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Endocrinology Vol. 149, No. 4 1480-1489
Copyright © 2008 by The Endocrine Society

Resistance to the Nitric Oxide/Cyclic Guanosine 5'-Monophosphate/Protein Kinase G Pathway in Vascular Smooth Muscle Cells from the Obese Zucker Rat, a Classical Animal Model of Insulin Resistance: Role of Oxidative Stress

I. Russo, P. Del Mese, G. Doronzo, L. Mattiello, M. Viretto, A. Bosia, G. Anfossi and M. Trovati

Diabetes Unit (I.R., P.D.M., G.D., L.M., M.V., G.A., M.T.), Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, 10043 Orbassano (Turin), Italy; and Department of Genetics, Biology, and Medical Chemistry (A.B.), University of Turin, 10126 Turin, Italy

Address all correspondence and requests for reprints to: Professor Mariella Trovati, M.D., Diabetes Unit, Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, 10043 Orbassano (TO), Italy. E-mail: mariella.trovati{at}unito.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Some in vivo and ex vivo studies demonstrated a resistance to the vasodilating effects of nitric oxide (NO) in insulin-resistant states and, in particular, obese Zucker rats (OZR). To evaluate the biochemical basis of this phenomenon, we aimed to identify defects of the NO/cGMP/cGMP-dependent protein kinase (PKG) pathway in cultured vascular smooth muscle cells (VSMCs) from OZR and lean Zucker rats (LZR) by measuring: 1) NO donor ability to increase cGMP in the absence and presence of inhibitors of soluble guanylate cyclase (sGC) and phosphodiesterases (PDEs); 2) NO and cGMP ability to induce, via PKG, vasodilator-stimulated phosphoprotein (VASP) phosphorylation at serine 239 and PDE5 activity; 3) protein expression of sGC, PKG, total VASP, and PDE5; 4) superoxide anion concentrations and ability of antioxidants (superoxide dismutase+catalase and amifostine) to influence the NO/cGMP/PKG pathway activation; and 5) hydrogen peroxide influence on PDE5 activity and VASP phosphorylation. VSMCs from OZR vs. LZR showed: 1) baseline cGMP concentrations higher, at least in part owing to reduced catabolism by PDEs; 2) impairment of NO donor ability to increase cGMP, even in the presence of PDE inhibitors, suggesting a defect in the NO-induced sGC activation; 3) reduction of NO and cGMP ability to activate PKG, indicated by the impaired ability to phosphorylate VASP at serine 239 and to increase PDE5 activity via PKG; 4) similar baseline protein expression of sGC, PKG, total VASP, and PDE5; and 5) higher levels of superoxide anion. Antioxidants partially prevented the defects of the NO/cGMP/PKG pathway observed in VSMCs from OZR, which were reproduced by hydrogen peroxide in VSMCs from LZR, suggesting the pivotal role of oxidative stress.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS KNOWN THAT nitric oxide (NO) exerts a major role in vascular biology, by regulating vascular tone and remodeling and influencing relevant steps of atherogenesis: in particular, it inhibits platelet adherence and aggregation; decreases leukocyte chemotaxis; promotes endothelial regeneration and angiogenesis; reduces vascular smooth muscle cell (VSMC) constriction, migration, and proliferation; and enhances VSMC apoptosis, as reviewed (1, 2, 3).

In target cells, NO activates a complex cascade of events. A pivotal role is exerted by activation of soluble guanylate cyclase (sGC), a heterodymeric enzyme formed by {alpha}- and β-subunits covalently bound to a prosthetic heme group (1). Two isoforms of the {alpha}- ({alpha}1 and {alpha}2) and of the β- (β1 and β2) subunits have been identified (1): the {alpha}1/β1 isoenzyme is the largely predominant form in vascular tissues (4). NO interacts with the heme group of sGC leading to cGMP synthesis (1). cGMP exerts its biological actions mainly by activating the cGMP-dependent protein kinase (PKG), a homodimeric serine-threonine protein kinase found in mammalian cells in three isozymic forms, i.e. I{alpha}, Iβ, and II: PKG I is the most abundant form in VSMCs and platelets (5).

The sequential activation of sGC and PKG plays a crucial role in NO action: actually, down-regulation of both enzymes impairs NO ability to modulate VSMC functions, leading to the excessive proliferation, constriction, and secretory activity observed in vascular disorders. In particular, PKG I-deficient mice show an impaired NO/cGMP-mediated VSMC relaxation both in vitro and in vivo (6, 7). The PKG-dependent effects on VSMC relaxation are mediated by phosphorylation of specific target proteins, leading to reduction of cytosolic calcium and/or calcium desensitization of contractile elements (7, 8). A substrate of PKG is the vasodilator-stimulated phosphoprotein (VASP), which PKG preferentially phosphorylates at serine 239 (9). The VASP phosphorylation at serine 239 not only is a marker of PKG activation but also mediates relevant biological actions of the NO/cGMP/PKG pathway, such as modulation of actin polymerization and cell-cell contacts (10); furthermore, in rat aorta VASP phosphorylation at serine 239 correlates with relaxation of the VSMC layer, being a reliable marker of cGMP vasodilatory activity (11).

In VSMCs, the NO/cGMP/PKG pathway is also regulated through phosphodiesterases (PDEs), enzymes catalyzing the hydrolysis of 3',5'-cyclic nucleotides to the inactive nucleoside 5'-monophosphates (12): PDE5 is the main PDE involved in cGMP catabolism in smooth muscle (13).

