Biomedical subjects

H K Kimelberg

Publications and source records attributed to H K Kimelberg.

At least 19 recordsLinked to original sources

PubMed1995

Hypoosmotic volume regulation and osmolyte transport in astrocytes is blocked by an anion transport inhibitor, L-644,711.

Cell volume, potassium content, and potassium influx were measured in rat cerebral astrocytes grown in primary culture following exposure to hypoosmotic medium containing either 3.2 mM or 50 mM potassium. Some solutions also contained 1 mM L-644,711, an anion transport inhibitor. L-644,711 inhibited volume regulation and taurine efflux induced by hypoosmotic exposure in medium containing either potassium concentration. L-644,711 also inhibited potassium uptake associated and not associated with the sodium/potassium pump. The correlation of reduced taurine efflux and volume decrease produced by L-644,711 exposure indicates the important role for this amino acid in hypoosmotic astrocyte volume regulation. However, the effects of L-644,711 on potassium transport indicate that multiple actions of this drug may be important factors in its effect on astrocyte volume regulatory mechanisms.

Amino Acids↗

PubMed1995

Cadmium chloride (CdCl2)-induced metallothionein (MT) expression in neonatal rat primary astrocyte cultures.

Metallothionein (MT) protein and mRNA levels were studied following exposure of rat neonatal primary astrocyte cultures to cadmium chloride (CdCl2). MT mRNA was probed on Northern blots with a 32P labeled synthetic cDNA probe specific for rat MT mRNA. The probe hybridizes to a single mRNA with a size appropriate for MT, approximately 550 bases. Expression of MT-I mRNA in astrocyte monolayers exposed to 2 x 10(-6) M CdCl2 for 6 h was increased approximately 5-fold (9.7 fg/micrograms total RNA) over MT-I mRNA levels in controls (2 fg/micrograms total RNA). MT-I mRNA could also be detected in untreated cells, suggesting constitutive MT expression in these cells. Western-blot analysis revealed a marked increase in MT protein levels upon exposure to CdCl2 (1 x 10(-6) M; 96 h). Consistent with the constitutive expression of MTs both at the mRNA level and protein level, we have also demonstrated a time-dependent increase in MT-immunoreactivity in astrocytes exposed to CdCl2. The present study suggests that astrocytes constitutively express MTs, and that MT-induction by CdCl2 may be an example of a generalized increase in MTs in response to heavy metal exposure, thus protecting astrocytes, and perhaps also indirectly, juxtaposed neurons from the neurotoxic effects of heavy metals.

Animals↗

PubMed1995

Receptors on astrocytes--what possible functions?

Receptors for transmitters, as varied as those expressed by neurons, have been described on primary astrocyte cultures prepared from new-born rats and mice. A variety of functional effects and considerable cell-to-cell and regional heterogeneity have been observed for such receptors in vitro. The various systems available for studying the presence and properties of receptors on astrocytes in situ, and the results from these studies, are discussed. Much fewer studies using these more difficult systems have been done. So far, some resemblances and differences between in situ and in vitro work have been observed. More of these in situ studies, to supplement the ongoing in vitro work, are needed to enable us to determine unequivocally which receptors are present on astrocytes, and their functions in vivo. If there is cell-to-cell and CNS regional heterogeneity in vivo comparable to that seen in vitro, these analyses will be very complex. To illustrate the importance and variety of receptor-linked functions, a number of suggestions are made in this commentary, based on current proposals for the roles of astrocytes. However, it is argued that we need to have a more complete understanding of astrocyte functions in vivo, before we can really understand the functional significance of astrocyte receptors.

Animals↗

PubMed1995

Astrocytic swelling due to hypotonic or high K+ medium causes inhibition of glutamate and aspartate uptake and increases their release.

