(Received for publication, February 2, 1995; and in revised form, May 17, 1995)
From the
Activation of muscarinic acetylcholine receptors (mAChR) in
human embryonic kidney (HEK) cells stably expressing the human m3
subtype leads to stimulation of both phospholipase C (PLC) and D (PLD).
mAChR-stimulated PLD was turned off after 2 min of receptor activation
with either the full (carbachol) or partial agonist (pilocarpine) and
remained completely suppressed for at least 4 h. Partial recovery was
observed 24 h after agonist removal. This rapid arrest of PLD response
was not due to a loss of cell surface receptors and was also not caused
by negative feedback due to concomitant activation of protein kinase C,
tyrosine phosphorylation, increase in cytosolic calcium, or activation
of G proteins. Furthermore, PLD stimulation by directly
activated protein kinase C and GTP-binding proteins was unaltered in
carbachol-pretreated cells. Finally, neither prevention of PLD
stimulation during carbachol pretreatment by genistein nor inhibition
of protein synthesis by cycloheximide, added before or after carbachol
challenge, resulted in recovery of mAChR-stimulated PLD. The short term
carbachol pretreatment nearly completely abolished agonist-induced
binding of guanosine 5`-O-(3-thiotriphosphate) to membranes or
permeabilized adherent cells. Full recovery of this response was
achieved after 4 h. Similar to transfected m3 mAChR, PLD stimulation by
endogenously expressed purinergic receptors was also fully blunted
after 2 min of agonist (ATP) treatment. Preexposure of HEK cells to
either receptor agonist partially, but not completely, reduced PLD
stimulation by the other agonist. In contrast to desensitization of PLD
stimulation, 2 min of carbachol treatment led to a sensitization, by up
to 2-fold, of mAChR-stimulated inositol phosphate formation. This
supersensitivity was also observed with pilocarpine, which acted as a
full agonist on PLC. On the basis of these results, we conclude that
the m3 mAChR stimulates PLD and PLC in HEK cells with distinct
efficiencies and with very distinct durations of each response. The
rapid and long lasting desensitization of the PLD response is
apparently not due to a loss of cell surface receptors or PLD
activation by GTP-binding proteins, but it may involve, at least
initially, an uncoupling of receptors from GTP-binding proteins and
most likely a loss of an as yet undefined essential transducing
component.
A large number of signal transduction systems utilizes the
PLC()-catalyzed breakdown of phosphoinositides, generating
the two second messengers, inositol 1,4,5-trisphosphate and
diacylglycerol. The former stimulates the release of calcium from the
endoplasmic reticulum, while the latter activates protein kinase
C(1, 2) . In recent years, activation of PLD has been
demonstrated for a variety of hormones, neurotransmitters, and growth
factors, including those that act on receptors that possess intrinsic
tyrosine kinase activity as well as those acting on receptors coupled
to heterotrimeric G proteins(3, 4, 5) . PLD
preferentially hydrolyzes the phospholipid phosphatidylcholine,
resulting in the formation of phosphatidic acid and choline.
Phosphatidic acid may act as an intracellular signaling agent by
itself(6, 7) . Upon deacylation and dephosphorylation
of phosphatidic acid, respectively, the extra- and/or intracellular
signaling molecules, lysophophatidic acid, and diacylglycerol are
formed. Lysophosphatidic acid apparently stimulates cells via a
distinct cell surface receptor coupled to G
proteins(8, 9) . Phosphatidylcholine-derived
diacylglycerol species may activate specific isoforms of protein kinase
C(10, 11) .
Receptor coupling mechanisms to PLC
enzymes have been recently elucidated in great detail. While tyrosine
kinase-linked receptors apparently phosphorylate and thereby activate
distinct PLC isozymes (PLC), receptors coupled to heterotrimeric G
proteins activate PLC
isozymes by either GTP-activated
subunits (
) or free
dimers (for reviews,
see Refs. 12 and 13). Much less clear are the mechanisms that couple
receptors to PLD. For the G protein-coupled receptors, it can be
hypothesized that G proteins are involved, although direct interaction
of PLD with heterotrimeric G proteins or G protein subunits has as yet
not been demonstrated. Recently, evidence has been provided that small
molecular weight G proteins, ADP-ribosylation factors, and Rho
proteins, can stimulate PLD in cell-free
preparations(14, 15, 16, 17) .
