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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Inhibitory Phosphorylation of Soluble Guanylyl Cyclase by Muscarinic m2 Receptors via Gβ-Dependent Activation of c-Src Kinase

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

Received October 11, 2007; accepted January 4, 2008.

	Abstract

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In gastrointestinal smooth muscle, cGMP levels in response to relaxant agonists are regulated by activation of phosphodiesterase 5 and inhibition of soluble guanylyl cyclase (sGC) in a feedback mechanism via cGMP-dependent protein kinase. The aim of the present study was to determine whether contractile agonists modulate cGMP levels by cross-regulating sGC activity. In gastric muscle cells, acetylcholine (ACh) stimulated Src activity and induced sGC phosphorylation. Concurrent stimulation of cells with ACh attenuated sGC activity and cGMP formation in response to the nitric oxide (NO) donor, S-nitrosoglutathione (GSNO). The effect of ACh on Src activity, sGC phosphorylation, and on GSNO-stimulated sGC activity and cGMP formation were blocked by the m2 receptor antagonist (methoctramine), pertussis toxin, and by inhibitors of phosphatidylinositol 3 kinase, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], or Src kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, in dispersed muscle cells and in cells expressing G_i minigene or Gβ-scavenging peptide, whereas the m3 receptor antagonist, N-(2-chloroethyl)-4-piperidinyl diphenylacetate, or expression of the G_q minigene had no effect. ACh also attenuated sGC activity and cGMP formation in response to the NO-independent activator, YC-1 [3-(5'-hydroxymethyl-2'furyl)-1-benzylindazole]. The pattern implied that phosphorylation of sGC by c-Src kinase inhibits NO-sensitive sGC activity, and the inhibition was not due to a decrease in the binding of NO but probably due to decrease in catalytic activity. We conclude that cGMP levels are cross-regulated by contractile agonists via a mechanism that involves c-Src-dependent phosphorylation of sGC, leading to inhibition of sGC activity and cGMP formation. The finding highlights a novel mechanism for attenuation of the NO/sGC/cGMP signal by G_i-coupled contractile agonists, in addition to their inhibitory effect on adenylyl cyclase and cAMP formation.

Soluble guanylyl cyclase (sGC) is an obligate heterodimer composed of and β subunits with two isoforms of each subunit, termed ₁, ₂, β₁, and β₂, respectively (Nakane et al., 1990; Hanafy et al., 2001; Friebe and Koesling, 2003; Koesling et al., 2004). Each subunit consists of three domains: an N-terminal heme-binding regulatory domain, a highly conserved C-terminal catalytic domain, and a central dimerization domain involved in formation of heterodimers (Buechler et al., 1991; Andreopoulos and Papapetropoulos, 2000; Hanafy et al., 2001; Krumenacker et al., 2004).

Soluble GC activity can be regulated by transcriptional, post-transcriptional, and post-translational mechanisms (Krumenacker et al., 2004; Pyriochou and Papapetropoulos, 2005). Expression of both ₁ and β₁ isoforms in several cell types has been shown to be regulated by cytokines, growth factors, reactive oxygen species, estradiol, and by increases in cAMP or cGMP (Filippov et al., 1997; Liu et al., 1997; Krumenacker et al., 2005; Cabilla et al., 2006; Gerassimou et al., 2007). cAMP- and cGMP-elevating agents decrease the expression of both sGC ₁ and β₁ subunits via a post-translational mechanism involving decrease in the expression of elav-like mRNA-binding protein human-antigen R, which stabilizes sGC mRNA expression (Klöss et al., 2005). On the posttranslational levels, sGC activity was shown to be regulated by phosphorylation and S-nitrosylation. The primary sequence of both ₁ and β₁ subunits possesses multiple putative sites for phosphorylation by PKA, PKG, and PKC. Phosphorylation of sGC at serine/threonine residues in vitro by PKC and PKA and in vivo by PKA leads to an increase in activity, whereas phosphorylation by PKG in vivo lead to decrease in activity (Zwiller et al., 1981, 1985; Louis et al., 1993; Murthy, 2001, 2004). PKG-induced dephosphorylation was shown to decrease the activity of sGC in chromaffin cells, whereas Ca²⁺-dependent dephosphorylation was shown to increase the activity in cardiac myocytes (Ferrero et al., 2000; Agulló et al., 2005). S-Nitrosylation of sGC resulting in the inhibition of NO-stimulated activity was shown in primary aortic smooth muscle cells, umbilical vein endothelial cells, and in isolated aorta (Sayed et al., 2007).

