AJP - Heart AJP: Advances in Physiology Education
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 292: H1953-H1960, 2007. First published January 19, 2007; doi:10.1152/ajpheart.01193.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/4/H1953    most recent
01193.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (23)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koenitzer, J. R.
Right arrow Articles by Kraus, D. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koenitzer, J. R.
Right arrow Articles by Kraus, D. W.

Hydrogen sulfide mediates vasoactivity in an O2-dependent manner

Jeffrey R. Koenitzer,1 T. Scott Isbell,2 Hetal D. Patel,1 Gloria A. Benavides,1,3 Dale A. Dickinson,3 Rakesh P. Patel,2 Victor M. Darley-Usmar,2 Jack R. Lancaster, Jr,4 Jeannette E. Doeller,3 and David W. Kraus1,3

Departments of 1Biology, 2Pathology, 3Environmental Health Sciences, and 4Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama

Submitted 31 October 2006 ; accepted in final form 7 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hydrogen sulfide (H2S) has recently been shown to have a signaling role in vascular cells. Similar to nitric oxide (NO), H2S is enzymatically produced by amino acid metabolism and can cause posttranslational modification of proteins, particularly at thiol residues. Molecular targets for H2S include ATP-sensitive K+ channels, and H2S may interact with NO and heme proteins such as cyclooxygenase. It is well known that the reactions of NO in the vasculature are O2 dependent, but this has not been addressed in most studies designed to elucidate the role of H2S in vascular function. This is important, since H2S reactions can be dramatically altered by the high concentrations of O2 used in cell culture and organ bath experiments. To test the hypothesis that the effects of H2S on the vasculature are O2 dependent, we have measured real-time levels of H2S and O2 in respirometry and vessel tension experiments, as well as the associated vascular responses. A novel polarographic H2S sensor developed in our laboratory was used to measure H2S levels. Here we report that, in rat aorta, H2S concentrations that mediate rapid contraction at high O2 levels cause rapid relaxation at lower physiological O2 levels. At high O2, the vasoconstrictive effect of H2S suggests that it may not be H2S per se but, rather, a putative vasoactive oxidation product that mediates constriction. These data are interpreted in terms of the potential for H2S to modulate vascular tone in vivo.

aorta; sulfide sensor; oxygen consumption; vasorelaxation; mitochondria

HYDROGEN SULFIDE (H2S) has recently been shown to have a role in mammalian cell signaling. It is produced in many tissues and has been detected in micromolar amounts in blood and brain (2, 12, 19). In physiological solution, the equilibrium H2S {leftrightarrow} H+ + HS– (reaction 1) {leftrightarrow} H+ + S2– (reaction 2) is rapidly established with pKa 6.9 and 14 for reactions 1 and 2, respectively (31), and the concentrations of H2S and HS– represent ~30% and 70%, respectively, of the total sulfide, whereas S2– concentration is negligible. Because at physiological pH the major sulfide species are H2S and HS– and because the exact molecular targets for sulfide are not fully known, any mention of H2S here refers to H2S and HS–. H2S is capable of inducing posttranslational protein modifications, including dithiol reduction (20) and ligand displacement from heme iron (29), similar in many respects to nitric oxide (NO). Also similar to NO, H2S is highly soluble in aqueous and lipid environments, can readily pass between cells, is rapidly oxidized, and is a potentially controlled product arising from amino acid metabolism. Of particular relevance to cardiovascular disease and neurodegeneration is the amino acid homocysteine, which, when metabolized, can lead to H2S production (12, 26). Elevated homocysteine levels are associated with oxidative stress, but an interesting prediction is that the accompanying decreased levels of H2S may exacerbate oxidative injury. The dynamic processes of H2S production and consumption (15) that control cellular H2S levels are responsive to cellular redox status (44), as well as O2 levels. Since H2S is a ligand for the O2 binding site of cytochrome c oxidase in the respiratory chain, high levels of H2S can lead to mitochondrial production of reactive oxygen species (17), another possible mechanism of H2S redox signaling. In addition, because the rate of chemical and biological H2S oxidation is highly O2 dependent, tissue H2S levels may reflect tissue oxygenation, highlighting the general dependence of H2S-mediated processes on O2.

In recent reports, H2S has been shown to directly modulate vascular responses. Intravascular administration of H2S results in a marked depression of mean blood pressure that is not attributable to a decline in heart rate (55). In support of a potential physiological role of H2S in vascular function, spontaneous hypertensive rats exhibit chronic elevated blood pressure, with serum H2S levels that are half of control levels (~40 µM) (50, 55), conditions that are readily reversed after NaHS injection (50). In addition, serum H2S levels are elevated during septic or endotoxic shock (23), perhaps contributing to the associated drop in blood pressure. Rings or strips of vascular smooth muscle or uterine smooth muscle exposed to NaHS exhibit a decrease in tension, with an IC50 of 125–150 µM for aorta under air-saturated conditions of ~200 µM O2 (22, 41, 55). Whereas the data support a role for H2S in modulating vascular function, it is unlikely that these H2S levels occur in vivo. Recent studies have highlighted the importance of O2 tension in modulating the interactions of NO with the mitochondrion and NO metabolites with hemoglobin (8, 10, 11). These findings led us to hypothesize that O2 level may be a critical factor controlling the vascular responses to H2S.

Mechanistic studies have shown that H2S-mediated vasorelaxation may involve a non-ATP-associated increased conductance of the ATP-sensitive K+ (KATP) channel (55). Additionally, vasoactive effects are not diminished by blockade of the NO/soluble guanylate cyclase (sGC) pathway (54, 55), indicating that H2S-mediated vasorelaxation operates independently of cGMP formation. Potentially vasoactive mechanisms that are O2 dependent include H2S interaction with heme proteins such as cyclooxygenase (COX) and the formation of sulfide oxidation products such as sulfite, thiosulfate, and sulfate. However, the O2 dependence of these reactions and their impact on the biological function of H2S have not been previously considered.

To better understand the kinetic and functional interactions between vessel tension, H2S, and O2, we have measured both dissolved gases in respirometry and vessel tension experiments. Because H2S, similar to NO, is rapidly oxidized, most standard analytic methods for measuring H2S are critically limited in terms of real-time physiological measurements. A novel polarographic H2S sensor (PHSS) has been developed in our laboratory to overcome these limitations (15, 28) and was used to measure H2S levels throughout the course of these experiments. Here we report that, in the rat aorta, low physiological H2S concentrations mediate rapid vasoactive responses, either contraction at air saturation O2 levels or relaxation at physiological O2 levels, underscoring the importance of O2 in mediating the biological effects of H2S.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals

All chemical reagents were purchased from Sigma unless otherwise noted.

