Pramanicin, an antifungal agent, raises cytosolic Ca2+ and causes cell death in vascular endothelial cells.
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 microM, 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 CC 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.
Kwan
Chiu-Yin
CY
Department of Medicine, HSC-4N40, Faculty of Health Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. kwancy@mcmaster.ca
Harrison
Paul H M
PH
Kwan
Tony K
TK
D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003600:Cytosol / Q000187:drug effects* / Q000378:metabolism*; D004285:Dogs; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D004852:Epoxy Compounds / Q000494:pharmacology*; D005260:Female; D007769:Lactams / Q000494:pharmacology*; D008297:Male
D000935:Antifungal Agents; D004852:Epoxy Compounds; D007769:Lactams; C432807:pramanicin; D002118:Calcium