibitor L-alpha-aminoadipate (AAA) potently reduced cystine uptake. Additionally, deferoxamine (100 microM) as well as ammonium chloride (5 mM), both of which inhibit glutamate uptake, also inhibited cystine uptake. Taken together, our findings indicate that astrocytes take up cystine through a similar, if not identical, system used to take up glutamate. Interference of cystine uptake by astrocytes through the glutamate transport system may have profound effects on the redox state and the structural and functional integrity of the CNS.

Effect of ammonia on GABA uptake and release in cultured astrocytes.

While the pathogenesis of hepatic encephalopathy (HE) is unclear, there is evidence of enhanced GABAergic neurotransmission in this condition. Ammonia is believed to play a major pathogenetic role in HE. To determine whether ammonia might contribute to abnormalities in GABAergic neurotransmission, its effects on GABA uptake and release were studied in cultured astrocytes, cells that appear to be targets of ammonia neurotoxicity. Acutely, ammonium chloride (5 mM) inhibited GABA uptake by 30%, and by 50-60% after 4-day treatment. GABA uptake inhibition was associated with a predominant decrease in Vmax; the Km was also decreased. Ammonia also enhanced GABA release after 4-day treatment, although such release was initially inhibited. These effects of ammonia (inhibition of GABA uptake and enhanced GABA release) may elevate extracellular levels of GABA and contribute to a dysfunction of GABAergic neurotransmission in HE and other hyperammonemic states.

Association between cell swelling and glycogen content in cultured astrocytes.

Treatment of cultured rat astrocytes with hypotonic media or with 1 mM glutamate for 90 min caused cell swelling and a significant increase in glycogen content. Conversely, treatment with hypertonic media caused cell shrinkage with a corresponding decrease in astrocyte glycogen, which was proportional to the increasing osmolality of the hypertonic media. The glutamate receptor antagonist, MK-801, lowered both the glutamate-induced swelling and glycogen increase. These findings demonstrate a correlation between changes in cell volume and astrocyte glycogen content. This may explain the increased astrocytic glycogen observed in many neuropathological conditions where astrocyte swelling occurs. Because glycogen represents the largest energy reserve in the central nervous system, a swelling-induced disturbance in glycogen metabolism may lead to abnormal glial-neuronal interactions resulting in impaired brain bioenergetics.

Effect of osmolality and anion channel inhibitors on myo-inositol efflux in cultured astrocytes.

Recent studies have shown that swelling-activated myo-inositol efflux from rat C6 glioma cells is mediated by a single transport mechanism and most likely by a volume-sensitive anion channel. In those studies, cells were acclimated in hypertonic medium and then swollen by returning the cells to isotonic medium. In the present study, myo-inositol efflux was determined in primary cultures of astrocytes by first incubating the cells in isotonic radiolabelled medium for 2 hr and then placing the cells in either unlabelled isotonic, hypertonic, or hypotonic medium and measuring release with time. Computer analyses of efflux data indicated a two-component system of myo-inositol efflux. The rate constants for the initial fast component for isotonic and hypotonic cells were 0.0398 +/- 0. 005 and 0.0631 +/- 0.0288 min(-1), respectively. The efflux rates of the slow component, while quite small, were severalfold greater with increasing hypotonic media as compared to the cells in isotonic medium. Several anion membrane transport inhibitors were tested to explore the swelling activated efflux mechanism of myo-inositol. Furosemide (0.5 mM), 1,9 dideoxyforskolin (0.1 mM), NPPB (0.1 mM), niflumic acid (0.5 mM), and SITS (0.5 mM) blocked the fast component of myo-inositol efflux by 17, 49, 55, 75, and 93%, respectively. Our results suggest that the fast component of myo-inositol efflux in primary cultures of astrocytes is mediated by anion transporters or channels and that myo-inositol flux contributes to cell volume regulation in cultures of primary astrocytes.

Effect of dibutyryl cyclic AMP on the kinetics of myo-inositol transport in cultured astrocytes.

