Modulation of soluble guanylate cyclase activity by phosphorylation

Neurochemistry International 45 (2004) 845–851

Modulation of soluble guanylate cyclase activity by phosphorylation Karnam S. Murthy∗ Departments of Physiology and Medicine, Medical College of Virginia, Virginia Commonwealth University Richmond, Richmond, VA 23298-0711, USA

Abstract The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting ␣- and ␤-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 ␮M) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP. © 2004 Elsevier Ltd. All rights reserved. Keywords: Protein kinase A; Protein kinase G; Cyclic nucleotides; Soluble guanylyl cyclase

1. Introduction Relaxation of the smooth muscle in the gastrointestinal tract by inhibitory neurotransmitters reflects the interplay of main neurotransmitters, nitric oxide (NO), and the neuropeptides, vasoactive intestinal peptide (VIP), and its homologue pituitary adenylate adenylate cyclase activating peptide (PACAP) (Murthy and Makhlouf, 1995; Murthy et al., 1998). These neurotransmitters initiate signaling pathways in the smooth muscle that leads to generation of cGMP and cAMP, and activation of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) (Murthy and Makhlouf, 1995; Murthy, 2001). The Abbreviations: SGC, soluble guanylyl cyclase; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; SNP, sodium nitroprusside; PKI, protein kinase inhibitor (14–22) amide; cBIMPS, 5,6-dichloro-1-␤-d-ribofuranosyl benzimidazole 3 ,5-cyclic monophosphothioate, Sp-isomer; KT5823, (8R,9S,11s)-(−)-9-methoxy-carbamyl8-methyl-2,3,9,10-tetrahydro-8,1 1-epoxy-1H,8H,1H,-2,7b, 11a-trizadizobenzo9a,g)cycloocta(c,d,e)-trinden-1-one; MEK, mitogen-activated protein kinase kinase ∗ Tel.: +1-804-828-8504; fax: +1-804-828-2500. E-mail address: [email protected] (K.S. Murthy). 0197-0186/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2004.03.014

concurrent generation of both cyclic nucleotides and cyclic nucleotide-dependent kinases are the physiological norm, and has considerable bearing on the regulation of cyclic nucleotide levels and phosphodiesterase (PDE) activities, and the activities of PKA and PKG (Dousa, 1999; Soderling and Beavo, 2000; Murthy, 2001; Murthy et al., 2002). PKA and/or PKG act to regulate the levels of cyclic nucleotide by inducing feedback desensitization of the receptors, inhibiting adenylyl and guanylyl cyclase activities, and stimulating PDE activities. Cyclic AMP levels in the smooth muscle of the gut reflect continued synthesis by adenylyl cyclase and breakdown by PDE3 and PDE4 (Ekholm et al., 1997; Hoffmann et al., 1998; Murthy et al., 2002). We have previously (Murthy et al., 2002) shown that PKA regulates cAMP levels in smooth muscle via stimulatory phosphorylation of PDE3A and PDE4, and the inhibitory phosphorylation of adenylyl cyclase V/VI. Concurrent generation of cGMP inhibits PDE3 activity and augments cAMP levels. Cyclic GMP levels in smooth muscle of the gut reflect continued synthesis by soluble guanylyl cyclase (sGC) and breakdown by PDE, presumably PDE5 (Wyatt et al., 1998; Loughney et al., 1998; Corbin and Francis, 1999). Solu-

