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Muscarinic Receptor Activation of Arachidonate-mediated Ca
21
Entry in HEK293 Cells Is Independent of Phospholipase C*
(Received for publication, July 22, 1998, and in revised form, September 9, 1998)
Trevor J. Shuttleworth‡ and Jill L. Thompson
From the Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry,
Rochester, New York 14642
Receptor-enhanced entry of Ca
21
in non-excitable
cells is generally ascribed to a capacitative mechanism
in which the activation of the entry pathway is specifi-
cally dependent on the emptying of agonist-sensitive
intracellular Ca
21
stores. Although such entry can be
clearly demonstrated under conditions of maximal or
near-maximal stimulation, it is uncertain whether such
a mechanism can operate during the oscillatory [Ca
21
]
i
signals that are frequently seen following stimulation
with low concentrations of agonists. In this study, we
report that the stimulation of human m3 muscarinic
receptors stably transfected into HEK293 cells results in
the appearance of a novel arachidonate-mediated Ca
21
entry pathway. We show that the generation of arachi-
donic acid and the activation of this pathway are specif-
ically associated with stimulation at the low agonist
concentrations that typically give rise to oscillatory
[Ca
21
]
i
signals. At such agonist concentrations, how-
ever, the generation of arachidonic acid is independent
of the simultaneous activation of the phospholipase C-
inositol 1,4,5-trisphosphate pathway. We further show
that the arachidonate-mediated Ca
21
entry demon-
strates characteristics that distinguish it from the cor-
responding capacitative pathway in the same cells and
therefore is likely to represent an entirely distinct path-
way that is specifically responsible for the receptor-
enhanced entry of Ca
21
during [Ca
21
]
i
oscillations.
Calcium signaling in non-excitable cells is composed of two
components: a release of calcium from intracellular stores and
an increased entry of calcium from the extracellular medium.
The role of inositol 1,4,5-trisphosphate (InsP
3
),
1
generated as a
result of the receptor activation of phospholipase C, in the
release of calcium from specific intracellular stores is well
established, but the nature of the calcium entry pathway and
its regulation is far from clear. To date, discussion of such
calcium entry has generally focused on the so-called “capacita-
tive model,” in which calcium entry is activated as a direct
consequence of the emptying of the intracellular calcium stores
and is independent of how this emptying is actually achieved
(1, 2). The precise nature of the mechanism for the activation of
capacitative entry is, as yet, unclear, but it may involve the
release and/or generation of a diffusible signaling molecule
within the cell that activates the plasma membrane channels
(“store-operated channels”) responsible for calcium entry. Al-
ternatively, a more direct molecular coupling between the
stores and the plasma membrane channels may occur (3). Al-
though such capacitative entry can be clearly demonstrated in
a wide variety of different cells, it is far from certain that such
a mechanism is the only one involved in the increase in calcium
entry in non-excitable cells following receptor activation (4, 5).
For example, many cells show an oscillatory [Ca
21
]
i
signal
when stimulated at low agonist concentrations (6, 7), and such
signals are associated with an enhanced entry of Ca
21
. How-
ever, evidence indicates that the activation of capacitative
Ca
21
entry generally requires significantly higher levels of
agonist-generated InsP
3
than does the release of Ca
21
from the
bulk of the agonist-sensitive internal stores (i.e. at low concen-
trations of InsP
3
, substantial release of Ca
21
from agonist-
sensitive stores can occur without any activation of capacita-
tive entry) (8–10). Consequently, it is far from clear that the
transitory (and/or incomplete) nature of calcium store depletion
during [Ca
21
]
i
oscillations would provide an adequate or ap-
propriate signal for the activation of calcium entry via a capac-
itative mechanism. Such considerations led us previously to
investigate the nature of receptor-activated increases in Ca
21
entry during [Ca
21
]
i
oscillations in cells from the exocrine
avian nasal gland. In these studies, we showed that such Ca
21
entry was non-capacitative in nature (11) and appeared to
involve a novel arachidonate-activated pathway (12).
In the experiments reported here, we extend our earlier
findings to another, more widely used and functionally less
highly specialized cell type, namely HEK293 cells. The specific
cell line chosen had been stably transfected with the human m3
muscarinic receptor (m3-mAChR), thereby avoiding possible
complications resulting from the presence of multiple musca-
rinic receptor subtypes. Using this cell line, we were able to
show that the receptor-mediated generation of arachidonic acid
is independent of the simultaneous activation of the PLC-InsP
3
pathway, indicating that the m3-mAChR is capable of coupling
both to the activation of PLC and the generation of arachidonic
acid in a separate but parallel manner. Furthermore, we show
that the arachidonate-mediated calcium entry pathway dem-
onstrates characteristics that distinguish it from the more well
known capacitative or store-operated entry of calcium.