The human m3 mAChR, stably expressed in HEK cells, interacts with
both PTX-insensitive G and PTX-sensitive G
proteins in these cells(18) . Receptor activation causes
efficient stimulation of both PLC and PLD in a PTX-insensitive
manner(18, 19) . We recently reported that PLD can be
stimulated in HEK cells by various mechanisms, apparently involving
protein kinase C, calcium-dependent events as well as tyrosine
phosphorylation, and that the coupling of m3 mAChR to PLD is largely
independent of concomitant PLC activation but rather involves a
tyrosine kinase-dependent mechanism(20) . During these studies,
we noted that, in contrast to PLC activation, mAChR-stimulated PLD
apparently rapidly decays(20) . In the present study, we
demonstrate that short term agonist treatment persistently desensitizes
m3 mAChR-stimulated PLD, whereas the receptor-induced stimulation of
PLC is sensitized. The long lasting loss of the PLD response is not due
to receptor internalization or loss of PLD activity, but it seems to be
due to a loss of an as yet undefined essential transducing component.
Protein levels were measured by
the method of Bradford (22) in separate culture dishes. All
experiments were performed in triplicate culture dishes and repeated as
indicated. Formation of [H]PtdEtOH is expressed
as percentage of total phospholipids. [
H]Inositol
phosphate formation is normalized for protein content and is given as
cpm/mg of protein.
In HEK cells stably expressing the human m3 mAChR, the mAChR
agonist carbachol causes a marked activation of PLD measured as
accumulation of its transphosphatidylation product,
PtdEtOH(18, 19, 20) . Interestingly, maximal
levels of labeled PtdEtOH were obtained already after 2 min of
incubation with carbachol (1 mM), followed by a stable plateau
up to at least 30 min of incubation (Fig. 1). The very rapid
cessation of carbachol-induced PtdEtOH formation was not due to the
activation with a supramaximally effective agonist concentration (1
mM). An identical time course of
[H]PtdEtOH formation, although at a lower level,
was obtained when the cells were treated with carbachol at a
half-maximally effective concentration (3 µM) (Fig. 1). To study whether the observed PLD desensitization is
the result of a negative feedback by carbachol-induced increase in
cytoplasmic calcium, activation of protein kinase C, or tyrosine
phosphorylation, the cells were pretreated with 20 µM BAPTA/AM, a condition completely preventing carbachol-induced
increase in cytosolic calcium, 0.1 µM staurosporine,
inhibiting PMA (0.1 µM) induced PLD activation by more
than 90%, or with the tyrosine kinase inhibitor, genistein (10
µM), causing about half-maximal inhibition of
carbachol-induced protein tyrosine phosphorylation in HEK
cells(20) . As illustrated in Fig. 2, none of these
pretreatments had any effect on the shape of the time course of
carbachol-induced PtdEtOH formation, although under all of these
conditions mAChR-stimulated PLD was reduced by 50-60%. The
BAPTA/AM treatment was performed in calcium-free medium, suggesting
that the observed reduction in PLD stimulation is due to the removal of
both extra- and intracellular calcium. The staurosporine-induced
inhibition of mAChR-stimulated PLD is apparently not due to inhibition
of protein kinase C, since it is not mimicked by specific protein
kinase C inhibitors(20) , but it may be due to inhibition of
tyrosine kinases by this rather unselective inhibitor(4) . The
use of the tyrosine kinase inhibitor, genistein, at a maximally
effective concentration was not possible in this type of experiments,
since under this condition genistein completely prevented the
carbachol-induced PLD activation(20) .
Figure 1:
Time course of mAChR-induced PtdEtOH
formation. Formation of [H]PtdEtOH was measured
in m3 mAChR-expressing HEK cells prelabeled with
[
H]oleic acid in the absence (
) and presence
of either 3 µM (
) or 1 mM carbachol
(
) for the indicated periods of time. Data are from one
representative experiment (mean ± S.D., triplicate culture
dishes), characteristic of six similar
ones.
Figure 2:
Influence of BAPTA/AM, staurosporine and
genistein on the time course of mAChR-induced PtdEtOH formation.
[H]Oleic acid-prelabeled HEK cells were
pretreated for 30 min without (Control) and with BAPTA/AM (20
µM), staurosporine (0.1 µM), or genistein (10
µM) as indicated. Thereafter, carbachol (1 mM)
induced [
H]PtdEtOH formation was measured in the
presence of ethanol (400 mM) for the indicated periods of
time. For the BAPTA/AM experiment, extracellular Ca
was additionally removed from the incubation medium. Data are
from one representative experiment (mean ± S.D., triplicate
culture dishes), characteristic of three separate
ones.
In order to exclude
ethanol and PtdEtOH, the PLD product, as causative agents for the rapid
desensitization, HEK cells were pretreated for 2 min with carbachol (1
mM) in the absence of ethanol and LiCl. After wash-out of
carbachol, the cells were immediately restimulated with carbachol in
the presence of ethanol and LiCl. As shown in Fig. 3,
mAChR-mediated stimulation of PLD was abrogated by prior cell treatment
with carbachol. In complete contrast, mAChR-stimulated PLC activity,
measured as accumulation of [H]inositol
phosphates, was not impaired but was significantly (p <
0.01, n = 12) enhanced (by 70-100%) upon
pretreatment of the cells with carbachol.