Astrocytic swelling occurs readily in ischemia and traumatic brain injury (TBI) as part of the cytotoxic or cellular edema response. Ischemia is known to produce large extracellular increases in both [K+] and excitatory amino acids (EAA) in vivo, and astrocytic swelling in vitro leads to marked release of EAA. In this study we compared the effect of swelling due to hypotonic media and high K+ medium on the uptake and release of EAA by rat primary astrocyte cultures in vitro. In both cases, there was a significant inhibition of uptake of [3H]L-glutamate and [3H]D-aspartate, and increased release of preloaded [3H]D-aspartate. The kinetics of the increased efflux was very different in response to hypotonic or high K+ media. In hypotonic medium there was a rapid initial release followed by a decline in the rate of release over time. This release was independent of whether Na+ was present. Upon exposure to high K+ medium there was a slow progressive increase in release of [3H]D-aspartate, which never showed any subsequent decline until the media was returned to normal [K+]. In high K+ media there was also an initial transient increase in [3H]D-aspartate release, which we attribute to reversal of the amino acid uptake system. The increased release due to hypotonic medium was not affected by a drop in temperature from 37 to 26 degrees C, while the increased release due to high K+ medium was completely inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Metallothionein induction in neonatal rat primary astrocyte cultures protects against methylmercury cytotoxicity.

Metallothionein (MT) protein and mRNA levels were monitored following exposure of rat neonatal primary astrocyte cultures to methylmercury (MeHg). MT-I and MT-II mRNAs were probed on northern blots with an [alpha-32P]dCTP-labeled synthetic cDNA probe specific for rat MT mRNA. MT-I and MT-II mRNAs were detected in untreated cells, suggesting constitutive MT expression in these cells. The probes hybridize to a single mRNA with a size appropriate for MT, approximately 550 and 350 bp for MT-I and MT-II, respectively. Expression of MT-I and MT-II mRNA in astrocyte monolayers exposed to 2 x 10(-6) M MeHg for 6 h was increased over MT-I and MT-II mRNA levels in controls. Western blot analysis revealed a time-dependent increase in MT protein synthesis through 96 h of exposure to MeHg. Consistent with the constitutive expression of MTs at both the mRNA level and the protein level, we have also demonstrated a time-dependent increase in MT immunoreactivity in astrocytes exposed to MeHg. The cytotoxic effects of MeHg were measured by the rate of astrocytic D-[3H]aspartate uptake. Preexposure of astrocytes to CdCl2, a potent inducer of MTs, completely reversed the inhibitory effect of MeHg on D-[3H]aspartate uptake that occurs in MeHg-treated astrocytes with constitutive MT levels. Associated with CdCl2 treatment was a time-dependent increase in astrocytic MT levels. In summary, astrocytes constitutively express MTs; treatment with MeHg increases astrocytic MT expression, and increased MT levels (by means of CdCl2 pretreatment) attenuate MeHg-induced toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Current concepts of brain edema. Review of laboratory investigations.

Klatzo's classification of brain edema into two types, vasogenic and cytotoxic, has been in general use since 1967. The former involves overall brain swelling due to fluid entry from the vasculature because of openings in the blood-brain barrier (BBB), whereas the latter refers to cell swelling without any loss of the normal impermeability of the BBB. This review principally covers new work that identifies the intracellular swelling of astrocytes as a major form of cytotoxic edema seen in many different kinds of brain injury. The term edema should be retained because of its familiarity; however, because such intracellular swelling is usually not a response to toxins, it is suggested that the term cellular edema is preferable to cytotoxic edema. The difficulties involved in measuring cellular edema clinically are discussed, and the belief that a "pure" form of either edema is unlikely to exist. It is emphasized that the mechanisms and direct consequences of vasogenic and cellular edema are so different that the connection is mainly semantic. Studies conducted in vitro have identified several potentially damaging secondary consequences of astrocytic swelling. One of the most important of these seems likely to be the increased release of excitatory amino acids from swollen astrocytes. Potential mechanisms for inhibition of the increased release of amino acids have been identified in vitro and could prove therapeutically useful.

Amino Acids↗

PubMed1994

Intracellular glutathione (GSH) levels modulate mercuric chloride (MC)- and methylmercuric chloride (MeHgCl)-induced amino acid release from neonatal rat primary astrocytes cultures.

Mercuric chloride (MC) and methylmercury (MeHg) were found to increase amino acid release from astrocytes. This suggests interaction with sulfhydryl (-SH) groups which are controlled by glutathione [GSH] levels. In the present study, we evaluated the effects of alterations in intracellular glutathione concentrations [GSH]i on the outcome of MC and MeHg treatment. [GSH]i were increased in a time-dependent fashion by incubating the astrocytes with 1 mM L-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine precursor. OTC attenuated the release of [2,3-3H]D-aspartic acid from astrocytes exposed to MC- (5 microM) and MeHg-(10 microM). MeHg-induced [3H]D-taurine release was also reduced by pretreatment of astrocytes with OTC. Treatment with BSO (50 microM) decreased [GSH]i in astrocytes, and increased [2,3-3H]D-aspartate release from MC- and MeHg-treated astrocytes, and [3H]D-taurine release from MeHg-treated cells. Neither OTC nor BSO when added to cultures in the absence of MC or MeHg had an effect on amino acid release by astrocytes. The current study underscores both the sensitivity of astrocytes to mercurials in terms of amino acid release and the relationship of these effects of astrocytic [GSH]i.

