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H K Kimelberg

Publications and source records attributed to H K Kimelberg.

At least 19 recordsLinked to original sources

Interactions of trimethyl tin (TMT) with rat primary astrocyte cultures: altered uptake and efflux of rubidium, L-glutamate and D-aspartate.

Studies were undertaken to assess the effects of trimethyl tin (TMT) on metabolic functions in primary neonatal rat cultured astrocytes. Concentrations as low as 10(-5) M TMT significantly inhibited the initial rate (1 min) of uptake of 86RbCl, used as a tracer for K+. TMT also markedly inhibited the initial rate (1 min) of Na(+)-dependent uptake of L-[3H]glutamate and D-[3H]aspartate, and stimulated the release of intracellular 86Rb+, -[3H]glutamate and D-[3H]aspartate in a dose-dependent fashion. These observations support the hypothesis that the astrocyte plasma membrane is potentially an important target for TMT's toxic effect and specifically that small concentrations of this organometal can inhibit the ability of astrocytes to maintain a transmembrane K+ gradient. This would be expected to compromise the ability of astrocytes to control extracellular K+ either by spatial buffering or active uptake, and exacerbate on-going swelling. Increased levels of glutamate and aspartate in the extracellular fluid upon release from astrocytes may play an important role in TMT neurotoxicity.

Animals

Uptake of [3H]serotonin and [3H]glutamate by primary astrocyte cultures. I. Effects of different sera and time in culture.

Na(+)-dependent, fluoxetine-sensitive high-affinity uptake of serotonin and Na(+)-dependent uptake of glutamate were studied in primary astrocyte cultures from 1-day-old rat neocortex. This uptake was independent of time in culture from 1 to 6 weeks. High-affinity serotonin uptake was decreased when cells were grown in horse serum as compared to fetal bovine serum and was almost absent when cells were grown in chemically defined medium. In contrast, glutamate uptake was unaffected by the composition of the medium in which the cultures were grown. The serum effect on serotonin uptake was not due to the greater level of serotonin in the fetal bovine serum and was only reversed by a change of serum over a time period of days.

Animals

Uptake of [3H]serotonin and [3H]glutamate by primary astrocyte cultures. II. Differences in cultures prepared from different brain regions.

Regional astrocyte cultures were derived by dissecting six regions; brain stem, cerebellum, mesencephalon, basal ganglia plus diencephalon, cerebral cortex, and hippocampus, from 3 to 4-day-old neonatal rat brains. Glial fibrillary acidic protein (GFAP) immunocytochemistry was used to confirm the astrocyte composition of the cultures. The percentage of GFAP (+) cells between regions varied from 75% to 100%. Once confluent these cultures were incubated with radiolabeled serotonin or glutamate for uptake and autoradiographic studies. For the different brain regions Na(+)-dependent, [3H] L-glutamate, and fluoxetine-sensitive [3H] 5-HT uptake varied markedly. The relative order of uptake for [3H] 5-HT was MS (mesencephalon) greater than CC (cerebral cortex) greater than BG + DI (basal ganglia + diencephalon) greater than HP (hippocampus) greater than BS (brain stem) greater than CB (cerebellum). For [3H] L-glutamate the order was HP greater than CC greater than BG + DI greater than MS = BS greater than CB. For [3H] 5-HT this essentially corresponds to the reported order of binding in situ of the [3H] 5-HT-specific uptake ligand [3H] citalopram. For [3H] L-glutamate regional variation of the uptake for the different cultures corresponds to the regional uptake reported for different regions of rat brain. Double-label studies with GFAP and radiolabeled neurotransmitters were also used to study uptake into GFAP(+) astrocytes by autoradiography. Flat GFAP cells with or without processes comprised 65-98% of the cultures and represented most of the uptake. The percentage of all GFAP(+) cells that were positive for uptake of ARG varied from 50% to 90% and also showed differences in grain density both intra- and inter-regionally. These differences in transmitter uptake by GFAP(+) astrocytes in primary culture, which are dependent on the region of origin and correspond to regional differences in situ, suggest that such uptake in vitro may reflect uptake by astrocytes in vivo. Implied in this is that uptake by astrocytes represents a significant component of serotonin uptake in vivo.

