(Received for publication, October 22, 1996, and in revised form, October 30, 1996)
From the Herein we present multiple lines of evidence
which demonstrate that depletion of internal calcium stores is both
necessary and sufficient for the activation of calcium-independent
phospholipase A2 during arginine vasopressin (AVP)-mediated
mobilization of arachidonic acid in A-10 smooth muscle cells. First,
AVP-induced [3H]arachidonic acid release was independent
of increases in cytosolic calcium yet was decreased by pharmacological
inhibition of the release of calcium ion from internal stores. Second,
thapsigargin induced the dramatic release of
[3H]arachidonic acid from A-10 cells at a similar rate as
the AVP-induced release of arachidonic acid, and the release of
arachidonic acid by either AVP or thapsigargin was entirely inhibited
by
(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Third, the magnitude of thapsigargin-induced
[3H]arachidonic acid release was entirely independent of
alterations in cytosolic calcium concentration. Fourth, A23187 resulted
in the BEL-inhibitable release of [3H]arachidonic acid
from A-10 cells even when ionophore-induced increases in cytosolic
calcium were completely prevented by calcium chelators. Fifth,
pretreatment of A-10 cells with a calmodulin antagonist
(N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, HCl)
resulted in the time-dependent decrease of subsequent
thapsigargin-induced [3H]arachidonic acid release.
Collectively, these results identify a novel paradigm which links
alterations in calcium homeostasis to the calmodulin-mediated
regulation of calcium-independent phospholipase A2 through
the depletion of internal calcium stores.
Alterations in cellular calcium homeostasis are a widely employed
and potent regulatory mechanism that modulates many functionally distinct physiologic processes (1-6). Historically, attention has
focused on alterations in cytosolic free calcium as the primary mechanism through which calcium mediates its downstream effects on
cellular biochemical events (7-9). However, recent studies have
identified intracellular calcium store depletion as an important mechanism mediating calcium signaling in activated cells (10-12). For
example, intracellular calcium store depletion regulates
transplasmalemmal calcium flux (13, 14), hormone secretion (15, 16),
and cellular proliferation (17-19). Although the importance of
intracellular calcium store depletion is now well established, the
precise biochemical mechanisms which link the depletion of
intracellular calcium stores to cellular signaling processes remains
enigmatic.
Eicosanoids are critical lipid second messengers in stimulated cells
whose production has traditionally been envisaged to result from
increases in cytosolic calcium leading to the activation of one or more
calcium-dependent phospholipases A2
(e.g. cPLA2, sPLA2)1 (20, 21). Recently, we
have demonstrated that the predominant phospholipase A2
activity in A-10 muscle cells is not activated by calcium ion
(i.e. it is a member of the iPLA2 family) (22, 23) and that this iPLA2 is physically associated with, and
functionally coupled to, the intracellular calcium signal transducer,
calmodulin (24). In this paradigm, iPLA2 exists in a
ternary complex with calcium and calmodulin in a catalytically inactive
state. Pharmacologic removal of calmodulin from the phospholipase
A2 ternary complex by W-7 in intact A-10 smooth muscle
cells results in the activation of iPLA2 and the release of
arachidonic acid (24). Accordingly, one mechanism potentially
responsible for the release of arachidonic acid in activated cells is
the depletion of calcium ion from specific subcellular loci and the
resultant release of calmodulin-mediated inhibition of
iPLA2.
A-10 smooth muscle cells contain arginine vasopressin (AVP) receptors
whose occupancy results in the activation of PLC (25), the generation
of IP3 (25, 26), and the subsequent release of calcium ion
from internal stores (25-27). In previous studies, we utilized
specific mechanism-based inhibition to demonstrate that AVP-induced
release of arachidonic acid in A-10 smooth muscle cells occurs through
the activation of iPLA2 (22). We now report that
AVP-induced release of arachidonic acid catalyzed by iPLA2 is mediated by the depletion of internal calcium stores and that depletion of internal calcium stores, even in the absence of receptor occupancy or elevations of cytosolic free calcium, is sufficient for
the activation of iPLA2.
A-10 cells derived from rat aortic smooth muscle
(ATTC no. CRL 1476) were obtained from ATTC.
(E)-6-(Bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL), thapsigargin, cyclopiazonic acid,
2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), and
N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7)
were obtained from Calbiochem.
