PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle

Karnam S. Murthy, Huiping Zhou, and Gabriel M. Makhlouf

Departments of Physiology and Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

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 [alpha -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 [alpha -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. [alpha -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Expression of phosphodiesterase (PDE) 3A in freshly dispersed smooth muscle cells. A: total RNA isolated from dispersed gastric muscle cells of rabbit was reverse transcribed, and cDNA was amplified with specific primers for PDE3A. Experiments were done in the presence (+) or absence (-) of reverse transcriptase (RT). No RT-PCR product was obtained with primers for PDE3B. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B: Western blot analysis of lysates prepared from dispersed smooth muscle cells of the circular muscle layer of the rabbit stomach (gastric) and small intestine (intestinal). The proteins were probed with polyclonal antibodies to PDE3A and PDE3B, and the bands were detected by enhanced luminescence. PDE3A but not PDE3B protein could be detected in both muscle layers.

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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Fig. 2.   Effect of cGMP and specific PDE3 and PDE4 inhibitors on basal cAMP-specific PDE activity. cAMP-specific PDE activity was measured in homogenates of dispersed gastric smooth muscle cells by using [3H]cAMP as substrate. Activity was measured in the presence of cGMP (1 µM), the PDE3 inhibitors milrinone (10 µM) or trequinsin (1 µM) (A), and the PDE4 inhibitor rolipram (10 µM) (B). The agents were added alone or in combination for 10 min. The results are expressed as cpm/mg protein. Values are means ± SE of 4-6 experiments. **Significant inhibition (P < 0.01) of basal PDE activity.

Basal cAMP-specific PDE activity was also inhibited (26 ± 2%) by the selective PDE4 inhibitor rolipram (10 µM). The effect of a combination of rolipram with cGMP, milrinone, or trequinsin was additive, eliciting 68 ± 4, 70 ± 3, and 67 ± 5% inhibition, respectively (Fig. 2). The results provide evidence for the existence of both cGMP-inhibited, cAMP-specific PDE3 activity and cAMP-specific PDE4 activity in gastric smooth muscle cells. PDE3 activity was about twofold greater than PDE4 activity. A small residual basal activity in the presence of PDE3 and PDE4 inhibitors could reflect the presence of other cAMP-specific PDE isoforms such as PDE7 and/or PDE8.

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-beta -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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Fig. 3.   Phosphorylation of PDE3A by cAMP-dependent protein kinase (PKA) in smooth muscle. Gastric smooth muscle cells labeled with 32P were incubated with forskolin (1 µM) or with the selective PKA activator 5,6-dichloro-1-beta -D-ribofuranosyl benzimidazole 3',5'-cyclic monophosphate SP-isomer (cBIMPS; 10 µM) for 1 min in the presence or absence of the PKA inhibitors myristoylated protein kinase inhibitor (PKI; 1 µM) and H-89 (1 µM) and the cGMP-dependent protein kinase (PKG) inhibitor KT-5823 (KT; 1 µM). Immunoprecipitates derived from 500 µg of protein using polyclonal PDE3A antibody were separated on SDS-PAGE. [32P]PDE3A was identified by autoradiography (A). Measured radioactivity of the bands is expressed as counts/min (cpm; C), and immunoblots (50 µg protein) of the bands are shown for a loading control (B). Values are means ± SE of 4 experiments. **Significant inhibition (P < 0.001) of forskolin- or cBIMPS-induced phosphorylation.

Isoproterenol, like forskolin, stimulated PKA-dependent phosphorylation of PDE3A that was inhibited by myristoylated PKI but was not affected by KT-5823 (Fig. 4). Sodium nitroprusside (SNP), which selectively stimulates cGMP and activates PKG in these muscle cells (21, 23), did not induce PDE3A phosphorylation, but it augmented isoproterenol-induced phosphorylation. PDE3A phosphorylation in the presence of SNP was inhibited by myristoylated PKI and H-89 but was not affected by KT-5823 (Fig. 4). The increase in isoproterenol-stimulated PDE3A phosphorylation in the presence of SNP reflected an increase in cAMP (see below), leading to greater activation of PKA.


