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Hydrogen sulfide (H2S) is a toxic gas that is found in contaminated environments. Many studies have shown that
H2S affected the nervous system and vascular and intestinal smooth muscle of
mammals[1_6]. Although toxic at high doses, there
was increasing evidence to suggest that
H2S has important physiological functions in the mammalian cardiovascular
system[4]. The production of
H2S from L-cysteine can be catalyzed by two pyridoxal-5กฏ-phosphate-dependent enzymes,
cystathionine b-synthase (CBS) and cystathionine
a-lyase (CSE), in mammalian tissue[7_9]. The expression of these two enzymes is
tissue-type specific[4]. H2S is directly produced in arterial and venous tissue by
CSE[10,11]. DL-propargylglycine (PPG) is a
specific inhibitor of CSE, which can suppress endogenous
H2S production[12].
H2S has been proposed to be an endogenous gaseous transmitter similar to 2 other vasoactive gases, NO (nitric oxide)
and CO (carbon monoxide). The vascular effect of
H2S is exerted by direct activation of
KATP channels and hyperpolarization
of the membrane potential of vascular smooth muscle cells
(VSMCs)[10]. It has been found that the vascular effect of
H2S was partially mediated by a functional endothelium, and its action was dependent on extracellular calcium entry, but is
independent of activation of the cGMP pathway, unlike NO and
CO[13]. Recently, H2S has been found to play a negative inotropic role
in the heart[14].
The baroreflex is the major method of blood pressure modulation. Whether
H2S affects the carotid sinus baroreflex (CSB)
remains to be clarified. The aim of our study was to observe the action of exogenous
H2S, derived from sodium hydrosulfide (NaHS), and endogenous
H2S on the isolated CSB and to elucidate the mechanism involved.
Materials and methods Drugs NaHS, Bay K8644 and PPG were purchased from Sigma. NaHS was used as a donor of
H2S. Glibenclamide (a KATP channel blocker) was purchased from the Tianjin Institute of Medical and Pharmaceutical Industry. Glibenclamide was
initially dissolved in dimethylsulfoxide (100 µmol/L). The final concentration of dimethylsulfoxide in the K-H solution was
0.01% (v/v). No change was observed in the CSB during perfusion with the final concentration of dimethylsul-foxide. Bay
K8644 (an L-type calcium channel agonist) was dissolved in 99% ethyl alcohol. No changes in the CSB were observed during
perfusion with ethyl alcohol (1:2000). PPG (an inhibitor of CSE) was dissolved in distilled water.
General surgical procedure Sprague-Dawley rats (male, 320±20 g), which were obtained from the Experimental Animal
Center of Hebei Province, were anesthetized with 25% urethane (1.0 g/kg, ip). In each rat, the trachea was cannulated for
ventilation, and the right femoral artery was cannulated for recording blood pressure (BP) with a transducer (MPU-0.5A;
Nihon Kohden). Body temperature was maintained at 37_38 °C throughout the experiment.
Perfusion of left isolated carotid sinus
The perfusion of isolated carotid sinus area was carried out using a previously
reported method with modifications[15]. Carotid sinus areas were fully exposed by turning the trachea and esophagus in the
rostral direction. The sternohyoideus muscles and superior laryngeal nerves were cut, then the bilateral aortic nerves, right
carotid sinus nerve, cervical sympathetic nerves and recurrent laryngeal nerves were all sectioned. The common, external
and internal carotid arteries and smaller arteries originating from these vessels were exposed and ligated, while carefully
leaving the left carotid sinus nerve undisturbed. Ligation of the occipital artery at its origin from the external carotid artery
excluded chemoreceptors from the isolated carotid sinus, thereby preventing chemoreceptor activation secondary to
decrease in carotid sinus pres-sure. Plastic catheters were inserted anterograde into the left common carotid artery (inlet tube)
and retrograde into the external carotid artery (outlet tube). The carotid sinus was then perfused with warm (37 °C)
oxygenated modified Krebs-Henseleit (K-H) solution (in mmol/L; NaCl 118.0, KCl 4.7,
CaCl2 2.5, MgSO4 1.6,
KH2PO4 1.2,
NaHCO3 25, glucose 5.6, pH 7.35_7.45) bubbled with 95%
O2 and 5% CO2. The intrasinus pressure (ISP) was monitored by using a
pressure transducer (MPU-0.5A; Nihon Kohden) connected to the inlet tube. The ISP was controlled by using a peristaltic
pump.
After perfusion of the left carotid sinus, the ISP was kept at 100 mmHg for 20 min and was then lowered to 0 mmHg rapidly.
From this point, the ISP was elevated to 250 mmHg via a pulsatile ramp by regulating the speed of the peristaltic pump, which
was automatically controlled by a program designed by our
laboratory[16]. It took 0.5 min for the ISP to be increased from 0 to
250 mmHg. The ISP and BP were simultaneously recorded on a polygraph (RM-6240; Chengdu Instrument Factory). This
process was repeated at an interval of 5 min to check the stability of the baroreflex. The reproducibility of the experimental
set-up was confirmed by the recurrent drop of BP in response to the increase in ISP.
Experimental protocols By perfusing the left carotid sinus with K-H solution and elevating the ISP, a functional curve for
the ISP-BP relationship was constructed, and the functional parameters of baroreflex, such as threshold pressure (TP),
saturation pressure (SP), equilibrium pressure (EP), peak slope (PS), reflex decrease of BP (RD), and operating range (OR)
were determined. TP was the ISP at which BP began to decrease in response to the increase of the ISP. SP was the ISP at
which BP just showed no further reflex decreases with an increase in the ISP. OR was calculated as SP minus TP.
