Effect of cholesterol on membrane microheterogeneity: a study using 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime distributions.

The effect of cholesterol on microheterogeneity of liposomes obtained from saturated and unsaturated phospholipids was studied by measuring the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Data obtained by frequency domain fluorometry have been analyzed either by discrete exponential or continuous lifetime distribution approaches. In egg phosphatidylcholine liposomes, the addition of cholesterol increases the lifetime value or the centre of the lifetime distribution. At high cholesterol concentration, good fits are obtained using a monomodal distribution analysis or single exponential component. At low cholesterol concentration an additional short component of low fractional intensity must be included to obtain a good fit. In dipalmitoylphosphatidylcholine, the addition of cholesterol decreases the long lifetime component centre value both in the gel and in the liquid-crystalline state. The DPH lifetime value is sensitive to the dielectric constant of the probe microenvironment, and cholesterol has been shown to modify water penetration in the bilayer. Using this information our data indicate that cholesterol affects the polarity of the microenvironment in liposomes of unsaturated phosphatidylcholine and saturated phosphatidylcholine in different ways. Although the major conclusions of this paper are obtained using changes of the distribution centre upon cholesterol addition, there are also preliminary indications that the lifetime distribution width decreases as cholesterol is added. We have interpreted this observation as being due to the homogenizing effect of cholesterol.

The effect of cholesterol on mlero~terogeneity of liposomes obtained from saturated and unsaturated phosphollpids was studied by measuring the fluorescence decay of L6-diphenyl-l,3,5-hexatriene (DPH).Data obtained by frequency domain fluomewlry have been analyzed either by discrete exponential or continuous lifetime distribution approaches.In ~khollue lipmmnes, the addition of cholesterol increases the lifetime value or the centre of the lifetime dba'lbutioa.At Idllh cholesterol concentration, good fits are obtained using a monomodal distribution analysis or single expouentlal cempenent.At low cholesterol concentration an additional short component of low fractional intensity must be Indudad to obtain a good fit.In dipalmituylphoSlJatidyicholine, the addition of cholesterol eeereL~s the long lifetime component centre value both in the gel and in the liquid-crystalline state.The DPH lifetime value is sensitive to the dldectrk cemhmt of the probe mlemenvbonment, and cholesterol ires been shown to modify water penetration in the bilayer.Using this infonltlen our dam indicate that cholesterol affects the polarity of the mieroenvironment in lipasmaes el umatt~ted phospbutidylcholine and saturated phusphatidylcholine in different ways.Although the major el this piper are obtained ualng changas of the dis~buthm een~'e upon cholesterol addition, there are also p¢olimhtaff iedleatlous that the lifetime distribution width decreases as cholesterol is added.We have interpreted this observation as bein 8 due to the homogenizing effeet of cholesterol.

ln~
Cholesterol is an important component of biological membranes, constituting up to 505 of the total lipid content.The involvement of cholesterol in physiological events such as homeoviscous adaptation [1] and in pathological processes such as atherosclerosis has stimulated the study of its structural and functional role in natural membranes and model systems.
A number of methodological approaches have been used to study different aspects of cholesterol effect on membrane organization.Spectroscopic studies on liposomes have shown an increase of 'rigidity' of phospho-Abbreviadons-DPH, 1,6-diphenyl-l,3,5-he.~atriene:DPPC, dipalmi.toyl~osphatidylcholine; F~, ¢8g phosphatidylcholine; PL, phospholipid; POl~P, 1,4-bis($-phenyl-2ooxazolyl)bcnzene; THF, tetrahydrofuran; FWHM, full width at half maximum.Cocresponden~: R. Fiofini, lstituto dl Biochimica, Facolfft di Medicina, Via Ranieri, 65, 60131 Ancona, Italy.lipid fatty acyl chains in the presence of cholesterol [2,3].Moreover, using differential scanning calorimetry a decrease of order has been detected in the gel phase [4].From the observations of the different effect of cholesterol in gel and liquid crystalline phases a possible role of this molecule has been proposed in which cholesterol controls an intermediate-state fluidity in natural membranes [5].In LM cells this hypothesis is supported by the disappearance of phase separation when cholesterol is added to previously cholesterol-depleted cells [6].A similar effect has been shown in cholesterol-depleted erythrocytes, in which there is a segregation of protein rich domains [7].In previous studies [8] using DPH fluorescence decay we have shown that, in the erythrocyte membrane, cholesterol depletion induces an increase of membrane microheterogeneity, as evaluated from the lifetime distribution width.We proposed that the origin of the lifetime distribution is due to different dielectric microenvironments in which the DPH molecules are located [9].Therefore the homogenizing effects of cholesterol observed in erythrocyte membrane have been related to changes in membrane water permeability [10].A possible role has been ascribed to the hydroxyl group, since different 3-OHblocked cholesterol derivatives are unable to affect some physicochemical properties of lipid bilayer [11]. in this respect, Demel et al. [12] have pointed out that the presence and the orientation of the oxygen function are the principal factors to observe a sterol-like effect in membranes.
Theoretical considerations and experimental evidence suggest that biological membrane are organized into domains of specific composition and properties [8,13]; however, this structurai aspect of membrane organization is at present difficult to define on a molecular basis due to methodological and technical limitations.
To investigate the role of cholesterol in controlling the degree of membrane heterogeneity we studied the modification induced by this molecule on liposomes of unsaturated and saturated phospholipids using the changes produced on the fluorescence decay of DPH The decay was measured using multifrequency phase fluorometry, which we have shown to be a suitable technique to describe membrane heterogeneity by means of the distribution analysis of complex excited state decay [8,91.The results are discussed in relation to the changes of membrane order as detected by steady-state anisotropy.