Animals and Tissue Preparation

Male and female Sprague-Dawley rats (250–350 g body wt) were housed in University of Alabama at Birmingham animal care facilities under 12:12-h light-dark cycles, with food and water available ad libitum. Anesthesia was induced by intraperitoneal injection of ketamine and xylazine (100 and 16 mg/kg, respectively), and thoracic aortas were immediately excised and placed in Krebs-Henseleit (K-H) buffer (pH 7.3 at 37°C) containing 50 µM diethylenetriaminepentaacetic acid (DTPA) to chelate trace metals that catalyze H2S oxidation. Aorta segments were cleaned of adventitious tissue and blood and then cut into seven to eight ~3-mm-long segments. Excised tail artery was also sectioned into 3-mm-long segments and used in respirometric experiments to assess O2 dependence of a model peripheral artery. All animal procedures were performed according to University of Alabama at Birmingham Institutional Animal Care and Use Committee-approved protocols.

H2S Measurement

Solution H2S concentration in the respirometer chamber or in the vessel organ bath (see below) was recorded with a PHSS (15) connected to a multichannel analyzer (Apollo 4000, WPI, Sarasota, FL). The PHSS is selective for H2S, responds rapidly to changing H2S concentrations, and has a lower detection limit near 10 nM (15). PHSS calibrations were made in each experimental chamber using anoxic stock solutions prepared daily as 10 mM H2S in 50 mM potassium phosphate buffer with 50 µM DTPA (pH 7.3 at 37°C), as previously described (15). Stock solutions of H2S can be conveniently made with the salt Na2S·9H2O (Sigma-Aldrich certificate of analysis for product S2006, lot 085K0667, is 101.1% by titration). This avoids the need for special gas-handling apparatus required for pure H2S gas, and the additional micromolar sodium, as Na2S, dissociates to form Na+ and the three sulfide species H2S, HS– and S2– in pH-dependent ratios and contributes negligibly to the ~145 mM sodium in the K-H buffer. Buffered anoxic stock solutions made with Na2S·9H2O are indistinguishable with respect to the molar sulfide content from those made with pure H2S gas, as determined with the 2,2'-dipyridyl disulfide assay (28, 42), given H2S solubility in saline buffer at 30°C (31), and are thus referred to as H2S stock solutions. We avoid using the sodium hydrosulfide hydrate NaSH·xH2O (Sigma-Aldrich certificate of analysis for product 161527, lot 01824CI, is 71.2% by titration, with thiosulfate and other sulfide oxidation products as contaminants), because the hydration quantity is variable and the formula weight is anhydrous. In addition, the purity of commonly available NaSH·xH2O is much less than that of Na2S·9H2O, so even anoxic stock solutions of fresh NaSH·xH2O contain approximately half the expected amount of H2S as well as substantial amounts of other sulfurs (see Fig. 7 in Ref. 15).

O2 and H2S Respirometry

Instrumentation. Respirometry experiments with intact rat vessel segments were performed in a closed dual-chamber oxygraph respirometer (Oroboros, Innsbruck, Austria; pH 7.3 at 37°C). The stirred (500 rpm) 2.5 ml of K-H buffer with 50 µM DTPA contained 20 mM HEPES, instead of NaHCO3, to eliminate CO2 degassing. Vessel segments, which exhibit adhesion, were held on an epoxy-coated stainless steel screen (75% porosity) 2 mm above the stirrer in the chamber, and the chamber stopper, which was made of polyether ether ketone (Victrex USA, Rockford, MI) and held the PHSS, was positioned 2 mm above the vessel segments.

Effects of O2. To determine the effects of O2 on vessel respiration, segments from aorta and tail artery were placed in the respirometer chamber and allowed to respire until the O2 concentration reached functional anoxia with no further O2 decline, usually <1 µM. To determine whether the critical O2 concentration and/or apparent Km changed with increased energetic demand, 100 nM phenylephrine was added to the respirometer chamber to cause 50–70% maximum contraction of the aorta as determined in tension experiments (see below). Phenylephrine-treated aortas are referred to as contracted and nontreated aortas as noncontracted aortas. On the basis of these experimental results, subsequent respirometric and tension experiments were performed at 40–60 µM O2, which has been determined to be above the hypoxic range for the aorta (present study; 24).

Effects of H2S. To determine the effects of H2S on vessel respiration, simultaneous H2S and O2 consumption rates of contracted and noncontracted aorta segments were measured in one respirometer chamber, while the other chamber without segments served as control. To establish a 60 µM O2 concentration, a 3-ml gas space above the liquid, adjusted with stopper height, was perfused with N2. When the desired O2 concentration was reached, the N2 stream was removed and the stopper was fully inserted.

To measure H2S consumption rates at specific H2S concentrations, aliquots of the H2S stock were injected into both chambers in a stepwise manner to give typically 5, 5, 10, 10, and 20 µM H2S, etc. The slope of the decreasing PHSS signal between injections provided a measure of the H2S consumption rate over a wide range of H2S concentrations. To carry out these experiments within the 40–60 µM O2 range, the respirometer stopper was lifted at 40 µM O2 and was lowered at 60 µM O2.

On completion of the experiment, the aorta segments were removed, blotted dry, and weighed. Rates of O2 and H2S consumption were corrected by subtraction of the respective background consumption rates recorded in the absence of aorta and then normalized to the aorta fresh weight. The contribution of background rate to total rate was dependent on tissue weight, which was chosen to keep experimental O2 levels fairly constant and above the critical O2 concentration (see below). Average aorta fresh weight per experiment was 26.5 ± 2.5 mg (n = 9). Background consumption rates were ~30% of the total rates. Consumption rates were plotted as functions of H2S concentration.

Vessel Tension: Effects of O2 and H2S

Isometric tension of intact rat thoracic aortic ring segments in response to H2S at specific O2 concentrations was determined according to established methodology using a vessel bioassay system (Radnoti, Monrovia, CA) (11, 24) modified to house the polarographic oxygen sensor and the PHSS for simultaneous readouts of O2 and H2S concentrations and vessel tension recordings. Data were acquired on a personal computer using AcqKnowledge software (Biopac Systems, Goleta, CA).

