No abstract

Bovine aortic endothelial cells were grown on microcarrier beads and were perfused with Krebs-Ringer solution. Endothelium-derived relaxing factor (EDRF) was bioassayed on a cascade of four strips of rabbit aorta, and prostacyclin was analyzed by RIA of 6-oxo-prostaglandin F1 alpha. The endothelial cells released EDRF and prostacyclin when stimulated with bradykinin and its analogues, or with ADP, ATP, arachidonic acid, and phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3). The detection of EDRF was potentiated by superoxide dismutase, and the relaxation of rabbit aortic strips induced by EDRF was antagonized by methylene blue. The release of EDRF and prostacyclin was inhibited by phorbol myristate acetate, R59022 (a diacylglycerol kinase inhibitor), and gentamycin. We suggest that the release of EDRF and prostacyclin is coupled and the initial common step is activation of a phospholipase C.

Incubation of neutrophils or mononuclear cells with washed platelets (all prepared from human venous blood) resulted in an inhibition of thrombin-induced platelet aggregation that was dependent on the number of nucleated cells added. The inhibition was potentiated by superoxide dismutase and reversed by oxyhemoglobin. In the case of neutrophils the inhibition was associated with an increase in cGMP, whereas with mononuclear cells both cAMP and cGMP were increased. The inhibitory activity of neutrophils or mononuclear cells was prevented by their preincubation with NG_ monomethyl-L-arginine methyl ester. L-Arginine reversed the action of NG-monomethyl-L-arginine methyl ester, whereas D-arginine was ineffective. Preincubation of the cells with catalase or mannitol did not prevent their inhibitory action on platelet aggregation. The inhibition of platelet aggregation was not due to platelet damage or to uptake of thrombin by neutrophils or mononuclear cells. It was overcome by increasing the concentration of thrombin and was absent in cell-free supernatants obtained from a suspension of neutrophils or mononuclear cells or from mixtures of platelets with neutrophils or platelets with mononuclear cells. These data provide evidence for the release of a nitric oxide-like factor from human neutrophils and mononuclear cells. In addition, evidence is provided that, as in stimulated murine macrophages and endothelial cells, the precursor of this factor is L-arginine.Activated leukocytes release factors such as superoxide anions (0-), hydrogen peroxide (H202), or hydroxy acids that increase (1-4) or decrease (5, 6) platelet aggregation induced by several agonists. Harrison et al. (7) demonstrated that human neutrophils inhibited platelet aggregation in platelet-rich plasma but erythrocytes did not. Furthermore, Rimele et al. (8) demonstrated that rat peritoneal leukocytes elicited by oyster glycogen released a substance that relaxed endothelium-denuded strips of rat aorta. This "neutrophilderived relaxing factor" (NDRF) had a pharmacological profile similar to that of endothelium-derived relaxing factor (EDRF). EDRF, which is released from vascular endothelium by a variety of agonists, induces vasodilatation (9) and inhibits platelet aggregation (10-12) and platelet adhesion (13,14) MATERIALS AND METHODSMaterials. The Krebs' solution was 137 mM NaCl/2.7 mM KCI/11.9 mM NaHCO3/0.3 mM NaH2PO4/0.8 mM MgSO4/ 5.6 mM glucose/1 mM CaCl2. The cell culture medium consisted of RPMI-1640 medium supplemented with Lglutamine (250 ,ug/ml), penicillin (85 units/ml), streptomycin (85 ,g/ml), and gentamicin (85 ug/ml). Human thrombin, hemoglobin (from bovine blood), superoxide dismutase (from bovine erythrocytes), catalase, mannitol, sodium hydrosulfite, L-arginine (free base), D-arginine (free base), and indomethacin were obtained from Sigma. 3-Isobutyl-1-methylxanthine was obtained from Aldrich. Kits for radioimmunoassay of guanosine 3',5'-(cyclic) monophosphate (cGMP) and adenosine 3',5'-(cyclic) monophosphate (cAMP) used cGMP or...

No abstract