In gastrointestinal smooth muscle, the frequency and amplitude of rhythmic contractions and the muscle tone are modulated by excitatory (e.g., acetylcholine) and inhibitory neurotransmitters (e.g., NO), and their releases often overlap. Acetylcholine (ACh) interacts with two muscarinic receptors present on the smooth muscle to activate distinct signaling pathways (Murthy et al., 2003a; Zhou et al., 2003; Huang et al., 2006; Murthy, 2006). Activation of various kinases in response to contractile agonists raises the possibility that these kinases could act to cross-regulate cGMP levels by modulating NO-sensitive sGC. In this study, we examined whether concurrent activation of contractile pathways by ACh regulates NO-sensitive sGC phosphorylation and activity. We have identified a novel mechanism for inhibition of sGC activity by acetylcholine acting via G_i-coupled m2 receptors. The mechanism involves activation of c-Src derived from the Gβ-dependent PI3 pathway.

	Materials and Methods

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Dispersion and Culture of 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 (Murthy and Makhlouf, 1995b, 1996; Murthy and Zhou, 2003; Murthy et al., 2003a). In brief, smooth muscle strips were incubated at 31°C for 20 min in HEPES medium containing type II collagenase (0.1%) and soybean trypsin inhibitor (0.1%). Muscle cells were harvested by filtration through 500 µM Nitex and centrifuged twice at 350g for 10 min.

Expression of G Minigene in Cultured Gastric Smooth Muscle Cells. Activation of G_i or G_q was blocked by the expression of cDNA encoding the last COOH-terminal 11 amino acids as described previously (Zhou and Murthy, 2004). The cDNA sequences were amplified by polymerase chain reaction and verified by DNA sequencing. Cultured rabbit gastric smooth muscle cells were transfected transiently with minigene plasmid DNA using Effectene transfection reagent. The cells were cotransfected with 1 µg of pGreen lantern-1 to monitor expression. Transfection efficiency (70%) was monitored microscopically by the expression of green fluorescent protein using fluorescein isothiocyanate filters.

Assay for Soluble GC Activity. Soluble GC activity was measured using [-³²P]GTP as the substrate (Murthy, 2001, 2004). Gastric muscle cells were treated with various concentrations of GSNO in the presence of isobutylmethylxanthine (IBMX; 1 mM) and zaprinast (10 µM). Crude membranes of muscle cells were incubated in a solution of 50 mM Tris-HCl, pH 7.4, 2 mM cGMP, 0.1 mM GTP, 1 mM IBMX, 5 mM MgCl₂, 100 mM NaCl, 5 mM creatine phosphate, 50 units/ml creatine kinase, and 0.5 mM [-³²P]GTP (approximately 0.2 µCi) for 15 min at 37°C. The reaction was terminated by the addition of 2% SDS/45 mM GTP/1.5 mM cGMP. [-³²P]GMP was separated from [-³²P]GTP by sequential chromatography on Dowex AG50W-4X and alumina columns. The results were expressed as picomoles of cGMP per milligram of protein.

Phosphorylation of sGC. Soluble GC phosphorylation was also measured using phosphotyrosine antibody in sGC immunoprecipitates. sGC immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with an antibody to the phosphotyrosine residue. After incubation with a secondary antibody, the proteins were visualized using enhanced chemiluminescence.