Dibutyryl cyclic AMP (dBcAMP) is known to induce maturation and differentiation in astrocytes. As myo-inositol is an important osmoregulator in astrocytes, we examined the effects of maturation and biochemical differentiation on the kinetic properties of myo-inositol transport. Treatment of astrocytes with dBcAMP significantly decreased the Vmax of myo-inositol uptake, but the effect on Km was not significant. The myo-inositol content of astrocytes was significantly decreased in cells treated for 5 days with dBcAMP as compared with untreated controls. Maximum suppression of myo-inositol uptake occurred 7 days after exposure of astrocytes to dBcAMP; this was gradually reversible when dBcAMP was removed from the medium. After exposure to hypertonic medium for 6 h, mRNA expression of the myo-inositol co-transporter was diminished by approximately 36% in astrocytes treated with dBcAMP as compared with untreated cells. It appears that myo-inositol transporters in astrocytes treated with dBcAMP are either decreased in number or inactivated during maturation and differentiation, suggesting that the stage of differentiation and biochemical maturation of astrocytes is an important factor in osmoregulation.

Effect of ammonia and methionine sulfoximine on myo-inositol transport in cultured astrocytes.

Ammonia causes astrocyte swelling which is abrogated by methionine sulfoximine (MSO). Since myo-inositol is an important osmolyte, we investigated the effects of ammonia and MSO on myoinositol flux in cultured astrocytes for periods up to 72 hours. Uptake of myo-inositol was significantly decreased by 26.7 (P < 0.05) and 39.3 (P < 0.006) percent after 48 hours of exposure to 5 or 10 mM ammonia, respectively. The maximum rate of uptake was 14.0+/-0.5 nmol/hour/mg protein which was reduced to 7.45+/-0.27 and 7.02+/-0.57 nmoles/hour/mg protein by 5 or 10 mM ammonia, respectively. The Kms by Michaelis-Menten equation for the control, and in the presence of 5, or 10 mM ammonia were 32.5+/-4.52, 44.4+/-5.82, and 39.3+/-7.0 microM, respectively. Kms by Hanes-Woolf plot for the control, 5, or 10 mM ammonia were 25, 45, and 40 microM, respectively. Treatment of astrocytes with either 5 or 10 mM NH4Cl for 6 hours caused a decrease in myo-inositol content by 66% and 58%, respectively. MSO (3 mM) partially diminished the ammonia-induced inhibition of myo-inositol uptake and decreased myo-inositol content by 31% after 24 hours. Additionally, ammonia increased myo-inositol efflux briefly through the fast efflux component but had little effect on myo-inositol efflux through the slow efflux component of astrocytes exposed to ammonia for up to 72 hours. Predominantly decreased myo-inositol influx coupled with brief efflux through the fast component may represent an adaptive response to diminish the extent of ammonia-induced astrocyte swelling.

Effect of benzodiazepines and neurosteroids on ammonia-induced swelling in cultured astrocytes.

Astroglial swelling occurs in acute hyperammonemic states, including acute hepatic encephalopathy. In these conditions, the peripheral-type benzodiazepine receptor (PBR), a receptor associated with neurosteroidogenesis, is up-regulated. This study examined the potential involvement of PBRs and neurosteroids in ammonia-induced astrocyte swelling in culture. At low micromolar concentrations, the PBR antagonist PK 11195, atrial natriuretic peptide, and protoporhyrin IX, which are known to interact with the PBR, attenuated (16-100%) the effects of ammonia, whereas the PBR agonists Ro5-4864, diazepam binding inhibitor (DBI51-70), and octadecaneuropeptide exacerbated (10-15%) the effects of ammonia. At micromolar concentrations, diazepam, which interacts with both the PBR and the central-type benzodiazepine receptor (CBR), increased swelling by 11%, whereas flumazenil, a CBR antagonist, had no effect. However, at 100 nM diazepam and flumazenil abrogated ammonia-induced swelling. The neurosteroids dehydroepiandrosterone sulfate, tetrahydroprogesterone, pregnenolone sulfate, and tetrahydrodeoxycorticosterone (THDOC), products of PBR stimulation, at micromolar concentrations significantly enhanced (70%) ammonia-induced swelling. However, at nanomolar concentrations, these neurosteroids, with exception of THDOC, blocked ammonia-induced swelling. We conclude that neurosteroids and agents that interact with the PBR influence ammonia-induced swelling. These agents may represent novel therapies for acute hyperammonemic syndromes and other conditions associated with brain edema and astrocyte swelling.

