O R I G I N A L A R T I C L E
Nitric Oxide and Hydrogen Sulfide Interact When Modulating
Gastric Physiological Functions in Rodents
Larisse Tavares Lucetti1•Renan Oliveira Silva1•Ana Paula Macedo Santana1• Bruno de Melo Tavares1•Mariana Lima Vale1•Pedro Marcos Gomes Soares2• Francisco Jose´ Batista de Lima Ju´nior1• Pedro Jorge Caldas Magalha˜es1• Fernando de Queiroz Cunha3• Ronaldo de Albuquerque Ribeiro1• Jand-Venes Rolim Medeiros4•Marcellus Henrique Loiola Ponte Souza1
Received: 28 March 2016 / Accepted: 7 November 2016 / Published online: 18 November 2016
ÓSpringer Science+Business Media New York 2016
Abstract
Aim The objective was to evaluate the effects of nitric oxide (NO) and hydrogen sulfide (H2S) donors and possible interactions between these two systems in modulating gastric function.
Methods Mice received saline, sodium nitroprusside (SNP), or sodium hydrosulfite (NaHS), and after 1 h, the animals were killed for immunofluorescence analysis of CSE or eNOS expressions, respectively. Other groups received saline, SNP, NaHS, Lawesson’s reagent (H2S donor), PAG?SNP, L-NAME, L-NAME?NaHS, or L-NAME?Lawesson’s reagent. Then, the gastric secre-tions (mucous and acid), gastric blood flow, gastric defense against ethanol, and gastric motility (gastric emptying and gastric contractility) were evaluated.
Results SNP and NaHS increased the expression of CSE or eNOS, respectively. SNP or Lawesson’s reagent did not alter gastric acid secretion but increased mucus production, and these effects reverted with PAG and L-NAME treat-ment, respectively. SNP or NaHS increased gastric blood flow and protected the gastric mucosa against ethanol injury, and these effects reverted with PAG andL-NAME treatments, respectively. SNP delayed gastric emptying
when compared with saline, and PAG partially reversed this effect. NaHS accelerate gastric emptying, and L-NAME partially reversed this effect. SNP and NaHS alone induced gastric fundus and pylorus relaxation. However, pretreatment with PAG orL-NAME reversed these relaxant effects only in the pylorus but not in the gastric fundus. Conclusion NO and H2S interact in gastric physiological functions, and this ‘‘cross-talk’’ is important in the control of mucus secretion, gastric blood flow, gastric mucosal defense, and gastric motility, but not in the control of basal gastric acid secretion.
Keywords Nitric oxideHydrogen sulfideGastric
functionsGasotransmitters
Introduction
The discovery that mammalian cells synthesize nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) initiated a new paradigm that led to a large number of studies on the roles of these molecules in human physiol-ogy and disease. NO was the first of a new class of sig-naling molecules, followed by carbon monoxide and hydrogen sulfide, now known as ‘‘gasotransmitters’’ [1].
NO and H2S share several common features of gaseous mediators; for example, they are small gaseous molecules, are freely permeable to cell membranes, and do not rely on membrane receptors to exert their functions. In addition, they are endogenously and enzymatically generated [2]. They are produced via separate pathways; i.e., NO is produced from L-arginine catalyzed by NO synthase (NOS) and can be clas-sified into two types. One is the constitutive type, which includes neuronal NOS (nNOS) and endothelial NOS (eNOS), and both can be continuously expressed under physiological
& Marcellus Henrique Loiola Ponte Souza [email protected]
1 Department of Physiology and Pharmacology, Federal University of Ceara´, Fortaleza, CE, Brazil
2 Department of Morphology, Federal University of Ceara´, Fortaleza, CE, Brazil
3 Department of Pharmacology, Ribeira˜o Preto Medical School, University of Sa˜o Paulo, Ribeira˜o Preˆto, SP, Brazil 4 Department of Biotechnology and Biodiversity Center
conditions. The other is the inducible type (iNOS), which is mainly expressed during injury and inflammation [3–5]. Endogenous H2S can be formed by the action of the enzymes cystathionine gamma-lyase (CSE), cystathionine beta syn-thase (CBS), cysteine aminotransferase (CAT), and 3-mer-captopyruvate sulfurtransferase (MST) [6–8]. NO and H2S are involved in several physiological functions, which include inflammatory and immune processes [9], as well as the control of gastric mucosal defense [10], vascular tone, and the control of gastrointestinal motility [11–13].
Recently, it was demonstrated that gasotransmitters might interact with each other. Although it was generally assumed that H2S and NO exert their effects via separate pathways, the literature has indicated there is ‘‘cross-talk’’ between H2S and NO, an early indication that NO interacts with H2S by the observation that low concentrations of H2S increase vascular relaxation induced by NO donors, such as SNP [14]. Studies showed that H2S increased HCO3 -secretion in stomach and this effect is dependent on the NO [15]. Other published data have shown that the endogenous production of H2S from rat aortic tissues is enhanced by NO donor treatment [16] and that H2S and NO are mutu-ally required to elicit angiogenesis and vasodilatation [17]. The paths that appear to modulate the ‘‘cross-talk’’ between NO and H2S in gastrointestinal (GI) tract function have not yet been fully elucidated and serve as fertile territory for future experiments. In this context, this study aimed to evaluate the effects of nitric oxide (NO) and hydrogen sulfide (H2S) and possible interactions between these two gasotransmitters in modulating gastric function.
Materials and Methods
Animals
Male Swiss mice (25–30 g) and male Wistar rats (200–250 g) were obtained from the Department of Phys-iology and Pharmacology, Federal University of Ceara´. The animals were housed in cages in a temperature-con-trolled environment under a 12-h light/12-h dark cycle and received food and water ad libitum. However, they were deprived of food for 18–24 h, but had free access to water, before the experiment. All treatments and surgical proce-dures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, National Institutes of Health (Bethesda, MD, USA) and were approved by the local ethics committee (Protocol No. 63/07).
