Departments of Physiology and Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298
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ABSTRACT |
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Regulation of adenylyl cyclase type
V/VI and cAMP-specific, cGMP-inhibited phosphodiesterase (PDE) 3 and
cAMP-specific PDE4 by cAMP-dependent protein kinase (PKA) and
cGMP-dependent protein kinase (PKG) was examined in gastric smooth
muscle cells. Expression of PDE3A but not PDE3B was demonstrated by
RT-PCR and Western blot. Basal PDE3 and PDE4 activities were present in
a ratio of 2:1. Forskolin, isoproterenol, and the PKA activator
5,6-dichloro-1--D-ribofuranosyl benzimidazole
3',5'-cyclic monophosphate, SP-isomer, stimulated PDE3A phosphorylation
and both PDE3A and PDE4 activities. Phosphorylation of PDE3A and
activation of PDE3A and PDE4 were blocked by the PKA inhibitors
[protein kinase inhibitor (PKI) and H-89] but not by the PKG
inhibitor (KT-5823). Sodium nitroprusside inhibited PDE3 activity and
augmented forskolin- and isoproterenol-stimulated cAMP levels; PDE3
inhibition was reversed by blockade of cGMP synthesis. Forskolin
stimulated adenylyl cyclase phosphorylation and activity; PKI blocked
phosphorylation and enhanced activity. Stimulation of cAMP and
inhibition of inositol 1,4,5-trisphosphate-induced Ca2+
release and muscle contraction by isoproterenol were augmented additively by PDE3 and PDE4 inhibitors. The results indicate that PKA
regulates cAMP levels in smooth muscle via stimulatory phosphorylation of PDE3A and PDE4 and inhibitory phosphorylation of adenylyl cyclase type V/VI. Concurrent generation of cGMP inhibits PDE3 activity and
augments cAMP levels.
protein kinase A; phosphodiesterase; cyclic nucleotides; relaxation
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INTRODUCTION |
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THE MAIN RELAXANT NEUROPEPTIDES of the gut, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP), initiate dual signaling cascades in smooth muscle that ultimately lead to concurrent generation of cGMP and cAMP (22, 23). Both VIP and PACAP interact with cognate seven-transmembrane VPAC2 receptors coupled via Gs to activation of adenylyl cyclase and with single-transmembrane natriuretic peptide clearance receptors coupled via Gi-1 and Gi-2 to sequential activation of endothelial nitric oxide synthase and soluble guanylyl cyclase (22, 27). cGMP preferentially activates cGMP-dependent protein kinase (PKG), whereas cAMP, which is produced in greater abundance than cGMP (~10-fold), preferentially activates cAMP-dependent protein kinase (PKA) and, at higher concentrations, cross activates PKG (5, 10, 14, 23). The extent of cAMP and cGMP accumulation, and the intensity and duration of their physiological effects depend on the rates of their synthesis by adenylyl cyclase and guanylyl cyclase, respectively, and the rates of their breakdown by various phosphodiesterases (PDEs; see Refs. 2, 3, 6, 8, 34).
Cyclic nucleotide PDEs comprise 11 families of related enzymes that differ in their genetic derivation, molecular structure, substrate selectivity, inhibitor sensitivity, and mechanisms of regulation (2, 3, 6, 8, 34). PDE4, PDE7, and PDE8 are highly specific for cAMP, and PDE5, PDE6, and PDE9 are highly specific for cGMP. PDE1, PDE2, PDE3, PDE10, and PDE11 exhibit dual specificity with greater or lesser preference for cAMP or cGMP. A conserved carboxyl terminal catalytic domain contains a histidine-rich motif [HD(X2)H(X)4N] and two consensus Zn2+-binding sites (2, 6, 34). A divergent amino terminal domain confers isoform-specific regulatory properties, such as calmodulin and cGMP binding, membrane association, and PKA- or PKG-dependent phosphorylation.
PDE3A is highly expressed in cardiac muscle, vascular and visceral smooth muscle, and platelets, whereas PDE3B, the product of a distinct gene, is expressed in adipocytes, hepatocytes, spermatocytes, and renal collecting duct epithelium (1, 16, 18, 28, 30, 31, 35). Multiple isoforms generated by alternative mRNA splicing or different promoters have been identified (2, 19). cAMP and cGMP bind with equally high affinity to PDE3; however, cGMP is poorly hydrolyzed and thus acts as an inhibitor of cAMP hydrolysis (2, 34). Concurrent generation of cAMP and cGMP is the physiological norm in smooth muscle of the gut (23, 25); it would be expected under these conditions that cGMP might increase cAMP levels by attenuating PDE3 activity.
