- Email: [email protected]
adenylyl cyclase pathway
Toxicology and Applied Pharmacology 289 (2015) 389–396
Contents lists available at ScienceDirect
Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap
H2S induces vasoconstriction of rat cerebral arteries via cAMP/adenylyl cyclase pathway Sen Li, Na-na Ping, Lei Cao ⁎, Yan-ni Mi, Yong-xiao Cao ⁎ Department of Pharmacology, Medical College, Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, China
a r t i c l e
i n f o
Article history: Received 16 October 2015 Revised 28 October 2015 Accepted 28 October 2015 Available online 31 October 2015 Keywords: β-Adrenoceptor cAMP Cerebral artery Constriction Hydrogen sulfide Sodium hydrosulfide
a b s t r a c t Hydrogen sulfide (H2S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H2S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H2S, concentration-dependently induced vasoconstriction on basilar artery, which was enhanced in the presence of isoprenaline, a β-adrenoceptor agonist or forskolin, an adenylyl cyclase activator. Administration of NaHS attenuated the vasorelaxant effects of isoprenaline or forskolin. Meanwhile, the NaHS-induced vasoconstriction was diminished in the presence of 8B-cAMP, an analog of cAMP, but was not affected by Bay K-8644, a selective L-type Ca2+ channel agonist. These results could be explained by the revised effects of NaHS on isoprenaline-induced cAMP elevation and forskolin-stimulated adenylyl cyclase activity. Additionally, NaHS-induced vasoconstriction was enhanced by removing the endothelium or in the presence of L-NAME, an inhibitor of nitric oxide synthase. L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the pre-contracted artery. In conclusion, H2S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Cerebral vascular tone is regulated by vascular receptors, such as adrenergic receptors, angiotensin receptors, 5-hydroxytryptamine (5-HT) receptors, and endothelium receptors, which mediate vasoconstriction or vasodilatation by their ligands, agonists or antagonists. In recent years, the gaseous transmitters, such as nitric oxide (NO) and carbon monoxide (CO), are also demonstrated to be mediators of the vascular tone. Hydrogen sulfide (H2S), a toxic gas with a strong characteristic rotten egg smell, has been well documented on its fatal intoxication (Burnett et al., 1977; Kabil and Banerjee, 2010; Smith and Gosselin, 1979). However, in recent years, H2S is accepted as the third gas transmitter after NO and CO for its similar vascular effects and biological features (Wang, 2002). These gases (NO, CO, H2S) together frequently make up a combination, so-called “gaseous triumvirate” to address the
Abbreviations: 5-HT, 5-hydroxytryptamine; 8B-cAMP, 8-bromoadenosine-3′, 5′-cyclic monophosphate; BayK, Bay K-8644; DMSO, dimethyl sulfoxide; EC50, the concentration that produce 50% of the maximal constriction; Emax, maximal constriction; Forsk, forskolin; H2S, hydrogen sulfide; HBVSMCs, human brain vascular smooth muscle cells; HEPES, hydroxyethyl piperazine ethanesulfonic acid; ISO, isoprenaline; L-NAME, NGnitro-L-arginine methyl ester hydrochloride; NaHS, sodium hydrosulfide; NO, nitric oxide. ⁎ Corresponding authors at: Department of Pharmacology, Medical College, Xi'an Jiaotong University, 76# Yanta West Road, Xi'an, Shannxi 710061, China. E-mail addresses: [email protected] (L. Cao), [email protected] (Y. Cao).
http://dx.doi.org/10.1016/j.taap.2015.10.021 0041-008X/© 2015 Elsevier Inc. All rights reserved.
physiological function (Li et al., 2009). NO and CO have been well established to play pivotal roles in the regulation of vascular homeostasis by acting as vasodilators that helps to maintain an equilibrium of vasomotion (Ashley et al., 2012, Ulrich and Huige, 2012). In comparison with NO and CO, H2S also can relax vessels, for example, isolated rat aorta (Ali et al., 2006; Hosoki et al., 1997; Kubo et al., 2007a; Zhao et al., 2001), gastric artery (Kubo et al., 2007b), coronary artery (Cheang et al., 2010), portal vein (Hosoki et al., 1997) and dilate perfused rat mesenteric (Cheng et al., 2004), and hepatic vascular beds (Fiorucci et al., 2005, Siebert et al., 2008). However, in the vascular system, the equilibrium between vasoconstrictor and vasodilator play a crucial role in maintaining the homeostasis of the vascular tone, structurally as well as functionally (Raij, 2001). Although H2S is generally considered as a vasodilator, its vasoconstrictive effect has been reported. H2S induces vasoconstriction in rat gastric artery (Kubo et al., 2007b), pulmonary artery (Olson et al., 2006), or human internal mammary artery (Webb et al., 2008). These data suggest that H2S possess a complex action in regulating vascular tone. We discovered that sodium hydrosulfide (NaHS), a donor of H2S, could induce a constriction of rat cerebral artery in experiments. Interestingly, this phenomenon is in line with the literature in which H2S elicits a vasoconstrictive effect either in rat or human vessels (Kubo et al., 2007b; Olson et al., 2006; Webb et al., 2001). Therefore, the present study was to explore the vasoconstrictive effect of NaHS in cerebral arteries and the possible mechanisms.
390
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
2. Materials and methods
2.5. Cell culture and cAMP level assay
2.1. Ethical approval
Human brain vascular smooth muscle cells (HBVSMCs) were purchased from ScienCell™ (Cat. No. 1100, Lot. No. 4706, Carlsbad, CA, USA) and cultured in DMEM supplemented with 20% fetal bovine serum, penicillin (80 U/ml) and streptomycin (0.08 mg/ml) in a humidified incubator (37 °C, 5% CO2). The HBVSMCs used for assays were from generations 4–8. The HBVSMCs were washed with PBS, and lysed with 0.1 M HCl. The cell lysates were aspirated and subjected to centrifugation at 1000 g for 10 min to collect the supernatant. The supernatant from 105 HBVSMCs was added to a well. The cAMP levels in the wells were assayed by the direct cAMP enzyme immunoassay kit (Cayman Chemical, USA).
The experimental protocol was approved by the Ethics Committee of Xi'an Jiaotong University. The investigation is conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). 2.2. Drugs and chemicals NaHS, 5-HT, acetylcholine, isoprenaline (ISO), forskolin (Forsk), 8bromoadenosine-3′,5′-cyclic monophosphate (8B-cAMP), Bay K-8644 (BayK), NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) and hydroxyethyl piperazine ethanesulfonic acid (HEPES) were purchased from Sigma Aldrich (St. Louis, USA). Dimethyl sulfoxide (DMSO) and fluo-3/AM were purchased from Biotium (Hayward, USA) and Solarbio (Beijing, China), respectively. NaHS was freshly prepared before each experiment. Forsk, BayK or fluo-3/AM were dissolved in DMSO, whereas other chemicals were dissolved in deionized water. The concentrations were expressed as the final molar concentration in the baths. 2.3. Artery preparation and functional study Male Sprague–Dawley rats weighing 250–350 g were purchased from the Experimental Animal Center of Xi'an Jiaotong University, China. The rats were housed under controlled temperature and humidity with water and food ad libitum. Rats were sacrificed by asphyxia induced by CO2 inhalation. Basilar artery was removed and placed in cold oxygenated Krebs buffer (mM: NaCl, 119; KCl, 4.6; MgCl2·6H2O, 1.2; CaCl2, 1.5; NaH2PO4·2H2O, 1.2; NaHCO3, 15; glucose, 5.6; pH 7.4). Wire myograph (Myo Technology A/S, Denmark) was used to record the isometric tension and to examine artery contractile properties in isolated cerebral artery segments. The segments of the basilar artery (2 mm in length), free of connective tissue, were mounted between two steel hooks in a 5 mL chamber containing gassed (95% O2 and 5% CO2) Krebs solution at 37 °C. Initial tension, equivalent to tension caused by 0.9 times the diameter of the vessel at 100 mm Hg (McPherson, 1992; Ping et al., 2014), was applied by using DMT Normalization v1.3 software (ADInstruments, UK) to each segment for a 60-min equilibration, where after, changes of the tension induced by drugs were recorded via LabChart 7.2 software (ADInstruments, UK). Arteries without endothelium were performed by rubbing the vessel lumen with a wire. The endothelium was identified as successful removal when the acetylcholine-induced artery relaxation was less than 30%. 2.4. Measurement of intracellular calcium ([Ca2+]i) of the artery [Ca2+]i levels of the cerebral artery were measured by spectrofluorometric method using fluo-3/AM as a calcium indicator similar to the previously described (Hashimoto et al., 2007; Sun et al., 2011). Segments of the basilar artery (3 mm in length) were fixed on the bottom of the chamber close to cover glass. The specimens were immediately immersed in HEPES-Krebs solution (mM: NaCl, 135; KCl, 5; MgSO4, 1.2; CaCl2, 2.5; glucose, 10; HEPES, 8.4) with fluo-3/AM (10 μM) for 30 min. A real-time confocal microscope (Olympus, FV1000, Japan) was used to take the fluorescent image. The segments were exposed to the drugs and the image frames were acquired continuously. The F488/F520 ratio was calculated from individual image using FV-ASW software (version 1.7, Olympus, Tokyo, Japan) and was used to indicate [Ca2+]i levels of the artery. It was considered that changes in the fluorescence ratio reflect the fluctuations in [Ca2+]i levels.
2.6. Adenylyl cyclase activity assay The HBVSMCs were suspended in lysis buffer (mM: Tris–HCl 25, EDTA 0.4, EGTA 1.0, sucrose 250, leupeptin 0.1, and PMSF 0.04, pH 7.4) for 10 min at 25 °C. Then the lysate was collected and subjected to centrifugation at 50,000 g for 30 min to obtain the membrane protein fractions (Mackay and Mochly-Rosen, 2001; Pan et al., 2008; Weber et al., 2005). A 400 μl reaction mixture (mM: ATP 1.0, NaCl 100, HEPES 50, 3-isobutyl-1-methylxanthine 0.5, MgCl2 6, GTP 0.001 and 20 μg of membrane protein fractions from 5 × 106 cells) was added to a well at 37 °C for 10 min. Reactions were stopped by addition of 0.6 ml of 10% trichloroacetic acid to the well. The productive rate of cAMP in each well was assayed by the cAMP EIA kit.
