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J. Biol. Chem., Vol. 279, Issue 45, 46631-46636, November 5, 2004

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Regulation of cGMP-dependent Protein Kinase Expression by Soluble Guanylyl Cyclase in Vascular Smooth Muscle Cells^*

Natasha C. Browner, Nupur B. Dey, Kenneth D. Bloch¶, and Thomas M. Lincoln||

From the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294, ^¶Cardiovascular Research Center and Cardiovascular Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, and Department of Physiology, University of South Alabama, Mobile, Alabama 36688

Received for publication, July 27, 2004 , and in revised form, August 18, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase, or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e. bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the ₁ and ₁ subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 h resulted in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The nitric oxide (NO)-cGMP signaling pathway plays a critical role in vascular function (1, 2). Cyclic GMP is a second messenger that is generated by cytoplasmic soluble guanylyl cyclases (sGCs),¹ which are activated by NO, or particulate guanylyl cyclases, which are activated by peptide hormones such as atrial natriuretic peptide. sGC is a heterodimer that consists of two subunits, termed and , which are linked by disulfide bonds, and has a prosthetic heme group covalently bound to the heterodimer (1, 2). NO activates cGMP synthesis by binding to the heme, leading to the conversion of GTP to cGMP. Although two isoforms of each subunit have been cloned from rat and bovine lung, the a₁b₁ heterodimer has been found in most tissues and cell types, including vascular smooth muscle cells (VSMC) (3–6).

NO and cGMP are involved in many physiological processes such as VSMC relaxation, apoptosis, proliferation, migration, and extracellular matrix production (7–10). An important cGMP receptor protein expressed in VSMC is cGMP-dependent protein kinase (PKG), which is ultimately responsible for mediating the effects of cGMP in vascular smooth muscle relaxation (11). There are two distinct genes encoding PKG in mammalian cells, the type I and type II genes (12, 13). Type I, which is the gene product expressed in VSMC, exists as two isoforms, PKG I and PKG I, generated by alternate mRNA splicing of the type I gene (13).

There is much current interest in the regulation of gene expression by NO and cGMP (14). However, less is known about the regulation of PKG and sGC gene expression in VSMC. This is particularly important because several studies demonstrate that sGC and PKG expression are altered in VSMC in culture. Indeed, we have suggested that the loss of PKG expression in cultured rat aortic VSMC may be responsible in part at least for the phenotypic modulation of the cells from a more differentiated, contractile phenotype to a dedifferentiated, synthetic phenotype (15). Several studies show that PKG expression is decreased in VSMC after balloon catheter injury in vivo (16, 17).

Recently our laboratory demonstrated that inflammatory cytokines and lipopolysaccharide (LPS), biological modulators associated with inflammation, suppressed PKG I gene expression in VSMC through a mechanism involving the expression of inducible, type II NO synthase (iNOS). Other studies show that sGC is down-regulated in various cells in response to various stimuli (18–20). In rat pulmonary artery SMC, for instance, NO donors were found to reduce sGC subunit mRNA levels, and this effect was inhibited by the addition of a selective sGC inhibitor (20). Additionally, sGC has been shown to be down-regulated in young and aging rats that are spontaneously hypertensive (21). The down-regulation of sGC and/or PKG would be expected to impair the ability of NO to regulate VSMC functions, potentially contributing to the excessive proliferation and secretory activity of VSMC in vascular disorders.

In the current study we have examined the role of sGC activity on PKG expression in cultured VSMC. We found in several lines of VSMC expressing high levels of PKG that pro-inflammatory cytokines and NO donor compounds were unable to induce cGMP synthesis or reduce PKG expression. This lack of responsiveness was determined to be due to loss of sGC activity and protein expression. Adenovirus-mediated gene transfer of ₁ and ₁ sGC subunits followed with NO treatment decreased PKG protein expression in VSMC.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin, and gentamicin were purchased from Invitrogen. Human recombinant IL-I and TNF- were purchased from R&D systems (Minneapolis, MN). LPS was obtained from Sigma. (Z)-1-[2-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA NONOate) were obtained from Alexis Biochemicals (San Diego, CA). Human aortic vascular smooth muscle cells (HuAoSMC) were obtained from Clonetics (San Diego, CA). Rat embryonic thoracic aortic VSMC (A7r5) were purchased from American Type Tissue Culture Collection (Manassas, VA). All other reagents were purchased from Fisher or VWR Scientific (West Chester, PA).

