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CARDIOVASCULAR

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

Departments of Biochemistry and Molecular Biology and Medicine, Division of Experimental Medicine, George Washington University Medical Center, Washington DC

The influence of selected -receptor blockers on iron overload and oxidative stress in endothelial cells (ECs) was assessed. Confluent bovine ECs were loaded with iron dextran (15 µM) for 24 h and then exposed to dihydroxyfumarate (DHF), a source of reactive oxygen species, for up to 2 h. 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 ECs were pretreated 30 min before iron loading with 1.25 to 10 µM D-propranolol, glutathione losses were attenuated 15 to 80%, with EC₅₀ = 3.1 µM. D-Propranolol partially inhibited the DCF intensity increase, but atenolol up to 10 µM was ineffective. At 2 h, caspase 3 activity was elevated 3.2 ± 0.3-fold (p < 0.01) in the iron-loaded and DHF-treated ECs, and cell survival, determined 24 h later, decreased 47 ± 6% (p < 0.01). Ten micromoles of 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-h iron loading resulted in a 3.6 ± 0.8-fold increase in total EC iron 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 intralysosomal pH; it is suggested that the lysosomotropic properties of D-propranolol retarded the EC iron accumulation and thereby conferred the protective effects against iron load-mediated cytotoxicity.

Address correspondence to: Dr. I. Tong Mak, Dept. of Biochemistry and Molecular Biology, Division of Experimental Medicine, George Washington University Medical Center, 2300 Eye Street, N.W. Ross Hall, Rm 443, Washington DC 20037. E-mail: itmak{at}gwu.edu