Animals

Serotonin uptake by astrocytes in situ.

Co-localization of glial fibrillary acidic protein (GFAP) and radioactivity was examined after intraventricular injection of [3H]5-HT in adult rat brains. Radioactivity localized over GFAP-positive astrocytes was seen, especially when image-enhancing techniques were applied to the data. Also slices prepared from astrogliotic hippocampi of rats pretreated with kainic acid showed a twofold increased uptake of [3H]5-HT compared to control slices. This indicates that the uptake of [3H]5-HT seen in primary astrocyte cultures also occurs for astrocytes in situ. Also, as with astrocyte cultures, only some of the GFAP(+) astrocytes in situ showed localization of radioactivity, supporting the concept of intraregional heterogeneity of astrocyte functions.

Animals

In vitro evidence supporting two mechanisms of action for the anion transport inhibitor L-644,711 in cerebral ischaemia.

L-644,711 is a novel anion channel inhibitor which has previously been shown to decrease brain injury in two related rabbit models of cerebral ischaemia. We hypothesize two mechanisms of action of L-644,711 for its salutary effects on cerebral ischaemia: inhibition of neutrophil function and prevention of excitotoxin release from astrocytes. We present in vitro evidence supporting these two mechanisms of action. L-644,711 demonstrated a dose dependent inhibition of fMLP-induced neutrophil aggregation and superoxide anion release. In addition, L-644,711 demonstrated a dose dependent inhibition of glutamate release from swollen astrocytes in primary culture. We conclude that L-644,711 may prevent brain injury in cerebral ischaemia by inhibiting neutrophil function and preventing release of glutamate from swollen astrocytes.

Animals

Manganese uptake and efflux in cultured rat astrocytes.

Astrocytes play a central role in manganese (Mn) regulation in the CNS. Using primary astrocyte cultures from neonatal rat brains, these studies demonstrate a specific high-affinity transport system for Mn2+. Saturation kinetics are clearly indicated by both 1/v versus 1/s plots (Km = 0.30 +/- 0.03 microM; Vmax = 0.30 +/- 0.02 nmol/mg of protein/min) and plots of v versus [s]. Several divalent cations (Co2+, Zn2+, and Pb2+) failed to inhibit the initial rate of 54Mn2+ uptake. In contrast, extracellular Ca2+ at 10 microM decreased 54Mn2+ uptake. Exchange with extracellular Mn2+ was not obligatory for the efflux of 54Mn2+ into extracellular medium because efflux occurred into Mn(2+)-free extracellular medium, but efflux of 54Mn2+ was enhanced when astrocytes were equilibrated in the presence of unlabeled Mn2+. Efflux of 54Mn2+ was biphasic with both a rapid and a slow component. Efflux was most rapid during the first 10 min of incubation, with 27.5 +/- 2.2% of 54Mn2+ transported extracellularly, and 37.2 +/- 1.2% of preloaded 54Mn2+ was retained by the astrocytes at 120 min. These studies show, for the first time, that mammalian astrocytes can transport Mn via a specific transport system.

Animals

Methods for determination of cell volume in tissue culture.

In this paper we present an overview of methods for determining cell volume in both suspension and monolayer cultures. Data from the use of selected methods such as the Coulter counter system for suspension cultures and radiolabelled intracellular markers for substratum-attached, monolayer cultures are presented. The advantages, limitations, and conditions under which the different methods can be used are discussed. It is pointed out that there is a need for more direct physical methods for measuring dynamic changes in the cell volume of monolayer cultures without removing the cells from the substratum. Data from a method applicable to such cultures that measures extracellular impedance are presented.

Animals

Reduction in ischemic brain injury in rabbits by the anion transport inhibitor L-644,711.

