Role of three isoforms of phospholipase A 2 in capacitative calcium influx in human T-cells Aziz Hichami 1 , Beenu Joshi 2 , Anne Marie Simonin 1 and Naim Akhtar Khan 1 1 UPRES Lipides & Nutrition, Universite ´ de Bourgogne 21000 Dijon, France; 2 Central Jalma Research Institute for Leprosy, Agra, UP, India The present study was conducted on human Jurkat T-cell lines in order to elucidate the role of phospholipase A 2 in capacitative calcium entry. We have employed thapsigar- gin (TG) that induces increases in [Ca 2+ ] i by emptying the calcium pool of endoplasmic reticulum, followed by capacitative calcium entry. We designed a Ca 2+ free/Ca 2+ reintroduction (CFCR) protocol for the experiments, conducted in Ca 2+ -free medium. By employing CFCR protocol, we observed that addition of exogenous arachi- donic acid (AA) stimulated TG-induced capacitative calcium influx. The liberation of endogenous AA and its autocrine action seems to be implicated during TG- induced capacitative calcium influx: TG potentiates the induction of constitutively expressed mRNA of four PLA 2 isoforms (type 1B, IV, V, VI), the inhibitors of the three PLA 2 isotypes (type 1B, V, VI) inhibit TG-induced release of [ 3 H]AA into the extracellular medium, and finally, these PLA 2 inhibitors do curtail TG-stimulated capacita- tive calcium entry in these cells. These results suggest that stimulation of three isoforms of PLA 2 by thapsigargin liberates free AA that, in turn, induces capacitative calcium influx in human T-cells. Keywords: Jurkat cells; thapsigargin; arachidonic acid. In T-lymphocytes, a biphasic rise in concentrations of free calcium, [Ca 2+ ] i , is elicited by the binding of antigen or polyclonal mitogens to the T-cell receptor [1,2]. Hence, the rise in [Ca 2+ ] i constitutes an essential triggering signal for T-cell differentiation and proliferation [3]. Raising [Ca 2+ ] i has been found to increase the activity of a transcription factor, the nuclear factor of activated T-cells (NF-AT), which in turn results in the expression of lacZ gene in transfected murine T-cells [4]. Calcium oscillations with repeated spikes for a period of 100 s are sufficient to activate the transcriptional factors in human T-cells [5]. In a study conducted on caged IP 3 molecules, the trains (from 0.3 to 1.5 s) of short ultraviolet pulses, which induced calcium oscillations, promoted the activity of NF-AT in RBL-2H3 lymphocytes [6]. According to the capacitative model of calcium entry, first, calcium is released via receptor activation from two intracellular stores; first, from endoplasmic reticulum (ER) and then, from the Ca 2+ -induced Ca 2+ -release (CICR) pool. Ca 2+ , in turn, is extruded into the extracellular medium. The cells refill their intracellular emptied pools via store-operated calcium (SOC) influx by opening calcium channels [7]. In Jurkat T-cells, SOC influx is brought about by opening of Ca 2+ release-activated Ca 2+ , CRAC [8] and Ca 2+ release-activated nonselective cation, CRANC [9], channels. Human Jurkat T-cells express nearly 10 000 CRAC channels per cell [3]. The refilling mechanism via CRAC and CRANC channels is regulated by the calcium influx factor (CIF), which is released into the extracellular medium during calcium release in Jurkat T-cells [9,10]. It has been demonstrated that human T-cells possess func- tional ER and CICR calcium pools [1,11]. The mechanisms by which SOC influx is brought about are still not well understood. For example, the nature of the signal trans- duction pathway by which store depletion is linked to the opening of plasma membrane Ca 2+ channels remains unknown. During the recent past, there has been an upsurge of information on the possible implication of phospholipase A 2 and polyunsaturated fatty acids in the regulation of immune cell functions [12–14], particularly the cell signal- ling mechanisms [15–19]. Several studies have reported that arachidonic acid (AA) blocks agonist-stimulated sustained rise in [Ca 2+ ] i [18–20]. On the other hand, some investigators have observed that AA both reduced and increased SOC influx, induced by store depletion in lymphocytes [21]. Keeping in view this discrepancy, the present study was conducted to ascertain the role of PLA 2 activation and hence released AA in the capacit- ative influx of calcium in Jurkat T-cells. These cells represent a good model to study the effects of AA per se as these lymphocytes can not metabolize this fatty acid via lipoxygenase and cyclooxygenase pathways [22,23]. Correspondence to N. A. Khan, UPRES Lipides & Nutrition, Universite ´ de Bourgogne, Faculte ´ des Sciences de la Vie, 6, Boulevard Gabriel, 21000 Dijon, France. Fax: + 33 3 80 39 63 30, Tel.: + 33 3 80 39 63 12, E-mail: Naim.Khan@u-bourgogne.fr Abbreviations: AA, arachidonic acid (20 : 4 n-6); ARC, arachidonate- regulated, calcium; BPB, 4-bromophenacyl, bromide; CFCR, Ca 2+ , free/Ca 2+ reintroduction; CRAC, Ca 2+ , release-activated Ca 2+ ; [Ca 2+ ] i , free, intracellular calcium concentrations; MAF, methyl- arachidonyl, fluorophosphonate; PLA 2 , phospholipase, A 2 ;SOC, store-operated, calcium. (Received 11 March 2002, revised 9 September 2002, accepted 16 September 2002) Eur. J. Biochem. 