PDE4 is mainly expressed in inflammatory cells and central neurons (2, 8, 20, 34). Its presence in vascular and visceral smooth muscle has been inferred from the ability of selective PDE4 inhibitors to enhance relaxation (15, 36, 37). Four PDE4 genes (PDE4A-D) encoding multiple isoforms generated by alternative mRNA splicing or alternate promoters have been identified (8, 20). PDE4 is susceptible to stimulatory serine phosphorylation by PKA in the regulatory domain and inhibitory or stimulatory serine phosphorylation by p42/44-mitogen-activated protein kinase in the catalytic domain (4, 7, 13, 17).
Our recent studies (21) have shown that both PKA and PKG activate PDE5 but that only PKG inhibits soluble guanylyl cyclase; concurrent generation of cAMP led to PKA-dependent activation of PDE5 and attenuation of cGMP levels. The balance of adenylyl cyclase and cAMP-specific PDE3 and PDE4 activities determines the levels of cAMP in gastrointestinal smooth muscle. Because PDE3 activity is inhibited by cGMP, concurrent generation of cGMP should influence the levels of cAMP. The regulation of adenylyl cyclase and of PDE3 and PDE4 by PKA and PKG and the effect of concurrent generation of cGMP on cAMP levels have not been characterized in gastrointestinal smooth muscle. Studies in other cell types (2, 4, 9, 13) have shown that PKA activates PDE3 and PDE4 and inhibits adenylyl cyclase but have not examined the effect of PKG or the influence of concurrent generation of cGMP on cAMP levels. In this study, we examined 1) whether PKA and/or PKG regulates the activity of adenylyl cyclase type V/VI expressed in gastric smooth muscle (24) and the activities of PDE4 and cGMP-inhibited PDE3 and 2) the extent to which concurrent generation of cGMP affects cAMP levels. The results indicate that PKA regulates cAMP levels via stimulatory phosphorylation of PDE3A and PDE4 and inhibitory phosphorylation of adenylyl cyclase type V/VI. Concurrent generation of cGMP enhances cAMP levels by attenuating PDE3A activity.
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MATERIALS AND METHODS |
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Preparation of dispersed gastric smooth muscle cells. Smooth muscle cells were isolated from the circular muscle layer of rabbit stomach by sequential enzymatic digestion, filtration, and centrifugation, as described previously (22-24). The partly digested strips were washed, and muscle cells were allowed to disperse spontaneously. The cells were harvested by filtration through 500-µm Nitex.
RT-PCR analysis of PDE3A and PDE3B. Specific primers were designed based on homologous sequences in human, rat, and mouse PDE3A and PDE3B. PDE3A primers were 5'-CATTCAGAATGGGACCACAA-3' (forward) and 5'-GTCTGCCACAGCTGCTAC-3' (reverse; corresponding to nucleotide numbers 964-983 and 1754-1771 in the open-reading frame of rat PDE3A, respectively); the expected size of the PCR product was 807 bp. PDE3B primers were 5'-GGAGGTGGAAATGGA-3' (forward) and 5'-GGGTGATCGATTAGTTAG-3' (reverse; corresponding to nucleotide numbers 988-1002 and 1540-1557 in the open-reading frame of rat PDE3B, respectively); the expected size of the PCR product was 569 bp. RNA (5 µg) prepared from dispersed rabbit gastric smooth muscle cells were reversibly transcribed. Reversibly transcribed cDNA (5 µl) was 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 using a QIAquick Gel Extraction Kit (Qiagen) and sequenced.
Western blot analysis for PDE3A and PDE3B. Cell homogenates were prepared from dispersed muscle cells and solubilized on ice for 1 h in 20 mM Tris · HCl (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, and 0.5% sodium cholate. Solubilized proteins (100 µg) were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated for 12 h at 4°C with antibodies (1:1,000) to PDE3A and PDE3B and then for 1 h with secondary antibody conjugated with horseradish peroxidase. The protein bands were visualized by enhanced chemiluminescence.