Thus, in VSMCs the cGMP concentrations reflect both synthesis by sGC and catabolism, predominantly by PDE5.

cGMP plays a role in its own breakdown by up-regulating PDE5 via two different mechanisms: by activating PKG, which phosphorylates PDE5, and binding to cGMP-specific binding sites in the PDE5 molecule (14). Both mechanisms are necessary because PKG does not phosphorylate PDE5 if cGMP is not bound to its specific binding sites (14). Permeable cGMP analogs, such as 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), are able to activate PKG but unable to bind the cGMP-specific sites in PDE5 molecules: for this reason, they do not activate PDE5 (14). They are able, however, to increase the PDE5 activation induced by NO donors because in this case, endogenous cGMP, increased by NO, binds the cGMP-specific sites in the PDE5 molecule, allowing PDE5 activation by PKG (14).

Owing to the wide spectrum of the NO actions in vascular biology (1, 2, 3), a reduced activation of the NO/cGMP/PKG pathway or a reduced response to it play relevant roles in hypertension, vascular remodeling, and atherosclerosis.

One of the mechanisms involved in the inactivation of the NO/cGMP/PKG pathway is oxidative stress, which interplays with not only NO ability to activate sGC (15) but also NO bioavailability (15) because superoxide anion quenches NO, transforming it into peroxynitrite (3).

Among the conditions characterized by reduced synthesis and action of NO, a relevant role is played by insulin resistance, the common soil of a cluster of metabolic, hemodynamic, thrombotic, and inflammatory features deeply involved in atherogenesis (16). Even though resistance to the vasodilating effects of NO donors in the insulin-resistant states has not been observed in many studies carried out in vivo, in type 2 diabetes, a classical condition of insulin resistance, a reduced vasodilating effect of NO donors (17, 18, 19, 20) and a positive correlation between insulin sensitivity and the vasodilating effect of NO (20) have been demonstrated. Resistance to the vasodilating action of NO donors has also been observed in another condition of human insulin resistance, the polycystic ovary syndrome (21), and in the classical animal model of insulin resistance, the obese Zucker fa/fa rats (OZR), which are homozygous for a nonfunctional leptin receptor gene and develop a phenotype similar to that of human metabolic syndrome (22).

OZR present defects in insulin signaling in vascular tissues (23) and cultured VSMCs in particular (24), an impaired insulin action on endothelial NO synthase expression in endothelial cells (25), and an impaired endothelium-dependent relaxation, as recently reviewed (22). Interestingly, an impairment of vascular responses to NO, the so-called endothelium-independent relaxation, has been demonstrated in OZR by many (26, 27, 28, 29, 30), even if not by all the studies (31, 32). As far as we know, the steps of the resistance to NO have not been investigated so far in VSMCs from OZR cultured in vitro. We previously observed that baseline NO production is similar in cultured aortic VSMCs from lean, insulin-sensitive Zucker fa/+ rats (LZR) and those from OZR (33).

The aim of the present study was to clarify whether in cultured VSMCs from OZR, there are defects at the following levels: 1) ability of NO to increase cGMP by activating sGC; 2) ability of NO/cGMP to activate VASP via PKG; and 3) ability of NO/cGMP to activate PDE5 via PKG. Finally, we aimed at evaluating whether oxidative stress plays a role in the defects of the NO/cGMP/PKG/VASP pathway in VSMCs from OZR, considering the occurrence of an increased oxidative stress in intact animals or vessels (26, 31, 34).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experiments have been carried out in cultured VSMCs from OZR and LZR. The study was approved by our institutional ethical committee, and all animal experimentation was conducted in accord with accepted standards of experimental animal use and care in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals 1996 (7th ed.; Washington, DC: National Academy Press, National Research Council Guide).

Chemicals
MEM, BSA, rabbit anti-sGC-{alpha}1 and -β1, monoclonal antirabbit IgG horseradish peroxidase-conjugate antibodies, ambroxol, 1H-[1,2,4]oxadiazolo [4,3-a]quinoxaline-1-one (ODQ), NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), 3-isobuthyl-1-methylxanthine (IBMX), zaprinast, sodium nitroprusside (SNP), 8-pCPT-cGMP, KT 5823, Rp-8pCPT-cGMP, Crotalus atrox snake venom, superoxide dismutase (SOD), catalase, amifostine, and lucigenin were purchased from Sigma-Aldrich (St. Louis, MO).

SpermineNONOate was from Alexis (San Diego, CA). Rabbit anti-VASP, mouse monoclonal anti-VASP phosphorylation at serine 239, rabbit anti-PKG-I{alpha}/β, and rabbit anti-PDE5 antibodies were from Calbiochem (La Jolla, CA).

Peroxidase-conjugated affine pure rabbit antimouse antibody was from Jackson ImmunoResearch (West Grove, PA). ECL-plus Western blotting detection system kit, 3H-cGMP, and diethylaminoethyl fast flow Sepharose columns were from Amersham Biosciences (Little Chalfont, UK).

Animals and VSMC cultures
Male OZR (n = 4) and LZR (n = 4), purchased from Charles River Laboratories (Calco, Italy), were fed with standard rodent chow and water ad libitum until 14 wk old and killed with CO2 after 12 h fasting. The aorta was isolated and processed for VSMC isolation, culture, and characterization as described (24, 33). For experiments, cells at the fourth to sixth passage were cultured until 80% confluence; then MEM with 10% fetal calf serum was removed and cells were cultured overnight in MEM containing 0.1% BSA. Fresh MEM/BSA 0.1% was added before experiments.

