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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...

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SUMMARY This review discusses the role of three mediators, synthesized by vascular endotheiial cells, that help to keep the surface of the normal endotheliuin nonthrombogenic. The first is prostacydin, a product of arachldonic add metabolism discovered hi 1976. This labile prostanoid, with a half-life of approximately 3 minutes, relaxes vascular smooth musde and inhibits the aggregation of blood platelets. Prostacydin and its analogues are currently being tested clinically for use hi cardiovascular diseases such as primary pulmonary hypertension. The second mediator discussed is endothelium-derived relaxing factor (EDRF), discovered in 1980, which also relaxes smooth muscle and inhibits the aggregation and adhesion of platelets. Substances that stimulate the release of EDRF include acetykhoUne, bradykinin, and adenosine 5'-diphosphate. EDRF is even more labile than prostacydin, with a half-life of about 6 seconds, and it has recently been identified as nitric oxide. Prostacydin and EDRF are released together following stimulation of endothelial receptors and synergize to inhibit platelet aggregation. 13-Hydroxy-9,11-octadecadienoic add, a third suggested mediator, is not released but acts from inside the cell to make the endothelial surface nonadhesive for circulating blood ceils. It is proposed that these three mediators form the endothelial defense mechanism against blood-borne cells and chemicals and that breakdown of this barrier results in diseases such as hypertension and atherosclerosis. (Hypertension 12: 530-548,1988) KEY WORDS • prostacyclin • endothelium-derived relaxing factor • thromboresistance • 13-hydroxy-9,ll-octadecadienoic add • endothelin • 6-keto-prostaglandin E,

Heme oxygenase (HO) is a microsomal enzyme that oxidatively cleaves heme to form biliverdin, with the release of iron and carbon monoxide (CO). HO not only controls the availability of heme for the synthesis of heme proteins but also is responsible for the generation of CO, which binds to the heme moiety of heme proteins thus affecting their enzymatic activity. Cyclooxygenase (COX) is a heme protein that catalyzes the conversion of arachidonic acid to prostaglandin H 2 , the precursor of prostanoids that participate in the regulation of vascular function. The goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells. Endothelial cells stably transfected with the human HO-1 gene exhibited a severalfold increase in human HO-1 mRNA levels, which was accompanied by an increase in HO activity and a marked decrease in prostaglandin (PG) E 2 and 6-keto-PGF 1␣ levels. Exposure of cells to CoCl 2 , an inducer of HO-1 gene expression, resulted in increases in HO-1 protein levels and HO activity. The increase in HO activity was associated with a subsequent decrease in COX activity, which returned to normal levels following normalization of HO activity. The addition of heme resulted in an increase in COX activity with an increase in PGE 2 and 6-keto-PGF 1␣ levels. The degree of HO-1 expression and, consequently, the level of cellular heme, were directly related to COX activity. These results demonstrate that the heme-HO system can function as a cellular regulator of the expression of vascular COX, thus influencing the generation of prostanoids, PGE 2 and PGI 2 , known to play a role in vascular homeostasis.

1 The effects of hydrogen peroxide (H202, 0.1-1 mM) on the tone of the rings of rabbit aorta precontracted with phenylephrine (0.2-0.3 gAM) were studied.2 H202 induced a concentration-dependent relaxation of both the intact and endothelium-denuded rings. However, in the presence of intact endothelium, H202-induced responses were 2-3 fold larger than in its absence, demonstrating the existence of endothelium-independent and endothelium-dependent components of the vasorelaxant action of H202. 3 The endothelium-dependent component of H202-induced relaxation was prevented by N0-nitro-Larginine methyl ester (L-NAME, 30 gM) or N0-monomethyl-L-arginine (300 JAM), inhibitors of nitric oxide synthase (NOS), in a manner that was reversible by L-, but not by D-arginine (2 mM). The inhibitors of NOS did not affect the responses of denuded rings.4 Methylene blue (10 JAM), an inhibitor of soluble guanylate cyclase, blocked H202-induced relaxation of both the intact and denuded rings. 5 H202 (1 mM) enhanced the efflux of cyclic GMP from both the endothelium-intact and denuded rings. The effect of H202 was 4 fold greater in the presence of intact endothelium and this endotheliumdependent component was abolished after the inhibition of NOS by L-NAME (30 JAM). 6 In contrast to the effects of H202, the vasorelaxant action of stable organic peroxides, tert-butyl hydroperoxide or cumene hydroperoxide, did not have an endothelium-dependent component. Moreover, they did not potentiate the efflux of cyclic GMP from the rings of rabbit aorta. 7 Exogenous donors of NO, specifically, 3-morpholinosydnonimine (SIN-1), glyceryl trinitrate or sodium nitroprusside were used to decrease the tone of denuded rings to the level induced by endogenous NO released from intact endothelium. This procedure did not influence the vasorelaxant activity of H202, showing that H202 does not potentiate the vasorelaxant action of NO within the smooth muscle. 8 Thus, H202-induced relaxation in the rabbit aorta has both endothelium-dependent and independent components. The endothelium-dependent component of the relaxant action of H202 is due to enhanced endothelial synthesis of NO.

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