Thomas Schlüter

Increase of bovine alveolar macrophage superoxide anion and hydrogen peroxide release by dusts of different origin.

The release of reactive oxygen intermediates (ROI) from bovine alveolar macrophages (BAM) after stimulation with heavy metal-containing dusts was investigated. BAM were obtained by postmortem lavages of bovine lungs. The dusts were collected from waste incineration, sewage sludge incineration, an electric power station, and from two different factories. Three quartz dusts were used as heavy metal-free controls. The dusts were fractionated by sieving and sedimentation and analyzed by electron microscopy, atomic absorption spectrometry (AAS), and atomic emission spectrometry with inductively coupled plasma (AES-ICP). Incubation of BAM with the dusts (12.5-1000 micrograms/ml medium) led to concentration-dependent increases in ROI release. The secretion of ROI was already seen after 15 min and lasted throughout the experiment up to 90 min, with the exception of a waste incinerator ash, which contained the highest contents of some heavy metals and where the release of ROI ceased after 60 min. We suggest that this dust exhibits simultaneously stimulating and inhibiting effects. The ratio of the secreted O2- and H2O2 varied, depending on the dust being investigated. The release of hydrogen peroxide correlated best, in descending order, with the content of iron, manganese, chromium, vanadium, and arsenic in the dusts.

Protection of mitochondrial integrity from oxidative stress by the triaminopyridine derivative flupirtine.

The suitability of the triaminopyridine derivative flupirtine, an analgesic drug with antioxidative property [Gassen, M., Pergande, G. and Youdim, M.B.H. (1998) Biochem. Pharmacol. 56, 1323–1329], for the preservation of mitochondrial integrity from oxidative stress‐induced damage was studied. Rat liver mitochondria were exposed to strong oxidative stress as generated by Fe2+ plus ascorbate. Peroxidation damage of membrane lipids was followed by the measurement of thiobarbituric acid reactive substances. Protein oxidation was estimated by electron spin resonance spectroscopy, after labeling of the ‘peroxidized’ mitochondria with 4‐maleimido‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl. We found that (i) low concentrations of flupirtine (10 μM) protect lipids and also proteins (with lesser efficiency) from attacks of reactive oxygen species; (ii) flupirtine remarkably delayed the decline of complex mitochondrial functions, such as the respiratory control or the Ca2+ retention capacity of mitochondria, under oxidative stress; and (iii) the ADP/ATP antiporter (ANT), a main component of the oxidative phosphorylation machinery as well as a core component of the permeability transition pore complex, seems to be a membrane protein particularly protected by flupirtine. In conclusion, the preservation of the Ca2+ buffer capacity of mitochondria and of the ANT activity against oxidative stress supports an antiapoptotic application of flupirtine.

Effect of flupirtine on cell death of human umbilical vein endothelial cells induced by reactive oxygen species.

Flupirtine (KATADOLON), known as a nonopiate centrally acting analgesic drug, was tested as to its potential to prevent apoptosis of human endothelial cells induced by reactive oxygen species (ROS). It was found that Flupirtine displayed no effect on viability and cell proliferation of human umbilical vein endothelial cells (HUVEC) up to a concentration of 10 microg/mL. Apoptosis, induced by ROS and generated by hypoxanthine/xanthine oxidase (EC 1.1.3.22) (HX/XOD) or t-butyl hydroperoxide, was reduced after preincubation with Flupirtine for 3 hr by 35% and 41%, respectively. The maximal cytoprotective effect against apoptosis was observed at a drug concentration of 1 to 3 microg/mL. Flow cytometric studies revealed that Flupirtine was able to decrease the number of necrotic cells as well as of apoptotic cells. Neither the simultaneous administration of Flupirtine with the apoptosis-inducing agent nor the preincubation of HUVEC with Flupirtine influenced the increase in the intracellular Ca2+ concentration [Ca2+]i caused by the production of ROS.

Effect of heavy metal ions on the release of reactive oxygen intermediates by bovine alveolar macrophages.

