J Membrane Biol DOI 10.1007/s00232-016-9872-7
Effects of Phospholipase A2 Inhibitors on Bilayer Lipid Membranes Mikhail V. Dubinin1,2 • Maxim E. Astashev2,3 • Nikita V. Penkov3 • Sergey V. Gudkov2,4,5 • Igor A. Dyachenko6,7 • Victor N. Samartsev1 • Konstantin N. Belosludtsev1,2
Received: 9 September 2015 / Accepted: 6 January 2016 Ó Springer Science+Business Media New York 2016
Abstract The work examines the effect of inhibitors of cytosolic Ca2?-dependent and Ca2?-independent phospholipases A2 on bilayer lipid membranes. It was established that trifluoroperazine (TFP) and, to a lesser extent, arachidonyl trifluoromethyl ketone (AACOCF3) and palmitoyl trifluoromethyl ketone (PACOCF3) were able to permeabilize artificial lipid membranes (BLM and liposomes). It was shown that AACOCF3 lowered the temperature of phase transition of DMPC liposomes, inducing disordering of the hydrophobic region of lipid bilayer. TFP disordered membranes both in the hydrophobic region and in the region of hydrophilic heads, this being accompanied by changes in the membrane permeability: appearance of a channel-like BLM activity and leakage of sulforhodamine B from liposomes. In contrast to AACOCF3 and TFP, PACOCF3 increased membrane orderliness in the hydrophobic region (heightened the temperature
& Mikhail V. Dubinin
[email protected] 1
Mari State University, pl. Lenina 1, Yoshkar-Ola, Mari El, Russia 424001
2
Institute of Theoretical and Experimental Biophysics RAS, Institutskaya 3, Pushchino, Moscow Region, Russia 142290
3
Institute of Cell Biophysics RAS, Institutskaya 3, Pushchino, Moscow Region, Russia 142290
4
Prokhorov General Physics Institute RAS, Vavilova 38, Moscow, Russia 119991
5
Lobachevsky State University of Nizhny Novgorod, pr. Gagarina 23, Nizhny Novgorod, Russia 603950
6
Branch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, pr. Nauki 6, Pushchino, Moscow Region, Russia 142290
7
Pushchino State Institute of Natural Sciences, pr. Nauki 3, Pushchino, Moscow Region, Russia 142290
of phase transition of DMPC liposomes) and in the region of lipid heads. The effectiveness of AACOCF3 and PACOCF3 as inductors of BLM and liposome permeabilization was considerably lower comparatively to TFP. As revealed by dynamic light scattering, incorporation of TFP, AACOCF3 and PACOCF3 into the membrane of liposomes resulted in the increase of the average size of particles in the suspension, presumably due to their aggregation or fusion. The paper discusses possible mechanisms of the influence of phospholipase A2 inhibitors on bilayer lipid membranes. Keywords Phospholipase A2 Liposomes Membrane permeabilization Membrane fusion Lipid pores Phase transitions Abbreviations PLA2 Phospholipase A2 AACOCF3 Arachidonyl trifluoromethyl ketone PACOCF3 Palmitoyl trifluoromethyl ketone TFP Trifluoroperazine BLM Bilayer lipid membrane LUV Large unilamellar vesicles DMPC 1,2-Dimiristoylphosphatidylcholine SRB Sulforhodamine B DLS Dynamic light scattering GP Generalized polarization DSC Differential scanning calorimetry TX-100 Triton X-100
Introduction Phospholipases A2 (PLA2) are enzymes catalyzing hydrolysis of membrane phospholipids at the sn-2 bond, which results in the release of free fatty acids and
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lysophospholipids. In the mammal organism, more than 30 enzymes possessing a PLA2 activity has been found. Most of phospholipases A2 falls into three families: secretory phospholipases A2, cytosolic Ca2?-dependent, and cytosolic Ca2?-independent phospholipases A2. According to modern views, cytosolic Ca2?-dependent phospholipases play an important role in the metabolism of arachidonic acid, a precursor of prostaglandins, and leukotrienes. Cytosolic Ca2?-independent phospholipases A2 are involved in the homeostasis of membrane lipids (and, hence, in the maintenance of membrane structure and packing) and in the energy metabolism of the cell. Secretory phospholipases A2 participate in various pro- and antiinflammatory biological phenomena, modulating the extracellular phospholipid environment (see review by Murakami et al. 2011; Dennis et al. 2011). A balanced operation of phospholipases A2 is important for stable functioning of the animal organism. However, the balance can be disturbed at some pathologies associated with oxidative stress—and then phospholipases A2 may become harmful for the cells of the organism. The