Modification of phospholipids in erythrocyte membranes by phospholipase D. A fluorescence and ESR spectroscopic study.

The conversion of more than 65% of the phospholipids in human erythrocyte membranes to phosphatidyl-methanol and phosphatidic acid by incubation with phospholipase D and methanol increased the dissociation constant of the fluorescence probe ANS compared to untreated membranes, but did not affect the number of binding sites and the limiting fluorescence enhancement at maximal binding (Imax). On the contrary, the cationic fluorescence probe dansylcadaverin showed additional binding sites without a change in Kd and an increase of Imax upon incubation with phospholipase D treated erythrocyte membranes compared to incubations of membranes with the original phospholipid pattern. The characteristic temperature-dependence of the quenching of the membrane protein fluorescence by a membrane-bound nitroxide-labeled stearic acid was not influenced by the modification of the phospholipids. A slight reduction of the order parameter, S, determined by ESR-spectroscopy with the same nitroxide spin-labeled fatty acid incorporated into modified membranes compared to controls was found at 40 degrees C, but not at 25 degrees C. The results were interpreted as an indication of membrane domains that retained their physical properties and lipid composition during the incubation with phospholipase D.

Identification of phosphatidyl-1-aminopropane-2-ol in rat liver after administration of 1-aminopropane-2-ol.

Phosphatidyl-1-aminopropane-2-ol has been detected in rat liver after intraperitoneal administration of 1-aminopropane-2-ol hydrochloride. The new glycerophospholipid was chromatographically identified by comparison with chemically and enzymatically synthesized phosphatidyl-1-aminopropane-2-ol and by gas-liquid chromatography of the polar group as trifluoroacetyl derivative.

Carbamoylcholine-induced accumulation of inositol mono-, bis-, tris- and tetrakisphosphates in isolated cardiac myocytes from adult rats.

The effect of carbamoylcholine on the phosphoinositide cycle in isolated ventricular myocytes from adult rats was studied. Separation of the phosphoinositides by high-performance thin-layer chromatography showed a constant ratio of incorporation of myo-[2-3H]inositol into phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate of cultured cardiac myocytes after at least 2 h. Carbamoylcholine caused a dose-dependent and time-dependent accumulation of inositol mono-, bis- and trisphosphates, which was antagonized by atropine. Using anion-exchange HPLC the existence of inositol 1,4,5-trisphosphate, inositol 1,3,4-triphosphate and inositol 1,3,4,5-tetrakisphosphate was confirmed in rat ventricular myocytes. Inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate accumulated within 20 s, while inositol 1,3,4-trisphosphate, inositol 1,4-bisphosphate and inositol monophosphate increased within 5 min.

Identification and characterization of insulin-like growth factor receptors on adult rat cardiac myocytes: linkage to inositol 1,4,5-trisphosphate formation.

