INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

NUMEROUS AGENTS ARE CAPABLE of activating guanylyl cyclases (GC) and increasing tissue levels of cGMP in smooth muscle. These include nitrovasodilators such as sodium nitroprusside (SNP) and S-nitroso-N- acetylpenicillamine (SNAP), particulate GC (pGC) activators such as atrial natriuretic peptide (ANP), and endogenous nitric oxide (NO) generated within the endothelium. Elevation of cGMP may affect cell function through a variety of mediators, including cGMP-gated ion channels, cGMP-regulated phosphodiesterases, and cGMP-dependent protein kinases [protein kinase G (PKG)] (40). There is strong evidence suggesting that relaxation of vascular smooth muscle by cGMP-elevating agents is mediated via activation of PKG (20). This protein kinase presumably regulates the activity of specific proteins involved in the relaxation process by catalyzing the transfer of the -phosphoryl group of ATP to the hydroxyl group of serine and threonine residues on target substrates (9). Several lines of evidence support this hypothesis (22, 33). However, the exact identities of those PKG substrates involved in intact vascular smooth muscle relaxation and the mechanism(s) by which intracellular calcium levels are reduced remain unclear.

The early literature describing PKG-mediated phosphorylation is composed mainly of in vitro studies using broken cell preparations (6, 10, 14, 39, 44). Although these studies provide useful information regarding possible PKG substrates, it is not always possible to extrapolate in vitro results to an intact tissue setting. However, only a few reports have appeared in the literature investigating PKG-mediated phosphorylation in intact tissues. In 1982, using intact rabbit aorta, Rapoport et al. (37) were the first to demonstrate that SNP induced a concentration-dependent increase in incorporation of ³²P into nine proteins and decrease in incorporation into two proteins. These patterns of phosphorylation were mimicked by 8-bromo-cGMP (8-BrcGMP), indicating that PKG may be involved. However, none of the proteins described in this study were identified. Since this original study, considerable research regarding the role of PKG-mediated phosphorylation in intact smooth muscle relaxation has focused on the effects of PKG on myosin light chain phosphorylation. Several pathways have been described whereby PKG can mediate a decrease in the phosphorylation of myosin light chains: inhibition of the calcium-dependent activation of myosin light chain kinase (30), stimulation of myosin phosphatase activity (16, 19, 21), and intermediate filament phosphorylation (15). Despite the evidence indicating that activation of PKG can result in dephosphorylation of myosin light chains, it is unclear whether this effect is a direct result of PKG-mediated phosphorylation or simply a result of the decrease in intracellular calcium that occurs during relaxation. Because PKG-mediated phosphorylation is well correlated with decreases in intracellular calcium levels and PKG has been shown to phosphorylate substrates that could mediate calcium uptake, such as the inositol trisphosphate (IP₃) receptor and phospholamban (7, 17), it is possible that this protein kinase mediates relaxation via activation of calcium-sequestering mechanisms rather than direct effects on myosin light chains.

More recently, in bovine carotid artery smooth muscle, PKG was shown to phosphorylate two 20-kDa proteins that share sequence homology with a heat-shock-related protein (HSP-20) (1, 3). Although the exact function of heat-shock protein (HSP) phosphorylation is unknown, it has been suggested that these proteins are important regulatory components of the actin-based cytoskeleton that can interact with intermediate filaments and, in turn, regulate vascular smooth muscle contraction and relaxation (26, 28, 38). Despite the many studies described above investigating PKG-mediated phosphorylation, the underlying mechanisms by which PKG leads to a relaxant response remain undefined. This is due in part to a difficulty in directly correlating PKG-mediated phosphorylation with smooth muscle relaxation.

In the present study, we provide quantitative estimation of SNP-induced, PKG-mediated phosphorylation in the intact smooth muscle of the rat aorta using high-resolution two-dimensional gel electrophoresis. Studies with the soluble GC (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) and with other agents such as a pGC activator (ANP) and a cGMP analog (8-BrcGMP) indicate that the substrates identified in our experiments are phosphorylated in a cGMP-dependent manner. To determine which of these substrates may be playing a physiological role in the relaxant response of the rat aorta, we also investigated PKG-mediated phosphorylation in the rat myometrium and rat vas deferens, which are not relaxed by SNP, despite significant elevations in cGMP and activations of PKG (13, 32). The results of these studies suggest that the failure of the uterus and vas deferens to relax in the face of cGMP elevation and PKG activation may be due to a lack of phosphorylation of the PKG substrates identified in the rat aorta. Relaxation of the rat myometrium by the nitrosothiol donor SNAP is accompanied by phosphorylation of several other proteins, but this phosphorylation occurs independently of cGMP elevation and is presumably mediated by a kinase other than PKG.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials. Acrylamide, urea, glycine, Tris-base, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and SDS were purchased from Roche Diagnostics (Laval, PQ, Canada). Ampholyte (Bio-Lyte) 3-10, N,N'-methylene-bis acrylamide, bromphenol blue, N,N,N',N'-tetramethylethylenediamine, and SDS-PAGE low-molecular-weight silver stain standards were purchased from Bio-Rad Laboratories (Mississauga, ON, Canada). [-³²P]ATP, BIOTRAK cGMP scintillation proximity assay kit, and cellulose sheets were obtained from Amersham Pharmacia Biotech (Baie d'Urfe, PQ, Canada). Bio-Max MS X-ray film, transcreen high-energy intensifying screens, and ³²P-labeled o-phosphoric acid were purchased from Mandel/DuPont-NEN (Boston, MA). Kodak Photoflow was purchased from a local photography supply store. BPDEtide and ANP were purchased from Bachem (Torrance, CA). KT-5823 and ODQ were obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Resolyte 4-8 was obtained from BDH (Toronto, ON, Canada). 4-(2-Aminoethyl)benzenesulfonyl fluoride was purchased from Calbiochem (San Diego, CA). Silver SNAP stain was purchased from Pierce (Rockford, IL). All remaining reagents were purchased from Sigma (Oakville, ON, Canada).