MATERIALS AND METHODS
Cultures of the human embryonic kidney cell line HEK293 that were
stably transfected with the human m3 muscarinic receptor (m3-HEK
cells) were obtained from Dr. Craig Logsdon (University of Michigan,
Ann Arbor, MI) (see Ref. 13 for details). These cells were cultured under
standard conditions in Dulbecco’s modified Eagle’s medium supple-
mented with 10% calf serum and antibiotics. Changes in [Ca
21
]
i
in
* This work was supported by NIGMS Grant GM 40457 from the
National Institutes of Health. The costs of publication of this article
were defrayed in part by the payment of page charges. This 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: Dept. of Pharmacol-
ogy and Physiology, P. O. Box 711, University of Rochester Medical
Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-2076;
Fax: 716-244-9283; E-mail: tshut@pharmacol.rochester.edu.
1
The abbreviations used are: InsP
3
, inositol 1,4,5-trisphosphate;
[Ca
21
]
i
, intracellular free calcium ion concentration; mAChR, musca-
rinic receptor; PLC, phospholipase C; PLD, phospholipase D; PLA
2
,
phospholipase A
2
; PKC, protein kinase C; BAPTA, 1,2-bis(2-aminophe-
noxy)ethane-N,N,N9,N9-tetraacetic acid; ETYA, 5,8,11,14-eicosatet-
raynoic acid; PMA, phorbol 12-myristate 13-acetate.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 49, Issue of December 4, pp. 32636–32643, 1998
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
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single individual cells were determined following loading with the flu-
orescent probe indo-1. Loading was achieved by incubation in 4
m
M
indo-1/AM for 12 min, followed by washing (three times) in saline. Cells
were then incubated for a further 30 min at 37 °C to allow for complete
hydrolysis of the acetoxymethyl ester. [Ca
21
]
i
was measured as the
ratio of the emitted fluorescence, measured as photon counts, at 405
and 485 nm using excitation at 350 nm as described previously (14, 15).
The technique utilized for the simultaneous determination of changes
in [Ca
21
]
i
and Mn
21
quench was as described previously (15). In the
experiments designed to eliminate the effects of increases in [Ca
21
]
i
,
cells were loaded with the Ca
21
chelator BAPTA by preincubation in
saline containing 15
m
M BAPTA/AM for 20 min. Preliminary experi-
ments showed that this was sufficient to completely abolish any detect-
able [Ca
21
]
i
signals in cells subsequently stimulated with concentra-
tions of carbachol up to 5
m
M.
Determinations of arachidonic acid release were essentially as de-
scribed previously (12). Briefly, m3-HEK cells were cultured in six-well
plates until ;50% confluent. 0.5
m
Ci/ml [
3
H]arachidonic acid was then
added to each well, and culture was continued overnight. The cells were
then washed three times with saline containing 0.2% fatty acid-free
bovine serum albumin prior to addition of agonist and/or drugs as
appropriate. Arachidonic acid released during a 5-min incubation was
determined by liquid scintillation counting of the supernatant and
normalized to the total counts in the cells, determined following solu-
bilization with 1.5 M NaOH. Total inositol phosphates were determined
in a similar manner. Cells cultured in six-well plates were incubated
overnight in the presence of 5
m
Ci/ml myo-[
3
H]inositol, followed by
washing three times in saline. To enhance detection of inositol phos-
phate generation and turnover, experiments were performed in the
presence of lithium (10 mM) following a 10-min preincubation in the
same concentration of lithium. At the end of the experimental period (5
min), the saline was removed and replaced with 1 ml of 0.5 M trichlo-
roacetic acid. After incubation for 15 min on ice, the trichloroacetic
acid-soluble fraction was extracted with ether and neutralized with
sodium bicarbonate, and the extract was applied to Dowex columns.
The loaded columns were washed with water, followed by 60 mM sodium
formate plus 5 mM borax, before eluting the inositol phosphates with 1.2
M ammonium formate in 100 mM formic acid. Samples of the eluted
inositol phosphates were counted by liquid scintillation. Total inositol
phosphate generation is expressed as percent total counts in the phos-
phoinositide pool, determined from counting samples of the trichloro-
acetic acid-insoluble tissue fractions following their digestion overnight
with 1.5 M NaOH.
Arachidonic acid, isotetrandrine, indomethacin, nordihydroguaia-
retic acid, 5,8,11,14-eicosatetraynoic acid (ETYA), and U73122 were
from BIOMOL Research Labs Inc., and the phorbol ester phorbol 12-
myristate 13-acetate (PMA) was from Calbiochem. [
3
H]Arachidonic acid
and [
3
H]inositol were from American Radiolabeled Chemicals.
RESULTS
m3-mAChRs Couple to Arachidonic Acid Generation to Acti-
vate Calcium Entry—We first determined whether activation
of the transfected m3 muscarinic receptor in HEK cells was
coupled to the generation of arachidonic acid, as described
previously for the native muscarinic receptor in avian nasal
gland cells (12). Fig. 1 shows that addition of the muscarinic
receptor agonist carbachol to cells prelabeled with [
3
H]arachi-
donic acid resulted in a marked increase in arachidonic acid
generation. This increase was dependent on agonist concentra-
tion and was clearly apparent even at agonist concentrations
close to the threshold for inducing detectable [Ca
21
]
i
signals in
these cells (;1
m
M carbachol). Concentrations of carbachol .5
m
M were not examined as, based on our earlier studies (12), we
believed that the receptor activation of arachidonic acid gener-
ation and the associated increase in calcium entry were specif-
ically associated with stimulation at low agonist concentra-
tions, when, typically, oscillatory [Ca
21
]
i
signals are seen.