Potassium-stimulated GABA release is a chloride-dependent but sodium- and calcium-independent process in cultured astrocytes.

Depolarization of cultured astrocytes by KCl stimulated gamma-aminobutyric acid (GABA) release in a dose-dependent manner. At 60 mM KCl, the stimulatory effect was calcium- and sodium- independent, and was not altered by the presence of beta-alanine. The potassium-evoked GABA release was inhibited by furosemide and 4-acetamido-4' -isothiocyano-2,2'-stilbene disulfonic acid (SITS), blockers of the chloride transporter across the plasma membrane, as well by chloride ion replacement with glucuronate. Other depolarizing agents, such as veratridine and ouabain, decreased basal GABA release; ouabain also inhibited the stimulatory effect of 60 mM KCl. The high K(+)-induced GABA release may affect CNS excitability and may represent an important aspect of glial-neuronal interactions.

Ionic mechanisms in glutamate-induced astrocyte swelling: role of K+ influx.

L-Glutamate (L-GLU) induced astrocyte swelling in a time- and concentration-dependent, as well as Na+- and Ca2+-dependent, and Cl(-)-independent manner. Swelling was prevented by MK-801, cystine, and ouabain. Since L-GLU swelling is ionically dependent, we determined the role of various ions in such swelling. Our results indicate that K+ uptake plays a major role in the mechanism of L-GLU-induced astrocyte swelling. Like swelling, K+ uptake is dependent on Ca2+ and Na+, but not on Cl-. Likewise, K+ uptake was inhibited by MK-801, cystine, and ouabain. The K+ channel blockers, Ba2+ and tetraethylammonium, partially prevented L-GLU-induced swelling. In addition to K+ channels, K+ influx may also be mediated through Na+/K+-ATPase, as its activity is increased by L-GLU uptake along with the required Na+. Taken together, the data suggest that K+ influx plays a key role in the mechanism of L-GLU-mediated astrocyte swelling.

Effect of osmolality and myo-inositol deprivation on the transport properties of myo-inositol in primary astrocyte cultures.

myo-Inositol uptake measured in primary astrocyte cultures was saturable in the presence of Na+ with a Km of 13-18 microM and a Vmax of 9.4 nmoles/mg protein/hour in myo-inositol-fed cells, indicating a high affinity transport system. In myo-inositol-deprived cells, Km was about 53 microM with a Vmax of 13.2 nmoles/mg protein/hour. Decreasing osmolality decreased the Vmax to about 1.9 nmoles/mg protein/hour whereas increasing osmolality increased Vmax about 5-fold, while Kms were essentially unchanged in myo-inositol fed cells. In cells deprived of myo-inositol, Vmax decreased in hypotonic medium and increased in hypertonic medium almost 10-fold, but with more than a doubling of the Km regardless of the osmolality. Glucose (25 mM) inhibited myo-inositol uptake 51% whereas the other hexoses used inhibited uptake much less. Our findings indicate that myo-inositol uptake in astrocytes occurs through an efficient carrier-mediated Na(+)-dependent co-transport system that is different from that of glucose and its kinetic properties are affected by myo-inositol availability and osmotic stress.

The rapid L- and D-aspartate uptake in cultured astrocytes.

Astrocytes in primary culture possess a rapid L-aspartate saturable transport system (K(m) = 93 microM; V(max) = 81 nmol/min/mg protein), which shows certain stereospecificity since V(max) was 36% lower for D-aspartate uptake. These are values obtained at short incubation time (15 seconds), to obtain approximate initial rate conditions. Metabolic energy inhibitors, rotenone and iodoacetate very potently inhibited the L- and D-aspartate uptake processes, indicating that the transport process is an active one. However, the accumulation of L-aspartate was "enhanced" by inhibitors of L-aspartate metabolism, such as the aspartate aminotransferase inhibitor, aminooxyacetate and L-methionine sulfoximine, an inhibitor of glutamine synthetase, whereas D-aspartate (a non-metabolizable analog of L-aspartate) uptake was not affected. The accumulated levels of L-aspartate in the presence of aminooxyacetate were similar to the levels of D-aspartate. These effects of L-aspartate metabolic inhibitors, suggest that due to metabolism of the the L-aspartate, short incubation time (eg., 15 seconds) is necessary to measure the initial rate of L-aspartate uptake, in order to obtain the "true" kinetic parameters.