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ble GC is a heme containing, heterodimeric protein consisting ␣- and ␤-subunits; each subunit has a C-terminal catalytic domain, a central dimerization domain and an N-terminal regulatory domain with prosthetic heme moiety (Nakane and Murad, 1994; Wedel et al., 1995; Hobbs, 1997; Koesling, 1999; Lee et al., 2000; Lucas et al., 2000; Friebe and Koesling, 2003). The heme moiety acts as an intracellular receptor for NO and the binding of NO induces a conformational change in the enzyme resulting in an increase in the catalytic activity. Although both ␣- and ␤-subunits contain their own catalytic domain, expression of both subunits and dimerization are required for basal and NO-stimulated catalytic activity. At least two isoforms for each subunit (␣1/␣2 and ␤1/␤2) has been cloned and their expression is tissue-specific. The most abundant and widely expressed heterodimer of sGC appears to be ␣1/␤1, and it has the most basal and NO-stimulated activity. Recent studies have shown that co-expression of ␤2 subunit of sGC with the ␣1/␤1 heterodimer in COS cell results in formation of ␣1/␤2 heterodimer and inhibition of NO-stimulated cyclase activity suggesting that ␤2 subunit can exchange with the ␤1 subunit and inhibit stimulation of cGMP formation (Gupta et al., 1997). Both ␣1 and ␤1 subunits of sGC have putative sites for phosphorylation for multiple kinases (Andreopoulos and Papapetropoulos, 2000; Zhang et al., 2002). Studies in rat vascular smooth muscle cells showed that pretreatment with cAMP or cGMP elevating agents reduced sGC expression and inhibited both basal and sodium nitroprusside (SNP)-stimulated cGMP formation (Papapetropoulos et al., 1995; Fillippov et al., 1997). The regulation of sGC activities has not been characterized in gastrointestinal smooth muscle. In the current study, we examined the characteristics of inhibitory regulation of sGC by phosphorylation in gastric smooth muscle. The results indicate that PKG-dependent phosphorylation inhibits sGC activity, thereby attenuating the levels of cGMP. Concurrent generation of cAMP also attenuates cGMP levels by inhibiting sGC activity via cross-activation of PKG. 2. Experimental procedure 2.1. Preparation of dispersed gastric smooth muscle cells Dispersed gastric smooth muscle cells were prepared by sequential enzymatic digestion, filtration and centrifugation as described previously (Murthy and Makhlouf, 1995, 1997; Murthy, 2001). Briefly, strips of circular muscle were dissected and incubated at 31 ◦ C for 30 min in HEPES medium with type II collagenase (0.1%) and soybean trypsin inhibitor (0.1%). The composition of the HEPES medium was (in mM) NaCl 120, KCl 4, KH2 PO4 2.6, MgCl2 , 0.6, HEPES 25, glucose 14, and 2.1% Eagle’s essential amino acid mixture. After the partly digested strips were washed twice with 50 ml of enzyme-free medium, the muscle cells were al-

lowed to disperse spontaneously for 30 min. The cells were harvested by filtration through 500-␮m Nitex (Tetko Inc., Briarcliff Manor, NY) and centrifuged twice at 350 × g for 10 min. 2.2. Assay for soluble guanylyl cyclase activity Soluble guanylyl cyclase activity was measured using [␣-32 P]GTP as substrate (Murthy, 2001). Crude homogenates of muscle cells were incubated for 15 min at 37 ◦ C in a medium consisting of 50 mM Tris–HCl (pH 7.4), 2 mM cGMP, 0.1 mM GTP, 1 mM isobutylmethylxanthine, 5 mM MgCl2 , 100 mM NaCl, 5 mM creatine phosphate, 50 U/ml creatine phosphokinase and 0.5 mM [␣-32 P]GTP (∼0.2 ␮Ci). The reaction was terminated by addition of 2% SDS, 45 mM GTP and 1.5 mM cGMP. [32 P]GMP was separated from [32 P]GTP by sequential chromatography on Dowex AG50W-4X and alumina columns. The results were expressed as picomoles of cGMP per mg protein. In experiments using phosphatases 1 and 2A, sGC immunoprecipitates were washed with a medium containing 50 mM Tris–HCl (pH 7.5), 0.5 mM EDTA, 5 mM ␤-mercaptoethanol, and 0.1% Triton X-100 and incubated for 20 min at 30 ◦ C with the purified protein phosphatase 1 (0.3 ␮g) and 2A (0.3 ␮g) in the presence or absence of okadaic acid (10 ␮M) and calyculin A (10 ␮M). The phosphatases were then removed by further washes with Tris–HCl medium and sGC phosphorylation measured (Murthy, 2001). 2.3. Phosphorylation of soluble GC (sGC) Phosphorylation of sGC was determined from the amount of 32 P incorporated into the enzyme after immunoprecipitation with specific antibody for sGC (Murthy, 2001). Ten milliliter of a suspension of dispersed smooth muscle cells (4 × 106 cells/ml) were prelabeled with 0.5 mCi/ml of [32 P]orthophosphate for 3 h. Samples (1.0 ml) were incubated with sodium nitroprusside (SNP) for 1 min in the presence or absence of the specific PKG inhibitors (Rp-cGMPS or KT5823), or the specific PKA inhibitors (myristoylated PKI or H-89). The reaction was terminated with an equal volume of lysis buffer (final concentrations: 1% Triton X-100, 0.5% SDS, 0.75% deoxycholate, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml leupeptin, 100 ␮g/ml aprotinin, 10 mM Na2 P2 O7 , 50 mM NaF, 0.2 mM Na3 VO4 ), and placed on ice for 30 min. The cell lysates were separated from the insoluble material by centrifugation at 13,000 × g for 15 min at 4 ◦ C, precleared with 40 ␮l of protein A-sepharose, and incubated with polyclonal PDE5 or sGC antibody for 2 h at 4 ◦ C, and with 40 ␮l of protein A-Sepharose for another 1 h. The immunoprecipitates were washed five times with 1 ml of wash buffer (0.5% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl, pH 7.4), extracted with Laemmli sample buffer, and boiled for 15 min, and then separated on 10% SDS-polyacrylamide gel electrophoresis.

