PLA 2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators

ELSEVIER J. Lipid Mediators Cell Signalling 15 (1997) 233-247

PLA, activity in Tetrahymena pyrlformis. Effects of inhibitors and stimulators

Pkter Kov&x, Gyiirgy Csaba*

Semmelweis Unicersity of’ Medicine, Department of Biology, I445 Budapest, Nagywirad tt? 4, P.O.B. 370, Hun~arv

Received 8 July 1996: revised 27 August 1996; accepted 28 August 1996

Abstract

Phospholipase A2 (PLA,) is an enzyme which participates in signalling mechanisms cleaving arachidonate from sn-2 position of glycerophospholipids. In this study we have verified the existence of a PLA,-like activity in the free living protozoan, Tetrahymena pyrijbrmis GL. This activity is Ca *+-independent, EDTA (10 mM) has no effect on its activity. Quinacrine (0.1 mM) and 4-bromophenacyl bromide (BPB; 0.1 mM) inhibited, melittin (20 pggjml) significantly stimulated the PLA, activity and the release of free arachidonic acid (AA) from 1-acyl 2-‘4C-arachidonyl-3-phosphatidylethanolamine substrate. Melittin stimulated PLA, hyperactivity is Ca2+ -dependent. There was no considerable alteration in the PLA, activity by stimulation of the activity by tyrosine kinase (with vanadate, H,O,), phospholipase C (PLC) (with phorbol 12, 13-dibutyrate) or G-proteins (with NaF, AlF,), thus in Tetrahymena PLA, activity seems to be independent of these-in Tetrahymena (also functioning)-signalling pathways. Treatment with quinacrine and BPB leads to decreased synthesis and disturbed breakdown of phospholipids and phosphoinositides. These findings suggest that PLA, activity is in connection with the phospholipid metabolism of Tetrahymena. Copyright 0 1997 Elsevier Science B.V.

Keywords: Phospholipase; Phospholipids; Tetrahymena

* Corresponding author. Tel.: + 36 1 210 2950; e-mail: [email protected]

0929-7855:97!$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved

PII SO929-7855(96)00559-7

234 P. Kowics, G. C~sahu / J. Lipid Mediators Cell Signalling 1.5 (1997) 2X-247

1. Introduction

The unicellular Tetruhymena pyrijbrmis GL utilizes many signalling pathways analogous to those of mammalian cells. Cyclic AMP (Csaba and Nagy, 1976; Kuno et al., 1979); cGMP (Kohidai et al., 1992; Kuno et al., 1979); guanylate cyclase-dependent Ca2 + -calmodulin (Muto et al., 1983; Kovacs et al., 1989); phosphatidylinositol (Kovacs and Csaba, 1990); glycosyl phosphatidylinositol (Kovacs and Csaba, 1994a, 1996b); protein kinase C-like systems (Hegyesi and Csaba, 1994) and phospholipase D (Kovacs et al., 1996a,b) were demonstrated in this organism.

Phospholipase A, (PLA,) hydrolyses fatty acids bound at the sn-2 position of glycerophospholipids. Predominantly the sn-2 position of the glycerophospholipids are esterified by arachidonic acid (AA). The liberation of arachidonate, which may be mediated directly by PLA, has a crucial importance in signal transduction by AA metabolites as the eicosanoids and prostaglandins. Besides the direct release of AA by PLA, there are pathways also for the indirect release of AA. Activation of either phospholipase A, (PLA,) or phospholipase C (PLC) coupled with secondary enzymes, lysophospholipase or diacylglycerol lipase would also result in the release of AA (Dennis, 1987). In Tetrahymena the incorporation of AA into the glycerophospholipids is very rapid. After treating the cells with “H-labelled AA, the radioactivity was recovered mostly in the phosphatidylethanolamine (PE), phos- phatidylcholine (PC), phosphatidylinositol (PI) and diacylglycerol (DAG) (Kovacs and Csaba, 1996a). Thus it can be suggested that AA in Tetrahymena plays an important role in the phospholipid metabolism and in close connection with this-in the signal transduction, and the activity of PLA, by all means has significance in this phenomenon. However some contradictory data are in connection with the presence of PLA, in Tetrahymena. Ramesha and Thompson (1984) found its activity in isolated cilia, while Arai et al. (1986) detected no appreciable PLA, activity in the Tetrahymena membrane.

