Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298
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ABSTRACT |
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This study examined the upstream
signaling pathways initiated by muscarinic m2 and m3 receptors that
mediate sustained ERK1/2- and p38 MAP kinase-dependent phosphorylation
and activation of the 85-kDa cytosolic phospholipase
(cPL)A2 in smooth muscle. The pathway initiated by m2
receptors involved sequential activation of Gi3,
phosphatidylinositol (PI)3-kinase, Cdc42, and Rac1, p21-activated
kinase (PAK1), p38 mitogen-activated protein (MAP) kinase, and
cPLA2, and phosphorylation of cPLA2 at
Ser505. cPLA2 activity was inhibited to the
same extent (61 ± 5 to 72 ± 4%) by the m2 antagonist
methoctramine, G
antibody, pertussis toxin, the PI3-kinase inhibitor
LY 294002, PAK1 antibody, the p38 MAP kinase inhibitor SB-203580, and a
Cdc42/Rac1 GEF (Vav2) antibody and by coexpression of dominant-negative
Cdc42 and Rac1 mutants. The pathway initiated by m3 receptors involved
sequential activation of G
q, PLC-
1, PKC, ERK1/2, and
cPLA2, and phosphorylation of cPLA2 at
Ser505. cPLA2 activity was inhibited to the
same extent (35 ± 3 to 41 ± 5%) by the m3 antagonist
4-diphenylacetoxy-N-methylpiperdine (4-DAMP), the
phosphoinositide hydrolysis inhibitor U-73122, the PKC inhibitor
bisindolylmaleimide, and the ERK1/2 inhibitor PD 98059. cPLA2 activity was not affected in cells coexpressing
dominant-negative RhoA and PLC-
1 mutants, implying that PKC was not
derived from phosphatidylcholine hydrolysis. The effects of ERK1/2 and
p38 MAP kinase on cPLA2 activity were additive and
accounted fully for activation and phosphorylation of
cPLA2.
cytosolic phospholipase A2; cytosolic phospholipase A2 phosphorylation; muscarinic receptors.
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INTRODUCTION |
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PHOSPHOLIPASE (PL)A2 comprises a large group of enzymes that catalyze the hydrolysis of the sn-2-fatty acyl ester bonds of membrane glycerol-phospholipids to yield fatty acid and lysophospholipid (2, 11, 18). The 85-kDa cytosolic PLA2 (cPLA2), a group IV enzyme, possesses several distinctive features including a preference for hydrolysis of arachidonate-containing phospholipids, a dependence on Ca2+ for translocation of the enzyme to specific membrane sites, and a susceptibility to regulatory phosphorylation by various protein kinases (10, 19). Stimulatory phosphorylation at Ser505 and Ser727, chiefly by mitogen-activated (extracellular signal-regulated) protein (MAP) kinases, p40/p42 MAP kinases (ERK1/2), and/or p38 MAP kinase, has been described in several cell systems (3, 4, 12, 16, 20, 31). We (25) have recently demonstrated inhibitory phosphorylation of the 85-kDa cPLA2 by cAMP- and cGMP-dependent protein kinases in intestinal smooth muscle.
Binding of two Ca2+ to Asp43 and Asp93 in the NH2-terminal C2 domain of cPLA2 is essential for the enzyme to gain access to phospholipid-containing membranes. Mutation of either one of these residues yields an inactive cPLA2 (5, 10, 31). Although, Ca2+ binding can occur at resting Ca2+ concentrations, the magnitude of the increase in Ca2+ levels determines the intracellular membrane (Golgi, endoplasmic reticulum, or perinuclear membrane) targeted by the enzyme (9).
Dual phosphorylation of Ser505 and Ser727 by ERK1/2 and/or p38 MAP kinase (mainly the 2a isoform) is required for activation of cPLA2 and acts synergistically with the increase in intracellular Ca2+ (1, 3, 4, 13). The enzyme has a consensus motif for phosphorylation by various MAP kinases, which includes Ser505. Mutation of either Ser505 or Ser727 reduces cPLA2 activity as much as mutation of both residues (13, 16). Both residues are phosphorylated on treatment of human platelets with thrombin or collagen; phosphorylation is entirely dependent on activation of p38 MAP kinase despite the concurrent activation of ERK1/2. p38 MAP kinase phosphorylates Ser505, and a distinct kinase, MAP kinase-interacting kinase 1 (MNK1), that is downstream of p38 MAP kinase, phosphorylates Ser727 (13). Phosphorylation and activation of human platelet cPLA2 by phorbol 12,13-dibutyrate, however, is mediated by ERK1/2 and probably an MNK1-related kinase.
