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N Suttorp

Publications and source records attributed to N Suttorp.

88 records · Page 5Linked to original sources
PubMed1987

Mechanism of leukotriene generation in polymorphonuclear leukocytes by staphylococcal alpha-toxin.

The effects of staphylococcal alpha-toxin on arachidonic acid metabolism in rabbit polymorphonuclear leukocytes (PMNs) were investigated and compared with those of the ionophore A23187 and the chemotactic tripeptide formylmethionyl-leucyl-phenylalanine (fMLP). Sublytic amounts of alpha-toxin stimulated the release of leukotriene B4 (LTB4) in PMNs in a dose-dependent manner. The toxin was several times more potent than fMLP but was not as effective as the ionophore. Preincubation of the toxin with neutralizing antibodies abolished the effect. Extracellular calcium was strictly required for eliciting LTB4 generation. Verapamil, a calcium channel blocker, inhibited fMLP-mediated LTB4 generation but had no effect on alpha-toxin- or A23187-exposed PMNs. Agents such as trifluoperazine and N-6(aminohexyl)-5-chloro-1-naphthalene sulfonamid that interfered with calmodulin activity, however, inhibited LTB4 generation in all cases. One minute after the addition of alpha-toxin, PMNs exhibited a severalfold enhancement in passive permeability to 45Ca2+. In addition, these cells became permeable to sucrose but not to inulin or dextran. The influx pattern was consistent with the previous observation that alpha-toxin creates discrete transmembrane channels in erythrocytes with an effective internal diameter of 2 to 3 nm. The results suggest that alpha-toxin triggers the arachidonic acid pathway in PMNs by facilitating calcium influx into the cells, possibly via transmembrane toxin pores that serve as calcium gates. Generation of arachidonic acid metabolites in PMNs by sublytic amounts of alpha-toxin may represent an important cellular reaction that generally occurs during infections with Staphylococcus aureus.

Animals↗
PubMed1987

Complement complex C5b-8 induces PGI2 formation in cultured endothelial cells.

The effects of the terminal complement sequence on prostacyclin (PGI2) generation in antibody-sensitized pulmonary arterial endothelial cells were examined. Whereas C5b-7 complement complexes induced no PGI2 formation, addition of purified complement component C8 resulted in a time- and dose-dependent burst of PGI2 release 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. In contrast, W7 [N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide] and trifluoperazine, inhibitors of calmodulin activity, all reduced the C5b-8-dependent PGI2 generation. None of the inhibitors used impaired Ca2+ flux into the cells. One minute after addition of C8 to endothelial cells carrying C5b-7 complexes, a six- to seven-fold enhanced passive influx of 45Ca2+ into the cells was noted. An enhanced passive influx was also observed for 51Cr O4(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.

Animals↗
PubMed1987

Endotoxin alters arachidonate metabolism in pulmonary endothelial cells.

Endotoxin and lipid A dose dependently (1 ng/ml to 10 micrograms/ml) and time dependently (6-24 h) stimulated the generation of large amounts of prostacyclin in cultured pig pulmonary artery endothelial cells. This effect occurred in the absence of cell detachment and overt cell damage. The presence of at least 1% serum was required but the activation of the complement cascade was not. Endotoxin-treated endothelial cells generated increased amounts of prostacyclin upon stimulation with A23187 and arachidonic acid. Endotoxin-induced activation of arachidonate metabolism could be reduced by 10(-10) M glucocorticoids but not by progesterone. It was further affected by inhibitors of protein and RNA synthesis and calmodulin function. In addition, exposure of endothelial cells to endotoxin resulted in an enhanced synthesis of cyclooxygenase and in a higher enzymatic capacity of prostacyclin synthase. The data indicate that endotoxin in concentrations occurring in the plasma of patients profoundly alters arachidonic acid metabolism in endothelial cells.

Animals↗
PubMed1987

Activation of the pulmonary arachidonic acid system and its consequences for hemodynamics and fluid balance.

The pulmonary vasculature and perivascular tissue is able to generate arachidonic acid metabolites with strong effects on vascular tone and permeability. Stimulators of the pulmonary arachidonic acid metabolism, which represents a highly potent mediator system, are components of the classical cascade systems, bacterial toxins, hypoxia, and a variety of other physiological and non-physiological factors. Upon excessive stimulation, the pulmonary circulation responds with vasoconstriction and extravasation. Since granulocytes are always involved, their contribution to the resulting vascular effects has to be considered.

Animals↗
PubMed1986

Noncytolytic terminal complement complexes may serve as calcium gates to elicit leukotriene B4 generation in human polymorphonuclear leukocytes.

