Cell Biology International 2000, Vol. 24, No. 10, 699–704doi:10.1006/cbir.2000.0552, available online at http://www.idealibrary.com on

INFLUENCE OF MEMBRANE FLUIDITY MODIFIERS ON LYSOSOMAL OSMOTICSENSITIVITY

LU YANG, GUO-JIANG ZHANG*, YI-GANG ZHONG and YAN-ZHEN ZHENG

Department of Cellular Biophysics, Institute of Biophysics, Academia Sinica, Beijing, 100101, P. R. China

Received 5 November 1999; accepted 22 March 2000

Since lysosomes are prone to osmotic lysis, we have examined the correlation between theirphysical state and sensitivity to osmotic challenge, using agents which modify membranefluidity. The latency loss of �-hexosaminidase after an incubation in hypotonic sucrose mediumwas followed under different conditions of membrane fluidity, recorded by steady-statefluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Increasing fluidity of the lysosomalmembranes 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 CHStreatments on osmotic sensitivity were reversible subsequently by reciprocal treatments of thelysosomes with CHS and BA, respectively. The results indicate that the physical state ofthe membrane does indeed affect lysosomal osmotic stability. � 2000 Academic Press

K: lysosomes; membrane fluidity; osmotic sensitivity.A: BA: benzyl alcohol; CHS: cholesteryl hemisuccinate; DPH:1,6-diphenyl-1,3,5-hexatriene.

*To whom correspondence should be addressed: Guo-jiang Zhang,Department of Cellular Biophysics, Institute of Biophysics, AcademiaSinica, Beijing, 100101, P. R. China. E-mail: zhangzl@sun5.ibp.ac.cn

INTRODUCTION

The lysosome acts as an intracellular ‘osmometer’,being susceptible to osmotic destabilization (Lloydand Forster, 1986). In the past, a number of studieshave focused on lysosomal osmotic stability, suchas the K+ entry-induced osmotic stress and theosmotic protection to lysosomes by their H+-ATPase-mediated proton translocation (Ruth andWeglicki, 1983; Yao and Zhang, 1996, 1997; Zhangand Yao, 1997). While the results indicate thatlysosomal integrity is very sensitive to osmoticchanges, little information is available on howlysosomal membrane fluidity per se affect thisproperty.

Membrane lipid fluidity can be modulated bya wide range of physiological variables, such asfatty acid composition (Kuo et al., 1990), aging(Miyamoto et al., 1990), alcohols (Edelforsand Ravn-Jonsen, 1990), phosphatidylethanol(Omodeo-Sale et al., 1991), sterols (Schuler et al.,1990), insecticides (Antunes-Madeira et al., 1989),

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diacylglycerols (Ortiz et al., 1988), drug-inducedcytochrome P-450 activity (Kawato et al., 1991)and ether lipids (Kaufman et al., 1990). Manydifferent physiological and biochemical propertiesof membranes, such as permeability andmembrane-bound enzyme activity, can be regu-lated by the changes in fluidity (Shinitzky, 1984).Since the mutual regulations between membranelipid fluidity and cellular activities play an import-ant role in cell life, clarifying the influence of thevarious factors which impinge on membranefluidity to affect the latter continues to be an activearea of investigation.

Lysosomal membrane fluidity changes undervarious conditions such as the onset of apoptosis,following episodes of lipid peroxidation, or theaccumulation of polyanions in lysosomes (Zhanget al., 2000). It was of interest to establish whetherthe membrane fluidity of lysosomes has majoreffects on the osmo-sensitivity of these organelles.Here we have changed lysosomal membranefluidity with benzyl alcohol (BA) and cholesterylhemisuccinate (CHS; see Zhang et al., 2000; Zhonget al., 2000), and examined the effects on osmoticsensitivity.

� 2000 Academic Press

700 Cell Biology International, Vol. 24, No. 10, 2000

MATERIALS AND METHODS

Chemicals

DPH, CHS and 4-methylumbelliferyl N-acetyl-�-D-glucosaminide were from Sigma (St Louis,MO, U.S.A.). The other chemicals used were ofanalytical grade from local sources.

