European Journal of Pharmacology 720 (2013) 276–285

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Pulmonary, gastrointestinal and urogenital pharmacology

Different regulatory effects of hydrogen sulfide and nitric oxide on gastric motility in mice Xu Huang a, Xiang-Min Meng a, Dong-Hai Liu a, Yi-Song Wu a, Xin Guo a, Hong-Li Lu a, Xue-Yan Zhuang a, Young-chul Kim b, Wen-Xie Xu a,n a Department of Physiology, Shanghai Jiao Tong University School of Medicine, 800 Dongchuan Road, Minhang, 328 Wenxuan Medical Building, Shanghai 200240, China b Department of Physiology, Chungbuk National University College of Medicine, 12 Gaeshin-dong, Hungduk-gu, Cheongju, Chungbuk 361-763, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 18 April 2013 Received in revised form 2 October 2013 Accepted 14 October 2013 Available online 21 October 2013

NO and H2S are gaseous signaling molecules that modulate smooth muscle motility. We aimed to identify expressions of enzymes that catalyze H2S and NO generation in mouse gastric smooth muscle, and determine relationships between endogenous H2S and NO in regulation of smooth muscle motility. Western blotting and immunocytochemistry methods were used to track expressions of neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in gastric smooth muscles. Smooth muscle motility was recorded by isometric force transducers. cGMP production was measured by a specific radioimmunoassay. We found that CBS, CSE, eNOS, and nNOS were all expressed in mice gastric antral smooth muscle tissues, and in cultured gastric antral smooth muscle cells. AOAA significantly inhibited smooth muscle contractions in the gastric antrum, which was significantly recovered by NaHS, while PAG had no significant effect. L-NAME enhanced contractions. NaHS at low concentrations increased basal tension but decreased it at high concentrations. SNP significantly inhibited the contractions, which could be recovered by NaHS both in the absence and presence of CuSO4. ODQ did not block NaHS-induced excitatory effect, while IBMX partially blocked this effect. cGMP production in smooth muscle was significantly increased by SNP but was not affected by NaHS. All these results suggest that endogenous H2S and NO appear to play opposite roles in regulating gastric motility and their effects may be via separate signal transduction pathways. Intracellular H2S/NO levels may be maintained in a state of balance to warrant normal smooth muscle motility. & 2013 Elsevier B.V. All rights reserved.

Keywords: Endogenous H2S Endogenous NO Gastric antral smooth muscle Motility

1. Introduction Nitric oxide (NO) has been called the first gasotransmitter molecule since it was verified to be an endothelium-derived relaxing factor, which functions as an intracellular second messenger (Wang, 2002). NO is produced from L-arginine catalyzed by NO synthase (NOS), and can be classified into two types. One is the constitutive type which includes neuronal NOS (nNOS) and endothelial NOS (eNOS). Both can be continuously expressed under physiological conditions. The other is the inducible type (iNOS), which is mainly expressed during injury and inflammation (Albrecht et al., 2003; Matsuda and Miller, 2010; Takahashi, 2003). In the gastrointestinal tract, NO is an important non-adrenergic non-cholinergic (NANC) inhibitory neurotransmitter which is

n

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0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.10.017

released in response to nerve stimulation and relaxes smooth muscles (Lefebvre et al., 1992; Lefebvre et al., 1995; Stark et al., 1991). Expression of NOS has also been identified in gastrointestinal smooth muscle cells (Chakder et al., 1997; Teng et al., 1998), although the functional significance is not clear. Hydrogen sulfide (H2S) is the third gasotransmitter besides NO and CO. It was regarded as a “toxic gas” until Abe and Kimura described that it may function as an endogenous neurotransmitter (Abe and Kimura, 1996; Wang, 2002). In the tissues, H2S is produced mainly by two enzymes: cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), using L-cysteine as the substrate (Łowicka and Bełtowski, 2007; Kimura, 2011; Wang, 2002). Besides serving as a neurotransmitter, H2S also regulates vascular tone and blood pressure, mediates both pro- and anti-inflammatory functions and performs some cytoprotective roles (Łowicka and Bełtowski, 2007; Kimura, 2011; Wang, 2002). In the gastrointestinal tract, sodium hydrogen sulfide (NaHS), a source of H2S, can reduce spontaneous or acetylcholine (ACh)-induced contraction of ileal

