INTRODUCTION

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DIETARY SALT has a profound effect on systemic and renal hemodynamic vascular responses, particularly the endogenous vasodilator nitric oxide (NO). A recent study showing that increased production of NO is an important part of adaptation to increased dietary sodium intake in healthy humans (2) agrees well with previous studies in rats (6, 13, 28, 29). In our earlier study (6), we found that the hypertensive effect of N-methyl-L-arginine (L-NMMA) was augmented in normotensive rats on 8.0% NaCl diet, suggesting an important role for NO in maintenance of blood pressure during high salt intake. Although the mechanism producing this effect is not yet clear, sodium loading leads to fluid retention and increased blood volume (12), potentially increasing shear stress on the endothelial cells; shear is a potent stimulus for NO release (19, 30). Indeed, as reviewed by Davies (11), the increase in the activity of the endothelial isoform of nitric oxide synthase (NOS3) is part of the physiological response to flow-induced shear stress. Modulation of the secretion of NO allows control of vasodilation and adaptation of the vessel lumen and wall to changes in flow.

Our previous studies demonstrated that dietary salt enhanced glomerular production of NO by increasing NOS3 expression. Interestingly, the increase in NOS3 was mediated through transforming growth factor (TGF)-1 (32). Dietary salt increased TGF-1 expression through a tetraethylammonium (TEA) chloride-sensitive mechanism (33), suggesting that the effect was related to shear stress (9, 11, 20-22, 30). We hypothesized that dietary NaCl intake also modulated NOS3 and TGF-1 in arterial endothelium. The current studies sought to determine whether aortic endothelial cell production of NO and TGF-1 was enhanced by an increase in dietary salt and further to identify the interactions involved.

	MATERIALS AND METHODS

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Animal preparation. Studies were conducted on 56 male Sprague-Dawley rats, 28 days of age, obtained from Charles River Laboratories (Wilmington, MA). Animals were chosen at this age because of our previous experience that showed renal function and blood pressure did not change in response to an increase in dietary salt during 2 wk of observation (6). Rats were housed under standard conditions and given 0.3% NaCl chow (AIN-76A containing 0.3% NaCl; Dyets, Bethlehem, PA) and water ad libitum for 4 days before the experiment was initiated. The animals were then either continued on a diet that contained 0.3% NaCl or were changed to 8.0% NaCl (AIN-76A containing 8.0% NaCl; Dyets). These diets were formulated to be identical in protein and electrolyte composition but differed in NaCl content. On the fourth and seventh days of study, rats were anesthetized with pentobarbital sodium, 50 mg/kg ip, and killed by exsanguination. Aortas were harvested under sterile conditions to obtain the protein extract for Western blotting, total RNA for Northern analysis, and for in vitro incubation studies, as described below. In some experiments, to determine the effect of dietary NaCl on blood pressure, as we have done previously (5-7), rats were anesthetized with Inactin (BYK Gulden, Hamburg, Germany), 100 mg/kg body wt ip. After tracheostomy was performed using PE-240 tubing, a PE-50 catheter was inserted in the right femoral artery, and systolic blood pressure was monitored for 15 min, using a computerized system (MacLab; Analog Digital Instruments, Dunedin, New Zealand).

