Hydrogen Sulfide Increases Nitric Oxide Production and Subsequent S-Nitrosylation in Endothelial Cells

Hydrogen sulfide (H2S) and nitric oxide (NO), two endogenous gaseous molecules in endothelial cells, got increased attention with respect to their protective roles in the cardiovascular system. However, the details of the signaling pathways between H2S and NO in endothelia cells remain unclear. In this study, a treatment with NaHS profoundly increased the expression and the activity of endothelial nitric oxide synthase. Elevated gaseous NO levels were observed by a novel and specific fluorescent probe, 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid methyl ester (FA-OMe), and quantified by flow cytometry. Further study indicated an increase of upstream regulator for eNOS activation, AMP-activated protein kinase (AMPK), and protein kinase B (Akt). By using a biotin switch, the level of NO-mediated protein S-nitrosylation was also enhanced. However, with the addition of the NO donor, NOC-18, the expressions of cystathionine-γ-lyase, cystathionine-β-synthase, and 3-mercaptopyruvate sulfurtransferase were not changed. The level of H2S was also monitored by a new designed fluorescent probe, 4-nitro-7-thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) with high specificity. Therefore, NO did not reciprocally increase the expression of H2S-generating enzymes and the H2S level. The present study provides an integrated insight of cellular responses to H2S and NO from protein expression to gaseous molecule generation, which indicates the upstream role of H2S in modulating NO production and protein S-nitrosylation.


Introduction
Gas molecules that are produced by cells have been discussed for several decades regarding their protective role in the vascular system. Recently, the diverse physiologic actions of carbon monoxide (CO), nitric oxide (NO), and hydrogen sulfide (H 2 S) and their role in preventing diseases through the mediation of gas-regulating and -sensing mechanisms have attracted a great deal of interest [1]. For example, NO plays an important role in the regulation of the cardiovascular function through a posttranslational protein Snitrosylation on the cysteine residue [2]. In our previous study, a mechanical shear flow is regarded as protective for endothelial cells (ECs), leading to a series S-nitrosylation of proteins [3]. Investigating the reported mechanisms of NO on EC protection, the NO-mediated S-nitrosylated proteins, such as F1F0-ATPase, reduced the generation of Ca 2+ and ROS in mitochondria during ischemia/reperfusion injury [4]. NO was also reported to be essential in the prevention of irreversible oxidative stress and finally provided protection from several diseases including cancer, diabetes, and neuron degeneration [5][6][7].
The toxic effects of hydrogen sulfide (H 2 S) on living organisms have been recognized for nearly 300 years.  In recent years, however, interest has been directed towards H 2 S as the third gaseous mediator, which has been shown to exhibit potent vasodilatory activity both in vitro and in vivo. This is assumed to be realized by opening vascular smooth muscle K ATP channels [8]. Of the three enzymes, cystathionine--lyase (CSE), cystathionine--synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) can utilize L-cysteine as a substrate to produce H 2 S. Deficiency of H 2 S-producing enzymes results in some disorders such as homocystinuria, which is characterized by mental retardation, skeletal abnormalities, increased urine homocysteine, increased risks of thromboembolism, and early onset of atherosclerosis [9][10][11]. H 2 S was also reported to protect against vascular remodeling from endothelial damage [12]. Recently, a signaling molecule for H 2 S was shown to regulate vascular relaxation and angiogenesis via potassium channel S-sulfhydration [13][14][15].
With a similar physiological function, it is interesting to discuss the interactions between H 2 S and NO in responding stimuli. In the reports cited above, H 2 S and NO in synergy might regulate smooth muscle relaxation and also mitochondrial integration [16,17]. H 2 S triggers late-phase preconditioning in the postischemic small intestine by an NO-and p38 MAPK-dependent pathway [18]. Despite H 2 S inhibiting NO production in lipopolysaccharide-stimulated macrophages, the H 2 S can also stimulate NO production from other cells [19,20].
Because of the technical difficulty in detecting gaseous molecules, in the current study, not only monitoring the regulations of theses enzymes but also quantifying the molecules of H 2 S and NO specifically with the new designed fluorescent probes. Therefore, we question here if H 2 S has any upstream role in the regulation of endothelial NO production.

Cell Culture and Drug
Treatments. The EAhy 926 cell line was kindly donated by Cora-Jean S. Edgell, University of North Carolina, Chapel Hill. EAhy 926 cells were cultured in DMEM supplemented with fetal bovine serum (FBS, 10%), streptomycin (100 g/mL), and penicillin (100 U/mL). ECs were replaced by the same medium containing 2% FBS and incubated overnight prior to the experimental NaHS and NOC-18 treatments.

Application of Fluorescent
Probes and Imaging Conditions. For NO detection, 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid methyl ester (FA-OMe) was designed [21]. ECs with NaHS treatment were coincubated with 10 M of FA-OMe for 4 h prior to imaging. The ECs were washed three times with PBS buffer and then bathed in 2 mL of PBS. The images were obtained by the fluorescence microscope ( ex 460 nm, em 524 nm; Axiovert 40 CFL, Zeiss). As for 4-nitro-7-thiocyanatobenz-2-oxa-1,3diazole (NBD-SCN) that was used for detecting H 2 S, the cells were incubated with 5 M NBD-SCN for 30 min and then subjected to fluorescence microscope ( ex 460 nm, em 550 nm) [22]. For confocal fluorescence images study, ECs were seeded at a density of 2 × 10 5 cells/well on cover glasses (24 × 24 mm 2 ) and grown for 24 h. The cells with 10 M of FA-OMe incubation were fixed with 4% formaldehyde solution for 20 min at room temperature. Cell nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI). Cover glasses containing fixed ECs were mounted in a mixture of PBS and glycerol (1 : 1) on a microscopic slide. The cells were observed using a laser scanning confocal imaging system (Olympus FluoView 300) consisting of Olympus BX51 microscope and a 20 mW output argon ion laser.