Cell Culture—Aortas from freshly sacrificed cattle were generously provided by Kimbrell Slaughterhouse (Saraland, AL) and Richardson Meat (Tuscaloosa, AL). Bovine aortic VSMC were prepared from the anterior region below the aortic arch. Briefly, cells were propagated in DMEM containing 10% FBS, 2.5 µg/ml amphotericin B, 200 µg/ml penicillin-streptomycin, and 50 µg/ml gentamicin and grown in a 5% CO₂ incubator at 37 °C. Most experiments were conducted using passaged bovine aortic VSMC (passage 2–10). HuAoSMC were grown according to the manufacturer's specifications and protocol. A7r5 cells were cultured in DMEM containing 10% FBS and 50 µg/ml gentamicin in a 10% CO₂ incubator at 37 °C. Rat aortic VSMC were isolated and cultured as previously described (22). Rat aortic VSMC were grown in DMEM containing 5% FBS, 5% calf serum, and 50 µg/ml gentamicin in a 10% CO₂ incubator at 37 °C. The routine subculturing procedure was to remove the cells using buffered trypsin and to plate the cells at as 35,000 cells/cm² plating density. Viability was assessed after each experiment using trypan blue exclusion.

Protein Extraction and Western Blot Analysis—Cultured VSMC were extracted with 20 mM potassium phosphate, pH 6.8, 150 mM NaCl, 1% Triton X-100, 10 µM leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 10 µM pepstatin A, and 10 mM benzamidine. After centrifugation, the supernatants (10 µg) were mixed with an equal volume of 2x sample loading buffer (125 mM Tris-HCl, pH 6.9, 4% SDS, 50% glycerol, 0.02% bromphenol blue and 1.4% -mercaptoethanol). The proteins were resolved by electrophoresis in 10% SDS-polyacrylamide gels and transferred to nitrocellulose. The blots were blocked in 5% nonfat milk and probed with anti-PKG (Stressgen, Carlsbad, CA), anti-sGC (Alexis Biochemicals, San Diego, CA), or anti--actin (Sigma) antibodies as previously described (22).

Isolation of RNA and Northern Analysis—Total cellular RNA was isolated from cell monolayers using Qiagen RNeasy Mini Kit (Valencia, CA) as instructed by the manufacturer's protocol. RNA (15 µg) was resolved on 1% formaldehyde agarose gels, and Northern analysis was performed as previously described (22). Briefly, membranes were then washed with 2x SSC and 0.1% SDS at room temperature, 2x SSC and 0.1% SDS at 42 °C, and 0.1x SSC with 0.1% SDS at 65 °C, consecutively. All values were normalized to glyceraldehyde-3-phosphate dehydrogenase values, which served as the housekeeping gene for quantitative analysis. The probes for bovine iNOS and glyceraldehyde-3-phosphate dehydrogenase were generated as purified insert DNAs and labeled using the Stratagene Random Prime DNA labeling kit (La Jolla, CA).

Amplification of Adenovirus—Adenoviruses (Ad) expressing the ₁ and ₁ subunits of sGC or the catalytic domain of PKG were generated as previously described (23). Briefly, Ad₁, Ad₁, Ad-Cat, and AdLacZ are E1-deleted replication-defective adenoviruses. Ad ₁ contains the cDNA encoding the 82-kDa ₁ subunit of sGC. Ad ₁ contains the cDNA encoding the 70-kDa ₁ subunit of sGC. Ad-Cat contains the coding sequence for the PKG-1 catalytic domain (residues 336–671 of the isoform). AdLacZ carries the lacZ gene encoding a nuclear-localizing variant Escherichia coli -galactosidase. Adenovirus was amplified based on a method developed by Becker (24). All peak fractions were combined, and 0.1% bovine serum albumin was added. The virus titer was analyzed by plaque assay, and aliquots were stored at –80 °C to prevent degradation.