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smooth muscles (Hosoki et al., 1997; Teague et al., 2002). H2S also causes concentration-dependent relaxation of pre-contracted smooth muscles in the mouse gastric fundus and distal colon (Dhaese and Lefebvre, 2009; Dhaese et al., 2010). Muscle contractions of the mouse colon and jejunum were also inhibited by application of NaHS (Gallego et al., 2008). All these results suggest that H2S mediates smooth muscle relaxation in the gastrointestinal tract. However, our previous studies showed that low-concentration of NaHS significantly enhanced smooth muscle tonic contraction in the gastric antrum (Han et al., 2011; Zhao et al., 2009). Since the excitatory effect of low-concentration or endogenous H2S has been shown in gastric smooth muscle (Han et al., 2011; Zhao et al., 2009), and the inhibitory effect of NO has also been reported (Lefebvre et al., 1992; Lefebvre et al., 1995; Stark et al. 1991), we wondered whether there is interaction between these two gaseous signal molecules in the regulation of smooth muscle motility. Many previous studies have demonstrated an interaction between NO and H2S in regulation of vascular tone (Hosoki et al., 1997; Ali et al., 2006; Kubo et al., 2007; Wang et al., 2008; Zhao et al., 2001), but few such studies have been performed in the gastrointestinal tract. The present study was designed to determine the effects of endogeneous H2S and NO, and their interaction on the motility of gastric smooth muscle. We were also interested in whether H2S and NO share the same signal transduction pathway. We aimed to elucidate the possible physiological significance of intracellular NO and H2S in the regulation of gastric motility.

2. Materials and methods 2.1. Tissue preparation Adult male ICR mice were obtained from the Experimental Animal Center of the Chinese Academy of Sciences, Shanghai, China. Animals were killed by cervical dislocation after fasting for 12 h with ad libitum access to water. The stomach was removed quickly and placed in aerated (95% O2 and 5% CO2) Krebs solution containing (mM): NaCl 121.9, KCl 5.9, NaHCO3 15.5, KH2PO4 1.2, MgSO4 1.2, D-glucose 11.5, and CaCl2 2.5. Stomachs were opened along the lesser curvature and the mucosa was removed. Full-thickness muscle strips (3  5 mm2) of antrum were cut along the circular axis. All experimental protocols performed were approved by the local Animal Care Committee, and conformed to the Guide for the Care and Use of Laboratory Animals published by the Science and Technology Commission of P.R.C. (STCC Publication no. 2, revised 1988). 2.2. Western blotting analysis Smooth muscle tissues of murine antrum obtained from the protocol described above were washed twice in cold phosphatebuffered saline (PBS, 0.01 M) and homogenized in RIPA lysis buffer (Beyotime, Jiangsu, China) supplemented with 1 mmol L  1 PMSF (Beyotime, Jiangsu, China) on ice. The homogenate was then centrifuged at 12,000  g for 5 min at 4 1C, and the protein concentration in the supernatant was determined using the BCA method (Thermo Scientific, Rockford, USA). An equal amount of protein (30 μg) of each sample was heat denatured at 100 1C for 10 min and separated by 10% SDS-PAGE gel electrophoresis, and then transferred to a PVDF membrane (Immobilon, Bedford, MA, USA). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline (TBS, pH 7.6) containing 0.5% Tween-20 for 2 h at room temperature and incubated with a primary rabbit anti-CBS polyclonal antibody (1:200, Santa Cruz Biotechnology, CA, USA), rabbit anti-eNOS polyclonal antibody (1:200, Santa Cruz Biotechnology), rabbit anti-nNOS monoclonal antibody (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-GAPDH monoclonal antibody (1:1000, Cell Signaling