Materials
Cholesterol was obtained from Sigma, St. Louis (MO), EPC and DPPC were obtained from Avanti Polar Lipids, Birmingham (AL).DPH was obtained from Molecular Probes, Eugene (OR).All the lipids and probe were used without further purification.

Methods
MultilameUar iiposomes were prepared by drying both pure lipids and mixtures under N 2, resuspending them in 10 mM Hepes/100 mM KC! (pH 7.4) and shaking the lipid suspension vigorously.
DPH previously dissolved in tetrahydrofuran was added to the sample to produce a probe to phospholipid molar ratio of 1 : 1000.The suspensions were incubated for 1 h in the dark at room temperature.The effect of very small addition of THF was separately studied.At the concentration used in this work no effect on the spectroscopic properties of DPH was detected.
The background fluorescence was checked prior to each measurement, and was less than 0.5% of ~he fluoro escence when DPH was added.
Steady-state fluorescence anisotropy measurements were performed with a Perkin Elmer Fluorescence Speetrophotometer MPF-66 equipped with Series 7000 Pro-fessional Computer.The sample was excited by polarized light at 360 nm and the n~orescence aniso~ropy was detected at 430 nm by u~mg a cut-off filter at 390 am.
DPH lifetime measurements were performed with the multifrequency phase fluorometer described by Oratton and Limkeman [14], equipped with an ISS-ADC interface for data collection and analysis.The wavelength of excitation was set at 325 nm (ultraviolet line of an HeCd Lieonix Model 4240NB laser) and a large range of modulation frequencies (from 2 to 70 MHz) were used.Data were acumulated at each modulation frequency until the standard deviations of the phase and modulation values were below 0.1 ° and 0.002, respectively.
All lifetime measurements were obtained using POPOP in the reference cell.The POPOP lifetime was 1.35 ns [15].The fluorescence was measured through a long-pass filter type RG 370 from Janos Technology (Townshed, VT) which showed negligible luminescence.Data were analyzed assuming either a sum of exponen~ tial or a continuous distribution of lifetime values [9].For both the exponentml and distribution analysis, the programs minimize the reduced chi-square defined by an equation reported eisewhere [16].

EPC liposomes
Table I shows the analysis using two exponentials of DPH fluorescence decay in EPC multilamellar liposomes at different cholesterol concentrations.In EPC liposomes (without cholesterol) a component of 7.44 ns with a fractional intensity of 0.96 and a short component of 1.84 ns were obtained.Both lifetime values increased with increasing cholesterol concentration.
The same set of data was also analyzed by using a sum of one of two continuous distributions of lifetime values characterized by a Lorentzian shape centered at a

DPPC
Since EPC is an highly unsaturated phospholipid species and under our experimental conditions it is in the liquid-crystalline state, we have studied the effect of cholesterol on saturated phosphaddylcholine (DPPC) in gel and liquid-crystalline phase.
In   the addition of cholesterol (16.6 mol% cholesterol) above and below the DPPC transition temperature (Tin).Below the transition temperature (220C) the presence of the cholesterol changes the DPH fluorescence lifetime value from 10.9 ns to 7.4 ns.The effect on the diLstribution width is minor with an increase from 0.830 to 0.997 corresponding to a !2% change.Above T,, the presence of cholesterol induces a very slight decrease of the lifetime value without any significant changes on the distribution width.

Steady.state anisotropy
The steady-state anisotropy of DPH in EPC and DPPC in the presence and absence of cholesterol are reported in Fig. 3 and Table Ill, respectively.Fig. 3 shows that the presence of increasing cholesterol concentrations induces an increase of anisotropy.Below the DPPC transition temperature the presence of cholesterol induces a significant decrease of the anisotropy, while above the transition the anisotropy increases (Table III).