Animals↗

PubMed1994

The role of sulfhydryl groups in D-aspartate and rubidium release from neonatal rat primary astrocyte cultures.

We have recently demonstrated that both methylmercury (MeHg) and mercuric chloride (MC) induce D-aspartate release from neonatal rat primary astrocyte cultures maintained in isotonic conditions. In the present study, we compare several other sulfhydryl-(-SH) selective alkylating reagents [methyl methanethiosulfonate (MMTS), N-ethylmaleimide (NEM), and iodoacetamide (IA)] in isotonic, as well as hypotonic conditions to discern the functional importance of -SH groups in [3H]D-aspartate and 86rubidium (86Rb) release from astrocytes. Treatment of astrocytes (5 min) in isotonic buffer with the hydrophobic reagent NEM (10 microM) caused a marked increase in 86Rb release but had no effect on [3H]D-aspartate release. Neither IA-, nor MMTS-treatment (both at 10 microM) induced increase in [3H]D-aspartate or 86Rb release in isotonic buffer. In hypotonic condition (-50 mM Na+), astrocytes were most sensitive to MC exposure (5 microM), exhibiting an increase in both [3H]D-aspartate and 86Rb efflux. The hydrophobic compounds MMTS and NEM, and the hydrophilic -SH modifying reagent, IA, attenuated the hypotonic-induced efflux of [3H]D-aspartate, in the absence of an effect on 86Rb release. These observations are consistent with a critical role for -SH groups both in basal (i.e. isotonic) and hypotonic-induced release of D-aspartate and Rb from astrocytes. Lack of uniformity of these effects may be attributed to site-specificity, related to the physicochemical properties of these -SH alkylating reagents.

Alkylating Agents↗

PubMed1994

Potassium and taurine release are highly correlated with regulatory volume decrease in neonatal primary rat astrocyte cultures.

Neonatal rat primary astrocyte cultures were swollen by exposure to hypotonic buffer. Using an electrical impedance method for determination of cell volume coupled with on-line measurements of efflux of radioactive ions or amino acids, we have investigated the role of K+ (using 86Rb), taurine, and D-aspartate (an analogue of glutamate) in regulatory volume decrease (RVD). Addition of 1 mM quinine, 10 microM nimodipine, 100 microM BAPTA-AM, 10 microM trifluoperazine, or a calcium-free buffer significantly (p < 0.0001) inhibited RVD. This was accompanied by inhibition of 86Rb release but an increase in D-[3H]-aspartate release, which was proportional to the degree to which RVD was inhibited. These results support a regulatory role for calcium in RVD and show that inhibition of calcium entry from the extracellular fluid, intracellular calcium sequestration, inhibition of calcium-activated K+ channels, and inhibition of calmodulin all inhibit RVD. Because D-[3H]aspartate efflux profiles increase as RVD is inhibited, it is unlikely that D-aspartate release is a main determinant of RVD. In contrast, [3H]taurine release was increased by 1 mM quinine and inhibited by 10 microM trifluoperazine. The net release of K+ and taurine is highly correlated with the degree of RVD, implicating a regulatory role for both K+ and taurine release in RVD.

Animals↗

PubMed1994

Astrocytes as mediators of methylmercury neurotoxicity: effects on D-aspartate and serotonin uptake.

