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Endocrinology Vol. 147, No. 12 5914-5920
Copyright © 2006 by The Endocrine Society

Activation of Endothelial Nitric Oxide Synthase by the Angiotensin II Type 1 Receptor

Hiroyuki Suzuki, Kunie Eguchi, Haruhiko Ohtsu, Sadaharu Higuchi, Sudhir Dhobale, Gerald D. Frank, Evangeline D. Motley and Satoru Eguchi

Cardiovascular Research Center and Department of Physiology (H.S., K.E., H.O., S.H., S.D., S.E.), Temple University School of Medicine, Philadelphia, Pennsylvania 19140; Department of Biochemistry (G.D.F.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and Department of Biomedical Sciences (E.D.M.), Meharry Medical College, Nashville, Tennessee 37208

Address all correspondence and requests for reprints to: Satoru Eguchi, M.D., Ph.D., FAHA, Cardiovascular Research Center and Department of Physiology, Temple University School of Medicine, 3420 North Broad Street, Philadelphia, Pennsylvania 19140. E-mail: seguchi{at}temple.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Enhanced angiotensin II (AngII) action has been implicated in endothelial dysfunction that is characterized as decreased nitric oxide availability. Although endothelial cells have been reported to express AngII type 1 (AT1) receptors, the exact role of AT1 in regulating endothelial NO synthase (eNOS) activity remains unclear. We investigated the possible regulation of eNOS through AT1 in bovine aortic endothelial cells (BAECs) and its functional significance in rat aortic vascular smooth muscle cells (VSMCs). In BAECs infected with adenovirus encoding AT1 and in VSMCs infected with adenovirus encoding eNOS, AngII rapidly stimulated phosphorylation of eNOS at Ser1179. This was accompanied with increased cGMP production. These effects were blocked by an AT1 antagonist. The cGMP production was abolished by a NOS inhibitor as well. To explore the importance of eNOS phosphorylation, VSMCs were also infected with adenovirus encoding S1179A-eNOS. AngII did not stimulate cGMP production in VSMCs expressing S1179A. However, S1179A was able to enhance basal NO production as confirmed with cGMP production and enhanced vasodilator-stimulated phosphoprotein phosphorylation. Interestingly, S1179A prevented the hypertrophic response similar to wild type in VSMCs. From these data, we conclude that the AngII/AT1 system positively couples to eNOS via Ser1179 phosphorylation in ECs and VSMCs if eNOS and AT1 coexist. However, basal level NO production may be sufficient for prevention of AngII-induced hypertrophy by eNOS expression. These data demonstrate a novel molecular mechanism of eNOS regulation and function and thus provide useful information for eNOS gene therapy under endothelial dysfunction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACCUMULATING EVIDENCE INDICATES that angiotensin II (AngII) has a critical role in the progression of vascular remodeling associated with hypertension, atherosclerosis, and restenosis after balloon injury. Thus, AngII stimulates growth and migration of vascular smooth muscle cells (VSMCs) through the G-protein-coupled AngII type 1 (AT1) receptor, and the AT1 receptor is responsible for most of the pathological effects of AngII (1, 2). In cardiovascular diseases, enhanced AngII/AT1 actions are frequently associated with endothelial dysfunction that is characterized by a reduced nitric oxide bioavailability (3). In this regard, not only vasoconstriction induced by AngII but also other pathological effects of AngII including VSMC growth and migration have been demonstrated to be blocked by NO in vitro (4, 5, 6). In vivo, endothelial NO synthase (eNOS) gene transfer reduces AngII-induced endothelial dysfunction (7). Moreover, eNOS gene transfer protected against atherosclerosis and postangioplasty restenosis (8, 9, 10, 11). Thus, NO could exert antagonistic effects against AngII/AT1 in cardiovascular diseases.

In addition to a well established mechanism of eNOS activation through Ca2+ and calmodulin (CaM) that is typically induced by several agonists targeting G-protein-coupled receptors (GPCRs), eNOS activity can be markedly enhanced by its phosphorylation at Ser1179/1177(bovine/human) (12). It has been demonstrated that the eNOS phosphorylation can be induced by several non-GPCR agonists as well as some GPCR agonists in particular, such as bradykinin (13). Interestingly, AngII has been shown to stimulate release of NO in cultured endothelial cells (14, 15) and several arteries in vivo (16, 17) through the AT1 receptors. Similar to that of VSMCs, endothelial AT1 seems to be coupled to Gq and thereby activates eNOS through a Ca2+/CaM-dependent mechanism (14, 15). However, detailed mechanisms of eNOS regulation by the AT1 receptor remain obscure partly because of the lack of stable AT1 receptor expression in cultured endothelial cells (18, 19).

