Bioch#nica et Biophysica Acta, 1127(1992) 131-140 © 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2760/92/$05.00

131

BBALIP 53970

Peroxidation and phospholipase A 2 hydrolytic susceptibility of liposomes consisting of mixed species of phosphatidylcholine and phosphatidylethanolamine Maria Giulia Salgo a, Francesco P. Corongiu b and Alex Sevanian a " Institute for To:dcology, Department of Pathology, Unicersity of Southern Califon~ia, Los Angeles CA (USA) and ~' Dipartimozto di Biologia Sperimentale. Sez Patologia Sperima~tale, Cagliari (Italy) (Received 13 February 1992) (Revised manuscript received 21 April 1992)

Key words: Uailanlellar vesicle; Lipid per'oxidation; Membrane; Unsaturated rally acid; Liposome preparatio,~; HPLC

The relationship between lipid peroxidation and phospholipase A 2 (PLA 2) hydrolytic activity was st:udied using unilamellar vesicles (liposomes) as model membranes. Hydrolytic specificity was examined using vesicles prepared with pure bovine heart phosphatidylcholine (PC), bovine heart phosphatidylethanolamine (PE), or mixtures of these phospholipids, using two preparative procedures, i.e., sonication or extrusion. Lipid peroxidation was induced by incubating vesicles with cumene hydroperoxide and hematin at 37°C. Determinations of the extent of peroxidation by means of diene conjugate content derived from second derivative spectra or by polarographic measurement of oxygen consumption rates provided a basis for comparing the extent of peroxidation of each phospholipid species to their subsequent hydrolysis by PLA 2 (from Crotalus adamanteus). The extent of hydrolysis was determined through the release of arachidonic acid from either PC or PE. The PE distribution among the outer vs. inner leaflet of the membrane bilayer was nearly equal in sonicated vesicles, whereas most of the phospholipid was incorporated into the inner leaflet in extruded vesicles. The proportion of PE found in the inner leafle~ progressively increased as the ratio of PE to PC increased in both sonicated and extruded vesicle preparations. Lipid peroxida~ion had no effect on PE distribution under the conditions examined. There was a clear preference for PC peroxidation for all vesicle compositions tested and PC was preferentially hydrolyzed by PLA 2. This effect is proposed to result from a perturbation of membrane structure following pcroxidation with assimilation of PC into PLA2-susccptible domains whereas PE peroxidation and hydrolysis is less affected in mixcd PC/PE vesicles. Lipid peroxidation imposes an additional hydrolytic susceptibility over the cffccts exerted through the mixing of these phospholipids which is based on structural changes rather than formation of specific substratcs for PLA 2.

Introduction Phospholipids (PLs) are important constituents of biological membranes and their physico-chemical properties in membrane arrangements have been the subject of extensive studies. The bilayer structure commonly assumed by PLs has been shown to be an integral part of biological membranes and a knowledge of the structural behavior of membrane lipids is essential for an understanding of membrane assembly, and function [1]. The molecular packing, structure and distribution, in mixed bilayers of PLs is determined by

Correspondence to: A. Sevanian, Institute for Toxicology, Department of Pathology, University of Southern California, Los Angeles, CA 90033, USA.

several factors [2] among which are: (a) the composition and degree of similarity of the PL acyl chains, particularly the degree of acyl chain unsaturation; (b) the nature, orientation, interaction and extent of hydration of the polar head groups; (c) the ambient temperature relative to the thermotropic phase transition of the component PLs; (d) the extent ef peroxidative degradation of the polyunsaturated membrane phospholipids [3,4]. Unilameilar vesicles, i.e., liposomes, have been widely used to study the structural organization of membrane PLs. The properties of these vesicles, which range in sizes from large multilamellar (MLV) to small unilamellar (SUV), as well as large unilamellar (LUV), vary widely depending on the PL composition and on the method of preparation. Two different procedures 'sonication' and 'extrusion' are widely used to prepare

132 SUV composed of different lipid species. Extrusion methods allow preparation of SUV from MLV obtained by simple hydration of dry PLs. Although similar vesicles can be prepared by sonication, this procedure is known to promote formation of hydroxyl radical from ultrasonic irradiation of water [5]. These reactive species are capable of inducing peroxidation of unsaturated lipids [6] and the resulting damage to PLs could impart intrinsic membrane disturbances during vesicle preparation, thus, affecting the packing and distribution of phospholipids. The generation of oxidized lipids within the membrane hydrophobic core is proposed to create disturbance among PL acyi chains, increase membrane rigidity [7] and promote PL flip-flop [8]. Oxidation of aqueous dispersions of PLs has been found to increase the gel to liquid-crystalline transition temperature creating multiphasic structural arrangements [9]. Several groups have shown that membrane alterations, induced by lipid peroxidation are associated with increased phospholipase A 2 (PLA2)activity [3,4,10,11]. Lipid peroxidation in P C / P E vesicles has been reported to induce PLA 2 hydrolytic activity towards PE initially, but with progressive oxidation, both PLs are hydrolyzed to similar degrees [7]. It is generally accepted that PLA 2 exhibits enhanced activity towards membranes which contain multiphasic PL arrangements [12,13]. The packing irregularities created by peroxidation of membrane lipids are also envisioned to facilitate membrane fusion [14] where hydrolytic activity of PLA 2 is known to be maximal, particularly at the phase transition temperature of membrane PLs [ 12-14]. The striking selectivity of PLA 2 to fluctuations in PL organization [13,15] and its responsiveness to membrane defects has prompted its use as a probe for oxidativ~ damage in membranes. In this study, we used PLA 2 to investigate the effects of sonication vs. extrusion techniques on the relative hydrolytic susceptibility of two structurally distinct PLs, namely PC and PE, as mixed species in LUV, and the extent to which these preparative techniques, varied as a function of the extent of PL mixing, affect lipid peroxidation among the component PLs. In addition, the relative susceptibility of PC and PE to PLA, following peroxidation was examined. Materials and Methods

