Mechanism of endothelial nitric oxide synthase phosphorylation and activation by tentacle extract from the jellyfish Cyanea capillata

Drugs and chemicals reagents

The HUVECs cell line was purchased from Zhongqiaoxinzhou Biotech (Shanghai, China). MTT assay kit and NOS assay kit were purchased from Beyotime (Jiangsu, China). Human NO ELISA assay kit was purchased from Sangon Biotech (Shanghai, China). eNOS inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME) and PI3-K inhibitor Wortmannin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies against phospho-Akt (Ser473), Akt, phospho-eNOS (Ser1177) and eNOS were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody against GAPDH was purchased from Abcam (Cambridge, MA, USA). HRP-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Beyotime (Jiangsu, China). Fluo-4 AM was purchased from Invitrogen (Carlsbad, CA, USA). The stock solution of 1 mM was prepared by adding dimethyl sulfoxide (DMSO) to solid powder. The working solution of 5 μM was prepared by adding serum free medium to the stock solution. 1 × HBSS (without phenol red, liquid, sterile-filtered) and 1 × HBSS (without phenol red,1.26 mM CaCl₂) were purchased from Sangon Biotech (Shanghai, China). L-type calcium channel blocker Nifedipine, PKA inhibitor H89, IP₃ receptor blocker Heparin and PKC inhibitor PKC 412 were purchased from Sigma-Aldrich (St. Louis, MO, USA). In the measurement of Ca²⁺ mobilization, Nifedipine solution of 100 μM was prepared by Ca²⁺-containing HBSS, whereas H89 solution of 10 μM, Heparin solution of 125 IU and PKC 412 solution of 10 μM were prepared by Ca²⁺-free HBSS.

TE preparation from the jellyfish C. capillata

Specimens of C. capillata were collected in June, 2014, in the Sanmen Bay, East China Sea, and identified by Professor Huixin Hong from the Fisheries College of Jimei University, Xiamen, China. The removed tentacles were preserved in plastic bags on dry ice and immediately shipped to Shanghai, where the samples were frozen at −70°C until use. TE was prepared following the method as described in previous reports (Bloom, Burnett & Alderslade, 1998; Wang et al., 2013b). Briefly, frozen tentacles were thawed at 4°C and immersed in filtered seawater at a mass/volume ratio of 1:1 to allow autolysis of the tissues for four days. The mixture was stirred for 30 min twice daily. The autolyzed mixture was centrifuged at 10,000× g for 15 min, thrice. The resultant supernatant was the TE. All procedures were performed at 4°C or in an ice bath. The TE was centrifuged at 10,000× g for 15 min to remove the sediments, followed by dialysis against phosphate buffered saline (PBS, 0.01 mol/L, pH 7.4) for over 8 h before use. The protein concentration in the preparations was determined using the method of Bradford.

Endothelial cell cultures

Human umbilical vein endothelial cells (HUVECs) were cultured in high glucose DMEM (Hyclone, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified incubator with 95% air and 5% CO₂.

Cell viability assay

Cell viability was determined by the MTT assay. Cells were plated in 96-well culture plates at a density of 10⁴ cells/ml. After incubation for 24 h, cell groups were respectively treated with various doses of TE (0–24 μg/ml) for 1 h or 6 h. Then, 20 μl MTT reagent (5 mg/ml) was added to each well. At 4 h later, the supernatants were removed and the formazan dye was subsequently dissolved in DMSO. The absorbance value at 490 nm was measured using a microplate reader (BioTek, Winooski, VT, USA).

Measurement of eNOS activity in HUVECs

Cells were serum-starved overnight in 96-well culture plates before measurements. In the dose–effect experiments, cells were incubated with different concentrations of TE (0–4 μg/ml) at 37°C for 1 h. In the time-effect experiments, cells were incubated with TE (1 μg/ml) for different time durations (0–180 min) at 37°C. After treatments, eNOS activity was measured according to manufacturer’s instructions (NOS assay kit). Briefly, cells were exposed to 100 μl reaction buffer solutions and subsequent 100 μl reaction solutions for 2 h with/without eNOS inhibitor (L-NAME). Then the plates were observed on a microplate reader (BioTek, Winooski, VT, USA) at excitation/emission wavelengths of 495/515 nm. The activity of eNOS was calculated by the following equation: relative activity (eNOS) = (RFU_stimulated − RFU_{inhibitor+stimulated})/(RFU_unstimulated − RFU_{inhibitor+unstimulated}).

Measurement of NO concentration in HUVECs

HUVECs were divided into three groups. In the dose–effect experiments, cells were incubated with different concentrations of TE (0–4 μg/ml) at 37°C for 1 h. In the time-effect experiments, cells were incubated with TE (1 μg/ml) for different time durations (0–180 min) at 37°C. In the third group, cells were incubated with eNOS inhibitor L-NAME or PI3-K inhibitor Wortmannin for 15 min before treatment with TE (1 μg/ml, 1 h). After incubation, culture supernatants were collected, and NO concentration was measured using a microplate reader (BioTek, Winooski, VT, USA) in the absorbance value at 450 nm according to manufacturer’s instructions (Human NO ELISA assay kit).

