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.     

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.     

The use of acridine orange cytofluorometry in the study of macrophage lysosomal exocytosis

We present a rather simple cytofluorometric technique for the study of exocytosis of lysosomal contents from individual cultured cells. It is based on the use of the lysosomotropic weak base acridine orange (AO) which, in its stacked form, as it occurs within lysosomes, emits red fluorescence when excited by blue light. Mouse peritoneal macrophages were cultured for 48 h and, after 2 h in serum-free medium, stained with AO. The cells were then exposed to F10-medium with or without newborn calf serum (NCS), zymosan A (Z) or cytochalasin B (CB) for different times at 20 or 37 degrees C. After staining, the macrophages showed no change in red fluorescence intensity, if stored at room temperature in the dark. If, however, the cells were kept in the incubator at 37 degrees C, the cells showed slightly decreasing red fluorescence intensity with time. This decrease was markedly potentiated by the presence of NCS, Z or CB, which are known to induce secretion of lysosomal enzymes from macrophages in vitro. Selective lysosomal enzyme release was confirmed biochemically during treatment with zymosan A. The technique presented here may be of value in further studies on the stimulation of, and the mechanisms behind, lysosomal exocytosis in cultured cells.     

Dimethylsulfoxide increases the survival and lysosomal stability of mouse peritoneal macrophages exposed to low-LET ionizing radiation and/or ionic iron in culture

Reactive oxygen-derived free radicals form excessively during irradiation of biological structures, but also normally in many cellular oxidative processes, albeit in small amounts. Unless scavenged by protective mechanisms, such radicals may induce peroxidation of polyunsaturated fatty acids resulting in membrane damage. The process may be catalysed to a considerable extent by transitional metals with the capacity to form redox systems, such as Fe3+ in equilibrium Fe2+. In the present study, it is shown that radiation by X-rays and/or exposure to ionic iron (Fe3+) causes decreased survival in parallel with lysosomal labilization of cultured mouse peritoneal macrophages (MPMs). The latter event was demonstrated as a reduced capacity of lysosomes in living MPMs to retain acridine orange during photo-oxidative stress caused by continuous exposure to blue light of short wavelength. The effects of X-irradiation, and/or lysosomal iron-loading, could be counteracted by the addition of the .OH-scavenging drug dimethylsulfoxide (DMSO) to the cell culture medium. The findings suggest that X-irradiation may damage certain sensitive G0 cells, such as Kupffer cells, serous cells of salivary glands and old macrophages, which normally have substantial concentrations of metals within their vacuolar apparatus, possibly by lysosomal damage involving .OH-mediated lipid peroxidation.     

Influence of membrane fluidity modifiers on lysosomal osmotic sensitivity

Since lysosomes are prone to osmotic lysis, we have examined the correlation between their physical state and sensitivity to osmotic challenge, using agents which modify membrane fluidity. The latency loss of beta-hexosaminidase after an incubation in hypotonic sucrose medium was followed under different conditions of membrane fluidity, recorded by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3, 5-hexatriene. Increasing fluidity of the lysosomal membranes with benzyl alcohol (BA) and greater rigidity caused by cholesteryl hemisuccinate (CHS) increased and decreased the enzyme latency loss, respectively. The effects of BA and CHS treatments on osmotic sensitivity were reversible subsequently by reciprocal treatments of the lysosomes with CHS and BA, respectively. The results indicate that the physical state of the membrane does indeed affect lysosomal osmotic stability.     

Effect of the segregation of neutral red, acridine orange and ammonium chloride by L cells (subline LSM) on lysosomal hydrolase activity

Kinetics of Neutral red (NR) and Acridine orange (AO) uptake by cultured L cells (subline LSM) has been studied. It was found that the uptake of both NR and AO, with their constant concentrations in the medium was characterized as a two-phase process. During 2 hours, these cells concentrated as much as 90% of the total amount of NR and AO taken up during the whole incubation period. The segregation and accumulation of NR, AO as well as NH4Cl took place in lysosomes. NR and AO concentrations within the cells exceed by 600 and 400 times, respectively, those in the medium. NR, AO and NH4+ accumulation in cells resulted in inhibition of the activity of the following lysosomal hydrolases: cathepsins B and D, acid lipase, N-acetyl-beta,D-glucosaminidase, beta-galactosidase, acid phosphatase and galactosyltransferase, the latter being a marker of Golgi apparatus. The effect of lysosomal enzyme activity inhibition on the cell economy, and a possible role of lysosomotropic agents as regulators of the lysosomal apparatus functional activity are discussed.     