Rat smooth muscle A-10 cells were
cultured in DMEM containing 20% fetal bovine serum at 37 °C as
described previously (22). Briefly, A-10 cells (passages 5-10) were
grown to confluence in DMEM containing 20% fetal bovine serum and
incubated with 0.5 µCi of [3H]arachidonic acid for
16 h at 37 °C. After radiolabeling, the cells were repeatedly
washed (× 2) in fresh DMEM containing 0.25% bovine serum albumin to
remove unincorporated radiolabeled arachidonic acid. For studies
employing BEL, cells were incubated with medium containing serum which
had been previously heat-inactivated (56 °C for 1 h) to prevent
enzymatic degradation of BEL by serum proteases during the
preincubation period. Cells were incubated in this solution containing,
in addition, either BEL (10 µM) or ethanol vehicle alone
(0.1% final volume) for 15 min at 37 °C. The medium was removed,
and the cells were incubated with fresh DMEM containing 0.25% bovine
serum albumin and the indicated agonists or antagonists at 37 °C for
the indicated times (typically 5 min). Fatty acids and phospholipids
were extracted by a modified Bligh-Dyer procedure employing
chloroform:methanol:distilled H2O (1:1:0.8; v/v) containing 2% acetic acid (final volume). The radiolabeled fatty acids were separated by TLC utilizing Silica Gel 60A plates with a mobile phase
comprised of chloroform:methanol:acetic acid:distilled H2O (90:8:1:0.8, v/v), visualized by iodine staining of a fatty acid standard, scraped, and quantitated by scintillation spectrometry. Radiolabeled phospholipids and fatty acid from the cells were extracted
by the Bligh-Dyer method, resolved by TLC employing a mobile phase of
chloroform:methanol:ammonium hydroxide (65:25:5; v/v), and quantitated
by scintillation spectrometry.
Measurements of cytosolic calcium concentrations
were conducted in Fura-2-loaded cells utilizing dual wavelength
fluorescence recording at excitation wavelengths of 340 nm and 380 nm
and an emission wavelength of 505 nm as described previously (22). The
small deflections in the fluorescence ratio which precede the
arrow in Figs. 1, 2, 3 reflect fluorescence changes due to mechanical agitation during addition of agonists.
As previously demonstrated, treatment of prelabeled A-10 smooth
muscle cells with AVP (1 µM) resulted in the robust
release of [3H]arachidonic acid which was ablated by
pretreatment with 10 µM BEL (BEL possesses a 1,000-fold
selectivity for the inhibition of iPLA2 versus
cPLA2 or sPLA2 (28) and does not attenuate
AVP-induced activation of PLC in A-10 cells) (22) (Fig.
1a). Since A-10 smooth muscle cells contain
receptors which are coupled to PLC and result in the
IP3-mediated mobilization of intracellular calcium stores
(26, 27), the effects of pharmacologic inhibition of intracellular
calcium pool depletion on AVP-mediated activation of smooth muscle cell
iPLA2 were investigated. Neomycin-mediated inhibition of
PLC (29) decreased AVP-induced [3H]arachidonic acid
release by 70% (Fig. 1a). Moreover, replacement of calcium
in the incubation media with EGTA (1 mM) and BAPTA-AM (100 µM) did not attenuate AVP-induced
[3H]arachidonic acid release, although alterations in
[Ca2+]i were completely prevented (Figs. 1,
b and c).
To determine whether intracellular calcium store depletion in the
absence of receptor occupancy and IP3 production is a
sufficient stimulus for iPLA2 activation, two independent
approaches were taken. In the first approach, three structurally
disparate inhibitors of sarco/endoplasmic reticular calciumATPases
(SERCA) were employed to deplete intracellular calcium pools (30, 31).
Thapsigargin-induced the robust release of
[3H]arachidonic acid from A-10 cells which was inhibited
by BEL. The magnitude of both the release of
[3H]arachidonic acid by thapsigargin and its inhibition
by BEL were identical when cells were stimulated in media containing
2.5 mM calcium, EGTA alone, or EGTA and BAPTA-AM (Fig.
2a). Treatment of smooth muscle cells with
thapsigargin in the presence of medium containing EGTA resulted in a
small increase in cytosolic calcium ion concentration which could be
completely chelated by BAPTA-AM (Fig. 2b). Although BAPTA-AM
completely prevented the modest thapsigargin-induced increments in
cytosolic calcium concentration, no alterations in thapsigargin-induced
release of [3H]arachidonic acid were manifest (Fig.
