Endogenous hydrogen sulfide (H2S) is involved in the regulation of vascular tone. We hypothesized that the lowering of calcium and opening of potassium (K) channels as well as calcium-independent mechanisms are involved in H2S-induced relaxation in rat mesenteric small arteries. Amperometric recordings revealed that free [H2S] after addition to closed tubes of sodium hydrosulfide (NaHS), Na2S, and GYY4137 [P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid] were, respectively, 14%, 17%, and 1% of added amount. The compounds caused equipotent relaxations in isometric myographs, but based on the measured free [H2S], GYY4137 caused more relaxation in relation to released free H2S than NaHS and Na2S in rat mesenteric small arteries. Simultaneous measurements of [H2S] and tension showed that 15 µM of free H2S caused 61% relaxation in superior mesenteric arteries. Simultaneous measurements of smooth muscle calcium and tension revealed that NaHS lowered calcium and caused relaxation of NE-contracted arteries, while high extracellular potassium reduced NaHS relaxation without corresponding calcium changes. In NE-contracted arteries, NaHS (1 mM) lowered the phosphorylation of myosin light chain, while phosphorylation of myosin phosphatase target subunit 1 remained unchanged. Protein kinase A and G, inhibitors of guanylate cyclase, failed to reduce NaHS relaxation, whereas blockers of voltage-gated KV7 channels inhibited NaHS relaxation, and blockers of mitochondrial complex I and III abolished NaHS relaxation. Our findings suggest that low micromolar concentrations of free H2S open K channels followed by lowering of smooth muscle calcium, and by another mechanism involving mitochondrial complex I and III leads to uncoupling of force, and hence vasodilation.
The three gasses hydrogen sulfide (H2S), nitric oxide (NO), and carbon monoxide have biologic effects at low concentrations and are toxic at high concentrations (Szabo, 2007; Sun et al., 2011). The reported physiologic levels of H2S vary greatly; in both humans and rodents, plasma levels ranging from below 1 µM and up to 300 µM have been reported (Olson, 2011). Part of this variance can probably be ascribed to the techniques used to measure H2S because it is often the total sulfur pool rather than the free H2S that is measured (Olson et al., 2014). Although it is not well understood which of the forms H2S, HS−, or S2− are relevant for the biologic effects, free H2S is thought to mediate most of the effect on vascular tone (Kimura, 2014).
The precise role of H2S in regulating blood pressure remains unclarified as knockout of cystathione gamma-lyase (CSE), which is considered the most important H2S producing enzyme in the cardiovascular system, has yielded disparate results. One CSE knockout mouse was found to be normotensive (Ishii et al., 2010); another CSE knockout mouse was reported to have substantially lower levels of plasma H2S as well as age-dependent hypertension (Yang et al., 2008). Although injection of a H2S salt transiently reduces mean arterial pressure (Ali et al., 2006; Yang et al., 2008), the effect on the vascular tone is complex. In non-mammalian species, H2S induces dose-dependent constriction and/or constriction followed by relaxation (Dombkowski et al., 2005). In arteries from rats, it has been suggested that low H2S concentrations cause contractions whereas high H2S concentrations induce relaxation of mesenteric, aorta, and gastric arteries as well as of human mammary arteries (Ali et al., 2006; Kubo et al., 2007; Webb et al., 2008; d’Emmanuele di Villa Bianca et al., 2011). These studies were based on the application of the H2S salts NaHS and Na2S, thought to yield high transient concentrations of H2S; therefore, the use of slow-release H2S donors such as P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid (GYY4137) has been advocated (Papapetropoulos et al., 2015). However, GYY4137 also causes both vasodilation and vasoconstriction (Li et al., 2008; Salomone et al., 2014). This suggests that the actual concentrations reaching the vascular smooth muscle are of major importance for the observed effect and mechanisms underlying H2S-induced vasodilation.
A number of other mechanisms underlying H2S-induced vasorelaxation have been suggested, including involvement of NO (Zhong et al., 2003; Cheang et al., 2010), release of calcitonin gene-related peptide from nerve-endings due to activation of transient receptor potential ankyrin-1 channels (Fernandes et al., 2013; White et al., 2013), and changes in intracellular pH through an effect on a 4.4-diisothiocyano-2.2ʹ-stilbenedisulfonic acid-sensitive Cl−/HCO3-exchanger (Lee et al., 2007; Kiss et al., 2008). Opening of potassium (K) channels leads to hyperpolarization and closing of voltage-dependent Ca2+-channels, decreasing the intracellular calcium concentration ([Ca2+]i), followed by vasodilation. Several studies have shown that H2S opens ATP-sensitive potassium-channels (KATP channels), leading to hyperpolarization of the vascular smooth muscle layer (Zhao and Wang, 2002; Tang et al., 2005; Webb et al., 2008; Martelli et al., 2013). Although opening of KATP channels has been widely accepted as a main mechanism underlying H2S-induced vasorelaxation, some studies have found only a small or no involvement of KATP channels (Kubo et al., 2007; Kiss et al., 2008; Cheang et al., 2010). Other potassium channels (K channels) have also been reported to play a role in H2S relaxation such as voltage-gated K channels (Cheang et al., 2010), KV7 channels (Schleifenbaum et al., 2010; Martelli et al., 2013; Hedegaard et al., 2014), and large conductance calcium-activated potassium channels (BKCa) (Jackson-Weaver et al., 2011, 2013; Li et al., 2012). It has been shown that the H2S donors NaHS and Na2S activate calcium sparks (Liang et al., 2012; Jackson-Weaver et al., 2013) and also that they reduce global [Ca2+] in cerebral arterioles (Liang et al., 2012). However, so far there have been no attempts to directly correlate vascular smooth muscle cell calcium to changes in vascular tone by simultaneous measurements of [Ca2+]i and relaxation.
