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J Biol Chem, Vol. 273, Issue 51, 34519-34526, December 18, 1998
From the Departments of Physiology and Medicine, Medical College of
Virginia, Virginia Commonwealth University,
Richmond, Virginia 23298-0711
Both cAMP- and
cGMP-dependent protein kinases inhibit agonist-stimulated
phospholipase C- Phospholipases A2
(PLA2s),1, which
catalyze the hydrolysis of the Sn2 fatty acyl bond of phospholipids to
yield free fatty acids and lysophospholipids, have been divided into
two main categories comprising up to nine groups (1, 2). One category
(Groups I, II, III, V, VII, and IX) includes the small molecular
weight, secretory PLA2s, which are
Ca2+-dependent except for Group VII
PLA2 (3-6). The other category includes large molecular
weight, Ca2+-dependent (Group IV) and
Ca2+-independent (Groups VI and VIII) cytosolic
PLA2s (7-9). The Ca2+-independent, Group VII
secretory PLA2 and the
Ca2+-dependent, Group VIII cytosolic
PLA2s are specific for platelet-activating factor and may
be viewed as platelet-activating factor acetyl hydrolases (2, 10). The
Ca2+-independent, Group VI cytosolic PLA2 is
involved in the continuous recycling of phospholipids and incorporation
of arachidonic acid (11, 12).
Group IV cytosolic PLA2 (cPLA2) possesses
several distinctive features, including a dependence on submicromolar
concentrations of Ca2+ essential for translocation of the
enzyme via a Ca2+ and phospholipid binding domain, a
preference for hydrolysis of arachidonate-containing phospholipids, and
a susceptibility to regulatory phosphorylation by mitogen-activated
protein kinase and protein kinase C (13-18). There is evidence that
phosphorylation by protein kinase C may be mediated indirectly by
mitogen-activated protein kinase (19). Phosphorylation by
mitogen-activated protein kinase on serine residues (chiefly
Ser505) or tyrosine residues is associated with increase in
cPLA2 activity (14, 17, 18, 20, 21). Phosphorylation by
cAMP- or cGMP-dependent protein kinase in vivo
has not been characterized.
Agonist-induced, G protein-dependent activation of
cPLA2 has been demonstrated in dispersed intestinal smooth
muscle cells and is the initial trigger for Ca2+
mobilization in muscle cells of the longitudinal layer (22). An initial
transient increase in cPLA2 activity occurs only in smooth
muscle cells from the intestinal longitudinal layer and coincides with
the initial Ca2+ transient. This is followed by a sustained
increase in cPLA2 activity, which is partly dependent on
activation of protein kinase C. The initial increase in
cPLA2 activity in longitudinal muscle leads to arachidonic
acid (AA)-induced stimulation of Ca2+ influx, which
triggers Ca2+ release from sarcoplasmic stores via
ryanodine receptor/Ca2+ channels and stimulates the
activity of ADP-ribosylcyclase. The resultant increase in cADP ribose
enhances Ca2+-induced Ca2+ release (22-24).
Unlike longitudinal smooth muscle, agonist-stimulated Ca2+
mobilization in smooth muscle of the circular layer is mediated by
phospholipase C- The initial steps in Ca2+ mobilization in vascular and
intestinal circular smooth muscle (i.e. activation of
PLC- Dispersion of Intestinal Smooth Muscle Cells--
Smooth muscle
cells were isolated from the longitudinal muscle layer of rabbit
intestine by sequential enzymatic digestion, filtration, and
centrifugation as described previously (22-24). Muscle strips were
incubated for 30 min at 31 °C in 15 ml of HEPES medium containing
0.1% collagenase (type II) and 0.1% soybean trypsin inhibitor with no
added Ca2+. The composition of the medium was 120 mM NaCl, 4 mM KCl, 2.6 mM
KH2PO4, 0.6 mM MgCl2,
25 mM HEPES, 14 mM glucose, and 2.1% Eagle's
essential amino acid mixture. The partly digested tissue was washed
with 100 ml of enzyme-free medium and reincubated for 30 min to allow
spontaneous dispersion of muscle cells. The cells were harvested by
filtration through 500-µm Nitex mesh, centrifuged twice for 10 min at
350 × g, and resuspended in HEPES medium containing 2 mM Ca2+.
AA-induced Contraction of Dispersed Smooth Muscle
Cells--
Contraction of dispersed muscle cells was measured by
scanning micrometry as described previously (22-24). An aliquot (0.5 ml) of cells (104 cells/ml) was added to 0.2 ml of medium
containing 1 µM AA in the presence of the cyclooxygenase
inhibitor indomethacin (10 µM) and the lipoxygenase
inhibitor nordihydroguaiaretic acid (10 µM). The reaction
was terminated after 30 s with acrolein. Inhibition of contraction
(i.e. relaxation) was measured in muscle cells maximally
contracted for 30 s with AA (1 µM). Relaxation was
expressed as the increase in the length of AA-contracted muscle cells
(mean resting muscle cell length, 118 ± 5 µm; mean length of
AA-contracted muscle cells, 83 ± 2 µm).
