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

Abstract

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 M) 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 2Ј,7Ј-dichlorofluorescin (DCF; 30 M), increased and peaked at 30 min; total glutathione decreased by 52 Ϯ 5% (p Ͻ 0.01) at 60 min. When the ECs were pretreated 30 min before iron loading with 1.25 to 10 M D-propranolol, glutathione losses were attenuated 15 to 80%, with EC 50 ϭ 3.1 M. D-Propranolol partially inhibited the DCF intensity increase, but atenolol up to 10 M was ineffective. At 2 h, caspase 3 activity was elevated 3.2 Ϯ 0.3-fold (p Ͻ 0.01) in the iron-loaded and DHF-treated ECs, and cell survival, determined 24 h later, decreased 47 Ϯ 6% (p Ͻ 0.01). Ten micromoles of D-propranolol suppressed the caspase 3 activation by 63% (p Ͻ 0.05) and preserved cell survival back to 88% of control (p Ͻ 0.01). In separate experiments, 24-h iron loading resulted in a 3.6 Ϯ 0.8-fold increase in total EC iron determined by atomic absorption spectroscopy; D-propranolol at 5 M reduced this increase to 1.5 Ϯ 0.4-fold (p Ͻ 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.

d-Propranolol protects against oxidative stress and progressive cardiac dysfunction in iron overloaded rats

“…Although l -propranolol's pharmacologic action as a β-adrenergic receptor blocker is well documented, it's antioxidant and lysosomotropic properties are not as well recognized (Kramer et al 1994, 2000, 2006; Mak et al 2006). We have previously shown that both the l (active blocker) and d (inactive) stereoisomer's of propranolol were equally potent as membrane chain breaking antioxidants in cardiomyocyte sarcolemmal preparations (decreased lipid peroxidation-derived carbon-centered free radical formation) exposed to an iron-catalyzed oxygen free radical system (Mak et al 1989; Mak and Weglicki 1992).…”

“…Beta-blockers that are more water soluble, like atenolol and sotalol, were not effective membrane chain breaking antioxidants and (or) cyto-protective agents (Mak and Weglicki 1988; Kramer et al 1991), and also argue against β-adrenergic receptor blockade as a major contributor to the observed beneficial effects of d -Pro. In oxidatively-stressed cellular (Kramer et al 1991; Mak et al 2006) and perfused heart models (Kramer et al 1994, 2006), the protective effects of d -Pro required prolonged treatment (>30 min) to allow active uptake. In addition, we (Dickens et al 2002) and others (Cramb 1986) have shown that lysosomes are principle sites of propranolol accumulation within cells, and that propranolol can preserve the stability of lysosomes when exposed to exogenous free radicals in vitro (Kramer et al 2006).…”

“…In addition, we (Dickens et al 2002) and others (Cramb 1986) have shown that lysosomes are principle sites of propranolol accumulation within cells, and that propranolol can preserve the stability of lysosomes when exposed to exogenous free radicals in vitro (Kramer et al 2006). Compounds containing basic amine groups (ethanolamine side chain) like propranolol, possess lysosomotropic properties and raise lysosomal pH (alkalinization) (Dickens et al 2002; Mak et al 2006). Since lysosomes are also a primary storage depot for cellular iron (Eaton and Qian 2002; Mak et al 2006), agents causing alkalinization may afford protection by enhancing iron binding to lysosomal ferritin (major tissue storage protein) (Berenshtein et al 2002), and diminish low molecular weight lysosomal iron (redox active) release during oxidative stress (Voogd et al 1992; Whittaker et al 1996).…”

“…Compounds containing basic amine groups (ethanolamine side chain) like propranolol, possess lysosomotropic properties and raise lysosomal pH (alkalinization) (Dickens et al 2002; Mak et al 2006). Since lysosomes are also a primary storage depot for cellular iron (Eaton and Qian 2002; Mak et al 2006), agents causing alkalinization may afford protection by enhancing iron binding to lysosomal ferritin (major tissue storage protein) (Berenshtein et al 2002), and diminish low molecular weight lysosomal iron (redox active) release during oxidative stress (Voogd et al 1992; Whittaker et al 1996). Indeed, we have shown that the protective effects of acute d -Pro against oxidative injury to iron-loaded rat peritoneal macrophages (lower release of iron and reactive oxygen species) (Komarov et al 2006), and to postischemic hearts from iron-loaded rats (improved cardiac function, and lower release of tissue iron, lactate dehydrogenase, conjugated dienes, and lysosomal NAGA) (Kramer et al 2006) were largely related to its membrane antioxidant and lysosomotropic properties.…”

Contribution of Catecholamine Reactive Intermediates and Oxidative Stress to the Pathologic Features of Heart Diseases

“…I/R is probably the most studied cardiac disease and several drugs have been evaluated using I/R models. The antioxidant properties of propranolol and other -adrenoceptor antagonists are controversial, as some in vitro and in vivo studies support them [421,657,658] while others do not [651,659,660]. Propranolol or pindolol did not show free radical scavenging properties relevant for cardioprotection in a coronary occlusion model of isolated hearts of rabbit [660].…”