Inhibition of the histamine-induced Ca2+ influx in primary human endothelial cells (HUVEC) by volatile anaesthetics.

BACKGROUND AND OBJECTIVE: Vasoactive substances such as histamine, acetylcholine or ATP increase the [Ca2+]i of endothelial cells, which leads to the activation of nitric oxide synthase (eNOS). The NO produced by this enzyme relaxes the underlying smooth muscle. Evidence suggests that eNOS activation is dependent on agonist-induced Ca2+ entry. Recently we have shown that in human endothelial cells (HUVEC), this Ca2+ entry is sensitive to isoflurane. The objective here was to study the mechanism by which volatile anaesthetics can depress the histamine-induced Ca2+ entry into HUVEC cells. METHODS: HUVECs on coverslips were loaded with the Ca2+ indicator Fluo-3 and inserted in a gastight, temperature-controlled perfusion chamber. Excitation was at 488 nm and fluorescence signals were monitored with a confocal laser scanning microscope (MRC1024, Biorad). Direct measurement of the Ca2+ influx was with Mn2+ as surrogate for calcium at 360 nm in cells loaded with Fura-2. RESULTS: Addition of histamine induces a biphasic [Ca2+]i increase consisting of Ca2+ release from internal stores and a Ca2+ influx from the external medium (plateau phase). The plateau phase was dose-dependently inhibited by enflurane and sevoflurane (13.7 resp. 21.9% inhibition by 1 MAC anaesthetic). Direct measurement of the Ca2+ influx using the Mn2+ quench of the Fura-2 fluorescence gave similar results. The inhibition of the anaesthetics was not reduced by inhibition of the cGMP pathway, inactivation of protein kinase C, depolarization of the cells or the presence of specific Ca2+-dependent K+ channel inhibitors. Interestingly, unsaturated fatty acids inhibit the histamine-induced Ca2+ influx in a similar way as the volatile anaesthetics. CONCLUSIONS: Volatile anaesthetics dose-dependently inhibit the histamine-induced Ca2+ influx in HUVECs by a mechanism that may involve unspecific perturbation of the lipid bilayer.

Effects of addition of derived 40 S subunits on translation rate and polysome profile of the reticulocyte lysate.

We have investigated the way in which the addition of exogenous 40 S subunits to a reinitiating cell-free translation system, prepared from reticulocytes, may affect translational parameters of the system. The disturbance of the system's subunit stoichiometry resulted in the following changes in the ribosome profile: (1) rapid exhaustion of the pool of native 60 S subunits; (2) appearance of humps on the peaks of the polysome profile, which probably represent unusually long-lived [40 S. polysomal] complexes; (3) at higher doses of exogenous particles, the amount of polysomes decreased. This latter effect reflected a corresponding decrease in the overall translation (i.e. initiation) rate. The phenomena are interpreted as follows: exogenous 40 S subunits combine with 60 S subunits, forming idle 80 S ribosomes. The shortage of 60 S subunits delays the utilization of [40 S. polysomal] complexes, which is compensated for by a pool increase of these complexes. At high 40 S subunit doses this compensatory mechanism fails, and the 60 S shortage begins to determine the overall translation rate. The observations underline that the various translational parameters of the lysate function in an optimally concerted manner, so that only small amounts of derived 40 S subunits are tolerated by the system for analysis.

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.

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.

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.

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.

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.

Inhibition of the elongation step of protein synthesis by vaccinia virus.

Vaccinia cores inhibit translation in cell-free protein synthesis systems at two stages: initiation; and, as shown here, elongation. The former effect tends to obscure the latter. Elongation control could, however, be revealed as follows: when, in a reticulocyte of L-cell lysate, initiation was blocked by a drug (edein), the residual [35S]methionine incorporation was severely reduced by the subsequent addition of vaccinia cores. The elongation block could also be demonstrated by analysis of ribosome profiles: treatment with edein alone permitted ribosomal run-off; treatment with either the elongation inhibition anisomycin or with cores preserved the polyribosomes.

Halothane inhibits the neurotoxin stimulated [14C]guanidinium influx through 'silent' sodium channels in rat glioma C6 cells.

