Phospholipase A
2
Is Involved in Thapsigargin-Induced
Sodium Influx in Human Lymphocytes
Jerzy-Roch Nofer,*
,1
Ralf Junker,* Michael Walter,* Udo Seedorf,† Gerd Assmann,*
,
†
Walter Zidek,‡ and Martin Tepel‡
*Institut fu ¨ r Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfa ¨ lische Wilhelms-Universita ¨t,
A. Schweitzer Str 33, 48-149 Mu ¨ nster, Germany; †Institut fu ¨ r Arterioskleroseforschung an der Universita ¨t Mu ¨ nster,
Domagk Str 3, 48-149 Mu ¨ nster, Germany; and ‡Universita ¨ tsklinik Marienhospital, Ruhr-Universita ¨ t Bochum,
Ho ¨lkeskampring 40 44-625 Herne, Germany
Received June 25, 1999, and in revised form November 2, 1999
Previously, we reported that emptying of intracellu-
lar Ca
2
pools with endoplasmatic Ca
2
-ATP-ase inhib-
itor thapsigargin leads to the Na
influx in human
lymphocytes (M. Tepel et al., 1994, J. Biol. Chem. 269,
26239 –26242). In the present study we examined the
mechanism underlying the thapsigargin-induced Na
entry. We found that the thapsigargin-induced in-
crease in Na
concentration was effectively inhibited
by three structurally unrelated phospholipase A
2
(PLA
2
) inhibitors, p-bromophenacyl bromide, 3-(4-oc-
tadecyl)-benzoylacrylic acid (OBAA), and bromoenol
lactone (BEL). The thapsigargin-induced Na
influx
could be mimicked by PLA
2
exogenously added to the
lymphocyte suspension. In addition, thapsigargin
stimulated formation of arachidonic acid (AA), the
physiological PLA
2
product. AA induced Na
entry in a
time- and concentration-dependent fashion. Both,
thapsigargin-induced Na
influx and AA liberation
were completely inhibited in the presence of tyrosine
kinase inhibitor genistein but not in the absence of
extracellular Ca
2
. Collectively, these data show that
thapsigargin-induced Na
entry is associated with ty-
rosine kinase-dependent stimulation of PLA
2
. © 2000
Academic Press
Key Words: sodium influx; thapsigargin; phospho-
lipase A
2
; arachidonic acid.
Thapsigargin, the sesquiterpen lacton tumor pro-
moter from Thapsia garganica, inhibits the dense tu-
bular Ca
2+
-ATP-ase and elevates cytoplasmic Ca
2+
con-
centration by causing Ca
2+
to be released from intra-
cellular stores (1–3). The interest in the mechanism of
thapsigargin action has grown after it has been shown
that, in addition to the mobilization of intracellularly
stored Ca
2+
, thapsigargin promotes the influx of extra-
cellular Ca
2+
(4 – 8). Despite extensive investigations
the mechanism of thapsigargin-stimulated Ca
2+
influx
remains obscure. In the hypothesis referred to as the
capacitative model, Ca
2+
influx is controlled by the
filling state of intracellular Ca
2+
pools (9, 10). Deple-
tion of these pools with thapsigargin may trigger Ca
2+
influx by means of diffusible messengers such as cGMP
(11–15), unidentified epoxygenase product (16), or a
putative calcium influx factor (CIF)
2
(17–19).
In addition to promoting Ca
2+
influx, depletion of
intracellular Ca
2+
pools with thapsigargin triggers ex-
tracellular Na
+
entry (20 –22). Whereas thapsigargin-
induced Ca
2+
influx was extensively studied in recent
years, little effort has been devoted toward under-
standing the mechanism underlying thapsigargin-in-
duced Na
+
entry. The present study reveals that acti-
vation of phospholipase A
2
(PLA
2
) promotes Na
+
entry
in thapsigargin-treated lymphocytes.
MATERIALS AND METHODS
Materials. P-Bromophenacyl bromide (BPB), arachidonic acid
(AA), genistein, TMB8, FURA-2-AM, and benzofuran isophtalate
1
To whom correspondence should be addressed. Fax: ++49-251-
834-7227.
2
Abbreviations used: CIF, calcium influx factor; PLA
2
, phospho-
lipase A
2
; BPB, p-bromophenacyl bromide; AA, arachidonic acid; PI,
propium iodide; BEL, bromoenal lactone; SPQ, 3-(6-methoxyquino-
lino)-propanesulfonate; OBAA, 3-(4-octadecyl)-benzoylacrylic acid;
HBSS, Hank’s balanced salt solution; SBFI-AM, benzofuran isoph-
talate acetoxymethyl ester; NPPB, 5-nitro-2-(3-phenylpropylamino)-
benzoate; I
CRAC
, transplasma membrane current; I
CRANC
, nonselective
cation current.
0003-9861/00 $35.00 213
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
Archives of Biochemistry and Biophysics
Vol. 374, No. 2, Febuary 15, pp. 213–221, 2000
doi:10.1006/abbi.1999.1588, available online at http://www.idealibrary.com on