Activation of c-Src. Activation of c-Src was measured by Western blot analysis using a phospho-Src (Tyr527) antibody. Dispersed muscle cells were treated with ACh in the presence or absence of a selective c-Src inhibitor, PP2 (1 µM), and solubilized on ice for 2 h in 20 mM Tris/HCl medium containing 1 mM dithiothreitol, 100 mM NaCl, 0.5% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 100 µg/ml aprotinin. The proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred electrophoretically to polyvinylidene difluoride membrane. The membranes were incubated for 12 h with phospho-Src (Tyr527) antibody and then for 1 h with horseradish peroxidase-conjugated secondary antibody. The bands were identified by enhanced chemiluminescence.

Radioimmunoassay for cGMP. Cyclic GMP production was measured by radioimmunoassay as described previously (Murthy, 2001, 2004). In brief, muscle cells (3 x 10⁶ cells) were treated with GSNO in the presence or absence of ACh for 5 min, and the reaction was terminated with 10% trichloroacetic acid. After extraction with water-saturated diethyl ether, the lyophilized aqueous phase was reconstituted in 500 µl of 50 mM Na acetate, pH 6.2. The samples were acetylated with triethylamine/acetic anhydride (2:1) for 30 min, and cGMP was measured in duplicate using 100-µl aliquots. The results were expressed as picomoles per milligram of protein.

Materials. [¹²⁵I]cGMP and [-³²P]GTP were obtained from NEN Life Sciences Products (Boston, MA); antibodies to c-Src, phospho-c-Src (Tyr527), and phosphotyrosine were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), The polyclonal sGC antibody and YC-1 were from Alexis Corporation (San Diego, CA). Western blotting and chromatography materials were obtained from Bio-Rad Laboratories (Hercules, CA). Collagenase and soybean trypsin inhibitor were obtained from Worthington Biochemical (Freehold, NJ). All other reagents were from Sigma-Aldrich (St. Louis, MO).

Statistical Analysis. All values are expressed as means ± S.E.; n represents the number of animal studies. Regression analysis was performed using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA). Statistical analysis was performed by unpaired Student's t test, and P < 0.05 was considered statistically significant.

	Results

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Activation of c-Src by m2 Receptors via PI3. In dispersed muscle cells, devoid of acetylcholinesterase, ACh (0.1 µM) induced activation of c-Src, measured as phosphorylation of tyrosine 527 using phosphospecific [phospho-Src (Tyr527)] antibody. The effect of ACh on c-Src activity was blocked by the m2 receptor antagonist methoctramine (0.1 µM), pertussis toxin (PTx; 400 ng/ml), PI3 inhibitor LY294002 (10 µM), or Src kinase inhibitor PP2 (1 µM), suggesting that activation of c-Src was mediated by the G_i-coupled m2 receptor via PI3 pathway (Fig. 1) (Zhou et al., 2003; Huang et al., 2006). The m3 receptor antagonist 4-DAMP (0.1 µM) had no effect on ACh-induced tyrosine phosphorylation of sGC (Fig. 1).