Cultured cardiac myocytes from adult Sprague-Dawley rats express both insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose 6-phosphate (IGF-II/Man6P) receptors and respond to IGF-I with a dose-dependent accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2]. Specific binding of [125I]IGF-I to isolated membranes from cultured cardiac myocytes amounted to 1-1.2%. Binding of [125I]IGF-I was inhibited by unlabeled IGF-I at nanomolar concentrations and insulin at much higher concentrations. These data suggest that IGF-I binds to its own receptor on rat cardiac myocytes. Competitive binding studies using isolated membranes from cardiac myocytes and [125I]IGF-II showed 2-4% specific binding. Binding of [125I]IGF-II was inhibited by IGF-II and much less potently by IGF-I and insulin. Immunoglobulin G (IgG) 3637 (an IgG directed against the IGF-II/Man6P receptor) partially inhibited binding of [125I]IGF-II whereas nonimmune IgG did not. Affinity cross-linking studies with [125I]IGF-II and cardiac myocyte membranes and subsequent analysis of the ligand-receptor complex using SDS-PAGE and autoradiography showed a radiolabeled band of approximately 250 kilodalton (kDa). The formation of the [125I]IGF-II-receptor complex was inhibited by incubation with IGF-II and IgG 3637 but not by insulin or nonimmune IgG. Western blotting of protein extracts from cultured cardiac myocytes was performed using IgG 3637 and an immunoperoxidase technique for the visualization of the IGF-II/Man6P receptor protein. A specific band at 220 kDa under nonreducing conditions was detected on the blots, providing further evidence for the expression of the IGF-II/Man6P receptor by cardiac myocytes. The effect of IGFs on the accumulation of inositol phosphates was measured by HPLC analysis of perchloric acid extracts from myo-[3H]inositol-labeled cultured cardiac myocytes. IGF-I (50 ng/ml) stimulated the accumulation both of Ins(1,4,5)P3 and Ins(1,4)P2 after 30 sec by 43% and 63%. IGF-II (up to 500 ng/ml) had no significant effect on inositol phosphate accumulation under the same conditions. However, in the presence of millimolar concentrations of Man6P, IGF-II (500 ng/ml) also increased Ins(1,4,5)P3 accumulation by 59%. We conclude that cardiac myocytes from adult rats express IGF receptors and respond to IGFs with the accumulation of Ins(1,4,5)P3 and Ins(1,4)P2. This effect seems to be mediated by an IGF-I receptor-specific pathway.

Metabolism of ether lysophospholipids in Leishmania donovani promastigotes.

Ether lysophospholipids, 1-O-[1'-14C]octadec-1'-enyl-sn-glycero-3-phosphoethanolamine (A) and -phosphocholine (B) as well as 1-O-[1'-14C]octadecyl-sn-glycero-3-phosphoethanolamine (C) and -phosphocholine (D) were taken up rapidly and metabolized extensively in Leishmania donovani promastigotes. Degradation to neutral lipids occurred first, followed by incorporation into phospholipids. Incubation of the cells with (A) or (B) revealed the stability of the O-[1-14C]octadec-1-enyl group up to 15 h, indicating the absence of any O-alk-1-enyl cleavage enzymes. Most of the radioactivity was found in 1-O-alkenyl-2-acyl-glycerophosphoethanolamine and 1-O-alkenyl-2,3-diacylglycerol. 1-O-Alkenyl-2-acyl-glycerophosphocholine was detected only after incubation with substrate (B). In contrast to the O-alk-1-enyl residue, the O-[1-14C]octadecyl moiety in substrate (C) and (D) could be converted into the O-[1-14C]octadec-1-enyl moiety or cleaved, yielding labelled acyl groups. Following 5 h incubation with substrate (C), most of the incorporated radioactivity was associated with 1-O-[1'-14C]octadec-1'-enyl-2-acyl-glycerophosphoethanolamine, 1-O-[1'-14C]octadecyl-2,3-diacylglycerol and 1-O-[1'-14C]octadecyl-2-acyl-glycerophosphoinositol. After 15 h minor amounts of label appeared in diacyl glycerophosphocholine. Similar labelling patterns were obtained with the choline analogue (D), except that 1-O-[1'-14C]octadecyl-2-acyl-glycerophosphocholine was found additionally. Incubations of the four labelled ether lysophospholipids with cell homogenates showed the presence of a lysophospholipase D and a phosphohydrolase. There was no specificity towards different ether residues or phosphobase moieties. Formation of alkyl- and alkenylglycerol, respectively, was stimulated by Mg2+ ions and the phosphohydrolase was inhibited by NaF. The results support the conclusion that the specific pattern of ether phospholipids in L. donovani cells is due to a pronounced specificity of the biosynthetic enzymes. Enzymes of the catabolic reactions are of low specificity or absent, such as plasmalogenases.

Cytotoxicity of ester and ether lysophospholipids on Leishmania donovani promastigotes.