Intracellular cGMP measurement
cGMP was measured as previously described (35) using the RIA from ImmunoBiological Laboratories (Hamburg, Germany). Sensitivity was less than 0.3 fmol per 0.1 ml; specificity was 100% for cGMP, 0.0004% for cAMP, and 0.0001% for GMP, GDP, ATP, and GTP; and intraassay coefficient of variation was 4.4%. Results were expressed as picomoles cGMP per milligram cell proteins.

Protein expression of sGC, PKG, total VASP, and extent of VASP phosphorylation at serine 239
Protein expression of sGC {alpha}1- and β1-subunits, PKG-I{alpha}/β, total VASP, and the extent of VASP phosphorylation at serine 239 was determined by Western blots, as described (24). We used the following primary antibodies: rabbit anti-{alpha}1 sGC subunit (1:10,000), rabbit anti-β1 sGC subunit (1:8,000), rabbit anti-PKG-I{alpha}/β (1:300), antitotal VASP (1:15,000), and monoclonal anti-VASP phosphorylation at serine 239 (1:1,000). We used as secondary antibodies antirabbit (1:10,000) or antimouse (1:50,000) conjugated to horseradish peroxidase. Blots were scanned and analyzed densitometrically by the image analyzer 1D Image Analysis software (Kodak, Rochester, NY).

Expression and activity of PDE5
Expression and activity of PDE5 were measured in immunoprecipitates by using rabbit anti-PDE5 according to Wyatt et al. (36). For Western blots, immunopellet was washed four times in immunoprecipitation buffer consisting of 50 mmol/liter Tris (pH 7.5), 200 mmol/liter NaCl, 5 mmol/liter EDTA, 0.1% Triton X-100, and 0.05% sodium dodecyl sulfate and processed as described in the previous paragraph. Membranes were incubated with rabbit anti-PDE5 (1:1,000) as primary antibody and antirabbit (1:10,000) horseradish peroxidase conjugated as secondary antibody.

For PDE5 activity, immunopellets, washed without detergents, were incubated for 15 min at 30 C in a 500-µl reaction mixture containing 100 mmol/liter 3-(N-morpholino) propanesulfonic acid buffer (pH 7.5), 10 mmol/liter ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.1 mol/liter magnesium acetate, 0.9 mg/ml BSA, 20 µmol/liter cGMP, and [3H]cGMP. Samples were then boiled for 3 min and then chilled for 3 min before the addition of 10 µl of 10 mg/ml C. atrox snake venom containing 5'-nucleotidase activity. Then samples were incubated at 30 C for 10 min with an equal volume of 20 mmol/liter Tris-HCl (pH 7.5) and finally added to diethylaminoethyl-Sephacel A-25 columns. Radioactivity in the effluent was counted and results were expressed as counts per minute per milligram of protein.

Superoxide anion (O2–) measurement
Superoxide anion (O2–) levels were measured by lucigenin-enhanced chemiluminescence method based on light emission from reaction between reduced lucigenin and O2–. Briefly, VSMCs, after 24 h serum starvation, were resuspended at 5 x 105 cells/ml into a luminometer cuvette containing phosphate buffer and maintained at 37 C for 10 min. After a 5-sec dark adaptation, lucigenin (final concentration 25 µmol/liter) was added into the cuvette and chemiluminescence was recorded 3 sec after the last injection over a 2-min period at 1-sec intervals by the luminescence reader (Lumat LB9507; Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Specificity of reaction for O2– was demonstrated by preincubating cells with extracellular SOD (300 U/ml). Chemiluminescence activity unit of measure is the relative light unit and intracellular O2– levels were expressed as relative light unit per cell.

Statistical analysis
Results are expressed as means ± SEM. Statistical comparison for concentration- and time-response experiments were performed using one-way ANOVA, followed by the Bonferroni analysis. Differences between groups were evaluated by using Student’s t test. The probability value used to identify significance is P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Baseline cGMP concentrations and cGMP response to NO donors
Baseline cGMP concentrations were about 3 times higher in VSMCs from OZR than LZR (n = 6, P = 0.0001), as pictured in Fig. 1Go.


Figure 1
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  		FIG. 1. A, cGMP concentrations in VSMCs from LZR (white bars) and OZR (black bars) incubated for 60 min with the NO donors SNP (10–100 µM) and SpermineNONOate (5–50 µM). Both NO donors elicited significant effects in VSMCs from LZR (n = 6, ANOVA: P = 0.0001 for both) and OZR (n = 6, ANOVA: P = 0.017 for SNP and 0.002 for SpermineNONOate). B, cGMP concentrations in VSMCs from LZR (white bars) and OZR (black bars) incubated for 240 min with 100 µM of the NO donor SNP. SNP elicited significant effects in VSMCs from LZR (n = 6, ANOVA: P = 0.0001) and OZR (n = 6, ANOVA: P = 0.003). Significance of values in VSMCs of LZR vs. OZR: *, P = 0.01; **, P = 0.005; ***, P = 0.0001. Significance vs. the respective baselines measured by Bonferroni analysis after ANOVA: bullet, P < 0.05.