Drugs and Solutions
Sodium nitroprusside (SNP, a nitric oxide donor), sodium hydrosulfide (NaHS, a hydrogen sulfide donor),
Lawesson’s reagent (a hydrogen sulfide donor),Nx -nitro-L-arginine methyl ester hydrochloride (L-NAME, a inhi-bitor of NO synthase), D,L-propargylglycine (PAG, inhi-bitor of cystathionine c-lyase enzyme) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Immunofluorescence Assay for CSE and eNOS Analyses
Initially, mice received saline, SNP (10 mg/kg, po) [45], or NaHS (150lmol/kg, po) [12,46]. One hour later, the animals were killed and the stomach was removed and fixed in 4% paraformaldehyde (PFA) in 0.2 M phosphate sodium buffer (PBS) for 2 h at room temperature. The tissues were cry-oprotected in 30% sucrose in 0.2 M PBS overnight at 4°C and
frozen in Tissue-Tek OCT compound 4583 (Sakura Finetechnical Co. Ltd., Tokyo, Japan). They were then sec-tioned on a Leica CM3050 cryostat (Leica Microsystems, Germany) at a thickness of 10lm. All of the sections were blocked with glycine 0.1 M in 5% BSA for 1 h at room temperature, washed and incubated overnight at 4°C with
mouse primary antibody anti-CSE, anti-CBS (Abnova Cor-poration; 1:200 dilution), or rabbit anti-eNOS (Santa Cruz Biotechnology, 1:200 dilution). Next, the slides were washed in 0.2 M PBS and incubated at room temperature for 1 h with a secondary antibody for CSE and CBS (Invitrogen, goat anti-mouse conjugated with Alexa Fluor 546; 1:400 dilution) or eNOS (Invitrogen, goat anti-rabbit conjugated with Alexa Fluor 488; 1:400 dilution). DAPI was used to label cell nuclei (Sigma-Aldrich). Sections were viewed using a confocal microscope (FV-1000; Olympus, Tokyo, Japan). Fluores-cence intensity was analyzed using FIJI-Image J software, and the number of pixels in the selected area was estimated from the total number of pixels in the entire image for quantification of CSE, CBS, or eNOS expression [18].
Gastric Acid Secretion
Gastric Wall Mucus
Mice were treated only with saline, SNP (10 mg/kg, po), or Lawesson’s reagent (27lmol/kg, po). In other experi-mental groups, the animals were pretreated with PAG (50 mg/kg, po) [12] orL-NAME (3 mg/kg, ip) for 30 min before treatment with the donors (SNP and Lawesson’s reagent, respectively). After 30 min, glandular segments of the stomach were collected, weighed, and transferred to 0.1% Alcian blue solution for 2 h. Excess dye was removed by washing the segments with 0.25 M sucrose for 1 h. The mucus–dye complex was extracted by placing the segments in 0.5 M MgCl2for 2 h. The dye extract was mixed with diethyl ether, centrifuged at 3500 rpm for 10 min, and absorbance of the supernatants was measured on a spec-trophotometer at 598 nm. Results are expressed as Alcian bluelg/g tissue, using a standard curve of Alcian blue [21].
Gastric Blood Flow
The gastric mucosal blood flow was evaluated with an ex vivo gastric chamber technique and measured with a laser-Doppler flowmeter (model BLF 21D, Transonic Systems, Ithaca, NY, USA), as described previously [22, 23]. Unlike other models in the gastric flow mea-surement, the rats, but not the mice, were used, due to the size of the chamber. Briefly, the stomachs of the rats were bathed with PBS (pH 7.4 at 37°C) and a laser-Doppler
probe was placed on the gastric corpus region. After a basal period of 15 min, the buffer was replaced by SNP (50 mg/ ml) or NaHS (30lmol/ml). In other experimental groups, a incubation with PAG (50 mg/kg, ip) orL-NAME (3 mg/kg, ip) was performed before of donors (SNP and NaHS, respectively). Blood flow was expressed as a percentage of the flow rate in the basal period.
Ethanol-Induced Gastric Damage
Mice received only SNP (10 mg/kg, po) or NaHS (150lmol/kg, po). In other experimental groups, the ani-mals were pretreated with PAG (50 mg/kg, po) orL-NAME (3 mg/kg, ip) 30 min before treatment with the donors (SNP and NaHS, respectively). After 30 min, gastric damage was induced by 50% ethanol (0.5 ml/25) [12]. The control group received only saline. One hour later, the animals were killed and the stomachs were removed and opened for analysis. Gastric damage was evaluated blindly using a computer planimetry program (Image JÒ). Samples from the stomach were removed for analysis of GSH levels and MDA concentration, as described below.
For the histopathologic study, a sample of the stomach was fixed in a formaldehyde solution 10%, where it stayed
for 24 h. Then the samples were transferred to a 70% alcohol solution, where they remained until the completion of the histological procedures. The organic material was embedded in paraffin and sectioned. There were five made cuts microns, placed on a slide, and stained with hema-toxylin–eosin to study the optical microscope coupled to the image acquisition system (LEICA). The samples were evaluated according to the criteria Laine & Weinstein (1988), which evaluates the loss of epithelial cells (scores 0–3), swelling of the mucosal surface (scores 0–4), bleeding (score 0–4), and infiltration of inflammatory cells (scores 0–3). The entire histopathologic evaluation was performed by a blind study by an experienced histopathologist (PMGS).
Glutathione (GSH) Assay
The samples were homogenized in 0.02 M EDTA. Next, aliquots (400ll) of the homogenate were mixed with distilled water (320ll) and 50% (w/v) trichloroacetic acid (80ll) and centrifuged at 3000 rpm for 15 min. Then, 400 ll of the supernatant was mixed with 0.4 M Tris buffer (800 ll; pH 8.9) and 0.01 M DTNB (20ll). Subsequently, the samples were stirred for 3 min and read on a spec-trophotometer at 412 nm [24]. Results are expressed as lg/g tissue.