In this study we address the effects of methylmercuric chloride (MeHgCl), a metal that is preferentially sequestered in astrocytes, on 5-HT and glutamate/aspartate uptake by rat primary astrocyte cultures. Quantitative autoradiography (ARG) combined with glial acidic fibrillary protein (GFAP) immunocytochemistry, as well as intact-cell (bulk) measurements of radiolabel uptake of these neurotransmitters were performed in 7- and 21-day-old primary astrocyte cultures. MeHg (10 microM for 30 min) treatment of astrocytes (21 days in culture) significantly inhibited the Na(+)-dependent and fluoxetine-sensitive [3H]5-HT uptake. D-aspartate uptake in 7- and 21-day-old cultures was even more sensitive to MeHg, leading to > 99% inhibition of D-aspartate uptake by astrocytes (30 min; 10 microM MeHg). These results imply that the Na(+)-dependent and fluoxetine-sensitive 5-HT uptake, as well as the Na(+)-dependent L-glutamate/D-aspartate uptake systems in primary astrocyte cultures are sensitive to low concentrations of MeHg. Since astrocytic removal of glutamate (and aspartate) and 5-HT from the extracellular space in situ is crucial to the maintenance of chemical homeostasis, MeHg-induced uptake inhibition of 5-HT and aspartate could have cytotoxic effects on neighboring neurons.

Animals↗

PubMed1994

Na(+)-dependent, fluoxetine-sensitive serotonin uptake by astrocytes tissue-printed from rat cerebral cortex.

Previous studies have established that rat primary astrocyte cultures prepared from several brain regions of 1-4-d-old rats exhibit high-affinity, Na(+)-dependent and fluoxetine-sensitive serotonin (5-HT) uptake with a Km for 5-HT of 0.4 microM and a Ki for fluoxetine of 23 nM, which correspond to the characteristics for this transport for other brain preparations. However, it is not known whether astrocytes in situ show such uptake. We addressed this question by performing 3H-5-HT uptake experiments on cortical astrocytes, within 4 hr of isolating them from 6- and 21-d-old rats by the tissue-print technique. Quantitative autoradiography was combined with GFAP and neurofilament (NF) immunocytochemistry to distinguish astrocytic from neuronal 3H-5-HT uptake. In composition, the tissue-printed (TP) cells and processes were 60-70% GFAP (+) and 10-15% NF(+). 3H-5-HT uptake (0.3 microM 5-HT, 3.4 microCi/ml) in both tissue-printed GFAP(+) astrocytes and NF(+) structures was sensitive to 1 microM fluoxetine and was also Na+ dependent. More than 90% of TP astrocytes from 6- and 21-d-old rats and 100% of NF(+) structures from 21-d-old rats showed positive 3H-5-HT uptake (defined as > or = 31 grains/10(3) microns2). The highest level of uptake (> or = 191 grains/10(3) microns2) was never observed in TP astrocytes but was exhibited by about half of the NF(+) structures. In other experiments were found that 3H-5-HT uptake by 6-d-old TP astrocytes was comparable to uptake by postnatal age-matched primary cultured astrocytes that were grown in fetal bovine serum (FBS). However, primary cultured astrocytes grown in horse serum showed lower uptake than that observed with FBS, a finding similar to previous results in cultures where 3H-5-HT uptake was measured per milligram of cell protein. These results imply that high-affinity, Na(+)-dependent and fluoxetine-sensitive 5-HT uptake occurs in rat cortical astrocytes in situ.

Animals↗

PubMed1993

Stimulation of D-aspartate efflux by mercuric chloride from rat primary astrocyte cultures.

Mercuric chloride (HgCl2; MC) was shown to increase D-aspartate release from preloaded astrocytes in a dose-dependent fashion. Two sulfhydryl (-SH) protecting agents, a cell membrane non-penetrating compound, reduced glutathione (GSH), and the membrane-permeable dithiothreitol (DTT), were found to inhibit the stimulatory action of MC on the efflux of radiolabeled D-aspartate. MC-induced D-aspartate release was completely inhibited by the addition of 1 mM DTT or GSH during the actual 5 min perfusion period with MC (5 microM). However, when added after MC treatment, this inhibition could not be sustained by GSH, while DTT fully inhibited the MC-induced release of D-aspartate. Neither DTT nor GSH alone had any effect on the rate of astrocytic D-aspartate release. Accordingly, it is postulated that the stimulatory effect exerted by MC on astrocytic D-aspartate release is associated with vulnerable -SH groups located within, but not on the surface of the cell membrane. Omission of Na+ from the perfusion solution did not accelerate MC-induced D-aspartate release, suggesting that reversal of the D-aspartate carrier can not be invoked to explain MC-induced D-aspartate release. Furthermore, MC did not appear to be associated with astrocytic swelling.

