Alteration of lipid order profile and permeability of plasma membranes from Trypanosoma cruzi epimastigotes grown in the presence of ketoconazole

Molecular and Biochemical Parasitology, 30 (1988) 185-196 Elsevier

185

MBP 01008

Alteration of lipid order profile and permeability of plasma membranes from Trypanosoma cruzi epimastigotes grown in the presence of ketoconazole Julio A. Urbina, Julio Vivas, Herlinda Ramos, Gisela Larralde, Zuleima Aguilar and Luisana Avil~n Centro de Biologia Celular, Escuela de Biologia, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela (Received 13 October 1987; accepted 4 April 1988)

Highly purified preparations of plasma membranes from control and ketoconazole-treated (1 ixM, 120 h) epimastigotes of Trypanosoma cruzi have been obtained by cell disruption using abrasion with glass beads, differential centrifugation and isopycnic centrifugation in continuous, self-generating Percoll gradients. The purity of the preparation was ascertained by the specific activity 125Ibound to the membranes obtained from enzymatically radiolabeled epimastigotes and by the a-methyl-mannoside sensitive binding of ~25I-concanavalinA. The membranes form closed vesicles of 0.2-0.4 txm in diameter which display Mg2÷ ATPase and acid phosphatase activities, but are devoid of 5'-nucleotidase and succinate-cytochrome c oxidoreductase; these vesicles can be strongly agglutinated by concanavalin A. The lipid order profiles of membranes from control and treated cells were compared with that present in egg phosphatidylcholine/ergoster01liposomes (84:16, mol/mol) by electron spin resonance spectroscopy of doxylstearic acid probes with the nitroxide group bound to carbon 5, 10, 12 and 16 of the stearic acid chain. Membranes from treated epimastigotes have a lipid order profile which resembles that of control plasma membranes near the polar surface (positions 5 and 10) but there is an abrupt decrease of order at position 12 and from there to the center of bilayer is highly disordered, even more than in pure lipid membranes. Consistent with these results, the leakage of L-[14C]glucosefrom membrane vesicles of ketoconazole-treated cells is much faster than that observed in vesicles obtained from control cells. These results indicate a strong alteration of the plasma membrane physical and biological properties due to the incubation of the parasite with the drug; this alteration is consistent with the accumulation of methylated precursors of ergosterol, which affects both lipid-lipid and lipid-protein interactions in the membrane. Key words: Trypanosoma cruzi; Epimastigote; Plasma membrane; Ketoconazole; Sterol synthesis; Ergosterol; Electron spin resonance; Passive permeability

Introduction T h e azole derivatives are p o t e n t a n t i m y c o t i c agents, several of t h e m orally active, which act by b l o c k i n g the synthesis of ergosterol in these cells Correspondence address: J.A. Urbina, Centro de Biologia Celular, Escuela de Biologia, Facultad de Ciencias, Universidad Central de Venezuela, Apartado 47860, Caracas 1041, Venezuela Abbreviations: Tris, tris-(hydroxymethyl)-aminomethane; Hepes, 4-(2-hydroxymethyl)-l-piperazinediethanesulfonicacid; PMSF, phenylmethylsulfonyl fluoride; EPC, egg yolk phosphatidylcholine; ESR, electron spin resonance; LIT, liver infusion-tryptose.

at the level of the c y t o c h r o m e P-450 d e p e n d e n t d e m e t h y l a t i o n at the C-14 of l a n o s t e r o l [1-3]. R e c e n t l y , it has b e e n s h o w n that these comp o u n d s are also very effective b o t h in vitro a n d in vivo against K i n e t o p l a s t i d parasites such as Leishrnania tropica [4,5], L. mexicana [6-9] a n d T r y p a n o s o m a cruzi [10-14]. T h e m e c h a n i s m of action of the drugs in the parasites seems to be similar to that r e p o r t e d for fungi [6-9,13,14] a n d this is consistent with the fact that these organisms synthesize ergosterol-like c o m p o u n d s a n d i n c o r p o r a t e t h e m as the m a i n sterols in their m e m b r a n e s [15-18]. A l t h o u g h the p r i m a r y effect of the azoles in these organisms is fairly well u n d e r s t o o d , the mo-

0166-6851/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

186

lecular basis of the growth arrest and eventual cell lysis which follow the block of the ergosterol synthesis are not so clear: an explanation frequently advanced is that the accumulation of C-14 methylated turn lead to the loss of vital cell components, a view supported by studies in model systems [19-21]. Other authors support the notion that the depletion of the endogenous ergosterol pool per se is the primary cause of cell death as it has been shown that 4-desmethyl sterols perform essential cellular functions, which seem to require relatively small amounts of these compounds but are highly specific with respect to the structure of the 4-desmethyl sterol required type by the different organisms [22-26]. We have recently presented evidence in favor of this second hypothesis [14] in the study of the concentration and time-dependence of effects of ketoconazole on T. cruzi epimastigotes, but we cannot rule out the possible membrane destabilizing effects of methylated ergosterol precursors (mainly trimethylated sterols, see refs. 13 and 14) which accumulate in these organisms. In this paper we present a comparative study of the lipid bilayer order profile and passive permeability properties of purified plasmatic membranes from control and ketoconazole-treated T. cruzi epimastigotes. The membranes from ketoconazole-treated epimastigotes present a highly disordered lipid bilayer from carbon 12 to the central region, when compared with control plasma membranes or artificial lipid membranes whose chemical compositions resemble that of the control membranes. The alteration in the lipid order is correlated with a large increase in the passive permeability of these membranes towards large hydrophilic molecules such as k-glucose, which must contribute to the loss of cell viability induced by the drug. Materials and Methods

