Fatty acid metabolism in Paramecium Oleic acid metabolism and inhibition of polyunsaturated fatty acid synthesis by triparanol

Fatty acid metabolism in Paramecium Oleic acid metabolism and inhibition of polyunsaturated fatty acid synthesis by triparanol

20 BBA 51697 FA’M’Y ACID METABOLISM IN PARAMEClUM OLEIC ACID METABOLISM AND ACID SYNTHESIS BY TRIPARANOL (Received January INHIBITION OF POLY...

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BBA 51697

FA’M’Y ACID METABOLISM

IN PARAMEClUM

OLEIC ACID METABOLISM AND ACID SYNTHESIS BY TRIPARANOL

(Received

January

INHIBITION

OF

POLYUNSATURATED

FATTY

9th, 1984)

Purume&m requires oieic acid for growth and can grow in media containing no other fatty acids. In the present study, we have shown that this ciliate utilized oleate mainly as a carbon and energy source, even though this fatty acid was the only substrate available for synthesis of polyunsaturated fatty acids. Culture growth was inhibited by the addition of the drug triparanol. Triparanol decreased the formation of polyunsaturated fatty acids from oleate by preventing desaturation to form the dienoic acid, linoleate. Triparauol inhibition resulted in an altered phospholipid fatty acyl composition, an increased fragility and an altered behavioral response of the celis to a depolarizing stimulation solution. Therefore, although most of the dietary oleate was not used by the cells for polyunsaturated fatty acid synthesis, the desaturation of oleic acid was critical for normal euiture growth, cell integrity and swimming behavior, all of which are expected to be dependent on normal membrane lipid composition.

The ciliated protozoan, Paramecium, has been a useful cellular model for the study of membrane electrical excitability [l]. The utility of Paramecium for biochemical studies was strengthened by the development of chemically defined media for the axenic mass cultivation of this ciliate [2]. During this development, Parumecium aurelia was shown to have a nutritional requirement for lipids. both sterol and unsaturated fatty acid. Only C-24 alkyd-substituted sterols possessing a A5 and/or a A7 unsaturation (e.g., stigmasterol) could support growth of Paramecium. Of the fatty acids tested, only oleic acid (cis-9-octadecenoic acid) was capable of satisfying the fatty acid requirement. In

* To whom correspondence

should be addressed at (present address): Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104, U.S.A. Abbreviation: DFMO, difluorometh~iornithin~. 0005-2760/84/$03.00

Q 1984 Elsevier Science Publishers

B.V.

prokaryotic systems, such fatty acid auxotrophs have been employed to study the role of lipids in membranes because the cellular esterified fatty acid composition can be modified by limiting the dietary lipids to selected fatty acids [3,4]. In contrast, recent studies of the lipid composition of Parumerium have shown that these cells have a wide assortment of fatty acids even when the ciliates are cultured with oleic acid as the only dietary fatty acid [S]. One of the principal characteristics of the fatty acid composition of Purumecium is a large amount of polyunsaturated fatty acids, especially in the electrically-excitable ciliary membrane [5,6]. This characteristic is one that P~~urneci~rn shares with other eIectrically-excitable cells such as mammalian photoreceptor cells where both the degree of unsaturation and the position of the double bonds in the polyunsaturated fatty acids are critical to the electrical response in visual excitation [7,8]. In the present study, the relationship between

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the fatty acid composition of Paramecium and its nutritional r~uiremcnt for oleic acid was established by studying the metabolism of radiolabeled oleic acid during growth in axenic culture. While it is perhaps best known as an inhibitor of mammalian cholesterol synthesis [9,10], triparanol has also been implicated by indirect evidence as an inhibitor of unsaturated fatty acid synthesis in another ciliate, ~et~a~yrne~a [11-131. The effects of triparanol were examined on fatty acid metabolism in Paramecium, which does not synthesize sterols and has a dietary requirement for sterols ]21. Material and Methods

