Biomedical subjects

H Sontheimer

Publications and source records attributed to H Sontheimer.

At least 37 records · Page 2Linked to original sources

PubMed1999

Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters and enhanced activity of cystine-glutamate exchange.

Elevated levels of extracellular glutamate ([Glu](o)) can induce seizures and cause excitotoxic neuronal cell death. This is normally prevented by astrocytic glutamate uptake. Neoplastic transformation of human astrocytes causes malignant gliomas, which are often associated with seizures and neuronal necrosis. Here, we show that Na(+)-dependent glutamate uptake in glioma cell lines derived from human tumors (STTG-1, D-54MG, D-65MG, U-373MG, U-251MG, U-138MG, and CH-235MG) is up to 100-fold lower than in astrocytes. Immunohistochemistry and subcellular fractionation show very low expression levels of the astrocytic glutamate transporter GLT-1 but normal expression levels of another glial glutamate transporter, GLAST. However, in glioma cells, essentially all GLAST protein was found in cell nuclei rather than the plasma membrane. Similarly, brain tissues from glioblastoma patients also display reduction of GLT-1 and mislocalization of GLAST. In glioma cell lines, over 50% of glutamate transport was Na(+)-independent and mediated by a cystine-glutamate exchanger (system x(c)(-)). Extracellular L-cystine dose-dependently induced glutamate release from glioma cells. Glutamate release was enhanced by extracellular glutamine and inhibited by (S)-4-carboxyphenylglycine, which blocked cystine-glutamate exchange. These data suggest that the unusual release of glutamate from glioma cells is caused by reduction-mislocalization of Na(+)-dependent glutamate transporters in conjunction with upregulation of cystine-glutamate exchange. The resulting glutamate release from glioma cells may contribute to tumor-associated necrosis and possibly to seizures in peritumoral brain tissue.

ATP-Binding Cassette Transporters↗

PubMed1999

Recording of intracellular Ca2+, Cl-, pH and membrane potential in cultured astrocytes using a fluorescence plate reader.

A multi-well fluorescence plate reader was used to determine changes in intracellular ionic concentrations and changes in transmembrane voltage in primary cortical or hippocampal astrocytes. Recordings were compared to those obtained using a traditional microscope-based imaging setup that utilizes a monochromatic light source for excitation and an intensified CCD camera for detection. Measurement of pHi with the ratiometric dye BCECF provided resolution similar to that of a microscopic approach. We also demonstrate using Fluo-3 that the measurement of glutamate-induced [Ca2+]i fluctuations are comparable to recordings on the microscope-based system when 25 cells were averaged. This is expected because the plate reader averages responses from many cells. Voltage changes induced by changes in K+(o) from 3 to 5 mM were readily resolvable with the plate reader using the potentiometric dye bis-oxynol, and overall sensitivity was similar to that of microscopically-determined voltage changes. We also found that the plate reader was capable of resolving GABA-induced Cl-(i) fluctuations using the Cl(-)-sensitive indicator MEQ. From these experiments we conclude that multi-well fluorescence plate readers can be used to effectively record changes in intracellular ion concentrations and transmembrane voltage of populations of cells affording time and amplitude resolution approaching that of conventional fluorescence imaging methods. In addition, plate reader-based fluorescence studies demonstrate the added capability to rapidly screen large numbers of samples.

Aniline Compounds↗

PubMed1999

Glioma cells release excitotoxic concentrations of glutamate.