269, 5557–5563 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03261.x MATERIALS AND METHODS Chemical products The culture medium RPMI 1640 and L -glutamine were purchased from Biowhitaker, Belgium. The Fura-2/AM was procured from Molecular Probes (Eugene, OR, USA). The SuperScript II Reverse Transcriptase, Platinum Taq DNA Polymerase, random primers, oligo(dT)18 and the oligonucleotides used as primers in the RT-PCR analysis were purchased from Invitrogene, Life Technologies (Cergy Pontoise, France). Agarose was from Promega (Char- bonnie ` re, France). Phospholipases A 2 inhibitors arachido- nyl trifluoromethyl ketone (AACOCF 3 ) and bromoenol lactone (BEL) were from Cayman Chemical (Ann Arbor, USA). Aristolochic acid and 4-bromo phenacyl bromide were from Sigma Chemicals (St Louis, MO, USA). Methyl arachidonyl fluorophosphonate was obtained from Cal- biochem (Orsay, France). [ 3 H]Arachidonic acid (specific activity, 217 CiÆmmol )1 ) was purchased from Amersham (Orsay, France). All other chemicals including arachidonic acid (20 : 4 n-6) were obtained from Sigma Chemicals (St. Louis, MO, USA). Cell culture The human (Jurkat) T-cells were kindly provided by Dr Bent Rubin, Head, UMR-CNRS Research Unit at CHR of Toulouse (France). The cells were cultured in RPMI-1640 medium supplemented with L -glutamine and 10% foetal calf serum at 37 °C in a humidified chamber containing 95% air and 5% CO 2 . Cell viability was assessed by the trypan blue exclusion test. Cell numbers were determined by haemocytometer. Measurement of Ca 2+ signalling The cells (2 · 10 6 ÆmL )1 ) were washed with NaCl/Pi (phos- phate buffered saline), pH 7.4. The composition of NaCl/P i was as follows: 3.5 m M KH 2 PO 4 ;17.02m M Na 2 HPO 4 ; 136 m M NaCl. The cells were then incubated with Fura-2/ AM (1 l M ) for 60 min at 37 °C in loading buffer which contained the following: 110 m M NaCl; 5.4 m M KCI; 25 m M NaHCO 3 ;0.8m M MgCl 2 ;0.4m M KH 2 PO 4 ; 20 m M Hepes-Na; 0.33 m M Na 2 HPO 4 ;1.2m M CaCl 2 , and the pH was adjusted to 7.4. After loading, the cells were washed three times (2000 g; 10 min) and remained suspended in the identical buffer. [Ca 2+ ] i was measured according to Grynkiewicz et al. [24]. The fluorescence intensities were measured in the ratio mode in PTI spectrofluorometer at 340 nm and 380 nm (excitation filters) and 510 nm (emission filters). The cells were continuously stirred throughout the experimentation. The test molecules were added into the cuvettes in small volumes with no interruptions in recordings. The intracellular concentration of free Ca 2+ , [Ca 2+ ] i , were calculated by using the following equation: [Ca 2+ ] i ¼ K d · (R ) R min )/(F max ) F) · (Sf 2 /Sb 2 ). A value of 224 n M for K d was added into the calculations. R max and R min values were obtained by addition of ionomycin (5 l M ) and MnCl 2 (2 m M ), respectively. All the experiments were performed at 37 °C. Arachidonic acid (AA) was dissolved in ethanol (0.1%, w/v) and used immediately or kept at )20 °C in ampoules, tightly sealed under a stream of nitrogen. We designed a Ca 2+ -free/Ca 2+ -reintroduction (CFCR) protocol for the experiments, conducted in Ca 2+ -free (0% Ca 2+ ) medium. Hence, we examined the role of AA on direct calcium influx. First, AA and then CaCl 2 was added into the cuvette. Arachidonic acid release The experiment on arachidonic acid incorporation and release was performed as described elsewhere [25]. In brief, Jurkat T-cells were serum-starved for 4 h before labelling with [ 3 H]-arachidonic acid (AA, 1.5 lCi per 3 · 10 8 cells) for 2 h in RPMI 1640 serum-free medium supplemented with 0.2% fatty acid-free BSA. At the end of the incubation, cells were washed twice with RPMI 1640 serum-free medium containing 0.2% BSA. The cells were resuspended in 500 lL RPMI 1640 medium supplemented with 0.5% BSA at a final concentration of 12 · 10 6 cellsÆmL )1 and treated or not (vehicle carrier contained dimethylsulfoxide, 0.1% v/v) with 5 or 15 l M of PLA 2 inhibitors or vehicle for 10 min with or without TG (5 l M ). Cells were centrifuged (1250 g; 3 min) and 0.4 