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CARDIOVASCULAR

Ca²⁺-Independent, Inhibitory Effects of Cyclic Adenosine 5'-Monophosphate on Ca²⁺ Regulation of Phosphoinositide 3-Kinase C2, Rho, and Myosin Phosphatase in Vascular Smooth Muscle

Department of Physiology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan (M.A.A., K.Y., N.S., Y.T.); Department of Veterinary Pharmacology, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan (S.O., K.S.); and Department of Health and Medical Sciences, Ishikawa Prefectural Nursing University, Kahoku, Japan (N.T.)

Received July 23, 2006; accepted November 15, 2006.

	Abstract

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We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca²⁺-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca²⁺-induced Ca²⁺-sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 (CPI-17). In the present study, we investigated whether and how cAMP affected Ca²⁺-dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20-kDa myosin light chain (MLC) phosphorylation without inhibiting Ca²⁺ mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca²⁺-mediated Rho activation is dependent on class II -isoform of phosphoinositide 3-kinase (PI3K-C2). Forskolin inhibited KCl-induced stimulation of PI3K-C2 activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr⁸⁵⁰ and CPI-17 at Thr³⁸ in manners dependent on both PI3K and Rhokinase, but not PKC. Forskolin, dbcAMP, and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca²⁺-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2, Rho, and Rho kinase in a manner independent of Ca²⁺ and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction.

The second messenger adenosine 3',5'-cyclic monophosphate cAMP mediates relaxation of smooth muscle (Somlyo and Somlyo, 1994). cAMP was shown previously to induce a reduction in the [Ca²⁺]_i through multiple mechanisms including inhibition of phospholipase C stimulation, inhibition of Ca²⁺ influx across the plasma membrane, and stimulation of Ca²⁺ extrusion from the cell interior, resulting in inhibition of MLCK activation and MLC phosphorylation (Somlyo and Somlyo, 2003). On the other hand, cAMP inhibited contraction in permeabilized smooth muscle (Ruegg et al., 1981; Nishimura and van Breemen, 1989), suggesting that cAMP also exerted a relaxing effect that was independent of the [Ca²⁺]_i-lowering effect. Subsequent investigations showed that cAMP as well as cGMP stimulated MLCP (Wu et al., 1996; Lee et al., 1997; Etter et al., 2001; Bonnevier et al., 2004), thereby mediating inhibition of MLC phosphorylation and contraction. More recent investigations (Sauzeau et al., 2000; Murthy et al., 2003; Wooldridge et al., 2004) suggested that cAMP might stimulate MLCP through mechanisms involving phosphorylation of Rho and/or MYPT1. Very recently, it was shown that forskolin (FSK) inhibited KCl-induced MYPT1 phosphorylation without inhibiting the intracellular Ca²⁺ increase and stimulated relaxation of Ca²⁺-induced contraction (Porter et al., 2006). However, it is unknown whether cAMP affects the unique Ca²⁺-dependent activation of PI3K-C2 and Rho in smooth muscle.

Membrane depolarization by high KCl induces an increase in the [Ca²⁺]_i through gating of the L-type voltage-dependent Ca²⁺ channel (Sato et al., 1988). cAMP is much less effective in inhibiting membrane depolarization-induced Ca²⁺ mobilization compared with receptor agonist-induced Ca²⁺ mobilization (Takuwa and Rasmussen, 1987; Sato et al., 1988). In the present study, we employed the experimental condition in which cAMP does not significantly inhibit membrane depolarization-induced Ca²⁺ mobilization but inhibits MLC phosphorylation and contraction in isolated rabbit VSM and investigated the effects of cAMP on Ca²⁺-dependent activation of PI3K-C2 and Rho and phosphorylation of MYPT1 and CPI-17. Our results show that without significantly inhibiting membrane depolarization-induced [Ca²⁺]_i increase, cAMP inhibits membrane depolarization-induced activation of PI3K-C2 and Rho, reversing phosphorylation of MYPT1 and CPI-17. These findings together with recent observations (Porter et al., 2006) indicate that cAMP reverses Ca²⁺-induced MLCP inhibition and suggest that the site of the cAMP action involves the activation of PI3K-C2.

