Fendiline and calmidazolium enhance the release of endothelium-derived relaxant factor and of prostacyclin from cultured endothelial cells

Abstract

We investigated the effects of fendiline, calmidazolium and trifluoperazine, compounds described as calmodulin antagonists, on the release of the endothelial autacoids prostacyclin (PGI₂) and endothelium-derived relaxant factor (EDRF). Cultured bovine aortic endothelial cells were grown on microcarrier beads and continuously superfused with Tyrode's solution. Samples collected from the superfusate were assayed for PGI₂ concentration (6-keto PGF_1α radioimmunoassay) and for EDRF activity (stimulation of soluble guanylate cyclase in vitro). Stimulation of endothelial cells by ATP (3 μM) resulted in a 6.9 ± 1.4-fold increase of PGI₂ concentration in the superfusate (p < 0.01) and an 8.6 ± 3.4-fold enhanced guanylate cyclase activity (p < 0.01). 1n the presence of calmidazolium (10 μM), the basal values of PGI₂ concentration increased 28-fold (p < 0.01) and the guanylate cyclase activity 10-fold (p < 0.01). Further enhancement of both was observed after additional administration of ATP. Fendiline (30 μM) did not affect autacoid release by non-stimulated cells. However, the ATP-induced release of PGI₂ and EDRF was more than doubled (p < 0.01) in the presence of this drug compared to ATP-stimulation alone. Trifluoperazine (10 μM) had no enhancing effect on EDRF release, and the ATP-induced release of PGI₂ was even significantly attenuated by 84 ± 12% (p < 0.01). Calmidazolium and fendiline were also applied to endothelial cells loaded with the fluorescent indicator of free calcium concentration (Ca _infi ^sup2+ ) indo-1. However, effects of calmidazolium on Ca _infi ^sup2+ could not be quantified since calmidazolium caused some leakage of indo-1 out of the cells. A smaller leakage was observed during the combined application of fendiline and ATP. The findings suggest that the characterization of these drugs as calmodulin antagonists may not adequately describe their spectrum of actions in intact endothelial cells. Calmidazolium enhanced the autacoid release probably by increasing cell membrane permeability, as indicated by the leakage of the large hydrophilic molecule indo-1. The enhancement of autacoid release by fendiline, possibly also due to an effect at the level of the membrane bilayer, may be relevant for clinically useful amplification of endothelium-mediated dilation.