Organism and growth conditions. The epimastigote form of the EP stock of T. cruzi was cultivated in liver infusion-tryptose (LIT) medium at 28°C with strong aeration (120 rpm in a rotary shaker), as described previously [27]. Only exponential-phase epimastigotes (1.5-2.0 × 10 s epimastigotes m1-1) were used; in this phase > 9 9 % of the cells are typical, highly motile epimasti-

gotes. In the study of the effect of ketoconazole on the plasma membrane the drug was added (1 ~M final concentration) at an initial cell density of 107 epimastigotes m1-1 and growth was allowed to proceed for 120 h. Ketoconazole was added as aqueous solutions titrated to pH 2.4 with HCI and sterilized by filtration through 0.22 ~m Millipore filters. Cell density was determined both with a Coulter counter (model ZBI) and by the use of a hemocytometer.

Isolation of plasma membranes. Plasma membranes from control and ketoconazole-treated epimastigotes were isolated and purified at 4°C, essentially as described previously for the preparation of plasma membrane vesicles from L. mexicana promastigotes [28]. Briefly, epimastigotes (10 ll cells) were harvested and washed twice by centrifugation (1000 × g for 10 min) with buffer A (75 mM Tris-HCl, 140 mM NaC1, 11 mM KC1, pH 7.4). After a further wash with buffer B (400 mM mannitol, 10 mM KCI, 3 mM magnesium acetate, 10 mM Hepes, pH 7.6, supplemented with 2 mM phenylmethylsulfonyl fluoride (PMSF) and 0.2 mg ml I of trypsin inhibitor) the pellet was mixed in an ice-cold mortar with glass beads (75-120 p~m in diameter, Sigma Chemical Company, St. Louis, MO) in a 4:1 (weight of beads/wet weight of pellet) ratio. The mixture was ground gently until 90% of cell rupture was observed by phase contrast microscopy (5-7 min for control cells and 1-3 min for ketoconazole-treated cells). The ground material was resuspended in buffer B (4 ml per g wet weight) and unbroken cells, cell debris and glass beads were removed by centrifugation at 1000×g for 10 min. The supernatant, defined as the whole cell homogenate, was subjected to centrifugation at 5000 × g for 20 min: the pellet was defined as the large granule fraction and the supernatant was subjected to centrifugation at 15 000 × g for 35 min yielding a pellet (small granule fraction) and a supernatant which was centrifuged at 105 000 × g for 50 min to give a pellet, the microsomal fraction. This last fraction was purified by centrifugation in a continuous, self-generating density gradient of 17% Percoll in 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4; for this purpose the microsomal fraction was resuspended in 25 ml of the above Percoll-con-

187 taining solution and the suspension was centrifuged in a fixed angle rotor (Sorvall SS-34) at 35 000 × g for 50 min. This produced a continuous, non-linear gradient of density ranging from 1.03 mg ml -~ at the top to 1.15 mg ml -~ at the bottom. The purified plasma membrane fraction was recovered in the range of 1.040-1.045 mg ml -~. Percoll was removed by centrifugation of the fraction layered on top of a 50% sucrose cushion at 105000 × g for 1 h; the membranes were recovered at the interface, resuspended in buffer B without mannitol, pelleted at 105 000 × g for 60 min and finally resuspended in the same buffer at a protein concentration of 5-10 mg ml -~, frozen in liquid nitrogen and stored at -80°C until use. Gradient densities were determined using density marker beads (Pharmacia Fine Chemicals, Uppsala).

Enzymatic radioiodination of epimastigotes. The radioiodination of intact cells was carried out essentially as described by Phillips and Morrison [29]. The epimastigotes were harvested and washed in buffer A as described above and resuspended to a final cell density of 2 × 108 cells ml -~. The incubation medium contained, in buffer A: 250 ~Ci m1-1 of lzsI, 25 IxM KI, 10 mM glucose, 0.2 mg ml -~ glucose oxidase, 0.2 mg m1-1 lactoperoxidase. Cells were incubated for 15 min at 4°C and the reaction stopped by the addition of Na2SzO 3 to a final concentration of 0.1 M. The cells were then washed four times with buffer A supplemented with 5 mM KI. After radioiodination the cells were subjected to the subcellular fractionation procedure described above and the radioactivity of the fractions determined in a Nuclear Chicago gamma counter. Omission of lactoperoxidase decreased iodine incorporation to 1.0-1.5% of the labeling observed in the presence of the enzyme. Cell viability of labeled cells using trypan blue indicated > 9 8 % of viable cells.