We hypothesized that lowering of calcium and opening of K channels as well as calcium-independent mechanisms are involved in H2S-induced relaxation. To investigate this hypothesis, we performed the following measurements: 1) the release of H2S from NaHS, Na2S, and GYY4137 was examined by the use of a H2S microsensor; 2) changes in [Ca2+]i were measured simultaneously with relaxation to determine whether lowering of [Ca2+]i contributes to H2S relaxation; 3) the phosphorylation of myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT-1) was measured; and 4) the involvement of different K channels and the mitochondrial electron chain complexes was investigated using selective blockers in rat small mesenteric arteries.
Materials and Methods
Solutions and Chemicals.
The following drugs were used: norepinephrine (NE), acetylcholine (ACh), 4-aminopyridine, antimycin A, NaHS, Na2S, tetraethylammonium (TEA), 4,4ʹ-diisothiocyano-2,2ʹ-stilbenedisulfonic acid (DIDS), linopirdine, MITO-TEMPO [(2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride monohydrate], L-NOARG (NG-nitro-l-arginine), retigabine, Rp-8-pCPT-cGMPS [(Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate], KT5720 [(9S,10S,12R)-2,3,9,10,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]-benzo-diazocine-10-carboxylic acid hexyl ester], XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride], potassium polysulfide (K2Sn), rotenone, sodium nitroprusside (SNP), and glibenclamide from Sigma-Aldrich (St. Louis, MO). ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) was obtained from Tocris (Bristol, United Kingdom), and Fura-2 AM and pluoronic F127 were purchased from Invitrogen (Taastrup, Denmark). Iberiotoxin was purchased from Latoxan, Valence, France. GYY4137 was synthesized as previously described elsewhere (Li et al., 2008).
The NaHS and Na2S solution was made fresh every day. To neutralize pH of the solution, hydrochloric acid was added until a pH of 7.35–7.45 was obtained. The composition of the physiologic salt solution (PSS) was NaCl 119 mM, NaHCO3 25 mM, glucose 5.5 mM, CaCl2 1.6 mM, KH2PO4 1.18 mM, MgSO4 1.17 mM, and EDTA 0.027 mM. The composition of the lysis buffer was 20 mM tris/HCL, 5 mM EGTA, 150 mM NaCl, 20 mM glycerophosphate, 10 mM NaF, 1% Triton X-100, 0.1% Tween-20, and 1x Halt Protease and Phosphatase Inhibitor Cocktail. The sample buffer composition was dithiothreitol 6 mM, Tris-HCl 350 mM, dithiothreitol and sodium lauryl sulfate 10%, glycerol 30%, and bromphenol blue 0.12%
Hydrogen Sulfide Measurements.
For measurement of the H2S concentration, a hydrogen sulfide microsensor (Unisense A/S, Aarhus, Denmark) was used. The microsensor is a miniaturized amperometric sensor consisting of an internal reference and a sensing and guard anode. H2S from the environment is driven by the external partial pressure and will penetrate through the sensor tip membrane into the alkaline electrolyte. Because the sensor is sensitive to temperature, we performed all calibrations and measurements at 37°C, as this temperature is physiologically appropriate. The sensor was calibrated below pH 4.0 because all the added Na2S would be on the H2S form; at pH 7.4 a larger part of the H2S is in the HS− form and thus is not measured by the sensor. Calibration was made by dilution of a stock solution of 1 M Na2S, which was dissolved in N2-flushed PSS in a closed tube. Measurements were performed in PSS (pH 7.4, 37°C) in closed tubes; after addition of the donors, H2S concentration was measured for 30 minutes. Because GYY4137 has been reported to release more H2S at low pH (Li et al., 2008), we also measured the release of H2S from GYY4137 at pH 3.0.
Microvascular Myograph Studies.