Northern hybridization analysis. Northern hybridization analysis was performed as we have reported previously (32-34). Briefly, total RNA from the aorta was isolated by the single-step method of acid guanidinium thiocyanate-phenol chloroform extraction (8). Concentration and purity were determined using spectrophotometry. Thirty-five micrograms of total RNA were electrophoresed in 1.0% agarose gels containing 2.2 M formaldehyde and 0.2 M MOPS, pH 7.0, and then transferred to a nylon membrane (Genescreen Plus hybridization transfer membrane; NEN Life Science Products, Boston, MA) using vacuum blotting (model 785; Bio-Rad Laboratory, Hercules, CA) for 2 h in 10× saline sodium citrate (SSC). Nucleic acids were cross-linked by ultraviolet irradiation (Stratagene, La Jolla, CA). The membranes were prehybridized for 20 min at 68°C with standard hybridization solution (QuikHyb; Stratagene). They were then hybridized at 68°C for 4 h with cDNA probes for NOS3 (bovine NOS3 cDNA generously provided by Dr. William C. Sessa, Yale University School of Medicine), rat TGF-1 (kindly provided by Dr. Thomas S. Winokur, University of Alabama at Birmingham), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; human GAPDH probe obtained through American Type Culture Collection, Rockville, MD). The probes were labeled with [³²P]dCTP by random oligonucleotide priming (Prime-a-Gene labeling system; Promega). The blots were washed two times in 2× SSC containing 0.1% SDS at room temperature for 15 min, then in 0.1× SSC containing 0.1% SDS at 60°C for 30 min. Membranes were exposed to XAR-5 film (Kodak) at 80°C. Membranes were stripped in standard fashion using solution containing 1 mmol/l Tris · HCl, pH 8.0, 0.1 mmol/l EDTA, and 0.1× Denhardt's at 75°C for 2 h. Autoradiographs were scanned using a densitometer (model 620 Video Densitometer; Bio-Rad). Density of the GAPDH band in the same lane was used to normalize mRNA loading. For quantification, density of the NOS3 and TGF-1 bands was individually divided by the density of band representing GAPDH in the same lane.

Western blot analysis. Aortic segments from rats on the 0.3 and 8.0% NaCl diets were washed with cold PBS and chilled in RIPA buffer (50 mmol/l Tris · HCl, 150 mmol/l NaCl, 1 mmol/l disodium EDTA, 0.1 mmol/l EGTA, 1.0% Nonidet P-40, 0.1% SDS, and 0.5% sodium deoxycholate). Phenylmethylsulfonyl fluoride (1 mmol/l), aprotinin (10 µg/ml), and leupeptin (10 µg/ml; all from Sigma Chemical) were added as the protease inhibitors. Tissues were homogenized (Omni-Mixer 17105; Omni, Waterbury, CT) in standard fashion. After several passages through a 26-gauge needle, the homogenates were centrifuged at 20,000 g for 45 min. The supernatants were collected, and total protein concentration was determined using a kit (Micro BCA Protein Assay Reagent Kit; Pierce, Rockford, IL). Samples were mixed with equal volumes of 2× SDS gel loading buffer (100 mmol/l Tris · HCl, pH 6.8, 4% SDS, 20% glycerol, 200 mmol/l dithiothreitol, and 0.2% bromphenol blue) and boiled for 5 min. Solutions containing 60 µg of total protein were resolved by electrophoresis in an 8% polyacrylamide gel (SDS-PAGE) and transferred to a nitrocellulose membrane. After 4 h of incubation in blocking buffer, which consisted of 5% milk in TBST (10 mmol/l Tris, 100 mmol/l NaCl, and 0.1% Tween 20, pH 7.5), membranes were probed with anti-human NOS3 monoclonal antibody (Transduction Laboratories, Lexington, KY), 1:1,000 dilution in blocking buffer, overnight at 4°C. We have used this commercial antibody successfully in a previous study to demonstrate NOS3 expression in the rat glomerulus (32). The membranes were then washed five times with Tris-buffered saline-Tween, and the bound primary antibody was identified using peroxidase-conjugated anti-mouse IgG (Bio-Rad), 1:2,000 dilution in blocking buffer, for 2 h at room temperature. Washed blots were developed using the enhanced chemiluminescence Western blotting system and were exposed to Hyperfilm (Amersham International, Buckinghamshire, UK). The films were scanned using a densitometer (model 620 Video Densitometer; Bio-Rad) to quantify NOS3.

In vitro incubation studies. After removal of adherent fat and connective tissue, aortas from rats on 0.3 and 8.0% NaCl diets were cut into 3- to 4-mm ring segments and placed in 48-well plates. The samples were washed two times with ice-cold PBS and initially incubated with serum-free medium (RPMI 1640; Life Technologies, Grand Island, NY) that contained 1 µmol/l calcium ionophore (A23187; Sigma Chemical). Some wells also contained 10 µg/ml rabbit polyclonal antibody that specifically neutralizes TGF- (catalog no. AB-100-NA; R&D Systems, Minneapolis, MN) or 10 µg/ml nonspecific rabbit IgG (Southern Biotechnology Associates, Birmingham, AL). After a 30-min incubation period, the medium was removed and refreshed with serum-free medium that contained A23187 along with neutralizing TGF- antibody or control rabbit IgG. All samples were incubated in 48-well plates for 24 h at 37°C. Medium was then harvested and assayed for nitrate and nitrite in standard fashion using nitrate reductase and Griess reagent, as described previously (7, 32). These studies were repeated using aortic segments that had the endothelium mechanically removed by gently rubbing the luminal surface of each ring segment with a sterile wooden probe. This classical technique of endothelial cell removal has been shown to preserve vasoconstrictive responses but eradicate endothelium-dependent relaxation of blood vessels in vitro (14).