AA-induced Ca2+ Influx--
Ca2+ influx
in dispersed muscle cells was measured from the initial uptake of
45Ca2+ in the presence of anti-mycin and
thapsigargin to prevent uptake into mitochondrial and sarcoplasmic
Ca2+ stores. Dispersed muscle cells were suspended in 10 ml
of HEPES medium containing 2 mM Ca2+ and
45Ca2+ (10 µCi/ml), with thapsigargin (2 µM) and anti-mycin (10 µM). The muscle
cells were treated for 60 s with a relaxant agent (Na nitroprusside (SNP) and isoproterenol) or protein kinase activator (cBIMPS and 8-pcCPT-cGMP), followed by addition of 1 µM
AA for 2 min. Samples (0.5 ml) were withdrawn at intervals for
measurement of 45Ca2+ cell content. The muscle
cells were centrifuged and washed twice with HEPES medium, and the
45Ca2+ cell content was measured and expressed
as cpm/106 cells.
Phosphorylation of cPLA2--
Phosphorylation of
cPLA2 was measured from the amount of
[32P]ATP incorporated into the enzyme after
immunoprecipitation with specific cPLA2 antibody. Dispersed
smooth muscle cells (10 ml, 4 × 106 cells/ml) were
prelabeled with 0.5 mCi/ml [32P]orthophosphate for 3 h. Samples (0.5 ml) were incubated with various relaxant agents for
60 s in the presence or absence of cAMP-dependent
protein kinase inhibitors (H-89 or myristoylated PKI) or
cGMP-dependent protein kinase inhibitor (KT-5823), and the
reaction was terminated with an equal volume of lysis buffer (final
concentrations, 1% Triton X-100, 0.5% SDS, 0.75% deoxycholate, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 100 µg/ml aprotinin, 10 mM
Na4P2O7, 50 mM NaF, 0.2 mM Na3VO4) and placed on ice for 30 min. The cell lysates were separated from the insoluble material by
centrifugation at 13,000 × g for 15 min at 4 °C,
precleared with 40 µl of protein A-Sepharose CL-4B, and incubated
with polyclonal rabbit cPLA2 antibody for 2 h at 4 °C and with 40 µl of protein A-Sepharose CL-4B for another
1 h. The immunoprecipitates were collected, washed five times with 1 ml of wash buffer (0.5% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4), extracted with Laemmli sample buffer,
boiled for 15 min, and separated on 10% SDS-polyacrylamide gel
electrophoresis. After transfer to polyvinylidene difluoride membranes,
32P-labeled cPLA2 was visualized by
autoradiography, and the amount of radioactivity in the band was measured.
Agonist-mediated Stimulation of cPLA2
Activity--
cPLA2 activity in dispersed muscle cells was
measured by an adaptation of the method of Damron et al.
(33) as described previously (22). Twenty ml of cell suspension
(106 cells/ml) were incubated with [3H]AA (1 µCi/ml) at 31 °C for 3 h. The cells were diluted with 50 ml
of HEPES medium, centrifuged at 350 × g for 15 min,
and then resuspended in 10 ml of fresh medium containing 10 µM indomethacin and 10 µM
nordihydroguaiaretic acid to inhibit AA metabolism via the lipoxygenase
and cyclooxygenase pathways. Duplicate samples (106
cells/0.5 ml) were incubated at 31 °C with 1 nM
cholecystokinin octapeptide (CCK-8) for 30 s, and the reaction was
terminated with 1.8 ml of chloroform/methanol/HCl (100:200:2, v/v/v).
The phases were separated with 0.6 ml of chloroform and 0.6 ml of 2 mM HCl. The organic phase was dried under nitrogen,
resuspended in 50 µl of chloroform/methanol (9:1), and spotted on
silica gel plates for thin layer chromatography using
hexane/ethylether/acetic acid (70:30:3.5). The radioactivity in spots
corresponding to AA was counted, and the results were expressed as
cpm/106 cells above basal levels.