In the present study, we hypothesized that AT1 positively couples to eNOS through Ser1179/1177 phosphorylation and thereby counteracts endothelial dysfunction and vascular remodeling induced by AngII. To test the hypothesis, bovine aortic endothelial cells (BAECs) that do not express endogenous AT1 receptors were infected with adenovirus encoding AT1, and AngII-induced eNOS phosphorylation and activation were investigated. Also, in AngII-stimulated rat aortic VSMCs expressing the endogenous AT1 receptor and infected with adenovirus encoding eNOS, we investigated eNOS phosphorylation, activation, and prevention of hypertrophy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
AngII was purchased from Sigma Chemical Co. (St. Louis, MO). N-Nitro-L-arginine methyl ester (L-NAME) was purchased from Calbiochem (La Jolla, CA). A selective AT1 receptor antagonist, RNH6270, and a selective Gq inhibitor, YM-254890 (20), were gifts from Sankyo Co., Ltd. (Tokyo, Japan) and Astellas Pharma Inc. (Tokyo, Japan), respectively. Antibodies raised against Ser1179/1177 (bovine/human)-phosphorylated eNOS and Thr497/495 (bovine/human)-phosphorylated eNOS were purchased from Cell Signaling Technology (Beverly, MA). The antibody used to detect total eNOS was purchased from BD Transduction Laboratories (Lexington, KY). Antibody raised against Ser157-phosphorylated vasodilator-stimulated phosphoprotein (VASP), Ser239-phosphorylated VASP, and total VASP were purchased from Calbiochem, Upstate Biotechnology (Lake Placid, NY), and Axxora, LLC (San Diego, CA), respectively. ERK2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell culture
Isolation and characterization of rat aortic VSMCs in culture were described previously (21). BAECs were purchased from Cambrex (Baltimore, MD). BAECs were chosen as a model system because they are one of the most frequently used cells to characterize regulation of eNOS phosphorylation by several GPCR agonists (22, 23, 24). Cells were subcultured in DMEM containing 10% fetal bovine serum, penicillin, and streptomycin as previously described (21). Cells at passages 3–12 at approximately 80% confluence in culture wells were made quiescent by incubation with serum-free medium for 2 d before the experiments.

Adenoviral Infection
Generation and characterization of replication-deficient adenovirus encoding rat AT1A (kindly provided by Dr. Junichi Sadoshima), bovine eNOS, and bovine S1179A-eNOS mutant (kindly provided by Dr. William Sessa) were described previously (25, 26). Each adenovirus titer [multiplicity of infection (MOI)] was determined by Adeno-X Rapid Titer Kit (BD Biosciences, San Diego, CA). VSMCs and BAECs were infected with adenovirus for 2 d as previously described (27). The infection efficiency was estimated to be 90–100% as defined by infection with adenovirus (25–100 MOI) encoding green fluorescent protein.

Immunoblotting
Cell lysates were subjected to SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane as previously described (28). The membranes were then exposed to primary antibodies overnight at 4 C. After incubation with the peroxidase-linked secondary antibody for 1 h at room temperature, immunoreactive proteins were visualized by a chemiluminescence reaction kit (Chemicon, Temecula, CA). The results were quantified by densitometry in the linear range of film exposure using CanoScan N670U (Canon, Lake Success, NY) and Un-Scan-It Gel 4.3 software (Silk Scientific, Orem, Utah). Results were expressed as percent increase in which the response to AngII is defined as 100% because the basal signals are more varied depending on film exposure than the stimulated signals.

Intracellular cGMP assay
BAECs were incubated with agonists at 37 C for 20 min in the presence of 0.5 mM methylisobutylxanthine (29), and intracellular cGMP was determined by an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI).

Protein assay
Protein assay to estimate protein synthesis induced by AngII was performed as previously described (30). Subconfluent VSMCs on 12-well culture plates were incubated with serum-free DMEM for 1 d and were infected with adenovirus encoding control empty vector or eNOS for 2 d. Adenovirus-infected cells were further incubated with or without 100 nM AngII (applied just once) for 3 d. After aspiration of the medium, cells were washed twice by ice-cold Hanks’ balanced salt solution, and total amount of cellular protein was measured using a BCA protein assay kit (Pierce, Rockford, IL) before and after the AngII stimulation.