Adenovirus Infection of Aortic SMC—Bovine aortic VSMC (passage 2–10) were plated at a 15,000 cells/cm² density and allowed to adhere overnight. Cells were infected with AdLacZ (200 m.o.i.) or co-infected with Ad₁ and Ad₁ (m.o.i. 50–100 each) for 4 h in DMEM without serum at 37 °C. Culture medium containing 10% FBS was added to each plate, and cells were allowed to grow for 48 h. Infection efficiency was measured using the Stratagene in situ -galactosidase immunohistochemical staining kit (La Jolla, CA) and by immunostaining for the ₁ subunit of sGC. Similar to previous studies using rat aortic SMC infected with PKG-containing adenovirus (15), we found that for sGC subunit expression, an m.o.i. of 100–200 was sufficient to infect >90% of the cells. For infection of rat aortic SMC with Ad-Cat, an m.o.i. of 200 was sufficient to infect 90% of the cells.

Cyclic Nucleotide Radioimmunoassay—cGMP levels were quantified using the radioimmunoassay method as previously described (25). Briefly, cells were cultured in 35-mm dishes and treated with cytokines or NO donors. Cells were extracted with 750 µl of 0.1 N HCl, 50% methanol. The extracts were lyophilized and resuspended in 500 µl of water for determination of cGMP levels. Tubes were counted for 1 min in a Cobrell Auto Gamma counter (Packard), and data were analyzed using Assay Zap software (Biosoft). Each plate was scraped with 0.1 N NaOH and assayed for protein concentration by the Bradford method. Sample values were compared with protein concentration and expressed as pmol of cGMP per mg of protein.

Soluble Guanylyl Cyclase Activity Assay—Intracellular activity of sGC was measured using a modified method developed by Murad and co-workers (4). Cell monolayers were rinsed twice with ice-cold phosphate-buffered saline and harvested with 1.0 ml of 50 mM triethanolamine-HCl buffer, pH 7.6. Samples were homogenized, sonicated for 20 s at 50% power, and centrifuged at 14,000 x g for 30 min at 4 °C. Sample supernatants were collected, and protein concentration was determined using the Bradford assay. Each reaction contained 30 µg of sample in a final volume of 100 µl of 1.0 M Tris-HCl, pH 7.5, containing 0.5 mM isobutylmethylxanthine, and 7.5 mM creatine phosphate containing 5 units of creatine phosphokinase. Each reaction was preincubated in the absence or presence of 10 µM DEA-NONOate for 10 min at 37 °C. Assays were initiated by the addition of 4 mM MgCl₂ and 1 mM GTP. The reactions were incubated at 37 °C for 10 min and terminated by the addition of 900 µl of ice-cold 50 mM sodium acetate buffer, pH 4.0. Samples were placed on ice for 5 min. Supernatants were collected, and a cGMP radioimmunoassay was performed.

Statistical Analysis—All data are expressed as the means ± S.E. Each experiment was performed in duplicate with n = 3. Statistical differences between values were evaluated by Student's t test or one-way analysis of variance. Statistical significance was then determined by Tukey's post-test using GraphPad Prism software with significant probabilities at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Effect of Cytokines on PKG Expression—Initial studies were conducted to identify smooth muscle cell model systems for examining the relationship between PKG expression and cytokine-dependent elevations in cGMP concentrations. In primary bovine aortic SMC, cytokines, such as IL-I and TNF-, as well as LPS increased intracellular cGMP levels and decreased PKG mRNA and protein expression (25). However, similar studies performed with bovine aortic SMC that had been passaged several times (2–9 passages) yielded entirely different results. As shown in Fig. 1, treatment with IL-I, TNF-, or LPS had no significant effect on PKG protein expression in passaged bovine aortic SMC. Similar results were obtained when this experiment was conducted in HuAoSMC and A7r5 cells (not shown). To determine the reasons for the differences observed between primary and passaged bovine aortic SMC, the effects of cytokines and LPS on the NO-cGMP-signaling pathway were more closely examined. As shown in Fig. 2, TNF-, IL-I, or LPS increased iNOS mRNA expression in passaged bovine aortic SMC. The fold-increase in iNOS mRNA expression was difficult to quantify because non-treated cells express little if any measurable iNOS. Nevertheless, these findings confirmed those of other laboratories and demonstrated that the cytokine signaling pathway that is responsible for iNOS expression was present in both the primary and the passaged VSMC (25, 26).