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After transfer to polyvinylidine difluoride membranes, 32 P-labeled sGC was visualized by autoradiography, and the amount of radioactivity in the bands was counted. A back phosphorylation approach was used to determine in situ phosphorylation of sGC (Murthy, 2001). Immunoprecipitates of sGC were washed with medium containing 50 mM Tris–HCl (pH 7.5), 5 mM MgCl2 and 0.1% Triton X-100, and then incubated with the purified PKG-I (0.5 ␮g) in the presence of 1 ␮M cGMP and 5-10 ␮Ci of [␥-32 P] ATP for 10 min at 30 ◦ C. The samples were treated with Laemmli sample buffer, and boiled for 15 min, and then separated on 10% SDS-polyacrylamide gel electrophoresis. After transfer to polyvinylidine difluoride membranes, 32 P-labeled sGC was visualized by autoradiography, and the amount of radioactivity in the bands was counted. The decrease in 32 P incorporation in the sGC band in samples treated with 1 ␮M SNP or 100 ␮M isoproterenol were compared to control phosphorylation. 2.4. Radioimmunoassay for cGMP Cyclic GMP production was measured by radioimmunoassay as described previously (Murthy and Makhlouf, 1995; Murthy, 2001). Briefly, muscle cells (3 × 10 cells) were treated with SNP for 1 min and the reaction terminated with 10% trichloroacetic acid. The samples were centrifuged and the supernatants extracted three times with water-saturated diethyl ether. The resulting aqueous phase was lyophilized and reconstituted in 500 ␮l 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 ␮l aliquots. The results were expressed as picomoles/mg protein. 2.5. Materials [␣-32 P]GTP, [32 P]orthophosphate, [␥-32 P]ATP, [125 I]GMP were obtained from Amersham Pharmacia Biotech (Piscataway, NJ); collagenase and soybean trypsin inhibitor from Worthington Biochemical Inc (Freehold, NJ); Western blotting and chromatography material and protein assay kit from Bio-Rad Laboratories (Hercules, CA); protein kinase A catalytic subunit, protein kinase G, protein phosphatase 1, 2A, and antibody to sGC from Alexis Corporation (San Diego, CA); cGMP, Crotalus atrox snake venom, and all other chemicals from Sigma Chemical Company (St. Louis, MO). 3. Results 3.1. Phosphorylation-dependent inhibition of sGC by cGMP-dependent protein kinase (PKG) We have previously shown that SNP stimulates cGMP formation and activates PKG in a concentration-dependent