We have investigated the effect of pharmacological agents on [14C]guanidinium ion influx through sodium channels in C6 rat glioma and N18 mouse neuroblastoma cells. The sodium channels of the N18 cells can be activated by aconitine alone, indicating that they are voltage-dependent channels. In contrast, sodium channels in the C6 cells require the synergistic action of aconitine and scorpion toxin for activation and are therefore characterized as so-called silent channels. The general anesthetic halothane used at clinical concentrations, specifically inhibited the ion flux through the silent sodium channel of C6 rat glioma cells. The voltage-dependent channels of the N18 cells were insensitive to halothane at the concentrations tested.

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.

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.

Preliminary characterization of an Na+,K+,Cl- co-transport activity in cultured human astrocytes.

We have studied the potassium uptake using 86Rb+ into monolayers of secondary cultures of human astrocytes prepared from cerebral hemispheres of a 4-month-old fetus. With the use of inhibitors we could attribute 30-40% of the 86Rb+ uptake to an Na+,K+-ATPase, 50-60% to an anion-cation co-transporter and 10% to potassium leak channels. The anion-cation co-transporter was dependent on the simultaneous presence of both sodium and chloride in the incubation medium and is therefore most likely an Na+,K+,Cl- co-transporter. This is the first evidence of such an Na+,K+,Cl- co-transport in human astrocytes.

The volatile anesthetic isoflurane suppresses spontaneous calcium oscillations in vitro in rat hippocampal neurons by activation of adenosine A1 receptors.

Primary cultures of rat hippocampal neurons were loaded with the Ca(2+)-indicator fluo-3 and studied with a confocal laser microscope. In Mg(2+)-free medium the cultures showed spontaneous synchronized calcium oscillations. These oscillations derived from excitatory signal transmission by N-methyl-D-aspartate and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptors and were modulated by gamma-aminobutyric acid(A) receptors. The oscillations were dose-dependently depressed by adenosine (IC50=2 microM) or by 2-chloro-N6-cyclopentyladenosine a specific adenosine A1 receptor agonist (IC50=40 nM). These effects were reverted by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific adenosine A1 receptor antagonist. The volatile anesthetic isoflurane also depressed these spontaneous calcium oscillations in a dose dependent manner (IC50=0.25 MAC, Minimum Alveolar Concentration). The isoflurane-induced inhibition was partly reversed in 29-38% of the neurons by DPCPX, indicating that the anesthetic activates this receptor possibly by increasing the extracellular concentration of adenosine.

The volatile anesthetic enflurane activates capacitative Ca2+ channels in rat glioma C6 cells.

(1) The volatile anaesthetics halothane and enflurane released Ca2+ from thapsigargin sensitive stores in rat glioma C6 cells, but only enflurane induced a significant Ca2+ influx. (2) The reason for this can be explained by a different inhibitory effect of the anesthetics on the capacitative Ca2+ influx. (3) Halothane inhibited the capacitative Ca2+ influx with an IC50 of 1.9 vol.%, whereas enflurane only marginally affected the ion flux through the channel.

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).

Indirect activation of adenosine A1 receptors in cultured rat hippocampal neurons by volatile anaesthetics.

BACKGROUND AND OBJECTIVE: Volatile anaesthetics depress excitatory signal transmission by potentiating the inhibitory action of GABAA receptors and there is strong evidence that this is related with anaesthesia. Using primary hippocampal cultures we analyzed the possibility that the volatile anaesthetics enflurane and sevoflurane depress excitatory signal transmission by activation of adenosine A1 receptors. METHODS: Primary rat hippocampal cultures on 4 cm poly-L-lysine coated glass coverslips were loaded with the Ca2+-indicator fluo-3 and incorporated in a gastight, temperature-controlled perfusion chamber. The intracellular Ca2+-concentration was monitored with a confocal laser-scanning microscope (BioRad) using the 488 nm laser line of a krypton-argon laser for excitation and the Lasersharp Acquisition software for analysis. RESULTS: Continuous perfusion in Mg2+-free medium generated spontaneous synchronized calcium oscillations, which were dose dependently depressed by the volatile anaesthetics enflurane and sevoflurane (0.25-1 minimum alveolar concentration). Addition of 100 nmol of 2-chloro-N6-cyclopentyladenosine, a specific adenosine A1 receptor antagonist, partly reversed the anaesthetic-induced inhibition of the oscillation amplitude of the oscillating cells. The effect of the anaesthetics was mimicked by the addition of S-(p-nitrobenzyl)-6-thioguanosine, an adenosine transport inhibitor and by the addition of 5-amino-5-deoxyadenosine, an inhibitor of adenosine kinase. CONCLUSIONS: The volatile anaesthetics sevoflurane and enflurane activate adenosine A1 receptors in primary rat hippocampal cultures. This effect is mediated by liberation of adenosine most likely by an interaction of the volatile anaesthetics with adenosine transport or key enzymes in adenosine metabolism.