Preparation of lysosomes

Rat liver lysosomes were isolated as previouslydescribed (Zhang and Yao, 1997). The preparedlysosomes were suspended in 0.25 sucrosemedium at 23.6 mg protein/ml and stored at 2–4�C.

Modulation of lysosomal membrane fluidity

Lysosomal membrane fluidity was modulated asrecently described (Zhang et al., 2000; Zhong et al.,2000). To increase fluidity, lysosomes were treatedwith 20 m BA at 37�C for the indicated time.To decrease the fluidity of BA-treated lysosomalmembranes, the lysosomes were subsequentlytreated with 0.5 m CHS for the given times. Todecrease membrane fluidity, lysosomes were incu-bated with 0.5 m CHS at 37�C for the indicatedtimes. To recover the fluidity of CHS-treated lyso-somal membranes, the lysosomes were subse-quently treated with 20 m BA for indicated time.Control samples in these two cases were incubatedat 37�C for the same time in the absence of BA orCHS.

Steady-state fluorescence anisotropy measurement

Lysosomal membrane fluidity was measured asdescribed recently (Zhang et al., 2000; Zhong et al.,2000). Briefly, the lysosomes (0.59 mg protein/ml)were incubated in DPH (4 �) labelling solutionat 37�C for 90 min. Fluorescence of DPH wasmeasured on a Hitachi 850 fluorescence spectro-photometer with excitation and emission at 350and 452 nm, respectively. Steady-state fluorescenceanisotropy (r) was calculated according to theequation:

r=(IVV�GIVH)/(IVV+2GIVH) (1)

where IVV and IVH are the fluorescence intensitiesmeasured with the excitation polarizer in the verti-cal position and the analyzing emission polarizermounted vertically and horizontally, respectively.G=IHV/IHH is the correction factor. Correction

for light scattering was carried out as described byLitman (Stubbs et al., 1976; Litman and Barenholz,1982) and Lentz (Lentz et al., 1979). As pointed byVan Blitterswijk et al. (1981), high degrees offluorescence anisotropy indicate higher degrees ofmembrane order or lower degrees of membranefluidity, and vice versa.

Assay of lysosomal osmotic sensitivity

The osmotic sensitivity of lysosome preparationswas assessed by examining integrity after an incu-bation in hypotonic sucrose medium by the methodof Zhang and Yao (1997). Briefly, lysosomalsample (56 �l for BA-treated lysosomes and con-trol, 62 �l for CHS-treated lysosomes and control)was incubated in 0.5 ml 0.1 sucrose medium(1.93 mg protein/ ml) at 2�C for 10 min, then a50 �l portion of this lysosomal suspension was usedfor the assay of lysosomal integrity.

Assay of lysosomal integrity

Lysosomal integrity was assessed as describedpreviously by measuring the activity oflysosomal �-hexosaminidase using 2 m4-methylumbelliferyl N-acetyl-�-D-glucosaminideas substrate (Yao and Zhang, 1997; Zhang andYao, 1997). The liberated 4-methylumbelliferonewas determined by measuring the fluorescence(excitation: 365 nm, emission: 444 nm) with aHitachi 850 fluorescence spectrophotometer.

Activities of the enzyme measured in the absenceand presence of Triton X-100 are designated thefree activity and the total activity, respectively.Percentage free activity was calculated as (freeactivity/total activity)�100. Lysosomal enzymelatency can be defined as [1�(free activity/totalactivity)]�100. Loss of lysosomal integrity wasdetermined as loss of lysosomal enzyme latency orincreased percentage free activity. The enzymelatency of freshly prepared lysosomes is about9%, which can be maintained for at least 3 h at2–4�C.

RESULTS

Modulation of lysosomal membrane fluidity

Lysosomal membrane fluidity was modulated bythe treatments with membrane fluidizer BA andrigidifier CHS. As shown in Figure 1, the degree offluorescence anisotropy (r) decreased and increasedafter the lysosomes were treated with BA and CHS,

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respectively. Increasing the treating time of bothBA and CHS produced larger changes in thefluorescence anisotropy (r). It indicates that themembrane fluidity was increased and decreased bythe treatments with BA and CHS, respectively. Thefluorescence anisotropy (r) of the BA-treated lyso-somes increased after the lysosomes were subse-quently treated with CHS for 20 min (Table 1),showing that the BA-induced membrane fluidiz-ation was partly reversed by the treatment withCHS. As demonstrated by the results, rigidificationof the CHS-treaded lysosomal membranes couldalso be reversed by the subsequent treatment withBA.