Elevated levels of extracellular glutamate ([Glu]o) cause uncontrolled Ca2+ increases in most neurons and are believed to mediate excitotoxic brain injury following stroke and other nervous system insults. In the normal brain, [Glu]o is tightly controlled by uptake into astrocytes. Because the vast majority of primary brain tumors (gliomas) are derived from astrocytes, we investigated glutamate uptake in glioma cells surgically isolated from glioma patients (glioblastoma multiforme) and in seven established human glioma cell lines, including STTG-1, D-54 MG, D-65 MG, U-373 MG, U-138 MG, U-251 MG, and CH-235 MG. All glioma cells studied showed impaired glutamate uptake, with a Vmax < 10% that of normal astrocytes. Moreover, rather than removing glutamate from the extracellular fluid, glioma cells release large amounts of glutamate, resulting in elevations of [Glu]o in excess of 100 microM within hours in a space that is 1000-fold larger than the cellular volume. Exposure of cultured hippocampal neurons to glioma-conditioned medium elicited sustained [Ca2+]i elevations that were followed by widespread neuronal death. Similarly, coculturing of hippocampal neurons and glioma cells, either with or without direct contact, resulted in neuronal death. Glioma-induced neuronal death could be completely prevented by treating neurons with the N-methyl-D-aspartate receptor antagonists MK-801/D(-)-2-amino-5-phosphonopentanoic acid or by depletion of glutamate from the medium. Interestingly, several phenylglycine derivatives including the metabotropic glutamate receptor agonist/antagonist (S)-4-carboxyphenylglycine (S-4CPG) potently and selectively inhibited glutamate release from glioma cells and prevented neurotoxicity. These data suggest that growing glioma tumors may actively kill surrounding neuronal cells through the release of glutamate. This glutamate release may also be responsible in part for tumor-associated seizures that occur frequently in conjunction with glioma. These data also suggest that neurotoxic release of glutamate by gliomas may be prevented by phenylglycine derivatives, which may thus be useful as an adjuvant treatment for brain tumors.

Animals↗

PubMed1999

Modulation of glioma cell migration and invasion using Cl(-) and K(+) ion channel blockers.

Human malignant gliomas are highly invasive tumors. Mechanisms that allow glioma cells to disseminate, migrating through the narrow extracellular brain spaces are poorly understood. We recently demonstrated expression of large voltage-dependent chloride (Cl(-)) currents, selectively expressed by human glioma cells in vitro and in situ (Ullrich et al., 1998). Currents are sensitive to several Cl(-) channel blockers, including chlorotoxin (Ctx), (Ullrich and Sontheimer; 1996; Ullrich et al; 1996), tetraethylammonium chloride (TEA), and tamoxifen (Ransom and Sontheimer, 1998). Using Transwell migration assays, we show that blockade of glioma Cl(-) channels specifically inhibits tumor cell migration in a dose-dependent manner. Ctx (5 microM), tamoxifen (10 microM), and TEA (1 mM) also prevented invasion of human glioma cells into fetal rat brain aggregates, used as an in vitro model to assess tumor invasiveness. Anion replacement studies suggest that permeation of chloride ions through glioma chloride channel is obligatory for cell migration. Osmotically induced cell swelling and subsequent regulatory volume decrease (RVD) in cultured glioma cells were reversibly prevented by 1 mM TEA, 10 microM tamoxifen, and irreversibly blocked by 5 microM Ctx added to the hypotonic media. Cl(-) fluxes associated with adaptive shape changes elicited by cell swelling and RVD in glioma cells were inhibited by 5 microM Ctx, 10 microM tamoxifen, and 1 mM TEA, as determined using the Cl(-)-sensitive fluorescent dye 6-methoxy-N-ethylquinolinium iodide. Collectively, these data suggest that chloride channels in glioma cells may enable tumor invasiveness, presumably by facilitating cell shape and cell volume changes that are more conducive to migration and invasion.

Adult↗

PubMed1999

Metabotropic glutamate receptor agonists reduce glutamate release from cultured astrocytes.