Ceil cultures. Unless otherwise indicated, Paramecium tetraurelia, strain Sls, was grown as described previously [5] in a chemically defined medium supplemented with 15 pg/ml monoolein as the sole source of dietary fatty acid. Culture growth curves were established by cell counts of formalin-fixed cells. Triparanol (MER-29, l-[ p(~-diethyla~noethoxy)phenyl]-l-(~-tolyl)-2-t pchlorophenyl)ethanol) was generously provided by Dr. W. Albrecht (Merrel-Dow Research Lab., Cincinnati, OH). Difluoromethylornithine (DFMO) was a gift from Dr. P. McCann (MerrelDow Research Lab.). Triparanol was added to culture media in ethanol with the final ethanol concentration never exceeding 0.05% (v/v). This concentration had no effect on the parameters measured in these studies as determined by control cultures grown in 0.05% ethanol. For analysis of cilia, cells were grown in a crude medium [S] because this medium supports higher culture densities and thus provides a greater yield of cilia. Cilia fractions were prepared as previously described [5] with one modification. The sucrose con~ntration in the solution used to wash triparanol-inhibited cells was increased from 150 to 200 mM to minimize lysis. [U-‘4C]Oleic acid (990 mCi/mmol; New England Nuclear Corp., Boston, MA) or [l-t4C]oleic acid (58 mCi/mmol; Amersham Corp., Arlington Heights, IL) was added to culture media in ethanol at activities of 2-10 pCi/50 ml culture media prior to the addition of ciliates. During culture growth, the radioactivity of the culture medium

was monitored by liquid scintillation spectrometry of appropriate aliquots to determine the decrease of radiolabel in the medium due to uptake and/or adsorption of the radiolabeled oleic acid by the cells. Cells were harvested by flow-through centrifugation as described previously [5]. Fatty acid analysis. Cellular or ciliary lipids were extracted and were separated into neutral and phospholipid fractions by employing adsorption column chromatography with 100-200 mesh silicic acid (Unisil, Clarkson Chemical Co., Williamsport, PA), as described in previous studies of the lipid composition of this ciliate [5,6,14]. Total saponifiable fatty acids were prepared from the phospholipid fractions by the micromethod of McGee and Allen [15] and the methyl ester derivatives were separated and quantified by gas-liquid chromatography (GC) [6]. Fatty acids were isolated from radiolabeled extracts using a 6 ft, l/8 in. (o.d.) stainless steel column equipped for preparative GC, packed with 10% Silar lOC, and operated isothermally at 170°C. The column was attached to a 94% splitter. Fatty acid methyl esters eluting from the column and corresponding to GC peaks at the flame ionization detector were collected by condensation of the sample on the walls of a Pasteur pipet inserted into the collection port. The condensate was eluted from the pipet with a toluene-based scintillation fluid directly into scintillation counting vials. Recoveries from preparative GC were 80-85%. ~e~aui~ru~ ~~a~~~~. Locomotory behavior of Paramecium, specifically the avoidance reaction, was analyzed [16] after subjecting cells to a depolarizing solution. Cells were equilibrated for a min~um of 6 h in a solution untying 0.5 mM CaCl, and 5 mM Mops (4-morpholinepropanesulfonic acid) buffer adjusted to pH 7.1 with NaOH. The backward swimming response was initiated by adding 10 ~1 of the cell suspension to 5 ml of a depolarizing solution that consisted of 0.5 mM CaCl,, 16 mM KC1 and 5 mM Mops buffer adjusted to pH 7.1 with NaOH. This solution contained sufficient KC1 to depolarize the cell membrane and resulted in immediate ciliary reversal. The duration of the response (backward swimming), from its initiation upon the cell’s contact with the depolarizing solution until normal forward swimming was resumed, was determined for

22

individual

cells observed under a dissecting microto the total duration of the response, the period of backward swimming, also referred to as continuous ciliary reversal, was timed and distinguished from the spinning and circling motions that intervened between continuous cihary reversal and the return of normal forward swimming. This intervening period will be referred to as the renormalization period. scope.