Preparation and tension measurements in rat aorta. Descending thoracic aortae were excised from rats immediately after death in a CO₂ inhalation chamber. Tissues were trimmed free of loosely adhering connective tissue and fat and cut into a helical strip to expose the endothelium. The endothelium was removed by gentle rubbing with a glass rod. Prepared muscle strips of ~10 × 4 mm were suspended in isolated organ baths at 37°C with a preload of 2 g in a physiological salt solution of the following composition (mM): 4.7 KCl, 140 NaCl, 10 HEPES, 1 MgCl₂, 1.5 CaCl₂, 0.2 NaH₂PO₄, and 10 glucose. Tissue baths were aerated with 95% O₂-5% CO₂, which maintained a pH of ~7.4. Isometric tension was recorded with force displacement transducers (model FT03C, Grass), which were connected to a polygraph recorder (model 7D, Grass), as described previously (31). At predetermined times after the addition of drug, tissues were frozen with liquid nitrogen-cooled clamps and stored at 80°C until assessment of cGMP levels and PKG activity. Percent inhibition of contraction was calculated by measuring the change in amplitude of the phenylephrine (PE)-induced contraction after addition of the drug.

cGMP estimation. Frozen smooth muscles from the above contractility experiments (10-20 mg) were placed in liquid nitrogen-cooled Teflon capsules (1-ml capacity; Hansen Industries; Richmond, BC, Canada) with a chilled metal pestle and pulverized in a ProMix, Dentsply dental amalgam mixer (30 s at high speed). Next, 0.75 ml of ice-cold TCA (6%, wt/vol) was added to the capsule, and the tissue was homogenized for another 30 s at high speed. The homogenate was removed and centrifuged at 2,000 g for 15 min at 4°C. TCA was extracted from the supernatant with four washes of ice-cold water-saturated ether (5 ml/wash). cGMP levels were measured using a commercially available scintillation proximity RIA kit (acetylation protocol; Amersham-Pharmacia). The TCA-insoluble pellet was stored at 80°C for protein estimation by methods previously described (23, 25). Tissue cGMP levels were calculated as picomoles of cGMP per milligram of protein.

cGMP-dependent protein kinase assay. Frozen aortic strips weighing ~40 mg were pulverized in Teflon capsules by a method similar to that described for cGMP extraction, except a homogenization buffer was substituted for TCA. The buffer contained 10 mM HEPES, 1 mM EDTA, 10 mM dithiothreitol, 1 mM IBMX, 125 mM KCl, 1 mM benzamidine, 10 µg/ml leupeptin, and 10 µg/ml pepstatin. The homogenate was centrifuged at 30,000 g for 5 min, and the supernatant was assayed for soluble PKG activity, as described elsewhere (13). Briefly, PKG activity was determined by measuring the transfer of the [-³²P]phosphoryl group of ATP to BPDEtide (RKISASEFDRPLR), which is a relatively specific substrate for PKG. The assay was carried out in a total volume of 70 µl containing 150 µM BPDEtide, 10 mM HEPES, 35 mM -glycerophosphate, 4 mM magnesium acetate, 200 µM [-³²P]ATP (2.5 µCi/tube), 5 µM synthetic protein kinase A inhibitor, and 0.5 mM EGTA in the absence or presence of 5 µM cGMP. The reaction was initiated by addition of 20 µl of the sample supernatant. The reaction was allowed to proceed for 10 min at 4°C and was stopped by spotting 50 µl of the reaction mixture onto 2 × 2 cm squares of phosphocellulose paper (Whatman P81). The paper was then washed four times in 0.5% o-phosphoric acid for 10 min each. The papers were dried and then transferred to scintillation vials containing 2.5 ml of liquid scintillant. Radioactivity was counted in a Beckman LS 6000TA liquid scintillation counter. PKG activity was expressed as picomoles of phosphate incorporated into substrate per minute per milligram of protein. PKG activation was assessed by calculating the activity ratio, which is a measure of the PKG activity in the absence of exogenously added cGMP (endogenous cGMP only) divided by the PKG activity in the presence of enough exogenous cGMP (5 µM) to maximally activate the kinase. Protein levels in the supernatant were estimated using a commercially available dye-binding assay previously described (4).

Two-dimensional gel electrophoresis: tissue preparation and radioactive labeling of proteins. Smooth muscle strips from rat aorta were prepared as described above. Uteri were removed from nonovariectomized, estrogen-primed progesterone-influenced rats, and myometrial strips were prepared by peeling the endometrium and circular muscle away from the longitudinal smooth muscle. This hormonal treatment has been shown previously to provide tissues that exhibit strong, persistent spontaneous contractions that are nonresponsive to marked increases in tissue levels of cGMP (13). Rat vas deferens were carefully removed, trimmed free of loosely adhering connective tissue, and cut open longitudinally to expose the lumen and remove all traces of sperm. All prepared muscle strips were placed in a well-oxygenated, low-phosphate physiological salt solution of the following composition (mM): 4.7 KCl, 140 NaCl, 10 HEPES, 1 MgCl₂, 1.5 CaCl₂, 0.2 NaH₂PO₄, and 10 glucose. ³²P-labeled P_i was then added to the buffer at a concentration of 500 µCi/ml to label cellular ATP pools. Tissues were incubated with radiolabeled phosphate for 3 h at 37°C with gentle agitation. Once cellular ATP pools were labeled, smooth muscle strips were treated as described in RESULTS. After the addition of the drug, individual smooth muscles were blotted on tissue paper and frozen with liquid nitrogen-cooled clamps and stored at 80°C for future use.

Sample preparation. Frozen smooth muscle strips labeled with ³²P were homogenized as described above with a homogenization buffer containing 10 mM EDTA, 10 mM HEPES, 1 mM benzamadine, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.001 mM protein kinase A inhibitor, 0.001 mM KT-5823, 100 mM sodium fluoride, 10 mM dithiothreitol, and 10 µg/ml leupeptin. The homogenate was removed and centrifuged for 1 h at 100,000 g. The supernatant from this spin made up the soluble protein fraction for our experiments. To concentrate the protein in the collected fractions, samples were added to Amicon Microcon 10 microconcentrators. Protein levels in the final filtrates were determined using the method of Lowry et al. (23) as modified by Markwell et al. (25). Once protein levels were determined, protein samples were prepared for isoelectric focusing (IEF) by addition of urea (9 M) and Resolyte 4-8 (2%).

