No abstract

Cholesterol is an essential component of lysosomal membranes. In this study, we investigated the effects of membrane cholesterol on the permeability of rat liver lysosomes to K+ and H+, and the organelle stability. Through the measurements of lysosomal beta-hexosaminidase free activity, membrane potential, membrane fluidity, intra-lysosomal pH, and lysosomal proton leakage, we established that methyl-beta-cyclodextrin (MbetaCD)-produced loss of membrane cholesterol could increase the lysosomal permeability to both potassium ions and protons, and fluidize the lysosomal membranes. As a result, potassium ions entered the lysosomes through K+/H+ exchange, which produced osmotic imbalance across the membranes and osmotically destabilized the lysosomes. In addition, treatment of the lysosomes with MbetaCD caused leakage of the lysosomal protons and raised the intra-lysosomal pH. The results indicate that membrane cholesterol plays important roles in the maintenance of the lysosomal limited permeability to K+ and H+. Loss of this membrane sterol is critical for the organelle acidification and stability.

No abstract

Loss of lysosomal integrity is a critical event for killing tumor cells in the photodynamic therapy of cancers. To elucidate the mechanism of photodamage induced lysosomal disintegration, we investigated the role of losing lysosomal proton translocation in latency loss of photosensitized lysosomes. Isolated rat liver lysosomes were light exposed in the presence of Methylene blue. Through monitoring lysosomal delta pH with Acridine orange and measuring its membrane potential with 3,3'-dipropylthiadicarbocyanine iodide, loss of Mg-ATP dependent proton translocation and decrease in electrogenicity of the proton pump were observed after lysosomes were photosensitized. When normal lysosomes were incubated for 60 min in K+ contained medium, percentage free activity of lysosomal enzyme beta-galactosidase increased, i.e. lysosomal latency decreased. In the presence of Mg-ATP, the latency loss of incubated lysosomes reduced. Addition of n-ethylmaleimide, a potent inhibitor of lysosomal H(+)-ATPase, abolished the effect of Mg-ATP on lysosomal latency. It suggests a role of proton translocation in protecting lysosomal integrity. Under the same conditions, Methylene blue photosensitized lysosomes increasingly lost latency of beta-hexosaminidase and beta-galactosidase with light exposure, presumably due to the photodamage induced loss of proton pumping. In contrast, the photosensitization did not decrease lysosomal latency in the absence of Mg-ATP, implying that lysosomal integrity might not be impaired via other photodamage effects under the conditions of this study. These results indicate that lysosomal integrity can be photodestructed via the loss of proton translocation.

Isotonic K2SO4 solution protected lysosomes osmotically during a 20 min incubation, but lost its protective effect if the lysosomes were initially photooxidized after sensitization with Methylene blue. Increasing K2SO4 concentration promoted the latency loss of photodamaged lysosomes, but did not impair the integrity of unirradiated lysosomes. The results indicate that the photodamage enhanced lysosomal ionic permeability, with osmotic imbalance over the lysosomal membrane. Out of the decreased latency induced by the photodamage, 32% was prevented by the addition of 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid to the incubation solution, suggesting that electroneutral K+/SO4(2-) co-uptake plays a role in the lysosomal destabilization. The photooxidation increased lysosomal H+/K+ exchange, which was confirmed by monitoring the H+ leakage with the pH sensitive probe p-nitrophenol and examining the K+ entry by membrane potential measurements. Addition of K2SO4 to a lysosomal suspension lowered the delta pH of photodamaged lysosomes, presumably due to an increase in the exchange of internal H+ for external K+. Out of the photodamage-induced lysosomal latency loss, 50-60% was prevented by either lowering the external pH or preincubating the lysosomes with methylamine to elevate their internal pH. The results suggest that the photodamage-promoted K+/H+ exchange plays a major role in lysosomal osmotic destabilization.