Malondialdehyde (MDA) Assay
The samples were homogenized in cold 1.15% KCl. Then, aliquots (250ll) of the homogenate were supplemented with 1% phosphoric acid (500ll) and 0.6% tert-butyl alcohol (aqueous solution). Next, this mixture was stirred and heated in a boiling water bath for 45 min. The mixture was then cooled immediately in an ice water bath, followed by the addition of 4 ml of n-butanol. This mixture was shaken, and the butanol layer was separated by centrifu-gation at 1200 rpm for 15 min. Next, samples were read on a spectrophotometer at 535 and 520 nm [25]. Results are expressed as nmol/g tissue.
Gastric Emptying
gastroduodenal, and ileocaecal junctions were isolated, removed, and divided into segments; i.e., stomach and intestine. Then, each segment was placed in a graduated cylinder that contained 0.1 N NaOH (10 ml), and the total volume was measured. Then, the samples were cut into small pieces and homogenized for 30 s. Twenty minutes later, 1 ml supernatant was removed and centrifuged for 10 min at 3000 rpm. Subsequently, 20% trichloroacetic acid (TCA) was added for protein precipitation and cen-trifuged for 20 min at 3000. Finally, 150ll of supernatant was supplemented with 200ll of 0.5 N NaOH and absorbance was determined on a plate reader at 540 nm. The results are expressed as fractional dye retention (%) in the stomach.
Gastric Fundus and Pylorus Contractility
Strips of stomach fundus or pylorus were removed from mice and mounted in an isolated organ bath system filled with Tyrode’s solution (in mM: NaCl 136, KCl 5, MgCl2 0.98, NaH2PO40.36, NaHCO311.9, CaCl22, and glucose 5.5) adjusted to pH 7.4 and aerated with carbogen mixture (95% O2, 5% CO2). These samples were attached by cotton thread to isometric force transducers (resting tension 1 g), which were connected to a data acquisition system (Pow-erLab 8/30; ADInstruments, Australia). Contractions induced by the addition of depolarizing solution with high K? concentration (80 mM) were performed to verify the viability of the preparations and to be used as a reference in the experimental protocols.
Statistical Analysis
The values are expressed as the mean±standard error of
the mean (SEM.). A Student’s unpaired two-tailed t test was used when comparing two groups and a one-way ANOVA and Student–Newman–Keuls test was used when comparing three or more groups. In the contractility experiments, a two-way ANOVA and Bonferroni’s test were used. Differences were considered to be significant whenP\0.05.
Results
The NO Donor Increased CSE Expression in the Gastric Mucosa
Figure1a shows CSE expression by immunofluorescence in the gastric mucosa of saline-treated animals. In addition, pretreatment with SNP (a NO donor) markedly increased CSE expression, extending from the gastric epithelium submucosa to the muscle layer (Fig.1b). Figure1e shows the percentage of marked areas (the number of pixels of
marked area) that were increased significantly (P\0.05) with SNP treatment (19.01±1.62%) compared to the
saline group (7.49±1.14%). It was not observed a mod-ification of CBS expression in the gastric tissue after SNP treatment (data not shown).
H2S Donor Increases eNOS Expression in the Gastric Mucosa
Treatment with NaHS (a H2S donor) increased eNOS expression (Fig.1d) in the gastric epithelium, blood ves-sels, and muscle layer, compared to the saline-treated animals (Fig.1c). In addition, Fig.1f shows the percentage of marked areas (the number of pixels of marked area) that showed a significant increase (P\0.05) with NaHS pre-treatment (9.92 ±0.77%) compared to the saline group (2.49±0.81%).
NO or H2S Donors Did Not Change Gastric Acid Secretion
Gastric acid secretion was evaluated 4 h after pylorus ligature. As shown in Table1, pretreatment with SNP (10 mg/kg, po) or Lawesson’s reagent (27lmol/kg, po, a H2S donor) did not alter the volume of gastric juice (410.0±59.7 and 457.5±57.5ll, respectively) or total acidity (32.0 ±2.4
and 49.2±9.3 mEq[H?]/l/4 h, respectively), compared to
the saline group (428.0±39.4ll and 43.0±7.4 mEq[H?]/
l/4 h, respectively). Furthermore, these parameters were not altered in mice treated with PAG?SNP (460.0±
63.7ll and 40.8±2.9 mEq[H?]/l/4 h, respectively) or
L-NAME?Lawesson’s reagent (496.0±55.1ll and 51.2±5.3 mEq[H?]/l/4 h, respectively), compared to
ani-mals receiving only the donors (Table1). In the addition, the treatment with only PAG (a CSE inhibitor) orL-NAME (a NOS inhibitor) did not alter these parameters (date not shown).
Mutual Necessity of NO and H2S for Gastric Wall Mucus Production
Figure2shows that treatment with SNP (10 mg/kg, po) or Lawesson’s reagent (27lmol/kg, po) promoted a signifi-cant increase (P\0.05) in gastric wall mucus production (0.086±0.004 and 0.063 ±0.002 Alcian bluelg/g tissue,
respectively), compared to the saline group (0.033±0.004
Alcian blue lg/g of tissue). However, pretreatment with PAG (0.065±0.010 Alcian blue lg/g of tissue) or
L-NAME (0.043±0.007 Alcian blue lg/g of tissue) reversed the effect of SNP or Lawesson’s reagent, respectively. In addition, PAG- or L-NAME-treated ani-mals showed no change in this analysis (0.035±0.005 and
NO and H2S Are Mutually Dependent in the Regulation of Gastric Blood Flow
As shown in Fig.3, the gastric blood flow (GBF) was increased significantly (P\0.05) in animals treated with SNP (50 mg/ml) or NaHS (30lmol/ml; 122.8±2.8%
basal; 23.7% of increase, and 125.4±1.5% basal; 26.3% of increase, respectively), compared to the control group (PBS, basal). However, previous incubation with PAG orL -NAME significantly (P\0.05) reversed the effect of these donors on GBF (107.0±1.4% basal and 104.0 ±1.0%
basal, respectively).
NO and H2S Donors Work Together to Prevent Ethanol-Induced Gastric Lesions by Reducing Oxidative Stress
Figure4 shows that ethanol administration promoted intense gastric damage (104.6±6.8 mm2, Fig.4a),
accompanied by significant reduction (P\0.05) of GSH levels (276.4±27.5lg/g tissue, Fig.4b) and an increase
in MDA concentrations (121.5±11.7 nmol/g tissue,
Fig.4c) compared to the saline group (592.6±26.4lg/g tissue and 29.97±1.4 nmol/g tissue, respectively).