Materials Phosphatidylcholine and phosphatidylethanolamine (bovine heart)were purchased from Avanti Polar Lipids (Alabaster-Alabama). Hematin (bovine blood), phospholipase A 2 (C adamanteus), ammonium molybdate and 2A,6,-trinitrobcnzenesulfonic acid (TNBS) were purchased from Sigma (St. Louis, MO). Cumene hgdrol~roxide (CuOOH, a,a-dimethylbenzylhydroperoxide) was from Mathelson Coleman and Bell (Los

Angeles, CA). 1-Palmitoyl-2-[1-14C]arachidonyl-a-PE (spec. act. 55 mCi/mmol) and 1-stearoyl-2-arachidonyl, [5,6,8,9,11,12,14,15- 3H]-a-PC (spec. act. 91 Ci/mmol) were obtained from New England Nuclear Research Products (Boston, MA). All PLs were kept in 2:1 chloroform (CHCl3)/methanol (MeOH) under argon at -20°C. 'Safety Solve' liquid scintillation cocktail was from Research Products International (Hount Prospect, IL).

Methods Vesicle preparation. Vesicles were prepared using bovine heart PC and PE by means of two different procedures: sonication and extrusion. These PLs were mixed in different ratios (i.e., 25" 75, 40: 60, 60: 40; PC/PE) or used in pure form. The fatty acyl composition of the PLs were as follows: paimitic (16:0 - 23%), stearic (18: 0 - 6%), oleic (18: l - 14%), linoleic (18: 2 - 38%), eicosatrienoic (20:3 - 3.5%) and arachidonic (20:4 - 8%). The PC and PE had the same fatty acyl compositions since PE was prepared by transphosphatidylation of PC. Sonication. The unlabeled PC and PE were mixed with the corresponding labeled PLs which were added at 0.03/zCi 3H and 0.003/zCi 14C per sample, vortexed and dried under a nitrogen stream and resuspended in a working buffer consisting of l0 mM Tris/150 mM KCI (pH 7.4), at a concentration of l0 mg/ml. The PL suspensions were sonicated for 10 rain in a cup-horn using a Heat System-Ultrasonic XL2020 sonifier at a power setting of 5. The temperature throughout this treatment was maintained at 30°C using a circulating bath. The resulting translucent preparation was centrifuged for 30 rain at 25 000 rpm in a Beckman L8-55 centrifuge using a 50 Ti Rotor maintained at 4°C. The supernatant was recovered for further use. Extrusion. The same preliminary procedure for suspending PLs in buffer was followed by subjecting the suspension to three freeze-thaw cycles [16]. The freeze-thaw procedure facilitates more homogeneous mixing of the two PL species. The PLs suspension was then extruded ten times through an extruder (Lipex Biomembranes, Vancouver, BC) using two layers of a 0.1/zm pore polycarbonate membrane, under an argon pressure of 1200 kPa. The resulting translucent preparation was used for further studies. Vesicle size distribution was checked by transmission electron microscopy using a previously described negative staining technique [17]. All grids were examined within 48 h of preparation and grid sections were examined randomly. PE distribution. The ratio of PE in the outer vesicle surface to the total PE content was determined using the method of Nordlund et al. with minor modifications [18]. An aliquot of the vesicle preparation containing 0.25/zmol of PE was diluted to a final volume