First dimension: IEF. First-dimension IEF tube gels were prepared according to the method described in detail by Hennan and Diamond (12). Briefly, an IEF gel mixture was prepared with 10 g of urea dissolved in 6.5 ml of deionized water and 3 ml of stock acrylamide (30% total monomer, 2.6% cross-linking monomer). Once dissolved, urea was deionized by gentle mixing with 200 mg of mixed bed resin for 1 h. This solution was filtered and degassed before the addition of 1 ml of a detergent solution (0.3 g of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 900 µl of deionized water, 100 µl of Nonidet P-40), 700 µl of Resolyte 4-8, and 300 µl of ampholyte 3-10. To start polymerization, 40 µl of 10% (wt/vol) ammonium persulfate (freshly prepared), and 20 µl of N,N,N',N'-tetramethylethylenediamine were added to the mixture. Gels were allowed to polymerize for 2 h. Next, the tube adaptors were attached to the cooling core, and the apparatus was placed in the electrophoresis tank filled with 4 liters of the lower anode buffer, i.e., 10 mM degassed phosphoric acid. Protein samples were added to the top of the capillary tubes (50 µg in 20 µl), with care taken not to disturb the unpolymerized region at the top of the tube gel. Samples were subsequently overlaid with 10 µl of overlay solution (8 M urea, 1% Resolyte 4-8) and then filled to the top with upper cathode buffer, i.e., 20 mM degassed NaOH. Once all samples were added, the upper buffer chamber was filled with the cathode buffer. IEF was carried out at 10°C with a constant voltage of 100 V for 30 min, 500 V for 1 h, and 1,000 V for 17 h. Extruded tube gels were lightly coated with transfer buffer containing 5% glycerol, 0.07 M Tris · HCl (pH 6.8), and 2.5% SDS and immediately frozen on dry ice.

Second dimension: SDS-PAGE. Frozen tube gels were thawed and equilibrated for 10 min in transfer buffer containing 1% -mercaptoethanol. Full-size 20-cm SDS-polyacrylamide gels 1.5 mm wide and various percentages of total monomer were prepared on the basis of the methods of Laemmli (18) as updated by Hennan and Diamond (12). Tube gels were carefully applied to the surface of the second-dimension gels, with care taken that no air bubbles appeared between the two gel surfaces. A small volume of 1% agarose-0.1% bromphenol blue in transfer buffer was used to cement the tube gels in place and to provide a dye line during electrophoresis. Second-dimension running buffer (2.5 liters) composed of 49.5 mM Tris-base, 383.6 mM glycine, and 6.9 mM SDS (0.2%) was carefully added to the upper buffer chamber to avoid dislodging the tube gels from the surface of the second-dimension gels. All remaining running buffer was placed in the lower electrophoresis tank. Electrophoresis was carried out at 4 mA/gel for 12 h followed by 20 mA/gel for ~6 h or until the dye line reached the bottom of the gel. Temperature during the run was maintained at ~10°C.

Detection methods and quantification. After electrophoresis, gels were placed into fixative (30% ethanol, 10% acetic acid) for 3 h and subsequently stained according to the protocol supplied by Pierce for the Gelcode SilverSnap stain kit. At the end of the stain protocol, gels were soaked in a 4% glycerol solution for 1 h and then dried overnight between two sheets of cellophane. Dried gels were trimmed free of excess cellophane and placed in X-ray cassettes. Autoradiographs were taken at 70°C over a 24-h period for aorta samples and over a 48-h period for myometrium and vas deferens samples. Bio-Max MS X-ray film and Bio-Max transcreen high-energy intensifying screens were used in this process. Autoradiographs were developed using an automated Kodak M35A X-OMAT processor. The degree of phosphorylation based on densitometry of the autoradiographs was quantitatively assessed using the BioImage Visage Computer System and a Videk digital camera. Integrated intensity is the background-corrected optical density integrated over all pixels in the spot. To account for experimental variations among autoradiographs within a single set of data, a quantitative ratio of the integrated intensities between the spots of two images was calculated. This quantitative ratio was then applied as a multiplicative factor to normalize any systematic differences in general darkness that existed between the phosphorylation patterns (29). This normalization step was carried out by the BioImage software system used for our densitometry analyses.

Statistical analysis. In the PKG assays of the rat aorta, PKG activity ratios in tissues treated with SNP were compared with ratios in untreated control tissues by Student's t-test. To compare the data obtained from the cumulative dose-response curves of SNP and SNAP in the absence and presence of ODQ in rat aorta, repeated-measures one-way ANOVA was used. All further comparisons of contractile responses and cGMP levels were carried out using a one-way ANOVA, followed by Student-Newman-Keuls multiple comparison test. To analyze the densitometry obtained from our two-dimensional gel electrophoresis studies, values were converted to percentage of corresponding control for statistical analysis. Because each individual set of results contains its own controls, we were able to calculate this conversion and eliminate some of the inherent variation in densitometry that can occur from one set of autoradiographs to the next. After conversion, all densitometry results were compared by one-way ANOVA, followed by Student-Newman-Keuls multiple comparison test. Values are means ± SE, and results were considered significant when P < 0.05 for all comparisons.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Contractility and cGMP levels in rat aorta preparations. The effects of SNP (1 and 30 µM), ANP (100 nM), 8-BrcGMP (100 µM), and SNAP (1 µM and 1 mM) on PE-induced contractions (1 µM) and cGMP levels in rat aorta are shown in Fig. 1. Representative traces from these experiments are also illustrated. SNP, ANP, 8-BrcGMP, and SNAP produced a complete relaxation of PE-induced contractions at the doses listed above. SNP (1 and 30 µM), ANP (100 nM), and SNAP (1 µM and 1 mM) generated significant increases in cGMP levels in association with their relaxant response. An additional experiment with 1 µM ANP produced complete relaxation of PE-induced contractions (105.7 ± 3.1%) and a further significant increase in the level of cGMP (to 4.3 ± 0.2 pmol/mg protein, n = 3). To determine the importance of cGMP in the relaxations mediated by SNP and SNAP, the specific inhibitor of sGC, ODQ (25 µM), was added to the bath 20 min before the addition of PE. ODQ completely reversed the relaxant response induced by 1 µM SNP and partially blocked the relaxation induced by 30 µM SNP and 1 µM SNAP (Fig. 1). ODQ had no effect on the relaxant response produced by 1 mM SNAP. Figure 1 shows that ODQ completely blocked the elevation of cGMP produced by all doses of SNP and SNAP, despite the fact that 30 µM SNP, 1 µM SNAP, and 1 mM SNAP produced significant relaxations. These results indicate that cGMP is an important mediator of the relaxant responses of SNP and SNAP in the rat aorta. However, they also suggest that at least a portion of SNP- and SNAP-induced relaxation in the rat aorta may be mediated via mechanisms independent of cGMP. cGMP-independent mechanisms mediate a larger portion of the relaxant response to SNAP than to SNP.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effects of sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine {SNAP, in the absence and presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ)}, atrial natriuretic peptide (ANP), and 8-bromo-cGMP (8-BrcGMP) on phenylephrine (PE)-induced contractions and cGMP levels in rat aorta. After equilibration, aortic strips were contracted with 1 µM PE for 5 min and then washed 4 times for a further 40 min. Tension was continually adjusted to maintain a 2-g preload. Next, muscles were again contracted with 1.0 µM PE and then relaxed with SNP (1 and 30 µM), SNAP (1 µM and 1 mM), ANP (100 nM), or 8-BrcGMP (100 µM). In several experiments, ODQ (25 µM) was added to the bath 20 min before addition of SNP and SNAP. Representative traces from these experiments as well as calculated percent relaxation values and corresponding cGMP levels are shown. Values are means ± SE, and the number of experiments is indicated for each agent. Arrow, addition of drug. *Significant difference from corresponding control. #Significant difference between the absence and presence of ODQ. All comparisons were made by one-way ANOVA followed by Student-Newman-Keuls multiple comparison test.