	Materials and Methods

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Materials. Forskolin, 1,9-dideoxy-forskolin (dd-forskolin), phosphatidylinositol, phorbol 12,13-dibutyrate (PDBu), dibutyryl cAMP (dbcAMP), and isoproterenol (ISO) were bought from Sigma-Aldrich (St. Louis, MO). LY294002 and GF109203X were purchased from Merck-Calbiochem Biosciences (Darmstadt, Germany). Y27632 was donated by WelFide Corporation (Osaka, Japan). LY294002, GF109203X, and Y27632 were dissolved in dimethyl sulfoxide. Forskolin and dd-forskolin were dissolved in ethanol. The solvent concentrations in the incubation buffers did not exceed 0.1%. Monoclonal anti-MLC antibody (MY-21) and anti-RhoA antibody (26C4) were purchased from Sigma-Aldrich and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Rabbit polyclonal anti-phospho (Thr³⁸)-CPI-17 (36-006), anti-CPI-17 (07-344), anti-phospho (Thr⁸⁵⁰)-MYPT1 (36-003), and phospho-MYPT1 (Thr⁶⁹⁵) (07-251) were bought from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal anti-MYPT1 antibody (PRB-457C) was bought from Covance (Berkeley, CA). Monoclonal antibody to PI3K-C2 (611046) for Western blotting was bought from BD Biosciences (San Jose, CA). Rabbit polyclonal anti-PI3K-C2 antibody was raised in our laboratory as described elsewhere (Wang et al., 2006). Glutathione S-transferase-Rhotekin was prepared as described previously (Sakurada et al., 2001).

Tissue Preparation and Tension Measurement. The animals were maintained in compliance with the guidelines of the Care and Use of Laboratory Animals of Kanazawa University. Japanese male white rabbits weighing approximately 2.0 kg were sacrificed by injecting overdoses (60 mg/kg body weight) of sodium pentobarbital into the ear vein (Sakurada et al., 2001). Descending thoracic aortae were removed and immediately placed into ice-cold modified Krebs-Henseleit buffer comprising 119 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.5 mM MgSO₄, 1.5 mM CaCl₂, 25 mM NaHCO₃, and 11 mM glucose and aerated with 95% O₂ and 5% CO₂. The aorta was cleaned off of adhering connective tissue and fat and cut into rings of about 4 to 5 mm length. The endothelial cells were removed by gentle rubbing of the intimal surface with a wooden stick. The rings were mounted under 1g resting tension in static incubation muscle chambers, equilibrated in the modified Krebs-Henseleit buffer at 37°C, and gassed with 95% O₂ and 5% CO₂. The tension generated was measured isometrically with force-displacement transducers (UM-203; Kishimoto Medical Instruments, Kyoto, Japan). Before test stimulation, the rings were precontracted several times with 60 mM KCl containing buffer followed by washing with the modified Krebs-Henseleit buffer, each time for 5 min at a 1-h interval until a stable contraction was obtained.

Measurement of the [Ca²⁺]_i in Aortic VSM. The method for determination of the [Ca²⁺]_i was previously described in detail (Sato et al., 1988, 2000). Briefly, rabbit thoracic aorta was removed and cut into helical strips, followed by removal of endothelium by gently rubbing with a cotton swab. VSM strips were loaded with the acetoxymethyl ester of fura-PE3 (TEFLABS, Austin, TX) by incubating in the normal HEPES-buffered solution comprising 125.4 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 1.5 mM CaCl₂, 11 mM glucose, and 10 mM HEPES, pH 7.4, in the presence of 10 µM fura-PE3-acetoxymethyl ester and 0.02% Cremophor EL for 4 to 6 h at room temperature in the dark room. VSM strips were placed in a tissue bath containing the HEPES-buffered solution aerated with 100% O₂ at 37°C. Strips were illuminated alternately with 340 and 380 nm of light, and the fluorescence strength of 500-nm emission at 340- and 380-nm excitation (F₃₄₀ and F₃₈₀, respectively) was detected with a CAF-100 spectrofluorometer (Japan Spectroscopic, Tokyo, Japan). A change in the [Ca²⁺]_i was determined by monitoring the ratio of F340/F380. KCl stimulated Mn²⁺ quenching of fura-PE3 fluorescence at 360-nm excitation in the presence of 0.5 mM Mn²⁺ in the buffer, indicating that KCl stimulated Ca²⁺ influx across the plasma membrane. VSM strips were first stimulated with 40 mM KCl in the absence of FSK, then treated or nontreated with 30 µM FSK for 20 min, and stimulated with KCl. Because the ratio of F340/F380 at the nonstimulated basal condition and KCl-stimulated condition was gradually reduced due to leak and bleaching of fura-PE3 during the observation periods, the ratio of F340/F380 at the second KCl stimulation was expressed as a percentage of the value at the first KCl stimulation.

