Hydrogen sulfide downregulates the aortic L-arginine/nitric oxide pathway in rats

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Hydrogen Sulfide Downregulates the Aortic L-Arginine/Nitric

Oxide Pathway in Rats

Bin Geng1,2*, Yuying Cui1, Jing Zhao1,2, Fang Yu2, Yi Zhu2, Geyang Xu2,

Zhiwen Zhang2, Chaoshu Tang1,2 , Junbao Du3

1. Institute of Cardiovascular Research, First Hospital of Peking University.

Beijing P.R. China

2. Department of Physiology, Peking University Health Science Center.

Beijing P.R. China

3. Department of Pediatric, First Hospital of Peking University. Beijing P.R.

China

*Corresponding Author: Bin Geng.

Institute of Cardiovascular Research, First Hospital of Peking University.

Beijing xishuku street 8, 100034. E-mail: [email protected],

Telephone: +86-010-82802183, Fax: +86-010-82802183.

of 34Articles in PresS. Am J Physiol Regul Integr Comp Physiol (July 18, 2007). doi:10.1152/ajpregu.00207.2006

Copyright © 2007 by the American Physiological Society.

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ABSTRACT

The aim of the present study was investigation the effect of H2S signaling by nitric

oxide (NO) in isolated rat aortas and cultured human umbilical vein endothelial cells

(HUVECs). Both administration of H2S and NaHS as well as endogenous H2S reduced

NO formation, eNOS activity, eNOS transcript abundance and L-Arg transport (all

P<0.01). The kinetics analysis of eNOS activity and L-Arg transport showed that H2S

reduced Vmax values (all P<0.01) without modifying Km parameters. Use of selective

NOS inhibitors verified that eNOS (versus iNOS and nNOS) was the specific target of

H2S regulation. H2S treatment (100 µmol/L) reduced Akt phosphorylation, and decreased

eNOS phosphorylation at Ser1177. H2S reduced L-Arg uptake by inhibition of a system

y+ transporter, and decreased the CAT-1 transcript. H2S treatment reduced protein

expression of eNOS, but not of nNOS and iNOS. Pinacidil (K ATP channel opener)

exhibited the similar inhibitory effects on the L-Arg/NOS/NO pathway. Glibenclamide

(KATP channel inhibitor) partly blocked the inhibitory effect of H2S and pinacidil. An in

vivo experiment revealed that H2S down-regulated the vascular L-Arg/eNOS/NO

pathway after intraperitoneal injection of NaHS (14 µmol/kg) in rats. Taken together, our

findings suggest that H2S downregulates the vascular L-Arg/NOS/NO pathway in vitro

and in vivo, and the KATP channel could be involved in the regulatory mechanism of H2S.

Keywords: Hydrogen sulfide, Nitric oxide, Nitric oxide synthase, L-Arg transport

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Hydrogen sulfide (H2S) is a well-known pungent gas, and its toxicology has been

extensively studied (40). Recently, H2S has been found to be endogenously generated in

various mammalian tissues by 2 pyridoxal-5’-phosphate-dependent enzymes such as

cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) (6), with L-cysteine

used as a main substrate. The gas may be a functional regulator in the nervous and

cardiovascular systems (21). In the cardiovascular system, CSE may be a key enzyme by

which H2S is generated (50). H2S causes a dose-dependent relaxation of the isolated rat

aorta (16, 50) and mesenteric arterioles (4); induces transient hypotension in anesthetized

rats in vivo (50), reduces proliferation of vascular smooth muscle cells (VSMCs) (8, 46),

and exerted a negative inotropic action of the heart in vivo and in vitro (13). Significantly,

there is evidence linking the endogenous CSE/H2S pathway to the pathogenesis of arterial

hypertension (45, 52), pulmonary hypertension induced by hypoxia (5), septic and

endotoxin shock (18), haemorrhagic shock (27), ischemic cardiac disease (2, 11),

ischemic brain damage (33) and hyperdynamic circulation in cirrhosis (10). Growing

evidence suggests that in addition to NO and CO (12), endogenous H2S may be a novel

cardiovascular gasotransmitter. As an endogenous ligand, H2S directly opens the KATP

channel in VSMCs and relaxes vascular smooth muscles in an endothelium-independent

manner (50), which is different from the effect of NO and CO.

Interestingly, the administration of sodium nitroprusside (SNP), an NO donor,

potentiates the vasorelaxation effects induced by H2S (16), and upregulats of H2S

production in rat vascular tissues in a concentration dependent manner (48). In contrast,

pretreatment with H2S attenuates the vasorelaxant effect induced by SNP in aortic rings

(49). In rats with septic and endotoxin shock, the plasma level of H2S is positively

correlated with that of NO (18) . In the brain, H2S acts as an endogenous peroxynitrite

scavenger (42). All these results suggest that NO and H2S signaling are irterrelated. Of

note, the expression of the H2S-generating enzyme CSE had been observed in VSMCs

(50), but not in the vascular endothelium, suggesting that H2S is generated in vascular

tissues mainly from VSMCs. Whether and how endogenous H2S from VSMCs affects the

endothelial NO production pathway is unknown. In the present study, we examined the

effects of H2S on NO production, endothelial NO synthase (eNOS) activity and

expression, and L-Arginine (L-Arg, substrate of NOS) transport in isolated rat aortas and

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cultured human umbilical vein endothelial cells (HUVECs) to explore the regulatory role

of H2S in the vascular L-Arg/NOS/NO pathway.

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MATERIALS AND METHODS

Animals

All animal care and experimental protocols complied with the Animal Management

Rules of the Ministry of Health of the People’s Republic of China (documentation

Number 55, 2001, Ministry of Health of P.R. China) and the guide for the Care and Use

of the Laboratory Animals of the First Hospital, Peking University. Male

Sprague-Dawley (SD) rats (250-300 g) were provided by the Animal Department, Health

Science Center of Peking University. All animals were maintained on normal rat chow

with unrestricted water and on a 12-h light/12 h dark cycle.

Materials

HUVECs (926 cell line) were purchased from the American Type Culture

Collection (ATCC, Manassas, USA). Tris-base, Dowex AG 50 w-x8 (Na+), NADPH,

tetrahydrobiopterin (BH4), calmodulin (CaM), flavin mononucleotide (FMN), flavin

adenine dinucleotide (FAD), L-Arg , L-canavanine, spermidine, pinacidil, glibenclamide,

N-ethylmaleimide (NEM), DL-propargylglycine (PAG), L-cysteine,

pyridoxal-5’-phosphate and sodium hydrosulfide (NaHS) were purchased from Sigma Co.

