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J Biol Chem, Vol. 273, Issue 49, 32636-32643, December 4, 1998
From the Department of Pharmacology and Physiology, University of
Rochester School of Medicine and Dentistry,
Rochester, New York 14642
Receptor-enhanced entry of Ca2+
in non-excitable cells is generally ascribed to a capacitative
mechanism in which the activation of the entry pathway is specifically
dependent on the emptying of agonist-sensitive intracellular
Ca2+ 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 [Ca2+]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 Ca2+ entry pathway. We show
that the generation of arachidonic acid and the activation of this
pathway are specifically associated with stimulation at the low agonist
concentrations that typically give rise to oscillatory
[Ca2+]i signals. At such agonist concentrations,
however, 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 Ca2+ entry demonstrates
characteristics that distinguish it from the corresponding capacitative
pathway in the same cells and therefore is likely to represent an
entirely distinct pathway that is specifically responsible for the
receptor-enhanced entry of Ca2+ during
[Ca2+]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
(InsP3),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 "capacitative 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.
Alternatively, a more direct molecular coupling between the stores and
the plasma membrane channels may occur (3). Although 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
[Ca2+]i signal when stimulated at low agonist
concentrations (6, 7), and such signals are associated with an enhanced entry of Ca2+. However, evidence indicates that the
activation of capacitative Ca2+ entry generally requires
significantly higher levels of agonist-generated InsP3 than
does the release of Ca2+ from the bulk of the
agonist-sensitive internal stores (i.e. at low
concentrations of InsP3, substantial release of
Ca2+ from agonist-sensitive stores can occur without any
activation of capacitative entry) (8-10). Consequently, it is far from
clear that the transitory (and/or incomplete) nature of calcium store depletion during [Ca2+]i oscillations would
provide an adequate or appropriate signal for the activation of calcium
entry via a capacitative mechanism. Such considerations led us
previously to investigate the nature of receptor-activated increases in
Ca2+ entry during [Ca2+]i
oscillations in cells from the exocrine avian nasal gland. In these
studies, we showed that such Ca2+ 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 muscarinic 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-InsP3 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 demonstrates characteristics that distinguish it from the more well known
capacitative or store-operated entry of calcium.
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
supplemented with 10% calf serum and antibiotics. Changes in
[Ca2+]i in single individual cells were
determined following loading with the fluorescent probe indo-1. Loading
was achieved by incubation in 4 µM 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. [Ca2+]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 [Ca2+]i and Mn2+ quench
was as described previously (15). In the experiments designed to
eliminate the effects of increases in [Ca2+]i,
cells were loaded with the Ca2+ chelator BAPTA by
preincubation in saline containing 15 µM BAPTA/AM for 20 min. Preliminary experiments showed that this was sufficient to
completely abolish any detectable [Ca2+]i signals
in cells subsequently stimulated with concentrations of carbachol up to
5 µM.
Determinations of arachidonic acid release were essentially as
described previously (12). Briefly, m3-HEK cells were cultured in
six-well plates until ~50% confluent. 0.5 µCi/ml
[3H]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 solubilization 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 µCi/ml
myo-[3H]inositol, followed by washing three
times in saline. To enhance detection of inositol phosphate 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 trichloroacetic 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
phosphoinositide pool, determined from counting samples of the
trichloroacetic acid-insoluble tissue fractions following their
digestion overnight with 1.5 M NaOH.
Arachidonic acid, isotetrandrine, indomethacin, nordihydroguaiaretic
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. [3H]Arachidonic
acid and [3H]inositol were from American Radiolabeled Chemicals.
m3-mAChRs Couple to Arachidonic Acid Generation to Activate 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
[3H]arachidonic acid resulted in a marked increase in
arachidonic acid generation. This increase was dependent on agonist
concentration and was clearly apparent even at agonist concentrations
close to the threshold for inducing detectable
[Ca2+]i signals in these cells (~1
µM carbachol). Concentrations of carbachol >5
µM were not examined as, based on our earlier studies
(12), we believed that the receptor activation of arachidonic acid
generation and the associated increase in calcium entry were specifically associated with stimulation at low agonist concentrations, when, typically, oscillatory [Ca2+]i signals are
seen.
To examine the effect of arachidonic acid on
[Ca2+]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 µM
arachidonic acid resulted, after a variable lag period of several tens
of seconds, in a slow increase in [Ca2+]i (Fig.
2A). Such an increase in
[Ca2+]i could result from an increase in calcium
entry from the extracellular medium or from release of calcium from
intracellular stores. To distinguish between these two alternatives,
lanthanum was used as a generic blocker of calcium entry channels. In
the presence of 100 µM La3+, addition of
exogenous arachidonic acid failed to increase
[Ca2+]i, and only when the La3+ was
removed was an increase in [Ca2+]i observed (Fig.