Effect of lactic acid on L-glutamate uptake in cultured astrocytes: mechanistic considerations.

Elevated levels of lactic acid can be deleterious to CNS tissue. Lactic acid is known to cause astroglial swelling and since glial swelling has been shown to inhibit L-glutamate (L-Glu) uptake, we examined whether one of the actions of lactic acid is to inhibit L-Glu uptake. Astrocyte cultures treated with lactic acid (25 mM; pH 6.1) showed an inhibition of L-Glu uptake by 65%. HCl (pH 6.1) also inhibited L-Glu uptake and this inhibition was potentiated by sodium lactate (25 mM). The inhibitory effect of lactic acid on L-Glu uptake was partially reversible and the reversibility was enhanced by hypothermia. Blocking glial swelling with D-mannitol, or treatment with antioxidants or hypothermia did not inhibit the effect of lactic acid on L-Glu uptake, indicating that swelling per se or free radicals, were not the factors in L-Glu uptake inhibition. Lactic acid induced a four-fold enhancement of L-Glu release and a seven-fold increase of K+ release. Our results suggest that lactic acid, by direct effect on pH, brings about a stimulation of K+ and L-Glu release which may be a factor in the inhibition of L-Glu uptake by lactic acid in astrocytes.

Effects of ammonia on L-glutamate uptake in cultured astrocytes.

The effect of ammonia on L-glutamate (L-GLU) uptake was examined in cultured astrocytes. Acute ammonia treatment (5-10 mM) enhanced L-[3H]GLU uptake by 20-42% by increasing the Vmax; this persisted for 2 days and than started to decline. Ammonia, however, did not affect the uptake of D-[3H]aspartate (D-ASP), a non-metabolizable analog of L-GLU, that uses the same transport carrier as L-GLU. Also, L-GLU uptake was not affected during the first 2 min of the assay. Thus, ammonia did not have an acute effect of L-GLU transport (translocation); rather, ammonia enhanced the accumulation or "trapping" of L-GLU or its by-products. Chronic ammonia treatment, on the other hand, inhibited L-GLU transport in astrocytes by approximately 30-45% and this was due to a decrease in Vmax, suggesting that the number of L-GLU transporters was decreased. This inhibitory effect was observed after 1 day of treatment and persisted for at least 7 days. The inhibition of L-GLU transport was partially reversible following removal of ammonia. The effects of ammonia on L-GLU transport and uptake may explain the abnormal L-GLU neurotransmission observed in hyperammonemia/hepatic encephalopathy, and the brain swelling associated with fulminant hepatic failure.

Ammonia stimulates the release of taurine from cultured astrocytes.

The effect of ammonia on the release of the neuroactive amino acids taurine (TAU), gamma-aminobutyric acid (GABA) and D-aspartate (D-ASP), an analog of L-glutamate (L-GLU), from cultured rat cortical astrocytes was studied. NH4Cl (1 and 5 mM) induced the release of TAU. TAU release was reduced when Na+ was removed, and was almost completely abolished when Cl- was omitted. In contrast, TAU basal release was enhanced upon removal of Na+ or Cl-. Ammonia inhibited the release of GABA and D-ASP. Ammonia-induced release of astroglial TAU may modify the neuronal excitability accompanying hyperammonemic conditions.

Ionic dependence of adenosine uptake into cultured astrocytes.

Adenosine uptake in cultured astrocytes is dependent on various ions and energy metabolism. The Na(+)-gradient plays an important role, since nigericin, ouabain, amiloride and substitution of Na+ with choline inhibited adenosine uptake. The proton-gradient was of importance, since carbonylcyanide m-chlorophenylhydrozone (CCCP) and omeprazole also inhibited adenosine uptake. Furthermore, adenosine uptake was dependent on Cl- anion. Substitution of Cl- with isethionate, as well as DIDS or furosemide inhibited adenosine uptake. Adenosine uptake was also sensitive to Ca2+ gradient, removal of extracellular Ca2+ and calcimycin inhibited adenosine uptake. Adenosine uptake was not dependent on extracellular K+ and was not affected by valinomycin. Although, K(+)-channel openers (BRL 34195 and nicorandil) as well as the K(+)-channel antagonist, glyburide, inhibited adenosine uptake, the inhibitory effect of BRL 34915 was not antagonized by glyburide. Rotenone and 2,4-dinitrophenol also inhibited adenosine uptake. Ionic dependence and metabolic energy dependence of adenosine uptake suggest that uptake is primarily an active process.