To examine the effect of arachidonic acid on [Ca
21
]
i
signaling
in m3-HEK cells, low concentrations of exogenous arachidonic
acid were added to otherwise unstimulated cells. Addition of as
little as 3–8
m
M arachidonic acid resulted, after a variable lag
period of several tens of seconds, in a slow increase in [Ca
21
]
i
(Fig. 2A). Such an increase in [Ca
21
]
i
could result from an
increase in calcium entry from the extracellular medium or
from release of calcium from intracellular stores. To distin-
guish between these two alternatives, lanthanum was used as
a generic blocker of calcium entry channels. In the presence of
100
m
M La
31
, addition of exogenous arachidonic acid failed to
increase [Ca
21
]
i
, and only when the La
31
was removed was an
increase in [Ca
21
]
i
observed (Fig. 2B). The failure to observe
any increase in [Ca
21
]
i
in the presence of La
31
confirms that
such an increase reflects an increase in Ca
21
entry. These data
also rule out the additional possibility that arachidonic acid
was inducing an increase in [Ca
21
]
i
by inhibition of the plasma
membrane calcium pump. At high concentrations (;1m
M or
higher), La
31
is also known to block the plasma membrane
Ca
21
-ATPase, but such an effect would be expected to further
increase [Ca
21
]
i
, not to block such an increase. To confirm that
arachidonic acid was indeed activating calcium entry, simulta-
neous determinations of changes in [Ca
21
]
i
and Mn
21
quench
were performed. In these, extracellular Mn
21
(0.2 mM) is used
as a surrogate for Ca
21
as, at low external concentrations, it
readily passes through many kinds of Ca
21
channels. On en-
tering the cytosol, the Mn
21
binds to and quenches the fluores-
cence of such probes as indo-1, and the rate of fluorescence
quenching can then be used as an indirect measure of the rate
of Mn
21
(and hence Ca
21
) entry, at least through those path-
ways permeable to Mn
21
. As shown in Fig. 2C, the arachido-
nate-induced increase in [Ca
21
]
i
was associated with a simul-
taneous increase in the rate of Mn
21
quench, indicating that
Ca
21
entry is increased.
To characterize the effects of the carbachol-induced genera-
tion of arachidonic acid and the consequent increase in Ca
21
entry on [Ca
21
]
i
signals in m3-HEK cells, we examined the
effect of the biscoclaurine alkaloid isotetrandrine. This sub-
stance acts as an apparently specific and readily reversible
inhibitor of agonist-induced arachidonic acid generation (16,
17), as demonstrated in our earlier studies on the avian nasal
gland (12). Consistent with those studies, isotetrandrine (10
m
M) had no effect on carbachol-induced inositol phosphate gen-
eration in m3-HEK cells, but completely blocked the simulta-
neous carbachol-induced generation of arachidonic acid (Fig.
3). Addition of 10
m
M isotetrandrine to cells demonstrating an
oscillatory [Ca
21
]
i
signal in response to low concentrations of
carbachol resulted in an immediate cessation of the oscilla-
tions, which subsequently rapidly returned with normal ampli-
tude and frequency on removal of the isotetrandrine (Fig. 4A).
This is identical to the effect seen in these cells if Ca
21
entry
was inhibited during an oscillatory [Ca
21
]
i
response. For ex-
ample, addition of La
31
to a cell during an oscillatory [Ca
21
]
i
FIG.1.Effect of carbachol on the release of arachidonic acid in
m3-HEK cells. Cells were incubated overnight in [
3
H]arachidonic acid
and, after washing, exposed to carbachol (CCh) at different concentra-
tions for 5 min in the presence of 0.2% fatty acid-free bovine serum
albumin (to act as a sink for released arachidonic acid (AA)), as de-
scribed under “Materials and Methods.” Values are the means 6 S.E.
(n 5 4).
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response to carbachol resulted in the immediate cessation of
the oscillations, which promptly returned on removal of the
La
31
(Fig. 4B). To confirm that exposure to isotetrandrine
during oscillations was indeed inhibiting Ca
21
entry, we again
used the Mn
21
quench approach. As previously reported by us
(11, 14) and by others (18, 19), oscillations in [Ca
21
]
i
were
associated with a constant enhanced rate of Mn
21
quench,
reflecting a constant Ca
21
entry. Fig. 4C shows that addition of
isotetrandrine induced an immediate inhibition of this rate of
Mn
21
quench, which was rapidly restored on removal of the
isotetrandrine. The observed effects on Mn
21
quench (and
hence, presumably Ca
21
entry) were temporally directly asso-
ciated with the inhibition of the [Ca
21
]
i
oscillations. It should
be noted that the acute sensitivity to inhibition of Ca
21
entry
during oscillatory [Ca
21
]
i
signals seen here is not a universal
feature in all cell types, some of which will continue to oscillate
for some time even after complete removal of extracellular
Ca
21
(e.g. Xenopus oocytes). Such variation most likely reflects
differences in surface-to-volume ratios and in the relative ac-
tivities of the Ca
21
pumps on the plasma membrane and on the
intracellular stores (as well as levels of cytosolic buffers, etc.).
