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  • H2S Is A Key Antisecretory Molecule Against Cholera Toxin Induced Diarrhoea in Mice

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    artigo científico: H 2 S is a key antisecretory molecule against cholera toxininduced diarrhoea in mice: Evidence for non-involvement of the AC/cAMP/PKA pathway and AMPK.

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    artigo científico: H 2 S is a key antisecretory molecule against cholera toxininduced diarrhoea in mice: Evidence for non-involvement of the AC/cAMP/PKA pathway and AMPK.

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    H2S Is A Key Antisecretory Molecule Against Cholera Toxin Induced Diarrhoea in Mice

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    artigo científico: H 2 S is a key antisecretory molecule against cholera toxininduced diarrhoea in mice: Evidence for non-involvement of the AC/cAMP/PKA pathway and AMPK.

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    Nii nid 762018) 152-165, ‘Contents lists available at ScienceDirect g2 138 Nitric Oxide ELSEVIER journal homepage: www.clsevier.com/locatelyniox HS is a key antisecretory molecule against cholera toxin-induced diarrhoea in mice: Evidence for non-involvement of the AC/cAMP/PKA. pathway and AMPK Francisca B.M. Sousa *', Luan K.M. Souza “-', Nayara A. Sousa *', Thiago S.L. Aratijo *', Simone de Aratijo ”*', Dvison M. Pacifico ‘, Irismara S. Silva “, Renan O. Silva °, Lucas A.D. Nicolau °, Fabiana M. Souza °, Marcelo C. Filgueiras *’, Jefferson S. Oliveira °, Marcellus H.LP. Souza °, Jand Venes R. Medeiros **'"~ ‘ otetnolgy end diversity Center Recarch BIOTEC Federal Universo Pa, Pn, Pk, Bait ina lnc Research Cente, NPM, ederai Unversity of Pe, Teresi, Pau Bait «Deparment of Barolo), Fal of Hele, dra Univesity Ceara Fatlzs, Cer, Bri 4 Physiology and Pharmacology, Federal University of Mnas Gea, Belo Moron Mins Geral, Bru < Deparment of silo) and Pharmacology, Federal Unters of ear Fortaleza, Car, Bazil "Laboratory of Experimental Pysopharmacoloy dra Univers of Pau, Parnas, Pat, Bail (on ARTICLE INFO ABSTRACT ‘re None Received 24 May 2017 Received in reise form 18 September 2017 Accepted 20 September 2017 Avaabe online 22 September 2017 Fyérogen sulphide (HS) sa gasotransmitir that participates in various physiological and pathophys fologiea! processes within the gastrointestinal tract We studied the effects and possible mechanism of {action oF H,S in secretory diarrhoea caused by cholera toxin (T)-The possible mechanisms of action of eS were investigated using an intestinal uid secetion model in isolated intestinal loops on anaes- ‘thetzed mice treated with CT, NHS and Lawesson's reagent and i-cysteine showed antisecretory activity ‘through reduction of intestinal uid secretion and loss of Cl induced by CT. Pretreatment with an in- Iibitor of eystathionine-y-yase (CSE). -propargylglycine (PAG), reversed the effect of cysteine and «caused severe intestinal secretion Co-teatment with PAG and a submaximal dose of CT increased n= Diane dacises testinal uid secretion, thus supporting the role of 1h inthe pathoptysioogy of cholera, CF increased holers the expression of CSE andthe production of HS Pretreatment with PAG didnot reverse the effet of SQ fw 22536 (an AC inhibitor}, bupivacaine (inhibitor of cAMP prodction), KT-5720 (a PKA inhibitor). and AAICAR an AMPK activator). The treatment with Forskoln does nat reverse the effets ofthe HS donors Coctreatment with either NaHS or Lawesson’s reagent and dorsomorphin (an AMPK inhibitor) didnot reverse the effect ofthe HS donors. HS has antisecretory activity and is an essential molecule for protection against the intestinal secretion induced by CT. Thus, HS donor drugs are promising candi {ates for cholera therapy. However, more studies are needed to elucidate the possible mechanism of axtion, ieywort (Gaseous mentors (© 2017 Elsevier ine. Al eights reserved 1. Introduction, Cholera, a disease characterised by acute secretory diarthoea, is, caused by intestinal infection from the gram-negative bacterium Vibrio cholerae serogroups O1 and 0139 [1 This disease causes * Coresponding