We (21) have previously shown that an early transient activation of cPLA2 and generation of arachidonic acid was confined to intestinal longitudinal muscle in which it caused arachidonic acid-dependent Ca2+ influx that led to Ca2+-induced Ca2+ release via sarcoplasmic ryanodine receptors/Ca2+ channels. Sustained activation of cPLA2 was present also in circular muscle (21). Recent studies (21, 27) on intestinal circular muscle suggested that sustained activation of cPLA2 was partly responsible for regulating capacitative Ca2+ influx. The effect appeared to be mediated by 4,5-epoxyeeicosatrienoic acid, a product of arachidonic acid metabolism via the monooxygenase pathway.
Upstream signaling pathways initiated by G protein-coupled receptors
that eventually result in ERK1/2- and/or p38 MAP kinase-dependent sustained phosphorylation and activation of cPLA2 in smooth
muscle, and other cell types have not been fully explored. The source of PKC responsible for ERK1/2 activity, and the relative contributions of ERK1/2 and p38 MAP kinase to cPLA2 phosphorylation and
activity appear to be receptor and G protein specific (14, 15,
32-34). In this study, we have used freshly dispersed and
cultured smooth muscle cells to identify the pathways initiated by
muscarinic m2 and m3 receptors that lead to phosphorylation and
activation of cPLA2. The pathway initiated by m2 receptors
involved sequential activation of Gi3
phosphatidylinositol (PI)3-kinase
Cdc42 and Rac1
p21-activated
kinase (PAK1)
p38 MAP kinase
cPLA2. The pathway
initiated by m3 receptors involved sequential activation of PLC-
1
PKC
ERK1/2
cPLA2. PKC derived from
phosphatidylcholine hydrolysis by PLC or by RhoA-dependent
phospholipase D (PLD) did not contribute to activation of
cPLA2. The effects of ERK1/2 and p38 MAP kinase on
cPLA2 activity were additive and accounted fully for
activation and phosphorylation of cPLA2.
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MATERIALS AND METHODS |
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Dispersion and culture of intestinal smooth muscle cells. Smooth muscle cells were isolated from circular muscle layer of rabbit intestine by sequential enzymatic digestion, filtration, and centrifugation as described previously (23, 25). For some experiments, the muscle cells were placed in culture in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum until they attained confluence (17).
Expression of dominant-negative RhoA, Cdc42, Rac1, and
PLC-1 cDNA in cultured smooth muscle cells.
Dominant-negative (DN) RhoA (RhoA-DN), Cdc42-DN, Rac1-DN, or
PLC-
1-DN cDNA was subcloned into the multiple cloning site
(EcoRI) of the eukaryotic expression vector pEXV, and a myc
tag was incorporated into the NH2 terminus. Recombinant
plasmid DNAs (2 µg each) were transiently transfected into smooth
muscle cells in primary culture by using Lipofectamine Plus reagent for
48 h. In some experiments, muscle cells were cotransfected with
Cdc42-DN and Rac1-DN or with RhoA-DN and PLC-
1-DN. The cells were
cotransfected with 1 µg of pGreen Lantern-1 to monitor expression.
Control cells were cotransfected with 2 µg of vector (pEXV) and 1 µg of pGreen Lantern-1 DNA. Transfection efficiency (~85%) was
monitored by the expression of green fluorescent protein by using FITC filters.
cPLA2 assay. cPLA2 activity in dispersed muscle cells was measured as described previously (21, 25). Twenty microliters of cell suspension (106 cells/ml) were incubated with [3H]arachidonic acid (1 µCi/ml) at 31°C for 3 h. After labeling, the cells were washed three times with HEPES medium to remove unincorporated [3H]arachidonic acid and then incubated in the presence or absence of ACh in 1 ml of medium. In experiments in which antagonist and inhibitors were used, the cells were preincubated for 10 min. At the end of incubation, the cells were centrifuged at 1,500 g for 10 min and the radioactivity in the supernatant was determined by liquid scintillation counting.
In vitro kinase assays for p38 MAP kinase and ERK1/2.
p38 MAPK and ERK1/2 activities were determined on immunoprecipitates
from cell extracts as described previously (17).