However, such differences should not be interpreted as indicat-
ing that Ca
21
entry plays no consistent or critical role in
oscillatory [Ca
21
]
i
signals as, even in Xenopus oocytes, changes
in Ca
21
entry profoundly influence the frequency of agonist-
induced [Ca
21
]
i
oscillations (20).
In intact cells, arachidonic acid frequently undergoes rapid
oxidation, resulting in the production of a variety of eicosanoids
that are, themselves, known to possess signaling activities. To
determine whether the observed effects were due to arachi-
donic acid itself or to one of the many products of arachidonic
acid metabolism, we examined the effects of inhibition of the
lipoxygenase pathway, using nordihydroguaiaretic acid, and
the cyclooxygenase pathway, using indomethacin. Addition of
either of these agents to carbachol-stimulated cells showing
oscillating [Ca
21
]
i
signals caused an increase in oscillation
frequency, which often progressively developed into a sus-
tained elevation of [Ca
21
]
i
(Fig. 5, A and B). If the activation of
Ca
21
entry observed was dependent on the metabolism of
arachidonic acid by these pathways, then their inhibition
would be expected to result in the reduction or cessation of the
[Ca
21
]
i
oscillations, much as seen above when arachidonic acid
generation was inhibited by isotetrandrine (Fig. 4). As such,
the responses observed are clearly incompatible with the pro-
posal that products of these metabolic pathways for arachi-
donic acid are responsible for the observed increases in Ca
21
entry during oscillations. The effects seen are, however, con-
sistent with a progressive increase in arachidonic acid levels in
stimulated cells subsequent to inhibition of the normal meta-
bolic pathways responsible for its degradation. Such increases
might be expected to further increase Ca
21
entry, resulting in
a sustained elevation of [Ca
21
]
i
, as observed. The direct effect
of arachidonic acid was further confirmed in experiments show-
ing that addition of the non-metabolizable arachidonic acid
FIG.2.Effect of exogenous arachidonic acid on [Ca
21
]
i
in un-
stimulated cells. A, indo-1-loaded cells were exposed to arachidonic
acid (arrow) at the concentrations indicated, and the changes in [Ca
21
]
i
(measured as the 405/485 nm fluorescence emission ratio) were deter-
mined (see “Materials and Methods” for details). Traces are superim-
posed for comparison purposes. B, an indo-1-loaded cell was exposed to
arachidonic acid (AA;8
m
M; arrow). La
31
(100
m
M) was present in the
extracellular medium during the period indicated (solid bar). C, shown
is the effect of exogenous arachidonic acid (8
m
M; added at the point
indicated) on the rate of Mn
21
quench in an indo-1-loaded cell. Mn
21
quenching of intracellular indo-1 (E), calculated from the corrected sum
of the two emitted fluorescences at 405 and 485 nm (Ftot), was deter-
mined simultaneously with the 405/485 nm fluorescence ratio (contin-
uous line) as described under “Materials and Methods.”
FIG.3.Effect of isotetrandrine on the carbachol stimulation of
increases in arachidonic acid release and total inositol phos-
phate generation. Arachidonic acid (AA) release (shaded columns)
and total inositol phosphate (IPs) generation (stippled columns) were
determined as described under “Materials and Methods” in the pres-
ence and absence of 1
m
M carbachol (CCh) and in the presence 1
m
M
carbachol plus 10
m
M isotetrandrine (isotet). Values are the means 6
S.E. (n 5 4). PI, phosphoinositide.
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analog ETYA to unstimulated cells caused an increase in
[Ca
21
]
i
similar to that seen with arachidonic acid (Fig. 5C). In
these experiments, it was noted that the effects of ETYA were
frequently more rapid in onset than those seen with arachi-
donic acid, but required somewhat higher concentrations (;20
versus ;5
m
M). Such data are consistent with the response to
exogenously added arachidonic acid being muted by metabo-
lism in the intact cell, yet showing some selectivity for arachi-
donic acid over its analog ETYA. Together, the above data
indicate that it is arachidonic acid itself that is the active
moiety responsible for the observed effects on Ca
21
entry. They
also suggest that both lipoxygenase and cyclooxygenase path-
ways are active in HEK293 cells and, in vivo, act to limit
increases in receptor-stimulated levels of arachidonic acid.
mAChR Activation Stimulates Arachidonic Acid Generation
Independently of PLC Activation—The m3-mAChR is known to
couple, via a member of the G
q
family of G proteins, to the
activation of phospholipase C and the generation of diacylglyc-
erol and inositol 1,4,5-trisphosphate. The former activates pro-
tein kinase C, whereas the latter is integral to the initiation of
[Ca
21
]
i
signals. To determine whether the observed increased
generation of arachidonic acid was a downstream effect of the
simultaneous activation of this PLC pathway, various ap-
proaches were employed. The possible involvement of InsP
3
-
mediated increases in [Ca
21
]
i
was examined by determining
arachidonic acid generation in cells loaded with the calcium
chelator BAPTA (as described under “Materials and Methods”)
prior to stimulation with carbachol. As shown in Fig. 6, the
carbachol-induced generation of arachidonic acid was com-
pletely unimpaired in such BAPTA-loaded cells, indicating that
this effect was not dependent on the receptor-activated [Ca
21
]
i
signals.