Flow Cytometry Assay.
After fluorescence microscope observation, the ECs were washed twice with PBS and detached by tryptic reaction. ECs were collected by centrifugation and then resuspended in PBS. The fluorescence was immediately measured by the Accuri C6 flow cytometer (BD, NJ, USA) with excitation and emission settings of 488 and 530 nm, respectively. The fluorescence strength was obtained from 1 × 10 4 cells and statistically calculated from three repeats.

Evaluation of Protein S-Nitrosylation.
The cell lysates (200 g) after NaHS treatment were blocked by methyl methanethiosulfonate (MMTS), reduced by ascorbate, and labelled by biotin according to reported guideline [23]. The biotinylated lysates were then subjected to a reductantfree SDS-PAGE and western blotted with streptavidin-HRP (1 : 3000) following a previous study [24].

NaHS Increased the Protein Level of eNOS and the Phosphorylation on Serine 1177
Residue. Endothelial nitric oxide synthase (eNOS) is responsible for endothelial nitric oxide (NO) production and the enzyme activity is reported to be highly affected by posttranslational phosphorylation on serine 1177 residue (S1177) [25]. In this study, with the treatment of different concentrations of NaHS (1∼100 M), we found that 50 M of NaHS can significantly enhance both the eNOS expression and the phosphorylation of the serine 1177 residue (peNOS 1177 ) (Figures 1(a) and 1(c)). This concentration conforms well with several vascular research articles [26]. At this concentration, the highest expression level of eNOS and peNOS 1177 was observed at 2 hours (Figures 1(b) and 1(d)).

Cellular NO Was Precisely Determined by Specific Fluorescent Probes.
In addition to the expression of eNOS, the levels of NO molecules were further measured by the specific fluorescent probe FA-OMe. This can distinguish NO and other reactive oxygen species (ROS) from reactive nitrogen species (RNS) [21]. After NaHS treatment, the NO level was increased from 34.7 ± 2.9% to 66.4 ± 3.8% at 2 hours ( Figures 2(a) and 2(b)). By using confocal microscopy, despite the fact that the basal fluorescence in the control treatment was difficult to see, the broadly distributed NO was observed in the cytosol and also in the nuclei (Figure 2(c)).

The Expression Profiles of AMPK and Akt in the Presence
of H 2 S. 5 AMP-activated protein kinase (AMPK) is an enzyme that plays a role in cellular energy homeostasis. Besides protein kinase B (Akt), AMPK is also reported to activate eNOS by phosphorylating Ser1177 in response to various stimuli [27]. In the current study, H 2 S increased the protein level of AMPK at 2 h and returned to a basal level at 12 h. However, sustainable expressions of Akt were observed from 0.5 to 12 h (Figures 3(a) and 3(b)). This indicated that H 2 S can stimulate eNOS activity through AMPK and Akt pathways. Similar findings were also reported recently [28,29].

H 2 S-Increased Bioavailability of NO That Can Enhance
Protein S-Nitrosylation. According to previous data, we confirmed that NO level got elevated by H 2 S. Since protein S-nitrosylation/denitrosylation is regarded as important in cardioprotection, its investigation of protein S-nitrosylation is, hence, important for applied medical purposes [2,3,30]. By using a modified biotin switch, we could identify at least 8 groups of increased S-nitrosoproteins and 2 groups of decreased S-nitrosoproteins (Figure 4). With the excellent performance in analyzing S-nitrosoproteins, mass spectrometry will be introduced in further identification of these proteins [24].

NO Did Not Reciprocally Increase the Expression of H 2 S-Generating Enzymes and the H 2 S Level.
After confirming that H 2 S can increase NO at the cellular level, we also examined whether NO can be synchronized at elevated H 2 S levels. As shown in Figure 5, three key enzymes are involved in the cellular H 2 S synthesis: CSE, CBS, and 3-MST. These were not changed by NOC-18 treatment (Figures 5(a) and 5(b)). Although several studies indicated that H 2 S-generating enzymes can also be exerted by NO [31], we could not find differences in our microscopic and flow cytometric analysis (46.0 ± 3.1% and 43.3 ± 4.8% separately) using NBD-SCN fluorescence probe (Figures 5(c) and 5(d)). The reported study demonstrated that H 2 S promotes NO production in ECs via the activation of a cascade of phosphorylation events, starting from p38 MAPK and Akt to eNOS, and this can be through NO-dependent or NO-independent mechanisms cascade. Thus, H 2 S may be a key regulator for angiogenic signalling pathways, whether they required NO or not [32]. This might indicate that NO works as a downstream gaseous transmitter in the endothelium.

Conclusion
In the present study, hydrogen sulfide increased nitric oxide production. This was not only concluded by studying related enzymes, but also confirmed directly by detecting the final products where NO levels were observed by a novel and specific fluorescent probe, FA-OMe, and quantified by flow cytometry. The level of H 2 S was also monitored by a new designed fluorescent probe, NBD-SCN, with high specificity. The present study provides an integrated insight of cellular responses to two gaseous molecules from protein expression to gaseous molecule generation, which indicates the upstream role of H 2 S in modulating NO production and protein S-nitrosylation.