In other experiments, aortic segments were cut in half. One-half of the aortic tissue had the endothelium mechanically removed. Washed aortic segments were placed in 48-well plates and were incubated with serum-free RPMI 1640 media for 24 h at 37°C. Medium was harvested and assayed for total and active TGF-1 using a kit (TGF-1 Emax TM ImmunoAssay System; Promega, Madison, WI), as described previously (33).

Statistical analysis. All data were presented as means ± SE. Significant differences among data sets were determined using either unpaired t-test or one-way ANOVA with standard post hoc testing (Statview, version 5.0; SAS Institute, Cary, NC), where appropriate. A P value of < 0.05 assigned statistical significance.

	RESULTS

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Whole animal data. Mean body weights of the animals on 8.0% NaCl diet on days 4 and 7 were 102 ± 7 and 122 ± 4 g, respectively; these data did not differ from mean body weights of animals on 0.3% NaCl on the same day of study (100 ± 14 g on day 4 and 116 ± 6 g on day 7). Blood pressures of animals were determined on day 7 using an intra-arterial catheter and direct blood pressure monitoring. As anticipated from our previous work (6), mean blood pressures did not differ between the groups (127 ± 4 mmHg on 8.0% NaCl diet vs. 130 ± 2 mmHg on 0.3% NaCl diet; P = 0.5038).

Expression of NOS3 was increased in aortic endothelium from rats on high-salt diet. Steady-state mRNA levels of NOS3 were determined by Northern analysis using aortic tissue from rats on days 4 and 7 of the protocol. Mean relative NOS3 expression increased 2.2-fold (0.987 ± 0.124 vs. 0.445 ± 0.079; P = 0.0103) on day 4 and 1.8-fold (0.642 ± 0.087 vs. 0.348 ± 0.05; P = 0.0263) on day 7 in aortic segments from rats on the 8.0% NaCl diet compared with the group on 0.3% NaCl diet (Fig. 1). Western blotting confirmed increased protein expression of NOS3 in aorta from rats on 8.0% NaCl diet compared with rats on 0.3% NaCl diet on day 4 (1.69 ± 0.27 vs. 0.79 ± 0.09; P = 0.0212) and day 7 (2.84 ± 0.13 vs. 1.22 ± 0.16; P = 0.0002) (Fig. 2).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Northern analysis showed an increase (P < 0.05) in steady-state levels of mRNA of the endothelial form of nitric oxide synthase (NOS3) on days 4 and 7 of study in freshly isolated segments of aorta from animals on 8.0% NaCl diet compared with animals on 0.3% NaCl diet (n = 4 rats in each group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Western blot of protein harvested from segments of aorta demonstrated an increase (P < 0.05) in NOS3 protein expression in animals on 8.0% diet compared with animals on 0.3% NaCl diet on both days of study (n = 4 rats in each group). Mr, relative molecular mass.

Expression of TGF-1 was increased in aortic endothelium of rats on high-salt diet. By Northern analysis, mean steady-state level of mRNA of TGF-1 in the aorta from rats on day 7 of the high-salt diet group was greater than in rats on 0.3% NaCl diet (0.59 ± 0.1 vs. 0.30 ± 0.03; P = 0.0312; Fig. 3). After a 24-h incubation of aortic ring segments, medium from the 8.0% NaCl group contained increased amounts of total TGF-1 (1.353 ± 0.07 vs. 0.522 ± 0.009 pg · day¹ · mg wet wt¹; P < 0.0001) and active TGF-1 (0.850 ± 0.005 vs. 0.200 ± 0.034 pg · day¹ · mg wet wt¹; P < 0.0001) compared with rats on 0.3% NaCl (Fig. 4). To determine the cell of origin of TGF-1 secretion in these experiments, endothelial cells were mechanically removed from aortic rings using a wooden probe. After denudation of the endothelium, production of active TGF-1 by aortic segments of rats on either diet fell to undetectable levels; total TGF-1 also fell to low levels and remained elevated in the high-salt group compared with rats on 0.3% NaCl diet (0.175 ± 0.038 vs. 0.020 ± 0.012 pg · day¹ · mg wet wt¹; P = 0.0081).