Aorta segments were mounted in baths containing 15 ml of K-H buffer with 50 µM DTPA bubbled with a mass flow-controlled (series 100, Sierra Instruments, Monterey, CA) mixture of N2 and air, each containing 5% CO2, to maintain O2 concentration at a specific level from 40 to 200 µM. To obtain an accurate measure of H2S concentration at the vessel, the PHSS was held within 5 mm of the aorta. After the aorta segments were mounted and allowed 30 min of equilibration, vessel response was tested for maximal contraction with 60 mM KCl. After relaxation was achieved by wash out of the KCl, the vessels were contracted with 100 nM phenylephrine, giving 50–70% maximum contraction. The aorta prepared by this method exhibited relaxation on addition of 10 µM acetylcholine, demonstrating the presence of a functional endothelium. Anoxic H2S stock was injected into vessel-containing baths to give concentrations of 5–40 µM, and the effect on vessel tension was recorded simultaneously with H2S concentration. Vessel tension at high (200 µM) and low (40 µM) O2 was also measured with and without 5 µM indomethacin, a pharmacological inhibitor of COX-1 and COX-2.

sGC Activity

Because H2S is a known low-spin ferric heme ligand (29) and can also reduce ferric heme iron in myoglobin to the ferrous state, the possibility that H2S binds to and/or reduces the ferric heme of sGC was considered. To test H2S interaction with sGC under physiologically relevant conditions, cGMP in aorta was measured according to Crawford et al. (9). Briefly, aorta segments similar in length to those used for tension measurements were tied individually with suture loops through the lumen for easy handling and suspended in five vessel baths equilibrated at 40 µM O2. After a 10-min equilibration period, 300 nM indomethacin was added to all baths. At 15 min, 100 nM phenylephrine, 100 µM N-monomethyl-L-arginine monoacetate (Alexis Biochemicals, San Diego, CA), an inhibitor of NO synthase, and 100 µM 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterases, were added to all baths. At 25 min, 10 µM 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-sensitive sGC, was added to baths 3 and 5. At 35 min, 100 nM sodium nitroprusside (SNP; Alexis Biochemicals, San Diego, CA), an NO donor, was added to baths 2 and 3, and 20 µM H2S was added to baths 4 and 5. After 45 min, aorta segments were blotted dry, weighed, and frozen in liquid nitrogen. cGMP was analyzed using an ELISA (Cayman Chemical, Ann Arbor, MI) following the manufacturer's protocols.

Data Analysis

The majority of data manipulations were performed with Oroboros DatLab software (Innsbruck, Austria). Statistical significance, determined by one-way ANOVA, was assigned at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of O2 on Mitochondrial Respiration in Isolated Vessel Preparations

To determine the O2 dependency of H2S effects on the tension of isolated vessels, it was first necessary to establish levels of O2 and H2S that are physiological, yet nonlimiting to the aerobic metabolism of the preparation. A decrease in vascular smooth muscle cell ATP levels could lead to vessel relaxation if there was concurrent increased KATP channel conduction. To ensure that ATP levels were not compromised as a result of hypoxia, it was important to determine vessel O2 consumption rate as a function of solution O2 concentration to determine the threshold of O2 limitation (Fig. 1). The O2 consumption rate of noncontracted rat aorta segments (typically 2 mm OD) was maintained at 1.3 pmol O2·s–1·mg fresh wt–1 at higher O2 levels to a threshold of ~25 µM O2, below which the rate of O2 consumption decreased in an O2-dependent fashion, consistent with inhibition of mitochondrial respiration (Fig. 1). This threshold level is called the critical O2 concentration (Table 1) (24). These data were analyzed to calculate an apparent Km, defined as the O2 concentration at half-maximal O2 consumption rate, near 10 µM (Table 1). Next we determined that contraction of the aorta segments with phenylephrine caused an approximately twofold increase in the rate of O2 consumption, to 2.8 pmol O2·s–1·mg fresh wt–1, with an unchanged critical O2 concentration and Km for O2 (Table 1). To compare the O2-mediated effects on respiration of aorta with those of a model peripheral artery with relatively little connective tissue compared with aorta and typically 1 mm OD, we measured the O2 consumption rate of contracted tail artery segments (Fig. 1). O2 consumption rate (3.5 pmol O2·s–1·mg fresh wt–1) was higher and critical O2 concentration and apparent Km values (near 18 and 3 µM, respectively) were lower in contracted tail artery than in contracted aorta segments (Table 1). On the basis of these results, subsequent experiments on contracted aorta segments were performed at O2 concentrations of ≥40 µM, which are reported as intravascular for arteries to arterioles (40, 46). This ensured that any measured aortic vasorelaxation was not the result of respiratory inhibition by O2 diffusion limitation.


Figure 1
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 1. Representative traces of O2 consumption rates in aorta with and without phenylephrine and in tail artery with phenylephrine as a function of O2 concentration. From such traces, critical O2 concentration (Table 1) was calculated as O2 concentration that yields an O2 consumption rate equivalent to 90% of maximum under each set of conditions. Apparent Km (Table 1) was calculated as O2 concentration at half-maximum O2 consumption.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Respiratory and tension characteristics of phenylephrine-contracted and noncontracted vessels

 
Aorta H2S and O2 Consumption

To determine the effects of H2S on aorta respiration over the concentration range that mediates vasorelaxation, rates of O2 and H2S consumption were measured as a function of H2S concentration in contracted and noncontracted aorta at 40–60 µM O2 (Fig. 2). In noncontracted aorta, an increase in H2S concentrations caused a commensurate decrease in O2 consumption rates, reaching near-complete inhibition at 90 µM H2S (Fig. 2A), most likely via blockade of cytochrome oxidase. In contrast, O2 consumption rates of contracted aorta with increased ATP demand were stimulated to a maximum at ~40 µM H2S, above which the O2 consumption rates began to decline. H2S consumption rates also showed differential response to H2S concentrations as contracted aorta exhibited enhanced H2S consumption rates, reaching a maximum near 40 µM H2S, compared with noncontracted aorta (Fig. 2B). On the basis of these results, tension experiments on contracted aorta at 40–60 µM O2 were performed at 5–40 µM H2S.