View larger version (28K): [in this window] [in a new window]   FIG. 1. Effect of cytokines and LPS on PKG expression in VSMC. Primary and passaged bovine aortic VSMC were stimulated with IL-I (10 ng/ml), TNF- (10 ng/ml), or LPS (100 µg/ml) for 48 h, and PKG protein levels were assessed by immunoblotting. Values shown are the means ± S.E. of at least three independent experiments. *, p < 0.01 versus control.

View larger version (45K): [in this window] [in a new window]   FIG. 2. Effect of cytokines and LPS on iNOS mRNA expression. Passaged bovine aortic VSMC were stimulated with IL-I (10 ng/ml), TNF- (10 ng/ml), or LPS (100 µg/ml) for 24 h, and iNOS mRNA levels were measured by Northern blot analysis. CTL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

View larger version (15K): [in this window] [in a new window]   FIG. 3. Measurement of intracellular cGMP levels in VSMC. Primary (panel A) and passaged (panel B) bovine aortic VSMC were treated for 24 h with IL-I (10 ng/ml), TNF- (10 ng/ml), or LPS (100 µg/ml). cGMP levels were measured using radioimmunoassay. Note the difference in the scales of the ordinate in panels A and B. Data are expressed as means ± S.E. of at least three experiments. p < 0.05 versus control.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Effect of NO donors on cGMP production in VSMC. Cells were treated for 3 min with DEA NONOate (1 or 5 µM) or for 5 min with SNP (10 µM) or SNAP (100 µM), and cGMP levels were measured using radioimmunoassay. Panel A represents passaged bovine aortic SMC (passage 5), panel B represents passaged human aortic SMC (HuA-oSMC), and panel C represents passaged rat aortic SMC (passage 16–21). Note the difference in the scales of the ordinate in panels A and B with that of panel C. Data are expressed as means ± S.E. of at least four experiments. **, p < 0.05 versus control. DEA, DEA NONOate.

View larger version (29K): [in this window] [in a new window]   FIG. 5. sGC activity and protein expression in primary and passaged VSMC. A, VSMC were grown to 80% confluence, and sGC activity was assessed. B, VSMC were grown to confluence, and sGC protein expression was quantified by immunoblotting. Data are expressed as means ± S.E. of at least three experiments. **, p < 0.05 versus control. PBA, pulverized bovine aorta. RASMC, rat aortic VSMC; BASMC, bovine aortic smooth muscle cells.

View larger version (28K): [in this window] [in a new window]   FIG. 6. Restoration of sGC expression and activity in passaged VSMC. A, passaged VSMC were infected with AdLacZ (m.o.i. 200) or coinfected with Ad1 and Ad1 (m.o.i. 5 or 100 each) and grown for 48 h. sGC and levels were measured by immunoblotting. B, passaged VSMC were infected with AdLacZ (m.o.i. 200) or co-infected with Ad1 and Ad1 (m.o.i. 50–100 each) and grown for 48 h. After stimulation with or without DEA-NONOate (10 µM) for 3 min, cGMP production was determined using radioimmunoassay. Data are expressed as means ± S.E. of at least four experiments. **, p < 0.05 versus control.

View larger version (21K): [in this window] [in a new window]   FIG. 7. Effect of chronic NO delivery on PKG expression in adenovirus infected VSMC. Passaged VSMC were infected with AdLacZ (m.o.i. 200) or coinfected with Ad1 and Ad1 (m.o.i. 100 each) and grown for 48 h. Cells were stimulated with DETA NONOate (10 µM) for 48 h, and protein levels were measured by immunoblotting. Data are expressed as means ± S.E. of at least four experiments. **, p < 0.01 AdLacZ versus Ad1 and Ad1 stimulated with NO.