Complement effects on human polymorphonuclear leukocytes (PMN) have generally been ascribed to the anaphylatoxin C5a, which induces degranulation, superoxide anion generation, migration, and cell aggregation via interaction with membrane receptors. We here report that complement activation on the surface of antibody-sensitized human PMN provokes generation of the potent lipid mediator leukotriene B4 (LTB4) in strict dependence on complement component C8, but in the absence of detectable C9. The kinetics of LT generation are rapid, comparable with those observed after challenge with the calcium-ionophore A23187. LTB4 release is a distinct event that is dissociable from cytotoxicity as assessed by lactate dehydrogenase (LDH) release (dependent on C9) and from superoxide generation (independent of C8 and C9). It is dose dependent on extracellular calcium and is not observed in the absence of calcium. It is inhibited by substances interfering with calcium-calmodulin function (trifluoperazine and W7), but not by blockers of physiologic calcium channels (nimodipine, verapamil, and D 888). Addition of purified C8 to cells bearing C5b-7 induces a severalfold increase in their passive permeability to 45calcium. Sieving experiments with the use of marker molecules of different sizes collectively indicate the existence of small hydrophilic channels consisting exclusively or predominantly of C5b-8 complexes, which allow passive transmembrane flux of small molecules with Mr less than 200. Thus, noncytolytic terminal complement complexes may serve as a biological bypass gate for calcium in PMN membranes, triggering the arachidonic acid cascade with generation of LTB4 at doses well below the threshold required to invoke overt cell damage.

Calcium↗
PubMed1986

Antioxidant defense mechanisms of endothelial cells: glutathione redox cycle versus catalase.

The importance of the glutathione (GSH) redox cycle and of catalase as intracellular antioxidant defense systems in cultured endothelial cells against an extracellular flux of H2O2, a critical mediator of polymorphonuclear leukocyte-induced oxidant injury of endothelial cells, was examined. The activities of different parts of the GSH redox cycle were impaired by 1,3-bis(2-chloroethyl)-1-nitrosourea, buthionine sulfoximine, diethyl maleate and 2-cyclohexene-1-one. Catalase activity was inhibited by 3-amino-1,2,4-triazole. After an impairment of the GSH redox cycle, but not of catalase, the susceptibility of pulmonary artery endothelial cells to an attack by H2O2 was dramatically increased independent of the source of extracellularly generated hydrogen peroxide (i.e., glucose oxidase or stimulated polymorphonuclear leukocytes). Exogenous catalase, d-alpha-tocopherol, and particularly Trolox, the chroman compound of tocopherol, but not phytol, the fatty acid side chain of tocopherol, provided almost complete protection of the endothelial cells against a H2O2-mediated attack. Additional fluorometric studies suggested that H2O2 is scavenged by the antioxidants before it hits the target cells.

Amitrole↗
PubMed1986

The glutathione redox cycle as a defense system against hydrogen-peroxide-induced prostanoid formation and vasoconstriction in rabbit lungs.

Leukocyte-derived oxidants have been described as causing vasoconstriction and edema formation in isolated lungs. In the present study, dose-dependent and reversible pressor responses were achieved reproducibly by injection of hydrogen peroxide (H2O2) into the pulmonary artery of blood-free, perfused, isolated rabbit lungs in a dose-dependent manner. The pressor responses were accompanied by an instantaneous release of thromboxane A2 and a more delayed but quantitatively larger release of prostaglandin I2 into the recirculating perfusion fluid. There was no release of potassium or LDH, indicating the absence of overt cell damage. The H2O2-induced pressor responses were blocked by indomethacin (cyclooxygenase inhibitor), imidazole (inhibitor of thromboxane synthetase), mepacrine (phospholipase inhibitor), and W7 and trifluoperazine (agents that interfere with calcium-calmodulin function). Treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) dose-dependently inhibited the lung glutathione reductase activity and augmented the metabolic (prostanoid release) and functional (vasoconstriction) responsiveness of the pulmonary vascular bed to H2O2. Application of 1-(2-chloroethyl)-1-nitrosourea (CCNU), a control to BCNU, and inhibition of catalase activity by aminotriazole did not increase the sensitivity to externally applied H2O2. We conclude that calcium-calmodium function and thromboxane generation may be involved in the pulmonary vasoconstrictive response to H2O2 and that the lung glutathione redox cycle is active in limiting the responsiveness of the pulmonary vascular bed to externally generated H2O2.