Concanavalin A and the in vitro induction in macrophages of vacuolation and lysosomal enzyme synthesis

Concanavalin A (ConA) induced extensive vacuolation in mouse peritoneal macrophages. Electron microscopic observations on thin sections reveal that the vacuoles are essentially empty except for minute vesicles attached to their inner periphery. The vacuoles consist of irregular structures and are heterogeneous in size distribution. ConA-induced vacuoles exhibit high acid phosphatase activity, suggesting fusion between vacuoles and lysosomes. Induction of acid phosphatase in ConA-treated macrophages was studied under several cultivation conditions. ConA-treated macrophage cultures responded in increase in acid phosphatase activity early after exposure to the lectin, a significant increase recorded already after 1 h. When cultivated in 1% serum medium for 48 h, ConA-treated macrophages exhibit twice the activity of acid phosphatase at zero time as well as that of non-treated control cultures. The effect of ConA on thioglycolate-stimulated mouse peritoneal macrophages was also studied. Vacuole formation resulting from lectin binding and internalization is discussed in terms of possible lectin effects on membrane fluidity, fusion capacity, surface to volume conservation during vacuole formation, fusion of vacuoles with lysosomes and intravacuolar lysosomal enzyme activities. The phenomenon of lysosomal enzyme induction as a result of ConA treatment is being correlated with enzyme induction due to other stimuli.     

Depletion of secondary lysosomes in mouse macrophages infected with Leishmania mexicana amazonensis: a cytochemical study

Leishmania amastigotes lodge and multiply within parasitophorous vacuoles, which can fuse with secondary lysosomes of the host macrophages. This study examines the effect of infection with amastigotes of L. mexicana amazonensis on the secondary lysosomes of mouse macrophage cultures. The cultures were stained for the activities of two lysosomal enzyme markers, acid phosphatase and arylsulfatase, and the light microscopic observations were supplemented by electron microscopy. Nearly all noninfected macrophages contained numerous stained secondary lysosomes. The number of such lysosomes was markedly reduced 24 h postinfection, and the reduction persisted for at least 10 days. Stained secondary lysosomes reappeared after the amastigotes were destroyed by exposure of the cultures to phenazine methosulfate or by placing them at 37.5 degrees C. The depletion of lysosomes shown by cytochemical methods may reflect a high rate of fusion of the lysosomes with the parasitophorous vacuoles, exceeding the rate of formation of new secondary lysosomes. Alternatively, the parasites may inhibit the synthesis of lysosomal hydrolases, or the assembly or formation of primary or secondary lysosomes.     

A novel method of imaging lysosomes in living human mammary epithelial cells

Cancer cells invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The ability to noninvasively label lysosomes and track lysosomal trafficking would be extremely useful to understand the mechanisms by which degradative enzymes are secreted in the presence of pathophysiological environments, such as hypoxia and acidic extracellular pH, which are frequently encountered in solid tumors. In this study, a novel method of introducing a fluorescent label into lysosomes of human mammary epithelial cells (HMECs) was evaluated. Highly glycosylated lysosomal membrane proteins were labeled with a newly synthesized compound, 5-dimethylamino-naphthalene-1-sulfonic acid 5-amino-3,4,6-trihydroxy-tetrahydro-pyran-2-ylmethyl ester (6-O-dansyl-GlcNH2). The ability to optically image lysosomes using this new probe was validated by determining the colocalization of the fluorescence from the dansyl group with immunofluorescent staining of two well-established lysosomal marker proteins, LAMP-1 and LAMP-2. The location of the dansyl group in lysosomes was also verified by using an anti-dansyl antibody in Western blots of lysosomes isolated using isopycnic density gradient centrifugation. This novel method of labeling lysosomes biosynthetically was used to image lysosomes in living HMECs perfused in a microscopy-compatible cell perfusion system.     

Mechanism of Lysophosphatidylcholine-Induced Lysosome Destabilization

Lysosomal destabilization is critical for the organelle and living cells. Phospholipase A₂ (PLA₂) was shown to be able to destabilize lysosomes under some conditions. By what mechanism the enzyme affects lysosomal stability is not fully studied. In this study, we investigated the effects of lysophosphatidylcholine (lysoPC), a PLA₂-produced lipid metabolite, on lysosomal ion permeability, osmotic sensitivity and stability. By measuring lysosomal β-hexosaminidase free activity, membrane potential, proton leakage and their enzyme latency loss in hypotonic sucrose medium, we established that lysoPC could increase the lysosomal permeability to both potassium ions and protons and enhance lysosomal osmotic sensitivity. These changes in lysosomal membrane properties promoted entry of potassium ions into lysosomes via K⁺/H⁺ exchange. The resultant osmotic imbalance across the membranes led to losses of lysosomal integrity. The enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. These results suggest that lysoPC may play a key role in PLA₂-induced lysosomal destabilization.     