View larger version (27K): [in this window] [in a new window]   FIG. 8. Effect of PKG catalytic domain expression on sGC in rat aortic SMC. Rat aortic SMC (passage 21) were infected with either adenovirus encoding the LacZ-coding sequence, AdLacZ (the Control) or adenovirus encoding the catalytic domain of PKG-I, Ad-Cat, and grown for 48 h. Cells were harvested, and sGC protein expression was quantified by immunoblotting. Panel A, representative immunoblot for sGC 1 and 1 subunits, with -actin as the loading control. Panel B, densitometric analysis of three separate experiments. SGC and refer to the 1 and 1 subunits of soluble guanylyl cyclase. Con, control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Examination of sGC activity and protein expression revealed that several lines of passaged VSMC express no detectable levels of sGC. Adenovirus-mediated gene transfer of a₁ and b₁ sGC subunits restored expression and NO-dependent cyclase activity in passaged bovine aortic SMC. These results suggest that the reason for the loss in the capacity of these cell lines to respond to cytokines and NO donors with increases in cGMP is due to the loss in expression of sGC protein. A significant finding of the current study is that after restoration of sGC expression to passaged bovine aortic SMC, we could now readily down-regulate the expression of PKG in these cells by stimulating sGC (Fig. 7). In primary bovine aortic SMC that have been in culture for less than 24 h, both sGC and PKG are expressed, presumably reflecting the physiologic balance between these two proteins. However, within one passage, sGC expression is suppressed, whereas PKG expression is intact. Conversely, in rat aortic SMC where PKG expression is very low but sGC expression and activity are high, transduction of constitutively active PKG down-regulates sGC 1 subunit expression. Chen et al. (31) also found that decreased expression of the ₁ subunit of sGC was sufficient to block NO-mediated cGMP formation in SMC from aged rats. The mechanism of such suppression is not well understood but may involve suppression of sGC subunit mRNA synthesis (18–20) or destabilization of sGC mRNA. de Frutos et al. (37) found that C-type natriuretic peptide and 8-Br-cGMP decreased sGC ₁ subunit in human mesangial cells by a proteasome-dependent mechanism. Recently, Kloss, Srivastava, and Mulsch (38) have shown that activation of cAMP-dependent protein kinase (protein kinase A) inhibits the expression of mRNA-stabilizing protein, HuR, in rat aortic smooth muscle. Similar effects could be operating for the NO/cGMP-mediated down-regulation of sGC.

Thus, the current study has revealed a new dynamic balance between the expression of sGC and PKG. In culture, rat aortic SMC lose PKG expression but express very robust sGC activity and protein levels. A7r5 cells, HuAoSMC, and passaged bovine aortic SMC retain PKG expression but lose sGC expression. These findings suggest that in culture at least, VSMC may develop these adaptive mechanisms to control the NO-cGMP pathway to increase survival of the cells in culture. It is generally acknowledged that an active NO/cGMP signaling pathway can be either growth inhibitory for VSMC (28) or can promote apoptosis in VSMC (9, 29). Endogenous mechanisms that disrupt the NO/cGMP/PKG pathway in culture may allow growth and survival of cultured VSMC.

Recent evidence suggests that sGC expression is decreased during vascular dysfunction. Kloss et al. (30) found that sGC expression is decreased in old and hypertensive strains of rats and that NO-dependent vasodilation was impaired. Decreased expression of the ₁ subunit of sGC also prevented NO-mediated inhibition of DNA synthesis from VSMC from older rats (31). Overexpression of endothelial NO synthase in transgenic mice was found to decrease sGC activity (32), and sGC was found to be desensitized by NO within seconds (33). Filippov et al. reported that NO decreases sGC mRNA and protein levels in pulmonary arterial SMC by a cGMP-dependent mechanism (20). These results suggest that PKG itself may inhibit sGC expression, analogous to the effects of sGC to suppress PKG expression.

