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N Suttorp

Publications and source records attributed to N Suttorp.

At least 19 recordsLinked to original sources
PubMed1992

Stimulation of PAF-synthesis in pulmonary artery endothelial cells by Staphylococcus aureus alpha-toxin.

Staphylococcus aureus alpha-toxin is a pore-forming exotoxin that probably represents a significant virulence factor in staphylococcal infections. Previous studies demonstrated that this agent stimulated arachidonate metabolism with subsequent formation of prostacyclin in endothelial cells and leukotriene B4 in granulocytes. We now examined the effect of alpha-toxin on the synthesis of platelet-activating factor (PAF) in cultured porcine pulmonary artery endothelial cells. PAF was labeled by bioincorporation of tritiated acetate and separated/quantitated using thin-layer chromatography, straight-phase-HPLC, or post-HPLC-bioassay. Alpha-toxin induced synthesis of small amounts of PAF in a time- and dose-dependent manner. The maximal amount of PAF elicited by alpha-toxin was approximately 7% of that observed after application of the ionophore A23187. Staphylococcal alpha-toxin but not the ionophore induced a rapid fall in cellular ATP-content. On kinetic grounds, the decrease in ATP-levels did not explain the differences in stimulated PAF-synthesis. Low amounts of PAF induced by the action of alpha-toxin on endothelial cells may contribute to the development of inflammatory lesions in infectious disease.

Adenosine Triphosphate↗
PubMed1991

Glutathione redox cycle is an important defense system of endothelial cells against chronic hyperoxia.

Exposure of cultured pulmonary artery endothelial cells to 95% O2 resulted in the following sequence of events: decrease in [3H]thymidine incorporation after 24 h; increase of intracellular glutathione (GSH) and loss of cellular protein after 48 h; increase of spontaneous and decrease of provoked prostacyclin formation as well as increased release of cellular LDH after 72 h. This oxygen toxicity model was used to study the following 2 questions. (1) What is the relative importance of the GSH redox cycle compared to catalase as antioxidative defense against hyperoxia? Endothelial cells were grown in selenium-depleted medium to inhibit glutathione peroxidase activity. Endothelial GSH biosynthesis was inhibited by buthionine sulfoximine. Catalase activity was reduced by aminotriazole. Endothelial cells with an impaired GSH redox cycle were easily killed by hyperoxia within 24 h, while inhibition of catalase did not enhance the susceptibility of endothelial cells to hyperoxia. (2) Can endothelial GSH content be increased by exogenous sulfhydryl reagents and does this result in an increase of endothelial cells' resistance to hyperoxia? Exogenous GSH, N-acetylcysteine, cysteine, and L-2-oxothiazolidine-4-carboxylate (L-2-oxo) increased intracellular GSH. All sulfhydryl reagents (with the exception of L-2-oxo) protected endothelial cells from hyperoxia. Concentrations of exogenous GSH and N-acetylcysteine that did not increase intracellular GSH reduced hyperoxia-induced endothelial cell injury. Thus the capacity of the GSH redox cycle rather than intracellular GSH levels or catalase determines endothelial cells' resistance to hyperoxia.

Animals↗
PubMed1991

Adenosine diphosphate-ribosylation of G-actin by botulinum C2 toxin increases endothelial permeability in vitro.

The endothelial cytoskeleton is believed to play an important role in the regulation of endothelial permeability. We used botulinum C2 toxin to perturb cellular actin and determined its effect on the permeability of endothelial cell monolayers derived from porcine pulmonary arteries. The substrate for botulinum C2 toxin is nonmuscle monomeric actin which becomes ADP-ribosylated. This modified actin cannot participate in actin polymerization and, in addition, acts as a capping protein. Exposure of endothelial cell monolayers to botulinum C2 toxin resulted in a dose- (3-100 ng/ml) and time-dependent (30-120 min) increase in the hydraulic conductivity and decrease in the selectivity of the cell monolayers. The effects of C2 toxin were accompanied by a time- and dose-dependent increase in ADP-ribosylatin of G-actin. G-Actin content increased and F-actin content decreased time- and dose-dependently in C2 toxin-treated endothelial cells. Phalloidin which stabilizes filamentous actin prevented the effects of botulinum C2 toxin on endothelial permeability. Botulinum C2 toxin induced interendothelial gaps. The effects occurred in the absence of overt cell damage and were not reversible within 2 h. The data suggest that the endothelial microfilament system is important for the regulation of endothelial permeability.

