Life cell imaging of amiodarone sequestration into lamellar bodies of alveolar type II cells

Abstract

Amiodarone is widely used to treat cardiac arrhythmias and is very effective in preventing these disorders. However, its use is limited by a wide range of adverse effects, mainly affecting the lungs, and ranging from mild shortness of breath to pulmonary fibrosis. Amiodarone has been shown to accumulate strongly in lung tissue, exceeding its plasma concentration by a hundredfold. However, the site of accumulation and the mechanisms of transport are not fully understood. In this study, we used live cell imaging of primary rat alveolar type II cells to show that amiodarone specifically accumulates in large amounts in lamellar bodies, the surfactant storage organelles. Fluorescence imaging and correlation, and colocalization studies combined with confocal Raman microscopy identified these organelles as a major target for sequestration. Accumulation was rapid, on the order of a few hours, while storage was much more persistent. Partial uptake was observed in chemically fixed, dead cells, or cells treated with bafilomycin A1. Not only was uptake pH dependent, but intraluminal pH, measured with lysosomotropic pH sensitive dyes, was also affected. From these observations and from the physicochemical properties of amiodarone, we propose that passive diffusion, ion-trapping and lipophilic interactions are the main mechanisms for intracellular bioaccumulation. Furthermore, we demonstrate that measurement of amiodarone autofluorescence is highly useful for tracking cellular uptake and sequestration.