2.7. Statistical analysis All data are presented as mean ± SE. The tensions of arterial ring segments were expressed as percentage of vasoconstriction precontracted by 10−5 M 5-HT. Relaxation responses were expressed as percentage of pre-constriction induced by agonists. Emax refers to the maximum constriction induced by an agonist. The EC50 is the agonist concentration that produces 50% of the Emax which was calculated from the straight line equation between the concentration above and below the midpoint of the concentration response curve. Oneway analysis of variance (ANOVA) with or without Bonferroni's post-test was used for multiple comparisons using Graphpad Prism 5.0 (GraphPad Software, La Jolla, USA). Statistical significance was set as P b 0.05.
3. Results 3.1. The vasoconstriction of NaHS on rat basilar artery To clarify the vasoactivity of H2S on basilar artery, cumulative NaHS (10−7–10–3.5 M) was applied to the artery at initial tension. The results showed that NaHS induced a concentration-dependent vasoconstriction on basilar artery with an Emax of 45.0 ± 3.0% and an EC50 of 20.8 ± 4.7 μM (Fig. 1A; Table 1). The vasoconstriction started to decrease when the concentration of NaHS increased to 10–3.5 M (Fig. 1A). To delineate this effect, the single concentration of NaHS was used. The results indicated that NaHS (10−5, 10−4, 10–3.5, 10−3 and 10−2 M) induced vasoconstrictions of 23.6 ± 1.0%, 40.6 ± 3.7%, 56.9 ± 3.9, 61.3 ± 4.0% and 80.0 ± 6.3%, respectively (Fig. 1B). Also, other H2S donors, Na2S and GYY4137 were used. The results showed that the contractile potency of the donors from low to high was GYY4137, Na2S, and NaHS with Emax of 4.5 ± 1.6%, 17.8 ± 3.0% and 45.0 ± 3.0%, respectively (Fig. 1A). Taken together, these data suggest that H2S induces a concentration-dependent vasoconstriction on basilar artery.
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
391
Fig. 1. The vasoconstriction of H2S on rat basilar artery. (A) Representative tracer (top) of cumulative concentration NaHS induced a concentration-dependent vasoconstriction, n = 14. Summarized analysis (bottom) of the effect of different H2S donors on rat cerebral basilar arteries, n = 6–14. (B) Representative tracer (top) and summarized analysis (bottom) of single concentration of NaHS induced vasoconstriction. n = 14. Mean ± SE. ⁎⁎P b 0.01.
3.2. The relationship of NaHS-induced vasoconstriction to β-adrenergic receptors β-Adrenergic receptor acts a part in regulating tension on cerebral artery (Tsukahara et al., 1986). The effect of β-adrenergic receptor on NaHS-induced vasoconstriction was studied. The accumulative concentrations of NaHS (10−7–10–3.5 M) were added into the baths in the presence or absence of isoprenaline, a β-adrenoceptor agonist. The results showed that isoprenaline shifted the concentration-vasoconstriction curve induced by NaHS towards the left in a non-parallel manner. The Emax was 92.3 ± 5.4% and 46.5 ± 3.1% in the presence of isoprenaline and control, respectively (P b 0.01, Fig. 2; Table 1), suggesting that isoprenaline enhances the NaHS-induced vasoconstriction on basilar artery. Furthermore, the effect of NaHS on isoprenaline-induced relaxation was determined. Given together with isoprenaline, the relaxant values of isoprenaline + NaHS 10−5, 10–4.5 and 10−4 M (61.0 ± 4.9%, 38.9 ± 6.2%, 31.3 ± 6.3%) were significantly lower than that of isoprenaline alone (83.8 ± 3.4%, P b 0.01, Fig. 3). These results suggest that NaHS attenuates the relaxant effect of isoprenaline on basilar artery. Taken together, these data suggested that the H2S-induced vasomotion appears primarily via modulation of the β-adrenergic receptors on cerebral artery.
3.3. The relationship of NaHS-induced vasoconstriction to adenylyl cyclase β-Adrenergic receptor relaxes artery through activating adenylyl cyclase. Further, the effect of adenylyl cyclase on NaHS-induced vasoconstriction was studied. The cumulative concentrations of NaHS (10−7–10–3.5 M) were added into the baths in the presence or absence
Table 1 Emax and EC50 values of NaHS-induced vasoconstrictions of cerebral arteries in different statuses.
Control Isoprenaline Forskolin 8B-cAMP (10−6 M) 8B-cAMP (10−5 M) Bay K-8644 Endothelium removal L-NAME
n
Emax (%)
EC50 (μM)
14 16 16 6 8 8 14 13
45.0 ± 3.0 92.3 ± 5.4⁎⁎ 80.3 ± 4.6⁎⁎ 11.6 ± 4.5⁎⁎ 3.1 ± 1.9⁎⁎
20.8 ± 4.7 17.6 ± 4.1 14.9 ± 3.9 22.3 ± 4.1 25.1 ± 3.9 22.3 ± 4.2 17.2 ± 5.6 8.9 ± 2.3⁎
47.0 ± 4.4 62.6 ± 4.7⁎⁎ 71.9 ± 3.9⁎⁎
Emax refers to the maximal constriction; EC50 refers to the concentration that produce 50% Emax. Mean ± SE; n = the number of rats. ⁎P b 0.05, ⁎⁎P b 0.01 vs. control.
Fig. 2. Effects of isoprenaline on NaHS-induced vasoconstriction of basilar arteries. Arterial rings were treated with isoprenaline (10−5 M) 10 min before the administration of NaHS at a concentration range of 10−7 to 10–3.5 M. NaHS-induced constrictions of the arteries were recorded. Representative tracer (top) and summarized analysis (bottom), Mean ± SE, n = 12–16. ⁎P b 0.05 and ⁎⁎P b 0.01 vs. control.
392
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
Fig. 3. NaHS (10−5, 10–4.5 and 10−4 M) attenuated the relaxant activity of isoprenaline (ISO, 10−5 M). Representative tracer (top) and summarized analysis (bottom), Mean ± SE, n = 8, ⁎⁎P b 0.01 vs. ISO.
of forskolin, an activator of adenylyl cyclase. The results showed that forskolin shifted the concentration-vasoconstriction curve induced by NaHS towards the left in a non-parallel manner. The Emax was 80.3 ± 4.6% and 45.5 ± 3.6% in the presence of forskolin and control, respectively (P b 0.01, Fig. 4; Table 1), suggesting that forskolin enhances the NaHS-induced vasoconstriction on basilar artery. Furthermore, the effect of NaHS on forskolin-induced relaxation was determined. Given together with forskolin, the relaxant values of forskolin + NaHS 10−5, 10–4.5 and 10−4 M (33.5 ± 3.3%, 19.2 ± 3.3%, 5.0 ± 3.1%) were significantly lower than that of forskolin alone (69.3 ± 2.2%, P b 0.01, Fig. 5). These results suggest that NaHS attenuates the relaxant effect of forskolin on basilar artery. Taken together, these data suggested that H2S act on adenylyl cyclase or its downstream effectors.
Fig. 4. Effect of forskolin on NaHS-induced vasoconstriction of basilar arteries. Arterial rings were treated with forskolin (10−7 M) 10 min before NaHS administration at concentrations of 10−7 to 10–3.5 M. NaHS-induced constrictions of the arteries were recorded. Representative tracer (top) and summarized analysis (bottom), Mean ± SE, n = 14–16. ⁎P b 0.05 and ⁎⁎P b 0.01 vs. control.
that Bay K-8644 did not affect the NaHS-induced concentrationvasoconstriction curve (Emax: 47.0 ± 4.4% with BayK, 45.3 ± 3.4% without BayK, P N 0.05, Fig. 7A; Table. 1). Additionally, pretreatment with Bay K-8644 failed to rise the [Ca2+]i of the artery elevated by NaHS (Fig. 7B). These data excluded the possibility that H2S directly act on L-type Ca2+ channels to induce the vasoconstriction.
3.4. Effect of 8B-cAMP on NaHS-induced vasoconstriction on basilar artery Adenylyl cyclase controls the synthetic rate of cAMP, a downstream effector relaxing vessels. To further delineate whether the action of H2S is involved with cAMP, the cumulative concentrations of NaHS (10−7–10–3.5 M) were added into the baths in the presence of 8B-cAMP, a non-hydrolysable cAMP analog. The results showed that 8B-cAMP shifted the concentration-vasoconstriction curve induced by NaHS towards the right in a non-parallel manner. The Emax was 11.6 ± 4.5%, 3.1 ± 1.9% and 45.5 ± 3.0% in the presence of 8B-cAMP (10−6,10−5 M) and control, respectively (P b 0.01, Fig. 6; Table 1), showing that 8BcAMP alleviates the contractile effect of NaHS on basilar artery. These data showed that the action of H2S may rely on reducing cAMP level of the cerebral artery. 3.5. Effect of Bay K-8644 on NaHS-induced vasoconstriction on basilar artery Previous works showed that the inhibition of L-type Ca2+ channel plays a role in the vasorelaxation of H2S in cerebral arteries (Streeter et al., 2012; Tian et al., 2012). To examine the involvement of this channel in the vasoconstrictive effect of H2S, after the basilar artery was incubated with Bay K-8644, a specific L-type Ca2 + channel agonist for 30 min, the cumulative concentrations of NaHS (10−7–10–3.5 M) were added into the baths to induce a vasoconstriction. The results showed
Fig. 5. NaHS (10−5, 10–4.5 and 10−4 M) attenuated the relaxant activity of forskolin (Forsk, 10–7.5 M). Representative tracer (top) and summarized analysis (bottom). Mean ± SE, n = 8, ⁎⁎P b 0.01 vs. Forsk.