We next examined the possible involvement of the PLC-
FIG.4.A, effect of isotetrandrine on carbachol-induced [Ca
21
]
i
oscil-
lations. A cell loaded with indo-1 was stimulated with carbachol (1
m
M)
to induce an oscillatory response in [Ca
21
]
i
, measured as changes in the
405/485 nm fluorescence ratio. During the period indicated (solid bar),
isotetrandrine (10
m
M) was added. B, effect of inhibiting Ca
21
entry by
addition of La
31
(500
m
M) on carbachol-induced [Ca
21
]
i
oscillations. C,
effect of isotetrandrine (10
m
M; solid bar) on the rate of Mn
21
quench (E,
●) in an oscillating cell. See Fig. 2 legend for details.
FIG.5.A and B, effect of inhibition of arachidonic acid metabolism on
carbachol-induced [Ca
21
]
i
oscillations. Indo-1-loaded cells were stimu-
lated with carbachol (1
m
M) to induce oscillations in [Ca
21
]
i
. During the
periods indicated (solid bars), either indomethacin (A;10
m
M), to inhibit
the cyclooxygenase pathway, or nordihydroguaiaretic acid (B;10
m
M), to
inhibit the lipoxygenase pathway, was present. C, effect of ETYA on
[Ca
21
]
i
in an unstimulated cell. An indo-1-loaded cell was exposed to
ETYA (20
m
M; added at the arrow), and changes in [Ca
21
]
i
were meas-
ured as the 405/485 nm ratio.
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mediated generation of diacylglycerol and the consequent acti-
vation of PKC in the observed mAChR-induced arachidonic
acid release. Direct stimulation of PKC activity by addition of
the phorbol ester PMA (preincubation for 5 min in the presence
of 0.1
m
M PMA) resulted in a modest, but significant, inhibition
of arachidonic acid release in unstimulated cells (0.14 6 0.01%
in PMA-treated cells versus 0.18 6 0.1% in control cells). More
significantly, pretreatment with PMA completely abolished the
normal mAChR-induced arachidonic acid release seen follow-
ing addition of carbachol (Fig. 7). These data indicate that the
principal effect of PKC activation appears to be to uncouple the
stimulation of arachidonic acid release by mAChRs. Although
the precise basis for this effect is unknown, these data clearly
indicate that the activation of PKC does not result in the
stimulation of arachidonic acid generation. Based on the above
data from BAPTA-loaded cells and PMA-treated cells, it seems
that neither the PLC-mediated increase in [Ca
21
]
i
nor the
activation of PKC is involved in the observed activation of
arachidonic acid release. Nevertheless, we were concerned that
the above experiments may not entirely eliminate a potential
involvement of the PLC pathway. For example, it has been
reported in several cell types that the stimulation of PKC can
exert a negative feedback on receptor-activated PLC activity. It
was therefore possible that the failure of PMA to stimulate
arachidonic acid release may have simply reflected the concom-
itant inhibition of PLC activity. Experiments on m3-HEK cells
confirmed that PMA (0.1
m
M with preincubation for 5 min) did
indeed significantly inhibit the carbachol-induced increase in
inositol phosphate generation, an effect consistent with a re-
duction of agonist-activated PLC activity (data not shown).
Alternatively, it is possible that the mAChR activation of
arachidonic acid generation requires an increase in both
[Ca
21
]
i
and PKC activity. We therefore carried out an addi-
tional series of experiments examining the effect of the drug
U73122 (21) on mAChR activation of arachidonic acid genera-
tion. Although not always entirely specific, U73122 has been
extensively used as an inhibitor of PLC activity, and consistent
with this, we found that in the presence of 10
m
M U73122,
carbachol-induced inositol phosphate generation was com-
pletely abolished (Fig. 8A). However, under identical condi-
tions, carbachol-induced arachidonic acid generation was un-
affected (Fig. 8B). These data confirm that the observed
receptor-activated arachidonic acid generation is not a down-
stream effect of any PLC activity.
Arachidonate-mediated Calcium Entry Is Distinct from Ca-
pacitative Entry—As discussed above, the activation of the
capacitative mechanism of Ca
21
entry is solely dependent on
the emptying of intracellular stores of calcium no matter how
this is achieved. However, under normal circumstances of re-
ceptor stimulation, the discharge of the intracellular Ca
21
stores occurs as a result of the PLC-mediated generation of
inositol 1,4,5-trisphosphate, which activates Ca
21
-permeable
channels on the stores. The demonstration that mAChR acti-
vation of arachidonic acid generation is not a downstream
effect of the simultaneous activation of PLC (see above) sug-
FIG.6.Effect of carbachol on the release of arachidonic acid in
BAPTA-loaded cells. Cells, loaded overnight with [
3
H]arachidonic
acid (AA), were washed and then loaded with sufficient BAPTA to
completely obliterate any detectable carbachol (CCh)-induced [Ca
21
]
i
signal by incubation in the presence of BAPTA/AM, as described under
“Materials and Methods.” Values are the means 6 S.E. (n 5 4).
