Selective phosphorylation of the IP3R-I in vivo
by cGMP-dependent protein kinase in smooth muscle
Karnam S.
Murthy and
Huiping
Zhou
Departments of Physiology and Medicine, Medical College of
Virginia, Virginia Commonwealth University, Richmond, Virginia
23298 - 0711
 |
ABSTRACT |
This study examined the expression of
inositol 1,4,5-trisphosphate (IP3) receptor
(IP3R) types and PKG isoforms in isolated gastric smooth
muscle cells and determined the ability of PKG and PKA to phosphorylate
IP3Rs and inhibit IP3-dependent
Ca2+ release, which mediates the initial phase of
agonist-induced contraction. PKG-I
and PKG-I
were expressed in
gastric smooth muscle cells, together with IP3-R-associated
cG-kinase substrate, a protein that couples PKG-I
to
IP3R-I. IP3R-I and IP3R-III were also expressed, but only IP3R-I was phosphorylated by PKA
and PKG in vitro and exclusively by PKG in vivo. Sequential
phosphorylation by PKA and by PKG-I
in vitro showed that PKA
phosphorylated the same site as PKG (presumably S1755) and
an additional PKA-specific site (S1589). In intact muscle
cells, agents that activated PKG or both PKG and PKA induced
IP3R-I phosphorylation that was reversed by the PKG
inhibitor
(8R,9S,11s)-(
)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizo-benzo9(a,g)cycloocta(c,d,e)-trinden-1-one. Agents that activated PKA induced IP3R-I phosphorylation in
permeabilized but not intact muscle cells, implying that PKA does not
gain access to IP3R-I in intact muscle cells. The pattern
of IP3R-I phosphorylation in vivo and in vitro was more
consistent with phosphorylation by PKG-I
. Phosphorylation of
IP3R-I in microsomes by PKG, PKA, or a combination of PKG
and PKA inhibited IP3-induced Ca2+ release to
the same extent, implying that inhibition was mediated by
phosphorylation of the PKG-specific site. We conclude that IP3R-I is selectively phosphorylated by PKG-I in intact
smooth muscle resulting in inhibition of IP3-dependent
Ca2+ release.
relaxation, gastric muscle, calcium release
 |
INTRODUCTION |
A CLOSE PARALLELISM
EXISTS between inhibition of agonist-induced inositol 1,4,5- trisphosphate (IP3)-dependent Ca2+ release by
cAMP- and cGMP-dependent protein kinases (PKA and PKG) and inhibition
of muscle contraction (i.e., relaxation). PKA and/or PKG can regulate
Ca2+ mobilization by acting on various molecular targets,
including IP3 generation (19, 33, 34) and
sarcoplasmic IP3 receptors (IP3R)/Ca2+ channels (9, 10),
which determine Ca2+ release, plasmalemmal and sarcoplasmic
Ca2+/ATPase pumps, which determine Ca2+ uptake
into intracellular stores or efflux from the cell (2, 14),
and plasmalemmal Ca2+ and K+ channels, which
regulate membrane polarity and Ca2+ influx via
voltage-dependent Ca2+ channels (25).
Activation of PKG or PKA inhibits agonist-induced Ca2+
release in intact visceral smooth muscle cells (17, 19)
and IP3-induced Ca2+ release in permeabilized
visceral smooth muscle cells (17). Inhibition of
agonist-induced Ca2+ release could result from inhibition
of IP3 formation, whereas inhibition of
IP3-induced Ca2+ release in permeabilized
muscle probably reflects phosphorylation of the IP3R by
either kinase. Studies in vascular smooth muscle suggest that both
kinases are capable of phosphorylating IP3R in vitro,
whereas only PKG phosphorylates IP3R in vivo (9, 10). Permeabilization may allow access of PKA to the
IP3R that is normally denied to this kinase in intact
smooth muscle cells.
Two PKG isoforms, PKG-I
and PKG-I
, have been implicated in
IP3R-I phosphorylation. Although both isoforms are
frequently colocalized, PKG-1
appears to be more widely distributed
in human, bovine, rabbit, and murine tissues (5, 7, 11, 29,
35). PKG-I
is 10-fold more sensitive to activation by cGMP
and is more susceptible to cross-activation by cAMP (12,
29). Both kinases recognize in vivo substrates by interaction
with their distinctive NH2-terminal leucine-zipper amino
acid sequences (1, 32). A 125-kDa protein,
IP3-R-associated cG-kinase substrate (IRAG), has been
recently identified that binds to both the IP3R and the
distinctive NH2-terminal sequence of PKG-1
(1,
28). In cells expressing IP3R-I, IRAG, and PKG-I
,
all three proteins are coimmunoprecipitated by antibodies to each.
Reconstitution studies in COS-7 cells suggest that phosphorylation of
IRAG at Ser696 is a prerequisite for IP3R
phosphorylation and inhibition of IP3-dependent
Ca2+ release by PKG-I
(28). However, recent
studies in native mouse aortic smooth muscle cells that normally
express IRAG and both PKG-I isoforms have raised doubts regarding the
functional role of PKG-I
(3). Transfection of PKG-I
into smooth muscle cells derived from PKG-I
- and -I
-deficient
mice restored the ability of PKG activators [nitric oxide (NO) donors
and 8-bromo-cGMP] to inhibit agonist-induced Ca2+
transients, whereas transfection of PKG-I
had no effect
(3).
In the present study, we have examined the expression of
IP3R types and PKG isoforms in freshly dispersed and
cultured gastric smooth muscle cells and determined the ability of PKG
and PKA to phosphorylate IP3Rs in vivo and inhibit
IP3-dependent Ca2+ release from isolated
sarcoplasmic microsomes. The results indicate that PKG and PKA
induce in vitro phosphorylation of IP3R-I expressed in
smooth muscle cells. IP3R-I phosphorylation in vivo is
mediated exclusively by PKG and is partly responsible for inhibition of IP3-dependent Ca2+ release.
 |
MATERIALS AND METHODS |
Preparation of dispersed gastric smooth muscle cells.
Dispersed gastric smooth muscle cells were prepared by sequential
enzymatic digestion, filtration, and centrifugation as described previously (16, 17, 19, 20). The partly digested strips were washed twice with 50 ml of enzyme-free medium, and the muscle cells were allowed to disperse spontaneously for 30 min. The cells were
harvested by filtration through 500-µm Nitex (Tetko, Briarcliff Manor, NY) and centrifuged twice at 350 g for 10 min. For
permeabilization, dispersed smooth muscle cells were treated for 5 min
with saponin (35 µg/ml) and resuspended in low-Ca2+ (100 nM) medium as previously described (16, 17). In some experiments, the cells were placed in culture in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum until they attained confluence.