Cumulative dose responses to SNP and SNAP in the absence and presence of ODQ. To further investigate the inability of an ODQ-mediated blockade of cGMP elevation to completely block SNP- and SNAP-induced relaxations, cumulative dose-response curves to SNP and SNAP were determined in the absence and presence of ODQ (25 µM). As shown in Fig. 2, ODQ produced a significant shift in the relaxant response induced by these cGMP-elevating agents. In Fig. 2A, ODQ markedly reduced the relaxation caused by 10 nM-1 µM SNP, although as the concentration of SNP increased above 1 µM, a large portion of the relaxant response continued to occur, despite sGC inhibition by ODQ. In Fig. 2B, ODQ only reduced SNAP-induced relaxation significantly at 100 nM SNAP. These results confirm the above conclusions that SNP- and SNAP-induced relaxations may be partially mediated by mechanisms independent of cGMP. They also indicate that SNAP-induced relaxation has a greater cGMP-independent component, making it less sensitive to sGC inhibition by ODQ.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Cumulative dose responses to SNP (A) and SNAP (B) in the absence and presence of ODQ (25 µM) on PE-induced contractions in rat aorta. After equilibration, aortic strips were contracted with 1 µM PE for 5 min and then washed 4 times for a further 40 min. Tension was continually adjusted to maintain a 2-g preload. Next, muscles were again contracted with 1.0 µM PE and then relaxed with increasing concentrations of SNP and SNAP. To investigate the effect of ODQ on the cumulative dose response, this soluble guanylate cyclase (sGC) inhibitor was added to the bath 20 min before the final PE contraction. Percent inhibition of contraction was determined by measuring the change in amplitude of the PE-induced contraction over time. Control, PE-contracted aortae were also monitored throughout the cumulative dose-response period to assess changes in the amplitude of contraction over time. In A, control, contracted muscles relaxed a total of 7.9 ± 4.2% (n = 4) over the length of the dose-response period. In B, control, contracted muscles relaxed a total of 9.8 ± 1.7% (n = 4) over the dose-response period. Values are means of 4 aortic strips from 4 different rats; error bars, SE. *Significant difference between the absence and presence of ODQ (P < 0.05). All comparisons were made using a one-way ANOVA.

Effect of SNP on PKG activity ratios in rat aorta. Total tissue levels of PKG and the effect of 30 µM SNP on PKG activity ratios were measured in rat aorta using the updated PKG assay described previously (13). Strips of rat aorta were suspended in tissue baths as described in EXPERIMENTAL PROCEDURES, allowed to equilibrate, and then contracted with 1 µM PE for a total of 7 min or contracted with PE for a total of 7 min with 30 µM SNP added for the last 3 min. As shown in Table 1, SNP produced a significant increase in the PKG activity ratio. No significant change in total PKG activity (i.e., in the presence of added cGMP) was observed between control and SNP-treated muscles. Thus the increase in the activity ratio with SNP was due to activation of specific PKG by increases in endogenous cGMP and not to an increase in nonspecific (cyclic nucleotide-independent) protein kinase activity.

Separation of proteins from intact smooth muscle using high-resolution two-dimensional gel electrophoresis. Figure 3 shows a typical silver stain of a soluble smooth muscle protein extract from an untreated, intact rat myometrial preparation. This extract was separated on an 11% total monomer second-dimension SDS-polyacrylamide gel. The sensitivity of detection for this silver stain is rated at ~0.25 ng. The molecular mass markers indicate that the proteins in this gel range from ~10 to 140 kDa. The isoelectric point (pI) ranges from ~4 to 7.8. Similar separations were also obtained in the rat aorta and rat vas deferens.

View larger version (161K): [in this window] [in a new window]   Fig. 3.   Representative 11% SDS-polyacrylamide gel of a soluble smooth muscle protein sample extracted from an intact rat myometrial preparation and separated using 2-dimensional gel electrophoresis. To determine the molecular masses of the proteins resolved, the molecular mass standards must be shifted upward by the depth of the well seen at top left. pI, isoelectric point.