Figure 3:
Desensitization of mAChR-stimulated PLD
and sensitization of PLC stimulation. m3 mAChR-expressing HEK cells
prelabeled with [H]oleic acid and myo-[
H]inositol were pretreated for 2
min in the absence of ethanol and LiCl without (Control) and
with 1 mM carbachol (Carbacholpretreated),
followed by wash-out of carbachol, as described under
``Experimental Procedures.'' Thereafter, basal and carbachol
(1 mM) stimulated formation of
[
H]PtdEtOH (leftpanel) and myo-[
H]inositol phosphates (rightpanel) were measured for 10 min in the presence of
ethanol (400 mM) and LiCl (10 mM) as indicated. Data
are from one experiment (mean ± S.D., triplicate culture
dishes), representative of six separate
experiments.
Activation of purinergic receptors endogenously expressed in HEK cells (25) by the agonist, ATP (1 mM), stimulated PLD activity to about 30% of the level observed with 1 mM carbachol (Fig. 4); however, with identical time kinetics (data not shown), suggesting that the ATP-stimulated PLD also rapidly desensitizes. Indeed, 2 min of ATP treatment followed by wash-out of the agonist completely prevented subsequent purinergic receptor-mediated PLD stimulation (Fig. 4B). Agonist-induced desensitization can be either homologous or heterologous. Therefore, mutual interactions between activation of mAChRs and purinergic receptors were studied. Carbachol pretreatment (1 mM for 2 min) abolished mAChR-stimulated PLD, while ATP-induced PLD stimulation was still present but significantly (p < 0.01, n = 9) reduced by about 50% (Fig. 4A). Vice versa, after ATP treatment (1 mM for 2 min), carbachol still effectively stimulated PLD, but the response was significantly (p < 0.01, n = 9) reduced by about 25% (Fig. 4B).
Figure 4:
Desensitization of mAChR- and purinergic
receptor-stimulated PLD. m3 mAChR-expressing HEK cells prelabeled with
[H]oleic acid were pretreated for 2 min in the
absence of ethanol without (Control) and with 1 mM carbachol (Carbacholpretreated) (A) or
without and with 1 mM ATP (ATPpretreated) (B), followed by wash-out of carbachol and ATP. Thereafter,
formation of [
H]PtdEtOH was measured for 10 min
in the presence of ethanol (400 mM) without (Basal)
and with 1 mM carbachol or 1 mM ATP as indicated.
Data are from one representative experiment (mean ± S.D.,
triplicate culture dishes), repeated three
times.
To study at which level the PLD response
to mAChR activation was blunted, several possibilities were examined.
First, we investigated whether activation of PLD itself by direct
activation of protein kinase C with the phorbol ester PMA and by direct
activation of heterotrimeric G proteins with
AlF was altered in carbachol-pretreated
intact cells. In contrast to the abolished mAChR stimulation,
pretreatment of the cells for 2 min with carbachol (1 mM) had
no effect on the PMA- and AlF
-induced
activation of PLD (Fig. 5A). Furthermore, pretreatment
of intact HEK cells with carbachol had no effect on stimulation of PLD
by the stable GTP analog GTP
S measured in permeabilized cells (Fig. 5B). Collectively, these data indicated that the
rapid desensitization of the mAChR-induced PLD stimulation was not due
to an inactivation of the PLD enzyme itself and its activation by
protein kinase C and G proteins.
Figure 5:
Influence of carbachol pretreatment on PLD
activation by PMA, AlF, and GTP
S.
[
H]Oleic acid-prelabeled HEK cells were
pretreated for 2 min in the absence of ethanol without (Control) and with 1 mM carbachol (Carbacholpretreated), followed by wash-out of carbachol.
Thereafter, [
H]PtdEtOH formation was measured in
intact cells (A) in the absence (Basal) and presence
of carbachol (1 mM, 10 min), AlF
(10 mM NaF plus 10 µM AlCl
, 60
min) or PMA (0.1 µM, 60 min) as indicated. In addition,
following digitonin permeabilization (B), basal and GTP
S
(100 µM) stimulated formation of
[
H]PtdEtOH was determined as described under
``Experimental Procedures.'' Data are from one representative
experiment (mean ± S.D., triplicate culture dishes),
characteristic of three similar ones.
Second, the rapid desensitization
of the mAChR-stimulated PLD could be due to a loss of cell surface
receptors. However, preexposure of the cells to 1 mM carbachol
for 2 min had no effect on binding of the hydrophilic mAChR antagonist
ligand [H]NMS, used at a receptor-saturating
concentration of 2 nM(26) , to intact HEK cells (Fig. 6A). These data indicated that the
desensitization of the PLD response was not due to m3 mAChR
internalization.