The position of lysosomes within the cell determines their luminal pH

We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H⁺–adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.     

Endocytic pathways and time sequence of lysosomal transfer of macromolecules in cultured mouse peritoneal macrophages

Summary A double-labeling protocol was used to study endocytic pathways and lysosomal transfer of exogenous macromolecules in cultured mouse peritoneal macrophages. After pulse-chase labeling of lysosomes with horseradish peroxidase (visualized cytochemically), the cells were exposed to native, anionic ferritin for 0–45 min at 37° C and then analysed by transmission electron microscopy. The results show that ferritin binds to the plasma membrane, accumulates in coated pits, and is rapidly taken up in small, smooth-surfaced endocytic vesicles. The latter carry the ferritin molecules directly to lysosomes, recognized by their peroxidase labeling, or fuse with each other to form larger endocytic vacuoles (endosomes) which in turn fuse with and empty their content into lysosomes. The first signs of transfer of ferritin into the lysosomes were seen after 5–10 min of exposure and after 25–30 min most of the lysosomes were labeled. Union of ferritin-labeled and other lysosomes was also noted, suggesting that the contents of the lysosomes were spread within the lysosomal compartment by fusion-fission processes. It is concluded that a multiplicity of structures is involved in the uptake and intracellular transport of exogenous macromolecules in macrophages and that the time sequence of lysosomal transfer of the interiorized material is highly variable.     

Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

Quantitative responses of the digestive-lysosomal system of winkles to sublethal concentrations of cadmium

The effects of the experimental exposure to sublethal concentrations of cadmium on the digestive gland lysosomal system of the marine prosobranch Littorina littorea have been studied by means of stereology in fresh frozen cryotome sections after demonstration of beta-glucuronidase activity. The volume density of secondary lysosomes was demonstrated to be independent of both the external concentration of the metal and the exposure-time. However, some punctual increases in this parameter have been related to the alternate renewal of the tissular population of this organelle. The lysosomal surface density showed a dose- and time-dependent significant decrease over the controls. The lysosomal surface to volume ratio increased over the time in the control series whilst decreased significantly in Cd-exposed animals at each sampling period. Lysosomal numerical density was strongly dependent on the external concentration of the metal, changes in this parameter showing the highest signification. It is concluded that sublethal exposure to cadmium leads to fusion of secondary lysosomes to give larger ones. This process is related to lysosomal membrane destabilisation, which could take place after the storage capacity of the organelles have been overloaded.     

Importance of the functional state of alveolar macrophages of the lungs for hygienic evaluation of protective reactions and cell damage due to atmospheric pollution

Total number of cells, their viability and ability to adhesion were examined in surface alveolar macrophages isolated from rat livers after exposure to sulphur dioxide during 2, 4 and 6 weeks (0.05, 0.5, 1.0 and 5.0 mg/m3); to nitrogen oxide during 5, 8 and 15 hours, 28 and 56 days (19 mg/m3) and to carbon monoxide during 2, 28 and 56 days (0.01% or 10 MAC). In the experiment with exposure to sulphur dioxide, the activity of enzymes of varying localization in the macrophages - soluble in the cytoplasm (lactate dehydrogenase) and connected with subcellular structures - lysosomes (beta-galactosidase, beta-glucosidase and acid phosphatase) was tested by means of biochemical methods in parallel with cytological examinations. Low concentrations of various chemical contaminants of the atmospheric air (sulphur dioxide, nitrogen oxides, carbon monoxide) have an unfavourable biological effect on rats, manifest in the impairment of local immunity, i.e., decreased number of alveolar macrophages, disturbance of their viability and reduced ability of the macrophages to adhesion. At the same time, sulphur dioxide induces enzyme disorganization in lactate dehydrogenase and in a number of lysosomal enzymes of the macrophages. These results serve as a basis for the recommendation of cytobiochemical methods of elaborating methodological approaches to the regulation of environmental factors. Alveolar macrophages as a constituent part of the mononuclear phagocytic system ensuring local non-specific and specific resistance of the organism form one of the most important cellular mechanisms of protection of the organism against the harmful effect of environmental factors including chemical contaminants of the atmospheric air (1, 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