Effect of SNP on protein phosphorylation in intact rat aorta smooth muscle. Representative autoradiographs showing soluble smooth muscle protein phosphorylation from untreated control tissues and from tissues treated with 1 µM PE for 7 min, 1 µM PE for 7 min plus 30 µM SNP for the last 3 min, and 25 µM ODQ for 20 min plus 1 µM PE for 7 min plus 30 µM SNP for the last 3 min are shown in Fig. 4. These gels represent the patterns of PKG-mediated phosphorylation separated on 11% second-dimension SDS-polyacrylamide gels. Additional gels of 7.5 and 15% total monomer were also used to better resolve some of the proteins of interest identified in Fig. 4 (autoradiographs not shown). Ten proteins, the phosphorylation levels of which are altered in the presence of SNP, have been identified on the autoradiographs (A-J). Corresponding molecular mass markers and pI markers have been added to indicate some of the characteristics of these proteins.

View larger version (114K): [in this window] [in a new window]   Fig. 4.   Representative autoradiographs of soluble protein phosphorylation in rat aortic smooth muscle separated on 11% 2nd-dimension SDS-polyacrylamide gels. Intact tissue strips were incubated with 32P for 3 h at 37°C and then left untreated to act as control or treated with 1 µM PE for 7 min, 1 µM PE for 7 min + 30 µM SNP for the last 3 min, and 25 µM ODQ for 20 min + 1 µM PE for 7 min + 30 µM SNP for the last 3 min. At the end of the treatment protocol, muscle strips were blotted on tissue paper and frozen between liquid nitrogen-cooled clamps. The soluble protein fraction from each tissue was separated using 2-dimensional gel electrophoresis. Gels were dried and exposed to X-ray film for 24 h. Ten proteins of interest to our study have been identified on the autoradiograph as A-J.

Effect of ANP, 8-BrcGMP, and SNAP on protein phosphorylation in intact rat aorta smooth muscle. To provide further evidence that the substrates identified in rat aorta during SNP-induced relaxation were, in fact, mediated by increases in cGMP and PKG activity, we next investigated the phosphorylation patterns induced by two other cGMP-elevating agents (ANP and SNAP) and a PKG activator (8-BrcGMP). Because ANP and 8-BrcGMP do not rely on sGC to increase cGMP and activate PKG, ODQ was not used in this group of experiments. Individual tissues were treated with 1 µM PE for 9 min plus ANP (10 µM) for the last 5 min and 1 mM 8-BrcGMP for 15 min, followed by 1 µM PE for 5 min. Autoradiographs were generated from 7.5, 11, and 15% total monomer second-dimension SDS-polyacrylamide gels. The same 10 proteins that were identified during SNP-induced relaxation showed altered phosphorylation in the presence of ANP and 8-BrcGMP (autoradiographs not shown).

Protein phosphorylation in the particulate fraction of rat aorta. In all the two-dimensional gel electrophoresis experiments in the rat aorta, we also investigated PKG-mediated phosphorylation in the particulate fraction. Despite achieving good solubilization of particulate fraction proteins, we were unable to identify any significant changes in protein phosphorylation from the autoradiographs (data not shown). Several in vitro investigations have demonstrated PKG-mediated phosphorylation of numerous membrane-bound proteins in smooth muscle (6, 39). However, we were unable to detect these phosphorylation events in the intact tissue setting.

Effects of SNAP and SNP on protein phosphorylation in intact myometrial smooth muscle. The rat myometrium can be classified as a "nonresponsive" smooth muscle, in that it does not relax in response to an elevation of cGMP and activation of PKG by agents such as SNP (8, 13). However, it does relax in the presence of SNAP through an unknown mechanism that may involve a cGMP-independent component (13). Although cGMP elevation does not appear to be responsible for SNAP-induced relaxation of rat myometrium, SNAP is an excellent elevator of cGMP in this tissue. Therefore, we utilized SNAP and SNP to investigate the patterns of PKG-mediated phosphorylation in the myometrium. In the introduction, it was proposed that if one or more of the PKG substrates identified in the aorta was absent or not phosphorylated during an elevation of cGMP and activation of PKG in the rat myometrium, that protein may be of particular importance in cGMP-mediated smooth muscle relaxation. Autoradiographs showing intact myometrial phosphorylation in a control, untreated tissue, tissues treated with 5 mM SNP for 5 min, tissues treated with 1 mM SNAP for 5 min, and tissues treated with 25 µM ODQ for 20 min, followed by 1 mM SNAP for 5 min are shown in Fig. 5. From these autoradiographs, we were unable to detect any SNAP- or SNP-induced changes in phosphorylation of the seven proteins identified as PKG substrates in the aorta. In fact, it appears as though the PKG substrates in the aorta are absent in the rat myometrium. In a comparison between control and SNAP-treated autoradiographs, four new proteins with phosphorylation levels that were altered in the presence of SNAP are identified and labeled K-N (Fig. 5). Interestingly, these increases in phosphorylation induced by SNAP (1 mM) were unaffected by an inhibition of sGC with ODQ (Fig. 5). The degree of phosphorylation of proteins K-N is quantitatively assessed in Table 5. SNAP resulted in a significant increase in ³²P incorporation into proteins K-M and also increased the phosphorylation of protein N, but because of inherent variability, the change in phosphorylation of protein N was not statistically significant. ODQ had no effect on SNAP-induced phosphorylation of proteins K-N, which remained significantly phosphorylated in the presence of the sGC inhibitor (Table 5). We previously showed that a blockade of cGMP elevation with ODQ does not block the ability of SNAP to induce relaxation (13). The apparent cGMP-independent phosphorylation of proteins K-N described above would suggest that the cGMP-independent component of SNAP-induced myometrial relaxation could involve activation of another kinase. In view of the fact that SNAP increased the phosphorylation of four new proteins in the rat myometrium, we also investigated whether these proteins were phosphorylated in the presence of SNP. As shown in Fig. 5, SNP did not produce any change in phosphorylation of proteins K-N. The quantitative assessments of phosphorylation confirm that no measurable change in phosphorylation occurred in the presence of SNP (Table 5). The patterns of phosphorylation induced by 8-BrcGMP were also investigated in the rat myometrium, but again we were unable to detect the PKG substrates identified in the aorta or changes in ³²P incorporation compared with controls (autoradiographs not shown). 8-BrcGMP did not increase the phosphorylation of proteins K-N (data not shown).