133 of 600/zl with the working buffer of Tris-KCi. After adding 200/zl of 0.8 M NaHCO 3 (pH 8.5), the sample was mixed and 5 ml of 1.5% TNBS was added followed by mixing and storage in tt:e dark for 20 min at room temperature. After this interval 100/zl of 1.2% Triton X-100 in 1.5 M HC! was added. The sample was mixed and stored in the dark and the absorbance at 410 nm was read within 1 h. The total PE content was determined in a similar manner. An aliquot of 0.25 /zmol was diluted to a final volume of 600 ~1 with the working buffer and to this was added 200/zl of 1.6% Triton X-100 in 0.8 M NaHCO 3 (pH 8.5). The sample was mixed and 20 /zl of 1.5% TNBS was added, remixed and kept in the dark for 20 rain at room temperature. After incubation, 400/zl of 0.4% Triton X-100 in 1.5 M HC! was added. Measurements were taken as described above using a HITACHI V-3110 spectrophotometer. Peroxidation of liposomes and treatment with PLA 2 Vesicles were subjected to lipid peroxidation by adding aliquots (2 rag) to borosilicate screw-cap tubes containing CuOOH (200 ~M) plus hematin (50 ~M) in a final volume of 1 mi working buffer. The samples were incubated for 30 min at 37°C in a shaking water bath. Control samples consisted of vesicles incubated in buffer only. The time-course and kinetics of lipid peroxidation were initially examined polarographically using a Gilson Instruments model 5/6H oxygraph (Gilson Medical Electronics, Middletown, Wl) equipped with a 1 ml Rank water jacketed oxygen electrode (Rank Brothers, Cambridge, UK), as described previously [19]. Immediately after incubation 200/zl aliquots were removed for measurement of lipid peroxidation by diene conjugate analysis [20,21]. The lipids were extracted using CHClffMeOH (2: 1), evaporated to dryness under nitrogen and redissolved in cyclohexane. The diene conjugates were determined by taking the second derivative absorption spectra over a wavelength range of 300 to 220 nm. Lipid peroxidation among the polyunsaturated fatty acids is evidenced by two sharp and distinct signals with minima at 242 and 233 nm due to absorption by cis/trans- and trans/trans- diene conjugate isomers, respectively. The sum of the absorptions at 233 and 242 nm was used to calculate the total diene conjugate content in samples. Linoleic acid hydroperoxide prepared as described previously [19] was used to develop a calibration curve. All the scans were taken using a Hitachi U-3110 spectrophotometer. The remaining samples were incubated for an additional 15 rain with PLA 2 (0.5 U/ml, equivalent to 2/zg protein) plus CaC! 2 (1 mM). The reaction was stopped by adding 5 ml of CHCI3/MeOH (2: 1), followed by brief centrifugation and recovery of the organic phase. A second extraction was made using only 3 ml of CHC! a. The organic phases were pooled, evaporated to dryness under nitrogen, the residue dissolved in 3 ml of CHC! 3

and applied to a 'diol' solid phase extraction column (Bond-Elute, Analytichem.). The chloroform fraction, containing free fatty acids, was recovered into scintillation vials for measurement of radioactivity. The columns were then eluted with 3 ml of MeOH and the eluent collected into test tubes representing the PL fraction. The PL fraction was diluted to a fixed volume and aliquots removed for measurements of radioactivity. All sample were transferred to scintillation vials, evaporated to dryness, suspended in 5 ml of SafetySolve and counts determined in an LS 7500 liquid scinti!!ation counter (Beckman). The remainder of the PL fractions were evaporated to dryness under nitrogen and dissolved in 100 gl of diethyl ether/CHCl 3 (2: 1) for analysis by HPLC. Determination of the kinetics and lag-phase for PLA2-dependent hydrolysis were made using 200 gl aliquots of the vesicle suspension immediately after peroxidation. These determinations were made polarographically using a previously described coupled enzyme assay [22]. The rate of hydrolysis was measured by means of lipoxygenase-catalyzed oxygen consumption upon the release of free polyunsaturated fatty acids lay PLA 2. Background rates of oxygen consumption, where vesicles were incubated with lipoxygenase only, were subtracted from the rates measured after addition of PLA 2 and 1 mM CaCi 2. HPLC analysis of PLs. Analysis of PLs was performed by high pressure liquid chromatography (HPLC) with ultraviolet detection using a Perkin-Ei'mer Series 4 Liquid Chromatograph coupled to a Beckman model 168 photodiode array detector interfaced to Beckman 'Gold System' chromatographics analytical software. The chromatographic conditions were adapted from the method of Kaduce et al. [23] and consisted of two normal phase columns (150 × 4.6 mm, Spherisorb silica, 3/~m) connected in series to a photodiode array detector which was set to monitor the ultraviolet absorbance of each eluting peak at 202 and 234 nm. An isocratic mobile phase consisting of acetonitrile/ methanol/sulfuric acid (100:2.75:0.075, v/v)was used with a programmed gradient flow rate of 2 ml/min for the first minute, a linear decrease in flow rate to 0.6 ml/min over 0.5 min, which was maintained for 1.5 min, followed by a step increase to 1 ml/min which was maintained for the remaining 16.5 rain of the run. Results

Size and structural characteristics of sonicated vs extruded liposomes Electron microscopy of sonicated and extruded vesicle, before and after induced lipid peroxidation showed non-significant differences in vesicle size. Sonicated vesicles tended to be smaller with a much broader range of particle sizes, whereas, extruded vesicles were

1N TABLE I

TABLE !II

PE distribution a in extruded liposomes

Measurements of peroxidation in son±cared vesicles as a function of phosphatidylcholine / phosphatidylethanolamine ratios

PC/PE

AtotaI

Aoutcr

out/tot

75/25 60/40 40/60

0.195+0.01 0.219±0.01 0.191 +0.009

0.078+0.004 0.067±0.003 0.036±0.002

0.4 +0.02 0.3 +0.01 0.18 +0.009

PC/PE

Treatment

60 2/6t a

Total CD b

100

ox c ox c ox c ox c

4.3+0.21 (130+ 6.5) 0.8+0.04 (24+ 1.2) 4.0±0.2 (120+ 6.0) 1.3 ± 0.06 (40-4- 2.0) 6.0±0.30 (181 ± 9.0) 0.6±0.03 (18+ 0.9) 6.6+0.33 (200+ 10) 2.2 ± 0.11 (66 ± 3.3)