The cytotoxic activity of four ester lysophospholipids, three ether lysophospholipids, and two radylglycerols on Leishmania donovani promastigotes was determined by measuring the inhibition of cell growth. The 1-acyl lysophospholipids reduced cell growth to 50% of controls at concentrations of 6.4-10.9 microM. In contrast, 1-O-alkenyl-sn-glycero-3-phosphoethanolamine, 1-O-hexadecyl-sn-glycero-3-phosphocholine, and 1-O-hexadecyl-sn-glycerol already showed a 50% inhibition of growth at concentrations between 2.1 and 2.8 microM. Moreover, the unnatural alkyl lysophospholipid analogue 1-O-octadecyl-2-methoxy-sn-glycero-3-phosphocholine was even 10-fold more toxic. Incubations of L. donovani promastigotes with radioactively labelled ether lysophospholipids revealed a rapid uptake of these compounds and their incorporation into cellular lipids at a non-toxic concentration of 1.0 microM. An accumulation of the lysophospholipids in the cell due to insufficient metabolism may be the cause of its cytotoxic effect. The sensitivity of L. donovani cells towards ether lysophospholipids was found to be similar to that reported for tumor cells.

Metabolism of 1-0-[1'-14C]octadecyl-sn-glycerol in Leishmania donovani promastigotes. Ether lipid synthesis and degradation of the ether bond.

A long-chain 0-alkylglycerol, 1-0-[1'-14C]octadecyl-sn-glycerol, was taken up and metabolized extensively in Leishmania donovani promastigotes grown on a lipid-free, semi-defined medium as well as on a lipid-free, synthetic medium in nearly identical ways. Cleavage of the ether bond was the main event even after short incubation times (1 h) and low precursor concentration (2 microM), as judged from the appearance of radioactive acyl moieties residing mainly in phosphatidylcholine. In addition, labeled plasmanyl inositol and plasmenyl ethanolamine were formed to a considerable extent. However, when administered in higher concentrations (25 microM), 1-0-alkyl-sn-glycerols as well as their enantiomers inhibited lipid metabolism dramatically, accompanied by rounding of the cells and appearance of cellular debris in the medium. This effect was not dependent on the aliphatic side chain (12:0, 16:0, 18:0, 18:1) of the 0-alkylglycerols. It is suggested that, by incorporation into cellular membranes, 0-alklglycerols affect factors responsible for the interaction of membranes and the cytoskeleton as well as enzymes of lipid metabolism.

Ether lipid synthesis from enantiomeric medium-chain and long-chain O-alkyl-sn-glycerols in Leishmania donovani promastigotes.

A medium-chain O-alkylglycerol, 1-O-[1'-14C]dodecyl-sn-glycerol, has been found to be incorporated into plasmenyl ethanolamine by Leishmania donovani promastigotes as revealed by radio gas-liquid chromatography; however, the ether bond of the administered O-alkyl-glycerol was cleaved extensively as judged from the occurrence of radioactive acyl moieties. The labelling pattern produced by the radioactive 'natural' 1-O-octadecyl-sn-glycerol was similar though the latter served as a slightly better substrate for plasmalogens. Experiments with the enantiomeric 3-O-alkyl-sn-glycerols in comparison revealed that these were poor substrates for plasmalogen synthesis, although they were taken up in identical amounts and cleaved even to a higher extent. Therefore, we conclude the 1-0-alkyl-sn-glycerols were utilized directly for plasmenyl ethanolamine synthesis. The incorporation of the dodecyl residue into plasmenyl ethanolamine did not affect the multiplication and shape of cells.

Cytotoxicity of dust constituents towards alveolar macrophages: interactions of heavy metal compounds.

