Dulbecco's modified Eagle's medium (DMEM)/high glucose and phenol red-free DMEM were purchased from HyClone (Logan, UT, USA). Charcoal stripped fetal bovine serum (FBS) was obtained from Gibco-BRL (Grand Island, NY, USA). Rabbit monoclonal antibodies against proliferating cell nuclear antigen (PCNA; 1:1,000; #13110), phosphorylated c-Jun amino N-terminal kinases (p-JNK; 1:1,000; #4668) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2; 1:2,000; #4370) were provided by Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal antibody against phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) was supplied by Signalway Antibody LLC (College Park, MD, USA). Rabbit monoclonal antibody against β-actin (1:10,000; JC-PA-βA1) and horseradish peroxidase-conjugated goat-anti-rabbit antibody (1:4,000; JC-PC012-1h) were purchased from Geneshare (Xi'an, Shannxi, China). NaCl, choline chloride, sorbital, mouse monoclonal anti-actin, α-smooth muscle-FITC antibody (anti-α-SM-actin antibody; F3777) and DAPI were obtained from Sigma-Aldrich (St. Louis, MO, USA). The Cell-Light™ 5-ethynyl-2′-deoxyuridine (EdU) imaging detecting kit was purchased from RiboBio (Guangzhou, China). SP600125 (a JNK inhibitor) was provided by Cell Signaling Technology. PD98059 (an ERK1/2 inhibitor), SB203580 (a p38 MAPK inhibitor) and anisomycin (a p38 MAPK activator) were supplied from Santa Cruz Biotechnology (Dallas, TX, USA). In addition, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT) was purchased from Amresco (Solon, OH, USA).

Rat VSMCs were purchased from CHI Scientific, Inc. (Jiangsu, China) and cultured in DMEM supplemented with 10% FBS at 37°C under 5% CO₂/95% air in a humidified incubator. Cells at passages 3 to 8 with a purity of >95% (determined by immunofluorescence staining for α-SM-actin) were used in the experiments. In order to obtain quiescent VSMCs, the cells were incubated in serum-free medium for 24 h. Subsequently, standard DMEM supplemented with 5% FBS was used as the control culture medium, in which the Na⁺ concentration was approximately 156 mM. High-sodium medium was prepared by the addition of NaCl (10, 20, 30, 40 and 50 mM,respectively; additional to the levels already present in the medium) to the control culture medium. Choline chloride and sorbital were used to examine the effects of chloridion (Cl⁻) and osmotic pressure on the proliferation of the VSMCs. High-Cl⁻ medium was prepared by the addition of choline chloride (10, 20, 30, and 50 mM, respectively; additional to the levels already present in the medium) to the control culture medium. High-osmotic pressure medium was prepared by the addition of sorbital (20, 40, 60, 80 and 100 mM, respectively; additional to the levels already present in the medium) to the control culture medium. For MTT assay, the VSMCs were treated for 12, 24 or 48 h. For EdU incorporation assay, the VSMCs were treated for 24 h. PCNA expression at the mRNA level was detected following treatment for 3, 6, 9 and 12 h. PCNA expression at the protein level was detected following treatment for 6, 15, or 24 h. Phosphorylation levels were detected after 15, 30, 60, 90 and 120 min of intervention. To examine the effects of 3 MAPK members on the proliferation of VSMCs induced by additional NaCl, the cells were pre-treated wtih SP600125 (a JNK inhibitor), PD98059 (an ERK1/2 inhibitor), SB203580 (a p38 MAPK inhibitor) and anisomycin (a p38 MAPK activator) for 30 min.

The VSMCs were cultured on sterile glass cover slips in 12-well plates. Following fixation with 4% paraformaldehyde, the VSMCs were permeabilized by 0.1% Triton X-100 for 30 min at room temperature and blocked with goat serum for 1 h at 37°C. The cells then covered with anti-α-SM-actin antibody were incubated at 4°C in a dark humidified chamber overnight. The samples were counter-stained with DAPI at room temperature for 10 min. The images were captured using NIS-Elements imaging software (Nikon, Tokyo, Japan).

