Proton trapping in the cellular acidic vacuolar compartment : lysosomal mechanisms in apoptosis/necrosis and iron chelation

University dissertation from Linköping : Linköpings universitet

Abstract: Under ischemic conditions, a number of cytotoxic metabolic products are formed. Reactive oxygen species are known to be important mediators of progressive ischemic cell injury, and the synergistic damage to cells caused by the combination of such oxygen species and redox-active iron is well appreciated. The acidic interior of lysosome leads to the trapping of substances with high pK4 values. A large variety of molecules, being weak bases, may thus concentrate within this acidic vacuolar compartment, potentially leading to both beneficial and detrimental effects. A major part of the intracellular pool of redoxactive iron is likely to be located in the lysosomal compartment, and iron chelators that are lysosomotropic due to high pK4 values may prove to be important pharmacological tools to protect the brain from oxidative stress. Among a variety of substances formed in the ischemic penumbra zone is the polyamine metabolite, 3-aminopropanal (3-AP), a substance of extreme neurotoxicity. 3-AP is a weak base and may theoretically exert its toxic action through induction of cell death after intralysosomal accumulation.On the 1774 mouse histiocytic lymphoma cell line, we used the common lysosomotropic agent NH3 to increase lysosomal pH, the lysosomotropic iron chelator, 5-[1,2] dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl)-amide (LAP) and the lysosomotropic iron binder, WR-1065, a metabolite of amifostine, as tools to determine that proton trapping within the lysosomal acidic vacuolar compartment plays an important role in oxidative stress-induced apoptosis. We also used another lysosomotropic agent, 3-AP, on the J774 cell line and on the SH-SY5Y human neuroblastoma cell line. The results indicate that proton trapping of this toxin within the lysosome might explain its toxicity to cells.Sulfide-silver cytochemical detection of iron revealed a pronounced decrease in the lysosomal content of redox-active iron following reduced acidity of the lysosome, and electron spin-resonance studies showed that no hydroxyl radicals [OH•] were formed from hydrogen peroxide under these conditions. This suggests that lysosomes contain most of the free, redox-active iron. In further support of this idea, the lysosomotropic agents LAP and WR-1065 were found to be 5000 and 2500 times more effective, respectively, in protecting cells from oxidative stress, compared with the well-known iron chelator desferrioxamine [DFO]. Evidence was obtained that LAP and WR-1065 exerted their effect on intralysosomal redox-active iron, and that the effect was linked to the acidity of the lysosome. Being weak bases (LAP, pKa = 8.0; WR-1065, pKa = 9.2), these compounds accumulate intralysosomally by proton trapping. The neurotoxic effect of 3-AP (pKa = 9.3) could be linked to a dose-dependent induction of cell death, most likely based on intralysosomal proton trapping of this molecule followed by lysosomal rupture. The lysosomal rupture seems to induce a chain of intracellular events (including generation of oxidative stress), leading to mitochondrial damage directly or indirectly caused by the release of lysosomal proteases.We conclude that the low pH of the lysosome may both serve to attract basic toxins, such as 3-AP, and promote the accumulation of protective agents, such as LAP and WR-1065. Prevention of lysosomal damage from both oxidants and neurotoxins by lysosomotropic agents has great potential therapeutic utility.

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