Immunoprecipitates were washed twice with a phosphorylation buffer
containing 10 mM MgCl2 and 40 mM HEPES (pH 7.4) and then
incubated for 5 min on ice with 5 µg of myelin basic protein. Kinase
assays were initiated by the addition of 10 µCi of
[-32P]ATP (3,000 Ci/mmol) and 20 µM ATP, followed by
incubation for 10 min at 37°C. [32P]myelin basic
protein was absorbed onto phosphocellulose discs, and free
radioactivity was removed by repeated washing with 75 mM phosphoric
acid. The extent of phosphorylation was determined from the
radioactivity on phosphocellulose discs by liquid scintillation.
PI3-kinase assay.
PI3-kinase was measured in smooth muscle cells as described previously
(17, 32). Dispersed muscle cells were treated for 5 min
with ACh in the presence or absence of
4-diphenylacetoxy-N-methylpiperdine (4-DAMP) or
methoctramine. After centrifugation for 5 min, 1 ml of lysis buffer was
added to the cells, and incubation was maintained for 20 min at 4°C.
The cell lysates were precleared by centrifugation, and an aliquot was
incubated with 5 µl of PI3-kinase antibody for 2 h at 4°C,
followed by incubation with 30 µl of protein A/G-Sepharose for 2 h at 4°C. The immunoprecipitates were washed with lysis buffer and
Tris · HCl buffer and incubated in a medium containing 1 mg/ml
phosphatidylinositol, 20 mM MgCl2, 10 µCi of
[-32P]ATP (3,000 Ci/mmol) and 20 µM ATP for 10 min
at 37°C. The organic phase containing phosphoinositol phosphates was
analyzed by thin layer chromatography and the spots were visualized by autoradiography.
Phosphorylation of cPLA2 at Ser505. Phosphorylation of cPLA2 was measured by Western blot by using a phospho-specific antibody for phosphorylation at Ser505. Dispersed smooth muscle cells were treated with ACh for different time periods in the presence or absence of 4-DAMP, methoctramine, bisindolylmaleimide, PD 98059, or SB-203580 and solubilized on ice for 1 h in a medium containing 20 mM Tris · HCl (pH 8.0), 1 mM dithiothreitol, 100 mM NaCl, 0.5% SDS, 0.75% deoxycholate, 1 mM PMSF, 10 µg/ml leupeptin, and 100 µg/ml aprotinin. The proteins were resolved by SDS-PAGE and electrophoretically transferred on to polyvinylidene difluoride membranes, which were incubated for 12 h with the phospho-specific antibody and then incubated for 1 h with a horseradish peroxidase-conjugated secondary antibody. The bands were identified by enhanced chemiluminescence.
Materials.
[3H]arachidonic acid and [-32P]ATP were
obtained from New England Nuclear Life Science Products; polyclonal
antibody to phospho-cPLA2 (Ser505) was obtained
from Cell Signaling Technology (Beverly, MA). Antibodies to PAK1,
ERK1/2, and P38 MAP kinase were obtained from Santa Cruz Biotechnologies, and all other reagents were from Sigma. RhoA-DN cDNA
was a gift of Dr. Andrea Todisco (University of Michigan). Cdc42-DN and
Rac1-DN were a gift of Dr. Lee Slice (University of California at Los
Angeles). PLC-
1 was a gift of Dr. Srinivas Pentyala (State
University of New York at Stony Brook).
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RESULTS |
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Phosphorylation and activation of cPLA2 by muscarinic
m2 and m3 receptors.
ACh induced a time-dependent increase in arachidonic acid release from
dispersed intestinal smooth muscle cells, which was sustained for a
period of 20 min (Fig. 1A).
Maximal arachidonic acid release was virtually abolished (91 ± 3% inhibition) by the selective cPLA2 inhibitor
arachidonyltrifluoromethyl ketone (AACOCF3), implying that
arachidonic acid release resulted from cPLA2 activity. cPLA2 activity (arachidonic acid release) measured at 5 min
was partly inhibited by the m3 receptor antagonist 4-DAMP (35 ± 3%) and the m2 receptor antagonist methoctramine (61 ± 6%) and
abolished by a combination of both antagonists (Fig. 1B).
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Inhibition of ACh-stimulated cPLA2 activity and
phosphorylation by PKC, ERK1/2, and p38 MAP kinase inhibitors.