athor. Ae Sao Sebsstie, 289, CEP: 6202-020, Paraiba, raat mal addres: jnvenes@ufpiedube QR Medes) epider 01016j.250e 201708007 ao. 8}0 2017 Elsevier ne A ight esr large epidemics worldwide and isa serious threat to public health, particularly in developing countries [2|. The main virulence factor responsible for the dehydration observed during cholera is cholera toxin (CT), which is secreted by V. cholerae into the small intestine 6 CCT causes severe diarrhoea through a direct effect on intestinal, epithelial cells. More specifically, CT binds to intestinal enterocytes via interaction of five identical B-subunits with the GMI ganglio- side receptor, which is then internalized through retrograde endocytosis [4], Within the cell, the A subunit causes constitutive FAM Sosa et ab / Nn Cue 75 (2018) 152-163 Py Abbreviations AMPK AMP-activated protein kinase AC Adenylyl cyclase cAMP Cyclic adenosine monophosphate CBS Cystathionine-f-synthetase CFTR Cystic fibrosis transmembrane conductance regulator CSE _Cystathionine-y-tyase CT Cholera toxin GMI ganglioside receptor GSH Glutathione reduced HS Hydrogen sulphide NaHS — Sodium hydrasulfide ORS Oral rehydration solution PAG —_propargylelycine PKA Protein kinase A activation of Adenylyl cyclase (AC) by inactivation of the stimula- {ory G protein Gso, resulting in elevated levels of intracellular cAMP [5], The increase in intracellular cAMP results inactivation of pro- tein kinase A (PKA) and subsequent Cl channel opening [cystic fibrosis transmembrane conductance regulator (CFTR)], causing an excessive secretion of CI- accompanied by the esmatic movement of a large quantity of water into the intestinal lumen (5. The fluid Joss is often so rapid and large that patients can die left untreated 7 ‘The treatment of cholera involves replacing water and lost, electrolytes, and consists of the administration of oral rehydration solution (ORS). Although this solution is effective for hydration and reduces mortality, ORS neither inhibits cholera toxin-mediated excessive secretion nor eliminates the infection from V. cholera [8 Consequently, ORS does not decrease diarrhoea in the short term, Because of these factors, curtently there are no pharmaco- logical approaches to treat cholera; treatment could be achieved by investigation of signalling molecules that can inhibit the increase of seeretion induced by cholera toxin. Among the molecules that play many important physiological and pathophysiological roles in hu- ‘man health, gaseous mediators such as hydrogen sulphide (HS) are of particular interest HS is a gaseous signalling molecule endogenously generated mainly from t-eysteine through the activity of the enzymes cys- tathionine-y-lyase (CSE) and cystathionine-f-synthetase (CBS), although there are alternative sources (e.g. cysteine aminottans- {erase and/or 3-metcaptosulfotransferase |. Studies have shown that 2S has important biological effects on the intestinal epithe- lium, local microcirculation, and inflammatory processes, and promotes changes in gastrointestinal smooth muscle |10—12). In addition, recent data have shown that H.S inhibits the AC/cAMP pathway [12,14] and promotes the activation of AMP-activated protein kinase (AMPK) (15], an important kinase that phosphory- lates the CFTR channel and inhibits the secretion of chloride ions in intestinal epithelial cells | 16), These effects indicate that HaS may have beneficial effects on the pathophysiology of cholera, whici involves increased activation of AC. Therefore, based on this back- sground information and owing to the absence of reports about the Tole of HS inthe secretory diarrhoea induced by cholera toxin, we evaluated the potential antisecretory effect of HyS in secretory diarhoea induced by the enterotoxin of. cholerae and the possible ‘mechanisms involved in this effect. 