The lysosomal proton pump is electrogenic

Lysosomes were purified approximately 40-fold from rat kidney cortex by differential and Percoll density gradient centrifugation. In a sucrose medium, the lysosomes quenched the fluorescence of the potential sensitive dye diS-C3-(5) (3,3'-dipropylthiocarbo-cyanine iodide) in a time-dependent manner, indicating that the dye accumulates within the lysosomal interior. After treatment of the lysosomes with valinomycin, the dye fluorescence displayed a logarithmic dependence upon the external K+ concentration; thus, the fluorescence signal provides a semiquantitative measure of the lysosomal membrane potential (delta psi). In the absence of valinomycin, lysosomal quenching of diS-C3-(5) fluorescence was partially reversed by agents which collapse the lysosomal pH gradient (ammonium sulfate, chloroquine, and K nigericin), suggesting that the proton gradient across the lysosomal membrane contributes to delta psi. A rapid increase in diS-C3-(5) fluorescence, indicative of an increase in delta psi, was observed upon the addition of Mg-ATP to the lysosomes. The ATP-dependent fluorescence change was inhibited by protonophores, K valinomycin, permeable anions, and N-ethylmaleimide, but was unaffected by ammonium sulfate, K nigericin, or sodium vanadate. Oligomycin had no effect at concentrations below 2 micrograms/ml; at higher concentrations, oligomycin partially inhibited the fluorescence response to Mg-ATP, but it also inhibited the fluorescence response to K valinomycin, suggesting that it had modified the permeability of the lysosomal membrane. Dicylohexylcarbodiimide behaved similarly to oligomycin. Mg-ATP also altered the lysosomal distribution of 86Rb+ (in the presence of valinomycin) and S[14C]CN-, consistent with an increase in the potential of the lysosomal interior of 40-50 mV. The results demonstrate that the lysosomal proton pump is electrogenic.     

Lead induced injury on in vitro cultured rat fibroblasts

Summary The mechanism for lead induced cellular injury was studied on in vitro cultured rat fibroblasts, treated with lead in a dose that caused demonstrable cellular alterations within a couple of days. The changes were studied by means of methods for the histochemical demonstration of heavy metals and lysosomal membrane latency.The lead, added to the cultivation medium, was quickly incorporated into presumed secondary lysosomes of the fibroblasts and caused regressive cellular changes such as cytoplasmic vacuolization, nuclear pycnosis and eventually cell death. The lead exposure resulted in reduced lysosomal membrane latency and signs of enzyme and lead leakage to the cytoplasm.The cell damage might be mediated through lysosomal membrane alteration resulting in reduced latency and presumably leakage of lytic enzymes.Supported by the Swedish Medical Research Council (grant no 12 X-2037).     

Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.     

A new fluorescent pH probe for imaging lysosomes in living cells

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5–4.1 with the pK_a value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no ‘alkalizing effect’ on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells.     

Acid sphingomyelinase-deficient macrophages have defective cholesterol trafficking and efflux

Cholesterol efflux from macrophage foam cells, a key step in reverse cholesterol transport, requires trafficking of cholesterol from intracellular sites to the plasma membrane. Sphingomyelin is a cholesterol-binding molecule that transiently exists with cholesterol in endosomes and lysosomes but is rapidly hydrolyzed by lysosomal sphingomyelinase (L-SMase), a product of the acid sphingomyelinase (ASM) gene. We therefore hypothesized that sphingomyelin hydrolysis by L-SMase enables cholesterol efflux by preventing cholesterol sequestration by sphingomyelin. Macrophages from wild-type and ASM knockout mice were incubated with [(3)H]cholesteryl ester-labeled acetyl-LDL and then exposed to apolipoprotein A-I or high density lipoprotein. In both cases, [(3)H]cholesterol efflux was decreased substantially in the ASM knockout macrophages. Similar results were shown for ASM knockout macrophages labeled long-term with [(3)H]cholesterol added directly to medium, but not for those labeled for a short period, suggesting defective efflux from intracellular stores but not from the plasma membrane. Cholesterol trafficking to acyl-coenzyme A:cholesterol acyltransferase (ACAT) was also defective in ASM knockout macrophages. Using filipin to probe cholesterol in macrophages incubated with acetyl-LDL, we found there was modest staining in the plasma membrane of wild-type macrophages but bright, perinuclear fluorescence in ASM knockout macrophages. Last, when wild-type macrophages were incubated with excess sphingomyelin to "saturate" L-SMase, [(3)H]cholesterol efflux was decreased. Thus, sphingomyelin accumulation due to L-SMase deficiency leads to defective cholesterol trafficking and efflux, which we propose is due to sequestration of cholesterol by sphingomyelin and possibly other mechanisms. This model may explain the low plasma high density lipoprotein found in ASM-deficient humans and may implicate L-SMase deficiency and/or sphingomyelin enrichment of lipoproteins as novel atherosclerosis risk factors.