View larger version (112K): [in this window] [in a new window]   Fig. 5.   Representative autoradiographs of soluble protein phosphorylation in rat myometrial smooth muscle separated on 11% 2nd-dimension SDS-polyacrylamide gels. Intact tissue strips were incubated with 32P for 3 h at 37°C and then left untreated to act as control or treated with 5 mM SNP for 5 min, 1 mM SNAP for 5 min, or 25 µM ODQ for 20 min followed by 1 mM SNAP for 5 min. At the end of the treatment protocol, muscle strips were blotted on tissue paper and frozen between liquid nitrogen-cooled clamps. The soluble protein fraction from each tissue was separated using 2-dimensional gel electrophoresis. Gels were dried and exposed to X-ray film for 48 h. Four proteins of interest to our study have been identified on the autoradiograph as K-N.

Dissociation between dose and detection limits. Early in our analysis of PKG-mediated phosphorylation in the rat aorta, we discovered an apparent dissociation between the concentration of SNP required to cause relaxation and the concentration of SNP required to show PKG-mediated phosphorylation of the proteins identified in Table 2. At 100 nM SNP, we were unable to detect significant changes in protein phosphorylation, although the smooth muscle was ~100% relaxed (Fig. 2A). In preliminary experiments at 1 µM SNP, PKG-mediated phosphorylation was detected in the same seven proteins described in Table 4 (data not shown). However, with the variability inherent in these experiments, we were unable to show statistical significance. This apparent dissociation also occurred with ANP, 8-BrcGMP, and SNAP. Despite having to employ higher doses in our two-dimensional gel electrophoresis experiments, the apparent increase in protein phosphorylation detected with 1 µM SNP indicates that the patterns of phosphorylation are not changed by increasing the dose. In addition, the use of ODQ to confirm that the identified proteins are cGMP dependent ensures that the phosphorylation we are observing is PKG mediated and not due to nonspecific kinase activation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A detailed analysis of the literature concerning the role of cGMP in mediating vascular smooth muscle relaxation confirms that the criteria necessary to determine that cGMP is involved in this effect have been reasonably well satisfied (27). In vascular smooth muscle, it is generally accepted that the cGMP-mediated component of relaxation involves activation of a specific PKG. Activation of PKG by relaxant drugs has been reported to occur in several vascular preparations (33), and recent experiments in our own laboratory have demonstrated that relaxation of rabbit aorta by nitrovasodilators is well correlated with elevation of cGMP and activation of PKG (31). In the present study, relaxation of rat aortic strips by 30 µM SNP was accompanied by a threefold activation of PKG. In contrast to these results in vascular preparations, elevation of cGMP and activation of PKG by SNP is not accompanied by relaxation in some types of smooth muscle, including rat vas deferens (32) and rat myometrium (8, 13), leading to the classification of the latter types of muscles as nonresponsive to cGMP elevation. PKG presumably elicits an effect in vascular smooth muscle by phosphorylating specific target proteins that somehow regulate tension in the smooth muscle. As noted in the introduction, the protein targets of PKG and the underlying mechanisms by which this kinase leads to a relaxant response have not been completely elucidated. The main objective of the present study was to investigate the substrates of PKG in intact smooth muscles using two-dimensional gel electrophoresis. To assess the physiological significance of any PKG substrates identified, experiments were performed to compare PKG-mediated phosphorylation in responsive vs. nonresponsive smooth muscles to determine whether the absence of crucial PKG substrates or different abilities of PKG to phosphorylate these substrates might explain the lack of relaxation in nonresponsive smooth muscles.

The results of our two-dimensional gel electrophoresis studies provide statistical verification of PKG- mediated phosphorylation in intact smooth muscles. Previous studies have shown changes in protein phosphorylation during an elevation of cGMP, but we have taken the additional steps to quantitatively assess the degree of phosphorylation using densitometry and to use statistical analyses to compare the levels between different treatment groups. In rat aorta, we identified 10 proteins with phosphorylation levels that are significantly altered in the presence of SNP (A-J, Table 2). By comparison of muscles undergoing different treatments, two of these phosphorylation events appeared to be calcium dependent (I and J) and seven others were identified as cGMP dependent, catalyzed by PKG (A-G). The PKG substrates were confirmed as cGMP dependent on the basis of studies with the sGC inhibitor ODQ. To our knowledge, this is the first demonstration of the ability of an sGC inhibitor to prevent the activation of PKG by cGMP-elevating agents such as SNP. As described in RESULTS, the apparent decrease in phosphorylation of protein H after SNP treatment probably represents a shift in the pI of this protein, which is then detected as protein B. Protein H is endogenously phosphorylated in our control gels and appears to be phosphorylated by PKG at a separate site, which leads to the pI shift (from 6.4 to 6.3) observed in our gels. Similar shifts in pI have been described during smooth muscle myosin light chain phosphorylation (42). In that study, the pI of myosin light chain changed from ~5.1 to 5.0 during a myosin light chain kinase-mediated addition of phosphate (42). To further confirm the cGMP-dependent nature of our seven PKG substrates, we also measured PKG-mediated phosphorylation with a pGC activator (ANP), a nitrosothiol donor (SNAP), and a cGMP analog (8-BrcGMP). Each of these PKG-activating agents resulted in patterns of PKG-mediated phosphorylation similar to those found with SNP. Thus the substrates identified above appear to be phosphorylated in a cGMP-dependent manner, presumably via activation of PKG. However, it is also possible that protein kinase A may be cross-activated by the high concentrations of 8-BrcGMP used in our tissues. Further studies are required to rule out this possibility. One piece of evidence that argues against this possibility is our earlier observation that high concentrations of 8-BrcGMP failed to inhibit spontaneous contractions in rat myometrium (13). If 8-BrcGMP were activating protein kinase A, we would have expected the myometrium to relax, since it is very sensitive to the relaxant effects of cAMP-elevating agents.