(St. Louis, MO, USA). L-[3H]-Arg (1.5 TBq/mmol) was from NEN Life Sci Products

(Boston, MA, USA); and eNOS antibody (SC-654), P-eNOS(Ser 1177)-R antibody

(SC-12972), iNOS antibody (SC-7271), nNOS antibody(SC-648),

phospho-Akt1/2/3(Ser473) antibody(Sc-7985R), Akt antibody(SC-8312) and β-tubulin

antibody (SC-5274) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,

USA).

H2S-saturated solution (0.09 mol/L at room temperature) was made by bubbling

with pure H2S gas (offered by Beijing XianHeYu CO. LTD, China) and stored at -70°C.

The specific primers used for sample loading calibration were synthesized by SBS

Co (Beijing, China) and were as follows: eNOS-S, 5'GTTTGTCTGCGGCGATGT-3';

eNOS-A, 5'AAGAAACAGGAAGCGGGTG-3'; cationic amino acid transporter-1

(CAT-1)- S, 5'-TGAAGACAGGGAGTGAGGG-3'; CAT-1-A ,

5'-TAGACAAACGGTAGCAGT-3'; GAPDH- S,

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5'GGATTTGGTCGTATTGGG3';GAPDH-A, 5'GGAAGATGGTGATGGGATT3'.

Other chemicals and reagents were analytical grade.

Organ culture of aortas in vitro

At the selected time, recipient rats were anesthetized with pentobarbital sodium (30

mg/kg, given intraperitoneally) and then decapitated quickly. The entire

thoraco-abdominal aorta was quickly removed and placed in cold, sterile

phosphate-buffered saline (PBS) (4°C). The organ culture of aortic tissues was carried

out as previously described (20), with some modification. Briefly, aortas were cleaned of

adherent adipose tissues and collateral vessels under sterile conditions. The aortic tissues

were placed in ice-cold PBS, cut into pieces of approximately 1 × 1 mm2 and transferred

to 24-well plates containing 1 mL of culture medium (RPMI 1640 with 0.5% [w/v]

bovine serum albumin, BSA) per well covering the tissue slices completely. Tissues w ere

cultured in a shaking bath (60 beats/min) at 37°C, in an atmosphere of 95% O2 and 5%

CO2. NaHS, the donor of H2S, or H2S gas buffer were added to the wells at a final

concentration of 50 µmol/L, then incubated for 2, 4 and 6 h respectively. For other groups,

1, 10, 100 and 1000 µmol/L NaHS or H2S gas buffer with or without other drugs

(L-cysteine 10 mmol/L; pyridoxal-5’-phosphate 2 mmol/L; DL-propargylglycine 2

mmol/L; pinacidil, 10 µmol/L, and glibenclimide, 10 µmol/L) were added to the wells,

which were incubated for 2 h. At the end of incubation, the NO production (content of

nitrate and nitrite in medium), tissue NOS activity and [3H]-L-Arg transport were

measured. The activity of lactate dehydrogenase (LDH) in the culture medium was

measured using a LDH-cytotoxic test kit (44) to determine the tissue viability.

H2S assay by sulfide-sensitive electrode

Measurement of H2S concentration in culture medium involved use of a

sulfide-sensitive electrode (Model 9616; Orion Research; Beverly, MA). In brief, sulfide

antioxidant buffer (SAOB) was added into standard sulfide solution or blood samples at a

ratio of 1:1 and then stirred thoroughly. Electrodes were rinsed in distilled water, blotted

dry, and placed into standards or samples. When a stable reading was displayed, the

millivolt value was recorded. The H2S concentration was calculated against the

calibration curve of the standard S2- solution (50).

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HUVEC culture

Confluent HUVECs (926 cell line) were put into medium 199 containing 5 mmol/L

D-glucose, 10% fetal calf serum, 5 mmol/L glutamine, 100 u/mL penicillin-streptomycin

and 0.03 mg/mL gentamycin at 37°C in a 5% CO2 atmosphere (43).Cell viability was

determined by trypan blue exclusion assay and by measurement of the activity of LDH in

the culture medium (44). Cell viability remained at more than 95% throughout the

experiments.

H2S (50 µmol/L) was added after changing to serum-free medium; cells were

incubated for 2 h at 37°C in a 95% air-5% CO2 atmosphere, and eNOS enzyme kinetics

and L-Arg transport dynamics were measured. For other groups, 100 µmol/L of H2S was

added and cells were incubated for 4 h before gene expression of cationic amino acid

transporter (CAT-1) and eNOS were analyzed by RT-PCR. The protein expression of

cellular phospho-Akt and total Akt were detected by Western blotting after incubation

with H2S (500 µmol/L), pinacidil (10 µmol/L) or glibenclamide (10 µmol/L) for 0.5 h;

phospho-eNOS and eNOS protein expression was measured after 2-h incubation. H2S

(100, 500 and 1000 µmol/L), and/or pinacidil (10 µmol/L) and glibenclamide (10 µmol/L)

were added after incubation for 6 h, eNOS, then iNOS and nNOS protein expression was

analyzed.

Assay of nitrate plus nitrite

L-Arg (200 µmol/L) was added to the organ and cell culture medium, which was

incubated for different times according to the experiment protocol; the media was then

collected. The assay was performed in a standard flat-bottomed 96-well polystyrene

microtitre plate, with PBS used as a control. An amount of 50 µL of nitrate reductase and

β-NADPH was added to each well, for final concentrations of 300 U/L and 25 µmol/L,

respectively. The plate was incubated at room temperature for 3 h. Excess β-NADPH was

consumed by the addition of 50 µL of PBS containing L-glutamic dehydrogenase,

α-ketoglutaric acid, and NH4Cl, (for final concentrations of 500 U/L, 4 mmol/L and 100

mmol/L, respectively) followed by 10-min incubation at 37oC. The nitrite concentration

was then measured by the addition of 50 µL Griess reagents, and the absorbance was read

at 540 nm by use of a plate reader after 10-min incubation at room temperature (14).

