Influence of cetiedil on erythrocyte membrane microviscosity and acetylcholinesterase activity

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INFLUENCE OF CETIEDIL ON ERYTHROCYTE MEMBRANE MICROVISCOSITY AND ACETYLCHOLINESTERASE ACTIVITY JEAN GIANNETTINI, MICHELE CHAUVET, MONIQUE D E L L ' A M I C O and MADELEINE BOURDEAUX* Ddpartement de Physique, UFR de Pharmacie, 27, Bd Jean Moulin, 13385 Marseille Cedex 5, France Received in final form 25 July 1991

SUMMARY Since one of the cellular targets of cetiedil, a vaso-erythroactive drug, is likely to be the erythrocyte membrane, we have studied the influence of this drug on erythrocyte membrane microviscosity and acetylcholinesterase activity. No effect was evidenced on microviscosity, as measured by fluorescence polarization of light emitted by DPH or T M A - D P H labelling of the lipid bilayer. Cetiedil, however, did lower acetylcholinesterase activity, but it did not directly inhibit this enzyme activity. It can therefore be considered as an amphiphilic drug that perturbs membrane properties without affecting the physical state of the erythrocyte membrane.

KEYWORDS:cetiedil, acetylcholinesterase activity, ghost microviscosity.

INTRODUCTION

Cetiedil (Fig. 1), ~-cyclohexyl-3-thiophenacetic acid 2-(hexahydro- 1H-azepin-1yl) ethyl ester, is a vaso-erythroactive drug which was first introduced as an anticholinergic vasodilatator for the treatment of vascular disease [1, 2]. The drug was subsequently shown to reduce the duration of painful sickle-cell crises [3, 4]. The rheological action of cetiedil, however, is still poorly understood. It does not act by preventing the gelling of deoxy-HbS, nor by increasing erythrocyte affinity for oxygen [5]. The various mechanisms evoked to explain cetiedil's antisickling effect directly or indirectly implicate the erythrocyte membrane with regard to its permeability towards cations and water [6-8] or to its enzymatic activity, especially Ca2+-ATPase activity [9, 10] or to its lipid and protein components [ 1 1]. Thus the major antisickling effect of the compound might be on the erythrocyte membrane [ 12]. Basically, lipids are major constituents of biological membranes and are *To whomall correspondenceshouldbe addressed. 1043-6618/92/010031 - 11/$03.00/0

© 1992The ItalianPharmacologicalSociety

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organized as a bilayer characterized by its microviscosity. This term refers to rotational and lateral diffusion rates of membrane components [13]. There is substantial evidence that alterations in membrane viscosity are triggered when membranes interact with various drugs [14-16]. Furthermore, changes in membrane microviscosity have been shown to influence a number of important membrane functions, including the activity of certain enzymes [17, 18] and water and cation permeability [19]. Many enzymatic activities, in particular ATPase activities, are associated with the erythrocyte membrane. As cetiedil has been shown to modify the cation content of dehydrated erythrocytes [8], we first considered investigating its effects on ATPase activity. Several studies, however, have been performed in this field [9, 10], and recently, Bilto et al. [8] concluded that cetiedil had no effect on the ATP content of dehydrated erythrocytes. We therefore chose acetylcholinesterase and sought to determine whether it is inhibited and, if so, by what mechanism: either directly, since cetiedil is known to be an inhibitor of acetylcholine release from Torpedo electric organ synaptosomes [20], or indirectly, i.e. as a consequence of erythrocyte membrane microviscosity changes. Fluorescent probes are currently used to follow membrane microviscosity by use of static polarization measurements. Therefore, the main objective of this study was to test the effect of cetiedil incubation on erythrocyte membranes: first on their microviscosity by measuring fluorescence polarization of the well known probes 1,6-diphenyl-l,3,5-hexatriene (DPH) and 1-(4-trimethylammonium phenyl)- 6-phenyl- 1,3,5-hexatriene (TMA-DPH), and second, on the acetylcholinesterase activity localized on the outer surface of the red cell membrane.

MATERIALS AND METHODS

Reagents All normapur grade chemical reagents were from Prolabo except analysis grade monosodium phosphate from Merck and spectrophotometry grade ethanol from Carlo Erba. Cetiedil citrate was a generous gift from Innothera (France). Its purity was greater than 98%. Microcrystalline cellulose (mean size 50 ym, Sigmacell 50) and a-cellulose were purchased from Sigma Chemical Co., as was DPH. Acetylthiocholine iodide, 5,5'-dithiobis 2-(nitrobenzoic acid) and bovine

iH2C02H CH--CO2CH2CH2--N'/--'-~.HO--C--COzHH20 ~/

!H2C02H

Fig. 1. Structure of cetiedil citrate.

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erythrocyte acetylcholinesterase (EC 3.1.1.7; specific activity 0.4 units/rag) were from Sigma. TMA-DPH was from Molecular Probes.