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Northern analysis demonstrated increased levels (P = 0.0312) of mRNA of transforming growth factor (TGF)-1 in aorta from rats on 8.0% NaCl diet compared with rats on 0.3% NaCl diet (n = 4 rats in each group).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Medium incubated with segments of aorta from animals on 8.0% NaCl diet contained increased (P < 0.05) amounts of total and active TGF-1 compared with 0.3% group (n = 4 rats in each group).

TGF-1 potentiated NO production in the aorta incubation from rats on high-salt diet. Increased amounts of metabolites of NO (nitrite and nitrate), termed NO_x, were observed in medium after a 24-h incubation with segments of aorta from rats on 8.0% NaCl diet compared with values obtained using aorta from the 0.3% NaCl group (Fig. 5). Nitrite alone was increased (16.8 ± 2.0 in 8.0% NaCl group vs. 4.4 ± 0.7 pmol · h¹ · mg aorta¹ in 0.3% NaCl group; P < 0.0001), but the ratio of nitrite to NO_x did not differ (0.78 ± 0.12 vs. 0.67 ± 0.08; P = 0.4748). Addition of a neutralizing antibody to TGF- to the medium during incubation decreased production of NO_x by the high-salt aortic segments; a nonspecific IgG had no effect on NO production.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Calcium-stimulated nitrite + nitrate (NOx) production was increased (23.8 ± 3.1 vs. 7.0 ± 1.5 pmol · h1 · mg aorta1; P < 0.0001) in medium incubated with segments of aorta on 8.0% NaCl diet compared with values obtained using aorta from the 0.3% NaCl group (n = 4 rats in each group). Addition of TGF- neutralizing antibody (10 µg/ml) to the medium at the start of incubation reduced NOx production by aortic segments from the 8.0% NaCl group (16.4 ± 2.2 vs. 23.8 ± 3.1 pmol · h1 · mg aorta1; P = 0.0346). Addition of a nonspecific IgG had no effect on NOx production (24.6 ± 2.0 vs. 23.8 ± 3.1 pmol · h1 · mg aorta1; P = 0.8475). In a separate series of studies, endothelium was mechanically removed from aortic ring segments, and experiment was repeated (dotted bars). The amount of NOx released in medium decreased to low levels that did not differ (P = 0.1882) among the groups.

	DISCUSSION

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Previous studies have shown that NO production in vivo is modulated by dietary salt, as determined by levels of nitrate and nitrite in the serum and urine (6, 13, 28, 29). Using L-NMMA, we demonstrated that this increase in NO production was important in blood pressure regulation (6). In a recent study, glomeruli from rats on a high-salt diet demonstrated increased expression of NOS3 and increases in NO production; interestingly, TGF-1 was also increased, and neutralizing antibodies to TGF- inhibited NOS3 expression and NO production (32). We therefore hypothesized that the amount of NaCl in the diet directly modulated NO production in the arterial wall. The present study demonstrated that expression of both NOS3 and TGF-1 was increased in aortas of rats on 8.0% NaCl chow. Removal of the endothelium abrogated active TGF-1 production. With the use of a neutralizing antibody to TGF-, we observed a decrease in NO production. Taken together, the studies showed that dietary salt modulated NOS3 and TGF-1 expression in the arterial wall; NOS3 expression was at least partially regulated by endothelial cell production of TGF-1.

The mechanism by which dietary salt modulated endothelial cell production of NO and TGF-1 was not defined in these experiments, but, because of the apparent involvement of the endothelium, shear stress may be involved. The endothelium is now recognized as an important physiological biomechanical sensor that responds to changes of flow. As reviewed by Davies (11), this monolayer of cells is intimately involved in determining the vascular effects of shear stress. In particular, endothelial cells in culture secrete NO when subjected to shear (9, 15); in turn, NO controls vasodilation and shear forces. In the setting of laminar flow, shear stress is proportional to the flow velocity and medium viscosity and inversely proportional to the third power of the internal radius. Dietary salt increases blood volume (12) and thus arterial flow, which enhances shear stress. For example, an increase in daily salt intake in healthy humans from 5 g (80 mmol) to 10 g (160 mmol) expands extracellular fluid volume by ~1.0-1.5 liters (1). Although other potential factors, such as subtle changes in electrolyte or calcium concentrations, were not excluded, shear stress is a plausible mechanism of the effect of dietary salt on function of the arterial endothelial cell.