How-ever, treatment with SNP or NaHS prevented gastric
Fig. 1 Regulation of CSE and eNOS expressions in the gastric
mucosa.aCSE immunoreactivity in normal gastric tissue (control); ban increase in the CSE expression with NO donor treatment (SNP; 10 mg/kg, po).eQuantification of the percentage of marked areas for CSE.ceNOS immunoreactivity in normal gastric tissue;dan increase
in the eNOS expression with H2S donor treatment NaHS (150lmol/ kg, po).fQuantification of the percentage of marked areas for eNOS. Photomicrographs of gastric mucosa (magnifications, 920). The results of quantification are expressed as the mean±SEM of at least 6 animals per group. *P\0.05 versus control group
Table 1 NO or H2S did not
alter gastric acid secretion Experimental group Volume (ll) Total acid (mEq[H?]/l/4 h)
Saline 428.0±39.4 43.0±7.4
SNP 410.0±59.7 32.0±2.4
PAG?SNP 460.0±63.7 40.8±2.9
Lawesson’s reagent 457.5±57.5 49.2±9.3
L-NAME?Lawesson’s reagent 496.0±55.1 51.2±5.3 Data shown are expressed as mean±SEM of at least 6 animals per group
damage (14.6±4.8 mm2 and 22.3±7.0 mm2, respec-tively), increased GSH levels (471.0±44.1lg/g tissue
and 795.0±53.3lg/g tissue, respectively), and decreased MDA concentrations (50.1±2.3 nmol/g tissue and 34.6±4.4 nmol/g tissue, respectively) induced by ethanol
administration. In addition, pretreatment with PAG (46.9±5.6 mm2 of lesion, 205.4±25.1lg/g tissue for GSH, and 113.8±6.7 nmol/g tissue for MDA) or L -NAME (88.9±16.9 mm2 of lesion, 534.6±33.2lg/g tissue for GSH, and 31.1±2.3 nmol/g tissue for MDA)
significantly reversed (P\0.05) these effects of SNP or NaHS, respectively (Fig.4). In addition to data, the treat-ment with L-NAME (NO inhibitor) (105.80±16.01) or PAG (H2S inhibitor) (91.43±15.72) did not change the
ethanol-induced gastric damage.
In Table2, it is observed that ethanol administration caused severe microscopic lesions, characterized by hemorrhage, edema and loss of epithelial cells compared to Gastric mucosa microscopy control animals. However, treatment of animals with SNP donors or NaHS decreased significantly these microscopic damages caused by etha-nol. Pretreatment with PAG or L-NAME significantly reversed the protection observed with SNP and NaHS in this order.
NO and H2S Donors Had Opposite Effects on Gastric Emptying
Treatment with SNP significantly increased (P\0.05) gastric retention at 20 min after administration of the test meal (62.2±5.5%, Fig.5), compared to the saline group (29.3±2.9%), an effect that was prevented in animals
pretreated with PAG (36.9 ±7.0%). However,
NaHS-treated mice had accelerated gastric emptying (20.6±1.1%) (P\0.05), an effect reversed by pretreat-ment withL-NAME (31.1±2.5%). In addition, the treat-ment with only PAG or L-NAME did not change this parameter (data not shown).
Gastric Fundus and Pylorus Contractility
SNP, which cumulatively added, induced concentration-dependent relaxation in the pylorus, reaching a maximal degree of relaxation at 100 lM, which was equivalent to 31.72±1.11% of a K? 80 mM contraction (P\0.05). This effect was reversed by pre-incubation with PAG 1 mM (12.8±3.52%; Fig.6a). In addition, SNP, which
was cumulatively added on the basal tonus of gastric strips, also produced relaxation dependent on concentration with a maximum relaxation equivalent to 103.5±18.0% of a K?
80 mM contraction. However, when the addition of SNP occurred in the presence of H2S synthesis inhibitor; i.e., PAG, the curve of relaxation was significantly different (P\0.05), with a higher maximum relaxant effect of 76.4±10.0% of K? 80 mM (Fig.6c). This can be understood as a partial loss in the ability of SNP to induce relaxation due to the presence of PAG 1 mM.
In other experiments, the NaHS also promoted relaxation of the pylorus with a cumulative concentration (10–1000lM) equivalent to 23.14±5.72% of a K?80 mM contraction with
a 1000lM concentration. However, the relaxing effect
Fig. 2 Effect of the interaction between NO and H2S in gastric
mucus production. Mice received saline (control), SNP (10 mg/kg, po; a NO donor), or Lawesson’s reagent (27lmol/kg, po; a H2S donor). In other experimental groups, the animals were pretreated with PAG (50 mg/kg, po) orL-NAME (3 mg/kg, ip) 30 min before treatment with the donors, where appropriate, and killed 30 min later. Results are expressed as the mean±SEM of at least 6 animals per group. *,dP\0.05 versus control group, #P\0.05 versus SNP group, XP\0.05 versus Lawesson’s reagent group (one-way ANOVA and Newman–Keuls test)
Fig. 3 Effect of the interaction between NO and H2S in the
regulation of gastric blood flow. SNP (50 mg/ml) or NaHS (30lmol/ml) applied topically increased gastric blood flow (mea-sured by laser-Doppler flowmetry), and a decrease in gastric blood flow was observed with PAG (50 mg/kg, ip) orL-NAME (3 mg/kg, ip), respectively. Results are expressed as the mean±SEM of at least 6 animals per group. Gastric blood flow is expressed as % increase from basal values. *,dP
induced by NaHS was significantly reduced (P\0.05) in the pylorus incubated with L-NAME 300lM (7.35±1.29%; Fig.6b). In addition, NaHS also relaxed the basal tonus of the
isolated gastric fundus when added in crescent concentrations, with the maximum relaxation being reached when it was equivalent to 53.1±9.0% of a K? 80 mM contraction.
