Biomedical subjects

K Koschel

Publications and source records attributed to K Koschel.

At least 19 recordsLinked to original sources

PubMed1998

Sphingosine-1-phosphate induces a Ca2+ signal in primary rat astrocytes and a Ca2+ signal and shape changes in C6 rat glioma cells.

Treatment of rat glioma C6 cells with the beta-adrenergic agonist L-isoproterenol leads to a rise in cAMP level and a subsequent change in cell morphology from an epithelial to an astrocytic type of appearance. This morphological response is reverted by the addition of sphingosine-1-phosphate (S1P) with an EC50 of 10 nM. In rat glioma C6 cells loaded with the Ca2+ indicator Fura-2, S1P evoked Ca2+ release from internal stores and Ca2+ influx from the external medium. Half-maximal stimulation of the Ca2+ increase was 10-20 nM. A similar Ca2+ signal was observed in primary rat astrocytes loaded with the Ca2+ indicator fluo-3. Pretreatment of the C6 cells with PMA (162 nM) prevented both the S1P-induced Ca2+ increase and the morphological reversion. Ca2+ ions therefore seem essential for the morphological reversion by S1P. Pretreatment of the cells with the Clostridium botulinum C3 exoenzyme did not affect the reversion of the morphological response by S1P, indicating that the small GTP-binding protein Rho is not involved in the S1P-induced reversion.

Adrenergic beta-Agonists↗

PubMed1995

Influence of cadmium ions on endothelin-1 binding and calcium signaling in rat glioma C6 cells.

Cadmium ions did not influence the binding of endothelin-1 (ET-1) to its receptor on the surface of rat glioma C6 and rat aorta A10 cells. This was studied (a) by the binding of 1251-ET-1 to intact cells in the absence or presence of cadmium (Cd2+) and (b) by analysis of the receptor/ET-1 complex after crosslinking with disuccinimidyl suberate (DSS) or ethylene glycol-bis-(succinimidyl succinate) (EGS) on SDS PAGE. Using Fura-2 and Quin-2 loaded C6 rat glioma cells, it was shown that Cd2+ ions strongly interfered with the ET-1 induced Ca(2+)-influx in C6 glioma cells (IC50 approximately 10 microM).

Animals↗

PubMed1995

Measles virus antisense sequences specifically cure cells persistently infected with measles virus.

Vectors expressing antisense mRNAs complementary to the measles virus (MV) nucleoprotein N or hemagglutinin H genes were used to transfect MV-permissive Vero cells, MV-nonpermissive C6 rat glioma cells, and C6 cells persistently infected with measles/SSPE virus (C6/SSPE cells). Transfected Vero cells infected with MV showed a drastically reduced yield of infectious virus (90-99.99%). In plaque assays, plaque numbers and plaque size were significantly reduced compared with untransfected Vero cells. With an unrelated control virus, VSV, no effects were seen in the transfected Vero cells, underlining the specificity for MV. Following stable transfection with MV antisense vectors, C6 rat glioma cells, which are normally suitable to establish persistently MV-infected lines, can no longer be infected with the virus. In this case also, VSV infection was not influenced. Furthermore, antisense transfection of already persistently infected C6/SSPE cells leads to a loss of MV-specific immunofluorescence, concomitant with a disappearance of viral RNA. Single cell clones from the antisense-transfected C6/SSPE cells appear to be totally free of virus in cocultivation with Vero cells, suggesting that they are really cured. The effectiveness of even low amounts of antisense sequences suggests that they are good candidates for antisense oligonucleotide therapy in tissue culture and might eventually also be useful for in vivo application.

Animals↗

PubMed1995

Downregulation of endothelin receptor mRNA synthesis in C6 rat astrocytoma cells by persistent measles virus and canine distemper virus infections.

Persistent infections of C6 rat astrocytoma cells with measles (subacute sclerosing panencephalitis [SSPE]) virus (C6/SSPE cells) or canine distemper virus (C6/CDV cells) cause a loss of endothelin-1 (ET-1) binding to its specific receptors (ETRA type) and subsequent ET-1-induced Ca2+ signaling. It was the aim of this study to investigate the underlying mechanism of this phenomenon in more detail. By using an RNase protection assay, it was found that ETRA mRNA disappears, whereas other cellular mRNA species, e.g., beta-actin mRNA, were not influenced. The data show that the loss of the ET-1 signaling pathway in C6/SSPE and C6/CDV cells is due to a receptor downregulation at the transcriptional level.