2B). The failure to observe any increase in
[Ca2+]i in the presence of La3+
confirms that such an increase reflects an increase in Ca2+
entry. These data also rule out the additional possibility that arachidonic acid was inducing an increase in
[Ca2+]i by inhibition of the plasma membrane
calcium pump. At high concentrations (~1 mM or higher),
La3+ is also known to block the plasma membrane
Ca2+-ATPase, but such an effect would be expected to
further increase [Ca2+]i, not to block such an
increase. To confirm that arachidonic acid was indeed activating
calcium entry, simultaneous determinations of changes in
[Ca2+]i and Mn2+ quench were
performed. In these, extracellular Mn2+ (0.2 mM) is used as a surrogate for Ca2+ as, at low
external concentrations, it readily passes through many kinds of
Ca2+ channels. On entering the cytosol, the
Mn2+ binds to and quenches the fluorescence of such probes
as indo-1, and the rate of fluorescence quenching can then be used as
an indirect measure of the rate of Mn2+ (and hence
Ca2+) entry, at least through those pathways permeable to
Mn2+. As shown in Fig. 2C, the
arachidonate-induced increase in [Ca2+]i was
associated with a simultaneous increase in the rate of Mn2+
quench, indicating that Ca2+ entry is increased.
To characterize the effects of the carbachol-induced generation of
arachidonic acid and the consequent increase in Ca2+ entry
on [Ca2+]i signals in m3-HEK cells, we examined
the effect of the biscoclaurine alkaloid isotetrandrine. This substance
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 µM) had no effect on
carbachol-induced inositol phosphate generation in m3-HEK cells, but
completely blocked the simultaneous carbachol-induced generation of
arachidonic acid (Fig. 3). Addition of 10 µM isotetrandrine to cells demonstrating an oscillatory
[Ca2+]i signal in response to low concentrations
of carbachol resulted in an immediate cessation of the oscillations,
which subsequently rapidly returned with normal amplitude and frequency on removal of the isotetrandrine (Fig.
4A). This is identical to the
effect seen in these cells if Ca2+ entry was inhibited
during an oscillatory [Ca2+]i response. For
example, addition of La3+ to a cell during an oscillatory
[Ca2+]i response to carbachol resulted in the
immediate cessation of the oscillations, which promptly returned on
removal of the La3+ (Fig. 4B). To confirm that
exposure to isotetrandrine during oscillations was indeed inhibiting
Ca2+ entry, we again used the Mn2+ quench
approach. As previously reported by us (11, 14) and by others (18, 19),
oscillations in [Ca2+]i were associated with a
constant enhanced rate of Mn2+ quench, reflecting a
constant Ca2+ entry. Fig. 4C shows that addition
of isotetrandrine induced an immediate inhibition of this rate of
Mn2+ quench, which was rapidly restored on removal of the
isotetrandrine. The observed effects on Mn2+ quench (and
hence, presumably Ca2+ entry) were temporally directly
associated with the inhibition of the [Ca2+]i
oscillations. It should be noted that the acute sensitivity to
inhibition of Ca2+ entry during oscillatory
[Ca2+]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 Ca2+
(e.g. Xenopus oocytes). Such variation most
likely reflects differences in surface-to-volume ratios and in the
relative activities of the Ca2+ 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 indicating that Ca2+ entry plays no
consistent or critical role in oscillatory
[Ca2+]i signals as, even in Xenopus
oocytes, changes in Ca2+ entry profoundly influence the
frequency of agonist-induced [Ca2+]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 arachidonic 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
[Ca2+]i signals caused an increase in oscillation
frequency, which often progressively developed into a sustained
elevation of [Ca2+]i (Fig.
5, A and B). If the
activation of Ca2+ 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
[Ca2+]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
proposal that products of these metabolic pathways for arachidonic acid are responsible for the observed increases in Ca2+ entry
during oscillations. The effects seen are, however, consistent with a
progressive increase in arachidonic acid levels in stimulated cells
subsequent to inhibition of the normal metabolic pathways responsible
for its degradation. Such increases might be expected to further
increase Ca2+ entry, resulting in a sustained elevation of
[Ca2+]i, as observed. The direct effect of
arachidonic acid was further confirmed in experiments showing that
addition of the non-metabolizable arachidonic acid analog ETYA to
unstimulated cells caused an increase in [Ca2+]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 arachidonic acid,
but required somewhat higher concentrations (~20 versus ~5 µM). Such data are consistent with the response to
exogenously added arachidonic acid being muted by metabolism in the
intact cell, yet showing some selectivity for arachidonic 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 Ca2+ entry. They also suggest that both
lipoxygenase and cyclooxygenase pathways 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 Gq family of G proteins, to the
activation of phospholipase C and the generation of diacylglycerol and
inositol 1,4,5-trisphosphate. The former activates protein kinase C,
whereas the latter is integral to the initiation of
[Ca2+]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
approaches were employed. The possible involvement of
InsP3-mediated increases in [Ca2+]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 completely unimpaired in such BAPTA-loaded cells,
indicating that this effect was not dependent on the receptor-activated
[Ca2+]i signals.