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Assay of NOS activity and eNOS enzyme kinetics

Aortic samples were fast frozen and ground into a fine powder in liquid nitrogen by

use of a mortar and pestle before sonicating in 5 volumes of homogenization buffer (50

mmol/L Tris-HCl, pH 7.2, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 0.1%

L-mercaptoethanol, 10 µg/mL leupeptin, 5 µg/mL pepstatin A, 1 mmol/L PMSF, 0.5

mmol/L dithiothreitol). NOS activity was measured by monitoring the conversion of

L-[3H]-Arg to L-[3H]-citrulline (23). For routine assay of total NOS (tNOS), 0.1 mg of

aortic tissue protein was incubated in a reaction buffer containing 1 mmol/L L-[3H]-Arg

(containing 1 mCi [3H]), 1 mmol/L NADPH, 300 IU/mL calmodulin, 5 µmol/L

tetrahydrobiopterin (BH4), 2 mmol/L CaCl2, 10 µmol/L flavine-adenine dinucleotide

(FAD) (31), and 10 µmol/L flavin mononucleotide (FMN), for 30 min at 37oC. For assay

of inducible NOS (iNOS) activity, the reaction buffer was Ca2+-free. The reaction was

stopped by adding 50 µL of ice cold Tris-HCl buffer (pH 5.5) containing 1 mmol/L

L-citrulline, 1 mmol/L EGTA and 1 mmol/L EDTA. The reaction substance was applied

to a 1 mL column of Dowex AG50WX-8 (Na+) to absorb L-[3H]-Arg. The NOS activity

was then quantified by determining the radioactivity on liquid scintillation spectroscopy

(Beckman, LS6500) and presented as nmol L-[3H]-citrulline/min/mg protein. eNOS

activity was calculated as the difference between total NOS activity and iNOS activity.

The measurement of eNOS enzyme kinetics in HUVECs was as previously

described (24), with minor modification. In brief, the cells were washed twice with

ice-cold buffer containing (in mmol/L) 100 NaCl, 25 NaH2PO4, and 80 Na2HPO4, pH 7.5.

The cells were pelleted and resuspended in ice-cold 50 mmol/L Tris-HCl buffer (pH 7.4)

containing l mmol/L EDTA, 5 mmol/L mercaptoethanol, 10 µg/mL pepstatin A, 10

µg/mL leupeptin, 90 µg/mL PMSF, and 1 µmol/L tetrahydrobiopterin. The cells were

disrupted by repeated freeze-thawing in liquid nitrogen. The cell lysate (50 µL) was

added to 50 µL of buffer as described above, except with different concentrations (0.1,

0.2, 0.4, 0.8, 1.6 and 3.2 mmol/L) of L-[3H]-Arg. The other reactions of eNOS activity

assay were further processed as described above.

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Assay of L-Arg uptake in aortic tissues and L-Arg transport kinetics in HUVECS

L-Arg uptake and transport kinetics analysis were performed by measured

intracellular incorporation L-[3H]-Arg. On completion of aortic incubation, the aortic

tissues were washed 3 times with ice-cold Krebs-Henseleit (KH) buffer (in mmol/L:

NaCl 131, KCl 5.6, NaHCO3 25, NaH2PO4 1, D-glucose 5, HEPES 20, CaCl2 2.5, MgCl2

1, pH 7.4). Aortic L-Arg uptake was measured by incubation with KH buffer containing

100 µmol/L L-Arg and 10 µCi/mL L-[3H]-Arg at 37oC for 15 min. Aortic tissues were

washed with ice-cold 10 mmol/L unlabeled L-Arg in PBS, then lysed with 200 µL

methanoic acid. Total radioactivity of L-[3H]-Arg was measured by liquid scintillation

counting (37).

Transport kinetics of L-Arg into endothelial cells was measured by the method of

Leoncini et al. (25), with minor modifications. Briefly, kinetic experiments were

performed in cells incubated for 15 min with KH buffer containing L-Arg (10 to 320

µmol/L) and 2 µCi/mL L-[3H]-Arg. Transport was terminated by removing the media and

washing the cells three times with ice-cold 10 mmol/L unlabeled L-Arg in PBS. Cells

were lysed with 0.5% Triton X-100 in 0.5 mol/L NaOH, and radioactive activity

[3H]-L-Arg taken by cells was assayed by liquid scintillation counting.

RT-PCR assay of gene expression of eNOS and CAT-1 in HUVECs

The mRNA levels of eNOS and CAT-1 were measured by RT-PCR, as described

previously by our lab (19). Total RNA from cells was extracted with Trizol reagent

(Applygen Technologies Inc. Beijing). RT-PCR was performed in a total volume of 25

µL. After denaturing at 95oC for 5 min, PCR was run at 94oC for 30 s, 61oC for 30 s, and

72oC for 40 s for 30 cycles. The PCR products were separated on a 1.5 % agarose gel and

stained with ethidium bromide. The optical density of the bands of eNOS cDNA (553 bp)

and CAT-1 cDNA (250 bp) was measured by use of the Gel Documentation System

(Bio-Rad, Hercules, CA). The PCR products were amplified again with the human

GAPDH primers at 94oC for 30 s, 55oC for 30 s, and 72oC for 30 s for 20 cycles, and the

optical density of the GAPDH band (205 bp) was measured. The ratio of eNOS or CAT-1

to GAPDH was considered as the relative amount of eNOS or CAT-1 gene expression.

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Western blot analysis

Protein isolation and Western blot analysis were carried out as previously described

(3). Briefly, a cellular lysate was prepared from the HUVECs and separated by

SDS-PAGE (on 7.5% gels). The proteins were transferred to nitrocellulose membranes

(Schleicher and Schuell; Dassel, Germany) by electroblotting (Bio-Rad). Proteins were

detected with polyclonal anti-eNOS, anti-P- eNOS, anti-iNOS, anti-nNOS, anti-Akt, and

anti-P- Akt antibodies and a monoclonal anti-tubulin antibody. After incubation with the

appropriate secondary antibodies conjugated to horseradish peroxidase, the membrane

was washed and color was developed by use of an enhanced chemiluminescence (7) kit

(Applygen Technologies Inc. Beijing).