Animals↗

PubMed1993

Lysophosphatidic acid reverts the beta-adrenergic agonist-induced morphological response in C6 rat glioma cells.

Elevation of cAMP changes the morphology of C6 rat glioma cells from a fibroblast to an astrocyte type of appearance. This change is prevented by the presence of serum from different species (chicken, mouse, rat, horse, adult bovine, fetal bovine, and human) in the cell culture medium. In this communication the component in serum responsible for this effect is identified as lysophosphatidic acid for the following reasons: First, lysophosphatidic acid alone at concentrations which are present in serum reverts the morphological response. Second, both lysophosphatidic acid and the component in serum no longer revert the morphological response after treatment with phospholipase B (E.C.3.1.1.5). Third, lysophosphatidic acid and serum produce an analogous intracellular Cai2+ signal in rat glioma C6 cells as measured by fluorescence spectrophotometry with the Ca2+ ion indicator Fura 2. Fourth, both the morphological response and the Cai2+ increase are prevented by pretreatment of the cells with 100 ng/ml phorbol 12-myristate 13-acetate. Finally, a maximal Cai2+ increase induced by FCS prevents the subsequent Cai2+ signal by lysophosphatidic acid. Interestingly, the morphological response is also reverted by Al3+ together with F- ions and also by lower n-alkanols such as ethanol and n-propanol suggesting that a regulatory GTP-binding protein is involved in the reversion. It is further shown that activation of the phosphatidylinositol 4,5-bisphosphate cleavage signal system is not responsible for the reversion of the morphological response.

Adrenergic beta-Agonists↗

PubMed1991

Loss of the endothelin signal pathway in C6 rat glioma cells persistently infected with measles virus.

Endothelin 1 causes a strong Ca2+ signal in C6 rat glioma cells as measured by fura-2 fluorescence. This endothelin 1-induced Ca2+ signal was not observed when the cells were persistently infected with a measles virus strain of subacute sclerosing panencephalitis (SSPE, strain Lec). Binding of 125I-labeled endothelin 1 to the C6/SSPE cells was less than 5% of the binding to the C6 control cells, suggesting that the impairment in signal transduction was due to a loss of binding sites for endothelin 1. Treatment of the C6/SSPE cells with measles antiserum resulted in the loss of expression of viral proteins located in the membrane as well as inside the cells (antigenic modulation), but it restored neither the endothelin 1-induced Ca2+ rise nor the 125I-endothelin 1 binding. Cocultivation of uninfected C6 cells with C6/SSPE cells (9:1 ratio) resulting in contact-mediated transmission of measles virus showed that the 125I-endothelin 1 binding activity was gradually lost as a consequence of persistent virus infection.

Animals↗

PubMed1991

Volatile anesthetics stimulate the phorbol ester evoked neurotransmitter release from PC12 cells through an increase of the cytoplasmic Ca2+ ion concentration.

PC12 cells preloaded with [3H]norepinephrine release this neurotransmitter at a slow rate (basal release). This rate is increased by the addition of phorbol myristate acetate (PMA), but not by a biologically inactive phorbol ester. This effect most likely is mediated by protein kinase C, since desensitization of this kinase abolished the stimulation of the neurotransmitter release by PMA. Unexpectedly, clinical concentrations of the volatile anesthetics halothane, enflurane, isoflurane and methoxyflurane stimulated the PMA evoked neurotransmitter release in good correlation with their anesthetic potency. Since the volatile anesthetics increased the cytoplasmic Ca2+ concentration of the PC12 cells in a dose dependent manner it seems very likely that the effect of the anesthetics on the PMA-evoked neurotransmitter release is mediated by this rise in Ca2+ concentration.

Adrenal Gland Neoplasms↗

PubMed1991

Effects of volatile anesthetics on cytoplasmic Ca2+ signaling and transmitter release in a neural cell line.

