Lysosomal perturbations in fish liver as indicators for toxic effects of environmental pollution

• 1.1. Lysomones play a key role in liver injury in fish caused by organic and inorganic xenobiotics. The lysosomal stability test was transferred to fish liver with the aim of testing responsive and practicable methods for biological-effects monitoring.
• 2.2. A two-step response of lysosomes in fish liver could be discerned, reflected by the activity (number and size of lysosomes) and the injury (membrane destabilisation) of the lysosomal detoxifying system.
• 3.3. Significant differences, with respect to lysosomal enlargement, membrane stability and pathological lipid accumulation, were found along a pollution gradient throughout the year.
• 4.4. The lysosomal tests clearly reflect the breakdown of the adaptive capacity of the fish liver to toxic injury. Therefore, a test battery measuring lysosomal perturbations should be recommended for the biological-effects monitoring.

     

Lysosomal and autophagic reactions as predictive indicators of environmental impact in aquatic animals

The lysosomal-autophagic system appears to be a common target for many environmental pollutants as lysosomes accumulate many toxic metals and organic xenobiotics, which perturb normal function and damage the lysosomal membrane. In fact, lysosomal membrane integrity or stability appears to be an effective generic indicator of cellular well-being in eukaryotes: in bivalve molluscs and fish, stability is correlated with many toxicological responses and pathological reactions. Prognostic use of adverse lysosomal and autophagic reactions to environmental pollutants has been explored in relation to predicting cellular dysfunction and health in marine mussels, which are extensively used as sensitive bioindicators in monitoring ecosystem health. Derivation of explanatory frameworks for prediction of pollutant impact on health is a major goal; and we have developed a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells and tissues. This model has also complemented the creation of a cell-based computational model for molluscan hepatopancreatic cells that simulates lysosomal, autophagic and other cellular reactions to pollutants. Experimental and simulated results have also indicated that nutritional deprivation-induced autophagy has a protective function against toxic effects mediated by reactive oxygen species (ROS). Finally, coupled measurement of lysosomal-autophagic reactions and modelling is proposed as a practical toolbox for predicting toxic environmental risk.     

溶酶体检测在海洋污染监测中的应用研究进展

溶酶体(lysosome)是真核细胞内重要的细胞器.近年来随着对溶酶体结构和功能研究的深入,溶酶体被认为是亚细胞水平上的有毒物质的靶点,在国外已广泛应用于海洋污染监测.本文概述了溶酶体标志酶、溶酶体 自噬系统和溶酶体膜的生物学特性,在此基础上介绍了利用溶酶体检测技术进行海洋污染监测的原理和方法.双壳贝类消化腺和鱼类肝脏最适于作为溶酶体检测的敏感器官;采用溶酶体膜稳定性测定(LMS)、溶酶体中性红保留时间测定(NRRT)、溶酶体形态测量(MM)、溶酶体标志酶免疫组化测定(Ih)和电镜(EM)观察等技术,能够指示海洋污染状况,因此溶酶体可作为生物标志物监测海洋环境污染.文中还分析了溶酶体检测的优缺点以及应注意问题,对其应用前景进行了展望.     

Loss of membrane cholesterol influences lysosomal permeability to potassium ions and protons

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 β-hexosaminidase free activity, membrane potential, membrane fluidity, intra-lysosomal pH, and lysosomal proton leakage, we established that methyl-β-cyclodextrin (MβCD)-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 MβCD 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.     

Elevated serum beta-glucuronidase reflects hepatic lysosomal fragility following toxic liver injury in rats.

The level of serum beta-glucuronidase increases in various pathological conditions, including liver disorders. The aim of this investigation was to study the changes in liver lysosomal membrane stability during experimentally induced hepatic fibrosis that may result in the elevation of serum beta-glucuronidase. Liver injury was induced by intraperitoneal injections of N-nitrosodimethylamine (NDMA) in adult male albino rats over 3 weeks. The progression of fibrosis was evaluated histopathologically as well as by monitoring liver collagen content. Lipid peroxides and beta-glucuronidase levels were measured in the liver homogenate and subcellular fractions on days 0, 7, 14, and 21 after the start of NDMA administration. Serum beta-glucuronidase levels were also determined. A significant increase was observed in beta-glucuronidase levels in the serum, liver homogenate, and subcellular fractions, but not in the nuclear fraction on days 7, 14, and 21 after the start of NDMA administration. Lipid peroxides also increased in the liver homogenate and the lysosomal fraction. The measurement of lysosomal membrane stability revealed a maximum lysosomal fragility on day 21 during NDMA-induced fibrosis. In vitro studies showed that NDMA has no significant effect on liver lysosomal membrane permeability. The results of this investigation demonstrated that lysosomal fragility increases during NDMA-induced hepatic fibrosis, which could be attributed to increased lipid peroxidation of lysosomal membrane. In this study, we also elucidated the mechanism of increased beta-glucuronidase and other lysosomal glycohydrolases in the serum during hepatic fibrosis.     