Figure 2
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 2. O2 and H2S consumption rates of contracted (open symbols) and noncontracted (filled symbols) aorta (O2 and H2S flux) as a function of H2S concentration at 40 and 60 µM O2. Each symbol represents a separate experiment. Bolus injections of 5–80 µM H2S were added to vessels in the respirometer chambers; higher values represent additive concentrations.

 
H2S-Dependent Vasoactivity

Effects of O2. Similar to reported results from experiments in air-equilibrated buffer (~200 µM O2) (55), vessels contracted with phenylephrine to 50–75% of maximum tension exhibited a biphasic response to H2S, with vasoconstriction at 5–100 µM H2S followed by vasorelaxation at ≥200–400 µM H2S (Fig. 3). However, at lower (40 µM) O2, H2S elicited a transient vasorelaxation at concentrations as low as 5 µM (Fig. 3). The short duration of the H2S-dependent relaxation is most likely the result of chemical or biological H2S oxidation, as well as the loss of H2S to the headspace above the vessel bath (15). The cumulative dose-response curves at high and low O2 (Fig. 4) indicate a 17-fold decrease in the EC50 for H2S-dependent vessel relaxation at 40 µM O2 compared with the EC50 at 200 µM O2 (Table 1).


Figure 3
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 3. Representative traces of aorta tension as a function of time. Aorta was exposed to bolus additions of H2S at 200 and 40 µM O2 in the presence and absence of 5 µM indomethacin. Vessel tension is expressed as fraction of maximum phenylephrine (PE)-induced contraction, which is defined as 1. At 40 µM O2, no differences were observed between vessels in the presence and absence of indomethacin (data not shown).

 

Figure 4
View larger version (31K):
[in this window]
[in a new window]

  		Fig. 4. Aorta tension as a function of H2S concentration. Aorta was exposed to 200 or 40 µM O2 in the presence and absence of 5 µM indomethacin. Data are from traces such as those in Fig. 3. Values are means (SD) of 6 experiments. *Significant differences (P < 0.5) between treatments and controls.

 
Reversibility of H2S-dependent vasorelaxation. To more directly define the relation between H2S and aorta tension at different O2 levels, the H2S sensor was placed directly in the vessel bath to give simultaneous measurements of vessel tension and H2S concentration. As contracted aortas at 40 µM O2 were exposed to bolus additions of 20 or 40 µM H2S, the vessels initially relaxed and then recontracted as the H2S was purged from the solution (Fig. 5). This could be repeated multiple times on the same vessel, indicating that H2S-mediated vessel relaxation is a reversible process. With data from these experiments and others performed at 70 µM O2, the relation between aorta tension and H2S concentration was determined (Fig. 6). The H2S EC50 for aorta tension as a function of O2 concentration, shown in the inset in Fig. 6, further illustrates the O2 dependency of H2S-mediated vasorelaxation.


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 5. Representative simultaneous traces of aorta tension and H2S concentration as a function of time. Aorta tension, expressed as fraction of maximum phenylephrine-induced contraction (black line, left y-axis), was recorded with bolus additions of 20 and 40 µM H2S (gray line, right y-axis) at 40 µM O2. Overlay of data shows direct, reversible, and limited response of vessel tension to H2S concentration. Data are summarized in Fig. 6.

 

Figure 6
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 6. Aorta tension at 40 (black symbols) and 70 µM (gray symbols) O2. Data for 40 µM O2 are from traces in Fig. 5. Effective H2S concentration for 50% maximum contraction (H2S EC50) at 40 and 70 µM O2 is shown at arrows. Inset: H2S EC50 values of aorta as a function of O2 concentration. Data represent 4–6 experiments; standard deviations are smaller than symbols.

 
Mechanisms of H2S-Mediated Vasorelaxation

H2S interactions with COX in intact aorta. Vessels described above were routinely treated with 5 µM indomethacin to inhibit production of vasoactive prostaglandins via the constitutive heme-containing COX-1 pathway. Because heme groups are potential biological targets for H2S reactivity, studies were also performed without indomethacin to determine whether COX-1 plays a role in the effects of H2S on the vasculature. In contrast to vessels treated with indomethacin, non-indomethacin-treated vessles exhibited no vasoconstriction in response to ≤200 µM H2S followed by relaxation at 200 and 400 µM H2S (Fig. 3). A summary of these data (Fig. 4) shows the significantly different response to H2S between aorta with and without indomethacin at high O2. At lower (40 µM) O2, the effect of indomethacin was not pronounced (Fig. 4), with no significant difference observed between vessels with and without indomethacin. These data suggest that products of the COX pathway may limit the vasoconstrictive effects of H2S at high O2.

H2S interaction with sGC. To determine whether H2S vasodilatory effects at 40 µM O2 are mediated via the NO-dependent sGC pathway, cGMP levels were measured in the presence of H2S, the NO donor SNP, and the sGC inhibitor ODQ (Fig. 7). Although cGMP levels were increased by SNP and inhibited by ODQ as expected, 20 µM H2S had no effect on cGMP activity, indicating that, under these conditions, H2S does not mediate vasodilation via sGC activity directly or via an indirect effect on NO biosynthesis.


Figure 7
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 7. cGMP levels of aorta under different conditions of NO stimulation of soluble guanylate cyclase activity. Vessels were exposed to 40 µM O2 in the presence of 5 µM indomethacin, 100 nM phenylephrine, 10 µM N-monomethyl-L-arginine monoacetate (L-NMMA), and 100 µM 3-isobutyl-1-methylxanthine (IBMX) and treated with 100 nM sodium nitroprusside (SNP), 10 µM 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) + SNP, 20 µM H2S (H2S), and ODQ + H2S. Values are means (SD) of 5 experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
H2S is newly recognized as a cell signal in the mammalian cardiovascular, neurological, hepatic, pancreatic, pulmonary, and renal systems (27, 30, 32). Although there have been no definitive studies showing endogenously produced H2S-mediated vasorelaxation in situ, it is generally accepted that H2S has vasoactive effects in the cardiovascular system. Here we demonstrate that H2S-mediated vasorelaxation is an O2-dependent process and that low (5–40 µM) H2S concentrations are sufficient to relax vessels under physiological O2 conditions.