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
393
To further examine whether the decrease of cAMP production by H2S is caused by inhibition of adenylyl cyclase, the effect of NaHS on forskolin-stimulated cAMP productive rate in the HBVSMCs was determined. Fig. 8B shows that forskolin stimulated adenylyl cyclase activity which was shown as the productive rate of cAMP from 0.41 ± 0.02 nmol/106 cells·min to 1.25 ± 0.04 nmol/106 cells·min (P b 0.01). NaHS at 10− 4 M significantly attenuated this effect (0.58 ± 0.07 nmol/106 cells·min; P b 0.01), suggesting that the reducing effect on cAMP by NaHS is due to the inhibition of adenylyl cyclase activity. 3.7. Effect of NaHS on vasoconstriction in the endothelium-denuded cerebral artery or in the presence of L-NAME
Fig. 6. Effect of 8B-cAMP on NaHS-induced vasoconstriction of basilar arteries. Arterial rings were treated with 8B-cAMP (10−6 or 10−5 M) 10 min before NaHS administration at concentrations of 10−7 to 10–3.5 M. NaHS-induced constrictions of arteries were recorded. Representative tracer (top) and summarized analysis (bottom), Mean ± SE, n = 6–8. ⁎⁎P b 0.01 vs. control.
3.6. Effect of NaHS on the elevated cAMP level and adenylyl cyclase activity in HBVSMCs To further determine whether a cAMP-dependent pathway was involved in the vasoconstriction induced by H2S, the effect of NaHS on isoprenaline-stimulated cAMP production in HBVSMCs was studied. Fig. 8A shows that isoprenaline significantly elevated the intracellular cAMP level, which was markedly attenuated by 10− 4 M NaHS (ISO: 10.9 ± 0.6 pmol/106 cells; NaHS + ISO: 7.2 ± 0.4 pmol/106 cells, P b 0.01). However, NaHS alone did not altered cAMP level (control: 2.3 ± 0.5 pmol/106 cells; NaHS: 2.0 ± 0.5 pmol/106 cells, P N 0.05; Fig. 8A).
The endothelium regulates vasomotion by releasing vasoconstrictive and vasodilatory agents. To test whether the endothelium participates the action of H2S, the effect of NaHS in both endothelium-intact and denuded cerebral artery rings were compared. The results showed that the Emax was 45.0 ± 3.0% and 62.6 ± 4.7% in endothelium-intact and denuded basilar arteries, respectively (P b 0.01, Fig. 9A; Table 1). NO, releasing from endothelium, is one of the main mediators regulating cerebrovascular tone. To verify whether NO affects the NaHSinduce vasoconstriction, L-NAME, a nitric oxide synthase inhibitor, was used. Fig. 9B showed that, L-NAME significantly enhanced the NaHS-induced vasoconstriction on the artery. The Emax was 71.9 ± 3.9% and 46.3 ± 5.6% in presence of L-NAME and control, respectively (P b 0.01, Fig. 9B; Table 1). The EC50 was 8.9 ± 2.3 μM and 20.8 ± 4.7 μM in presence of L-NAME and control, respectively (P b 0.05, Table 1). Additionally, the effect of NaHS on the relaxation caused by forskolin in the presence and absence of L-NAME was compared. The results showed that, when given together with NaHS, forskolin-induced vasorelaxation was significantly less than that caused by forskolin alone. L-NAME failed to abolish this effect (For the administration of Forsk + NaHS, without L-NAME: 19.2 ± 3.3%, with L-NAME: 9.7 ± 1.7%, P b 0.01 for L-NAME vs without L-NAME; Fig. 10), suggesting that cAMP, at least partly, mediates the vasoconstriction induced by NaHS separately. 4. Discussion A H2S donor is commonly used to replace H2S in experiments. There are three main donors of H2S commercially available: NaHS, Na2S and
Fig. 7. Effect of Bay K-8644 on NaHS-induced vasoconstriction of basilar arteries. (A) Arterial rings were treated with Bay K-8644 (10−5 M) 30 min before NaHS administration at concentrations of 10−7 to 10–3.5 M. NaHS-induced constrictions of the arteries were recorded. Mean ± SE, n = 8. (B) The effects of NaHS on Ca2+ fluorescence intensity in basilar arteries. Arterial rings were treated with Bay K-8644 (10−5 M) 30 min before the administration of 10−4 M NaHS. Changes in the fluorescence ratio reflect fluctuations in [Ca2+]i levels in cerebral arteries. Mean ± SE, n = 5–6.
394
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
Fig. 8. Effect of NaHS on elevated cAMP accumulation and adenylyl cyclase activity in HBVSMCs. (A) NaHS (10−4 M) was added after a 5-min isoprenaline (ISO, 10−5 M) treatment. Mean ± SE, n = 8–10. ⁎⁎P b 0.01. (B) NaHS (10−4 M) was added to HBVSMCs 10 min after forskolin (Forsk, 10–7.5 M). Mean ± SE, n = 6–8, ⁎⁎P b 0.01.
GYY4137 (Chitnis et al., 2013; Njie-Mbye et al., 2010; Streeter et al., 2012). The effects of these donors on rat cerebral arteries were investigated. The results showed the contractile potency of the donors from low to high was GYY4137, Na2S, and NaHS. The difference of contractile potency among the donors may be from the varying rate of H2S releasing from donors. GYY4137 releases H2S slowly. When incubated in aqueous buffer (pH 7.4, 37 °C), GYY4137 (1 mmol/L, i.e., 100 nmol incubated) releases H2S very slowly with a rate of 4.17 ± 0.5 nmol/25 min. In contrast, H2S generation from NaHS is fast. Real-time assessment of H2S release from NaHS by amperometry showed peak signal generation (for H2S) within 5 to 8 s (Li et al., 2008). Because of stronger potency, NaHS was selected as the donor of H2S in the present investigation. It is reported that the actual H2S concentration in solution may be less than 30% of the stated NaHS concentration (Li et al., 2008). Therefore, the concentrations of NaHS 3–300 μM, at which constricted the arteries, are equivalent to 1–100 μM H2S, which is close to the physiological H2S concentration (1–160 μM) in mammalian blood and tissues (Kubo et al., 2007a). In other word, the H2S concentration produced by the donor NaHS used here is close to the physiological range. Nevertheless, it was reported that this concentration of NaHS also induced a relaxation in middle cerebral artery pre-contracted by U46619 or KCl (Streeter et al., 2012; Tian et al., 2012), which differs from our present results. However, this constrictive status for the cerebral arteries is too violent to be attained in the physiological condition. Actually, compared to the artery pre-contracted by an agonist, the vasoconstrictive effect of H2S on artery in basal tension is much closer to the physiological status. The results showed that NaHS do induce vasoconstriction on the cerebral
basilar arteries in a concentration-dependent manner. This H2S-induced vasoconstrictive effect may have more physiological significance than its relaxant effect. However, there is a difference of the vasoconstriction in two difference manners. The maximal vasoconstriction induced by NaHS in the single concentration manner was stronger than that in cumulative concentration manner (Fig. 1). The exact reason is unclear. We speculate that NaHS-induced vasomotion may be related to the status of vessels. In cumulative concentration experiment, the vasoconstriction induced by relatively higher concentration of NaHS is attained on the base of the vasoconstriction induced by the former concentrations, but, in the single concentration experiment, the vasoconstrictions was all attained on the base of normal initial tension. β-Adrenergic receptor of cerebral artery is considered to play an important role in controlling cerebral blood flow by exerting vasomotor effects on cerebral blood vessels (Tsukahara et al., 1986). β-Adrenergic receptor stimulation activates GTP-binding protein alpha subunits, which in turn increases intracellular cAMP level and therefore activates PKA. Afterwards, PKA phosphorylates the myosin light chain kinase and renders its inaction. This causes the myosin light chain to remain unphosphorylated and thus induces a vasorelaxant response. On the contrary, inhibition of β-adrenergic receptor can produce contractile function in the smooth muscle cells. So the action mechanism of H2S was studied by stimulating different enzymes/channel proteins in the signaling cascade of β-adrenergic receptor system. Our results indicated that NaHS significantly attenuated the effects of isoprenaline/forskolin (Figs. 3, 5). NaHS-induced vasoconstriction on cerebral artery was
Fig. 9. NaHS-induced concentration-constriction curves in endothelium-denuded and endothelium-intact cerebral arterial rings (A, n = 13–14), or in presence and absence of L-NAME in endothelium-intact cerebral arterial rings (B, n = 8–13). Mean ± SE, ⁎⁎P b 0.01 vs. endothelium-intact or control.
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
395
Unexpectedly, the exogenous H2S-induced vasoconstriction was potently enhanced in endothelium-denuded arteries (Fig. 9A), as well as in the presence of L-NAME (Fig. 9B). Obviously, these enhancements may be due to the deletion of NO-induced vasodilation, which is different with the result on aortic rings by Ali et al. (2006). Besides, the concentration of H2S in the presence of L-NAME (Fig. 9B) was close to those concentrations found in blood plasma and the vascular tissues (Kubo et al., 2007a). Taken together, these findings suggested that H2S might be a physiological mediator for moderating the relaxant effect of NO (Zhong et al., 2003) on cerebral artery. Further, it was found that L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the precontracted artery (Fig. 10). This finding supports the involvement of additional mechanism on the NaHS-induced vasoconstriction. In other words, H2S-induced vasoconstriction is partially antagonized by NO, not that H2S necessarily acts via NO. Nevertheless, it is definitely that more research on the cross-talk between H2S and NO on cerebral artery are needed. 5. Conclusions Fig. 10. L-NAME enhances the antagonizing effect of NaHS on forskolin-induced vasodilation in pre-contracted cerebral arteries. Arterial rings were pre-contracted with 5-HT (10−5 M) or 5-HT + L-NAME (10−4 M), and the effects of Forsk (10–7.5 M) or Forsk + NaHS (10−4 M) on relaxation were recorded. Representative tracer (top) and summarized analysis (bottom), Mean ± SE, n = 9. ⁎⁎P b 0.01.