71+3.5 21± 1.0 110±5.5 30 ± 1.5 126±6.3 32±1.6 76:!:3.8 40 ± 2.0

65/35

more uniform in size and shape, tending to be more rounded in appearance. In all instances the size range of vesicles are representative of LUV. The inner vs. outer membrane bilayer distribution of PLs is shown in Tables ! and II. 50 to 65 percent of the PE is located in the outer leaflet in sonicated vesicles, whereas, only 18 to 40 percent is found in the outer leaflet in extruded vesicles, thus, greater asymmetry in inner vs. outer membrane leaflet distribution of PLs is found when vesicles are prepared by extrusion. The amount of PE located in the outer leaflet of the vesicles was also found to be inversely proportional to the relative content of PE in the total vesicle preparation. The distribution of PE was unrelated to the intrinsic level of lipid peroxidation in extruded or son±cared vesicles after preparation and there was no effect on inner vs. outer bilayer PE distribution after induction of peroxidation (data not shown).

Diane conjugate analysis and PLA 2 hydrolytic activity Lipid peroxidation as reflected by diane conjugate ~CD) analysis showed that sonicated vesicles displayed higher background levels of peroxidation than extruded vesicles (Tables I!! and IV). For example, in vesicles prepared using pure PC the CD content was approx. 2-fold higher in sonicated vs. extruded vesicles. Moreover, in all cases extruded vesicles were more resistant to peroxidation. The extent of peroxidation, as measured by oxygen consumption, corresponded to the TABLE II P£ dt¢tribmion " in sonicated lilmsomes PC/PE

Al,,~l

Ao,1~r

out/tot

65/~ 50150

0.246 ± 0.01 0.225 ± 0.01 0.275 ± 0.01

0.160 ± 0.008 0.123 ± 0.006 0.145 ± 0.007

0.65 ± 0.03 0.54 ± 0.02 0.52 ± 0.02

40/60

" In both Tables I and Ii the relative amounts of PE located in the outer vs. inner bilayer leaflet are expressed on the basis of absorbance ( A ) o f the trinitmbenzylsulfonate (TNBS) reaction with PE as described in the text. The total absorbance represents the total amount of PE-TNBS chmmophore measured (A total),whereas the PE content in the outer leaflet of intact vesicles is represented by Aout~. The percent of total PE in the outer leaflet is presented in the column entitled (out/tot). The results are expressed as the mean and standard error obtained from three experiments where each sample was analyzed in duplicat(:.

50/50 40/60

'~ The rates of oxygen consumption measured polarographically over a 30 rain interval at 37°C are expressed as the computed rate in nmol/min per mg phospholipid. Total oxygen consumption (nmoles/30 rain) is indicated in parentheses. The actual ratios of P C / P E were obtained by HPLC analysis of samples immediately after preparation. b The formation of total CD after 30 rain incubation at 37°C is expressed as nmol/mg phoxpholipid. The measurements were taken as the second derivative absorption spectra by adding the second derivative absorption units at 233 and 242 nm to obtain the total CD. A calibration curve was constructed using purified linoleic acid hydroperoxide as a standard. The molar extinction coefficients at 233 and 242 nm were assumed to be similar (2.54.104) and the calibration curves were generated for each absorbance based on a constant ratio of these two isomers in the standard. See text for further details concerning measurement CD. The results are expressed as the mean and standard error obtained from two independent experiments involving duplicate determinations for each sample/condition.

levels of CD formed in each sample. The rates of oxygen consumption, or total oxygen consumed during the 30 min incubation interval as indicated in parentheses in Tables Ill and IV, tended to be higher in vesicles containing higher proportions of PE. The rates of oxygen consumption shown for control samples are assumed to represent autoxidation of phospholipids. In extruded vesicles (Table IV) there was no apparent relationship between the proportion of PE and the extent of peroxidation as indicated by both the CD and oxygen Consumption measurements. The extent of peroxidation was significantly less in extruded vesicles as compared to sonicated vesicles. In Tables III and IV a close relationship is found between the extent of peroxidation as measured by CD and oxygen consumption. When the total oxygen consumption (shown in parentheses) is compared to CD the molar ratios for each measurement are in the range of 1 : 1 to 2: 1 for control samples, whereas for samples subjected to peroxidation the ratios of oxygen consumption vs. CD are in the range of 2:1 to 4: I. Fig. I shows the time-course of PLAz hydrolysis of a sonicated vesicle preparation consisting of pure PC. Linear rates of hydrolysis were found over a 15 min interval after which hydrolytic activity subsided. During the 15 min period a significant difference in the extent

135 TABLE iV

Measurements of peroxidation in extruded vesicles as a function of phosphatidylcholine / phosphatidylethanolamine ratios

20

Hydrolysis of PC in PC/PE sonicoted liposomes 1:::3control + PLA2 i ~ ox + PLA2

10

PC/PE

602 ~St a

Treatment

100/0

ox c ox c ox c ox c ox c

75/25 60/40 40/60 0/100

Total CD b

5.3+0.3 (160+ 0.7+0.03 (20± 6.0+0.3 (180± 0.6±0.03 (18± 3.0+0.15 (90± 0.3:!:0.01 (10± 4.3+0.21 (130± 1.2+0.06 (35+ 6.7 + 0.33 (200 ± 0.8 + 0.04 (24 ±

8) 1) 9) I) 4) 0.5) 6) 1.7) 10) 1.2)