View larger version (15K): [in this window] [in a new window]   Fig. 1. Activation of c-Src by m2 receptors. Gastric muscle cells were treated with ACh (0.1 µM) in the presence or absence of the m2 receptor antagonist methoctramine (0.1 µM), the m3 receptor antagonist 4-DAMP (0.1 µM), the PI3 inhibitor LY294002 (10 µM), or the Src kinase inhibitor PP2 (1 µM) for 5 min, and activation of c-Src (p-c-Src) was measured using the phospho-Src (Tyr527) antibody. PTx (400 ng/ml) was added for 60 min and then stimulated with ACh. Immunoblot analysis showed equal amounts of loaded protein. Values are expressed as means ± S.E. of four experiments. Statistical analysis was performed by unpaired Student's t test. **, P < 0.001 significant increase in c-Src activation by ACh.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Activation of c-Src by m2 receptors via Gβi. Cultured muscle cells expressing vector alone (control), Gi minigene, Gq minigene, or Gβ-scavenging peptide were treated with GSNO (1 mM) or GSNO plus ACh (0.1 µM) for 5 min, and activation of c-Src (p-c-Src) was measured using phospho-Src (Tyr527) antibody. Immunoblot analysis showed equal amounts of loaded protein. Values are expressed as means ± S.E. of four experiments. Statistical analysis was performed by unpaired Student's t test. **, P < 0.001, significant increase in c-Src activation by ACh.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Phosphorylation of sGC by ACh. Gastric muscle cells were treated with different concentrations of ACh for 5 min, and sGC phosphorylation was measured using phosphotyrosine antibody in sGC immunoprecipitates [p-sGC(Tyr)]. Immunoblot analysis showed equal amounts of loaded protein (data not shown). Values are expressed as means ± S.E. of four experiments. Regression analysis was performed using GraphPad Prism 4. Statistical analysis was performed by unpaired Student's t test. *, P < 0.05; **, P < 0.001, significant increase in sGC phosphorylation by ACh.

View larger version (8K): [in this window] [in a new window]   Fig. 4. Inhibition of GSNO-stimulated sGC activity and cGMP formation by ACh. Gastric muscle cells were treated with different concentrations of ACh in the presence of GSNO (1 mM) for 5 min. Soluble GC activity was measured in crude homogenates by the conversion of [32GTP] to [32P]cGMP as described under Materials and Methods. cGMP levels were measured in dispersed muscle cells by radioimmunoassay. Basal values (basal, 2.6 ± 0.3 pmol/mg protein and GSNO alone, 22.1 ± 2.4 pmol/mg protein for sGC; basal, 0.23 ± 0.04 and GSNO alone, 0.76 ± 0.07 pmol/mg protein for cGMP) were subtracted, and the results were expressed as picomoles per milligram of protein above basal levels. Values are expressed as means ± S.E. of four experiments. Regression analysis was performed using GraphPad Prism 4. Statistical analysis was performed by unpaired Student's t test. *, P < 0.05; **, P < 0.001, significant decrease in GSNO-induced sGC activity and cGMP formation by ACh.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Phosphorylation of sGC by m2 receptors via c-Src. Gastric muscle cells were treated with ACh (0.1 µM) in the presence or absence of the m2 receptor antagonist methoctramine (0.1 µM), the m3 receptor antagonist 4-DAMP (0.1 µM), the PI3 inhibitor LY294002 (10 µM), or the Src kinase inhibitor PP2 (1 µM) for 5 min, and sGC phosphorylation was measured using the phosphotyrosine antibody in sGC immunoprecipitates [p-sGC-(Tyr)]. PTx (400 ng/ml) was added for 60 min and then stimulated with ACh. Immunoblot analysis showed equal amounts of loaded protein. Values are expressed as means ± S.E. of four experiments. **, P < 0.001 significant increase in sGC phosphorylation by ACh.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Inhibition of GSNO-stimulated sGC activity by m2 receptors via c-Src. Gastric muscle cells were treated with GSNO (1 mM) or ACh (0.1 µM) alone and GSNO plus ACh in the presence or absence of the m2 receptor antagonist methoctramine (0.1 µM), the m3 receptor antagonist 4-DAMP (0.1 µM), the PI3 inhibitor LY294002 (10 µM), or the Src kinase inhibitor PP2 (1 µM) for 5 min. PTx (400 ng/ml) was added for 60 min and then stimulated with ACh. Soluble GC activity was measured in crude homogenates by the conversion of [32GTP] to [32P]cGMP as described under Materials and Methods and Results and expressed as picomoles per milligram of protein. Inset, effect of methoctramine (methoc) on ACh-induced inhibition of sGC activity was concentration-dependent. Values are expressed as means ± S.E. of four experiments. *, P < 0.05; **, P < 0.001, significant decrease in GSNO-stimulated sGC activity.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Inhibition of GSNO-stimulated cGMP formation by m2 receptors via c-Src. Gastric muscle cells were treated with GSNO (1 mM) or ACh (0.1 µM) alone and GSNO plus ACh in the presence or absence of the m2 receptor antagonist methoctramine (0.1 µM), the m3 receptor antagonist 4-DAMP (0.1 µM), the PI3 inhibitor LY294002 (10 µM), or the Src kinase inhibitor PP2 (1 µM) for 5 min. PTx (400 ng/ml) was added for 60 min and then stimulated with ACh. cGMP levels were measured in dispersed muscle cells by radioimmunoassay, and results were expressed as picomoles per milligram of protein. Inset, effect of methoctramine (methoc) on ACh-induced inhibition of cGMP formation was concentration-dependent. Values are expressed as means ± S.E. of four experiments. *, P < 0.05; **, P < 0.01, significant decrease in GSNO-stimulated cGMP formation.