FIG.7. Effect of PMA on carbachol-stimulated arachidonic
acid release. Cells were loaded overnight with [
3
H]arachidonic acid as
described under “Materials and Methods.” Experimental cells were
then exposed to 100 n
M PMA for 5 min prior to stimulation with
carbachol (CCh) at the concentrations indicated. Data are presented as
the carbachol-induced increase in arachidonic acid (AA) release over the
corresponding controls in the absence (shaded columns) or presence
(stippled columns) of PMA. Values are the means 6 S.E. (n 5 4).
FIG.8. Effect of U73122 on carbachol-induced inositol phos-
phate generation (A) and arachidonic acid release (B). Total
inositol phosphate generation and arachidonic acid release were meas-
ured as described under “Materials and Methods.” Prior to stimulation
with carbachol (CCh) at the concentrations indicated, experimental
cells were exposed to U73122 (10
m
M) for 10 min. Data are presented as
the carbachol-induced increase in total inositol phosphate generation or
arachidonic acid release over the corresponding controls in the absence
(shaded columns) or presence (stippled columns) of U73122. Values are
the means 6 S.E. (n 5 4 (arachidonic acid (AA)) and n 5 3 (total inositol
phosphates (IPs))). PI, phosphoinositide.
Arachidonate-mediated Ca
21
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gests that the arachidonate-activated Ca
21
entry is unlikely to
be part of a capacitative mechanism. This is further supported
by the fact that isotetrandrine, which, as shown, is an effective
inhibitor of receptor-activated arachidonic acid generation, has
no effect on the capacitative entry of Ca
21
induced by thapsi-
gargin (data not shown). Similar data were obtained in our
earlier studies on avian nasal gland cells (12) and have been
reported for the related compound tetrandrine in adrenal glo-
merulosa cells (22). However, this does not entirely preclude
the possibility that arachidonic acid is involved in some way in
the capacitative mechanism. For example, the complete deple-
tion of the stores induced by thapsigargin may result in such a
profound stimulation of the capacitative mechanism that
isotetrandrine is not able to reverse it significantly. Alterna-
tively, a single type of Ca
21
entry channel (store-operated
channel) may be capable of being activated by the capacitative
signal, whatever its nature, or by arachidonic acid depending
on the circumstances. To preclude these possibilities, we exam-
ined the effect of reducing extracellular pH on the Ca
21
entry
activated by thapsigargin and by arachidonic acid. Modest
reductions in extracellular pH have been shown to have a
profound inhibitory effect on capacitative Ca
21
entry in a range
of different cell types (23–26), and in the case of the store-
operated channel I
CRAC
, this has been shown to reflect a direct
action of extracellular protons on the channel (27).
2
As shown
in Fig. 9A, reducing the pH of the superfusing saline to 6.7
induced a marked decrease in the sustained [Ca
21
]
i
seen in
thapsigargin-treated cells, consistent with an inhibition of ca-
pacitative entry. This effect was readily reversed on restoration
of normal extracellular pH. An identical reduction in extracel-
lular pH was, however, completely without effect on the similar
sustained [Ca
21
]
i
seen in cells exposed to exogenous arachi-
donic acid (Fig. 9B). This marked difference in the effect of
extracellular pH indicates that the Ca
21
entry channel acti-
vated by arachidonic acid is unlikely to be the same as that
activated by thapsigargin-induced depletion of intracellular
stores.
DISCUSSION
In the studies reported here, we have shown that activation
of the m3-mAChR stably transfected in HEK293 cells results in
the generation of arachidonic acid and that this specifically
activates a Ca
21
entry pathway that is critical to the regulation
of the oscillatory [Ca
21
]
i
signals generated by low concentra-
tions of appropriate agonists. Although detailed concentration-
response curves for the generation of arachidonic acid were not
determined, it is clear that this response shows a sensitivity to
muscarinic agonists that is very similar to that demonstrated
by the activation of PLC and is therefore consistent with a
potential role in the regulation or generation of [Ca
21
]
i
signals.
We have also shown that inhibition of the generation of arachi-
donic acid results in the immediate inhibition of receptor-en-
hanced Ca
21
entry and the cessation of the oscillatory [Ca
21
]
i
response. The data further indicate that this is an effect of
arachidonic acid itself and not of a product of the commonly
recognized metabolic pathways for arachidonic acid. Regarding
our data on the activation of Ca
21
entry by exogenous applica-
tion of arachidonic acid, such application has been reported to
produce a variety of effects on [Ca
21
]
i
signals in a many differ-
ent cells, including effects on Ca
21
release from intracellular
stores (28) as well as on Ca
21
entry (29). However, most of
these studies involved the use of very high concentrations of
arachidonate (.50
m
M), raising the possibility of a variety of
nonspecific effects. In contrast, the effects on Ca
21
entry we
have observed are seen at very low concentrations of exog-
enously added arachidonic acid (typically ;5
m
M). Given that
extracellular application via the general perfusing medium is
probably only a crude mimic of the specific receptor-mediated
intracellular generation of arachidonic acid, this is likely to
represent a significant underestimate of the true sensitivity of
the Ca
21
entry pathway to this substance.