Specific labeling of cell surfaces with ~2~l-concanavalin A. 125I-concanavalin A was prepared using the I O D O G E N method [30]: the reactive was deposited in glass vials and then a solution of concanavalin A (10 mg m1-1) and 125I (50 txCi m1-1, 5 ixM KI) in buffer A was added to the vials and incubated for 20 min at 25°C, with gentle shak-

ing. The radiolabeled protein was purified by gel filtration chromatography on Sephadex G-25-80. The pure protein was stored at - 2 0 ° C in the presence of 1 mM CaC12 until use. For the specific labeling of cells, washed epimastigotes (108 cells m1-1) were incubated in the presence of 20 tzg ml-~ of 125I-concanavalin A for 30 min at 4°C, to avoid a lectin-induced agglutination of the cells [31]. The cells were then harvested and washed four times with buffer A by centrifugation at 1000×g for 10 min and then fractionated as described above except that in the Percoll density gradient step the osmolarity was adjusted with 0.25 M glycerol to avoid the competitive effect of sucrose on the concanavalin A binding; this required increase of the Percoll concentration to 32%. Controls, in which the incubation of the cells with the radioiodinated lectin was carried out in the presence of 0.1 M o~-methylmannoside, bound 0.5-1.0% of the radioactivity in all fractions.

Electron microscopy. Pellets of all subceUular fractions obtained by centrifugation were fixed with 3% glutaraldehyde in buffer A and post-fixed in 1% OsO~. The fixed samples were dehydrated with a graded series of ethanol solutions; final dehydration was accomplished by the use of propylene oxide. The pellets were embedded in Epon and thin sections were stained in sequence with 1% lead citrate and 1% uranyl acetate and examined in a Hitachi H-500 electron microscope. Enzymatic assays. Succinate-cytochrome c oxidoreductase was assayed according to Sottocasa et al. [32]. Acid phosphatase was measured by the release of p-nitrophenol from p-nitrophenylphosphate according to Gottlieb and Dwyer [33]. Mg2+ATPase was assayed by a coupled reaction assay; the reaction medium contained 2 mM ATP, 3 mM MgCI2, 20 mM Hepes, 1 mM PEP, 5 units ml-I of both pyruvate kinase and lactic dehydrogenase, 0.2 mM N A D H , p H 7.5 and the subcellular fraction (40-60 I~g ml-l). The oxidation of N A D H was followed spectrophotometrically at 340 nm. In some assays oligomycin was included at a final concentration of 100 ~g (mg protein)-1. For the assay of ouabain-sensitive Na ÷, K ÷ ATPase, 100 mM NaCI and 100 mM KCI were included in the reaction medium and assays were

188 carried out in the presence and absence of 1 mM ouabain. Oligomycin and ouabain were added as ethanol solutions; the final ethanol concentration in the assay medium was 0.5% (v/v) and had no effect on the enzymatic activity. 3' and 5'-nucleotidase were assayed as described previously [28].

Concanavalin A-induced agglutination of subcellular fractions. The agglutination of subcellular fractions induced by concanavalin A at 25°C was quantitated by turbidimetry as described before [31]. Typically, the subcellular fraction (100 p,g ml-1 in buffer A) was incubated with the desired concentration of the lectin, ranging from 0 to 60 ixg m1-1, and the agglutination process was followed by the increase in the absorbance at 600 nm as a function of time.

to optimize signal to noise ratio without spectral distortion. Spectra were analyzed and order parameters (S) calculated according to Griffith and Jost [36].

Passive permeability studies. Plasma membrane vesicles (5-10 mg m1-1) from control and ketoconazole-treated cells were incubated for 24 h in buffer B without mannitol containing 4 mM L[14C]glucose (10 mCi mmol-1). They were then diluted 100-fold in buffer B without mannitol plus 4 mM L-glucose, incubated for different times at 28°C and then samples of 2 ml of the suspension were rapidly filtered through 0.45 ixm Millipore filters. The filters were washed with 15 ml of the incubation buffer at 0°C and the radioactivity retained in them was determined by liquid scintillation spectrometry.

Preparation of artificial lipid vesicles. Single-walled large lipid vesicles (liposomes) were prepared by the reverse phase method of Szoka and Papahadjopoulos [34] as described previously [35].

Spin labeling of membranes and electron spin resonance measurements. For spin labeling the doxylstearic acid derivatives were added directly from stock ethanolic solutions to plasma membranes or lipid vesicles (10 mg ml 1 in buffer A) to give a molar ratio of phospholipids to probe of 100:1, with a final ethanol concentration of < 1 % (v/v); the membranes are incubated at 4°C overnight and electron spin resonance (ESR) spectra were recorded in a Varian E-104 spectrometer at 25°C using special aqueous sample cells. Modulation amplitude and microwave power were adjusted

Chemical analysis. Proteins were determined by the Folin phenol method of Lowry et al. [37], with bovine serum albumin as standard. Total phospholipids were measured by the organic phosphate method of Ames and Dubin [38] and sterols according to Stadman [39]. Results

Table I shows the specific radioactivity of subcellular fractions obtained from surface-radiolabeled epimastigotes. A purified membrane fraction recovered in the 1.040-1.045 mg m1-1 region of the Percoll density gradient had a specific activity 9-10 times higher than that of the whole cell homogenate in the case of enzymatic radioiodi-

TABLE I Specific radioactivities of subcellular fractions of T. cruzi epimastigotes labeled by enzymatic radioiodination or by ~251-concanavalin A binding Subcellular fraction Homogenate Large granule fraction Small granule fraction Microsomal fraction Purified membranes "cpm (rag protein) -~.