BACKGROUND AND PURPOSE: We studied the anion transport inhibitor L-644,711, which is known to reduce astrocyte swelling and excitotoxin release in primary astrocyte culture, in two models of thromboembolic stroke to assess its capacity to influence ischemic brain injury. METHODS: New Zealand White rabbits were used in this study. The two models include autologous clot embolized to the brain via the carotid artery, with one model using a transient period of systemic hypotension. Cerebral blood flow was determined by the hydrogen clearance method, intracranial pressure was measured with a fiberoptic transducer, and infarct size was assessed with triphenyltetrazolium chloride staining of the coronally sectioned brain. Both models received a 2-hour infusion of L-644,711 (total dose, 12 mg/kg) beginning 20 minutes before embolization. RESULTS: In both the normotensive (p less than 0.01) and the hypotensive (p less than 0.05) model, treatment with L-644,711 resulted in a significant reduction in infarct size and a significant improvement in regional cerebral blood flow (p less than 0.03, normotensive model, and p less than 0.05, hypotensive model). Raised intracranial pressure, unique to the hypotensive model, was abolished by the administration of L-644,711 (p less than 0.05). A hyperglycemic response associated with embolization, also unique to the hypotensive model, was significantly reduced by the administration of L-644,711 (p less than 0.05). CONCLUSIONS: The ability of L-644,711 to limit brain injury in two related models of thromboembolic stroke suggests a potential therapeutic role for anion channel blockers in cerebral ischemia.

Animals

Astrocytic edema in CNS trauma.

The occurrence of astrocytic swelling in response to CNS trauma is reviewed. This response occurs rapidly and appears earlier than the reactive astrocytic response to neuronal and axonal injury. Astrocytic swelling is viewed as an exaggerated pathologic extension of normal astrocytic functions, such as regulation of extracellular ion levels and brain pH. Potential deleterious consequences of swelling, such as failure of ion homeostasis mechanisms, impaired uptake of neurotransmitters, and release of excitotoxic amino acids, are discussed. Possible pharmacologic interventions are reviewed showing that inhibition of such swelling or prevention of some of its potential deleterious consequences by an anion transport inhibitor, L-644,711, may have beneficial effects in head trauma.

Animals

Interactions of methylmercury with rat primary astrocyte cultures: methylmercury efflux.

Methylmercury (MeHg) efflux from rat astrocyte cultures was studied to complement our previous studies on uptake of MeHg in these cells. Exchange with extracellular MeHg was not obligatory for the efflux of [203Hg]MeHg into the extracellular media, because efflux occurred into MeHg-free extracellular media, but stimulation of [203Hg]MeHg net efflux was shown when astrocytes were equilibrated in the presence of 'cold' MeHg and graded concentrations of L-cysteine. Net efflux of MeHg was most rapid for the first 5 min, and approximately 20% of preloaded [203Hg]MeHg was lost from the astrocytes by 60 min. Uptake of [203Hg]MeHgCl was maximal by 30 min and did not increase when the loading period was extended up to 4 h. However, the total amount of intracellular 203Hg that was available for net efflux gradually decreased as the duration of the preloading period increased. MeHg net efflux from astrocytes was unchanged when [203Hg]MeHgCl preloaded astrocytes were equilibrated in hypotonic buffer, suggesting that unlike ions and amino acids swollen astrocytes remain impervious to MeHg efflux. Thus, the main MeHg efflux transport system is apparently specific for the MeHg-L-cysteine conjugate and represents transport by the same neutral amino acid System L that facilitates its uptake.

Animals

Effects of mercury and lead on rubidium uptake and efflux in cultured rat astrocytes.

Astrocytes readily sequester lead and mercury (8, 10, 19, 22). Accordingly, studies were undertaken to assess the effects of lead and mercury on homeostatic functions in neonatal rat brain primary astrocyte cultures. Both inorganic and organic mercury, but not lead, significantly inhibited the initial rate (5 min) of uptake of 86RbCl, used as a tracer for K+, at concentrations of 10-100 microM. Mercury and to a lesser extent lead also stimulated the efflux of intracellular 86Rb+ at 10-500 microM. These observations suggest that the astrocyte plasma membrane may be an important target for lead and mercury, and that relatively low concentrations of these heavy metals should inhibit the ability of astrocytes to maintain a transmembrane K+ gradient.

Animals

The use of astrocytes in culture as model systems for evaluating neurotoxic-induced-injury.

