Interaction between hydrogen sulfide/cystathionine γ-lyase and carbon monoxide/heme oxygenase pathways in aortic smooth muscle cells¹

Introduction

In the late 1980s, nitric oxide (NO) was first demonstrated as an endogenous gasotransmitter^[1] with a variety of vital functions including vasorelaxation, suppression of cell proliferation and inhibition of platelet aggregation^[2–4]. Such findings led the biomedical study into a new stage. Several years later, carbon monoxide (CO) catalyzed by heme oxygenase (HO) was confirmed as another endogenous gasotransmitter^[5] which also showed great importance in the modulation of vascular structure and functions. The studies on NO and CO have made it easier to explain the mechanisms and pathogenesis of diseases. However, the regulation of functions, as well as structures of systems under physiological or pathophysiological conditions, remains unclear. The most hopeful approach is to look for new gasotransmitters. Hydrogen sulfide (H₂S) has been generally considered a toxic gas found in the contaminated environmental atmosphere^[6]. In recent years, however, more and more studies have suggested that endogenous H₂S is another gasotransmitter^[7]. H₂S is produced endogenously from cysteine by pyridoxal-5'-phosphate-dependent enzymes, including cystathionine β-synthase and/or cystathionine γ-lyase (CSE)^[8]. The enzymes were found highly expressed in the brain, ileum and vessels. In the late 1990s, H₂S was proven to be a neuromodulator in the brain, as well as a tone regulator in smooth muscles^[9]. Endogenous H₂S is responsive to neuronal excitation by the Ca²⁺/calmodulin-mediated pathway, enhancing the NMDA receptor-mediated responses by induced cyclic adenosine monophosphate (cAMP), modifying long-term potentiation (LTP, a synaptic model of memory). More interestingly, Hosoki et al found that H₂S could be produced in the portal vein, thoracic aorta, tail artery, mesenteric artery and pulmonary artery^[10]. Recently, H₂S was found to exert an inhibitory effect on vascular smooth muscle cell proliferation^[11] and promote its apoptosis^[12]. H₂S was also found to play a role in the regulation of cardiac function and vasorelaxation by activating the K_ATP channel, a distinctive way that is different from NO and CO^[13–14].

However, gasotransmitters in vascular tissues might not exist independently, whereas they might have interactions^[15]. NO was found to be derived from vascular endothelial cells, H₂S is endogenously generated from vascular smooth muscle cells, while HO exists in both vascular endothelial cells and smooth muscle cells. This suggests that NO, CO and H₂S might represent the most rapid communicative pathway between vascular endothelial cells and smooth muscle cells, being involved in regulating the biological actions. Vascular endothelial cells and smooth muscle cells are the main place for the production of gasotransmitters. At the same time, they are the target cells of gasotransmitters. The above facts provide the basis for the likelihood of interactions among gasotransmitters. Our previous study demonstrated that exogenous supplements of H₂S could alleviate hypoxia-induced elevation of pulmonary arterial pressure. At the same time, H₂S might impact the increased CO production in pulmonary arteries on a chronic hypoxic animal model. However, the above results were derived from hypoxic pulmonary hypertensive rats; whether there are any impacts of H₂S on the CO pathway and vice versa in aortic smooth muscle cells (ASMC) under physiological conditions and its mechanisms remains unclear. Therefore, the present study was undertaken to examine the possible interaction between H₂S/CSE and CO/HO pathways in ASMC.