Actins↗
PubMed1991

Escherichia coli hemolysin is a potent inductor of phosphoinositide hydrolysis and related metabolic responses in human neutrophils.

Escherichia coli hemolysin (Hly) is a proteinaceous pore-forming exotoxin that probably represents a significant virulence factor in E. coli infections. We investigated its influence on human polymorphonuclear neutrophils (PMN), previously identified as highly susceptible targets. Hly provoked rapid secretion of elastase and myeloperoxidase, generation of superoxide, and synthesis of platelet-activating factor (PAF) and lyso-PAF. Concomitantly, marked phosphatidylinositol (PtdIns) hydrolysis with sequential appearance of the inositol-phosphates, inositol-phosphates, inositol triphosphate, diphosphate, and monophosphate, respectively, and formation of diacylglycerol, occurred. The metabolic responses displayed distinct bell-shaped dose dependencies, with maximum events noted at low toxin concentrations of 0.1-0.5 hemolytic units per milliliter. PtdIns hydrolysis and metabolic responses elicited by Hly exceeded those evoked by optimal concentrations of formylmethionyl-leucyl phenylalanine, PAF, leukotriene B4, A23187, or staphylococcal alpha-toxin. The toxin-induced effects were sensitive toward modulators of PMN stimulus transmission pathways (pertussis toxin, the kinase C inhibitor H7, and phorbol myristate acetate "priming"). We conclude that the marked capacity of low doses of Hly to elicit degranulation, respiratory burst, and lipid mediator generation in human PMN probably envolves signal transduction via PtdIns hydrolysis.

Bacterial Proteins↗
PubMed1991

Inhibition of cytoskeletal rearrangement by botulinum C2 toxin amplifies ligand-evoked lipid mediator generation in human neutrophils.

Botulinum C2 toxin, a binary toxin that ADP-ribosylates nonmuscle G-actin, was used as a selective tool to evaluate the role of actin-dependent cytoskeletal rearrangement in ligand-evoked lipid mediator generation. Human neutrophils (PMN) were preincubated with varying concentrations of the toxin for 30 min. Lipoxygenase products of arachidonic acid were measured by chromatographic techniques in the presence of exogenous arachidonic acid to probe PMN 5-lipoxygenase activity. Formation of platelet-activating factor (PAF) was assayed by the bioincorporation of [3H]acetate. Stimulation was performed with the soluble chemotactic ligands formyl-methionyl-leucyl-phenylalanine (FMLP) and PAF, as well as opsonized zymosan. PMN pretreatment with C2 toxin in the range between 200/400 and 800/1600 ng/ml C2I/II caused a dose-dependent suppression of the basal F-actin content and of stimulus-induced actin assembly. Phosphoinositide hydrolysis (measured as liberated inositol phosphates) and PAF generation in response to FMLP and exogenous PAF were markedly increased at these toxin doses. Minor C2 toxin concentrations (range, approximately 25/50 to 200/400 ng/ml C2I/II) were sufficient to amplify stimulus-induced formation of leukotriene B4 and its omega-oxidation products, nonenzymatic hydrolysis products of leukotriene A4, and 5-hydroxyeicosatetraenoic acid (5-HETE). With increasing toxin doses, leukotriene generation declined and 5-HETE became the predominant metabolite. In contrast to the soluble ligands, the zymosan-effected generation of PAF and leukotrienes was dose-dependently inhibited by C2 toxin concentrations of greater than 200/400 ng/ml, paralleled by a loss of motile and phagocytotic functions in these cells. We conclude that selective inhibition of actin assembly amplifies PAF and 5-lipoxygenase product formation in response to soluble chemoattractants with distinct dose dependences. The augmentation of PAF generation may be linked to amplified second messenger levels at higher doses of C2 toxin, whereas the sensitivity of the 5-lipoxygenase metabolism to low concentrations may indicate toxin effect on a small, functionally specified, actin pool. The present data support an important role of cytoskeletal rearrangement in temporal and/or spatial limitation of chemoattractant-evoked PMN activation.