mL of supernatant was added to 2 mL scintillation cocktail for counting in a liquid scintillation analyzer (Packard 1900 TR, France). RNA isolation and semiquantitative RT-PCR analysis Total RNA from cultured Jurkat T-cells was purified using trizol reagent (Invitrogene Life Technologies, Cergy Pon- toise, France) according to the manufacturer’s instructions. Oligonucleotide primer pairs, used for mRNA analysis by RT-PCR, were based on the sequences of the human genes, as described elsewhere [26]. The cDNA was either used immediately for PCR or stored at )20 °C until use. The conditions for the PCR amplification and the assays have been described elsewhere [26]. Human b-actin mRNA primers were used as internal control to normalize the data. Reaction products were electrophoresed on a 1% agarose gel impregnated with ethidium bromide. The RNA pattern was visualized by UV transillumination. Statistical analysis Results are shown as mean ± SEM. Statistical analysis of data was carried out using STATISTICA (version 4.1, Statsoft, Paris, France). The significance of the differences between mean values was determined by analysis of variance one way, followed by a least-significant-difference (LSD) test. RESULTS AA facilitates capacitative Ca 2+ influx in TG-stimulated cells The increases in [Ca 2+ ] i can also be achieved by employing thapsigargin [27]. According to the capacitative model of calcium homeostasis, an increase in [Ca 2+ ] i is responsible for the extrusion of free calcium into the extracellular medium, and this phenomenon is followed by SOC influx to refill the intracellular pool [7]. The thapsigargin 5558 A. Hichami et al.(Eur. J. Biochem. 269) Ó FEBS 2002 (TG)-induced Ca 2+ spike is followed by a lowered sustained response that is, indeed, the phase of SOC capacitative refilling. In CFCR protocol, addition of AA and then CaCl 2 to 0%Ca 2+ medium potentiated the thapsigargin-induced capacitative calcium influx in a dose dependent manner in Jurkat T-cells (Fig. 1). The increases by the addition of arachidonic acid at 1 l M ,5l M and 10 l M were, respect- ively, 8.01 ± 0.01, 16.0 ± 0.10 and 35.2 ± 3.15. The inset of Fig. 1 shows that AA (10 l M ) in 100% Ca 2+ medium induced a significant increase in [Ca 2+ ] i in compar- ison with that in 0% Ca 2+ medium (30 ± 2.10 n M , 100% Ca 2+ medium vs. 4.1 ± 0.56 n M ,0%Ca 2+ medium, P <0.001). TG induces the release of AA and the expression of mRNA of different PLA 2 isotypes In [ 3 H]AA loaded cells, TG induced the release of [ 3 H]AA into the extracellular medium (Fig. 2). We employed aristolochic acid and 4-bromo phenacyl bromide (BPB) which are the inhibitors of sPLA 2 , i.e. type IB/typ V. Arachidonyl trifluoromethyl ketone (AACOCF 3 ) and bromoenol lactone (BEL) are the respective inhibitors of type IV and type VI PLA 2 . Methyl-arachodonyl fluoro- phosphonate (MAF) inhibits type IV and type VI PLA 2 with the same selectivity. We observed that aristolochic acid, BPB and BEL, but not AACOCF 3 , inhibited the release of [ 3 H]AA, induced by TG (Fig. 2). MAF inhibited the TG- induced release [ 3 H]AA almost with the same order of magnitude as BEL. Figure 3 shows that Jurkat T-cells constitutively express the mRNA of four PLA 2 isotypes (type IB, type V, type IV and type VI). Interestingly, addition of TG stimulated the induction of the four PLA 2 isotypes in Jurkat T-cells. PLA 2 inhibitors that inhibit AA release diminish the TG-induced capacitative calcium entry Figure 4 shows that prior addition of aristolochic acid, and BEL inhibited the sustained TG-stimulated capacitative calcium entry in these cells. Interestingly, AACOCF 3 failed to significantly curtail the TG-induced capacitative calcium entry in these experiments (Fig. 4). The decreases of delta calcium by BEL and MAF were, respectively, 15 ± 1.02 n M and 14 ± 1.2 n M vs. control 35 ± 2.10 n M .BPB and aristolochic acid also diminished TG-induced capacit- ative calcium entry in human T-cells (aristolochic acid, 30 ± 1.10 n M and BPB, 29 ± 1.04 n M ). AA-induced calcium influx is contributed by opening the calcium channels We were tempted to assess whether AA-induced calcium influx is contributed by the opening of calcium channels. We employed ionomycin at 100 n M as in the continuous presence of this ionophore at this concentration, the internal calcium stores are short-circuited and recovery of an elevated calcium is almost entirely due to extracellular calcium intrusion [28]. Figure 5 shows that addition of AA, during the ionomy- cin-induced spike, evoked an additive sustained response in [Ca 2+ ] i in Jurkat T-cells. Hence, ionomycin-induced sus- tained response was 80 ± 4.2 