RT-PCR analysis of IP3R types, PKG isoforms, and
IRAG.
Specific primers were designed for IP3R-I, -II, and -III
based on homologous sequences in human, bovine, and rat cDNAs, for IRAG
based on homologous sequences in bovine and human IRAG cDNAs, for
PKG-I
based on the sequence of rabbit PKG-I
cDNA, and for PKG-I
based on homologous sequences in human, bovine, and mouse cDNAs. The sequences of the primers are listed in Table
1.
RNA (5 µg) prepared from cultured rabbit gastric smooth muscle cells
was reversibly transcribed and amplified by PCR under standard
conditions (2 mM MgCl2, 200 µM dNTP, and 2.5 units of Taq polymerase) in a final volume of 50 µl containing 100 ng of each primer. The PCR products were separated by electrophoresis in 1.2% agarose gel in the presence of ethidium bromide, visualized by
ultraviolet fluorescence, and recorded by a ChemiImager 4400 Fluorescence system. PCR products were purified by gel extraction kit
and sequenced.
Western blot analysis of IP3R.
The expression of IP3Rs was determined by Western blot as
described previously (18) using homogenates prepared from
freshly dispersed gastric smooth muscle cells. Proteins were resolved by 7.5% SDS-PAGE and elelctrophoretically transferred to
nitrocellulose membranes. The membranes were incubated for 12 h at
4°C with specific antibodies for IP3R-I, -II, or -III and
then for 1 h with secondary antibody. The bands were identified by
enhanced chemiluminescence.
In vivo phosphorylation and immunoprecipitation of
IP3R-I.
Phosphorylation of IP3R-I was determined from the amount of
32P incorporated after immunoprecipitation with specific
IP3R-I antibody as previously described for other proteins
(18). Ten milliliters of smooth muscle cell suspension
(4 × 106 cells/ml) were incubated with
[32P]orthophosphate for 4 h at 31°C. Samples (1 ml) were reincubated with PKA or PKG activators for 1 min in the
presence or absence of the specific PKG or PKA inhibitors. The reaction
was terminated with an equal volume of lysis buffer. 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, and incubated overnight with IP3R-I antibody
at a final concentration of 5 µg/ml. Protein A/G-sepharose was then
added for 1 h, and the mixture was centrifuged for 5 min. The
immunoprecipitates were washed four times with the lysis buffer and
resuspended in Laemmli buffer and boiled for 15 min. The proteins were
resolved by SDS-PAGE and transferred onto polyvinylidene difluoride
membranes. [32P]IP3R-I was visualized by
autoradiography, and the amount of radioactivity in the bands was
counted. Immunoblot analysis was performed on the membranes after
autoradiography to determine comigration of IP3R-I with the
corresponding radioactive bands.
In vitro phosphorylation of IP3R-I.
IP3R-I immunoprecipitates were washed five times with lysis
buffer and phosphorylated in the presence of exogenous PKG-I
holoenzyme (0.5 µM) or the catalytic subunit of PKA (0.5 µM). Phosphorylation with the catalytic subunit of PKA was performed in a
medium containing 20 mM Tris · HCl (pH 7.4), 10 mM
MgCl2, 1 mM EGTA, 10 µM cAMP, 6 mM
p-nitrophenylphosphate, 12 mM
-glycerophosphate, and 20 µM sodium vanadate. Phosphorylation with the purified holoenzyme of
PKG-I
was performed in the presence of PKI14-22
amide and 10 µM cGMP instead of cAMP. Phosphorylation was initiated by the addition of [
-32P]ATP (12 Ci/mmol) and
terminated after 30 min by the addition of 40 mM EDTA and an equal
volume of Laemmli buffer. The proteins were resolved, and the
immunoblots were analyzed as described above for in vivo phosphorylation.
In experiments involving sequential phosphorylation by PKA and PKG,
IP3R-I was initially phosphorylated in the presence of one
kinase and nonradioactive ATP for 30 min, after which the immunoprecipitates were washed with Tris buffer saline containing Tween
20. A second incubation was then initiated by addition of the other
kinase in the presence of [
-32P]ATP, and the mixture
was incubated for 30 min. The samples were analyzed for
IP3R-I phosphorylation as described above.
Back phosphorylation of IP3R.
A back-phosphorylation approach was also used to determine in vivo
phosphorylation of IP3R-I (10).
IP3R-I immunoprecipitates derived from control smooth
muscle cells and cells treated with activators of PKA or PKG were
phosphorylated in vitro using [
-32P]ATP in the
presence of exogenous PKG-I
holoenzyme as described in In
vitro phosphorylation of IP3R-I. The amount of
32P incorporated into the immunoprecipitated
IP3R-I derived from control muscle cells was taken as
100%, and the amount of 32P incorporated into the
immunoprecipitated IP3R-I derived from cells treated with
PKA or PKG activators was calculated as a percentage of the control
value. The decrease in 32P incorporation after treatment
with PKA and PKG activators reflected endogenous phosphorylation by PKA
or PKG.
Ca2+ release from smooth muscle
microsomes.
The effect of IP3R-I phosphorylation on
IP3-induced Ca2+ release was measured in
microsomes prepared from freshly dispersed smooth muscle cells and
preloaded with 45Ca2+ as described previously
(16). One-milliliter samples (0.5 mg) of the microsomal
suspension were incubated for 60 min at 31°C with
45Ca2+ (10 µCi/ml), ATP (1 mM), and ATP
regenerating system when a steady state of Ca2+ uptake was
reached (16). After the addition of IP3,
100-µl samples were removed at 15 s and added to 25 µl of
quench medium consisting 0.633 M formalin and 50 mM EDTA (pH 7.0). The
samples were centrifuged at 13,000 g for 5 min, and the
pellets were washed twice with the same medium and extracted with 50 µl of tricholoroacetic acid for measurement of
45Ca2+. Before the addition of IP3
in some experiments, the microsomes were treated with the PKG-I
holoenzyme (0.5 µM) in the presence of 10 µM cGMP or with the
catalytic subunit PKA (0.5 µM) for 15 min.
Materials.