Rho Assay. Aortic rings contracted isometrically were frozen by quickly immersing into liquid nitrogen. Frozen rings were homogenized with a homogenization buffer comprising 50 mM Tris/HCl, pH 7.2, 500 mM NaCl, 10 mM MgCl₂, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 20 µg/ml each of leupeptin and aprotinin, and 1 mM phenylmethylsulfonyl fluoride (PMSF), as described previously (Sakurada et al., 2001). The homogenates were centrifuged at 15,000 rpm at 4°C for 10 min, and the resultant supernatants (300 µg of protein) were incubated with glutathione S-transferase-Rhotekin immobilized onto glutathione-Sepharose 4B beads (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 4°C for 30 min. Portions of the supernatants were employed for a protein assay by Lowry's method and total Rho assay by Western blotting using anti-RhoA antibody. The beads were washed twice with a washing buffer comprising 50 mM Tris/HCl, pH 7.2, 1% Triton X-100, 150 mM NaCl, and 10 mM MgCl₂. RhoAs bound to beads were solubilized with Laemmli's SDS sample buffer and boiled for 5 min, followed by separation on a 15% acrylamide gel, electrotransfer onto polyvinylidene difluoride membrane, and detection using anti-RhoA antibody and alkaline phosphatase-conjugated secondary antibody. Specific bands were visualized with alkaline phosphatase colorigenic reaction, and densities of bands were quantitated with a densitometry using the Quantity One software (Bio-Rad, Hercules, CA). The amount of GTP-bound RhoA (GTP-Rho) was normalized for total amount of RhoA in each sample, and the quantitative data of normalized amounts of GTP-Rho are expressed as multiples over a value in unstimulated tissues, which is expressed as 1.0.

Determinations of MYPT1 and CPI-17 Phosphorylation. Aortic rings contracted isometrically were fixed in acetone dry ice slurry containing 20 mM dithiothreitol and 10% trichloroacetic acid and washed in acetone containing 10 mM dithiothreitol at room temperature, as described previously (Wang et al., 2006). Fixed tissue were homogenized in a homogenization buffer comprising 20 mM Tris/HCl, pH 7.5, 100 mM NaF, 1 mM Na₃Vo₄, 0.1% SDS, 2 mM EGTA, 0.5% Nonidet P-40, 20 µg/ml each of leupeptin and aprotinin, and 1 mM PMSF. A portion of homogenates was taken for determination of a protein concentration by Lowry's method. The homogenates were mixed with 4 times concentrated Laemmli's SDS sample buffer and boiled for 5 min. The samples (40 µg of protein) were separated on 8% SDS-PAGE, followed by Western blot analysis using phosphoprotein-specific antibodies and antibodies that recognize both phospho- and nonphosphoproteins. The amounts of phospho-MYPT1 and phospho-CPI-17 quantitated by densitometry were normalized for total amounts of MYPT1 and CPI-17 in each sample, and the quantitative data of normalized amounts of the phosphoproteins were expressed as multiples over a value in unstimulated tissues, which is expressed as 1.0.

Measurement of MLC Phosphorylation. Contracted aortic rings were fixed as described for determinations of MYPT1 and CPI-17 phosphorylation and solubilized in the urea sample buffer. For determination of total MLC phosphorylation, the extracts were separated on urea-glycerol PAGE, followed by Western blotting using anti-MLC antibody (MY-21) (Noda et al., 1995; Nagumo et al., 2000). Densities of bands corresponding to nonphosphorylated, monophosphorylated, and diphosphorylated forms of MLC were quantitated, and the ratio of monophosphorylated and diphosphorylated forms of MLC over total MLC were calculated. Because the amount of diphosphorylated MLC was small (less than 3% of total MLC) in KCl-stimulated muscle, it was difficult to quantitatively evaluate the amount of diphosphorylated MLC by the urea-glycerol PAGE and Western blotting. Therefore, we employed Western blot analysis using anti-diphosphorylated (Thr¹⁸ and Ser¹⁹) MLC-specific antibody, which was more sensitive than the urea-glycerol PAGE method, for quantitative determination of MLC diphosphorylation. Three parts of urea-extracted samples were mixed with 1 part of 4 times concentrated Laemmli's SDS sample buffer, and the extracts were separated on 15% SDS-PAGE, followed by Western analysis using anti-MLC antibody (MY-21) and anti-diphosphorylated MLC-specific antibody (Sakurada et al., 1998), which was donated by Dr. Seto (Asahi Chemical Industry, Fuji, Japan). For quantitation of MLC diphosphorylation, a density of a band detected by anti-diphosphorylated MLC antibody was corrected by MLC protein amounts, and the results were expressed as multiples over a value in the nontreatment.