n M whereas arachidonic acid-induced response was 165 ± 5.4 n M , if the latter agent Fig. 1. Effects of extracellular calcium on arachidonic acid (AA)-facilitated capacitative calcium (SOC) influx in Jurkat T-cells. Cells (4 · l0 6 per assay) were loaded with the fluorescent probe, Fura-2/AM, as described in Materials and methods. The experiments were performed in 0% Ca 2+ medium. The arrow heads indicate the time when the test molecules, thapsigargin, TG (1 l M ),AA(from0to10l M ) and CaCl 2 (1.5 m M ) were added into the cuvette. During TG-induced steady-state of capacitative calcium influx, AA and then CaCl 2 were sequentially added or not into the cuvette without interruptions in the recordings. The control trace shows the recording observed in the absence of AA and CaCl 2 . The figure shows the single traces of observations which were reproduced (n ¼ 10), independently. The inset shows the experiment conducted in the absence of thapsigargin but in 0% and 100% Ca 2+ medium (n ¼ 11). Ó FEBS 2002 Capacitative calcium influx in human T-cells (Eur. J. Biochem. 269) 5559 was added during the spike of ionomycin. In order to probe the role of different calcium channels, implicated in AA- induced calcium influx, we employed, tyrphostin A9 (TA9), an inhibitor of CRAC channels, diltiazem and x-conotoxin, the respective inhibitors of L -type and N -type calcium channels. We observed that these agents did not diminish the AA-induced increases in [Ca 2+ ] i in these cells (results not shown). AA interacts extracellularly In order to assess whether AA acts extracellularly during Ca 2+ influx, we used the fatty acid free BSA at a final concentration of 0.2% (w/v) as this concentration of BSA has been shown to compete with polyunsaturated fatty acids, bound to the plasma membrane [17]. Figure 6 shows that addition of BSA during the peak response of AA Fig. 2. Effects of phospholipase A 2 inhibitors on TG-induced [ 3 H]arachidonic acid release. Serum-starved Jurkat T-cells (3 · 10 8 ) were labelled for two hours with 1.5 lCi of [ 3 H]AA. The cells were then treated with thapsigargin (1 l M ) in the presence or absence of 5 or 15 l M of AACOCF 3 , aristolochic acid, BEL, BPB, MAF or with vehicle control (dimethylsulfoxide, 0.1% final concentration) for 10 min. Cells were harvested as described in Materials and methods. Results are expressed as mean ± SEM of three independent experiments. Data are expressed as a percentage of the control, which was considered 100%. Data are significantly different as compared to vehicle control (\P < 0.01) and TG-stimulated cells (\\P < 0.001). Fig. 3. Effects of TG on the induction of mRNA of different phospho- lipase A 2 isoforms in Jurkat T-cells. Cells were treated for two hours with or without TG (1 l M ). Total RNA was isolated and analyzed by RT-PCR using specific primers for human PLA 2 -IB, -V, -IV and -VI as describedinMaterialsandmethods.b-actinmRNAwasusedasan internal standard. The lower panel shows the histograms of three in- dependent experiments. Data are significantly different as compared to respective constitutively expressed mRNA levels (\P < 0.001). Fig. 4. Effect of phospholipase A 2 inhibitors on TG-induced capacitative calcium influx in Jurkat T-cells. Cells (4 · l0 6 per assay) were loaded with the fluorescent probe, Fura-2/AM, as described in Materials and methods. The experiments were performed in 100% Ca 2+ medium. The arrow heads indicate the time when the test molecules, thapsi- gargin, TG (1 l M ) or PLA 2 inhibitors, all at 15 l M , i.e. AACOCF 3 , aristolochic acid, BEL, BPB, MAF, were added into the cuvette. The control trace (none) shows the recording observed in the absence of PLA 2 inhibitors. The figure shows the single traces of observations which were reproduced (n ¼ 11), independently. 5560 A. Hichami et al.(Eur. J. Biochem. 269) Ó FEBS 2002 abruptly diminished the AA-induced rise in [Ca 2+ ] i in Jurkat T-cells (AA-induced spike response, 40 ± 4.2 n M vs. BSA-induced inhibition after the addition, 20 ± 2.1 n M ). Addition of BSA alone exerted no significant perturbation in the Fura-2 fluorescence (results not shown). DISCUSSION Our observations that arachidonic acid (AA) induces calcium influx in Jurkat T-cells are in accordance with the reports of several investigators who have shown that this fatty acid induces calcium influx in different cell lines [17, 28–31]. To shed light on whether exogenous AA evoked capacitative calcium influx, we employed thapsigargin (TG) and conducted experiments in 0% Ca 2+ buffer. In these experiments, calcium was replaced by EGTA. In 0% Ca 2+ buffer, addition of AA alone did not induce any increases in [Ca 2+ ] i in these cells. In the CFCR protocol, in the presence of thapsigargin, addition of AA and then exogenous Ca 2+ exerted dose dependent effects on the increases in [Ca 2+ ] i in Jurkat T-cells. These observations suggest that AA evokes capacitative calcium influx in these cells. To elucidate whether TG liberates the endogenous AA that may account for the TG-induced capacitative calcium influx, we first loaded cells with [ 3 H]AA and then incubated in the presence of TG. We observed that TG significantly induced the liberation of free AA into the extracellular environment. Ohuchi et al. [32] Have also shown that TG induces an increase in the release of [ 3 H]AA; however, the PLA 2 isoform implicated is not well known, though Tornquist et al [33] have demonstrated that cPLA 2 might be responsible for the liberation of AA in FRTL-5 cells. We employed the inhibitors of different isoforms of PLA 2 .We observed that TG seemed to act on the activation of three PLA 2 isotypes as aristolochic acid and BPB, the inhibitors of type IB and type V, and BEL, an inhibitor of type VI, inhibited significantly the TG-induced [ 3 H]AA release. The type IV-PLA 2 does not seem to play a role in the release of AA as its inhibitor, AACOCF 3 , failed to significantly diminish the TG-induced release of [ 3 H]AA in Jurkat T-cells. MAF, an inhibitor of type IV and type VI, also inhibited the release of [ 3 H]AA but its effect does not seem additive as compared to BEL. Whether TG exerts its action at the transcriptional level, we detected the expression of mRNA, encoding for these four PLA 2 isotypes. We observed that, in RT-PCR, Jurkat T-cells constitutively express the mRNA of four PLA 2 isotypes, i.e. type 1B, type IV, type V and type VI. In fact, the different phospholipases detected in our study belong to secretory (type IB and type V, sPLA 2 ) and cytosolic (calcium-dependent-type IV, cPLA 2 and calcium-independent-type VI, iPLA 2 ) families. Our results on the constitutive expression of these mRNA of different PLA 2 isoforms are in accordance with our recent report [26]. Our results on the detection of type IV cPLA 2 corroborate the findings of Boilard and Surette [34] who have recently shown that this isotype of PLA 2 is phospho- rylated after anti-CD3 stimulation in human T-lympho- cytes. Addition of TG potentiates the induction of mRNA of these four PLA 2 isotypes. The mechanism of action of TG on the induction of these enzymes is not well under- stood. Several investigators have shown that the generation of [Ca 2+ ] i oscillations by some agonists also accompanies PLA 2 -mediated AA release [33,35], though PLA 2 can be activated independently of increases in [Ca 2+ ] i , probably via receptor coupling of this enzyme [36]. Though TG stimu- lated the induction of expression of mRNA of four PLA 2 isotypes, only three of them seem to be implicated in capacitative calcium influx as the aristolochic acid and BPB (inhibitors of sPLA 2 ) and BEL (inhibitor of iPLA 2 ), but not AACOCF 3 (inhibitor of cPLA 2 ), curtailed the sustained TG-induced capacitative calcium entry in these cells. MAF (inhibitor of iPLA 2 and cPLA 2 ) diminished the TG-induced calcium with the same order of magnitude as BEL. The stimulus-induced release of AA by the action of iPLA 2 , though does not fit with the role of iPLA 2 in phospholipid remodelling, but it seems to be a specific feature of these cells as we have reported recently that an inhibitor of this isoform significantly diminished the release of AA, induced by phorbol 12-myristate 13-acetate and ionomycin in Jurkat Fig. 6. Effects of addition of BSA on AA-evoked increases in [Ca 2+ ] i in Jurkat T-cells. Cells (4 · l0 6 per assay) were loaded with the fluores- cent probe, Fura-2/AM, as described in Materials and methods. The arrow heads indicate the time when the test molecules, fatty acid free BSA (0.2% w/v) and AA (10 l M ) were added into the cuvette without interruptions in the recordings. The figure shows the single traces of observations that were reproduced (n ¼ 12), independently. Fig. 5. Effects of arachidonic acid (AA) on ionomycin-induced calcium influx in Jurkat T-cells. Cells (4 · l0 6 per assay) were loaded with the fluorescent probe, Fura-2/AM, as described in Materials and methods. The arrow heads indicate the time when the test molecules, ionomycin (100 n M ) and AA (10 l M ), were added into the cuvette without interruptions in the recordings. The figure shows the single traces of observations which were reproduced (n ¼ 8), independently. Ó FEBS 2002 Capacitative calcium influx in human T-cells (Eur. J. Biochem. 269) 5561 T-cells [26]. Similarly, Roshak et al. [37] have also reported that iPLA 2 is expressed in human peripheral blood lymphocytes and Jurkat T-cells, and it plays an important role in T-cell proliferation as its depletion by antisense treatment resulted in marked suppression of cell division. As the addition of AA during the ionomycin-induced response exerted an additive prolonged effect, we can state that AA is opening the calcium channels, probably specific to this fatty acid. The AA-stimulated calcium influx is not mediated via classical mechanisms as TA9, an inhibitor of CRAC channels [38], and diltiazem and x-conotoxin, the respective inhibitors of L-type and N-type calcium channels, failed to block AA-induced calcium influx in these cells (results not shown). Our hypothesis on the presence of AA-specific calcium influx is contributed by the recent reports [39,40] that have demonstrated the arachidonate- regulated calcium (ARC) current, specific to this fatty acid in HEK293 cells. The ARC current, evoked by AA from 8 to 10 l M in patch clamp experiments, can be blocked by La 3+ at 50 l M in these cells [39]. Similarly, in our study, we observed that AA-induced capacitative calcium influx was inhibited by the preaddition of La 3+ (results not shown). As the specific inhibitors of ARC channels are not available, it is difficult to provide a direct evidence for the implication of these channels during capacitative calcium influx in Fura-2 loaded cells. Our results on AA-evoked capacitative calcium influx are in contradiction with the observations of Gamberuchi et al. [19] who have reported that AA, in place of stimulating, inhibits thapsigargin-induced capacitative calcium influx in Jurkat T-cells. The difference in the observations can be explained by the fact that these investigators determined the increases in [Ca 2+ ] i at a single excitation wavelength, 340 nm. In fact, this approach is not very precise as, during the increases in [Ca 2+ ] i , there is usually spectral displace- ment from 340 nm to another wavelength of the excitation spectrum when using Fura-2 [24]. However, in our study, we excited the probe, in the ratio mode, at two wavelengths, i.e. 340 nm and 380 nm, in the excitation spectrum and this method provides accurate results by eliminating any spectral displacement during determinations the increases in [Ca 2+ ] i [24]. In our study, AA seems to act extracellularly as addition of fatty acid free BSA abruptly diminished the Ca 2+ rise, evoked by the former. BSA is known to possess high affinity binding sites for free fatty acids. Hence, it seems that BSA detaches the plasma membrane bound-AA and, thereby, contributes to the lowered response of this polyunsaturated fatty acid. How AA directly opens or modulates the calcium channels is not known. However, a direct action of arachidonic acid on N-methyl- D -aspartate receptor-chan- nels has been proposed as the channel protein shares an amino acid sequence homology with fatty acid binding proteins, FABP [41]. Whether ARC channels also possess such binding sites that will bear homology with FABP remains to be shown. Nonetheless, our study is consistent with our recent report in which docosahexaenoic acid, a polyunsaturated fatty acid, affects the calcium channels in an albumin reversible manner [42]. The present study demonstrates that AA facilitates TG- induced capacitative calcium entry. The sequence of events will be as follows: thapsigargin fi PLA 2 activation fi AA release fi SOC capacitative influx. Hence, arachi- donic acid will, probably, act via opening of ARC channels. Though AA does act on the capacitative calcium entry, its role in the modulation of the other T-cell signalling mechanisms cannot be ruled out as the PLA 2 inhibitors almost completely inhibit the release of AA. This hypothesis can be supported by our recent observations in which we have shown that free AA potentiates okadaic acid-stimu- lated activation of mitogen-activated protein kinases in Jurkat T-cells [43]. Our study is certainly of physiological relevance as under some pathophysiological conditions like cardiac ischemia, the concentrations of AA are increased up to 50 l M [44]. Several studies have demonstrated that PLA 2 , during T-cell activation, can catalyze the liberation of free arachidonic acid [34,45], and hence, free AA can modulate the proliferation and clonal selection of T-cells during an antigenic challenge. In fact, polyunsaturated fatty acids have been considered as immunomodulators and one can envisage that free AA in vivo can modulate T-cell activation in health and disease. ACKNOWLEDGEMENTS Authors are thankful to the Region Bourgogne (France) for the sanction of a contingent grant. REFERENCES 1. Donnadieu, E., Bismuth, G. & Trautmann, A. (1992) Calcium fluxes in T-lymphocytes. J. Biol. Chem. 267, 25864–25872. 2. Lewis, R.S. & Cahalan, M.D. (1989) Mitogen-induced oscillations of cytosolic Ca 2+ and transmembrane Ca 2+ current in human leukemic T-cells. Cell Regul. 1, 99–112. 3. Lewis, R.S. & Cahalan, M.D. (1995) Potassium and calcium channels in lymphocytes. Annu. Rev. Immunol. 13, 623–653. 4. Negulescu, P.A., Shastri, N. & Cahalan, M.D. (1994) Intracellular calcium dependence of gene expression in single T-lymphocytes. Proc. Natl Acad. Sci. USA 91, 2873–2877. 5. Dolmetsch, R.E., Xu, K. & Lewis, R.S. (1998) Calcium oscilla- tions increase the efficiency and specificity of gene expression. Nature 392, 933–936. 6. Li, W., Llopis, J., Whitney, M., Zlokarnik, G. & Tsein, R.Y. (1998) Cell-permeant caged InsP3 ester shows that Ca 2+ spike frequency can optimize gene expression. Nature 392, 936–941. 7. Putney, J.W. Jr (1997) Type 3 inositol 1,4,5-trisphosphate receptor and capacitative calcium entry. Cell Calcium 21, 257–261. 8. Zweifach, A. & Lewis, R.S. (1993) Mitogen regulated Ca 2+ cur- rent of T-lymphocytes is activated by depletion of intracellular Ca 2+ stores. Proc.NatlAcad.Sci.USA90, 6295–6299. 9. Su, Z., Csutora, P., Huntaon, D., Shoemaker, R.L., Marchase, R.B. & Blalock, J.E. (2001) A store-operated nonselective cation channel in lymphocytes is activated by Ca 2+ influx factor and diacylglycerol. Am.J.Physiol.CellPhysiol.280, C1284–C1292. 10. Ramdriamampita, C. & Tsien, R.Y. (1993) Emptying of intracellular Ca 2+ stores releases a novel small messenger that stimulates Ca 2+ influx. Nature 364, 809–814. 11. Guse, A.H., Da Silva, C.P., Berg, I., Skapenko, A.L., Weber, K., Heyer, P., Hohenegger, M., Ashamu, G.A., Schulze-Koops, H., Potter, B.V.L. & Mayer, G.W. (1999) Regulation of calcium sig- nalling in T lymphocytes by the second messenger cyclic ADP- ribose. Nature 398, 70–73. 12. Calder, P.C. (1999) Dietary fatty acids and immune system. Lipids 34, S137–S140. 13. Denys, A., Hichami, A. & Khan, N.A. (2001) Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase (ERK1/ ERK2) signalling in human T-cells. J. Lipid Res. 42, 2015–2020. 5562 A. Hichami et al.(Eur. J. Biochem. 269) Ó FEBS 2002 14. Khan, N.A. & Hichami, A. (2002) Role of n-3 polyunsaturated fatty acids in the modulation of T-cell signalling. In: Recent Advances in Research in Lipids (ed. G. Pandali). Transworld Publications, India. in press 15. Mcmurray, D.N., Jolly, C.A. & Chapkin, R.S. (2000) Effects of dietary n-3 fatty acids on T cell activation and T cell receptor- mediated signaling in a murine model. J. Infect. Dis. 182 (Suppl.), S103–S107. 16. Triboulot, C., Hichami, A., Denys, A. & Khan, N.A. (2001) Dietary (n-3) polyunsaturated fatty acids exert antihypertensive effects by modulating calcium signalling in T-cells of rats. J. Nutr. 131, 2364–2369. 17. Chow, S.C. & Jondal, M. (1990) Polyinsatured free fatty acids stimulate an increase in cytosolic Ca 2+ by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca 2+ pool in T-cells through a mechanism independent of phosphoinositide turnover. J. Biol. Chem. 265, 902–907. 18. Chow, S.C., Sisfontes, L., Jondal, M. & Bjo ¨ rkhem, I. (1991) Modification of membrane phospholipid fatty acyl composition in a leukemic T-cell line: effects on receptor mediated intracellular Ca 2+ release. Biochim. Biophys. Acta 1092, 358–366. 19. Gamberuchi, A., Fulceri, R. & Benedetti, A. (1997) Inhibition of store-dependent capacitative Ca 2+ influx by unsaturated fatty acids. Cell Calcium 21, 375–385. 20. Breittmayer, J.P., Pelassy, C., Cousin, J.L., Bernard, A. & Aussel, C. (1993) The inhibition by fatty acids of receptor mediated cal- cium movements in Jurkat T-cells is due to increased calcium extrusion. J. Biol. Chem. 268, 20812–20817. 21. Khurodova, A.B. & Astashkin, E.I. (1994) A dual effect of ara- chidonic acid on Ca 2+ transport system in lymphocytes. FEBS Lett. 353, 167–170. 22. Goldyne, M.E., Burrish, G.E., Paubelle, P. & Borgeat, P. (1984) Arachidonic acid metabolism among human mononuclear leu- kocytes. Lipoxygenase-related pathways. J. Biol. Chem. 259, 8815–8819. 23. Kurland, J.L. & Bockman, R. (1978) Prostaglandin E production by human blood monocytes and mouse peritoneal macrophages. J. Exp. Med. 147, 952–957. 24. Grynkiewicz, G.M., Ponie, M. & Tsein, R.Y. (1985) A new gen- eration of Ca 2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450. 25. Hichami, A., Boichot, E., Germain, N., Legrand, A., Moodley, I. & Lagante, V. (1995) Involvement of cyclic AMP in the effects of phosphodiesterase IV inhibitors on arachidonate release from mononuclear cells. Eur. J. Pharmacol. 291, 91–97. 26. Tessier, C., Hichami, A. & Khan, N.A. (2002) Implication of three isoforms of PLA 2 in human T-cell proliferation. FEBS Lett. 520, 111–116. 27. Thastrup, O., Cullen, P.J., Drobak, B.K., Hanley, M.R. & Dawson, A.P. (1990) Thapsigargin, a tumor promoter, discharges intracellular Ca 2+ stores by specific inhibition of the endoplasmic reticulum Ca 2+ -ATPase. Proc. Natl Acad. Sci. USA 87, 2466– 2470. 28. Pollock, W.K., Sage, S.O. & Rink, T.J. (1987) Stimulation of Ca 2+ efflux from fura-2 loaded platelets activated by thrombin or phorbol myristate acetate. FEBS Lett. 210, 132–136. 29. Hoffmann, P., Richards, D., Hoffmann-Heinroth, I., Mathias, P., Wey, H. & Toraason, M. (1995) Arachidonic disrupts calcium dynamics in neonatal rat cardiac myocytes. Cardiovas. Res. 30, 889–898. 30. Roudbaraki, M., Drouhault, R., Bacquart, T. & Vacher, P. (1992) Arachidonic acid-induced hormone released in somatotropes: involvement of calcium. Neuroendocrinology 63, 244–256. 31. Soliven, B., Takeda, M., Shandy, T. & Nelson, T.J. (1993) Arachidonic acid and its metabolites increase Ca 2+ iinculturedrat oligodendrocytes. Am.J.Physiol.CellPhysiol.264, C632–C640. 32. Ohuchi, K., Sugawara, T., Watanabe, M., Hirasawa, N., Tsurufuji, S., Fujiki, H., Sugimura, T. & Christensen, S.B. (1987) Stimulation of arachidonic acid metabolism in rat perito- neal macrophages by thapsigargin, a non-(12-O-tetra- decanoylphorbol-13-acetate) (TPA)-type tumor promotor. J. Cancer Res. Clin. Oncol. 113, 319–324. 33. Tornquist, K., Ekokoski, E. & Forss, L. (1994) Thapsigargin- induced calcium entry in FRTL-5 cell: possible dependence on phospholipase A 2 activation. J. Cell Physiol. 160, 40–46. 34. Boilard, E. & Surette, M.E. (2001) Anti-CD3 and concanavalin A- induced human T cell proliferation is associated with an increased rate of arachidonate-phospholipid remodeling. Lack of involve- ment of group IV and group VI phospholipase A 2 in remodeling and increased susceptibility of proliferating T cells to CoA- independent transacyclase inhibitor-induced apoptosis. J. Biol. Chem. 276, 18321–18326. 35. Tsumoda, Y. & Owayng, C. (1993) Differential involvement of phospholipase A 2 /arachidonic acid and phospholipase C/phos- phoinositol pathways during cholecystokinin receptor activated Ca 2+ oscillations in pancreatic acini. Biochem. Biophys. Res. Commun. 194, 1194–1202. 36. Shuttleworth, T.J. (1996) Arachidonic acid activates the noncap- acitative entry of Ca 2+ during [Ca 2+ ]i oscillations. J. Biol. Chem. 271, 21720–21725. 37. Roshak, A.K., Capper, E.A., Stevenson, C., Eichman, C. & Marshall, L.A. (2000) Human calcium-independent phospho- lipase A 2 mediates lymphocyte proliferation. J. Biol. Chem. 275, 35692–35698. 38. Marhaba, R., Mary, F., Pelassy, C., Stanescu, A.T., Aussel, C. & Breittmayer, J.P. (1996) Tyrphostin A9 inhibits calcium release- dependent phosphorylations and calcium entry via calcium release- activated channel in Jurkat T-cells. J. Immunol. 157, 1468–1473. 39. Mignen, O. & Suttleworth, T.J. (2000) I ARC , a novel arachidonic- regulated, noncapacitative Ca 2+ entry channel. J. Biol. Chem. 275, 9114–9119. 40. Mignen,O.,Thomson,J.L.&Suttleworth,T.J.(2001)Reciprocal regulation of capacitative and arachidonate regulated non-capa- citative Ca 2+ entry channel. J. Biol. Chem. 276, 35676–35683. 41. Petrou, S., Ordwa, R.W., Singer, J.J. & Walsh, J.V. Jr (1993) A putative fatty acid binding domain of the NMDA receptor. Trends Biol. Sci. 18, 41–42. 42. Bonin, A. & Khan, N.A. (2000) Regulation of calcium signalling by docosahexaenoic acid in human T cells: implication of CRAC channels. J. Lipid Res. 41, 277–284. 43. Denys, A., Hichami, A. & Khan, N.A. (2002) Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase enzyme activity in human T-cells. Mol. Cell. Biochem. 232, 143–148. 44. Nakamura, K., Ichihara, K. & Abiko, Y. (1989) Effect of pro- pranolol on accumulation of NEFA in the ischemic perfused rat heart. Eur. J. Pharmacol. 160, 61–69. 45. Le Gouvello, S.L., Colrad, O., Theodorou, I., Bismuth, G., Tar- antino, N. & Debre, P. (1990) CD2 triggering stimulates a phos- pholipase A 2 activity beside the phospholipase C pathway in human T lymphocytes. J. Immunol. 144, 2359–2364. Ó FEBS 2002 Capacitative calcium influx in human T-cells (Eur. J. Biochem. 269) 5563 . Role of three isoforms of phospholipase A 2 in capacitative calcium in ux in human T-cells Aziz Hichami 1 , Beenu Joshi 2 , Anne Marie Simonin 1 and. phospholipase A 2 in capacitative calcium entry. We have employed thapsigar- gin (TG) that induces increases in [Ca 2+ ] i by emptying the calcium pool of endoplasmic