Animals↗
PubMed1985

Pseudomonas aeruginosa cytotoxin stimulates prostacyclin production in cultured pulmonary artery endothelial cells: membrane attack and calcium influx.

The effects of highly purified Pseudomonas aeruginosa cytotoxin were investigated on cultured pulmonary artery endothelial cells. This toxin dose-dependently (7.5-60 micrograms/ml) and time-dependently (20-75 minutes) stimulated the release of radiolabeled arachidonic acid and metabolites and the synthesis of prostacyclin in the absence of overt cell damage (no enhanced lactate dehydrogenase [LDH] release). Preincubation of the toxin with neutralizing antibodies abolished the effect. The toxin response on endothelial cells required extracellular calcium but not magnesium and was accompanied by a calcium influx. Interference with intracellular calcium function by TMB 8 or with (calcium)-calmodulin function by trifluoperazine and W7 dose-dependently reduced the cytotoxin mediated synthesis of prostacyclin. Calcium channel blockers (nimodipine, diltiazem, verapamil, D 888), however, were ineffective in this system. Following addition of cytotoxin to endothelial cells, an increased passive permeability for small marker molecules (potassium, 45calcium, 3H-sucrose), but for large ones (3H-inulin, 3H-dextran, LDH) was noted, suggesting that cytotoxin creates discrete hydrophilic transmembrane lesions of about 0.5-1.5 nm in diameter. These data are compatible with the notion that Pseudomonas aeruginosa cytotoxin triggers the arachidonic acid pathway in cultured pulmonary artery endothelial cells by calcium influx and suggest that this calcium influx may proceed through toxin created transmembrane lesions.

Animals↗
PubMed1985

Staphylococcal alpha-toxin-induced PGI2 production in endothelial cells: role of calcium.

Studies in erythrocytes indicate that staphylococcal alpha-toxin generates discrete transmembrane channels with an effective diameter of 2-3 nm. In cultured, confluent, pig pulmonary arterial endothelial cells we studied the triggering of the arachidonic acid cascade and its dependence on calcium influx, possibly through toxin-created pores. In endothelial cells alpha-toxin time dependently (5-30 min) and dose dependently (0.1-8 micrograms/ml) stimulated the release of radiolabeled arachidonic acid and prostacyclin (PGI2) production in similar amounts as the calcium ionophore A23187 (10 microM). Preincubation of alpha-toxin with neutralizing antibodies abolished the effect. The toxin response was strictly dose dependent on extracellular calcium but not on magnesium. The toxin effect was accompanied by an up to 10-fold increased passive permeability of pulmonary arterial endothelial cells for 45Ca. Interference with calcium-calmodulin function (trifluoperazine, W7) dose dependently reduced production of PGI2, but blockers of physiological calcium channels (verapamil, nimodipine, nisoldipine, and diltiazem) did not. In contrast to the effect of the ionophore A23187, the toxin effect was accompanied by a release of potassium, but in neither system was there a release of lactate dehydrogenase. In addition, alpha-toxin but not ionophore-exposed endothelial cells showed an increased passive influx of small radiolabeled markers (45Ca and [3H]sucrose) but not of large markers [( 3H]inulin and [3H]dextran). These data are consistent with the concept that alpha-toxin triggers the arachidonic acid cascade in pulmonary arterial endothelial cells by calcium influx and suggest that this calcium influx may proceed through toxin-created transmembrane channels.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Lung cell oxidant injury: decrease in oxidant mediated cytotoxicity by N-acetylcysteine.

Lung cell damage mediate by polymorphonuclear leukocyte (PMN) reactive oxygen metabolites has been suggested as a pathophysiologic mechanism in a variety of acute and chronic pulmonary disease states, while oxidant injury may be a non-specific cytotoxic mechanism. Reducing agents therefore represent one therapeutic direction for decreasing lung cell injury in several clinical circumstances. N-Acetylcysteine (NAC) is a known antioxidant which can be distributed in soluble form to multiple intrapulmonary sites. We have therefore examined a possible role for NAC against oxidant injury in a controlled in vitro model for oxygen metabolite cytotoxicity. Our data suggest that extracellular NAC is able to protect lung cells against PMN mediated oxidant injury. Pre-exposure of lung cells to NAC results in decreased susceptibility to oxidant damage by increasing intracellular antioxidant defense systems. An increase in extracellular and/or intracellular resistance to toxic oxygen metabolites by NAC may be one approach to the prevention of in vivo lung oxidant injury.

Acetylcysteine↗
PubMed1984

[Molecular basis for the pathogenicity of S. aureus alpha-toxins].