Enzymatic radioiodination Specific radioactivity' 4 020 14 900 11 100 18 700 35 500

% 100 48 25 7.5 5.0

~25I-ConAbinding Specific radioactivity" 6 400 20 000 20 000 41 500 82 000

% 100 45 24 10 10

189

nation of the epimastigotes and 12-14 times higher in the case of epimastigotes labeled with 125I-concanavalin A. The recoveries are 5 and 10% of the total radioactivity in the purified membrane fraction, indicating that a substantial proportion of the cell surface cosediments with other, heavier, subcellular fractions. The radioactivity associated with the microsomal fraction corresponds completely to surface membranes as shown by the single, symmetric radioactivity peak in the Percoll density gradient (Fig. 1). The purified membrane fraction consists of closed, single-walled vesicles whose diameters fall mostly in the 0.2-0.4 Ixm range and do not contain attached microtubules (Fig. 2); these vesicles display an almost ideal osmotic behaviour (Urbina, J.A. and Cohen, B.E., unpublished). The vesicular preparation is strongly agglutinated by concanavalin A in a saturable and ot-methylmannoside sensitive fashion.

The agglutination is essentially completed in 20 min. The concentration of the lectin required to attain the maximal effect is 11.5 Ixg ml 1; at 60 Ixg ml-1 the response is already saturated. Morphological analysis of the agglutinated membranes shows large clumps of aggregated and, in some cases, fused vesicles. In the presence of 0.1 M of the sugar hapten the phenomenon is completely abolished (data not shown). Specific activities of several marker enzymes in the different subcellular fractions are presented in Table II; it can be appreciated that the purified membrane fraction is enriched in oligomycin-insensitive Mg2÷ ATPase and acid phosphatase, but is devoid of succinate-cytochrome c oxidoreductase, which shows that it is free of mitochondrial contamination. No activities of Na ÷, K ÷ activated ouabain-sensitive Mg 2÷ ATPase, 3' or 5'nucleotidase were detected in any of the subcel-

cpm/~g prot t 80

/

6(>

5O-

5O-

20.

I(3-

,--.--.-.--,--,--.--.-.--,--,--.-.,-, I

(TOP)

2

3

4

5

6

7

8

9

I0

II

IZ

FRACTION N U M B E R ~

13

14.

(BO'I-fOM)

Fig. 1, Distribution of radioactivity in an autoforming continuous Percoll density gradient loaded with microsomal fraction from T. cruzi epimastigotes labeled with ]25I-concanavalin A and centrifuged at 35 000 × g for 50 min. The two profiles correspond to cells labeled with the lectin in the absence (e) or presence (o) of 0.1 M {x-methylmannoside. Fraction 1 corresponds to the top of the gradient (density 1.03 mg ml -~) while fraction 14 is the bottom (density 1.15 mg ml 1).

Fig. 2. Electron micrograph of a thin section of purified plasma membrane vesicles from T. cruzi epimastigotes. Membranes were obtained from the Percoll density gradient, washed free of Percoll, pelleted, fixed and stained as described in Materials and Methods. Bar corresponds to 0.4 Ixm.

190

T A B L E 11 Specific activities of marker enzymes in subcellular fractions of T. cruzi epimastigotes Subcellular fraction

Homogenate Large granule fraction Small granule fraction Purified membranes

Specific activity (nmol min ~ (mg protein) ~) Succinate-cytochrome c oxidoreductasc

Acid phosphatase

ATPase a - OL/+ OL

0.76 1.64 2.37 0.00

4.0 1.2 4.2 16.9

6.8/5.7 3.9/2.8 5.7/4.4 19.5/19.5

"ATPase was assayed in the absence ( - O L ) or in the presence ( + O L ) of 150 Ixg oligomycin (mg protein) t. For other details, see Materials and Methods.

lular fractions under our assay conditions. The phospholipid/protein ratio in the purified membranes is 0.24 I~mol phospholipid (mg of protein) -~, while the phospholipid/sterol molar ratio is 4.1; no significant differences were found in this respect between membranes from control and ketoconazole-treated epimastigotes. A gross alteration of the properties of the cell envelope in the treated cells is indicated by the dramatic increase of their fragility in the abrasion procedure used for cell disruption (see Materials and Methods). To explore in more detail the modification of the physical properties of the cellular membranes resulting from the exposure of the epimastigotes to ketoconazole, we investigated the order profiles of the hydrophobic region of membranes obtained from control and drug-treated cells and compared them with that found in EPC/ergosterol (84:16, mol/mol) large, unilamellar lipid vesicles. Fig. 3 and Table III contain quantitative information of this order profile from the three preparations. The information is obtained from the maximum (2Amax) splittings of the ESR spectra of doxyl-stearic acid probes incorporated in these membranes, with the paramagnetic nitroxide group bound to carbon 5, 10, 12 and 16 of the stearate chain, from which approximate order parameters can be obtained. Although these order parameters (Table III) are not corrected for membrane polarity effects they are useful for comparison of the lipid order at comparable bilayer depths in different preparations [36]. Plasma membranes from control epimastigotes are significantly more ordered than the pure lipid membranes near the polar surface (po-