Treatment of cultured muscle cells with ACh also caused phosphorylation of sGC and inhibition of GSNO-stimulated cGMP formation. The effect of ACh was blocked in cells expressing the Gβ-scavenging peptide or G_i minigene but not by the G_q minigene (Fig. 8). The function of G_i3-coupled m2 receptors was shared by the other G_i-coupled receptors (Murthy and Makhlouf, 1995a, 1996; Murthy et al., 1996). Somatostatin acting via G_i1-coupled sst3 receptor, [D-Pen, D-Pen]enkephalin, acting via G_i2-coupled -opioid receptors and cyclopentyl adenosine acting via G_i3-coupled adenosine A₁ receptors inhibited GSNO-stimulated sGC activity (basal, 2.6 ± 0.3 pmol/mg protein; GSNO, 23.1 ± 2.5 pmol/mg protein), and the extent of inhibition by somatostatin (71 ± 9% inhibition), [D-Pen, D-Pen]enkephalin (69 ± 8% inhibition), and N⁶-cyclopentyladenosine (75 ± 10% inhibition) was closely similar to that elicited by ACh (64 ± 8% inhibition).

View larger version (18K): [in this window] [in a new window]   Fig. 8. sGC phosphorylation and inhibition of GSNO-stimulated cGMP formation by m2 receptors via Gβi-dependent activation of c-Src. Cultured muscle cells expressing vector alone (control), Gi minigene, Gq minigene, or Gβ-scavenging peptide were treated with GSNO (1 mM) or GSNO plus ACh (0.1 µM) for 5 min. sGC phosphorylation was measured using phosphotyrosine antibody in sGC immunoprecipitates [p-sGC-(Tyr)]. cGMP levels were measured by radioimmunoassay, and results are expressed as picomoles per milligram of protein. Values are expressed as means ± S.E. of four experiments. **, P < 0.001, significant increase in sGC phosphorylation by ACh and significant inhibition of GSNO-stimulated cGMP formation by ACh.

Cotreatment of cells with ACh also caused a decrease in sGC activity and cGMP formation induced by YC-1, an NO-independent activator of sGC (Fig. 9). YC-1 binds to an allosteric site on the enzyme, leading to the activation of enzyme activity and cGMP formation (Friebe and Koesling, 2003). These results suggest that inhibition of sGC activity by tyrosine phosphorylation was not due to a decrease in NO binding to the enzyme but probably due to a decrease in catalytic activity. Because cGMP levels are measured in the presence of IBMX and the phosphodiesterase 5 inhibitor zaprinast, decreases in cGMP levels are not due to stimulation of phosphodiesterase 5 activity.