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Technology, MA, USA), or mouse anti-CSE monoclonal antibody (1:200, Santa Cruz Biotechnology) in 5% nonfat dry milk in TBST at 4 1C overnight. Membranes were then washed three times, each 15 min with TBST and incubated with HRP-labeled goat anti-rabbit (1:1000, Santa Cruz Biotechnology) or goat anti-mouse secondary antibody (1:1000, Santa Cruz Biotechnology) at room temperature for 2 h. After washing, the membranes were treated with BeyoECL reagents (Beyotime, Jiangsu, China) and exposed to Kodak film. The image was scanned and band densities were quantified with Quantity One image software; protein expression was normalized to expression of GAPDH. 2.3. Cell culture and immunocytochemistry Cell culture was prepared from the murine gastric antrum using the explant technique described by Qi et al. (2008). Antral smooth muscle tissues were isolated and cut into small pieces before placement into a 6-well plate in DMEM (Gibco, Grand Island, NY, USA) containing 20% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin. Tissues were then incubated at 37 1C with 5% CO2 supplied in an incubator. After one week's culture, cells displayed triangular- or flat-shaped morphology. Double-labeling immunocytochemical technique was employed to determine the expressions of CBS, CSE, eNOS and nNOS in the cultured smooth muscle cells (SMCs). Cells between passages 1–2 were transferred to polylysine-coated sterile glass cover-slips. After 2–3 days culture, the cells were washed twice with 0.01 M PBS and fixed with 4% paraformaldehyde for 20 min on ice. After rinsing with PBS three times, the cells were incubated in PBS containing 10% normal goat serum for 30 min, followed by incubation with rabbit anti-CBS polyclonal antibody (1:50, Abcam (Hong Kong) Ltd., Hong Kong), rabbit anti-eNOS polyclonal antibody (1:100, Abcam (Hong Kong) Ltd., Hong Kong), rabbit anti-nNOS monoclonal antibody (1:500, Cell Signaling Technology, USA), or mouse anti-CSE monoclonal antibody (1:200, Santa Cruz Biotechnology) mixed with mouse anti-smooth muscle α-actin monoclonal antibody (1:200, Santa Cruz Biotechnology) or rabbit anti-smooth muscle α-actin monoclonal antibody (1:250, Epitomics, Burlingame, CA, USA) at 4 1C overnight. After washing, the cells were incubated at room temperature with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:100, Jackson Immuno Research, West Grove, PA, USA) mixed with DyLight 594conjugated goat anti-rabbit IgG (1:100, ImmunoReagents Inc, Raleigh, NC, USA) for 30 min. Nuclei were stained with DAPI for 5 min at room temperature. Cells for which the primary antibodies were omitted in the same procedure were used as controls. After thorough washing with PBS, cells were visualized under a fluorescence microscope (BX3, Olympus, Tokyo, Japan). 2.4. Mechanical experiments The muscle strips obtained from the tissue preparations were mounted along the circular axis in a 7-ml organ bath containing oxygenated Krebs solution maintained at 37 1C. One end of the strip was fixed to an isometric force transducer connected to the RM6240C biological signal processing system (Chengdu Equipment Factory, Chengdu, China) to record the mechanical activity. A tension of 0.15 g was applied to the tissues and they were equilibrated for 1 h before the experiments when rhythmic spontaneous contractions were recorded. 2.5. cGMP content Smooth muscle tissues were obtained from the gastric antrum with the mucosa disassociated, and then incubated with NaHS in concentrations of 20 μM, 60 μM or 100 μM or sodium nitroprusside (SNP, 0.05 μM) at 37 1C for 5 min, followed by quick freezing with

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liquid nitrogen. Samples were homogenized in 10 volumes of 6% trichloroacetic acid (TCA) and centrifuged at 12,000g for 20 min; supernatants were then extracted three times with equal volumes of water-saturated diethyl ether, and lyophilized. The contents of cGMP in the samples were determined by the radioimmunoassay described by Cui et al. (2000).

2.7. Statistical analysis Data was analyzed using Origin 7.5 software and expressed as means 7SEM. Data recordings of multiple groups were evaluated using one-way ANOVA followed by a post-hoc Bonferroni test, while Student's t test was used to evaluate two data sets. P o0.05 was considered statistically significant.

2.6. Drugs Drugs used in this experiment including NaHS, SNP, Nω-nitro-Larginine methyl ester (L-NAME), aminooxyacetic acid (AOAA), DL-propargylglycine (PAG), 1H-[1,2,4]Oxadiazolo[4,3-α]quinoxalin1-one (ODQ) and 3-Isobutyl-1-methylxanthine (IBMX) were all purchased from Sigma (St. Louis, MO, USA). ODQ and IBMX were dissolved in DMSO and diluted to the desired concentration using extracellular solutions. The final concentration of DMSO was not more than 0.05%.

3. Results 3.1. Expressions of catalyzing enzymes of NO and H2S in gastric smooth muscles To determine whether NO and H2S can be produced endogenously in the murine gastric antrum, we identified NOS expression, including eNOS and nNOS, which can continuously catalyze the

Fig. 1. Expressions of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in the gastric smooth muscles. A: Expressions of eNOS and nNOS in smooth muscle tissues of mouse gastric antrum were detected by Western blotting. Enzyme expressions were normalized to that of GAPDH. B: Expressions of eNOS and nNOS in cultured smooth muscle cells of mouse gastric antrum were detected by the immunocytochemistry method. (Scale bar¼ 100 μm) Ba: control. Bb: Double-labeling immunocytochemistry result shows that α-actin þ smooth muscle cells were also eNOS þ . Bc: α-actin þ /nNOS þ smooth muscle cells.