[
-32P]ATP and [32P]orthophosphate were
obtained from Amersham Pharmacia Biotech (Piscataway, NJ); collagenase
and soybean trypsin inhibitor were from Worthington Biochemical
(Freehold, NJ); 8-(4-chlorophenylthio)guanosine 3',5'-cyclic
monophosphate (8-pCPT-cGMP) and
5,6-dichloro-1-
-D-ribofuranosyl benzimidazole
3',5-cyclic monophosphothioate, Sp-isomer (cBIMPS) were from Alexis
Biochemicals (San Diego, CA); IP3 was from Calbiochem (San
Diego, CA); Western blotting and chromatography material and protein
assay kit were from Bio-Rad Laboratories (Hercules, CA); antibody to
IP3R types I, II, and III were from Santa Cruz (Santa Cruz,
CA). All other chemicals were obtained from Sigma (St. Louis, MO).
 |
RESULTS |
Expression of IP3R types, PKG isoforms, and IRAG in
gastric smooth muscle.
PKG-I
and PKG-I
were detected by RT-PCR in primary cultures of
gastric smooth muscle cells and after first passage using specific
primers based on conserved sequences in rabbit for PKG-I
and in
human, bovine, and mouse for PKG-I
(Fig.
1).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1.
Expression of PKG isoforms and
IP3-R-associated cG-kinase substrate (IRAG) in cultured
gastric smooth muscle cells. RNA isolated from primary cultures of
rabbit gastric smooth muscle cells was reverse transcribed and cDNA
amplified using specific primers for PKG-I and PKG-I
(A) and IRAG (B). Two sets of primers were used
for IRAG, derived from the NH2-terminal (lanes 2 and 3) and middle (lanes 4 and 5)
regions of IRAG. Experiments were done in the presence (+) or absence
( ) of RT. Transcript sizes were 474 and 406 bp for PKG-I and
PKG-I and 524 and 378 bp for IRAG. Identical results were obtained
in gastric smooth muscle cells after first passage (data not shown).
MW, molecular weight.
|
|
IRAG was also detected by RT-PCR in primary cultures of rabbit smooth
muscle cells and after first passage using specific primers based on
conserved sequences in human and bovine, including a primer based on
the sequence that specifically binds PKG-I
(Fig. 1). The partial
amino acid sequence was 91-92% similar to human and bovine IRAG.
Both IP3R-I and -III but not IP3R-II were
detected by RT-PCR in primary cultures of gastric smooth muscle cells
and after first passage using primers based on conserved sequences in
human, bovine, and rat (Fig. 2). Western
blot analysis of lysates derived from freshly dispersed smooth muscle
cells confirmed expression of IP3R-I and -III but not -II
(Fig. 2). The partial amino acid sequence of IP3R-I was
92-94%, and that of IP3R-III was 90-94%, similar to those of human and rat receptors.

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 2.
Expression of inositol 1,4,5- trisphosphate
(IP3) receptor (IP3R) types in cultured gastric
smooth muscle cells. A: RNA isolated from primary cultures
of rabbit gastric smooth muscle cells was reverse transcribed and cDNA
amplified using specific primers for IP3R-I, -II, and -III.
Experiments were done in the presence (+) or absence of ( ) of RT.
Transcript sizes were 194 bp for IP3R-I and 400 bp for
IP3R-III. No RT-PCR product was obtained with primers for
IP3R-II. Identical results were obtained in gastric smooth
muscle cells after first passage (data not shown). B:
Western blot analysis of lysates prepared from dispersed rabbit gastric
smooth muscle cells and probed with polyclonal antibodies to
IP3R-I, -II, and -III. Western blot confirmed expression of
IP3R-I and IP3R-III but not
IP3R-II. Selective phosphorylation of IP3R-I
but not IP3R-III occurred in cells treated with 1 µM
sodium nitroprusside.
|
|
In vitro phosphorylation of IP3R-I by PKA and PKG.
Exogenous PKG-I
holoenzyme and the catalytic subunit of PKA
phosphorylated IP3R-I in immunoprecipitates obtained from
lysates of dispersed smooth muscle cells (Fig.
3). Although the extent of
IP3R-I phosphorylation was greater with PKA than with PKG, phosphorylation by both kinases in combination was not greater than
that by PKA alone (Fig. 3). The relationship between phosphorylation by
PKA and PKG was further examined by the sequential addition of the two
kinases. Incubation with nonradioactive ATP in the presence of PKA for
30 min prevented further phosphorylation of IP3R-I by PKG.
In contrast, incubation with nonradioactive ATP in the presence of PKG
for 30 min did not prevent additional phosphorylation of the receptor
by PKA (Fig. 4). The extent of additional
phosphorylation by PKA was similar to the difference between
phosphorylation by PKA and PKG added separately (cf. Figs. 3 and 4).
The pattern implied that PKA phosphorylated the same residue(s) as PKG
as well as additional PKA-specific residue(s).

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 3.
In vitro phosphorylation of IP3R-I by PKA and
PKG. A: IP3R-I was immunoprecipitated and
phosphorylated in the presence of [ -32P]ATP by
PKG-I holoenzyme alone for 30 min (filled bar) or sequentially by
PKG-I for 30 min and by the catalytic subunit of PKA for 30 min
(hatched bar). B: immunoprecipitated IP3R-I was
phosphorylated by the catalytic subunit of PKA alone for 30 min (filled
bar) or sequentially by the catalytic subunit of PKA for 30 min and by
PKG-I for 30 min (hatched bar). Basal phosphorylation represents
32P incorporation in the absence of PKA and PKG.
[32P] p-IP3R-I was identified by autoradiography, and the
radioactivity in the bands was expressed as counts/min (cpm).
Immunoblots of the IP3R-I bands are shown for loading
control. Values are means ± SE of 4 experiments.
**P < 0.01 significant increase in phosphorylation
over that induced by PKG-I alone.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Combined phosphorylation of IP3R-I by PKA and
PKG. IP3R-I immunoprecipitates were phosphorylated with
nonradioactive ATP in the presence of the catalytic subunit of PKA for
30 min followed by phosphorylation with [ -32P]ATP in
the absence (lane 1) or presence of PKG-I holoenzyme for
another 30 min (lane 2). IP3R-I was
phosphorylated with nonradioactive ATP in the presence of PKG-I
holoenzyme for 30 min followed by phosphorylation with
[ -32P]ATP in the absence (lane 3) or
presence of the catalytic subunit of PKA for 30 min (lane
4). [32P]IP3R-I was identified by
autoradiography and radioactivity in the bands was expressed as cpm.
Immunoblots of the IP3R-I bands are shown for loading
control. Values are means ± SE of 4 experiments.
**P < 0.01 significant increase in 32P
incorporation by PKA.
|
|
In vivo phosphorylation of IP3R-I by PKG.