Arachidonate 5-Lipoxygenase↗
PubMed1990

Role of actin and myosin in the control of paracellular permeability in pig, rat and human vascular endothelium.

1. We have investigated the endothelial actomyosin system with particular emphasis on its possible role in actively opening a paracellular route for permeability. 2. Actin and myosin comprised 16% of total endothelial protein with a molar actin/myosin ratio of 16.2 which is close to the actin/myosin ratio of muscle (studies on freshly isolated pig pulmonary arterial endothelial cells, PAEC). 3. By immunocytochemistry at the light and electron microscope levels the bulk of actin and myosin was colocalized in close vicinity to the intercellular clefts of both micro- and macrovascular endothelial cells in situ and in vitro. 4. Calcium-ionophore-induced rise in permeability of human umbilical venous endothelial cells (HUVEC) and PAEC monolayers grown on filters in a two-chamber permeability system was caused by opening of intercellular gaps. Gap formation depended on the rise in intracellular Ca2+ and could be blocked by the calmodulin-binding drugs trifluperazine (TFP) and W7. 5. In skinned monolayers of cultured PAEC and in isolated sheets of HUVEC gap formation was shown to require ATP and occurred only when free myosin binding sites were available on endothelial actin filaments (experiments with myosin subfragment 1 modified by N-ethylmaleimide, S1-NEM). 6. These experiments suggest that actin and myosin in endothelial cells play a central role in regulating the width of the intercellular clefts, thereby controlling the paracellular pathway of vascular permeability.

Actins↗
PubMed1990

Effects of Escherichia coli hemolysin on endothelial cell function.

Escherichia coli hemolysin is considered an important virulence factor in extraintestinal E. coli infections. The present study demonstrates that cultured pulmonary artery endothelial cells are susceptible to attack by low concentrations of E. coli hemolysin (greater than or equal to 0.05 hemolytic units/ml; greater than or equal to 5 ng/ml). Sublytic amounts of hemolysin increased the permeability of endothelial cell monolayers in a time- and dose-dependent manner. The hydraulic conductivity increased approximately 30-fold and the reflection coefficient for large molecules dropped from 0.71 to less than 0.05, indicating a toxin-induced loss of endothelial barrier function. The alterations of endothelial monolayer permeability were accompanied by cell retraction and interendothelial gap formation. In addition, E. coli hemolysin stimulated prostacyclin synthesis in endothelial cells. This effect was strictly dependent on the presence of extracellular Ca2+ but not of Mg2+. An enhanced passive influx of 45Ca2+ and 3H-sucrose but not of tritiated inulin and dextran was noted in toxin-treated cells, indicating that small transmembrane pores comparable to those detected in rabbit erythrocytes had been generated in endothelial cell membranes. These pores may act as nonphysiologic Ca2+ gates, thereby initiating different Ca2+-dependent cellular processes. We conclude that endothelial cells are highly susceptible to E. coli hemolysin and that two major endothelial cell functions are altered by very low concentrations of hemolysin.

Animals↗
PubMed1990

Staphylococcal alpha-toxin-induced vascular leakage in isolated perfused rabbit lungs.