In addition

Results Culture growth Paramecium cultures exhibited typical growth characteristics when supplemented with monoolein (Fig. 1A). In the absence of a fatty acid source, cell division ceased and the onset of culture death occurred within 48 h. Culture growth was reduced in the presence of 1 PM triparanol and complete inhibition was observed with 2 FM triparanol. Cells grown in another chemically defined culture medium [S] containing phosphatidylethanolamine as a fatty acid source and a mixture of fatty acids (including some polyunsaturated fatty acids), were unaffected by triparanol at concentrations up to 2 PM (Fig. 1B). Since the two culture media differed only with respect to the lipids, these results suggested that triparanol inhibition could be modified by dietary lipids. A concentration of 10 PM triparanol was also required for complete inhibition of culture growth in the crude medium. Oieic acid metabolism The level of [‘4C]oleic acid, added in trace amounts, decreased in the culture medium during culture growth (Fig. 2). The decrease began immediately upon inoculation of the medium with Paramecium and continued through day 5, by which time the remaining radioactivity had been reduced by more than 80%. The total cellular fatty acid content was determined to examine further the fate of cellular oleic acid and to allow an estimate of the net synthesis of saturated fatty acids and polyunsaturated fatty acids during culture growth. The total cellular oleic acid increased during the first 3 days of growth (Fig. 3), apparently reflecting the uptake or adsorption of the oleie acid in the medium. As the oleic acid content

D

2

CULTURE

4 AGE

6

8

(DAYS)

Fig. I. inhibition of culture growth in the monoolein defined medium (A) and the defined medium containing phosphatidylethanolamine and free fatty acids (B). A, the normal growth (0) of Paramecium cultures was inhibited by 1 pM (I) and 2 pM (0) triparanol. Culture death was rapid in the absence of any oleic acid ( X) in the medium. B, normal growth (0) was inhibited by 6 PM (A), 9 PM (o), 2nd 10 pM (x) triparanol when cells were grown in the medium containing a fatty acid source that included pol~nsaturated fatty acids (ph~phatidyiethanolamine and free fatty acids). These cultures required higher concentrations of inhibitor before decreases in cell density were detected.

of the growth medium neared depletion during the log phase of growth, the cellular level of oleic acid began to drop sharply. When compared to the growth curve in Fig. IA, these events were noted to immediately precede the onset of stationary phase. Cellular saturated fatty acids, which were principally palmitic (16 : 0) and stearic (18 : 0) acids, increased during the early part of culture growth, then leveled without substantial decreases thereafter (Fig. 3). Cellular polyunsaturated fatty acids increased throughout culture aging. Since

23

CULTURE CULTURE

AGE

(DAYS1

Fig.2.Depletion of radiolabeled oleic acid from the growth medium. Cells were cultured in 50 ml of the monoolein defined medium entailing 2 &i {U-‘4C)oleic acid. The disappearance of radioactivity from the medium began at the first time-point examined and continued with a time course that reflected culture growth. The onset of stationary phase folfowed the depletion of exogenous radiolabeled oleic acid from the medium.

neither pol~nsaturated fatty acids nor saturated fatty acids were present in the culture medium, increases in these fatty acids represented net synthesis. Since the decrease in the radiolabeled oleic acid present in the culture rn~urn during growth was regarded as uptake and/or adsorption by the cells, recoveries of radiolabel in the cellufar lipids and the residual (lipid-extracted) cell mass were examined (Table I). The percent recovery of radiolabel in these fractions decreased dr~at~c~ly with culture age. The radioactivity not recovered in either the growth medium, cellular Iipids or residual cell mass was assumed lost at t4C02 due to metabolism of the substrate presumably through the t~c~boxyI~c acid cycle. Recovery of the radiolabel innately after addition of the cells to the culture medium was greater than 95%; therefore we concluded that the inability to recover 95% of the radiola~l during culture growth was not due to the meth~o~ogy used. Pr~uctio~ of r4C0, from oleic acid under these conditions was verified

AGE (DAYS)

Fig. 3. Changes in the cellular content of fatty acids during growth in the monoolein defined medium. Total lipids were prepared from all cells in 50-ml cultures at different ages and the fatty acids were isolated after mild alkaline meth~ol~sis of the total lipids. These values can be compared with the removai of oleic acid from the culture medium during culture growth (Fig. 2). The uptake of oleic acid (0) and the synthesis of saturated (@) and polyunsaturated ( x ) fatty acids by the cells are shown with respect to culture growth.