PI3K-C2 Kinase Assay. Contracted aortic rings were rapidly frozen in liquid nitrogen and homogenized in a homogenization buffer comprising 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 10 mM NaF, 1% Nonidet-40, 1 mM Na₃VO₄, 5 mM EDTA, 20 µg/ml each of leupeptin and aprotinin, and 1 mM PMSF (Wang et al., 2006). The homogenates were clarified by centrifugation at 15,000 rpm at 4°C for 10 min. Portions of the supernatants were taken for determination of protein contents by Lowry's method and PI3K-C2 protein amounts by Western blotting. The resultant supernatants (400 µg of protein) were subjected to immunoprecipitation using specific polyclonal anti-PI3K-C2 antibody. The anti-PI3K-C2 immunoprecipitates were incubated with 50 µl of the kinase assay buffer [20 mM Tris/HCl, pH 7.5, 100 mM NaCl, 0.5 mM EGTA, 20 mM MgCl₂, 10 µM ATP, 1.8 MBq/ml [-³²P]ATP (GE Healthcare), 200 µg/ml phosphatidylinositol] at 25°C for 30 min. The reaction was terminated by adding 100 µl of CH₃OH/CHCl₃/HCl [2:1:0.02 (v/v)]. Extracted lipid fraction was separated by thin-layer chromatography using Silica gel 60 plates and the solvent system of CH₃OH/CHCl₃/25% NH₄OH/H₂O [43:38:5:7 (v/v)]. Radioactivity in the spot corresponding to phosphatidylinositol 3-phosphate was quantitated by using a FujiBAS Bioimage analyzer 2000 (Fuji, Tokyo, Japan). The activity of PI3K-C2 was normalized for the PI3K-C2 protein amount in each sample, and the quantitative data of normalized amounts of phosphatidylinositol 3-phosphate were expressed as multiples over a value in unstimulated tissues, which is expressed as 1.0.

Statistics. All data are shown as mean ± S.E.M. One- or two-way analysis of variance was followed by Dunnett's test to determine the statistical significance of differences between mean values, except Fig. 1C and Fig. 4A, where unpaired Student's t tests were performed for the comparison between two groups. For all statistical comparisons, p < 0.05 was considered significant.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Inhibition of KCl-induced contraction and MLC phosphorylation but not [Ca2+]i increase by forskolin and dibutyryl cAMP. De-endothelialized rabbit aortic rings were pretreated with 30 µM FSK, 30 µM dd-FSK, or 5 mM of the cell-permeable cAMP analog N6, dbcAMP for 20 min, and stimulated with 40 mM KCl. Isometric tension (A), MLC total phosphorylation (B), and [Ca2+]i (C) were determined. A, tension was expressed as a percentage of 60 mM KCl-induced maximal tension. Total MLC phosphorylation (the ratio of the sum of monophosphorylated and diphosphorylated MLC over total MLC) was determined as described under Materials and Methods. C, [Ca2+]i and tension were simultaneously monitored in fura-PE3-loaded aortic VSM strips and expressed as a percentage of the control response as described in detail under Materials and Methods. Values are means ± SE. of four to eight determinations. , statistical significance at the level of p < 0.01 compared with the group of "None."

View larger version (27K): [in this window] [in a new window]   Fig. 4. Inhibition of KCl-induced contraction, Rho activation, MYPT1 phosphorylation, PI3K-C2, and MLC diphosphorylation by isoproterenol. Aortic rings were pretreated with 100 µM ISO in the presence of 30 µM phentolamine for 10 min or nonpretreated and then stimulated with 40 mM KCl for 5 min. The isometric tension was recorded, and aortic rings were rapidly frozen and analyzed for amounts of GTP-Rho (B), MYPT1 phosphorylation at Thr850(C), PI3K-C2 activity (D), and MLC diphosphorylation, as described in Figs. 2 and 3. Values are means ± SE. of four determinations. and , statistical significance at the level of p < 0.05 and p < 0.01, respectively, compared with "None." and , statistical significance at the level of p < 0.05 and p < 0.01, respectively, compared with "KCl."

	Results

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We examined the effect of FSK on KCl-induced increases in the [Ca²⁺]_i by simultaneously monitoring fura-PE3 fluorescence and tension in VSM. KCl (40 mM) induced a rapid and sustained increase in the [Ca²⁺]_i as reported previously (Sato et al., 1988). Pretreatment with FSK (30 µM) inhibited KCl-induced contraction by approximately 70% but did not significantly affect KCl-induced rise in the [Ca²⁺]_i at either the peak value or the value in 10-min stimulation (Fig. 1C). The observations suggest that FSK induces inhibition of KCl-induced contraction and MLC phosphorylation largely through a [Ca²⁺]_i-independent effect under the experimental condition.