This study followed the recommendations in the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health and the ARRIVE Guidelines. Wistar rats (10–12 weeks) were euthanized by cervical dislocation followed by exsanguination, and the mesentery bed was removed and placed in ice-cold PSS. Third branch mesenteric arteries were dissected and mounted on wire myographs (as previously described elsewhere) for recordings of isometric tension (Mulvany and Halpern, 1977). The 1.5–2.0 mm long arterial rings, with diameters of approximately 200–300 µm, were mounted on 40-µm stainless steel wires and kept in PSS at 37°C and bubbled with 5% CO2/21% O2/74% N2.
After 30 minutes of equilibration, the arteries were normalized. Experiments were performed on arteries stretched to 90% of L100, where L100 is defined as the circumference of the relaxed artery exposed to a transmural pressure of 100 mmHg. Before conducting experiments, the viability of the arterial segment was tested. The mesenteric arteries were contracted twice by NE (10 µM). To test the presence of functional endothelium, the arteries were contracted with NE (3 µM) before ACh (10 µM) was added. Arteries were only included if they developed an active force corresponding to a transmural pressure of 100 mmHg and relaxed a minimum of 60% to ACh (10 µM).
Simultaneous Measurements of H2S and Relaxation.
For simultaneous measurements of force and H2S concentration, a segment of the mesenteric superior artery was mounted on two 100-µm thick steel wires in a single chamber myograph as previously described elsewhere for simultaneous measurements of force and NO concentration (Simonsen et al., 1999). A H2S-sensitive microelectrode with tip diameter of 50–80 µm (Unisense A/S, Aarhus, Denmark) was first calibrated as described earlier; by use of a micromanipulator, it was introduced into the lumen of the artery while another sensor was placed in the organ bath. Mesenteric superior arteries were normalized and tested as described for small mesenteric arteries, only difference being that 1 µM of NE was used to contract the arteries. The tension and electrode current were recorded in Labscribe (World Precision Instruments, Hitchen, United Kingdom), which allowed simultaneous measurements of H2S and relaxation. Changes in the coating of the electrode resulted in abrupt changes in basal electrode current, and in these cases the electrode was discarded.
To investigate the mechanism of H2S-induced vasodilation, arteries with endothelium were incubated with either vehicle or inhibitors before concentration–response curves (CRCs) were constructed for NaHS, Na2S, or GYY4137 in preparations contracted with NE. The control and examination of drugs were run in parallel, and only one CRC was constructed for each vasodilator per animal.
To investigate the involvement of K channels in H2S-induced relaxation, CRCs for NaHS were performed on NE-contracted arteries incubated with blockers of different K channels: glibenclamide (10 µM), TEA (1 mM and 3 mM), iberiotoxin (100 nM), 4-AP (0.5 mM), linopirdine (10 µM), and XE991 (10 µM) were incubated with arteries for 30 minutes before dilation was induced by NaHS.
To investigate other pathways suggested to be involved in NaHS relaxation, CRCs for NaHS were performed on NE-contracted arteries incubated with L-NOARG (100 µM), ODQ (3 µM), KT5720 (200 nM), and Rp-8-pCPT-cGMPS (20 µM) (incubated with arteries for 30 minutes), and rotenone (1 µM), antimycin A (1 µM), Tempol [4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl] (300 µM), MITO-TEMPO (10 µM), and DIDS (100 µM) (incubated with arteries for 15 minutes). Rotenone (1 µM), antimycin A (1 µM), Tempol (300 µM), and MITO-TEMPO (10 µM) were also investigated in arteries contracted with 60 mM K (K60PSS). To obtain comparable levels of contraction in arteries incubated with rotenone or antimycin A, NE was added on top of high-potassium physiologic saline solution (KPSS).
Simultaneous Measurements of Intracellular Calcium Levels and Tension.
Measurements of intracellular calcium levels were performed in mesenteric small arteries as previously described elsewhere (Rodriguez-Rodriguez et al., 2008). After mounting, equilibration, and normalization, the arteries were loaded with Fura-2 AM (8 µM) and loading mix (Pluronic F127) in the dark for 2 hours. The myograph was placed on a Zeiss inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) for dual-excitation wavelength fluorometry. After 30 minutes of washout, the arteries were illuminated with light alternating at 340 and 380 nm, and the intensity of the emitted fluorescence was collected at 510 nm. At the end of the experiment, ionomycin (10 μM) was added to PSS to achieve maximal saturation of Fura-2 with calcium. Then the fluorescence was quenched using nominally calcium-free PSS and MnCl2 (15 mM) to measure the background signal, which was subtracted from the recordings.