Fig. 4 Evaluation of the interaction between NO and H2S donors on
oxidative stress in gastric damage by ethanol-induced. The mice were treated with saline, SNP (10 mg/kg, po), or NaHS (150lmol/kg, po). In other experimental groups, the animals were pretreated with PAG (50 mg/kg, po) orL-NAME (3 mg/kg, ip) 30 min before of treatment with donors, where appropriate. After 30 min, gastric damage was induced by 50% ethanol and killed 1 h later.a50% ethanol-induced
gastric damage, b GSH levels, c MDA concentration. Results are expressed as the mean±SEM of at least 6 animals per group. wP
\0.05 versus saline group, *,dP\0.05 versus 50% ethanol group, #P\0.05 versus SNP?50% ethanol group, XP\0.05 versus NaHS?50% ethanol group (one-way ANOVA and New-man–Keuls test)
Table 2 Histopathologic evaluation
Experimental group Hemorrhagic
(score 0–4)
Edema (score 0–4)
Epithelial cells loss (score 0–3)
Inflammatory cells (score 0–4)
Control (saline) 0 (0–0) 0 (0–1) 1 (0–1) 0 (0–1)
Saline?ethanol 4 (3–4) 2 (2–3) 3 (2–3) 1 (0–1)
NPS?ethanol 0 (0–0)** 0 (0–1)* 1 (0–2)* 0 (0–1)
PAG?NPS?ethanol 2 (2–3)# 1 (0–3) 2 (1–3)# 1 (1–2)
NaHS?ethanol 0 (0–0)* 0 (0–1)* 1 (0–2)* 0 (0–0)
L-NAME?NAHS?ethanol 3 (2–4)W 3 (2–3)W 2 (1–3) 0 (0–0) Values denote median with minimum and maximum, respectively
Nonparametric Kruskal–Wallis, followed by Dunn’s test
However, with the previous addition of the NO synthase inhi-bitor L-NAME, the maximum relaxant effect achieved was almost the same; i.e., 47.3±8.0% of K?80 mM (Fig.6d).
Discussion
NO and H2S are important gaseous transmitters produced under physiological conditions that regulate several bio-logical processes, such as blood flow, mucus secretion, vascular tone, and inflammation, thereby maintaining nor-mal gastric functions [27]. Recent studies have shown evidence of a possible ‘‘cross-talk’’ between these two mediators, which interact with each other, influencing enzymatic expression, bioavailability, and reactivity [28]. In this regard, it has been shown a mutual dependence of NO and H2S in the regulation of different biological pro-cesses, such as angiogenesis and endothelium-dependent vasorelaxation [17], cytoprotection of ischemia/reperfusion (I/R) injury [29], and protection against pressure overload-induced heart failure [30] under conditions of acute oxidative stress in vitro [31]. However, there was no definitive evidence suggesting that there was ‘‘cross-talk’’ between these two molecules in gastric physiological functions.
In the present study, we evaluated the effects of NO and H2S donors and possible interactions between these gas-eous mediators on gastric physiological functions, which demonstrated that they interact among themselves for maintenance of stomach homeostasis, and this ‘‘cross-talk’’
Fig. 5 Effect of the interaction between NO and H2S on gastric
emptying. Mice were treated with saline, SNP (10 mg/kg, po), or NaHS (150lmol/kg, po). In other experimental groups, the animals were pretreated with PAG (50 mg/kg, po) orL-NAME (3 mg/kg, ip) 30 min before of treatment with donors, where appropriate. Thirty minutes later, they received phenol red (0.75 mg/ml) and were killed 20 min later. The results are expressed as fractional dye retention (%) in the stomach. Results are expressed as the mean±SEM of at least 6 animals per group. *,dP\0.05 versus control group, #P\0.05 versus SNP group,XP\0.05 versus NaHS group (one-way ANOVA and Newman–Keuls test)
Fig. 6 Effect of the interaction between NO and H2S in gastric
fundus and pylorus contractility. After 1 h of equilibration, we performed a cumulative curve concentration–response with SNP or NaHS.a,b Pyloric sphincter relaxation induced by SNP or NaHS, respectively, which was reversed by pre-incubation with PAG (a) or
is important in the control of mucus production, blood flow, mucosal defense (using an ethanol-induced gastric damage model; a dependent effect, at least in part due to the reduction of oxidative stress), and gastric motility, but not in the control of basal gastric acid secretion.
Initially, we performed an immunofluorescence assay for evaluation of CSE and eNOS expression in the gastric mucosa. These enzymes are produced constitutively in gastric tissue, which are important for maintaining integrity and normal physiological processes [11, 32]. Our results demonstrated that treatment with SNP (a NO donor) increased CSE expression in the gastric mucosa. Likewise, pretreatment with NaHS (a H2S donor) increased eNOS expression, compared to the saline-treated animals. In this context, Altaany et al. (2014) demonstrated that NaHS increases eNOS activity in vitro by inducing S-sulfhydra-tion, an important posttranslational modification processes, resulting in increased NO bioavailability [33]. Corroborant with our data, NaHS andL-cysteine (a substrate for CSE) have been shown to be associated with increased eNOS expression in the gastric mucosa [34]. Reciprocally, NO can regulate the endogenous concentration of H2S and SNP increased CSE expression and activity in cultured vascular smooth muscle cells (SMCs) [14]. In addition, another study showed that L-arginine (a substrate for NO) aug-mented CSE mRNA as well as its activity in lung tissues [35]. In this sense, based on the literature and according to our results, strong evidence suggests that there is a com-mon signaling pathway between NO and H2S and possible ‘‘cross-talk’’ culminates with the maintenance of gastric physiological functions.
In order, to show that this communication is possible, the animals were treated with NO or H2S donors (i.e., SNP, NaHS, or Lawesson’s reagent). Other experimental groups were pretreated with enzymatic antagonists (i.e.,L-NAME or PAG), and gastric functional parameters were evaluated, such as gastric acid secretion, mucus production, blood flow, oxidative stress (ethanol-induced gastric damage, GSH and MDA levels), and gastric motility (gastric emp-tying and contractility).