Cell proliferation was firstly investigated by MTT assay according to published literature (23). Briefly, the VSMCs were treated with additional NaCl (10–50 mM), choline chloride (10–50 mM) or sorbital (20–100 mM) in DMEM supplemented with 5% FBS for 12, 24 or 48 h. The cells were then incubated with MTT solution (0.1 mg/ml) for 4 h. The formed formazan crystals were dissolved in 150 μl/well dimethyl sulfoxide (DMSO). The absorbance was recorded at a wavelength of 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). All experiments were performed at least 3 times. The relative proliferation of the cells was calculated as the absorbance of treated cells/control cells ×100%.

Following synchronization with serum-free medium for 24 h, the cells were treated with or without additional NaCl (30 mM) in DMEM supplemented with 5% FBS for 24 h. The EdU incorporation assay was performed according to the manufacturer's instructions (RiboBio). In brief, the VSMCs were incubated with 50 μM EdU for 2 h. Following fixation with 4% paraformaldehyde and permeabilization in 1.0% Triton X-100, the cells underwent EdU staining. The cell nuclei were counterstained with Hoechst 33342. EdU-positive nuclei were determined under a fluorescence microscope (Olympus BX51; Olympus, Tokyo, Japan). The cell proliferation rate was calculated as the proportion of nucleated cells incorporating EdU in 5 high-power fields per well.

Quantification was carried out as previously described (24). Briefly, total RNA was extracted from the cells using TRIzol reagent and reverse transcribed using the cDNA synthesis kit (Fermentas, Burlington, CA, USA). Quantitative (real-time) PCR (qPCR) was performed using SYBR^® select Master Mix on an iQ5 Multicolor Real-Time PCR Detection system (Bio-Rad). The primer sequences used for PCNA were as follows: sense, 5′-ACCTCACCAGC ATGTCCAA-3′ and antisense, 5′-CATAGTCTGAAACTTTC TCTTGATTTG-3′. Beta-glucuronidase (GUSB) was used as a housekeeping gene and the primer sequences were: sense, 5′-CTCTGGTGGCCTTACCTGAT-3′ and antisense, 5′-CAGA CTCAGGTGTTGTCA TCG-3′. The relative expression level of the target gene was then determined using a comparative method (2^−ΔΔCT).

Western blot analysis was performed as previously described (24). Briefly, the VSMCs were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. The protein content was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equivalent amounts of protein were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for electrophoresis and then transferred onto PVDF membranes (Bio-Rad). The blots were incubated with primary antibodies against PCNA, p-JNK, p-ERK1/2, p-p38 MAPK and β-actin, and then with horseradish peroxidase-conjugated goat-anti-rabbit antibody. The protein signals were detected using chemiluminescence. All densitometric data for the target genes were corrected with β-actin as a loading control.

Data are expressed as the means ± standard deviation (SD). Comparisons among 3 or more groups were analyzed by one-way analysis of variance (ANOVA), whereas the Student's t-test was used for comparisons between 2 groups. A value of P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA).

In the present study, we used α-SM-actin as a marker of contractile VSMCs to identify the phenotype and purity of the commercially available VSMCs (25). The cultured VSMCs exhibited a spindle-like shape. Immunofluorescence staining revealed an abundance of green myonemes in the cytoplasm. The purity of the obtained cells was >95% (data not shown).