Materials--
[3H]arachidonic acid (210 Ci/mmol),
carrier-free [32P]Pi, and
45Ca2+ were obtained from NEN Life Science
Products; indomethacin, nordihydroguaiaretic acid, and PKI were from
Biomol; polyclonal antibodies to cPLA2 were from Santa
Cruz; HEPES was from Research Biochemicals; thapsigargin and H-89 were
from Calbiochem; CCK-8 and vasoactive intestinal peptide (VIP) were
from Bachem; KT-5823 was from Kamiya Biomedical (Thousand Oaks, CA);
8-pcCPT-cGMP and cBIMPS were from Alexis Corp. (San Diego, CA); and all
other reagents were from Sigma.
Phosphorylation of cPLA2 in Intestinal Smooth Muscle by
cAMP- and cGMP-dependent Protein Kinases--
Selective
activators of cGMP-dependent protein kinase (8-pcCPT-cGMP)
and cAMP-dependent protein kinase (cBIMPS) and three relaxant agents were used to determine the ability of cAMP- and cGMP-dependent protein kinases to phosphorylate
cPLA2 in smooth muscle: SNP, which stimulates cGMP and
selectively activates cGMP-dependent protein kinase at
concentrations <1 µM; isoproterenol, which stimulates cAMP and preferentially activates cAMP-dependent protein
kinase at low concentrations (<1 µM) but can
cross-activate cGMP-dependent protein kinase at higher
concentrations; and VIP, which stimulates both cAMP and cGMP in gastric
smooth muscle and activates cAMP- and cGMP-dependent
protein kinases at all concentrations (32, 34-36). The
cAMP-dependent protein kinase inhibitors H-89 and
myristoylated PKI and the cGMP-dependent protein kinase
inhibitor KT-5823 were used to evaluate the involvement of each kinase
in cPLA2 phosphorylation. Previous studies (32) of cAMP-
and cGMP-dependent protein kinase activity in dispersed
muscle cells had shown that at concentrations of
Both 8-pcCPT-cGMP and cBIMPS increased cPLA2
phosphorylation in dispersed muscle cells by 406 ± 58%
(p < 0.01) and 343 ± 40% (p < 0.01), respectively. Phosphorylation induced by 8-pcCPT-cGMP was
abolished by KT-5823 (98 ± 2% inhibition) but was not affected by H-89 or myristoylated PKI, whereas phosphorylation induced by cBIMPS
was abolished by H-89 (97 ± 2% inhibition) and myristoylated PKI
(99 ± 3% inhibition) but was not affected by KT-5823 (Fig. 1). SNP, isoproterenol (1 µM), and VIP also increased cPLA2
phosphorylation by 515 ± 67% (p < 0.01),
262 ± 23% (p < 0.01), and 459 ± 52%
(p < 0.01), respectively (Fig.
2). The phosphorylation induced by SNP
was abolished by KT-5823 (98 ± 2% inhibition) but was not affected by H-89 or myristoylated PKI, whereas phosphorylation induced
by 1 µM isoproterenol was abolished by H-89 (99 ± 4% inhibition) and myristoylated PKI (98 ± 3% inhibition) but
was not significantly affected by KT-5823. Phosphorylation induced by 1 µM VIP was weakly inhibited by myristoylated PKI and H-89
(17 ± 3% inhibition; p < 0.02) but was not
affected by KT-5823; however, a combination of H-89 and KT-5823
virtually abolished cPLA2 phosphorylation (87 ± 7%
inhibition). Because VIP activates both cAMP- and
cGMP-dependent protein kinases, minimal inhibition by each
kinase inhibitor separately and virtually complete inhibition by a
combination of both inhibitors suggest interplay of the two kinases
when activated concurrently.
Inhibition of cPLA2 Activity by cAMP- and
cGMP-dependent Protein Kinases--
Previous studies have
shown that activation of cPLA2 by contractile agonists
(e.g. CCK-8) is biphasic, with an initial peak of activity
during the first minute followed by a sustained phase of activity that
is partly mediated by protein kinase C (22). Phosphorylation of
cPLA2 was not present during the initial phase (1 min) of
cPLA2 activity but increased significantly during the sustained phase (10 min after treatment with CCK-8). Preincubation of
the cells with the protein kinase C inhibitor calphostin C (1 µM) abolished the increase in cPLA2
phosphorylation during the sustained phase and, as shown previously
(22), inhibited cPLA2 activity by 40% (Fig.
3).