For detection of MLC, phosphorylated MLC (pMLC), MYPT, and phosphorylated MYPT (pMYPT), mesenteric small arteries were frozen in ice-cold acetone with trichloroacetic acid (10%) and dithiothreitol (10 mM) and then placed in a −80°C freezer for 24 hours before being washed 3 times with acetone with dithiothreitol (10 mM). Before homogenization with pellet pestles (Sigma, St. Louis, MO), the samples were heated to 50°C for 10 minutes in 15 µl of 50% lysis buffer and 50% sample buffer. After homogenization they were sonicated for 45 seconds and centrifuged for 10 minutes at 10.000 rpm at 4°C. The sampling in acetone and direct dissolving in sample buffer is incompatible with our protein measurements. The same volume was added to the gels for the blotting, and the results expressed as a ratio of phosphorylated to unphosphorylated protein.
A linear relation is required for quantitative protein expression (Eaton et al., 2013). We have previously observed that the densitometric measurements of MYPT-1 correlated linearly with pMYPT and MLC with pMLC. Therefore, pMYPT and pMLC were expressed as ratios of their respective unphosphorylated proteins.
All samples were run simultaneously to minimize any differences in transfer. Samples and a prestain marker (Bio-Rad Laboratories, Hercules, CA) were loaded onto the gel. Immunoblotting was performed as described previously elsewhere (Hedegaard et al., 2014). The following antibodies were applied: MYPT 1:6000 (Sc-25618; Santa Cruz Biotechnology, Dallas, TX), pMYPT 1:500 (05-773; Millipore, Billerica, MA), MLC 1:1000 (3672S; Cell Signaling Technology, Beverly), pMLC 1:2000 (3671S; Cell Signaling Technology), and secondary anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology) 1:4000.
Data Calculation and Analysis.
All recordings and calculations were performed by PowerLab data system and Chart 5.5 (ADInstruments, Oxfordshire, United Kingdom) or Labscribe (World Precision Instruments, Hitchin, United Kingdom). The mechanical responses of the vessel segments were measured as active wall tension (ΔT), which is the changes in force (ΔF) divided by twice the segment length (2l). The CRCs were compared with controls by a two-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test. Differences between means were analyzed by unpaired two-tailed t test. One-way ANOVA followed by Bonferroni post-test or a Student t test was used to analyze difference between mean relaxation to one dose of NaHS or SNP. P < 0.05 was considered statistically significant for all tests. All graphs and statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA). The results are given as mean ± S.E.M.
Release of H2S from NaHS, Na2S, and GYY4137.
The release of free H2S from the donors was examined by use of a microsensor in closed containers containing PSS. The H2S microsensor responded with changes in current to micromolar concentrations of Na2S, and the output current of the probes correlated linearly with the concentrations of Na2S (Fig. 1A). The amount of H2S released from 300 µM of NaHS in PSS at pH 7.4 was 43 µM (14%); during the 30 minutes of measurements the concentration slowly decreased to 33 µM (11%) (Fig. 1B). The corresponding curve for 300 µM Na2S in PSS at pH 7.4 had a similar appearance, with a fast release of H2S reaching a maximum of 50 µM (17%) after 2 minutes, and a slow decrease resulting in a final concentration of 39 µM (13%) after 30 minutes (Fig. 1C). Addition of 300 µM GYY4137 to PSS at pH 7.4 failed to change the electrode current, whereas 300 µM GYY4137 added to PSS at pH 3.0 gave a slower release of H2S than NaHS and Na2S, reaching a maximum after 6 minutes, after which a stable level of H2S at 3 µM (1%) was obtained that remained stable through the next 25 minutes (see Fig. 1D).
Effect of Exogenous H2S.
To test the response of mesenteric arteries to different H2S donors, CRCs for NaHS, Na2S, and GYY4137 were obtained in arteries contracted to NE. NE induced stable contractions; however, in some cases they were oscillatory around a stable mean tension as previously described for rat mesenteric small arteries (see Fig. 2, A and B) (Peng et al., 2001). NaHS and Na2S induced contraction starting from 10 µM (Fig. 2A), and only at higher concentrations (100 µM) was the contraction followed by relaxation; at 3 mM the arteries relaxed 100% (see Fig. 2, A and C). NaHS induced comparable relaxations in NE- and U46619 (9,11-dideoxy-11α, 9α-epoxymethano-prostaglandin F2α)-contracted mesenteric arteries (n = 6, results not shown). GYY4137 induced relaxations starting at a concentration of 30 µM, and maximum relaxation was reached at 300 µM; no constriction was observed with GYY4137 (see Fig. 2, B and C). Based on the added amounts, the compounds caused equipotent relaxations; NaHS gave EC50 values of 188 ± 33 µM (n = 13), Na2S of 187 ± 52 µM (n = 5), and GYY4137 of 107 ± 21 µM (n = 6). Impurities in NaHS have been suggested as an explanation for low sensitivity, but our results show that NaHS and Na2S induced equipotent and also reproducible relaxations in mesenteric arteries. Based on the estimated concentration of free H2S showing that a maximum of, respectively, 14% of NaHS, 17% of Na2S, and 1% of GYY4137 (at pH = 3.0) is present on the H2S form, the curves depicted in Fig. 2D are obtained. Based on the measured free H2S, GYY4137 caused more relaxation in response to released free H2S with EC50 values of 1.3 ± 0.2 µM (n = 6), whereas NaHS and Na2S were equipotent, yielding EC50 values of 21 ± 4 µM for NaHS (n = 13) and 34 ± 12 µM for Na2S (n = 5) in rat mesenteric small arteries.