Lipofuscin Accumulation in Cultured Retinal Pigment Epithelial Cells Causes Enhanced Sensitivity to Blue Light Irradiation

Lipofuscin accumulates with age within secondary lysosomes of retinal pigment epithelial (RPE) cells of humans and many animals. The autofluorescent lipofuscin pigment has an excitation maximum within the range of visible blue light, while it is emitting in the yellow-orange area. This physico-chemical property of the pigment indicates that it may have a photo-oxidative capacity and, consequently, then should destabilize lysosomal membranes of blue-light exposed RPE. To test this hypothesis, being of relevance to the understanding of age-related macular degeneration, cultures of heavily lipofuscin-loaded RPE cells were blue-light–irradiated and compared with respect to lysosomal stability and cell viability to relevant controls. To rapidly convert primary cultures of RPE, obtained from neonatal rabbits, into aged, lipofuscin-loaded cells, they were allowed to phagocytize artificial lipofuscin that was prepared from outer segments of bovine rods and cones. Following blue-light irradiation, lysosomal membrane stability was measured by vital staining with the lysosomotropic weak base, and metachromatic fluorochrome, acridine orange (AO). Quantifying red (high AO concentration within intact lysosomes with preserved proton gradient over their membranes) and green fluorescence (low AO concentration in nuclei, damaged lysosomes with decreased or lost proton gradients, and in the cytosol) allowed an estimation of the lysosomal membrane stability after blue-light irradiation. Cellular viability was estimated with the delayed trypan blue dye exclusion test. Lipofuscin-loaded blue-light–exposed RPE cells showed a considerably enhanced loss of both lysosomal stability and viability when compared to control cells. It is concluded that the accumulation of lipofuscin within secondary lysosomes of RPE sensitizes these cells to blue light by inducing photo-oxidative alterations of their lysosomal membranes resulting in a presumed leakage of lysosomal contents to the cytosol with ensuing cellular degeneration of apoptotic type. The suggested mechanism may have bearings on the development of age-related macular degeneration. © 1997 Elsevier Science Inc.     

β-Glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are β-glucuronidase (β-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that β-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas β-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, β-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that β-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed. This study was funded by the University of the Basque Country through a grant to Consolidated Research Groups. U.I. is a recipient of a pre-doctoral fellowship from the Basque Government.     

Lysosomal and Autophagic Reactions as Predictive Indicators of Environmental Impact in Aquatic Animals

The lysosomal-autophagic system appears to be a common target for many environmental pollutants as lysosomes accumulate many toxic metals and organic xenobiotics, which perturb normal function and damage the lysosomal membrane. In fact, lysosomal membrane integrity or stability appears to be an effective generic indicator of cellular well-being in eukaryotes: in bivalve molluscs and fish, stability is correlated with many toxicological and pathological endpoints. Prognostic use of adverse lysosomal and autophagic reactions to environmental pollutants has been explored in relation to predicting cellular dysfunction and health in marine mussels, which are extensively used environmental sentinels. Derivation of explanatory frameworks for prediction of pollutant impact on health is a major goal; and we have developed a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells and tissues. This model has also complemented the creation of a cell-based computational model for molluscan hepatopancreatic cells that simulates lysosomal, autophagic and other cellular reactions to pollutants. Experimental and simulated results have also indicated that nutritional deprivation - induced autophagy has a protective function against toxic effects mediated by reactive oxygen species (ROS). Finally, coupled measurement of lysosomal-autophagic reactions and modelling is proposed as a practical toolbox for predicting environmental risk.

Addendum to:

Environmental Prognostics: An Integrated Model Supporting Lysosomal Stress Responses as Predictive Biomarkers of Animal Health Status

M.N. Moore, J.I. Allen and A. McVeigh

Mar Environ Res 2005; In press     

Studies on the subcellular localization and properties of bis(monoacylglyceryl)phosphate biosynthesis in rat liver.