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production of NO, and the two kinds of H2S-producing enzymes CBS and CSE, in the smooth muscle tissues of murine antrum using the Western blotting and immunocytochemistry methods. We found that all four enzymes were expressed in antral smooth muscle tissues using the Western blotting method (Figs. 1 and 2A). To determine whether NO and H2S can be generated by the SMCs, the expressions of eNOS, nNOS, CBS and CSE in cultured SMCs of the gastric antrum were identified using the double-labeling immunocytochemistry technique. After culture, the triangular- or flat-shaped cells which were immunopositive for a putative SMC marker, α-actin, were also positively stained for eNOS, nNOS, CBS and CSE (Figs. 1 and 2B). The results suggest that both NO and H2S can be generated endogenously in the gastric SMCs. 3.2. Effects of AOAA, PAG and L-NAME on gastric smooth muscle contractions Since nNOS, eNOS, CBS, and CSE were all expressed in GI smooth muscle tissues, we would like to test whether endogenous NO and H2S affect gastric smooth muscle contractions. AOAA, an inhibitor of CBS, (Gil et al., 2011; Hosoki et al., 1997; Zhao et al.,

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2003) PAG, an inhibitor of CSE, (Abeles and Walsh, 1973; Martin et al., 2010; Washtien and Abeles, 1977) and L-NAME, an inhibitor of NOS were used in this study. After equilibration, spontaneous rhythmic contractions were recorded in gastric antral smooth muscle strips. AOAA (1 mM) significantly reduced both amplitude (100% before vs 57.40 75.93% after administration of AOAA, Fig. 3Aa, Ba) and basal tension (from 0 g to  0.09 70.02 g; Fig. 3Aa, Bb; P o0.05, n ¼19), which was partially recovered by NaHS (Fig. 3 Ab, Bc, Bd; P o0.05 vs AOAA, n ¼8). PAG (1 mM) had no significant effect on both amplitude (100% before vs 99.37 72.77% after administration of PAG, Fig. 3Ac, Be) and basal tension (from 0 g to  0.006 70.003 g; Fig. 3 Ac, Bf; P 40.05, n ¼5) of the contractions. L-NAME (200 μM) increased amplitude (100% before vs 129.71 74.39% after administration of L-NAME; Fig. 3 Ad, Bg; P o0.05, n¼ 15) and had no effect on basal tension (from 0 g to  0.006 70.003 g; Fig. 3 Bh; P 40.05, n ¼ 15). These results suggested that endogenous NO and H2S have opposite effects on gastric smooth muscle motility, with H2S as an excitatory regulator and NO as an inhibitory one. CBS may be the main catalyzing enzyme of generating H2S in mouse gastric antrum, which is in line with our previous study (Han et al., 2011).

Fig. 2. Expressions of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the gastric smooth muscles. A: Expressions of CBS and CSE in smooth muscle tissues of mouse gastric antrum were detected by Western blotting. Enzyme expressions were normalized to that of GAPDH. B: Expressions of CBS and CSE in cultured smooth muscle cells of mouse gastric antrum were detected by the immunocytochemistry method. (Scale bar ¼100 μm) Ba: control. Bb: Double-labeling immunocytochemistry result shows that α-actin þ smooth muscles were also CBS þ . Bc: α-actin þ /CSE þ smooth muscle cells.

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Fig. 3. Effects of AOAA, PAG and L-NAME on mouse gastric smooth muscle contractions. A: AOAA (1 mM) significantly decreased, PAG (1 mM) had no significant effect on, and L-NAME (100 μM) increased smooth muscle contractions in the gastric antrum. Aa: Raw traces of effect of AOAA. Ab: AOAA-induced inhibition of contractions was significantly recovered by low concentrations NaHS. Ac: Raw traces of effect of PAG. Ad: Raw traces of effect of L-NAME. B: Summary of effects of AOAA, PAG and L-NAME on gastric smooth muscle contractions. Ba, Bb: Summary of effects of AOAA on amplitude and basal tension of gastric antrum contractions (nPo 0.05, n¼ 19). Bc, Bd: Summary of effects of low concentrations NaHS on AOAA-induced inhibition of gastric antrum contractions (nPo 0.05 vs AOAA, n¼ 8). Be, Bf: Summary of effects of PAG on amplitude and basal tension of gastric antrum contractions (P 40.05, n¼ 5). Bg, Bh: Summary of effects of L-NAME on amplitude (nP o 0.05, n ¼15) and basal tension (P 40.05, n¼ 15) of gastric antrum contractions.