To correlate the actual H2S concentrations to the relaxations, simultaneous measurements of tension and H2S concentrations were obtained for NaHS and GYY4137. NaHS (1–1000 µM) induced concentration-dependent increases in the current of the H2S microsensors and simultaneously relaxed the mesenteric artery (Fig. 3B). At 300 µM NaHS, the H2S concentration measured with the intraluminal and extraluminal H2S-sensitive microsensors was, respectively, 15 ± 1.6 µM and 6.5 ± 0.5 µM, and the artery relaxed 61 ± 1.9 µM (n = 3). GYY4137 failed to induce any increase in current of the H2S microsensors; at 300 µM GYY4137, the H2S concentration measured with the intraluminal and extraluminal H2S-sensitive microsensors was, respectively 0.7 ± 0.8 µM and 0.04 ± 0.1 µM (Fig. 3C). GYY4137 did not yield detectable H2S at pH 7.4 in closed containers or in the myograph; therefore, NaHS was used for the further investigation of the mechanisms underlying H2S-induced relaxation in rat mesenteric arteries.
Effect on Smooth Muscle Calcium Levels of NaHS.
Simultaneous measurements of tension and intracellular calcium levels were conducted. Norepinephrine induced stable increases in intracellular calcium and contraction (Fig. 4A). Increasing the concentration of NaHS resulted in slight increases in calcium, while the artery relaxed at concentrations from 100 µM to 1 mM. At concentrations equal to or above 1 mM, NaHS simultaneously reduced the intracellular calcium concentration and tension (Fig. 4, A and B). In arteries contracted with high extracellular potassium, the fall in calcium was abolished, and the maximum relaxation was reduced compared with the NaHS relaxation in NE-contracted preparations (Fig. 4, B and C).
The lowering of calcium in NE-contracted arteries shows that H2S-induced vasodilation involves mechanisms dependent on changes in intracellular calcium levels. The calcium-dependent mechanisms likely involve the K channels because relaxation was attenuated in potassium-contracted arteries. Another part of the relaxation is independent of changes in calcium, as we still observed some relaxation in the potassium-contracted arteries where no change in calcium was observed.
Effect of K Channels Blockers on NaHS Relaxation.
To investigate the involvement of K channels in H2S-induced relaxation, CRCs for NaHS were performed on arteries incubated with blockers of different K channels. None of the blockers changed contraction levels to NE (Supplemental Table 1). Glibenclamide (10 µM) failed to inhibit NaHS-induced relaxation (Fig. 5A), but it significantly reduced relaxations induced by pinacidil, an opener of KATP channels (Supplemental Fig. 1). This suggests that K channels other than KATP channels are involved in NaHS relaxation. Inhibition with TEA (1 mM and 3 mM), which has been suggested to inhibit BKCa (Nelson and Brayden, 1993) and KV7 channels expressed in Chinese hamster ovary cells with IC50 values of 3–5 mM (Hadley et al., 2000), yielded a significant reduction of NaHS-induced relaxation (Fig. 5B); the higher concentration of TEA (3 mM) had a larger effect on NaHS relaxation than 1 mM. TEA (1 mM) failed to reduce SNP relaxation (n = 4, results not shown) or GYY4137-induced relaxation (n = 5–6, results not shown). Iberiotoxin, a selective blocker of BKCa, failed to inhibit NaHS relaxation (Fig. 5C) and the same was observed when inhibiting voltage-dependent K channels by 4-aminopyridine (Fig. 5D).
XE991 (10 µM), an inhibitor of KV7 channels, reduced the relaxations induced by retigabine, an opener of KV7 channels (n = 5 P = 0.004, results not shown). XE991 failed to change the CRCs for NaHS, but it significantly reduced the relaxation to 300 µM NaHS (Fig. 5E). Another blocker of KV7 channels, linopirdine, inhibited the CRCs for NaHS, giving further support for the involvement of KV7 channels in NaHS relaxation (Fig. 5F).
Effect of NaHS on Phosphorylation of MLC and MYPT-1.