Cell volume measurement
After the pretreatments described in the protein assay, quiescent VSMCs in six-well plates were stimulated with 100 nM AngII (applied just once) for 3 d. The cells were washed with Hanks’ balanced salt solution and trypsinized. The cells were then suspended in PBS, and cell volume was measured by a Coulter counter (Beckman Coulter, Fullerton, CA) (31). The data were analyzed with Coulter Z2 AccuComp (R).

Living cell number assay
Living cell number was measured using a CellTiter 96 AQueous cell proliferation assay kit (Promega, Madison, WI) following the manufacturer’s protocol as previously described (30). In brief, after the pretreatments described in the protein assay, quiescent VSMCs in 96-well plates were stimulated with 100 nM AngII (applied just once) for 3 d. After incubation with MTS solution provided by the kit, viable cells were determined at 490-nm absorbance with a 96-well plate reader.

Statistical analysis of data
Unless stated otherwise, results are representative of at least three separate experiments giving similar results. Densitometry data were analyzed using the Student’s t test. The mean ± SEM was measured with a significance level of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Endogenous eNOS activation via AT1 in BAECs
To examine endogenous eNOS activation via the AT1 receptor, BAECs were infected with the adenovirus encoding AT1 or control empty vector and stimulated with 100 nM AngII. In BAECs expressing the AT1 but not in control cells, AngII stimulated eNOS phosphorylation at Ser1179. The phosphorylation started from 1 min after stimulation and decreased thereafter but was still significant at 5 min (Fig. 1AGo). AngII did not alter eNOS phosphorylation at Thr497, a negative regulatory site, during these time periods (data not shown). The eNOS Ser1179 phosphorylation by AngII was completely abolished by an AT1 receptor blocker (ARB) (Fig. 1BGo) as well as by a Gq inhibitor (Fig. 1CGo), confirming that eNOS activation was mediated by the Gq-coupled AT1 receptor. To examine whether the eNOS phosphorylation by AngII is associated with eNOS activation in BAECs, we measured intracellular cGMP production upon AngII treatment. As shown in Fig. 1DGo, AngII treatment stimulated intracellular cGMP production in BAECs infected with AT1-encoded adenovirus, which was inhibited by ARB or a NOS inhibitor, L-NAME. These data not only clearly confirm the previous reports of eNOS activation by AngII through the AT1 receptor but also suggest an involvement of the eNOS phosphorylation in the activation.


Figure 1
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  		FIG. 1. Gene-transferred AT1 stimulation by AngII in BAECs leads to eNOS activation. A, BAECs infected with adenovirus encoding AT1 (100 MOI) or control empty vector (100 MOI) for 48 h were stimulated with AngII (100 nM) for indicated durations; B, BAECs infected with adenovirus encoding AT1 were pretreated with or without RNH6270 (10 µM), an ARB, for 30 min and stimulated with AngII (100 nM) for 1 min; C, BAECs infected with adenovirus encoding AT1 were pretreated with or without YM-254890 (1 µM), a Gq inhibitor, for 10 min and stimulated with AngII (100 nM) for 1 and 5 min, and phosphorylation of eNOS at Ser1179 was determined by immunoblot analysis; D, BAECs infected with adenovirus encoding AT1 were pretreated with RNH6270 (10 µM) or L-NAME (100 µM), an eNOS inhibitor, for 30 min and stimulated with AngII (100 nM) for 20 min. cGMP was measured as described in Materials and Methods. Data shown are the mean ± SEM from three independent experiments. *, P < 0.05 compared with the basal control; {dagger}, P < 0.05 compared with the stimulated control.

 
eNOS activation by AngII in VSMCs
To examine the possible eNOS regulation by AngII in VSMCs under eNOS gene therapy, the effect of exogenous eNOS gene delivery was first titrated with endogenous eNOS expression in BAECs. Infection of adenovirus encoding the eNOS gene to VSMCs induced expression of eNOS protein concentration dependently (Fig. 2AGo). Ten to 25 MOI of the adenovirus infection induced eNOS protein expression in VSMCs comparable to the endogenous eNOS expression in BAECs.