Animals↗

PubMed1993

Use of 51Cr cell labelling to distinguish between release of radiolabelled amino acids from primary astrocyte cultures being due to efflux or cell damage.

Continuous perfusion methods are widely used to monitor release of substances, particularly transmitters, from brain cell cultures growing as monolayers. However, if stimuli used to produce release also cause loss or lysis of cells, the appearance of label in the perfusate due to such effects will be indistinguishable from release. Using a perfusion method we have studied release of preloaded, radiolabelled amino acids from primary astrocyte cultures due to a variety of stimuli; hypotonic or high K+ media, activation of beta-receptors or swelling-induced release due to isosmotic ethanol. In this study primary astrocyte cultures were simultaneously labelled with Na2(51)CrO4 and allowed to take up radiolabelled D-aspartate or taurine. It was found that while all of the above methods caused release of radiolabelled amino acids none caused release of 51Cr into the perfusion fluid. In contrast, perfusion with 0.05% (v/v) Triton X-100 did lead to release of 51Cr. Thus a variety of means of inducing swelling or shape changes in astrocytes causes true release of radiolabelled amino acids and simultaneously monitoring 51Cr release seems a convenient means of distinguishing such release from cell loss or lysis.

Amino Acids↗

PubMed1993

Lead increases inositol 1,4,5-trisphosphate levels but does not interfere with calcium transients in primary rat astrocytes.

Alteration of receptor-mediated signal transduction pathways by inorganic lead (Pb) has been postulated to contribute to the neurotoxicity of this environmental toxicant, some of these effects involving astrocytes. As Pb is known to mimic Ca2+ in various biological systems or alter Ca(2+)-mediated cellular processes, we analyzed the effect of Pb exposure on alpha 1 receptor activated astrocytic phosphoinositide metabolism and Ca2+ responses in primary astrocyte cultures prepared from cerebral cortex of 1-day-old rats. Exposure to norepinephrine (NE; 10-100 microM) resulted in a significant increase in astrocytic inositol 1,4,5-trisphosphate levels, concomitant with an increase in intracellular Ca2+ levels. Fifteen minute exposure to Pb (10 microM lead acetate) significantly increased inositol 1,4,5-trisphosphate generation compared with controls, both in the presence and absence of NE. However, the inositol 1,4,5-trisphosphate-mediated Ca2+ transients following NE stimulation was unaltered in the presence of Pb (1-100 microM). NE-evoked intracellular Ca2+ responses, both in the presence and absence of extracellular Ca2+ did not differ between control and Pb-treated astrocytes. Additional studies failed to demonstrate the occurrence of Pb influx into astrocytes within the first 12 min of exposure such that Ca2+ responses would be directly affected. It therefore appears unlikely that astrotoxic effects of Pb are mediated via direct changes in intracellular Ca2+ transients.

Animals↗

PubMed1993

Detection of 5-hydroxytryptamine2 receptors by radioligand binding, northern blot analysis, and Ca2+ responses in rat primary astrocyte cultures.

Radioligand binding, Northern blot analysis, and changes in [Ca2+]i were used to study serotonin [5-hydroxytryptamine (5HT)] receptor subtypes in primary cultures of astrocytes from neonatal rat cerebral cortex. Radioligand binding studies revealed the presence of 5HT2, but not the 5HT1 or 5HT3 receptor subtypes. Radioligand binding was also used to show the presence of serotonin uptake sites, which had previously been shown to be present by [3H]-5HT uptake, and also alpha 1-adrenergic receptors as has previously been reported by binding studies. Northern blot analysis of cortical astrocyte mRNA demonstrated the presence of transcripts for 5HT2 receptors, but failed to identify mRNA for 5HT1a or 5HT1c receptors. Thus, results from Northern blot analysis correlated with the radioligand binding data which showed only 5HT2 receptors. Equilibrium saturation studies, using 125[I]-LSD to label 5HT2 receptors, yielded a KD of 9 nM and a Bmax of 177 fmol/mg protein. Radioligand binding studies or primary astrocyte cultures prepared from other brain regions also showed the presence of alpha 1-adrenergic, 5HT2 receptor, and 5HT-uptake sites, but no detectable 5HT1a receptors, which were the only 5HT1 receptors studied. Studies demonstrating 5HT-induced, spiperone- and ketanserin-sensitive increases in free [Ca2+]i as measured by FURA-2, showed that the 5HT2 receptors were functional in these cells. These data provide clear evidence for the existence of both 5HT2 receptors and 5HT-uptake sites in the same primary astrocyte cultures from neonatal rat cerebral cortex, with no detectable evidence of 5HT1a or 5HT1c subtypes.