There are a number of studies using cultured VSMC to define pathways that may be involved in the expression of genes that determine specific phenotypes of the cells. In culture, for instance, rat aortic SMC rapidly lose contractile function and become highly proliferative and secretory. Coincident with this change in phenotype, there is a decrease in the expression of PKG. Restoration of PKG expression to these cells by transfection or adenoviral gene transfer causes the cells to assume a more contractile phenotype again (15). On the other hand, cultured bovine aortic SMC and HuAoSMC express abundant contractile phenotype markers such as smooth muscle myosin heavy chain. Likewise, these cells still express PKG and still have basal levels of cGMP present (although sGC expression is negligible). Thus, it is tempting to speculate that the endogenous PKG, which has been shown by Corbin and co-workers (34) to exist in a partially active state, may be responsible in part at least for the expression of contractile phenotype markers in these cells. Loss of PKG expression, on the other hand, would lead to the loss in ability of the cells to maintain contractile phenotype markers even with robust sGC activity present.

Other studies have demonstrated the importance of regulation of PKG expression in the development of vascular disease. Pfeifer et al. (35) have shown that deletion of the PKG gene in mice resulted in hypertension and increased platelet adhesion and aggregation. Other studies have shown that PKG expression is decreased after vascular injury (16, 17). Anderson et al. (17) demonstrated lowered PKG expression in balloon-injured porcine coronary arteries (17). These findings were similar to those reported by Sinnaeve et al. (16) in rat carotid arteries. Furthermore, adenovirus-mediated gene transfer of the PKG I catalytic domain reduced neointima formation in rats after balloon catheter injury (16). Because loss of sGC or PKG leads to enhanced development of vascular disease, it is possible that restoration of sGC or PKG function might effect disease progression.

Although the results of these studies are consistent with an antiproliferative, proapoptotic role for the NO/cGMP/PKG pathway in vascular SMC, a recent report by Wolfsgruber et al. (36) suggests that the NO/cGMP/PKG pathway may also be pro-proliferative for vascular SMC. Both an in vivo mouse model and in vitro studies with cultured mouse aortic SMC were used to demonstrate such a role for PKG. It is not known, however, whether the high concentration of cGMP analogs used in this study could alter expression of enzymes in this pathway, making the cells more susceptible to growth factor-induced proliferation. Because the results of this study opposes many other studies regarding the role of the NO/cGMP/PKG pathway in vascular SMC growth, more studies will need to be done to more clearly assess the real biological role of cGMP and PKG on SMC growth and gene regulation.

In summary, the aim of the current study was to examine molecular mechanism involved in the coordinated expression of sGC and PKG in VSMC. We found that pro-inflammatory cytokines and LPS decrease PKG protein expression in primary bovine aortic VSMC. However, PKG protein expression was not suppressed by cytokines or LPS in several lines of passaged VSMC. Detailed analysis of these passaged cell lines revealed that these lose expression and activity of sGC with cell passage. In contrast, rat aortic VSMC lose expression of PKG but maintain sGC activity and expression. Using adenovirus-mediated gene transfer, sGC expression was restored in passaged bovine aortic VSMC. Additionally, chronic NO treatment of sGC-transduced cells resulted in a decrease in PKG expression. These results demonstrate that there is a coordination of sGC and PKG expression in VSMC, and our findings suggest an important role in the regulation of the NO-cGMP signaling pathway in the development of survival mechanism of VSMC in vitro.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants HL53426 and HL66164. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. of Physiology, University of South Alabama, Medical Science Bldg. Rm. 3024, Mobile, AL 36688. Tel.: 251-460-6428; Fax: 251-460-6464; E-mail: tlincoln{at}usouthal.edu.

¹ The abbreviations used are: sGC, soluble guanylyl cyclase; VSMC, vascular smooth muscle cells (VSM); PKG, cGMP-dependent protein kinase; Cat, active catalytic domain of PKG-I; LPS, lipopolysaccharide; iNOS, inducible NO synthase; TNF-, tumor necrosis factor-; IL-I, interleukin I; DETA NONOate, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; DEA NONOate, 2-(N,N-diethylamino)-diazenolate-2-oxide; HuAoSMC, human aortic smooth muscle cells; A7r5, rat embryonic thoracic aortic smooth muscle cells; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; Ad, adenovirus; SNAP, S-nitroso-N-acetylpenicillamine; SNP, sodium nitroprusside; m.o.i., multiplicity of infection.