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fashion in dispersed gastric smooth muscle cells (Murthy and Makhlouf, 1995). Here, we show that SNP stimulated sGC phosphorylation and inhibited sGC activity. SNP stimulated sGC phosphorylation by four-fold within 60 s (basal: 529 ± 75 cpm; SNP: 2567 ± 297 cpm). Stimulation of sGC phosphorylation was concentration-dependent with an EC50 of ∼1 nM (Fig. 1A). The selective PKG inhibitors, KT5823 and Rp-cGMPS blocked sGC phosphorylation (Fig. 1A); the selective PKA inhibitors, H-89 (1 ␮M) and myristoylated PKI (1 ␮M), the selective PKC inhibitor, bisindolylmaleimide (1 ␮M), the selective MEK inhibitor, PD98059 (10 ␮M), and the p38 MAP kinase inhibitor, SB203580 (1 ␮M) had no effect (data not shown; Murthy, 2001). In previous studies, we have shown that at concentrations of 0.1–1 ␮M, KT5823 selectively inhibited PKG activity, whereas H-89 and PKI selectively inhibited PKA (Murthy and Makhlouf, 1995; Murthy, 2001). In contrast, selective activators of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS or isoproterenol (1 ␮M) had no effect on sGC phosphorylation (7 ± 8% above basal; not significant) (Fig. 1B). A higher concentration of isoproterenol (100 ␮M) capable of generating cAMP levels that cross-activate PKG (Jiang et al., 1992; Jin et al., 1993; Murthy and Makhlouf, 1995) stimulated sGC phosphorylation that was selectively inhibited by Rp-cGMPS, but was not affected by myristoylated PKI (Fig. 1B). 3.2. Feedback inhibition of soluble guanylyl cyclase (sGC) activity by PKG Our recent studies have shown that PKA, but not PKG, can phosphorylate, and thus attenuate the activity of Type V/VI adenylyl cyclase expressed in gastric smooth muscle, and block further formation of cAMP (Murthy et al., 2002). In this study, we examined the whether sGC phosphorylation by PKG attenuate the cyclase activity. SNP acting as a NO donor stimulated sGC activity by 183 ± 24% above basal level within 60 s. Inhibition of PKG activity by KT5823 or Rp-cGMPS increased significantly (p < 0.01) SNP-stimulated sGC activity (Fig. 2A). 3.3. Attenuation of cGMP Levels by PKG-dependent inhibition of sGC The effect of sGC phosphorylation on SNP-stimulated cGMP levels was also examined in the presence or absence of PKG inhibitors. As previous studies (Murthy, 2001) in gastric smooth muscle cells had shown that PKG regulates cGMP levels by stimulatory phosphorylation cGMP-specific PDE5, the effect of PKG inhibitors was examined in the presence of phosphodiesterase inhibitors (1 mM IBMX and 10 ␮M zaprinast). SNP stimulated cGMP levels in a concentration-dependent fashion with an EC50 of ∼1 nM (Fig. 2B). Inhibition of sGC phosphorylation by KT5823 or Rp-cGMPS significantly augmented SNP-stimulated cGMP levels (Fig. 2B). The results imply that phosphorylation of

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Fig. 2. (A) Inhibition of soluble guanylyl cyclase (sGC) activity by PKG-dependent phosphorylation. Gastric muscle cells were treated with SNP (1 ␮M) in the presence or absence of the PKG inhibitor, KT5823 (1 ␮M) or Rp-cGMPS (1 ␮M). Soluble GC activity was measured by the conversion of [32 P]GTP to [32 P]GMP as described in Section 2.2. All studies were done in the presence of 1 mM IBMX and 10 ␮M zaprinast. Results were expressed as picomoles of cGMP/mg protein. (**) Significant increase over that of SNP alone, P < 0.01. (B) Augmentation of SNP-induced cGMP levels by selective inhibition of PKG. Gastric smooth muscle cells were incubated with various concentrations of SNP (1 pM to 1 ␮M) for 1 min in the presence or absence of selective inhibitors of PKG (Rp-cGMPS: 1 ␮M or KT5823: 1 ␮M). cGMP levels were measured by radioimmunoassay and the results expressed as picomoles/mg protein. Suppression of PKG with KT5823 or Rp-cGMPS significantly (P < 0.01) augmented SNP-stimulated cGMP formation. Values are means ± S.E. of four–five experiments. Fig. 1. Concentration-dependent phosphorylation of sGC induced by SNP in smooth muscle. (A) Gastric smooth muscle cells labeled with 32 P were incubated with various concentrations of SNP (1 pM to 1 ␮M) for 1 min in the presence or absence of selective inhibitors of PKG (Rp-cGMPS: 1 ␮M or KT5823: 1 ␮M). 32 P-labeled sGC was identified by autoradiography, and the measured radioactivity expressed as cpm/mg protein above basal levels (basal: 525±72 cpm/mg protein). (B) Gastric smooth muscle cells labeled with 32 P were incubated with selective activators of PKA (cBIMPS: 10 ␮M or isoproterenol: 1 ␮M) or 100 ␮M isoproterenol that cross-activate PKG for 1 min in the presence or absence of selective inhibitor of PKG (Rp-cGMPS: 1 ␮M) or PKA (Myristoylated PKI: 1 ␮M). 32 P-labeled sGC was identified by autoradiography, and the measured radioactivity expressed as cpm/mg protein. Values are means ± S.E. of four experiments.