2a). Thus, thapsigargin-induced release of
[3H]arachidonic acid is independent of alterations of
cytosolic calcium ion concentration and is directly correlated with
thapsigargin-induced depletion of intracellular calcium pools.
Furthermore, the time course of thapsigargin-induced release of
[3H]arachidonic acid was similar to that manifest by
treatment of A-10 smooth muscle cells with AVP (Fig. 2c).
Treatment of A-10 smooth muscle cells with two other structurally
distinct SERCA inhibitors, cyclopiazonic acid (10 µM)
(32) and BHQ (50 µM) (33), also resulted in similar
increases in [3H]arachidonic acid release. These
increases were entirely inhibited by BEL and the magnitude of the
cyclopiazonic acid and BHQ-mediated [3H]arachidonic acid
release was independent of alterations in
[Ca2+]i which were ablated by BAPTA-AM
treatment utilizing medium containing EGTA (data not shown).
Collectively, these findings demonstrate that the depletion of
intracellular calcium stores in A-10 smooth muscle cells by SERCA
inhibitors results in the activation of smooth muscle cell
iPLA2 independent of alterations in cytosolic calcium ion
concentration.
In the second approach, the effects of intracellular calcium pool
depletion on the activation of smooth muscle cell iPLA2 were investigated employing the calcium ionophore, A23187 (34). This
compound mediates the passive transport of calcium ion across both
plasma and intracellular membranes down existing [Ca2+]
gradients, and thus can induce the depletion of calcium ion from
intracellular sequestration sites. Treatment of A-10 smooth muscle
cells with A23187(10 µM)for 5 min in the presence of 2.5 mM extracellular calcium resulted in the robust release of
[3H]arachidonic acid which was entirely inhibited by BEL
(Fig. 3a). Treatment of A-10 smooth muscle
cells with A23187 (10 µM) in the presence of
extracellular EGTA (5 mM) resulted in similar increases in[3H]arachidonic acid release (which was
BEL-inhibitable) as those manifest in the presence of extracellular
calcium ion (Fig. 3a). Moreover, A23187-induced release of
[3H]arachidonic acid in the presence of external EGTA was
not altered by BAPTA-AM, although BAPTA-AM completely prevented
A23187-induced increases in cytosolic calcium ion (Fig.
3b).Collectively, these findings demonstrate that: 1) A23187
stimulation of A-10 smooth muscle cells mediates the release of
[3H]arachidonic acid through iPLA2; 2) the
A23187-induced release of [3H]arachidonic acid does not
depend on either the entry of extracellular calcium ion or
ionophore-induced increases in cytosolic calcium ion concentration; and
3) iPLA2 is exquisitely sensitive to alterations in
intracellular calcium ion compartmentation.
Recently, calmodulin has been demonstrated to be physically associated
with, and functionally coupled to, iPLA2 (24). To determine
if the physical association of calmodulin with iPLA2 is
causally related to the activation of iPLA2 by
intracellular calcium pool depletion, we exploited the principle of
pharmacologically directed radiolabeled phospholipid pool depletion.If
Ca2+-store depletion activated latent iPLA2
activity through dissociation of the calmodulin iPLA2
complex, then prior exposure of cells to W-7 would deplete the
[3H]arachidonic acid content of phospholipid pools which
serve as substrates for iPLA2 and would result in decreased
[3H]arachidonic acid release after subsequent exposure to
thapsigargin. The results demonstrated that as the duration of
pretreatment with W-7 increased, A-10 cells released progressively
smaller amounts of [3H]arachidonic acid in response to
subsequent thapsigargin treatment (Fig. 4). This
suggests that activation of iPLA2, induced either by
Ca2+-store depletion or by a calmodulin antagonist, results
from the hydrolysis of a common pool of phospholipids.
Collectively, the results of the present study demonstrate that what
has been nominally termed a "calcium-independent" phospholipase A2 based on in vitro activity assays is actually
profoundly modulated by alterations in calcium homeostasis in its
natural context in vivo. Moreover, these studies provide a
rationale and experimental proof which integrates the receptor-mediated
activation of phospholipase C with the subsequent phospholipase
A2-mediated release of arachidonic acid through the
IP3-mediated depletion of internal calcium stores. A
substantial body of literature has utilized the A23187-induced release
of arachidonic acid as a bona fide indicator of the participation of
calcium-dependent phospholipases A2 in
arachidonic acid release (35-37). These results demonstrate that the
interpretation of those experiments is more complex than previously
anticipated.