Initially, we performed an evaluation of gastric acid secretion. Investigations have shown that NO or H2S can negatively modulate gastric acid secretion output when stimulated by distention [34,36] or pentagastrin [37]. This effect is dependent, in part, on two mechanisms; directly on parietal cells or indirectly by inhibiting the release and/or action of histamine. For example, studies showed that gastric distension increases acid secretion primarily through the perception by sensory neurons of mechanical stimulation and consequently through the efferent vago-cholinergic pathway, and endogenous NO reverses this process by suppressing the release of histamine from ECL cells [38]. On the other hand, our results showed that SNP
or Lawesson’s reagent did not alter the volume of gastric juice or total acidity compared to the saline group in both basal conditions (unstimulated), an event that was not changed by pretreatment with the antagonists PAG or L -NAME, respectively. Another paper shows that H2S increases HCO3(-) secretion in the stomach of rats and
this effect depends on NO and prostaglandins, but not ATP-sensitive K(?) channels [15]. Moreover, the
treat-ment using only the antagonists also did not change this parameter. These data seem to be contradictory; however, when assessing the circumstances in which the gastric acid secretion was analyzed, we can see that the published studies carried out their analyses in stimulated conditions. On the other hand, our results are from analysis in physi-ological conditions. Together, these data suggest that NO and H2S regulate gastric acid secretion only in stimulated conditions.
Sequentially, we investigated the effect NO or H2S and the possible interaction between these gaseous transmitters on gastric mucus production. Mucus is an important gastric mucosa defense mechanism, which forms an adherent gel layer that promotes a pH gradient in the epithelial surface of the stomach, preventing the penetration of acid and pepsin [39]. Studies have reported that NO donors stimu-late mucus secretion in the gastric and colonic mucosa, improving the barrier function [40,41]. Likewise, a gen-erator of H2S increased mucus secretions, thereby increasing the protection of gastric acid-induced duodenal damage [42]. In accordance with the literature, our results showed that NO or H2S donors stimulated gastric mucus production. In addition, we demonstrated for the first time a mutual necessity between these two gaseous mediators in mucus production, because CSE and NOS inhibitor reversed the effect of NO and H2S donors, respectively.
dependent in GBF regulation to maintain vascular home-ostasis. With this line of reasoning, we demonstrated that H2S promotes cardioprotection in cardiac arrest and car-diopulmonary resuscitation, a missing benefit in eNOS deficient mice [46].
Another important gastric physiological function is the capacity to defend against mucosal injuries. To study this, we used ethanol-induced gastric damage to understand the relationship between NO and H2S in this model. Our research group recently showed that NO donors prevent gastric lesions induced by ethanol [13] and alendronate, with severity aggravated by the inhibition of NO synthesis [47]. Correspondingly, H2S donors prevented ethanol- and alendronate-induced gastric damage [12, 48] and nons-teroidal anti-inflammatory drugs (NSAIDs), which was exacerbated with a CSE inhibitor [49]. However, at the present time, there are no studies in the literature showing a possible relationship between these two mediators in gas-tric injury models. In the present study, we found that ethanol-induced gastric damage, with intense oxidative stress, reduced GSH levels and increased MDA concen-trations. On the other hand, previous administration of SNP or NaHS prevents ethanol-induced gastric damage, with restoration of GSH levels and MDA concentrations to values similar to normal. In addition, our results demon-strated that inhibition of CSE or eNOS reversed the gas-troprotection of SNP and NaHS and alterations in GSH and MDA levels, respectively. Therefore, our results suggest that NO and H2S work together to prevent ethanol-induced gastric lesions by reducing oxidative stress. These results are consistent with the findings on gastric mucus produc-tion and regulaproduc-tion of gastric blood flow, which showed a mutual necessity between NO and H2S.
It has been shown that intense production of free radicals, reduction of blood flow, and a decrease in gastric adhered mucus are major factors that are important for loss of gastric mucosa integrity [13,50]. We can therefore infer that the effect of NO and H2S donors in preventing gastric damage is, at least in part, dependent gastric mucus production for the rise in biosynthesis of PGE2[10] and regulation of blood flow, since the mucosal microcirculation is essential delivery of oxygen nutrients, and removal of free radicals, which are extremely toxic molecules into the cells [39].
In other experimental designs, we evaluated the effect of NO and H2S, and the interrelation between these systems in the regulation of gastric motility, mainly on gastric emp-tying of liquid meals in awake mice and contractility of the gastric fundus and pylorus (basal tonus). In this study, our results showed that SNP promoted delayed gastric empty-ing, which was prevented in animals pretreated with PAG. It has been shown that NO plays an inhibitory role that is important in gastrointestinal motility patterns by mecha-nisms that are dependent on cGMP [51,52]. Additionally,
nitroglycerin (a NO donor) slows gastric emptying in humans by mechanism dependent, in part, on inhibiting pyloric motility [53]. Corroborant with these results, a previous study showed that sildenafil, a phosphodiesterase-5 inhibitor, delays gastric emptying and gastrointestinal transit of liquid in rats [54]. Thus, the effect of SNP in gastric emptying could be due to an increase in gastric compliance and/or an increase in antro-pyloric-duodenal resistance. In the present study, we also showed that SNP promoted pylorus and gastric fundus relaxation, which was prevented partially by pre-incubation with PAG. These results are in accordance with the literature, which showed that SNP caused a concentration-dependent relaxant response in the gastric fundus, supporting NO being involved in the inhibition of gastrointestinal smooth muscle by non-cholinergic (NANC) nerves [55]. Based on these findings, we can infer that fundus relaxation has primary importance in the decrease in SNP-induced gastric com-pliance, which could explain the ability of NO donors to decrease gastric emptying, and this effect is, at least in part, dependent on H2S.