sGC results in inhibition of sGC activity and decrease in cGMP generation. 3.4. Effect of phosphatase inhibitors on sGC phosphorylation and activity Inhibition of phosphatase activity by okadaic acid (10 nM to 10 ␮M) increased sGC phosphorylation in a concentration-dependent fashion (Fig. 3A). The increase in phosphorylation induced by 10 ␮M okadaic acid inhibited SNP-stimulated sGC activity by 32 ± 4% (P < 0.02) (SNP: 4.9 ± 0.3 pmol/mg protein above basal levels;

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Fig. 3. (A) Concentration-dependent phosphorylation of sGC by okadaic acid. Gastric muscle cells were treated with okadaic acid (10 nM to 10 ␮M) and phosphorylation of sGC was measured in cells labeled with 32 P as described in Section 2.3. Immunoprecipitates using polyclonal antibody to sGC were separated on SDS-PAGE. 32 P-labeled soluble GC was identified by autoradiography and the measured radioactivity was expressed as cpm/mg protein above basal levels (basal: 489 ± 69 cpm/mg protein). Values are means ± S.E. of three experiments. (B) Inhibition of SNP-induced sGC activity by okadaic acid. Gastric muscle cells were treated with SNP (1 ␮M) in the presence or absence of okadaic acid (10 ␮M). Soluble GC activity was measured by the conversion of [32 P]GTP to [32 P]GMP as described in Section 2.2. All studies were done in the presence of 1 mM IBMX and 10 ␮M zaprinast. Results were expressed as picomoles of cGMP/mg protein. Values are means ± S.E. of three experiments. (**) Significant decrease over that of SNP alone, P < 0.02.

SNP plus okadaic acid: 3.3 ± 0.4 pmol/mg protein above basal levels) (Fig. 3B). The ability of PKG to stimulate sGC phosphorylation directly was examined in vitro by a back-phosphorylation technique. Immunoprecipitates of sGC from resting muscle cells were treated with purified PKG-I or the catalytic subunit of PKA in the presence of [␥-32 P]ATP. Immunoprecipitates of sGC derived from cells stimulated with SNP (1 ␮M) or isoproterenol (100 ␮M) were treated in similar fashion and the extent of 32 P incorporation into sGC compared with that obtained in unstimulated cells. sGC was phosphorylated by SNP- and isoproterenol-stimulated PKG (Fig. 4). In contrast, activation of PKA did not stimulate sGC phosphorylation (Fig. 4). The role of phosphatases in the dephosphorylation of sGC was corroborated by studies with purified phosphatase 1. Addition of protein phosphatase 1 (PP1) to sGC im-

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Fig. 4. Back phosphorylation of sGC by PKG in vitro. Gastric muscle cells were treated with SNP (1 ␮M) or isoproterenol (Isop; 100 ␮M) for 60 s in the presence or absence of selective inhibitor of PKG (Rp-cGMPS; 1 ␮M) or PKA (myristoylated PKI; 1 ␮M). Soluble GC was immunoprecipitated and back-phosphorylated using purified PKG-I as described in Section 2.3. 32 P-labeled soluble GC was identified by autoradiography and the measured radioactivity was expressed as cpm/mg protein. Values are means ± S.E. of three experiments.

munoprecipitates inhibited sGC phosphorylation by 71±6% (P < 0.01), and the inhibition was completely reversed by (10 ␮M) (SNP: 2965 ± 378 cpm/mg protein above basal levels; SNP plus PP1: 865 ± 141 cpm/mg protein above basal levels, and SNP plus PP1 and okadaic acid: 2785 ± 349 cpm/mg protein above basal levels).