0

ej

48.0+2.4 10.0+0.5 40.5±2 10.0_+0.5 56.0±2.8 10.5±0.52 36.3 ± 1.8 14.0±0.7 60.0 ± 3.0 20.0 ± 1.0

12 m

8 4 (.J n 40

a The rates of oxygen consumption measured polarographically over a 30 rain interval at 37°C are expressed as the computed rate in nmol/min per mg phospholipid. Total oxygen consumption (nmol/30 rain) is indicated in parentheses. The actual ratios of PC/PE were obtained by HPLC analysis of samples immediately after preparation. b The formation of total CD after 30 min incubation at 37 °C is expressed as nmol/mg phospholipid. The measurements were taken as the second derivative absorption spectra by adding the second derivative absorption units at 233 and 242 nm to obtain the total CD. A calibration curve was constructed using purified linoleic acid hydroperoxide as a standard. The molar extinction coefficients at 233 and 242 nm were assumed to be similar (2.54.104) and the calibration curves were generated for each absorbance based on a constant ratio of these two isomers in the standard. See text for further details concerning measurement CD. The results are expressed as the mean and standard error obtained from two independent experiments involving duplicate determinations for each sample/condition.

of hydrolysis was found betweei~ oxidized and control vesicles. A similar time-course wag: found for vesicles prepared using mixtures of PC and PE and a maximum level of hydrolysis was also observed at 15 rain (data

12 • .... • oxidized

O

16

.=

20

50

65 % o! PC

1OO

Hydrolysis of PE in PC/PE sonlcated Ilposomes r--qcontroI + PLA2 ox + PLA2

~< 12 .m m m

R t-

4

O.

35

50 60 % of PE Fig. 2 and 3. The percent of phospholipid hydrolysis for PC and PE is shown for sonicated vesicles as a function of PC/PE ratios. PC hydrolysis is expressed as a percent recovery of [3H]arachidonic acid released following incubation with PLA 2 and vesicles containing the indicated mixtures of PC and PE along with 0.03 p,Ci l-stearoyl-2[3H]arachidonoyl-PC (Fig. 2). PE hydrolysis was expressed as percent recovery of [14C]arachidonic acid released following incubation with PLA 2 and vesicles containing the indicated mixtures of PC and PE along with 0.003 #Ci l-palmitoyl-2[ 14C]arachidonoyl-PC (Fig. 3). The incubation mixture contained 2 mg phospholipids, 0.5 U PLA 2 and 1 mM Ca2. in 10 raM/150 mM Tris.KCl (pH 7.4) buffer. Pure PE vesicles were not analyzed since it was not possible to sonicate this phospholipid. The mean and S.E. are shown for three independent experiments involving duplicate analysis for each sample/condition.

O control

9

10

o

3

o

~

;o

s'~

20

Incubation time (min) Fig. 1. Time-course of hydrolysis by PLA2 vesicle consisting of sonieated PC only containing 0.03 ~,Ci t-stearoyl-2-[SH]arachi donoyI-PC. Hydrolytic activity is expressed on the basis of percent recovery of [H]arachidonic acid released from PC over a 20 rain interval. Hydrolysis was measured by incubating the samples at 37°C in a mixture containing 2 mg of PC, 0.5 U PLA 2 and I mM Ca2+, in 10 raM/150 mM Tris-KCi (pH %4) buffer.

not shown). Based on these findings, all subsequent results are presented as percent PL hydrolysis measured at the 15 min interval. Figs. 2 and 3 describe the relationship between the extent of peroxidation (as described in Table 111) and PLA2-mediated hydrolysis of PC and PE in peroxidized and control sonicated vesicles measured by the release of labeled flee arachidonic acid. In control samples PC hydrolysis increased in direct relation to the proportion of PE and PC was preferentially hydrolyzed. Moreover, PE hydrolysis was low in vesicles

containing a low proportion of this PL, however, the extent of hydrolysis increased in direct proportion to the relative content of PE. The increase in PE hydroly-

136 sis paralleled the increase in total diene conjugate content. In peroxidized vesicles PC remained a better P ~ 2 substrate than PE, however, the degree of hydrolytic susceptibility became similar when the relative proportion of these lipids was equal (i.e., 50:50). Hydrolytic susceptibility of PC vs. PE in extruded ~ l e s is shown in Figs, 4 and 5. PC was more readily hydmlyzed than P E and its extent of hydrolysis was facilitated by increasing the proportion of PE, regardless of whether the vesicles were peroxidized or not. the other hand, hydrolysis of PE was inversely related to the proportion of PC in vesicles. Although the hydrolytic susceptibility of PC was similar for extruded and sonicated vesicles, the effect of PE addition

1.000 -

O,025 :',

,-,

".. "- ii "....,.."...,:' ',..." :..

O.TSO .*

I

'- , , .

...........