On the other hand, NaHS accelerated gastric emptying, an effect reversed in animals pretreated withL-NAME. Our results also showed that NaHS promoted a relaxation of the pylorus and gastric fundus, which was prevented in the pylorus incubated withL-NAME, but not the fundus. In this context, our research group showed that H2S donors enhanced gastric emptying and induced relaxation of the pyloric sphincter by mechanisms that involved the activa-tion ofKATPchannels and afferent neurons [56]. Based on these results, we propose that H2S donors can decrease antro-pyloric-duodenal resistance by inducing pyloric sphincter relaxation, which could explain the ability of H2S donors to enhance gastric emptying and that this effect is dependent on cross-talk with NO.
Conclusions
In summary, NO and H2S interact in the regulation of gastric physiological functions, and this ‘‘cross-talk’’ is important in the control of mucus secretion, gastric blood flow, gastric mucosal defense, and gastric motility, but not in the control of basal gastric acid secretion. Thus, we infer that mutual necessity of these gasotransmitters plays a role of paramount importance in maintaining a state of equi-librium in gastric physiological functions.
Acknowledgements The authors gratefully acknowledge the
Compliance with ethical standards
Conflict of interest None.
References
1. Martin F, Kenneth RO. Embracing sulfide and CO to understand nitric oxide biology.Nitric Oxide. 2013;35:2–4.
2. Wang R. Two’s company, three’s a crowd: can H2S be the third endogenous gaseous transmitter?FASEB J. 2002;16:1792–1798. 3. Albrecht EW, Stegeman CA, Heeringa P, Henning RH, van Goor H. Protective role of endothelial nitric oxide synthase.J Pathol. 2003;199:8–17.
4. Matsuda NM, Miller SM. Non-adrenergic non-cholinergic inhi-bition of GI smooth muscle and its intracelular mechanism(s).
Fundam Clin Pharmacol. 2010;24:261–268.
5. Takahashi T. Pathophysiological significance of neuronal nitric oxide synthase in the GI tract.J Gastroenterol. 2003;38:421–430. 6. Lowicka E, Bełtowski J. Hydrogen sulfide (H2S)—the third gas of interest for pharmacologists.Pharmacol Rep. 2007;59:4–24. 7. Kimura H. Hydrogen sulfide: its production, release and
func-tions.Amino Acids. 2011;41:113–121.
8. Wallace JL, Wang R. Hydrogen sulfide-based therapeutics: exploiting a unique but ubiquitous gasotransmitter.Nat Rev Drug Discov. 2015;14:329–345.
9. Dal-Secco D, Cunha TM, Freitas A, et al. Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: role of ATP-sensitive potassium channels.J Immunol. 2008;181: 4287–4298.
10. Magierowski M, Jasnos K, Kwiecien S, et al. Endogenous pros-taglandins and afferent sensory nerves in gastroprotective effect of hydrogen sulfide against stress-induced gastric lesions.PLoS One. 2015;10:e0118972.
11. Fiorucci S, Distrutti E, Cirino G, Wallace JL. The emerging roles of hydrogen sulfide in the gastrointestinal tract and liver. Gas-troenterology. 2006;131:259–271.
12. Medeiros JV, Bezerra VH, Gomes AS, et al. Hydrogen sulphide prevents ethanol-induced gastric damage in mice: role of KATP channels and capsaicin-sensitive primary afferent neurons. J Pharmacol Exp Ther. 2009;330:764–770.
13. Santana AP, Tavares BM, Lucetti LT Jr, et al. The nitric oxide donor cis-[Ru(bpy)2(SO3)NO](PF6) increases gastric mucosa protection in mice—involvement of the soluble guanylate cyclase/KATPpathway.Nitric Oxide. 2015;45:35–42.
14. Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide.Biochem Biophys Res Commun. 1997;237:527–531. 15. Takeuchi K, Ise F, Takahashi K, Aihara E, Hayashi S.
H2S-induced HCO3
-secretion in the rat stomach-involvement of nitric oxide, prostaglandins, and capsaicin-sensitive sensory neurons.Nitric Oxide. 2015;30:157–164.
16. Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of H(2)S as a novel endogenous gaseous K(ATP) channel opener.
EMBO J. 2001;20:6008–6016.
17. Coletta C, Papapetropoulos A, Erdelyi K, et al. Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation. Proc Natl Acad Sci USA. 2012;109:9161–9166.
18. Magalha˜es PA, de Brito TS, Freire RS, et al. Metabolic acidosis aggravates experimental acute kidney injury.Life Sci. 2016;146: 58–65.
19. Reitman S. Gastric secretion. In: Frankel S, Reitman S, Son-nenwirth AC, eds.Gradwohl’s Clinical Laboratory Methods and Diagnosis. London: Mosby; 1970:1949–1958.
20. Carvalho ACS, Guedes MM, Sousa AL, et al. Gastroprotective effect of mangiferin, a xanthonoid from mangifera indica, against gastric injury induced by ethanol and indomethacin in rodents.
Planta Medica (Stuttgart). 2007;73:1372–1376.
21. Corne SJ, Morrissey SM, Woods RJ. A method for the quanti-tative estimation of gastric barrier mucous. J Physiol. 1974; 242:116–117.
22. Ancha H, Ojeas H, Tedesco D, Ward A, Harty RF. Somatostatin-induced gastric protection against ethanol: involvement of nitric oxide and effects on gastric mucosal blood flow. Regul Pept. 2003;110:107–113.
23. Camara PRS, Ferraz GJN, Franco-Penteado CF. Ablation of primary afferent neurons by neonatal capsaicin treatment reduces the susceptibility of the portal hypertensive gastric mucosa to ethanol-induced injury in cirrhotic rats. Eur J Pharmacol. 2008;589:245–250.
24. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent.
Anal Biochem. 1968;25:1192–1205.
25. Mihara M, Uchiyama M. Determination of malonaldehyde pre-cursor in tissues by thiobarbituric acid test. Anal Biochem. 1978;86:271–278.
26. Reynell PC, Spray GH. The simultaneous measurement of absorption and transit in the gastrointestinal tract of the rat. J Physiol. 1978;15:361–371.
27. Farrugia G, Szurszewski JH. Carbon monoxide, hydrogen sulfide, and nitric oxide as signaling molecules in the gastrointestinal tract.Gastroenterology. 2014;147:303–313.