FSK Inhibits Membrane Depolarization-Induced Activation of Rho and PI3K-C2 and Phosphorylation of MYPT1 and CPI-17. KCl (40 mM) induced a maximal 4-fold increase in the amount of the GTP-bound, active form of Rho (GTP-Rho). FSK abolished KCl-induced increases in the amount of GTP-Rho at every time point examined (Fig. 2A). The addition of dbcAMP also abolished KCl-induced Rho activation (Fig. 2B). We recently showed that PI3K-C2 is involved in Ca²⁺-mediated Rho activation in VSM (Wang et al., 2006). Therefore, we explored the possible inhibitory effect of FSK on KCl-induced PI3K-C2 stimulation. KCl induced stimulation (approximately 2.5-fold) of PI3K-C2 activity (Fig. 2C). FSK nearly abolished KCl-induced PI3K-C2 activation. These observations suggest that the site of the FSK action in inhibiting VSM contraction involves PI3K-C2.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Inhibition of KCl-induced Rho activation and PI3K-C2 by forskolin. Aortic rings were pretreated with 30 µM FSK or 5 mM dbcAMP for 20 min or nonpretreated and then stimulated with 40 mM KCl for indicated time periods (A) or for 10 min (B and C). Aortic rings were rapidly frozen and analyzed for amounts of GTP-Rho (A and B) and PI3K-C2 activity (C) by the pull-down assay and the immunoprecipitation kinase assay, respectively, as described under Materials and Methods. Values are means ± SE. of four to six determinations. and , statistical significance at the level of p < 0.01 compared with "None" and "KCl," respectively.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Inhibition of KCl-induced phosphorylation MYPT1 and CPI-17 by forskolin. Aortic rings were pretreated with 30 µM FSK for 20 min or nonpretreated and then stimulated with 40 mM KCl for indicated time periods. Aortic rings were rapidly frozen and analyzed for phosphorylation of MYPT1 and CPI-17 by Western blotting using respective antiphosphoprotein-specific antibodies. MYPT1 phosphorylation at Thr850 (A), MYPT1 phosphorylation at Thr695 (B), and CPI-17 phosphorylation at Thr38 (C). D, rings were pretreated with 5 µM GF109203X (GFX), 30 µM FSK, 100 µM LY 294002 (LY), or 10 µM Y27632 (Y) or nonpretreated for 30 min and then stimulated with 40 mM KCl for 10 min. MLC diphosphorylation at Ser18 and Thr19 was determined by Western blot analysis using anti-diphosphorylated MLC-specific antibody. Values are means ± SE. of four to six determinations. and , statistical significance at the levels of p < 0.05 and p < 0.01, respectively, compared with the value in the nonstimulated rings; and , statistical significance at the level of p < 0.05 and p < 0.01, respectively, compared with "KCl."

Membrane Depolarization-Induced Phosphorylation of CPI-17 and MYPT1 Are PI3K- and Rho Kinase-Dependent. CPI-17 phosphorylation was shown previously to be mediated by both Rho kinase and protein kinase C in vascular and other smooth muscle types (Eto et al., 2001; Niiro et al., 2003), and membrane depolarization was suggested to stimulate phospholipase C, activating protein kinase C (Slish et al., 2002). KCl membrane depolarization-induced CPI-17 phosphorylation was not inhibited by the protein kinase C inhibitor GF109203X (5 µM) (Fig. 5A). However, GF109203X abolished PDBu-induced CPI-17 phosphorylation (Fig. 5B), confirming that GF109203X was effective in inhibiting protein kinase C-mediated CPI-17 phosphorylation. Instead, either the Rho kinase inhibitor Y27632 or the PI3K inhibitor LY294002 abolished KCl-induced CPI-17 phosphorylation (Fig. 5A). In contrast, PDBu-induced CPI-17 was insensitive to Y27632. These observations together indicate that membrane depolarization-induced CPI-17 phosphorylation is dependent on PI3K and Rho kinase, different from PDBu-induced CPI-17 phosphorylation. Similarly, either LY294002 or Y27632 but not GF109203X abolished KCl-induced MYPT1 phosphorylation at Thr⁸⁵⁰ and Rho activation (Fig. 5, C and D). LY294002 and Y27632, but not GF109203X, markedly inhibited KCl-induced contraction (Fig. 5E). Furthermore, either LY294002 or Y27632, but not GF109203X, abolished KCl-induced diphosphorylation of MLC (Fig. 3D). These observations suggest that the PI3K/Rho/Rho kinase-dependent pathway, but not protein kinase C, plays an essential role in membrane depolarization-induced CPI-17 and MYPT1 phosphorylation, consequent MLC phosphorylation and contraction.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Inhibition of KCl-induced phosphorylation of CPI-17 and MYPT1, Rho stimulation, and contraction by a PI3K inhibitor and a Rho kinase inhibitor but not a protein kinase C inhibitor. Aortic rings were pretreated with 5 µM GF109203X, 100 µM LY 29400, or 10 µM Y27632 for 30 min and then stimulated with 40 mM KCl for 10 min or indicated time periods or with 1 µM PDBu for 60 min. Aortic rings were rapidly frozen and analyzed for CPI-17 Thr38 phosphorylation (A and B), MYPT1 Thr850 phosphorylation (C), and amounts of GTP-Rho (D) by Western blotting using respective anti-phosphoprotein-specific antibodies or the pull-down assay. E, tension. Values are means± SE. of four to six determinations. , statistical significance at the level of p < 0.01, compared with the value in the nonstimulated rings; , statistical significance at the level of p < 0.01, compared with KCl or PDBu.