Figure 2
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  		FIG. 2. Gene-transferred eNOS in VSMCs is activated by AngII through the AT1 receptors. A, VSMCs were infected with increasing amount of adenovirus encoding eNOS (10–100 MOI) for 48 h, and eNOS expression was compared with BAECs; B–E, VSMCs were infected with adenovirus encoding eNOS (25 MOI): B, VSMCs expressing eNOS were stimulated with AngII (100 nM) for indicated durations; C, VSMCs expressing eNOS were stimulated with AngII in indicated concentrations for1 min; D, VSMCs expressing eNOS were pretreated with an ARB, RNH6270 (10 µM), for 30 min or a Gq inhibitor, YM-254890 (1 µM), for 10 min and stimulated with AngII (100 nM) for 1 min, and phosphorylation of eNOS at Ser1179 was determined using immunoblot analysis with the phosphospecific antibody; E, VSMCs expressing eNOS were pretreated with RNH6270 (10 µM) or L-NAME (100 µM) for 30 min and stimulated with AngII (100 nM) for 20 min. cGMP was measured as described in Materials and Methods. Data shown are the mean ± SEM from three independent experiments. *, P < 0.05 compared with the basal control; {dagger}, P < 0.05 compared with the stimulated control.

 
To test whether AngII activates the gene-transferred eNOS, VSMCs infected with adenovirus encoding eNOS or control empty vector were stimulated with AngII (100 nM). In eNOS-expressing but not control VSMCs (data not shown), AngII stimulated eNOS phosphorylation at Ser1179 in a time-dependent manner. The eNOS phosphorylation started at 1 min and persisted thereafter up to 20 min (Fig. 2BGo). Also, treatment of eNOS-expressing VSMCs with AngII for 1 min concentration-dependently stimulated eNOS Ser1179 phosphorylation, which was detected as low as 0.1 nM and was maximal at 100 nM (Fig. 2CGo). Again, AngII did not alter eNOS phosphorylation at Thr497 (data not shown). The phosphorylation of eNOS at Ser1179 induced by AngII was completely inhibited by ARB or the Gq inhibitor (Fig. 2DGo). Also, AngII stimulated cGMP production in VSMCs infected with eNOS adenovirus. The cGMP production was completely inhibited by ARB or L-NAME as well (Fig. 2EGo). Taken together, these data clearly indicate that AngII activates exogenously introduced eNOS via the AT1 in VSMCs.

eNOS Activation by AngII is dependent on the Ser1179 phosphorylation
To test the involvement of eNOS phosphorylation at Ser1179 in AngII-induced eNOS activation, the effect of an eNOS mutant, S1179A-eNOS in which Ser1179 is substituted with alanine (25), on cGMP production was examined in VSMCs. Although less than the wild-type eNOS, basal cGMP production was increased in VSMCs infected with S1179A-eNOS adenovirus (Fig. 3AGo). However, in contrast to wild-type eNOS, AngII could not further stimulate cGMP production in VSMCs expressing this mutant (Fig. 3BGo). We confirmed a comparable infection efficiency of each adenovirus encoding eNOS or the mutant (Fig. 3CGo) as well as expression efficiency of each eNOS (Fig. 3DGo) in VSMCs. These data indicate that the Ser1179 phosphorylation is indispensable for the eNOS activation by AngII through the AT1 receptor.


Figure 3
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  		FIG. 3. cGMP production induced by AngII in VSMCs expressing eNOS is dependent on eNOS phosphorylation at Ser1179. A, VSMCs were infected with adenovirus encoding wild-type eNOS or its mutant, S1179A-eNOS, for 48 h, and basal cGMP production for 20 min was measured as described in Materials and Methods. Data shown are the mean ± SEM from 3 independent experiments. *, P < 0.05 compared with the basal control. B, VSMCs were infected with adenovirus encoding wild-type eNOS or its mutant, S1179A-eNOS, for 48 h, and stimulated with AngII (100 nM) for 20 min. cGMP was measured as described in Materials and Methods. Data shown are the mean ± SEM from three independent experiments. *, P < 0.05 compared with the basal control. C, Photomicrograph of green fluorescent protein detection in VSMCs infected with adenovirus encoding green fluorescent protein together with wild-type eNOS or S1179A-eNOS for 48 h. D, Cell lysates prepared for the cGMP experiments (B) were evaluated for expression of eNOS and ERK2 by immunoblotting.