Phosphatidylglycerol conversion to bis(monoacylglyceryl)phosphate by rat liver homogenate was studied and maximum rates of synthesis were observed at pH 4.4. The distribution of bis(monoacylglyceryl)P synthetase in rat liver subcellular fractions was determined, and evidence is presented establishing the lysosomes as the site of bis(monoacylglyceryl)P synthesis. In addition to phosphatidylglycerol, 1-acyl- and 2-acyllysophosphatidylglycerol also served as precursors for bis(monoacylglyceryl)P with lysosomes as an enzyme source. Bis(monoacylglyceryl)P synthesis did not require high energy intermediates or cofactors. The possibility that a lysosomal phospholipase A with acyl transferase activity catalyzes the formation of bis(monoacylglyceryl)P was investigated. Heat stability and inhibitor studies suggested that this is probably not the case. Lysosomes were shown to be unable to synthesize phosphatidylglycerol, and lipid analyses showed that lysosomes do not contain phosphatidylglycerol or lysophosphatidylglycerol. Bis(monoacylglyceryl)P synthesis in the cell may require the interaction of lysosomes with a phosphatidylglycerol-containing membrane.     

Enhancement of lysosomal proton permeability induced by photooxidation of membrane thiol groups

Effects of photooxidation of membrane thiol groups on lysosomal proton permeability were studied by measuring intralysosomal pH with fluorescein isothiocyanate-dextran and monitoring proton leakage with p-nitrophenol. Methylene blue-mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linking of the membrane proteins, which was established by the measurement of residual membrane thiol groups with 5,5'-dithio-bis(2-nitrobenzoic acid) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The cross-linking of proteins could be abolished by subsequent treatment of the photodamaged lysosomes with dithiothreitol, indicating that the proteins were linked via disulfide bonds. In addition, the photodamage of lysosomes raised the intralysosomal pH and caused leakage of the lysosomal protons, which could also be reversed by subsequent dithiothreitol treatment. This indicates that lysosomal proton permeability can be increased by photooxidation of the membrane thiol groups and recovered to the normal level by reduction of the groups.     

Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes

Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and beta-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40-GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.     

Glycoprotein catabolism in rat liver. Lysosomal digestion of iodinated asialo-fetuin

(125)I-labelled asialo-fetuin, administered intravenously, rapidly accumulates in rat liver and the radioactivity is subsequently cleared from the liver within 60min. Plasma radioactivity reaches a minimum between 10 and 15 min after injection and rises slightly during the period of liver clearance. Free iodide is the only radioactive compound found in plasma during this latter period. Fractionation of rat liver at 5 and 13min after injection of (125)I-labelled asialo-fetuin supports the hypothesis that asialo-glycoprotein is taken into liver by pinocytosis after binding to the plasma membrane and is then hydrolysed by lysosomal enzymes. At 5min, radioactivity was concentrated 23-fold in a membrane fraction similarly enriched in phosphodiesterase I, a plasma-membrane marker enzyme, whereas at 13min the radioactivity appeared to be localized within lysosomes. Separation of three liver fractions (heavy mitochondrial, light mitochondrial and microsomal) on sucrose gradients revealed the presence of two populations of radioactive particles. One population banded in a region coincident with a lysosomal marker enzyme. The other, more abundant, population of radioactive particles had a density of 1.13 and contained some phosphodiesterase, but very little lysosomal enzyme. These latter particles appear to be pinocytotic vesicles produced after uptake of the asialo-fetuin bound by the plasma membrane. Lysosomal extracts extensively hydrolyse asialo-fetuin during incubation in vitro at pH4.7 and iodotyrosine is completely released from the iodinated glycoprotein. Protein digestion within lysosomes was demonstrated by incubating intact lysosomes containing (125)I-labelled asialo-fetuin in iso-osmotic sucrose, pH7.2. The radioactive hydrolysis product, iodotyrosine, readily passed through the lysosomal membrane and was found in the external medium. These results are not sufficient to account for the presence of free iodide in plasma, but this was explained by the observation that iodotyrosines are deiodinated by microsomal enzymes in the presence of NADPH.     

Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content

Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.     

ErbB2-associated changes in the lysosomal proteome

Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far depended on purification methods based on either density gradient centrifugations or magnetic purification of iron-loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar-type H(+) -ATPase. Quantitative MS-based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal membrane proteome was significantly altered by the ectopic expression of an active form of the ErbB2 oncogene, which renders the cells highly metastatic. The furthermost ErbB2-associated changes included increased levels of CD63, S100A11 and ferritin heavy chain. Overall, our data introduce the antibody-based purification of lysosomes as a suitable method for the characterization of lysosomes from a variety of pathological conditions with altered lysosomal density and stability.     