Figure 6:
Influence of carbachol pretreatment on
mAChR number and mAChR-induced GTPS binding. m3 mAChR-expressing
HEK cells were pretreated for 2 min with (filledcolumns) and without (opencolumns) 1
mM carbachol. After wash-out of carbachol, binding of
[
H]NMS to intact cells was determined as
described under ``Experimental Procedures'' (A).
Data are from two experiments (mean ± S.D., quadruplicate
culture dishes). In addition, in membranes of control and
carbachol-pretreated cells, agonist-induced binding of
[
S]GTP
S was determined (B). Data
are from three separate experiments (mean ± S.D.) each performed
in triplicate. Basal [
S]GTP
S binding was
0.90 ± 0.05 pmol and 0.77 ± 0.10 pmol/mg protein in
membranes of control and carbachol-pretreated cells,
respectively.
Third, desensitization of mAChR-induced PLD
activation might be due to a receptor-G protein uncoupling. Therefore,
agonist-induced binding of GTPS to membranes of cells pretreated
or not for 2 min with carbachol (1 mM) was studied.
Pretreatment of intact cells with carbachol caused an almost complete
abrogation of agonist-induced GTP
S binding to membranes of these
cells (Fig. 6B). Similar data were obtained when we
measured GTP
S binding to permeabilized adherent cells pretreated
with carbachol (Fig. 7A). These data suggested that the
rapid mAChR desensitization of PLD activation may be due to uncoupling
of the receptor from the G protein(s) mediating the mAChR action. This
hypothesis was further examined by measuring the time courses of
recovery of agonist-stimulated GTP
S binding and PLD activation
following pretreatment of intact cells with carbachol (1 mM for 2 min). After 4 h of culturing of the cells in agonist-free
medium, the carbachol-induced binding of GTP
S measured in
permeabilized adherent HEK cells was fully restored (Fig. 7A). However, mAChR-stimulated PLD activity
remained completely abolished during this time scale (Fig. 7B). Stimulation of PLD by PMA and
AlF
was not altered (data not shown).
After 24 h of culturing of the cells in agonist-free medium, PLD
stimulation by carbachol was restored by about 50%, with the
carbachol-induced PtdEtOH formation being 0.13 ± 0.02 and 0.23
± 0.02% of total phospholipids in carbachol-pretreated and
untreated control cells, respectively (mean ± S.D., n = 3, from one experiment, representative for two
independent ones). mAChR-stimulated PLC was sensitized by prior short
term carbachol treatment, up to about 2-fold (Fig. 7C).
This supersensitivity to carbachol disappeared after 2 h of cell
culturing in agonist-free medium.
Figure 7:
Time courses of recovery of mAChR-induced
GTPS binding and stimulation of PLD and PLC. A, HEK cells
were pretreated for 2 min with 1 mM carbachol. After wash-out
of carbachol, the carbachol (1 mM) induced GTP
S binding
was determined at the indicated periods of time in permeabilized
adherent cells as described under ``Experimental
Procedures.'' The opensymbol at 0 h indicates
the agonist-induced GTP
S binding to permeabilized cells not
pretreated with carbachol. Basal GTP
S binding was 46 ± 3.6
and 31.5 ± 2.2 fmol/mg of protein in untreated and
carbachol-pretreated cells, respectively. B and C,
HEK cells prelabeled with [
H]oleic acid and myo-[
H]inositol were pretreated for 2
min with 1 mM carbachol in the absence of ethanol and LiCl.
After wash-out of carbachol, the carbachol (1 mM) induced
formation of [
H]PtdEtOH (B) and myo-[
H]inositol phosphates (C)
was measured at the indicated periods of time for 10 min in the
presence of ethanol (400 mM) and LiCl (10 mM). Opensymbols at 0 h indicate the agonist-induced
stimulation of PLD and PLC in cells not pretreated with carbachol.
Basal accumulation of [
H]PtdEtOH was unchanged.
Basal accumulation of myo-[
H]inositol
phosphates was 5.8 ± 1.7
10
and 9.7 ±
1.5
10
cpm/mg of protein in untreated and
carbachol-pretreated cells, respectively. Data are from one experiment
(mean ± S.D., triplicate culture dishes). Identical results were
obtained in two separate experiments.
Fourth, we assessed whether PLD
stimulation during short term agonist treatment was a prerequisite for
the long lasting loss of the mAChR response. Therefore, HEK cells were
pretreated with genistein (100 µM for 30 min) before a
2-min challenge with carbachol, followed by a 4-h recovery in the
absence of either agent. The genistein pretreatment completely
inhibited acute carbachol and AlF stimulation of PLD as reported before(20) , but it did
not prevent the desensitization of mAChR-stimulated PLD (Fig. 8). Independent of whether short term carbachol treatment
was performed in the absence or presence of genistein,
receptor-mediated stimulation of PLD, measured after a 4-h recovery
period, was reduced by more than 80%. On the other hand,
AlF
stimulation of PLD was not affected
by prior carbachol alone treatment and reduced by about 40% in cells
pretreated with genistein and carbachol.