decreased by 8B-cAMP (Fig. 6), but was not influenced by BayK (Fig. 7). These evidences suggested that the contractile effect of H2S on cerebral artery is, at least partially, associated with the reduction of cAMP level. The result seems to be contradictive with the fact that isoprenaline/ forskolin also can enhance the H2S-induced vasoconstriction instead of antagonizing it (Figs. 2, 4). To assure this, we examined the effect of NaHS on the elevated cAMP level in the HBVSMCs. The results showed that H2S alone did not altered cAMP level, but markedly attenuated isoprenaline-elevated cAMP levels (Fig. 8A), which can enhance the vasoconstriction of H2S. That is to say, H2S and cAMP are working together to increase the tone, not to simply antagonize each other. The mechanisms involved in reducing cAMP level by NaHS have yet to be further investigated, particularly in the activities of adenylyl cyclase and phosphodiesterase. The former controls the rate of cAMP synthesis, whereas the latter controls its hydrolysis. Both inhibition of adenylyl cyclase or activation of phosphodiesterase can decrease cAMP level in vascular smooth muscle cells. We examined the effect of NaHS on adenylyl cyclase activity of HBVSMCs. It was found that NaHS markedly attenuated forskolin-stimulated adenylyl cyclase activity in the HBVSMCs (Fig. 8B), indicating that H2S-induced vasoconstriction is through the inhibition of cAMP/adenylyl cyclase pathway. Additionally, in Fig. 8A, NaHS only has a tendency to decrease the cAMP level of the in vitro HBVSMCs, but without significant differences. Since adenylyl cyclase is the target site of NaHS in cerebral vascular smooth muscle cells, this experimental phenomena can be explained. The adenylyl cyclase activity of the in vitro HBVSMCs is relative low, which makes the effect of NaHS in inhibiting this enzyme not easy to be revealed. While, with the increasing activity by isoprenaline, adenylyl cyclase is relatively easier to be inhibited by NaHS. However, without measuring the activity of phosphodiesterase, it is hard to conclude that NaHS-induced vasoconstriction is only resulted from inhibition of adenylyl cyclase. Further studies are warranted. Previously, most scholars regarded the decease of NO as the general cause of H2S-induced vasoconstriction, through either forming a novel inactive nitrosothiol molecule with NO (Ali et al., 2006; Webb et al., 2008) or scavenging NO by a bicarbonate-dependent pathway (Liu and Bian, 2010). Therefore, to evaluate whether NO alters NaHS-induced vasoconstriction on cerebral artery, endothelium removal and incubation with L-NAME, a nitric oxide synthase inhibitor were investigated.
H2S induces vasoconstriction of cerebral arteries in normal initial tension. The effect is via inhibiting adenylyl cyclase to reduce cAMP level in cerebral vascular smooth muscle cells. The present study shows a novel and important profile of H2S effect on cerebral artery, which provides a new view on understanding the physiological regulation of cerebrovascular tone. Conflict of interest None. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgments This investigation was supported by the Specialized Research Fund for the Doctoral Program of Higher Education (20100201110054) and National Natural Science Foundation of China (81173059). References Ali, M.Y., Ping, C.Y., Mok, Y.Y., Ling, L., Whiteman, M., Bhatia, M., 2006. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br. J. Pharmacol. 6, 625–634. Ashley, A., Wang, L., Wang, R., 2012. The role of carbon monoxide as a gasotransmitter in cardiovascular and metabolic regulation. In: Hermann, A., Sitdikova, G., Weiger, T. (Eds.), Gasotransmitters: Physiology and Pathophysiology. Springer-Verlag, Berlin, pp. 37–70. Burnett, W.W., King, E.G., Grace, M., Hall, W.F., 1977. Hydrogen sulfide poisoning: review of 5 years' experience. Can. Med. Assoc. J. 11, 1277–1280. Cheang, W.S., Wong, W.T., Shen, B., Lau, C.W., Tian, X.Y., Tsang, S.Y., 2010. 4-Aminopyridinesensitive K+ channels contributes to NaHS-induced membrane hyperpolarization and relaxation in the rat coronary artery. Vasc. Pharmacol. 3-4, 94–98. Cheng, Y., Ndisang, J.F., Tang, G., Cao, K., Wang, R., 2004. Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats. Am. J. Physiol. Heart Circ. Physiol. 5, 2316–2323. Chitnis, M.K., Njie-Mbye, Y.F., Opere, C.A., Wood, M.E., Whiteman, M., Ohia, S.E., 2013. Pharmacological actions of the slow release hydrogen sulfide donor GYY4137 on phenylephrine-induced tone in isolated bovine ciliary artery. Exp. Eye Res. 350-354. Fiorucci, S., Antonelli, E., Distrutti, E., Rizzo, G., Mencarelli, A., Orlandi, S., 2005. Inhibition of hydrogen sulfide generation contributes to gastric injury caused by antiinflammatory nonsteroidal drugs. Gastroenterology 4, 1210–1224. Hashimoto, T., Ohata, H., Nobe, K., Sakai, Y., Honda, K., 2007. A novel approach for the determination of contractile and calcium responses of the basilar artery employing realtime confocal laser microscopy. J. Pharmacol. Toxicol. Methods 1, 79–86. Hosoki, R., Matsuki, N., Kimura, H., 1997. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem. Biophys. Res. Commun. 3, 527–531.
396
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
Kabil, O., Banerjee, R., 2010. Redox biochemistry of hydrogen sulfide. J. Biol. Chem. 29, 21903–21907. Kubo, S., Doe, I., Kurokawa, Y., Nishikawa, H., Kawabata, A., 2007a. Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide: contribution to dual modulation of vascular tension. Toxicology 232, 138–146. Kubo, S., Kajiwara, M., Kawabata, A., 2007b. Dual modulation of the tension of isolated gastric artery and gastric mucosal circulation by hydrogen sulfide in rats. Inflammopharmacology 15, 288–292. Li, L., Whiteman, M., Guan, Y.Y., Neo, K.L., Cheng, Y., Lee, S.W., 2008. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide. Circulation 18, 2351–2360. Li, L., Hsu, A., Moore, P.K., 2009. Actions and interactions of nitric oxide, carbon monoxide and hydrogen sulphide in the cardiovascular system and in inflammation–a tale of three gases! Pharmacol. Ther. 3, 386–400. Liu, Y.H., Bian, J.S., 2010. Bicarbonate-dependent effect of hydrogen sulfide on vascular contractility in rat aortic rings. Am. J. Physiol. Cell Physiol. 4, 866–872. Mackay, K., Mochly-Rosen, D., 2001. Arachidonic acid protects neonatal rat cardiac myocytes from ischaemic injury through epsilon protein kinase C. Cardiovasc. Res. 1, 65–74. McPherson, G.A., 1992. Assessing vascular reactivity of arteries in the small vessel myograph. Clin. Exp. Pharmacol. Physiol. 12, 815–825. Njie-Mbye, Y.F., Bongmba, O.Y., Onyema, C.C., Chitnis, A., Kulkarni, M., Opere, C.A., 2010. Effect of hydrogen sulfide on cyclic AMP production in isolated bovine and porcine neural retinae. Neurochem. Res. 3, 487–494. Olson, K.R., Dombkowski, R.A., Russell, M.J., Doellman, M.M., Head, S.K., Whitfield, N.L., 2006. Hydrogen sulfide as an oxygen sensor/transducer in vertebrate hypoxic vasoconstriction and hypoxic vasodilation. J. Exp. Biol. 20, 4011–4023. Pan, T.T., Neo, K.L., Hu, L.F., Yong, Q.C., Bian, J.S., 2008. H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes. Am. J. Physiol. Cell Physiol. 1, 169–177. Ping, N.N., Cao, L., Xiao, X., Li, S., Cao, Y.X., 2014. The determination of optimal initial tension in rat coronary artery using wire myography. Physiol. Res. 1, 143–146. Raij, L., 2001. Workshop: hypertension and cardiovascular risk factors: role of the angiotensin II-nitric oxide interaction. Hypertension 2 (Pt 2), 767–773. Siebert, N., Cantre, D., Eipel, C., Vollmar, B., 2008. H2S contributes to the hepatic arterial buffer response and mediates vasorelaxation of the hepatic artery via activation of KATP channels. Am. J. Physiol. Gastrointest. Liver Physiol. 6, 1266–1273.
Smith, R.P., Gosselin, R.E., 1979. Hydrogen sulfide poisoning. J. Occup. Med. 2, 93–97. Streeter, E., Hart, J., Badoer, E., 2012. An investigation of the mechanisms of hydrogen sulfide-induced vasorelaxation in rat middle cerebral arteries. Naunyn Schmiedeberg's Arch. Pharmacol. 10, 991–1002. Sun, T., Liu, R., Cao, Y.X., 2011. Vasorelaxant and antihypertensive effects of formononetin through endothelium-dependent and -independent mechanisms. Acta Pharmacol. Sin. 8, 1009–1018. Tian, X.Y., Wong, W.T., Sayed, N., Luo, J., Tsang, S.Y., Bian, Z.X., 2012. NaHS relaxes rat cerebral artery in vitro via inhibition of l-type voltage-sensitive Ca2+ channel. Pharmacol. Res. 2, 239–246. Tsukahara, T., Taniguchi, T., Shimohama, S., Fujiwara, M., Handa, H., 1986. Characterization of beta adrenergic receptors in human cerebral arteries and alteration of the receptors after subarachnoid hemorrhage. Stroke 2, 202–207. Ulrich, F., Huige, L., 2012. Nitric oxide: biological synthesis and functions. In: Hermann, A., Sitdikova, G., Weiger, T. (Eds.), Gasotransmitters: Physiology and Pathophysiology. Springer-Verlag, Berlin, pp. 1–36. Wang, R., 2002. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter? FASEB J. 13, 1792–1798. Webb, J.G., Yates, P.W., Yang, Q., Mukhin, Y.V., Lanier, S.M., 2001. Adenylyl cyclase isoforms and signal integration in models of vascular smooth muscle cells. Am. J. Physiol. Heart Circ. Physiol. 4, 1545–1552. Webb, G.D., Lim, L.H., Oh, V.M., Yeo, S.B., Cheong, Y.P., Ali, M.Y., 2008. Contractile and vasorelaxant effects of hydrogen sulfide and its biosynthesis in the human internal mammary artery. J. Pharmacol. Exp. Ther. 2, 876–882. Weber, N.C., Toma, O., Wolter, J.I., Obal, D., Mullenheim, J., Preckel, B., 2005. The noble gas xenon induces pharmacological preconditioning in the rat heart in vivo via induction of PKC-epsilon and p38 MAPK. Br. J. Pharmacol. 1, 123–132. Zhao, W., Zhang, J., Lu, Y., Wang, R., 2001. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J. 21, 6008–6016. Zhong, G., Chen, F., Cheng, Y., Tang, C., Du, J., 2003. The role of hydrogen sulfide generation in the pathogenesis of hypertension in rats induced by inhibition of nitric oxide synthase. J. Hypertens. 10, 1879–1885.