sitions 5 and 10); this is most probably due to the penetration of intrinsic proteins in the lipid bilayer of these protein-rich membranes (see above). However, in both cases order decreases monotonically with increasing distance from the head-group and the two membranes are very similar in this respect at the center of the bilayer. The lipid order in membranes from ketoconazoletreated epimastigotes is similar to that of membranes from control cells near the polar surface but at position 12 there is an abrupt decrease of order and from there to the center the bilayer is highly disordered, even more than in the case of the pure lipid membranes. The drop in the lipid order at position 12 in the membranes derived from the treated cells is highly significant and

6O

25

~5

;

-~

~

;

,;

,',

I

,2

L

,3

I

,4

i

,'5 ,6

i

,'7 ,s

CARBON ATOM NUMEER Fig. 3. Maximum (external) hyperfine splinings of the ESR spectrum of stearic acid spin labels incorporated in plasma membrane vesicles from control T. cruzi epimastigotes (m), ketoconazole-treated epimastigotes ( A ) and large unilamellar EPC/ergosterol (84:16, mol/mol) lipid vesicles (o) as a function of the position of the nitroxide group in the stearic acid chain. Membrane vesicles and liposomes were prepared and spin labeled as described in Materials and Methods.

191 TABLE III Order parameters (S) of the nitroxide group of stearic acid spin probes incorporated in plasma membranes from control and ketoconazole-treated T. cruzi epimastigotes and large unilameUar EPC/ergosterol lipid vesicles" Carbon atom number 5

10

12

16

Membranes from control cells

0.75

0.59

0.19

0.14

Membranes from ketoconazole-treated cells

0.70

0.53

0.10

0.10

EPC/ergosterol (84:16, mol/mol) lipid vesicles

0.50

0.32

0.18

0.04

aOrder parameters were calculated from the maximum hyperfine splittings (2Amax)of the ESR spectra as described by Griffith and Jost [36].

c o r r e s p o n d s to a d e c r e a s e in t h e o r d e r p a r a m e t e r of t h e n i t r o x i d e g r o u p in t h a t p o s i t i o n f r o m 0.2 in c o n t r o l e p i m a s t i g o t e s to 0.1 in t h e k e t o c o n a z o l e t r e a t e d cells ( T a b l e I I I ) . W e also i n v e s t i g a t e d t h e p a s s i v e p e r m e a b i l i t y p r o p e r t i e s o f t h e m e m b r a n e vesicles o b t a i n e d f r o m c o n t r o l a n d k e t o c o n a z o l e - t r e a t e d cells; Fig. 4 shows t h e r e l e a s e o f L-[a4C]glucose f r o m b o t h t y p e s o f s t r u c t u r e s . W e s t u d i e d t h e p e r m e a t i o n of

this u n n a t u r a l i s o m e r to c h e c k that t h e m o v e m e n t was through the lipid bilayer and not via the D-glucose t r a n s p o r t s y s t e m p r e s e n t in t h e e p i m a s tigotes, which d o e s n o t r e c o g n i z e t h e L - i s o m e r ( J . A . U . , u n p u b l i s h e d o b s e r v a t i o n s ) . T h e vesicles f r o m c o n t r o l cells clearly s h o w at least t w o p o p ulations: l e a k y vesicles with a h a l f - t i m e for release of t h e c a r b o h y d r a t e o f 12 m i n , which c o m prises 55% of the t o t a l i n t e r n a l v o l u m e o f t h e

60" 5c~4o" ~--2o-

rr

6-

4-

z

I o

40

50

60

--~ TIME (minutes)

Fig. 4. L-[14C]glucoserelease from plasma membrane vesicles obtained from control (o) and ketoconazole-treated T. cruzi epimastigotes (e). Plasma membranes vesicles (7 mg m1-1) were loaded for 24 h with L-[14C]glucoseand then diluted and rapidly filtered at the indicated times. The amount of radioactivity retained at the onset of the experiment, after filtering and washing, is 39 300 dpm (mg protein) -1 for vesicles from control cells and 12500 dpm (mg protein) -I for vesicles from ketoconazole-treated cells.

192 preparation, and tightly sealed vesicles with a halftime for release of 114 min. In contrast, the vesicles from ketoconazole-treated cells retain after filtration and wash (30 s of exposure to the release medium) only 32% of the labeled compound when compared, on the basis of protein content, with the vesicles from control cells, indicating a population of highly leaky vesicles which completely release the radiolabeled compound in the above indicated period of time and account for two thirds of the total internal volume; a second population of vesicles has a halftime for L-glucose release of 25 min. Discussion