Endothelial cell signal transduction mechanisms produced from shear stress remain incompletely understood (11). However, one early event in this process is opening of a shear-activated potassium channel (22), which hyperpolarizes the endothelium and increases cytoplasmic calcium (9, 17, 27). Ohno and associates (20) demonstrated shear-induced expression of TGF-1 in endothelial cells in culture. Blockade of the shear-activated potassium channel with TEA produced dramatic reductions in gene transcription and protein activity of TGF-1 (20). Our previous studies reproduced these phenomena in isolated glomeruli from animals on 8.0% NaCl diet (33). In the present experiments using aortic segments, removal of the endothelium decreased active TGF-1 production to undetectable levels. Thus the high-salt diet directly effected arterial endothelial cell function and increased TGF-1 production by these cells.

Expressional regulation of NOS3 by shear stress controls endothelial cell production of NO in vivo (18, 26) and in vitro (19, 30). We recently found that, in vivo, dietary salt increased glomerular steady-state levels of mRNA of NOS3, which correlated linearly with TGF-1 mRNA expression. Although the magnified production of NOS3 and TGF-1 in response to dietary salt was prevented by TEA, suggesting the effect was mediated through shear stress, experiments that used anti-TGF- neutralizing antibodies showed that the increase in glomerular NOS3 expression was dependent on concomitant expression of TGF-1 (32). Inoue and associates (15) have shown that TGF-1 increased steady-state mRNA levels and protein expression of NOS3 in a dose-dependent manner in bovine aortic endothelial cells in culture. These investigators further demonstrated that TGF-1 upregulates mRNA expression of NOS3 through activation of a nuclear factor-1 binding site in the promoter region (15). In our present study, addition of a neutralizing antibody to TGF-1 decreased, but did not completely inhibit, NO production, suggesting that in the arterial wall of animals on a high-salt diet, both expression of TGF-1 and an additional component of shear stress promoted NOS3 expression.

NO plays a critical role in the maintenance of blood pressure homeostasis during changes in salt intake (2, 6, 13, 28, 29) and is involved in vascular remodeling (24). Overexpression of NOS3 in myocytes inhibited smooth muscle cell proliferation in vitro and in balloon-injured carotid arteries of rats (4). Inhibition of endogenous renal synthesis of NO with nitro-L-arginine methyl ester increased blood pressure and facilitated collagen I gene expression and fibrogenesis in afferent arterioles and glomeruli of hypertensive rats (3). In spontaneously hypertensive rats, inhibition of NO production in the setting of a high-salt diet markedly increased renal injury (31). However, by potentially facilitating peroxynitrite formation and lipid peroxidation (10), high salt intake could accelerate atherosclerosis through augmented NO production in conditions that promote superoxide formation.

TGF-1 is a pleiotropic growth factor that may also be beneficial in the setting of a high-salt diet. Not only is NOS3 expression increased, but the antiproliferative and hypertrophic effects of this growth factor on vascular smooth muscle may provide additional benefit (23). In addition, pretreatment with TGF-1 preserved endothelial cell function and had a cardioprotective effect in rat hearts subjected to ischemia-reperfusion injury (16). However, TGF-1 is also a fibrogenic growth factor that can have profound effects on the arterial wall. For example, overexpression of TGF-1 produced a reversible cellular- and matrix-rich neointima in the arterial wall and also promoted a remarkable transdifferentiation of vascular smooth muscle cells in the media (25).

In summary, dietary salt, without altering blood pressure, enhanced aortic endothelial cell production of active TGF-1 and NO synthesis through increased NOS3 expression. Given the important effects of NOS3 and TGF-1 on the arterial wall, it is intriguing to consider that, in the proper setting, dietary salt may facilitate vascular damage through modulation of endothelial cell function.