 
To evaluate the possible influence of NO on baseline cGMP, we measured cGMP concentrations after a 30-min preincubation with 1 mM of the NOS inhibitor L-NMMA, and we observed that L-NMMA did not modify baseline cGMP concentrations in VSMCs from LZR (n = 6: 0.60 ± 0.05 vs. 0.67 ± 0.07 pmol/mg protein, P = NS) and OZR (n = 6: 1.67 ± 0.08 vs. 1.83 ± 0.08 pmol/mg protein, P = NS). Thus, also in the presence of L-NMMA, baseline cGMP was higher in VSMCs from OZR (P = 0.0001).

In VSMCs from LZR, a 60-min incubation with 10–100 µM SNP and 5–50 µM SpermineNONOate dose-dependently increased cGMP (n = 6, ANOVA: P = 0.0001 for both): significant effects were observed for 50 and 100 µM SNP and for all SpermineNONOate concentrations. In VSMCs from OZR, a 60-min incubation with 10–100 µM SNP and 5–50 µM SpermineNONOate dose-dependently increased cGMP (n = 6, ANOVA: P = 0.017 for SNP and P = 0.002 for SpermineNONOate): significant effects, however, were observed only for 100 µM SNP and 50 µM SpermineNONOate (Fig. 1AGo).

SNP (100 µM) time-dependently increased cGMP in both LZR (n = 6, ANOVA: P = 0.0001) and OZR (n = 6, ANOVA: P = 0.003), as pictured in Fig. 1BGo. In LZR, significant effects were observed at all times from 5 to 240 min; in OZR significant effects were observed only at 30 and 60 min; furthermore, the SNP-induced cGMP increase was much lower in VSMCs from OZR at the different times (P = 0.002–0.0001). For instance, at 60 min, cGMP values, expressed as percent of respective baselines, were 286 ± 25 vs. 119 ± 7% in VSMCs from LZR vs. OZR, respectively (P = 0.0001).

Differences in the cGMP responses to NO were not due to different protein expression of the {alpha}1- and β1-subunits of sGC because they were similar in VSMCs from LZR and OZR (n = 6, P = NS), as pictured in Fig. 2AGo.


Figure 2
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  		FIG. 2. A, Western immunoblottings of {alpha}1 and β1-subunits of sGC in VSMCs from lean and obese Zucker rats. Blots are representative of six different experiments. B, cGMP concentrations in VSMCs from lean (white bars) and obese (black bars) Zucker rats with or without a 60-min incubation with 100 µM of the NO donor SNP, in the absence or presence of a 30-min preincubation with the sGC inhibitors ODQ (50 µM) and Ambroxol (50 µM) (n = 6). Significance of values in VSMCs of LZR vs. OZR: *, P = 0.0001. Significance vs. respective baselines: bullet, P = 0.05; bulletbullet, P = 0.02; bulletbulletbullet, P = 0.002; bulletbulletbulletbullet, P = 0.0001. Significance vs. the respective values with SNP: §, P = 0.0001.

 
A 30-min preincubation with the sGC inhibitors ODQ 50 µM and Ambroxol 50 µM did not change baseline cGMP in VSMCs from LZR but reduced baseline cGMP in VSMCs from OZR (n = 6, P = 0.002 for ODQ and P = 0.023 for Ambroxol), as pictured in Fig. 2BGo: also in the presence of these inhibitors, however, baseline cGMP remained higher in VSMCs from OZR (n = 6, P = 0.0001). sGC inhibitors, as expected, completely blunted the increase of cGMP induced by a 60-min incubation with 100 µM SNP in VSMCs from both LZR and OZR, as pictured in Fig. 2BGo.

PKG protein expression and PKG activation elicited by SNP and 8-pCPT-cGMP, evaluated as VASP phosphorylation at serine 239
We also observed that protein expression of PKG-I {alpha}/β and total VASP were similar in VSMCs from LZR and OZR (n = 6, P = NS), as pictured in Fig. 3AGo.


Figure 3
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  		FIG. 3. A, Western immunoblottings of PKG-I{alpha}/β and the total VASP in VSMCs from lean and obese Zucker rats. Blots are representative of six different experiments. B, Western immunoblottings of VASP phosphorylation at serine 239 and their densitometric analysis measured in VSMCs from lean (white bars) and obese (black bars) Zucker rats with or without a 60-min incubation with 100 µM of the NO donor SNP or 100 µM of the cGMP analog 8-pCPT-cGMP in the absence or presence of a 30-min preincubation with the PKG inhibitors KT5823 (2.5 µM) and Rp-8-pCPT-cGMP (50 µM) (n = 6). Significance of values in VSMCs of LZR vs. OZR: *, P = 0.01; **, P = 0.002. Significance vs. the respective values with SNP or 8-pCPT-cGMP: §, P = 0.0001.