pathologies can be related to disorders in the metabolism of arachidonic acid (which may lead to such diseases as asthma, multiple sclerosis, and rheumatoid arthritis) or to disturbance of the membrane structure (which eventually may result in the cell death) (Murakami et al. 2011; Tripathi et al. 2013; Mironova et al. 2011). Today, every family of phospholipases A2 is known to have its own inhibitors. Some of those inhibitors are highly specific toward phospholipases (arachidonyl trifluoromethyl ketone, AACOCF3; palmitoyl trifluoromethyl ketone, PACOCF3; YM-26734; bromoenol lactone); other inhibitors act less specifically and manifest their effects at comparatively higher concentrations (bromophenacyl bromide; trifluoroperazine, TFP; aristolochic acid; etc.) Taking into account the pathophysiological role of phospholipases A2, testing their inhibitors as potential drugs seems perspective. Indeed, PLA2 inhibitors prevent the development of cell death and exert a therapeutic effect upon treatment of many diseases related to the disorders in the metabolism of arachidonic acid and to cancerogenesis (Mironova et al. 2011; Tripathi et al. 2013; Murakami et al. 2011; Mironova et al. 2015). On the other hand, some inhibitors can suppress not only phospholipases A2 but other enzymes as well, membrane transport proteins being among these enzymes. Moreover, specifics of the chemical structure of many inhibitors (more precisely, their hydrophobicity) suggest that they will directly interact with lipid membranes. It can be assumed that this will be accompanied by the disturbance of the molecular order of membranes and changes in their permeability to various ions and larger molecules. Such changes can, in their turn, cause the death of cells. This effect can be especially
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pronounced when the inhibitors are used at high concentrations (Broekemeier et al. 2002). For example, trifluoroperazine was shown to cause lysis of erythrocytes, which was attributed to the disorder of the membrane structure (Malheiros et al. 1998; Hendrich et al. 2001). Thus, the objective of the present work was to study the interaction of PLA2 inhibitors with bilayer lipid membranes. We tested trifluoroketonic substances (AACOCF3, PACOCF3, and TFP), which are the most conventional inhibitors of cytosolic Ca2?-dependent and Ca2?-independent phospholipases A2 (Dennis et al. 2011). It was shown that (1)
(2)
(3)
AACOCF3 and TFP lowered the temperature of phase transition of DMPC liposomes and decreased the orderliness of membrane in the region of phospholipid heads, whereas PACOCF3 had the opposite effects; TFP induced permeabilization of bilayer lipid membranes (BLM) and liposomes, with the effectiveness of AACOCF3 and PACOCF3 being much lower. Incorporation of TFP, AACOCF3, and PACOCF3 in the liposomal membrane resulted in the increase of the average vesicle size.
Materials and Methods Chemicals Medium components, all inorganic chemicals, sulforhodamine B, laurdan and lecithin were purchased from SigmaAldrich; DMPC, brain total lipid extract and cardiolipin were purchased from Avanti Polar Lipids. In the work, commercially available inhibitors of phospholipases A2 were used: arachidonyl trifluoromethyl ketone (AACOCF3), palmitoyl trifluoromethyl ketone (PACOCF3; Tocris), and trifluoroperazine (TFP; Sigma-Aldrich). The structures of PLA2 inhibitors used in this work are shown in Fig. 1. Phospholipase A2 inhibitors were introduced into the suspension of liposomes or BLM cell as ethanolic solutions. The final concentrations of ethanol in the medium did not exceed 1 % (v/v). The concentration of stock inhibitor solutions was 30 mM. Preparation of Large Unilamellar Vesicles Large unilamellar vesicles (LUV) were prepared by an extrusion technique, as described (Agafonov et al. 2003). Dry egg-phosphatidylcholine (0.75 mg) was hydrated for several hours with periodic vortexing in 0.75 ml of buffer containing 40 mM sulforhodamine B (SRB), 10 mM Tris– HCl (pH 8.5), and 50 lM EGTA. After five cycles of
M. V. Dubinin et al.: Effects of Phospholipase A2 Inhibitors on Bilayer Lipid Membranes
vesicles were added to a buffer containing 40 mM KCl, 50 lM EGTA, and 10 mM Tris–HCl (pH 8.5) (the final concentration of lipid, 50 lM), and laurdan fluorescence was measured before and after various experimental additions. Acoustic Measurements
