Platelet-activating factor (PAF) is generated by endothelial cells, polymorphonuclear leukocytes, and macrophages after activation by appropriate receptor agonists, but much of the PAF remains intracellular. We have investigated whether PAF formation is important for the subsequent generation of icosanoids and superoxide anions by these cells. The generation of prostacyclin and leukotriene B4 were measured by radioimmunoassay, superoxide anion was measured by reduction ofcytochrome c, and PAF was measured by bioassay. In each cell type, PAF formation preceded or accompanied icosanoid generation. Bradykinin-induced prostacyclin generation in endothelial cells was markedly reduced by the PAF receptor antagonists WEB 2086 or CV 6209. In guinea pig adherent macrophages in vitro, basal prostacyclin generation and that induced by endotoxin and fMet-Leu-Phe were inhibited by either WEB 2086 (1-100 FM) or CV 6209 (0.1-10 FM). In isolated rabbit polymorphonuclear leukocytes, fMetLeu-Phe stimulated the generation of both leukotriene B4 and superoxide anion. WEB 2086 and CV 6209 caused concentration-dependent inhibition of both these markers of leukocyte activation. These observations lead us to suggest that PAF may be a second messenger in leukocytes and endothelial cells.

1. The actions of N-nitro-L-arginine (NOLA) on the release of nitric oxide (NO) from arterial endothelial cells was studied in rat isolated thoracic aortic rings and by bioassay of NO derived from cultured bovine aortic endothelial cells. 2. NOLA (3-10 mumol/L) caused concentration-dependent inhibition of acetylcholine-induced relaxation of phenylephrine-contracted rat aortic rings, which is dependent on the release of NO from the endothelium. The inhibitory actions of NOLA could be prevented by pre- and co-incubation with L-arginine (1 mmol/L). 3. Endothelium-independent relaxation induced by sodium nitroprusside was not affected by NOLA. 4. The release of NO from bovine aortic endothelial cells, induced by bradykinin (10 nmol/L), was detected by bioassay on pre-contracted rabbit aortic strips. NOLA (1-3 mumol/L, given through the cell column) reduced or abolished the release of NO, but did not affect relaxations of the bioassay tissues induced by glyceryl trinitrate or authentic NO. 5. These data indicate that NOLA potently inhibits the biosynthesis of NO from L-arginine, and thus prevents its release from arterial endothelial cells. It may be a useful pharmacological tool for probing the significance of NO biosynthesis in cardiovascular function.

Bovine culture endothelial cells (BAECs) generate platelet-activating factor (Pat) following activation by bradykinin (Bk O.1nM), the ionophore, A23187 (31iM), and ATP (10piM), but Paf is not released from the cells.These stimuli also elicit generation of prostacyclin (PGI2). The specific and competitive Paf receptor antagonists, WEB 2086 (0.1-1.OpM) and CV 6209 (0.01-0.1 pM), inhibited Bk-, A23187-and, to a lesser extent, ATP-induced PGI2 generation but had no effect on basal PGI2 generation. These data suggest a role for intracellular Paf in signal transduction.

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