Several agents that activate PKA, PKG, or both kinases were used alone
and in conjunction with selective inhibitors of PKA (myristoylated PKI14-22 amide) or PKG
[(8R,9S,11s)-(
)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizo-benzo9(a,g)cycloocta(c,d,e)-trinden-1-one (KT5823)] to determine the ability of PKA and PKG to phosphorylate IP3R-I in vivo. Direct measurement of PKA and PKG
activities in these muscle cells have shown that, at concentrations of
1 µM and less, myristoylated PKI selectively inhibits PKA activity, whereas KT5823 selectively inhibits PKG activity (15, 17, 18). The kinase activators included selective activators of PKG
[8-pCPT-cGMP and sodium nitroprusside (SNP)], selective activators of
PKA (cBIMPS and a low concentration of isoproterenol, <1 µM), and
activators of both PKA and PKG [vasoactive intestinal peptide (VIP)
and high concentrations of forskolin or isoproterenol, >1 µM]
(8, 15, 17). As shown previously, VIP interacts with distinct receptors to stimulate cAMP and cGMP and activate both kinases
(17, 20); high concentrations of forskolin and
isoproterenol generate high levels of cAMP that activate PKA and
cross-activate PKG (8, 17).
Two sets of parallel studies were performed: in one set, the cells were
first labeled with 32P and then treated with various agents
before immunoprecipitation of IP3R-I. In a parallel set of
studies, the cells were first treated with the same agents and
immunoprecipitated IP3R-I was then treated with
[
-32P]ATP and PKG-I
holoenzyme to induce complete
IP3R-I phosphorylation. Maximum back phosphorylation
(100%) occurred in untreated cells. Endogenous (i.e., in vivo)
phosphorylation by PKG activators resulted in a decrease in
32P-labeling during back phosphorylation.
SNP and 8-pCPT-cGMP increased IP3R-I phosphorylation by
414 ± 37 and 358 ± 46% above basal level (basal: 510 ± 46 cpm). Phosphorylation by both SNP and 8-pCPT-cGMP was virtually
abolished by KT5823 but was not affected by myristoylated PKI (Fig.
5A). Identical results were
obtained using back phosphorylation to determine PKG-induced
phosphorylation of IP3R-I (Fig. 5B). The effect
of SNP using the technique of back phosphorylation was concentration dependent (Fig. 6).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5.
PKG-dependent phosphorylation of IP3R-I by
SNP and 8-pCPT-cGMP. (a) Gastric smooth muscle cells were labeled with
32P and then treated with SNP (1 µM) or
8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate
(8-pCPT-cGMP; 10 µM) in the presence or absence of myristoylated PKI
(1 µM) or
(8R,9S,11s)-( )-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizo-benzo9(a,g)cycloocta(c,d,e)-trinden-1-one
(KT5823; 1 µM). [32P]IP3R-I was
immunoprecipitated, and radioactivity in the bands was expressed as
cpm. Immunoblots of the IP3R-I bands are shown for loading
control. B: back phosphorylation technique: smooth muscle
cells were first treated with sodium nitroprusside (SNP) or 8-pCPT-cGMP
in the presence or absence of myristoylated PKI or KT5823; after
immunoprecipitation, IP3I-R was phosphorylated in vitro in
the presence of PKG-I holoenzyme and [ -32P]ATP.
Control cells were treated in similar fashion without SNP or
8-pCPT-cGMP. The difference between 32P incorporation in
control cells (100%) and cells treated with SNP or 8-pCPT-cGMP
represents the extent of endogenous (in vivo) phosphorylation by the 2 agents. Radioactive bands are not shown for back phosphorylation.
Values are means ± SE of 4 experiments each in A and
B. With both techniques, a significant increase in
phosphorylation (P < 0.01) was selectively suppressed
by the PKG inhibitor KT5823.
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 6.
Concentration-dependent phosphorylation of
IP3R-I by SNP. Phosphorylation of IP3R-I was
determined by the technique of back phosphorylation as described in
MATERIALS AND METHODS and the legend to Fig. 5. As shown in
the radioactive bands, maximal IP3R-I back phosphorylation
was observed in the basal state (B). Increasing in vivo phosphorylation
with SNP concentration was reflected by a decrease of radioactivity in
the bands. Immunoblots of the IP3R-I bands are shown for
loading control. Values are means ± SE of 5 experiments.
|
|
The selective PKA activator cBIMPS had no effect on IP3R-I
phosphorylation, whereas forskolin, which cross-activates PKG, increased IP3R-I phosphorylation by 342 ± 43% above
basal level; the increase induced by forskolin was abolished by KT5823
but was not affected by myristoylated PKI (Fig.
7A). Similarly, 1 µM
isoproterenol had no effect on IP3R-I phosphorylation,
whereas 100 µM isoproterenol, which cross-activates PKG, increased
IP3R-I phosphorylation by 320 ± 38% above the basal
level; the increase was virtually abolished by KT5823 but was not
affected by myristoylated PKI (Fig.
8a). VIP (1 µM), which
activates both PKA and PKG, increased IP3R-I
phosphorylation by 304 ± 25% above the basal level; the increase
was abolished by KT5823 but was not affected by myristoylated PKI (Fig.
9A). With each of these agents
(cBIMPS, forskolin, isoproterenol, and VIP) identical results were
obtained using the technique of back phosphorylation (Figs.
7B, 8B, and 9B). These results
indicated that, in vivo, only PKG is capable of phosphorylating
IP3R-I, raising the possibility that PKA, which is capable
of phosphorylating the receptor in vitro, may not gain access to the
receptor in vivo. This notion was examined in permeabilized smooth
muscle cells. In these cells, selective activation of PKA by cBIMPS or a low concentration of isoproterenol (1 µM) increased phosphorylation of IP3R-I by 300 ± 23 and 345 ± 39% above the
basal level (basal: 612 ± 86 cpm); phosphorylation by both agents
was selectively inhibited by myristoylated PKI but was not affected by
KT5823 (Fig. 10).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 7.
PKG-dependent phosphorylation of IP3R-I by
forskolin. A: gastric smooth muscle cells were labeled with
32P and then treated with forskolin (10 µM) or the PKA
activator 5,6-dichloro-1- -D-ribofuranosyl benzimidazole
3',5-cyclic monophosphothioate, Sp-isomer (cBIMPS; 10 µM) in the
presence or absence of myristoylated PKI (1 µM) or KT5823 (1 µM).