28. Pae HO, Lee YC, Jo EK, Chung HT. Subtle interplay of endogenous bioactive gases (NO, CO and H(2)S) in inflamma-tion.Arch Pharm Res. 2009;32:1155–1162.
29. King AL, Polhemus DJ, Bhushan S, et al. Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase-nitric oxide dependent.Proc Natl Acad Sci USA. 2014;1113:182–187. 30. Kondo K, Bhushan S, King AL, et al. H2S protects against pressure overload-induced heart failure via up-regulation of endothelial nitric oxide synthase. Circulation. 2013;127:1116– 1127.
31. Al-Magableh MR, Kemp-Harper BK, Ng HH, Miller AA, Hart JL. Hydrogen sulfide protects endothelial nitric oxide function under conditions of acute oxidative stress in vitro.Naunyn Sch-miedebergs Arch Pharmacol. 2014;387:67–74.
32. Guth PH. Current concepts in gastric microcirculatory patho-physiology.Yale J Biol Med. 1992;65:677–688.
33. Altaany Z, Ju Y, Yang G, Wang R. The coordination of S -sulfhydration,S-nitrosylation, and phosphorylation of endothelial nitric oxide synthase by hydrogen sulfide. Sci Signal. 2014;7: ra87.
34. Mard SA, Askari H, Neisi N, Veisi A. Antisecretory effect of hydrogen sulfide on gastric acid secretion and the involvement of nitric oxide.BioMed Res Int. 2014;2014:1–7.
35. Shi L, Du JB, Pu DF, Qi JG, Tang CS. Regulation of endogenous cystathionine-gamma-lyase gene expression in high pulmonary flow by nitric oxide precursor.Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2006;22:343–347.
36. Kato S, Kitamura M, Roman P, Takeuchi K, Takeuchi K. Role of nitric oxide in regulation of gastric acid secretion in rats: effects of NO donors and NO synthase inhibitor. Br J Pharmacol. 1998;123:839–846.
protective reflex mediated by cerebral nitric oxide. Proc Natl Acad Sci. 1996;93:14839–14844.
38. Kitamura M, Sugamoto S, Kawauchi S, Kato S, Takeuchi K. Modulation by endogenous nitric oxide of acid secretion induced by gastric distention in rats: enhancement by nitric oxide synthase inhibitor.J Pharmacol Exp Ther. 1999;291:181–187.
39. Laine L, Takeuchi K, Tarnawski A. Gastric mucosal defense and cytoprotection: benchto bedside.Gastroenterology. 2008;135:41–60. 40. Brown JF, Keates AC, Hanson PJ, Whittle BJ. Nitric oxide generators and cGMP stimulate mucus secretion by rat gastric mucosal cells.Am J Physiol. 1993;265:G418–G422.
41. Schreiber O, Petersson J, Walde´n T, et al. iNOS dependent increase in colonic mucus thickness in DSS-colitic rats. PLoS ONE. 2013;8:e71843.
42. Magierowski M, Jasnos K, Kwiecien´ S, Brzozowski T. Role of hydrogen sulfide in the physiology of gastrointestinal tract and in the mechanism of gastroprotection. Postepy Hig Med Dosw. 2013;67:150–156.
43. de Souza GF, Taladriz-Blanco P, Velloso LA, de Oliveira MG. Nitric oxide released from luminal S-nitroso-N-acetylcysteine increases gastric mucosal blood flow.Molecules. 2015;20:4109– 4123.
44. Yang G, Wu L, Jiang B, et al. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase.
Science. 2008;322:587–590.
45. Capettini LSA, Cortes SF, Silva JF, Alvarez-Leite JI, Lemos VS. Decreased production of neuronal NOS-derived hydrogen per-oxide contributes to endothelial dysfunction in atherosclerosis.Br J Pharmacol. 2011;164:1738–1748.
46. Minamishima S, Bougaki M, Sips PY, et al. Hydrogen sulfide improves survival after cardiac arrest and cardiopulmonary resuscitation via a nitric oxide synthase 3-dependent mechanism in mice.Circulation. 2009;120:888–896.
47. Silva RO, Lucetti LT, Wong DV, et al. Alendronate induces gastric damage by reducing nitric oxide synthase expression and
NO/cGMP/KATP signaling pathway. Nitric Oxide. 2014; 40:22–30.
48. Nicolau LA, Silva RO, Damasceno SR, et al. The hydrogen sulfide donor, Lawesson’s reagent, prevents alendronate-induced gastric damage in rats.Braz J Med Biol Res. 2013;46:708–714. 49. Fiorucci S, Antonelli E, Distrutti E, et al. Inhibition of hydrogen
sulfide generation contributes to gastric injury caused by anti-in-flammatory nonsteroidal drugs.Gastroenterology. 2005;129:1210– 1224.
50. Nakagiri A, Sunamoto M, Takeuchi K, Murakami M. Evidence for the involvement of NADPH oxidase in ischemia/reperfusion-induced gastric damage via angiotensin II.J Physiol Pharmacol. 2010;61:171–179.
51. Russo A, Fraser R, Adachi K, Horowitz M, Boeckxstaens G. Evidence that nitric oxide mechanisms regulate small intestinal motility in humans.Gut. 1999;44:72–76.
52. Huang X, Meng XM, Liu DH, et al. Different regulatory effects of hydrogen sulfide and nitric oxide on gastric motility in mice.
Eur J Pharmacol. 2013;720:276–285.
53. Sun WM, Doran S, Jones KL, et al. Effects of nitroglycerin on liquid gastric emptying and antropyloroduodenal motility.Am J Physiol. 1998;275:G1173–G1178.
54. Rosalmeida MC, Saraiva LD, da Grac¸a JR, et al. Sildenafil, a phosphodiesterase-5 inhibitor, delays gastric emptying and gas-trointestinal transit of liquid in awake rats. Dig Dis Sci. 2003;10:2064–2068.
55. Vetri T, Bonvissuto F, Marino A, Postorino A. Nitrergic and purinergic interplay in inhibitory transmission in rat gastric fun-dus.Auton Autacoid Pharmacol. 2007;27:151–157.