Fig. 1 Chemical structures of PLA2 inhibitors: TFP (a), PACOCF3 (b), AACOCF3 (c)
freezing/thawing at -20/?30 °C, the suspension was pressed 11 times through a 0.1 lm polycarbon membrane using an Avanti microextruder (Avanti Polar Lipids, USA). All operations except freeze/thawing were carried out at room temperature. After extrusion, liposomes were applied onto a Sephadex G-50 column to remove external SRB. The buffer for gel filtration was 40 mM KCl, 50 lM EGTA, and 10 mM Tris–HCl; pH 8.5. SRB was selfquenched inside LUV. Accordingly, release of SRB was estimated from the increase of SRB fluorescence (unquenching) in buffer containing 40 mM KCl, 50 lM EGTA, and 10 mM Tris–HCl (pH 8.5) as described (Agafonov et al. 2003). Fluorescence was measured using a USB-2000 spectroscopy system at excitation and emission wavelengths of 565 and 586 nm, respectively.
Measurements of Laurdan Generalized Polarization (GP) Laurdan is a fluorescent molecule that detects changes in membrane phase properties through its sensitivity to water molecules presented in the environment in the bilayer. Fluorescence of laurdan was measured using Cary Eclipse spectrofluorimeter. Excitation wavelength for laurdan was 355 nm; emission wavelengths were 440 and 500 nm. The generalized polarization (GP) was defined as GP = (I440 - I500)/(I440 ? I500) where I440 and I500 are the emission intensities at 440 and 500 nm, respectively (Parasassi et al. 1990). GP values can theoretically assume values from ?1 (being most ordered) and -1 (being least ordered). In the experiments with laurdan-containing lecithin liposomes (laurdan/lipid molar ratio, 1:200), the suspension of
The parameters of the main phase transition in the membrane of dimyristoylphosphatidylcholine (DMPC) LUV were measured with a temperature-scanning differential ultrasonic fixed-length interferometer (Kharakoz et al. 2007; Astashev et al. 2014). The measured quantity was the relative change in sound absorption caused by the dispersed lipid of the concentration, c: [A] = (ak - a0k0)/c. Scan rates were *.3 K/min on cooling runs. LUV were obtained as described above except that (1) DMPC was used instead of egg-phosphatidylcholine, (2) the concentration of DMPC in samples was higher (4 mM), and (3) all operations were carried out at 35 °C. The medium contained 10 mM Tris–HCl buffer (pH 8.5), 50 lM EGTA, and 40 mM KCl. The phase transition point was calculated as a midpoint between the half-maximum points. Dynamic Light Scattering The size of particles in the LUV suspension was measured by dynamic light scattering (DLS) at 25 °C using a Zetasizer Nano ZS device (Malvern Instruments Ltd.). The back-scattered light from a 4 mW He/Ne laser (632.8 nm) was collected at an angle of 173°. LUV was prepared as described above. The concentration of phospholipid in the samples was 50 lM. The medium contained 10 mM Tris– HCl buffer (pH 8.5), 50 lM EGTA, and 40 mM KCl. The acquisition time for a single autocorrelation function was 12 s. The resulting autocorrelation functions were averaged values from 12 measurements. The intensity-weighted size distributions were calculated using the following parameters: a refractive index of solution, 1.330; a viscosity value of solution, 0.8882 cP. Effect of PLA2 Inhibitors on the Ion Conductivity of BLM The ion-transport activity of PLA2 inhibitors was measured using the BLM technique (Mironova et al. 1994). Brain total lipid extract (20 mg/ml) and 10 % cardiolipin were dissolved in hexane and small aliquots were applied on a 1-mm aperture in the thin Teflon film, separating the two compartments of the measuring cell. The cell was filled with a medium containing 10 mM Tris–HCl buffer (pH 8.5), 50 lM EGTA, and 40 mM KCl. Ag–AgCl electrodes provided the electrical contact between the buffer and the operational amplifier (AD 711C, Analog Device, US). The
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data acquisition system included a 986 PC with a L-791 DAC/ADC board (L-Card, Moscow, Russia) and used adapted WINEdr 3.2.6 software (J.Dempster, Strathclyde Electrophysiology Software). Experiments were carried out at 20–22 °C. Statistical Analysis The data were analyzed using GraphPad Prism 5 and Excel software and were presented as mean ± SEM of 3–10 experiments. Statistical differences between means were determined by a two-tailed t test, with p \ 0.05 as the criterion of significance.