As described above, the nonvascular smooth muscles of the rat myometrium and rat vas deferens do not relax in response to SNP-induced increases in cGMP and PKG activity. Our two-dimensional gel electrophoresis results provide the first evidence as to whether downstream PKG-mediated phosphorylation (or lack of phosphorylation) can be used to explain the lack of relaxation in these types of smooth muscle. In the rat myometrium, we were unable to identify any of the seven PKG substrates identified in the rat aorta. In fact, it is possible that these PKG substrates are not present in the myometrium, inasmuch as no traces of them could be detected from our gels. Endogenous protein phosphorylation was slightly lower in certain areas of the autoradiographs from myometrial preparations. Therefore, we performed preliminary experiments where 150 µg of protein from myometrial extracts were separated using two-dimensional gel electrophoresis. Normally, only 50 µg of protein were used in our experiments, but despite having triple the amount of protein in these gels, no traces of the PKG substrates were found. Endogenous phosphorylation levels in these high-protein experiments were well above those observed in the rat aorta, indicating that our lack of protein detection was not simply due to low levels of phosphorylation. Because our myometrial tissues were not precontracted before the addition of the cGMP-elevating agents above, we were also unable to show changes in the phosphorylation of the calcium-dependent proteins, I and J. However, low levels of phosphorylation of these proteins indicate that they are present in this smooth muscle. The possibility was considered that our lack of PKG substrate detection could be a result of lower total levels of PKG. However, a comparison of total PKG activities in the rat aorta and rat myometrium indicates that the kinase levels are similar in these two smooth muscles (Table 2) (13). Another possible explanation for the absence of PKG-mediated phosphorylation may reside in variations in the levels of phosphatases. If phosphatases are more concentrated in the myometrium than in the aorta, this could explain the absence of PKG-mediated phosphorylation in this nonresponsive tissue. However, we have no information on the relative amounts of phosphatases present in these tissues. Our preliminary experiments with increased protein loads tend to eliminate this possibility, since phosphorylation levels in these autoradiographs were well above those observed in the aorta. However, no detectable increases in PKG-mediated phosphorylation were observed. With the assumption that phosphorylation of one or more of the proteins we identified in the rat aorta is responsible for relaxation, the lack of relaxation in the myometrium could be attributed to the absence of such protein(s).

In our analysis of the changes in phosphorylation induced by SNAP in the myometrium, we identified four new phosphorylated proteins that were not found in the rat aorta. These phosphorylation events were not altered in the presence of ODQ, suggesting that they may occur in a cGMP-independent manner. Although the elevation of cGMP by SNAP in the myometrium is not completely reversed by ODQ, it is markedly reduced, which should result in some level of reduction in PKG-mediated phosphorylation. Because the levels of phosphorylation of these four proteins are similar to, or even further elevated, in the presence of ODQ, it seems likely that these phosphorylation events occur independently of cGMP and PKG. It has already been shown that SNAP-induced relaxation in the myometrium is unaffected by a significant blockade of cGMP elevation by ODQ (13). Thus our finding of cGMP-independent, SNAP-induced phosphorylation correlates well with these earlier results. The fact that we have detected cGMP-independent protein phosphorylation in the presence of SNAP suggests that the mechanisms of relaxation caused by this NO donor may involve activation of another kinase. However, the identity of this kinase is unknown. NO has been shown to increase the activity of p38 mitogen-activated protein kinase (5). However, cGMP and PKG were shown to be required for this effect (5). It is possible that the low level of cGMP still present in the myometrium during sGC inhibition with ODQ could allow sufficient PKG activation to permit activation of the p38 kinase. However, further investigation is required to implicate such a pathway in smooth muscle.

Rat vas deferens has also been reported to be a nonresponsive smooth muscle, in that it does not relax in response to increases in the tissue levels of cGMP and PKG activity induced by SNP (32). Interestingly, the vas deferens does relax in the presence of 8-BrcGMP (41), and one of the objectives of the present study was to determine whether this relaxant response involves activation of PKG. None of the seven proteins previously identified in the rat aorta as PKG substrates were phosphorylated during 8-BrcGMP-induced relaxation or during cGMP elevations induced by SNP. One calcium-dependent protein, possibly a myosin light chain, did show a significant decrease in phosphorylation in the presence of 8-BrcGMP, which is consistent with the fact that this agent can cause relaxation of PE-contracted rat vas deferens. Unlike the myometrium, some of the rat aorta PKG substrates appear to be present in our rat vas deferens autoradiographs. In particular, protein H is easily identified from these autoradiographs, although no change in the densitometry of this spot was observed between control and relaxed smooth muscles. Similar to our results in the rat myometrium, the overall levels of phosphorylation seem to be lower in the rat vas deferens than in the rat aorta. As a result, preliminary experiments with higher protein loads of rat vas deferens extracts were also analyzed using our two-dimensional gel electrophoresis method. Despite the higher levels of protein used, no drug-induced changes in phosphorylation were observed in these autoradiographs. However, the presence of some of the PKG substrates identified in the aorta was more clearly resolved. If the proteins identified in the rat aorta are, in fact, present in the rat vas deferens, the inability of PKG to phosphorylate these substrates in an intact tissue may be due to the fact that it cannot gain access to the compartments in which these substrates are located. However, it is also possible that one or more of the substrates are not present in the vas deferens. Further investigations would be required to differentiate between these possibilities.

As mentioned in RESULTS, a dissociation between the concentration of SNP required to cause relaxation and the concentration of SNP required to show PKG-mediated phosphorylation of proteins A-J was discovered early in our investigations of PKG-mediated phosphorylation in the rat aorta. Attempts to increase the sensitivity of detection of our method were unsuccessful, and, as a result, the majority of our two-dimensional gel electrophoresis studies were performed with doses of SNP above those required to cause relaxation. Despite having to use higher doses, we believe that our detailed analysis of protein phosphorylation with several different activators of PKG in the absence and presence of the sGC inhibitor ODQ provides a reasonable assessment of the cGMP-dependent, PKG-mediated phosphorylation in intact smooth muscle. Although it is possible that high doses of NO donors can be toxic to cells and initiate pathways independent of cGMP elevation, the short duration of incubation with these agents used in our studies and the confirmation of the PKG substrates as cGMP-dependent using ODQ eliminate the likelihood that such events contribute to the patterns of phosphorylation observed in our studies.

Although no attempts were made to clearly identify the proteins of interest found in our rat aorta experiments, the literature does provide some possible identities. The two proteins exhibiting calcium-dependent phosphorylation, I and J, have molecular mass of ~21 kDa and pI of ~5.2 and 5.3, respectively. Numerous reports investigating in vivo smooth muscle phosphorylation have identified proteins with similar molecular mass and pI that exhibit a decrease in phosphorylation during relaxation (15, 16, 19, 21, 30, 43). In the experiments of Paglin et al. (30) and Ishibashi et al. (15), these proteins were identified as myosin light chains by their comigration with appropriate standard proteins extracted from rabbit aorta and bovine trachea, respectively. In other experiments, these two proteins were identified as myosin light chains on the basis of their molecular mass and pI, as described by Silver and Stull (42). Given the similarities between our results and those reported in the studies described above, we have concluded that proteins H and I in our studies are probably myosin light chains.