Assay for PDE3 and PDE4 activity. Muscle cells (3 × 106 cells/ml) were homogenized and assayed for cAMP-specific PDE activity by using [3H]cAMP as substrate, as described previously (4, 17). Reactions were carried out for 15 min at 30°C in a medium containing 20 mM Tris · HCl (pH 7.5), 20 mM imidazole (pH 7.5), 5 mM MgCl2, 15 mM Mg acetate, 0.2 mg/ml BSA, and 2 µM [3H]cAMP. 5'-[3H]AMP was converted to [3H]adenosine by the nucleotidase action of Crotalus atrox snake venom (25 µg). Samples were applied to DEAE-Sephacel A-25 columns equilibrated with 20 mM Tris · HCl (pH 7.5). The radioactivity in the effluent was counted, and the results were expressed as counts per minute (cpm) per milligram protein. PDE3 activity was measured in the presence of the PDE4 inhibitor rolipram (10 µM). PDE4 activity was measured in the presence of the PDE3 inhibitor milrinone (10 µM).
Phosphorylation of PDE3A. Phosphorylation of PDE3A was measured from the amount of 32P incorporated in the enzyme after immunoprecipitation with specific PDE3A antibody. Ten milliliters of smooth muscle cell suspension (3 × 106 cells/ml) were incubated with [32P]orthophosphate for 4 h at 31°C. Samples (1 ml) were then incubated with various agents for 1 min in the presence or absence of the PKA inhibitors [H-89 or myristoylated protein kinase inhibitor (PKI)] or the PKG inhibitor (KT-5823). Cell lysates were separated by centrifugation at 13,000 g for 5 min at 4°C, precleared with 40 µl of protein A-Sepharose, and incubated with PDE3A antibody for 2 h at 4°C and with 40 µl of protein A-Sepharose for another 1 h. The immunoprecipitates were extracted with Laemmli sample buffer, boiled for 15 min, and separated by electrophoresis on 10% SDS-PAGE. After transfer to polyvinylidene difluoride membranes, [32P]PDE3A was visualized by autoradiography, and the amount of radioactivity in the band was measured. The results were expressed as cpm.
Assay for adenylyl cyclase activity.
Adenylyl cyclase activity was measured by using
[-32P]ATP as substrate, as described previously
(22, 32). Crude homogenates of dispersed gastric muscle
cells were incubated for 15 min at 37°C in 50 mM Tris-HCl (pH 7.4), 2 mM cAMP, 0.1 mM ATP, 1 mM IBMX, 5 mM MgCl2, 100 mM NaCl, 5 mM creatine phosphate, 50 U/ml creatine phosphokinase, and 0.5 mM
[
-32P]ATP (~ 0.2 µCi). The reaction was terminated
by addition of 2% SDS, 45 mM ATP, and 1.5 mM cAMP.
[32P]cAMP was separated from [32P]ATP by
sequential chromatography on Dowex AG50W-4X and alumina columns. The
results were expressed as picomoles of cAMP per milligram protein per minute.
Phosphorylation of adenylyl cyclase type V/VI. Our previous studies had shown that the adenylyl cyclase isozyme expressed in rabbit gastric and intestinal smooth muscle is adenylyl cyclase types V/VI (24). Phosphorylation of this isozyme was measured from the amount of 32P incorporated after immunoprecipitation with a specific polyclonal antibody to adenylyl cyclase types V/VI, as described above for PDE3A.
Measurement of cAMP by RIA. cAMP levels were measured by RIA as described previously (23, 24, 26). Dispersed muscle cells (3 × 106 cells) were stimulated with isoproterenol or forskolin for 1 min, and the reaction was terminated with 10% trichloroacetic acid. After extraction, the lyophilized aqueous phase was reconstituted in 500 µl of 50 mM sodium acetate (pH 6.2) for assay, and the samples were acetylated with triethylamine-acetic anhydride (2:1) for 30 min. cAMP was measured in duplicate by using 100-µl aliquots, and the results were expressed as picomoles per milligram protein.