The interactions between different heavy metal compounds which affect their cytotoxicity towards rabbit alveolar macrophages were investigated. The cells were exposed in vitro to combinations of As3+, Cd2+, Hg2+, Ni2+, or V5+ with different concentrations of another heavy metal compound. Toxicity was determined as the depression of zymosan-induced release of superoxide anion radicals. Significant antagonisms occurred in the combinations Cd2+/Zn2+, Hg2+/As3+, and Hg2+/Se4+, while significant synergisms were exhibited by the combinations Cd2+/Cu2+, Cd2+/Sn2+, Hg2+/Cu2+, Ni2+/Cd2+, Ni2+/Cu2+, Ni2+/Sn2+ and V5+/Cu2+. In the combinations As3+/Zn2+, Hg2+/Cd2+ and Hg2+/Zn2+, both kinds of interactions were observed depending on the concentrations of the heavy metal compounds. An interpretation of the measured heavy metal interactions with reference to the toxicity of heavy metal-containing dusts is attempted.

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).

Synthesis of a fluorescent analogue of phosphatidylcholine.

A fluorescent analogue of phosphatidylcholine was synthesized by acylation of 1-oleoyl-sn-glycero-3-phosphocholine with 6-N-(tert-butyloxycarbonyl)aminocaproic acid anhydride employing the catalyst 4-pyrrolidinopyridine. Removal of the protective group by treatment with HCl in chloroform was followed by subsequent reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) to form the fluorescent analogue of phosphatidylcholine, 1-oleoyl-2-(NBD)aminocaproyl-sn-glycero-3-phosphocholine, in good yield and with high isomeric purity.

Biosynthetic preparation of radioactively labelled ethanolamine plasmalogen (1-O-[1'-14C]octadec-1'-enyl-2-acyl-sn-glycero-3-phosphoethanola mine) using a protozoan cell culture.

Ethanolamine plasmalogen radiolabelled mainly in the O-alkenyl moiety was prepared from cell suspension cultures of the flagellate Leishmania donovani previously incubated with [1-14C]octadecanol over one growth period. The optimal concentration of [1-14C]octadecanol for labelling was shown to be 1 microM, when 60% of total lipid radioactivity appeared in the 1,2-diradyl-sn-glycero-3-phosphoethanolamine fraction, with an overall yield of approx. 35%. Analysis of this fraction revealed that 93% of the label was present in O-octa-dec-1-enyl, 3% in O-alkyl and 4% in acyl moieties. A specific radioactivity of approx. 14 mCi/mmol was determined. Raising the culture medium concentration of [1-14C]octadecanol to 2 microM yielded a product with a specific radioactivity of 25 mCi/mmol.

Conversion of radiolabelled ethanolamine plasmalogen into the dimethylethanolamine and choline analogue via transphosphatidylation by phospholipase D from cabbage.

Starting from biosynthetically prepared ethanolamine plasmalogen 14C-labelled in the O-alkenyl moiety, choline and dimethylethanolamine plasmalogen were prepared by transphosphatidylation utilizing phospholipase D from cabbage. Investigation of the time course of the reaction showed that transphosphatidylation was simultaneously accompanied by hydrolysis of both the substrate and the desired product, resulting in a maximum of product yield after 1-3 h under the reaction conditions investigated. Optimal reaction conditions gave yields of 40% and 62% (of total radioactivity) respectively for the purified choline and dimethylethanolamine derivatives.

Measurement of phospholipase A2 and 1-alkylglycerophosphocholine acetyltransferase activities in stimulated alveolar macrophages by HPLC analysis of NBD-labeled ether lipids.