Contents lists available at ScienceDirect
Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap
H2S induces vasoconstriction of rat cerebral arteries via cAMP/adenylyl cyclase pathway Sen Li, Na-na Ping, Lei Cao ⁎, Yan-ni Mi, Yong-xiao Cao ⁎ Department of Pharmacology, Medical College, Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, China
a r t i c l e
i n f o
Article history: Received 16 October 2015 Revised 28 October 2015 Accepted 28 October 2015 Available online 31 October 2015 Keywords: β-Adrenoceptor cAMP Cerebral artery Constriction Hydrogen sulfide Sodium hydrosulfide
a b s t r a c t Hydrogen sulfide (H2S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H2S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H2S, concentration-dependently induced vasoconstriction on basilar artery, which was enhanced in the presence of isoprenaline, a β-adrenoceptor agonist or forskolin, an adenylyl cyclase activator. Administration of NaHS attenuated the vasorelaxant effects of isoprenaline or forskolin. Meanwhile, the NaHS-induced vasoconstriction was diminished in the presence of 8B-cAMP, an analog of cAMP, but was not affected by Bay K-8644, a selective L-type Ca2+ channel agonist. These results could be explained by the revised effects of NaHS on isoprenaline-induced cAMP elevation and forskolin-stimulated adenylyl cyclase activity. Additionally, NaHS-induced vasoconstriction was enhanced by removing the endothelium or in the presence of L-NAME, an inhibitor of nitric oxide synthase. L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the pre-contracted artery. In conclusion, H2S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Cerebral vascular tone is regulated by vascular receptors, such as adrenergic receptors, angiotensin receptors, 5-hydroxytryptamine (5-HT) receptors, and endothelium receptors, which mediate vasoconstriction or vasodilatation by their ligands, agonists or antagonists. In recent years, the gaseous transmitters, such as nitric oxide (NO) and carbon monoxide (CO), are also demonstrated to be mediators of the vascular tone. Hydrogen sulfide (H2S), a toxic gas with a strong characteristic rotten egg smell, has been well documented on its fatal intoxication (Burnett et al., 1977; Kabil and Banerjee, 2010; Smith and Gosselin, 1979). However, in recent years, H2S is accepted as the third gas transmitter after NO and CO for its similar vascular effects and biological features (Wang, 2002). These gases (NO, CO, H2S) together frequently make up a combination, so-called “gaseous triumvirate” to address the
Abbreviations: 5-HT, 5-hydroxytryptamine; 8B-cAMP, 8-bromoadenosine-3′, 5′-cyclic monophosphate; BayK, Bay K-8644; DMSO, dimethyl sulfoxide; EC50, the concentration that produce 50% of the maximal constriction; Emax, maximal constriction; Forsk, forskolin; H2S, hydrogen sulfide; HBVSMCs, human brain vascular smooth muscle cells; HEPES, hydroxyethyl piperazine ethanesulfonic acid; ISO, isoprenaline; L-NAME, NGnitro-L-arginine methyl ester hydrochloride; NaHS, sodium hydrosulfide; NO, nitric oxide. ⁎ Corresponding authors at: Department of Pharmacology, Medical College, Xi'an Jiaotong University, 76# Yanta West Road, Xi'an, Shannxi 710061, China. E-mail addresses: [email protected] (L. Cao), [email protected] (Y. Cao).
http://dx.doi.org/10.1016/j.taap.2015.10.021 0041-008X/© 2015 Elsevier Inc. All rights reserved.
physiological function (Li et al., 2009). NO and CO have been well established to play pivotal roles in the regulation of vascular homeostasis by acting as vasodilators that helps to maintain an equilibrium of vasomotion (Ashley et al., 2012, Ulrich and Huige, 2012). In comparison with NO and CO, H2S also can relax vessels, for example, isolated rat aorta (Ali et al., 2006; Hosoki et al., 1997; Kubo et al., 2007a; Zhao et al., 2001), gastric artery (Kubo et al., 2007b), coronary artery (Cheang et al., 2010), portal vein (Hosoki et al., 1997) and dilate perfused rat mesenteric (Cheng et al., 2004), and hepatic vascular beds (Fiorucci et al., 2005, Siebert et al., 2008). However, in the vascular system, the equilibrium between vasoconstrictor and vasodilator play a crucial role in maintaining the homeostasis of the vascular tone, structurally as well as functionally (Raij, 2001). Although H2S is generally considered as a vasodilator, its vasoconstrictive effect has been reported. H2S induces vasoconstriction in rat gastric artery (Kubo et al., 2007b), pulmonary artery (Olson et al., 2006), or human internal mammary artery (Webb et al., 2008). These data suggest that H2S possess a complex action in regulating vascular tone. We discovered that sodium hydrosulfide (NaHS), a donor of H2S, could induce a constriction of rat cerebral artery in experiments. Interestingly, this phenomenon is in line with the literature in which H2S elicits a vasoconstrictive effect either in rat or human vessels (Kubo et al., 2007b; Olson et al., 2006; Webb et al., 2001). Therefore, the present study was to explore the vasoconstrictive effect of NaHS in cerebral arteries and the possible mechanisms.
390
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
2. Materials and methods
2.5. Cell culture and cAMP level assay
2.1. Ethical approval
Human brain vascular smooth muscle cells (HBVSMCs) were purchased from ScienCell™ (Cat. No. 1100, Lot. No. 4706, Carlsbad, CA, USA) and cultured in DMEM supplemented with 20% fetal bovine serum, penicillin (80 U/ml) and streptomycin (0.08 mg/ml) in a humidified incubator (37 °C, 5% CO2). The HBVSMCs used for assays were from generations 4–8. The HBVSMCs were washed with PBS, and lysed with 0.1 M HCl. The cell lysates were aspirated and subjected to centrifugation at 1000 g for 10 min to collect the supernatant. The supernatant from 105 HBVSMCs was added to a well. The cAMP levels in the wells were assayed by the direct cAMP enzyme immunoassay kit (Cayman Chemical, USA).
The experimental protocol was approved by the Ethics Committee of Xi'an Jiaotong University. The investigation is conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). 2.2. Drugs and chemicals NaHS, 5-HT, acetylcholine, isoprenaline (ISO), forskolin (Forsk), 8bromoadenosine-3′,5′-cyclic monophosphate (8B-cAMP), Bay K-8644 (BayK), NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) and hydroxyethyl piperazine ethanesulfonic acid (HEPES) were purchased from Sigma Aldrich (St. Louis, USA). Dimethyl sulfoxide (DMSO) and fluo-3/AM were purchased from Biotium (Hayward, USA) and Solarbio (Beijing, China), respectively. NaHS was freshly prepared before each experiment. Forsk, BayK or fluo-3/AM were dissolved in DMSO, whereas other chemicals were dissolved in deionized water. The concentrations were expressed as the final molar concentration in the baths. 2.3. Artery preparation and functional study Male Sprague–Dawley rats weighing 250–350 g were purchased from the Experimental Animal Center of Xi'an Jiaotong University, China. The rats were housed under controlled temperature and humidity with water and food ad libitum. Rats were sacrificed by asphyxia induced by CO2 inhalation. Basilar artery was removed and placed in cold oxygenated Krebs buffer (mM: NaCl, 119; KCl, 4.6; MgCl2·6H2O, 1.2; CaCl2, 1.5; NaH2PO4·2H2O, 1.2; NaHCO3, 15; glucose, 5.6; pH 7.4). Wire myograph (Myo Technology A/S, Denmark) was used to record the isometric tension and to examine artery contractile properties in isolated cerebral artery segments. The segments of the basilar artery (2 mm in length), free of connective tissue, were mounted between two steel hooks in a 5 mL chamber containing gassed (95% O2 and 5% CO2) Krebs solution at 37 °C. Initial tension, equivalent to tension caused by 0.9 times the diameter of the vessel at 100 mm Hg (McPherson, 1992; Ping et al., 2014), was applied by using DMT Normalization v1.3 software (ADInstruments, UK) to each segment for a 60-min equilibration, where after, changes of the tension induced by drugs were recorded via LabChart 7.2 software (ADInstruments, UK). Arteries without endothelium were performed by rubbing the vessel lumen with a wire. The endothelium was identified as successful removal when the acetylcholine-induced artery relaxation was less than 30%. 2.4. Measurement of intracellular calcium ([Ca2+]i) of the artery [Ca2+]i levels of the cerebral artery were measured by spectrofluorometric method using fluo-3/AM as a calcium indicator similar to the previously described (Hashimoto et al., 2007; Sun et al., 2011). Segments of the basilar artery (3 mm in length) were fixed on the bottom of the chamber close to cover glass. The specimens were immediately immersed in HEPES-Krebs solution (mM: NaCl, 135; KCl, 5; MgSO4, 1.2; CaCl2, 2.5; glucose, 10; HEPES, 8.4) with fluo-3/AM (10 μM) for 30 min. A real-time confocal microscope (Olympus, FV1000, Japan) was used to take the fluorescent image. The segments were exposed to the drugs and the image frames were acquired continuously. The F488/F520 ratio was calculated from individual image using FV-ASW software (version 1.7, Olympus, Tokyo, Japan) and was used to indicate [Ca2+]i levels of the artery. It was considered that changes in the fluorescence ratio reflect the fluctuations in [Ca2+]i levels.
2.6. Adenylyl cyclase activity assay The HBVSMCs were suspended in lysis buffer (mM: Tris–HCl 25, EDTA 0.4, EGTA 1.0, sucrose 250, leupeptin 0.1, and PMSF 0.04, pH 7.4) for 10 min at 25 °C. Then the lysate was collected and subjected to centrifugation at 50,000 g for 30 min to obtain the membrane protein fractions (Mackay and Mochly-Rosen, 2001; Pan et al., 2008; Weber et al., 2005). A 400 μl reaction mixture (mM: ATP 1.0, NaCl 100, HEPES 50, 3-isobutyl-1-methylxanthine 0.5, MgCl2 6, GTP 0.001 and 20 μg of membrane protein fractions from 5 × 106 cells) was added to a well at 37 °C for 10 min. Reactions were stopped by addition of 0.6 ml of 10% trichloroacetic acid to the well. The productive rate of cAMP in each well was assayed by the cAMP EIA kit.