Measurement of 45Ca2+ release in dispersed smooth muscle cells. 45Ca2+ release was measured in permeabilized muscle cells as described previously (23, 26, 29). Permeabilized muscle cells were suspended in a medium containing 100 nM Ca2+, 10 µM antimycin, and 10 µCi/ml 45Ca2+. ATP (1.5 mM) and the ATP-regenerating system (5 mM creatine phosphate and 10 U/ml creatine kinase) were added, and steady-state Ca2+ uptake was measured after 60 min. Inositol 1,4,5-trisphosphate (IP3; 1 µM) was added, and 45Ca2+ release was determined after 15 s. Isoproterenol (10 nM), alone or with milrinone (10 µM) and/or rolipram (10 µM), was added 60 s before IP3. IP3-induced 45Ca2+ release was expressed as the decrease in steady-state 45Ca2+ cell content (2.64 ± 0.32 nmol/106 cells). Inhibition of Ca2+ release by isoproterenol was expressed as the decrease in IP3-induced Ca2+ release.
Measurement of relaxation in dispersed muscle cells. Inhibition of IP3-induced contraction (i.e., relaxation) by isoproterenol was expressed as the decrease in maximal cell contraction induced by 1 µM IP3 (24, 26). Briefly, an aliquot (0.5 ml) of cell suspension was added to 0.2 ml of HEPES medium containing IP3 alone, IP3 plus isoproterenol, or IP3 plus isoproterenol, milrinone, and/or rolipram. The mean cell length of 50 muscle cells treated with various agents was measured by scanning micrometry and was compared with the length of untreated muscle cells (mean control cell length: 104 ± 5 µm).
Materials.
[-32P]ATP, [32P]orthophoshate,
[125I]cAMP, and [3H]cAMP were obtained from
Amersham Pharmacia Biotech (Piscataway, NJ); collagenase and soybean
trypsin inhibitor were from Worthington Biochemical (Freehold, NJ);
Western blotting and chromatography material were from Bio-Rad
Laboratories (Hercules, CA); adenylyl cyclase type V/VI antibody was
from Santa Cruz Biotechnology (Santa Cruz, CA); C. atrox
snake venom and all other chemicals were from Sigma Chemical (St.
Louis, MO). PDE3A and PDE3B antibodies were a gift from Drs. Vincent
Manganiello and Young Choi from the Pulmonary/Critical Care Medicine
Branch, National Heart, Lung, and Blood Institute, National Institutes
of Health.
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RESULTS |
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Expression of PDE3A in gastric smooth muscle.
PDE3A was detected by RT-PCR in gastric smooth muscle cells by using
primers based on conserved sequences of human, rat, and mouse PDE3A
(Fig. 1). The amplified partial DNA
sequence of rabbit PDE3A (GenBank accession no. AY057938) was 84%
similar to the human sequence and 81-83% similar to rat and mouse
sequences. The primary amino acid sequence was 89% similar to the
human, rat, and mouse sequences. PDE3B could not be detected by RT-PCR by using primers based on conserved sequences of human, rat, and mouse
PDE3B. Western blot analysis confirmed expression of PDE3A but not
PDE3B (Fig. 1). The large number of PDE4 isoforms and splice variants
and the lack of PDE4 antibody precluded analysis of PDE4 expression.
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Basal PDE3 and PDE4 activities in smooth muscle.
Basal cAMP-specific PDE activity in homogenates of dispersed smooth
muscle cells (measured by using [3H]cAMP as substrate,
2,805 ± 189 cpm/mg protein) was inhibited to the same extent by 1 µM cGMP (40 ± 5%) and by the selective PDE3 inhibitors
milrinone (10 µM; 42 ± 3%) and trequinsin (1 µM; 43 ± 6%; Fig. 2). The effect of a combination
of cGMP with either milrinone or trequinsin was not additive (44 ± 4 and 45 ± 5% inhibition; Fig. 2).
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Phosphorylation of PDE3A by PKA.
Analysis of PDE3A immunoprecipitates obtained from muscle cells labeled
with 32P showed selective phosphorylation of PDE3A by PKA.
Forskolin increased PDE3A phosphorylation by 301 ± 31%
(P < 0.01); the increase in phosphorylation was
inhibited by the PKA inhibitors myristoylated PKI (1 µM) and H-89 (1 µM) but was not affected by the PKG inhibitor KT-5823 (1 µM; Fig.