The goal of our present work was to study the following: (1) the PLA, activity in Tetrahymenu pyrzjiormis, (2) the role of stimulation and inhibition of supposed PLA, activity and (3) the possible connections of PLA, activity with other components of signal transduction. Tha data gained could help to clear the similarities or dissimilarities between mammalian and unicellular signalling mechanisms.

2. Materials and methods

2.1. Muterids.

Quinacrine, BPB, melittin, phorbol 12, 13-dibutyrate (PDBu), aprotinin and phospholipid chromatographic standards (PI, PIP, PIP,, PA, PE) were purchased from Sigma (St. Louis, MO). ‘2P-Na-orthophosphate (specific activity 7 GBq/mM) was obtained from Izinta (Budapest, Hungary). My0-[2-~Hl inositol (specific activity 633 GBq/mM), l-acyl-2-[l-‘4C]arachidonyl L-3-phosphatidylethanolamine (spe-

P. Koc&s, G. C’saha / J. Lipid Mediators Cell Signding 15 (1997) 233-247 235

cific activity 1.85 GBq/mM) were obtained from Amersham (Buckinghamshire, UK). Dowex AG 1-X8 anion exchange resin (200-400 mesh, formate form) was purchased from BioRad (Richmond, CA). Ultima Gold XR scintillation fluid was obtained from Packard (Groningen). Silica gel G thin layer chromatography plates were obtained from Merck (Darmstadt). Tryptone and yeast extract were obtained from Difco (Michigan). All other chemicals used were of analytical grade available from commercial sources.

2.2. Tetrahymena cultures

In the experiments Tetrahymena pyrij&mis GL strain was tested in the logarith- mic phase of growth. The cells were cultivated at 28°C in 0.1% yeast extract containing 1% tryptone medium. Before the experiments the cells were washed with fresh culture medium, and were resuspended at a concentration of 5 x lo6 cells/ml.

2.3. Assay of PLA, activity

Preparation of cell homogenate. Washed cells were suspended in 20 mM Tris- HCl buffer (pH 7.4) and disrupted with a vibra cell sonifier (Sonics and Materials, Danbury, CT) for two periods of 15 s, with a 30 s interval. Almost all cells were disrupted by this procedure.

The standard incubation mixture for the assay of PLA, activity contained: ~ 100 ~1 CaCl, or 100 ~1 EDTA (10 mM); ~ 10 ~1 aprotinin (protease inhibitor) (0.1 mgiml); - 10 /11 1-acyl 2-‘4C-arachidonyl-L-3-phosphatidylethanolamine (10 PCi); - 27 111 20 mM Tris-HCl buffer, pH 7.4, or 27 ~1 of agonists or antagonists; ~ 100 ~1 cell homogenate (200 pg protein).

The following agonists and antagonists were used: - melittin (PLA, activator), 20 pg/ml; ~ sodium orthovanadate, 0.1 mM; H,Oz, 1 mM; H,O, + sodium orthovanadate

(tyrosine kinase activators); ~ sodium fluoride, 10 mM; AlF, (10 mM NaF + 10 PM AlCl,) (G-protein

activators); ~ PDBu, 5 rug/ml (PKC activator); _ EDTA, 10 mM (Ca’+ -chelator); ~ Quinacrine, 0.1 mM; and BPB, 0.1 mM (PLA* inhibitors).

The reaction was stopped by the addition of 1 ml chloroform/methanol (1:2, v:v) and the lipids were extracted twice by the method of Bligh and Dyer (1959). The combined chloroform phase was evaporated by N, stream to dryness, dissolved in two drops of chloroform/methanol (2: 1, v:v) and subjected to thin layer chromatography. The chromatography plates were developed by hexane:diethylether:formic acid (90:60:9, v:v:v) solvent. After development, 0.5 cm strips were scraped into scintillation vials and the radioactivity was measured by a Beckmann scintillation counter.