The aortic alterations of L-Arg/NOS/NO pathway by injection H2S to in vivo rats

Rats were intraperitoneally injected with NaHS (3.5 µmol/kg, intraperitoneally each

30 min, for a total of 4 injections, total H2S 14 µmol/kg, n=6 rats) or normal saline (n=6

control rats). Two hours after the first injection, rats were anesthetized by intraperitoneal

injection with pentobarbital sodium (30 mg/kg). The blood was collected in heparinized

syringes from the abdominal aortas and transferred to tubes. The plasma was separated to

assay nitrate plus nitrite content. The aortic tissues were isolated, and its NOS activity

and L-Arg uptake were measured according to the above methods.

Statistical analysis

The results are expressed as means ± SD. Comparisons across more than 2 groups

were analyzed by one -way ANOVA followed by the Student-Newman-Keuls test. A

two-tailed P value<0.05 was considered statistically significant.

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RESULTS

H2S inhibited NO generation of aortic tissues

After L-Arg administration to cultured aortic tissues, the level of nitrate plus nitrite,

both stable products of NO, continuously accumulated in the medium up to 6 h (Fig. 1A).

After administration of H2S (50 µmol/L) and NaHS (50 µmol/L), the NO products in the

medium were decreased, by 63% and 47% (P<0.01) after 2 h, 48% and 30% (P<0.01)

after 4 h, and 32 % and 19% (P <0. 05) after 6 h, respectively, compared with that in

controls (Fig. 1A). According to the Fig. 1A results, we selected 2 h as a time of best

inhibitory effect, and observed the effects of various concentrations (from 1 to 1000

µmol/L) of H2S on NO release at this time. Administration of both H2S and NaHS

significantly reduced NO production in a concentration-dependent manner after a 2-h

incubation (Fig. 1B). The IC50 value was 19.5 µmol/L for H2S (95% confidence interval

[CI], 7.06-53.09 µmol/L) and 15.9 µmol/L for NaHS (95% CI, 7.99-31.59 µmol/L).

Although the IC50 of H2S was a little higher than that of NaHS, the inhibitory effect of

H2S was stronger than that of NaHS (Fig. 1C). The results showed that exogenous H2S

inhibits vascular NO production in vitro.

We felt it important to explore the effect of endogenously generated H2S.

Administration of L-cysteine (L-Cys, the substrate of CSE) and pyridoxal-5’-phosphate

(PLP, a co-factor of CSE) increased H2S release from incubated aortic tissues with H2S

concentration in the culture medium reaching 37.5±2.7 µmol/L (Fig.1D); baseline H2S

concentration in the culture medium was undetectable. Plasma H2S concentration was

48.96±10.59µmol/L according to the Zhao W reported (53). At the same time, L-Cys and

PLP treatment reduced NO production by 43% as compared with that in controls (P<0.01,

Fig. 1E). The effect of L-Cys and PLP treatment on NO production was blocked by

preincubation with an inhibitor of CSE, DL-propargylglycine (PAG) (Fig.1E), which

prevented endogenous H2S formation (the H2S concentration in culture medium could not

be detected by sensitive sulfur electrode, as shown in Fig.1D).

The KATP channel is an important molecular target of H2S in cardiovascular tissues

(4, 47, 50). To assess the role of KATP channel in H2S-induced decrease of NO production,

the KATP channel opener pinacidil and KATP channel inhibitor glibenclamide were used.

Pinacidil reduced NO production by 34% and H2S (100 µmol/L) level by 30% (P<0.01)

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(Fig. 1F). The activity of both agents was largely blocked by pretreatment with

glibenclamide (10 µmol/L). These results suggest that the KATP channel is involved in

inhibition of H2S-induced NO generation.

H2S downregulated aortic eNOS activity and changed its kinetics

eNOS is the principle form of NO synthase in normal arterial endothelium. On

incubation aortic tissue slices with H2S (50 µmol/L) and NaHS (50 µmol/L), reduced

eNOS activity by 56% and 47% (P<0.01) after 2 h, 39% and 27% (P<0.01) after 4 h, and

12% and 11% (P>0.05, Fig. 2A) after 6 h, respectively, compared with that in controls. In

addition, H2S and NaHS (from 1 to 1000 µmol/L) markedly decreased aortic eNOS

activity in a concentration-dependent manner after 2 h incubation (Fig. 2B). The IC50

values of H2S and NaHS for eNOS activity were 11.49 µmol/L (95% CI, 9.53-19.87

µmol/L) and 31.97 µmol/L (95% CI, 11.93-85.70 µmol/L), respectively. H2S was

stronger in inhibiting eNOS activity than NaHS (Fig. 2C). The results are similar to those

with NO generation, which suggests that the H2S reduced vascular NO generation at least

in part, by inhibiting eNOS activity.

The kinetics of eNOS regulation by H2S were studied in HUVEC cultures.

Incubation of HUVEC with NaHS (50 µmol/L, 2 h) inhibited the maximal activity of

eNOS (Vmax, 75.1±2.9 vs control 113.0±4.0 pmol/min/mg protein, P<0.01) without

modifying the Km value for L-Arg (33.6±4.4 vs control 39.3±3.0 µmol/L, P>0.05, Fig.

2D), thus significantly decreasing the enzyme activity efficienc y (ratio of Vmax to Km:

2.24±0.66 vs 2.88±1.33, P<0.01). We investigated the effect on aortic eNOS activity of

endogenously generated H2S by adding L-Cys and PLP to HUVECs. eNOS activity was

reduced by 48% (P<0.01; Fig. 2E), an effect that was attenuated by pretreatment with

PAG (a CSE inhibitor) (48). L-canavanine, an selective iNOS inhibitor (26) and

spermidine an nNOS inhibitor, alone or together, did not affect the inhibition effects of

H2S (100 µmol/L) on NOS activity (Fig. 2F), which indicates that H2S specifically

regulates eNOS activity. Like H2S, pinacidil inhibited eNOS activity, which was not

blocked by the iNOS or nNOS inhibitors used. However, glibenclamide blocked

inhibition of eNOS activity by H2S and pinacidil. Collectively, the results suggest that a

KATP channel is involved in the impact of H2S on eNOS activity.

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Both Ca2+-dependent and -independent pathways are involved in the regulation of

eNOS activity. There is evidence that H2S modulates Ca2+ influx in cardiovascular cells

(1) suggesting that H2S may inhibit eNOS activity through a Ca2+-dependent pathway.