The initial peak of cPLA2 activity induced by CCK-8
(431 ± 41 cpm arachidonic acid/106 cells in the
presence of cyclooxygenase and lipoxygenase inhibitors) was inhibited
by 1 µM isoproterenol (36 ± 8% inhibition;
p < 0.01) and by the selective
cAMP-dependent protein kinase activator cBIMPS (54 ± 2%; p < 0.001). The inhibition of cPLA2
activity by either agent was completely reversed by H-89 and PKI but
was not affected by KT-5823 (Fig. 4). The
initial peak of CCK-stimulated cPLA2 activity was also
inhibited by SNP (75 ± 5%, p < 0.01) and the selective cGMP-dependent protein kinase activator
8-pcCPT-cGMP (51 ± 2%, p < 0.001); the
inhibition by either agent was completely reversed by KT-5823 but was
not affected by H-89 or PKI (Fig. 5). A
higher concentration of isoproterenol (100 µM),
which activates cAMP-dependent protein kinase and
cross-activates cGMP-dependent protein kinase, inhibited
CCK-stimulated cPLA2 activity by 83 ± 4%
(p < 0.001); the inhibition was partly reversed by
H-89 and PKI to 55 ± 3 and 56 ± 6%, respectively, and by
KT-5823 to 66 ± 5% and was completely reversed by a combination
of KT-5823 with either H-89 or PKI (Fig.
6). Similarly, VIP (1 µM),
which activates both cAMP- and cGMP-dependent protein
kinases, inhibited CCK-stimulated cPLA2 activity by 88 ± 4% (p < 0.001); the inhibition was partly reversed
by H-89 and PKI to 58 ± 6 and 55 ± 7%, respectively, and
to a lesser extent by KT-5823 (73 ± 6%) and was completely reversed by a combination of both inhibitors (Fig. 6).
Selective Inhibition of AA-induced Ca2+ Influx by
cGMP-dependent Protein Kinase--
We have previously
shown that endogenous formation of AA stimulates Ca2+
influx in intestinal longitudinal muscle cells; the effect could be
reproduced by addition of nanomolar concentrations of AA (22). In the
present study, the rate of Ca2+ influx was measured from
the initial uptake of 45Ca2+ (2869 ± 510 cpm/106 cells) induced by exogenous AA in the presence of
cyclooxygenase and lipoxygenase inhibitors. AA-induced Ca2+
influx was not affected by selective activation of
cAMP-dependent protein kinase with cBIMPS (3115 ± 321 cpm/106 cells) or 1 µM isoproterenol
(2816 ± 636 cpm/106 cells). A higher concentration of
isoproterenol (100 µM) inhibited AA-induced
Ca2+ influx by 84 ± 4% (p < 0.001);
the inhibition was completely reversed by KT-5823 but was not affected
by H-89 (Fig. 7), implying that
inhibition was dependent on cross-activation of
cGMP-dependent protein kinase by higher concentrations of
isoproterenol.
Consistent with selective inhibition of Ca2+ influx by
cGMP-dependent protein kinase, SNP and 8-pcCPT-cGMP
inhibited AA-induced Ca2+ influx by 83 ± 4%
(p < 0.001) and 62 ± 8% (p < 0.01), respectively; the inhibition by both SNP and 8-pcCPT-cGMP was
completely reversed by KT-5823 but was not affected by H-89 (Fig.
8).
Inhibition of AA-induced Muscle Contraction by cAMP- and
cGMP-dependent Protein Kinases--
AA-stimulated
Ca2+ influx in intestinal longitudinal smooth muscle cells
leads to Ca2+-induced Ca2+ release from
sarcoplasmic stores in intestinal longitudinal smooth muscle cells; the
resultant increase in [Ca2+]i triggers an initial
muscle contraction (22-24). AA-induced muscle contraction (36.2 ± 2.9 µm decrease in muscle cell length) was inhibited by activators
of cGMP-dependent protein kinase (SNP and 8-pcCPT-cGMP) and
cAMP-dependent protein kinase (1 µM
isoproterenol and cBIMPS) (Figs. 9 and
10). The inhibition of contraction
(i.e. relaxation) induced by SNP and 8-pcCPT-cGMP was
selectively blocked by KT-5823 (Fig. 9), whereas the inhibition of
contraction induced by 1 µM isoproterenol and cBIMPS was
selectively blocked by H-89 (Fig. 10). Thus, although
cAMP-dependent protein kinase had no effect on AA-induced
Ca2+ influx, it inhibited contraction by acting at one or
more loci distal to AA-induced Ca2+ influx.
Agonist-induced activation of PLC- Ca2+ mobilization in smooth muscle from the intestinal
longitudinal layer, however, differs markedly in that it is initiated by G protein-dependent activation of cPLA2 and
generation of arachidonic acid; the latter triggers Ca2+
influx and induces Ca2+ release from sarcoplasmic stores
via ryanodine receptors/Ca2+ channels (22, 23). Minimal
amounts of IP3 are produced in this smooth muscle, which is
virtually devoid of IP3 receptors/Ca2+ channels
(23, 26). In this study we show that by analogy with other types of
smooth muscle, the initial steps in Ca2+ mobilization in
longitudinal smooth muscle, i.e. activation of cPLA2 and arachidonic acid-induced Ca2+ influx,
are inhibited by cAMP- or cGMP-dependent protein kinase. It
is possible that more distal targets involved in Ca2+
mobilization in intestinal longitudinal muscle, such as ryanodine receptors/Ca2+ channels, Ca2+-ATPase pumps, and
plasmalemmal Ca2+ and K+ channels, are also
susceptible to regulatory phosphorylation by cAMP- and
cGMP-dependent protein kinases, but these were not examined
in the present study.