	Discussion

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An increase in the intracellular cAMP or cGMP in response to various physiological stimuli including hormones, neurotransmitters, and autacoids induces relaxation in VSM contracted by excitatory receptor agonists and membrane depolarization. The relaxing actions of both cAMP and cGMP involve stimulation or deinhibition of MLCP (Somlyo and Somlyo 2003), leading to Ca²⁺ desensitization of the contractile proteins. Rho and Rho kinase, the major signaling pathway to negatively regulate MLCP, is activated by the receptor-coupled, G_12/13-mediated mechanism as well as the recently discovered Ca²⁺-mediated, PI3K-C2-dependent one (Pfitzer, 2001; Somlyo and Somlyo, 2003; Hartshorne et al., 2004; Takuwa et al., 2005). Receptor-coupled Rho signaling pathway is effectively inhibited by both cAMP and cGMP. On the other hand, whether and how the latter Ca²⁺-mediated Rho activation and MLCP inhibition are regulated by cAMP remained unknown. The present results, together with the recent observations by Porter et al. (2006) that FSK inhibited KCl-induced MYPT1 phosphorylation and promoted relaxation of Ca²⁺-induced contraction, demonstrate that cAMP can inhibit the Ca²⁺-mediated Rho activation and consequent MLCP suppression without reducing the [Ca²⁺]_i in intact VSM, that the cAMP inhibition is likely mediated through prevention of Rho kinase-dependent phosphorylation of MYPT1 and CPI-17, and that the site of the cAMP inhibition involves the Rho regulator PI3K-C2 (Fig. 6).

View larger version (28K): [in this window] [in a new window]   Fig. 6. The model depicting the sites of cAMP actions in the Ca2+-induced, PI3K-C2-dependent Rho signaling. KCl membrane depolarization induces stimulation of Ca2+ influx across the plasma membrane through the voltage-dependent Ca2+ channels (VDCCs). Stimulation of Ca2+ influx induces PI3K-C2 stimulation, which leads to Rho activation. Stimulation of Ca2+ influx results in MLCP inhibition (Ca2+-induced Ca2+-sensitization) through mechanisms involving Rho- and Rho kinase-dependent phosphorylation of MYPT1 and CPI-17. Ca2+-induced MLCK activation and MLCP inhibition synergistically induces efficient MLC phosphorylation and contraction. The adenylate cyclase (AC) activator FSK and the -adrenergic agonist ISO stimulates cAMP production, which exerts inhibitory effects on Ca2+-induced PI3K-C2 stimulation, which at least in part contributes to cAMP-induced Rho inhibition and MLCP deinhibition. In addition to cAMP inhibition of PI3K-C2, previous studies showed that cAMP also inhibits Rho by phosphorylating it at Ser188, and deinhibits MLCP by phosphorylating MYPT1 (please see Discussion). PM, plasma membrane; MLC-P, phosphorylated MLC; MYPT1-P, phosphorylated MYPT1; CPI17-P, phosphorylated CPI-17.