 
A 60-min incubation with both SNP (100 µM) and the cell-permeable cGMP analog 8-pCPT-cGMP (100 µM) induced a significant increase of the extent of VASP phosphorylation at serine 239 in VSMCs from LZR and OZR (n = 6, P = 0.0001 for both), blunted by a 30-min preincubation with the PKG inhibitors KT 5823 (2.5 µM) and Rp-8pCPT-cGMP (50 µM): the effects of SNP and 8-pCPT-cGMP, however, were smaller in VSMCs from OZR (P = 0.002 and 0.01, respectively), as pictured in Fig. 3BGo, demonstrating that in VSMCs from OZR, the ability of the NO/cGMP pathway to phosphorylate VASP at serine 239 with a PKG-dependent mechanism is impaired. To verify the hypothesis that this impairment could be attributable to PKG desensitization by the elevated cGMP concentrations occurring in VSMCs from OZR, we reproduced this increase by a 24 h-incubation of VSMCs from LZR with the unselective PDE inhibitor IBMX (100 µM). In these experimental conditions, baseline cGMP values rose from 0.71 ± 0.02 to 2.2 ± 0.14 pmol/mg protein (P = 0.0001); the extent of VASP phosphorylation at serine 239 in response to a 60 min-incubation with 100 µM 8-pCPT-cGMP, however, was similar in the absence and in the presence of IBMX: in particular, the ratio between phosphorylated and not phosphorylated VASP was 1.4 ± 0.05 without and 1.5 ± 0.04 with IBMX (P = NS).

Role of phosphodiesterase inhibition on cGMP concentrations
We further explored the role of cGMP catabolism in the modulation of baseline and SNP-stimulated cGMP concentrations, as pictured in Fig. 4Go. For this purpose, we measured cGMP with and without a 30-min preincubation with the unselective PDE inhibitor IBMX (1 mM) and the PDE5 selective inhibitor Zaprinast (50 µM). As pictured in Fig. 4Go, A and B, IBMX increased cGMP concentrations in VSMCs from LZR and OZR (n = 17, P = 0.0001 for both). The increase, however, was smaller in OZR than LZR: actually, cGMP values with IBMX were 243 ± 132 vs. 689 ± 132% of the respective baselines (P = 0.002). Furthermore, we observed that, in the presence of IBMX, SNP increased cGMP concentrations in VSMCs from LZR (n = 17, P = 0.0001) and OZR (n = 17, P = 0.004): the increase, again, was smaller in VSMCs from OZR than LZR ({delta} values: 1.3 ± 0.42 vs. 4.2 ± 0.84 pmol/mg protein, P = 0.004). Similar results were obtained with the PDE5 selective inhibitor Zaprinast (data not shown).


Figure 4
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  		FIG. 4. cGMP concentrations in VSMCs from lean (white bars) (A) and obese (black bars) (B) Zucker rats incubated for 60 min with 100 µM of the NO donor SNP in the absence or presence of a 30-min preincubation with the phosphodiesterase inhibitor IBMX (1 mM) (n = 17). Significance vs. baseline: bullet, P = 0.05; bulletbullet, P = 0.0001; significance vs. IBMX: §, P = 0.004; §§, P = 0.0001.

 
In VSMCs from OZR, the smaller increase of baseline cGMP in the presence of IBMX indicates a defective baseline PDE activity, whereas the smaller increase of cGMP in response to SNP when cGMP catabolism was blunted by IBMX indicates a defective cGMP synthesis by sGC.

To better explore the putative defects in cGMP catabolism, we measured PDE5 activity because, as already mentioned, PDE5 is the most represented PDE in VSMCs.

PDE5 protein expression and PDE5 activity at baseline and in response to SNP without or with 8-pCPT-cGMP: role of PKG
Protein expression of PDE5 was similar in VSMCs from LZR and OZR (n = 3, P = NS), as pictured in Fig. 5AGo: baseline PDE5 activity, however, was lower in VSMCs from OZR than those from LZR (n = 7, P = 0.0001), as pictured in Fig. 5BGo.


Figure 5
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  		FIG. 5. A, Western immunoblottings of PDE5 in VSMCs from lean and obese Zucker rats. Blots are representative of three different experiments. B, PDE5 activity measured in VSMCs from lean (white bars) and obese (black bars) Zucker rats, with or without a 60-min incubation with 100 µM of the NO donor SNP or 100 µM of the cGMP analog 8-pCPT-cGMP in the absence or presence of a 30-min preincubation with the PKG inhibitor KT5823 (1 µM) (n = 7). Significance of values in VSMCs of LZR vs. OZR: *, P = 0.0001. Significance vs. baseline: bullet, P = 0.0001; Significance vs. SNP: §, P = 0.01; §§, P = 0.0001. Significance vs. SNP + 8-pCPT-cGMP: #, P = 0.0001.

 
PDE5 activity was increased in response to a 60-min incubation with 100 µM SNP in VSMCs from both LZR and OZR (P = 0.0001 for both): the increase, however, was greater in VSMCs from LZR (P = 0.0001), as pictured in Fig. 5BGo. The SNP-induced PDE5 activation was mediated by PKG, being completely abolished by a 30-min preincubation with 1 µM of the PKG inhibitor KT5823. Similarly, when 100 µM SNP were coincubated for 60 min with 100 µM 8-p-CPT-cGMP, we observed a further increase of the SNP-induced PDE5 activation in VSMCs from LZR (n = 7, P = 0.0001) and OZR (P = 7, P = 0.01): the increase, however, was much smaller in OZR (P = 0.0001). Also, this enhancement was PKG mediated, being blunted by the PKG inhibitor.

Role of oxidative stress in the activation of the NO/cGMP/PKG/VASP pathway
VSMCs from OZR showed higher baseline concentrations of superoxide anion than VSMCs from LZR (n = 10, P = 0.0001), as pictured in Fig. 6AGo. As expected, a 90-min preincubation with 300 U/ml SOD decreased the elevated superoxide anion values measured in VSMCs from OZR from 626.4 ± 10.5 to 362.5 ± 33.97 relative light units x cell (P = 0.0001).