Agonist-induced phosphorylation of cPLA2 in various cell
types is accompanied by increase in cPLA2 activity, which
appears to be mediated by protein kinase C-dependent and
-independent activation of mitogen-activated protein kinase (19-21,
43-45). In the present study we show a delayed, protein kinase
C-dependent, stimulatory phosphorylation of
cPLA2 induced by the contractile agonist CCK and
demonstrate for the first time an inhibitory phosphorylation induced by
relaxant agonists (SNP, isoproterenol, and VIP) and activators of
cAMP-dependent protein kinase (cBIMPS) and
cGMP-dependent protein kinase (8-pcCPT-cGMP). The different
patterns of phosphorylation by contractile and relaxant agonists
suggest that the residues phosphorylated by cAMP- and
cGMP-dependent protein kinases are distinct from the
residues (chiefly Ser505) phosphorylated by protein
kinase C-dependent mechanisms (14, 17-20).
Phosphorylation of cPLA2 induced by relaxant agents that
preferentially activated cAMP-dependent protein kinase
(i.e. low concentrations of isoproterenol) was blocked by
the selective cAMP-dependent protein kinase inhibitors H-89
and myristoylated PKI, whereas phosphorylation induced by relaxant
agents that preferentially activated cGMP-dependent protein
kinase (i.e. SNP) was blocked by the selective
cGMP-dependent protein kinase inhibitor KT-5823. Phosphorylation induced by agents that activated both cAMP- and cGMP-dependent protein kinases (i.e. VIP) was
partially blocked by cAMP- and cGMP-dependent protein
kinase inhibitors and abolished by a combination of both inhibitors
(32).
The pattern of inhibition of cPLA2 activity by cAMP- and
cGMP-dependent protein kinases paralleled the pattern of
phosphorylation of cPLA2, suggesting that inhibition of
activity was probably mediated by phosphorylation. The initial peak (1 min) of cPLA2 activity elicited by the contractile agonist
CCK-8 was inhibited by both cAMP- and cGMP-dependent
protein kinases. The inhibition induced by cBIMPS and low
concentrations of isoproterenol was selectively blocked by the
cAMP-dependent protein kinase inhibitors H-89 and
myristoylated PKI, whereas the inhibition induced by 8-pcCPT-cGMP and
SNP was selectively blocked by the selective cGMP-dependent
protein kinase inhibitor KT-5823. Inhibition induced by VIP and high
concentrations of isoproterenol, which activate both cAMP- and
cGMP-dependent protein kinases, was partially blocked by cAMP- and cGMP-dependent protein kinase inhibitors and
completely blocked by a combination of both inhibitors (32).
The next step in Ca2+ mobilization in intestinal
longitudinal smooth muscle, i.e. arachidonic acid-induced
Ca2+ influx (22-24), was inhibited by agents that
activated cGMP-dependent protein kinase only
(i.e. SNP, 8-pcCPT-cGMP, and high concentrations of
isoproterenol). The molecular target of cGMP-dependent
protein kinase in this instance could be either Cl The present study addressed only the initial steps in Ca2+
mobilization and showed that inhibition of the effector enzyme
cPLA2, which mirrors inhibition of PLC- *
This work was supported by Grants DK-28300 and DK-15564 from
the NIDDK, 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:
PLA, phospholipase
A; p8-pcCPT-cGMP, 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic
monophosphate; cBIMPS, 5,6-dichloro-1-
Differential Regulation of Phospholipase A2
(PLA2)-dependent Ca2+ Signaling
in Smooth Muscle by cAMP- and cGMP-dependent Protein
Kinases
INHIBITORY PHOSPHORYLATION OF PLA2 BY CYCLIC
NUCLEOTIDE-DEPENDENT PROTEIN KINASES*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(PLC-) activity and inositol
1,4,5-trisphosphate-dependent Ca2+
release in vascular and visceral smooth muscle. In smooth muscle of the
intestinal longitudinal layer, however, the initial steps in
Ca2+ mobilization involve activation of cytosolic
PLA2 (cPLA2) and arachidonic acid
(AA)-dependent stimulation of Ca2+ influx. The
present study examined whether cAMP- and cGMP-dependent protein kinases are capable of regulating these processes also. Agents
that activated cAMP-dependent protein kinase
(5,6-dichloro-1--D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate (Sp-isomer) and isoproterenol), cGMP-dependent protein kinase