To provide new insights into the effects of volatile agents on the basic regulatory events involved in cytoplasmic free Ca2+ ([Ca2+]i) and stimulus-secretion coupling, the well-characterized clonal rat pheochromocytoma cell line PC12 was chosen as an experimental model. This cell line possesses nicotinic and muscarinic receptors, L-type voltage-operated channels (VOCs), and receptor-operated Ca2+ channels (ROCs). A PC12 variant, defective in nicotinic response, made it possible to study the influx-independent inositol trisphosphate-mediated intracellular Ca2+ release that is triggered by muscarinic receptor stimulation. [Ca2+]i was measured with the fluorescent Ca2+ indicator fura-2. Dopamine and norepinephrine secretion were determined by high-performance liquid chromatography. High K+ and nicotinic-receptor-induced [Ca2+]i increase and catecholamine secretion were inhibited by halothane, enflurane, isoflurane, and methoxyflurane in a dose-dependent manner; half-maximal inhibition (IC50) occurred within the clinically relevant concentration range. The inhibition was reversible after wash-out of anesthetic; was not restricted to dihydropyridine-sensitive L-type VOCs; and could not be overcome by increasing extracellular Ca2+. The inhibitory mechanisms of volatile anesthetics therefore differed from those of classical organic Ca2(+)-channel blockers, a difference also reflected by the differing Hill coefficients found for both substance groups. In contrast, the muscarinic-receptor-evoked internal Ca2+ release remained unimpaired, and secretion even increased under anesthetic exposure. In conclusion, the current study provides evidence that volatile anesthetics depress the Ca2+ influx through at least two independent Ca2+ channels, one of which proved insensitive to the dihydropyridine Ca2(+)-channel blocker nifedipine. This is particularly noteworthy, since dihydropyridine-insensitive N-type VOCs, so far found exclusively in neurons, are assumed to play a dominant role in synaptic transmission, which, although resistant to dihydropyridine inhibition, is effectively blocked by volatile anesthetics.

Anesthetics↗

PubMed1990

Lack of selectivity between anesthetic stereoisomers for an inhibitory site which is located on a membrane protein.

Lack of selectivity towards anesthetic stereoisomers is one of the few criteria available for the identification of an anesthetic target site. Until now this criterion has not been tested for anesthetics that directly interact with sensitive membrane proteins which are considered the targets of anesthetic action. In this communication we show that stereoisomers of 2-butanol and 2,4-pentanediol did not differ in their inhibitory potency for a site located on the Na+/K+/Cl- co-transporter protein. This suggests that an inhibition site on a membrane protein can fulfill the criterion of lack of stereoisomer selectivity.

Anesthetics↗

PubMed1990

Lipid solubility is not the sole criterion for the inhibition of a Ca2(+)-activated K+ channel by alcohols.

The effect of a homologous series of n-alkanols (C2-C8) on the 86Rb+ influx through charybdotoxin sensitive Ca2(+)-activated K+ channels of rat glioma C6 cells was investigated. The lipid solubility of the n-alkanols was not the sole determinant of the inhibitory potency of these substances for ion flux inhibition. 1-Hexanol for example was about 8-times less potent than one would expect on the basis of its lipid solubility. The introduction of a second OH-group in this molecule (giving 1,6-hexanediol) or a structural shift in the OH-group of 1-hexanol from position 1 to 3 strongly increased the potency of the alcohol. The above data cannot be explained by a pure lipid site of action of the alcohols. Therefore it seems likely that direct effects on protein are involved in the inhibitory action of some of the alcohols.

Alcohols↗

PubMed1990

Thrombin reverts the beta-adrenergic agonist-induced morphological response in rat glioma C6 cells.

Treatment of rat glioma C6 cells with a beta-adrenergic agonist leads to a rise in cAMP level and a subsequent change in cell morphology from an epithelial to an astrocyte type of appearance. This morphological change is reverted by the addition of thrombin. In 10-15 min the cells acquire their normal epithelial morphology. The reversion by thrombin is inhibited by hirudin, but not by antithrombin III (an inhibitor of the proteolytic action of thrombin). Using the intracellular Ca2(+)-indicator fura-2, we observed that the addition of thrombin to the glioma cells generated a Ca2(+)-signal which was inhibited by pretreatment of the cells with hirudin or with 1 mM neomycin. These results suggest that thrombin uses the phospholipid-inositol pathway to counteract the morphological response, which was induced by activation of the cAMP pathway.

Animals↗

PubMed1989

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.

Anesthetics↗

PubMed1989

Coupling of viral membrane proteins to phosphatidylinositide signalling system.