2. Materials and methods 21, Chemicals and drugs All drugs were purchased from Sigma Chemical Company, St. louis, MO, USA. 5-Aminoimidazole-4-carboxamide 1-f-o-ribofur- anoside, (acadesine; N1-(f-v-ribofuranosyl)-5-aminoimidazole-4- ‘carboxamide: AICAR), 9-tetrahydro-2-furanyl)-9H-purin-6-amine (9-THE-Ade: SQ 22536), Forskolin and bupivacaine HCL were dis- solved in 0.9% saline and administered directly on the loops at concentrations of 1 mM, 0.01 M, 20 jM and 100 uM, respectively. (9840S, 12R}-2,39,10,11,12-Hexahydro-10-hydroxy-9-methyl-1- (0x0-9,12-epoxy-IH-diindolo| 1.2.3-fg:32'.V-Kl)pyrrolo[3.4-i] [1.6] benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) and 6-|4- (2-piperidin-1-ylethoxy)phenyl|-3-pyridin-4-yipytazolo [1.5-alpy- rimidine (dorsomorphin; compound C) were dissolved in PBS and administered directly to the loops at 1 ug and 30 uM, respectively. These doses were selected from published studies and the previous ‘work of our research group [17-20 -cysteine (HS precursor), 2 propargylglycine (PAG: inhibitor of CSE) and NaHS (H2S donor) ‘were dissolved in 0.9% saline and were administered by gavage [21], Lawesson’s reagent (HS donor) was suspended in 1% carboxymethylcellulose and also administered orally. Cholera toxin ‘was dissolved in PBS and administered directly tothe loops (1 12). Al other chemicals and reagents were of analytical grade and ob- tained from standard commercial suppliers ‘The pl! of the studied solutions are in the range of 4~5, More specifically the pH of NaHS was 4.67, -cysteine 5.38 and Lawesson's reagent 4.12, which was adjusted to pH 3. The pH adjust of the Lawesson’s reagent solution was necessary in view of the fact that the literature shows that GYY4I37, or morpholin-4-ium 4 ‘methoxyphenyl(morpholino) phosphinodithioate, a Lawesson's reagent derivative, has a greater release at acidic pH like pH 3.0 and very slowly release under physiological conditions (pH 7.4) [22.23] 22, Animals Al in vivo work was subject to internal ethical review and conducted in aecordance with Home Office requirements under the ‘Animals Scientific Procedures Act (1986), with the approval ofthe Local Ethical Committee (No. 079/2015, Ethics Committee in Research of the Federal University of Piaui, Brazil. Swiss mice of Doth sexes (weight: 25~30 g) were used in this study. They were maintained in cages under laboratory conditions at a temperature of (23 1) °C under a 12 h lightidark cycle with free access to a standard pellet diet and drinking tap water ad libitum, Mice were randomly assigned into groups of six to eight. They were deprived ‘of food for 24 h before the experiments, but sil allowed free access to water. 23. Cholera toxin-induced fluid secretion in closed intestinal loops ‘The antisecretory effect of HpS was measured by assay of the intestinal fluid secretion induced by cholera toxin inocdlation, as previously described by Ref. (24), with some modifications. Mice ‘were treated by gavage with -cysteine (10 oF 50 mg kg"), NaHS (3, 9 or 27 jumol kg’ '), Lawesson’s reagent (3, 9 or 27 mol kg"). or saline (25 mL ke). Another group received o-propareylelycine (PAG) 100 mg kg" by gavage, an inhibitor of CSE, 30 min before administration of t-cysteine (50 mg kg. by gavage). After 30 min, the mice were intraperitoneally anaesthetized with a combination ‘of xylazine hydrochloride (8 mg kg") and ketamine (40 mg kg") and a median laparotomy was performed to expose the small i testine. A portion of the jejunum was isolated and closed with double ties to form an intestinal loop measuring approximately, st BM Soa a. i aie 76 (2008) 152-163 2-.em. Intestinal loops were inoculated with 100. ofphosphate- buffered saline (PBS) (for the negative control group treated with saline) oF 100 ul of CT dissolved in PBS at a dose of 1 a/loop forthe ‘groups treated with saline (positive contro for intestinal secretion), Leesteine alone, PAG + cysteine, and H,5 donors. The intestinal loops were returned to the abdominal cavity, the abdominal incl- sion was closed with sutures, and the mice were allowed to recover from the anaesthesia. Four hours after injecting anaesthesia, the rice were euthanized, and the closed loops were rapidly removed from the abdominal cavity. The fluid secretion was measured indirectly as the ratio of loop weight ro length expressed in gem Tissue samples of isolated intestinal loops were collected and weighed for immediate analysis of H,S tissue levels. Other sample was fixed in 10% formalin immediately after their collection