A problem in the preparation of the subcellular fractions of organisms of the order Kinetoplastida is the presence of a highly resistant cytoskeleton which requires the use of drastic conditions for cell disruption; this usually leads to complete fragmentation of cell organelles and concomitant cross-contamination. The problem has been circumvented previously using hypotonic conditions followed by cell homogenization in the presence or absence of non-ionic detergents [40,41], vesiculation in acidic media [42] or controlled sonic vibration [43,44]. However, most of these methods involve lengthy centrifugations in sucrose density gradients to attain reasonably purified membrane preparations and the crucial step of cell disruption is not easily reproduced. We developed a simple and fast procedure which relies on controlled cell disruption with glass beads and centrifugation in autoforming, non-viscous Percoll density gradients which yield a highly purified membrane preparation, almost ideal for structural and transport studies. We have successfully applied this method to prepare plasma membrane vesicles from L. mexicana promastigotes in a study of the mechanism of action of polyene antibiotics [28]. One problem in the characterization of plasma membrane from T. cruzi epimastigotes is the lack of classical marker enzymes in them; we, as others [40,42], have not been able to detect in this organism Na ÷, K ÷ activated, ouabain-sensitive Mg 2+ ATPase nor 3' or 5'-nucleotidase, which are, however, very active in Leishmania [28,45]. The only report on the

presence of 5'-nucleotidase in T. cruzi is that of Nagakura et al. [44] in which a 95-fold purification of the membrane-bound enzyme is claimed, a claim we have not been able to confirm. The presence of acid phosphatase in cell surface has been reported for this organism and Leishmania promastigotes [28,43,44,46]. Finally, the oligomycin-insensitive Mg 2+ ATPase present in the purified membranes could probably play a role in carbohydrate active transport processes in this and related organisms (J.A.U., unpublished; see also ref. 47); a detailed biochemical and biophysical characterization of this enzyme will be reported elsewhere. Due to the lack of well-established marker enzymes, an independent criterium of purity is the increase in the purification procedure of the specific radioactivity of the preparation starting with epimastigotes surface-radiolabeled enzymatically with 125I or with ]2sIconcanavalin A, as it is known that these cells are agglutinated by the lectin [31,48]. The >10-fold increase in specific radioactivity using both criteria and the lack of significant mitochondrial contamination in the purified membrane fraction suggest a highly purified surface membrane preparation, a fact confirmed by the specific agglutination of this preparation by concanavalin A. The properties of the membrane fraction described in this paper are similar to that reported by Zingales et al. [43], but the time required to obtain it is only a fraction of that required using the method devised by these authors. The yield, in terms of the percentage of total cell protein, is rather low but certainly comparable to that reported by these and other authors [40,43,44]. We have shown previously [14] that ketoconazole at 1 IxM produces only a very slight effect on the growth rate of T. cruzi epimastigotes immediately after the addition of the drug, despite the fact that it produces a rapid and complete inhibition of the cell's ergosterol biosynthesis. However, after 120 h growth stops rather suddenly and cell lysis begins to take place. We showed in the same study that growth inhibition coincides with the depletion of the endogenous ergosterol pool which could lead to specific metabolic defects [22] but we could not rule out a general modification of the physical properties of the membrane due to its altered lipid composition. Thus we investi-

193

gated in this work some physical properties of the plasma membranes of drug-treated epimastigotes at the onset of the growth arrest. The altered lipid order profile in the plasma membranes of ketoconazole-treated cells as compared with those from control cells and phosphatidylcholine/ergosterol vesicles is clearly consistent with the accumulation of C-14 methylated precursors of ergosterol in these cells [13,14], as it is known from ESR studies of doxyl-stearate probes incorporated in model membranes that the presence of an alkylation at C-14 of the steroid nucleus disrupts drastically the lipid packing precisely at the C-12 position of the acyl chains [21]. This is the first report which demonstrates directly the alteration of the lipid packing in membranes as a major cellular effect of ketoconazole in treated cells. Such perturbation of the lipid packing, which has been confirmed in model systems by microviscosity measurements using fluorescence depolarization and glucose permeability [19,20], must underlie a serious alteration of the passive permeability properties of the membranes. This prediction was confirmed by the results of the study of the passive permeability properties of the membrane vesicles from control and treated cells which indicate a large increase in the rate of the L-glucose release from the vesicles of the treated ceils when compared with those obtained from control cells. We have also studied these two membrane preparations using an osmotic method with rapid mixing described previously [28]; the results show that the vesicles from the treated cells retain the full osmotic response of the vesicles from the control cells but are much more permeable to a series of non-electrolytes and potassium salts (Ramos, H., Cohen, B.E. and Urbina, J.A.,

in preparation). Thus, the decreased retention of the radiolabeled carbohydrate in the vesicles from treated cells is not due to the lack of closed membranous structures of the appropriate size in the preparation but to the very large permeability of the intravesicular solute. Taken together these facts demonstrate that after 120 h of treatment with ketoconazole a rapid loss of large polar molecules takes place in the treated cells, which should lead to an irreversible metabolic arrest. Similar permeability alterations have been observed when cholesterol is replaced by some of its natural and synthetic alkyl derivatives in artificial lipid membranes [19,22]. In conclusion, the results of the present study demonstrate that the treatment of T. cruzi epimastigotes with the antimycotic imidazole ketoconazole produces profound alterations in the composition and properties of its plasmatic membranes, particularly in their lipid order profile and related passive permeability properties, which are essential for the maintenance of the cellular activities. These alterations could by themselves be the primary causes for the loss of cell viability, although specific metabolic defects due to the depletion of the ergosterol pool could also be involved [14,22].