C6 rat glioma cells persistently infected with subacute sclerosing panencephalitis virus (C6/SSPE) were treated with measles antiserum and purified anti-measles IgG. This stimulated phosphoinositide breakdown and an increase in inositol phosphates. In uninfected C6 cells, however, only fetal calf serum (FCS), but not measles antiserum could induce inositol polyphosphate production.

Animals↗

PubMed1989

The sodium channels of the neuroblastoma x glioma 108 CC 15 hybrid cell change their sensitivity for volatile and local anesthetics upon continuous passage.

We have studied the ion flux through the sodium channels of low passage number (less than 50 p.) and high passage number (greater than 150 p.) neuroblastoma x glioma hybrid cells using [14C] guanidinium and specific neurotoxins to induce channel opening and closing. The sodium channels of low passage number hybrid cells could be opened by veratridine alone, suggesting the presence of voltage dependent channels in agreement with electrophysiological studies reported in the literature. The sodium channels of the high passage number hybrid cells, however, needed the synergistic action of veratridine and scorpion toxin for activation suggesting that these channels are "silent". The [14C] guanidinium ion flux through the sodium channels of the high passage number hybrid cells was inhibited by significantly lower concentrations of the volatile anesthetics (halothane, isoflurane and enflurane) and the local anesthetics (tetracaine and bupivacaine) than the comparable flux through the sodium channels of the low passage number hybrid cells.

Anesthetics↗

PubMed1988

Presence of a charybdotoxin sensitive Ca2+-activated K+ channel in rat glioma C6 cells.

A study was made of the 86Rb+ influx and efflux through Ca2+-activated K+ channels of intact rat glioma C6 cells after addition of a Ca2+ ionophore to the incubation medium. Half-maximal activation of the channels was obtained at a cytoplasmic Ca2+ concentration of approximately 400 nM. The 86Rb+ ion flux through the Ca2+-activated K+ channels was insensitive to apamin, but was inhibited by low concentrations of charybdotoxin (IC50 = 1.6 nM). This is the first evidence for the presence of charybdotoxin-sensitive Ca2+-activated K+ channels in glial cells.

Animals↗

PubMed1987

Characterization of an Na+/K+/Cl- co-transport in primary cultures of rat astrocytes.

The furosemide- and bumetanide-sensitive component of the 86Rb+ uptake into primary cultures of rat astrocytes was fully dependent on the simultaneous presence of Na+ and Cl- in the incubation mixture and is therefore most likely an Na+/K+/Cl- co-transporter. As expected for such a co-transporter, its activity is insensitive to 0.1 mM amiloride and to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and of the tested anions, only Br- could partly replace Cl-. The K0.5 values for K+, Na+ and Cl- activation were 2.7, 35 and 40 mM, respectively. The activity of the co-transporter was stimulated 1.5-times in hyperosmolar (500 mosM) medium.

Amiloride↗

PubMed1987

Volatile anesthetics depress the depolarization-induced cytoplasmic calcium rise in PC 12 cells.

In the rat pheochromocytoma cell line PC 12, the effects of four volatile anesthetics (halothane, isoflurane, enflurane, methoxyflurane) on the K+-evoked intracellular calcium [( Ca2+]i) rise were investigated using the Ca2+-sensitive fluorescence dye fura-2. The [Ca2+]i rise was depressed, at clinical concentrations, by all anesthetics with almost identical aqueous IC50 values. The study extends to neuronal cells the observation made previously in cardiac tissue that volatile anesthetics may interfere with Ca2+ fluxes through voltage-gated channels.

Adrenal Gland Neoplasms↗

PubMed1987

General anesthetics can competitively interfere with sensitive membrane proteins.

It is not known whether proteins or lipids are the primary target of anesthetic action. The resolution of this problem is hampered by the fact that it is not possible to investigate the biological activity of integral membrane proteins in the absence of lipids. However, certain characteristics of membrane protein function inhibition by anesthetics cannot be explained on the basis of an indirect inhibition by disturbance of the lipid bilayer and, therefore, most likely are the result of a direct anesthetic-protein interaction. This is the case (i) when the anesthetics competitively interfere with the binding of an endogenous ligand to the membrane protein and (ii) when the size of the anesthetic molecule is of importance for the potency and/or mechanism of inhibition. The present study shows that this is true for a membrane transport system, the Na+/K+/Cl- cotransport in glial-type cells.

1-Butanol↗