Differential control and calcium‐dependence of production of endothelium‐derived relaxing factor and prostacyclin by pig aortic endothelial cells

Abstract

1 Production of endothelium-derived relaxing factor (EDRF) by primary cultures of pig aortic endothelial cells was assessed indirectly by measuring endothelial cyclic GMP content, and prostacyclin production was measured by radioimmunoassay of 6-keto prostaglandin F_1α (6-keto PGF_1α). 2 The resting level of cyclic GMP fell significantly following removal of extracellular calcium (1 mM EGTA present), but elevations of cyclic GMP content induced by sodium azide (10 μm) or atriopeptin II (10 nm) were similar in the absence and presence of extracellular calcium. 3 Haemoglobin (10 μm) reduced the resting level of cyclic GMP in the presence, but not the absence of extracellular calcium. M&B 22,948 (100 μm), superoxide dismutase (30 u ml⁻¹), bradykinin (0.1 μm), ATP (10 μm) and ionophore A23187 (0.1 μm) each induced an increase in endothelial cyclic GMP content that was reduced in the absence of extracellular calcium. 4 In cascade bioassay experiments using endothelial cells on microcarrier beads and perfused in columns, continuous infusion of bradykinin (0.1 μm) induced release of EDRF, assayed on rabbit aortic rings, that was maximal after 2 min and still detectable up to about 16 min. 5 In the presence of extracellular calcium, the time course of bradykinin (0.1 μm)-stimulated production of EDRF, assessed as endothelial cyclic GMP content was maximal within 1 min, declined thereafter, but was still significant after 30 min. Production of 6-keto PGF_1α, measured simultaneously, rose rapidly but was complete within 3 min. 6 In the absence of extracellular calcium the resting endothelial content of cyclic GMP fell, but resting production of 6-keto PGF_1α was unaffected. The rise in cyclic GMP content stimulated by bradykinin (0.1 μm) was lower than when calcium was present and was complete within 3 min, but the magnitude and time course of bradykinin (0.1 μm)-stimulated production of 6-keto PGF_1α was unaffected. 7 In the presence of TMB-8 (100 μm) resting endothelial content of cyclic GMP rose slightly, but production of 6-keto PGF_1α fell. The bradykinin (0.1 μm)-stimulated increase in cyclic GMP content was augmented, but the stimulation of 6-keto PGF_1α production was blocked. Results from cascade bioassay experiments confirmed that TMB-8 (100 μm) did not inhibit bradykinin-induced production of EDRF. 8 The data suggest that resting production of EDRF but not prostacyclin is dependent upon the presence of extracellular calcium. Bradykinin-stimulated production of EDRF is sustained and requires the presence of extracellular calcium, but stimulated production of prostacyclin is transient and may result from discharge of an intracellular pool of calcium. 9 The vascular endothelial cell appears therefore to control differentially production of EDRF and prostacyclin.