Samples

Human blood samples from healthy donors were collected in heparin coated tubes. Blood was processed immediately. Centrifugations were performed at 4°C. Red blood cell suspensions were prepared from a 0.3 ml blood sample according to the Beutler et al. technique [21]; blood was filtered through 1 ml of a microcrystalline and a-cellulose resin (50/50 w:w) packed into a syringe. Elution was performed by using 12ml of isotonic NaC1 solution. After 10min centrifugation at 2500g, the erythrocyte pellet was suspended in 3 ml HEPES buffer pH 7.4 to obtain erythrocyte suspensions. To prepare ghosts, we haemolysed packed erythrocytes by hypotonic stress with 7.5 mM phosphate buffer pH 8, as described by Dodge et al. [22]. The erythrocyte ghosts obtained after three washing steps, each followed by centrifugation at 20 000 g, were haemoglobin-free. They were resuspended in 3 ml HEPES buffer pH 7.4. Negative stain electron micrographs of erythrocyte ghosts were fixed under the following conditions: a drop of ghost suspension was applied onto the grid and drawn off with filter paper; a drop of 1% (w/v) uranyl acetate was added immediately and allowed to dry for 30 min. Samples were examined with a Jeol JEM 1200 EX electron microscope at 80 kV.

Cetiedil incubation

A 125 mM solution stock of cetiedil citrate in ethanol was prepared and stored at -20°C until use within a month. Dilutions of this stock solution were prepared in alcohol extemporaneously. An amount of 6-12/.tl of the solution was added to 3 ml of erythrocyte or ghost suspension so as to obtain a final cetiedil concentration in the 83-250/.tM range. Samples were incubated for 30 rain at 37°C. The same volume of ethanol without cetiedil was added to incubation test tubes to prepare blanks. After centrifugation, buffer supernatants of erythrocyte and ghost suspensions were recovered and analysed for their cetiedil content by UV spectrophotometry on a Kontron 820 apparatus. Ghosts were prepared from pellets of erythrocyte suspensions as previously described.

Ghost labelling

Ten microlitres of a 2 mM DPH stock solution in dimethyl formamide, were vigorously shaken with 10 ml of polarization buffer (5 mM NaHzPO4, 5 mM KC1, 145 mM NaC1 pH 7.4). This suspension was mixed to an equal volume of ghost suspension in the same buffer containing about 1 mmol of phospholipids per ml, as checked by the method of Amic et al. [23]. This mixture was maintained in the dark for 30 rain at 37°C under gentle magnetic stirring. Incorporation of DPH was assessed by spectrofluorometry on a Kontron SFM25 apparatus. The suspension was then diluted in the polarization buffer so that its absorbance was less than 0.1

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Pharmacological Research, Vol. 25, No. 1, 1992

at 365 nm, the DPH excitation wavelength, in order to avoid any filter effect. T M A - D P H labelling was performed under identical experimental conditions, except that the stock solution used was 5 raM. Fluorescence measurements The fluorescence polarization of the light emitted at (425_+8) nm was measured on a SLM 4800 spectrofluorometer equipped with thermostated cells. Measurements were done at 37°C under gentle stirring. The apparatus displays the average of ten measurements. The results are the mean of nine calculations from parallel (L0 and perpendicular (I±) components of ~he light emitted, taking into account the balancing factor. Unlabelled controls under the same conditions as samples were studied to correct for the scattered light contribution, as described by Kuhry et al. [24]. Data are expressed as stationary anisotropies, r, defined as the ratio LrI=/l,+2I±. Microviscosities, in poises, were evaluated for DPH measurements from the equation: fT=2.4r/O.365-r,

as proposed by Shinitzky and Barenholtz [25]. Twenty samples and corresponding blanks without cetiedil were measured. Fluorescence lifetimes, z-, were obtained by observing at 3 0 M H z the modulation of the fluorescence emission of the samples relative to that of a glycogen scattering suspension. All other conditions were identical with those for fluorescence polarization measurements except that the excitation polarizer was in the vertical position and the emission polarizer set at 54.7°C to eliminate the effects of depolarizing rotation on the observed fluorescence lifetime. Acetylcholinesterase activity measurements Ghost acetylcholinesterase activity was determined at 37°C as described by Ellman et al. [26]. The assay mixture (2.5 ml) contained 0.5 mM acetylthiocholine, 35/tM 5,5'-dithiobis-(2-nitrobenzoic acid) and 7 flg/ml of membrane proteins in 0.1 M NaHzPO4 buffer, 1.5 mM NaHCO3, pH 8. Some experiments were performed with a free commercial acetylcholinesterase solution (0.350u/ml), of which 100/21 was taken to measure activity as described just above. Statistical analyses The paired t-test was used to compare blanks and samples. A P value of less than 5% was considered as significant.