Figure 6
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  		FIG. 6. A, Superoxide anion concentrations in VSMCs from lean (white bars) and obese (black bars) Zucker rats (n = 10). B and C, cGMP concentrations in VSMCs from lean (white bars) and obese (black bars) Zucker rats incubated for 60 min with 100 µM of the NO donor SNP in the absence or presence of a 30-min preincubation with the antioxidant mixture SOD (300 U/ml) and catalase (250 U/ml) (n = 6) and amifostine (50 µM) (n = 6). Significance of values in VSMCs of LZR vs. OZR: *, P = 0.0001. Significance vs. baseline: bullet, P = 0.05; bulletbullet, P = 0.0001. Significance vs. SOD/catalase or amifostine: §, P = 0.0001.

 
To assess the role of oxidative stress in the abnormalities observed in VSMCs from OZR, we designed experiments with antioxidants and, as pictured in Fig. 6Go, B and C, we observed that a 30-min preincubation with the antioxidant mixture SOD (300 U/ml)/catalase (250 U/ml) and the reactive oxygen species scavenger amifostine (50 µM) decreased baseline cGMP in VSMCs from OZR (n = 6, P = 0.0001) but not LZR (n = 6, P = NS) and increased the cGMP response to SNP in VSMCs from OZR (n = 6, P = 0.0001) but not LZR (n = 6, P = NS). In particular, cGMP values after SNP incubation without and with SOD/catalase were 276 ± 12 vs. 300 ± 26% of baseline, respectively, in VSMCs from LZR (n = 6, P = NS) and 116 ± 21 vs. 190 ± 17%, respectively, in VSMCs from OZR (n = 6, P = 0.02). Similar results have been obtained with amifostine. Antioxidants therefore partially reversed the increase of baseline cGMP concentrations and the impairment of cGMP response to SNP observed in OZR.

Furthermore, as pictured in Fig. 7Go, incubation with SOD/catalase and amifostine increased phosphorylation of VASP at serine 239 in response to both SNP (n = 6, P = 0.0001) and 8-pCPT-cGMP (n = 6, P = 0.0001) in VSMCs from OZR but not LZR (n = 6, P = NS). Antioxidants therefore restored the impairment of VASP phosphorylation at serine 239 in response to the NO/cGMP pathway observed in OZR.


Figure 7
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  		FIG. 7. Western immunoblottings of total VASP and VASP phosphorylation at serine 239 (pVASP; representative of six different blots) and their densitometric analysis measured in VSMCs from lean (white bars) and obese (black bars) Zucker rats with or without a 60-min incubation with 100 µM of the NO donor SNP or 100 µM of the cGMP analog 8-pCPT-cGMP in the absence or presence of a 30-min preincubation with the antioxidant mixture SOD (300 U/ml) and catalase (250 U/ml) (n = 6) and with amifostine (50 µM) (n = 6). Significance of values in VSMCs of LZR vs. OZR: *, P = 0.0001. Significance vs. SNP or 8-pCPT-cGMP: §, P = 0.0001.

 
To better clarify the role of oxidative stress, we tried to reproduce in VSMCs from LZR the abnormalities observed in OZR by incubating them with hydrogen peroxide (H2O2).

In particular, to further explore the influence of oxidative stress on baseline cGMP, we incubated VSMCs from LZR with 0.5 and 5 µM H2O2 for 24 h, and we observed that H2O2 dose-dependently increased cGMP (n = 4, ANOVA, P = 0.0001), with significant effects for both concentrations, as pictured in Fig. 8AGo.


Figure 8
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  		FIG. 8. A, cGMP values measured in VSMCs from lean Zucker rats without or with a 24-h incubation with hydrogen peroxide (H2O2; 0.5 and 5 µM) (n = 4). Hydrogen peroxide induced a significant increase of cGMP values (ANOVA, P = 0.0001). B, PDE5 activity measured in VSMCs from lean Zucker rats in response to a 60-min coincubation with 100 µM of the NO donor SNP and 100 µM of the cGMP analog 8-pCPT-cGMP without or with a 24-h incubation with hydrogen peroxide (5 µM) (n = 3). C, Extent of VASP phosphorylation at serine 239 (pVASP) measured in VSMCs from lean Zucker rats in response to a 60-min incubation with 100 µM of the cGMP analog 8-pCPT-cGMP without or with a 24-h incubation with hydrogen peroxide (0.5 and 5 µM) (n = 6). Hydrogen peroxide induced a significant decrease of VASP phosphorylation (ANOVA, P = 0.0001). Significance vs. baseline measured with the Bonferroni analysis after ANOVA in A: bullet, P = 0.05. Significance vs. baseline in B: bulletbullet, P = 0.0001. Significance vs. SNP + 8-pCPT-cGMP in B: §, P = 0.0001. Significance vs. 8-pCPT-cGMP measured with the Bonferroni analysis after ANOVA in C: #, P = 0.05.

 
To evaluate the role of oxidative stress on cGMP catabolism, we measured baseline PDE5 activity in VSMCs from LZR incubated for 24 h with 5 µM H2O2, and we observed, as pictured in Fig. 8BGo, that H2O2 inhibited PDE5 activity (n = 3, P = 0.0001); furthermore, a 24-h preincubation with 5 µM H2O2 blunted the increase of PDE5 activity induced by a 60-min coincubation with 100 µM SNP and 100 µM 8-pCPT-cGMP.

