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VASCULAR BIOLOGY
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Submitted 6 April 2008 ; accepted in final form 5 September 2008
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ABSTRACT |
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3–30 µM H2S) concentration-dependently reversed the vasodilation caused by isoprenaline and salbutamol, two β-adrenoceptor agonists, and forskolin, a selective adenylyl cyclase activator, in phenylephrine-precontracted rat aortic rings. Pretreatment with NaHS (10–100 µM) for 5 min also significantly attenuated the vasorelaxant effect of salbutamol and forskolin. More importantly, NaHS (5–100 µM) significantly reversed forskolin-induced cAMP accumulation in vascular smooth muscle cells. However, NaHS produced significant, but weaker, vasoconstriction in the presence of NG-nitro-L-arginine methyl ester (100 µM), a nitric oxide synthase inhibitor, or in endothelium-denuded aortic rings. Blockade of ATP-sensitive potassium channels with glibenclamide (10 µM) failed to attenuate the vasoconstriction induced by H2S. Taken together, we demonstrated for the first time that the vasoconstrictive effect of H2S involves the adenyly cyclase/cAMP pathway. hydrogen sulfide; smooth muscle cell; adenosine 3',5'-cyclic monophosphate; nitric oxide; vascular contractility
Endogenous H2S is synthesized from L-cysteine, a sulfur-containing amino acid, mainly by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-
-synthase (CSE). CBS and CSE are widely distributed in tissues; however, CBS is a predominant source of H2S in the central nervous system, whereas CSE is highly expressed in the cardiovascular system (the aorta, mesenteric artery, pulmonary artery, tail artery, and portal vein). In some other tissues such as liver and kidney, both enzymes contribute to generation of endogenous H2S (10).
H2S has shown great importance in modulating the vascular tone in various vascular systems, and the signaling mechanisms involved are complicated where some have yet to be clarified. Immunohistochemical studies and RT-PCR revealed that CSE is expressed in vascular smooth muscle cells but not in the endothelial cells (17). In earlier studies, H2S (>60 µM) was found to dilate blood vessels and decrease mean arterial pressure in anesthetized rats by activating ATP-sensitive K+ (KATP) channels and hyperpolarizes membrane potential of vascular smooth muscle cells (17). Subsequent studies demonstrated H2S induces relaxation in isolated rat aorta and mesenteric artery at higher concentrations (4, 6, 16, 17). We recently reported that H2S relaxes smooth muscle cells via induction of acidification due to activation of Cl–/HCO3– exchanger (9).
Interestingly, NaHS may also exhibit contractile activity. It is recently reported that H2S at lower concentrations (50–100 µM) induces vasoconstriction in rat aortic rings by forming a novel nitrosothiol molecule with endothelial NO (1, 14). Low concentrations of H2S reversed the vasorelaxant effects of endothelium-dependent vasodilators such as acetylcholine (ACh) and histamine. In vivo, low doses of sodium hydrosulfide (NaHS) increase the mean arterial pressure in anesthetized rats (1). In another recent study, it is suggested that this contractile activity of H2S is attributed to the direct inhibition of recombinant endothelial NO synthase (eNOS) (8). It was also found that H2S exhibits both contractile and relaxant effects in rat pulmonary artery precontracted with norepinephrine (5, 12). These data suggest that H2S at different concentrations may have different effects in modulating the vascular tone which involves different signaling mechanisms.
cAMP is an intracellular second messenger for signal transduction in modulating vasodilation. Normal intracellular concentration of cAMP is 10–7 M. An extracellular signal can change the intracellular cAMP level by >20-fold in a few seconds. cAMP is synthesized from ATP by adenylyl cyclase (AC) located at the cell membrane. Binding of an agonist to the β-adrenoceptor in the cell membrane activates AC via the stimulatory guanine nucleotide-binding protein (Gs) and leads to elevations in intracellular cAMP. Increase in intracellular cAMP activates the cAMP-dependent protein kinase (PKA), which in turn phosphorylates the myosin light chain kinase and renders it inactive. This causes the myosin light chain to remain unphosphorylated and thus induces a vasorelaxant response. Inhibition of the cAMP/PKA pathway may therefore produce contractile function in the smooth muscle cells. cAMP decomposition to AMP is catalyzed by the enzyme phosphodiesterase (PDE). PDE is activated by phosphorylation catalyzed by PKA, which in turn is activated by cAMP. The cAMP/PKA pathway therefore plays an important role in regulation of the vascular tone.