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Fig. 4.   Augmentation of isoproterenol (Iso)-induced PDE3A phosphorylation by sodium nitroprusside (SNP). Gastric smooth muscle cells labeled with 32P were incubated with Iso (1 µM) and/or SNP (0.1 nM) for 1 min in the presence or absence of the PKA inhibitors myristoylated PKI (1 µM) and H-89 (1 µM) and the PKG inhibitor KT-5823 (KT; 1 µM). Immunoprecipitates, derived from 500 µg of protein by using polyclonal PDE3A antibody, were separated on SDS-PAGE. [32P]PDE3A was identified by autoradiography (A). Measured radioactivity of the bands is expressed as cpm (C), and immunoblots (50 µg protein) of the bands are shown for a loading control (B). Values are means ± SE of 4 experiments. **Significant inhibition (P < 0.001) of Iso-induced phosphorylation. pi pi Significant augmentation (P < 0.01) of Iso-induced phosphorylation.

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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Fig. 5.   Inhibition of forskolin-stimulated PDE3 and PDE4 activity by PKI. Dispersed gastric smooth muscle cells were treated with forskolin (1 µM) for 1 min. PDE3 and PDE4 activities were measured in homogenates of dispersed gastric muscle cells by using [3H]cAMP as substrate. PDE3 activity was determined as cAMP-specific activity measured in the presence of the PDE4 inhibitor rolipram (10 µM), and PDE4 activity was determined as cAMP-specific activity measured in the presence of the PDE3 inhibitor milrinone (10 µM). The effect of myristoylated PKI added 10 min before on forskolin-stimulated PDE activity in the presence or absence of PDE inhibitors also was determined. Both basal and forskolin-stimulated PDE activities decreased in the presence of rolipram and milrinone. Myristoylated PKI abolished the increase in PDE3 or PDE4 activity induced by forskolin. Results are expressed as cpm/mg protein. Values are means ± SE of 4 experiments. **Significant inhibition (P < 0.01) of basal PDE activity induced by rolipram or milrinone. pi pi Significant increase (P < 0.01) above basal activity induced by forskolin.



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Fig. 6.   Inhibition of cBIMPS- and Iso-stimulated PDE3 and PDE4 activity by PKI. Dispersed gastric smooth muscle cells were treated with the selective PKA activator cBIMPS (10 µM; A) or with Iso (1 µM; B) for 1 min. PDE3 activity was determined as cAMP-specific activity measured in the presence of the PDE4 inhibitor rolipram (10 µM), and PDE4 activity was determined as cAMP-specific activity measured in the presence of the PDE3 inhibitor milrinone (10 µM). The effect of myristoylated PKI added 10 min before on cBIMPS- or Iso-stimulated PDE activity in the presence or absence of PDE inhibitors also was determined. The results are similar to those obtained with forskolin (Fig. 5) and are expressed as cpm/mg protein. Values are means ± SE of 4 experiments. **Significant inhibition (P < 0.01) of basal PDE activity induced by rolipram or milrinone. pi pi Significant increase (P < 0.01) above basal activity induced by cBIMPS or Iso.

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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Fig. 7.   Inhibition of PDE3 activity by cGMP. Dispersed gastric muscle cells were treated with forskolin (1 µM) for 1 min, alone and in the presence of SNP (0.1 µM), SNP + ODQ (guanylyl cyclase inhibitor; 1 µM), or SNP + KT-5823 (1 µM). PDE3 activity was measured in the presence of rolipram. SNP-stimulated cGMP inhibited PDE3 activity; the inhibition was reversed by ODQ and intensified by KT-5823. Results are expressed as cpm/mg protein above basal levels (basal: 2,096 ± 220 cpm/mg protein). Values are means ± SE of 4-6 experiments. **Significant inhibition (P < 0.01) of forskolin-stimulated PDE3 activity.

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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Fig. 8.   Augmentation of forskolin-stimulated cAMP levels by cGMP. A: cAMP was measured in dispersed muscle cells treated with forskolin for 1 min in the presence or absence of 0.1 µM SNP. B: cAMP also was measured in cells treated with 1 µM forskolin and various concentrations (0.01-10 µM) of zaprinast, SNP, or SNP plus the PDE5 inhibitor zaprinast (1 µM). SNP significantly increased forskolin-stimulated cAMP levels. Inhibition of PDE5 by zaprinast caused a further increase in cAMP levels. Results are expressed as pmol/mg protein above basal levels (basal cAMP: 3.95 ± 0.42 pmol/mg protein). Values are means ± SE of 4 experiments. pi P < 0.05 and pi pi P < 0.01, significant augmentation of forskolin-induced cAMP levels.