0.1030

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Fig. 1. Effects of BA and CHS treatments on the membrane fluidity of lysosomes. Assessment of lysosomal membrane fluidityby measuring DPH fluorescence anisotropy and the treatments of lysosomes with BA and CHS, as described in Materials andMethods. (A) Lysosomes treated with 20 m BA at 37�C for indicated time. (B) Lysosomes treated with 0.5 m CHS at 37�C forindicated time. Values are means�SD of four measurements.

Table 1.Reversion of the effects of BA and CHS treatments on the fluorescence anisotropy of lysosomes

Treatment of lysosomes Anisotropy (r) P

Controla 0.147�0.002 —20 m BA 30 minb 0.115�0.001 <0.00120 m BA 30 min, then 0.5 m CHS 30 minc 0.130�0.001 <0.001Controld 0.147�0.002 —0.5 m CHS 40 mine 0.164�0.002 <0.0010.5 m CHS 40 min, then 20 m BA 15 minf 0.138�0.001 <0.001

For reversing the effect of BA treatment, lysosomes were treated with 20 m BA for 30 min, thentreated with 0.5 m CHS for 30 min or not during a 60 min incubation at 37�C. For reversing the effectof CHS treatment, lysosomes were treated with 0.5 m CHS for 40 min, then treated with 20 m BA for15 min or not during a 55 min incubation at 37�C. After the treatments, membrane fluidity of thetreated lysosomes was assessed by measuring DPH fluorescence anisotropy (r). All procedures weredetailed in Materials and Methods. Values are means�SD, n=4. Statistical analysis was performedusing Student’s t-test. Note: b vs a, c vs b, e vs d, f vs e.

Effects of lysosomal membrane fluidity on theosmotic sensitivity

Lysosomal membranes are generally impermeableto sucrose (Reeves, 1984). The degree of enzymelatency loss in hypotonic sucrose medium canreflect the lysosomal osmotic sensitivity. As shownin Figure 2A, free �-hexosaminidase activity wasincreased with prolonged BA treatment. Since thelysosomes treated with BA or CHS could maintaintheir latency in isotonic sucrose for more than20 min (data not shown), the lysosomes wereimpermeable to sucrose, BA and CHS. Thereforethe latency loss of the treated lysosomes in

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hypotonic sucrose medium was not caused bythe entry of the solutes. Based on the data inFigure 1A, the results suggest that increasing thefluidity of lysosomal membranes increases theirosmotic sensitivity. This conclusion was supportedby the evidence presented in Table 2. The increasein free enzyme activity of BA-treated lysosomescould be sharply reversed by the subsequent appli-cation of CHS, thereby confirming the finding. Incontrast, decreased membrane fluidity, induced bythe treatment with CHS, stabilized the lysosomesto osmotic challenge. As shown in Figure 2B, freeenzyme activity decreased with increasing CHS-treating time, which shows that the lysosomestreated with CHS were less sensitive to the hypo-tonic stress. Moreover, the CHS effect was reversed

by the subsequent application of BA (Table 2).These results further suggest that making thelysosomal membrane stiffer decreases osmoticsensitivity.

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Fig. 2. Effects of BA and CHS treatments on lysosomal osmotic sensitivity. (A) lysosomes treated with 20 m BA for indicatedtime during a 30 min incubation at 37�C. (B) Lysosomes treated with 0.5 m CHS for indicated time during a 40 min incubationat 37�C. After incubation, the treated lysosomes were suspended in 0.1 sucrose medium (1.93 mg protein/ml) at 2�C for 10 minbefore free betasign-hexosaminidase activity was assessed. Values are means�SD of four measurements.