Animals↗

PubMed1993

The role of sulfhydryl groups and calcium in the mercuric chloride-induced inhibition of glutamate uptake in rat primary astrocyte cultures.

Inhibition by mercuric chloride (MC) of the astrocytic uptake of the excitotoxic neurotransmitter L-glutamate (L-GLU) has been postulated to contribute to MC neurotoxicity. In the present study, we analyzed the ability of two sulfhydryl (SH)-protecting agents: a cell membrane non-penetrating compound-reduced glutathione (GSH), and the membrane permeable dithiothreitol (DTT), to reverse the inhibitory action of MC on the initial rate of uptake of radiolabelled GLU (100 microM) in primary cultures of rat astrocytes. MC at 5 microM concentration reduced the uptake to 46% of control when present in the incubation medium during the 5 min of actual uptake, and to 27% of control when astrocytes were preincubated for 30 min in HEPES buffer containing MC prior to GLU uptake measurements. GLU uptake inhibition caused by 30 min preincubation with MC was partly relieved by the addition of 1 mM DTT during the actual 5 min uptake period. However, this inhibition could not be reversed by 1 mM GSH. Accordingly, it is postulated that the inhibitory effect exerted by MC on GLU uptake is associated with vulnerable SH groups located within, but not on the surface of the cell membrane. Neither 5 microM N-ethylmaleimide (NEM) nor 5 microM or 25 microM iodoacetate (IA) affected GLU uptake, indicating steric hindrance of the access of these two sulfhydryl reagents to the SH groups critical for the uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Methylmercury-induced alterations in excitatory amino acid transport in rat primary astrocyte cultures.

To determine whether methylmercury (MeHg) has the potential to interfere with homeostatic functions in neonatal rat cortical primary astrocyte cultures, the effects of MeHg on the uptake and efflux of both L-glutamate and D-aspartate were examined. Uptake of both of these excitatory amino acids (EAAs) was significantly (P < 0.05) reduced in the presence of MeHg concentrations as low as 10(-5) M. Efflux of both glutamate and aspartate from preloaded astrocytes was also increased by MeHg in a dose- and time-dependent fashion. Since in our earlier studies we had found that MeHg causes dose-dependent astrocytic swelling, which could have been the mechanism of the increased efflux, we examined whether blockage of conductive ion fluxes, which have been implicated in astrocytic swelling, could reverse the MeHg-induced increase in L-glutamate and D-aspartate efflux. Three compounds which inhibit the hypotonic-media-induced efflux of EAA, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), L-644,711 (a derivative of ethacrynic acid) and furosemide were tested at their maximal concentrations for their ability to reverse MeHg-induced EAA efflux. Only furosemide (5 mM) could sustain the reversal for the entire 120 min duration of the efflux measurement. Since hypotonic-media swelling-induced release of EAAs is inhibited by these anion inhibitors (in the following rank order: L-644,711 > SITS > furosemide), we conclude that different mechanisms account for EAA release from primary astrocyte cultures during MeHg exposure as compared to hypotonic media-induced efflux.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1993

Effect of ascorbate on Na(+)-independent and Na(+)-dependent uptake of [3H]norepinephrine by rat primary astrocyte cultures from neonatal rat cerebral cortex.

We have previously reported that primary astrocyte cultures prepared from neonatal rat brains show Na(+)-dependent, tricyclic antidepressant-sensitive, high-affinity uptake of [3H]norepinephrine ([3H]NE). Other workers, however, using primary astrocyte cultures from neonatal mice, have failed to find such uptake. This prompted us to examine possible reasons for the variability of the uptake in primary astrocyte cultures such as growth conditions and the effect of ascorbic acid. The presence of ascorbic acid increased the Na(+)-dependent uptake of NE by inhibiting the Na(+)-independent component. Na(+)-dependent uptake in rat cultures occurs when either fetal bovine or horse serum are present in the growth media, but not in a serum-free growth medium. Other workers have shown a species difference such that, even under optimal uptake conditions where rat astrocyte cultures exhibit Na(+)-dependent [3H]NE uptake, mouse astrocyte cultures do not.

Animals↗