To further evaluate the role of oxidative stress on PKG activation in response to cGMP, we incubated VSMCs from LZR for 24 h with 0.5 and 5 µM H2O2, and we observed a dose-dependent inhibition of the VASP phosphorylation at serine 239 induced by a 60-min incubation with 100 µM 8-pCPT-cGMP (n = 6, ANOVA P = 0.0001, with significant effects exerted by the two H2O2 concentrations).

Taken together, the results of experiments carried out with H2O2 confirm that oxidative stress is involved in the defects concerning baseline cGMP concentrations and activity of both PDE5 and PKG.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that VSMCs from OZR show multiple defects in the NO/cGMP/PKG pathway in comparison with VSMCs from LZR, partially reversible by antioxidants.

The first phenomenon we observed was the strikingly higher baseline cGMP concentrations in VSMCs from OZR, reverted by antioxidants. We tried to clarify the mechanisms involved, and we concluded that a decreased cGMP catabolism certainly plays a relevant role. In particular, we concluded: 1) experiments carried out with the NO synthase inhibitor L-NMMA ruled out the hypothesis of a sGC hyperactivation in response to an increased production of endogenous NO, a phenomenon that, on the other hand, we already previously excluded (33); 2) baseline cGMP concentrations remained higher in VSMCs from OZR also in the presence of effective sGC inhibition, suggesting the occurrence of a defective cGMP catabolism; and 3) baseline PDE5 activity was lower in VSMCs from OZR than those from LZR, and the IBMX-induced increase of cGMP was smaller in VSMCs from OZR than those from LZR, suggesting a defective PDE activity in VSMCs from OZR, in agreement with the demonstration of a similar defect in soleus skeletal muscle from the same rats (37). We also observed that oxidative stress is deeply involved in the increased baseline cGMP values in VSMCs from OZR because in these cells superoxide anion concentrations were elevated and antioxidants reduced baseline cGMP concentrations. When we induced oxidative stress by hydrogen peroxide in VSMCs from LZR, we observed both an increase of cGMP concentrations and an inhibition of PDE5 activity. However, even if a defective PDE5 activity, at least in part attributable to oxidative stress, seems to play a relevant role in the increased baseline cGMP concentrations in VSMCs from OZR, we cannot exclude the simultaneous occurrence of an increased baseline sGC activity: actually, hydrogen peroxide has been described to activate sGC in vascular smooth muscle (38, 39). Interestingly, in our experimental conditions, sGC inhibitors did not reduce baseline cGMP concentrations in VSMCs from LZR, in agreement with observations carried out by other researchers in rat aortic vascular smooth muscle (40) but reduced baseline cGMP concentrations in VSMCs from OZR, thus diminishing, without blunting, the difference in baseline cGMP concentrations between VSMCs from LZR and OZR. Thus, it is likely that oxidative stress contributes to the increased baseline cGMP concentrations in VSMCs from OZR by simultaneously inducing a decrease of PDE activity and an increase of sGC activity.

A second phenomenon we observed was the deep impairment of the NO donor ability to increase cGMP concentrations in VSMCs from OZR, partially reverted by antioxidants. Because no difference has been observed in sGC subunit protein expression between VSMCs from OZR and LZR, we ruled out the hypothesis that a putative reduction of sGC activity could be due to reduction of sGC content. Therefore, because cGMP concentrations in response to NO depend on the balance between synthesis by sGC and catabolism by PDEs (1, 14), and in VSMCs from OZR, the NO ability to increase PDE5 activity is deeply impaired, a phenomenon that per se would increase cGMP levels, the observation that in the same cells, the cGMP enhancement in response to SNP is impaired suggests a defective ability of NO to activate sGC, in agreement with the demonstration of a similar defect in soleus muscle of the same rats (37). This phenomenon could be attributed, at least in part, to a reduced bioavailability of NO, quenched by the increased concentrations of superoxide anion (3).

A third phenomenon we observed was the reduced ability of NO and cGMP to activate PKG in VSMCs from OZR. The results obtained with the cGMP analog are very important because those obtained with SNP could also be interpreted on the basis of a reduced NO bioavailability due to the high superoxide anion levels.

PKG activation in response to SNP or the cGMP analog was studied by measuring VASP phosphorylation at serine 239 and demonstrating that it is blunted by two different PKG inhibitors: i.e. by not only KT5823, the use of which in intact cells have been questioned (41), but also Rp-8-pCPT-cGMP. We tried to investigate the mechanisms involved in the reduced PKG activation in VSMCs from OZR, and we concluded that an increase of oxidative stress is the more convincing explanation. Actually, two different antioxidants reverted the impairment of VASP phosphorylation in response to NO and cGMP in VSMCs from OZR, whereas induction of oxidative stress in VSMCs from LZR by hydrogen peroxide deeply inhibited the extent of the cGMP-induced VASP phosphorylation. The hypothesis that the high cGMP concentrations observed in VSMCs from OZR can induce PKG desensitization has not been confirmed because the extent of VASP phosphorylation in response to 8-pCPT-cGMP was not modified in VSMCs from LZR in which a 24-h incubation with low IBMX concentrations reproduced the elevated baseline cGMP concentrations observed in VSMCs from OZR. Finally, PKG and total VASP expression were similar in VSMCs from OZR and LZR, ruling out the hypothesis that the defective activation of PKG was due to a reduced protein content of PKG itself or its target molecule VASP.