Figure 8:
Influence of genistein on PLD stimulation
and desensitization. HEK cells prelabeled with
[H]oleic acid were pretreated without (Control) and with genistein (100 µM) for 30 min
followed immediately by measurement of [
H]PtdEtOH
accumulation in the absence (Basal) and presence of carbachol
(1 mM, 10 min) or AlF
(10 mM NaF plus 10 µM AlCl
, 60 min) as
indicated. In addition, control (Carbachol) and
genistein-pretreated cells (Genistein + Carbachol) were treated for 2 min with 1 mM carbachol
in the absence of ethanol followed by wash-out of carbachol and
genistein. After a 4-h recovery, basal and carbachol- or
AlF
-stimulated
[
H]PtdEtOH accumulation was determined as
described above. Data are from one experiment (mean ± S.D.,
triplicate culture dishes).
Fifth, the desensitization
of mAChR-stimulated PLD may be due to activation of G proteins not
directly involved in PLD stimulation. We have previously reported that
the m3 mAChR can couple to both PTX-insensitive G proteins and PTX-sensitive G
proteins in HEK
membranes(18) . Although PLD stimulation is PTX-insensitive (18, 20) (Fig. 9A), the concomitant
activation of G
by the m3 mAChR could induce a signal
resulting in the long lasting PLD desensitization. However, as
demonstrated in Fig. 9B, PTX pretreatment (100 ng/ml
for 16 h) did not affect desensitization of mAChR-stimulated PLD. As in
nonintoxicated cells, following 2 min of treatment with 1 mM carbachol, subsequent carbachol-induced PLD stimulation was
completely suppressed in PTX-pretreated cells, even after 4 h of
culturing of the cells in agonist-free medium.
Figure 9:
Influence of PTX on desensitization of
mAChR-stimulated PLD. m3 mAChR-expressing HEK cells were treated for 16
h without (Control) and with 100 ng/ml PTX as described under
``Experimental Procedures.'' A,
[H]PtdEtOH formation was measured in the absence (Basal) and presence of 1 mM carbachol for 10 min in
control and PTX-pretreated cells. B, control and
PTX-pretreated HEK cells were treated for 2 min with 1 mM carbachol in the absence of ethanol followed by wash-out of
carbachol. After a 4-h recovery, basal and carbachol-stimulated
[
H]PtdEtOH formation was determined as described
above. Data are from one representative experiment (mean ± S.D.,
triplicate culture dishes). Similar results were obtained in two
separate experiments.
Finally, to study
whether synthesis of a protein causing a long lasting inhibition of PLD
stimulation is induced by carbachol, the effect of the protein
synthesis inhibitor, cycloheximide, on mAChR-stimulated PLD and its
desensitization was studied. For this, cells were pretreated for 1 h
with 350 µM cycloheximide, which inhibited
[H]leucine incorporation in HEK cells by 95%
(data not shown). In these cells, a similar time course of
[
H]PtdEtOH formation, although at a lower level
(about 50%), was obtained as in untreated controls (Fig. 10A). Furthermore, treatment of HEK cells
immediately after a 2-min carbachol challenge with cycloheximide (350
µM) for 4 h did not result in recovery of mAChR-stimulated
PLD. In the short term carbachol-pretreated cells, agonist-induced
[
H]PtdEtOH formation was reduced to about 6 and
9% of initial response after 4 h of treatment with and without
cycloheximide, respectively (Fig. 10B).
Figure 10:
Influence of protein synthesis inhibition
on desensitization of mAChR-stimulated PLD. A, formation of
[H]PtdEtOH in response to 1 mM carbachol
was measured for the indicated periods of time in
[
H]oleic acid-prelabeled HEK cells pretreated for
1 h without (
) and with (
) 350 µM
cycloheximide. Data are from one experiment (mean ± S.D.,
triplicate culture dishes). B, carbachol (1 mM)
stimulated formation of [
H]PtdEtOH was measured
for 10 min in the presence of ethanol in untreated HEK cells (Con) and in cells pretreated for 2 min with 1 mM
carbachol in the absence of ethanol, followed by wash-out of carbachol
and an additional 4 h of treatment without (Carbpretreated) and with 350 µM cycloheximide (Carb + CHXpretreated). Data are from
one representative experiment (mean ± S.D., triplicate culture
dishes), characteristic of three similar
ones.
The
discrepancy between the largely reduced GTPS binding and the even
enhanced PLC response in the carbachol-pretreated cells suggested that
there exists a large signal reserve in the stimulatory PLC pathway.