TABLE I RECOVERY QF RADIOLABELED ING GROWTH OF P~~~~~I~~ MON~~EIN DEFINED RADIUM

OLEIC ACID DURCUtTURES IN THE

The recovery of radioactivity originating from [U-‘4C]oieic acid, as % of the initial radioactivity (2 @i) added to the culture medium, was determined for four locations: the culture medium, the cellular lipids, the ~pid-extracts cell residue (residual cell mass), and the radioactivity that was not recovered in these studies and was presumed converted to r* CO,. The indicated days represent the time after the monook& defined medium, contaiuing the radiolabeied oleic acid, was inoculated with Parofflecium. The values are representative of two to four experiments. Location

Culture medium Cellular lipids Residual cell mass Not recovered

4; Distribution of radioactivity on day 1

3

6

8’1.5 6.3 3.1 3.1

18.7 6.3 25.0 50.0

6.2 3.1 18.8 71.9

24

in other studies $171.Of the radio~abeled ofeic acid that had been removed Erom the medium after 24 k of gr~wtk, 50% was recovered in the celfular lipid extract (Table I>. The recovery of the radiolabel in eeltular lipids decreased to 3% by the sixth day, by which time the culture medium was virtually depleted of radiolabeled oleic acid. The majority (SS-90%) of the radiolabel that was recovered in cellular lipids after growth of the culture to stat~~ary phase was associated with the ester~~ed fatty acids of the phosph~li~id fraction. Oleic acid (18 : l(9)), Iinoteic acid (I8 : 2(9,X2)), Iin~Ien~~ acid (18 : 3(6,9,12)) and ara~hidoni~ acid (20 : 4(5~~,1~,14~~,were the ~redo~na~t ph~s~h~~~p~d fatty acids rad~~~abe~ed under these ~ond~ti~~s (Table XI). AXIof the other major fatty acids were labeled to a lesser extent. The lower relative specific activities of the saturated fatty acids would be expected if randomization of the radiolabel QCcurred due to ~-oxidation of oleic acid. Shorter term (24 h) in~~bat~~~s resulted in the rapid appearance of label in oleic acid and [email protected]

TABLE JJJ

Specific activities were 2.t .lU- ’ pCi/mg lipid (control) itId 2.2~Joe3 pCi/mg lipid (triparanoif. Values are the means-t_ S.D. of four determinations. Fatty acids were isolated by preparative gas chromatography after mild alkaline methanoly sis of total ph~p~oiipids from ceIJs incubated for 48 h in the monooIein defined medium suppJeme~ted with 2 &i [Ut4CJoJeic acid in the presence (Z PM) or absence of triparanot. Fatty acid Control 14:o 16:O

16:1(9)+16: J(7) 18:O 18:J(9)+18:1(11) 18: 2(9,12) 18: 3(6,9X) 20: 4~5,8~Jl,~4~ Others a

a Sumof ali other fatty TABLE II

Fatty acids were isolated by preparative gas cbros~to~apb~ after mild alkaline methanoJysis of total phospholipids from cells grown S days in the monoolein defined medium suppiemented with 2 pCi &J-r4CJoJeic acid. radioactivity values are percentages of the totaf ~~co~orated into the ~hospbotip~~ fatty acids; values represent the means&-SD. of five determinations. Relative spec. act. vatues are from preparative gas cbromatogra~b~~ cpm/peak area; the values represent means &SD. of five det~~~~at~ons; n.d., not determined. Fatty acid 14:0+14:t 16:O 15 : 1(9)-b16: l(7) 18:O 18: l~~)~l~:l~ll~ 18: 2(9,t2) 18 : 3(6,9X) 20: 3(8,J l,i4) 20: qs,s,~l,~4~ 20: 5(5,8,11,~4~~7~ Cfthers ’

f6 Rad~~cti~ty 3,0+1.0 b.Ofi 3.1 1.4*0.4 0.8 & 0.2 l&3*1.4

25.8 * 3.4 17.4 _4r3.6 3.5 rt:0.7 21.3 & 6.3 1.5&0.8 3.art: I.4

Relative s&xx. act,

J.710.9 0.7 & 0.4 1.310.6 0.3$:0.2 2.4qQ.4 3.2*0.3 2.9kO.3 n.& 2.6~U.5 n.d. nd.