A further demonstration of the reduced ability of NO and cGMP to activate PKG in VSMCs from OZR is the impaired PKG-dependent PDE5 activation in response to SNP and 8-pCPT-cGMP, in the presence of a normal PDE5 protein expression. Actually, as we mentioned in the introductory text, PDE5 is a target of the NO/cGMP/PKG pathway because it is activated by cGMP by a mechanism involving both cGMP binding to specific sites and PKG activation (14): thus, the lower baseline PDE5 activity in VSMCs from OZR in the presence of increased baseline cGMP concentrations suggests the inability of cGMP to exert one of its biological actions. This phenomenon has been further confirmed by the deep impairment of PDE5 activation in response to the NO/cGMP pathway.

Also in this case oxidative stress plays a pivotal role: actually, incubation of VSMCs from LZR with hydrogen peroxide reduced both baseline PDE5 activity and the increase of PDE5 activity in response to SNP and 8-pCPT-cGMP.

The impairment of cGMP-induced increase of VASP phosphorylation and PDE5 activity observed in VSMCs from LZR incubated with hydrogen peroxide demonstrate that oxidative stress reduces the PKG activation in response to cGMP.

Thus, in VSMCs from OZR, oxidative stress plays a role both in the enhanced baseline cGMP concentrations and the reduced ability of NO to activate the cGMP/PKG cascade, involving both VASP phosphorylation and PDE5 activation.

The comparison of the results obtained in our investigation with literature studies allows some considerations.

First of all, the increased superoxide anion concentrations we found in VSMCs from OZR cultured in vitro are in agreement with the increased oxidative stress described in these animals both in vivo and ex vivo (26, 31, 34).

Furthermore, our observation that antioxidants do not modify the NO/cGMP/PKG/VASP pathway in VSMCs from LZR but restored the defects of the same pathway in VSMCs from OZR agrees with the observation that reactive oxygen species scavengers increase dilator responses of arterioles to SNP in OZR but not LZR (27).

Finally, the demonstration that the increased oxidative stress occurring in VSMCs from OZR plays a role in the decreased NO ability to activate the cGMP/PKG/VASP system agrees with the observation that treatment of rat aortic rings with the antioxidant vitamin C in a condition of increased oxidative stress improves relaxation and increases VASP phosphorylation in response to a NO donor (42).

The multiple defects of the NO/cGMP/PKG pathway activation in VSMCs from OZR we observed in this in vitro study could at least in part account for the impairment of endothelial-independent vasodilation observed in the majority of studies addressing in these animals the responses to NO donors both in vivo and in isolated vessels (26, 27, 28, 29, 30). They may also account for the impairment of endothelial-dependent vasodilation (22) because defects in NO action amplify the consequences of defects in NO production.

But the effects of NO in VSMCs are not confined to vasorelaxation, also implying inhibition of migration and proliferation, enhancement of apoptosis, and angiogenesis (1, 2, 3); thus, the defects observed in this study could have deep implications in vascular biology, accounting for the increased atherogenetic risk observed in the insulin-resistant states.

An intriguing question is whether the multiple abnormalities of the NO/cGMP/PKG pathway described in VSMCs cultured in vitro demonstrate that VSMCs from OZR present intrinsic defects independently of circulating factors increased in vivo as a consequence of obesity, i.e. free fatty acids, cytokines, adipokines, and hyperinsulinemia compensatory to insulin resistance (16) or whether they are due to the influence of in vivo environment somehow retained, for a kind of memory, in the early passages of primary cell cultures. On the other hand, it has been described that cultured VSMCs, even within one passage, present some adaptive modifications concerning the sGC/PKG pathway with respect to VSMCs that have been in culture for less than 24 h (43), further supporting the well-known concept that results obtained in cultured cells are influenced by the culture condition itself.

In any case, the present study shows that increased oxidative stress characterizes cultured VSMCs from the insulin-resistant OZR, which also show well-defined alterations of insulin signaling (24), underlining the close interrelationships between oxidative stress and insulin resistance (44). Finally, because OZR are an interesting animal model of the metabolic syndrome (22), the occurrence of similar alterations of the NO/cGMP/PKG pathway in cultured VSMCs from OZR and platelets from insulin-resistant subjects affected by central obesity (45, 46, 47, 48) suggests that insulin resistance is characterized by defects in the NO/cGMP/PKG pathway in different cell types, which could represent another piece of the insulin resistance syndrome.


   Footnotes
 
First Published Online December 13, 2007

Abbreviations: IBMX, 3-Isobuthyl-1-methylxanthine; L-NMMA, NG-monomethyl-L-arginine; LZR, lean Zucker rats; NO, nitric oxide; ODQ, 1H-[1,2,4]oxadiazolo [4,3-a]quinoxaline-1-one; OZR, obese Zucker rats; 8-pCPT-cGMP, 8-(4-chlorophenylthio)-cGMP; PDE, phosphodiesterase; PKG, cGMP-dependent protein kinase; sGC, soluble guanylate cyclase; SOD, superoxide dismutase; SNP, sodium nitroprusside; VASP, vasodilator-stimulated phosphoprotein; VSMC, vascular smooth muscle cell.

Received July 6, 2007.

Accepted for publication December 6, 2007.


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 Introduction
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 Results
 Discussion
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