Therefore, we studied the effects of the partial mAChR agonist,
pilocarpine(27) , on GTP
S binding and on PLC and PLD
activities. Pilocarpine, at a receptor-saturating concentration of 1
mM, increased GTP
S binding to permeabilized HEK cells by
about 75% of that induced by 1 mM carbachol (61 ± 4.0 versus 79 ± 4.1 fmol/mg protein, n =
8). With regard to activation of PLD, pilocarpine exhibited about 50%
maximal efficiency compared with carbachol (Fig. 11A).
However, similar to the full agonist, carbachol, pretreatment of HEK
cells for 2 min with pilocarpine (1 mM) completely abrogated
subsequent pilocarpine- or carbachol-stimulated PLD. On the other hand,
pilocarpine was a full agonist in activating PLC, both in control cells
and in carbachol-pretreated cells. Similar to carbachol, inositol
phosphate accumulation induced by pilocarpine was almost 2-fold higher
in carbachol-pretreated than in control cells (Fig. 11B).
Figure 11:
Comparison of carbachol- and
pilocarpine-induced stimulation of PLD and PLC. Formation of
[H]PtdEtOH (A) and myo-[
H]inositol phosphates (B)
was measured in the presence of LiCl (10 mM) and ethanol (400
mM) in control HEK cells prelabeled with
[
H]oleic acid and myo-[
H]inositol for 10 min in the
absence (Basal) and presence of 1 mM carbachol or 1
mM pilocarpine as indicated. In addition, after treatment of
[
H]oleic acid-prelabeled HEK cells for 2 min with
1 mM pilocarpine in the absence of ethanol (Pilocarpinepretreated) and of myo-[
H]inositol-prelabeled cells for 2
min with 1 mM carbachol in the absence of LiCl (Carbacholpretreated), followed by wash-out of the agonists, basal
and carbachol- or pilocarpine-stimulated formation of
[
H]PtdEtOH (A) and myo-[
H]inositol phosphates (B)
was measured in the presence of ethanol (400 mM) and LiCl (10
mM), respectively. Data are from one experiment (mean ±
S.D., triplicate culture dishes), representative of three separate
experiments.
Activation of m3 mAChR stably expressed in HEK cells stimulates both PLC and PLD(18, 19, 20) . In addition, receptor activation leads to a rapid increase in cytosolic calcium concentration and protein tyrosine phosphorylation(20) . We recently reported that PLD activity in HEK cells can be stimulated by various mechanisms, apparently involving GTP-binding proteins, calcium, and protein kinase C as well as tyrosine phosphorylation(20) . On the other hand, mAChR-mediated PLD stimulation is apparently independent of concomitant PLC stimulation, including activation of protein kinase C and increase in cytosolic calcium, but it most likely involves PTX-insensitive G proteins and a tyrosine kinase-dependent mechanism(20) . Data presented in this report corroborate this apparent PLC independence of receptor-stimulated PLD.
Recently, rapid desensitization of receptor-stimulated PLD has been reported for various receptors, including m1 and m3 mAChR, in various cellular systems(28, 29, 30, 31, 32) . However, in contrast to m3 mAChR-expressing HEK cells, receptor stimulation of PLD in these cells was protein kinase C-dependent, as demonstrated by the use of inhibitors and/or protein kinase C down-regulation(28, 29, 30, 31, 32) . Since mAChR-stimulated PLC and PLD in HEK cells exhibited distinct kinetics, with rapid cessation of PtdEtOH accumulation in contrast to prolonged accumulation of inositol phosphates(20) , we studied the apparent desensitization of PLD stimulation and possible mechanisms underlying this refractoriness. As demonstrated here, mAChR-stimulated PLD in HEK cells is rapidly and completely desensitized upon receptor activation, whereas PLC stimulation is not impaired but sensitized. The mechanisms possibly underlying the desensitization of the PLD response were analyzed at various levels.
First, we considered that cellular
responses and factors elicited by mAChR activation, in addition to PLD
stimulation, may act as negative feedback inhibitors. However, neither
prevention of cytosolic calcium increase by BAPTA/AM pretreatment, nor
inhibition of protein kinase C by staurosporine, nor inhibition of
mAChR-induced tyrosine phosphorylation by genistein affected the time
kinetics of carbachol-stimulated PtdEtOH formation. Furthermore,
down-regulation of protein kinase C by prolonged PMA treatment and
inhibition of protein kinase C with the selective inhibitor, calphostin
C, did not substantially reduce nor resulted in increased
agonist-induced PtdEtOH accumulation(20) . Finally, although m3
mAChR stimulation of PLD is PTX-insensitive in HEK cells,
agonist-activated receptor has been shown to activate G proteins in membranes of these cells(18) , raising the
possibility that G
and G
-derived signals are
involved in desensitization of PLD stimulation. However, PTX treatment
affected neither direct PLD stimulation nor desensitization of this
response by m3 mAChR. Collectively, these data suggest that
desensitization of mAChR-stimulated PLD is not due to cellular
responses and factors independent of receptor-induced and G
protein-mediated PLD stimulation.