[32P]IP3R-I was immunoprecipitated, and
radioactivity in the bands was expressed as cpm. Immunoblots of the
IP3R-I bands are shown for loading control. B:
back phosphorylation: the cells were first treated with forskolin or
cBIMPS in the presence or absence of myristoylated PKI or KT5823. After
immunoprecipitation, IP3I-R was phosphorylated in vitro in
the presence of PKG-I holoenzyme and [ -32P]ATP.
Control cells were treated in similar fashion without forskolin or
cBIMPS. Radioactive bands are not shown for back phosphorylation.
Values are means ± SE of 4 experiments each in A and
B. No IP3R-I phosphorylation was observed with
cBIMPS. With both techniques, significant increase in phosphorylation
(P < 0.01) by forskolin was selectively suppressed by
KT5823.
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 8.
PKG-dependent phosphorylation of IP3R-I by
high concentrations of isoproterenol. A: gastric smooth
muscle cells were labeled with 32P and then treated with 1 or 100 µM isoproterenol or in the presence or absence of
myristoylated PKI (1 µM) or KT5823 (1 µM).
[32P]IP3R-I was immunoprecipitated, and
radioactivity in the bands was expressed as cpm. Immunoblots of the
IP3R-I bands are shown for loading control. B:
back phosphorylation: the cells were first treated with isoproterenol
in the presence or absence of myristoylated PKI or KT5823, and
IP3I-R immunoprecipitates were phosphorylated in vitro in
the presence of PKG-I holoenzyme and [ -32P]ATP.
Control cells were treated in similar fashion without isoproterenol.
Radioactive bands are not shown for back phosphorylation. Values are
means ± SE of 4 experiments each in A and
B. With both techniques, significant increase in
phosphorylation (P < 0.01) observed with 100 µM
isoproterenol was selectively suppressed by KT5823.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 9.
PKG-dependent phosphorylation of IP3R-I by
VIP. (a) Gastric smooth muscle cells were labeled with 32P
and then treated with vasoactive intestinal peptide (VIP; 1 µM) in
the presence or absence of myristoylated PKI (1 µM) or KT5823 (1 µM). [32P]IP3R-I was immunoprecipitated,
and radioactivity in the bands was expressed as cpm. Immunoblots of the
IP3R-I bands are shown for loading control. B:
back phosphorylation: the cells were first treated with VIP in the
presence or absence of myristoylated PKI or KT5823; after
immunoprecipitation, IP3I-R was phosphorylated in vitro in
the presence of PKG-I holoenzyme and [ -32P]ATP.
Control cells were treated in similar fashion without VIP. Radioactive
bands are not shown for back phosphorylation. Values are means ± SE of 4 experiments each in A and B. With both
techniques, a significant increase in phosphorylation
(P < 0.01) by VIP was selectively suppressed by
KT5823.
|
|

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 10.
PKA-dependent IP3R-I phosphorylation by
isoproterenol (1 µM) and cBIMPS in permeabilized smooth muscle cells.
Dispersed smooth muscle cells were labeled with 32P and
permeabilized with saponin for 5 min. The cells were then treated with
isoproterenol (1 µM) or cBIMPS (10 µM) in the presence or absence
of myristoylated PKI (1 µM) or KT5823 (1 µM).
[32P]IP3R-I was immunoprecipitated, and
radioactivity in the bands was expressed as cpm. Immunoblots of the
IP3R-I bands are shown for loading control. Values are
means ± SE of 4 experiments. Significant increase in
phosphorylation (P < 0.01) by both isoproterenol and
cBIMPS was selectively suppressed by myristoylated PKI.
|
|
Inhibition of IP3-induced
Ca2+ release by IP3R-I.
The effect of IP3R-I phosphorylation on
IP3-induced Ca2+ release was determined in
microsomes derived from freshly dispersed gastric smooth muscle cells.
After the microsomes were loaded with 45Ca2+,
they were treated with PKG-1
holoenzyme (0.5 µM) or the catalytic subunit of PKA (0.5 µM) for 15 min. Treatment with either kinase induced IP3R-I phosphorylation. The addition of
IP3 for 15 s elicited rapid concentration-dependent
Ca2+ release (EC50 = 1 nM
IP3) that was inhibited to the same extent by PKA
or PKG (EC50 = 1 µM IP3 with either PKG
or PKA), suggesting that phosphorylation of a common site by either
kinase was responsible for inhibition of Ca2+ release (Fig.
11). An increase in phosphorylation
induced by a higher concentration of the catalytic subunit of PKA (1 µM) or a combination of PKA and PKG-I
did not elicit greater
inhibition of Ca2+ release (50% inhibition of release by 1 µM IP3), implying that the site phosphorylated by
PKG-I
mediated inhibition of IP3R-I activity. Treatment
of the microsomes for 15 min with a selective antibody directed against
the COOH terminal of IP3R-I but not antibodies directed
against the COOH terminals of IP3R-II or IP3R-III strongly
inhibited IP3-induced Ca2+ release (Fig.
12) (23).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 11.
Phosphorylation-dependent inhibition of Ca2+
release from smooth muscle microsomes. Microsomes prepared from
dispersed gastric smooth muscle cells were loaded with
45Ca2+ for 60 min when a steady state of
Ca2+ uptake was attained (16). The microsomes
were treated for 15 min with PKG-I holoenzyme (0.5 µM) or the
catalytic subunit of PKA (0.5 µM), followed by the addition of
IP3 for 15 s. Ca2+ release was determined
from the decrease in steady-state microsomal
45Ca2+ content. Results were expressed as
%maximal release. Inset: phosphorylation of
IP3R-I in microsomes by PKG and PKA. Values are means ± SE of 5 experiments.
|
|

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 12.
Selective inhibition of IP3-induced
Ca2+ release from smooth muscle microsomes by
IP3R-I antibody. Ca2+ release from smooth
muscle microsomes by IP3 (1 µM) was determined as
described in the legend to Fig. 11. In some experiments, the microsomes
were first incubated with selective antibodies to IP3R-I,
-II, or -III for 15 min. Values are means ± SE of 4 experiments.
**P < 0.01 significant inhibition of
IP3-induced Ca2+ release by IP3R-I
antibody.
|
|
 |
DISCUSSION |
IP3Rs belong to a family of tetrameric
Ca2+ channels that open on binding of IP3.
Three receptor types have been identified (IP3R-I, -II, -III) that
exhibit similar structures consisting of an endoplasmic
membrane-spanning Ca2+ channel domain flanked by a large
cytosolic IP3-binding domain and a smaller cytosolic
COOH-terminal domain (25, 26, 36). A regulatory domain,
NH2 terminal to the Ca2+ channel, contains
putative consensus sequences for phosphorylation by PKG
(S1755) and PKA (S1755 and S1589)
(4, 6, 9, 31, 37, 38, 40). These sequences (GRRES1755L and ARRDS1589V) appear to be
restricted to IP3R-I, which is most abundant and has the
widest central and peripheral distributions but is often coexpressed
with IP3R-II and/or -III (6, 38).