We next examined the possible involvement of the PLC-mediated
generation of diacylglycerol and the consequent activation 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 µM PMA) resulted in a
modest, but significant, inhibition of arachidonic acid release in
unstimulated cells (0.14 ± 0.01% in PMA-treated cells
versus 0.18 ± 0.1% in control cells). More
significantly, pretreatment with PMA completely abolished the normal
mAChR-induced arachidonic acid release seen following 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 [Ca2+]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
concomitant inhibition of PLC activity. Experiments on m3-HEK cells
confirmed that PMA (0.1 µM with preincubation for 5 min)
did indeed significantly inhibit the carbachol-induced increase in
inositol phosphate generation, an effect consistent with a reduction 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 [Ca2+]i
and PKC activity. We therefore carried out an additional series of
experiments examining the effect of the drug U73122 (21) on mAChR
activation of arachidonic acid generation. 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 µM U73122, carbachol-induced inositol phosphate
generation was completely abolished (Fig.
8A). However, under identical
conditions, carbachol-induced arachidonic acid generation was
unaffected (Fig. 8B). These data confirm that the observed
receptor-activated arachidonic acid generation is not a downstream
effect of any PLC activity.
Arachidonate-mediated Calcium Entry Is Distinct from Capacitative
Entry--
As discussed above, the activation of the capacitative
mechanism of Ca2+ entry is solely dependent on the emptying
of intracellular stores of calcium no matter how this is achieved.
However, under normal circumstances of receptor stimulation, the
discharge of the intracellular Ca2+ stores occurs as a
result of the PLC-mediated generation of inositol 1,4,5-trisphosphate,
which activates Ca2+-permeable channels on the stores. The
demonstration that mAChR activation of arachidonic acid generation is
not a downstream effect of the simultaneous activation of PLC (see
above) suggests that the arachidonate-activated Ca2+ 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 Ca2+ induced by
thapsigargin (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 glomerulosa 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 depletion 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. Alternatively, a single type of Ca2+ 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
examined the effect of reducing extracellular pH on the
Ca2+ entry activated by thapsigargin and by arachidonic
acid. Modest reductions in extracellular pH have been shown to have a
profound inhibitory effect on capacitative Ca2+ entry in a
range of different cell types (23-26), and in the case of the
store-operated channel ICRAC, 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
[Ca2+]i seen in thapsigargin-treated cells,
consistent with an inhibition of capacitative entry. This effect was
readily reversed on restoration of normal extracellular pH. An
identical reduction in extracellular pH was, however, completely
without effect on the similar sustained [Ca2+]i
seen in cells exposed to exogenous arachidonic acid (Fig.
9B). This marked difference in the effect of extracellular pH indicates that the Ca2+ entry channel activated by
arachidonic acid is unlikely to be the same as that activated by
thapsigargin-induced depletion of intracellular stores.
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
Ca2+ entry pathway that is critical to the regulation of
the oscillatory [Ca2+]i signals generated by low
concentrations 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 [Ca2+]i signals.
We have also shown that inhibition of the generation of arachidonic
acid results in the immediate inhibition of receptor-enhanced
Ca2+ entry and the cessation of the oscillatory
[Ca2+]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 Ca2+ entry by
exogenous application of arachidonic acid, such application has been
reported to produce a variety of effects on
[Ca2+]i signals in a many different cells,
including effects on Ca2+ release from intracellular stores
(28) as well as on Ca2+ entry (29). However, most of these
studies involved the use of very high concentrations of arachidonate
(>50 µM), raising the possibility of a variety of
nonspecific effects. In contrast, the effects on Ca2+ entry
we have observed are seen at very low concentrations of exogenously
added arachidonic acid (typically ~5 µM). 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
Ca2+ entry pathway to this substance.