Astrocytes are thought to control extracellular glutamate concentrations ([Glu]o) in the brain, thereby protecting neurons from excitotoxic injury. We investigated the effects of metabotropic glutamate receptor (mGluR) agonists on glutamate transport and [Glu]o in primary hippocampal astrocytic cultures. Acute or chronic exposure of astrocytes to the mGluR agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) or its active isomer 1S,3R-ACPD reduced [Glu]o in a time- and dose-dependent manner (44.5 +/- 3.6% reductions of [Glu]o in astrocytes from P0-P10 rats and 65.9 +/- 4.1% from rats P20 by 100 microM 1S,3R-ACPD, EC50 approximately 5 microM). 1S,3R-ACPD effects developed slowly (median effective at approximately 60 min) and persisted for several hours after agonist removal. ACPD-pretreated astrocytes established lower steady-state [Glu]o levels. ACPD effects persisted in the presence of the glutamate uptake inhibitors D,L-threo-beta-hydroxyaspartate (THA) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC) but were impaired by disruption of the transmembrane Na+, K+, or H+ gradients. In addition, 1S,3R-ACPD had no effects on intracellular glutamate content and did not directly block glutamate transport. Furthermore, ACPD effects could be mimicked by glutamate per se and several other compounds presumed to be mGluR agonists, although (S)-3,5-dihydroxyphenylglycine (DHPG), (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), and L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) were without effect. These data suggest that glutamate and certain mGluR agonists may regulate [Glu]o by modulating the transmembrane equilibrium of glutamate transport, especially by attenuating glutamate release.

Animals↗

PubMed1999

Differential inhibition of glial K(+) currents by 4-AP.

Spinal cord astrocytes express four biophysically and pharmacologically distinct voltage-activated potassium (K(+)) channel types. The K(+) channel blocker 4-aminopyridine (4-AP) exhibited differential and concentration-dependent block of all of these currents. Specifically, 100 microM 4-AP selectively inhibited a slowly inactivating outward current (K(SI)) that was insensitive to dendrototoxin (< or = 10 microM) and that activated at -50 mV. At 2 mM, 4-AP inhibited fast-inactivating, low-threshold (-70 mV) A-type currents (K(A)) and sustained, TEA-sensitive noninactivating delayed-rectifier-type currents (K(DR)). At an even higher concentration (8 mM), 4-AP additionally blocked inwardly rectifying, Cs(+)- and Ba(2+)-sensitive K(+) currents (K(IR)). Current injection into current-clamped astrocytes in culture or in acute spinal cord slices induced an overshooting voltage response reminiscent of slow neuronal action potentials. Increasing concentrations of 4-AP selectively modulated different phases in the repolarization of these glial spikes, suggesting that all four K(+) currents serve different roles in stabilization and repolarization of the astrocytic membrane potential. Our data suggest that 4-AP is an useful, dose-dependent inhibitor of all four astrocytic K(+) channels. We show that the slowly inactivating astrocytic K(+) currents, which had not been described as separate current entities in astrocytes, contribute to the resting K(+) conductance and may thus be involved in K(+) homeostatic functions of astrocytes. The high sensitivity of these currents to micromolar 4-AP suggests that application of 4-AP to inhibit neuronal A-currents or to induce epileptiform discharges in brain slices also may influence astrocytic K(+) buffering.

4-Aminopyridine↗

PubMed1998

Passive glial cells, fact or artifact?

Astrocytes that are recorded in acute tissue slices of rat hippocampus using whole-cell patch-clamp, commonly exhibit voltage-activated Na+ and K+ currents. Some reports have described astrocytes that appear to lack voltage-activated currents and proposed that these cells constitute a subpopulation of electrophysiologically passive astrocytes. We show here that these cells can spontaneously change during a recording unmasking expression of previously suppressed voltage-activated currents, suggesting that such cells do not represent a subpopulation of passive astrocytes. Superfusion of a low Ca2+/EGTA solution was able to reversibly suppress voltage-activated K+ currents in cultured astrocytes. Currents were restored upon addition of normal bath Ca2+. These effects of Ca2+ on both outward and inward K+ currents were dose- and time-dependent, with increasing concentrations of Ca2+ (from 0 to 800 micrometers) leading to a gradual unmasking of voltage-dependent outward and inward K+ currents. The transition from an apparently passive cell to one exhibiting prominent voltage-activated currents was not associated with any changes in membrane capacitance or access resistance. By contrast, in cells in which low access resistance or poor seal accounted for the absence of voltage-activated currents, improvement of cell access was always accompanied by changes in series resistance and membrane capacitance. We propose that spillage of pipette solution containing low Ca2+/EGTA during cell approach in slice recordings and/or poor cell access, lead to a transient masking of voltage-activated currents even in astrocytes that express prominent voltage-activated currents. These cells, however, do not constitute a subpopulation of electrophysiologically passive astrocytes.