Western blotting

In the time-effect group, HUVECs were treated with TE (1 μg/ml) for different time durations (0-60 min). In PI3K/Akt-dependent signaling pathways, HUVECs were treated with TE (1 μg/ml) for 30 min in the presence or absence of Wortmannin. In calcium-dependent signaling pathways, HUVECs were treated with TE (1 μg/ml) for 30 min in the presence of extracellular Ca²⁺-containing, Nifedipine, extracellular Ca²⁺-free, H89, Heparin and PKC 412, respectively.

After treatments, cells were lysed on ice in RIPA buffer with protease inhibitor (1% PMSF). The protein content of the lysate was measured by the method of Bradford. Equal amounts of protein per sample were loaded on 10% SDS-PAGE gels and then transferred to the nitrocellulose membranes. The membranes were subsequently blocked with 5% non-fat dry milk in TBST (3 g Tris-base, 8 g NaCl, 0.2 g KCl, 0.05% Tween-20, dilute with water to 1,000 ml, pH 7.4) for 2 h at room temperature. Then, the samples were incubated overnight at 4°C with primary antibodies as follows: p-Akt (1:1,000), Akt (1:1,000), p-eNOS (1:500), eNOS (1:500) and GAPDH (1: 5,000), with gentle shaking. The ECL method was used with secondary antibodies (HRP-conjugated anti-rabbit IgG and anti-mouse IgG) at a dilution of 1:5,000 for 2 h at room temperature. After that, membranes were exposed using a chemiluminescent detection system (Syngene G: Box; Syngene, Cambridge, UK). Quantitative densitometric analyses of immunoblots were performed using an ImageJ software (Ver. 1.48), and the relative ratio was calculated.

Measurement of Ca²⁺ mobilization

HUVECs were cultured in a confocal dish (Coverglass Bottom Dish; Corning, Inc., Corning, NY, USA) and serum-starved overnight before use. Cells were loaded with Fluo-4 AM working solution (5 μM) at 37°C in the dark for 30 min, then washed three times with Ca²⁺-free HBSS to remove excess extracellular dye.

To characterize the possible contribution of extracellular calcium, the cells were pre-treated with 1.26 mM Ca²⁺-containing HBSS with/without Nifedipine (100 μM) for 15 min before the addition of TE (10 μg/ml final concentration). To examine the possible participation of intracellular calcium stores, the cells were pre-treated with Ca²⁺-free HBSS in the presence or absence of H89 (10 μM), Heparin (125 IU) or PKC 412 (10 μM) for 15 min before the addition of TE (10 μg/ml final concentration). All the concentrations of the inhibitors were screened out and proved effective in similar preparations of the preliminary experiments.

The fluorescence of Ca²⁺ mobilization in HUVECs were monitored using a laser scanning confocal microscope (Olympus FV 1000; Olympus, Tokyo, Japan) with excitation and emission wavelengths of 488 nm and 526 nm, respectively. Fluorescence images were recorded as time-series mode per 10 s intervals (100 images altogether). TE was added after the first five images were collected. Fluorescence intensities were obtained from the data set of images using FV10-ASW 3.1 (Olympus, Tokyo, Japan).

Statistical analysis

In the experiments, the images shown were the representatives of at least three experiments performed on different experimental days. Data were presented as mean ± SEM. Analysis of variance (ANOVA) and Student’s t-test were used in statistical evaluation of the data as appropriate. P-values less than or equal to 0.05 were considered significant.

Effects of TE on the viability of HUVECs

As shown in Fig. 1, when HUVECs were treated with various doses of TE for 1 h, the viability was decreased at the concentrations of 12–24 μg/ml in a dose-dependent manner, and the IC₅₀ was 15.55 μg/ml. However, TE at 0.5–8 μg/ml, did not significantly influence the cell viability after 1 h treatment. Similarly, after treatment with TE for 6 h, the viability of cells was decreased at the concentrations of 4–24 μg/ml in a dose-dependent manner, and the IC₅₀ was 14.88 μg/ml. But TE at 0.5–2 μg/ml did not significantly influence the cell viability, either. Therefore, the relatively safe dose of TE less than 8 μg/ml was selected for the next study.

Effect of TE on the eNOS activity and NO production in HUVECs

Treatments with various doses of TE (0–4 μg/ml) increased the activity of eNOS in a concentration-dependent manner within 1 h in HUVECs: a significant increase was induced at the concentration of 0.5 μg/ml and the peak at 4 μg/ml (Fig. 2A). Similarly, TE-induced NO production was also concentration-dependent within 1 h: a significant increase was induced at the concentration of 1 μg/ml and the peak at 4 μg/ml (Fig. 2C). Therefore, the dose of 1 μg/ml was used in the next time-effect experiments. It was also showed a significant time-dependent increase was induced by TE (1 μg/ml) in the activity of eNOS (Fig. 2B) and NO production (Fig. 2D), which reached the maximum at about 1 h and maintained until 3 h. Therefore, the concentration of 1 μg/ml and the time of 1 h were selected for the next study on PI3K/Akt/eNOS signaling pathways.