Second, the desensitization was not due to mAChR down-regulation or internalization. Identical kinetics of PtdEtOH accumulation were obtained with a supramaximally and a half-maximally effective concentration of carbachol. Moreover, under conditions where PLD stimulation was completely abrogated, mAChR-stimulated PLC was not impaired. Most important, as observed in Chinese hamster ovary cells transfected with the human m3 mAChR(26) , binding of the hydrophilic mAChR antagonist ligand NMS to intact HEK cells preexposed for 2 min to carbachol, a condition causing complete desensitization of the PLD response, was not affected. In the other cellular systems exhibiting rapid desensitization of receptor-stimulated PLD, participation of receptor internalization or down-regulation was either not studied (28, 29, 30, 32) or was even made likely, e.g. for thrombin and bombesin receptors in CCL39 and Swiss 3T3 fibroblasts, respectively (28, 31) .
Third, the desensitization might be due to PLD products or may occur at the PLD level. The PLD product, PtdEtOH, and its co-substrate, ethanol, could be excluded as causative agents, as full desensitization was obtained in the absence of ethanol and concomitant PtdEtOH formation. The natural PLD product, phosphatidic acid, as causative compound was also made unlikely by the observation that in cells pretreated with genistein, which completely inhibited acute agonist-induced PLD stimulation(20) , mAChR-stimulated PLD was desensitized by a 2-min carbachol treatment. A PLD substrate depletion was made improbable by experiments using a low carbachol concentration that exhibited the same decay of PtdEtOH formation as observed with a maximally effective concentration. Furthermore, treatment of cells with the partial m3 mAChR agonist, pilocarpine, caused the same long lasting desensitization of PLD stimulation as the full receptor agonist, carbachol. Finally, in cells completely insensitive to mAChR stimulation, PLD was fully sensitive to activation by various other stimuli. PMA-stimulated protein kinase C, apparently not involved in m3 mAChR-stimulated PLD in HEK cells(20) , caused an identical increase in PLD activity in desensitized cells as in naive control cells. A similarly unaltered phorbol ester responsiveness despite full desensitization of receptor-stimulated PLD was also reported in other cells(28, 30, 32) . However, since the G protein-linked m3 mAChR and PMA-activated protein kinase C are independent activators of PLD activity in HEK cells, it is feasible that different PLD isozymes (33) or different pools of PLD substrate (34) are utilized.
Since activated receptors and G
proteins apparently stimulate PLD via a common pathway(20) ,
PLD stimulation by directly activated GTP-binding proteins was examined
in mAChR refractory HEK cells. Stimulation of PLD activity in intact
and permeabilized HEK cells, respectively, by
AlF, an activator of heterotrimeric G
proteins, and the stable GTP analog GTP
S, which can activate
heterotrimeric and small molecular weight G proteins, was not affected
by prior carbachol challenge. In contrast, in CCL39 fibroblasts
expressing the human m1 mAChR, desensitization of PLD stimulation was
heterologous, i.e. both mAChR and thrombin receptor as well as
AlF
stimulation of PLD was blunted upon
preexposure to carbachol(28) , suggesting that the
desensitization is due to a modification of more distal components in
the PLD signal transduction cascade. In 1231N1 astrocytoma cells,
treatment with carbachol resulted as well in heterologous
desensitization of receptor-stimulated PLD (m3 mAChR and thrombin
receptor)(32) . Since stimulation of PLD by GTP
S in cell
sonicates was not reduced by prior carbachol treatment (no data on
AlF
stimulation), it was concluded that G
proteins are not uncoupled from PLD(32) . However, recent data
suggest that GTP
S stimulation of PLD activity in cell-free
preparations is mainly due to small molecular weight G proteins
(ADP-ribosylation factors and Rho
proteins)(14, 15, 16, 17) . Thus, it
remains to be seen whether or not PLD stimulation by heterotrimeric G
proteins is unaltered concomitant with the abrogated
receptor-stimulated PLD in 1231N1 astrocytoma cells. In HEK cells,
rapid and complete desensitization of PLD stimulation was not only
observed with the transfected m3 mAChR but also upon activation of
endogenously expressed purinergic receptors. Short term (2 min)
exposure of the cells to either mAChR or purinergic receptor agonist,
followed by agonist wash-out and immediate restimulation with the other
receptor agonist, demonstrated that desensitization of
receptor-mediated PLD stimulation is, at least in part, also
heterologous in this cell type, although no complete
cross-desensitization was observed as in other cellular
systems(28, 32) . This partial heterologous
desensitization may be due to receptor-G protein uncoupling induced,
for example, by activated protein kinase C or G protein-coupled
receptor kinase(s)(35) .