The prevailing thought that astrocytes function predominantly as passive metabolic or even physical support for neurons has faded over the last 20 years. Today these stellar shaped cells are credited with an expanded role, playing key functions in CNS development, homeostasis, and pathology. In probing their expanded roles, primary astrocyte culture systems have proven to be an indispensable tool. Astrocytes have been implicated in both a defensive and facilitatory capacity for many toxic injuries. Evidence for a protective role of astrocytes in modulating CNS toxicity is afforded by observations that the toxicity of glutamate to cortical neurons is diminished upon astrocytic enrichment of the cell culture (Rosenberg and Aizenman, 1989). In cultures of rat cerebral cortex in which astrocyte proliferation is stringently suppressed, glutamate neurotoxicity occurs at low glutamate concentrations similar to those which are normally found in the extracellular space in the hippocampus. In the presence of excess astrocytes, concentrations of glutamate one-hundred fold higher are required to produce equivalent neurotoxicity (Rosenberg and Aizenman, 1989). Astrocytes can facilitate the action of neurotoxins via a modulating process which takes place within the astrocyte or by a direct cytotoxic effect. Whereas primary astrocyte cultures remain unaffected by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; Marini et al., 1989), they function prominently in the selective destruction of dopaminergic neurons of the nigrostriatal pathway in humans, other primates and rodents (Davis et al. 1979; Langston et al., 1983; Burns et al., 1983; Langston et al., 1984; Heikkila et al., 1984; Jarvis and Wagner, 1985). Thus, while MPTP by itself is not toxic to cerebellar cells in co-culture with cerebellar astrocytes, MPTP is toxic to the granule cells (Marini et al, 1989). This is thought to be due to an astrocyte-mediated conversion of MPTP to its highly polar and toxic metabolite, 1-methyl-4-phenylpyridinium ion (MPP+; Chiba et al. 1984). There is compelling evidence that astrocytes respond directly or indirectly to a number of other neurotoxins. Direct cytotoxic effects on astrocytes constitute the major morphologic feature in hyperammonemia (Norenberg, 1981), a condition implicated as an etiologic factor in several CNS disorders. In addition, a predisposition of astrocytes for methylmercury uptake (Aschner et al., 1990 a,b) offers a possible explanation for the observed neurotoxicity of this heavy metal, since a direct toxic effect on astrocytes would result in failure of astrocyte homeostatic functions, indirectly resulting in neuronal impairment, injury and death.

Ammonia

Interactions of methylmercury with rat primary astrocyte cultures: inhibition of rubidium and glutamate uptake and induction of swelling.

The ability of astrocytes to sequester MeHg may indicate an astrocyte-mediated role in MeHg's neurotoxicity. Hence, studies were undertaken to assess the effects of MeHg on metabolic functions in cultured astrocytes. MeHg (10(-5) M) significantly inhibited the initial rate (5 min) of uptake of 86RbCl, used as a tracer for K+. 86RbCl uptake was also sensitive to the omission of medium Na+. MeHg (10(-5) M) also markedly inhibited the initial rate of uptake (1 min) of the Na(+)-dependent uptake of [3H]L-glutamate. A second neurotoxin, MnCl2 (0-5 x 10(-4) M), did not alter [3H]glutamate or 86RbCl uptake. MeHg, but not MnCl2, also stimulated the release of intracellular 86Rb+ in a dose-dependent fashion. This effect could be prevented by the administration of MeHg as the glutathione conjugate. These observations support the hypothesis that the astrocyte plasma membrane is an important target for MeHg's toxic effect and specifically that small concentrations of this organometal inhibit the ability of astrocytes to maintain a transmembrane K+ gradient. This would be expected to compromise the ability of astrocytes to control extracellular K+ either by spatial buffering or active uptake, resulting in cellular swelling. We therefore studied volume changes in astrocytes using uptake of [14C]3-O-methyl-D-glucose, in attached cells in response to exposure to MeHg. Exposure to MeHg (0-5 x 10(-4) M) caused a marked increase in the cell volume that was proportional to concentrations of MeHg.

3-O-Methylglucose

Swelling-induced changes in electrophysiological properties of cultured astrocytes and oligodendrocytes. I. Effects on membrane potentials, input impedance and cell-cell coupling.