View larger version (11K): [in this window] [in a new window]   Fig. 9. Inhibition of YC-1-stimulated sGC activity and cGMP formation by ACh. Gastric muscle cells were treated with YC-1 (10 µM) in the presence or absence of ACh (0.1 µM). Soluble GC activity was measured in crude homogenates by the conversion of [32GTP] to [32P]cGMP as described under Materials and Methods and Results, expressed as picomoles per milligram of protein. cGMP levels were measured in dispersed muscle cells by radioimmunoassay, and results were expressed as picomoles per milligram of protein. Values are expressed as means ± S.E. of four experiments. *, P < 0.01; **, P < 0.001 significant decrease in YC-1-stimulated sGC activity and cGMP formation by ACh.

	Discussion

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In the gastrointestinal tract, NO acting as an inhibitory nonadrenergic noncholinergic neurotransmitter regulates smooth muscle tone. In smooth muscle, the NO receptor, NO-sensitive (soluble) GC, plays an important role in the generation of cGMP and activation of PKG. PKG, in turn, acts on various targets, and its action culminates in the reduction intracellular Ca²⁺ and/or myosin light phosphorylation, a prerequisite for muscle relaxation (Murthy and Makhlouf, 1995b; Murthy and Zhou, 2003; Murthy et al., 2003b; Murthy, 2006). sGC knockout mice exhibit dysfunctional motility with significantly reduced transit time and constipation akin to the PKG knockout mice, clearly emphasizing the importance of sGC/cGMP/PKG pathway in the maintenance of normal peristalsis (Pfeifer et al., 1998; Friebe et al., 2007).

A distinctive feature of gastrointestinal smooth muscle is the rhythmic contractions and relaxation during physiological peristalsis. Various constituents in the signaling pathway that mediate contraction in smooth muscle of the gut could modulate cGMP levels by regulating sGC activity. The present study characterized the cross-regulation of GSNO (NO)-induced sGC activity and cGMP formation by concurrent activation of muscarinic receptors by ACh in smooth muscles and provided evidence that coactivation of m2 receptors attenuated GSNO-stimulated sGC activity and cGMP formation. The mechanism involves G_i3-dependent activation of c-Src by m2 receptors via the Gβ/PI3 pathway, leading to tyrosine phosphorylation of sGC and inhibition of GSNO-stimulated sGC activity and cGMP formation. The evidence for the mechanism is based on: 1) ACh-induced activation of Src and tyrosine phosphorylation of sGC were blocked selectively by the m2 receptor antagonist (methoctramine), PTx and inhibitors of PI3 (LY294002), or Src kinase (PP2) in freshly dispersed muscle cells and in cells expressing Gβ-scavenging peptide or G_i minigene; and 2) concurrent stimulation of cells with ACh inhibited GSNO-stimulated sGC activity and cGMP formation, and the effect of ACh was blocked selectively by methoctramine, PTx, LY294002, or PP2 in freshly dispersed muscle cells and in cells expressing Gβ-scavenging peptide and G_i. Methoctramine-sensitive receptors were uncoupled selectively from G_i3 by PTx (Murthy and Makhlouf, 1997; Murthy et al., 2003a). Unexpectedly, the m3 receptor antagonist potentiated the effect of ACh on sGC phosphorylation and inhibition of sGC activity, suggesting that normally, activation of m3 receptors would act to reverse the effect of m2 receptors. Although the mechanism for such protection was not studied, it is possible that activation of m3 receptors could lead to stimulation of tyrosine phosphatase, resulting in dephosphorylation of GC. Inhibition of sGC activity by phosphorylation would require a mechanism for restoring the enzyme activity by dephosphorylation.