² N. C. Browner, N. B. Dey, K. D. Bloch, and T. M. Lincoln, unpublished observations.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Wong, S. K., and Garbers, D. L. (1992) J. Clin. Investig. 90, 299–305[Medline] [Order article via Infotrieve]
2. Denninger, J. W., and Marletta, M. A. (1999) Biochim. Biophys. Acta 1411, 334–350[Medline] [Order article via Infotrieve]
3. Harteneck, C., Koesling, D., Soling, A., Schultz, G., and Bohme, E. (1990) FEBS Lett. 272, 221–223[CrossRef][Medline] [Order article via Infotrieve]
4. Buechler, W. A., Nakane, M., and Murad, F. (1991) Biochem. Biophys. Res. Commun. 174, 351–357[CrossRef][Medline] [Order article via Infotrieve]
5. Nakane, M., Saheki, S., Kuno, T., Ishii, K., and Murad, F. (1988) Biochem. Biophys. Res. Commun. 157, 1139–1147[CrossRef][Medline] [Order article via Infotrieve]
6. Koesling, D., Herz, J., Gausepohl, H., Niroomand, F., Hinsch, K. D., Mulsch, A., Bohme, E., Schultz, G., and Frank, R. (1988) FEBS Lett. 239, 29–34[CrossRef][Medline] [Order article via Infotrieve]
7. Garg, U. C., and Hassid, A. (1989) J. Clin. Investig. 83, 1774–1777[Medline] [Order article via Infotrieve]
8. Sarkar, R., Meinberg, E. G., Stanley, J. C., Gordon, D., and Webb, R. C. (1996) Circ. Res. 78, 225–230[Abstract/Free Full Text]
9. Pollman, M. J., Yamada, T., Horiuchi, M., and Gibbons, G. H. (1996) Circ. Res. 79, 748–756[Abstract/Free Full Text]
10. Murad, F. (1997) Circulation 95, 1101–1103[Free Full Text]
11. Schlossmann, J., Feil, R., and Hofmann, F. (2003) Ann. Med. 35, 21–27[CrossRef][Medline] [Order article via Infotrieve]
12. Wernet, W., Flockerzi, V., and Hofmann, F. (1989) FEBS Lett. 251, 191–196[CrossRef][Medline] [Order article via Infotrieve]
13. Francis, S. H., Woodford, T. A., Wolfe, L., and Corbin, J. D. (1988) Second Messengers Phosphoproteins 12, 301–310[Medline] [Order article via Infotrieve]
14. Pilz, R. B., and Casteel, D. E. (2003) Circ. Res. 93, 1034–1046[Abstract/Free Full Text]
15. Boerth, N. J., Dey, N. B., Cornwell, T. L., and Lincoln, T. M. (1997) J. Vasc. Res. 34, 245–259[Medline] [Order article via Infotrieve]
16. Sinnaeve, P., Chiche, J. D., Gillijns, H., Van Pelt, N., Wirthlin, D., Van De Werf, F., Collen, D., Bloch, K. D., and Janssens, S. (2002) Circulation 105, 2911–2916[Abstract/Free Full Text]
17. Anderson, P. G., Boerth, N. J., Liu, M., McNamara, D. B., Cornwell, T. L., and Lincoln, T. M. (2000) Arteriosclerosis, Arterioscler. Thromb. Vasc. Biol. 20, 2192–2197[Abstract/Free Full Text]
18. Ujiie, K., Hogarth, L., Danziger, R., Drewett, J. G., Yuen, P. S., Pang, I. H., and Star, R. A. (1994) J. Pharmacol. Exp. Ther. 270, 761–767[Abstract/Free Full Text]
19. Takata, M., Filippov, G., Liu, H., Ichinose, F., Janssens, S., Bloch, D. B., and Bloch, K. D. (2001) Am. J. Physiol. Lung Cell. Mol. Physiol. 280, 272–278
20. Filippov, G., Bloch, D. B., and Bloch, K. D. (1997) J. Clin. Investig. 100, 942–948[Medline] [Order article via Infotrieve]