We showed that the PI3K inhibitor LY294002 as well as wortmannin suppressed KCl-induced, Ca²⁺-dependent stimulation of PI3K-C2, Rho, MYPT1, and MLC phosphorylation and contraction (Wang et al., 2006; the present study). In differentiated vascular smooth muscle cells, selective PI3K-C2 down-regulation by a specific short interfering RNA duplex suppressed Ca²⁺-dependent phosphorylation of MYPT1 and MLC and abolished further inhibition of these responses by LY294002. These observations suggested PI3K-C2 as a target for LY294002 in vascular smooth muscle. On the other hand, it was reported previously that LY294002 but not wortmannin inhibited Ca²⁺ influx through both voltage-dependent and receptor-coupled Ca²⁺ channels in smooth muscle and nonmuscle cells overexpressing a Ca²⁺ channel (Takayama et al., 1996; Tolloczko et al., 2004; Welling et al., 2005). We observed that LY294002 at its lower concentration (10 µM), which was shown previously to inhibit the voltage-dependent Ca²⁺ channel (Welling et al., 2005), failed to inhibit KCl-induced Ca²⁺ entry in rabbit aortic smooth muscle (S. Ohkura and K. Sato, unpublished observation). These observations may suggest that the inhibitory effect of LY on Ca²⁺ channels could be different among smooth muscle types and animal species probably because of differences in Ca²⁺ channel isotypes and accessory proteins. However, it was not possible to determine the effect of LY294002 at higher concentrations on KCl-induced [Ca²⁺]_i increase in the present study because LY294002 highly interfered with fura3 fluorescence. We cannot exclude that inhibition of Ca²⁺ entry by LY contributed to LY inhibition of PI3K-C2 and Rho, particularly at its higher concentrations.

A recent study by Huang et al. (2006) demonstrated that stimulation of isolated intestinal smooth muscle cells by G_i-coupled receptor agonists induced activation of integrin-linked kinase (ILK) through a PI3K-dependent mechanism and that ILK mediated phosphorylation of CPI-17 and MLC. The study showed that a relatively low concentration (10 µM) of LY294002 nearly abolished agonist-induced phosphorylation of CPI-17 and MLC although the responsible PI3K isoform was not identified. On the other hand, we observed that this low concentration of LY294002 failed to inhibit membrane depolarization-induced Rho activation, phosphorylation of MYPT1, CPI-17 and MLC, and contraction in intact VSM, but a higher concentration of LY294002 inhibited these Ca²⁺-dependent responses (Wang et al., 2006). Our observations are quite consistent with the notion that PI3K-C2, which is clearly less sensitive to LY294002 than the other PI3K isoforms, is responsible for the mediation of these Ca²⁺-dependent responses. In addition, ILK activation is not dependent on Ca²⁺, whereas activation of both PI3K-C2 and Rho by membrane depolarization is dependent on Ca²⁺. The observations by Huang et al. (2006) and us may suggest that more than single PI3K isoforms participate in MLCP regulation through different mechanisms in smooth muscle type- and stimulus-specific manners.

Accumulating evidence shows that cAMP and cGMP exert stimulating or deinhibiting effects on MLCP by acting at more than a single sites (Somlyo and Somlyo, 2003). Protein kinase (PK) A and PKG were shown to phosphorylate RhoA at Ser¹⁸⁸ and to inhibit RhoA activity through enhancement of RhoA and guanine nucleotide dissociation inhibitor interaction in various cell types including smooth muscle (Sauzeau et al., 2000; Murthy et al., 2003; Bonnevier et al., 2004). Antagonism of Rho and Rho kinase by cyclic nucleotides may also be caused by PKA- and PKG-mediated G₁₃ phosphorylation that induces inhibition of G₁₃-mediated Rho activation (Manganello et al., 2003). PKG was also suggested to inhibit geranyl-geranylation of Rho, a maturation process of Rho that is required for the Rho translocation to the plasma membrane (Begum et al., 2002). The Ca²⁺-mediated Rho activation mechanism, together with G_12/13-coupled mechanism, contributes to excitatory receptor agonist-induced Rho activation in VSM (Sakurada et al., 2003; Takuwa et al., 2005). Therefore, the cAMP-induced PI3K-C2 inhibition may at least partially contribute to cAMP-induced Rho inhibition in receptor agonist-stimulated VSM. Both cAMP and cGMP also appear to stimulate MLCP independently of inhibiting Rho and Rho kinase because the cyclic nucleotides inhibit the activity of MLCP in Triton X-100-permeabilized smooth muscle in which Rho and Rho kinase signaling is disrupted (Ruegg et al., 1981). The Rho mechanism-independent stimulation of MLCP by the cyclic nucleotides may involve MLCP stimulation by direct PKG binding to MLCP (Surks et al., 1999), MYPT1 phosphorylation by PKG and PKA (Wooldridge et al., 2004), and telokin phosphorylation by these kinases (Walker et al., 2001). Telokin expression is more abundant in phasic type of smooth muscle and relatively lower in tonic smooth muscle. We have shown the operation of Ca²⁺- and PI3K-C2-mediated Rho activation in tonic VSM (Wang et al., 2006). However, it is yet unknown how this Ca²⁺- and PI3K-C2-mediated Rho activation mechanism contributes to Rho regulation in other smooth muscle types. It is likely that mechanisms by which cAMP induces MLCP stimulation might be different among various smooth muscle types.