B Sum of ail other fatty acids present in tess than 0.5%.

acids present in less than 0.5%.

u~sat~~at~d fatty acids with Iess than 2% of the rad,i~a~tiv~ty present in the saturated fatty acids. The majesty (more than 80%) of the rad~~~ab~~ recovered in the ~~~~~~arlipids after 24 k incuhation was present in the neutral lipid fract~un” presumably in triacylglycerols and free fatty acids fl41.

Cells were incubate in culture medium containing trace amounts of [r4G]ofeic acid in the presence (2 PM) or absence of t~~aran~~. During incubations of 24-48 h, tke amount of label taken up by the eelIs and the specific activities of both the total lipid and the ~kospk~~i~id fractions were not significantly different in the two groups of cells. These conditions permitted direct cornpa& son of the distribution of the radiolabel in the fatty acids of the ~k~sph#~~p~d fractions (Table HI). Compared to control cells, cells incubated with triparau~~ retains almost twice the amount of rad~olabe1 in oleic acid with comparable reductions in the label ~~c~~~rated into the major poiyunsaturated fatty acids. Analyses of the ~k~sph~~~~~d Fatty acid cam-

25

position of triparanol-treated cells (Table IV) showed reductions in esterified polyunsaturated fatty acids, which were consistent with the decreased capacity of inhibited cells to synthesize polyunsaturated fatty acids from oleic acid. Since the fatty acid composition of Paramecium phospholipids is known to change with culture age [6], the fatty acid compositions of phospholipids from cells after 1 (cell density control) and 5 (culture age control) days of growth were determined to compare with those of triparanol-inhibited cells (Table IV). The fatty acid composition of control cells changed during culture growth mainly as a result of relative increases in polyunsaturated fatty acid concentrations. This was evident by comparing the sums of total phospholipid polyunsaturated fatty acids in cells from younger vs. older cultures (Table IV). Most striking among the alterations in the fatty acid composition of triparanol-inhibited cells was the lowered concentration of polyunsaturated fatty acid, which was only

12% of the total phospholipid fatty acids. In contrast, control cells ranged from 36% polyunsaturated fatty acids at day 1 to 52% at day 5. The higher concentration of saturated fatty acids in triparanol-inhibited cells was reflected in the increased ratio of saturated to unsaturated fatty acids of their phospholipids. The cellular content (pg/106 cells) of phospholipid fatty acids after 5 days exposure to triparanol was at the level present in normal cultures at early growth stages (day 1). Thus, not only was there an effect on the higher percentage of saturated fatty acids (reduction in polyunsaturated fatty acids), but triparanol inhibition affected the absolute amounts of fatty acids within the cells after 5 days of culture age. This suggested that (1) saturated fatty acid synthesis was not inhibited by triparanol, and (2) the reduced polyunsaturated fatty acids among the fatty acids available in the cell affected their esterification into phospholipids. Cells inhibited by triparanol had significant amounts of 20 : l(11) (Ta-

TABLE IV EFFECT

OF CULTURE

Fatty acid

AGE AND TRIPARANOL

ON PARAMECIUM

PHOSPHOLIPID

Weight ‘R, Control

14:o 16:0 16: 1(7)+16: l(9) 18:0 18:1(9)+18:1(11) 18 : 2(9,12) 18 : 3(6,9,12) 20 : l(11) 20 : 3(8,11,14) 20: 4(5,8,11,14) 20 : 5(5,8,11,14,17) Others a Short/long b Sat./unsat. ’ H Polyunsaturated d Fatty acid content’ ’ b ’ d =

FAT”N ACID COMPOSITION

TriparanoI(2 PM)

Day 1

Day 5

Day 5

4.3+ 2.3 21.9* 4.2 5.9+ 3.2 7.7+ 2.8 23.0* 4.2 12.1* 2.5 10.2* 4.2 trace l.lk 0.6 11.7* 4.2 0.6* 0.4 1.7 6.5 0.5 35.7 1043 *133

3.7* 1.1 19.8f 4.1 3.4* 1.7 5.6* 1.6 13.2k 2.0 18.4k 2.4 13.6+ 3.2 trace 0.8* 0.4 18.3+ 2.5 l.l+ 0.6 2.1 4.0 0.5 52.2 506 k52

5.3* 1.6 29.9* 2.6 5.0* 1.0 18.6zt 5.3 24.5k 5.6 4.3* 2.6 2.0* 1.3 3.lf 1.3 0.8+ 0.4 4.8* 2.1 0.2* 0.1 1.5 10.1 1.2 12.1 1302 +127

Sum of all other fatty acids present in less than 0.5%. 46 fatty acids zz18 C length/% fatty acids > 18 C length. % saturated fatty acids/% unsaturated fatty acids. Sum of polyunsaturated fatty acids. /.&g/106 cells.