2.7. Statistical analysis All data are presented as mean ± SE. The tensions of arterial ring segments were expressed as percentage of vasoconstriction precontracted by 10−5 M 5-HT. Relaxation responses were expressed as percentage of pre-constriction induced by agonists. Emax refers to the maximum constriction induced by an agonist. The EC50 is the agonist concentration that produces 50% of the Emax which was calculated from the straight line equation between the concentration above and below the midpoint of the concentration response curve. Oneway analysis of variance (ANOVA) with or without Bonferroni's post-test was used for multiple comparisons using Graphpad Prism 5.0 (GraphPad Software, La Jolla, USA). Statistical significance was set as P b 0.05.
3. Results 3.1. The vasoconstriction of NaHS on rat basilar artery To clarify the vasoactivity of H2S on basilar artery, cumulative NaHS (10−7–10–3.5 M) was applied to the artery at initial tension. The results showed that NaHS induced a concentration-dependent vasoconstriction on basilar artery with an Emax of 45.0 ± 3.0% and an EC50 of 20.8 ± 4.7 μM (Fig. 1A; Table 1). The vasoconstriction started to decrease when the concentration of NaHS increased to 10–3.5 M (Fig. 1A). To delineate this effect, the single concentration of NaHS was used. The results indicated that NaHS (10−5, 10−4, 10–3.5, 10−3 and 10−2 M) induced vasoconstrictions of 23.6 ± 1.0%, 40.6 ± 3.7%, 56.9 ± 3.9, 61.3 ± 4.0% and 80.0 ± 6.3%, respectively (Fig. 1B). Also, other H2S donors, Na2S and GYY4137 were used. The results showed that the contractile potency of the donors from low to high was GYY4137, Na2S, and NaHS with Emax of 4.5 ± 1.6%, 17.8 ± 3.0% and 45.0 ± 3.0%, respectively (Fig. 1A). Taken together, these data suggest that H2S induces a concentration-dependent vasoconstriction on basilar artery.
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
391
Fig. 1. The vasoconstriction of H2S on rat basilar artery. (A) Representative tracer (top) of cumulative concentration NaHS induced a concentration-dependent vasoconstriction, n = 14. Summarized analysis (bottom) of the effect of different H2S donors on rat cerebral basilar arteries, n = 6–14. (B) Representative tracer (top) and summarized analysis (bottom) of single concentration of NaHS induced vasoconstriction. n = 14. Mean ± SE. ⁎⁎P b 0.01.
3.2. The relationship of NaHS-induced vasoconstriction to β-adrenergic receptors β-Adrenergic receptor acts a part in regulating tension on cerebral artery (Tsukahara et al., 1986). The effect of β-adrenergic receptor on NaHS-induced vasoconstriction was studied. The accumulative concentrations of NaHS (10−7–10–3.5 M) were added into the baths in the presence or absence of isoprenaline, a β-adrenoceptor agonist. The results showed that isoprenaline shifted the concentration-vasoconstriction curve induced by NaHS towards the left in a non-parallel manner. The Emax was 92.3 ± 5.4% and 46.5 ± 3.1% in the presence of isoprenaline and control, respectively (P b 0.01, Fig. 2; Table 1), suggesting that isoprenaline enhances the NaHS-induced vasoconstriction on basilar artery. Furthermore, the effect of NaHS on isoprenaline-induced relaxation was determined. Given together with isoprenaline, the relaxant values of isoprenaline + NaHS 10−5, 10–4.5 and 10−4 M (61.0 ± 4.9%, 38.9 ± 6.2%, 31.3 ± 6.3%) were significantly lower than that of isoprenaline alone (83.8 ± 3.4%, P b 0.01, Fig. 3). These results suggest that NaHS attenuates the relaxant effect of isoprenaline on basilar artery. Taken together, these data suggested that the H2S-induced vasomotion appears primarily via modulation of the β-adrenergic receptors on cerebral artery.
3.3. The relationship of NaHS-induced vasoconstriction to adenylyl cyclase β-Adrenergic receptor relaxes artery through activating adenylyl cyclase. Further, the effect of adenylyl cyclase on NaHS-induced vasoconstriction was studied. The cumulative concentrations of NaHS (10−7–10–3.5 M) were added into the baths in the presence or absence
Table 1 Emax and EC50 values of NaHS-induced vasoconstrictions of cerebral arteries in different statuses.
Control Isoprenaline Forskolin 8B-cAMP (10−6 M) 8B-cAMP (10−5 M) Bay K-8644 Endothelium removal L-NAME
n
Emax (%)
EC50 (μM)
14 16 16 6 8 8 14 13
45.0 ± 3.0 92.3 ± 5.4⁎⁎ 80.3 ± 4.6⁎⁎ 11.6 ± 4.5⁎⁎ 3.1 ± 1.9⁎⁎
20.8 ± 4.7 17.6 ± 4.1 14.9 ± 3.9 22.3 ± 4.1 25.1 ± 3.9 22.3 ± 4.2 17.2 ± 5.6 8.9 ± 2.3⁎
47.0 ± 4.4 62.6 ± 4.7⁎⁎ 71.9 ± 3.9⁎⁎
Emax refers to the maximal constriction; EC50 refers to the concentration that produce 50% Emax. Mean ± SE; n = the number of rats. ⁎P b 0.05, ⁎⁎P b 0.01 vs. control.
Fig. 2. Effects of isoprenaline on NaHS-induced vasoconstriction of basilar arteries. Arterial rings were treated with isoprenaline (10−5 M) 10 min before the administration of NaHS at a concentration range of 10−7 to 10–3.5 M. NaHS-induced constrictions of the arteries were recorded. Representative tracer (top) and summarized analysis (bottom), Mean ± SE, n = 12–16. ⁎P b 0.05 and ⁎⁎P b 0.01 vs. control.
392
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
Fig. 3. NaHS (10−5, 10–4.5 and 10−4 M) attenuated the relaxant activity of isoprenaline (ISO, 10−5 M). Representative tracer (top) and summarized analysis (bottom), Mean ± SE, n = 8, ⁎⁎P b 0.01 vs. ISO.
of forskolin, an activator of adenylyl cyclase. The results showed that forskolin shifted the concentration-vasoconstriction curve induced by NaHS towards the left in a non-parallel manner. The Emax was 80.3 ± 4.6% and 45.5 ± 3.6% in the presence of forskolin and control, respectively (P b 0.01, Fig. 4; Table 1), suggesting that forskolin enhances the NaHS-induced vasoconstriction on basilar artery. Furthermore, the effect of NaHS on forskolin-induced relaxation was determined. Given together with forskolin, the relaxant values of forskolin + NaHS 10−5, 10–4.5 and 10−4 M (33.5 ± 3.3%, 19.2 ± 3.3%, 5.0 ± 3.1%) were significantly lower than that of forskolin alone (69.3 ± 2.2%, P b 0.01, Fig. 5). These results suggest that NaHS attenuates the relaxant effect of forskolin on basilar artery. Taken together, these data suggested that H2S act on adenylyl cyclase or its downstream effectors.
Fig. 4. Effect of forskolin on NaHS-induced vasoconstriction of basilar arteries. Arterial rings were treated with forskolin (10−7 M) 10 min before NaHS administration at concentrations of 10−7 to 10–3.5 M. NaHS-induced constrictions of the arteries were recorded. Representative tracer (top) and summarized analysis (bottom), Mean ± SE, n = 14–16. ⁎P b 0.05 and ⁎⁎P b 0.01 vs. control.
that Bay K-8644 did not affect the NaHS-induced concentrationvasoconstriction curve (Emax: 47.0 ± 4.4% with BayK, 45.3 ± 3.4% without BayK, P N 0.05, Fig. 7A; Table. 1). Additionally, pretreatment with Bay K-8644 failed to rise the [Ca2+]i of the artery elevated by NaHS (Fig. 7B). These data excluded the possibility that H2S directly act on L-type Ca2+ channels to induce the vasoconstriction.
3.4. Effect of 8B-cAMP on NaHS-induced vasoconstriction on basilar artery Adenylyl cyclase controls the synthetic rate of cAMP, a downstream effector relaxing vessels. To further delineate whether the action of H2S is involved with cAMP, the cumulative concentrations of NaHS (10−7–10–3.5 M) were added into the baths in the presence of 8B-cAMP, a non-hydrolysable cAMP analog. The results showed that 8B-cAMP shifted the concentration-vasoconstriction curve induced by NaHS towards the right in a non-parallel manner. The Emax was 11.6 ± 4.5%, 3.1 ± 1.9% and 45.5 ± 3.0% in the presence of 8B-cAMP (10−6,10−5 M) and control, respectively (P b 0.01, Fig. 6; Table 1), showing that 8BcAMP alleviates the contractile effect of NaHS on basilar artery. These data showed that the action of H2S may rely on reducing cAMP level of the cerebral artery. 3.5. Effect of Bay K-8644 on NaHS-induced vasoconstriction on basilar artery Previous works showed that the inhibition of L-type Ca2+ channel plays a role in the vasorelaxation of H2S in cerebral arteries (Streeter et al., 2012; Tian et al., 2012). To examine the involvement of this channel in the vasoconstrictive effect of H2S, after the basilar artery was incubated with Bay K-8644, a specific L-type Ca2 + channel agonist for 30 min, the cumulative concentrations of NaHS (10−7–10–3.5 M) were added into the baths to induce a vasoconstriction. The results showed
Fig. 5. NaHS (10−5, 10–4.5 and 10−4 M) attenuated the relaxant activity of forskolin (Forsk, 10–7.5 M). Representative tracer (top) and summarized analysis (bottom). Mean ± SE, n = 8, ⁎⁎P b 0.01 vs. Forsk.