The importance of alterations in calcium homeostasis and
iPLA2 activation in the receptor-mediated release of
arachidonic acid in many tissues is now well established (38-41).
However, the mechanism through which calcium can modulate
calcium-independent phospholipase A2 during signal
transduction has previously represented a fundamental paradox. The
present results provide a novel paradigm which links alterations in
calcium homeostasis to the activation of iPLA2 activity
through the calcium·calmodulin-mediated regulation of
iPLA2 and the depletion of internal calcium stores. Thus,
iPLA2 can function as a sensor of the filling state of
internal calcium stores, and this sensor function is mediated by the
calcium-dependent association of iPLA2 with
calmodulin.
Finally, we point out that depletion of internal calcium pools has been
causally linked to the activation of a plasma membrane calcium channel
through an as yet unidentified second messenger (42-45). The
demonstration of the coupling of iPLA2 activation with
calcium pool depletion suggests that this second messenger is likely
either a direct product of the iPLA2 reaction
(e.g. arachidonic acid or lysolipids) or a downstream
product whose synthesis is initiated by iPLA2 activation.
It is our hope that the identification of the coupling of calcium pool
depletion with the activation of iPLA2 will serve as a
biochemical foundation to identify the second messenger(s), which
facilitates the activation of store-operated plasma membrane calcium
channels.
Division of Bioorganic Chemistry and
Molecular Pharmacology,
Materials
Fig. 1.
Arginine-vasopressin-mediated release of
[3H]arachidonic acid from A-10 smooth muscle cells is
attenuated by neomycin and is independent of alterations in
intracellular calcium ion. a, A-10 muscle cells were
prelabeled with 0.5 µCi of [3H]arachidonic acid for
16 h as described under "Experimental Procedures." Next, cells
were pretreated with either BEL (10 µM) or vehicle alone
for 15 min prior to exposure to AVP (1 µM) for 5 min in the absence or presence of neomycin (500 µM). The results
are expressed as the percentage of [3H]arachidonic acid
released from radiolabeled cellular phospholipids. b, A-10
muscle cells were prelabeled with 0.5 µCi of
[3H]arachidonic acid for 16 h as described above.
Next, the cells were pretreated with BEL (10 µM) or
vehicle alone for 15 min and treated with BAPTA-AM (100 µM) for 30 min as indicated prior to exposure to AVP (1 µM) in medium containing calcium (2.5 mM) or EGTA (1 mM). The results are expressed as the percentage of
[3H]arachidonic acid released from radiolabeled cellular
phospholipids. c, A-10 smooth muscle cells were loaded with
Fura 2-AM (5 µM) for 30 min and treated with BAPTA-AM
(100 µM) for 30 min as indicated. The cells were placed
in KRB buffer containing either calcium (2.5 mM) or EGTA (1 mM) and monitored by dual-wavelength fluorescence as
described under "Experimental Procedures" during exposure to AVP (1 µM) (indicated by the arrow). The results are
expressed as the 340/380 nm ratio, which is an index of intracellular
calcium ion concentration and are representative of multiple
independent single cell recordings.
[View Larger Version of this Image (15K GIF file)]
Fig. 2.
Thapsigargin-induced intracellular calcium
store depletion activates the iPLA2-mediated release of
[3H]arachidonic acid from A-10 smooth muscle cells.
a, A-10 smooth muscle cells were prelabeled with 0.5 µCi of
[3H]arachidonic acid for 16 h as described under
"Experimental Procedures." Next, the cells were pretreated with BEL
(10 µM) or vehicle alone for 15 min and BAPTA-AM (100 µM) for 30 min as indicated prior to exposure to
thapsigargin (5 µM) in medium containing either calcium
(2.5 mM) or EGTA (1 mM). The results are
expressed as the percentage of [3H]arachidonic acid
released from radiolabeled cellular phospholipids. b, A-10
smooth muscle cells were loaded with Fura 2-AM (5 µM) for
30 min and treated with BAPTA-AM (100 µM) for 30 min as
indicated. The cells were placed in KRB buffer containing either
calcium (2.5 mM) or EGTA (1 mM) and monitored
by dual-wavelength fluorescence as described under "Experimental
Procedures" during exposure to thapsigargin (5 µM) (as
indicated by the arrow). The results are expressed as the
340/380 nm ratio, which is an index of intracellular calcium ion
concentration and are representative of multiple independent single
cell recordings. c, A-10 smooth muscle cells were
preincubated with 0.5 µCi of [3H]arachidonic acid for
16 h as described under "Experimental Procedures." The cells
were exposed to AVP (1 µM) (), thapsigargin (5 µM) (
), or vehicle alone (
) for 0, 5, 10, and 15 min. The medium was removed, and the [3H]arachidonic acid
was extracted and quantitated as described under "Experimental
Procedures." The results are expressed as the percentage of
[3H]arachidonic acid released from radiolabeled
intracellular phospholipids.