The data we have presented demonstrate the receptor-acti-
vated generation of arachidonic acid, together with potential
pathways for its rapid removal. We have shown that low con-
centrations of arachidonic acid activate a Ca
21
entry pathway
and that inhibition of its generation produces a parallel inhi-
bition of the receptor-activated entry of Ca
21
seen during
[Ca
21
]
i
oscillations. All of these features are consistent with the
critical experimental criteria widely recognized as defining a
substance as a second messenger and lead us to conclude that
arachidonic acid is the intracellular signal responsible for the
mAChR activation of Ca
21
entry during [Ca
21
]
i
oscillations.
We have previously reported a similar arachidonate-dependent
entry of Ca
21
during oscillatory [Ca
21
]
i
responses to musca-
rinic receptor activation in the exocrine cells of the avian nasal
gland (12). The demonstration of this same signaling pathway
in these very different cell types suggests that it is likely to
have a widespread distribution. This is further supported by
recent reports indicating the presence of an arachidonate-acti-
vated Ca
21
entry that is involved in maintaining [Ca
21
]
i
oscil-
2
R. Penner, personal communication.
FIG.9.Comparison of the effect of reducing the extracellular
pH on thapsigargin- and arachidonate-induced sustained eleva-
tions in [Ca
21
]
i
. A, an indo-1-loaded cell was exposed to thapsigargin
(1
m
M) to discharge the intracellular Ca
21
stores and to activate capac-
itative Ca
21
entry (as reflected in a sustained increase in [Ca
21
]
i
).
During the periods indicated (solid bars), extracellular pH was reduced
to 6.7. B, indo-1-loaded cells were exposed to either 1
m
M thapsigargin
(E)or8
m
M arachidonic acid (●) to induce a sustained increase in
[Ca
21
]
i
. At the point indicated (solid bar), extracellular pH was reduced
to 6.7.
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21
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lations induced by cholecystokinin B receptors transfected into
Chinese hamster ovary cells (30). An arachidonate-activated
Ca
21
entry that is believed to be involved in basic fibroblast
growth factor-induced responses in Balb/c 3T3 fibroblasts has
also been recently described (31), and low concentrations of
exogenous arachidonic acid have been shown to activate Ca
21
-
permeable, voltage-insensitive cation channels in chromaffin
cells (32). Direct regulation of ion channels by fatty acids,
including arachidonic acid, has been shown for a variety of
different channel types (33, 34) and includes both stimulatory
and inhibitory effects. More important, although the full char-
acterization of the arachidonate-mediated pathway we have
described must await further studies, we have shown that it
possesses characteristics that distinguish it from the more well
known capacitative or store-operated pathway. For example, in
our previous study of this pathway in avian nasal gland cells
(12), we showed that its activation was apparently independent
of any InsP
3
-induced release of Ca
21
from intracellular stores.
In fact, the data indicated that, at the low agonist concentra-
tions that typically give rise to oscillatory [Ca
21
]
i
signals, gen-
erated levels of InsP
3
alone were not adequate to induce any
detectable release of Ca
21
from the stores, and such release
was absolutely dependent on the additional effect of the arachi-
donate-mediated entry of Ca
21
. However, the possibility re-
mained that the entry we observed was, in fact, dependent on
a capacitative mechanism that was activated by the emptying
of a small specific subset of the overall agonist-sensitive store
whose discharge was undetectable in our experiments. Pub-
lished evidence suggests this is unlikely because, as noted
earlier, data from a variety of different cells indicate that if
such a subset exists, its discharge would require higher con-
centrations of InsP
3
than those required to empty the bulk of
the agonist-sensitive stores in the cell (8–10). Nevertheless, in
the study reported here, we have made an additional critical
observation that supports our hypothesis that the arachido-
nate-mediated Ca
21
entry is entirely distinct from the capaci-
tative pathway activated as a result of the depletion of intra-
cellular stores. We showed that, consistent with reports from
various different cell types, such capacitative entry in HEK293
cells is acutely sensitive to reductions in extracellular pH. In
marked contrast, the Ca
21
entry activated by arachidonic acid
in the same cells is entirely insensitive to such changes in
extracellular pH. Given that the observed effects on the capac-
itative entry are reported to reflect a direct effect on the store-
operated Ca
21
channel (at least in the case of I
CRAC
), such a
clear difference indicates that the arachidonate-activated path-
way must be distinct from that activated by store depletion.
This conclusion is further supported by our demonstration that
the receptor-mediated increase in arachidonic acid generation
we have observed is independent of the simultaneous activa-
tion of the PLC-InsP
3
pathway (see below). Clearly, such a
finding is inconsistent with the idea that the muscarinic recep-
tor stimulation of the arachidonate-mediated pathway is down-
stream of any InsP
3
-mediated release of stored Ca
21
.