3). Selective activation of PKA by the
cAMP analog 5,6-dichloro-1--D-ribofuranosyl
benzimidazole 3',5'-cyclic monophosphate, SP-isomer (cBIMPS; 10 µM),
also increased PDE3A phosphorylation by 278 ± 26%
(P < 0.01), and the increase was inhibited by
myristoylated PKI and H-89 (Fig. 3). Direct measurements of PKA and PKG
activities in these muscle cells (23) have shown that, at
concentrations of 1 µM and less, PKI and H-89 selectively inhibit PKA
activity, whereas KT-5823 selectively inhibits PKG activity.
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Activation of PDE3 and PDE4 by PKA.
Forskolin (10 µM) stimulated basal cAMP-specific PDE activity
(basal: 2,853 ± 210 cpm/mg protein; forskolin: 4,069 ± 306 cpm/mg protein; P < 0.01; Fig.
5). Basal and forskolin-stimulated PDE activities decreased from their control levels in the presence of
rolipram or milrinone. The residual increase in PDE activity induced by
forskolin was abolished by myristoylated PKI (Fig. 5). Myristoylated
PKI had no effect on basal PDE3 activity (2,762 ± 301 cpm/mg
protein). Basal activity in the presence of both milrinone and rolipram
decreased to 442 ± 46 cpm/mg protein; this activity was not
augmented by forskolin (566 ± 90 cpm/mg protein) or inhibited by
myristoylated PKI (550 ± 44 cpm/mg protein). Similar results were
obtained upon activation of cAMP-specific PDE with isoproterenol or
cBIMPS (Fig. 6).
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Inhibition of PDE3 activity by cGMP.
SNP (0.1 µM) increased basal cGMP levels by 52 ± 5% and
inhibited forskolin-stimulated PDE3 activity (i.e., cAMP-specific PDE
activity measured in the presence of the PDE4 inhibitor rolipram) by
46 ± 4% (Fig. 7). The soluble
guanylyl cyclase inhibitor ODQ completely reversed SNP-induced
inhibition of PDE3 activity, whereas KT-5823 caused further inhibition
(74 ± 5%) of PDE3 activity (Fig. 7). The effect of KT-5823
implied that inhibition of PKG-stimulated PDE5 activity led to an
increase in SNP-stimulated cGMP levels (21) and to greater
inhibition of PDE3 activity by cGMP.
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Augmentation of cAMP levels by cGMP-mediated inhibition of PDE3.
Concurrent stimulation of cGMP by SNP (0.1 µM) increased
forskolin-stimulated cAMP levels (Fig.
8). The effect of SNP was concentration
dependent (Fig. 8) and was not augmented further by milrinone, implying
that it reflected inhibition of PDE3 activity. The ability of SNP to
increase cAMP levels was greatly enhanced by the PDE5 inhibitor
zaprinast (Fig. 8), providing further evidence that SNP augments cAMP
levels by inducing cGMP-mediated inhibition of PDE3 activity.
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Augmentation of cAMP levels by PDE3 and PDE4 inhibitors.
Milrinone (10 µM) and trequinsin (1 µM) increased basal cAMP levels
by 15 ± 4 and 19 ± 5%, respectively, whereas rolipram (10 µM) had no effect (Fig. 9). The cAMP
response to a near-threshold concentration (10 nM) of isoproterenol
(12 ± 4% above the basal level; P < 0.01) was
significantly augmented to 26 ± 4% (P < 0.02) by rolipram and 72 ± 7% (P < 0.01) by
trequinsin (Fig. 9). The cAMP response in the presence of a combination
of rolipram and trequinsin was increased further to 135 ± 11%,
representing a ninefold increase in the cAMP response over the response
to isoproterenol alone (Fig. 9). The cAMP response to 10 nM
isoproterenol (12 ± 4%) was increased to 74 ± 16% by SNP,
reflecting inhibition of PDE3 activity by SNP-stimulated cGMP. The
increase induced by SNP was similar to that induced by trequinsin
(72%; see Fig. 9).
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Feedback phosphorylation and inhibition of adenylyl cyclase by PKA.
We have recently shown the existence of an inhibitory feedback
mechanism mediated by PKG-specific phosphorylation and inhibition of
soluble guanylyl cyclase (21). Here we show a similar
inhibitory feedback mechanism mediated by PKA-specific phosphorylation
and inhibition of adenylyl cyclase. Forskolin stimulated the
phosphorylation of adenylyl cyclase type V/VI in a
concentration-dependent fashion (Fig.
