Amiodarone and desethylamiodarone increase intrasynaptosomal free calcium through receptor mediated channel

Long term amiodarone (AM) therapy has been associated with several side effects including neurotoxicity. Since AM alters Ca²⁺ regulated events, we have studied its effects on the compartmentation of free Ca²⁺ in the synaptosomes as an attempt to understand the mechanism of AM and its metabolite, desethylamiodarone (DEA)-induced neurotoxicity. Intact brain synaptosomes were prepared from male Sprague-Dawley rats. Both AM and DEA produced a concentration dependent increase in intrasynaptosomal free Ca²⁺ concentration ([Ca²⁺]_i) to micromolar levels. The increase in [Ca²⁺]_i was not transient and a steady rise was observed with time. Omission of Ca²⁺ from the external medium prevented the AM- and DEA-induced rise in [Ca²⁺]_i suggesting that AM and DEA increased the intracellular [Ca²⁺]_i due to increased influx of Ca²⁺ from external medium. AM- and DEA-induced increase in intrasynaptosomal [Ca²⁺]_i was neither inhibited by a calcium channel blocker, verapamil, nor with a Na⁺ channel blocker, tetrodotoxin. However, the blockade of [Ca²⁺]_i rise by AM and DEA was observed with MK-801, a receptor antagonist indicating that AM and DEA induced rise in [Ca²⁺]_i is through receptor mediated channel. Both AM and DEA also inhibited N-methyl-D-aspartic acid (NMDA)-receptor binding in synaptic membranes in a concentration dependent manner, DEA being more effective, indicating that AM and DEA compete for the same site as that of NMDA and confirm the observation that these drugs increase intrasynaptosomal [Ca²⁺]_i through receptor mediated channel. ⁴⁵Ca accumulation into brain microsomes and mitochondria was significantly inhibited by AM and DEA, but without any effect on the Ca²⁺ release from these intracellular organelles. Also, both these drugs did not interfere with inositol 1,4,5-trisphosphate induced Ca²⁺ release from microsomes even at 10 μM concentration. These results clearly indicate that both AM and DEA increase intrasynaptosomal [Ca²⁺]_i by an action on receptor mediated channel in plasma membrane, but not due to the release of Ca²⁺ from intracellular storage sites. This initial rise in [Ca²⁺]_i, together with other changes in Ca²⁺ homeostasis, might be responsible for AM and DEA-induced neurotoxicity.