Results Effect of PLA2 Inhibitors on the Phase State of DMPC Liposomal Membranes Figure 2 shows temperature dependencies of specific sound absorption by a suspension of dimyristoyl phosphatidylcholine (DMPC) liposomes in the presence and absence of PLA2 inhibitors. A sharp increase in sound absorption upon temperature raising reflects a change in the phase state of lipids in the membrane (transition from the solid-crystalline (gel) to liquid-crystalline state), with the point of maximal absorption corresponding to the temperature of phase transition. As known from the literature data, the temperature of phase transition of DMPC is 23.9 °C (Kharakoz et al. 2007; Marsh 2012). The result of our measurements is close to the literature data (24.1 °C). As seen from figure, 400 lM PACOCF3 (*9 mol % of lipid)
Fig. 2 Effect of PLA2 inhibitors on the temperature dependence of specific sound absorbtion in the DMPC suspension. Medium composition: 10 mM Tris–HCl; 50 lM EGTA; 40 mM KCl (pH 8.5). 1, pure 4 mM DMPC; 2, DMPC ? 0.4 mM PACOCF3; 3, DMPC ? 0.4 mM AACOCF3; 4, DMPC ? 0.4 mM TFP
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increases temperature of phase transition by 2.7 °C, solidifying the membrane. AACOCF3, on the contrary, lowers the temperature by 3.3 °C and widens the range of phase transition temperatures, making the membrane more fluid (Fig. 2). TFP has a similar effect (Fig. 2): the temperature of phase transition decreases by 3.0 °C and the range of transition temperatures widens. This result is in agreement with the data obtained earlier by the DSC technique that TFP lowers the temperature of phase transition of phospholipid membranes (Hendrich et al. 2001). Laurdan Fluorescence Study To get more information on the effect of PLA2 inhibitors on the phase state of phospholipid bilayer, we performed a series of experiments with laurdan, a fluorescent probe whose spectral characteristics depend on the degree of order of lipid bilayer in the region of hydrophilic heads, which, in its turn, depends on the phase state of the membrane (Parasassi and Gratton 1995). On the basis of laurdan fluorescence spectra, we calculated values of generalized polarization (GP), as described in the Materials and Methods section. As seen in Fig. 3, the addition of PACOCF3 to the suspension of lecithin liposomes resulted in the growth of GP. This indicates that in the presence of the inhibitor, viscosity of liposomal membranes increased. AACOCF3 practically did not influence GP. At the same time, TFP considerably lowered viscosity of liposomal membranes (GP decreased significantly after the addition of TFP). Effect of PLA2 Inhibitors on the Conductivity of Bilayer Lipid Membranes As it is known, substances that alter the phase state of membranes, their viscosity and the molecular order of lipid bilayer can induce permeabilization of the membranes for ions and larger molecules (Anel et al. 1993; Antonov et al. 2005; Belosludtsev et al. 2015). To test if PLA2 inhibitors are able to change permeability of bilayer lipid membranes, we studied their effects on the conductivity of BLM and permeability of liposomes for the fluorescent probe sulforhodamine B. In our experiments, we applied the current relaxation technique to measure the inhibitor-mediated electric current across planar BLM under voltage-clamp conditions. As seen in Fig. 4, application of voltage (50 mV) to a planar BLM (formed from a mixture of total brain lipids and cardiolipin) in the absence of phospholipase A2 inhibitors results in a jump of electric current followed by its relaxation. The addition of 50 lM PACOCF3 to BLM did not alter permeability of the membrane. The addition of 50 lM AACOCF3 led to an increase in the steady-state
M. V. Dubinin et al.: Effects of Phospholipase A2 Inhibitors on Bilayer Lipid Membranes
Fig. 3 Changes of the parameter GP of the liposomal membrane in the presence of PLA2 inhibitors. The liposomes were formed from lecithin; the lipid concentration was 0.05 mM and the laurdan/lecithin ratio was 1:200. Medium composition: 10 mM Tris–HCl; 50 lM EGTA; 40 mM KCl (pH 8.5). Additions: 30 lM TFP, 30 lM AACOCF3, and 30 lM PACOCF3. The experiments were conducted at 25 °C. *p \ 0.05
current from *1 pA up to 2 pA. At the same time, the addition of 30 lM TFP led to the appearance of a channellike conductance (the level of current was about 3–5 pA; Fig. 4). Effect of PLA2 Inhibitors on the Permeability of Liposomes to Sulforhodamine B On the basis of the previous series of experiments, one can conclude that TFP (and, to a lesser extent, AACOCF3) increases permeability of bilayer lipid membrane for ions. To test if TFP and other PLA2 inhibitors are able to permeabilize membrane for larger molecules, we conducted experiments with lecithin unilamellar liposomes loaded with the fluorescent dye sulforhodamine B (SRB; Mr = 580.7 kDa). As seen in Fig. 5, the addition of TFP to liposomes resulted in a slow, gradual increase of SRB fluorescence. It took several minutes for fluorescence to reach a plateau. This experiment indicates that TFP induces a leak of SRB from liposomes. As seen in Table 1, AACOCF3 was much less effective comparatively to TFP. At the concentration of AACOCF3 45 lM (*50 mol% of total lipid), the release of SRB from liposomes reached only 13 %. PACOCF3 was even less effective: at the same concentration of the inhibitor, the release of the dye was only 7.5 %. A substantial release of SRB from liposomes upon the addition of PACOCF3 was observed only at much higher concentrations of the inhibitor (100 lM and above). The effect of TFP on the permeability of liposomes was investigated in more detail. Figure 6 shows a dependence of SRB release from liposomes on the concentration of TFP. As seen in figure, the dye begins to leak from liposomes at the concentration of TFP 15 lM. Figure 7 represents a dependence of TFP-induced liposome permeabilization on pH of the incubation medium. One can
Fig. 4 Effect of AACOCF3 (a), PACOCF3 (b), and TFP (c) on the electric current through a bilayer lipid membrane (BLM). Where indicated, 50 lM AACOCF3, 50 lM PACOCF3, or 30 lM TFP were added to the both compartments prior to the measurements. The incubation mixture contained 10 mM Tris–HCl, 50 lM EGTA, and 40 mM KCl (pH 8.5). At t = 8–11 s, 50 mV voltage was applied to the BLM
see that lowering pH results in the decrease of SRB release from the vesicles. Effect of PLA2 Inhibitors on the Size of Liposomes The ability of PLA2 inhibitors to induce nonspecific permeabilization of bilayer lipid membranes can be
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Fig. 5 Release of SRB from liposomes upon the addition of phospholipase A2 inhibitors (Inh). Medium composition: 10 mM Tris–HCl, 50 lM EGTA, 40 mM KCl (pH 8.5). The concentration of phospholipid was *40 to 50 lM. Additions: 1, 45 lM PACOCF3; 2, 45 lM AACOCF3; 3, 30 lM TFP. Common addition: 0.1 % TX-100
associated, on the one hand, with a change of the phase state of the membrane and appearance of membrane defects (lipid pores) and, on the other hand, with a detergent-like effect, which implies disintegration and micellization of vesicles. To test if PLA2 inhibitors can act as detergents, we examined their effect on the sizes of lecithin unilamellar vesicles by the method of dynamic light scattering. Figure 8 shows how a suspension of lecithin liposomes is distributed over their size in the presence and absence of PLA2 inhibitors. As seen in figure, TFP increases the size of lecithin liposomes. At the same time, the effect of the two other inhibitors is much weaker. As seen in Table 2, both AACOCF3 and PACOCF3 slightly increase the average hydrodynamic diameter of liposomes. Thus, it can be concluded that all the inhibitors tested cause aggregation and/or fusion of liposomes but not their disintegration and micellization.
Table 1 Effect of PLA2 inhibitors on the release of SRB from liposomes (% of total entrapped) Inhibitor
SRB release (% of total entrapped)
30 lM AACOCF3
7.7 ± 1.7
45 lM AACOCF3
12.6 ± 0.9
30 lM PACOCF3
4.5 ± 0.5
45 lM PACOCF3
7.5 ± 0.2
100 lM PACOCF3
16.5 ± 0.6
200 lM PACOCF3
59.6 ± 1.8
30 lM TFP
45.5 ± 4.3
Medium composition: 10 mM Tris–HCl, 50 lM EGTA, 40 mM KCl (pH 8.5)
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Fig. 6 Dependence of SRB release from LUV on the concentration of TFP. Medium composition: 10 mM Tris–HCl, 50 lM EGTA, 40 mM KCl (pH 8.5)
Fig. 7 A pH-dependence of the SRB release from LUV induced by the TFP addition. Medium composition: 10 mM Tris–HCl, 50 lM EGTA, 40 mM KCl. The concentration of TFP was 30 lM
Discussion Today, a large number of PLA2 inhibitors is known. Most of them exert their inhibitory effect in the submicromolarmicromolar range of concentrations—yet in many studies, PLA2 inhibitors are used at concentrations of 10-6–10-4 M (Broekemeier et al. 1985; Street et al. 1993; Riendeau et al. 1994; Hoyt et al. 1997; Li and Cathcart 1997; Broekemeier et al. 2002). Therefore, one can suppose that PLA2 inhibitors will have some nonspecific effects and, indeed, PLA2 inhibitors were reported to suppress other enzymes and signal systems (Johnson and Wittenauer 1983; Font et al. 1990; Doualla Bell Kotto Maka et al. 1990; Dabbeni Sala and Palatini 1990; Ichikawa et al. 1991; Riendeau et al. 1994; Laver et al. 1997; Li and Cathcart 1997; Jan et al. 2000; Bate et al. 2004; Leis and Windischhofer 2008). It should be noted that most of PLA2 inhibitors are hydrophobic, often lipid-like, compounds (Dennis et al.
M. V. Dubinin et al.: Effects of Phospholipase A2 Inhibitors on Bilayer Lipid Membranes
Fig. 8 Changes in the size of vesicles observed upon the addition of PLA2 inhibitors to the suspension of lecithin liposomes. Medium composition: 10 mM Tris–HCl; 50 lM EGTA; 40 mM KCl (pH 8.5). The concentration of phospholipid was *50 lM. 1, LUV; 2, LUV ? 50 lM AACOCF3; 3, LUV ?75 lM PACOCF3; 4, LUV ? 30 lM TFP
Table 2 Size of lecithin liposomes under various experimental conditions Experiment
Particle diameter (nm)
50 lM LUV
143.35 ± 0.64
50 lM LUV ? 30 lM TFP
218.97 ± 7.36
50 lM LUV ? 50 lM AACOCF3
152.4 ± 2.19
50 lM LUV ? 75 lM PACOCF3
171.7 ± 13.19
Medium composition 10 mM Tris–HCl, 50 lM EGTA, 40 mM KCl (pH 8.5)
2011), which means they will incorporate in the lipid bilayer of biological membranes and affect their properties. The compounds used in our research are lipophilic trifluoromethyl derivatives, inhibiting cytosolic Ca2?-dependent and Ca2?-independent phospholipases A2. In the current work, we have investigated how these PLA2 inhibitors affect some key features of bilayer lipid membranes. As follows from the data obtained, TFP and AACOCF3 are able to permeabilize membranes. It should be stressed that if AACOCF3 increases membrane permeability at a relatively high content in the membrane (50 mol% of total lipid), TFP causes membrane permeabilization at concentrations comparable to those used for inhibition of phospholipase activity (Broekemeier et al. 2002). The ability of AACOCF3 to increase nonspecific permeability of bilayer phospholipid membranes probably relates to the presence of an unsaturated fatty acid tail. Long-chain unsaturated fatty acids are known to permeabilize liposomal membranes at high concentrations (Muranushi et al. 1981; Ehringer et al. 1990), and this effect is generally considered to be associated with disordering of
the hydrophobic region of lipid bilayer. As a result, membrane defects (lipid pores) will appear in the disordered areas, increasing nonspecific permeability of the membrane (Muranushi et al. 1981; Ehringer et al. 1990). As seen in Fig. 2, the addition of AACOCF3 to DMPC liposomes leads to the decrease of the temperature of lipid phase transition, which may indicate an ability of the inhibitor to lower microviscosity of the membrane. The ability of PACOCF3 to make membranes more rigid (ordered) is associated with the presence of a saturated fatty acid tail. Similar to saturated long-chain fatty acids (Anel et al. 1993), PACOCF3 increases orderliness of the membrane—both in the hydrophobic region and the region of lipid heads. This should stabilize the membrane and suppress the leakage of the dye from liposomes. One can be suppose that the release of a small fraction of the dye observed upon the addition of PACOCF3 to liposomes is primarily related to the incorporation of a large number of the inhibitor molecules into the membrane, which would initially result in a perturbation of lipid bilayer. Raising the concentration of PACOCF3 to 100 lM led to a significant release of SRB from liposomes, which could be associated with morphological changes of the membranes or their micellization. A similar effect was observed in the case of long-chain fatty acids, when their content in the membrane exceeded 60 mol% (Mabrey and Sturtevant 1977). It should be noted that bromoenol lactone—another inhibitor of the Ca2?