To further investigate the role of calcium levels or calcium desensitization in mesenteric arteries, we determined the phosphorylation levels of MLC and of MYPT-1 (Fig. 6). A reduced amount of pMLC/MLC was observed in arteries exposed to NaHS (1 mM) compared with control arteries contracted with NE (3 µM) (Fig. 6B), corresponding to our calcium measurements, where we see a decline in intracellular calcium levels in response to 1 mM NaHS (Fig. 4B). To investigate the role of calcium desensitization, MYPT-1 phosphorylation was determined. In arteries treated with the Rho kinase inhibitor Y27632 (4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide) (1 µM), which served as a control for our experiment, we observed inhibition of MYPT-1 phosphorylation (Fig. 6C), but no difference was observed in pMYPT-1/MYPT-1 in arteries exposed to NaHS (1 mM) compared with control arteries contracted with NE (3 µM). In both in NE- and K-contracted arteries, 1 mM NaHS relaxed the arteries compared with control, but Y27632 (1 µM) failed to change the contraction even though it reduced phosphorylation of MYPT-1 (Fig. 6D). A higher concentration of Y27632 (10 µM) inhibited contraction without further change in MYPT phosphorylation (n = 3, results not shown).
Investigation of Protein Kinase Inhibitors and Rotenone on NaHS Relaxation.
Following previously published results, we investigated a series of pathways potentially involved in NaHS relaxation. The effect of the Cl−/HCO3-exchanger inhibitor DIDS was found to inhibit the CRC not only for NaHS, but also for SNP and ACh (n = 6, results not shown), suggesting that the effect is either unspecific or dependent on the presence of NO. However, the NaHS relaxations were unaltered in the presence of 100 µM L-NOARG, a NO synthase inhibitor (n = 6, results not shown).
To investigate the role of the cyclic nucleotide pathways in H2S relaxation, the cGMP and cAMP pathways were blocked. ODQ (3 × 10−6 M), an inhibitor of guanylyl cyclase, markedly reduced relaxations induced by the NO donor SNP but failed to change NaHS relaxation (Fig. 7, A and B). RP-8-cPTP-cGMPS (20 µM), an inhibitor of PKG, also inhibited SNP-induced relaxation but failed to change NaHS relaxation (Fig. 7, A and B). RP-8-cPTP-cGMPS and ODQ were also found to inhibit relaxation by polysulfides (K2Sn) (Supplemental Fig. 2). KT5720 (200 nM), an inhibitor of protein kinase A, inhibited forskolin-induced relaxation but failed to reduce NaHS relaxation (Fig. 7, C and D).
NaHS has been proposed to be converted by sulfide quinone oxoreductase, leading to sulfation of the mitochondrial complexes. Therefore, rotenone and antimycin A were added, which are inhibitors of, respectively, complex I and complex III. In vessels constricted with high extracellular potassium, NaHS 3 × 10−4 M induced 57% ± 3% relaxation (n = 12), which was inhibited in the presence of rotenone (1 µM) and antimycin A (1 µM) to 7.8% ± 3.8% and 0.4% ± 0.4%, respectively; these relaxations were not inhibited by the superoxide mimetic Tempol (300 µM) or the mitochondria-specific superoxide mimetic MITO-TEMPO (10 µM) (Fig. 8A). In NE-contracted arteries, rotenone and antimycin A also inhibited relaxation from NaHS (Fig. 8B), but the superoxide mimetics failed to affect relaxation. SNP (10−4 M), an NO donor, relaxed the NE-contracted arteries to the same level in the absence or presence of the inhibitors (Fig. 8C).
The main findings of our study are that low micromolar concentrations of free H2S lead to relaxations involving the lowering of smooth muscle [Ca2+]i. This is supported by the observation that high extracellular K and inhibition of KV7 channels caused inhibition of NaHS-induced vasodilation, suggesting that low micromolar concentrations of free H2S lead to relaxations involving K channels followed by lowering of [Ca2+]i and of MLC phosphorylation in NE-contracted preparations. Moreover, in preparations activated with high extracellular K, NaHS induced relaxations independent of changes in [Ca2+]i and MLC phosphorylation, probably via direct inhibition of the mitochondrial electron transport leading to force suppression. The latter observation was supported by the observation that NaHS relaxations were sensitive to inhibitors of mitochondrial complex I and III.
Evaluation of H2S Concentration and Relaxation.
The H2S concentration measured with microelectrodes in our study reflects the free [H2S], which is the amount added to the bath where the electrode is placed minus clearance by degradation and diffusion. The addition of 300 µM NaHS and Na2S salts to a closed container resulted in sustained free H2S concentrations of 14%–17%, but taking pH 7.4 into account the measured levels correspond to the expected levels of free H2S. In the myograph bath, the free H2S concentrations were also lower compared with those in a closed container. In pressurized rat mesenteric arteries, 10–300 µM of added NaHS induced relaxation (White et al., 2013); in our study, 100–1000 µM NaHS induced relaxations. Although there are differences in pressure versus isometric mounted small arteries (Buus et al., 1994; Wesselman et al., 1997), our electrode measurements underline that it is pivotal to measure the H2S concentration in the experimental setup used. Our simultaneous measurements of the H2S concentration and relaxation showed a direct relationship of free H2S concentration to relaxation when Na2S was added.