3.3. Interaction between H2S and NO on smooth muscle contractions Since the synthases of H2S and NO are co-expressed in gastric smooth muscles and their inhibitors had opposite effects on gastric motility, we were interested in the interaction between these two gaseous molecules. To verify the effects of endogenous NO and H2S on smooth muscle contractions, interactions between

exogenous NO and H2S on gastric smooth muscle motility were also observed. NaHS and SNP were used as donors of H2S and NO, respectively. After equilibration, spontaneous rhythmic contractions were recorded from the smooth muscle strips of gastric antrum. NaHS significantly increased tonic contractions of the gastric smooth muscles at low concentrations (from 60 to 100 μM, Fig. 4 Aa, Ba;

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Fig. 4. Interaction between exogenous H2S and NO on spontaneous contractions in mouse gastric antrum. A: Effects of NaHS and SNP on gastric smooth muscle contractions and their interaction. Aa: Raw traces of effects of NaHS. Ab: Raw traces of effects of SNP. Ac: Raw traces of interaction between NaHS and SNP. B: Summary of the effects of NaHS and SNP on gastric smooth muscle contractions and their interaction. Ba: NaHS at low concentrations (60 and 100 μM) significantly enhanced basal tension (nPo 0.05 vs control, n ¼20) of mouse antrum contractions but inhibited it at high concentrations (200 and 400 μM) (nPo 0.05 vs control, n¼ 20). Bb: SNP significantly inhibited the basal tension of the contractions (nPo 0.05 vs control, n¼ 13). Bc: NaHS at low concentrations recovered SNP-induced inhibition of basal tension (nP o 0.05 vs SNP, n¼ 10). Bd: NaHS at low concentrations (60 and 100 μM) had no significant effect on the amplitude of the contractions (P40.05 vs control, n¼ 20) but inhibited the amplitude at high concentrations (200 and 400 μM) (nPo 0.05 vs control, n¼ 20). Be: SNP significantly inhibited the amplitude of the contractions (nPo 0.05 vs control, n¼ 13). Bf: NaHS at low concentrations recovered SNP-induced inhibition of amplitude (nPo 0.05 vs SNP, n ¼10).

P o0.05 vs control, n ¼ 20) but not the amplitude of phasic contraction (Fig. 4 Aa, Bd; P4 0.05 vs control, n ¼20). However, both amplitude and basal tension were decreased by high concentrations of NaHS ( 4100 μM, Fig. 4 Aa, Ba, Bd; P o0.05 vs control, n ¼20). SNP significantly reduced both amplitude and

basal tension of gastric smooth muscle contractions (Fig. 4 Ab, Bb, Be; Po0.05 vs control, n ¼13). Amplitudes were decreased from 100% of control to 73.80 75.87%, 51.11 76.23%, 42.387 5.10% and 8.64 73.59% by SNP at 0.01, 0.05, 0.1 and 0.5 μM, respectively (Fig. 4 Ab, Be; P o0.05 vs control, n ¼13). The inhibitory effect of

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SNP was significantly recovered by NaHS (Fig. 4 Ac, Bc, Bf; Po 0.05 vs SNP, n ¼10). These results demonstrated that exogenous NO and low concentrations of H2S have opposite effects on gastric smooth muscle contractions. The opposing effects of NO and H2S may be important in regulation of gastric smooth muscle tone under the physiological condition. Intracellular NO and H2S generation may be maintained in a state of equilibrium to allow normal tonic contraction of gastric smooth muscles. 3.4. Effect of CuSO4 on interaction between H2S and NO on smooth muscle contractions It has been reported that NO and H2S can form a novel nitrosothiol, which may underlie the contractive effect of low concentrations of H2S on precontracted rat aortic rings (Ali et al., 2006; Whiteman et al., 2006). That is, H2S may induce contractions of rat aorta via regulating the concentration of vasodilator NO. To determine whether the interaction between NO and H2S on the smooth muscle contraction results from the formation of the nitrosothiol, we observed their interaction in the absence and presence of CuSO4, which can convert nitrosothiol to a mixture of nitrite and nitrate. We observed that SNP-induced inhibition was significantly recovered by low concentration of NaHS both in the absence (P o0.05 vs SNP, n ¼8; Fig. 5 Aa, Ba, Bb) and presence of CuSO4 (200 nM, P o0.05 vs SNP, n ¼8; Fig. 5 Ab, Ba, Bb). These results suggested that the interaction between NO and H2S was not a result of formation of a novel nitrosothiol. 3.5. Effect of cGMP signal pathway on NaHS-induced excitatory effect As reported previously, the gaseous signaling molecules NO and CO take effect through activation of soluble guanylate cyclase (sGC) and subsequent increase of intracellular cGMP (Matsuda and Miller, 2010). We were interested in whether the excitatory effect of H2S on gastric smooth muscle motility is via the cGMP signal pathway. cGMP can be synthesized by the catalyzation of sGC and