Discussion
Using a double-exponential analysis of the DPH fluorescence decay we have shown that cholesterol affects both long-and short-component lifetime values.The small fractional intensity associated with this short component makes its interpretation difficult.However, its sensitivity to increasing cholesterol concentration, as well as the abrupt change of its fractional intensity observed at 33.3 mol% cholesterol, is compatible with the proposal of Karnovsky et al. [17] that the short component represents DPH molecules located in polar membrane regions.
The two-component distributional analysis is very similar to that obtained using two exponentials.Moreover, the distributional approach shows a narrowing distribution width of the long component as the concentration of cholesterol is increased.The increase in EPC of the average lifetime value with increasing cholesterol concentration could be related to the effect of this molecule in reducing the penetration of water [10].Through this action, cholesterol could also induce a more homogeneous environment in the membrane 201 hydrophobic core.Since the DPH lifetime is single exponential in isotropic solvents at room temperature and is directly related to the dielectric constant of its surroundings [18], the homogeniTing effect of cholesterol can also induce a decrease of microenvironments heterogeneity.The results using EPC liposomes are in agreement with our previous observations [8] of higher values for the main lifetime component in erythrocyte membranes with respect to the phospholipid extracts.Moreover, in erythrocyte membranes depleted of cholesterol we also observed an increase of the distribution width.
The effect of cholesterol in inducing a higher degree of membrane homogeneity seems to be important from a structural point of view, since it is already apparent after an addition of one cholesterol molecule per 22 EPC molecules (4.7 tool% cholesterol).This concentration is also the minimum cholesterol content which induces changes of cooperative behaviour observed by differential scanning calorimetry [19].
Under our experimental conditions the increase of membrane order as measured by DPH steady-state anisotropy is also evident.
It has been shown that cholesterol is incorporated into the structure of aqueous lamellar phospholipids even at the higher concentrations used in this study [20].The polar hydroxyl group of cholesterol enables it to orient in the membrane bilayer parallel to the phospholipid molecules [21].It has been suggested that cholesterol exerts its influence by the hydration of the -OH group, ordering the water near the membrane surface [22].Moreover, cholesterol decreases lipid chain mobility, thus reducing structural defects and therefore the space in wlfich water molecules can be accommodated [23].Kao et al. [24] have shown that the presence of cholesterol reduces the apparent polarity of the hydrocarbon region of the phospholipid bilayer.In unsaturated phosphatidylcholine, Davenport et al. [25] have shown that DPH is located in the central part of the bilayer.Therefore, in our experimental conditions it cannot be excluded that cholesterol favours this tendency so that the probe i~ located in a more hydrophobic and homogeneous environment.The aforementioned aspects of cholesterol interactions with membrane phospholipids could explain both the decrease of DPH lifetime value and distributional width, further supporting its effect in reducing the degree of membrane heterogeneity.As mentioned before, the homogenizing effect could be directly related to the decrease of water penetration; however, the reduction of the number of structural defects could be of importance in this respect.The ~ffects exerted by cholesterol are dependent upon the phase in whict, the molecules ar,~ introduced.The presence of cholesterol induces a decrease of order as detected by steady-state anisotropy in the gel phase of DPPC and an increase above the transition in agree-ment with calorimetric data [4].However, the disordering effect in the gel phase induces also a decrease in long lifetime component value.In our interpretation, the decrease of lifetime value depends on the increase of the dielectric constant of the medium.This hypothetical increase of water permeability below the t.ransition temperature is it~ agreement with data obtained on phosphatidylcholines by De Gier et at.[26].
Our results indicate that the effect of cholesterol on membrane heterogeneity is related to membrane composition.The effect of cholesterol on unsaturated phosphatidylcholine is different from that exerted on saturated species, indicating that the double bonds in the fatty acid chains are very important in determining structural defects which can be related to the degree of water penetration.
At high cholesterol concentration the lifetime analysis sh,~ws that there is only one narrow distribution component, which indicates a homogeneous environment for the DPH molecules.
The study of the effect of cholesterol on saturated and unsaturated phosphatidylcholine by using the changes of the fluorescence properties of DPH has enabled us to define an homogenizing effect of cholesterol due to a decrease in water permeability on unsaturated phosphatidylcholine in analogy with the observation made on natural membrane [8].This effect is shown by the changes of the average lifetime values both using double-exponential or distributional analysis.

Fig
Fig, I. (A) Distribution analyds of DPH at 22'~C in DPPC multi-ian~iar ~osomcs, The gecoveged distribution has a centre at 10.43 ns acd a FWHM of 0.770 ns.(B) Distribution analysis of DPH at 22°C in a DM~/cholesterol mixtu~ (16,6 mol~ cholesterol).The recovered distribution has a centre at 7.444 ns and a FWHM of 0.99/ns.

Fig. 3 .
Fig. 3. Fluorescence anisotropy of DPH in EPC as a function of cholesterol concentrations in multilamellar liposomes.

TABLE I
Double.exponential analysis of the fluorescence emission decay of DPtl in egg PC and egg PC/cholesterol mixtures

TABLE ! 1
One and two component dstributions analysis of fluorescence emission decay in egg PC and egg PC/cholest, ,el mixturesAbbreviations: C, centre of the distribution in nanoseconds; W, full width at half maximum in nanoseconds (FWHM); f, fractional intensity; X 2, reduced chi.square.
Figs.I and 2 are reported the lifetime distributions obtained in DPPC multilamellar liposomes with

TABLE I11I Fluorescence
anisotropy of DPH in DPPC aad DPPC/cholesterol mixture below (21 °C) and above (43°C) the main transition temperature