The proteins identified as A and B in our experiments with molecular mass of 22,000 and 21,000 and pI of 5.8 and 6.3, respectively, may have been identified as PKG substrates previously (3, 37). Bergh et al. (3) identified two proteins with molecular mass of 20 kDa that showed mobilities in two dimensions similar to those of proteins A and B. In subsequent experiments, these proteins were identified as HSP-20. Rapoport et al. (37) also identified two proteins with molecular mass of 24,000 and pI of 6.5 and 6.2 that showed increased phosphorylation after SNP-induced relaxation. Although the molecular mass and pI are slightly different in this study, an analysis of the autoradiographs of Rapoport et al. (37) indicates that these proteins may be the same as those described above in our experiments and by Bergh et al. (3). Considering these results, it is possible that proteins A and B in our studies are HSP. If PKG does mediate the phosphorylation of HSP during intact smooth muscle relaxation, it is unknown what function these activated proteins may have. Interestingly, in the study of Bergh et al. (3), the HSP were reported to be phosphorylated in a vascular smooth muscle relaxed by SNP but not in a vascular smooth muscle that did not relax in response to SNP. It is important to note that Bergh et al. (3) were unable to demonstrate activation of PKG in either of these tissues. It has been suggested that HSP are important regulatory components of the actin-based cytoskeleton that can interact with intermediate filaments and, in turn, regulate vascular smooth muscle contraction and relaxation (1). Other HSP, such as _B-crystallin, are colocalized in the Z-band of cardiac muscle, the counterpart of which in smooth muscle is dense bodies (2). Phosphorylation of HSP in dense bodies of smooth muscle may lead to a blockade of cross-bridge attachments and, as a consequence, relaxation (11). In cell culture, Pryzwansky and colleagues (36) reported that PKG phosphorylates, and is anchored to, the soluble protein vimentin in vascular smooth muscle cells. However, we were unable to show PKG-mediated vimentin phosphorylation in the soluble protein fraction of our intact smooth muscle experiments. It is possible that PKG-mediated phosphorylation in cell culture differs significantly from that in the intact tissue setting.

Numerous studies have identified possible PKG substrates in the particulate fraction of smooth muscle. Casnellie et al. (6) demonstrated particulate fraction PKG-mediated phosphorylation of four proteins in vascular smooth muscle cell fractions treated with cGMP (250, 130, 85, and 75 kDa). In rat aorta smooth muscle cells, Sarcevic et al. (39) showed similar in vitro particulate fraction PKG-mediated phosphorylation of three proteins (225, 132, and 11 kDa) in the presence of ANP. Despite achieving good solubilization of our particulate fractions, we were unable to detect any significant changes in phosphorylation of membrane-bound proteins. The high-molecular-mass proteins described above (250 and 225 kDa) were subsequently identified as splice variants of the IP₃ receptor. More recent studies have demonstrated that the IP₃ receptor is, in fact, a substrate of PKG in intact vascular smooth muscle cells (17). Because of the high molecular mass of this protein (~260 kDa), we were unable to resolve it on our gels. As a result, we cannot eliminate the PKG-mediated phosphorylation of this protein as a possible mediator of intact smooth muscle relaxation. From these comparisons, it is clear that until substrates of PKG have been demonstrated to be phosphorylated in the intact tissue setting, their role in smooth muscle relaxation is subject to controversy.

To accompany our analysis of PKG-mediated phosphorylation in the rat aorta, contractile responses were assessed in response to treatment protocols similar to those described in our two-dimensional gel electrophoresis experiments. Because numerous recent reports have provided evidence for the presence of cGMP-independent components in NO-mediated smooth muscle relaxation (34, 35, 45), we performed a number of experiments utilizing ODQ to investigate the role of cGMP in the NO-mediated relaxation of the rat aorta. SNP, SNAP, ANP, and 8-BrcGMP produced complete relaxations of PE-induced contractions in our rat aorta experiments. Coincident with their relaxant responses, SNP, SNAP, and ANP generated significant increases in cGMP levels. ODQ completely blocked the elevation of cGMP induced by SNP and SNAP and concomitantly reversed a large portion of the relaxant responses to these agents. These data suggest that cGMP is an important mediator of the relaxant responses of SNP and SNAP in the rat aorta. However, a partial relaxation still occurred at the higher concentrations of SNP and SNAP, despite a complete blockade of cGMP elevation, indicating that at least a portion of the relaxant responses to these agents may be mediated via mechanisms independent of cGMP. Cumulative dose-response curves to SNP and SNAP (in the absence and presence of ODQ) further demonstrated the dissociation between cGMP elevation and relaxation in the rat aorta. For both agents, the dissociation becomes more apparent as the concentration of drug is increased. A comparison between the dissociations that occur with SNP and SNAP reveals that SNAP seems to have a larger cGMP-independent component to its relaxant response. Because SNAP has been reported to be a better NO donor than SNP (24), this may be the reason for its larger NO-mediated, cGMP-independent component. Nevertheless, despite the presence of an apparent cGMP-independent component in SNP- and SNAP-induced relaxation of rat aorta, cGMP is still responsible for a predominant part of the relaxant responses in this tissue. The complete relaxations induced by ANP and 8-BrcGMP in Fig. 1, which occur without the generation of NO, also support this conclusion.

In summary, PKG-mediated phosphorylation of seven proteins was found to accompany relaxation of intact rat aorta strips by cGMP-elevating agents and 8-BrcGMP. We were unable to detect phosphorylation of any of these proteins in rat myometrium and vas deferens after treatment with concentrations of SNP that markedly elevated cGMP levels and activated PKG in these tissues. The lack of PKG-mediated phosphorylation may explain the absence of relaxation seen in the latter tissues. Further experiments are required to identify which protein(s) is involved in the relaxation of vascular smooth muscle.