The present study was therefore designed to investigate the involvement of the cAMP/PKA signaling pathway in the vasoconstrictive effect of H2S. For the first time, we found that low concentrations of H2S significantly reversed or reduced the relaxant activity of various endothelium-independent vasodilators.
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METHODS |
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NaHS was used as a H2S donor and was freshly prepared on the day of every experiment. In normal physiological solution, one-third of NaHS exists as H2S, and the other two-third exists as HS– (2). As used in numerous studies, NaHS has been widely used as a source of exogenous H2S.
Animals. Male Sprague-Dawley rats 6–7 wk old (250–300 g) were used for all animal experiments under the approval of the Institutional Animal Care and Use Committee of the National University of Singapore.
Cell culture.
A7r5 cells, derived from rat BD1X embryonic rat aortic smooth muscle, were purchased from American Type Culture Collection (Rockville, MD). The cells were cultured in DMEM supplemented with 10% (wt/vol) FBS and 1% (wt/vol) antibiotics (streptomycin and penicillin) at 37°C with 95% oxygen and 5% CO2. The culture medium was changed every other day. Experiments were performed when cells reached 80% confluence between passages 5 and 10.
Measurement of rat aorta contractility. The rats were anesthetized with pentobarbital sodium (50 mg/kg ip) followed by administration of heparin (1,000 units). The thoracic aorta was excised immediately and placed in oxygenated (95% O2-5% CO2) Krebs solution (composition in mM: 118 NaCl, 5.4 KCl, 1.3 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 1.7 glucose). The aorta was cleaned of adventitious tissues and blood and cut into ring segments of 1–2 mm in length. The ring segments were then mounted between two wire hooks and suspended in organ bath chambers containing 10 ml of oxygenated Krebs solution (pH 7.4) maintained at 37°C. The segments were allowed to equilibrate for 1 h under a resting tension of 1.5–2.0 g. Any changes in tension were recorded through force transducers connected to a PowerLab system (ADInstruments, Bella Vista, NSW, Australia) running the Chart v5.1 software.
After 1 h equilibration, the vessel response was tested for maximal contraction with 1.68 µM PE. The PE was then washed out, allowing the vessel to return to the initial basal tone before each experiment. Subsequently, the rings were precontracted with PE at a concentration of 100 nM, which was found to produce 70–80% of the maximum contraction (1.68 µM PE) in preliminary experiments (data not shown). In some experiments, the aortic segments were denuded of endothelium by gently rubbing the lumen with cotton string. The presence of endothelium was confirmed by adding 200 nM ACh, which was able to produce 80% relaxation on PE-precontracted rings, whereas a relaxation of <10% indicated the aortic segments were successfully denuded.
Two different types of experimental protocols were employed in this study. In the first protocol, PE-precontracted rings were relaxed to 80% in each case by 10 µM isoprenaline, 2 µM salbutamol, and 50 nM forskolin (concentrations were predetermined in preliminary experiments). When the relaxation response reached a stable state after 5–10 min, increasing concentrations of NaHS (1–200 µM) were added to the bath solution to study the effect of NaHS on each of the vasodilators applied. Results are shown as percent of contraction or relaxation with respect to each vasodilator.
In the second protocol, the ability of salbutamol (2 µM) and forskolin (50 nM) to relax PE-precontracted rings to 80% was reobserved either alone or with NaHS at specific concentrations of 10, 50, and 100 µM. In this case, NaHS was added either before PE precontraction or together with the vasodilator where NaHS was added to the vasodilator in an Eppendorf tube, and the mixture was then added directly to the organ bath solution. Results are shown as percent relaxation of the PE-induced contraction.
To study the signaling mechanisms, glibenclamide (10 µM, an inhibitor of K+ ATP channels) and L-NAME [100 µM, an inhibitor of nitric oxide synthase (NOS)] were added to the bath solution 30 and 5 min, respectively, before PE precontraction. The successful inhibition of NOS by L-NAME (100 µM, 5 min) was verified by observing a marked reduction in ACh-induced relaxation in the presence of L-NAME.
Assay of cAMP. The concentrations of cAMP in A7r5 aortic smooth muscle cells were determined using a cAMP enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). The assay is based on the competition between free cAMP and a cAMP-acetylcholinesterase (AChE) conjugate for a limited number of cAMP-specific rabbit antibody binding sites. The concentration of the conjugate is held constant while the concentration of cAMP varies. Therefore, the amount of conjugate that binds to the rabbit antibody will be inversely proportional to the concentration of cAMP in the well.