Effect of TE on the PI3K/Akt/eNOS signaling pathway in HUVECs

We next examined the phosphorylation of Akt and eNOS after the stimulation with TE (1 μg/ml). As shown in Fig. 3A, Akt phosphorylation was significantly induced by TE from 15 to 45 min, with the maximum phosphorylation occurring at about 15 min, while TE seemed to have no effect on the level of total Akt. Similarly, eNOS phosphorylation was also induced by TE from 15 to 60 min, with the maximum phosphorylation at about 30 min, while TE had no effect on the level of total eNOS, either. These results indicated that TE could activate the Akt/eNOS pathways. On the other hand, since Akt had been reported to phosphorylate eNOS via PI3-kinase, we then pretreated HUVECs with the PI3-K inhibitor Wortmannin (10 μM) before TE to investigate whether this upstream signaling pathway was involved. As shown in Fig. 3B, the inhibition of PI3-kinase completely blocked the TE-induced Akt/eNOS phosphorylation, thus demonstrating the requirement for this kinase during the Akt/eNOS activation by TE.

Effect of PI3K/Akt/eNOS pathway inhibition on the TE-induced NO production

As shown in Fig. 4, both eNOS inhibitor L-NAME and PI3-K inhibitor Wortmannin resulted in a significant reduction in TE-induced NO production, which indicated that PI3K/Akt/eNOS mediated the release of NO induced by TE, suggesting an important role of the PI3K/Akt/eNOS pathway in TE-induced NO release.

Effects of TE on intracellular Ca²⁺ concentration changes in HUVECs

To explore the effects of TE upon the Ca²⁺ concentration in HUVECs, we incubated the cells with the Ca²⁺ indicator dye Fluo-4 AM, and then stimulated them with TE whilst the time-series images of intracellular Ca²⁺ levels were detected. As shown in Fig. 5, TE could induce a rapid rise in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), which peaked within 1 min and sustained for about 10 min. Secondly, to determine the involvement of extracellular calcium, we next used the L-type calcium blocker Nifedipine (in Ca²⁺-containing HBSS) and Ca²⁺-free HBSS instead of the normal HBSS for the examination of TE-induced [Ca²⁺]_i changes. Our results showed that neither Nifedipine nor extracellular Ca²⁺-free had influences on the TE-induced [Ca²⁺]_i rise, which indicated that TE-induced [Ca²⁺]_i rise did not come from the extracellular Ca²⁺ influx, but mainly from the intracellular stored Ca²⁺ release. Therefore, to further explore the source of Ca²⁺, we next investigated the effects of PKA inhibitor H89, IP₃ receptor blocker Heparin and PKC inhibitor PKC 412 on the [Ca²⁺]_i elevation. Our results showed that the TE-induced [Ca²⁺]_i rise was significantly attenuated either by Heparin or by PKC 412, but not affected by H89, suggesting that the IP₃R and PKC signaling play major roles in the TE-induced [Ca²⁺]_i elevation.

Effect of calcium signaling on the TE-induced Akt/eNOS activation

Since eNOS is also activated by a rapid increase in the intracellular Ca²⁺, we next explored the role of Ca²⁺ in the activation of Akt/eNOS pathway in response to TE. As shown in Fig. 6, we indeed found the evidence for a critical role of Ca²⁺ in the TE-induced Akt/eNOS activation. Firstly, we found that TE (in Ca²⁺-containing HBSS) could induce the phosphorylation of both Akt and eNOS, while the phosphorylation level was dramatically decreased by the removal of extracellular calcium, indicating that Ca²⁺ was essential to the TE-induced Akt/eNOS activation. Secondly, to further elucidate the contribution of extracellular and intracellular calcium to the effects of TE-induced Akt/eNOS activation, we similarly performed the Akt/eNOS activity assays in the presence of L-type calcium channel blocker Nifedipine (100 μM), PKA inhibitor H89 (10 μM), IP₃ receptor blockers Heparin (125 IU) and PKC inhibitor PKC 412 (10 μM), respectively. As shown in Fig. 6, the phosphorylation of Akt/eNOS was completely attenuated by the incubation with Heparin or PKC 412, which indicated that the Akt/eNOS activation might be associated with Ca²⁺ release from the ER through IP₃R and PKC pathways. This result was in consistent with that of the calcium fluorescence assay. However, the expressions of p-Akt/p-eNOS were not affected by Nifedipine or H89, which indicated that the Akt/eNOS activation was not due to the influx of extracellular Ca²⁺ via L-type calcium channel or through PKA-dependent Ca²⁺ signaling.