Before administration of the drugs, the K-H solution was used as a control. Four experimental treatments were used. (1)
To test the effect of NaHS on carotid baroreflex
(n=18), the ISP was fixed at 100 mmHg for 20 min with K-H solution as a
control, and the baroreflex parameters were measured. Then K-H solution containing NaHS (25, 50, or 100 µmol/L) was used
to perfuse the isolated carotid sinus for 50 min, then the parameters were measured again. Finally, the carotid sinus was
perfused with K-H solution to wash out the NaHS. (2) To test the effect of glibenclamide (Gli; 20 µmol/L) on the actions of
NaHS (n=6), baroreflex parameters were examined following the application of NaHS before and after pretreatment with Gli for
20 min. (3) To test the effect of Bay K8644 (500 nmol/L) on the actions of NaHS
(n=6), baroreflex parameters were examined
following the application of NaHS before and after pretreatment with Bay K8644 for 15 min. (4) To test the effect of PPG (200
µmol/L) on the carotid baroreflex (n=6), after the baroreflex parameters of the control were recorded, PPG was added into a
K-H solution and used to perfuse the isolated carotid sinus, then washed out. Finally, the carotid sinus was perfused with
K-H solution containing NaHS (50 µmol/L) to check the carotid baroreflex activity.
Data analysis All data are expressed as mean±SD. The differences between groups of means were assessed by one-way
ANOVA and further analyzed using the Student-Newman-Keuls test.
P<0.05 was considered statistically significant.
Results Effects of NaHS on carotid sinus baroreflex
By perfusing the left carotid sinus with K-H solution and elevating the ISP
from 0 to 250 mmHg, BP was reflex decreased. NaHS induced obvious changes in baroreflex parameters, which appeared
approximately 30 min after perfusing the isolated carotid sinus with K-H solution containing NaHS, and disappeared 30_60
min after washout. Compared with the control group, NaHS increased RD and PS in a concentration-dependent manner, and
decreased TP and EP, shifting the functional curve of the baroreflex downward and to the left (Table 1, Figure
1). The functional curve was fit using Origin 6.0 procedures. The effects described indicate that exogenous NaHS exerts a
facilitatoryeffect on the carotid baroreflex (Figure 2).
Effects of Gli and Bay K8644 on the actions of NaHS
Neither Gli (20 µmol/L) nor Bay K8644 (500 nmol/L) induced any
changes in the functional parameters of the baroreflex, but both completely blocked the effects of NaHS (Table 2).
Effect of PPG on carotid sinus baroreflex
PPG inhibited the CSB in male rats and shifted the functional curve of the
baroreflex upward and to the right (Table 3).
Discussion
The present study showed that H2S could facilitate the CSB in a dose-dependent manner. By perfusing the left isolated
carotid sinuses of rats with exogenous
H2S derived from NaHS, the functional curve of the CSB was shifted downward and
to the left, with increases in PS and RD and decreases in TP, indicating the facilitatory action of
H2S on the CSB.
It has been shown that H2S has cardiovascular effects, and that
H2S can relax isolated aortic tissue
in vitro in a KATP channel-dependent manner. Furthermore, in isolated VSMCs,
H2S can directly increase
KATP channel currents and hyperpolarize
membrane[10]. So H2S is considered to be an important endogenous vasoactive factor and the first identified gaseous
opener of KATP channels in vascular smooth muscle cells. It is known that
KATP channels exist in vascular smooth
muscle[17]. A previous study from our laboratory reported that the opener of
KATP channels can facilitate the carotid
baroreflex[18]. On the basis of these data, we added gliben-clamide (20 µmol/L), a
KATP channel blocker, to the perfusate and found that it completely
eliminated the effect of H2S on the carotid baroreflex. Such results suggest that the opening of the
KATP channel is involved in the action of
H2S on the CSB.
Brayden recently reported that KATP channel opening could hyperpolarize smooth muscle, which led to closure of the
voltage-dependent Ca2+ channels, thereby causing a reduction in intracellular
Ca2+ and vasodilation[19]. It is also known that
distention of the carotid sinus can activate mechanosensitive ion channels, which would enhance the activity of the
baroreceptors[20]. In the present study, pretreatment with L-type calcium channel agonist Bay K8644 completely abolished the
facilitating effect of H2S on CSB. Based on the above observations, it can be concluded that
H2S facilitates CSB through opening
KATP channels and further closing calcium channels in VSMCs.
The results so far discussed only relate to the actions of exogenous
H2S. In order to determine the function of
endogenous H2S, PPG (an inhibitor of CSE) was used in our experiment. Zhao reported that PPG might be a membrane-permeable
drug, and that it had the potential to be used to study the physiological function of endogenously produced
H2S[12]. In the present study, PPG shifted the functional curve of CSB upward and to the right, with decreases in PS and RD, and increases
in TP. These results indicate that endogenous
H2S may have an activating role with respect to CSB activity
in vivo.
It is known that arterial baroreceptors play an important role in the short-term modulation of CSB activity. Facilitating the
CSB would tend to decrease arterial blood pressure, which could antagonize the hypertension caused by other stimulators.
Some diseases, such as hypoxic pulmonary
hypertension[21], result from a deficiency in endogenous
H2S production, so the modulation activity of the CSB is suppressed and the arterial blood pressure will increase. Deficits in the
H2S/CSE system are responsible for the development of spontaneous hypertension accompanying aorta
remodeling[22,23]. Exogenous supply of
an H2S donor may exert a protective effect in the pathogenesis of hypertension.
In summary, H2S facilitated the carotid baroreflex, which may result from the opening of
KATP channels and further closing of the calcium channels. It is possible that
H2S activates the carotid baroreflex in physiological conditions.
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