degraded by phosphodiesterase (PDE). In this study, we used ODQ, a sGC inhibitor, and IBMX, a non-selective PDE inhibitor, to verify involvement of the cGMP pathway in the effects of NaHS. SNP induced-inhibition of gastric antral smooth muscle contraction was blocked by ODQ (10 μM; P o0.05, n ¼6; Fig. 6 Aa, Ab, Ba), while enhancement of contractions by NaHS was not affected by ODQ (Fig. 6 Ac, Ad, Bb; P 40.05, n ¼4). IBMX (20 μM) partially inhibited NaHS-induced enhancement of the basal tension (Fig. 6 Ae, Bc; Po 0.05, n ¼9). We also directly observed the effect of NaHS on cGMP production in gastric smooth muscle tissue. cGMP content was not significantly affected by NaHS (20–100 μM, P 40.05, n ¼8), but was increased by SNP (0.05 μM, Po 0.05, n ¼8; Fig. 6C). These results indicate that intracellular cGMP is not involved in NaHSinduced increase of basal tension in gastric antral smooth muscle.

4. Discussion NO and H2S are important gaseous signaling molecules that modulate smooth muscle motilities in the GI tract and cardiovascular system. As an NANC neurotransmitter, NO may directly activate potassium channels or indirectly open channels through the activation of sGC and subsequent increase of cGMP (Jin et al., 2000; Koh et al., 1995; Kwon et al., 2000; Lefebvre et al., 1995; Li et al., 2000; Van Crombruggen and Lefebvre, 2004). However, the effects of H2S on smooth muscles are somewhat complicated. In vascular tissues, intravenous bolus injection of H2S decreased blood pressure, which was antagonized by the blocker of KATP channels (Zhao et al., 2001). The result suggests that H2S relaxes vascular smooth muscles by activating KATP channels. Subsequent in vitro experiments confirmed this hypothesis (Ali et al., 2006; Zhao et al., 2001). These results were observed in the aorta of Sprague–Dawley (SD) rat; however different outcomes were found in the aorta of Wistar rat and mouse, in which blockade of KATP channels only partially inhibited, or had no effect on, H2S-induced relaxation (Kubo et al., 2007). In the aorta, the relaxation effect of H2S only occurred at high concentrations; in contrast, low

Fig. 5. Effect of CuSO4 on the interaction between H2S and NO on gastric smooth muscle contraction. A: Raw traces of effect of CuSO4 on the interaction between H2S and NO on smooth muscle contraction in the gastric antrum. Aa: NaHS (100 μM) recovered SNP-induced inhibition of smooth muscle contractions in the gastric antrum. Ab: NaHS recovered SNP-induced inhibition of smooth muscle contractions even in the presence of CuSO4 (200 nM). B: Summary of effect of CuSO4 on the interaction between H2S and NO on smooth muscle contraction in the gastric antrum. Ba: NaHS recovered SNP-induced inhibition of the amplitude of contractions both in the absence (nPo 0.05 vs SNP, n¼ 8) and in the presence (#P o 0.05 vs SNP, n¼ 8) of CuSO4. Bb: NaHS recovered SNP-induced inhibition of the basal tension of contractions both in the absence (nPo 0.05 vs SNP, n ¼8) and in the presence (#Po 0.05 vs SNP, n¼ 8) of CuSO4.