TABLE EFFECTS

V OF CULTURE

AGE AND INHIBITORS

ON THE DURATION

OF CILIARY

REVERSAL

IN PARAMEC’ICI’M

Cells were grown in the monoolein defined culture medium. Values are means* SD. of 15-50 detern~ination~. Day 1 control VS. day j control: total response, P i 0.05; CCR, P < 0.10. Day S triparanoi vs. day 5 control: total response, P ic 0.01: CCR. P CT0.01. Day 5 triparanoi vs. day I control: total response, P < 0.05; CCR, P i 0.05 (two-sided t-tests).

Inhibitor

None None TriparanoI(2 PM) a Triparanol(2 p M) DFMO (25 mM) a Cells from day 5 cultures

Culture

Duration

age (days)

Total response

1 5 0.25 5 5

72.0 f: 31.2i 41.8& 122.21 30.8k

were incubated

---.-.--___

(s)

-

(TR) 30.6 7.7 9.1 9.5 1.8

in the adaptation

solution

ble IV), which is present in only trace amounts under control conditions as an elongation product of oleic acid (Rhoads, Honer-Schmidt and Kaneshiro, unpublished data). Neither alterations in the phospholipid class composition nor changes in the neutral lipid class composition, including the sterol/sterol ester ratio, were detected in triparanol-inhibited cells.

Comparison of the behavioral responses of triparanol-inhibited cells with those of control cells indicated a difference (Table V). Control cells from day 1 cultures differed from those in day 5 cultures in total response time and, to a lesser extent, in the duration of continuous ciliary reversal. Thus the avoidance behavior was slightly different in cells at different culture ages. The total response, continuous ciliary reversal and renormalization times of cells inhibited by triparanol all differed from day 1 as well as day 5 controls. Cells inhibited by triparanol exhibited total response times up to double that of the longest total response observed in control cells. The duration of the renorm~zation period was the component of the behavioral response that was affected to the greatest extent in ~p~~ol~in~bited cells. Two other experimental observations are relevant for the interpretation of these results. First, growth of cells could be inhibited by 25 mM DFMO (i~bits ornithine decarboxylase and

Continuous ciliary reversal (CCR)

Renormalization (TR - CCR)

23.2+ 7.3 ll.Ort;2.9 13.7 I, 3.3 28.2 & 9.5 12.7 + 2.4

48.8 20.2 28.1 94.0 18.1

(see text) in the presence

of inhibitor

-

for 6 h.

polyamine synthesis) without concomitant alterations of behavioral responses (Table V) or phospholipid fatty acid compositions (data not shown). Thus, the observed alterations in the behavioral responses by Paramecium are not associated with inhibition of culture growth per se, but instead are related more specifically to the inhibition of oleic acid desaturation by triparanol. Second, only under prolonged in~bition by triparanol, i.e., after 4-5 days in culture with triparanol, were behavioral changes observed. After 24 h in the presence of triparanol, values were not different from control cultures (Table V). Therefore, triparanol does not have a direct and immediate effect on the cell’s avoidance behavior; the behavioral response alterations observed appear to be secondary to the effects of the in~bitor on the cellular phospholipid fatty acid composition. Since the behavioral response is mediated by the ciliary membrane, the effects of triparanol on the fatty acid composition of the cilia were determined (Table VI). The triparanol-inhibited cells were more susceptible to lysis during the deciliation procedure than were cells from control cultures. The fragility of these cells may again be a secondary ma~festation of triparanol’s action. Because of cell lysis, cilia were isolated from cells grown in the enriched medium in the presence of 6.8 PM triparanol for 5 days. Under these conditions, culture growth was only partially inhibited (6750 cells/ml). Control culture in this medium was 30~ cells/ml; these culture conditions and

