Differential role of extra‐ and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells

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Abstract
1 The effects of extracellular Ca2+ on the release of endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2), and on the intracellular free calcium concentration ([Ca2+]i), were studied in cultured bovine aortic endothelial cells. 2 Receptor-mediated stimulation of endothelial cells with bradykinin (10 nM) elicited a transient release of EDRF (assayed by its stimulant effect on purified soluble guanylate cyclase) and of PGI2 (measured by radioimmunoassay for 6-keto prostaglandin F1α). 3 Bradykinin (10 nM) also increased [Ca2+]i (measured with the fluorescent probe indo-1) from 125 ± 11 nM to 631 ±59 nM, with the same time course as for autacoid release. 4 In Ca2+-free medium, [Ca2+]i was still increased by bradykinin but declined faster (within 1 min) to resting levels than in the presence of extracellular Ca2+. 5 PGI2 release was almost completely abolished in Ca2+-free medium. The intracellular calcium antagonist TMB-8 evoked a similar inhibition of PGI2 release. 6 In contrast, bradykinin-induced EDRF release was not significantly affected by TMB-8 but was completely abolished in Ca2+-free medium. 7 When endothelial cells were stimulated with the receptor-independent drug thimerosal (an inhibitor of the enzyme acyl-CoA-lysolecithin-acyl-transferase; 5 μM), a long-lasting release of EDRF (>90 min) and PGI2 (>20 min) was observed. 8 In contrast to bradykinin stimulation, thimerosal-induced autacoid release was associated with only a slight increase of [Ca2+]i to 201 ± 13 nM after 40min. 9 After removal of extracellular Ca2+ from thimerosal-stimulated endothelial cells, [Ca2+]i was little affected during the observation time of 90 s. EDRF release was completely abolished within 90 s whereas PGI2 release was unchanged. 10 We conclude that EDRF production is directly controlled by extracellular Ca2+ during both receptor-dependent and independent stimulation. This effect of extracellular Ca2+ is not mediated by changes in [Ca2+]i. In contrast, PGI2 release is closely correlated to [Ca2+]i in bradykinin-stimulated endothelial cells. However, the results obtained during thimerosal stimulation indicate that there is not necessarily a tight coupling between the absolute level of [Ca2+]i and the amount of PGI2 released.