Acknowledgements This work was supported by the Consejo Nacional de Investigaciones Cientificas y Tecnologicas, Caracas, Venezuela, grant DDCT-SAL-9 and in part by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, grant No. 77-0308.

References 1 Sud, I.J.S. and Feingold, D.S. (1981) Mechanisms of action of antimycotic imidazoles. J. Invest. Dermatol. 76, 438-441. 2 Van den Bossche, H., Bellens, D., Cools, W., Gorrens, J., Marichal, P., Verhoeven, H., Willemsens, G., De Coster, D., Beerens, D., Haelterman, C., Coene, M.-C., Lauwers, W. and Le Jeune, L. (1986) Cytochrome P-450: target for itraconazole. Drug Dev. Res. 8, 287-298. 3 Van Cutsem, J. and Van Gerven, F. (1986) The in vivo antifungal activity of broad-spectrum azoles. Drug Dev. Res. 8,309-316.

4 Berman, J.D. (1981) Activity of imidazoles against Leishmania tropica in human macrophage cultures. Am. J. Trop. Med. Hyg. 30,566-569. 5 Wienrauch, L., Livshin, R. and El-On, J. (1983) Cutaneous leishmaniasis treatment with ketoconazole. Cutis 32, 288-294. 6 Berman, J.D., Holz, Jr., G.G. and Beach, D.H. (1984) Effects of ketoconazole on growth and sterol biosynthesis of Leishmania mexicana promastigotes in culture. Mol. Biochem. Parasitol. 12, 1-13. 7 Goad, L.J., Holz, Jr., G.G. and Beach, D.H. (1985) Ster-

194

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

ols of ketoconazole-inhibited Leishmania mexicana mexicana promastigotes. Mol. Biochem. Parasitol. 15,257-279. Berman, J.D., Goad, J.L., Beach. D.H. and Holz, Jr., G.G. (1986) Effects of ketoconazole on sterol biosynthesis by Leishmania mexicana mexicana amastigotes in murine macrophage tumor cells. Mol. Biochem. Parasitol. 20, 85-92. Urcuyo, F.G. and Zaias, N. (1982) Oral ketoconazole in the treatment of leishmaniasis. Int. J. Dermatol. 21, 414-416. Docampo, R., Moreno, S.N.J., Turrens, J.F., Katzin, A.M., Gonzales-Cappa, S.M. and Stoppani, A.O.M. (1981) Biochemical and ultrastructural alterations produced by miconazole and econazole in Trypanosoma cruzi. Mol. Biochem. Parasitol. 3, 16%180. McCabe, R.E., Remington, J.S. and Araujo, E.G. (1984) Ketoconazole inhibition of intracellular of Trypanosoma cruzi and protection against lethal infection with the organism. J. Infect. Dis. 150, 594-601. Raether, W. and Seidenath, H. (1984) Ketoconazole and other antimycotic azoles exhibit pronounced activity against Trypanosoma cruzi, Plasmodium berghei and Entamoeba hystolytica in vivo. Z. Parasitenkd. 70, 135-138. Beach, D.H., Goad, L.J. and Holz, Jr., G.G. (1986) Effects of ketoconazole on sterol biosynthesis by Trypanosoma cruzi epimastigotes. Biochem. Biophys. Res. Commun. 136, 851-856. Larralde, G., Vivas, J. and Urbina, J.A. (In press) Concentration and time dependence of the effects of ketoconazole on growth and sterol synthesis by Trypanosoma (Schizotrypanum) cruzi epimastigotes. Acta Cient. Venez. Meyer, H. and Holtz, Jr., G.G. (1966) Biosynthesis of lipids by kinetoplastid flagellates. J. Biol. Chem. 241, 5000-5007. Korn, E.D., Von Brand, T. and Tobie, E.J. (1969) The sterols of Trypanosoma cruzi and Crithidia fasciculata. Comp. Biochem. Physiol. 30,601-610. Dixon, H., Ginger, C.D. and Williamson, J. (1972) Trypanosome sterols and their metabolic origins. Comp. Biochem. Physiol. 41B, 1-18. De Moraes Fagundes, L.J., Angluster, J., Gilbert, B. and Roitman, I. (1980) Synthesis of sterols in Herpetomonas samuelpessoai: influence of growth conditions. J. Protozool. 27,238-241. Lala, A.K., Lin, H.K. and Bloch, K. (1978) The effect of some alkyl derivatives of cholesterol on the permeability properties and microviscosities of model membranes. Biorg. Chem. 7,437-445. Dahl, C., Dahl, J.S. and Bloch, K. (1980) Effect of alkylsubstituted precursors of cholesterol on artificial and natural membranes and on the viability of Mycoplasma capricolum. Biochemistry 19, 1462-1467. Dahl, C. (1981) Effect of sterol structure on acyl chain ordering in phosphatidylcholine vesicles: A deuterium nuclear magnetic resonance and electron spin resonance study. Biochemistry 20, 7158-7161. Bloch, K.E. (1983) Sterol structure and membrane function. CRC Crit. Rev. Biochem. 14, 47-92.