(8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate and Na
nitroprusside), or both kinases (vasoactive intestinal peptide and
isoproterenol >1 µM) induced phosphorylation of
cPLA2 and inhibition of agonist-stimulated cPLA2 activity. Phosphorylation and inhibition of
cPLA2 activity by cAMP- and cGMP-dependent
protein kinases were blocked by the corresponding selective
inhibitors (cAMP-dependent protein kinase, N-[2(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide
hydrochloride (H-89) and myristoylated protein kinase inhibitor
(14-22) amide; cGMP-dependent protein kinase,
(8R,9S,11S)-(
)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H,-2,7b,11a-trizadizobenzo(a,g)cycloocta(c,d,e)-trinden-1-one (KT-5823)). In contrast, AA-stimulated Ca2+ influx was
inhibited by agents that activated cGMP-dependent protein kinase only;
the inhibition was selectively blocked by KT-5823. The study provides
the first evidence of inhibitory phosphorylation of cPLA2
in vivo by cAMP- and cGMP-dependent protein kinases. Inhibition of cPLA2 activity and AA-induced
Ca2+ influx partly account for the ability of
cAMP-dependent protein kinase and/or cGMP-dependent protein kinase to
cause relaxation. Their importance resides in their location at the
inception of the Ca2+ signaling cascade.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 and/or -3, resulting in
inositol 1,4,5-trisphosphate (IP3)-dependent
Ca2+ release (25, 26). Only small amounts of
IP3 are formed in longitudinal smooth muscle, which does
not possess high affinity IP3 receptor/Ca2+
channels (23, 26).
and stimulation of Ca2+ release) are inhibited by
cAMP- and cGMP-dependent protein kinases (27-32). It is
not known, however, whether the initial steps in Ca2+
mobilization in intestinal longitudinal smooth muscle (i.e.
activation of cPLA2 and stimulation of Ca2+
influx) are influenced by cAMP- and cGMP-dependent protein
kinases. In this study, we have examined the initial steps in
Ca2+ signaling in longitudinal intestinal smooth muscle to
determine the ability of cAMP- and cGMP-dependent protein
kinases (a) to phosphorylate cPLA2 and influence
its activity and (b) to modulate AA-induced stimulation of
Ca2+ influx. The results provide the first evidence of
inhibitory phosphorylation of cPLA2 by both cAMP- and
cGMP-dependent protein kinases and demonstrate their
ability to regulate the initial step in Ca2+ signaling. The
next step, i.e. AA-stimulated Ca2+ influx, is
selectively inhibited by cGMP-dependent protein kinase.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 µM,
H-89 and KT-5823 were selective inhibitors of cAMP- and
cGMP-dependent protein kinases, respectively.
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Fig. 1.
Phosphorylation of cPLA2 by
selective activators of cAMP-dependent protein kinase
(cBIMPS) and cGMP-dependent protein kinase (8-pcCPT-cGMP)
in smooth muscle. Intestinal smooth muscle cells labeled
with 32P were incubated with cAMP-dependent
protein kinase inhibitors H-89 (1 µM) and myristoylated
PKI (1 µM) or the cGMP-dependent protein
kinase inhibitor KT-5823 (1 µM) for 10 min and treated
with cBIMPS (10 µM; upper panel) or
8-pcCPT-cGMP (10 µM; lower panel) for 1 min.
Immunoprecipitates using polyclonal cPLA2 antibody were
separated on SDS-polyacrylamide gel electrophoresis,
32P-labeled cPLA2 was identified by
autoradiography, and the measured radioactivity was expressed as
cpm/mg protein. Values are means ± S.E. of three experiments.
**, inhibition of phosphorylation, p < 0.01.
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Fig. 2.
Phosphorylation of cPLA2 by
relaxant agents in smooth muscle. Intestinal smooth muscle cells
labeled with 32P were incubated with
cAMP-dependent protein kinase inhibitors H-89 (1 µM) and myristoylated PKI (1 µM) or the
cGMP-dependent protein kinase inhibitor KT-5823 (1 µM) for 10 min and treated with isoproterenol
(ISOP; 1 µM), SNP (0.1 µM), or
VIP (1 µM) for 1 min. Immunoprecipitates using polyclonal
cPLA2 antibody were separated on SDS-polyacrylamide gel
electrophoresis, 32P-labeled cPLA2 was
identified by autoradiography (upper panel), and the
measured radioactivity was expressed as cpm/mg protein (lower panel).