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
393
To further examine whether the decrease of cAMP production by H2S is caused by inhibition of adenylyl cyclase, the effect of NaHS on forskolin-stimulated cAMP productive rate in the HBVSMCs was determined. Fig. 8B shows that forskolin stimulated adenylyl cyclase activity which was shown as the productive rate of cAMP from 0.41 ± 0.02 nmol/106 cells·min to 1.25 ± 0.04 nmol/106 cells·min (P b 0.01). NaHS at 10− 4 M significantly attenuated this effect (0.58 ± 0.07 nmol/106 cells·min; P b 0.01), suggesting that the reducing effect on cAMP by NaHS is due to the inhibition of adenylyl cyclase activity. 3.7. Effect of NaHS on vasoconstriction in the endothelium-denuded cerebral artery or in the presence of L-NAME
Fig. 6. Effect of 8B-cAMP on NaHS-induced vasoconstriction of basilar arteries. Arterial rings were treated with 8B-cAMP (10−6 or 10−5 M) 10 min before NaHS administration at concentrations of 10−7 to 10–3.5 M. NaHS-induced constrictions of arteries were recorded. Representative tracer (top) and summarized analysis (bottom), Mean ± SE, n = 6–8. ⁎⁎P b 0.01 vs. control.
3.6. Effect of NaHS on the elevated cAMP level and adenylyl cyclase activity in HBVSMCs To further determine whether a cAMP-dependent pathway was involved in the vasoconstriction induced by H2S, the effect of NaHS on isoprenaline-stimulated cAMP production in HBVSMCs was studied. Fig. 8A shows that isoprenaline significantly elevated the intracellular cAMP level, which was markedly attenuated by 10− 4 M NaHS (ISO: 10.9 ± 0.6 pmol/106 cells; NaHS + ISO: 7.2 ± 0.4 pmol/106 cells, P b 0.01). However, NaHS alone did not altered cAMP level (control: 2.3 ± 0.5 pmol/106 cells; NaHS: 2.0 ± 0.5 pmol/106 cells, P N 0.05; Fig. 8A).
The endothelium regulates vasomotion by releasing vasoconstrictive and vasodilatory agents. To test whether the endothelium participates the action of H2S, the effect of NaHS in both endothelium-intact and denuded cerebral artery rings were compared. The results showed that the Emax was 45.0 ± 3.0% and 62.6 ± 4.7% in endothelium-intact and denuded basilar arteries, respectively (P b 0.01, Fig. 9A; Table 1). NO, releasing from endothelium, is one of the main mediators regulating cerebrovascular tone. To verify whether NO affects the NaHSinduce vasoconstriction, L-NAME, a nitric oxide synthase inhibitor, was used. Fig. 9B showed that, L-NAME significantly enhanced the NaHS-induced vasoconstriction on the artery. The Emax was 71.9 ± 3.9% and 46.3 ± 5.6% in presence of L-NAME and control, respectively (P b 0.01, Fig. 9B; Table 1). The EC50 was 8.9 ± 2.3 μM and 20.8 ± 4.7 μM in presence of L-NAME and control, respectively (P b 0.05, Table 1). Additionally, the effect of NaHS on the relaxation caused by forskolin in the presence and absence of L-NAME was compared. The results showed that, when given together with NaHS, forskolin-induced vasorelaxation was significantly less than that caused by forskolin alone. L-NAME failed to abolish this effect (For the administration of Forsk + NaHS, without L-NAME: 19.2 ± 3.3%, with L-NAME: 9.7 ± 1.7%, P b 0.01 for L-NAME vs without L-NAME; Fig. 10), suggesting that cAMP, at least partly, mediates the vasoconstriction induced by NaHS separately. 4. Discussion A H2S donor is commonly used to replace H2S in experiments. There are three main donors of H2S commercially available: NaHS, Na2S and
Fig. 7. Effect of Bay K-8644 on NaHS-induced vasoconstriction of basilar arteries. (A) Arterial rings were treated with Bay K-8644 (10−5 M) 30 min before NaHS administration at concentrations of 10−7 to 10–3.5 M. NaHS-induced constrictions of the arteries were recorded. Mean ± SE, n = 8. (B) The effects of NaHS on Ca2+ fluorescence intensity in basilar arteries. Arterial rings were treated with Bay K-8644 (10−5 M) 30 min before the administration of 10−4 M NaHS. Changes in the fluorescence ratio reflect fluctuations in [Ca2+]i levels in cerebral arteries. Mean ± SE, n = 5–6.
394
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
Fig. 8. Effect of NaHS on elevated cAMP accumulation and adenylyl cyclase activity in HBVSMCs. (A) NaHS (10−4 M) was added after a 5-min isoprenaline (ISO, 10−5 M) treatment. Mean ± SE, n = 8–10. ⁎⁎P b 0.01. (B) NaHS (10−4 M) was added to HBVSMCs 10 min after forskolin (Forsk, 10–7.5 M). Mean ± SE, n = 6–8, ⁎⁎P b 0.01.
GYY4137 (Chitnis et al., 2013; Njie-Mbye et al., 2010; Streeter et al., 2012). The effects of these donors on rat cerebral arteries were investigated. The results showed the contractile potency of the donors from low to high was GYY4137, Na2S, and NaHS. The difference of contractile potency among the donors may be from the varying rate of H2S releasing from donors. GYY4137 releases H2S slowly. When incubated in aqueous buffer (pH 7.4, 37 °C), GYY4137 (1 mmol/L, i.e., 100 nmol incubated) releases H2S very slowly with a rate of 4.17 ± 0.5 nmol/25 min. In contrast, H2S generation from NaHS is fast. Real-time assessment of H2S release from NaHS by amperometry showed peak signal generation (for H2S) within 5 to 8 s (Li et al., 2008). Because of stronger potency, NaHS was selected as the donor of H2S in the present investigation. It is reported that the actual H2S concentration in solution may be less than 30% of the stated NaHS concentration (Li et al., 2008). Therefore, the concentrations of NaHS 3–300 μM, at which constricted the arteries, are equivalent to 1–100 μM H2S, which is close to the physiological H2S concentration (1–160 μM) in mammalian blood and tissues (Kubo et al., 2007a). In other word, the H2S concentration produced by the donor NaHS used here is close to the physiological range. Nevertheless, it was reported that this concentration of NaHS also induced a relaxation in middle cerebral artery pre-contracted by U46619 or KCl (Streeter et al., 2012; Tian et al., 2012), which differs from our present results. However, this constrictive status for the cerebral arteries is too violent to be attained in the physiological condition. Actually, compared to the artery pre-contracted by an agonist, the vasoconstrictive effect of H2S on artery in basal tension is much closer to the physiological status. The results showed that NaHS do induce vasoconstriction on the cerebral
basilar arteries in a concentration-dependent manner. This H2S-induced vasoconstrictive effect may have more physiological significance than its relaxant effect. However, there is a difference of the vasoconstriction in two difference manners. The maximal vasoconstriction induced by NaHS in the single concentration manner was stronger than that in cumulative concentration manner (Fig. 1). The exact reason is unclear. We speculate that NaHS-induced vasomotion may be related to the status of vessels. In cumulative concentration experiment, the vasoconstriction induced by relatively higher concentration of NaHS is attained on the base of the vasoconstriction induced by the former concentrations, but, in the single concentration experiment, the vasoconstrictions was all attained on the base of normal initial tension. β-Adrenergic receptor of cerebral artery is considered to play an important role in controlling cerebral blood flow by exerting vasomotor effects on cerebral blood vessels (Tsukahara et al., 1986). β-Adrenergic receptor stimulation activates GTP-binding protein alpha subunits, which in turn increases intracellular cAMP level and therefore activates PKA. Afterwards, PKA phosphorylates the myosin light chain kinase and renders its inaction. This causes the myosin light chain to remain unphosphorylated and thus induces a vasorelaxant response. On the contrary, inhibition of β-adrenergic receptor can produce contractile function in the smooth muscle cells. So the action mechanism of H2S was studied by stimulating different enzymes/channel proteins in the signaling cascade of β-adrenergic receptor system. Our results indicated that NaHS significantly attenuated the effects of isoprenaline/forskolin (Figs. 3, 5). NaHS-induced vasoconstriction on cerebral artery was
Fig. 9. NaHS-induced concentration-constriction curves in endothelium-denuded and endothelium-intact cerebral arterial rings (A, n = 13–14), or in presence and absence of L-NAME in endothelium-intact cerebral arterial rings (B, n = 8–13). Mean ± SE, ⁎⁎P b 0.01 vs. endothelium-intact or control.
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
395
Unexpectedly, the exogenous H2S-induced vasoconstriction was potently enhanced in endothelium-denuded arteries (Fig. 9A), as well as in the presence of L-NAME (Fig. 9B). Obviously, these enhancements may be due to the deletion of NO-induced vasodilation, which is different with the result on aortic rings by Ali et al. (2006). Besides, the concentration of H2S in the presence of L-NAME (Fig. 9B) was close to those concentrations found in blood plasma and the vascular tissues (Kubo et al., 2007a). Taken together, these findings suggested that H2S might be a physiological mediator for moderating the relaxant effect of NO (Zhong et al., 2003) on cerebral artery. Further, it was found that L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the precontracted artery (Fig. 10). This finding supports the involvement of additional mechanism on the NaHS-induced vasoconstriction. In other words, H2S-induced vasoconstriction is partially antagonized by NO, not that H2S necessarily acts via NO. Nevertheless, it is definitely that more research on the cross-talk between H2S and NO on cerebral artery are needed. 5. Conclusions Fig. 10. L-NAME enhances the antagonizing effect of NaHS on forskolin-induced vasodilation in pre-contracted cerebral arteries. Arterial rings were pre-contracted with 5-HT (10−5 M) or 5-HT + L-NAME (10−4 M), and the effects of Forsk (10–7.5 M) or Forsk + NaHS (10−4 M) on relaxation were recorded. Representative tracer (top) and summarized analysis (bottom), Mean ± SE, n = 9. ⁎⁎P b 0.01.
