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First published online June 27, 2008
Journal of Experimental Biology 211, 2205-2213 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.016766
Oxygen dependency of hydrogen sulfide-mediated vasoconstriction in cyclostome aortas
1 Indiana University School of Medicine–South Bend, 1234 Notre Dame
Avenue, South Bend, IN 46617, USA
2 School of Biological Sciences, University of Canterbury, Private Bag 4800,
Christchurch 8020, New Zealand
3 Department of Biology, Saint Mary's College, Notre Dame, IN 46556, USA
* Author for correspondence (e-mail: kolson{at}nd.edu)
Accepted 10 April 2008
 |
Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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-lyase. Oxygen consumption
(
O2) of hagfish
DA was constant between 15 and 115 mmHg PO2 (1
mmHg=0.133 kPa), decreased when PO2 <15
mmHg, and increased after PO2 exceeded 115
mmHg. 10 µmol l–1 H2S increased and 
100
µmol l–1 H2S decreased
O2. Consistent
with the effects on HVC, cysteine increased and amino-oxyacetate decreased
O2. These
results show that H2S is a monophasic vasoconstrictor of specific
cyclostome vessels and because hagfish lack vascular NO, and vascular
sensitivity to H2S was enhanced at low
PO2, it is unlikely that H2S
contractions are mediated by either H2S–NO interaction or an
oxidation product of H2S. These experiments also provide additional
support for the hypothesis that the metabolism of H2S is involved
in oxygen sensing/signal transduction in vertebrate vascular smooth
muscle.
Key words: hypoxic vasoconstriction, oxygen sensing, vascular smooth muscle
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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) and it is now generally accepted that in mammals this
response is unique to the pulmonary circulation, whereas hypoxic vasodilation
(HVD) is the prominent response of systemic vessels
(Weir and Archer, 1995).
Although HVC and HVD may be modulated by endothelial-derived and/or
circulating substances
(Félétou et al.,
1995; Jacobs and Zeldin,
2001; Kerkhof et al., 2001;
Liu et al., 2001;
Aaronson et al., 2002;
Deussen et al., 2006), the
basic responses are intrinsic to the vascular smooth muscle cell
(Madden et al., 1992). In
non-mammalian vertebrates, HVC has been observed in both systemic and
respiratory conductance vessels (Olson et
al., 2001; Russell et al.,
2001; Smith et al.,
2001; Russell et al.,
2007) and HVC appears to be an intrinsic response of vascular
smooth muscle cells in the cyclostome dorsal aorta as well
(Olson et al., 2001). HVD has
only recently been systematically examined in non-mammalian vertebrates
(Russell et al., 2007) and
while it is common in systemic vessels it is not necessarily the predominant
response.
How vascular smooth muscle cells `sense' hypoxia and transduce this into a
mechanical response, either HVC or HVD, is unknown. We recently proposed that
the metabolism of H2S is involved in the O2-sensing
signal transduction process. Our model is based on the balance between
constitutive cellular production of vasoactive hydrogen sulfide
(H2S) and its oxidation to inactive products by available
O2 (Olson et al.,
2006). Furthermore, this model appears to be applicable to both
HVC and HVD and evidence for a H2S-mediated hypoxic relaxation has
even been observed in the trout urinary bladder
(Dombkowski et al., 2006).
There is also relatively little information on the mechanism through which
H2S elicits mechanical responses in the vasculature.
H2S-mediated vasodilation has been demonstrated in mammalian
systemic vessels and at least part of this response is due to H2S
opening of ATP sensitive potassium (KATP) channels on the vascular
smooth muscle cell and to release of nitric oxide (NO) from the endothelium
(Zhao et al., 2001;
Zhao and Wang, 2002;
Wang et al., 2004).
H2S-mediated vasoconstriction has been demonstrated in mammalian
pulmonary vessels (Olson et al.,
2006) and in a variety of both pulmonary and systemic vessels from
non-mammalian vertebrates (Dombkowski et
al., 2005). Although it is unlikely that H2S
contractions are mediated through either KATP channels or
endothelial-derived vasoconstrictor substances, the mechanism(s) of
H2S-mediated vasoconstriction is unknown.
Recently, Koenitzer et al. (Koenitzer
et al., 2007) examined the effects of H2S on rat
thoracic aortas at high (200 µmol l–1, 
150 mmHg) and
low (40 µmol l–1, 30 mmHg) partial pressures of
O2 and showed that vascular relaxation was more sensitive to
H2S at low oxygen concentration ([O2]) and that
H2S-mediated contractions were present at high, but not low
[O2]. They postulated that the decreased sensitivity of the
H2S-mediated vasorelaxation at high [O2] was due to the
combined effect of rapid oxidation (and therefore inactivation) of
vasodilatory H2S plus the generation of a vasoconstrictor oxidation
product of H2S that would compete with the H2S
relaxation. Thus H2S does not directly produce vasoconstriction.
The identity of this oxidation product was not determined.
There are other possible explanations for the results of Koenitzer et al.
(Koenitzer et al., 2007) that
seem equally or more plausible. First, Koenitzer et al.
(Koenitzer et al., 2007) only
examined rat aortas and these vessels relax when exposed to either hypoxia or
lower (and perhaps more physiological?) concentrations of H2S and
thus a contraction would not normally be expected. Second, our theory of
H2S metabolism in vascular O2 sensing predicts that as
[O2] falls, endogenous [H2S] increases. Thus at low
PO2 we would expect greater sensitivity to
exogenous H2S when applied against a background of elevated
endogenous H2S, consistent with the observations of Koenitzer et
al. (Koenitzer et al., 2007).
Furthermore, we also think that H2S directly produces
vasoconstriction in vessels that exhibit hypoxic vasoconstriction (e.g.
hagfish and lamprey aortas) because it seems unlikely to us that cellular
concentrations of an oxidation product of H2S would be increasing
when PO2 is falling.
In the present study we examined the interaction between [O2]
and [H2S] in the dorsal aorta of the most ancient extant craniate,
the hagfish. This vessel was chosen because it has a mono-phasic,
[O2]-dependent HVC that is endothelium independent, and does not
involve KATP channels, products of lipoxygenase, cyclooxygenase,
cytochrome P450 enzyme activity, or 
-adrenergic, muscarinic,
nicotinic, purinergic or serotoninergic receptors
(Olson et al., 2001). If our
hypotheses that H2S directly produces vasoconstriction and that
endogenous H2S increases when PO2
falls is correct, we expect to see an increased sensitivity of
H2S-constriction at low [O2], not an unmasking of
H2S relaxation. We also provide additional evidence for
H2S metabolism in the O2 sensing mechanism by examining
the contribution of its precursor, cysteine, and the effects of inhibitors of
H2S synthesis on HVC. The effects of H2S, cysteine and
enzyme inhibitors on vessel O2 consumption were measured to
determine whether H2S exposure increased
O2 and
inhibitors decreased it. For comparison, we examined the O2
sensitivity of H2S contraction in lamprey aortas. These vessels are
identical to hagfish aortas in their response to hypoxia
(Olson et al., 2001) and
evidence for the role of H2S in O2 sensing has been
described (Olson et al.,
2006).
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Sea lamprey (Petromyzon marinus L.; 130–450 g) were captured by the US Geological Survey, Biological Resources Division, in Michigan during the spring-summer spawning migration and airlifted to Indiana University School of Medicine–South Bend (IUSM-SB). They were housed in 500 l rectangular tanks in aerated, flowing well water (15°C), and exposed to a 12 h:12 h light:dark photoperiod. They were not fed. Lamprey were anesthetized in benzocaine (1:5000, wt:vol), and the vessels were dissected out and placed in Cortland buffered saline at 4°C until use.
Salmon (Oncorhynchus tshawytscha Walbaum) were obtained from a nearby hatchery, anaesthetized with 22 p.p.m. AQUI-STM in their holding tanks and then killed by pithing the brain and proximal spinal cord. The ventral aorta and afferent branchial arteries were rapidly excised and stored at 4°C in freshwater salmon Ringer's solution; the dorsal aorta is firmly attached to the vertebral column and cannot be removed intact. Storage and preparation of the salmon vessels for respirometry was identical to that described below for hagfish.
Experimental procedures were approved by the University of Canterbury's Animal Ethics Committee and the IUSM-SB IACUC.
Myography
Vessels were cut transaxially into 3–4 mm long segments, mounted on
280 µm diameter stainless-steel hooks and suspended in 20 ml,
water-jacketed (12°C) smooth muscle chambers and bubbled with room air.
Tension was measured with Grass FT03C force-displacement transducers (Grass
Instruments, West Warwick, RI, USA) and collected electronically using Biopac
model MP35 (Biopac Systems Inc., Goleta, CA, USA), or measured with MLT0210
isometric force transducers (ADInstruments, Castle Hill, Waverley, NSW,
Australia) using Powerlab® systems with bridge amplifiers (ADInstruments).
Data were archived at 2 Hz on notebook computers. A resting tension of
500±50 mg (Olson et al.,
2001) was applied to the vessels for 45–60 min prior to
experimentation. Vessels were maximally contracted with the acetylcholine
analog carbamylcholine chloride (carbachol, 10 µmol l–1;
Fig. 1) until tension plateaued
(30–45 min), then rinsed four times with buffer and resting tension
re-established over the ensuing 60 min. They were then contracted a second
time with 10 µmol l–1 carbachol, the rinse repeated, and
the vessels were allowed to stabilize and resting tension re-established for
the next 1–2 h. The tension produced by the second application of
carbachol was used as the reference contraction for subsequent
experiments.
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Effect of H2S on buffer pH
The dissolution of Na2S in water produces H2S and
HS– (collectively referred to in this study as
H2S) and increases pH. Because extracellular alkalinity can
contract vascular smooth muscle independently of other exogenous stimuli
(Smith et al., 2006), the
buffering capacity of Hepes samples over the range of 1 µmol
l–1 to 10 mmol l–1 H2S was
measured in triplicate using an Orion 911600 semi-micro pH electrode (Beverly,
MA USA) and a PHM 84 pH meter (Radiometer, Copenhagen, Denmark).
H2S dose-dependent responses
Cumulative H2S dose–response curves (1 µmol
l–1 to 1 mmol l–1) were obtained for
otherwise unstimulated, normoxic (bubbled with room air; 21% O2)
dorsal aortas and efferent branchial arteries. To determine if H2S
relaxed vessels, a second series of experiments were conducted with the
vessels pre-contracted with 150 mmol l–1 KCl or 0.3 µmol
l–1 carbachol prior to the H2S doses. In pilot
studies, H2S had no effect on ventral aortas or afferent branchial
arteries (N=4) and these vessels were not examined further.
Effect of PO2 on H2S responses
The effect of graded hypoxia on the H2S dose response of hagfish
dorsal aortas was examined by initially aerating groups of vessels with either
100% room air (PO2=157 mmHg), 6% air/94%
N2 (PO2=10 mmHg), or 100%
N2 (PO2<1 mmHg) for 20–30
min prior to and during the H2S treatments (1 mmHg=0.133 kPa). The
air/N2 mixture was controlled with a Wöstoff type 1M 300/a-F
gas mixing pump (H. Wöstoff, Bochum, Germany). The
PO2 was measured in one myograph chamber using
a Microelectrodes MI-730 oxygen electrode and meter (Bedford, NH).
The effect of moderate hypoxia on the H2S dose response of
lamprey dorsal aortas was examined using the following protocol. Vessels were
contracted twice with 80 mmol l–1 KCl, washed twice after
each contraction, and then vigorously bubbled with 100% N2 to
produce a maximal HVC. After recovery (normoxia) the flow of N2 was
reduced to produce HVC that was 20±6% of the maximal HVC. This is
equivalent to a bath PO2 of 20–30 mmHg
(Olson et al., 2001).
Cumulative doses of H2S (10 nmol l–1–1 mmol
l–1) were applied during this moderate hypoxia.
Involvement of H2S mechanisms in hagfish hypoxic vasoconstriction
The involvement of H2S in hagfish HVC was examined by measuring
the response of hypoxia-contracted (100% N2) aortas to serial
additions of the substrate for H2S synthesis: L-cysteine
(0.1, 1 and 10 mmol l–1), amino-oxyacetate (AOA; 0.1, 1 and 4
mmol l–1), an inhibitor of cystathionine β-synthase
(CBS), D,L-propargylglycine (PPG; 0.1, 1 and 4 mmol
l–1), an inhibitor of cystathionine -lyase, or
hydroxylamine (0.01, 0.1 and 1 mmol l–1), a general inhibitor
of pyridoxyl 5'-phosphate-dependent enzymes. Following a standard
carbachol (10 µmol l–1) contraction the vessels were
thoroughly washed and gassed with 100% N2 for 20–30 min until
the hypoxic contraction stabilized. Cumulative doses of cysteine or inhibitors
were applied during the hypoxic contraction followed by a final application of
10 µmol l–1 carbachol. Vessels were not washed prior to
the final carbachol. The effects of hypoxia, cysteine, inhibitors and final
carbachol were normalized relative to the reference carbachol contraction.
Oxygen consumption by hagfish dorsal aortas
Hagfish dorsal aortas used in the oxygen consumption
(O2) experiments
were stored and maintained in hagfish Hepes buffer containing gentamicin
sulfate (200 µg ml–1) to reduce the potential for
micro-organisms to contribute to
O2
(Rudin et al., 1970). Four to
six aortas, 3–8 mm long, were freed of any remaining connective tissue
and fat and threaded onto a 280 µm diameter stainless steel wire frame.
They were then placed into 1 ml of either air-saturated or reduced
[O2] (PO2 60 mmHg) Hepes buffer
in model RC300 respirometers (Strathkelvin Instruments, Glasgow, Scotland)
fitted with IL 1302 oxygen electrodes and maintained at 12°C. The
electrode signal was fed into a Strathkelvin model 781 O2 meter and
then via a Powerlab 4SP to a notebook running Chart 5 software (both
ADInstruments). Treatments were introduced into the respirometer via
a small slot in the electrode holder with a 50 µl Hamilton syringe
(Hamilton Co., Reno, Nevada, USA) fitted with a length of polythene tubing
(Portex Ltd, Hythe, Kent, England; o.d. 0.61 mm, i.d. 0.28 mm). Oxygen
consumption was determined from the equation:
 |
O2 is the
rate of oxygen consumption (mmol O2 mg–1
min–1), 
PO2 is the
change in PO2 during the treatment period (in
mmHg), O2 is the solubility of O2 in seawater (in
mmol O2 l–1 mmHg–1; hagfish
plasma has very similar ionic composition), V is the volume of the
respirometer (in l), t is the time (in s) between
PO2 measurements and M is the mass of
the vessels in the respirometer (in mg).
The relationship between PO2 and
O2 was measured
in vessels that were allowed to deplete the oxygen content from air saturation
(PO2 155 mmHg) down to zero. These
experiments showed that the vessels could efficiently regulate their
O2 between a
PO2 of 15 and 115 mmHg (see Results).
Subsequent experiments on the effects of H2S, cysteine and
inhibitors of H2S production were performed between 40 and 60 mmHg
PO2 where
O2 was otherwise
independent of PO2.
A cumulative H2S
concentration–O2
response was established for 1 µmol l–1–1 mmol
l–1 H2S in both un-contracted and carbachol (100
µmol l–1)-contracted vessels. The effects of the
H2S precursor, L-cysteine (1 and 1 mmol
l–1) and inhibitors of H2S production, AOA and HA
(both 10 µmol l–1–1 mmol l–1), on
O2 were also
determined.
Oxygen consumption by salmon vessels
In the initial studies on hagfish vessels it became evident that
contracting the vessels with carbachol had no effect on oxygen consumption.
This was unexpected as it has been shown that contracting mammalian vessels
increases oxygen consumption (Koenitzer et
al., 2007). To determine whether this was an actual physiological
difference or an experimental artifact, we repeated the studies with ventral
aortas and afferent branchial arteries isolated from chinook salmon using the
general protocol described above for hagfish vessels.
Chemicals
The composition of hagfish Hepes-buffered saline was (in mmol
l–1): 497.95 NaCl, 8.05 KCl, 5.10 CaCl2, 9.00
MgCl2, 3.04 MgSO4, 3.00 Hepes
[N-(2-hydroxyethyl)piperazine-N=–(2-ethane-sulfonic
acid)] acid form, 6.99 Hepes sodium salt, 5.55 glucose, pH 7.8. The
composition of lamprey Cortland saline was (in mmol l–1): 124
NaCl, 3 KCl, 2 CaCl2, 0.57 MgSO4, 12 NaHCO3,
0.09 NaH2PO4, 1.8 Na2HPO4, 5.5
glucose, pH 7.8. The composition of salmon Ringer was (in mmol
l–1; 136.89 NaCl; 2.11 KCl; 0.99 MgCl2; 1.30
CaCl2; 3.00 Hepes acid form; 6.99 Hepes sodium salt; 0.30 sodium
glutamate; 0.40 L-glutamine; 0.02 sodium aspartate; 0.05
DL-carnitine; 10.00 glucose, pH 7.6. AOA was purchased from ACROS
Organics (Morris Plains, NJ, USA) all other chemicals were purchased from
Sigma Chemical Co. (St Louis, MO, USA).
Calculations
Concentration response curves were expresses as a percentage of the maximal
response. Vessel responses were normalized to the second carbachol contraction
produced prior to experimentation. At the end of an experiment the vessel was
blotted on paper toweling, weighed and vessel tension was normalized to wet
mass, i.e. mg tension g–1 wet mass. Because the hypoxic
responses of individual vessels were reproducible
(Olson et al., 2001), each
vessel served as its own control and treatment effects were statistically
examined by paired t-test or repeated measures tests. Results are
presented as mean ± s.e.m. Student's t-test and analysis of
variance (ANOVA) were used for comparisons between vessels. Significance was
assumed when P
0.05.
Significant differences in rates of
O2 and responses
to drugs were determined using a repeated measures ANOVA. Where significant
differences were calculated between means, Tukey's post-hoc tests
showed which means were significantly different from each other. Paired
Student's t-tests were used to detect differences between
carbachol-treated and -untreated vessels in the
O2 data
(controls and at each concentration of H2S). Significance was
assumed when P0.05. All analyses were performed in Prism 4.00
(Graphpad software, San Diego, CA, USA).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Effects of H2S on pH
The effects of increasing concentrations of H2S on pH of hagfish
Hepes buffer is shown in Fig.
2. Buffering was very efficient between 1 µmol
l–1 and 1 mmol l–1 H2S and
increased less than 0.3 pH unit between 1 mmol l–1 and 3 mmol
l–1 H2S. However, pH increased nearly 2.5 units
between 3 and 10 mmol l–1 H2S. H2S
contractions appeared to be independent of pH between 1 µmol
l–1 and 1 mmol l–1 H2S, but
concentrations above 1 mmol l–1 alkalinized the medium and
this appeared to greatly augment H2S contractions. H2S
concentrations were limited in subsequent experiments to 1 mmol
l–1 in order to minimize the possibility of pH interference
and to avoid complications that might result from changes in ionic strength or
composition due to increasing the buffering capacity, or titration.
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|
Effect of PO2 on H2S responses of hagfish and lamprey dorsal aortas
Hagfish dorsal aortas bubbled with 100% N2 were significantly
more sensitive to low H2S concentrations than aortas bubbled with
room air and the H2S dose–response curve of hypoxic (anoxic)
vessels appeared to have two components
(Fig. 3).
H2S-mediated contractions of hagfish aortas bubbled with 6% air/94%
N2 (data not shown) were not significantly different from aortas
bubbled with room air.
H2S produced dose-dependent contractions of lamprey dorsal aortas (Fig. 3). Moderate hypoxia increased H2S sensitivity between 10 and 300 µmol l–1 H2S (P value at 30 µmol l–1 H2S was 0.053). Hypoxia did not affect the magnitude of the 1 mmol l–1 H2S contraction which in hypoxia was 41±5% and in normoxia 43±4% of a 80 mmol l–1 KCl contraction. H2S at concentrations between 10 nmol l–1 to 1 µmol l–1 did not affect either normoxic or hypoxic vessels (not shown).
Involvement of H2S mechanisms in hagfish hypoxic vasoconstriction
In these experiments vessels were contracted with 10 µmol
l–1 carbachol, washed, then continuously contracted with 100%
N2 aeration. During the hypoxic contraction, the vessels were given
cumulative additions of cysteine or inhibitors and this was followed, without
washing the vessels, by 10 µmol l–1 carbachol.
The effect of L-cysteine, a substrate for H2S synthesis, on hypoxic contractions of hagfish dorsal aortas is shown in Fig. 4, top left panel. 100 µmol l–1 cysteine produced a consistent, but statistically insignificant increase in the force of the N2 contraction. Increasing cysteine to 1 mmol l–1 significantly (P<0.05) contracted the vessels to approximately double that in the original N2 contraction. Raising cysteine to 10 mmol l–1 produced an immediate relaxation back to the pre-cysteine (N2) level (P<0.05). The carbachol (10 µmol l–1) contraction at the end of the experiment, in the presence of N2 and 10 mmol l–1 cysteine, was not significantly different from the reference carbachol contraction (90±14% of reference, N=7).
|
The effect of the cystathionine β-synthase (CBS) inhibitor, amino-oxyacetate (AOA), on hypoxic vasoconstriction of the dorsal aorta is shown in Fig. 4 top right panel. Hypoxic contractions were unaffected by 100 µmol l–1 and 1 mmol l–1 AOA. 4 mmol l–1 AOA completely inhibited the hypoxic contraction (P<0.05). The carbachol (10 µmol l–1) contraction at the end of the experiment, in the presence of 4 mmol l–1 AOA, was not significantly different from the reference carbachol contraction (111±12% of reference, N=8).
As shown in Fig. 4, lower
left panel, the cystathionine -lyase (CSE) inhibitor, propargyl
glycine (PPG; between 100 µmol l–1 and 4 mmol
l–1) had no effect on the hypoxic contraction. A carbachol
(10 µmol l–1) contraction at the end of the experiment, in
the presence of PPG, was similarly unaffected (104±7% of reference,
N=8).
Hydroxylamine (HA), an uncoupler of pyridoxyl 5'-phosphate-dependent enzymes including CBS and CSE, at 10 µmol l–1 significantly increased the force of the N2 contraction (Fig. 4, lower right panel). Increasing HA to 100 µmol l–1 and 1 mmol l–1 produced slight, but statistically insignificant, further increases in tension. The carbachol (10 µmol l–1) contraction at the end of the experiment, in the presence of HA, was not significantly different from the reference carbachol contraction (103±12% of reference, N=8).
Vessel O2 consumption
The relationship between PO2 and oxygen
consumption (O2)
in uncontracted and carbachol pre-contracted hagfish dorsal aortas is shown in
Fig. 5A.
O2 was well
maintained around 2.4 pmol mg–1 min–1
between 15 and 115 mmHg PO2 but doubled between
115 and 155 mmHg and fell to zero as PO2
approached zero.
O2 fell to 90%
of the regulated rate at a PO2 of 12 mmHg and
the PO2 at which the regulated
O2 fell to half
(P50) was 3 mmHg. Pre-treating hagfish aortas with 100 µmol
l–1 carbachol did not significantly affect
O2.
O2 was also
well-regulated in unstimulated salmon vessels between 15 and 115 mmHg
PO2 but the rate of oxygen consumption per unit
tissue mass was five times that of hagfish aortas
(Fig. 5B). Pre-treating salmon
vessels with 100 µmol l–1 carbachol nearly doubled
O2 at all but
the lowest PO2.
|
O2 are
summarized in Fig. 6. 10
µmol l–1 H2S significantly stimulated
O2 whereas 100
µmol l–1 and 1 mmol l–1 significantly
inhibited O2. A
12-fold increase in
O2 was produced
by 10 mmol l–1 cysteine; in many experiments 1 mmol
l–1 cysteine often appeared to increase
O2 as well,
although this was not statistically significant.
O2 was inhibited
by either 10 mmol l–1 AOA or 10 mmol l–1 HA.
Other concentrations of AOA (10 µmol l–1–1 mmol
l–1) and HA (10 µmol l–1–1 mmol
l–1) did not significantly affect
O2.
Pre-contraction with 100 µmol l–1 carbacol did not
significantly affect
O2 in vessels
treated with 10 µmol l–1, 100 µmol l–1
or 1 mmol l–1 H2S
(Fig. 6), 10 mmol
l–1 cysteine, 10 mmol l–1 AOA or 10 mmol
l–1 HA (N=5 for all; data not shown), although
O2 of vessels in
10 µmol l–1 H2S was significantly greater than
O2 of vessels in
100 µmol l–1 H2S
(Fig. 6).
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|
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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H2S as a vasoconstrictor
As shown in Fig. 3, the
H2S sensitivity in both hagfish and lamprey aortas increased when
PO2 was decreased. Hagfish vessels bubbled with
100% N2 responded to 1 µmol l–1 H2S,
whereas 100 µmol l–1 H2S was required to
contract vessels bubbled with room air
(Fig. 3). Similarly, the
apparent H2S thresholds for hypoxic
(PO2 20–30 mmHg) and normoxic
lamprey vessels were 10 and 30 µmol l–1 H2S,
respectively (Fig. 3).
Furthermore, the H2S dose–response curves for both animals
were left-shifted in low PO. Thus although the effect of
hypoxia on the H2S response of lamprey vessels was less dramatic
than that of hagfish vessels (probably because the hypoxia was less severe),
the basic responses were, nevertheless, quite similar. As described below,
these results support the hypothesis that H2S has direct
vasoconstrictory activity in specific vessels.
Koenitzer et al. (Koenitzer et al.,
2007) observed a bi-phasic effect of H2S on rat aortas;
low H2S concentrations produced dilation and high concentrations (1
mmol l–1) produced contraction. They also found that
H2S-mediated dilation of rat aortas became more sensitive to
H2S at low PO2. These authors
(Koenitzer et al., 2007)
suggested that the H2S-mediated vasoconstriction of aortas bubbled
with room air was due to an oxidation product of H2S, not
H2S itself, and that the reason rat aortas became more sensitive to
H2S during hypoxia was because in the absence of this putative
oxidation product there was no offsetting constrictory stimulus to compete
with the direct H2S dilation. Our findings argue against these
hypotheses. First, H2S only constricted hagfish and lamprey dorsal
aortas and therefore the increased sensitivity observed at low
PO2 could not be due to removal of a competing
(in this case dilatory) process. Second, it seems unlikely that production of
this hypothetical vasoconstrictory oxidation product of H2S would
increase when the vessels are bubbled with 100% N2. An alternative,
and we think more plausible, explanation for the increased H2S
sensitivity, and one that is consistent with our
(Olson et al., 2006)
hypothesis of H2S involvement in HVC (see below), is that when
PO2 falls endogenous H2S increases.
Therefore, less exogenous H2S is required for vasoconstriction.
Ali et al. (Ali et al.,
2006) and Kubo et al. (Kubo et
al., 2007) observed the opposite effects of Koenitzer et al.
(Koenitzer et al., 2007), i.e.
low H2S concentrations (<200 µmol l–1)
contracted, and elevated H2S concentrations (200–1600 µmol
l–1) relaxed rat aortic rings. They attributed the low-dose
H2S contraction to H2S combining with NO and thereby
removing the tonic NO-mediated vasodilation
(Ali et al., 2006), or directly
inhibiting endothelial nitric oxide synthase
(Kubo et al., 2007). This also
is unlikely to occur in either hagfish or lamprey dorsal aortas because, (1)
there is at present no evidence for endothelial NO production by either of
these vessels (Olson et al.,
2001), (2) exogenous NO produces a modest contraction in the
ventral aorta of the hagfish, Myxine glutinosa
(Evans and Harrie, 2001), and
(3) NO synthesis from L-arginine and O2 would be
expected to be reduced during prolonged hypoxia. Our studies suggest that
H2S may directly constrict specific vessels and that this response
is an intrinsic property of the smooth muscle cells. Clearly, however,
variations in this response can be achieved through H2S
interactions with other vasoregulatory mechanisms.
H2S metabolism in O2 sensing
Our model of the role of H2S metabolism in oxygen sensing and/or
signal transduction appears to accommodate both hypoxic vasoconstriction (HVC)
and hypoxic vasodilation (HVD) in vertebrate smooth muscle (Olson et al.,
2007). This model is based on the balance between H2S production by
vascular tissue and its inactivation through oxidation, and it provides a
simple and rapid mechanism that couples the concentration of a vasoactive
molecule directly to PO2. The model is
supported by observations that the responses of a wide variety of vessels
(either constriction, dilation or multi-phasic) to hypoxia and H2S
are identical, H2S is constitutively produced by blood vessels,
cysteine the metabolic precursor of H2S, augments HVC and
inhibitors of H2S production inhibit HVC and HVD. The present study
provides additional support for the involvement of H2S in HVC in
hagfish vessels.
Similarity of vascular responses to H2S and hypoxia
The responses of New Zealand hagfish vessels to H2S are in many
respects similar to those produced by hypoxia. H2S and hypoxia
(Olson et al., 2001), appear
to be exclusively vasoconstrictory in hagfish dorsal aortas and efferent
branchial arteries because they consistently contracted both un-stimulated and
pre-contracted vessels. Conversely, neither H2S nor hypoxia
produced a sustained response in ventral aortas or afferent branchial
arteries. H2S and hypoxic
(Olson et al., 2001)
contractions of aortas and efferent branchial arteries were also unaffected by
pre-contraction with KCl. Furthermore, because KCl pre-contraction presumably
depolarizes smooth muscle cells it is likely that the mechanism of
H2S excitation is independent of cell depolarization; evidence for
depolarization-independent HVC in these vessels has also been presented
previously (Olson et al.,
2001). This is in contrast to the H2S-mediated
relaxation of rat aorta (Zhao et al.,
2001; Zhao and Wang,
2002) and trout efferent branchial arteries
(Dombkowski et al., 2004) where
elevated KCl partially inhibits the response. Collectively, these findings
suggest that H2S contraction and HVC have a common, or at least
similar, excitation pathway in hagfish vessels. This is consistent with other
studies (Olson et al., 2001;
Dombkowski et al., 2004;
Dombkowski et al., 2005;
Olson et al., 2006;
Russell et al., 2007) that
have shown that the vascular response to hypoxia is identical to that of
H2S irrespective of whether this response is contraction,
relaxation, multi-phasic, or, as in the case of hagfish ventral aorta and
afferent branchial arteries, no response at all. We have also observed
identical hypoxic and H2S responses in trout urinary bladder
(Dombkowski et al., 2006) and
thus hypoxia and H2S appear to have a common, or at least similar
excitation pathway in vertebrate smooth muscle in general.
Metabolic coupling of HVC to H2S production
Cysteine, which is presumed to be the precursor of H2S
production in animals (Julian et al.,
2002) increases the magnitude of HVC at lower concentrations
(Fig. 4) suggesting that it
increases tissue production of H2S. This is consistent with a
cysteine-enhanced HVC in lamprey dorsal aortas and bovine pulmonary arteries
and enhanced HVD observed in rat thoracic aortas
(Olson et al., 2006). Further
elevation of the cysteine concentration (10 mmol l–1)
inexplicably reduced the HVC in hagfish dorsal aorta. This may be due to a
feedback-type inhibitory effect of cysteine on H2S production, as
we (R.D., S. Head, N. Whitfield and K.O., unpublished observation) have also
observed elevated cysteine (10 mmol l–1 or 100 mmol
l–1) inhibition of H2S production in homogenized
bovine heart or trout vessels, respectively, which is consistent with feedback
inhibition. Alternatively, cysteine at a concentration of 10 mmol
l–1 may be toxic to smooth muscle cells. However, the fact
that carbachol contractions on top of 10 mmol l–1 cysteine
were not significantly different from the reference contractions, suggests
that cytotoxicity of cysteine at 10 mmol l–1 is unlikely, and
the reason for this inhibition remains to be identified.
The effects of inhibitors of H2S production provide additional
evidence for H2S signaling in hypoxic responses. As shown in
Fig. 4, amino-oxyacetate (AOA),
an inhibitor of cystathionine β-synthase (CBS) completely inhibited HVC
in hagfish aortas, whereas the cystathionine -lyase (CSE) inhibitor,
propargyl glycine (PPG) was ineffective
(Fig. 4). This suggests that
HVC in the hagfish dorsal aorta is dependent upon H2S synthesis
via CBS. This is in contrast to mammalian systemic vessels where CSE,
but not CBS, catalyzes H2S production
(Hosoki et al., 1997;
Zhao et al., 2003). We
(Olson et al., 2006) have also
shown that inhibition of CSE, but not CBS, blocked HVD in rat aortas, whereas
inhibition of CBS, but not CSE, blocked HVC in bovine pulmonary arteries.
Interestingly, in trout, H2S produces a tri-phasic
relaxation-contraction-relaxation
(Dombkowski et al., 2004) and
these vessels appear to possess both CBS and CSE (G. Yang, R. Wang and K.O.,
unpublished observation). These studies not only support the hypothesis of
H2S as a vascular O2 sensor but they also provide
additional evidence that different enzymes for H2S production, CBS
and CSE, may mediate HVC and HVD, respectively in different vessels.
Contractions produced by carbachol while hagfish dorsal aortas were exposed
to AOA or PPG were not significantly different from the reference contraction
produced by carbachol in the absence of inhibitors. Thus the inhibitory effect
of AOA on HVC could not be due to general inhibition of the contractile
apparatus. Separate H2S and ligand-mediated responses have also
been observed in other vessels (Dombkowski
et al., 2004; Olson et al.,
2006), indicating that the pathway for H2S activation
is not shared with some of the more common ligand-mediated mechanisms.
It is not clear why hydroxylamine potentiated HVC in hagfish aortas,
although a non-specific effect seems likely. Hydroxylamine inhibits at least
100 enzymes that use pyridoxyl 5'-phosphate as a co-enzyme
(Kery et al., 1999;
Tang et al., 2005) including
CBS and CSE. Although we expected it to act in a manner similar to AOA and
inhibit vasoconstriction, it was the most potent constrictor tested, on a
molar basis. Interestingly, despite turning the vasa vasorum brown, probably
as a result of an action on heme groups
(Canty and Driedzic, 1987;
Nichols and Weber, 1989), and
contracting the vessels, the vessels remained viable and the response to the
final carbachol exposure was not diminished.
Effect of PO2 on O2 consumption
As shown in Fig. 5, hagfish
aortas display a remarkable ability to maintain O2 consumption
(O2) over a wide
range of ambient PO2 (15–115 mmHg).
It is not clear why the vessels lose their regulatory ability when
PO2 exceeds 115 mmHg, but this may be near
the maximum PO2 these vessels encounter in the
wild, i.e. in air-saturated seawater (inspired
PO2 of 156 mmHg), arterial
PO2 was 109 mmHg
(Forster et al., 1992). When
PO2 falls below 15 mmHg,
O2 also falls.
The PO2 over which
O2 decreases is
quite similar to the PO2 at which HVC increases
(dashed line in Fig. 5).
The PO2 at which
O2 begins to
decrease in hagfish dorsal aortas (15 mmHg) is somewhat less than the 20 mmHg
critical PO2
(PO2 at which
O2 was reduced
by 5%) in isolated rat aortas (Koenitzer
et al., 2007). However, the
O2 for rat
aortas [78 pmol mg–1 min–1
(Koenitzer et al., 2007)] is
32.5 times greater than the
O2 for hagfish
aortas (2.4 pmol mg–1 min–1). Even assuming
a Q10 of 2.4, the 25°C temperature difference between our study
and that of Koenitzer et al. (Koenitzer et
al., 2007) would only account for a sixfold difference in
O2. In fact,
these differences would probably be even greater if the O2
solubility coefficients were accounted for; mammalian (human) plasma at
37°C is 1.26 µmol l–1 mmHg–1 and
seawater (with the same osmolarity of hagfish plasma) at 12°C is 1.72
µmol l–1 mmHg–1
(Boutilier et al., 1984).
Koenitzer et al. (Koenitzer et al.,
2007) also showed that
O2 more than
doubled when rat aortas were contracted with phenylephrine. We did not find
any difference in
O2 between
un-contracted and contracted hagfish aortas, perhaps because
O2 was so low to
begin with, or, more likely, because once hagfish aortas are contracted they
are able to maintain tension with little additional energy expenditure. The
latter point may be related to the hypoxia tolerance of hagfish vessels where
hypoxic contractions can be sustained for 8 h of continuous aeration with 100%
N2 (Olson et al.,
2001). Many non-mammalian vertebrates, especially the more
`primitive' ones are considerably more hypoxia tolerant than mammals because
of their ability to downregulate cellular metabolism and balance ATP demand
with ATP supply (Boutilier,
2001). Hypoxia tolerance varies across hagfish species and
interestingly, E. cirrhatus does not voluntarily tolerate an ambient
PO2 of less than 45 mmHg (82 µmol
l–1) at 11°C (Forster, 1992). Clearly, the lack of an
increase in O2
was not due to the technique used as carbachol nearly doubled
O2 in
salmon.
Despite the elevated metabolic rate of rat aortas, the tension (in mg
tension mg–1 wet mass) produced by KCl contraction of rat
aortas, which varies from 240 (Olson et
al., 2001) to 720 (Resende et
al., 2004) is only 2.5–7.5 times greater than a KCl
contraction of hagfish dorsal aorta (94±12, N=4; data from
this study). Thus is appears in rat vessels that either more oxygen is
consumed for non-contractile-related activities, or that force development is
energetically less efficient.
Relationship between O2 consumption and H2S production
In many organisms, O2 consumption is affected by H2S.
At low [H2S], O2 often increases because of the use of
H2S in mitochondrial ATP synthesis or for H2S
detoxification; at elevated [H2S], O2 consumption often
decreases because of H2S inhibition of mitochondrial cytochrome
c oxidase (Grieshaber and
Völkel, 1998), or perhaps even a general metabolic depression
(Blackstone et al., 2005).
H2S also affects
O2 in
un-contracted hagfish aortas (Fig.
6) in a manner consistent with that described by Grieshaber and
Völkel (Grieshaber and Völkel,
1998). An increase in
O2 is also
predicted by our (Olson et al.,
2006) model of H2S oxidation by blood vessels as a
mechanism to inactivate H2S during normoxia. Higher (100 µmol
l–1 and 1 mmol l–1) [H2S]
inhibits O2
(Fig. 3) but not tension
development (Figs 2,
3). This likely reflects the
inherently low energy cost of force development, consistent with our
observation that
O2 does not
change even during maximal carbachol contraction and it also provides a
mechanism for sustaining HVC even when mitochondrial energy production is
compromised.
The increase in
O2 produced by
cysteine and the decrease in
O2 produced by
AOA (Fig. 6) are also
consistent with a positive and negative effect on H2S production by
hagfish dorsal aortas. It is not clear why 10 mmol l–1
cysteine appeared to decrease tension, yet increase
O2. This
suggests that H2S oxidation continues, although the mechanism that
causes contraction is subject to feedback inhibition. However, other
explanations are also plausible, i.e. the experimental conditions were
different (anoxia in myograph studies, PO2
40-50 mmHg in H2S studies), this cysteine concentration is
near the threshold for both processes, or there are temporal differences in
responses. The effects of hydroxylamine on
O2 do not
correlate with its effects on tension and may also be nonspecific as it
inhibits many other enzymes (Zollner,
1989).
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). (B) H2S
dose–response curves for lamprey dorsal aortas bubbled with room air
(filled circles; N=8) or during moderate hypoxia (open circles;
N=8). Hypoxic vessels were significantly (*) more
sensitive to H2S. Values are means ± s.e.m.
