Báo cáo Y học: Role of three isoforms of phospholipase A2 in capacitative calcium influx in human T-cells pot

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:
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.

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Ó FEBS 2002 Capacitative calcium influx in human T-cells (Eur. J. Biochem. 269) 5563