Materials and methods

Smooth muscle cell culture Male Sprague-Dawley rats, weighing 200–220 g, were killed by anesthetic overdose (intraperitoneal injection of pentobarbital sodium). The thoracic aorta were stripped of adventitia and removed to culture media sterilely. The ASMCs were isolated and cultured according to the method used by Hirata et al^[16]. Briefly, cells were maintained in DMEM containing 5 mmol/L glucose, 10% FBS, and antibiotics in a CO₂ incubator at 37 ^oC. The subcultures of ASMC from passage 4 were used in the experiments and cells were cultured for 24 h in serum-free DMEM before each experiment. The cells were divided into 4 groups according to different treatments, and each group included at least 6 wells. (1) the control group, where cells were continuously cultured in serum-free DMEM; (2) the zinc protoporphyrin (ZnPP, an inhibitor of HO) group, where 20 mmol/L ZnPP was added; (3) the propargylglycine (PPG, an inhibitor of CSE) groups, where 2 mmol/L, 4 mmol/L and 10 mmol/L of PPG were added, respectively; and (4) the sodium hydrosulfide (NaHS, a H₂S donor) groups, where 1×10^-5 mol/L, 1×10^-4 mol/L and 1×10^-3 mol/L of NaHS were added, respectively. Each of the above groups was further divided into 6 h, 12 h, 18 h and 24 h subgroups. ZnPP (Sigma, St Louis, MO,USA) was dissolved in 50 mmol/L sodium carbonate. PPG (Sigma, St Louis, MO, USA) and NaHS (Sigma, St Louis, MO, USA) were dissolved in sterile water.

Measurement of carboxyhemoglobin (HbCO) in conditioned medium To examine the relative amount of CO being released from the ASMC into the medium, hemoglobin (50 µmol/L) was added to the cells during the last hour of incubation, and HbCO was measured by a spectrophotometric method^[17]. Medium 0.5 mL was mixed with 1 mL of the hemoglobin solution, which was derived from a mixture of 0.25 mL of fresh-packed erythrocytes from rats and 50 mL of 0.25 mol/L ammonia solution. Then, 0.1 mL of the sodium dithionite was added. The difference of absorbance at 568 and 581 nm was read via the double-wavelength spectrophotometer. Both the wavelengths were selected according to the absorbance spectrum of HbCO and oxygenated hemoglobin (HbO₂), at which the difference of absorbance of HbCO was largest and the difference of absorbance of HbO₂ was 0. The percentage of HbCO was then calculated from a standard curve derived by mixing different proportions of 2 hemoglobin solutions containing 100% HbCO and 100% HbO₂. We obtained these solutions by gassing hemoglobin solutions with pure oxygen and CO, respectively. For our spectrophotometer, the following relationship was obtained from the standard curve: %HBCO=[(OD₅₆₈- OD₅₈₁)-0.03858]/0.0498.

Measurement of H₂S in conditioned medium H₂S was measured with a sulfide sensitive electrode (Model 9616; Orion Research; Beverly, MA, USA) as described previously^[18]. In brief, sulfide antioxidant buffer was added into standards or samples at a ration of 1:1 and then stirred thoroughly. Electrodes were rinsed in distilled water, blotted dry and placed into standards and samples. When a stable reading was displayed, the millivolt value was recorded. The H₂S concentration was calculated against the calibration curve of the standard H₂S solution.

Western blotting Cultured cells (3×10⁶) were harvested and lysed in a lysis buffer (0.5 mol/L EDTA, 1 mol/L Tris-HCl, pH 7.4, 0.3 mol/L sucrose, 1 µg/mL antipain hydro-chloride, 1 mmol/L benzamidine hydrochloride hydrate, 1 µg/mL leupeptin hemisulfate, 1 mmol/L 1,10-phenanthroline monohydrate, 1 µmol/L pepstatin A, 0.1 mmol/L phenyl-methylsulfonyl fluoride and 1 mmol/L iodoacetamide). The extracts were clarified by centrifugation at 14 000×g for 15 min at 4 ^oC. SDS-PAGE and Western blot analysis were performed according to experimental protocol. The primary antibody dilutions were as follows: 1:2000 for HO-1 (Sigma, St Louis, MO, USA), 1:50 for CSE and 1:1000 for β-actin (TBD, Tianjin, China). HRP-conjugated secondary antibody was used at 1:5000. The immunoreactions were visualized by electrochemiluminescence (ECL) and exposed to X-ray film (Kodak Scientific Imaging film, X-Omat Blue XB-1, USA). Membranes were stripped by incubation in a buffer containing 100 mmol/L β-mercaptoethanol, 2% SDS, and 62.5 mmol/L Tris-HCl, pH 6.8.