Translation of vaccinia virus and cellular mRNA in cell-free systems prepared from uninfected and vaccinia virus infected L929 cells.

Cell-free translation systems were prepared from uninfected and vaccinia infected (3 and 5 hours post-infection) L929 cells. The systems were made mRNA dependent in order to translate exogenous mRNA mixtures. The overall rate of protein synthesis was similar in the three translation systems. However, one-dimensional electrophoresis showed that the systems differed in terms of the translation efficiency for individual mRNAs. This could be demonstrated with each of the following mRNA mixtures: early vaccinia mRNA synthesized by vaccinia cores in vitro, mRNA isolated from polysomes of vaccinia infected HeLa cells ("late" vaccinia mRNA) and cytoplasmic ascites mRNA. When the above mentioned groups of mRNAs were allowed to compete for translation in the cell-free systems and their products were analyzed on one-dimensional gels, the following order of translational efficiency was observed: the most prominent species of vaccinia early mRNA (other species could not be judged) were translated better than some late vaccinia mRNA species which in turn were slightly more efficiently translated than cellular mRNAs.

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.

Regulation of ribosomal protein S6 phosphorylation in heat-shocked HeLa cells.

Decreases in energy charge, ribosomal protein phosphorylation and rate of protein synthesis are well-documented facets of the cellular response to hyperthermia in non-vertebrates. We have tried to reproduce this response pattern in 32P-labelled HeLa cells in order to investigate the hypothetical causal relationship between these effects. In HeLa cells shifted from 36 degrees C to 42 degrees C, dephosphorylation of S6 and inhibition of protein synthesis, owing to a decreased initiation rate, were observed, but could not have been mediated by changes in the cells' general energy charge since the ATP and GTP levels were not reduced. In addition, we found that the hyperthermic translation block developed faster than the overall dephosphorylation of S6, showing that S6 dephosphorylation cannot be responsible for the translation block unless site-specific effects play a critical role.

Are highly phosphorylated 40-S subunits preferentially utilized during protein synthesis in a cell-free system from HeLa cells?

It has been concluded from circumstantial evidence obtained with HeLa cells in vivo that the phosphorylation of ribosomal protein S6 increases the affinity of 40S particles for mRNP [Duncan, R. and McConkey, E. H. (1982) Eur. J. Biochem. 123, 535-538; Thomas, G., Martin-Pérez, J., Siegmann, M. and Otto, A.M. (1982) Cell 30, 235-242]. This conclusion needs to be tested in vitro in a reinitiating cell-free translation system from growth-competent cells. We have prepared such a system from HeLa cells and have compared the capacity of homologous 40S subunits of various degrees of phosphorylation to enter the existing polysome pool. The 40S subunits' degree of phosphorylation was manipulated by exposing aliquots of growth-stimulated HeLa cells to hyperthermia (see accompanying paper). 40S subunits from heat-shocked and control cells, despite differences in S6 phosphorylation level as verified by two-dimensional electrophoresis, did not differ with respect to their recruitment into the existing polysome fraction. Owing to the reinitiation activity of the translation system, assay times could be kept sufficiently short, to avoid any serious interference by the S6 phosphatase activities of the system. Our results suggest that increased S6 phosphorylation by itself is not sufficient to accelerate the participation of 40S subunits in protein synthesis.