Additional NaCl was added to the basal medium in order to verify whether Na⁺ directly promotes the proliferation of VSMCs at higher concentrations. First, MTT assay was used to evaluate cell proliferation. Following incubation for 24 h, the addition of 20–40 mM NaCl to the cell medium markedly promoted the proliferation of the VSMCs compared with the untreated control group (Fig. 1A). Furthermore, this induction of cell proliferation by the high sodium levels was the most significant when 30 mM NaCl were added. To further clarify whether the proliferative effects of NaCl on VSMCs are due to Na⁺ itself, choline chloride and sorbital were also employed in the present study. Choline chloride at various concentrations, which was used as Cl⁻ intervention, did not increase the proliferation of the VSMCs (Fig. 1B). Sorbital, which was used as osmotic intervention, exhibited no significant effect on the proliferation of the VSMCs (Fig. 1C) at low concentrations (20 and 40 mM). Indeed, sorbital inhibited the proliferation of the VSMCs when used at a concentration of >60 mM. As shown in Fig. 1D, the addition of NaCl (30 mM; additional to the levels already in medium) to the cell medium promoted the proliferation of the VSMCs following incubation for 24 and 48 h, but not for 12 h. These results indicated that Na⁺ at relatively high concentrations per se, rather than Cl⁻ or osmotic pressure promoted the proliferation of VSMCs.

EdU, a thymidine analogue, is incorporated into cellular DNA during cell proliferation (26). Thus, in this study, we used the EdU incorporation assay to further confirm the effects of high sodium levels on the proliferation of VSMCs. In accordance with the results of MTT assay, EdU incorporation assay revealed that addition of NaCl (30 mM; additional to the levels already in medium) to the cell culture medium for 24 h increased the percentage of EdU-positive nuclei compared with the control group (Fig. 2), thus indicating an increase in DNA synthesis in the ultured VSMCs which was induced by NaCl.

PCNA, the eukaryotic DNA sliding clamp, confers high processivity to replicative DNA polymerase, which is recognized as a marker of cell proliferation (27,28). We thus measured the mRNA and protein expression levels of PCNA in the cells cultured in medium with the addition of high level of sodium. As shown in Fig. 3A, the mRNA expression of PCNA was significantly increased following the addition of 30 mM Na⁺ to the cell culture medium (additional to the levels already in medium) for 6 and 9 h. In addition, the protein expression of PCNA was transiently, but markedly increased following the addition of Na⁺ to the medium for 15 h (Fig. 3B).

JNK, ERK1/2 and p38 MAPK are the members of MAPK family, and they play important roles in cell proliferation (29,30). Thus, in order to elucidate the underlying mechanisms responsible for the proliferative effects of additional Na⁺ on VSMCs, we measured the phosphorylation levels of JNK, ERK1/2 and p38 MAPK by western blot analysis. As shown in Fig. 4, the expression levels of p-JNK (Fig. 4A and B), p-ERK1/2 (Fig. 4C and D) and p-p38 MAPK (Fig. 4E and F) were significantly increased following the addition of 30 mM Na⁺ to the cell culture medium (additional to the levels already in medium) for 30 min, and such an increase was maintained until 120 min post-treatment.

To further investigate the exact roles of MAPK members in the high-sodium induced proliferation of VSMCs, specific inhibitors of JNK and ERK1/2 were used in this study. As shown in Fig. 5, both SP600125, a JNK inhibitor (Fig. 5A), and PD98059, an ERK1/2 inhibitor (Fig. 5B), almost completely inhibited PCNA expression induced by the addition of Na⁺ (30 mM; additional to the levels already in medium). The JNK inhibitor, SP600125, also decreased the expression of p-ERK1/2 which was induced by the addition of Na⁺ (30 mM; additional to the levels already in medium) (Fig. 5C). However, the ERK1/2 inhibitor, PD98059, did not significantly affect the phosphorylation level of JNK (Fig. 5D). These results indicated that high sodium levels induced the expression of PCNA through JNK/ERK1/2 pathway, and that JNK was located upstream of ERK1/2.

In the present study, we also used SB203580 (a p38 MAPK inhibitor) in order to examine the role of p38 MAPK in the high-sodium induced proliferation of VSMCs. Surprisingly, SB203580 markedly increased PCNA expression at the protein level (Fig. 6A). Conversely, anisomycin, the activator of p38 MAPK, inhibited PCNA expression (Fig. 6B). These results suggested that activated p38 MAPK played an opposite role to JNK and ERK1/2 in the high-sodium induced proliferation of VSMCs.