Statistical analysis The results were expressed as Mean±SD. ANOVA was used to compare the mean values, including HbCO level in the cultured ASMC medium and HO-1 expression by the ASMC in the various groups. Post-hoc analysis was then used to compare the data between the different groups. Independent t-test was used to estimate the differences in H₂S content in the cultured ASMC medium and CSE expression by the ASMC between the control group and the ZnPP group. P<0.05 was considered statistically significant.

Results

Impact of endogenous CO on the H₂S/CSE pathway To examine the impact of endogenous CO on H₂S release by the ASMCs in the medium, the ASMCs were treated with ZnPP at a concentration of 20 µmol/L. After ZnPP treatment for 6 h, 12 h,18 h and 24 h, the H₂S content in the medium markedly increased by 11.81%, 18.79%, 41.58% and 12.19%, respec-tively, as compared with that in the medium without ZnPP treatment (all P<0.01) as seen in Table 1. The results also showed that the peak time point of the augmentation was at 18 h after the treatment of ZnPP.

Table 1 H₂S contents (µmol/L) in the medium of ASMC cultured in the presence or absence of ZnPP. n=8. Mean±SD. ^bP<0.05 vs control group.
Full table

After the treatment of ZnPP at a concentration of 20 µmol/L with cultured ASMC for 6 h, 12 h,18 h, and 24 h, CSE expression by ASMC markedly increased by 35.73%, 31.94%, 58.22% and 16.78%, respectively, compared with the controls (all P<0.01). The peak time point of the augmentation was also at 18 h after the the ZnPP treatment (Figure 1).

Figure 1 Effect of ZnPP on CSE protein expression by ASMC. After the treatment of ZnPP at a concentration of 20 µmol/L with cultured ASMC for 6 h, 12 h, 18 h, and 24 h, CSE expression by ASMC markedly increased. (A) Representative immunoblots of CSE in the ASMC extracts from the control group and the ZnPP treatment group; and (B) CSE expression by ASMC normalized with β-actin expression. ^bP<0.05 vs control group.

Impact of endogenous H₂S on the CO/HO pathway To examine the impact of endogenous H₂S on CO release by medium of cultured ASMC, the ASMCs were treated with PPG at a concentration of 2 mmol/L, 4 mmol/L and 10 mmol/L, respectively. After PPG treatment at each concentration for 6 h and 12 h, there were no significant changes in CO release by cultured ASMC as seen in Table 2 (P>0.05). The CO release started to be augmented by 4.02% following the 24 h treatment with PPG at 2 mmol/L, but was clearly augmented by 7.11% and 7.78% following 18 h and 24 h treatment with PPG at 4 mmol/L, respectively (P<0.01). Also, in the presence of PPG at 10 mmol/L, CO release markedly increased by 5.38% and 15.57% compared with the controls at 18 h and 24 h, respectively (all P<0.01; Table 2).

Table 2 Impact of PPG on HbCO content (%) in the supernatant of cultured ASMC. n=8. Mean±SD. ^bP<0.05 vs control group. ^eP<0.05 vs PPG 2 mmol/L group. ^hP<0.05 vs PPG 4 mmol/L group.
Full table

After PPG treatment at each concentration for 6 h, there were no significant changes in HO-1 expression by cultured ASMC (P>0.05). The HO-1 expression by cultured ASMC started to be strengthened by 34.95% following 24 h treatment with PPG at a concentration of 2 mmol/L, but was evidently strengthened by 169.19% and 54.42% after 18 h and 24 h treatment with PPG at 4 mmol/L, respectively(P<0.01). In the presence of PPG at 10 mmol/L, HO-1 expression by cultured ASMC markedly increased by 60.29%, 102.31% and 243.36% compared with the controls at 12, 18 h and 24 h, respectively (all P<0.01). The results also showed that PPG increased HO-1 expression by cultured ASMC at 24 h in a dose-dependent manner (P<0.01; Figure 2).