Animals↗

PubMed1998

Use of chlorotoxin for targeting of primary brain tumors.

Gliomas are primary brain tumors that arise from differentiated glial cells through a poorly understood malignant transformation. Although glioma cells retain some genetic and antigenic features common to glial cells, they show a remarkable degree of antigenic heterogeneity and variable mutations in their genome. Glioma cells have recently been shown to express a glioma-specific chloride ion channel (GCC) that is sensitive to chlorotoxin (CTX), a small peptide purified from Leiurus quinquestriatus scorpion venom [N. Ullrich et al, Neuroreport, 7: 1020-1024, 1996; and N. Ullrich and H. Sontheimer, Am. J. Physiol. (Cell Physiol.), 270: C1511-C1521, 1996]. Using native and recombinant 125I-labeled CTX, we show that toxin binding to glioma cells is specific and involves high affinity [dissociation constant (Kd)=4.2 nM] and low affinity (Kd=660 nml) binding sites. In radioreceptor assays, 125I-labeled CTX binds to a protein with Mr=72,000, presumably GCC or a receptor that modulates GCC activity. In vivo targeting and biodistribution experiments were obtained using 125I- and (131)I-labeled CTX injected into severe combined immunodeficient mice bearing xenografted gliomas. CTX selectively accumulated in the brain of tumor-bearing mice with calculated brain: muscle ratios of 36.4% of injected dose/g (ID/g), as compared to 12.4% ID/g in control animals. In the tumor-bearing severe combined immunodeficient mice, the vast majority of the brain-associated radioactivity was localized within the tumor (tumor:muscle ratio, 39.13% ID/g; contralateral brain:muscle ratio, 6.68%ID/g). Moreover, (131)I-labeled CTX distribution, visualized through in vivo imaging by gamma ray camera scans, demonstrates specific and persistent intratumoral localization of the radioactive ligand. Immunohistochemical studies using biotinylated and fluorescently tagged CTX show highly selective staining of glioma cells in vitro, in situ, and in sections of patient biopsies. Comparison tissues including normal human brain, kidney, and colon were consistently negative for CTX immunostaining. These data suggest that CTX and CTX-conjugated molecules may serve as glioma-specific markers with diagnostic and therapeutic potential.

Animals↗

PubMed1998

Lysophosphatidic acid stimulates actomyosin contraction in astrocytes.

Lysophosphatidic acid (LPA) is an extracellular signaling molecule that can enter the central nervous system following injury or diseases that disrupt the blood-brain-barrier. Using a combination of time-lapse microscopy, immunocytochemistry, and biochemical techniques, we demonstrate that LPA stimulates profound changes in astrocyte morphology that are due to effects on the actomyosin cytoskeleton. Flat astrocytes in primary culture display prominent actin stress fibers. Treatment with the myosin light chain kinase inhibitor, ML-9, causes stress fiber dissolution and dramatic morphology changes including rounding of the cell body and the formation of processes. LPA can stabilize actin stress fibers and inhibit the morphology changes in ML-9-treated cells. Furthermore, this activity is dependent upon activation of the GTP-binding protein Rho as evidenced by the ability of C3 exoenzyme, a specific inhibitor of Rho, to block the effect. Phosphorylation of the regulatory light (RLC) chain initiates conformational changes in myosin II that result in the formation of myosin filaments and the recruitment of actin into contractile stress fibers. LPA-induced stabilization of stress fibers is accompanied by increases in phosphorylation of the RLC of myosin. Furthermore, astrocytes grown on flexible silicone undergo rapid contraction in response to LPA treatment. The forces generated by these cells manifest themselves as increased wrinkling in the silicone. The observed contraction and accompanying increases in regulatory light chain phosphorylation suggest that LPA-induced signaling cascades in astrocytes regulate actin/myosin interactions.