for Subsequent assessment of intestinal CSE expression by immuno- histochemistry. The intestinal contents accumulated in each closed loop were collected separately to allow measurement of chloride ion (CL) concentration In another set of experiments, we evaluated the direct effect of HS donors against CT induced diarehoea via intzaluminal adm istration using an isolated intestinal loop model. The mice were anaesthetized and the loops were inoculated with 50 uL of the NaHS (27 uM) or Lawesson’s reagent (27 ND, and 5 min later, the loops were inoculated again with 100 of the CT (1 /loop). The intestinal laops were returned to the abdominal cavity and Oud secretion was measured as described above. We also evaluated the effect of on physiological secretion in the same model of intestinal loop isolate, with the treatment of the mice by gavage with NaHS 27 umol kg! or Lawesson's reagent 27 yimol kg and the loops were inoculated with 100 of PBS. 24. Effect of PAG pretreatment on fluid secretion in closed intestinal loops with a submaximal cholera toxin dose Mice were treated by gavage with saline (25 mL kg“) or PAG (100 mg kg"). After 30 min, the animals were anaesthetized and diarhoea induction in the isolated intestinal loop was conducted as described above. Intestinal loops were inoculated with 100 ul of the PBS for the negative control group treated with saline alone, and 100 nl of the CTat a dose of 0.5 pglloop for the groups treated with saline and PAG. The loops were returned to the abdominal Cavity and the abdominal incsion was closed with sutures. Four hours after anaesthesia, the mice were euthanized and the closed loops were rapidly removed from the abdominal cavity. The Nuid secretion was measured was measured as described in item 23, 25. Determination of chloride ion concentration in intestinal fluid Secretion Fluid secretion accumulated in each closed loop of the animals pretreated by, gavage with .-cysteine 90 mg kg ', NaHS 27 mol kg", Lawesson’s reagent 27 ymol kg! or saline 255 ml kg" and inoculated inthe loops with PAS (for the negative control group treated with saline) or CT fr the other groups, ob- tained previously in item 23 described, were used to determine the intestinal concentration of chloride ions. This analyse was per- formed by standard procedures according to the manufacturer's instructions (abtest, Lagoa Santa, MG, Brazil). Briefly, the super- natant ofthe samples was collected by centrifugation at 4000 rpm for 10 min. Next the secretion was diluted ina volume rato of 1:2 with distilled water, The samples were mixed with specific reagent kts for 2 min. After this ime period, the absorbance was measured at 470 nim on a spectrophotometer and the values obtained were expressed in mEq Lt 26. Evaluation of intestinal absorption on closed loops The effect of HS on intestinal absorption was determined following the method previously described by Ref. [25]. Initially, the animals and intestinal loops were prepared as previously described, The loops were inoculated with 200 yl. of PRS (negative control for absorption), 200 iL of PBS containing 10 mM glucose (positive control for absorption), or 200 iL of PBS containing 1- cysteine (50 mg kg"), 200 yl of NaHS 27 jM, and 200 yd of Law- esson's reagent 27M. The intestinal loops were returned to the abdominal cavity and the abdominal laparotomy median was closed with sutures. At 30 min after inoculation of the loops the animals were euthanized, the abdominal cavity was reopened, and the closed intestinal lops were removed. The loops were weighed with and without the luminal contents, and the absorbed mass was calculated by subtraction of the two values. The percentage fluid absorption was measured indirectly from the loop weight to length ratio. 