ACh-stimulated cPLA2 activity was inhibited to the same
extent (37 ± 4 to 41 ± 5%) by the ERK1/2 inhibitor PD
98059, the PKC inhibitor bisindolylmaleimide, or a combination of both
inhibitors (Fig. 2). cPLA2
activity was more potently inhibited (72 ± 4%) by the p38 MAP
kinase inhibitor SB-203580 and abolished by a combination of SB-203580
with either PD 98059 or bisindolylmaleimide (Fig. 2). cPLA2
phosphorylation was affected in similar fashion by these inhibitors
with SB-203580 causing more profound inhibition of phosphorylation
(65 ± 6%) than either PD 98059 (27 ± 4%) or
bisindolylmaleimide (32 ± 6%) and virtually abolishing
phosphorylation in combination with either PD 98059 or
bisindolylmaleimide (Fig. 2).
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Activation of ERK1/2 via m3 receptors and p38 MAP kinase via m2
receptors.
ACh-stimulated ERK1/2 activity (97 ± 7% above basal level) was
abolished by 4-DAMP, PD 98059, and bisindolylmaleimide, but was not
affected by methoctramine or SB-203580 (Fig.
4), whereas ACh-stimulated p38 MAP kinase
activity (215% ± 21% above basal level) was abolished by
methoctramine and SB-203580 but was not affected by 4-DAMP, PD 98059, or bisindolylmaleimide (Fig. 4). Results from direct measurement of the
MAP kinases confirmed the conclusions derived from measurement of
cPLA2 in the presence of MAP kinase inhibitors and
muscarinic receptor antagonists. Inhibition of ERK1/2 activity by
bisindolylmaleimide supported the notion that ERK1/2 was downstream of
PKC in the pathway leading to activation of cPLA2 by m3
receptors.
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Source of PKC for m3 receptor-dependent activation of ERK1/2 and
cPLA2.
Earlier studies on intestinal smooth muscle cells have shown that
agonist-stimulated PKC activity is derived initially from phosphoinositide hydrolysis by PLC- and subsequently from
phosphatidylcholine hydrolysis by a RhoA-dependent PLD and a
phosphatidylcholine-specific PLC (23, 29). ACh-stimulated,
PKC-dependent ERK1/2 activity was not affected by expression of a
RhoA-DN in cultured smooth muscle cells, or coexpression of Cdc42-DN
and Rac1-DN (Fig. 5). We considered the
possibility that in cells expressing RhoA-DN, PKC could be derived from
activation of PLC-
1, which is normally repressed by RhoA and is
derepressed in cells expressing RhoA-DN. However, cPLA2
activity was not affected in cells expressing RhoA-DN, PLC-
1-DN, or
coexpressing RhoA-DN and PLC-
1-DN (Fig.
6). The functionality of these mutants is
supported by the fact that expression of RhoA-DN inhibits
ACh-stimulated RhoA activity, whereas expression of Cdc42-DN and/or
Rac1-DN inhibits ACh-stimulated PAK1 activity (29, 30).
Expression of PLC-
-DN inhibits Ca2+-stimulated PLC-
activity (26).
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Role of Cdc42, Rac1, and PAK1 in m2 receptor-dependent activation of p38 MAP kinase and cPLA2. Our recent studies (30) have shown that m2 receptors are coupled to sequential activation of Cdc42/Rac1 and PAK1. ACh-stimulated PAK1 activity was partly inhibited in smooth muscle cells expressing Cdc42-DN or Rac1-DN and abolished in cells coexpressing both mutants. PAK1 activity was not affected by SB-203580 implying that PAK1 is upstream of p38 MAP kinase (30). In the present study, ACh-stimulated p38 MAP kinase activity was abolished in cultured smooth muscle cells coexpressing Cdc42-DN and Rac1-DN but was not affected in cells expressing RhoA-DN (Fig. 5). Activation of p38 MAP kinase by Cdc42 and Rac1 was probably mediated by PAK1 (6). Treatment of permeabilized smooth muscle cells for 60 min with PAK1 antibody (5 µg/ml) inhibited m2 receptor-dependent cPLA2 activity (85 ± 7% inhibition) but had no effect on m3 receptor-dependent activity (6 ± 8% inhibition).
ACh-stimulated cPLA2 activity was strongly inhibited (64 ± 5%) in cultured smooth muscle cells coexpressing Cdc42-DN and Rac1-DN (Fig. 8) but was not affected in cells expressing RhoA-DN (vector alone, 4976 ± 549; RhoA-DN, 4,798 ± 662 counts/min (cpm)/mg protein above basal level). Residual cPLA2 activity in cells coexpressing Cdc42-DN and Rac1-DN was abolished by 4-DAMP, bisindolylmaleimide, and PD 98059 but was not affected by SB-203580, implying that it reflected the m3 receptor-dependent response mediated by PKC and ERK1/2 (Fig. 8).