Staphylococcal alpha-toxin is produced by most strains of S. aureus and is considered a major pathogenic factor of these bacteria. The toxin is produced as a water-soluble molecule of MW 34000. Binding to a membrane target is accompanied by the formation of ring-structured hexamers with outer and inner diameters of 10 and 2-3 nm, respectively. The toxin rings carry lipid-binding surfaces that allow for insertion into and firm embedment within the membrane. Small transmembrane channels are thus generated that can induce a variety of pathological cellular changes. Large doses of toxin will generally cause cell lysis and death. However, sub-cytolytic toxin doses can also elicit major pathophysiological reactions. When introduced into the circulation of an isolated and perfused rabbit lung, the toxin causes steep rises in the pulmonary artery pressure, and lung edema results as a consequence of increases in vascular permeability occurring in parallel. These processes are the result of the activation of the arachidonic acid cascade by alpha-toxin in the lung. Studies using cultured endothelial cells as targets subsequently led to a hypothesis that would explain how membrane channel formation by a toxin could be linked to the observed arachidonic acid cascade activation. In essence, we propose that the toxin pores serve as non-physiological calcium channels, and that calcium influx triggers the observed reactions. It is probable that many other pathophysiological processes including inflammatory tissue reactions derive from such secondary effects of toxin action.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Decreased bactericidal function and impaired respiratory burst in lung macrophages after sustained in vitro hyperoxia.

Lung macrophages (LM) play a crucial role in pulmonary bacterial defense. High inspired oxygen concentrations are used in a variety of diseases and "oxygen toxicity" could impair antibacterial function. We therefore examined the effect of sustained in vitro hyperoxia on LM bactericidal function, and on generation of two bactericidal oxygen metabolites. The LM were cultivated under aerobic (PO2 approximately 140 mmHg) or hyperoxic (PO2 approximately 630 mmHg) conditions for 48 h, and then incubated with Staphylococcus aureus labeled with 3H thymidine for 30 min. Incubated monolayers were processed for measurement of total bacterial uptake and for number of viable intracellular bacteria. Superoxide anion (O2-) and hydrogen peroxide (H2O2) generation was determined in similarly cultivated cells stimulated with opsonized zymosan. The results indicate that the bacterial killing capacity of oxygen-cultivated LM is significantly decreased (p less than 0.001). In addition, a significant (p less than 0.001) decrease in generation of O2- and H2O2 was noted after exposure to high oxygen tensions. The data suggest that decreased bactericidal function after sustained hyperoxia may be due to an impairment of a specific bactericidal mechanism, i.e., an impaired "respiratory burst."

Animals↗
PubMed1982

Lung cell oxidant injury. Enhancement of polymorphonuclear leukocyte-mediated cytotoxicity in lung cells exposed to sustained in vitro hyperoxia.

The oxidant damage of lung tissue during in vivo hyperoxic exposure appears to be amplified by neutrophils that release toxic amounts of oxygen metabolites. In our studies cloned lung epithelial cells (L2 cells), lung fibroblasts, and pulmonary artery endothelial cells were cultured under either ambient (Po(2) approximately 140 torr) or hyperoxic (Po(2) approximately 630 torr) conditions for 48 h (24 h for endothelial cells). After cultivation, phorbol myristate acetate- or opsonized zymosan-stimulated neutrophils were added to the cultivated monolayers for 4 h, and lung cell damage was quantitated using (51)Cr release as an index. The data show that stimulated neutrophils are able to injure the three lung cell lines tested, with endothelial cells being highly susceptible to this injury and L2 cells being slightly more susceptible than lung fibroblasts. The studies also demonstrate that all three lung cell lines exposed to sustained hyperoxia are more susceptible to neutrophil-mediated cytotoxicity than their time-matched air controls. Hydrogen peroxide was the main toxic oxygen metabolite because catalase (2,500 U/ml) completely protected the target cells. Equivalent quantities of hydrogen peroxide generated by glucose oxidase instead of by neutrophils gave a similar degree of target cell injury. Superoxide dismutase at high concentrations (250 mug/ml) provided some protection. Other systems that detoxify oxygen metabolites were without protective effect. These findings indicate that the increase in susceptibility of lung cells to neutrophil-mediated oxidant damage is a toxic effect of hyperoxia on lung cells. This specific manifestation of oxygen damage provides insight into the integration between primary mechanisms (oxygen exposure) and secondary mechanisms (release of oxygen metabolites by neutrophils) with respect to the cellular basis for pulmonary oxygen toxicity.

Animals↗