Calcium dependence of hypoosmotically induced potassium release in cultured astrocytes.

A major mechanism in cell volume regulation after hypoosmotic stress is K+ release. Our studies show that in astrocytes, K+ release during hypoosmotic stress is a Ca(2+)-dependent process. Agents that increase intracellular Ca2+, such as ionomycin and ouabain, potentiated hypoosmotically stimulated K+ release, while compounds that block Ca2+ entry during hypoosmotic stress, such as nimodipine, bepridil, and MK-801, inhibited hypoosmotically stimulated K+ release. Similarly, chelation of intracellular Ca2+ blocked hypoosmotically induced K+ release. Caffeine and U-73122 also inhibited K+ efflux under hypoosmotic conditions, suggesting that intracellular Ca2+ release from Ca(2+)-induced Ca2+ release stores and inositol trisphosphate-sensitive intracellular Ca2+ stores play a role in the mechanism of K+ release. Blocking the activity of calmodulin, and of CaM kinase, attenuated hypoosmotically induced K+ release. Our findings indicate that entry of extracellular Ca2+ and Ca2+ release from intracellular stores play a key role in the activation of K+ release under hypoosmotic conditions and thus in cell volume regulation.

Stimulation of calcium uptake in cultured astrocytes by hypoosmotic stress--effect of cyclic AMP.

To investigate the role of Ca2+ in astrocyte volume regulation, we determined Ca2+ fluxes following hypoosmotic stress and how these fluxes were modified by cyclic AMP. In isoosmotic conditions treatment with dibutyryl cyclic AMP (dBcAMP) caused almost a twofold increase in 45Ca2+ uptake. Efflux studies of 45Ca2+ in dBcAMP-treated cells showed three Ca2+ compartments while only two Ca2+ compartments were identified in non-dBcAMP-treated cells. Following hypoosmotic stress a twofold stimulation of 45Ca2+ uptake occurred in both non-dBcAMP-treated and dBcAMP-treated astrocytes. Stimulation of Ca2+ uptake begins at approximately 270 mOsm and is half-maximally stimulated at approximately 100 mOsm. This uptake is partly mediated through L-type 'slow' inactivating Ca2+ channels. Hypoosmotic stress also induces the release of Ca2+ from intracellular stores. The influx of extracellular Ca2+ and efflux of intracellular Ca2+ appear to be important factors in volume regulation after hypoosmotic stress. Cyclic AMP plays an important role in modulating hypoosmotically stimulated Ca2+ uptake.

Effect of hypoosmotic stress on peripheral-type benzodiazepine receptors in cultured astrocytes.

Astrocytes appear to be the primary source of peripheral benzodiazepine (PBZD) receptors in brain. The function of this receptor is not well understood. Since there is evidence that this receptor may be involved in cell volume control, we examined the effect of hypoosmotic stress on the regulation of the PBZD receptors in homogenates of cultured astrocytes derived from neonatal rat cerebral cortex. Exposure of astrocytes that were maintained in the presence of dibutyryl cAMP (dBcAMP) to hypoosmotic medium (200 mOsm) for 24 h resulted in 27 and 57% increased in the number of [3H]PK 11195 and [3H]Ro5-4864-binding sites, respectively, as compared with isoosmotic media (320 mOsm). This receptor upregulation is osmolarity- and time-dependent. However, hypoosmotic stress had no effect on PBZD receptor-binding in astrocytes that were maintained in the absence of dBcAMP. Under isoosmotic conditions, dBcAMP appears to regulate [3H]Ro5-4864 but not [3H]PK 11195-binding sites, a finding which further supports a partial distinction between the binding sites labeled with these ligands. The modulation of PBZD receptors by hypoosmotic stress suggests a possible role for these receptor sites in astrocyte volume control.