236 P. Kovrics, G. Csuba /J. Lipid Mediators Cell SipaIling 15 (1997) 233-247

2.4. Assay of 32P incorporation into the phospholipids of Tetrahymena

In these experiments the following groups were tested: (1) control, untreated groups; (2) 0.1 mM BPB treated groups; (3) 0.1 mM quinacrine treated groups.

Tetrahymena cultures (25-25 ml) were treated for 30 min with the concentrations mentioned above of BPB or quinacrine, then 12 MBq 32P Na-orthophosphate was added to the cells in the further presence of BPB or quinacrine. After 5, 15, 30 and 60 min 5 ml samples were taken from the cultures. The phospholipids were separated according the method of &chard et al. (1989). The phospholipid samples were analyzed on silica gel chromatography plates (solvent system, chloroform:acetone:methanol:acetic acid:water; 40: 15: 13: 12:8, v:v:v:v:v). After development the chromatograms were covered with Kodak TMG X-ray film. After exposure ( z 18 h) and development of radiograms, the radioactivity of individual phospholipid spots were analyzed by laser densitometer (Ultroscan XL, Pharmacia- LKB, Uppsala, Sweden). The individual phospholipids were expressed as 100% of total incorporated 32P. The phospholipids were identified by a parallel run of authentic standards of PI, PIP, PIP,, PE and PA.

2.5. Assq~ of breakdown of 32P Iubelled phospholipids

Tetruhymenu cultures (90-90 ml) were treated with 54 MBq 32P Na-orthophosphate for 90 min. After the treatment the cultures were washed thoroughly with fresh culture medium, and then were transferred into 90 ml fresh culture medium. From this cell suspension three groups were formed: (1) control group (no further treatment); (2) 0.1 mM BPB treated group; (3) 0.1 mM quinacrine treated group.

Samples of 5 ml were taken after 1, 5, 15, 30 and 60 min. The isolation and analysis of 32P-containing phospholipids was done according to the method described above.

2.6. Assay of incorporution qf’ ‘H inositol into the inositol phospholipids and formation (release) of inositol phosphates

The method for measurement of PI hydrolysis in Tetruhymena was similar to that described by McArdle et al. (1988). Tetruhymena cultures (20 ml) were treated with 1 pCi/ml ‘H-inositol for 2 h. After washing three times with 20 mM LiCl containing culture medium the cells were inoculated into LiCl containing culture medium, and divided into three groups (2-2 ml): (1) control groups; (2) 0.1 mM BPB treated groups; (3) 0.1 mM quinacrine treated groups.

P. Koch, G. Csaba /J. Lipid Mediators Cell S&palling 15 (1997) 233-247 231

After 30 min the reaction was stopped by the addition of 2 ml chloroform/ methanol (1:2, v:v). The organic (lower) and aqueous phases were separated by centrifugation.

An aliquot (3 ml) of upper phase was taken from each sample for analysis of inositol phosphates by Dowex chromatography. The samples were applied to 1 ml Dowex AG 1-X8 resin, and the inositiol phosphate metabolites were eluted by H,O (free inositol); 5 mM sodium tetraborate/60 mM NH4 formate (glycero phosphoinositol); 0.2 M NH, formate/O.l M formic acid (inositol 4-monophosphate-IP); 0.4 M NH, formate/O.l M formic acid (inositol 4,5-bisphosphate-IP,) and 1 M NH, formate/O. 1 M formic acid (inositol 1,4,5-trisphosphate-IP,), respectively. The radioactivity of eluted materials were determined by the use of Ultima Gold XR scintillation fluid and Beckmann scintillation counter.

The organic (chloroform) phase was dried under N, stream and chro- matographed as described above in Section 2.4. From developed chromatograms, 0.5 cm areas were scraped directly into scintillation vials. The radioactivity present on each area was determined by measurement using a Beckman scintillation counter.

The experiments on control cells were performed both in the presence and the absence of LiCl.

2.7. Statistid treutment qf’ data

In each experiment, the experimental data represent the means of quadruplicate experiments. Student’s t-test was used for calculations, with P < 0.05 accepted as the level of statistical significance.