4. Discussion The emergence of NO as the main mediator of visceral smooth muscle relaxation has focused attention on the role of cGMP and cGMP-dependent protein kinase in this process. The levels of cAMP and cGMP in gastrointestinal smooth muscle are determined by the synthetic activities of adenylyl cyclase and soluble guanylyl cyclase, respectively, and the degradative activities of specific PDEs, mainly cAMP-preferring PDE3 and cAMP-specific PDE4, and cGMP-specific PDE5 (Corbin and Francis, 1999; Dousa, 1999; Soderling and Beavo, 2000). The ability of PKA to phosphorylate and thus attenuate the activity of adenylyl cyclase Type V/VI has been demonstrated in various tissues (Iwami et al., 1995), including gastric smooth muscle where phosphorylation and inhibition of activity were shown to be PKA-specific (Murthy et al., 2002). Here, we provide evidence that feedback phosphorylation of sGC and attenuation of its activity is PKG-specific. SNP induced sGC

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phosphorylation and stimulated sGC activity. Rp-cGMPS or KT5823 abolished phosphorylation and augmented sGC activity, implying that feedback inhibition of sGC activity by PKG-dependent phosphorylation contributed to cessation of cGMP formation. Inhibition of sGC activity was direct and PKG-specific as shown in vitro by back-phosphorylation studies. Although both isoforms of PKG-I (PKG-I␣ and PKG-I␤) were expressed in smooth muscle (Murthy and Zhou, 2003), the results of back phosphorylation with PKG-I␣ holoenzyme were similar to those obtained by activating endogenous PKG-I in vivo suggesting selective involvement of PKG-I␣ in sGC phosphorylation. The evidence supporting the regulation of sGC by PKG-dependent phosphorylation can be summarized as follows: (1) SNP, but not selective PKA activators, stimulated sGC phosphorylation that was blocked by the selective PKG inhibitors Rp-cGMPS and KT5823 but were not affected by the PKA inhibitor myristoylated PKI, PKC inhibitor bisindolylmaleimide or MEK inhibitor PD98059. (2) SNP-stimulated sGC activity and cGMP generation was significantly augmented by PKG inhibitors suggesting that phosphorylation of sGC led to the decrease in catalytic activity (3) Pretreatment of cells with PKG activator so as to induce endogenous phosphorylation resulted in a decrease in 32 P-labeling of sGC during back phosphorylation. Activation of PKA had no effect on 32 P-labeling during back phosphorylation. (4) High concentrations of isoproterenol, which crossactivate PKG, stimulated phosphorylation of sGC. Phosphorylation of sGC by isoproterenol was blocked in the presence PKG inhibitors providing further evidence that phosphorylation was PKG specific. Both ␣1 and ␤1 subunits of sGC contain putative sites for phosphorylation for multiple kinases and may represent potential target sites contributing to the regulation of sGC catalytic activity (Andreopoulos and Papapetropoulos, 2000; Zhang et al., 2002). Our results in smooth muscle cells appear to be at variance with those of Ferrero et al. in chromaffin cells (Ferrero et al., 2000). The experimental conditions, however, were different. The present study examined initial effects within 1 min of addition of SNP or analogues, whereas in the studies of Ferraro et al. (Ferrero et al., 2000), the measurements were made after incubations lasting up to 30 min, leaving open the possibility that additional time-dependent pathways may have been activated that led to stimulation of sGC activity. Previous studies have demonstrated that phosphorylation of sGC by cAMP-dependent protein kinase and protein kinase C increases the responsiveness of that enzyme to NO (Zwiller et al., 1981, 1985; Louis et al., 1993). The precise mechanism of this process has not been determined. In summary, sGC activity in smooth muscle can be regulated by a feedback inhibition of via PKG-dependent phos-

phorylation, which is analogous to PKA-mediated inhibition of the AC V/VI (Iwami et al., 1995; Murthy et al., 2002) and PKC-mediated inhibition of the phospholipase C-␤ pathway (Strassheim and Williams, 2000).

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