Whether a Ca2+-independent pathway is involved in the inhibition of eNOS activity by

H2S is unknown. We detected phosphorylation of Ser1177, which activates eNOS by a

Ca2+-independent pathway (17), in eNOS protein. Incubation with H2S (500 µmol/L) for

2 h inhibited the phosphorylation of eNOS Ser1177 without changing eNOS expression

(Fig. 2G; P<0.01). Pinacidil also slightly inhibited eNOS phosphorylation. Pretreatment

with glibenclamide (10 µmol/L) partially blocked the effects of H2S and pinacidil on

eNOS phosphorylation (Fig. 2G). The results confirmed the role of a KATP channel in the

regulation of H2S in eNOS activity.

Akt (also named protein kinase B) mediates eNOS phosphorylation of Ser1177 in

vitro and in vivo, and plays a role in both calcium-dependent and -independent eNOS

activation (15). H2S treatment for 30 min significantly reduced Akt phosphorylation

without changing total Akt protein expression (Fig. 2H). Pinacidil also reduced Akt

phosphorylation, and glibenclamide blocked the effects of H2S and pinacidil. This

suggests that Akt signaling involves in the regulation of H2S to eNOS phosphorylation,

and which partially modulates by opening KATP channels.

H2S inhibited L-[3H]-Arg uptake by aortic tissues and changed L-[3H]-Arg

transport kinetics in HUVECs

To determine the impact of H2S on transcellular transport of L-Arg, we measured

L-[3H]-Arg uptake in aortic tissues. H2S (50 µmol/L) and NaHS (50 µmol/L) decreased

L-[3H]-Arg uptake by 58% and 50% (P<0.01) after 2 h, 39% and 30% (P<0.01) after 4 h,

and 12% and 8% (P>0.05) after 6 h, respectively (Fig. 3A). In addition, incubation with

H2S and NaHS for 2 h inhibited the L-[3H]-Arg uptake in a concentration-dependent

manner (1 to 1000 µmol/L; Fig. 3B). The IC50 values were 84.08 µmol/L (95% CI:

27.28-259.20 µmol/L) and 39.15 µmol/L (95% CI: 10.46-146.50 µmol/L) for H2S and

NaHS, respectively, the inhibitory effect of H2S being stronger than that of NaHS (Fig.

3C).

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To explore the regulation of L-Arg transport activity by H2S, we investigated the

kinetics of L-Arg transport in HUVECs. NaHS (50 µmol/L, 2 h) decreased the Vmax of

L-[3H]-Arg transport (17.5±1.2 vs control 20.6±1.1 pmol/105 cells/min, P<0.01) without

modifying the Km value (84.4±14.8 vs 73.0±10.4 µmol/L, P>0.05) and thus reduced

significantly the transport efficiency (Vmax / Km : 0.21±0.08 vs 0.28±0.11, P<0.01) (Fig.

3D).

In addition, administration of L-Cys and PLP inhibited L-Arg uptake, which was

blocked by PAG pretreatment, as shown in Fig. 3E. The results suggest that H2S, whether

exogenously administered or endogenously generated, inhibits L-Arg transport.

HUVECs transport arginine through two Na+-independent systems. System y+ is

sensitive to N-ethylmaleimide (NEM) and referable to the expression of CAT1 and

CAT2B. System y+L is referable to the expression of y+LAT2, y+LAT1, and 4F2hc (35).

To identify which type of transporter system acts as a main target of H2S, we investigated

L-Arg uptake with or without NEM (0.5 mmol/L, pretreatment 20 min). NEM reduced

L-Arg uptake by 32% compared with control (Fig.3F, P<0.01). NEM pretreatment plus

H2S (100 µmol/L) did not change the L-Arg influx compared with the effect of NEM

alone (Fig. 3F; P>0.05). The KATP channel opener pinacidil slightly reduced L-Arg

uptake (12% decrease compared to control) as did H2S, but NEM intensified the

inhibitory effects of pinacidil on L-Arg uptake compared with NEM treatment alone

(Fig.3F, P<0.01). Simultaneously, glibenclamide blocked in part the inhibitory effects of

both H2S and pinacidil.

H2S down-regulated CAT-1 and eNOS gene transcript level in HUVECs

To determine whether H2S modulates the transcript level of CAT-1, mRNA

expression of the L-Arg transporter and of eNOS was determined by RT-PCR in HUVEC

after incubation for 4 h with H2S (100 µmol/L). Transcript levels of CAT-1 and eNOS

were 40% and 43%, respectively (all P<0.01, Fig. 4) less than that in controls.

H2S inhibited eNOS but did not change iNOS and nNOS protein expression in

HUVECs

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To confirm whether H2S modulates eNOS, eNOS protein expression was assayed by

Western blots in HUVES after 6-h H2S incubation. H2S (100, 500 and 1000 µmol/L)

significantly inhibited eNOS protein expression, but not in a concentration-dependent

manner, and the inhibitory effect was partially blocked by glibenclamide preincubation

(10 µmol/L, Fig. 5A). Fig. 5B confirmed the above phenomena and showed

pinacidil-induced inhibition of eNOS protein expression and block of this by

glibenclamide.

Induction of iNOS protein expression by H2S was not observed (Fig. 5C). nNOS is

another constitutive NOS that is expressed in endothelial cells. Neither H2S nor pinacidil

altered nNOS expression (Fig. 5D).

NaHS inhibited L-Arg/NOS/NO pathway in rats in vivo

To demonstrate the in vivo effects of H2S on the vascular L-Arg/NOS/NO pathway,

we analyzed plasma levels of nitrite and nitrate after intraperitoneal administration of 14

µmol/kg NaHS in rats. At 2 h after NaHS injection, the plasma content of nitrate plus

nitrite was 21% (24.44±0.80 vs 31.00±2.66 µmol/L less than in controls, P<0.01, Fig.

6A). Aortic eNOS activity was reduced by 42% (23.06±5. 08 vs 39.50±8.67

pmol/min/mg protein, P<0.01, Fig. 6B), and L-Arg uptake decreased by 30%

(88.35±14.31 vs 125.40 ± 18.96 pmol/min/mg protein, P<0.01, Fig. 6C). iNOS activity

was not induced by H2S injection in the in vivo experiments (Fig. 6B).