3. Results

3.1. PLA, activity in Tetruhymenu

On the basis of formation of free 14C-labelled AA from the 1-acyl 2-[1-‘4C] L-3-phosphatidylethanolamine by Tertruhymena homogenate it is obvious that this unicellular organism shows PLA, activity. Ca2+ ions were not required for the degradation of radioactive substrate, because their activity was not inhibited by EDTA (Fig. 1)

Quinacrine treatment (0.1 mM) resulted in a significant decrease of PLA, activity and likewise, 0.1 M BPB treatment decreased the amount of liberated AA. This decrease was smaller than in the quinacrine treated ones (Fig. 1).

Melittin treatment resulted in a very high elevation of PLA, activity, but this PLA,-hyperactivation requires the presence of Ca2 + and the activity of this enzyme during melittin treatments in the presence of EDTA was significantly lower than in the absence of EDTA (Table 1).

Treatment with the tyrosine kinase activators sodium orthovanadate and H,O, did not cause the liberation of free AA, but their combination reduced considerably

238 P. Kouics, G. Csaba / J. Lipid Mediators Cdl SignaIling 15 (1997) 233-247

- without cell homogenate 25ocQ-

--~-- control AA

EDTA -2M)o- 2

\

klxKl-

-- - - Quinacrine I#. BPB i

: 1

c 5 .- z %Ooo- g

f? ’ \ I( :: I/

xz *- -0 ?k 8’ fJ! SW_ Y /

I

I/ \ 5 / i

cl- “s,_ I _ , _

I , I ,,*,,I.,,,, 0 2 4 6 a # P

migration distance (cm)

Fig. 1. The effect of quinacrine, BPB and EDTA on the PLAz activity of Tetrahymena. Migration distance of the free AA is 6.5 cm (Rf 0.54). The experiments were done four times with a representative experiment shown.

PLA, activity. The G-protein activator fluorides (NaF and ALF,), and the PKC activator PDBu did not cause significant changes in PLA, activity (Table 1).

3.2. Incorporation of 32P into the phospholipids of Tetrcthymenu

Treatments with 0.1 M quinacrine totally abolished the incorporation of 32P into the phospholipids of Tetrahymena, while 0.1 M BPB treatment resulted in a

Table 1 PLA, activity after different treatments of Tetrahymma related to the control as 100’%1

Group Activity (‘XI)

Control Vanadate TOP4 M H,O, I mM Vanadate + H,Oz Melittin 20 /lg/ml EDTA + melittin NaF AlF, PDBu 5 pg/ml

100 93.36 f 3.8 98.53 * 4. I 48.55 i 6.2*

381.48 4 13.5.5* 184.0 4 s.41*,** 96.58 + 3.25 94.03 14.7

105.72 & 6.38

The data represent the means + SD of four independent experiments * pcO.01 to control. ** p < 0.01 to melittin treated.

P. Kowks, G. Csaha I J. Lipid Mediators Cell Signalling 15 (1997) 233-247 239

# 1 - control - PIP,

- - - control - PIP .’ I - - - control - PI

BPB - PIP,

, /’

.’ ,’

,’

BPB - PIP ,,’

,’ BPB - PI /’ _’

, _ , 7 *’

,’ _---

--- -S S

..~ S _.

, I, I-, 1. I. I I

D 20 30 40 50 60

time of incubation (min)

Fig. 2. The effect of BP9 on the incorporation of “P into the inositol phospholipids of Tetrahymena. The data represent the mean i_ SD of four independent experiments. S = p < 0.01 to the control.

reduced 32P incorporation into the phospholipids. (Quinacrine treatments in 0.1 mM concentration and in the time periods used were not toxic to Tetruhymena according to the microscopic control of the behaviour of the treated cells).

In presence of BPB, 32P incorporation into the inositol phospholipids (PI, PIP and PIP,) was slower than that in the controls; the radioactivity appeared after 15 min (PI and PIP) or 30 min (PIP,) and in the controls these inositol phospholipids contain incorporated 32P already after 5 min. The amount of incorporated 32P in inositol phospholipids was significantly lower in the BPB treated cells compared to the controls in the whole incubation period (Fig. 2).

32P incorporation into PE and PA was significantly reduced in BPB treated groups within the first 30 min of treatments, but after 60 min the difference between the BPB treated and control cells was not significant (Fig. 3).