To determine the effects of H2S on cAMP concentration in the smooth muscle cells, different concentrations of NaHS were added to the cell culture medium 10 min before the addition of 10 µM forskolin. The cell cultures were then incubated for another 10 min at 37°C with 95% oxygen and 5% CO2. The treated cells were washed with PBS and lysed with 0.1 M HCl after first removing the culture media. After short incubation, the cell lysates were centrifuged at 1,000 g for 10 min, and the supernatants were collected. To increase the sensitivity of the assay, the samples were acetylated before the assay.
Microplates precoated with mouse monoclonal antibody were preprovided. The samples were pipetted into the wells and incubated with the cAMP-specific antibody and AChE conjugate for 18 h at 4°C. After incubation, the well contents were removed, and the wells were washed with wash buffer to remove any unbound reagents. Subsequently, a reagent that contains the substrate to AChE conjugate was added to the wells. The enzymatic reaction generated a distinct yellow color inside the wells, and the plate was read on a microplate reader at a wavelength of 415 nm. The intensity of this color is proportional to the amount of cAMP conjugate bound to the well, which is inversely proportional to the amount of free cAMP present in the well during the incubation.
Statistical analysis. Data are shown as means ± SE with the number of experimental observations indicated in parenthesis. Aortic ring segments were obtained from at least three rats in every experiment. Statistical analysis was carried out using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Statistical significance was set at P < 0.05.
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RESULTS |
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80% (Fig. 1, A and B). When applied cumulatively from 5 to 100 µM, NaHS caused vasoconstriction in both ACh- and isoprenaline-relaxed aortic rings in a concentration-dependent manner (Fig. 1C). The maximum effect was reached when the concentration of NaHS was at 50–100 µM. When the concentration of NaHS was increased to 200 µM, the vasoconstrictive effect of H2S diminished. These data imply that, besides NO, cAMP may also be an important contributor in the vasoconstriction caused by H2S.
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Effect of H2S on vascular contractility in aortic rings relaxed by forskolin. To examine the involvement of AC in the signaling pathway of β2-adrenoceptor activation, we observed the vasoconstrictive effect of H2S in the presence and absence of forskolin, an activator of AC. As shown in Fig. 3A, forskolin (50 nM) also markedly relaxed aortic rings precontracted by PE. This effect was concentration-dependently reversed by NaHS (10–50 µM) when applied cumulatively. Given together with forskolin, NaHS at a similar concentration range also significantly attenuated the vasorelaxant effect of forskolin in a concentration-dependent manner (Fig. 3B).
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In the second series of experiments, we examined whether L-NAME could abolish the effect of NaHS with a pretreatment protocol. As shown in Fig. 6A, given together with NaHS (100 µM), L-NAME failed to abolish the vasoconstrictive effect of NaHS.
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Vasoconstrictive effect of H2S in endothelium-denuded aortic rings. To further confirm our findings, we observed the effect of H2S in endothelium-denuded aortic rings. We first compared the vasorelaxant effect of forskolin in both endothelium-intact and denuded aortic rings. The data shows that forskolin-mediated vasodilation is independent of the endothelium (Fig. 7A). The vasoconstrictive effect of H2S was therefore observed in endothelium-denuded aortic rings. As shown in Fig. 7B, NaHS at 50–100 µM was still able to significantly, but in a weaker manner, induce vasoconstriction in forskolin (50 nM)-relaxed endothelium-denuded aortic rings. This further supports our hypothesis that the vasoconstrictive effect of NaHS at low concentrations is partially endothelium-independent.
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DISCUSSION |
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To examine whether cAMP mediates H2S-induced vasoconstriction, we first observed the vasoconstrictive effect of H2S in β-adrenoceptor agonists-induced vasorelaxation. We found that NaHS at a concentration range of 10–100 µM (yields 3–30 µM H2S) concentration-dependently reversed the vasorelaxation caused by the β-adrenoceptor agonists isoprenaline and salbutamol in PE-precontracted rat aorta. These data suggest that H2S may modulate β-adrenergic function in vascular vessels.