ADP Ribose Transferases↗

PubMed1998

Astrocytes protect neurons from neurotoxic injury by serum glutamate.

Serum is used widely for culturing neurons and glial cells, and is thought to provide essential, albeit undefined, factors such as hormones, growth factors, and trace elements that promote the growth of cells in vitro. Moreover, serum can have profound effects on cell proliferation, differentiation, and cell morphology, and may even influence cell fate decisions. Despite the overall growth-promoting influence of serum on cell culture, frequent media changes have been shown to be detrimental to neuronal cultures, significantly reducing the yield of viable neurons. The reason for this loss of neurons by frequent media changes has been puzzling. We demonstrate that bovine and horse sera, the most popular serum complements for CNS cell culture, are a significant source for glutamate, supplying glutamate at concentrations sufficient to kill primary cultured hippocampal neurons. By using the bioluminescence detection method, we determined the glutamate concentration [Glu] in several batches of fetal bovine (calf) sera (FBS) to be close to 1 mM, and that of horse sera to be approximately 0.3 mM. Thus 10% serum supplement to culture media results in [Glu] of 30-100 microM due to serum alone. We subsequently produced glutamate depleted media (GDM) by using primary cultures of hippocampal astrocytes to absorb glutamate from media containing 10% FBS. Within 3 h, astrocytes reduced the [Glu] in the medium from approximately 90 microM to less than 1 microM. Sister cultures of hippocampal neuron that underwent frequent media changes with GDM or GDM + partial untreated media demonstrated that GDM significantly increase neuronal survival (10-fold at 21 DIV). Subsequent exposure to glutamate provided by either untreated serum or by equivalent doses of exogenous glutamate added to GDM led to dose-dependent neuronal cell death. The relative sensitivity of hippocampal neurons to glutamate increased with increasing culture age from initial ED50 values of > 100 microM (< 6 DIV) to approximately 6 microM in cultures maintained for 3 weeks or longer. The relative sensitivity to exogenous glutamate was at least 2-fold higher in neurons cultured in GDM than in sister cultures maintained in media containing untreated serum. The death of neurons exposed to untreated media was blocked by the NMDA receptor antagonist MK-801. These experiments suggest that the vulnerability of neurons to media changes can be solely explained by excitotoxicity resulting from serum-borne glutamate. Moreover, we propose that use of GDM may be advantageous for culturing hippocampal neurons and may eliminate the possible selection for glutamate resistant neurons. The use of GDM could be particularly important for studies of excitotoxicity; our study predicts that the ED50 for neuronal culture with regular serum will be artificially high and may not adequately reflect the in vivo state.

Animals↗

PubMed1998

Glial glutamate transport as target for nitric oxide: consequences for neurotoxicity.

Overwhelming evidence suggest that accumulations of extracellular glutamate are toxic to neurons. It has also been proposed that astrocytes protect neurons from glutamate toxicity by removal of glutamate from extracellular space. By using co-cultures of hippocampal neurons and astrocytes, we studied the influence of astrocytes on neuronal excitotoxicity. Moreover, we evaluated the role of nitric oxide and pro-inflammatory cytokines on astrocytic glutamate transport.

Animals↗

PubMed1998

Expression of voltage-activated chloride currents in acute slices of human gliomas.

Using whole-cell patch-clamp recordings, we identified a novel voltage-activated chloride current that was selectively expressed in glioma cells from 23 patient biopsies. Chloride currents were identified in 64% of glioma cells studied in acute slices of nine patient biopsies. These derived from gliomas of various pathological grades. In addition, 98% of cells acutely isolated or in short-term culture from 23 patients diagnosed with gliomas showed chloride current expression. These currents, which we termed glioma chloride currents activated at potentials >45 mV, showed pronounced outward rectification, and were sensitive to bath application of the presumed Cl- channel specific peptide chlorotoxin (approximately 600 nM) derived from Leiurus scorpion venom. Interestingly, low grade tumours (e.g., pilocytic astrocytomas), containing more differentiated, astrocyte-like cells showed expression of glioma chloride currents in concert with voltage-activated sodium and potassium currents also seen in normal astrocytes. By contrast, high grade tumours (e.g., glioblastoma multiforme) expressed almost exclusively chloride currents, suggesting a gradual loss of Na+ currents and gain of Cl- currents with increasing pathological tumour grade. To expand on the observation that these chloride currents are glioma-specific, we introduced experimental tumours in scid mice by intracranial injection of D54MG glioma cells and subsequently recorded from tumour cells and adjacent normal glial cells in acute slices. We consistently observed expression of chlorotoxin-sensitive chloride channels in implanted glioma cells, but without evidence for expression of chloride channels in surrounding "normal" host glial cells, suggesting that these chloride channels are probably a glioma-specific feature. Finding of this novel glioma specific Cl- channel in gliomas in situ and it's selective binding of chlorotoxin may provide a way to identify or target glioma cells in the future.

Adolescent↗

PubMed1998

Properties of human glial cells associated with epileptic seizure foci.

We studied physiological properties of glial cells from acute slices of biopsies from patients operated for intractable mesio-temporal lobe epilepsy using whole-cell patch-clamp recordings. Cells were filled with Lucifer Yellow (LY) during recordings to allow morphological reconstruction and immunohistochemical cell identification. Seizure-associated astrocytes had complex, arborized, highly branched processes giving them a stellate appearance, and cells stained intensely for the intermediate filament GFAP as previously reported for 'reactive' astrocytes. GFAP-positive astrocytes from epilepsy biopsies consistently expressed voltage-activated, TTX-sensitive Na+ channels that showed fast activation and inactivation kinetics. Unlike comparison astrocytes, derived from tissues that were not associated with seizure foci, these astrocytes expressed Na+ channels at densities sufficient to generate slow action potentials (spikes) in current clamp studies. In these cells, the ratio of Na+ to K+ conductance was consistently 3-4-fold higher than in comparison human or control rat astrocytes. Four of 17 astrocytes from epilepsy patients versus 14/14 from control rat hippocampus and four of five in comparison human tissue showed a lack of inwardly rectifying K+ currents, which in normal astrocytes are implicated in the control of extracellular K+ levels. These results suggest that astrocytes surrounding seizure foci differ in morphological and physiological properties, and that glial K+ buffering could be impaired at the seizure focus, thus contributing to the pathophysiology of seizures.

Adult↗

PubMed1998

Astrocytes from human hippocampal epileptogenic foci exhibit action potential-like responses.

PURPOSE: We studied Na+ channel expression and the ability to generate action potential (AP)-like responses in primary cultures of human astrocytes by whole cell patch-clamp recording techniques. METHODS: Tissue samples from 22 patients with various classifications of temporal lobe epilepsy (TLE) were plated to form separate astrocyte cultures from three regions; the hippocampus, parahippocampus, and anterolateral temporal neocortex. RESULTS: The resting membrane potential of seizure focus astrocytes (MTLE, mesial TLE) was significantly depolarized (approximately -55 mV) as compared with cortical astrocytes (-80 mV). Hippocampal astrocytes from other substrates for TLE (MaTLE, mass-associated TLE; PTLE, paradoxical TLE) in which the hippocampus is not the seizure focus displayed resting membrane potentials similar to those of neocortical astrocytes (approximately -75 mV). Astrocytes from the seizure focus (MTLE) displayed much larger tetrodotoxin (TTX)-sensitive Na+ currents with -66-fold higher Na+ channel density (113.5 +/- 17.41 pA/pf) than that of comparison neocortical astrocytes (1.7 +/- 3.7 pA/pf) or than that of the hippocampal and parahippocampal astrocytes of the MaTLE and PTLE groups. As a consequence of this higher channel density, seizure focus astrocytes were capable of generating AP-like responses. However, at the resting potential, most Na+ channels are inactive and no spontaneous firing was observed. In contrast, astrocytes in the comparison neocortex from all groups and the hippocampus and parahippocampus from the MaTLE and PTLE groups could not fire AP-like responses even after large current injections. CONCLUSIONS: The function of Na+ channels in these astrocytes is unclear. However, the marked differences in seizure focus astrocytes as compared with cortical and nonseizure focus hippocampal astrocytes suggest a more active role for astrocytes associated with hyperexcitable neurons at a seizures focus.

Action Potentials↗

PubMed1998

Spinal cord astrocytes display a switch from TTX-sensitive to TTX-resistant sodium currents after injury-induced gliosis in vitro.

Two distinct morphological subtypes of astrocytes have been shown to express Na+ currents that differ biophysically and pharmacologically. Using an in vitro model for reactive gliosis, we recently reported marked changes in Na+ and K+ channel expression by astrocytes induced to proliferate. Using this in vitro assay in which a confluent monolayer of astrocytes is mechanically scarred to induce gliosis, we now demonstrate that sodium currents of scar-associated cells, in addition to doubling in current density, also switch from being tetrodotoxin-sensitive(TTX-S, IC50 8 nM) to being approximately 40-fold more TTX-resistant (TTX-R,IC50 314 nM). These changes occurred within 6 h after injury and were not associated with any notable changes in cell morphology. Changes in biophysical properties were analyzed for the two current types. The activation curve for TTX-R currents demonstrated a significant depolarized shift versus that of TTX-S currents (P </= 0. 003), and TTX-R currents have more depolarized V1/2 of activation (-33 vs. -23 mV). The V1/2 of inactivation was slightly, but not significantly, more depolarized for TTX-R currents as compared to TTX-S (-63 vs. -68 mV). Most notably, TTX-R currents showed significantly slower inactivation kinetics at depolarized voltage potentials than TTX-S sodium currents (0.76 vs. 1.128 ms, at -10 mV; P < 0.0004).

Astrocytes↗

PubMed1998

Electrophysiological properties of human astrocytic tumor cells In situ: enigma of spiking glial cells.

To better understand physiological changes that accompany the neoplastic transition of astrocytes to become astrocytoma cells, we studied biopsies of low-grade, pilocytic astrocytomas. This group of tumors is most prevalent in children and the tumor cells maintain most antigenic features typical of astrocytes. Astrocytoma cells were studied with the use of whole cell patch-clamp recordings in acute biopsy slices from 4-mo- to 14-yr-old pediatric patients. Recordings from 53 cells in six cases of low-grade astrocytomas were compared to either noncancerous peritumoral astrocytes or astrocytes obtained from other surgeries. Astrocytoma cells almost exclusively displayed slowly activating, sustained, tetraethylammonium (TEA)-sensitive outward potassium currents (delayed rectifying potassium currents; IDR) and transient, tetrodotoxin (TTX)-sensitive sodium currents (INa). By contrast, comparison glial cells from peritumoral regions or other surgeries showed IDR and INa, but in addition these cells also expressed transient "A"-type K+ currents and inwardly rectifying K+ currents (IIR), both of which were absent in astrocytoma cells. IIR constituted the predominant conductance in comparison astrocytes and was responsible for a high-resting K+ conductance in these cells. Voltage-activated Na+ currents were observed in 37 of 53 astrocytoma cells. Na+ current densities in astrocytoma cells, on average, were three- to fivefold larger than in comparison astrocytes. Astrocytoma cells expressing INa could be induced to generate slow action potential-like responses (spikes) by current injections. The threshold for generating such spikes was -34 mV (from a holding potential of -70 mV). The spike amplitude and time width were 52.5 mV and 12 ms, respectively. No spikes could be elicited in comparison astrocytes, although some of them expressed Na+ currents of similar size. Comparison of astrocytes to astrocytoma cells suggests that the apparent lack of IIR, which leads to high-input resistance (>500 MOmega), allows glioma cells to be sufficiently depolarized to generate Na+ spikes, whereas the high resting K+ conductance in astrocytes prevents their depolarization and thus generation of spikes. Consistent with this notion, Na+ spikes could be induced in spinal cord astrocytes in culture when IIR was experimentally blocked by 10 microM Ba2+, suggesting that the absence of IIR in astrocytoma cells is primarily responsible for the unusual spiking behavior seen in these glial tumor cells. It is unlikely that such glial spikes ever occur in vivo.