3.3. Breakdoolvn of’ 32P labelled phospholipids of Tetrcthymenu

On the breakdown of 32P labelled phospholipids, BPB and quinacrine had different effects. In the case of PI and PIP,, quinacrine generally resulted in a decreased level, while BPB resulted in an elevated level; on the PIP level the effect of two drugs was a slightly similar, but here also the quinacrine treatment resulted in a lower level. Opposite to the controls the PIP level shows an elevation in the last 30 min both in the BPB and quinacrine treated groups (Fig. 4).

The different effect of BPB and quinacrine was seen also on the PE, PA and PC levels during the breakdown of phospholipids. In the case of PA and PE, quinacrine treatment resulted in a higher value, while in PC lower values than that of the

240 P. Kovcics, G. Csaha /J. Lipid Mediators Cell Signalling 15 (1997) 233-247

control (Fig. 5) resulted. In general BPB treatments caused an opposite effect. In spite of these facts, apart from some exceptions, the alteration of the phospholipid ratio was similar when compared to that of the controls.

3.4. Incorporation of ‘H-inositol into the inositol phospholipids of Tetruhymena

Treatment with 0.1 mM quinacrine resulted in reduced ‘H-inositol incorporation into PI and PIP, but in PIP, we measured higher radioactivity after the treatments (Fig. 6). BPB treatment resulted in only a minimal decrease in the inositol incorporation into PI, but in PIP a significant decrease was measurable. The radioactivity in PIP, was at the control level.

3.5. Formation of inositol phosphates

After 30 min incubation of the ‘H-inositol labelled Tetruhymena population in LiCl-containing culture medium ,higher radioactivity levels in glycerophosphoinosi- tol, IP, IP, and IP, were always measured, than in the LiCl-free culture medium (Fig. 7).

Quinacrine treatment resulted in a significantly higher amount of IP than we found in the control cultures, however other inositol phosphates and glycero phosphatidylinositol were measurable at the level which almost corresponds to the controls. BPB treatments did not cause changes in the amount of ‘H-inositol labelled inositol phosphates (Fig. 8).

30

I -control - PE ---control - PA

n 25

A.

BPB - PE

----BPB - PA ET

” 1: \/‘,,,,,,,,*.’ F 8 _ u ,’

.r 3

/

,’

5 u- s, I’ >I

?f .r_ _ _ - - - ?~

% 5 Y.f. s ;‘_

__-I _---


Fig. 3. The effect of BPB on the incorporation of “P into the PA and PE of Tetrahymena. The data represent the mean + SD of four independent experiments. S =p -C 0.01 to the control.

P. Kovrics, G. Csaba /J. Lipid Mediators Cell Signalling 15 (1997) 233-247 241

4. Discussion

Many lipids or lipid-derived products generated by phospholipases acting on phospholipids in membranes are implicated as mediators and second messengers in

lime of incubation (min)

b

o,‘l--. 1 1 ’ 1 ’ 0 ll w 30 40 50 60


45 pI c

IS

,_I ,__’

_’

7.0 ,rz

’ ’ I ’ ’ ’ L ’ 0 0 20 aI 40 50 60


Fig. 4. (a,b and c) The effect of quinacrine and BPB on the breakdown of 3”P-labeled inositol phospholipids of Tetralzymma. The data represent the mean + SD of four independent experiments. S = P -c 0.01 to the control; Z = p < 0.05 to the control.

242 P. Kouics, G. Csaba 1 J. Lipid Mediators Cell Signalling I5 (1997) 233-247

signal transduction. Receptor-mediated activation of PI-specific PLC (PI-PLC) catalyses the cleavage of PIP, to second messengers DAG and IP, (reviewed in Rana and Hokin, 1990). Phospholipase D (PLD) catalyses the cleavage of PC to PA and free choline (reviewed in Waite, 1987). The agonist stimulated (and antagonist-inhibited)

~ control - ~ quinacrine 0.1 mM

0 0 20 3.3 40 50 60


\~ ‘~

n- ~<i z / ,

” 0 20 30 40 50 @I


._ ?ij ,’

,I. I z“.. z 25 j/ ,l’ -.