The importance of phospholipases in cellular signaling and 1-alkylglycerophosphocholine acetyltransferase in the formation of platelet-activating factor (PAF) has stimulated demand for methods to measure these enzyme activities in inflammatory cells. Most of the assays currently used rely on radiolabeled substrates. We have synthesized NBD-labeled ether lipids as substrates for measuring enzyme activities of the PAF cycle and of lysosomal phospholipase A2 (PLA2). The fluorescent lipids were incubated with homogenates of stimulated bovine alveolar macrophages. The generated products were separated from the substrates by HPLC on a normal phase and monitored with a fluorescence detector. NBD-lyso-PAF was well accepted by acetyl- and acyltransferases of the cell-free preparations, which metabolized the substrate into NBD-PAF and NBD-alkyl-acylglycerophosphocholines. Homogenates of stimulated cells showed an enhanced production of NBD-PAF. The increased formation of the biological mediator was dependent on the nature of the stimuli and the time of stimulation. Lysosomal PLA2 was measured with 1-O-(12-NBD-aminododecyl)-2-acyl-sn-glycero-3-phosphocholine as substrate. By varying the pH and the calcium concentration, it was possible to distinguish between the cytosolic PLA2 and the lysosomal PLA2 activity. Optimal conditions for the determination of the lysosomal PLA2 were obtained at pH 4.5 and in the presence of EDTA. Stimulation with particulate agonists induced an enhancement of the lysosomal PLA2 activity in macrophages.

Mechanisms of lipid peroxidation in human blood plasma: a kinetic approach.

There is strong evidence that the oxidation of plasma lipoproteins plays an important role in atherogenesis. The exact mechanisms by which lipoprotein oxidation occurs in the presence of other plasma constituents, however, remains unclear. To investigate the role of different antioxidants for this process, we studied the oxidation of human plasma supplemented in vitro with physiological amounts of major plasma antioxidants alpha-tocopherol, ubiquinol-10 ascorbate, urate, bilirubin and albumin. The plasma was diluted 2-fold and oxidized by 3.75 mM Cu(II). The concentrations of the antioxidants, fatty acids, linoleic acid hydroperoxides and oxycholesterols in oxidizing plasma were measured. The oxidation was characterized by three consecutive phases similar to the known lag, propagation, and decomposition phases of low density lipoprotein oxidation. The rate of the initiation of oxidation as calculated from antioxidant consumption rates was raised by supplementation with alpha-tocopherol or ascorbate. The oxidation rate in the lag phase was lowered by supplementation with any of the antioxidants, whereas in the propagation phase the oxidation rate was slightly higher in supplemented than in unsupplemented plasma. The kinetic chain length in the lag phase was less than one in supplemented plasma and about one in unsupplemented plasma. The chain length in the propagation phase was between three and six for all plasma samples. A higher rate of urate consumption and a reduced rate of alpha-tocopherol consumption were found in plasma supplemented with ascorbate in comparison with unsupplemented plasma. These data suggest that: (i) the reduction of Cu(II) by alpha-tocopherol and ascorbate is a major initiating event in Cu(II)-catalyzed oxidation of human plasma; (ii) the following lag phase is caused by radical-scavenging effects of all antioxidants with alpha-tocopherol as a major lipophilic and urate as a major hydrophilic scavenger; (iii) interactions between antioxidants, such as regeneration of ascorbate by urate and of alpha-tocopherol by ascorbate, take place during the lag phase; (iv) in the absence of added antioxidants the oxidation in the lag phase can occur via a chain reaction; and (v) in the propagation phase the oxidation is not inhibited by antioxidants and occurs autocatalytically.

Comparative studies of induction and repair of DNA double-strand breaks in X-irradiated alveolar macrophages and resting peripheral blood lymphocytes using constant-field gel electrophoresis.

Induction and repair of X-ray-induced DNA double-strand breaks (dsbs) was compared for normal broncho-alveolar macrophages and human peripheral blood lymphocytes, using CHO cells as a reference cell model. The cells, upon their separation, were processed in a similar manner. After X-irradiation, cell lysis and proteinase K treatment, the DNA samples were subjected to constant-field gel electrophoresis (CFGE) followed by fluorimetric densitometry for quantification of released DNA. Induction of dsbs after X-ray doses of 5-100 Gy was found to show no gross differences for all cell systems used. Repair of dsbs was studied after X-ray dose of 60 Gy for up to 24 h after irradiation. The repair curves obtained proved to be similar for bronchoalveolar macrophages and CHO cells (97% of all dsbs rejoined after 24 h). However, in blood lymphocytes from normal subjects and from bone marrow recipients, dsb repair proceeded rapidly only for 0.5-1 h post-irradiation, being followed by the gradual degradation of DNA at longer intervals. The kinetics of DNA degradation correlated with cytological features of pyknosis and necrosis.