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DISCUSSION

Recently, the novel endogenous gas H2S has been recognized as another

cardiovascular gasotransmitter that exerts important cardiovascular effects similar to

those of NO and CO. However, H2S, as opposed to NO and CO, is specifically released

from VSMCs but not endothelium, opens the KATP channel, induces cellular membrane

hyperpolarization and causes vessel dilation in an autocrine/paracrine manner (40, 41, 50),

which differs from the effect of NO and CO. Some studies have reported that NO

enhanced endogenous H2S production (48) and its vascular dilation effect (16), whereas

H2S inhibited vascular relaxation induced by NO (49). In the present study, we found that

endogenous H2S down-regulated the vascular L-Arg/NOS/NO pathway. This was

mediated in part through the KATP channel. Taken together these results suggest a delicate

crosstalk between endothelial and VSMCs by a complex interaction of the

gasotransmitters NO and H2S (Fig.7).

The physiological serum concentration of H2S in Sprague Dawley (SD) rats has

been reported to be 45.6+/-14.2 µmol/L (50). In the present study, an exogenously

administered physiological concentration of H2S or its donor, NaHS, inhibited NO

release from cultured aortic tissues. The inhibitory effect of H2S weakened with

incubation time but remained detectable for 6 h or more, consistent with anticipated

changes of H2S concentration in the medium (as a gasotransmitter, H2S could be

metabolized in the cell quickly (44). According to the concentration-dependent inhibitory

effects of H2S, the IC50 value of H2S was calculated as approximately half of the

physiological serum concentration. Endogenous H2S generation from aortic tissues is 3.6

nmol/min/g wet tissue (48). SNP (from 10 to 1000 nmol/L) increased aortic H2S

production in a concentration-dependent manner; 1 µmol/L SNP can increase H2S by 2

nmol/min/g wet tissue (48). One possibility is a negative feedback regulation in which

accumulated NO enhances H2S release from VSMCs during the short term and, over H2S

inhibits NO formation, achieving dynamic balance in the ultimate.

H2S endogenously generated by L-Cys plus PLP reduced NO production by 43%.

Pretreatment with PAG, a CSE inhibitor (51) and pinacidil, a non-selective KATP channel

opener, decreased aortic NO production, similar to the effect of H2S. Glibenclamide, a

non-selective inhibitor, blocked the inhibitory effects of H2S and pinacidil. Collectively,

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these data suggest that endogenously generated H2S downregulates vascular NO

production and opening of the KATP channel could be one of possible mechanisms.

NO is generated from the conversion of L-Arg to L-citrulline by the enzymatic

action of NADPH-dependent NO synthases (NOS). NOS exists in 3 distinct isoforms , a

constitutive neuronal NOS (NOS I or nNOS), an inducible NOS (NOS II or iNOS), and a

constitutive endothelial NOS (NOS III or eNOS) (1). In the vaculature, NO is produced

mainly in the endothelium by constitutively expressed eNOS. The present results suggest

that exogenous H2S or its donor NaHS reduced eNOS activity within 4 h in a

concentration (from 1 to 1000 µmol/L)-dependent manner. The IC50 values for H2S

(11.49 umol/L) were approximately 26% of the physiological serum H2S concentration,

and lower than that of H2S-inhibited NO production (19.5 umol/L). The kinetic study

showed that H2S did not modify the enzyme affinity to L-Arg (represented as Km value),

but decreased the maximum value of catalytic velocity (Vmax), indicating that H2S

reduces eNOS catalytic efficiency. H2S endogenously generated by administration of

L-Cys plus PLP also inhibited eNOS activity.

To verify the specificity of H2S-mediated inhibition of eNOS activity, we

investigated the effect of single or combined administration of the selective iNOS

inhibitor L-canavanine and nNOS inhibitor spermidine. These treatments did not change

the inhibitory effect of H2S on constitutive NOS activity. Although iNOS activity could

be induced by stimuli such as cytokines, reactive oxygen species or some drugs (1), we

detected very low iNOS activity in normal cultured aortic tissues, and neither H2S nor

NaHS incubation affected iNOS activity during the entire incubation period. These

results suggest that H2S is specifically inhibition eNOS activity. Additionally, H2S

inhibits eNOS transcript level and protein expression. These findings suggest that eNOS

is an important regulatory target of H2S-affected NO generation.

L-Arg transport is another key step to limiting NO generation besides NOS catalysis

(8). In the present study, exogenous administration of both H2S and NaHS significantly

reduced L-[3H]-Arg transporter activity within 4 h and returned to control levels after 6 h.

Inhibition was concentration-dependent in the 1 to 1000 umol/L range, and the inhibitory

effect of H2S (from 12.4% to 55.3%) was higher than that of NaHS (from 3.6% to 38.8%).

The IC50 values were 84 µmol/L for H2S, and approximated 182% of physiological serum

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concentrations. Intracellular levels of L-Arg are not generally considered rate-limiting for

eNOS catalysis (51). Consumption of intracellular L-Arg initiates and stimulates its

uptake by cells (30), at this time, NO generation is dependent on L-Arg uptake. These

may explain why the IC50 of H2S for L-Arg uptake was higher than that for NO

generation. Kinetic analysis of the L-Arg transport showed that H2S inhibited the Vmax of

L-[3H]-Arg uptake, but did not influence its Km value. Furthermore, administration of

L-Cys plus PLP increased endogenous H2S production and reduced L-Arg transport.

L-Arg enters mammalian cells through several membrane-bound cationic aminoacid

transporters: systems y+, b0, +, B0, +, and y+L (8). Previous studies have demonstrated

that Arg transport in the vascular endothelium is mainly attributed to system y+, and to

some extent to system y+L (35). NEM, a system y+ selective inhibitor, reduced L-Arg

influx per se. After pretreatment with NEM, H2S did not change L-Arg influx suggesting

that the NEM-sensitive system y+ transporter activity contributes to the regulation of

L-Arg uptake by H2S. In addition, the present study found that H2S also decreased the

CAT1 (encoding the system y+ transporter) transcript level by 34%. All the above results

suggest that endogenous H2S inhibits L-Arg transport under physiological conditions, and

that the system y+ transporter is the main pathway regulated by H2S.