alpha-Toxin, the major, pore-forming exotoxin of Staphylococcus aureus, caused acute hypertension when perfused through blood-free rabbit lungs (21). This reaction is mediated by pulmonary thromboxane generation, for which toxin-induced calcium flux into target cells with subsequent stimulation of arachidonic acid metabolism is predominantly responsible. In the present study, we investigated the effects of alpha-toxin on the integrity of the lung microvasculature. Thromboxane generation was inhibited in all experiments to suppress the development of pulmonary hypertension. Application of low alpha-toxin concentrations (5 to 40 ng/ml) induced protracted, severe vascular leakage in a dose-dependent manner. After a lag period of 40 to 120 minutes, gravimetrically determined capillary filtration coefficients progressively increased to greater than 10-fold values, and this was followed by pronounced weight gain of the isolated organs. These physiologic alterations were paralleled by dose- and time-dependent structural changes documented by electron microscopic examination of perfusion-fixed lungs. Increasing electron density of microvascular endothelial cell nuclei and subsequently of their cytoplasma was noted, followed by detachment of these cells from the mutual endoepithelial basal lamina. Edema was localized in the blood-gas exchange area, in contrast to hydrostatically induced lung fluid accumulation. These results identify pulmonary microvascular endothelium cells as highly susceptible targets for attack by alpha-toxin. Given a similar sensitivity of human endothelial cells, alpha-toxin might directly contribute to the pathogenesis of acute respiratory failure under conditions of severe infection with Staphylococcus aureus.

Animals↗
PubMed1989

Pharmacological and biochemical studies of cytotoxicity of Clostridium novyi type A alpha-toxin.

The actions of apparently homogeneous alpha-toxin from Clostridium novyi type A were studied in order to develop an in vitro system which closely mimics its in vivo effects and to search for the mode of poisoning. Time to death (by intravenous injection of mice) was inversely related to dose, with a detection limit of about 200 ng/kg of body weight at 100 h. Injections of 2.5 ng or more into the rat paw led to a slowly (maximum after about 30 h) developing, dose-dependent edema which was useful as a quantitative in vivo assay based on volumetry. Vascular leakage was due to gap formation between endothelial cells. Similarly, endothelial cells cultured from pig pulmonary artery lost their "cobblestone" arrangement after a dose-dependent lag period of some hours after poisoning. The morphological changes were accompanied by depression of uptake or incorporation of [3H]uridine. A quantitative in vitro assay was established on the inhibition of [3H]uridine incorporation. As in animals, the action of alpha-toxin started with a few nanograms per milliliter and proceeded slowly for at least 1 day but became resistant to antitoxin within 2 h of exposure. The toxin action is not limited to endothelial cells, since chicken embryonic cells, a mouse fibroblast line (L-929), and a rat phaeochromocytoma line (PC-12) behaved similarly. Alpha-toxin was found to differ from other bacterial toxins investigated whose modes of action are already known.

Adenosine Diphosphate Ribose↗
PubMed1989

Thromboxane-mediated hypertension and vascular leakage evoked by low doses of Escherichia coli hemolysin in rabbit lungs.

Escherichia coli hemolysin has been implicated as a pathogenicity factor in extraintestinal E. coli infections including sepsis. In the present study the effects of intravascular administration of hemolysin were investigated in isolated blood-free perfused rabbit lungs. Low concentrations of the toxin in the perfusate (0.05-5 hemolytic units/ml, corresponding to approximately 5-500 ng/ml), caused a dose- and time-dependent release of potassium, thromboxane A2, and prostaglandin I2, but not of lactate dehydrogenase, into the recirculating medium, as well as a dose-dependent liberation of the prostanoids into the bronchoalveolar space. These events were paralleled by a dose-dependent pulmonary hypertension, and studies with different inhibitors collectively indicated that the vasoconstrictor response was mediated predominantly by pulmonary thromboxane generation. In addition, E. coli hemolysin elicited a protracted, dose-dependent increase in the lung capillary filtration coefficient, which was independent of the prostanoid-mediated pressor response and resulted in severe pulmonary edema formation. We conclude that E. coli hemolysin can elicit thromboxane-mediated pulmonary hypertension combined with severe vascular leakage in isolated lungs in the absence of circulating inflammatory cells and humoral mediator systems, mimicking the key events in the development of acute respiratory failure in states of septicemia.

Animals↗
PubMed1989

Role of Ca2+ and Mg2+ for endothelial permeability of water and albumin in vitro.