 
VASP is a 46-kDa protein that is phosphorylated preferentially at Ser239 by cGMP-dependent protein kinase (cGK)/protein kinase G (PKG) and at Ser157 by PKA or PKC (32, 33). Phosphorylation of VASP at Ser157 but not Ser239 leads to a shift in molecular mass to 50 kDa in SDS-PAGE (32). To eliminate a possible indirect inhibition of the AngII signal by the S1179A mutant, phosphorylation of VASP by AngII was further examined in VSMCs infected with eNOS, S1179A, or the control vector adenovirus. As shown in Fig. 4Go, AngII stimulated phosphorylation of VASP at Ser157 and thus caused the partial shift of total VASP in VSMCs regardless of the infection. In the nonstimulated condition, enhanced Ser239 phosphorylation was detected in 46-kDa (Ser157-nonphosphorylated) VASP in VSMCs infected with eNOS or S1179A but not the control vector. However, in the AngII-stimulated condition, the total amount of Ser239-phosphorylated VASP in 46-kDa (Ser157-nonphosphorylated) and 50-kDa (Ser157-phosphorylated) was further enhanced only when intact eNOS was expressed in VSMCs. These data can be interpreted as such: 1) eNOS or S1179A expression had no inhibitory effect on Gq/PKC-dependent VASP Ser157 phosphorylation by AngII, 2) S1179A restores an eNOS ability to produce comparable basal level NO production to enhance VASP Ser239 phosphorylation, and 3) the AngII response to activate eNOS and subsequent cGK/PKG-dependent VASP Ser239 phosphorylation depends on the eNOS Ser1179 phosphorylation. Also, some dissociation between basal cGMP production and VASP Ser239 phosphorylation by wild-type eNOS or S1179A-eNOS could be a result of distinct experimental procedures used for the measurements.


Figure 4
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  		FIG. 4. VASP phsophorylation at Ser239 and Ser157 in VSMCs expressing eNOS or its mutant, S1179A-eNOS. A, VSMCs infected with adenovirus encoding wild-type eNOS, S1179A-eNOS or control empty vector were stimulated with AngII for indicated durations, and phosphorylation of VASP at Ser239 and Ser157 were detected using phosphospecific antibodies; B, signal intensities of the VASP phosphorylation with or without AngII stimulation for 10 min. Data shown are the mean ± SEM from three independent experiments. *, P < 0.05 compared with the basal control; #, P < 0.05 compared with the stimulated control.

 
Effects of eNOS gene delivery on the AngII-induced hypertrophic response
eNOS gene delivery has been shown to prevent vascular hypertrophy. Thus, we have studied whether the exogenously introduced eNOS activation by AngII has a role in prevention of VSMC hypertrophy induced by AngII. As shown in Fig. 5Go, AngII-induced increases in cell protein content and cell volume were completely abrogated in VSMCs infected with wild-type eNOS adenovirus (Fig. 5Go, A and B). AngII had no significant effect on living cell number, and this was not affected by eNOS adenovirus (data not shown). Thus, eNOS gene delivery does not induce cell death i.e. apoptosis under these conditions. Interestingly, mutation of Ser1179 did not alter the inhibitory effect of eNOS on AngII-induced VSMC hypertrophy. These data suggest that the mechanism of the inhibition of hypertrophy by eNOS gene delivery might be explained by the enhanced basal cGK activity resulting from enhanced basal cGMP production as also evident in the VASP Ser239 phosphorylation.


Figure 5
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  		FIG. 5. Effect of eNOS gene transfer on AngII-induced hypertrophic response in VSMCs. VSMCs infected with adenovirus encoding wild-type eNOS, S1179A-eNOS, or control empty vector for 48 h were stimulated with AngII for 72 h. Cell protein (A) and cell volume (B) were measured as described in Materials and Methods. Data shown are the mean ± SEM from three independent experiments. *, P, < 0.05 compared with the basal control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major novel findings of the present study are that 1) AngII activates eNOS to produce NO/cGMP by stimulating eNOS Ser1179 phosphorylation in BAECs and VSMCs, if eNOS and AT1 coexist, 2) the eNOS activation and subsequent NO/cGMP production in VSMCs induced by AngII is accompanied by enhanced VASP phosphorylation at Ser239, 3) adenovirus encoding eNOS protected VSMC from hypertrophy induced by AngII, and 4) inhibition of AngII-induced VSMC hypertrophy by eNOS gene transfer does not require eNOS Ser1179 phosphorylation but depends on enhanced basal NO production.