E 0.500 e.,

O.250

0.0%

lb

l'S

~o °°°°

Time (mln)

1,000

0.025

Hydrolylls of PC In PC/PE extruded Ilposomes

2O

0,750

f

÷ PLA = ~tro, ,,.P,A

1

T E 0 500 ° o

O.g50 '~,

8

0,000~)

40

°[

60

75 % of PC

100

Hydrolysis of PE in I ~ / R [ extruded Ilposomes

L-~control + PLA I ~ + PLA

!: o,, 2~

40

nl n./ eo

loo

% o f RE Fil~ 4 and 5, The percent of phospholipid hydrolysis for PC and PE is shown for extruded vesicles as a function of PC/PE ratios, PC hydrolysis is expressed as a percent recovery of [3H]arachidonic acid relemed ~s41owi~ incubation with PLA= and vesicles containing the

indicated mixtures of PC and PE alonll with 0,03/tC~ I-steamyl,2, [sHJarachidonoyl,PC (Fill, 4), PE hydrolysiswas expressed as percent recoveW of [t4Clarachidoni¢ acid released following incubation with PLA 2 and verities containing the indicated mixtures Qf PC and PE aloall with 0,t~03 /tC'i l,palmitoyl,2,[t*C]arachidonoy|,pc (Fig. 5). The incubation mixture contained 2 m8 phospholipids, 0.5 U PLA 2 and i mM C,az+ in 10 raM/150 mM Tris-KCI (pH 7.4) buffer. The mean and S,E, are shown for three independent exper~ents invoivinn duplicate analysis for each sample/conditkm,

5

10 Time (rain)

15

2

:)000

Fig, 6. (a) HPLC chmmatogram of sonicated oxidized PC/PE (50:50) vesicles The peaks elated at 7.14 rain and 8.09 rain are two different species of PE. The peaks eluted at 10.55 rain and 14.99 rain are two different species of PC. (b) HPLC chromatosram of extruded unoxidized PC/PE (40: 60)vesicles. The peaks eluted at 7.13 rain and 8.66 rain represent PE and peaks at 10.62 rain and 13.51 represent PC. A portion of the last peak elutins around 20 rain corresponds to lysoPL The cumulated areas for all peaks determined at 234 nm were used to calculate the amount of CD in the sample.

to extruded vesicles was not as marked as its effect in sonicated vesicles. In vesicles prepared with pure PC or PE, the extent of hydrolysis and CD content were in good agreement although the tendency for PE to undergo hydrolysis by PLA 2 following peroxidation was less than PC. In general, extruded vesicles were more resistant to PLA 2 hydrolytic activity than sonicated vesicles. TABLE V HPLC measurement of the relative levels of peroxidation in PC vs. PE sonicated vesicles

PC/PE 65/35 50/50 40/60

ox c ox c ox c

PC

PE

0.01 0.006 0.03 0.01 0.03 0.02

0,002 N.D. 0,01 N.D. 0,004 0,001

137 TABLE VI

HPLC measurement of the relative levels of peroxidation in PC~ PE extruded vesicles Peak area ratios were determined by measuring the 234 nm absorbance vs. 202 nm absorbance corresponding to PC and PE following the resolution of these phospholipids by HPLC. The integrated areas for 234 nm were divided by the integrated areas for 202 nm absorbance to obtain the area ratios shown. The ratios represent the relative levels of peroxidation for each phospholipid after extraction from vesicles containing the PC/PE ratios shown in the table. N.D., it was at times difficult to quantitate the 234 nm area for PE peaks and the results are thus presented as not determined (N.D.). The calculated area ratios never deviated more than 10% from values shown. The values shown are the mean of two independent experiments involving a single analysis of each sample. PC/PE 7.;/25

ox

c 60/40

ox

40/60

c ox c

PC

PE

0.03 N.D. 0.04 0.01 0.05 N.D.

0.01 N.D. 0.01 0.01 0.03 0.01

HPLC analysis of PLs Using pure PL standards to calibrate the instrument response, HPLC analysis of the PLs extracted from vesicles did not necessarily yield the ratio of PC and PE desired for a given vesicle preparation. The chromatographic data showed that the desired ratios of P C / P E were best achieved by the sonication procedure. Chromatograms obtained from extruded liposomes showed that there was a constant decrement of PE regardless of the initial ratios of PC vs. PE used for vesicle preparation. The loss was probably due to poor mixing between the PLs and a consequent trapping of PE on the polycarbonate membrane during extrusion. The freeze-thaw procedure, performed before extrusion, enabled a more reproducible vesicle preparation with the desired F C / P E ratio. Fig. 6a and b shows HPLC chromatograms of sonicated and extruded/unoxidized vesicles. The cumulated areas for all peaks detected at 202 nm were used to calculate the P C / P E ratios in the samples. The cumulated areas for all peaks determined at 234 nm were used to calculate the amount of CD which can be compared to values shown in Tables V and VI. The total integrated area representing 234 absorbance for PC was greater than the corresponding area for PE in extruded and in sonicated vesicles indicating that PC was the more peroxidized PL. In sonicated vesicles the peaks corresponding to PC oxidation products increased in direct proportion to the PE content despite the lower overall peroxidation when vesicles were prepared with increasing proportions of PE (Table V). In extruded preparations, it was at times difficult to detect any 234 nm peaks corresponding to PE, whereas 234 nm absorbing peaks corresponding to PC were still

evident. There was no apparent relationship between PE content and the extent of peroxidation of either PC or PE which contrasted with the results obtained with sonicated vesicles. The only exception to this was found for unoxidized vesicles containing high proportions of PE wherein PC, but not PE, peroxidation was facilitated. On the other hand, vesicles consisting of pure PE (possible only for extruded vesicles) were quite readily oxidized and in fact displayed rates of peroxidation comparable to the peroxidation rates measured for pure PC vesicles. Extruded vesicles also contained a small amount of lysoPLs, which may be accounted for by the freeze-thaw procedure since lysoPLs were always absent in sonicated preparations as well as extruded vesicles which were prepared without fieezethawing. Discussion