10), whereas SNP (0.1 µM) had no
effect. The increase in adenylyl cyclase phosphorylation induced by
forskolin was abolished by myristoylated PKI. In contrast,
forskolin-stimulated adenylyl cyclase activity was significantly
enhanced by PKI at all concentration levels (Fig.
11), implying that phosphorylation
resulted in inhibition of activity. Thus PKA initiates dual feedback
inhibitory mechanisms that lead to a decrease in cAMP synthesis via
inhibitory phosphorylation of adenylyl cyclase and an increase in cAMP
degradation via stimulatory phosphorylation of PDE3 and PDE4.
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Modulation of cAMP-mediated cellular responses by PDE3A and PDE4.
In permeabilized gastric smooth muscle cells, IP3 (1 µM)
stimulated Ca2+ release, measured as the decrease in
steady-state 45Ca2+ cell content during the
first 15 s after stimulation (steady-state 45Ca2+ content: 2.64 ± 0.32 nmol/106 cells, decrease in 45Ca2+
cell content: 0.82 ± 0.04 nmol/106 cells), and
induced smooth muscle cell contraction, measured as the mean decrease
in control muscle cell length (mean control cell length: 104 ± 5 µm, mean decrease in cell length: 33 ± 4 µm). A low
concentration of isoproterenol inhibited Ca2+ release by
10 ± 2% and muscle cell contraction by 20 ± 3% (Fig. 12). Inhibition of Ca2+
release and muscle cell contraction increased significantly in the
presence of either milrinone or rolipram and increased further in the
presence of a combination of milrinone and rolipram (Fig. 12).
Inhibition of Ca2+ release and muscle cell contraction in
the presence of both inhibitors increased by fourfold and twofold,
respectively.
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DISCUSSION |
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A distinctive feature of gastrointestinal smooth muscle is the
concurrent production of cGMP and cAMP during physiological relaxation,
which engages a variety of regulatory mechanisms mediated by cyclic
nucleotide-dependent protein kinases, PDEs, and cyclases. The present
study investigated the mechanisms by which PKA and PKG regulate the
activities of adenylyl cyclase and cAMP-specific PDE3 and PDE4 in
dispersed gastric smooth muscle cells, as well as the effect of
concurrent generation of cGMP. The results demonstrate 1)
PKA-specific phosphorylation of PDE3A and activation of PDE3A and PDE4
and 2) PKA-specific phosphorylation and inhibition of adenylyl cyclase type V/VI, the only types expressed in gastric smooth
muscle (24). The results also indicate that concurrent production of cAMP and cGMP leads to a further increase in cAMP levels
resulting from cGMP-induced inhibition of PDE3. The evidence is
summarized below, and the conclusions are depicted schematically in
Fig. 13.
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1) RT-PCR and Western blot analysis showed that PDE3A but not PDE3B is expressed in gastric smooth muscle. It is not known which of the numerous isoforms of PDE4 is (or are) expressed in these muscle cells.
2) Basal PDE4 activity (measured in the presence of milrinone or trequinsin) and basal PDE3 activity (measured in the presence of rolipram) were present in a ratio of 1:2. The effect of cGMP on basal activity paralleled that of milrinone or trequinsin and was additive to that of rolipram.
3) Forskolin, isoproterenol, and the selective PKA activator cBIMPS stimulated PDE3A phosphorylation and activity that were inhibited by the selective PKA inhibitors myristoylated PKI and H-89 but were not affected by the selective PKG inhibitor KT-5823. SNP did not stimulate PDE3A phosphorylation but augmented isoproterenol-stimulated phosphorylation; the increase in phosphorylation induced by SNP reflected an increase in cAMP levels resulting from cGMP-mediated inhibition of PDE3.
4) PDE3 and PDE4 activities stimulated by forskolin, isoproterenol, and cBIMPS were mediated by PKA and abolished by myristoylated PKI.
5) Forskolin-stimulated PDE3 activity was inhibited by SNP, and the inhibition was reversed by blockade of cGMP synthesis with ODQ, implying that PDE3 activity was inhibited by cGMP (Fig. 7). The inhibition of PDE3 activity by SNP-stimulated cGMP caused an increase in forskolin- and isoproterenol-stimulated cAMP levels, as shown in Figs. 8 and 9, and an increase in PKA-stimulated phosphorylation of PDE3, as shown in Fig. 4; thus, a concurrent increase in cGMP led to a decrease in PDE3 activity, an increase in cAMP levels, and an increase in PKA-dependent phosphorylation of PDE3. The pattern implied that cGMP-dependent inhibition of PDE3 activity predominated over its activation by PKA.