Relatively limited information has been available regarding eNOS regulation by the AT1 receptor (14, 15). Although previous studies have reported beneficial effects of eNOS gene delivery toward the vasculature (8, 9, 10, 11), the possible regulation of exogenously introduced eNOS in VSMCs remains unstudied. Here, we explored the importance of eNOS Ser1179 phosphorylation for eNOS activation both in BAECs and VSMCs through the AT1 receptor. eNOS phosphorylation at Ser1179 in the reductase domain is believed to enhance electron flux and improve sensitivity to Ca2+/CaM by slowing CaM dissociation (12). Thus, our findings are in good agreement with earlier publications (14, 15) demonstrating the importance of a Ca2+/CaM-dependent mechanism in eNOS activation by AngII. Although our findings suggest a critical role of the Ser1179 phosphorylation in AngII-induced eNOS activation, it should be noted that phosphorylation of Ser1179 is not always a predictor of eNOS activity as shown in uterine artery endothelial cells (34). Also, in COS7 cells, ATP-stimulated eNOS activity was only partially decreased with S1179A mutant (34). Quite recently, involvement of sphingosine kinase in AngII-induced eNOS activation has been reported (35). However, possible roles of sphingosine kinase in our model system remain to be determined.

The functional significance of the interaction between AT1 and AngII receptor type 2 (AT2) is an important issue to investigate in light of current clinical applications of AngII inhibitors toward cardiovascular diseases (36). Although antagonistic signals and functions of the AT2 receptors have been dominantly characterized in the past (37), recent publications have presented several examples of collaborative functions among these receptors. Not only AT1 but also AT2 has been shown to induce cardiac myocyte hypertrophy (38, 39). In VSMCs infected with retrovirus encoding AT2, AngII stimulated collagen synthesis through both AT1 and AT2 receptors (40). Moreover, AT2 appears to stimulate NO production in part through kininogen and bradykinin (41). Activation of the AT2 receptor also leads to eNOS Ser1179 phosphorylation (42). Although we did not address the possible AT1/AT2 interaction in regulation of eNOS in the present study partly because of the lack of AT2 receptor expression in our experimental systems, this interesting issue should be developed more in future experiments in select cells expressing both receptors natively.

cGMP activates cGK/PKG to phosphorylate its well characterized substrate, VASP, at Ser239 (32). In this regard, the VASP phosphorylation at Ser239 has been recognized as a useful biomarker to monitor endothelial function (43, 44). Our data showed that phosphorylation of VASP Ser239 was further enhanced by AngII in VSMCs expressing wild-type eNOS. It is likely that activation of exogenously introduced eNOS via AT1 should counteract AngII functions such as vasoconstriction, which should contribute to an improvement of endothelial function observed under eNOS gene therapy. To support this notion, NO production through the AT1 receptor has been shown to counteract the vasoconstrictor effect of AngII (16, 17). Surprisingly, inhibition of AngII-induced hypertrophy was equally observed in VSMCs expressing eNOS as well as in VSMCs expressing S1179A-eNOS, which cannot be further activated by AT1. It has been demonstrated that expression of S239A-VASP significantly increased proliferation. Also, this rendered VSMCs less sensitive to growth inhibition by a NO donor (45). Taken together with enhanced basal cGMP production and VASP Ser239 phosphorylation by S1179A, we interpret that the enhanced basal level of NO production is sufficient enough to counteract the hypertrophic response of AngII in S1179A-expressing VSMCs.

There are certain limitations in the present study. Our findings are limited in subcultured cells with eNOS or AT1 transduced by adenovirus, which may or may not bear a relationship to in vivo situations. Therefore, additional in vivo studies must be conducted to support the in vitro findings presented here. In conclusion, we demonstrated that the AngII/AT1 system could positively couple to the eNOS via Ser1179 phosphorylation to produce NO in BAECs and VSMCs, if eNOS and AT1 coexist. The NO production can counteract against AngII-induced endothelial dysfunction under eNOS gene therapy. However, the basal level NO production and not the phosphorylation-dependent NO production is sufficient for prevention of AngII-induced hypertrophy. These data demonstrate a novel molecular mechanism of eNOS regulation and function and thus provide useful information for gene targeting of the eNOS system.


   Footnotes
 
This work was supported by National Institute of Health Grants HL076770 (S.E.), 2G12RR03032 (E.D.M.), and HL076575 (G.D.F.).

Disclosure statement: The authors have nothing to disclose.

First Published Online September 15, 2006

Abbreviations: AngII, Angiotensin II; ARB, AT1 receptor blocker; AT1, AngII type 1; BAEC, bovine aortic endothelial cell; CaM, calmodulin; cGK, cGMP-dependent protein kinase; eNOS, endothelial nitric oxide synthase; GPCR, G-protein-coupled receptor; L-NAME, N-nitro-L-arginine methyl ester; PKG, protein kinase G; VASP, vasodilator-stimulated phosphoprotein; VSMC, vascular smooth muscle cell.

Received June 20, 2006.

Accepted for publication September 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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