This study shows that both IP3R-I and -III are expressed in
gastric smooth muscle cells and that only IP3R-I is
susceptible to phosphorylation by PKA and PKG in vitro and exclusively
by PKG in vivo. Sequential phosphorylation by PKA and PKG in vitro shows that PKA phosphorylates the same site as PKG (presumably S1755) and an additional PKA-specific site
(S1589). Komalavilas and Lincoln (9, 10)
previously reached similar conclusions in their studies of PKA- and
PKG-dependent phosphorylation of IP3R-I in cerebellar and
vascular smooth muscle tissues. In the present study, phosphorylation
of IP3R-I in microsomes by PKG or PKA inhibited
IP3-induced Ca2+ release to the same extent;
combined phosphorylation by PKA and PKG did not cause further
inhibition of Ca2+ release, implying that inhibition was
mediated by phosphorylation of the PKG-specific site
(S1755).
Two experimental strategies that yielded similar results were used to
characterize PKA- and PKG-specific phosphorylation. In one, PKG and PKA
were selectively activated in the presence or absence of specific
kinase inhibitors in cells prelabeled with 32P and
IP3R-I was immunoprecipitated to determine the extent and specificity of phosphorylation. In the other, unlabeled cells were
treated in similar fashion and immunoprecipitated IP3R-I was subsequently phosphorylated by PKG-I
holoenzyme. PKG was selectively activated by the cGMP analog 8-pCPT-cGMP and SNP, whereas
PKA was selectively activated by the cAMP analog cBIMPS and low
concentrations of isoproterenol (1 µM). PKG and PKA were also
activated concurrently by 1) VIP, which interacts with VPAC2 receptors to stimulate cAMP formation and with the natriuretic peptide
clearance receptors to stimulate sequentially NO and cGMP formation
(20); and 2) high concentrations of forskolin
(10 µM) or isoproterenol (100 µM), which stimulates cAMP formation in amounts that activate PKA and cross-activate PKG (8, 13, 17). These experiments showed clearly that activation of PKA alone by cBIMPS or 1 µM isoproterenol did not induce
IP3R-I phosphorylation in vivo. When PKA was activated
concurrently with PKG by VIP, forskolin, or 100 µM isoproterenol,
IP3R-I phosphorylation was selectively inhibited by KT5823,
implying that it was mediated by PKG. Previous studies in vascular
smooth muscle also showed that IP3R-I phosphorylation
induced by forskolin reflected cross-activation of PKG, because
phosphorylation was strongly inhibited by KT5823 and was only weakly
inhibited by KT5720, a preferential inhibitor of PKA (10).
When used at low concentrations (1 µM and less), KT5823 and
myristoylated PKI inhibit selectively PKG and PKA, respectively, as
shown in previous studies by direct measurement of PKA and PKG
activities in gastric smooth muscle cells (15, 17).
In permeabilized smooth muscle cells, unlike intact cells, PKA
phosphorylated IP3R-I, and the phosphorylation was reversed by a selective PKA inhibitor (Fig. 10). As shown previously,
IP3-induced Ca2+ release in permeabilized
gastric smooth muscle cells was inhibited by PKG and PKA, and the
inhibition was reversed by selective PKG and PKA inhibitors,
respectively (17). In light of the present study, we
propose that permeabilization enables PKA to gain access to and
phosphorylate IP3R-I and thus inhibit Ca2+ release.
Both isoforms of PKG-I (PKG-I
and PKG-I
) were identified by
RT-PCR in cultured gastric smooth muscle cells. Although both PKG-I
and -I
are often coexpressed, the former appears to be the
predominant isoform in the peripheral tissues of most mammalian species, including human, bovine, rabbit, and mouse (5, 7, 11,
29, 35). As noted above, PKG-I
is more sensitive to activation by cGMP and is susceptible to cross-activation by cAMP (12, 29). The two isoforms recognize in vivo substrates by binding them to distinct NH2-terminal leucine-zipper amino
acid sequences. PKG-I
binds the "regulatory myosin light chain
phosphatase-targeting subunit" and skeletal muscle troponin T via its
NH2-terminal sequence (32, 39), whereas
PKG-I
binds a recently identified substrate, IRAG, via its distinct
NH2-terminal sequence (1, 28). IRAG appears to
couple PKG-I
to IP3R-I: reconstitution studies in COS-7
cells suggest that phosphorylation of IRAG at Ser696 is a
prerequisite for IP3R-I phosphorylation and inhibition of IP3-dependent Ca2+ release by PKG-I
(28). However, more recent studies in native mouse aortic
smooth muscle cells that normally express both PKG-I
and -I
have
raised doubts regarding the functional role of PKG-I
in inhibition
of IP3-dependent Ca2+ release (3).
Transfection of PKG-I
into smooth muscle cells derived from mice
deficient in both PKG-I isoforms restored the ability of PKG activators
(NO donors and 8-bromo-cGMP) to inhibit agonist-induced
Ca2+ transients, whereas transfection of PKG-I
had no
effect (3).
Although IRAG was expressed in gastric smooth muscle cells, the
evidence suggests that PKG-I
rather than PKG-I
and IRAG was
involved in IP3R-I phosphorylation and
IP3-dependent Ca2+ release. The results
obtained with forskolin and high concentrations of isoproterenol imply
that PKG-I
was involved in IP3R-I phosphorylation and
IP3-dependent Ca2+ release (17),
because this isoform is preferentially cross-activated by cAMP.
Furthermore, the results of back phosphorylation with PKG-I
holoenzyme were identical to those obtained by activating endogenous
PKG-I in vivo. In permeabilized smooth muscle cells, IP3R-I
could be specifically phosphorylated by PKA using a selective activator
of PKA (cBIMPS) or a low concentration of isoproterenol: phosphorylation of IP3R-I by PKA did not require IRAG,
which does not bind to PKA. Phosphorylation of IP3R-I in
microsomes by PKG-I
holoenzyme or the catalytic subunit of PKA
inhibited Ca2+ release induced by the addition of various
concentrations of IP3, implying that phosphorylation of the
receptor was the proximate cause of inhibition of Ca2+ release.