27. Immunohistochemistry for cystathionine~y-tyase (CSE) Isolated bowel samples ofthe animals pretreated by gavage with, saline 25 mL kg? and inoculated in the loops with PBS or CT, obtained previously in item 2.3 described, were used to determine the expression of CSE. For this formalin-fixed paraffin-embedded intestinal loop tissues were sliced into sections of 5 jim. After eparaffinisation and rehydration, the sections were placed in ‘Target Retrieval Solution ($1700, DAKO), pH 90, for 30 min in a 95 °C water bath. Endogenous peroxidase was blocked with 3% 11,02 for 20 min to reduce nonspecific binding, and then incubated with an anti-CSE primary antibody (Abnova Corporation; 1:200 dilution) for 1 hat 25 °C. Next, the sections were incubated for 30 min with a polymer (EnVision + Dual Link System-HRP; K4061, DAKO), The antibody binding sites were visualized by incubation with 3.3"-diaminobenzidine (DAB) (K3467, DAKO) solution. Intestinal tissue cells were identified based on their specific ‘morphology, localisation and staining behaviour. The total cell count was obtained at 40X magnification. Each microscope slide comprised two specimens of the intestinal segment. The immu- rnopositive cells from the intestinal vili or crypts and other intes- tinal layers from each intestinal segment were counted using alight ‘microscope by a blinded pathologist. 28. Measurement of HS in bowel tissue Isolated bowel samples of the animals pretreated by gavage with, saline 25 mL kg’ and inoculated in the loops with PBS or CT, ‘obtained previously in item 2.3 described, were used tothe levels of HES by the method described in Ref. [26]. This method allows the indicect quantitation of HS levels in tissues by the use of an anti- ‘oxidant bufter diluted in the sample of interest. This buffer contains sodium salicylate, ascorbic acid, and NaOH, which convert all HyS into the S- ion, which can be measured by a sulphide ion micro- electrode. Using this protocol, the intestinal loops were weighed and homogenized in diluted antioxidant buffer. The homogenate was inserted into the sulphide ion microelectrode (Lazar Research Laboratories, Los Angeles, CA, USA) connected to a pH meter (model 66230MJenco instruments, San Diego, CA, USA) and the levels of were measured following the manufacturer's recommendations. ‘The standard solutions for the calibration curve were prepared using NaHS as the sulphide donor. FAM Sosa et ab / Nn Cue 75 (2018) 152-163 5 29, Roles of adenylate cyclase (AC), cAMP, and protein kinase A (PKA) in the antisecretory effect of Fas ‘To identify the roles of AC, cAMP, and PKA in the antisecretory effect of HS, mice were treated by gavage with PAG (100 mg kg") or saline (25 mL kg). After 25 min, the animals were anaes- thetized and a median laparotomy was performed to expose the small intestine. A portion of the jejunum was isolated and loops ‘were directly inoculated with 50 pL of SQ 2536 (0.01 Mjloop}, bupivacaine (100 jM/loop), or KT-5720 (1 yg/loop), which are in- Inbitors of AC, cAMP, and PKA, respectively. After 5 min, intestinal loops were then inoculated with 100 yl of PBS for the negative control group treated with saline alone and 100 jLof CT (1 jglloop) for all other groups. The intestinal loops were returned to the abdominal cavity and the fluid secretion was measured as ‘described in item 2.3. Although bupivacaine is frequently used with local anaesthetic, it can also inhibit cAMP production as observed in a bupivacaine cardiotoxicity assay (17) ‘To validate previous studies and to investigate the role of AC on, the antisecretory effect of H,S, we used forskolin, an AC activator [27], The mice were anaesthetized and a median laparotomy was performed to expose and isolate the small intestinal loop as described above. The loops were inoculated with 50 ul of forskolin 20 Mor 100 jLof PBS, ter 5 min, the loops were inoculated with 50 ul of NaHS 27 uM or 50 pL of Lawesson’s reagent 27 uM. Finally, 5 min later, the intestinal loops were inoculated with 100 yL of PBS {or the negative control group and 100 uL of CT (1 ugiloop) for all the other groups. The intestinal loops were retumed (© the abdominal cavity. After 4h, the mice were euthanized, and closed loops were removed in order to evaluate the fluid secretion as previously in item 23. 