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the increase of osmolarity of the deciliation solution allowed us to obtain sufficient amounts of an enriched cilia fraction from the cells, since they were less prone to lysis as compared to cells from cultures totally inhibited from growth by the drug. The fatty acid composition of the cilia phospholipids from cells in cultures partially inhibited by triparanol had reduced polyunsaturated fatty acid and elevated saturated fatty acid contents when compared to control cilia. Thus, the alterations that occurred in whole cell phospholipid fatty acids in the presence of triparanol were also apparent in the lipids of cilia. It is likely that if it were possible to obtain cilia fractions from cells in cultures that were totally inhibited by triparanol, the differences would be even more striking. On the assumption that the inhibition of culture growth and the alterations in the behavioral response to depolarizing stimuli were due to the in~bition of pol~nsaturat~ fatty acid synthesis

by triparanol, attempts were made to overcome these effects by supplementation of the culture medium with polyunsaturate fatty acids. However, the toxicity of free polyunsaturated fatty acids prevented the addition of pol~saturated fatty acids to the culture medium in sufficient amounts to have an effect on the phospholipid fatty acid composition. Supplementatoin with phospholipids isolated from day 5 or day 7 cultures of paramecium cells was also toxic to the cells, as was supplementation of the culture medium with polyunsaturated fatty acid-containing triacylglycerols (trilinolein, trilinolenin, triarac~donin~ with or without monoste~~, palmitate and monoolein, and with polyunsaturated fatty acids bound to bovine serum albu~n. Lipids were added to culture media with or without the antioxidant, butylated hydroxytoluene. Concentrations of butylated hydroxytoluene up to 0.04 mg/ ml did not decrease culture growth.

TABLE VI FATTY ACID COMPOSITION OF CILIARY PHOSPHOLIPIDS FROM CELLS GROWN IN THE PRESENCE OR ABSENCE OF TRIPARANOL Cells were grown for 5 days in the crude, enriched medium in the presence (4.8 @i) or absence of triparanol. Other fatty acids present were in amounts less than 0.5%. Culture densities (cells/ml) were 30000 (control) and 6750 (triparanol). Values are means+ S.D. of four (control) and three (triparanol) determinations. Fatty acid

14:o 1S:O 16:0 16: 1(9)+16: l(7) 17:o 18:O lS:lt9)~18:1(11) 18 : 2(9,12) 18 : 3(6,9,12) 20: I(lI) 20 : 2(8,11) 20 : 3(8,11,14) 20 : qs,8,11,14) 20: 5(5,8,11,14,17) 22 : 4(7,10,13,16) % Saturated 46 Polyunsaturated

Weight % Control

Triparanol

l.Of0.3 0.6 rtO.2 1X3$2.0 0.8 rtO.2 trace 3.6f1.7 6.4&4.5 15.9f: 5.6 2.3Jc1.4 0.5~O.I l.Of0.4 1.5 f 0.4 34.9 f 8.8 10.1* 2.5 0.8 f 0.3 20.8 67.3

1.5*0.1 0.7fO.l 22.7 f 1.7 0.8 f 0.1 0.5 * 0.1 10.0&1.8 16.2k2.1 IO.3 f 2.0 3.3 + 1.6 0.710s 2.7*0.2 0.7 * 0.1 19.7fl.3 4.3*0.3 trace 35.0 43.2

Cultures of Paramecium accumulate dietary oleic acid and utilize this fatty acid directly for the synthesis of polyunsaturated fatty acids and phospholipids. Oleic acid also serves as an energy and carbon source. As the supply of oleic acid diminishes, ~-o~dation of this fatty acid becomes the dominant mode of its utilization by the cell. Thus, while dietary oleic acid is plentiful there is cell and culture growth but, as the supply of oleate becomes limiting, it is used primarily for maints nance rather than proliferation of the cells. These results are consistent with those obtained by Fok et al. [I81 and Kane&&o et al. [171, who showed that Paramecium culture growth is directly correlated with the dietary supply of lipids and that a number of processes that occur in these cells during stationary phase are triggered by the depletion of exogenous lipids [19]. Triparanol selectively interferes with the processing of oleic acid by Paramecium. Other effects on the lipid composition were not observed. Further, triparanol affected neither the uptake of dietary oleate nor its oxidation, i.e., its role as an energy and carbon source. This may account for the viability of cells for more than 5 days in the presence of triparanol in the defined medium with