Increased PLA 2 activity during lipid peroxidation involving a preferential hydrolysis of oxidized PLs has been proposed as a secondary mechanism of defense against membrane oxidative stress [3]. In biological membranes oxidant stress is often manifested by lipid peroxidation. Measurements of the extent of lipid peroxidation by means of oxygen consumption rates and formation of CDs suggest that the primary products are PL hydroperoxides which may be subject to further propeigation and decomposition reactions. Although the extent of these secondary reactions was not determined for this study, the results suggest that propagation reactions contributed significantly under conditions where peroxidation was imposed by incubating vesicles with CuOOH and hematin. The conditions for peroxidation were mild in the sense that the overall levels of peroxidation products increased over control samples 2-4-fold during the 30 rain treatment period. This amounted to less than 15% of the initial unsaturated PL content. Formation of PL hydroperoxides was estimated by the HPLC isolation of both PC and PE with increased specific absorbance of CD. Moreover, the ratios for oxygen consumption vs. CD in the range of (2-4): 1 indicated that hydroperoxide propagation and decomposition reactions were involved under these treatment conditions as described in previous studies [19]. The extent of PL hydrolysis by PLA 2 was directly correlated to the accumulation of these peroxidation products. Thus, the increased PLA2 activity represents a direct measurement of the extent of substrate hydrolysis and is not a determination of the enzyme's kinetics. The use of vesicles containing various proportions of PC vs. PE was intended to determine the extent to which lipid peroxidation affected specific PLs and the degree to which this was associated with their specific hydrolysis. It has been demonstrated in artificial PL

mixtures that the introduction of PE to vesicle preparations consisting of PC p ~ u c e s a concentration-dependent change in the properties of vesicles [24]. Each lipid in pure form exists in a discrete structural state when prepared as vesicles. In unilamellar vesicles PE has been reported to arrange as inverted, stacked rods (hexagonal l l ) i n its liquid/crystalline state while PC largely ~ u m e s a normal bilayer structure [25], When these lipi~ are mixed~ transitions ranging from bilayer to h ~ n a i arrangements are observed as defined through3SP.NMR spectroscopy [26]. Pl.s entering these non-bilayer domains tend to e ~ r i e n c e rapid transbilayer movement or flip-flop [27]. This is particularly evident in unsaturated PE-rich systems [28,29]. PLs in biological membranes are distributed asymmetrically across the bilayer, although most PLs may be found in both the outer and inner sides of the membrane bilayer. Strong evidence for this asymmetric distribution has been obtained for erythrocytes. Previous studies have shown that PC and sphingomyelin occupy the outer monolayer whereas PE, phosphatidylinositol (PI) and phosphatidylserine (PS) are localized in the inner monolayer [18]. Similarly, PE has been shown to be ~symmetrically distributed, being localized primarily on the inner half of biological and artificial membranes [30], This asymmetry of PLs is due at least in part to the three-dimensional structure of the phospholipid and preferred conformation and packing in membranes [1] which influences its interaction with other PLs in the inner vs outer leaflet of the bilayer. Transbilayer lipid movement 'flip-flop', is regarded as a slow process and the extent to which this accounts for asymmetry remains unclear. Hirata proposed that the transbilayer movement of PC and PE can be facilitated by sequential methylation of PE (originally in the inner bilayer) to PC: although other mechanisms may also facilitate this process[31]. Based on previous and extensive studies demonstrating distinct structural arrangements for PC and PE in membranes [1,~24-26], the effects of peroxidation on P ~ z hydrolytic activity were compared to the known structural effects imposed by mixing these structurally distinct PLs. Our findings confirm those of Nordlund et al. [18] who showed that liposomes prepared by sonieation of PC and PE mixtures contained a preferred distribution of PE into the outer leaflet of the bilayer and that increasing the proportion of PE resulted in the assimilation of relatively larger amounts into the inner leaflet. This is not the case, however, for liposomes prepared by extrusion. The results in Tables ! and l] reveal rather distinct differences in bilayer distribution of PC and PE where most of the PE is located in the inner leaflet. This marked difference in PE distribution may be related to the size of vesicles in each type of preparation and/or to the size distribution which was more uniform for extruded vesicles.