The effect of injection of NaHS on the L-Arg/NOS/NO pathway was also examined

in vivo in rats.According to our previous work, injection of NaHS (14 µmol/kg) after 2 h

increased plasma H2S concentration by 67% over baseline (52). In the present study, the

plasma level of nitrate plus nitrite was decreased by 21%, and aortic eNOS activity by

42% and L-Arg by 30 % 2 h after injection of NaHS. These in vivo results were similar to

those of the in vitro experiments and support the notion that H2S down-regulates the

vascular L-Arg/NOS/NO pathway (i.e., inhibited L-Arg uptake, eNOS activity and NO

generation). Venous injection of H2S can directly induce vasodilation, and cause transient

hypotension (16, 50), and we have previously reported that intraperitoneal bolus injection

of NaHS reduces areterial blood pressure. Increasing plasma H2S level from 39µmol/L to

79 µmol/L reduced arterial blood pressure by 22 mmHg (from 134 mmHg to 112 mmHg);

but plasma H2S concentration increased from 79 µmol/L to 123 µmol/L, while arterial

blood pressure fell by 6 mmHg (from 112 mmHg to 106 mmHg) (52). The present in

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vivo studies showing inhibition of the L-Arg/NOS/NO pathway by H2S may explain in

part this phenomenon.

Our findings support the concept that eNOS is an important target molecule of H2S,

affecting its activity as well as gene and/or protein expression. Several mechanisms are

known to regulate eNOS activity, including the interaction of eNOS with caveolin-1, heat

shock protein 90 (Hsp90), or membrane phospholipids, and enzyme translocation and

phosphorylation (1). Ser1177 appears to be the most important site of eNOS

phosphorylation and is affected by most, if not all, of the diverse stimuli that promote

eNOS activation. Phosphorylation of eNOS-Ser1177 increases eNOS sensitivity to

Ca2+/calmodulin binding and leads to eNOS activation (28). Our study found that H2S

downregulated eNOS phosphorylation at Ser1177. This result implies that H2S inhibits

eNOS activity partly through inhibition eNOS phosphorylation at Ser 1177.

Akt-mediated-phosphorylation of eNOS-Ser1177 is a crucial step of eNOS activation

induced by estrogen, insulin, VEGF, statins and shear stress (15). H2S reduced Akt

phosphorylation without influencing total Akt protein, suggesting that the Akt pathway

may contribute to regulation of H2S by eNOS-Ser1177 phosphorylation. H2S is an

endogenous opener of the KATP channel in many cells types, e.g. (39, 47, 50). The KATP

channel inhibitor glibenclamide blocked the effects of H2S on eNOS activity (Fig.2G-H),

eNOS phosporylation and Akt phosphorylation. Glibenclamide also blocked inhibition of

eNOS phosphorylation by the non-selective KATP channel opener pinacidil. These data

imply that H2S may open KATP channels, followed by reduced Akt phosphorylation and

inhibition of eNOS phosphorylation, leading to downregulation of eNOS activity.

Our study found that H2S also decreased eNOS transcript abundance. Numerous

physiological and pathophysiological stimuli have been identified to modulate eNOS

expression. Reactive oxygen species, as a signal molecule, play an important role in

eNOS transcription and posttranslational regulation. Effects of H2O2 on eNOS

transcription and posttranslational modification were reported in bovine and human

endothelial cells. Chronic oxidative stress caused by excessive H2O2 production in vivo

evokes a compensatory response involving increased eNOS transcript (36). Our previous

work showed that H2S reduced oxidative radical release and scavenged H2O2 directly

(11). H2S may reduce the H2O2 signal in eNOS transcription and posttranslation, and

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thereby reduce eNOS transcription. Fig.5D revealed that KATP channel opening by H2S

and pinacidil inhibited eNOS protein expression, effects that were blocked by

glibenclamide. These data suggest that KATP channels are involved in the inhibition of

eNOS protein expression by H2S. However, the precise mechanisms of inhibition of

eNOS protein expression by H2S need to be further investigated.

L-Arg transporter could be additional target molecules of H2S regulation. L-Arg

transport from plasma into cells is mediated by several different classes of CATs. System

y+ activity, selective for cationic amino acids only, is encoded by 4 genes (CAT1, CAT2,

CAT3, and CAT4) representing different isoforms (35). Systems y+ (CAT-1) and y+L

(heavy chain subunit-4F2hc) have been detected in the endothelium (9). Transport of

cationic amino acids via CAT-1 is voltage dependent with alterations of membrane

potential affecting both Vmax and Km values for influx (22). L-Arg transport is sensitive

to the membrane potential; it is stimulated by drugs that cause membrane

hyperpolarization and inhibited by those that cause membrane depolarization (51). H2S

opens the KATP channel, and induces membrane hyperpolarization (50), which may

contribute to L-Arg transport activity, and furthermore, results in an accumulation of the

intracellular pool of L-Arg available for eNOS. However, opening KATP channels alone is

not enough to explain the mechanisms of H2S-mediated L-Arg influx. Administration of

pinacidil reduced L-Arg influx as did H2S, but NEM could not block the inhibitory

effects of pinacidil. Glibenclamide blocked the effect of pinacidil to a lesser extent than

H2S, suggesting that the mechanisms involved in the H2S regulation of L-Arg influx may

be more complex.

Administration of pinacidil reduced L-Arg influx as did H2S, but NEM could not

block the effects of pinacidil. Glibenclamide blocked almost completely the inhibition

effects of pinacidil, but only slightly blocked the effects of H2S, which suggests that the

mechanisms involved in the H2S regulation of L-Arg influx are complex. Opening KATP

channels alone is not enough to explain the mechanisms of H2S-mediated L-Arg influx.

Oh et al. found that H2S could inhibit NO production in LPS-stimulated macrophages

through a mechanism that involves heme oxygenase-1(HO-1)/carbon monoxide (CO)

(29). H2S increased pulmonary arterial HO-1 gene transcription and protein expression

(32). CO, derived from VSMCs and endothelium, has vasodilatory and antiproliferative

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effects and acts as a competitive antagonist for NO-mediated sGC activation or displaces

internal stores of NO. As a heme ligand, CO potentially inhibits NOS activity and

reduces NO production (34, 38).

Taken together, these results suggest that the gasotransmitter family, including NO,

CO and H2S, derived from vascular tissues in a paracrine/autocrine manner, interacts in

the regulation of vascular homeostasis. The interaction of these gasotransmitters in

distinct locations of the blood vessels could maintain a dynamic balance and form a

regulatory ″network″ (41). Imbalances of the network regulation may contribute to

pathogenesis of cardiovascular diseases. Research into the network regulation of

gasotransmitters should reveal a novel prospect for understanding the mechanism of

cardiovascular diseases and a novel target for prevention and treatment.