Figure 2 Effect of PPG on HO-1 expression by ASMC. After the treatment of PPG at different concentrations with ASMC for 6 h, 12 h, 18 h, and 24 h, HO-1 expression by ASMC increased, respectively. (A) Representative immunoblots of HO-1 in the ASMC extracts from the control group and the PPG treatment group. Lane 1: control group; lane 2: PPG 2 mmol/L group; lane 3: PPG 4 mmol/L group; lane 4: PPG 10 mmol/L group. (B) HO-1 expression relative to control, with control taken as 1. ^bP<0.05 vs control group. ^eP<0.05 vs PPG 2 mmol/L group. ^hP<0.05 vs PPG 4 mmol/L group.

Impact of H₂S donor on the CO/HO pathway To examine the impact of H₂S on CO release by cultured ASMC, the ASMCs were treated with NaHS, a H₂S donor, at a concentration of 1×10^-5 mol/L, 1×10^-4 mol/L, and 1×10^-3 mol/L, respectively (Table 3). After treatment for 6 h, NaHS at 1×10^-4 mol/L and 1×10^-3 mol/L decreased HbCO content in the supernatant of cultured ASMC by 3% and 5.09% (all P<0.05), respectively. After treatment for 12 h, NaHS at each concentration decreased HbCO content in the supernatant of cultured ASMC by 2.74%, 5.47%, and 8% (all P<0.05) in a dose-dependent manner. After treatment for 18 h and 24 h, NaHS at 1×10^-5 mol/L and 1×10^-4 mol/L did not change the HbCO content in the supernatant (all P>0.05), while NaHS at 1×10^-3 mol/L reduced the HbCO content in the supernatant significantly by 5.37% and 8.22% (all P<0.05), respectively.

Table 3 Impact of NaHS on HbCO content (%) in the supernatant of cultured ASMC. n=8. Mean±SD. ^bP<0.05 vs control group. ^eP<0.05 vs NaHS 1×10^-5 mol/L group. ^hP<0.05 vs NaHS 1×10^-4 mol/L group.
Full table

After NaHS treatment at each concentration for 6 h and 12 h, HO-1 expressions by cultured ASMC decreased in a dose-dependent manner (all P<0.05). The results also showed that NaHS at 1×10^-3 mol/L inhibited HO-1 expression by cultured ASMC at all time points of the treatments, while NaHS at 1×10^-5 mol/L and 1×10^-4 mol/L inhibited HO-1 expression by cultured ASMC at 6 h and 12 h of the treatment (Figure 3).

Figure 3 Effect of NaHS on HO-1 expression by ASMC. After the treatment of NaHS at different concentration with cultured ASMC for 6 h, 12 h, 18 h, and 24 h, HO-1 expression by ASMC decreased, respectively. (A) Representative immunoblots of HO-1 in the ASMC extracts from the control group and the NaHS treatment group. Lane 1: control group; lane 2: NaHS 1×10^-5 mol/L group; lane 3: NaHS 1×10^-4 mol/L group; lane 4: NaHS 1×10^-3 mol/L group. (B) HO-1 expression relative to control, with control taken as 1. ^bP<0.05 vs control group. ^eP<0.05 vs NaHS 1×10^-5 mol/L group. ^hP<0.05 vs NaHS 1×10^-4 mol/L group.

Discussion

Hydrogen sulfide (H₂S) has been recognized as a toxic gas in water pollution and industrial air pollution with a strong odor for long time. More interestingly, Hosoki et al^[10] found that H₂S could be produced in the portal vein, thoracic aorta, tail artery, mesenteric artery and pulmonary artery. The vascular smooth muscle was relaxed by the treatment of H₂S in a dose-dependent manner, including intact endothelia and removed endothelia, and in synergy with NO. Endogenous H₂S in rat vascular tissues, as a vascular relaxant factor, could maintain the basal blood pressure balance under physiological conditions^[9]. It could also suppress the proliferation of cultured vascular smooth muscle cells (vascular smooth muscle cells) through the mitogen-activated protein kinase pathway in a dose-dependant manner in vitro^[11]. Based on its endogenous metabolism and physiological functions, H₂S is well positioned in the novel family of endogenous gaseous transmitters. More and more evidence is proving that H₂S might be the third endogenous signaling gasotransmitter besides NO and CO^[7]. CO acts as a second gasotransmitter that shares some of the physiochemical properties of NO^[15]. Like NO, CO can bind to the iron atom of the heme moiety associated with soluble guanylyl cyclase, thereby activating the enzyme and increasing intracellular cGMP produc-tion. CO is produced endogenously by various cell types as a byproduct of heme catabolism, in which HO catalyzes the degradation of heme into equimolar amounts of biliverdin, iron and CO^[5].