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concentrations of H2S caused contraction of pre-contracted tissues (Ali et al., 2006; Kubo et al., 2007). In the GI tract, NaHS caused relaxation in pre-contracted guinea pig ileum by KATP channels-independent mechanisms, (Teague et al., 2002) while NaHS-induced relaxation in human and rat colon tissues were significantly reduced by glybenclamide, a blocker of KATP channels, and apamin, a blocker of small-conductance calcium-activated

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potassium (SK) channels. These results indicate the involvement of KATP channels and SK channels in relaxation. SK channels, but not KATP channels, are also involved in NaHS-induced relaxation in mouse colon (Gallego et al., 2008). Our previous studies showed that exogenous H2S has a dual effect on gastric motility in guinea pigs and mice depending on its concentration. At low concentrations, H2S increases basal tension of smooth muscle contractions

Fig. 6. Effects of ODQ and IBMX on SNP and NaHS-regulated smooth muscle contraction. A: Raw traces of effects of ODQ and IBMX on SNP and NaHS-regulated smooth muscle contraction. Aa: SNP inhibited smooth muscle contraction in gastric antrum. Ab: ODQ (10 μM) blocked SNP-induced inhibition. Ac: NaHS enhanced smooth muscle contraction. Ad: ODQ had no significant effect on NaHS-induced enhancement. Ae: IBMX (20 μM) partially inhibited effects of NaHS. B: Summary of effects of ODQ and IBMX on SNP and NaHS-regulated contraction. Ba: Summary of ODQ on SNP-induced inhibition of gastric antrum contraction. Bb: Summary of ODQ on NaHS-induced enhancement of gastric antrum contraction. Bc: Summary of IBMX on NaHS-induced enhancement of gastric antrum contraction. C: NaHS at 20–100 μM had no significant effect on intracellular cGMP (P40.05, n ¼8), while SNP at 0.05 μM increased intracellular cGMP (nP o0.05, n ¼8).

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by inhibiting voltage-dependent potassium channels, while at high concentrations H2S inhibits the phasic contractions by activating KATP channels (Han et al., 2011; Zhao et al., 2009). In this study, we found that CBS, CSE, eNOS, and nNOS, the enzymes that catalyze generation of NO and H2S, were all expressed in the smooth muscle tissues of murine gastric antrum and in cultured gastric antrum SMCs. These results suggest that the enzymes that catalyze generation of NO and H2S are co-existed in gastric antral smooth muscle cells. In another word, NO and H2S could be generated endogenously and continuously in the gastric antral smooth muscle cells. Therefore, it is necessary to observe the interaction between endogenous NO and H2S on gastric smooth muscle motility. In the present study, we observed the effects of AOAA, an inhibitor of CBS (Gil et al., 2011; Hosoki et al., 1997; Zhao et al., 2003), PAG, an inhibitor of CSE, (Abeles and Walsh, 1973; Martin et al., 2010; Washtien and Abeles, 1977) and L-NAME, an inhibitor of NOS, on smooth muscle contraction in mouse antrum. We found that AOAA and L-NAME showed opposite effects on the spontaneous contraction in gastric antral smooth muscle, i.e., AOAA reduced the amplitude and basal tension of the contraction, while L-NAME enhanced the amplitude. PAG had no significant effect on the contraction (Fig. 3). These results suggest that the two important gaseous signal molecules—endogenous NO and H2S, play opposite roles in the regulation of gastric smooth muscle motility, with H2S as an excitatory regulator and NO as an inhibitory one. Our results also demonstrated that PAG had no significant effect on the gastric motility. This is in line with the observation by Martin et al. (2010) that PAG did not affect the generation of H2S in the mouse stomach. Together with our present results, we speculate that CSE may not be the main enzyme that catalyzes the generation of H2S in the mouse gastric antrum. Although AOAA has been reported to inhibit CBS (Gil et al., 2011; Hosoki et al., 1997; Zhao et al., 2003), AOAA is also widely used as an inhibitor of several pyridoxal phosphate-dependent enzymes (John and Charteris 1978). Martin et al. (2010) has demonstrated that AOAA reduces the generation of H2S in the mouse stomach. In the present study, we observed that AOAAinduced inhibitory effect on gastric antral smooth muscle contraction was significantly recovered by exogenous NaHS (Fig. 4). These results suggest that AOAA-induced inhibitory effect on gastric smooth muscle contraction is partially via inhibiting CBS to generate endogenous H2S, i.e., endogenous H2S acts as an excitatory modulator. To further determine the effects of endogenous NO and H2S on gastric smooth muscle contraction, we also observed the effects of exogenous NO and H2S on these contractions and the interaction between them. Our previous studies (Han et al., 2011; Zhao et al., 2009) showed that NaHS, a donor of exogenous H2S, had a dual effect on gastric antral smooth muscle motility (Fig. 4). That is, NaHS at low concentrations (r100 μM) increased the basal tension of gastric antral smooth muscle contraction, while inhibited the contraction at high concentrations (Z200 μM). As the physiological concentration of H2S is no more than 160 μM (Abe and Kimura, 1996; Martin et al., 2010), we speculate that the endogenous H2S maybe mainly play an excitatory role in modulating gastric antral smooth muscle motility. However, SNP, a donor of exogenous NO, only displayed inhibitory effects on both amplitude and basal tension of spontaneous rhythmic contractions. Interestingly, the SNP-induced inhibitory effect was significantly recovered by NaHS (Fig. 4). These results further confirmed the opposite effects of endogenous NO and H2S on gastric smooth muscle contractions. In the vascular tissues, the contractile effect of H2S is associated with NO. As an endothelium-derived relaxing factor, NO relaxes smooth muscles in vascular tissue. H2S is thought to induce contractile effects by inhibiting eNOS or combining with NO to

form a novel nitrosothiol to reduce NO release (Ali et al., 2006; Kubo et al., 2007; Whiteman et al., 2006). In the present study, we also tested whether the interaction between NO and H2S in regulation of gastric smooth muscle motility was due to the formation of the nitrosothiol. The results demonstrated that CuSO4, which can convert nitrosothiol to a mixture of nitrite and nitrate, had no significant effect on the interaction between NO and H2S (Fig. 5). The results suggest that this mechanism is not responsible for the interaction between these two gaseous signal molecules in the gastric antrum. Previous reports suggest that H2S-induced smooth muscle relaxation is mainly due to the opening of KATP channels (Ali et al., 2006; Gallego et al., 2008; Zhao et al., 2009; Zhao et al., 2001), although some of the responses are KATP channelindependent (Hosoki et al., 1997; Kubo et al., 2007; Teague et al., 2002). However, the mechanism of H2S-induced contractile function is a little bit complicated. Our previous studies have shown that voltage-dependent potassium channels are involved in the contractile response of H2S in the gastric antrum, indicating a direct effect by H2S on smooth muscle cells (Han et al., 2011; Zhao et al., 2009). However, NaHS is suggested to stimulate endogenous capsaicin sensitive nerves in the urinary bladder, resulting in tachykinin release and contraction of the smooth muscles (Patacchini et al., 2004). Both NO and CO generate their effects through intracellular second messengers, especially the cGMP signaling pathway (Matsuda and Miller, 2010). H2S increases intracellular cGMP by inhibiting PDE activity (Coletta et al., 2012). Therefore, we were also interested in whether H2Sinduced excitatory effect on gastric smooth muscle contractions was through the cGMP pathway. As cGMP can be generated by sGC and degraded by PDE, we used ODQ, an inhibitor of sGC, and IBMX, an inhibitor of PDE, in this study. We found that ODQ had no significant effect on NaHS-induced excitatory effect, while IBMX partially inhibited this effect (Fig. 6). However, NaHS did not significantly affect cGMP production (Fig. 6). These results suggest that H2S-induced excitatory effect on gastric motility may not result from the change of intracellular cGMP. The partial inhibition of IBMX on NaHS-induced contraction may be due to the effect of IBMX itself, but not via cGMP pathway. Many studies have focused on the relationship between NO and H2S in regulating smooth muscle motility so far. In vascular smooth muscle, NO and H2S had a synergistic effect on relaxation (Hosoki et al., 1997; Zhao et al., 2001; Zhong et al., 2003) but also had opposite effect (Zhao and Wang, 2002). In the GI tract, the synergistic relaxant effect by NO and H2S was also seen in the guinea pig ileum (Teague et al., 2002). Our present study showed opposite effects of endogenous NO and H2S on gastric smooth contraction. NO may act as an inhibitory mediator to relax smooth muscle by activating calcium-activated potassium channels (Koh et al., 1995; Matsuda and Miller, 2010; Van Crombruggen and Lefebvre, 2004), whereas H2S acts as an excitatory mediator to induce tonic contraction by inhibiting voltage dependent potassium channels (Han et al., 2011; Zhao et al., 2009). But the mechanism underlying the interactions still needs to be clarified. In summary, we identified expressions of CBS, CSE, eNOS and nNOS in mouse gastric smooth muscles. AOAA, a blocker of CBS, significantly inhibited gastric smooth muscle contractions, which was significantly recovered by low concentration NaHS, but PAG, a blocker of CSE, had no significant effect on them. L-NAME, a blocker of NOS, enhanced the contractions. NaHS at low concentrations enhanced tonic contractions, while SNP inhibited smooth muscle contractions, which was also significantly recovered by NaHS both in the absence and presence of CuSO4. SNP significantly increased cGMP production in gastric smooth muscle tissue, however, NaHS had no effect. Our results suggest that endogenous H2S and NO play opposite roles in regulating gastric motility, but

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