decreased by 8B-cAMP (Fig. 6), but was not influenced by BayK (Fig. 7). These evidences suggested that the contractile effect of H2S on cerebral artery is, at least partially, associated with the reduction of cAMP level. The result seems to be contradictive with the fact that isoprenaline/ forskolin also can enhance the H2S-induced vasoconstriction instead of antagonizing it (Figs. 2, 4). To assure this, we examined the effect of NaHS on the elevated cAMP level in the HBVSMCs. The results showed that H2S alone did not altered cAMP level, but markedly attenuated isoprenaline-elevated cAMP levels (Fig. 8A), which can enhance the vasoconstriction of H2S. That is to say, H2S and cAMP are working together to increase the tone, not to simply antagonize each other. The mechanisms involved in reducing cAMP level by NaHS have yet to be further investigated, particularly in the activities of adenylyl cyclase and phosphodiesterase. The former controls the rate of cAMP synthesis, whereas the latter controls its hydrolysis. Both inhibition of adenylyl cyclase or activation of phosphodiesterase can decrease cAMP level in vascular smooth muscle cells. We examined the effect of NaHS on adenylyl cyclase activity of HBVSMCs. It was found that NaHS markedly attenuated forskolin-stimulated adenylyl cyclase activity in the HBVSMCs (Fig. 8B), indicating that H2S-induced vasoconstriction is through the inhibition of cAMP/adenylyl cyclase pathway. Additionally, in Fig. 8A, NaHS only has a tendency to decrease the cAMP level of the in vitro HBVSMCs, but without significant differences. Since adenylyl cyclase is the target site of NaHS in cerebral vascular smooth muscle cells, this experimental phenomena can be explained. The adenylyl cyclase activity of the in vitro HBVSMCs is relative low, which makes the effect of NaHS in inhibiting this enzyme not easy to be revealed. While, with the increasing activity by isoprenaline, adenylyl cyclase is relatively easier to be inhibited by NaHS. However, without measuring the activity of phosphodiesterase, it is hard to conclude that NaHS-induced vasoconstriction is only resulted from inhibition of adenylyl cyclase. Further studies are warranted. Previously, most scholars regarded the decease of NO as the general cause of H2S-induced vasoconstriction, through either forming a novel inactive nitrosothiol molecule with NO (Ali et al., 2006; Webb et al., 2008) or scavenging NO by a bicarbonate-dependent pathway (Liu and Bian, 2010). Therefore, to evaluate whether NO alters NaHS-induced vasoconstriction on cerebral artery, endothelium removal and incubation with L-NAME, a nitric oxide synthase inhibitor were investigated.
H2S induces vasoconstriction of cerebral arteries in normal initial tension. The effect is via inhibiting adenylyl cyclase to reduce cAMP level in cerebral vascular smooth muscle cells. The present study shows a novel and important profile of H2S effect on cerebral artery, which provides a new view on understanding the physiological regulation of cerebrovascular tone. Conflict of interest None. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgments This investigation was supported by the Specialized Research Fund for the Doctoral Program of Higher Education (20100201110054) and National Natural Science Foundation of China (81173059). References Ali, M.Y., Ping, C.Y., Mok, Y.Y., Ling, L., Whiteman, M., Bhatia, M., 2006. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br. J. Pharmacol. 6, 625–634. Ashley, A., Wang, L., Wang, R., 2012. The role of carbon monoxide as a gasotransmitter in cardiovascular and metabolic regulation. In: Hermann, A., Sitdikova, G., Weiger, T. (Eds.), Gasotransmitters: Physiology and Pathophysiology. Springer-Verlag, Berlin, pp. 37–70. Burnett, W.W., King, E.G., Grace, M., Hall, W.F., 1977. Hydrogen sulfide poisoning: review of 5 years' experience. Can. Med. Assoc. J. 11, 1277–1280. Cheang, W.S., Wong, W.T., Shen, B., Lau, C.W., Tian, X.Y., Tsang, S.Y., 2010. 4-Aminopyridinesensitive K+ channels contributes to NaHS-induced membrane hyperpolarization and relaxation in the rat coronary artery. Vasc. Pharmacol. 3-4, 94–98. Cheng, Y., Ndisang, J.F., Tang, G., Cao, K., Wang, R., 2004. Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats. Am. J. Physiol. Heart Circ. Physiol. 5, 2316–2323. Chitnis, M.K., Njie-Mbye, Y.F., Opere, C.A., Wood, M.E., Whiteman, M., Ohia, S.E., 2013. Pharmacological actions of the slow release hydrogen sulfide donor GYY4137 on phenylephrine-induced tone in isolated bovine ciliary artery. Exp. Eye Res. 350-354. Fiorucci, S., Antonelli, E., Distrutti, E., Rizzo, G., Mencarelli, A., Orlandi, S., 2005. Inhibition of hydrogen sulfide generation contributes to gastric injury caused by antiinflammatory nonsteroidal drugs. Gastroenterology 4, 1210–1224. Hashimoto, T., Ohata, H., Nobe, K., Sakai, Y., Honda, K., 2007. A novel approach for the determination of contractile and calcium responses of the basilar artery employing realtime confocal laser microscopy. J. Pharmacol. Toxicol. Methods 1, 79–86. Hosoki, R., Matsuki, N., Kimura, H., 1997. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem. Biophys. Res. Commun. 3, 527–531.
396
S. Li et al. / Toxicology and Applied Pharmacology 289 (2015) 389–396
Kabil, O., Banerjee, R., 2010. Redox biochemistry of hydrogen sulfide. J. Biol. Chem. 29, 21903–21907. Kubo, S., Doe, I., Kurokawa, Y., Nishikawa, H., Kawabata, A., 2007a. Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide: contribution to dual modulation of vascular tension. Toxicology 232, 138–146. Kubo, S., Kajiwara, M., Kawabata, A., 2007b. Dual modulation of the tension of isolated gastric artery and gastric mucosal circulation by hydrogen sulfide in rats. Inflammopharmacology 15, 288–292. Li, L., Whiteman, M., Guan, Y.Y., Neo, K.L., Cheng, Y., Lee, S.W., 2008. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide. Circulation 18, 2351–2360. Li, L., Hsu, A., Moore, P.K., 2009. Actions and interactions of nitric oxide, carbon monoxide and hydrogen sulphide in the cardiovascular system and in inflammation–a tale of three gases! Pharmacol. Ther. 3, 386–400. Liu, Y.H., Bian, J.S., 2010. Bicarbonate-dependent effect of hydrogen sulfide on vascular contractility in rat aortic rings. Am. J. Physiol. Cell Physiol. 4, 866–872. Mackay, K., Mochly-Rosen, D., 2001. Arachidonic acid protects neonatal rat cardiac myocytes from ischaemic injury through epsilon protein kinase C. Cardiovasc. Res. 1, 65–74. McPherson, G.A., 1992. Assessing vascular reactivity of arteries in the small vessel myograph. Clin. Exp. Pharmacol. Physiol. 12, 815–825. Njie-Mbye, Y.F., Bongmba, O.Y., Onyema, C.C., Chitnis, A., Kulkarni, M., Opere, C.A., 2010. Effect of hydrogen sulfide on cyclic AMP production in isolated bovine and porcine neural retinae. Neurochem. Res. 3, 487–494. Olson, K.R., Dombkowski, R.A., Russell, M.J., Doellman, M.M., Head, S.K., Whitfield, N.L., 2006. Hydrogen sulfide as an oxygen sensor/transducer in vertebrate hypoxic vasoconstriction and hypoxic vasodilation. J. Exp. Biol. 20, 4011–4023. Pan, T.T., Neo, K.L., Hu, L.F., Yong, Q.C., Bian, J.S., 2008. H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes. Am. J. Physiol. Cell Physiol. 1, 169–177. Ping, N.N., Cao, L., Xiao, X., Li, S., Cao, Y.X., 2014. The determination of optimal initial tension in rat coronary artery using wire myography. Physiol. Res. 1, 143–146. Raij, L., 2001. Workshop: hypertension and cardiovascular risk factors: role of the angiotensin II-nitric oxide interaction. Hypertension 2 (Pt 2), 767–773. Siebert, N., Cantre, D., Eipel, C., Vollmar, B., 2008. H2S contributes to the hepatic arterial buffer response and mediates vasorelaxation of the hepatic artery via activation of KATP channels. Am. J. Physiol. Gastrointest. Liver Physiol. 6, 1266–1273.
Smith, R.P., Gosselin, R.E., 1979. Hydrogen sulfide poisoning. J. Occup. Med. 2, 93–97. Streeter, E., Hart, J., Badoer, E., 2012. An investigation of the mechanisms of hydrogen sulfide-induced vasorelaxation in rat middle cerebral arteries. Naunyn Schmiedeberg's Arch. Pharmacol. 10, 991–1002. Sun, T., Liu, R., Cao, Y.X., 2011. Vasorelaxant and antihypertensive effects of formononetin through endothelium-dependent and -independent mechanisms. Acta Pharmacol. Sin. 8, 1009–1018. Tian, X.Y., Wong, W.T., Sayed, N., Luo, J., Tsang, S.Y., Bian, Z.X., 2012. NaHS relaxes rat cerebral artery in vitro via inhibition of l-type voltage-sensitive Ca2+ channel. Pharmacol. Res. 2, 239–246. Tsukahara, T., Taniguchi, T., Shimohama, S., Fujiwara, M., Handa, H., 1986. Characterization of beta adrenergic receptors in human cerebral arteries and alteration of the receptors after subarachnoid hemorrhage. Stroke 2, 202–207. Ulrich, F., Huige, L., 2012. Nitric oxide: biological synthesis and functions. In: Hermann, A., Sitdikova, G., Weiger, T. (Eds.), Gasotransmitters: Physiology and Pathophysiology. Springer-Verlag, Berlin, pp. 1–36. Wang, R., 2002. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter? FASEB J. 13, 1792–1798. Webb, J.G., Yates, P.W., Yang, Q., Mukhin, Y.V., Lanier, S.M., 2001. Adenylyl cyclase isoforms and signal integration in models of vascular smooth muscle cells. Am. J. Physiol. Heart Circ. Physiol. 4, 1545–1552. Webb, G.D., Lim, L.H., Oh, V.M., Yeo, S.B., Cheong, Y.P., Ali, M.Y., 2008. Contractile and vasorelaxant effects of hydrogen sulfide and its biosynthesis in the human internal mammary artery. J. Pharmacol. Exp. Ther. 2, 876–882. Weber, N.C., Toma, O., Wolter, J.I., Obal, D., Mullenheim, J., Preckel, B., 2005. The noble gas xenon induces pharmacological preconditioning in the rat heart in vivo via induction of PKC-epsilon and p38 MAPK. Br. J. Pharmacol. 1, 123–132. Zhao, W., Zhang, J., Lu, Y., Wang, R., 2001. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J. 21, 6008–6016. Zhong, G., Chen, F., Cheng, Y., Tang, C., Du, J., 2003. The role of hydrogen sulfide generation in the pathogenesis of hypertension in rats induced by inhibition of nitric oxide synthase. J. Hypertens. 10, 1879–1885.