Protein kinase C activity and phosphoprotein pattern in stimulated alveolar macrophages.

Bovine alveolar macrophages (BAM) were stimulated with quartz dusts, metal oxide-coated silica particles, and zymosan. To investigate the role of protein kinase C (PKC) in the mechanism of agonist-induced activation 12-O-tetradecanoyl phorbol 13-acetate (TPA), staurosporine, and the PKC specific inhibitor GF 109203X were applied. PKC activity was determined by means of a continuous fluorescence assay [1]. The assay is based on the measurement of fluorescence decrease caused by phosphorylation of an acrylodan-labelled MARCKS peptide, a specific substrate of PKC. The PKC fluorescence assay was verified with the purified enzyme, but it could not be adapted to cytosolic and membrane homogenates of BAM, as it is sensitive to the activity of proteases. PKC-mediated protein phosphorylation in intact BAM was achieved by mapping the [32P]phosphoproteins with an optimized horizontal 2D electrophoresis technique with subsequent autoradiography and image analysis. Agonist- and time-dependent changes of phosphoprotein patterns in BAM were detected and analysed.

Protein phosphorylation in alveolar macrophages after stimulation with heavy metal-coated silica particles.

The aim of our study was to determine agonist- and time-dependent phosphorylation of distinct proteins in bovine alveolar macrophages after activation with quartz dusts and heavy metal-coated silica particles. Silica particles were coated with vanadium oxide or cadmium oxide. Separation of proteins was achieved by one-dimensional and optimized horizontal two-dimensional polyacrylamide gel electrophoresis. Protein phosphorylation was either determined by [32P] labeling and subsequent autoradiography or Western blotting using monoclonal antibodies against phosphotyrosine. One of the phosphorylated proteins was identified as the heat shock protein HSP-27. Stimulation with cadmium oxide-coated silica particles and PMA led to a significant increase of phosphorylated HSP-27. Additionally cadmium oxide-coated silica particles induced tyrosine phosphorylation of six main proteins. Stimulation with vanadium oxide-coated silica particles drastically increased the amount of tyrosine phosphorylated proteins while this effect was not observed with uncoated silica particles.

Agonist-stimulated alveolar macrophages: apoptosis and phospholipid signaling.

Bovine alveolar macrophages (BAM) were labeled with [3H]-choline or [3H]-ethanolamine and exposed to quartz dust, metal oxide-coated silica particles, Escherichia coli-derived lipopolysaccharide (LPS) or tumor promotor 12-O-tetradecanoyl phorbol 13-acetate (PMA). The activation of phospholipases A2, C and D (PLA2, PLC and PLD) acting on phosphatidylcholine and phosphatidylethanolamine was determined by high performance liquid chromatography (HPLC) separation and liquid scintillation counting of water- and lipid-soluble phospholipid metabolites. Exposure of BAM to quartz dust, metal oxide-coated silica particles, and LPS led to a transient PLD activation while treatment with PMA caused a prolonged rise in PLD activity. LPS and quartz dust induced a short-term increase of PLC cleavage products. All agonists caused a transient activation of PLA2. To induce apoptosis, BAM were stimulated with C8-ceramide, calcium-ionophore 23187, or gliotoxin. Apoptosis was investigated by qualitative and quantitative methods like flow cytometry, propidium iodide/Hoechst 33258 double staining, Cell Death Detection ELISA, and electrophoretical detection of DNA fragmentation. All three agonists led to apoptosis of BAM in a time- and concentration-dependent manner. After stimulation with gliotoxin an increase in ceramide and a drastic decrease in sphingosine-1-phosphate levels were observed, suggesting an involvement of these sphingolipids in gliotoxin-mediated apoptosis.

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.