Table 2.Reversion of the effects of BA and CHS treatments on the osmotic sensitivity of lysosomes

Treatment of lysosomes %Free�-hexosaminidase

activity

P

Controla 50.2�1.3 —20 m BA 30 minb 90.1�0.9 <0.00120 m BA 30 min, then 0.5 m CHS 30 minc 68.1�0.4 <0.001Controld 48.5�0.5 —0.5 m CHS 40 mine 38.6�0.8 <0.0010.5 m CHS 40 min, then 20 m BA 15 minf 61.2�0.9 <0.001

Treatments of lysosomes with BA and CHS were as described in Table 1. After the treatments, thelysosomes were suspended in 0.1 sucrose medium (1.93 mg protein/ml) at 2�C for 10 min. Then,�-hexosaminidase free activity was assessed. All procedures were detailed in Materials and Methods.Values are means�SD, n=4. Statistical analysis was performed using Student’s t-test. Note: b vs a,c vs b, e vs d, f vs e.

DISCUSSION

We have previously shown that lysosomal osmoticsensitivity can be assessed by measuring the enzymelatency loss in hypotonic sucrose medium (Zhangand Yao, 1997). The prerequisite of this assessmentis lysosomal impermeability to sucrose. In order toclarify whether treatments of lysosomes with BAand CHS changed their impermeability towardsucrose (and also whether BA and CHS treatments

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affected lysosomal integrity), latency of treatedlysosomes was examined after 20 min incubation inisotonic sucrose. Since the lysosomal latency wasmaintained throughout the whole period of incu-bation (data not shown), we conclude that imper-meability of lysosomes to sucrose was not affectedby the treatments and that the lysosomes wererelatively impermeable to BA and CHS. Therefore,changes in the latency of the BA or CHS-treatedlysosomes in 0.1 sucrose medium were not, infact, attributable to a solute entry-induced osmoticstress but to an increased osmotic sensitivity medi-ated by membrane changes. The effects of mem-brane fluidity on the lysosomal osmotic sensitivitywas confirmed by the observations that changes inthe osmotic sensitivity of the BA- and CHS-treatedlysosomes could be reversed in either direction bythe subsequent treatment with the opposite agent.This ‘criss-cross’ experiment convincingly demon-strates that very sensitive regulation at the mem-brane level must occur, which is reflected byalteration in water permeability. This is consistentwith previous findings that increases and decreasesin the membrane fluidity can cause increases anddecreases in their water permeability, respectively(Carruthers and Melchior, 1983; Worman andField, 1985; Worman et al., 1986).

Lysosomes incubated at 37�C in isotonic sucrosemedium gradually lose latency by their enzymeaction (Ruth and Weglicki, 1978). Since the lyso-somal samples of Figure 2 were incubated at 37�Cand the incubation time of Figure 2B was morethan that of Figure 2A, free enzyme activity of theuntreated lysosomes of the former is higher thanthat of the latter.

Lysosomal permeability to various moleculesand ions can also be assessed using the osmoticprotection method (Lloyd and Forster, 1986;Forster and Lloyd, 1988). As explained by Lloyd, apermeant solute affords stabilization initially butsince it penetrates into the lysosomes, a progressiveosmotic imbalance develops, resulting in an influxof water and a bursting of the lysosomes. Theextent of lysosomal disintegration in the suspensionreflects their permeability to the solute. Actually,the loss of lysosomal latency correlates with theirpermeability toward both solute and water. Toassess the permeability of lysosomes to a solute,their water permeability must be maintained. Thus,changes in the lysosomal latency depend only onthe variations in their permeability to the assessedsolute. Since membrane fluidity can affect lyso-somal water permeability, the physical state oflysosomal membranes has to be considered inthe osmotic protection measurement to avoid

inaccurate estimation of their permeability to theassessed solute.

In this study, we established that the greater andlesser membrane fluidity of lysosomes can increaseand decrease their osmotic sensitivity, respectively.Since membrane fluidity can be modulated by awide range of physiological variables and thelysosomal membrane fluidity is liable to changeunder various conditions (Zhang et al., 2000), theinfluence of membrane fluidity on the lysosomalosmotic sensitivity probably has a biological sig-nificance. How physiological variables affectingmembrane fluidity influence lysosomal osmoticstability remains for further study.

ACKNOWLEDGEMENT

This study was supported by the project 39770194from NSFS.

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