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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Fig. 9.   Augmentation of Iso-stimulated cAMP levels by PDE3 and PDE4 inhibitors. cAMP was measured in dispersed gastric muscle cells treated with Iso (10 nM), SNP (10 nM), the PDE3 inhibitors milrinone (10 µM) or trequinsin (1 µM), and the PDE4 inhibitor rolipram (10 µM), either alone or in combination. Iso and PDE3 inhibitors stimulated cAMP. SNP, PDE3, and PDE4 inhibitors, alone and in combination, significantly augmented Iso-stimulated cAMP levels. Results are expressed as pmol/mg protein above basal levels (4.12 ± 0.53 pmol/mg protein). Values are means ± SE of 4 experiments. pi pi Significant increase (P < 0.01-P < 0.001) in Iso-stimulated cAMP.

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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Fig. 10.   PKA-dependent phosphorylation of adenylyl cyclase types V/VI (AC V/VI). Gastric smooth muscle cells labeled with 32P were incubated with various concentrations of forskolin (0.01-10 µM) in the presence or absence of the selective PKA inhibitor myristoylated PKI (1 µM). Immunoprecipitates using polyclonal antibody to AC V/VI were separated on SDS-PAGE. [32P]AC V/VI was identified by autoradiography (A). Measured radioactivity in the bands is expressed as cpm (C), and immunoblots (50 µg protein) of the bands are shown for a loading control (B). Values are means ± SE of 3 experiments. **Significant inhibition (P < 0.01) of forskolin-induced phosphorylation.



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Fig. 11.   Feedback inhibition of adenylyl cyclase activity by PKA. Gastric muscle cells were treated with various concentrations of forskolin (0.01-10 µM) in the presence or absence of myristoylated PKI (1 µM). Adenylyl cyclase activity was measured by the conversion of [32P]ATP to [32P]cAMP, as described in MATERIALS AND METHODS. Results are expressed as pmol cAMP/mg protein above basal levels (basal: 6.2 ± 0.54 pmol/mg protein). Values are means ± SE of 4 experiments. pi pi Significant augmentation (P < 0.01) of forskolin-induced adenylyl cyclase activity.

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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Fig. 12.   Inhibition of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release and muscle contraction by Iso and by PDE3 and PDE4 inhibitors. Ca2+ release and muscle contraction were measured as described in MATERIALS AND METHODS 15 s after addition of IP3 (1 µM) to permeabilized muscle cells. The cells were first treated for 1 min with 10 nM Iso in the presence or absence of the PDE3 inhibitor milrinone (10 µM) and/or the PDE4 inhibitor rolipram (10 µM). Results are expressed as percentage inhibition of IP3-induced Ca2+ release (0.82 ± 0.04 nmol/106 cells) or muscle cell contraction (33 ± 4 µm). Values are means ± SE of 4 experiments. pi pi Significant increase (P < 0.01) in isoproterenol-induced inhibition of Ca2+ release and muscle contraction.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 13.   Regulation of cyclic nucleotide levels by PKA and/or PKG-dependent activation of PDEs and inhibition of cyclases. cAMP levels are regulated by PKA via activation of cAMP-specific PDE3A and PDE4 and feedback inhibition of AC V/VI. cGMP levels are regulated by PKG via activation of cGMP-specific PDE5 and feedback inhibition of soluble guanylyl cyclase (sGC). During concurrent generation of cAMP and cGMP, PDE3 is inhibited by cGMP, leading to enhancement of cAMP levels, whereas PDE5 is further activated by PKA, leading to attenuation of cGMP levels. In the presence of cGMP, the affinity of cAMP for PKG is greatly enhanced, making PKG the dominant effector of both cAMP and cGMP. Dotted arrows denote the processes that emerge during concurrent generation of cAMP and cGMP.

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

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.


    FOOTNOTES

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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