Pramanicin, an antifungal agent, raises cytosolic Ca 2+ and causes cell death in vascular endothelial cells

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The effects of a newly discovered antifungal agent, pramanicin, on cytosolic Ca 2+ and cell viability of cultured bovine pulmonary artery endothelial cells and on endothelium-dependent relaxation of dog carotid arterial rings were investigated by digital dynamic fluorescence ratio imaging and morphological and contractility studies, respectively. Pramanicin 100 μM, previously shown to cause maximal endothelium-dependent and NO-mediated vascular relaxation, induced a small transient elevation of cytosolic Ca 2+ concentration in Ca 2+-free medium; subsequent introduction of 1 mM Ca 2+ caused a steady, nonsaturating increase of Ca 2+, which could be brought down to the basal level by the addition of EGTA. At the single cell level, the elevation of cytosolic Ca 2+ initiates from the cell periphery and progresses toward the central region. When added to the plateau phase of phenylephrine-induced contraction, pramanicin induced a slow endothelium-dependent relaxation, which could be reversed with the NO synthase inhibitor, l-NOARG. When preincubated with vascular tissue, pramanicin resulted in an irreversible loss of endothelial function characterized by the lack of carbachol-induced relaxation. Pramanicin caused cell injury characterized by plasmalemmal bleb formation, leading to cell death characterized by Trypan blue staining of the nuclei in cultured vascular endothelial cells in a concentration- and time-dependent manner. Such pramanicin-induced cell death was not associated with Ca 2+-mediated or NO-mediated mechanisms. The time course of Ca 2+ elevation corresponds with that of pramanicin-induced relaxation of precontracted arterial rings, whereas the time course of endothelial cell death corresponds to that of pramanicin-induced loss of endothelial function as assessed by carbachol-induced relaxation. The pramanicin analogue, PMC-A, a by-product of the biosynthesis of pramanicin, in which the epoxy group is replaced by a CC bond, caused little endothelial-dependent relaxation, but it was able to cause endothelial cell dysfunction, albeit to a lesser extent compared to pramanicin, suggesting a role of the epoxy group in pramanicin for its vasorelaxant effect.