cAMP inhibited KCl-triggered, Ca²⁺-induced stimulation of PI3K-C2. It is unknown how Ca²⁺ exactly induces PI3K-C2 activation and how stimulation of PI3K-C2 leads to Rho activation. Because Ca²⁺ is not required for the activity of PI3K-C2 despite the presence of C2 domain in PI3K-C2 molecule (Arcaro et al., 2000), it is unlikely that Ca²⁺ directly activates PI3K-C2. How cAMP brings about inhibition of Ca²⁺-induced PI3K-C2 stimulation in a Ca²⁺-independent manner also remains to be clarified.

Membrane depolarization induced a severalfold increase in Thr⁸⁵⁰ phosphorylation and a much smaller increase in Thr⁶⁹⁵ phosphorylation of MYPT1 (Fig. 3, A and B). Membrane depolarization also induced an increase in CPI-17 phosphorylation at Thr³⁸ (Fig. 3C). FSK inhibited all of phosphorylation of these MLCP-regulatory proteins, although FSK inhibition of MYPT1 Thr⁶⁹⁵ phosphorylation was partial. These results suggest that the mechanism for cAMP-induced MLCP inhibition involves inhibition of phosphorylation of both MYPT1 and CPI-17. Previous studies (Eto et al., 2001) showed that excitatory receptor agonist-induced CPI-17 phosphorylation was dependent on both Rho kinase and protein kinase C. In the present study, membrane depolarization-induced phosphorylation of CPI-17 as well as MYPT1 was abolished by the Rho kinase inhibitor Y27632 but not the nonselective protein kinase C inhibitor GF109203X (Fig. 5, A and C). These results are similar to that observed previously in GTPS-stimulated permeabilized VSM (Niiro et al., 2003). In contrast, phorbol ester-induced CPI-17 phosphorylation was abolished by GF109203X but not Y27632 (Fig. 5B). These observations suggest that membrane depolarization-induced MLCP inhibition is caused by Rho kinase-mediated phosphorylation of both MYPT1 and CPI-17. The signaling mechanism to induce CPI-17 phosphorylation at Thr³⁸ appears to be different, depending on contractile stimuli.

It is known that inhibition of MLCP is associated with elevation of diphosphorylation level of MLC (Noda et al., 1995; Somlyo and Somlyo, 2003; Hartshorne et al., 2004). We observed that membrane depolarization induced an increase in MLC diphosphorylation (Fig. 3D), consistent with the notion that membrane depolarization induces MLCP inhibition. FSK and ISO abolished membrane depolarization-induced MLC diphosphorylation as well as total phosphorylation, which was well in agreement with the facts that FSK and ISO suppressed Rho activation and phosphorylation of MYPT1 and CPI-17.

In conclusion, the present study demonstrates that cAMP exerts inhibitory effects on Ca²⁺-mediated Rho activation and resultant MLCP suppression in a manner independent of the Ca²⁺-lowering effect of cAMP. The actions of cAMP on Rho and MLCP appear to involve inhibition of the Rho regulator PI3K-C2. These results indicate the novel mechanism of cAMP-induced inhibition of contraction.

	Acknowledgements

 
We thank Yumiko Ohta and Chiemi Hirose for assistance in the experiments and preparing the manuscript.

	Footnotes

 
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan, the Japan Society for the Promotion of Science, and Novartis Pharma.

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

doi:10.1124/jpet.106.111443.

ABBREVIATIONS: MLCK, myosin light-chain kinase; MLCP, myosin phosphatase; MYPT1, myosin-targeting protein 1; CPI-17, 17-kDa PKC-potentiated inhibitory protein of PP1; VSM, vascular smooth muscle; MLC, 20-kDa myosin light chain; PI3K-C2, phosphoinositide 3-kinase class II isoform; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; FSK, forskolin; dd-forskolin, 1,9-dideoxy-forskolin; PDBu, phorbol 12,13-dibutyrate; dbcAMP, dibutyryl cAMP; ISO, isoproterenol; GF109203X, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide; Y27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; ILK, integrin-linked kinase; PK, protein kinase.

Address correspondence to: Dr. Yoh Takuwa, Department of Physiology, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan. E-mail:ytakuwa{at}med.kanazawa-u.ac.jp

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