Effects of O2 on Vessel Respiration

The aorta has been extensively studied as a model vessel for the evaluation of mechanisms by which vasoactive compounds mediate their effects. The O2 consumption rate and apparent Km reported here for noncontracted aorta are similar to values reported previously (5, 35). Aorta O2 consumption rate increased on addition of phenylephrine, whereas apparent Km and critical O2 remained nearly constant, indicating that the change in tissue geometry was not sufficient to change O2 availability under these conditions. In the tail artery, a peripheral vessel with a smaller diameter, smaller wall thickness, and greater proportion of smooth muscle cells than collagen compared with aorta, O2 consumption rate was higher, yet apparent Km and critical O2 were lower, as expected, supporting the concept that peripheral arteries and arterioles normally operate at lower O2 concentrations than the aorta with no hypoxia until O2 drops below 20 µM (40, 46).

Effects of H2S on Vessel Respiration

The range of reported endogenous H2S levels in tissues is quite large (from undetectable to >300 µmol/kg) (47), and the range of exogenous H2S levels used experimentally is equally large (up to 500 µM) (30). Because H2S is a potential inhibitor of mitochondrial respiration by ligation to the ferric heme iron of cytochrome a3 in complex IV (4, 34), it was important to show that the exogenous H2S concentrations necessary to cause vasorelaxation in this study were not inhibitory to respiration. Respiratory inhibition could result in ATP depletion, thus mediating relaxation via KATP channel opening (7). However, H2S has been shown to increase KATP channel conductance, even when ATP is elevated (55), thereby mediating vasorelaxation independent of any decrease in cellular ATP. We demonstrate here that when contracted aorta with increased ATP demand is provided sufficient H2S to cause relaxation, respiration is stimulated, not inhibited. This increased O2 consumption is accompanied by a nearly identical increase in H2S consumption, indicating that H2S oxidation is enhanced in contracted aorta. H2S, which is a source of reducing equivalents, has been shown to be a rapidly metabolized respiratory substrate in several tissue and mitochondrial preparations from a variety of animals, both vertebrates (3, 52) and invertebrates (13, 21, 39). In fact, H2S-oxidizing enzymes, which deliver electrons directly to the electron transport chain, have been identified in prokaryotes and eukaryotes, with gene sequence homologies extending to mammals (45, 48). When these tissue and mitochondrial preparations are provided with noninhibiting H2S levels and are stimulated with ADP or made to do work, O2 and H2S consumption rates increase in a parallel fashion, and ATP is produced. We argue that, on the basis of the similarity in reported and present results, phenylephrine-contracted aorta may also metabolize H2S to produce ATP.

The possibility has also been raised that H2S could uncouple mitochondria by diffusion past cytochrome oxidase into the mitochondria matrix, where it deprotonates to HS– and H+ (36). However, we have shown with isolated gill mitochondria that H2S stimulates coupled O2 consumption and ATP production (39). Although H2S is a weak acid, with pK near 7, it is not a classic uncoupler, because HS– is membrane impermeant. Therefore, although protons would be carried into the matrix and the small {Delta}pH would decrease, the anions would accumulate in the matrix and would enhance the potential between metal and solution, thereby maintaining the proton-motive force (33). We suggest that, instead of uncoupling, H2S would more likely inhibit cytochrome oxidase, as it does in noncontracted aorta. Because H2S does not inhibit O2 consumption in contracted aorta, it is highly unlikely that the vessels are ATP deprived. The mechanism of H2S-induced vasorelaxation is most likely direct interaction of H2S with the KATP channel, as proposed by several groups (7, 55) and supported by data showing glibenclamide inhibition of H2S-mediated aorta vasorelaxation (unpublished observations).

Effects of O2 on H2S-Mediated Vasoactivity

Although we have replicated the findings of Zhao et al. (55) and Olson et al. (37), demonstrating that 200–300 µM H2S was required to cause rat aorta vasorelaxation at 200 and 900 µM O2, respectively, we have also observed that lower H2S concentrations cause vasoconstriction at high O2 concentrations, under which H2S is rapidly oxidized. H2S-mediated vasoconstriction at high O2 has been observed in a variety of nonmammalian vertebrates, often as part of a multiphasic response (37), and may be facilitated by one or more H2S oxidation products, rather than by H2S alone. Although the vasoactivity of known biological H2S oxidation products, such as sulfite, sulfate, or thiosulfate, has not been examined thoroughly, Wills et al. (49) showed that sodium meta(bis)sulfate is a vasoconstrictor of rat vessels. By repeating these experiments at the physiological O2 concentrations found in the vasculature (40, 46), we demonstrate that much less H2S is required to cause vasorelaxation. This O2-mediated shift in vasorelaxation EC50 for H2S may reflect the fact that, at high O2 concentrations, more of the added H2S is oxidized, leading to less H2S available for vasorelaxation. Additionally, the generated H2S oxidation products at high O2 may drive a competing vasoconstrictive reaction.

An interesting prediction is that O2-dependent H2S levels and/or effects will be manifest differently in the pulmonary vasculature, where O2 levels are much higher and where hypoxia induces vasoconstriction, and several groups have shown that H2S has a role in pulmonary vascular tone (37) and that changes in H2S levels have been linked to hypoxic pulmonary hypertension (25, 53). Other vasoactive H2S interactions, such as those between H2S and NO (1), and the effects of H2S on the production of vasoactive prostaglandins by heme-containing COX may also be O2 dependent.

We propose that 15 µM H2S, the EC50 at 40 µM O2, is more representative of physiological H2S concentrations than 200–300 µM H2S, which is effective in rat aorta vasorelaxation at high O2 (37, 55). If the peripheral arteries operate at <40 µM O2, as indicated by the respirometric experiments, then their H2S EC50 would most likely be comparably lower. Vascular free H2S may be even lower. Preliminary determinations of free H2S concentrations in whole rat blood, measured within minutes of extraction using a flow-through PHSS system, indicate that normal levels are ≤5 µM (unpublished data). Indeed, as with NO, the level of H2S required within the vasculature for vasorelaxation will be difficult to measure in vivo because of the extreme dilution once H2S has left the cell, as well as cellular H2S consumption. We previously reported that intact aorta produces H2S, measured under low (5 µM) O2 conditions with the PHSS, at a sustained steady-state level near 1 µM for >30 min when provided with substrate (15), perhaps via a feedback-regulated dynamic equilibrium between cellular H2S production and consumption mechanisms. Because the H2S inhibition constant for isolated cytochrome oxidase is submicromolar (34), these processes, along with diffusion and equilibria between bound and free H2S, most likely serve to regulate intracellular and extracellular H2S concentrations at noninhibitory levels.