Plasma concentrations of H2S have been reported to vary from 0.1 to 100 µM in patients (Goslar et al., 2011). Taking into account that a maximum of 14%–17% of the added NaHS or Na2S was observed to be in the free H2S form, this significantly lowers the concentration of free H2S needed to induce vasodilation, giving us an estimated EC50 of 34 and 21 µM. Furthermore, the simultaneous measurements showed that 15 µM of H2S at the luminal side caused 61% relaxation of the superior mesenteric artery. These findings suggest that the concentration range of free H2S generated by the addition of NaHS salt in our study appears relevant for regulation of vascular tone.
The slow-releasing H2S donor GYY4137 at 0.1 mM relaxes aorta and ophthalmic arteries by 50% (Li et al., 2008; Salomone et al., 2014), and GYY4137 increases the plasma levels of total sulfides after intravenous and intraperitoneal injection (Li et al., 2008). However, in vitro GYY4137 only increased the free H2S concentrations measured by the use of a microsensor in acidic conditions (Li et al., 2008). Our findings support the previous observations; at pH 7.4 the H2S levels were below the detection limit of the electrode, although GYY4137 causes relaxation equipotent to that induced by the NaHS and Na2S salts in mesenteric arteries. Together with the observation that l-cysteine is required to measure the release of H2S (Martelli et al., 2013), our findings suggest that the GYY4137-induced release of free H2S may also depend on the presence of tissue thiols or it only releases other sulfide species (e.g., HS− and S2−). Moreover, it should be explored whether a sulfide-independent mechanism may contribute to GYY4137 relaxations.
Involvement of K Channels and Calcium Lowering in H2S-Induced Vasodilation.
Opening of K channels leads to reduced [Ca2+]i and vasodilation. NaHS and Na2S were found to activate calcium sparks and reduce global [Ca2+]i (Liang et al., 2012; Jackson-Weaver et al., 2013). In the present study, simultaneous measurements of [Ca2+]i in NE-contracted preparations revealed that NaHS lowered [Ca2+]i and induced relaxation and, as expected based on the calcium measurements, phosphorylation of MLC decreased compared with controls. However, in preparations contracted with high extracellular potassium, NaHS induced less relaxation without any corresponding changes in [Ca2+]i in rat mesenteric arteries. These findings suggest K channels are involved in NaHS-induced relaxation.
KATP channels (Zhao and Wang, 2002; Tang et al., 2005; Webb et al., 2008), KV7 channels (Schleifenbaum et al., 2010; Martelli et al., 2013; Hedegaard et al., 2014), and BKCa (Liang et al., 2012; Jackson-Weaver et al., 2013) have been suggested to be involved in H2S vasodilatation. Glibenclamide, 4-aminopyridine (a blocker of voltage-gated K channels), and iberiotoxin (a blocker of BKCa channels) failed to affect NaHS relaxation in our study. However, we found that TEA (1–3 mM) reduced NaHS relaxation; this concentration range was previously found to inhibit KV7 channels (Hadley et al., 2000). In agreement with these findings XE991 and linopirdine, blockers of KV7 channels, inhibited NaHS relaxation, suggesting that KV7 channels are involved in NaHS relaxation. However, the reductions in NaHS relaxation by XE991, linopirdine, and TEA were markedly less than observed in KPSS versus NE-contracted preparations, suggesting that other K channel subtypes may also contribute to NaHS relaxation.
Involvement of Calcium-Independent Mechanisms in H2S-Induced Vasodilation.
Vascular tone is dependent on the relative activities of MLC kinase and MLCP, where activation of MLC kinase or inhibition of MLCP can increase phosphorylation of MLC and thereby initiate vascular smooth muscle contraction (Somlyo and Somlyo, 2003). Inhibition of MLCP, but not Rho-kinase, reduced the H2S-induced relaxation of mouse gastrointestinal smooth muscle (Dhaese and Lefebvre, 2009); in the pig bladder, H2S lowered tension without changing the calcium concentration (Fernandes et al., 2013). In our study, H2S initially evoked vasodilation while the intracellular calcium levels increased or were maintained at the same level. These results suggest that besides a lowering of [Ca2+]i by high NaHS concentrations, calcium desensitization is involved in NaHS relaxation of rat mesenteric small arteries. However, the phosphorylated MYPT-1/MYPT-1 ratio did not change after exposure to NaHS in the mesenteric arteries, suggesting that other mechanisms than MLCP are involved in the calcium desensitization of the vascular smooth muscle cell contractile apparatus.
