Received for publication October 26, 2005.
Revised February 1, 2006.
Accepted for publication February 1, 2006.

D-Propranolol Attenuates Lysosomal Iron Accumulation and Oxidative Injury in Endothelial Cells

Abstract

The influence of selected -receptor blockers on iron (Fe) overload and oxidative stress in endothelial cells (EC) was assessed. Confluent bovine EC were loaded with Fe-dextran (15 µM) for 24 hrs and then exposed to dihydroxyfumarate (DHF), a source of reactive oxygen species, for up to 2 hrs. Intracellular oxidant formation, monitored by fluorescence of 2'7' dichlorofluorescin (DCF, 30 µM), increased and peaked at 30 min; total glutathione decreased by 52±5% (p<0.01) at 60 min. When the EC were pre-treated 30 min before Fe loading with 1.25-10 µM d-propranolol, glutathione losses were attenuated 15-80% with EC₅₀ =3.1 µM.. D-Propranolol partially inhibited the DCF intensity increase, but atenolol up to 10 µM was ineffective. At 2 hrs, caspase 3 activity was elevated 3.2±0.3-fold (p<0.01) in the Fe-loaded and DHF-treated EC, and cell survival determined 24 hrs later, decreased 47±6% (p<0.01). Ten µM d-propranolol suppressed the caspase 3 activation by 63% (p<0.05), and preserved cell survival back to 88% of control (p<0.01). In separate experiments, 24 hr Fe-loading resulted in a 3.6±0.8-fold increase in total EC Fe determined by atomic absorption spectroscopy; d-propranolol at 5 µM reduced this increase to 1.5±0.4-fold (p<0.01) of controls. Microscopic observation by Perls' staining revealed that the excessive iron accumulated in vesicular endosomal/lysosomal structures which were substantially diminished by d-propranolol. We previously showed that propranolol could readily concentrate into the lysosomes and raise the intra-lysosomal pH; it is suggested that this lysosomotropic properties of d-propranolol retarded the EC Fe accumulation, and thereby conferred the protective effects against Fe-load-mediated cytotoxicity.

Key words: D-propranolol, Endothelial cells, Iron-overload, Lysosomal iron, Lysosomotropic, Oxidative stress