The data we have presented demonstrate the receptor-activated
generation of arachidonic acid, together with potential pathways for
its rapid removal. We have shown that low concentrations of arachidonic
acid activate a Ca2+ entry pathway and that inhibition of
its generation produces a parallel inhibition of the receptor-activated
entry of Ca2+ seen during [Ca2+]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
Ca2+ entry during [Ca2+]i
oscillations. We have previously reported a similar arachidonate-dependent entry of Ca2+ during
oscillatory [Ca2+]i responses to muscarinic
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-activated Ca2+ entry that
is involved in maintaining [Ca2+]i oscillations
induced by cholecystokinin B receptors transfected into Chinese hamster
ovary cells (30). An arachidonate-activated Ca2+ 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 Ca2+-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
characterization 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
InsP3-induced release of Ca2+ from
intracellular stores. In fact, the data indicated that, at the low
agonist concentrations that typically give rise to oscillatory
[Ca2+]i signals, generated levels of
InsP3 alone were not adequate to induce any detectable
release of Ca2+ from the stores, and such release was
absolutely dependent on the additional effect of the
arachidonate-mediated entry of Ca2+. However, the
possibility remained 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. Published 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 concentrations of
InsP3 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 arachidonate-mediated
Ca2+ entry is entirely distinct from the capacitative
pathway activated as a result of the depletion of intracellular 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
Ca2+ 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 capacitative entry are reported to reflect a direct effect on the store-operated Ca2+ channel (at
least in the case of ICRAC), such a clear difference indicates that the arachidonate-activated pathway 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
activation of the PLC-InsP3 pathway (see below). Clearly,
such a finding is inconsistent with the idea that the muscarinic
receptor stimulation of the arachidonate-mediated pathway is downstream
of any InsP3-mediated release of stored
Ca2+.
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 diacylglycerol. 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 transfected into HEK293 cells
has been shown to completely desensitize within 2 min of stimulation,
even at the low concentrations 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 µM 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 generation. 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 arachidonic 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 PLA2
releasing arachidonic acid from appropriate phospholipids. Two main
types of cytosolic PLA2 with distinct properties and structures are currently identified: the "classic" type IV
Ca2+-dependent cPLA2 and the more
recently characterized type VI Ca2+-independent
PLA2 (iPLA2) (44-46). This latter group is
thought to be principally involved in general membrane phospholipid
remodeling (47), whereas it is the type IV
Ca2+-dependent cPLA2 that has
usually been shown to be associated with the receptor activation of
arachidonic acid release (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 cPLA2 has been generally reported to involve two
distinct processes: a Ca2+-dependent
translocation of the cPLA2 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 modulated by PKC (48). Alternatively, PKC may
itself directly phosphorylate the cPLA2 to increase its activity. The relative importance of the
Ca2+-dependent translocation and the
phosphorylation steps in the activation of cPLA2 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 [Ca2+]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 cPLA2 have generally utilized
maximal (or near-maximal) concentrations of the relevant agonist.
Consequently, although the data presented do not exclude the presence
of these activation pathways in HEK293 cells, it is clear that neither
a Ca2+-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 supported 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
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
Ca2+ entry required to drive receptor-activated oscillatory
[Ca2+]i signals in these and other cells, the
identification of the mechanism responsible for coupling of muscarinic
receptors to arachidonic acid generation at these physiologically
relevant levels of stimulation is clearly of considerable importance.
We thank Dr. Craig Logsdon for generously
providing the HEK293 cells stably transfected with the human m3
muscarinic receptor.
*
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. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
InsP3, inositol 1,4,5-trisphosphate; [Ca2+]i, intracellular free calcium ion concentration; mAChR, muscarinic
receptor; PLC, phospholipase C; PLD, phospholipase D; PLA2, phospholipase A2; PKC, protein kinase C; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid; ETYA, 5,8,11,14-eicosatetraynoic acid; PMA, phorbol 12-myristate
13-acetate.
2
R. Penner, personal communication.
Muscarinic Receptor Activation of Arachidonate-mediated
Ca2+ Entry in HEK293 Cells Is Independent of Phospholipase
C*
and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (53K):
[in a new window]
Fig. 1.
Effect of carbachol on the release of
arachidonic acid in m3-HEK cells. Cells were incubated overnight
in [3H]arachidonic acid and, after washing, exposed to
carbachol (CCh) at different concentrations 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 described under
"Materials and Methods." Values are the means ± S.E.
(n = 4).
View larger version (20K):
[in a new window]
Fig. 2.
Effect of exogenous arachidonic acid on
[Ca2+]i in unstimulated cells. A,
indo-1-loaded cells were exposed to arachidonic acid (arrow)
at the concentrations indicated, and the changes in
[Ca2+]i (measured as the 405/485 nm fluorescence
emission ratio) were determined (see "Materials and Methods" for
details). Traces are superimposed for comparison purposes.