We have studied the membrane potential responses of cultured oligodendrocytes and astrocytes during changes in the osmolarity of the bathing solution. Oligodendrocytes responded with a de- or hyperpolarization when changing to hypo- or hyperosmolar medium, consistent with dilution or concentration of intracellular [K+], respectively. Astrocytes responded with a similar hyperpolarization in hyperosmolar medium that could also be explained by an increase in intracellular [K+]. In hypoosmolar medium, however, a much larger depolarization of astrocytes was observed. These data imply that in astrocytes, in contrast to oligodendrocytes, swelling activates specific channels, which might be involved in volume control. Dual-impalement studies in single cells were used to measure effects of medium hypotonicity on cell input resistance, while dual-impalement studies on neighboring cells were used to measure changes in cell-cell coupling. In oligodendrocytes an increase in apparent cell input resistance and electrical coupling was found. In cultured astrocytes there was no change in apparent cell input resistance but an apparent decrease in cell-cell electrical coupling when cultures were exposed to the lowest medium osmolarity examined (-100 mM NaCl). However, because of the unavoidable contribution of cell-cell electrical coupling to the measurement of membrane impedance by current injection into a single cell a lack of change in apparent cell resistance could be due to offsetting changes in cell-cell coupling.

Animals

Swelling-induced changes in electrophysiological properties of cultured astrocytes and oligodendrocytes. II. Whole-cell currents.

Using whole cell patch-clamp recordings we have found that swelling cultured cerebrocortical astrocytes or mouse spinal cord oligodendrocytes by perfusing them with hypotonic medium induced inward currents at the normal resting potential of -60 mV. The currents in the oligodendrocytes were always less than for astrocytes. We examined the reversal potentials of these responses by rapidly jumping the holding potential to different values and measuring the currents. We found that the hypotonic medium-induced conductance increase was always preceded by a conductance decrease in the case of oligodendrocytes, but only sometimes preceded by a conductance decrease in cultured astrocytes. The reversal potential of the conductance increase for astrocytes was around -40 mV, while the conductance decrease had a more negative reversal potential of -60 mV or less. For oligodendrocytes the reversal potential for the conductance increase was around -50 mV while the conductance decrease had a reversal potential of -90 mV or less. This suggests that K+ conductance decreased in the initial phase, while the conductance increase was due to additional channel openings. Ion substitution experiments in the case of the astrocytes showed that the reversal potential was shifted to a more positive value when medium K+ was increased, but was unaffected when Na+ was substituted by N-methyl-D-glucamine or Cl- by D-glucuronate, when corrected for liquid junction potential changes. Thus, the channels opened in these cells are likely to include non-specific cation channels. It is of interest that the two cells show a difference in their responses, and in the case of astrocytes these are likely to be involved in the regulatory volume decrease processes documented in these cells.

Animals

Methylmercury uptake in rat primary astrocyte cultures: the role of the neutral amino acid transport system.

The significance of the dense labeling pattern of methylmercury (MeHg) over astrocytes in areas of damaged cortex remains obscure, and the extent to which individual neurons are altered by MeHg accumulation in astrocytes is unknown. As a first step in understanding the relationship between the astrocyte and the mechanisms of MeHg's neurotoxicity, studies were directed at how MeHg is transported into cultured astrocytes. Uptake of [203Hg]MeHg in primary astrocyte cultures from neonatal rat cerebral cortex following incubations with MeHgCl conformed to a simple diffusion process. Uptake of [203Hg]MeHg by astrocytes exhibited the kinetic criteria of a specific transport system when added to the media as the L-cysteine conjugate. Saturation kinetics, substrate specificity and inhibition, and trans-stimulation were demonstrated in the presence of this SH-containing amino acid. Cysteine-mediated uptake of MeHg was inhibited by the coadministration of L-methionine, and 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid. 2-Methylaminoisobutyric acid was ineffective in inhibiting the uptake of the MeHg-cysteine conjugate. Preloading of the astrocytes with glutamate was moderately effective in trans-stimulating the uptake of MeHg-cysteine conjugates, while in the absence of cysteine, uptake of [203Hg]MeHg was unchanged. These results indicate the presence in astrocytes of a neutral amino acid carrier transport System L, capable of selectively mediating cysteine-MeHg uptake. The substrate specificity and high affinity of this transport system resemble the properties of the System L neutral amino acid transport across the blood-brain barrier in the rat. Cellular uptake of MeHg-cysteine conjugates was not inhibited by preincubation of astrocytes with 100 microM N-ethylmaleimide or NaF.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids