Pharmacological Reports 66 (2014) 373–379
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Pharmacological Reports
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Original research article
Comparison of the effects of methanethiol and sodium sulphide on
uterine contractile activity
Ana Mijušković a,*, Zorana Oreščanin-Dušić a, Aleksandra Nikolić-Kokić a, Marija Slavić a,
Mihajlo B. Spasić a, Ivan Spasojević b, Duško Blagojević a
a
b
Department of Physiology, Institute for Biological Research ‘‘Siniša Stanković’’, University of Belgrade, Belgrade, Serbia
Life Science Department, Institute for Multidisciplinary Research, University of Belgrade, Belgrade, Serbia
A R T I C L E I N F O
Article history:
Received 12 March 2013
Received in revised form 4 November 2013
Accepted 19 December 2013
Available online 13 April 2014
Keywords:
Methanethiol
Hydrogen sulfide
Relaxation
SOD
Reactive oxygen species
A B S T R A C T
Background: Our aim was to investigate the effect of methanethiol (CH3SH) on contractility of rat uterus
and activities of redox-active enzymes, and to compare them with the effect of sodium sulphide (Na2S), a
hydrogen sulphide (H2S/HS ) donor.
Methods: Uteri were isolated from virgin Wistar rats, divided into six groups, controls (untreated uteri
allowed to contract spontaneously and in the presence of Ca2+(6 mM)), CH3SH treated (spontaneously
active and Ca2+ induced) and Na2S treated (spontaneously active and Ca2+ induced). Underlying
antioxidative enzyme activities (superoxide dismutase – SOD, glutathione peroxidase – GSHPx,
glutathione reductase – GR) in CH3SH- or Na2S-treated uteri were compared to controls.
Results: Our experiments showed that CH3SH and Na2S provoked reversible relaxation of both
spontaneous and Ca2+-induced uterine contractions. The dose–response curves differed in shape, and
CH3SH curve was shifted to higher concentration compared to H2S/HS . The effects of Na2S fitted
sigmoid curve, whereas those of CH3SH fitted linearly. CH3SH provoked increased SOD activity and
decreased GR activity. However, Na2S (H2S/HS ) provoked an increase in SOD activity exclusively in
Ca2+-stimulated uteri, while the activity of GSHPx was increased in both types of active uteri.
Conclusion: Our results imply that CH3SH may have a constructive role in the control of muscle function
and metabolism. Observed differences between CH3SH and H2S/HS could be attributed to a larger
moiety that is present in CH3SH compared to H2S, but they are more likely to be a consequence of the
specific actions of HS , in relation to its negative charge.
ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp.
z o.o. All rights reserved.
Introduction
Methanethiol (CH3SH) is produced in human (mammalian)
organism via several different mechanisms. A considerable day-today variation in CH3SH concentration was found in morning breath
of healthy examinees, while significantly higher concentration was
observed in the group of female subjects compared to males [1].
These results indicate some possible physiological function of
CH3SH. The metabolism of CH3SH is intertwined with hydrogen
Abbreviations: CH3SH, methanethiol; SOD, superoxide dismutase; GR, glutathione
reductase; GSHPx, glutathione peroxidase; H2O2, hydrogen peroxide; H2S/HS ,
hydrogen sulphide/hydrogen sulphide anion; Na2S, sodium sulphide; H2S,
HS donor; O2 , superoxide anion radical; ROS, reactive oxygen species.
* Corresponding author.
E-mail addresses: [email protected], [email protected]
(A. Mijušković).
sulfide (H2S), since the later can be methylated to CH3SH by thiolS-methyltransferase [2,3].
H2S represents an endogenous signalling molecule with various
roles. Sodium hydrosulphide (NaHS), a donor of H2S, is known to
relax guinea pig and rat ileum smooth muscles, as well as thoracic
aorta and portal vein [4,5]. Pertinent to our study, NaHS relaxes
isolated pregnant rat uterine strips in vitro [6], demonstrating the
role of H2S as an uterine relaxant. The production of H2S and the
presence of enzymes responsible for its endogenous production
(cystathionine beta-synthase and cystathionine gamma-lyase)
have been demonstrated in rat uterus [7]. H2S is a week acid
(7.0 and 12.9 are pKa values of the first and second steps of
dissociation, respectively). At the physiological pH of 7.4, the ratio
of HS :H2S is approximately 4:1 [8]. It appears unlikely that
molecular and anionic forms have pharmacologically identical
actions, including the regulation of contractility [9]. For example, it
seems plausible that, because of its negative charge, HS shows
http://dx.doi.org/10.1016/j.pharep.2013.12.012
1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
374
A. Mijušković et al. / Pharmacological Reports 66 (2014) 373–379
higher affinity towards metalloproteins (such as antioxidative
enzymes) compared to H2S. CH3SH shows a lower pKa (10.4)
compared to H2S, and under physiological conditions, CH3SH exists
exclusively in non-ionic form. It has a larger moiety compared to
H2S. In spite of this, CH3SH is clearly more similar to the molecular
form than to the ionic form – HS . Hence, CH3SH may represent a
useful tool for discrimination between different actions of H2S
and HS .
It is noteworthy that there may be an interplay between the
redox sensitivity of uteri [10,11,12], and the effects of thiols. H2S
can react with H2O2 or its precursor – superoxide [13], while on the
other hand, both H2S and CH3SH inhibit H2O2-removing enzyme
catalase [14]. Since the relaxatory effects of H2O2 on uterus have
been documented [10], it was important to examine the effects of
H2S and CH3SH on antioxidant enzymes together with their effect
on uterine contractility.
Whilst the pharmacology of H2S has been extensively studied
over the last decade, the effects of CH3SH are scarcely known. The
aim of our study was to examine the effects of CH3SH on uterine
smooth muscle contractions and on antioxidative system, which
could be involved in (redox) regulation of contractility. The effects
were compared to the actions of H2S/HS pool in order to try to
distinguish the effects of molecular form (H2S) from those of ionic
form (HS ).
Materials and methods
Experimental model
All protocols for handling rats were approved by the Local
Animal Care Committee of the Institute for Biological Research
(Belgrade, Serbia) in accordance with the recommendations
provided in the European Convention for the Protection of
Vertebrate Animals used for Experimental and Other Scientific
Purposes. Virgin female Wistar rats (225 g, 10–12 weeks of age)
were used in these experiments. The animals were kept under
standard laboratory conditions (12 h light, 12 h dark and
21 2 8C). All rats were housed in individual cages and given
standard diet and tap water ad libitum. Female rats were staged in
their estrus cycle, as determined by examination of a daily vaginal
lavage [15], and than sacrified by decapitation. Dissection and
experimentation were performed as described in [16]. In brief, the
uterine horns were rapidly excised and carefully cleaned of all fat and
surrounding connective tissue, rinsed in De Jalon’s solution and used
immediately. Uteri were divided into six groups (n = 6 per group):
Group 1: Control, untreated spontaneously active uteri; Group 2:
Control, untreated CaCl2 activated uteri; Group 3: CH3SH treated
spontaneously active uteri; Group 4: CH3SH treated CaCl2 activated
uteri; Group 5: Na2S treated spontaneously active uteri; Group 6:
Na2S treated CaCl2 activated uteri.
a force transducer (Experimetria, Budapest, Hungary). The
chambers contained De Jalon’s solution (containing in g/L): NaCl
9.0, KCl 0.42, and NaHCO3 0.5, CaCl2 0.06 and glucose 0.5;
maintained at 37 8C and aerated with a gas mixture of 95% oxygen
and 5% carbon dioxide. Uteri were allowed to equilibrate during
one hour, at 1 g tension by imposing a resting tension, until a stable
resting tone was acquired. After equilibration with washes every
15 min, tissues were allowed to contract either spontaneously or
they were challenged with Ca2+ (6 mM), and those responses were
used to normalize the tissue response from experiment to
experiment. Changes in isometric force were recorded on a TSZ04-E Tissue Bath System (Experimetria, Budapest, Hungary).
Spontaneously active or Ca2+-induced tissue was exposed to a
cumulative increase in concentrations of CH3SH or Na2S. Concentration–response curves of 100–600 mM CH3SH and 20–200 mM
Na2S were obtained by adding agent directly to the organ bath. In
each experiment, appropriate controls were run under similar
experimental conditions using uterus horns obtained from the
same rat. After treatment, samples were immediately frozen in
liquid N2 and then stored at 80 8C until further analysis.
Tissue preparation for antioxidant enzyme activity assays
In order to prepare the samples for analytical procedures,
thawed uteri (treated with CH3SH, Na2S and untreated – controls)
were homogenized at 0–4 8C in five volumes of ice-cold 0.25 M
sucrose, 1 mM EDTA and 0.05 M Tris–HCl buffer, pH 7.4. All
procedures were performed on ice. The homogenates were
centrifuged for 60 min at 105,000 g, 4 8C and the resulting
supernatants were used for determining total protein concentration and enzyme activities (using a Shimadzu UV-160 spectrophotometer, Shimadzu Scientific Instruments, Kyoto, Japan).
Spectrophotometric measurements
Na2S (Merck, Germany) has been commonly used in numerous
studies as an H2S/HS donor. It was freshly prepared on the day of
every experiment. A recent study has reported that sulfide is
rapidly removed from the plasma in vivo but it remains in both
Krebs and HEPES buffer in vitro in a recirculated system [3]. High
purity CH3SH (TraceCERT) was purchased from Fluka (Buchs,
Switzerland). All other chemicals were obtained from Sigma–
Aldrich (St Louis, MO, USA) and dissolved in deionized water.
Superoxide dismutase (SOD; EC 1.15.1.1) activity was determined by the epinephrine method [17]. This method is based on
the capacity of SOD to inhibit autoxidation of adrenaline to
adrenochrome. Reaction mixture consisted of 3 10 4 M adrenaline, 1 10 4 M EDTA and 0.05 M Na2CO3, pH 10.2. One unit of
SOD was defined as the amount of protein causing 50% inhibition of
the autoxidation of adrenaline at 26 8C.
Glutathione peroxidase (GSHPx; EC 1.11.1.9) activity was
measured following the spectrophotometric method of [18] based
on the measurement of NADPH consumption (i.e., NADPH
oxidation by glutathione reductase, 500 U/mg protein, Sigma) at
340 nm. The reaction mixture consisted of 50 mM potassium
phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM GSH, 1 mM sodium
azide, 1 IU mL 1 glutathione reductase, 0.2 mM NADPH and 3 mM
t-butyl hydroperoxide.
Glutathione reductase (GR; EC 1.6.4.2) activity was assayed as
described by [19]. This assay is based on NADPH oxidation
concomitant with GSH reduction. Reaction mixture consisted of
0.5 M sodium phosphate buffer (pH 7.5), 0.1 mM EDTA, 0.1 mM
NADPH, and 0.1 mM GSSG. Enzyme activity is expressed in units
(GSHPx and GR) per mg of soluble protein. One unit of enzyme
activity was defined as the amount of enzyme required to
transform 1 mmol of substrate per min under the above described
assay conditions. Soluble protein concentration was determined
using the method of [20] with bovine serum albumin used as
standard.
Organ bath studies
Statistical analysis
Uteri were mounted separately in 10-mL organ chambers with
one end tied to a tissue holder and the other to a wire connected to
Statistical analyses were performed according to the protocols
described by [21]. The effects of treatments on uterine contractions
Chemicals
A. Mijušković et al. / Pharmacological Reports 66 (2014) 373–379
375
Fig. 1. Representative original trace of spontaneous active uterus (A) and Ca2+ activated uterus (B), at different concentrations of CH3SH (100–600 mM) or Na2S (20–200 mM).
were calculated as percentages of control contractions. Each data
value is expressed as mean SD. The effect of CH3SH and Na2S on
uterine contractility was tested by two-way ANOVA (factors: CH3SH
and Na2S concentration and type of activation) and regression
analysis (statistically significant if p < 0.05) on logarithmically
transformed data. Dose–response curves for Na2S treatment were
sigmoid in shape and fitted according to Boltzmann functions (the
concentration axis was linear), and the Na2S dose required for half-
Fig. 2. Dose–response sigmoid fit curve for CH3SH and Na2S-induced relaxation of
spontaneous and Ca2+ induced activity of the isolated rat uterus. Data are expressed
as mean values SD (n = 7). Differences were tested by two-way ANOVA (factors:
treatment (T) and dose (D)). The sigmoid fits of Na2S were performed according to the
Boltzmann equation and ED50 were expressed (fits were compared by F-test and
showed no differences, and neither did the ED50 compared by t-test). The effects of
CH3SH were expressed linear (significant fit, p < 0.001 and p < 0.01) and analyzed by
regression analysis; R values and SD were showed (lines were not different according
to the fit comparison by F-test).
maximal effect (ED50) was calculated. Sigmoid curves were compared
using F-test. ED50 values were compared using t-test (significance:
p < 0.05). The effect of CH3SH was linear and calculated by linear
regression statistics. According to dose–response curve, single doses
for testing effects of CH3SH and Na2S were chosen, 600 and 200 mM
respectively. Contractions were analyzed for amplitude, frequency,
and area under the curve, (AUC, in arbitrary units, au) per minute. The
effects were tested by two-way ANOVA (the type of effect
and recovery as factors) and post hoc compared by Tukey’s HSD.
The activities of antioxidant enzymes were compared using
Fig. 3. Representative original trace of spontaneous active uterus (A) and Ca2+
activated uterus (B), exposed to CH3SH (600 mM) or Na2S (200 mM).
376
A. Mijušković et al. / Pharmacological Reports 66 (2014) 373–379
one-way ANOVA followed by Tukey’s HSD post hoc test (significance:
p < 0.05).
Results
Effects on uteri
Both CH3SH and Na2S caused reversible dose–dependent
relaxation of both spontaneous and Ca2+-induced contractions in
isolated rat uteri (Fig. 1), the effects being statistically significant
(Fig. 2, two-way ANOVA, p < 0.001). CH3SH (100–600 mM)
concentration–dependent response showed a significant 3-fold
rightward shift compared to Na2S (20–200 mM) dose–dependent
curve (Fig. 2). CH3SH fitted linearly. These differences imply that
the investigated agents might have different mechanisms of action.
Spontaneous and Ca2+-induced active uteri reacted similarly to
Na2S (there was no significant difference between ED50 values; the
shapes of sigmoidal curves were not different when calculated by
F-test). Linear fits of CH3SH effects were preserved in both
spontaneous and Ca+-induced active uteri (there were no
differences between R factors; fitted lines were not different
according to F-test). There was a marked reversal/recovery of the
inhibitory effect of both CH3SH and Na2S (Fig. 3). Amplitude,
frequency of contractions, and AUC were differently affected
(Fig. 4).
Antioxidant enzyme activities
Higher SOD (p < 0.01) and lower GR activity (p < 0.05) were
found in spontaneously active rat uteri after treatment with
CH3SH, compared to control uteri incubated for an equivalent time
(Fig. 5A and C). Treatment with CH3SH had no effect on GSHPx
Fig. 4. Frequency, amplitude and AUC of contractions of spontaneous active uterus (A) and Ca2+ activated uterus (B) in response to CH3SH (600 mM) or Na2S (200 mM).
A. Mijušković et al. / Pharmacological Reports 66 (2014) 373–379
Fig. 5. Changes in antioxidative enzyme activity in spontaneously active uterus:
untreated, (n = 6), CH3SH (n = 6) and Na2S treated (n = 6). (A) SOD specific activity
(F = 9.7; p < 0.01); (B) GSHPx specific activity (F = 10.4; p < 0.01); (C) GR specific
activity (F = 5.06; p < 0.05). Data are expressed as mean SEM. Differences were
tested by one-way ANOVA and post hoc compared by Tukey’s HSD test (different letters
above error bars show significant differences calculated by post hoc tests).
activity in spontaneously contracting rat uteri (Fig. 5B). There were
no significant changes in SOD activity in spontaneously active uteri
after Na2S treatment (Fig. 5A). On the other hand, Na2S treatment
led to an increase in GSHPx activity (p < 0.01) (Fig. 5B), while GR
activity was similar to controls (Fig. 5C).
The treatment of Ca2+-induced active uteri with CH3SH,
resulted in higher SOD and lower GR activity (p < 0.001) compared
to controls (Fig. 6A and C). There were no changes in GSHPx activity
in uteri treated with CH3SH (Fig. 6B). It is noteworthy that a
significant increase in SOD and GSHPx activity (p < 0.001) was
found in Na2S treated calcium induced uteri (Fig. 6A and B). There
were no changes in GR activity in uteri treated with CH3SH
(Fig. 6C).
Discussion
This is the first study to examine the pharmacological effects of
CH3SH and to compare the actions of CH3SH and H2S/HS .
Hydrogen sulfide has been shown to play a key role in the control of
smooth muscle tension. Robinson and co-workers have shown that
GYY4137 (H2S-donor) relaxes tonic contractions of rat uterus [22].
The role of endogenous H2S in the control of uterine contractility
has recently been shown [23]. However, the data about the effect of
CH3SH are lacking. Our results showed that both CH3SH and H2S/
HS caused a reversible concentration-dependent relaxation of the
377
Fig. 6. Changes in antioxidative enzyme activity in Ca2+ activated rat uteri:
untreated, (n = 6), CH3SH (n = 6) and Na2S treated (n = 6). (A) SOD specific activity
(F = 21.5; p < 0.001); (B) GSHPx specific activity (F = 17.5; p < 0.001); (C) GR
specific activity (F = 13.4; p < 0.001). Data are expressed as mean SEM. Differences
were tested by one-way ANOVA and post hoc compared by Tukey’s HSD test (different
letters above error bars show significant differences calculated by post hoc tests).
uteri, independent of the type of activation (spontaneous or Ca2+induced). The dose–response curves differed in shape and CH3SH
curve is shifted to higher concentration values compared to H2S/
HS . This implies that H2S/HS pool represents a more efficient
relaxant than CH3SH.
Both compounds affected the amplitude, as well as the
frequency of contractions. Decrease in contraction frequency
might be related to effects on pacemaker cells. However, concept of
pacemaker mechanism that initiates uterine action potentials is
still unknown [24,25]. The sarco-endoplasmic reticulum (SER) has
been shown to be involved in the pacemaking of the smooth
muscles [26,27]. SER constitutes the principal Ca2+ store that
participates in the initial rapid increase in [Ca2+]i via two different
types of Ca2+ release channels, i.e., the inositol 1,4,5-trisphosphate
receptors (IP3Rs) and the ryanodine receptors (RyRs). SER also
participates in the subsequent decrease in [Ca2+]i by removing Ca2+
from the cytoplasm and refilling the internal Ca2+ stores by the
action of SER Ca2+ pumps (SERCAs). Spontaneous contractions
might be initiated by spontaneous pacemaker activity, although
pacemaker cells are not fully defined [28,8]. H2S has been shown to
decrease intracellular Ca2+ transients that underlie spontaneous
contractions [22]. Our results imply that CH3SH and H2S/HS could
alter pacemaker activity, with different potencies, which might
be attributed to their different abilities to modulate calcium
signalling.
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It is well established that calcium signalling might be
modulated by redox-dependent mechanisms. Reagents that
oxidize thiols activate cardiac RyR channels [29], but inhibit
SERCA [30]. Bearing in mind the reductive and nucleophilic
properties of H2S/HS , reverse effects might be possible, resulting
in inhibition of RyR and activation of SERCA. This could further lead
to decreased intracellular Ca2+ and relaxation. However, this needs
further clarification.
The autoxidation of sulfhydryl compounds with accompanying
reduction of oxygen to give O2 is a well recognized source of
superoxide anion [31], which was shown to induce relaxation
[32]. Their autoxidation is negligible at low pH levels (such as in
our stock solution), but it is greatly accelerated at higher pH, as at
7.4 in our De Jalon’s solution. pH dependence is consistent with a
previously proposed mechanism for thiol oxidation by [33] in
which the reactive thiolate anion HS acts as a nucleophile
towards the oxidant. It has been shown that the addition of HS
enhances superoxide scavenging activity of CuZnSOD by about
twofold [34]. In our experimental settings, the increase in SOD
activity following the incubation with Na2S was observed only in
Ca2+-stimulated muscles. This increment might be explained by
increased concentration of its substrate O2 due to the said
ability to generate it, but also by the ability of H2S to react with
different ROS, superoxide radical anion and hydrogen peroxide
[35,36,37] giving false higher activity. It is plausible that
metalloproteins, particularly those that contain heme, represent
specific targets of HS since heme proteins coordinating to sulfide
ligands in the iron (III) oxidation state could have either
specialized (low polarity) environments or allow only limited
access to the HS /S2 binding site [38]. Contrary to that direct
action, molecular form of H2S and CH3SH may induce relaxation
through indirect mechanisms. The ability of both CH3SH and H2S
to inhibit catalase [39] implies that the relaxation mechanisms
could involve the relaxatory effects of H2O2 [10]. The effects of
CH3SH on the set of redox-active enzymes in our study implicate
that the incubation with CH3SH could result in increased
intracellular levels of H2O2, because of the increased H2O2
production by SOD and suppressed removal by GSH. An important
aspect underlying signalling properties of H2O2 is its ability to
target proteins containing oxidation susceptible cysteine residues
critical for protein function. It has been demonstrated that H2O2
oxidizes a vascular thiol target activating Kv channels leading to
subsequent relaxation [40]. Mechanism of Kv channels-dependant H2O2-relaxatory effect on rat smooth muscle contractility
was recently shown [41]. On the other hand, H2S/HS seem to
promote GSH-mediated H2O2 removal by increasing GSHPx
activity. The critical finding that H2S pool was significantly more
efficient in provoking the relaxation of uteri compared to CH3SH
implies that the involvement of H2O2 in the studied effects could
be applicable to CH3SH but minor or absent for H2S.
Taking into consideration the absolute rate constant for the
hydroxyl radical reactions with CH3SH [42], together with the
observed effects on antioxidative enzyme activities, it is
implicated that CH3SH in certain concentrations may have some
physiological role in rat uterus. This study provides novel data
about the metabolic actions of CH3SH, as well as on the
mechanisms of H2S/HS activity. Similar structural properties
of CH3SH and H2S are useful for telling apart the effects of H2S and
HS . Understanding the biological effects of these active thiols
requires an understanding of their distinct chemistry, which
remains to be clarified.
Conflict of interest
There are no conflicts of interests associated with this
publication.
Funding
This work was supported by a grant from the Ministry of
Science and Technological Development of the Republic of Serbia,
project No.: 173014 ‘‘Molecular mechanisms of redox signalling in
homeostasis: adaptation and pathology’’, University of Belgrade.
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