B, an indo-1-loaded cell was exposed to arachidonic acid
(AA; 8 µM; arrow). La3+
(100 µM) was present in the extracellular medium during
the period indicated (solid bar). C, shown is the
effect of exogenous arachidonic acid (8 µM; added at the
point indicated) on the rate of Mn2+ quench in an
indo-1-loaded cell. Mn2+ quenching of intracellular indo-1
( 
), calculated from the corrected sum of the two emitted
fluorescences at 405 and 485 nm (Ftot), was determined
simultaneously with the 405/485 nm fluorescence ratio (continuous
line) as described under "Materials and Methods."
View larger version (46K):
[in a new window]
Fig. 3.
Effect of isotetrandrine on the carbachol
stimulation of increases in arachidonic acid release and total inositol
phosphate 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
presence and absence of 1 µM carbachol (CCh)
and in the presence 1 µM carbachol plus 10 µM isotetrandrine (isotet). Values are the
means ± S.E. (n = 4). PI,
phosphoinositide.
View larger version (22K):
[in a new window]
Fig. 4.
A, effect of isotetrandrine on
carbachol-induced [Ca2+]i oscillations. A cell
loaded with indo-1 was stimulated with carbachol (1 µM)
to induce an oscillatory response in [Ca2+]i,
measured as changes in the 405/485 nm fluorescence ratio. During the
period indicated (solid bar), isotetrandrine (10 µM) was added. B, effect of inhibiting
Ca2+ entry by addition of La3+ (500 µM) on carbachol-induced [Ca2+]i
oscillations. C, effect of isotetrandrine (10 µM; solid bar) on the rate of Mn2+
quench ( , 
) in an oscillating cell. See Fig. 2 legend for
details.
View larger version (18K):
[in a new window]
Fig. 5.
A and B, effect of inhibition
of arachidonic acid metabolism on carbachol-induced
[Ca2+]i oscillations. Indo-1-loaded cells were
stimulated with carbachol (1 µM) to induce oscillations
in [Ca2+]i. During the periods indicated
(solid bars), either indomethacin (A; 10 µM), to inhibit the cyclooxygenase pathway, or
nordihydroguaiaretic acid (B; 10 µM), to
inhibit the lipoxygenase pathway, was present. C, effect of
ETYA on [Ca2+]i in an unstimulated cell. An
indo-1-loaded cell was exposed to ETYA (20 µM; added at
the arrow), and changes in [Ca2+]i
were measured as the 405/485 nm ratio.
View larger version (46K):
[in a new window]
Fig. 6.
Effect of carbachol on the release of
arachidonic acid in BAPTA-loaded cells. Cells, loaded overnight
with [3H]arachidonic acid (AA), were washed
and then loaded with sufficient BAPTA to completely obliterate any
detectable carbachol (CCh)-induced
[Ca2+]i signal by incubation in the presence of
BAPTA/AM, as described under "Materials and Methods." Values are
the means ± S.E. (n = 4).
View larger version (33K):
[in a new window]
Fig. 7.
Effect of PMA on carbachol-stimulated
arachidonic acid release. Cells were loaded overnight with
[3H]arachidonic acid as described under "Materials and
Methods." Experimental cells were then exposed to 100 nM
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 ± S.E. (n = 4).
View larger version (34K):
[in a new window]
Fig. 8.
Effect of U73122 on carbachol-induced
inositol phosphate generation (A) and arachidonic acid
release (B). Total inositol phosphate generation and
arachidonic acid release were measured as described under "Materials
and Methods." Prior to stimulation with carbachol (CCh) at
the concentrations indicated, experimental cells were exposed to U73122
(10 µM) 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 ± S.E. (n = 4 (arachidonic acid (AA)) and n = 3 (total
inositol phosphates (IPs))). PI,
phosphoinositide.
View larger version (19K):
[in a new window]
Fig. 9.
Comparison of the effect of reducing the
extracellular pH on thapsigargin- and arachidonate-induced sustained
elevations in [Ca2+]i. A, an
indo-1-loaded cell was exposed to thapsigargin (1 µM) to
discharge the intracellular Ca2+ stores and to activate
capacitative Ca2+ entry (as reflected in a sustained
increase in [Ca2+]i). During the periods
indicated (solid bars), extracellular pH was reduced to 6.7. B, indo-1-loaded cells were exposed to either 1 µM thapsigargin ( ) or 8 µM arachidonic
acid () to induce a sustained increase in
[Ca2+]i. At the point indicated (solid
bar), extracellular pH was reduced to 6.7.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Dept. of Pharmacology
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{at}pharmacol.rochester.edu.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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