Values are means ± S.E. of three experiments. Inhibition of
phosphorylation: **, p < 0.01; *,
p < 0.05.
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Fig. 3.
Phosphorylation of cPLA2 by
protein kinase C-dependent mechanisms in smooth
muscle. Intestinal smooth muscle cells labeled with
32P were incubated with CCK-8 (1 nM) for 1 or
10 min in the presence or absence of calphostin C (1 µM).
32P-Labeled cPLA2 was identified by
autoradiography (upper panel), and the measured
radioactivity was expressed as cpm/mg of protein. Results are
means ± S.E. of four experiments. **, inhibition of
phosphorylation, p < 0.01.
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Fig. 4.
Inhibition of agonist-stimulated
cPLA2 activity by selective activation of
cAMP-dependent protein kinase. Muscle cells labeled
with [3H]AA were incubated for 60 s with 1 µM isoproterenol (ISOP) or 10 µM
cAMP-dependent protein kinase activator cBIMPS in the
presence and absence of H-89, myristoylated PKI, or KT-5823, followed
by addition of 1 nM CCK-8 for 30 s. Formation of
[3H]AA was expressed as cpm/106 cells above
basal levels (basal levels in the presence or absence of kinase
inhibitors ranged from 343 ± 58 to 374 ± 46 cpm/106 cells). Results are means ± S.E. of four
experiments. *, inhibition of cPLA2 activity,
p < 0.05.
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Fig. 5.
Inhibition of agonist-stimulated
cPLA2 activity by selective activation of
cGMP-dependent protein kinase. Muscle cells labeled
with [3H]AA were incubated for 60 s with SNP (0.1 µM) or 8-pcCPT-cGMP (10 µM) in the presence
and absence of H-89, myristoylated PKI, or KT-5823, followed by
addition of 1 nM CCK-8 for 30 s. Formation of
[3H]AA was expressed as cpm/106 cells above
basal levels. Results are means ± S.E. of four experiments.
**, inhibition of cPLA2 activity, p < 0.01.
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Fig. 6.
Inhibition of agonist-stimulated
cPLA2 activity by combined activation of
cGMP-dependent protein kinase and
cAMP-dependent protein kinase. Muscle cells labeled
with [3H]AA were incubated for 60 s with 100 µM isoproterenol (ISOP) or 1 µM
VIP in the presence and absence of cAMP-dependent protein
kinase inhibitors (H-89 and myristoylated PKI),
cGMP-dependent protein kinase inhibitor (KT-5823), or both
cAMP- and cGMP-dependent protein kinase inhibitors,
followed by addition of 1 nM CCK-8 for 30 s. Formation
of [3H]AA was expressed as cpm/106 cells
above basal levels. Results are means ± S.E. of four experiments.
**, inhibition of cPLA2 activity, p < 0.01.
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Fig. 7.
Selective inhibition of AA-induced
Ca2+ influx by cross-activation of
cGMP-dependent protein kinase with high concentrations of
isoproterenol. Ca2+ influx in dispersed muscle cells
was measured from the initial uptake of 45Ca2+
in the presence of thapsigargin and anti-mycin. Muscle cells were
incubated with 45Ca2+ (10 µCi/ml) and treated
for 60 s with 1 µM (upper panel) and 100 µM isoproterenol (ISOP; lower
panel) in the presence and absence of H-89 or KT-5823 followed by
addition of AA (1 µM) for 2 min.
45Ca2+ cell content was measured at intervals
for 2 min and expressed as cpm/106 cells above basal levels
(basal levels in the absence and presence of H-89 or KT-5823 ranged
from 3519 ± 214 to 3753 ± 251 cpm/106 cells).
Results are means ± S.E. of three experiments. **, inhibition
of Ca2+ influx, p < 0.01.
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[in a new window]
Fig. 8.
Inhibition of AA-induced Ca2+
influx by selective activation of cGMP-dependent protein
kinase. Ca2+ influx in dispersed muscle cells was
measured from the initial uptake of 45Ca2+ in
the presence of thapsigargin and anti-mycin. Muscle cells were
incubated with 45Ca2+ (10 µCi/ml) and treated
for 60 s with SNP (0.1 µM) and 8-pcCPT-cGMP (10 µM) in the presence and absence of H-89 or KT-5823
followed by addition of AA (1 µM) for 2 min.
45Ca2+ cell content was measured at intervals
for 2 min and expressed as cpm/106 cells above basal
levels. Results are means ± S.E. of three experiments. **,
inhibition of Ca2+ influx, p < 0.01.
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Fig. 9.