‘< ,’ -.,_ 5 $6 ..__ -6 --._ ‘. s M- ‘IS

Cl 0 20 w 40 53 60 tame of incubation (min)

Fig. 5. (a,b and c) The effect of quinacrine and BPB on the breakdown of “P labeled phospholipids of Tetrahymena. The data represent the mean f SD of four independent experiments. S =p < 0.01 to the control; Z = p < 0.05 to the control.

P. K&cs, G. Csaha i .I. Lipid Mediators Cell Signalling 15 (1997) 233-247 243

4m r - -- -. quinacrine 0.1 mM

BPB 0.1 mM

0 2 4 6 a D P

migration distance (cm)

Fig. 6. The effect of quinacrine and BPB on the incorporation of ‘H inositol into inositol phospholipids of Tetrallymma. The experiments were done four times with a representative experiment shown.

breakdown of PIP, in Tetruhymenu strengthen our supposition about the presence and function of PI-PLC in Tetrahymenu (Kovics and Csaba, 1990, 1996a,b). Similarly in the presence of butanol the agonist stimulated (and antagonist inhibited) formation of phosphatidylbutanol indicated the presence of PLD in Tetruhymenu (Kovacs et al., 1996a;,1996b). On the basis of intensive AA metabolism in Tetrahymenu (Kovacs and Csaba, 1996a) and studies on the phospholipases A, and A, (Florin-Christensen et al., 1986; Arai et al., 1986; Ramesha and Thompson, 1984) we assumed that the AA metabolism is catalysed by PLA,-like activity in this unicellular organism.

The aim of this work was to obtain more information about the PLA, activity, and the connection of this activity with phospholipid and inositol phosphate metabolism in Tetrahymena. For the study of these problems materials influencing PLA, metabolism (quinacrine, BPB, melittin), and materials influencing other second messenger pathways (vanadate, H,Oz, phorbol 12,13-dibutyrate, NaF and AlF,) have been used.

The experiments unequivocally demonstrated a PLA,-like activity in a Tetrahy- menu p~w$rmis homogenate (Fig. 1). This activity was Ca* + independent, as in the presence of EDTA the production of free AA resembled that of Ca2+ containing samples.

The anti-malarial drug quinacrine inhibits the liberation of AA from phospholipids by both PLC and PLA, (Billah and Lapetina, 1982). BPB, a known active-site directed histidine reagent, has been found to inhibit different types of PLA,-s (Roberts et al., 1977). The bee venom peptide, melittin hyperactivates PLAz (Haberman, 1972). These results were gained by using mammalian cells for studies.

In our experiments, quinacrine significantly inhibited PLA, activity in Tetruhy- menu, and BPB also reduced the liberation of AA. The reduced PLAz activity in

244 P. Koucks. G. Gaba /J. Lipid Mediators Cell SiXnalling 15 (1997) 233-247

BPB treated cells may give an explanation to the reduced responsiveness to hormonal treatments of Tetruhymena (KovBcs and Csaba, 1995).

Quinacrine in platelets inhibited both PLC and PLA2 activity (Billah and Lapetina, 1982). Because both enzymes are able (PLC coupled with lysophospholipase (Dennis, 1987)) to release AA from phospholipids, we also examined the effect of quinacrine on the phospholipid breakdown, where PLC plays cardinal role.

During the breakdown of “P-1abelled phospholipids the amount of PIP, in the quinacrine treated Tetrahymenu was lower than that of the control (Fig. 4), thus we can exclude the possibility that quinacrine caused inactivation of PLC also during the liberation of AA. However an other possibility of formation of the lower PIP, level in the quinacrine treated cells is the inhibition of IP conversion to inositol (inhibition of inositol monophosphatase) and this could lead to a decrease in the concentration of phosphatidyl inositols (Fig. 4).

Lithium inhibits the activity of inositol monophosphatase also in Tetrahymena. The level of IP is higher in presence of LiCl than in the plain culture medium (Fig. 7). but in presence of quinacrine the IP level is significantly higher than that in the controls (Fig. 8). This could be another reason for the decreased PIP, level by the

MO-

WI-

MO-

?% Eu-

& : 100-

c- 15 @J-

tj

ii! 6o: .-

P 40.