Data from a wide range of different cell types suggest that
there are generally two principal sources of receptor-mediated
increases in arachidonic acid generation. The first involves the
action of diacylglycerol/monoacylglycerol lipases on diacylglyc-
erol. This, in turn, is mainly derived from the hydrolysis of
phosphatidylcholine as a result of receptor-induced increases
in either PLD activity (via phosphatidic acid) (35, 36) or PLC
(37, 38). In this context, it has previously been reported that
the m3-mAChR stably transfected into HEK293 cells is capable
of coupling to a PLD (39, 40). However, several characteristics
of this response in the m3-mAChR-transfected HEK293 cells
indicate that this is unlikely to be the basis for the observed
carbachol-induced increase in arachidonic acid generation.
First, the activation of PLD by the m3-mAChR stably trans-
fected into HEK293 cells has been shown to completely desen-
sitize within 2 min of stimulation, even at the low concentra-
tions of agonist used in this study (40). This desensitization is
apparently not a result of the loss of cell-surface receptors, but
reflects a rapid and sustained uncoupling of the receptors from
the activation of PLD. Second, it has been shown that addition
of the phorbol ester PMA induces a pronounced stimulation of
PLD activity in m3-HEK cells (40, 41) as well as in many other
mammalian cells (42, 43). Examination of the published data
indicate that 0.1
m
M PMA increased PLD activity to levels
;4-fold higher than those seen with a maximal concentration
of carbachol (40). This is clearly in marked contrast to our data
showing that, at the same concentration, PMA had a marked
inhibitory effect on the carbachol-induced arachidonic acid gen-
eration. Based on these findings, we conclude that the reported
characteristics of the PLD response in HEK293 cells are not
consistent with our findings on the mAChR-activated arachi-
donic acid generation and that the PLD-mediated generation of
diacylglycerol is unlikely to account for the observed mAChR-
induced arachidonic acid response.
The alternative mechanism for receptor-induced increases in
arachidonic acid generation involves the action of a cytosolic
PLA
2
releasing arachidonic acid from appropriate phospholip-
ids. Two main types of cytosolic PLA
2
with distinct properties
and structures are currently identified: the “classic” type IV
Ca
21
-dependent cPLA
2
and the more recently characterized
type VI Ca
21
-independent PLA
2
(iPLA
2
) (44–46). This latter
group is thought to be principally involved in general mem-
brane phospholipid remodeling (47), whereas it is the type IV
Ca
21
-dependent cPLA
2
that has usually been shown to be
associated with the receptor activation of arachidonic acid re-
lease (48). In this respect, a particularly significant finding of
the studies reported here is that the increase in arachidonic
acid generation we have observed was shown to be independent
of the simultaneous activation of PLC. Receptor activation of
cPLA
2
has been generally reported to involve two distinct pro-
cesses: a Ca
21
-dependent translocation of the cPLA
2
to the
membrane to allow interaction with its phospholipid substrate
and a phosphorylation that is usually mediated via a mitogen-
activated protein kinase, whose activity may, in turn, be mod-
ulated by PKC (48). Alternatively, PKC may itself directly
phosphorylate the cPLA
2
to increase its activity. The relative
importance of the Ca
21
-dependent translocation and the phos-
phorylation steps in the activation of cPLA
2
appears to vary in
different cell types and under different circumstances (48). In
marked contrast, the studies we have presented here clearly
show that neither an increase in [Ca
21
]
i
nor the activation of
PKC (either individually or together) was required for the
observed carbachol-induced generation of arachidonic acid. In
this regard, it is perhaps important to point out that most
studies investigating the receptor-activation of cPLA
2
have
generally utilized maximal (or near-maximal) concentrations of
the relevant agonist. Consequently, although the data pre-
sented do not exclude the presence of these activation path-
ways in HEK293 cells, it is clear that neither a Ca
21
-dependent
translocation nor a PKC-dependent phosphorylation step can
play any significant role in the activation of arachidonic acid
generation under the specific conditions employed, i.e. at low
concentrations of muscarinic agonists. This was further sup-
ported by the data obtained using the drug U73122, which
confirmed that the mAChR stimulation of arachidonic acid
generation we observed is not downstream of PLC activation.
Moreover, because the HEK293 cell line utilized here possesses
only a single muscarinic receptor subtype (m3), the data show
Arachidonate-mediated Ca
21
Entry in HEK293 Cells32642
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that this receptor is capable of coupling both to the activation of
PLC and the generation of arachidonic acid in a separate but
parallel manner. We therefore conclude that the two signaling
pathways are independently activated by the m3-mAChR.
Given the critical role that such arachidonic acid generation
plays in the activation of the Ca
21
entry required to drive
receptor-activated oscillatory [Ca
21
]
i
signals in these and other
cells, the identification of the mechanism responsible for cou-
pling of muscarinic receptors to arachidonic acid generation at
these physiologically relevant levels of stimulation is clearly of
considerable importance.
Acknowledgment—We thank Dr. Craig Logsdon for generously pro-
viding the HEK293 cells stably transfected with the human m3 mus-
carinic receptor.
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Arachidonate-mediated Ca
21
Entry in HEK293 Cells 32643
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Trevor J. Shuttleworth and Jill L. Thompson
Phospholipase C
HEK293 Cells Is Independent of
Entry in
2+
Arachidonate-mediated Ca
Muscarinic Receptor Activation of
CELL BIOLOGY AND METABOLISM:
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