Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells

Abstract

A cascade superfusion technique has been developed for the differential bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) released from porcine aortic endothelial cells cultured on microcarriers, packed into a column and perfused. Bradykinin (Bk; 20-100 nM) released prostacyclin (9.6 +/- 1.5 nM per 10(6) cells; mean +/- s.e.mean, n = 9) and prostaglandin E2 (PGE2; 2.1 +/- 0.6 nM per 10(6) cells) from the column measured by relaxation of strips of bovine coronary artery (BCA) and rabbit mesenteric or coeliac artery, respectively. The presence of these prostanoids in the effluent was confirmed by specific radioimmunoassays. A23187 (500-2000 nM) also released both prostacyclin and PGE2 from the cells. This release was long-lasting and not reproducible. Bk (20-100 nM) and A23187 (30-300 nM) released EDRF from the column. This was detected in a cascade of four rabbit aortic strips (RbA), denuded of endothelium and contracted with U46619 or phenylephrine. The relaxation of the RbA strips caused by EDRF was progressively attenuated down the cascade (half-life less than 7s) and was not affected by indomethacin. EDRF and prostacyclin could be differentially bioassayed in a cascade of alternating RbAs and BCAs as prostacyclin did not relax RbAs and the time delay to the BCAs destroys EDRF. EDRF could be bioassayed on its own when the endothelial cells were treated with indomethacin. 5-Hydroxytryptamine 0.2, noradrenaline 1.0, platelet-activating factor (Paf-acether) 1.0, formylmethionyl-leucyl-phenylalanine 1.0, acetylcholine 0.5, bethanecol 0.5, adenosine diphosphate 0.25 and angiotensin II 0.1 microM did not release either prostanoids or EDRF from the column.