XJ-

glyx-c@cs~rasitd IP ‘Pz ‘P3

Fig. 7. The effect of LiCl on the inositol phosphate formation of Tetrahymena. The data represent the mean + SD of three independent experiments. S =p -c 0.01 to the control; X=p < 0.05 to the control.

P. Korirc.s, G. Csaba I J. Lipid Mediators Cd Signalling 15 (1997) 233 -247 245

I

control quinauine BPB

Fig. 8. The effect of quinacrine and BPB on the formation of inositol phosphates of Tetrahymena. The data represent the mean of three independent experiments. S = p < 0.01 to the control.

inhibition of 32P incorporation (or phospholipid synthesis) in the case of quinacrine treatments. The presence of quinacrine in the incorporation of “P into phospholipids was totally abolished. The quinacrine treatments inhibited also the incorporation of 3H-inositol into the inositol phospholipids (Fig. 6). These phenomena are interesting, because quinacrine in the 0.777 mM range did not show significant inhibition of lipid synthesis measured by acetate incorporation in the cell free system of Tetrahynena pyriformis CL (Pan et al., 1974).

The effect of quinacrine is also manifested during the phospholipid breakdown in the ratio of PA and inositol phospholipids (PI, PIP and PIP,). Apart from data measured in 60 min of experiments the PA levels are higher, while those of inositol phospholipids are lower in the quinacrine treated groups compared to controls (Figs. 4 and 5). It can be supposed that quinacrine inhibits the activity of PI- and PIP-kinases, thus in the lipid cycle the accumulation of PA takes place (Berridge and Irvine, 1984). In connection with inhibition of the lipid cycle it is presumable that CDP-diacylglycerol inositol phosphatidate transferase activity decreases, which results in an accumulation of the members of the inositol phosphate cycle (mostly in the amount of IP).

Melittin (20 pg/ml) significantly elevated the amount of free AA, like in mam- mals, where the PLA, activity can be hyperstimulated with this bee venom peptide (Haberman, 1972). This melittin stimulated PLA, activity, in contrast to basic PLA, activity, is Cal+ dependent. These results reveal a close correlation between

246 P. Kocdcs, G. Csubu i J. Lipid Mediators Cd Signal&g 15 (1997) 233-247

the extent of PLA, hyperactivation and Ca2+ mobilization, suggesting a causal relationship, the phenomenon of which is similar to that of the ras-transformed cells (Sharma, 1993).

The activation of G-proteins with fluorides, the activation of tyrosin kinase with vanadate and the stimulation of phospholipase C with phorbol butyrate, did not cause alteration of PLA, activity, thus we can conclude that in our in vitro system these components of signal transduction have no contacts with PLA2 activity, in spite of the fact, that these treatments influence in vivo the phospholipid metabolism in Tetruhymenu (Kovacs and Csaba, 1994b).

The supposed role of PLA, in phosphoinositide and phospholipid metabolism may be responsible for disturbing the signal transduction of Tetrahymena. Our earlier findings indicated that disturbances of these signalling pathways inhibit the normal physiological reactions to the hormonal treatments in Tetrahymena (Csaba, 1985; Kovacs and Csaba, 1995; Kovacs et al., 1988).

The experiments extended the list of components of mammalian signalling mechanisms which is also present at a unicellular level and call to the attention the interrelations between different ways of signal transduction in Tetrmhymena (Csaba, 1994).

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Berridge, M.J. and Irvine, R.F. (1984) Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312, 3155321.

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Bligh, E.G. and Dyer, W.J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 91 I-917.

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Florin-Christensen, J., Florin-Christensen, M., Rasmussen, L., Knudsen, J. and Hansen, H.O. (1986) Phospholipase A, and triacylglycerol lipase: two novel enzymes from Tetrahymma extracellular medium. Comp. Biochem. Physiol. 85B, 1499155.

Haberman, E. (1972) The biochemistry and pharmacology of bee and wasp venom. Science 117, 314-322.

Hegyesi, H. and Csaba. G. (1994) Calcium-dependent protein kinase is present in Tetrcr/zymena. Cell Biochem. Funct. 12, 221-226.

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PLA 2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators

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