N-desethylamiodarone modulates intracellular calcium concentration in endothelial cells

Abstract.

The main in-vivo metabolite of amiodarone, N-desethylamiodarone (DEAM), possesses clinically relevant class-III 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 µM) both in the presence and absence of extracellular Ca²⁺. In the presence of extracellular Ca²⁺, the response of [Ca²⁺]_i to DEAM (10 µM) 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 µM). The initial rate of rise in [Ca²⁺]_i was decreased by blocking mitochondrial Ca²⁺ release with cyclosporine A (1 µM). Under Ca²⁺-free conditions, the response of [Ca²⁺]_i to DEAM (10 µM) 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 µM). 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.