Peroxidation of either sonicated or extruded vesicles, on the other hand, produced no effect on PE distribution at all ratios of PC/PE examined. This indicates that the differential effects of peroxidation and PLA 2 hydrolysis observed in these studies were not directly related to the bilayer distribution of the PLs, nor could the higher levels of peroxidation in sonicated vesicles adequately account for differences in PE distribution as compared to extruded vesicles. However, one can not exclude the possibility that other structural arrangements of PLs may account for differential susceptibility to peroxidation and/or PLA, hydrolysis of sonicared vs. extruded vesicles. Previous studies have shown that PL peroxidation increases the anisotropy, microheterogeneity and structural disordering of phospholipids in membrane preparations [17,32,33]. We have recently found that liposomes prepared by extrusion of soybean PC displayed a more uniform flourescence lifetime distribution for the fluorescent probe, diphenylhexatriene, than sonicated preparations, indicating a more ordered organization for extruded Pl.s (data not shown). The ordered packing of PLs, along with lower levels of peroxidation products formed during preparation, may be responsible for the greater stability and asymmetric PL distribution found for extruded vs. sonicated vesicles. The PL asymmetry in extruded vesicles may minimize the extent of PC-PE interactions. This could reduce the potentially disruptive effects of PE on PC and stabilize the PC organization in the membrane rendering the PLs more resistant to peroxidation and PLA 2 hydrolysis. A disruption of PL packing order may, thereby, be viewed as a common mechanism by which peroxidation [34] and PLA, activity [17] are facilitated. For all the vesicle PL compositions tested there was a clear preference for PC oxidation and hydrolysis. This preferential susceptibility of PC was not due to its greater peroxidation index since the fatty acyl composition of both PL species was identical. This was specifically the case for arachidonic acid which was used as the means for measuring PLA2.mediated hydrolysis. Increased PC hydrolysis as a function of either increasing the proportion of PE or lipid peroxidation was previously reported to be related to the structure and/or PL localization in mixed PC/PE vesicles [17]. The results presented in this paper disfavor the possibility that the preferred hydrolysis of the component PLs is related to their inherent peroxidizabilit~,. Moreover, vesicles prepared with either pure PC or PE are equally susceptible to peroxidation (see Table IV). The preferential hydrolysis of PC in mixed PL vesicles may be accounted for by the following observations. The preferred peroxidation of PC over PE in mixed vesicles was evident in preparations where PC was clearly peroxidized with minimal peroxidation of PE (Tables V and VI). Under conditions where PE per-

139 oxidation was found, increased hydrolysis of PE was also measured (compare Table VI vs. Fig. 5 and Table V vs. Fig. 3). Localization of PC largely into the outer leaflet of the vesicle increases its relative accessibility to P L A 2 (since the enzyme is added to the medium in which the vesicles are suspended). Increased PE hydrolysis as a function of increasing the proportion of PE in vesicles is consistent with larger amounts of PE being incorporated into the outer portion of the vesicle. This, however, may be offset by the greater proportion of PE located into the inner leaflet. Since peroxidation had no apparent effect on PE distribution, there is no reason to expect that greater amounts of PE would be subjected to hydrolysis at the outer surface of vesicles through a flip-flop process. Nevertheless, the methods employed in this study do not measure the rates of PL flip-flop but rather the steady state quantity of PE vs. PC on the outer portion of the bilayer. Thus, only the effects of vesicle preparation or peroxidation on the general distribution of PL were observed. The mixing of PE and PC increases the susceptibilII. 7" of PC tO PLA 2 hydrolysis (see Fig. 4). This effect may be due to disruptions in the bilayer arrangements of PC vesicles [26,35], however, other factors may also be important. Our findings show that in addition to the mixing of these PL species, which is reported to produce multiphasic organization [26-29] and increased PLA 2 hydrolytic susceptibility, the peroxidation of these PLs imposes an additional measure of hydrolytic susceptibility. PC has been reported to be an activator and substrate for PLA 2 [36]. In mixed vesicles both PC and PE are PLA 2 substrates but the lower hydrolytic rates for PE may be due to its relatively low effect on enzyme activation which appears unaffected by lipid peroxidation (e.g., Figs. 4 and 5). This was supported by measurements of the kinetic lag-phase for full catalytic activity of PLA 2 [37] which indicated no differences due either to PL composition or extent of peroxidation. For extruded liposomes an apparent lag-time of 30-40 s was found for all vesicle preparations. An even shorter lag-time (10-20 s) was found for sonicated liposomes (data not shown). Since no differences were found following peroxidation, the lag-time differences between extruded and sonicated vesicles may be due to reasons other than the content of lipid peroxidation products. The only apparent factor which may account for this is the difference in assymetry between extruded vs. sonicated vesicles. Activation of PLA 2 following membrane lipid peroxidation appears to fit a general hypothesis that the enzyme is preferentially activated when there are disturbances or fluctuations in structural domains [13,38,39]. The disruptive effects of peroxidation products tend to increase the hydrolytic susceptibility of a variety of PL species and in this report a preferred hydrolysis of PC is apparent. Based on this

general hypothesis the present findings suggest that creation of structural interfaces composed of distinct PL species, and assimilation of PC into these structural domains, specifically increases PC hydrolysis relative to PE. The latter PL, which prefers nonbilayer configurations, tends to facilitate PC hydrolysis when tbo two phospholipids are mixed with minimal effects o~ its own hydrolytic susceptibility. Lipid peroxidation imposes an additional hydrolytic susceptibility over the effects exerted through the mixing of these PLs wherein PC peroxidation and hydrolysis is facilitated. These findings indicate that increased peroxidat~ion manifested in a specific lipid species (PC) is associated with its increased susceptibility to PLA 2 induced hydrolysis.

Acknowledgements The authors wish to thank Drs. Eunjoo Kim, Mary l.ou Wratten, Laurie McLeod and Ishmael Ordonez for their participation in these studies and thoughtful contributions during the preparation of the manuscript. This study was supported by funds provided by the National Institutes of Health (grant number HL45206).

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