21. Ruetten, H., Zabel, U., Linz, W., and Schmidt, H. H. (1999) Circ. Res. 85, 534–541[Abstract/Free Full Text]
22. Dey, N. B., Boerth, N. J., Murphy-Ullrich, J. E., Chang, P. L., Prince, C. W., and Lincoln, T. M. (1998) Circ. Res. 82, 139–146[Abstract/Free Full Text]
23. Sinnaeve, P., Chiche, J. D., Nong, Z., Varenne, O., Van Pelt, N., Gillijns, H., Collen, D., Bloch, K. D., and Janssens, S. (2001) Circ. Res. 88, 103–109[Abstract/Free Full Text]
24. Becker, T. C., Noel, R. J., Coats, W. S., Gomez-Foix, A. M., Alam, T., Gerard, R. D., and Newgard, C. B. (1994) Methods Cell Biol. 43, 161–189[Medline] [Order article via Infotrieve]
25. Cornwell, T. L., Arnold, E., Boerth, N. J., and Lincoln, T. M. (1994) Am. J. Physiol. 267, 1405–1413
26. Beasley, D., and Eldridge, M. (1994) Am. J. Physiol. 266, R1197–1203[Medline] [Order article via Infotrieve]
27. Cornwell, T. L., Soff, G. A., Traynor, A. E., and Lincoln, T. M. (1994) J. Vasc. Res. 31, 330–337[Medline] [Order article via Infotrieve]
28. Jeremy, J. Y., Rowe, D., Emsley, A. M., and Newby, A. C. (1999) Cardiovasc. Res. 43, 580–594[Free Full Text]
29. Chiche, J. D., Schlutsmeyer, S. M., Bloch, D. B., de la Monte, S. M., Roberts, J. D., Jr., Filippov, G., Janssens, S. P., Rosenzweig, A., and Bloch, K. D. (1998) J. Biol. Chem. 273, 34263–34271[Abstract/Free Full Text]
30. Kloss, S., Bouloumie, A., and Mulsch, A. (2000) Hypertension 35, 43–47[Abstract/Free Full Text]
31. Chen, L., Daum, G., Fischer, J. W., Hawkins, S., Bochaton-Piallat, M. L., Gabbiani, G., and Clowes, A. W. (2000) Circ. Res. 86, 520–525[Abstract/Free Full Text]
32. Yamashita, T., Kawashima, S., Ohashi, Y., Ozaki, M., Rikitake, Y., Inoue, N., Hirata, K., Akita, H., and Yokoyama, M. (2000) Hypertension 36, 97–102[Abstract/Free Full Text]
33. Bellamy, T. C., Wood, J., Goodwin, D. A., and Garthwaite, J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2928–2933[Abstract/Free Full Text]
34. Smith, J. A., Reed, R. B., Francis, S. H., Grimes, K., and Corbin, J. D. (2000) J. Biol. Chem. 275, 154–158[Abstract/Free Full Text]
35. Pfeifer, A., Klatt, P., Massberg, S., Ny, L., Sausbier, M., Hirneiss, C., Wang, G. X., Korth, M., Aszodi, A., Andersson, K. E., Krombach, F., Mayerhofer, A., Ruth, P., Fassler, R., and Hofmann, F. (1998) EMBO J. 17, 3045–3051[CrossRef][Medline] [Order article via Infotrieve]
36. Wolfsgruber, W., Feil, S., Brummer, S., Kuppinger, O., Hofmann, F., and Feil, R. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 13519–13524[Abstract/Free Full Text]
37. de Frutos, S., Saura, M., Rivero-Vilches, F. J., Rodriguez-Puyol, D., and Rodriguez-Puyol, M. (2003) Biochim. Biophys. Acta 1643, 105–112[Medline] [Order article via Infotrieve]
38. Kloss, S., Srivastava, R., and Mulsch, A. (2004) Mol. Pharmacol. 65, 1440–1451[Abstract/Free Full Text]

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