-independent phospholipase A2, which has a completely different structure—increased orderliness of lipid bilayer in the region of hydrophilic heads, like PACOCF3, but did not permeabilize liposomes (data not shown). On the basis of the results obtained, one can conclude that PLA2 inhibitors affect membranes variously, making them either more fluid or more ordered. It was suggested earlier that inhibition of phospholipase A2 should increase microviscosity of the membrane, since the release of arachidonic acid was suppressed (Schaeffer et al. 2005). However, the authors used PACOCF3 as the inhibitor, which, as follows from the results of the present work, would have lower membrane fluidity by itself. Perhaps, if another inhibitor of phospholipase A2 was used (AACOCF3 or TFP), membrane orderliness would not increase under those conditions. As demonstrated in the present work, TFP is a powerful agent capable to permeabilize artificial lipid membranes (BLM and liposomes). In contrast to AACOCF3, TFP disorders membranes both in the hydrophobic region and the region of hydrophilic heads. This should increase the chance of appearance of fluctuating lipid pores, which is indicated in our experiments by the channel conductivity of BLM and the slow leakage of SRB in the presence of TFP. It is known that TFP is able to induce phase separation in
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phosphatidylcholine membranes (Hendrich et al. 2001). Separation of membrane molecules into domains which differ by their phase state can also increase nonspecific permeability of phospholipid membranes (Agafonov et al. 2007; Belosludtsev et al. 2014). It should be noted that the effect of TFP is more pronounced at alkaline pH values. pKa2 of TFP is 8.1 (Malheiros et al. 1998), which means that below 8.0 TFP is predominantly in the protonated, charged form. It was supposed that charged TFP molecules would be less likely to enter the membrane interior, lowering the ability of TFP to fluidify lipid bilayer and form a domain membrane structure (Hendrich et al. 2001). The TFP-induced permeabilization of membranes can also be underlain by its ability to promote membrane fusion. It was found earlier that TFP induced fusion of erythrocytes (Malheiros et al. 1998). As seen in Fig. 8, the addition of TFP to liposomes in the absence of any protein molecules results in the increased dynamic light scattering, which can be interpreted as the increase of the size of vesicles (due to their fusion) or as their aggregation. It can be assumed that it is the effect of fusion of liposomes, since their aggregation would not lead to the release of the fluorescent dye. Application of PLA2 inhibitors is a convenient method for studying the functions of phospholipases and the metabolism of arachidonic acid under both in vitro and in vivo conditions. However, the data obtained in this work raise the question: what structures are primarily affected by PLA2 inhibitors in the cell? Is it the enzyme or the lipid matrix? Judging from the literature data on the values of IC50 for PLA2 inhibitors, it seems obvious that the primary target for these compounds is phospholipase A2 (Dennis et al. 2011). It should be noted, however, that the concentrations of PLA2 inhibitors used in the experiments are often 1–2 orders of magnitude higher than the concentrations that would suppress the activity of the enzyme. Therefore, a membranotropic effect of PLA2 inhibitors cannot be excluded as well. It should be taken into account when PLA2 inhibitors and their concentrations are chosen for research purposes. Acknowledgments The work was supported by the grants from the Russian Foundation for Basic Research to K.N. Belosludtsev (15-3450346, 15-04-03081-a) and to V.N. Samartsev (14-04-00688-a) by the Government of Russian Federation (Project No. 14.Z50.31.0028) and by the Ministry for Education and Science of Russian Federation to V.N. Samartsev (State Order no. 1365).
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