Although more abundant, the functions of m2 receptors are considered as inert because smooth muscle contraction was exclusively mediated by G_q/G₁₃-coupled m3 receptors (Murthy et al., 2003a). Muscarinic m2 receptors were shown previously to inhibit adenylyl cyclase activity via the subunit of G_i3 in response to G_s-coupled VIP receptors (Murthy and Makhlouf, 1997). In the present study, the m2 receptors were shown to inhibit, additionally, sGC activity via the β subunit-dependent activation of Src kinase and Src kinase-mediated phosphorylation of sGC. Thus, activation of m2 receptors decreases cAMP and cGMP levels stimulated by the inhibitory transmitters (VIP and NO) to facilitate the contractile function of m3 receptors. This is the first demonstration that activation of Src kinase by contractile agonists attenuates cGMP signaling by inhibiting sGC activity. The inhibitory function of G_i3-coupled m2 receptors was shared by other G_i-coupled receptors such as G_i1-coupled somatostatin 3 receptors, G_i2-coupled -opioid receptors, and G_i3-coupled adenosine A₁ receptors, suggesting that inhibition of sGC activity by m2 receptors is not unique for this receptor but represents a common mechanism and underscores the importance of cross-regulation of cGMP levels by contractile agonists. Inhibition of sGC activity and cGMP levels by contractile agonists reduces the inhibitory tone and facilitates smooth muscle for optimal contraction. The results are also important in the context of several studies characterizing the role of inhibitory transmitters in muscle tissues precontracted with contractile agonists that activate c-Src.

Phosphorylation-dependent regulation of sGC activity was demonstrated both in vitro and in vivo in various cell types. Studies in gastric smooth muscle have shown that sGC was phosphorylated by PKG in a feedback mechanism leading to a decrease in sGC activity and attenuation of cGMP levels (Murthy, 2001, 2004). The results presented in this study suggest an alternate mechanism through which contractile agonists can decrease cGMP levels by inhibitory phosphorylation of sGC, and this mechanism would probably be exploited by any stimulus that leads to Src activation. Our results are consistent with the studies of Meurer et al. (2005) demonstrating that inhibitors of protein tyrosine phosphatases and reactive oxygen species such as H₂O₂ induced tyrosine phosphorylation of sGC β1 subunit at Tyr192 in PC12 cells, rat aortic smooth muscle cells, and aortic tissue, but in variance with the studies of Chen et al. (2001) demonstrating that overexpression of the protein tyrosine phosphatase Src homology phosphatase-1 inhibited basal and sodium nitroprusside-stimulated cGMP formation. Although not studied, identification of the specific Src kinase that phosphorylate sGC should provide some insight into which agonist might regulate sGC activity and what other physiological processes might be affected. It has been shown that β1 subunit of sGC contains consensus motif (¹⁸⁸EEDFYEDLD) for phosphorylation by Src-like kinases in its sequence (Meurer et al., 2005). Phosphorylation of sGC at Tyr192 exposes a docking site for SH2 domains, recruits other Src-like kinase, and promotes multiple phosphorylation of the enzyme. In addition to the post-translational regulation of sGC activity, sGC expression and smooth muscle relaxation were regulated by proinflammatory cytokines cytokines such as IL-1β (K.S. Murthy, unpublished data).

In summary, we describe here for the first time a novel mechanism for cross-regulation of the NO signal by G protein-coupled contractile agonists. The mechanism involves activation of G_i-coupled receptors, stimulation of Src kinase via Gβ, and phosphorylation of sGC. Regulation of sGC by contractile agonists may ensure that opposing actions of cGMP-stimulated relaxations do not compromise a committed response of smooth muscle to contraction.

	Footnotes

 
This study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (Grant DK 28300).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.132928.

ABBREVIATIONS: VIP, vasoactive intestinal peptide; NO, nitric oxide; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; sGC, soluble guanylyl cyclase; ACh, acetylcholine; PI3, phosphatidylinositol 3-kinase; GSNO, S-nitrosoglutathione; IBMX, isobutylmethylxanthine; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; YC-1, 3-(5'-hydroxymethyl-2'furyl)-1-benzylindazole; PTx, pertussis toxin; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; 4-DAMP, N-(2-chloroethyl)-4-piperidinyl diphenylacetate; ANP, atrial natriuretic peptide.

Address correspondence to: Dr. Karnam S. Murthy, Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298. E-mail: skarnam{at}hsc.vcu.edu

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