6) PDE3 and PDE4 inhibitors augmented isoproterenol-stimulated cAMP levels, and their effects were additive.
7) Forskolin activated adenylyl cyclase type V/VI and stimulated its phosphorylation in a concentration-dependent fashion. PKI abolished phosphorylation and augmented adenylyl cyclase activity, implying that feedback inhibition of adenylyl cyclase activity by PKA-dependent phosphorylation impeded further formation of cAMP. Phosphorylation and inhibition of adenylyl cyclase were PKA specific.
8) Isoproterenol-induced inhibition of IP3-dependent Ca2+ release and muscle cell contraction were enhanced by PDE3 and PDE4 inhibitors; the effects of the inhibitors were additive.
Thus cAMP levels in gastric smooth muscle are regulated by PKA-dependent feedback stimulation of PDE3A and PDE4 activity and inhibition of adenylyl cyclase V/VI activity. These results may be usefully contrasted with those obtained in a parallel study of PDE5, which is also expressed in gastric smooth muscle (21). Both PKA and PKG phosphorylated and activated PDE5, whereas only PKG phosphorylated and inhibited soluble guanylyl cyclase. When both cyclic nucleotides were present, cAMP attenuated the levels of cGMP via PKA-dependent activation of PDE5, whereas cGMP enhanced the levels of cAMP by inhibiting PDE3 activity (Fig. 8). An equally important consequence of the concurrent production of cAMP and cGMP was a change in the relative abundance and the characteristics of activation of PKA and PKG (5). The increase in cAMP levels resulted in greater PKA activity. Although cGMP levels were attenuated, the binding of cGMP to autophosphorylated PKG greatly enhanced the affinity of cAMP for PKG. cAMP, which is generated in ~10 times greater abundance than cGMP, became the main activator of PKG and greatly enhanced its activity (5, 12, 23, 33). At concentrations <1 µM, isoproterenol stimulates cAMP and activates PKA without cross activating PKG in vascular and visceral smooth muscle, including gastric smooth muscle (10, 23). In the present study, isoproterenol (10 nM) inhibited IP3-dependent Ca2+ release and muscle contraction; both effects were augmented additively by PDE3 and PDE4 inhibitors (Fig. 12) and, as shown previously (23), were mediated by PKA. In permeabilized smooth muscle cells, PKA can gain access to and directly phosphorylate the IP3 receptor, causing inhibition of IP3-dependent Ca2+ release (11, 25). In intact muscle cells, however, PKA inhibits Ca2+ release indirectly by inhibiting phosphoinositide hydrolysis and IP3 formation (26). PKG, however, inhibits Ca2+ release in both intact and permeabilized muscle cells, directly by phosphorylating the IP3 receptor and indirectly by inhibiting IP3 formation (11, 25, 26). Although both PKA and PKG activities are enhanced when both cAMP and cGMP are present, PKG largely mediates the inhibition of IP3-dependent Ca2+ release and muscle contraction (14, 23). It is worth emphasizing that concurrent generation of cAMP and cGMP is the physiological norm in smooth muscle of the gut and probably other visceral smooth muscle. In the gut, the resultant interplay of cyclic nucleotide-activated protein kinases and PDEs attenuates cGMP and enhances cAMP. Inhibitory feedback mechanisms that target the cyclases and arrest further synthesis are more specific, leading to PKA-mediated inhibition of adenylyl cyclase and PKG-mediated inhibition of soluble guanylyl cyclase (21). Although both PKA and PKG activities are enhanced, PKG becomes the main determinant of function, i.e., inhibition of Ca2+ mobilization and muscle contraction. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. Vincent C. Manganiello, Chief, Section on Biochemical Physiology, Pulmonary/Critical Care Medicine Branch, National Heart, Lung, and Blood Institute for helpful comments and a critical review of the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-28300.
Address for reprint requests and other correspondence: K. S. Murthy, P.O. Box 908711, 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/ajpcell.00373.2001
Received 3 August 2001; accepted in final form 31 October 2001.
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