Forskolin and isoproterenol have been the agents of choice in
evaluating the role of cAMP in mediating smooth muscle relaxation. This
and other studies (8, 17) clearly show that, in intact smooth muscle cells, this cyclic nucleotide acts, at least in part, by
cross-activating PKG-I to induce IP3R-I phosphorylation and
inhibition of IP3-dependent Ca2+ release. Lower
levels of cAMP that do not cross-activate PKG probably inhibit
agonist-induced Ca2+ release in intact smooth muscle cells
by inhibiting IP3 formation. Our recent studies in gastric
smooth muscle cells indicate that although PKA and PKG do not directly
phosphorylate and inhibit PLC-
1, they can reduce the ability of
G
q to activate PLC-
1 (21). Both kinases
phosphorylate RGS4, which further accelerates the hydrolysis of
G
q-bound GTP (21). Thus inhibition of
agonist-induced Ca2+ release is the net outcome of at least
two processes: a proximal process involving inhibition of
IP3 formation that can be evoked by both PKG and PKA and a
more distal process involving phosphorylation of IP3R-I and
inhibition of IP3-induced Ca2+ release that is
PKG-specific. The precise contribution of each process has not been determined.
It is worth emphasizing that inhibition of Ca2+
mobilization is relevant only to the initial transient contraction
mediated by Ca2+/calmodulin-dependent activation of myosin
light chain kinase and phosphorylation of MLC20. Sustained
MLC20 phosphorylation and contraction are largely
Ca2+ independent and reflect inhibition of MLC phosphatase
via at least two pathways that involve activation of RhoA and its
associated kinase (22, 30). Relaxation of sustained
contraction results from inhibition of RhoA activity by both PKA and
PKG (22, 27).
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-28300.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
K. S. Murthy, P.O. Box 980711, Medical College of Virginia,
Virginia Commonwealth Univ., 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.00401.2002
Received 18 September 2002; accepted in final form 5 November 2002.
 |
REFERENCES |
1.
Ammendola, A,
Geiselhoringer A,
Hofmann F,
and
Schlossmann J.
Molecular determinants of the interaction between the inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) and cGMP kinase I
.
J Biol Chem
276:
24153-24159,
2001[Abstract/Free Full Text].
2.
Corwell, TL,
Pryzwansky KB,
Wyatt TA,
and
Lincoln TM.
Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells.
Mol Pharmacol
40:
923-931,
1991[Abstract].
3.
Feil, R,
Gappa N,
Rutz M,
Schlossmann J,
Rose CR,
Konnerth A,
Brummer S,
Kuhbandner S,
and
Hofmann F.
Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase I isoforms.
Circ Res
90:
1080-1086,
2002[Abstract/Free Full Text].
4.
Ferris, CD,
Cameron AM,
Bredt DS,
Huganir RL,
and
Snyder SH.
Inositol 1,4,5-trisphosphate receptor is phosphorylated by cyclic AMP-dependent protein kinase at serines 1755 and 1589.
Biochem Biophys Res Commun
175:
192-198,
1991[ISI][Medline].
5.
Francis, SH,
and
Corbin JD.
Cyclic-nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action.
Crit Rev Clin Lab Sci
36:
275-328,
1999[ISI][Medline].
6.
Haug, L,
Jensen V,
Hvalby O,
Walaas I,
and
Ostvold AC.
Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic nucleotide-dependent kinases in vitro and in rat cerebellar slices in situ.
J Biol Chem
274:
7467-7473,
1999[Abstract/Free Full Text].
7.
Huber, A,
Trudrung P,
Storr M,
Franck H,
Schusdziarra V,
Ruth P,
and
Allecher H.
Protein kinase G expression in the small intestine and functional importance for smooth muscle relaxation.
Am J Physiol Gastrointest Liver Physiol
274:
G629-G637,
1998.
8.
Jiang, H,
Colbran JL,
Frnacis SH,
and
Corbin JD.
Direct evidence for cross-activation of cGMP-dependent protein kinase by cAMP in pig coronary arteries.
J Biol Chem
267:
1015-1019,
1992[Abstract/Free Full Text].
9.
Komalavilas, P,
and
Lincoln TM.
Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic GMP-dependent protein kinase.
J Biol Chem
269:
8701-8707,
1994[Abstract/Free Full Text].
10.
Komalavilas, P,
and
Lincoln TM.
Phosphorylation of inositol 1,4,5-trisphosphate receptor: cGMP-dependent protein kinase mediated cAMP- and cGMP-dependent phosphorylation in the intact rat aorta.
J Biol Chem
271:
21933-21938,
1996[Abstract/Free Full Text].
11.
Kumar, R,
Joyner RW,
Komalavilas P,
and
Lincoln TM.
Analysis of expression of cGMP-dependent protein kinase in rabbit heart cells.
J Pharmacol Exp Ther
291:
967-975,
1999[Abstract/Free Full Text].
12.
Landgraf, W,
Hullin R,
Gobel C,
and
Hofmann F.
Phosphorylation of cGMP-dependent protein kinase increases the affinity for cAMP.
Eur J Biochem
154:
113-117,
1986[Abstract].
13.
Lincoln, TM,
Cornwell TL,
and
Taylor AE.
cGMP-dependent protein kinase mediates the reduction of Ca2+ by cAMP in vascular smooth muscle cells.
Am J Physiol Cell Physiol
258:
C399-C407,
1990[Abstract/Free Full Text].
14.
Lincoln, TM,
Dey N,
and
Sellak H.
cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression.
J Appl Physiol
91:
1421-1430,
2001[Abstract/Free Full Text].
15.
Murthy, KS.
Cyclic AMP inhibits IP3-dependent Ca2+ release by preferential activation of cGMP- primed PKG.
Am J Physiol Gastrointest Liver Physiol
281:
G1238-G1245,
2001[Abstract/Free Full Text].
16.
Murthy, KS,
Grider JR,
and
Makhlouf GM.
Receptor-coupled G proteins mediate contraction and Ca2+ mobilization in isolated intestinal muscle cells.
J Pharmacol Exp Ther
260:
90-97,
1992[Abstract].
17.
Murthy, KS,
and
Makhlouf GM.
Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cells.
Am J Physiol Cell Physiol
268:
C171-C180,
1995[Abstract/Free Full Text].
18.
Murthy, KS,
and
Makhlouf GM.
Differential inhibition of PLA2-dependent Ca2+ influx in smooth muscle by cA-kinase and cG-kinase.
J Biol Chem
273:
34519-34526,
1998[Abstract/Free Full Text].
19.
Murthy, KS,
Severi C,
Grider JR,
and
Makhlouf GM.
Inhibition of IP3 production and IP3-dependent Ca2+ mobilization by cyclic nucleotides in isolated gastric muscle cells.