Mechanisms of H2S-Mediated Vasorelaxation

It has been proposed that H2S-mediated vasorelaxation is primarily the result of hyperpolarization due to H2S directly opening KATP channels (7, 55), with a small proportion of the relaxation attributed to other mechanisms, including those dependent on NO. NO and H2S have been shown to operate synergistically (1, 22), perhaps at the level of the H2S-producing enzymes cystathionine-beta-synthase and cystathionine-{gamma}-lyase (CGL). Cystathionine-beta-synthase contains a redox-sensitive hexacoordinate heme remote from the active site (44), and one of its axial ligands is the sulfhydryl of a cysteine residue that can be displaced by NO or carbon monoxide (43). CGL contains 12 cysteine residues that, if bearing free -SH groups, are potential targets for S-nitrosation-type regulation (55). The presence of S-nitrosothiols in vascular tissue may also contribute to vessel tone, if the bound NO group is effectively reduced and liberated from the S-nitrosothiol (18). We have determined that H2S catalyzes the reactivation of NO-inhibited GAPDH in cultured cells, as well as the stoichiometric release of NO from S-nitrosoglutathione in a highly O2-dependent manner (unpublished observations), further substantiating the interaction between H2S and NO. Vascular smooth muscle cells cultured in the presence of an NO donor express increased production of CGL and H2S (55). Interestingly, smooth muscle cell proliferation is limited by exogenous H2S (16), and the overexpression of CGL commensurate with increased endogenous H2S production limits cell proliferation via activation of the ERK-p21 signaling cascade (51). H2S, when given to mice in their breathable air, has recently been shown to cause a reversible suspended-animation-like state (6), perhaps as a result of mitochondrial inhibition, in addition to cell protection against oxidative damage.

We report no significant increase in tissue cGMP levels after the administration of H2S, confirming that H2S operates via cGMP-independent mechanisms (54, 55). However, we did see COX-dependent effects of H2S in vivo. The results of our vessel tension studies with and without indomethacin have implicated H2S interaction with the COX pathway at 200 µM O2, but not at lower O2 levels. Preliminary results with intact aorta and with human umbilical vein endothelial cells indicate that H2S directly affects COX activity only at high O2. The relation between H2S and the COX pathway may involve interactions between H2S and the COX-produced vasoactive prostaglandins, as well as between H2S and the heme-containing COX enzymes. However, regardless of potential O2-dependent mechanistic interactions between H2S and COX, the COX pathway does not appear to play a major role in H2S-mediated vasorelaxation under normal physiological conditions.

In conclusion, H2S is a potent vascular signal with O2-dependent vasoactivity. In rat aorta, H2S concentrations that mediate rapid contraction at high O2 levels cause rapid relaxation at lower O2 levels. At high O2, the effect of H2S at physiological concentrations is vasoconstrictive, suggesting that not H2S but, rather, a putative oxidation product is also vasoactive and mediates constriction. These results indicate that the role of H2S as a vascular signal and the predominant vasoactive mechanism is highly O2 dependent, with vasorelaxation being the response at physiological H2S and O2. We suggest that all studies assessing H2S biology must account for O2 effects as well.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by American Heart Association Grant-in-Aid 0455296B and National Institutes of Health Grants 08R GM-073049A (to D. W. Kraus), HL-70146 (to R. P. Patel) and HL71189 and HL74391 (to J. R. Lancaster). T. S. Isbell is supported by a cardiovascular training fellowship.


   ACKNOWLEDGMENTS
 
We acknowledge the help of Asaf Stein (University of Alabama at Birmingham Biomedical Engineering).


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. W. Krauss, Dept. of Environmental Health Sciences, Univ. of Alabama at Birmingham, 1665 Univ. Blvd., Birmingham, AL 35294-0022 (e-mail: dwkraus{at}uab.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ali MY, Ping CY, Mok YYP, Ling L, Whiteman M, Bhatia M, Moore PK. Regulation of vascular nitric oxide in vitro and in vivo: a new role for endogenous hydrogen sulphide? Br J Pharmacol 149: 625–634, 2006.[CrossRef][Web of Science][Medline]
  2. Bao L, Vicek C, Paces V, Kraus JP. Identification and tissue distribution of human cystathionine beta-synthase mRNA isoforms. Arch Biochem Biophys 350: 95–103, 1998.[CrossRef][Web of Science][Medline]
  3. Bartholomew TC, Powell GM, Dodgson KS, Curtis CG. Oxidation of sodium sulphide by rat liver, lungs and kidney. Biochem Pharmacol 29: 2431–2437, 1980.[CrossRef][Web of Science][Medline]
  4. Beauchamp RO Jr, Bus JS, Popp JA, Boreiko CJ, Andjelkovich DA. A critical review of the literature on hydrogen sulfide toxicity. Crit Rev Toxicol 13: 25–97, 1984.[CrossRef][Medline]
  5. Bernal-Mizrachi C, Gates AC, Weng S, Imamura T, Knutsen RH, DeSantis P, Coleman T, Townsend RR, Muglia LJ, Semenkovich CF. Vascular respiratory uncoupling increases blood pressure and atherosclerosis. Nature 435: 502–506, 2005.[CrossRef][Medline]
  6. Blackstone E, Morrison M, Roth MB. H2S induces a suspended animation-like state in mice. Science 308: 518, 2005.[Abstract/Free Full Text]
  7. Brayden JE. Functional roles of KATP channels in vascular smooth muscle. Clin Exp Pharmacol Physiol 29: 312–316, 2002.[CrossRef][Web of Science][Medline]
  8. Brookes PS, Kraus DW, Shiva S, Doeller JE, Barone MC, Patel RP, Lancaster JR Jr, Darley-Usmar VM. Control of mitochondrial respiration by NO*, effects of low oxygen and respiratory state. J Biol Chem 278: 31603–31609, 2003.[Abstract/Free Full Text]
  9. Crawford JH, Chacko BK, Pruitt HM, Piknova B, Hogg N, Patel RP. Transduction of NO-bioactivity by the red blood cell in sepsis: novel mechanisms of vasodilation during acute inflammatory disease. Blood 104: 1375–1382, 2004.[Abstract/Free Full Text]
  10. Crawford JH, Isbell TS, Huang Z, Shiva S, Chacko BK, Schechter AN, Darley-Usmar VM, Kerby JD, Lang JD Jr, Kraus D, Ho C, Gladwin MT, Patel RP. Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation. Blood 107: 566–574, 2006.[Abstract/Free Full Text]
  11. Crawford JH, White CR, Patel RP. Vasoactivity of S-nitrosohemoglobin: role of oxygen, heme and NO oxidation states. Blood 101: 4408–4415, 2003.[Abstract/Free Full Text]
  12. Dello Russo C, Tringali G, Ragazzoni E, Maggiano N, Menini E, Vairano M, Preziosi P, Navarra P. Evidence that hydrogen sulphide can modulate hypothalamo-pituitary-adrenal axis function: in vitro and in vivo studies in the rat. J Neuroendocrinol 12: 225–233, 2000.[CrossRef][Web of Science][Medline]
  13. Doeller JE, Gaschen BK, Parrino VV, Kraus DW. Chemolithoheterotrophy in a metazoan tissue: sulfide supports cellular work in ciliated mussel gills. J Exp Biol 202: 1953–1961, 1999.[Abstract]
  14. Doeller JE, Grieshaber MK, Kraus DW. Chemolithoheterotrophy in a metazoan tissue: thiosulfate production matches ATP demand in ciliated mussel gills. J Exp Biol 204: 3755–3764, 2001.[Abstract/Free Full Text]
  15. Doeller JE, Isbell TS, Benavides G, Koenitzer J, Patel H, Patel RP, Lancaster JR Jr, Darley-Usmar VM, Kraus DW. Polarographic measurement of hydrogen sulfide production and consumption by mammalian tissues. Anal Biochem 341: 40–51, 2005.[CrossRef][Web of Science][Medline]
  16. Du J, Hui Y, Cheung Y, Bin G, Jiang H, Chen X, Tang C. The possible role of hydrogen sulfide as a smooth muscle cell proliferation inhibitor in rat cultured cells. Heart Vessels 19: 75–80, 2004.[CrossRef][Web of Science][Medline]
  17. Eghbal MA, Pennefather PS, O'Brien PJ. H2S cytotoxicity mechanism involves reactive oxygen species formation and mitochondrial depolarisation. Toxicology 203: 69–76, 2004.[CrossRef][Web of Science][Medline]
  18. Feelisch M, Rassaf T, Mnaimneh S, Singh N, Bryan NS, Jourd'Heuil D, Kelm M. Concomitant S-, N-, and heme-nitrosylation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB J 16: 1375–1382, 2002.
  19. Goodwin LR, Francom D, Dieken FP, Taylor JD, Warenycia MW, Reiffenstein RJ, Dowling G. Determination of sulfide in brain tissue by gas dialysis/ion chromatography: postmortem studies and two case reports. J Anal Toxicol 13: 105–109, 1989.[Web of Science][Medline]
  20. Granlund-Edstedt M, Johansson E, Claesson R, Carlsson J. Effect of sulfide ions on complement factor C3. Infect Immun 59: 696–699, 1991.[Abstract/Free Full Text]
  21. Grieshaber MK, Volkel S. Animal adaptations for tolerance and exploitation of poisonous sulfide. Annu Rev Physiol 60: 33–53, 1998.[CrossRef][Web of Science][Medline]
  22. Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem Biophys Res Commun 237: 527–531, 1997.[CrossRef][Web of Science][Medline]
  23. Hui Y, Du J, Tang C, Bin G, Jiang H. Changes in arterial hydrogen sulfide H2S content during septic shock and endotoxin shock in rats. J Infect 47: 155–160, 2003.[CrossRef][Web of Science][Medline]
  24. Isbell TS, Koenitzer JR, Crawford JH, White CR, Kraus DW, Patel RP. Assessing NO-dependent vasodilation using vessel bioassays at defined oxygen tensions. Methods Enzymol 396: 553–568, 2005.[Web of Science][Medline]
  25. Jin H, Cong B, Zhao B, Zhang C, Liu X, Zhou W, Shi Y, Tang C, Du J. Effects of hydrogen sulfide on hypoxic pulmonary vascular structural remodeling. Life Sci 78: 1299–1309, 2006.[CrossRef][Web of Science][Medline]
  26. Julian D, Statile JL, Wohlgemuth SE, Arp AJ. Enzymatic hydrogen sulfide production in marine invertebrate tissues. Comp Biochem Physiol A 133: 105–115, 2002.[CrossRef][Medline]
  27. Kimura H. Hydrogen sulfide as a biological mediator. Antioxid Redox Signal 7: 781–787, 2005.[CrossRef][Web of Science][Medline]
  28. Kraus DW, Doeller JE. Sulfide consumption by mussel gill mitochondria is not strictly tied to oxygen reduction: measurements using a novel polarographic sulfide sensor. J Exp Biol 207: 3667–3679, 2004.[Abstract/Free Full Text]
  29. Kraus DW, Wittenberg JB. Hemoglobins of the Lucina pectinata/bacteria symbiosis. I. Molecular properties, kinetics and equilibria of reactions with ligands. J Biol Chem 265: 16043–16053, 1990.[Abstract/Free Full Text]
  30. Leffler CW, Parfenova H, Jaggar JH, Wang R. Carbon monoxide and hydrogen sulfide: gaseous messengers in cerebrovascular circulation. J Appl Physiol 100: 1065–1076, 2006.[Abstract/Free Full Text]