Short-term incubations of bovine alveolar macrophages (BAM) with metal-containing dusts induce the release of reactive oxygen intermediates (ROI). Incubations of BAM (90 min) with dissolved metal compounds (0.1-100 microM) combined with quartz dusts were performed to investigate the effects of single elements on BAM stimulation. As(III), as well as the calcium antagonists, Ni(II) and Ce(III), inhibited the secretion of superoxide anions (O2-) and hydrogen peroxide (H2O2). O2- concentrations were lowered by Mn(II) and Fe(II). Increased ROI concentrations were observed with V(IV) (O2- and H2O2) and Fe(III) (O2-). The addition of Cd(II), Cr(III) and V(V) showed no effect on the dust-induced respiratory burst. The influence of insoluble heavy metal compounds on ROI secretion by BAM were studied with metal oxide-coated silica particles. In most cases the release of ROI was not affected by the chemical modification of the particle surface. Coating with CuO markedly lowered the concentrations of O2- and H2O2, whereas vanadium(IV) oxide considerably increased both ROIs. Although most of the investigated metal compounds did not alter ROI secretion our present results with V(IV) and Fe(III) confirm our recent statistical evaluation of the effects of heavy metal-containing dusts on ROI secretion (Berg et al., 1993, J. Toxicol. Environ. Health 39, 341).

Serotonin-induced secretion of von Willebrand factor from human umbilical vein endothelial cells via the cyclic AMP-signaling systems independent of increased cytoplasmic calcium concentration

Endothelial cells are able to synthesize von Willebrand factor (vWf) protein, which is then either secreted in a constitutive way or stored within specific cellular secretory granules, the Weibel-Palade bodies. Stimulated secretion of vWf from these organelles is thought to be induced by agonists causing a transient increase in cytoplasmic calcium concentrations. Serotonin (5-hydroxytryptamine, 5-HT), a local transmitter substance released by activated platelets, has also recently been shown to induce the secretion of vWf. In experiments with human umbilical vein endothelial cells (HUVEC), we found that the 5-HT-induced secretion occurred without a significant increase in cellular calcium levels. The 5-HT 1(D) subtype-specific receptor agonist sumatriptan also induced the release of vWf without causing a calcium signal in HUVEC. Stimulation of endothelial cells with the adenylate cyclase inhibitor, MDL-12 A330, led to the secretion of vWf as well. Simultaneous addition of submaximal concentrations of histamine and 5-HT to HUVEC potentiated the effects of either agonist. Together, these results suggest that in HUVEC 5-HT-induced secretion of vWf is mediated by a decrease in cyclic AMP levels and is independent of changes in cytoplasmic calcium levels.

Mechanisms of particle-induced activation of alveolar macrophages

Bovine alveolar macrophages were exposed in vitro to quartz dusts, metal-containing dusts or silica particles coated with a single metal oxide. The release of reactive oxygen intermediates (ROI) was measured in short-term incubations (90 min). The secretion of both ROI was markedly enhanced by silica particles coated with vanadium oxide and lowered by copper oxide-coated particles. The particle-induced ROI release was significantly decreased by the inhibition of protein kinase C (PKC) as well as phospholipase A2, suggesting the involvement of both enzymes in the NADPH oxidase activation. Quartz dusts induced a transient increase of free cytosolic calcium ion concentration, slight intracellular acidification, and depolarization of the plasma membrane. In the presence of EGTA or verapamil the rise of [Ca2+]i was diminished, suggesting an influx of extracellular calcium ions. The PKC inhibitor GF 109203X did not inhibit the quartz-induced calcium rise, while both the cytosolic acidification and depolarization were prevented. BSA-coating of the quartz particles abolished the calcium influx as well as the decrease of pHi, and possibly hyperpolarized the plasma membrane.

Non-esterified polyunsaturated fatty acids distinctly modulate the mitochondrial and cellular ROS production in normoxia and hypoxia

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07286.x

Silica induces changes in cytosolic free calcium, cytosolic pH, and plasma membrane potential in bovine alveolar macrophages