Mechanism of cytosol phospholipase C and sphingomyelinase-induced lysosome destabilization

Lysosomal disintegration may cause apoptosis, necrosis and some diseases. However, mechanisms for these events are still unclear. In this study, we measured lysosomal beta-hexosaminidase free activity, membrane potential and intralysosomal pH. The results revealed that the cytosolic extracts of rat hepatocytes could increase the lysosomal permeability to both potassium ions and protons, and osmotically destabilize lysosomes via K(+)/H(+) exchange. The effects of cytosol on lysosomes could be completely abolished by D609, which inhibited both phospholipase C and sphingomyelinase, and partly prevented by sphingomyelinase inhibitor Ara-AMP, but not by the inhibitors of PLA(2). Moreover, purified phospholipase C could destabilize the lysosomes while phospholipase A(2) and phospholipase D did not produce such effects. The cytosolic phospholipases hydrolyzed lysosomal membrane phospholipids by 50%, which could be prevented by D609. Disintegration of the cytosol-treated lysosomes biphasically depended on the cytosolic [Ca(2+)]. The cytosol did not disintegrate lysosomes below 100 nM or above 10 muM cytosolic [Ca(2+)], but markedly destabilized lysosomes at about 340 nM [Ca(2+)]. The results suggest that cytosolic phospholipase C and sphingomyelinase may be responsible for the alterations in lysosomal stability by increasing the ion permeability.     

In vitro response of hepatic lysosomes to endogenous and exogenous cationic compounds

An investigation of the effect of four cationic compounds on rat liver lysosomes was carried out. Lysosomes from homogenized rat liver were isolated by differential centrifugation at 0-5 degrees C in 0.3 M sucrose. These lysosomes were then incubated for 1 hr at 37 degrees C in 0.25 M glycine solution containing widely varied concentrations of the test agent. The lysosomes were resedimented and the N-acetyl-beta-glucosaminidase (NAG) activity was measured in the supernatant and in the remaining pellet after disruption. Spermine, ferric ion, mepacrine, and gentamicin all produced dose-dependent effects on these lysosomes. Low concentrations of these compounds inhibited the release of NAG into the supernatant while high concentrations enhanced the release of NAG. This effect of these cationic compounds on the lysosomal membrane may be a mechanism by which they produce cellular toxicity with the organ or tissue selectivity being related to the distribution of the cation.     

Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry

The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time -a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift -was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonie medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 μM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.     

Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry. Effect of silver lactate and hypotonic conditions

The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time - a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift - was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonic medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 microM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.     

Isolation of highly purified rat liver lysosomal membranes using two Percoll gradients

Normal rat liver lysosomal membranes in the form of membrane vesicles have been purified using Percoll density gradient centrifugation. Lysosomes (density = 1.111) were purified approximately 63 +/- 12-fold (mean +/- standard deviation, n = 5) using a gradient of Percoll made isotonic with sucrose and buffered to pH 7.0. These lysosomes were then exposed to 10 mM methionine methyl ester, pH 7.0, the uptake of which resulted in swelling and breakage of the lysosomes with subsequent vesicle formation. These vesicles (density = 1.056) were further separated from residual mitochondrial and plasma membrane enzyme activities using a second Percoll density gradient. Marker enzyme analysis and electron microscopy indicated that the lysosomal membranes were essentially free of both beta-hexosaminidase, a soluble lysosomal enzyme, and contaminating organelles. The specific activity of lysosomal ATPase in the lysosomal membranes was fourfold greater than in the intact lysosomes.     

Influence of membrane physical state on lysosomal potassium ion permeability

Lysosomal permeability to potassium ions is an important property of the organelle. Influence of the membrane physical state on the potassium ion permeability of isolated lysosomes was assessed by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and monitoring the lysosomal proton leakage with p-nitrophenol. The membrane fluidity of lysosomes was modulated by treatment with membrane fluidizer benzyl alcohol and rigidifier cholesteryl hemisuccinate. Changes in the membrane order were examined by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The measurements of membrane potential and proton leakage demonstrated that the permeability of lysosomes to potassium ions increased with rigidification of their membranes by cholesteryl hemisuccinate treatment at 37 degrees C, and decreased with fluidization of their membranes by benzyl alcohol treatment at 2 degrees C. The changes in ion permeability could be recovered by fluidizing the rigidified membranes and rigidifying the fluidized membranes. The results suggest that the physical states of lysosomal membranes play an important role in the regulation of their K(+) permeability.