title: N-desethylamiodarone modulates intracellular calcium concentration in endothelial cells.
abstract: The main in-vivo metabolite of amiodarone, N-desethylamiodarone (DEAM), possesses clinically relevant class-II antiarrhythmic and vasodilator activities. Vasodilation by DEAM is endothelium dependent and involves a sustained and biphasic increase in cytosolic free Ca2+ concentration ([Ca2+]i). The aims of this study were to explore the mechanisms mediating the DEAM-induced increase in [Ca2+]i in endothelial cells and to determine whether this increase in [Ca2+]i was associated with altered cell proliferation. Cultured bovine aortic endothelial cells were loaded with the Ca2+-sensitive fluorescent dye Fura-2/AM, and [Ca2+]i measured spectrofluorimetrically. DEAM increased [Ca2+]i concentration dependently (EC50 approximately 6 microM) both in the presence and absence of extracellular Ca2+. In the presence of extracellular Ca2+, the response of [Ca2+]i to DEAM (10 microM) consisted of an initial rise to a plateau followed by a second increase to micromolar levels. The initial plateau was reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (200 nM) and by the antioxidant ascorbic acid (100 microM). The initial rate of rise in [Ca2+]i was decreased by blocking mitochondrial Ca2+ release with cyclosporine A (1 microM). Under Ca2+-free conditions, the response of [Ca2+]i to DEAM (10 microM) was also biphasic, consisting of an initial transient peak and a second slow increase. When extracellular Ca2+ was restored, [Ca2+]i rose to micromolar concentrations. The initial peak was abolished by thapsigargin, but not altered by ascorbic acid or cyclosporine A. Both the second [Ca2+]i increase and that due to restoring extracellular Ca2+ were reduced by ascorbic acid but not affected by thapsigargin or cyclosporine A. The DEAM-induced generation of free radicals and sustained increase in [Ca2+]i might alter cell proliferation and endothelial cell proliferation was indeed concentration-dependently inhibited by DEAM (IC50 approximately 2.5 microM). In conclusion, the DEAM-induced [Ca2+]i increase in endothelial cells is due to Ca2+ influx from the extracellular space and to Ca2+ release from endoplasmic reticulum and mitochondria and involves enhanced generation of free radicals.
authors.affiliation: Institut für Pharmakologie und Toxikologie, Universitätsklinikum Carl Gustav Carus, TU Dresden, Germany. himmel@rcs.urz.tu-dresden.de
authors.forename: H M
authors.initials: HM
authors.lastname: Himmel
authors.forename: D
authors.initials: D
authors.lastname: Dobrev
authors.forename: M
authors.initials: M
authors.lastname: Grossmann
authors.forename: U
authors.initials: U
authors.lastname: Ravens
mesh_terms: D000638:Amiodarone / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014665:Vasodilator Agents / Q000494:pharmacology
chemical_list: D000889:Anti-Arrhythmia Agents; D004791:Enzyme Inhibitors; D014665:Vasodilator Agents; D019284:Thapsigargin; D016572:Cyclosporine; D000252:Calcium-Transporting ATPases; C036116:desethylamiodarone; D000638:Amiodarone; D002118:Calcium