In large arteries, inhibition of phosphodiesterase type 5 was found to be involved in NaHS relaxation (Bucci et al., 2012), and NaHS was also recently suggested to contain polysulfides and by oxidation of protein kinase G to cause relaxation in mouse mesenteric arteries (Stubbert et al., 2014). However, in our study ODQ and RP-8-cPTP-cGMPS, an inhibitor of protein kinase G, inhibited SNP relaxation and relaxation induced by the polysulfide K2Sn while NaHS relaxation was unaltered, suggesting that polysulfides and NaHS cause relaxation through different pathways. Nor did we observe any effect on NaHS relaxation when protein kinase A was inhibited by KT5720. These findings do not exclude an interaction of H2S with phosphodiesterases or protein kinase A and G, but they suggest that other mechanisms are involved in NaHS-induced uncoupling of calcium from the contractile apparatus in mesenteric small arteries.
In perfused trout gills, it has been suggested that sulfide induces vasoconstriction by a mechanism involving mitochondrial complexes I, II, and IV, and by enhancing the formation of radical oxygen species (ROS) (Skovgaard and Olson, 2012). The interaction of H2S with the mitochondria is complex, as lower concentrations may stimulate the mitochondrial electron transfer through a mechanism involving metabolism by sulfide quinone oxoreductase (Szabo et al., 2014), whereas higher concentrations of H2S may inhibit mitochondrial electron transfer by interaction with cytochrome C (Goubern et al., 2007; Módis et al., 2013). In preparations contracted with high extracellular potassium to exclude the contribution of K channels, the mitochondrial complex I and III inhibitors rotenone and antimycin A both abolished NaHS relaxation in rat mesenteric arteries, but rotenone and antimycin A did not change the relaxation induced by exogenously added NO and hence the bioavailability. Moreover, NaHS relaxations were not inhibited in the presence of the superoxide mimetic Tempol or the mitochondrial superoxide scavenger MITO-TEMPO. Although ROS may lead to relaxation in mesenteric arteries, these findings do not suggest that ROS contribute to NaHS relaxation. Inhibition of cytochrome C by NaHS would lead to decreased ATP synthesis (Módis et al., 2013). Therefore, one may speculate that the lower ATP levels resulting from NaHS inhibition of the mitochondria lead to reduced actin-MLC cross-bridge turnover or to an increased smooth muscle AMP to ATP ratio followed by activation of AMP kinase and vascular relaxation (Rubin et al., 2005). That NaHS relaxation was also reduced by rotenone and antimycin A in NE-contracted preparations suggests that both K channels and a mitochondrial pathway contribute to NaHS relaxation in mesenteric small arteries.
Our findings suggest that low micromolar concentrations of free H2S lead to relaxations involving K channels followed by lowering of smooth muscle calcium. Moreover, in preparations activated with high extracellular potassium, NaHS induced relaxations independent of the changes in [Ca2+]i and MLC phosphorylation, probably by direct inhibition of the mitochondrial electron transport, leading to force suppression. The effect of H2S on both KV7 channels and the mitochondrial pathways may contribute to a cardioprotective effect of low concentrations of H2S.
The authors thank Henriette Gram Johansen and Heidi Knudsen for technical assistance.
Participated in research design: Hedegaard, Gouliaev, Skovgaard, Simonsen.
Conducted experiments: Gouliaev, Winther, Arcanjo, Aalling, Renalton, Skovgaard, Simonsen.
Contributed new reagents or analytic tools: Wood, Whiteman.
Performed data analysis: Hedegaard, Gouliaev, Winther, Arcanjo, Aalling, Renalton, Skovgaard, Simonsen.
Wrote or contributed to the writing of the manuscript: Hedegaard, Gouliaev, Skovgaard, Simonsen.
- Received June 24, 2015.
- Accepted October 21, 2015.
The work was supported by a grant from the Danish Research Council (to A.G. and M.A.), grants from the Villum Kann Rasmussen Foundation, Korning Foundation, and L’Oréal (to N.S.); the Danish Heart Foundation (to E.R.H.), the Korning Foundation, (to E.R.H.), and the Karen Elise Jensen Foundation (to E.R.H.); U.S. is part of the LiPHOS (Living Photonics).
E.R.H. and A.G. share first authorship.
- analysis of variance
- large conductance calcium-activated potassium channels
- intracellular calcium concentration
- concentration–response curve
- cystathionine gamma-lyase
- 4,4ʹ-diisothiocyano-2,2ʹ-stilbenedisulfonic acid
- P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid
- hydrogen sulfide
- ATP-sensitive potassium channel
- high-potassium physiologic saline solution
- (9S,10S,12R)-2,3,9,10,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]-benzo-diazocine-10-carboxylic acid hexyl ester
- (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride monohydrate
- myosin light chain
- myosin light chain phosphatase
- myosin phosphatase target subunit 1
- sodium hydrosulfide
- nitric oxide
- physiologic saline solution
- radical oxygen species
- (Rp)-8-(para-chlorophenylthio)guanosine-3ʹ,5ʹ-cyclic monophosphorothioate
- sodium nitroprusside
- 9,11-dideoxy-11α, 9α-epoxymethano-prostaglandin F2α
- 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics