Volatile anesthetics inhibit the ion flux through Ca2+-activated K+ channels of rat glioma C6 cells

Ca2+-activated K+ channels in rat glioma C6 cells were investigated using monolayers of these cells in petri dishes. The ion flux through the channels was studied with 86Rb+ after addition of a Ca2+-ionophore to the incubation medium. Both the influx and efflux of 86Rb+ through these Ca2+-activated K+ channels were inhibited by the general anesthetic halothane (at clinical concentrations). Other volatile anesthetics such as isoflurane, enflurane and methoxyflurane also inhibited the Ca2+-activated K+ channels at clinical concentrations. Inhibition of these channels by general anesthetics could have profound effects on signal transmission in the brain.     

Mouse brain synaptosomal sodium channels: activation by aconitine, batrachotoxin, and veratridine, and inhibition by tetrodotoxin

Batrachotoxin, veratridine and aconitine, activators of the voltage-dependent sodium channel in excitable cell membranes, increase the rate of 22Na+ uptake by mouse brain synaptosomes. Batrachotoxin was both the most potent (K0.5, 0.49 microM) and most effective activator of specific 22Na+ uptake. Veratridine (K0.5, 34.5 microM) and aconitine (K0.5, 19.6 microM) produced maximal stimulations of 22Na+ uptake that were 73% and 46%, respectively, of that produced by batrachotoxin. Activation of 22Na+ uptake by veratridine was completely inhibited by tetrodotoxin (I50, 6 nM ), a specific blocker of nerve membrane sodium channels. These results identify appropriate conditions for measuring sodium channel-dependent 22Na+ flux in mouse brain synaptosomes. The pharmacological properties of mouse brain synaptosomal sodium channels described here are distinct from those previously described for sodium channels in rat brain synaptosomes and mouse neuroblastoma cells.     

Pharmacological and Electrophysiological Characterization of Lithium Ion Flux Through the Action Potential Sodium Channel in Neuroblastoma × Glioma Hybrid Cells

Interaction of Li+ with the voltage-dependent Na+ channel has been analyzed in neuroblastoma X glioma hybrid cells. The cells were able to generate action potentials in media containing Li+ instead of Na+. The uptake of Li+ into the hybrid cells was investigated for the pharmacological analysis of Li+ permeation through voltage-dependent Na+ channels. Veratridine and aconitine increased the uptake of Li+ to the same degree (EC50 30 microM). This increase was blocked by tetrodotoxin (IC50 20 nM). Veratridine and aconitine did not act synergistically; however, the veratridine-stimulated influx was further enhanced by the toxin of the scorpion Leiurus quinquestriatus (EC50 0.06 micrograms/ml). This stimulation was also blocked by tetrodotoxin. Thus, the voltage-dependent Na+ channel of the hybrid cells accepts both Li+ and Na+ in a similar manner.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

C6 glioma cells retrovirally engineered to express IL-18 and Fas exert FasL-dependent cytotoxicity against glioma formation

The decreased antitumor immune response significantly contributes to the progression of glioma. To evaluate whether the antitumor immunity is restored by stable co-expression of IL-18 and Fas receptor, we retrovirally introduced these two genes into rat C6 glioma cells. We found that IL-18-transduced glioma cells secreted IL-18 and induced PBMC IFN-gamma production in vitro. We also found that Fas-transduced glioma cells were susceptible to Fas-mediated apoptosis. In vivo, we found that IL-18 expression and Fas expression synergistically inhibited C6 cell tumorigenesis with the glioma cells being subcutaneously injected in rat flank. Furthermore, we found that co-expression of IL-18 and Fas also produced a marked survival advantage with the rats being intracerebrally implanted with the glioma cells. Finally, we demonstrated that FasL-dependent PBMC cytotoxicity participated in the anti-glioma immunity induced by IL-18 and Fas expression. Taken together, these findings demonstrate that increasing IL-18 production in tumor microenvironment and prompting functional Fas receptor expression of tumor cells could enhance FasL-dependent cytotoxic antitumor immunity.     

Electrical properties of a clonal cell line as determined by measurement of ion fluxes

The electrical properties of the clonal muscle cell line L6 can be revealed by the measurement of ion fluxes. Under many circumstances, this technique provides a useful alternative to electro-physiology. In myoblasts, sodium uptake through voltage-dependent ionophores can be stimulated by veratridine and inhibited by tetrodotoxin. In myotubes which result from fusion of myoblasts, these voltage-dependent sodium channels appear to increase in number, paralleling the development of the action potential. Furthermore, in myotubes (but not myoblasts) carbamylcholine is able to stimulate a sodium influx through ionophores which are inhibitable by curare (dTC) but not tetrodotoxin (TTX). This demonstrates the presence of acetylcholine receptors on the fused cells. The cells also have a manganese-inhibitable calcium channel which appears to be voltage dependent and may be responsible for the calcium-dependent component of the action potential. Depolarizing concentrations of potassium in the medium stimulate calcium uptake both in the presence and absence of sodium. Veratridine and carbamylcholine also stimulate calcium influx, but both require the presence of sodium. This indicates that the depolarization necessary for opening the calcium channel is dependent upon sodium influx in these latter cases. Myoblasts and myotubes appear to have these channels in about equal numbers.     

Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system

The electrical properties of the clonal muscle cell line L6 can be revealed by the measurement of ion fluxes. Under many circumstances, this technique provides a useful alternative to electro-physiology. In myoblasts, sodium uptake through voltage-dependent ionophores can be stimulated by veratridine and inhibited by tetrodotoxin. In myotubes which result from fusion of myoblasts, these voltage-dependent sodium channels appear to increase in number, paralleling the development of the action potential. Furthermore, in myotubes (but not myoblasts) carbamylcholine is able to stimulate a sodium influx through ionophores which are inhibitable by curare (dTC) but not tetrodotoxin (TTX). This demonstrates the presence of acetylcholine receptors on the fused cells. The cells also have a manganese-inhibitable calcium channel which appears to be voltage dependent and may be responsible for the calcium-dependent component of the action potential. Depolarizing concentrations of potassium in the medium stimulate calcium uptake both in the presence and absence of sodium. Veratridine and carbamylcholine also stimulate calcium influx, but both require the presence of sodium. This indicates that the depolarization necessary for opening the calcium channel is dependent upon sodium influx in these latter cases. Myoblasts and myotubes appear to have these channels in about equal numbers.     

Ion channels and membrane potential in stimulus-secretion coupling in adrenal paraneurons

Cultured bovine adrenal medulla cells have been shown to contain several different ion channels (Na+, Ca2+, acetylcholine receptor regulated) whose activation leads to the secretion of catecholamines. The pharmacology of these ion channels and their interactions during secretion have been examined. The mechanisms of agonist-induced calcium influx are of particular interest since this is an early obligatory event during secretion from the adrenal medulla. Data obtained on catecholamine release and 45Ca2+ uptake indicate that both voltage-dependent and voltage-independent calcium influx mechanisms operate in cultured bovine adrenal medulla cells. The significance of these results in understanding the mechanism of action of the physiological stimulus acetylcholine (Ach) will be discussed. The alkaloid channel neurotoxins D-600, batrachotoxin, veratridine, and aconitine were shown to exert a noncompetitive inhibitory effect on Ach-induced ion flux in adrenal medulla cells, presumably through an interaction with the nicotinic receptor regulated channel. Lipid-soluble neurotoxins may interact with multiple ion channels in nerve and muscle membrane.     

Inhibition of plasma membrane Ca2+-atpase pump activity in cultured c6 glioma cells by halothane and xenon

We have compared the effect of two inhalational anesthetics, halothane and xenon, on Ca²⁺-ATPase (PMCA) pumping activity in plasma membrane vesicles prepared from cultured rat C6 glioma cells. Halothane, at concentrations ranging from 0.5 to 1.75 vol% (equivalent to 0.5 to 1.6 MAC), significantly inhibited Ca²⁺ uptake (transport) by plasma membrane vesicles in a dose-related fashion. Xenon, at partial pressures ranging from 0.5 to 1.5 atm (equivalent to 0.5 to 1.6 MAC), similarly inhibited PMCA pumping activity. Additive effects on suppression of PMCA pump activity were observed when C6 cell plasma membrane vesicles were exposed to increasing partial pressures of xenon in the presence of halothane (1 vol%). Halothane also inhibited PMCA pumping in cells from two other lines of neural origin, B104 (rat neuroblastoma) and PC12 (rat pheochromocytoma). Studies described in this report support the thesis that PMCA in cells of neural origin is inhibited by quite different inhalational anesthetics at clinically relevant concentrations.     

Formation of second messengers in response to activation of ion channels in excitable cells

1. Depolarization of excitable cells of the central nervous system results in the formation of the second messengers cyclic AMP, cyclic GMP, inositol phosphates, and diacylglycerides. 2. Depolarization-evoked accumulation of cyclic AMP in brain preparations can be accounted for mainly by the release of adenosine, which subsequently interacts with stimulatory adenosine receptor linked to adenylate cyclase. 3. Depolarization-evoked formation of cyclic GMP in brain preparations is linked to activation of voltage-dependent calcium channels, presumably leading to activation of guanylate cyclase by calcium ions. 4. In brain slices depolarization-evoked stimulation of phosphoinositide breakdown and subsequent formation of inositol phosphates and diacylglycerides are linked to activation of voltage-dependent calcium channels, which are sensitive to dihydropyridines, presumably leading to activation of phospholipase(s) C by calcium ions. 5. In the synaptoneurosome preparation depolarization-evoked stimulation of phosphoinositide breakdown does not involve activation of dihydropyridine-sensitive calcium channels and, instead, appears to be regulated primarily by the intracellular concentration of sodium ions. Thus, agents that induce increases in intracellular sodium--such as toxins that open or delay inactivation of voltage-dependent sodium channels; ouabain, an inhibitor of Na+/K+ ATPase that transports sodium outward and a sodium ionophore--all stimulate phosphoinositide breakdown. Mechanistically, increases in intracellular sodium either might directly affect phospholipase(s) C or might lead to influx of calcium ions through Na+/Ca2+ transporters. 6. Depolarization-evoked stimulation of cyclic AMP formation and phosphoinositide breakdown can exhibit potentiative interactions with responses to receptor agonists, thereby providing mechanisms for modulation of receptor responses by neuronal activity. 7. Since all these second messengers can induce phosphorylation of ion channels through the activation of specific kinases, it is proposed that depolarization-evoked formation of second messengers represents a putative feedback mechanism to regulate ion fluxes in excitable cells.     

Tubulin constancy during morphological differentiation of mouse neuroblastoma cells

Clonal cell lines N18 and N103 of the mouse neuroblastoma C1300 possess an undifferentiated neuroblast morphology under optimal growth conditions; however, when deprived of serum, N18 can be induced to extend long neurites. Although initial neurite outgrowth is rapid, very long fibers are found only after several days. Both initial outgrowths and established neurites contain microtubules; however, the number and density of these polymerized tubules increase markedly during this time. Optimum conditions have been established for assessing the colchicine-binding activity of neuroblastoma sonicates. A time-decay colchicine-binding assay was used to make a comparative study of the tubulin content of both undifferentiated and differentiated N18 as well as the nondifferentiating N103 and the rat glioma C6. Both morphologies of clone N18 possessed similar concentrations of tubulin (130-140 pmol/10(6) cells). Although cells of clone N103 contain 20% less tubulin than N18 cells, this is considerably more tubulin than is present in the glioma C6 (30 pmol/10(6) cells) which has a similar generation time. Quantitative densitometry of neuroblastoma extracts electrophoresed on SDS-polyacrylamide gels confirmed the constancy of tubulin. Radiolabeled proteins from neuroblastoma cells subjected to both growth conditions show that neurite outgrowth does not create a disproportionate demand for tubulin synthesis. Thus, the morphological differentiation of neuroblastoma cells probably reflects the regulation of tubulin storage and microtubule polymerization.     

Functional cross-talk of Ca2+-mobilizing endothelin receptors in C6 glioma cells

There are conflicting results concerning the receptor subtype(s) involved in calcium-mediated endothelin signaling in the glial cells. In order to elucidate the role of endothelin A and B receptors in these processes, we have studied the effect of a complex spectrum of endothelin receptor ligands on intracellular calcium concentration changes in proliferating and differentiated C6 rat glioma cells. Cell differentiation was induced by dibutyryl-cAMP and assessed by the glial fibrillar acidic protein content. Intracellular calcium changes were measured in cell suspensions using fluorescent probe Fura-2. The specific endothelin B receptor agonists sarafotoxin S6c and IRL-1620 did not influence the intracellular calcium concentration. However, calcium changes induced by endothelin-1 and especially by endothelin-3 after the pretreatment of cells with one of these endothelin B receptor specific agonists were significantly enhanced even above the values attained by the highest effective endothelin concentrations alone. Such endothelin B-receptor ligand-induced sensitization of calcium signaling was not observed in differentiated C6 cells. Moreover, endothelin-induced calcium oscillations in differentiated C6 cells were less inhibited by BQ-123 and BQ-788 than in their proliferating counterparts. In conclusion, the specific activation of endothelin B receptor in C6 rat glioma cells does not affect intracellular calcium per se, but probably does so through interaction with the endothelin A receptor. The pattern and/or functional parameters of endothelin receptors in C6 rat glioma cells are modified by cell differentiation.     

INTERCELLULAR COMMUNICATION IN RAPIDLY PROLIFERATING AND DIFFERENTIATED C6 GLIOMA CELLS IN CULTURE

Glial cells in the brain are known to provide structural and functional supports to neurons. To sustain such a supportive role, they have developed cell-to-cell communicating gap junctional channels. The authors studied the effect of dbcAMP on gap junctional channels mediated communication in C6 cells, a rat glioma cell line. Quantitative assessment of coupled cells under microscope after microinjection of a fluorescent dye was taken as a measure of junctional permeability. An enhanced coupling between cells was observed following dbcAMP treatment and this elevated coupling was found to be dependent on the duration of exposure of cells to dbcAMP. The studies have focused on a subtle shift in the spatial organization of the functional channels to the processes of dbcAMP induced differentiated cells from the cell cytoplasms and membranes of dbcAMP untreated cells. Immunofluorescence study with affinity purified antibody against gap junction further confirmed the spatial distribution of gap junctional protein(s) in the processes and also showed an increase in the density of the protein at the intercellular spaces in dbcAMP induced differentiated C6 glioma cells.     

Antioxidant defense against antidepressants in C6 and 1321N1 cells

The effects of pretreatment with the antioxidants reduced glutathione (GSH), ascorbate (ASC), Trolox (TROL), and combined ascorbate and Trolox (ASC/TROL) exposure on the acute (24 h) toxicities (EC50 value) of the antidepressants amitriptyline, imipramine (tricyclic antidepressants), fluoxetine (a selective serotonin reuptake inhibitor; SSRI), and tranylcypromine (a monoamine oxidase inhibitor; MAOI) were determined in the rat (C6) glioma and human (1321N1) astrocytoma cell lines using the neutral red uptake assay. The effects of pretreatment with buthionine-[S, R]-sulfoximine (BSO), and manipulation of intracellular cyclic AMP (cAMP) using isoproterenol (beta-receptor agonist), 3-isobutyl-1-methylxanthine (IBMX; a phosphodiesterase inhibitor), and dibutyryl cyclic AMP (dBcAMP; cAMP analogue) on antidepressant toxicity were also determined. Protective responses were observed after antioxidant treatments and manipulation of cAMP in both C6 cells pretreated with dBcAMP (+dBcAMP) and 1321N1 cells not pretreated with dBcAMP (-dBcAMP), with a few exceptions in 1321N1 cells (-dBcAMP). Some protective responses occurred in C6 cells (-dBcAMP) and 1321N1 cells (+dBcAMP) after isoproterenol and combined IBMX/isoproterenol pretreatment but not after just IBMX pretreatment. Pretreatment with BSO enhanced toxicity with the exception of fluoxetine. The antidepressants caused increases in intracellular GSH in the C6 cells at subcytotoxic concentrations, with decreases in GSH occurring at higher concentrations. Cytotoxicity of the antidepressants may be partly mediated through oxidative stress with alterations in signal transduction pathways.     

Inhibition of voltage-dependent sodium channels by Ro 31-8220, a 'specific' protein kinase C inhibitor

We find that several protein kinase C (PKC) inhibitors, previously considered to be specific, directly inhibit voltage-dependent Na(+) channels at their useful concentrations. Bisindolylmaleimide I (GF 1092037), IX (Ro 31-8220) and V (an inactive analogue), but not H7 (a non-selective isoquinolinesulfonamide protein kinase inhibitor), inhibited Na(+) channels assessed by several independent criteria: Na(+) channel-dependent glutamate release and [(3)H]batrachotoxinin-A 20-alpha-benzoate binding in rat cortical synaptosomes, veratridine-stimulated 22Na(+) influx in CHO cells expressing rat CNaIIa Na(+) channels and Na(+) currents measured in isolated rat dorsal root ganglion neurons by whole cell patch-clamp recording. These findings limit the usefulness of the bisindolylmaleimide class PKC inhibitors in excitable cells.     