Blockade of relaxation in dispersed smooth
muscle cells by cGMP-dependent protein kinase
inhibitors. Contraction of dispersed intestinal smooth muscle
cells treated with AA for 30 s was measured by scanning
micrometry. Inhibition of AA-induced contraction (i.e.
relaxation) by SNP (0.1 µM) or 8-pcCPT-cGMP (10 µM) measured in the presence and absence of H-89 and/or
KT-5823 was expressed as the mean change in muscle cell length (µm).
Results are means ± S.E. of three experiments. **, blockade
of relaxation, p < 0.01.
View larger version (14K):
[in a new window]
Fig. 10.
Blockade of relaxation in dispersed smooth
muscle cells by cAMP-dependent protein kinase
inhibitors. Contraction of dispersed intestinal smooth muscle
cells treated with AA for 30 s was measured by scanning
micrometry. Inhibition of AA-induced contraction (i.e.
relaxation) by isoproterenol (1 µM) or cBIMPS (10 µM) measured in the presence and absence of H-89 and/or
KT-5823 was expressed as the mean change in muscle cell length (µm).
Results are means ± S.E. of three experiments. **, blockade
of relaxation, p < 0.01.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and generation of
IP3 are the initial steps in Ca2+ mobilization
in most cell types. In vascular and visceral smooth muscle, including
smooth muscle of the intestinal circular layer (26-30, 37), relaxant
agents inhibit Ca2+ mobilization by activating
cAMP-dependent protein kinase and/or cGMP-dependent protein kinase. The kinases act on various
molecular targets to inhibit Ca2+ release from sarcoplasmic
stores and Ca2+ influx into the cell, and to stimulate
Ca2+ efflux from the cell. The targets include the effector
enzyme PLC-, the sarcoplasmic IP3
receptor/Ca2+ channel, plasmalemmal, and sarcoplasmic
Ca2+-ATPase pumps, and plasmalemmal Ca2+ and
K+ channels, all of which are affected by both cAMP- and
cGMP-dependent protein kinases, except for the sarcoplasmic
Ca2+-ATPase pump, which is selectively inhibited by
cGMP-dependent protein kinase (31, 32, 38-42). Both
PLC- and IP3 receptors/Ca2+ channels are
readily phosphorylated by cGMP- and cAMP-dependent protein
kinases, resulting in inhibition of IP3 formation and IP3-dependent Ca2+ release (30-32,
42).
channels or voltage-sensitive Ca2+ channels. Previous
studies (46) have shown that arachidonic acid activates primarily
Cl channels, resulting in membrane depolarization and
opening of voltage-sensitive Ca2+ channels. Consistent with
this notion, depolarization and Ca2+ influx induced by
contractile agonists was abolished by cPLA2 inhibitors and
stilbene Cl channel blockers; nifedipine, on the other
hand, abolished Ca2+ influx but only partially inhibited
depolarization, implying that Ca2+ influx was only a minor
contributor to membrane depolarization. Depolarization and
Ca2+ influx induced by nanomolar concentrations of
exogenous arachidonic acid was also abolished by stilbene
Cl channel blockers (46).
in other smooth
muscle cell types, was mediated by cAMP- and cGMP-dependent
protein kinases, whereas inhibition of AA-induced Ca2+
influx was exclusively mediated by cGMP-dependent protein
kinase. Blockade of either step by cAMP-dependent protein
kinase and/or cGMP-dependent protein kinase should lead to
a decrease in [Ca2+]i and inhibition of muscle
contraction. A decrease in [Ca2+]i, however,
could also result from inhibition of Ca2+-induced
Ca2+ release, stimulation of Ca2+ uptake into
intracellular stores, and Ca2+ extrusion from the cell.
Furthermore, inhibition of muscle contraction could also reflect
phosphorylation by cAMP- and cGMP-dependent protein kinases
of targets distal to those involved in Ca2+ mobilization,
such as myosin light chain kinase and/or phosphatase (37, 47). The
importance of the inhibitory processes examined in the present study
resides in their location at the start of the signaling cascade.
FOOTNOTES
To whom correspondence should be addressed: P. O. Box 980711, Medical College of Virginia, Richmond, VA 23298-0711. Tel.: 804-828-9601; Fax: 804-828-2500.
-D-ribofuranosylbenzimidazole
3',5'-cyclic monophosphothioate, Sp-isomer; H-89, N-[2(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide
hydrochloride; KT-5823, (8R,9S,11S)-()-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H,-2,7b,11a-trizadizobenzo(a,g)cycloocta(c,d,e)-trinden-1one; PKI, protein kinase inhibitor (14-22) amide; cPLA, cytosolic PLA; AA, arachidonic acid; IP3, inositol
1,4,5-trisphosphate; SNP, sodium nitroprusside; CCK-8, cholecystokinin octapeptide; VIP, vasoactive intestinal peptide.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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