28

monoolein, in contrast to the relatively rapid onset of cell and culture death in the absence of an oleic acid source. Yet, culture growth that occurred in the presence of oleic acid was inhibited by triparanol. Since the inhibition of culture growth by triparanol was not as great in media that contained polyunsaturated fatty acids, and since the only alterations in the cellular lipids was the reduced concentration of polyunsaturated fatty acids, it appears that the inhibition of culture growth was due to a suboptimal fatty acid composition unfavorable for membrane synthesis (or assembly) and cell division. The effect of triparanol on the prolonged renormalization period following membrane depolarization and continuous ciliary reversal seems to be a result of decreased phospholipid pol~nsaturated fatty acid, particularly that of the ciliary membrane. The requirement for prolonged exposure to the inhibitor before these secondary effects were detected may be due to (1) the time required for the alterations to become severe enough to alter membrane lipid-lipid or lipid-protein interactions, or (2) the time required for the alterations to specifically affect ciliary membranes. It has been reported that, in Tetruhymenu, newly synthesized lipids appeared in the lipids of the cilia and pellicle membranes only after a considerable delay in time 1201. By the time alteratoins in behavior are observed, the cilia phospholipids of triparanol-inhibited cells have been substantially reduced in polyunsaturated fatty acids. Earlier reports of Tetruhymena inhibition by triparanol are consistent with the proposed action of this drug on Paramecium fatty acid desaturation. In Tetru~ymen~ the levels of the major polyunsaturated fatty acids, linoleic and linolenic acids, were reduced in the presence of triparanol, and dietary supplementation of linolenic acid was effective in increasing culture growth in the presence of triparanol [13]. As in Paramecium, these polyunsaturated fatty acids are synthesized from oleic acid by Tetrahymena 1211. However, in Tetrahymena this pathway essentially terminates with linolenic acid, whereas in Paramecium the major end product is arachidonic acid. Our inability to reverse the triparanol inhibition in Paramecium by supplementation with arachidonate and other

long-chain polyunsaturated fatty acids may he because of the greater toxicity of high c~~ncentratiolls of these lipids. it is also possible that the ratios of the supplements added to media in these studies were a factor in their toxicity. Nonetheless. using radiolabeIed oleic acid, the present study has directly demonstrated a reduction of the incorporation of oleic acid into polyunsaturated fatty acids in the presence of triparanol. It has recently been reported that triparanol decreased phosphatidylserine and cholesterol concentrations in rat lens tissues [22]. In those studies, secondary effects of triparanol inhibition were also noted. They included decreased (Na i + K * )ATPase and ouabain-insensitive ATPase activities. Since the lens tissues accumulated Ca” ’ under conditions of triparano1 inhibition, this suggests that an outward-directed Ca’“-ATPase/ pump may have also been affected. A similar effect on Ca2+-ATPase activity in the ciliary membrane of Paramecium could explain the changes in the hehavioral response observed when cells were grown with triparanol. The renormalization period that follows depolarization-induced Ca2 ’ influx and ciliary reversal is thought to involve such an enzyme (pump) for reducing Cal+ concentrations in the cilia back to normal levels [I]. Alterations of phospholipid fatty acids that result in less fluid membrane bilayers, particularly in microdomains containing enzymes with Ca’+ pump activity. is a probable cause of the increase in time required for triparanol-inhibited cefls to return to normal forward swimming after backward swimming was induced. Ca2+ -ATPase activities in Paramecwm ciliary membranes have been identified and partially characterized 124-263. Future studies examining the effects of fatty acid compositions of the membrane on the enzymes’ function(s) are expected to help clarify the triparanol effect on the behavioral response of ~~rarne~~urn. Acknowledgements The authors wish to tions to preliminary metabolism by the late was supported in part (GM 20910) and N.S.F.

acknowledge the contribuexperiments on oleate Otmar Honer. This work by grants from the N.I.H. (PCM77-19088).

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