Am J Physiol Gastrointest Liver Physiol
264:
G967-G974,
1993[Abstract/Free Full Text].
20.
Murthy, KS,
Teng BQ,
Jin JG,
and
Makhlouf GM.
G protein-dependent activation of smooth eNOS meditated by natriuretic peptide-C receptor.
Am J Physiol Cell Physiol
275:
C1409-C1416,
1998[Abstract/Free Full Text].
21.
Murthy, KS,
Zhou H,
and
Makhlouf GM.
Inhibition of G
q-dependent phosphoinositide hydrolysis by PKA and PKG is mediated by phosphorylation of RGS4 and GRK2 (Abstract).
Gastroenterology
122:
A21,
2002.
22.
Murthy, KS,
Zhou H,
Grider JR,
and
Makhlouf GM.
Inhibition of RhoA-dependent sustained smooth muscle contraction by cAMP- and cGMP-dependent protein kinase (Abstract).
Gastroenterology
120:
A201,
2001.
23.
Nakade, S,
Maeda N,
and
Mikoshiba K.
Involvement of C-terminus of the inositol 1,4,5-trisphosphate receptor in Ca2+ release analyzed using region-specific monoclonal antibodies.
Biochem J
277:
125-131,
1991[ISI][Medline].
24.
Patel, S,
Joseph SK,
and
Thomas AP.
Molecular properties of inositol 1,4,5-trisphosphate receptors.
Cell Calcium
25:
247-264,
1999[ISI][Medline].
25.
Quignard, JF,
Frapier JM,
Harricane MC,
Albat B,
Nargeot J,
and
Richards S.
Voltage-gated calcium channel currents in human coronary myocytes. Regulation by cGMP and nitric oxide.
J Clin Invest
99:
185-193,
1997[Abstract/Free Full Text].
26.
Ross, CA,
Danoff SK,
Schell MJ,
Snyder SH,
and
Ullrich A.
Three additional inositol 1,4,5-trisphosphate receptors: molecular cloning and differential localization in brain and peripheral tissues.
Proc Natl Acad Sci USA
89:
4265-4269,
1992[Abstract].
27.
Sauzeau, V,
Le Jeune H,
Cario-Toumaniantz C,
Smolenski A,
Lohmann SM,
Beroglio J,
Chardin P,
Pacaud P,
and
Loriand G.
Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle.
J Biol Chem
275:
21722-21729,
2000[Abstract/Free Full Text].
28.
Schlossmann, J,
Ammendola A,
Ashman K,
Zong X,
Huber A,
Neubauer G,
Wang G,
Allescher H,
Korth M,
Wilm M,
Hofmann F,
and
Ruth P.
Regulation of intracellular calcium by a signaling complex of IRAG, IP3 receptor and cGMP kinase.
Nature
404:
197-201,
2000[ISI][Medline].
29.
Soff, G,
Cornwell T,
Cundiff D,
Gately S,
and
Lincoln T.
Smooth muscle cell expression of type I cGMP-dependent protein kinase is suppressed by continuous exposure to nitrovasodilators, theophylline, cGMP, and cAMP.
J Clin Invest
100:
2580-2587,
1997[Abstract/Free Full Text].
30.
Somlyo, AP,
and
Somlyo AV.
Signal transduction by G proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II.
J Physiol
522:
177-185,
2000[Abstract/Free Full Text].
31.
Supattapone, S,
Danoff S,
Theibert A,
Joseph SK,
Steiner J,
and
Snyder S.
Cyclic AMP-dependent phosphorylation of a brain inositol trisphosphate receptor decreases its release of calcium.
Proc Natl Acad Sci USA
85:
8747-8750,
1998.
32.
Surks, HK,
Mochizuki N,
Kasai Y,
Geogescu SP,
Tang KM,
Ito M,
Lincoln TM,
and
Mendelsohn ME.
Regulation of myosin phosphatase by a specific interaction with cGMP-dependent protein kinase I.
Science
286:
1583-1587,
1999[Abstract/Free Full Text].
33.
Szewczak, SM,
Behar J,
Bellet C,
Hillemeier B,
Rhim Y,
and
Biancani P.
VIP-induced alterations in cAMP and inositol phosphates in the lower esophageal sphincter.
Am J Physiol Gastrointest Liver Physiol
259:
G239-G244,
1990[Abstract/Free Full Text].
34.
Tachado Akthar, RA SD,
Zhou CJ,
and
Abdel-Latif AA.
Effects of isoproterenol and forskolin on carbachol-and fluroaluminate-induced polyphosphoinositide hydrolysis, inositol trisphosphate production and contraction bovine iris-sphincter smooth muscle: interaction between cAMP and IP3 second messenger system.
Cell Signal
4:
61-75,
1992[ISI][Medline].
35.
Tamura, N,
Itoh H,
Ogawa Y,
Nakagawa O,
Harada M,
Chun T,
Suga S,
Yoshimasa T,
and
Nakao K.
cDNA cloning and gene expression of human type I cGMP-dependent protein kinase.
Hypertension
27:
552-557,
1996[Abstract/Free Full Text].
36.
Taylor, CW,
Genazzani AA,
and
Morris SA.
Expression of inositol trisphosphate receptors.
Cell Calcium
26:
237-251,
1999[ISI][Medline].
37.
Tertyshnikova, S,
Yan X,
and
Fein A.
cGMP inhibits IP3-induced Ca2+ release in intact rat megakaryocytes via cGMP- and cAMP-dependent protein kinases.
J Physiol
512:
89-96,
1998[Abstract/Free Full Text].
38.
Thrower, EC,
Hager RE,
and
Ehrlich BE.
Regulation of Ins (1,4,5)P3 receptor isoforms by endogenous modulators.
Trends Pharmacol Sci
22:
580-586,
2001[ISI][Medline].
39.
Yuasa, K,
Michibata H,
Omori K,
and
Yanaka N.
A novel interaction of cGMP-dependent protein kinase I with troponin T.
J Biol Chem
274:
37429-37434,
1999[Abstract/Free Full Text].
40.
Wojcikiewicz, R,
and
Luo S.
Phosphorylation of inositol 1,4,5-trisphosphate receptors by cAMP-dependent protein kinase: type I, II and III receptors are differentially susceptible to phosphorylation and are phosphorylated in intact cells.
J Biol Chem
273:
5670-5677,
1998[Abstract/Free Full Text].
Am J Physiol Gastrointest Liver Physiol 284(2):G221-G230
0193-1857/03 $5.00
Copyright © 2003 the American Physiological Society