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A S Bender

Publications and source records attributed to A S Bender.

At least 19 recordsLinked to original sources
PubMed2000

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.

Ammonia↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Ammonia↗
PubMed1999

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.

Animals↗
PubMed1998

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.

Ammonia↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Aminooxyacetic Acid↗
PubMed1996

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↗
PubMed1994

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.

Adenosine↗
PubMed1994

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.

Ammonium Chloride↗
PubMed1994

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.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

Astrocyte swelling in liver failure: role of glutamine and benzodiazepines.

We examined the effect of methionine sulfoximine (MSO) and peripheral benzodiazepine (BZD) ligands on ammonia-induced swelling of primary astrocyte cultures. Swelling was completely abolished by co-treatment with MSO, an inhibitor of glutamine synthetase. We also established that many of the effects caused by ammonia, including the reduction in K+ uptake, increase in Cl- uptake and reduction in myo-inositol uptake were diminished by co-treatment with MSO. Agonists of the peripheral-type BZD receptor aggravated ammonia-induced swelling, whereas a peripheral BZD receptor blocker, PK 11195, diminished the extent of swelling. Our findings implicate glutamine and the peripheral-type benzodiazepine receptor in the pathogenesis of the edema associated with fulminant hepatic failure.

Ammonia↗
PubMed1994

The role of K+ influx on glutamate induced astrocyte swelling: effect of temperature.

We studied factors involved in glutamate-stimulated astrocyte swelling and the role of temperature in this process. Glutamate-induced astrocyte swelling was reduced by 74% when temperature was lowered from 37 degrees C to 24 degrees C. Reduction in temperature to 24 degrees C also resulted in inhibition of glutamate-stimulated K+ uptake. When both extracellular and intracellular Ca2+ were removed with BAP-TA/AM (50 microM) and 0.1 mM EGTA and in the absence of extracellular Ca2+, glutamate was not able to stimulate the K+ uptake or swelling. Dantrolene (20 microM), MK-801 (0.5 mM) and ouabain (0.5 mM) inhibited glutamate-induced K+ uptake as well as the glutamate effects on swelling. These findings show the importance of Ca2+ in the mechanism of glutamate swelling and suggests that glutamate induces swelling through stimulation of K+ influx and that the diminished effect of glutamate swelling with decrease in temperature may be due to a decrease in K+ uptake.

Animals↗