Recently, the rapid development of systems biology makes us aware that only a single molecule or its pathway cannot regulate the cardiovascular functions independently^[19]. It was discovered that H₂S is formed in VSMC, while CO is generated mainly in VSMC, but also in vascular endothelial cells, acting on vascular cells in an autocrine/paracrine manner. Previous studies have also demonstrated that gasotransmitters always interact. For instance, previous studies have indicated that endogenous CO/HO and NO/nitric oxide synthase (NOS) interacted with each other, as did NO/NOS and H₂S/CSE pathways, therefore playing important roles in regulating the homeostasis of the cardiovascular system^[20,21]. However, how CO/HO and H₂S/CSE pathways interact is still not clear.

For the purpose of understanding if there are any interactions between CO on the H₂S pathway in ASMC under physiological conditions, we performed the above studies culturing ASMC treated with the CO/HO inhibitor, H₂S/CSE inhibitors and H₂S donor, respectively. We noticed that there was an impact of endogenous CO on the H₂S/CSE pathway in ASMC. We observed that after the ASMC were treated with ZnPP, an inhibitor of HO^[22], the H₂S content in the medium markedly increased compared to the controls. The results demonstrated that endogenous CO could down-regulate H₂S production under physiological conditions. The mechanisms, however, were not fully understood. As we know, CSE was the main enzyme in ASMC catalyzing the endogenous production of H₂S. Therefore, we speculated that the induced ASMC CSE expression by ZnPP treatment might be involved in the mechanism by which endogenous CO regulated the H₂S/CSE pathway. In our study, we found that CSE expression by ASMC treated with ZnPP markedly increased compared with that untreated with ZnPP. How-ever, further studies should be conducted to understand the impact of endogenous CO on the CSE gene expression and modulations.

To understand if there are any impacts of H₂S on the CO pathway in ASMC under physiological condition, we performed the present study and found that after treatment with PPG^[23], an inhibitor of CSE in cultured ASMC, CO release by ASMC was augmented. Moreover, NaHS, a H₂S donor, decreased HbCO content in the supernatant of cultured ASMC. The above results indicated that H₂S could markedly inhibit CO release by ASMC under physiological conditions. To examine the possible mechanisms by which H₂S regulated endogenous CO production, we employed Western blot to detect HO-1 expression by ASMC. Our data showed that PPG strengthened the HO-1 expression by cultured ASMC, while NaHS inhibited the HO-1 expression by cultured ASMC. The results revealed that the mechanisms by which H₂S regulated endogenous CO production involved at least the inhibition of HO-1 expression by cultured ASMC. However, further studies dealing with the influence of endogenous H₂S on HO-1 transcription in addition to its translation should be done.

The interaction between CO and H₂S may have potential physiological significance. Both CO and H₂S were produced in VSMC and have distinct characteristics, including fast production, rapid diffusion, extensive action and a shorter life time. They also share similar biological functions. For vascular regulation, both were demonstrated to relax the vessels and inhibit the VSMC proliferation. However, they are different in many aspects, including their signaling pathways. The coordinated regulation of these species suggests that they all are used in cell signaling. These reactions establish “cross-talk” between 2 VSMC-derived gaso-transmitters. Such adaptive mechanisms are thought to be of evolutionary importance^[24]. However, investigations on the interactions among gasotransmitters and other vasoactive substances need to be further conducted.

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