Since both heterotrimeric and small
molecular weight G proteins are apparently involved in mAChR
stimulation of PLD in HEK cells (20) , ()and since
stimulation of PLD by directly activated G proteins was not
desensitized, we considered that receptor activation of G proteins may
be defective in the mAChR refractory cells. Indeed, short term
carbachol treatment almost completely abolished agonist-induced binding
of GTP
S to G proteins, as determined in membrane preparations and
permeabilized adherent cells. However, in measuring the recovery after
removal of carbachol, a complete dissociation of these two events was
observed. Whereas agonist-stimulated GTP
S binding was fully
restored after 4 h of culturing in agonist-free medium,
mAChR-stimulated PLD remained completely suppressed and only partially
(by about 50%) recovered after 24 h. It has to be noted here that,
although m3 mAChR stimulation of PLD and PLC in HEK cells is
PTX-insensitive, receptor-induced binding of GTP
S is to a large
extent to PTX-sensitive G
-type G proteins(18) .
Thus, although overall receptor-stimulated GTP
S binding was fully
restored, the data cannot exclude that receptor coupling to the
PTX-insensitive G protein(s) specifically mediating the PLD response is
defective even up to 24 h after removal of the desensitizing agonist.
In CCL39 fibroblasts, m1 mAChR-stimulated PLD was fully restored 30
min after removal of the agonist(28) , suggesting that rapid
processes, e.g. receptor internalization-externalization
and/or phosphorylation-dephosphorylation, are involved in
desensitization of receptor-stimulated PLD. On the other hand, the very
long lasting desensitization, 24 h, of m3 mAChR-stimulated PLD in
HEK cells suggested that either an inhibitory factor is formed upon
receptor activation or that the activated receptor induces the loss or
permanent inactivation of an essential coupling component. To study
whether the synthesis of an inhibitory protein responsible for the long
lasting inhibition of PLD stimulation is induced by carbachol, cells
were treated with the protein synthesis inhibitor, cycloheximide,
either before or immediately after carbachol challenge. However,
neither treatment prevented the desensitization of mAChR-stimulated
PLD. Thus, we would rather like to suggest that an essential
transducing component is degraded or persistently redistributed and
that new synthesis of this component apparently involved in m3 mAChR
coupling to the PLD-specific G protein(s) is required for
resensitization of the receptor response.
In contrast to the rapid
and long lasting desensitization of receptor-stimulated PLD, mAChR
stimulation of PLC was not desensitized following short term agonist
treatment of HEK cells. On the contrary, 2 min of preexposure to
carbachol led to an about 2-fold increase in receptor-mediated inositol
phosphate formation. This supersensitivity disappeared 2 h after
agonist removal. The mAChR agonist, pilocarpine, which acted as a
partial agonist with regard to PLD stimulation and G protein
activation(27) , behaved as a full agonist in activation of
PLC. Both in naive and in carbachol-pretreated cells, with a largely
reduced agonist-induced GTPS binding but a 2-fold enhanced PLC
response, pilocarpine stimulated inositol phosphate formation to the
same extent as the full receptor agonist, carbachol. These data suggest
that the G proteins mediating m3 mAChR stimulation of PLC, most likely
G
(18) , are very efficiently activated by the
receptor and/or that only a rather small fraction of these G proteins
needs to be activated to cause a full receptor-stimulated PLC. The
apparent PLC supersensitivity observed in HEK cells preexposed to
agonist contrasts with data reported in Chinese hamster ovary cells
transfected with and stably expressing the same human m3
mAChR(26) . In these cells, 5 min of treatment with carbachol
led to an about 50% reduction in initial (first 10 s) inositol
trisphosphate accumulation, while at later time points, no effect of
agonist preexposure was observed. Thus, it remains to be studied
whether in HEK cells the very initial phase of inositol trisphosphate
accumulation is reduced by agonist pretreatment as observed in Chinese
hamster ovary cells. Nevertheless, the 2-fold enhanced accumulation of
inositol phosphates, being mainly inositol monophosphate (data not
shown), observed in agonist-pretreated HEK cells indicates that at
least one of the phases, initial or late, of receptor-stimulated PLC is
supersensitive. Finally, the very distinct time kinetics of loss of PLC
supersensitivity and recovery of PLD stimulation suggest that these two
processes involve distinct molecular mechanisms.
In conclusion, the data presented here indicate that stimulation of PLD in HEK cells by the transfected m3 mAChR and the endogenously expressed purinergic receptor is very rapidly and fully desensitized. As extensively studied on mAChR-stimulated PLD, this rapid desensitization is not due to a loss of cell surface receptors or PLD activation by G proteins, but it may involve an initial receptor uncoupling from the responsive G proteins. On the other hand, the long lasting desensitization of receptor-stimulated PLD is rather due to a loss of an as yet undefined essential transducing component. In contrast to the fully desensitized PLD response, m3 mAChR stimulation of PLC was sensitized by agonist preexposure, indicating that regulation of these two phospholipases by agonist-activated m3 mAChR involves very distinct molecular mechanisms.