23 Dahl, J.S., Dahl, C. and Bloch, K. (1980) Sterols in membranes: growth characteristics and membrane properties of Mycoplasma capricolum cultured on cholesterol and lanosterol. Biochemistry 19, 1467-1472. 24 Rodriguez, R.J., Taylor, F.R. and Parks, L.W. (1982) A requirement for ergosterol to permit growth of yeast sterol auxotrophs and cholestanol. Biochem. Biophys. Res. Commun. 106, 435-441. 25 Pinto, W.J. and Nes, W.R. (1983) Stereochemical specificity for sterols in Saccharomyces cerevisiae. J. Biol. Chem. 258, 4472-4476. 26 Guyer, W. and Bloch, K. (1983) The sterol requirement of Tetrahymena setosa HZ-1. J. Protozool. 30, 625-629. 27 De Main, A. and Urbina, J.A. (1984) Trypanosoma (Schizotrypanum) cruzi: Terminal oxidases in two growth phases in vitro. Acta Cient. Venez. 35, 136-141. 28 Cohen, B.E., Ramos, H., Gamargo, M. and Urbina, J.A. (1986) The water and ionic permeability induced by polyene antibiotics across plasma membrane vesicles from Leishmania sp. Biochim. Biophys. Acta 860, 57-65. 29 Phillips, D.R. and Morrison, M. (1971) Exposed proteins on the intact erythrocyte. Biochemistry 10, 1766-1771. 30 Fraker, P.J. and Speck, J.C. (1978) Protein and cell membrane iodination with a sparingly soluble chloramide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem. Biophys. Res. Commun. 80, 849-854. 31 Vivas, J. and Urbina, J.A. (1982) Energy-dependent lectin-induced agglutination in Trypanosoma (Schizotrypanum) cruzi epimastigotes. Acta Cient. Venez. 33, 396-401. 32 Sottocasa, G.L., Kuylenstierna, B., Ernster, L. and Bergstrand, A. (1967) An electron-transport system associated with the outer membrane of liver mitochondria. J. Cell Biol. 32,415-438. 33 Gottlieb, M. and Dwyer, D.M. (1983) Evidence for distinct 5'- and 3'-nucleotidase activities in the surface membrane fraction of Leishmania donovani promastigotes. Mol. Biochem. Parasitol. 7, 303-317. 34 Szoka, F. and Pappahadjopoulos, D. (1979) Procedure for the preparation of liposomes with large internal aqueous space and high capture by reverse phase evaporation. Proc. Natl. Acad. Sci. USA 75, 4194-4198. 35 Cohen, B.E. (1986) Concentration- and time-dependence of amphotericin-B induced permeability changes across ergosterol-containing liposomes. Biochim. Biophys. Acta 857, 117-122. 36 Griffith, O.H. and Jost, P.C. (1976) Lipid spin labels in biological membranes. In: Spin Labeling. Theory and Applications (Berliner, L.J., ed.), pp. 453-523, Academic Press, New York and London. 37 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193,265-275. 38 Ames, B.N. and Dubin, D.T. (1960) The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J. Biol. Chem. 235,769-775. 39 Stadman, T.C. (1957) Preparation and assay of cholesterol and ergosterol. Methods Enzyrnol. 3,392-394. 40 Pereira, N.M., Timm, S.L., da Costa, S.C.G., RebeUo,

195

41

42

43

44

M.A. and de Souza, W. (1978) Trypanosoma cruzi: Isolation and characterization of membrane and flagellar fractions. Exp. Parasitol. 46, 225-234, Dwyer, D.M. (1980) Isolation and partial characterization of surface membranes from Leishmania donovani promastigotes. J. Protozool. 27, 176-182. Da Silveira, J.F., Abrahamsohn, P.A. and Colli, W. (1979) Plasma membrane vesicles isolated from epimastigote forms of Trypanosoma cruzi. Biochim. Biophys. Acta 550, 222-232. Zingales, B., Carniol, C., Abrahamsohn, P.A. and Colli, W. (1979) Purification of an adenylyl cyclase-containing plasma membrane fraction from Trypanosoma cruzi. Biochim. Biophys. Acta 550, 233-244. Nagakura, K., Tachibana, H., Kaneda, Y. and Sekine, T. (1986) Subcellular fractionation of Trypanosoma cruzi;

45

46

47

48

isolation and characterization of plasma membranes from epimastigotes. Tokai J. Exp. Clin. Med. 11, 23-29. Gottlieb, M. and Dwyer, D.M. (1983) Evidence for distinct 5'- and Y-nucleotidase activities in the surface membrane fraction of Leishmania donovani promastigotes. Mol. Biochem. Parasitol. 7, 303-317. Gottlieb, M. and Dwyer, D.M. (1981) Leishmania donovani: Surface membrane acid phosphatase activity of promastigotes. Exp. Parasitol. 52, 117-128. Zilberstein, D. and Dwyer, D.M. (1985) Protonmotive force-driven active transport of D-glucose and L-proline in the protozoan parasite Leishmania donovani. Proc. Natl. Acad. Sci. USA 82, 1716-1720. Alves, M.J.M. and Colli, W. (1974) Agglutination of Trypanosoma cruzi by Concanavalin A. J. Protozool. 21, 575-578.