Action Potentials↗

PubMed1997

Electrophysiological changes that accompany reactive gliosis in vitro.

An in vitro injury model was used to examine the electrophysiological changes that accompany reactive gliosis. Mechanical scarring of confluent spinal cord astrocytes led to a threefold increase in the proliferation of scar-associated astrocytes, as judged by bromodeoxyuridine (BrdU) labeling. Whole-cell patch-clamp recordings demonstrated that current profiles differed absolutely between nonproliferating (BrdU-) and proliferating (BrdU+) astrocytes. The predominant current type expressed in BrdU- cells was an inwardly rectifying K+ current (KIR; 1.3 pS/pF). BrdU- cells also expressed transient outward K+ currents, accounting for less than one-third of total K+ conductance (G). In contrast, proliferating BrdU+ astrocytes exhibited a dramatic, approximately threefold reduction in KIR (0.45 pS/pF) but showed a twofold increase in the conductance of both transient (KA) (0.67-1. 32 pS/pF) and sustained (KD) (0.42-1.10 pS/pF) outwardly rectifying K+ currents, with a GKIR:GKD ratio of 0.4. Relative expression of GKIR:GKD led to more negative resting potentials in nonproliferating (-60 mV) versus proliferating astrocytes (-53 mV; p = 0.015). Although 45% of the nonproliferating astrocytes expressed Na+ currents (0.47 pS/pF), the majority of proliferating cells expressed prominent Na+ currents (0.94 pS/pF). Injury-induced electrophysiological changes are rapid and transient, appearing within 4 hr postinjury and, with the exception of KIR, returning to control conductances within 24 hr. These differences between proliferating and nonproliferating astrocytes are reminiscent of electrophysiological changes observed during gliogenesis, suggesting that astrocytes undergoing secondary, injury-induced proliferation recapitulate the properties of immature glial cells. The switch in predominance from KIR to KD appears to be essential for proliferation and scar repair, because both processes were inhibited by blockade of KD.

Animals↗

PubMed1997

Bovine serum albumin and lysophosphatidic acid stimulate calcium mobilization and reversal of cAMP-induced stellation in rat spinal cord astrocytes.

We report that lysophosphatidic acid (LPA) stimulates dynamic calcium (Ca2+) fluctuations and morphological rearrangements in astrocytes derived from neonatal rat spinal cord. Addition of 10 microM LPA elicited single Ca2+ transients, or biphasic oscillations and sustained increases in intracellular Ca2+ ([Ca2+]i). The biphasic Ca2+ response consisted of an initial release from intracellular stores, often followed by a sustained elevation or continued oscillations that required Ca2+ flux across the cell membrane. The type of Ca2+ response, but not the overall magnitude, was dependent on LPA concentration. Higher concentrations (> 10 microM) often elicited sustained increases in [Ca2+]i, while lower concentrations stimulated oscillations or single Ca2+ transients. It has previously been established that agents that elevate cyclic adenosine monophosphate (cAMP) induce flat astrocytes to adopt a more stellate morphology. LPA can completely reverse this morphological change at a half-maximal concentration of 215 nM. Inhibiting LPA-induced [Ca2+]i fluctuations using BAPTA-AM to buffer [Ca2+]i and EGTA in the bath to prevent transmembrane flux had little effect on the ability of LPA to reverse stellation. LPA is found bound to serum albumin, in which crude preparations have been shown to induce various physiological responses in a number of cell types. Many of the activities have been attributed to albumin-associated lipid factors including LPA. We show that lipid factors associated with BSA can mimic the effect of LPA in both Ca2+ mobilization and reversal of cAMP-induced stellation.

Animals↗