R E S U L T S AND D I S C U S S I O N Our first aim was to check if cetiedil was actually retained in erythrocyte membranes and, if so, how much was retained. For this purpose, cetiedil

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concentration was measured by spectrophotometry in the supernatant from centrifugation of incubating medium. Figure 2 presents the UV absorption spectrum of cetiedil citrate in HEPES buffer and the calibration curve at 238 nm from which an absorption coefficient of 5257 M-1 cm -1 was calculated. The quantity of cetiedil retained in erythrocyte membanes or ghosts was calculated from the difference in absorbance between supernatant and the cetiedil solution used, taking into account the dilution factor. Results are reported in Table I. If one assumes 5.7×10-~°mg protein per ghost [11], the binding to red blood cells was much higher than that to ghosts. We then checked if handling to prepare haemoglobin-free ghosts from erythrocytes was able or not to remove cetiedil. This control was performed on ghosts; results are presented in Table I. It appears that some drug remained associated to ghosts after three hypotonic buffer washing steps. The binding of cetiedil to ghosts has been shown to be non-covalent and completely reversible as assessed by an overnight dialysis in buffer solution with a buffer to sample volume ratio of at least 1000 [11]. Our experimental procedure was far from these conditions and it seemed likely that cetiedil association to membranes persisted in part during ghost preparation from erythrocytes.

(a)

(b)

0.800

•0.600 / d O.400 O. 4 0 0

/

0.200

0

I

I

5

I0

I

15

[Cetiedil] [/0 -5 M] 0.500

0. 200

O. IO0

0.0

250

500 k (nm)

Fig. 2. (a) Absorption UV spectrum of a 80,UM cetiedil solution in HEPES buffer, pH=7.4. (b) Calibration curve at 1=238 nm.

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Ghosts were examined by electron microscopy; no difference was evidenced between ghosts treated or not with cetiedil (Fig. 3). To check DPH and T M A - D P H labelling, we recorded the fluorescence spectra over probe incubation time (Fig. 4). Ghosts were identically labelled by DPH when cetiedil was associated or not. For T M A - D P H , cetiedil hindered labelling, but to a very slight extent. This result was considered as proof of drug location on the ghost surface. Ghosts were then studied by fluoresence techniques. Variations in anisotropy can result from changes both in membrane

5~m

.......

Fig. 3. Electron microscopy observations of erythrocyte ghosts: (a) without cetiedil; (b) incubated 30 min with a 250 pM cetiedil solution in HEPES buffer pH=7.4.

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microviscosity and in the lifetime of the excited probe. Thus anisotropy and lifetime were determined (Table II). The DPH values are in good agreement with the data of Plasek and Jarolim [27] who reported an r value of 0.217 and those of Fiorini et al. [28] who gave a r value of l l . 5 x 1 0 - 9 s for ghosts labelled in comparable conditions. However, no significant differences (P=0.45) due to cetiedil treatment were evidenced. The location of DPH must be considered to explain this result. Indeed, if one takes into account the 30-min time lapse between

Table I Quantity of cetiedil retained in ghosts or intact cell membranes incubated for 30 min at various drug concentrations in HEPES buffer pH=7.4 A Cetiedil concentration in incubating medium (/~M)

83

150

170

B 200

250

Cetiedil associated with membranes after incubation (¢tmol/mg prot.)

0.16 0.20 0.23 0.28 0.38

Cetiedil associated with ghosts after washing (/.tmol/mg prot.)

0.12 0.13 0.15 0.17 0.22

83

250

2.82 7.21

Incubation A: with ghost suspensions. Ghosts were then submitted to three washing steps in 4.5 ml of 7.5 mM phosphate buffer pH=7.4; B, with erythrocyte suspensions.

F 1

F

j

Arbitrary units

Arbitrary units 70-

7O

60-

60

50-

50

40

40

3O

3O

20

20

10

10

0

i"

I

400

450

,

~

5 0 0 A(nm)

0

I

400

450

500

A(nm)

Fig. 4. Fluoresence spectra of DPH (left) or TMA-DPH (right) labelled ghosts: . . . . without cetiedil; - - - previously incubated with a 250/.tM cetiedil solution in HEPES buffer. (A) Labelling time 5 min; (B) labelling time 30 min.