  31. Millero FJ. The thermodynamics and kinetics of the hydrogen sulfide system in natural waters. Marine Chem 18: 121–147, 1986.[CrossRef]
  32. Moore PK, Bhatia M, Moochhala S. Hydrogen sulfide: from the smell of the past to the mediator of the future? Trends Pharmacol Sci 24: 609–611, 2003.[CrossRef][Medline]
  33. Nicholls DG, Ferguson SJ. Bioenergetics 3. New York: Academic, 2002, p. 65–66.
  34. Nicholls P. The effect of sulphide on cytochrome aa3. Isosteric and allosteric shifts of the reduced {alpha}-peak. Biochim Biophys Acta 396: 24–35, 1975.[Medline]
  35. Nunez C, Victor VM, Tur R, Alvarez-Barrientos A, Moncada S, Esplugues JV, D'Ocòn P. Discrepancies between nitroglycerin and NO-releasing drugs on mitochondrial oxygen consumption, vasoactivity and the release of CO. Circ Res 97: 1063–1069, 2005.[Abstract/Free Full Text]
  36. O'Brien J, Vetter RD. Production of thiosulphate during sufphide oxidation by mitochondria of the symbiont-containing bivalve Solemya reidi. J Exp Biol 149: 133–148, 1990.[Abstract/Free Full Text]
  37. Olson KR, Dombkowski RA, Russell MJ, Doellman MM, Head SK, Whitfield NL, Madden JA. Hydrogen sulfide as an oxygen sensor/transducer in vertebrate hypoxic vasoconstriction and hypoxic vasodilation. J Exp Biol 209: 4011–4023, 2006.[Abstract/Free Full Text]
  38. Olson KR. Vascular actions of hydrogen sulfide in nonmammalian vertebrates. Antioxid Redox Signal 7: 804–812, 2005.[CrossRef][Web of Science][Medline]
  39. Parrino V, Kraus DW, Doeller JE. ATP production from the oxidation of sulfide in gill mitochondria of the ribbed mussel Geukensia demissa. J Exp Biol 203: 2209–2218, 2000.[Abstract]
  40. Shibata M, Ichioka S, Kamiya A. Estimating oxygen consumption rates of arteriolar walls under physiological conditions in rat skeletal muscle. Am J Physiol Heart Circ Physiol 289: H295–H300, 2005.[Abstract/Free Full Text]
  41. Sidhu R, Singh M, Samir G, Carson RJ. L-Cysteine and sodium hydrosulphide inhibit spontaneous contractility in isolated pregnant rat uterine strips in vitro. Pharmacol Toxicol 88: 198–203, 2001.[CrossRef][Web of Science][Medline]
  42. Svenson A. A rapid and sensitive spectrophotometric method for determination of hydrogen sulfide with 2,2'-dipyridyl disulfide. Anal Biochem 107: 51–55, 1980.[CrossRef][Web of Science][Medline]
  43. Taoka S, Banerjee R. Characterization of NO binding to human cystathionine beta-synthase: possible implications of the effects of CO and NO binding to the human enzyme. J Inorg Biochem 87: 245–251, 2001.[CrossRef][Web of Science][Medline]
  44. Taoka S, Ohja S, Shan X, Kruger WD, Banerjee R. Evidence for heme-mediated redox regulation of human cystathionine beta-synthase activity. J Biol Chem 273: 25179–25184, 1998.[Abstract/Free Full Text]
  45. Theissen U, Hoffmeister M, Grieshaber M, Martin W. Single eubacterial origin of eukaryotic sulfide:quinone oxidoreductase, a mitochondrial enzyme conserved from the early evolution of eukaryotes during anoxic and sulfidic times. Mol Biol Evol 20: 1564–1574, 2003.[Abstract/Free Full Text]
  46. Tsai AG, Johnson PC, Intaglietta M. Oxygen gradients in the microcirculation. Physiol Rev 83: 933–963, 2003.[Abstract/Free Full Text]
  47. Ubuka T. Assay methods and biological roles of labile sulfur in animal tissues. J Chromatogr B Biomed Appl 781: 227–249, 2002.[CrossRef]
  48. Vande Weghe JG, Ow DW. A fission yeast gene for mitochondrial sulfide oxidation. J Biol Chem 274: 13250–13257, 1999.[Abstract/Free Full Text]
  49. Wills MH, Johns RA, Stone DJ, Moscicki JC, Difazio CA. Vascular effects of 2-chloroprocaine and sodium metabisulfite on isolated rat aortic rings. Reg Anesth 14: 271–273, 1989.[Web of Science][Medline]
  50. Yan H, Du J, Tang C. The possible role of hydrogen sulfide on the pathogenesis of spontaneous hypertension in rats. Biochem Biophys Res Commun 313: 22–27, 2004.[CrossRef][Web of Science][Medline]
  51. Yang G, Cao K, Wu L, Wang R. Cystathionine {gamma}-lyase overexpression inhibits cell proliferation via a H2S-dependent modulation of ERK1/2 phosphorylation and p21Cip/WAK-1. J Biol Chem 279: 49199–49205, 2004.[Abstract/Free Full Text]
  52. Yong R, Searcy DG. Sulfide oxidation coupled to ATP synthesis in chicken liver mitochondria. Comp Biochem Physiol B Biochem Mol Biol 129: 129–137, 2001.[CrossRef][Medline]
  53. Zhang C, Du J, Bu D, Yan H, Tang X, Tang C. The regulatory effect of hydrogen sulfide on hypoxic pulmonary hypertension in rats. Biochem Biophys Res Commun 302: 810–816, 2003.[CrossRef][Web of Science][Medline]
  54. Zhao W, Wang R. H2S-induced vasorelaxation and underlying cellular and molecular mechanisms. Am J Physiol Heart Circ Physiol 283: H474–H480, 2002.[Abstract/Free Full Text]
  55. Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of H2S as a novel endogenous KATP channel opener. EMBO J 20: 6008–6016, 2001.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. Yusof, K. Kamada, T. Kalogeris, F. S. Gaskin, and R. J. Korthuis
Hydrogen sulfide triggers late-phase preconditioning in postischemic small intestine by an NO- and p38 MAPK-dependent mechanism
Am J Physiol Heart Circ Physiol, March 1, 2009; 296(3): H868 - H876.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
K. R. Olson
Hydrogen sulfide and oxygen sensing: implications in cardiorespiratory control
J. Exp. Biol., September 1, 2008; 211(17): 2727 - 2734.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
B. Andruski, D.-M. McCafferty, T. Ignacy, B. Millen, and J. J. McDougall
Leukocyte trafficking and pain behavioral responses to a hydrogen sulfide donor in acute monoarthritis
Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2008; 295(3): R814 - R820.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
N. L. Whitfield, E. L. Kreimier, F. C. Verdial, N. Skovgaard, and K. R. Olson
Reappraisal of H2S/sulfide concentration in vertebrate blood and its potential significance in ischemic preconditioning and vascular signaling
Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2008; 294(6): R1930 - R1937.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/4/H1953    most recent
01193.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (23)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koenitzer, J. R.
Right arrow Articles by Kraus, D. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koenitzer, J. R.
Right arrow Articles by Kraus, D. W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.