[View Larger Version of this Image (15K GIF file)]
Fig. 3.
A23187-induced depletion of intracellular
calcium stores activates iPLA2-mediated release of
arachidonic acid. a, A-10 smooth muscle cells were
prelabeled with 0.5 µCi of [3H]arachidonic acid for
16 h as described under "Experimental Procedures." Cells were
preincubated with either BEL (10 µM) or vehicle alone for
15 min and BAPTA-AM (100 µM) for 30 min as indicated
prior to exposure to A23187 (10 µM) for 5 min in medium
containing either calcium (2.5 mM) or EGTA (1 mM). The results are expressed as the percentage of
[3H]arachidonic acid released from radiolabeled
intracellular phospholipids. b, A-10 smooth muscle cells
were loaded with Fura 2-AM (5 µM) for 30 min and treated
with BAPTA-AM (100 µM) for 30 min as indicated. The cells
were placed in KRB buffer containing either calcium (2.5 mM) or EGTA (1 mM) and monitored by
dual-wavelength fluorescence as described under "Experimental
Procedures" during exposure to A23187 (10 µM) (as
indicated by the arrow).The results are expressed as the
340/380 nm ratio, which is an index of intracellular calcium ion
concentration and are representative of multiple independent single
cell recordings.
[View Larger Version of this Image (14K GIF file)]
Fig. 4.
The calmodulin antagonist W-7 depletes
[3H]arachidonic acid from the phospholipid pools which
are the coupled to the activation of iPLA2 by depletion of
intracellular calcium stores. A-10 smooth muscle cells were
prelabeled with 0.5 µCi of [3H]arachidonic acid for
16 h as described under "Experimental Procedures." Next, the
cells were incubated with W-7 (50 µM) or vehicle alone for 0, 5, 15, or 30 min. The medium was then removed, and the [3H]arachidonic acid content in the medium was
quantified. W-7-induced [3H]arachidonic acid release was
calculated as the [3H]arachidonic acid content of medium
that had bathed W-7-exposed cells minus that of medium that had bathed
vehicle-exposed cells. This value was expressed as a percentage of
radiolabeled phospholipids (). Next, the cells that had been exposed
to W-7 for selected intervals were then placed in fresh medium and
stimulated with 5 µM thapsigargin for 5 min. The content
of [3H]arachidonic acid in the medium was determined and
expressed as a percentage of radiolabeled phospholipids (
). This
value is plotted as a function of the W-7 pretreatment interval that had preceded stimulation with thapsigargin.
[View Larger Version of this Image (17K GIF file)]
*
This research was supported by National Institutes of Health
Grant PO1 HL57278-01. The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Division of
Bioorganic Chemistry and Molecular Pharmacology, Washington University School of Medicine, 660 South Euclid, Box 8020, St. Louis, MO 63110. Tel.: 314-362-2690; Fax: 314-362-1402.
1
The abbreviations used are: cPLA2,
cytosolic calcium-dependent phospholipase A2;
iPLA2, calcium-independent phospholipase A2;
sPLA2, secretory phospholipase A2; AVP,
arginine-vasopressin; BAPTA-AM,
1,2-bis-(o-aminophenoxy)ethane-N,N,N,N
-tetraacetic acid tetra(acetoxymethyl) ester; BEL,
(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one; IP3, inositol 1,4,5 trisphosphate; KRB, Krebs-Ringer
bicarbonate buffer; PLC, phospholipase C; SERCA, sarco/endoplasmic
reticular calcium ATPases; W-7,
N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, HCl;
BHQ, 2,5-di-(t-butyl)-1,4-hydroquinone; DMEM, Dulbecco's modified Eagle's medium.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.