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outset o f probe labelling and fluorescence measurements, D P H was likely to be located in the h y d r o p h o b i c inner core o f the m e m b r a n e bilayer [27]. It seems reasonable to suppose that the amphiphilic drug is located not too deep in the m e m b r a n e leaflet, but rather near the head groups. Thus cetiedil would not be able to perturb the probe m i c r o e n v i r o n m e n t and would then have no influence on D P H fluorescence lifetime and anisotropy values. It seemed o f interest to use another fluorescent probe, T M A - D P H , which is k n o w n to label the m i c r o e n v i r o n m e n t o f the h y d r o p h o b i c / p o l a r interface o f the membranal outer leaflet [29, 30]. Surprisingly, results obtained for T M A - D P H were similar to those for D P H (Table II): once again, no r and r differences were evidenced between ghosts treated or not with cetiedil (P--0.40). Our conclusion was that although cetiedil is retained in erythrocyte ghosts, it does not perturb their fluidity as tested by D P H or T M A - D P H fluorescence measurements. These results do not agree with those o f Narasimhan and Fung [11] who used very high ( 4 5 - 5 4 mM) cetiedil concentrations because of the relatively low sensitivity o f their E P R studies.

Table II Fluorescence lifetime, z, and anisotropy, r, of DPH (A,B) and T M A - D P H (C) labelled erythrocyte membranes incubated 3 0 m i n at various cetiedil concentrations A [Cetiedil] (/aM) 2"(10 -9 S) t"

~/(poises)

0

B

83

250

C

0

83

0

250

11.4+0.3 -11.5_+0.5 11.2_+0.3 11.3_+0.4 5.0_+0.2 4.8_+0.4 0.218_+ 0 . 2 1 9 _ + 0 . 2 1 8 _ + 0 . 2 2 4 _ + 0 . 2 2 3 _ + 0.285_+ 0.283+ 0.002 0.003 0.002 0.003 0.002 0.002 0.004 3.58+0.06 3.61-+0.05 3.58+0.05 3.84-+0.06 3.80_+0.05

Incubation A, with erythrocyte suspensions; B,C, with ghost suspensions. Fluorescence measurements were performed in the following conditions: 0=-37°C, 2,~= (365-+8) nm, 2om=(428+8)rim. Microviscosity was calculated according to Shinitzky and Barenholtz [25]. The results, given with their standard deviation, are the mean of 20 experiments.

Table III Inhibition of ghost acetylcholinesterase activity by cetiedil incubation at various drug concentrations A 150

B

[Cetiedil](/aM)

83

130

170

200

250

83

130

150

170

200

250

Inhibition (%)

ND

ND 24+3 54+2 60+2 58+3

ND

ND

ND

ND 20+2 33+3

Incubation was done: A, with erythrocyte suspensions; B, with ghost suspensions. The results are the mean of 20 experiments. SE were calculated for a 95% confidence interval. ND, not detectable.

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Nevertheless cetiedil incubation produced a partial loss of ghost acetylcholinesterase activity (Table III). This inhibition was higher when incubation was performed on erythrocyte suspensions rather than on ghost suspensions. This is consistent with the more pronounced binding to erythrocytes mentioned above (Table I). Acetylcholinesterase, the role of which remains unknown at the erythrocyte membrane level, is an amphipathic globular protein anchored in the membrane by a covalently attached glycoinositol phospholipid [31, 32], while its hydrophilic domain is oriented exclusively on the extracellular side of the membrane. Thus, its activity can be considered as a proof of erythrocyte membrane surface integrity. Recent work has shown that some amphiphilic drugs such as local anaesthetics inhibited acetylcholinesterase activity at concentrations at which they did not perturb the physical state of the membrane lipid environment [33]. It was necessary to test the direct effect of cetiedil on acetylcholinesterase activity. A free commercial enzyme was used for this purpose, as described in Materials and Methods. Adding cetiedil to a final concentration of up to 250 pM did not produce any enzymatic inhibition. This finding ruled out any direct effect on the enzyme or its substrate. In conclusion cetiedil can be classified into the group of drugs with membraneperturbing properties that affect acetylcholinesterase activity independently of the effect on the physical state of the erythrocyte membrane. The mechanism by which the enzymatic activity is lowered is not clear and would necessitate further investigation. Presently, it may be thought that cetiedil, as an amphiphilic drug located at the membrane surface, hinders substrate approach to acetylcholinesterase in a non-specific manner. Nevertheless, the concentrations of cetiedil required for in vitro acetylcholinesterase activity inhibition are much higher than that needed for in vivo beneficial effects in abbreviating the duration of acute painful crises of sickle cell anaemia [12]. A similar disparity between the drug concentration required for in vitro antisickling effect and that concentration (0.25/./M) [34] achievable in vivo has already been noted [12] and was largely discussed by Orringer et aI. [35]. From the pharmacological point of view, it is not certain that erythrocyte acetylcholinesterase activity inhibition by cetiedil is of some relevance. ACKNOWLEDGEMENTS The authors are grateful to Dr Comminges from Innothera (Arcueil, France) for giving them cetiedil citrate. They wish to thank R. Gochgagarian (Service de microscopie 61ectronique, Facult6 de Mddecine) for recording the electron micrographs, M. Vidalin for typing the manuscript and H. Bouteille for his graphical work.

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