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Role of Gi3 and PI3-kinase in m2 receptor-dependent
activation of cPLA2 activity.
ACh stimulated PI3-kinase activity, which was abolished by the
selective PI3-kinase inhibitor LY 294002 and by methoctramine but was
not affected by 4-DAMP (Fig. 10).
ACh-stimulated cPLA2 activity was inhibited to the same
extent (59 ± 5 to 62 ± 4%) by pretreatment of dispersed
smooth muscle cells for 60 min with PTx (400 ng/ml) or for 10 min with
LY 294002 (10 µM), and by pretreatment of saponin-permeabilized
smooth muscle cells with a common G antibody (10 µg/ml) (Fig. 11).
Inhibition of cPLA2 activity by PTx, G
antibody, and LY
294002 was similar to that elicited by methoctramine (Fig.
11). The combination of methoctramine
with PTx, G
antibody, or LY 294002 was not additive, whereas
the combination of 4-DAMP with each of the three agents abolished
ACh-stimulated cPLA2 activity (Fig. 11). The inhibitory
effects of PTx and G
antibody implied m2 receptor-dependent
cPLA2 activity was mediated by G
i3. The
inhibitory effect of LY 294002 implied that G
was linked via
PI3-kinase to downstream effectors (Cdc42/Rac1
PAK1
p38 MAP
kinase
cPLA2).
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DISCUSSION |
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This study identified the upstream signaling pathways initiated by
muscarinic m2 and m3 receptors that lead to sustained phosphorylation and activation of cPLA2 in smooth muscle. Our earlier
studies (24, 30) have shown that m2 receptors, the
predominant species in smooth muscle (~80% of the total), are
coupled via Gi3 to inhibition of adenylyl cyclase and
via G
i3 to transient activation of PLC-
3 and
sustained, sequential activation of Cdc42/Rac1 and PAK1. The latter
mediates inhibitory phosphorylation of myosin light chain kinase
(30) and, as shown in the present study, stimulation of
p38 MAP kinase and activation of cPLA2. Furthermore, the
present study shows that PI3-kinase acts as the link between G
i3 and the downstream pathway.
Previous studies have also shown that m3 receptors, like other
Gq coupled receptors (30), are coupled via
Gq to activation of PLC-
1 and phosphoinositide
hydrolysis, and via G13 and RhoA to sustained activation of
Rho kinase and PLD. phosphatidylcholine hydrolysis by PLD yields
phosphatidic acid, which is dephosphorylated to diacylglycerol, causing
sustained activation of PKC (23). Phosphatidylcholine
hydrolysis by PLC also yields diacylglycerol and contributes to
sustained activation of PKC (23). Neither pathway,
however, was responsible for sustained PKC-dependent, ERK1/2-mediated
activation of cPLA2, which appeared to depend on the
initial PKC activity derived from phosphoinositide hydrolysis. This
pathway contributes only a fraction, about one-third, of ACh-stimulated
cPLA2 activity. The remainder is mediated via m2 receptors.
The linkage between m2 receptors and cPLA2 involved
sequential activation of Gi3 and PI3-kinase;
cPLA2 activity was inhibited to the same extent by
methoctramine, PTx, a common G
antibody, and the PI3-kinase
inhibitor LY 294002 (Figs. 1 and 11). When m3 receptor-mediated
responses were blocked with 4-DAMP, treatment with PTx, G
antibody,
or LY 294002 abolished ACh-stimulated cPLA2 activity. It is
worth noting that blockade of every step in the pathway initiated by m2
receptors (G
i3
PI3-kinase
Cdc42/Rac1
PAK1
p38 MAP kinase
cPLA2) elicited the same
degree of inhibition (61 ± 5 to 72 ± 4%) of
cPLA2 activity or phosphorylation, whether the step
involved m2 receptor antagonism with methoctramine (Fig. 1), uncoupling
of m2 receptors from Gi3 with PTx (Fig. 11A),
neutralization of G
activity with G
antibody (Fig.
11A), inhibition of PI3-kinase activity with LY 294002 (Fig.