ACKNOWLEDGMENTS

This work was supported by The Major State Basic Research Development Program of

the People’s Republic of China (no. 2006CB503807) National Natural Science

Foundation of People’s Republic China (no. 30400151), National Natural Science

Foundation for Distinguished Young Scholars (no. 30425010) and Grant no.

20030001035 from the Ministry of Education, China.

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Figure 1: H2S inhibited NO release in isolated aortic tissues. (A) H2S gas buffer or NaHS buffer (both 50 micromol/L) were added to cultured aortic tissues, incubated for 2, 4 and 6 h, and level of nitrite as NO production was measured. (B) NO production was measured after incubation with different concentrations of H2S and NaHS (1 to 1000 micromol/L) for 2 h. (C) Inhibitory effects of the H2S and NaHS on NO production. Control as 100%. (D) The H2S concentration in culture medium and plasma, measured by sensitive-sulfide electrode. (E) NO production was measured after increase in endogenous H2S levels on administration of L-Cys plus PLP. The inhibitory effect of L-Cys plus PLP on NO generation was blocked by PAG, an inhibitor of CSE (the key enzyme of endogenous H2S generation). (F) The NO production was measured after treatment with H2S (100 micromol/L) and pinacidil (10 micromol/L), or glibenclamide (10 micromol/L) pretreatment. All values are means S.D. a: P<0.05 H2S versus control; b: P<0.05 NaHS versus control; ** : P<0.01 versus control (N=6).

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• Figure 2: H2S downregulates eNOS activity without changing iNOS activity. (A) Alterations in aortic eNOS and iNOSactivities induced by H2S and NaHS (50 micromol/L) after 2, 4 and 6 h of incubation. (B) Effect of different concentrations of H2S and NaHS (1-1000 micromol/L) on aortic eNOS and iNOS activities after 2-h treatment. (C) Inhibitory effect of H2S and NaHS on eNOS activity. Control as 100%. (D) eNOS enzyme activity assay at various concentrations of L-Arg (0.1-3.2 mmol/L) in HUVECs with or without treatment with physiological concentrations of H2S (50 micromol/L). (E) Effect on eNOS and iNOS activities of endogenous H2S generated by treatment with L-Cys plus PLP. (F) The alteration of nNOS and iNOSactivity after treatment with H2S (100 micromol/L) and pinacidil (10 micromol/L), and the effects of the use of the selective iNOS inhibitor-L canavanine (2.5 mmol/L), the nNOS selective inhibitor spermidine (0.5 mmol/L) and the non-selective KATP channel inhibitor glibenclamide (10 micromol/L). (G) H2S (500micromol/L) and pinacidil (10 micromol/L) down-regulate eNOS phosphorylation at Ser1177, but do not change eNOS protein expression after 2-h incubation; glibenclamide(Gli) blocked the inhibitory effects in part (N=3). (H) H2S (500 micromol/L) and pinacidil (10 micromol/L) reduced phosphorylation of Akt and glibenclamide blocked the inhibitory effects. All values are means S.D. a: P<0.05 H2S versus control; b: P<0.05 NaHS versus control; *P<0.05 versus control; **: P<0.01 versus control; #:P<0.05 versus H2S treatment,&: P<0.05 versus pinacidil. $: P<0.01 versus control iNOS activity (N=5).

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Figure 3: H2S inhibited L-Arg transport. (A) After incubation with H2S and NaHS (50 micromol/L) for 2, 4 and 6 h, aortic L-Arg transport was measured by the isotope tracer method. (B) Aortic tissues were treated with various concentrations of H2S and NaHS (1-1000 micromol/L) for 2 h, and the aortic L-Arg uptake was assessed. (C) Correction statistics are given for concentrations of H2S and NaHS and inhibition proportion of L-Arg uptake. Control as 100%. (D) L-Arg transport activity assay at various concentrations of L-Arg (0.1-3.2 mmol/L) in HUVECs with or without H2S (50 micromol/L). (E) Alterations in L-Arg transport induced by endogenous H2S production. (F) The effect of the nonselective KATP channel opener pinacidil (10 micromol/L, Pina) on L-Arg uptake; and pretreatment with the system y+ transporter inhibitor NEM, the alteration of L-Arg uptake by H2S and pinacidil. All values are means S.D. a: P<0.05 H2S versus control; b: P<0.05 NaHS versus control; **: P<0.01 versus control, and #: P<0.05 versus H2S treatment. (N=5).

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Figure 4: H2S inhibited CAT-1 and eNOS gene expressions in HUVECs. (A) After 4 h of incubation with H2S (50micromol/L), cells underwent RT-PCR, separation on a 1.5 % agarose gel and staining with ethidium bromide; bands of eNOS mRNA (553 bp), CAT-1 mRNA (250 bp) and GAPDH mRNA (205 bp) are shown. M is the DNA marker. (B) The optical densities of the bands were measured by use of the Gel Documentation System. The mRNA ratio of eNOS and CAT-1 to GAPDH was considered the relative amount of eNOS and CAT-1 mRNA (N=3). **: P<0.01 versus control.

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Figure 5: H2S regulates eNOS, iNOS and nNOS protein expression. (A) eNOS protein expression was revealed by Western blotting after incubation with H2S (100, 500 and 1000 micromol/L) for 6 h. Glibenclamide (Gli), a KATP channel inhibitor blocked the inhibitory effects of H2S. Tubulin expression was detected as a protein control. (B)The effects of pinacidil on eNOS protein expression. (C) iNOS protein expression was analyzed after H2S treatment (100, 500 and 1000 micromol/L), and TNF (30 ng/ml) as a positive control. (D) nNOS protein expression was measured after treatment with H2S, pinacidil and glibenclamide. *: P<0.05 versus control, **: P<0.01 versus control; #: P<0.05 versus H2S treatment and $: P<0.05 versus pinacidil (N=3).

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Figure 6: H2S inhibited NO production in vivo. Six rats were intraperitoneally injected with NaHS (14 micromol/kg) for 2 h, and 6 controls with normal saline. (A) Nitrate plus nitrite plasma concentration after 2 h. (B) Aortic eNOS and iNOS activities are shown. (C) L-[3H]-Arg uptake in aortic tissues. ** P<0.01 versus control.

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Figure 7: Summary of the interaction between H2S and NO in artery

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Hydrogen sulfide downregulates the aortic L-arginine/nitric oxide pathway in rats

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