氟哌啶醇和（R—）—NPA抑制C6神经胶质瘤细胞的钾电流

本工作用全细胞膜片箝方法,观察了氟啶醇（ｈａｌｏｐｅｒｉｄｏｌ以下简称ＨＡＬＯ）和Ｒ（－）－Ｐｒｏｐｙｌｎｏｒａｐｏｍｏｒｐｈｉｎｅ（以下简称ＮＰＡ）对Ｃ６神经胶质瘤细胞（Ｃ６ｇｌｉｏｍａｃｅｌｌｓ）的电压依赖性钾电流的作用,并初步分析了它们的作用机制。结果表明,ＨＡＬＯ和ＮＰＡ都能抑制Ｃ６细胞的Ｋ＾＋Ｊ电流中的慢成分,而对快成分的作用有所不同。它们的抑制作用不是由多巴胺Ｄ２受体介导的,也不是通过     

Characterization of pancreatic islet Ca2+-ATPase

Distribution of three isoenzymes of brain enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11) (alpha alpha, alpha gamma and gamma gamma forms) in clonal cell lines of neuroblastoma (NS20Y and N18TG-2), glioma (C6BU-1), and hybrid cells NG108-15, NCB20, Nbr10A, Nbr20A, N4G-B-a and N4G-C-a) was examined with a sensitive enzyme immunoassay system, that uses a rabbit antibody to rat brain enolase alpha alpha or gamma gamma. All cell lines tested were found to possess the enolase which contains gamma subunit (a neuron-specific protein), although the alpha alpha enolase (non-neuronal enolase) was the dominant from in these cells. A clonal rat glioma (C6BU-1) cell contained about 40, 1 and 0.07 microgram/mg protein of alpha alpha, alpha gamma and gamma gamma enolases, respectively, at the confluent stage. Inclusion of 1 mM dibutyryl cyclic AMP or 10 micrometers prostaglandin E1 plus 1 mM theophylline in the culture medium of a hybrid cell (NG108-15, mouse neuroblastoma x rat glioma) resulted in a more than 2-fold increase in the concentrations of alpha gamma and gamma gamma in the cell within a few days, with little change in the alpha alpha enolase concentration. A similar increase in the concentration of gamma subunit by the nucleotide (but not by prostaglandin E1 plus theophylline) was also observed in the glioma cell (C6BU-1) line. The results suggest that the gamma subunit or the neuron-specific protein can be regulated in NG108-15 and C6BU-1 cells in a cyclic AMP-dependent fashion.     

The presence of voltage-gated sodium, potassium and chloride channels in rat cultured astrocytes

Patch-clamp recording from the plasmalemma of rat cultured astrocytes reveals the presence of both voltage-dependent sodium and voltage-dependent potassium conductances. These conductances are similar but not identical to the corresponding conductances in the axolemma. Whereas the h infinity relation of the sodium channels has the same voltage dependence as in the nodal axolemma, the peak current-voltage relation is shifted by about 30 mV along the voltage axis in the depolarizing direction. It is speculated that the glial cells synthesize sodium and potassium channels for later insertion into the axolemma of neighbouring axons. The astrocytes also express a plasmalemmal voltage-dependent anion conductance that is turned on at about -40 mV (that is, near the resting potential of the cultured astrocytes). The channels involved are large enough to be just permeable to glutamate but not to ascorbate. It is suggested that the conductance of this channel for chloride plays a physiological role in the spatial buffering of potassium by glial cells.     

Modulation of L-type Ca2+ channels by gbeta gamma and calmodulin via interactions with N and C termini of alpha 1C

Neuronal voltage-dependent Ca(2+) channels of the N (alpha(1B)) and P/Q (alpha(1A)) type are inhibited by neurotransmitters that activate G(i/o) G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of Gbetagamma subunit of G proteins to the channel. Since cardiac and neuronal L-type (alpha(1C)) voltage-dependent Ca(2+) channels are not modulated in this way, they are presumed to lack interaction with Gbetagamma. However, here we demonstrate that both Gbetagamma and calmodulin directly bind to cytosolic N and C termini of the alpha(1C) subunit. Coexpression of Gbetagamma reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca(2+), and is eliminated by EGTA. The N and C termini of alpha(1C) appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of Gbetagamma. Deletion of the N terminus profoundly impairs the Ca(2+)/calmodulin-dependent inactivation. We propose that Gbetagamma and calmodulin regulate the L-type Ca(2+) channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of alpha(1C).