11A), inactivation of Cdc42/Rac1 by expression of
dominant-negative mutants (Fig. 8) or with specific inhibitor (Vav2
antibody) (Fig. 9), immunoneutralization of PAK1, and inhibition of p38
MAP kinase activity with SB-203580 (Fig. 2).
Unexpectedly, the PKC responsible for m3 receptor-dependent sustained
activation of ERK1/2 and cPLA2 was generated from
phosphoinositide hydrolysis, which in smooth muscle cells occurs
predominantly in the first 2 min after receptor activation before
reverting to a low suprabasal level (23). Our previous
studies in intestinal smooth muscle have shown that sustained PKC
activity is predominantly generated via phosphatidylcholine hydrolysis
via RhoA-activated PLD (23, 29). Expression of RhoA-DN in
these cells abolishes RhoA and PLD activities (29, 30) but
had no effect on ACh-stimulated cPLA2 activity. We
considered the possibility that inactivation of RhoA could suppress its
inhibitory effect on phosphoinositide-specific PLC-1 leading to
generation of PKC; however, coexpression of RhoA-DN and PLC-
1-DN had
no effect on ACh-stimulated cPLA2 activity. We concluded
that inhibition of ERK1/2 activity by U-73122 and bisindolylmaleimide
reflected inhibition of PKC derived from phosphoinositide hydrolysis by
PLC-
1.
In their studies of iris smooth muscle, Husain and Abdel Latif
(14, 15) reported agonist-dependent differences in the contribution of ERK1/2 and p38 MAP kinase to phosphorylation and activation of cPLA2. Both ERK1/2 and p38 MAP kinase were
involved in phosphorylation and activation of cPLA2 by the
muscarinic agonist carbachol (15). Upstream pathways
initiated by m3 and m2 receptors were not examined, except for the
observation that both MAP kinases were dependent on PKC, in contrast
with the present study in which only ERK1/2 activation was dependent on
PKC. With PGF2 and endothelin (14, 15),
however, although ERK1/2 and p38 MAP kinase were activated, only the
latter appeared to be involved in phosphorylation and activation of
cPLA2. Studies (11, 12, 18, 31) on peritoneal
macrophages have demonstrated the ability of Ca2+
(Ca2+ ionophores) or MAP kinase-dependent phosphorylation
(phorbol esters and phosphatase inhibitors) to activate
cPLA2 independently or synergistically. In thrombin- or
collagen-stimulated human platelets, activation of cPLA2 by
Ca2+ appears to predominate over activation induced by
cPLA2 phosphorylation via p38 MAP kinase (3, 4,
16). The extent of Ca2+ mobilization determined the
specific intracellular membranes targeted by cPLA2
(9).
Although other investigators (1) have detected a negative control of ERK1/2 by p38 MAP kinase, we were unable to detect interplay between ERK1/2 and p38 MAP kinase in the present study. Inhibition of either enzyme had no effect on their activation by ACh or on their ability to activate cPLA2.
We have previously shown that an early transient activation of cPLA2 and generation of arachidonic acid was confined to intestinal longitudinal muscle in which it caused arachidonic acid-dependent Ca2+ influx that led to Ca2+-induced Ca2+ release via sarcoplasmic ryanodine receptors/Ca2+ channels. Sustained activation of cPLA2 was present also in circular muscle (21 and present study). Recent studies on intestinal circular muscle suggested that sustained activation of cPLA2 was partly responsible for regulating capacitative Ca2+ influx. The effect appeared to be mediated by 4,5-epoxyeeicosatrienoic acid, a product of arachidonic acid metabolism via the monooxygenase pathway (21, 27).
In summary, we have characterized the upstream signaling pathways
initiated by muscarinic m2 and m3 receptors that lead to phosphorylation and activation of cPLA2. The study shows
that the pathways initiated by m2 receptors involved sequential
activation of Gi3
PI3-kinase
Cdc42 and Rac1
PAK1
p38 MAP kinase
cPLA2, whereas the pathways
initiated by m3 receptors involved sequential activation of PLC-
1
PKC
ERK1/2
cPLA2. Phosphorylation and activation of cPLA2 were independently and additively
mediated by m3 receptor-dependent ERK1/2 and m2 receptor-dependent p38 MAP kinase.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-15564.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. S. Murthy, P.O. Box 980711, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298 (E-mail: skarnam{at}hsc.vcu.edu).
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.
10.1152/ajpgi.00345.2002
Received 16 August 2002; accepted in final form 13 November 2002.
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