The mineral‐dust induced activation of pulmonary phagocytes is thought to be involved in the induction of severe lung diseases. The activation of bovine alveolar macrophages (BAM) by silica was investigated by flow cytometry. Short‐term incubation (10 min) of BAM with silica gel and quartz dust particles induced increases in the cytosolic free calcium concentration ([Ca2+]i), decreases in intracellular pH (pHi), and increases in plasma membrane potential (PMP). The extent of these changes was concentration dependent, related to the type of dust and was due to Ca2+ influx from the extracellular medium. An increase in [Ca2+]i was inhibited, when extracellular Ca2+ was removed. Furthermore the calcium signal was quenched by Mn2+ and diminished by the calcium channel blocker verapamil. The protein kinase C specific inhibitor bisindolylmaleimide II (GF 109203 X) did not inhibit the silica‐induced [Ca2+]i rise. In contrast, silica‐induced cytosolic acidification and depolarization were inhibited by GF 109203 X but not by removal of extracellular calcium. Addition of TiO2 particles or heavy metal‐containing dusts had no effect on any of the three parameters. Our data suggest the existence of silica‐activated transmembrane ion exchange mechanisms in BAM, which might be involved in the specific cytotoxicity of silica by Ca2+‐dependent and independent pathways.

Activation of ion-conducting pathways in the inner mitochondrial membrane – an unrecognized activity of fatty acid?

The effect of non‐esterified myristate (C14:0) or dodecyl sulfate was studied on passive swelling of rat liver mitochondria suspended in hypotonic alkaline KCl medium in the absence of the potassium ionophore valinomycin. Both compounds rapidly initiated large‐amplitude swelling. However, they failed to initiate swelling when the mitochondria were suspended in hypotonic alkaline sucrose medium. In contrast to myristate or dodecyl sulfate, the non‐ionic detergent Triton X‐100 initiated swelling of mitochondria in both of the media. The following findings indicate that the inner mitochondrial membrane (IMM) is permeabilized by myristate to K+ and Cl− in a specific manner. (i) Swelling initiated by myristate did not respond to cyclosporin A, (ii) the protonophoric uncoupler FCCP was unable to mimic the myristate effect on swelling, and (iii) myristate‐induced Cl−‐permeation (measured with KCl medium plus valinomycin) was inhibited by N,N′‐dicyclohexylcarbodiimide, quinine or ATP. Myristate‐ or dodecyl sulfate‐initiated swelling was paralleled by the lowering of endogenous Mg2+ content. Both effects, stimulation of swelling and depletion of endogenous Mg2+ are correlated with each other. Similar effects have been reported previously for the carboxylic divalent cation ionophore calcimycin (A23187). The A23187‐induced swelling has identical inhibiting characteristics on Cl−‐permeation with respect to N,N′‐dicyclohexylcarbodiimide, quinine and ATP as the myristate‐stimulated swelling. Therefore, we conclude that non‐esterified fatty acids increase the permeability of mitochondria to K+ and Cl− at alkaline pH by activating Mg2+‐dependent ion‐conducting pathways in IMM.

Rapid screening of possible cytotoxic effects of particulate air pollutants by measurement of changes in cytoplasmic free calcium, cytosolic pH, and plasma membrane potential in alveolar macrophages by flow cytometry

BACKGROUND Inhalable particulate dusts are involved in the genesis of several lung diseases. Besides the well-known toxic dusts, i.e., asbestos and quartz, heavy metal-containing pollutants are considered as possible harmful substances. In the present study, we compared the effect of silica chemically coated with certain metal oxides and dusts from industrial productions on cell physiological parameters of bovine alveolar macrophages (BAM). METHODS The cytosolic free calcium concentration, [Ca2+](i), the intracellular pH (pH(i)), and the plasma membrane potential (MP) of BAM were measured by flow cytometry. The dust-induced secretion of reactive oxygen species (ROS) was measured enzymatically. RESULTS Compared with control incubations with pure silica, the dust-induced secretion of ROS by BAM was not affected when the particles were coated with Cr(2)O(3), NiO, and Fe(3)O(4), whereas VO(2)-coated dust induced a marked increase in ROS release. This effect was not correlated to changes in [Ca2+](i), pH(i), or MP. On the other hand, Cr(2)O(3)-coated silica caused alterations in all of the three latter parameters. The same pattern of changes has been reported previously for quartz dusts (Tárnok et al.: Anal Cell Pathol 15:61-72, 1997). CONCLUSIONS We conclude that cell physiological measurements by flow cytometry could extend the palette of tools to evaluate possible toxic effects of environmental dust samples.