It is interesting to note that posttreatment with NaHS (1–100 µM, particularly at 50 µM) produced stronger vasoconstriction (Fig. 2A) compared with that caused by pretreatment with NaHS (Fig. 2B). The different responses to pretreatment and posttreatment with NaHS may result from the different remaining concentrations of H2S in the organ bath at the observing time point in the two experimental situations. In the pretreatment experiments, the concentration of H2S might have been greatly reduced due to constant bubbling of O2 and CO2 in the organ bath chamber. A large amount of H2S might have escaped from the bath solution after waiting for addition of PE (5 min) and salbutamol (10 min). The concentration of NaHS in pretreatment experiments may be lower than those in posttreatment experiments where the effect of NaHS was observed immediately after addition of the drug.
Because activation of β-adrenoceptors stimulates the AC/cAMP pathway, we further examined whether H2S regulates the effects of forskolin, an AC activator, on vascular contractility and intracellular cAMP level in smooth muscle cells. It was found that administration of NaHS (10–100 µM) either before, during, or after addition of forskolin all attenuated/reversed the vasorelaxant effect of forskolin. More importantly, H2S (5–100 µM) also attenuated forskolin-induced cAMP accumulation. With these lines of pharmacological evidence, we propose that the contractile effect of H2S observed in isolated rat aorta is, at least partially, associated with reducing cAMP level.
The precise mechanisms involved have yet to be further investigated, particularly in the activities of AC and PDE. The former controls the rate of cAMP synthesis, whereas the latter controls its hydrolysis. Both inhibition of AC and stimulation of PDE may decrease cAMP level in the vascular smooth muscle cells. We found in the present study that H2S attenuated the elevated cAMP accumulation caused by forskolin-stimulated AC activity. However, without measuring the activities of AC and PDE, it is still far away to conclude that this is resulted from inhibition of AC. Further studies on the activities of these enzymes upon H2S treatment are warranted.
To date, nine types of AC, of which type III, IV, V, VI, and VIII are expressed in vascular vessels (13), and 11 distinct PDE families (11) have been identified. Each isoform exhibits distinct tissue, cell, and subcellular expression patterns. They are hence likely to participate in discrete signal transduction pathways and thus in discrete physiological and pathophysiological processes, e.g., penile erection, asthma, pulmonary hypertension, atherosclerosis, heart failure, and diabetes. Consequently, investigations of the effect of H2S on isoforms of AC/PDE are of both fundamental and pharmacological interest.
It is interesting to note that H2S regulates cAMP production in different ways in various systems. It was found that H2S increases cAMP production in brain cells (7), but inhibits the AC/cAMP pathway in cardiac myocytes (15). Several mechanisms are probably involved in this discrepancy. First, the primary action site of H2S may not be at AC and PDE. H2S may regulate activities of these enzymes indirectly via stimulating different signaling mechanisms in various cells. Second, H2S may act on different isoforms of these enzymes in brain and cardiovascular tissues as discussed above and therefore cause different effects on cAMP production. More works are warranted to examine the exact mechanisms involved.
The cross talk between H2S and NO is still not clear. H2S has been reported to either enhance (16) or to attenuate (6) the relaxant effect of NO in the rat aorta. Recently, it has been reported that H2S might suppress the vasodilator effect of NO through two distinct endothelium-dependent mechanisms via inhibiting eNOS and scavenging of NO with the formation of nitrosothiol compound (1, 8, 14), resulting in contractile responses in isolated rat aorta. However, in the present study, we demonstrated for the first time that the vasoconstrictive response of H2S was not completely abolished in the presence of L-NAME as well as in those in endothelium-denuded vascular rings. These observations strongly support the involvement of additional endothelium-independent mechanisms in the vasoconstrictive activity of H2S.
The most well-accepted endothelium-independent mechanism involved in H2S vasorelaxation to date is via the opening of KATP channels in the vascular smooth muscle cells. The vasodilatory effect of H2S was attenuated when vessels were incubated in a high-K+ medium. Patch-clamp studies have also demonstrated that H2S increases KATP-dependent current and induces hyperpolarization in isolated vascular smooth muscle cells (17). Our data demonstrated that glibenclamide, a KATP channel inhibitor, attenuated the H2S-induced vasodilation at higher concentrations (>100 µM) but had no significant effect on the contractile responses, implying the involvement of other mechanisms independent of KATP channels. In other words, the vasoconstrictive activity of H2S does not involve KATP channel activity.
In conclusion, we demonstrated for the first time the vasoconstrictive function of H2S in the aortic rings not only through decreasing NO production but also through inhibiting cAMP accumulation. Our study added a brand new line to the study on the vascular action of H2S.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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