The permeability of endothelial monolayers grown on a polycarbonate filter membrane and continuously exposed to a hydrostatic pressure of 10 cm H2O was studied. The ionophores A23187 and ionomycin dose dependently (0.1-10 microM) enhanced the hydraulic conductivity of sealed endothelial cell monolayers 10 to 15-fold, at the same time the reflection coefficient of albumin dropped from 0.75 to 0.2. The effects of A23187 were dependent on extracellular Ca2+. In the absence of Ca2+, however, Mg2+ could substitute for Ca2+. Phase contrast- and scanning electron-microscopy showed that the A23187-induced effects were accompanied by gap-formation in the intercellular clefts. These gaps probably are the sites which allowed enhanced fluid exchange. Ionophore-induced effects on permeability could be modified by antagonists of calmodulin function and of arachidonate liberation and metabolism. The data suggest that alterations of endothelial Ca2+-homeostasis induce metabolic events which result in an increased permeability of an endothelial monolayer.

Albumins↗
PubMed1989

Terminal complement complex and endothelial cells.

The effects of the terminal complement sequence on two endothelial cell functions (prostacyclin (PGI2) generation and permeability of an endothelial cell monolayer) were examined in antibody-sensitized pulmonary artery endothelial cells. Whereas C5b-7 complement complexes induced on PGI2 formation and no enhanced endothelial permeability, addition of purified complement component C8 resulted in a time- and dose-dependent burst of PGI2 release and in a substantially increased permeability of an endothelial cell monolayer in the absence of overt cell damage. Formation of the complete terminal complement complex C5b-9 enhanced PGI2 release but was accompanied by cytolysis. Extracellular Ca2+ was required for C5b-8 dependent PGI2 formation. Three different blockers of physiological calcium channels failed to suppress the observed stimulatory effect. One minute after addition of C8 to endothelial cells carrying C5b-7 complexes, a six to sevenfold enhanced passive influx of 45Ca2+ into the cells was noted. An enhanced passive influx was also observed for 51CrO4(2-), 3H-aminobutyric acid, and 3H-sucrose, but not for 3H-inulin and 3H-dextran. These data together suggest that complement C5b-8 complexes may serve as Ca2+-bypass gates in endothelial cells, the ensuing influx of Ca2+ leading to subsequent activation of the arachidonic acid pathway and the actin-myosin system.

Arachidonic Acid↗
PubMed1988

Effect of staphylococcal alpha-toxin on intracellular Ca2+ in polymorphonuclear leukocytes.

Staphylococcal alpha-toxin, a channel-forming protein, stimulates leukotriene B4 formation in rabbit polymorphonuclear leukocytes (PMN) (N. Suttorp, W. Seeger, J. Zucker-Reimann, L. Roka, and S. Bhakdi, Infect. Immun. 55:104-110, 1987). The concept was advanced that transmembrane toxin pores act as Ca2+ gates allowing passive Ca2+ influx into the cell, thus initiating stimulus response coupling. A critical step in this hypothesis is the demonstration of an increase in the cytosolic free Ca2+ concentration [( Ca2+]i). [Ca2+]i and membrane-associated Ca2+ were therefore monitored in quin-2- or chlorotetracycline-loaded PMN exposed to alpha-toxin. The effects of the Ca2+ ionophore ionomycin and the chemotactic tripeptide formylmethionyl-leucylphenylalanine (fMLP) were studied in parallel. All stimuli increased [Ca2+]i in dose- and time-dependent manner. In the presence of an EDTA excess there was a decrease of [Ca2+]i due to an efflux of Ca2+ in alpha-toxin- and ionomycin-treated cells, while addition of fMLP still induced an increase of [Ca2+]i. In the presence of verapamil, a Ca2+ channel blocker, [Ca2+]i was reduced after stimulation with fMLP but not with alpha-toxin or ionomycin. Addition of fMLP and ionomycin but not of alpha-toxin to PMN resulted in a rapid and substantial mobilization of membrane-associated Ca2+. The collective data demonstrate that exposure of PMN to staphylococcal alpha-toxin results in an increase in [Ca2+]i which is due to an influx of extracellular Ca2+ and not to a mobilization of intracellularly stored Ca2+. The concept of initiating stimulus response coupling by Ca2+ influx through transmembrane pores may be generally applicable to other channel-forming cytolysins.

Animals↗