2.10, Assay ofeyelic AMP levels Isolated bowel samples ofthe mice treated with saline and PBS (100 tjloop), saline and cholera toxin (1 wg/loop), NaHS 27 mol kg? and cholera toxin and Lawesson’s reagent 27 umol kg”! and cholera toxin, were used to determine the levels fof CAMP. After completion of the treatment described above, the tissue samples were rapidly frozen in liquid nitrogen. Frozen tissue samples were homogenized in 10% trichloroacetic acid at 2-8 °C, followed by centrifugation at 2000g for 15 min at 4°C Pellets were neutralized in 1 N NaOH for protein determination by the Bio-Rad method, with bovine serum albumin as standard, The supernatant ‘was recavered and washed four times with 5 vol of water-saturated liethyl ether. The remaining aqueous extract was dried and frozen at ~80 °C to cAMP assays, For determination of cAMP levels in ‘issue samples, an enzyme immunoassay kit from Amersham Bi sciences (Piscataway, NJ) was used. Briefly, antibodies 10 cAMP ‘were added to each sample and allowed to incubate at 3-5 °C for 2h. Next, samples were treated with cAMP-peraxidase conjugate nd allowed to incubate for 60 min, followed by washing. Imme- ately after the final wash, enzyme substrate was added and ‘samples were incubated at room temperature for 6 min. Reactions ‘were terminated with 100 jl of 1 M H2S04 and optical densities ‘were determined in a plate reader at 450 nm. 2.11, Role of AMP-activated protein kinase (AMPK) in the ‘anisecretory effect of Has To evaluate the role of AMPK in the antisecretory effect of HS, alternative experimental protocols were used involving AICAR, an AMPK activator, or dorsomorphin, an AMPK inhibitor. In the experiment with AICAR, mice were treated by gavage with PAG (100 mg kg") or saline (2.5 mL kg"). After 25 min, the animals ‘were anaesthetized and a median laparotomy was performed to ‘expose the small intestine. A portion of the jejunum was isolated and loops were directly inoculated with S0 ul of AICAR 1 mM or 100 iL of PBS. After 5 min, the intestinal loops were then inocu lated with 100 yl. of PBS for the negative control group treated ‘with saline alone and 100 ul.of CT(1 ygiloop) forall other groups. The intestinal loops were returned to the abdominal cavity and the fuid secretion was measured apreviously described in item 23. In the second experimental protocol. dorsomorphin was used, For this, the mice were anaesthetized and a median laparotomy was pperformed to expose and isolate the small intestinal loop as described above. A portion of the jejunum was isolated and loops ‘were inoculated with $0 ul of dorsomorphin 30 4M or 100 ul of PBS. After 5 min, the intestinal loops were then inoculated with 50 uLof NaHS or Lawesson's reagent (both at 27 uM). Finally after a further 5 min, the intestinal loops were inoculated again with 100 ul of PBS for the negative control group treated with saline alone and 100 ui of CT (1ug/toop) forall the other groups. The in- testinal loops were returned to the abdominal cavity and the fluid secretion was measured apreviously described in item 2.3. 2.12, Data analysis ‘The data and statistical analyses comply with the recommen- dations on experimental design and analysis in pharmacology. Data ‘were expressed as mean + SEM, and sample size in this study varied between n = 6-8, Statistical analysis was performed using, GraphPad Prism (Version 5.0) software. Statistical significance of the differences between groups was determined by one-way analysis of variance (ANOVA) followed by multiple comparisons Using the Student-Newman-Keuls test. To analyse the HS levels in intestinal tissues, the number of immunostained cells per field in the immunohistochemical assays were compared by a non- parametric test (t-test) followed by the Mann-Whitney test. P values of <0.05 were considered to be statistically significant in all the analyses 3. Results 3. H,S decreases intestinal fluid accumulation in cholera roxin- treated intestinal clased loops {A significant decrease (p < 0.001) in the accumulation of in- testinal fluid secretion was observed in the groups treated with the Nal 3 mol kg” (0.078 + 0.008 g cm"), NaHS 9 jmol kg" (0.049 + 0005 g cm') and NaHS 27 mol kg! (0.047 + 0.009 cm), Lawesson’s reagent 3 ymol kg-! (0.088 + 0.006 g em), Lawesson's reagent 9 yimol kg! (0.063 + 0.005 g em-!) and tawesson’s reagent 27 ymol kg (0.047 + 0.006 g em”) (Fi. 1A, Fig. 1B) and the precursor of HS synthesis, -eysteine 10 mg kg ',(0071 + 011 g em") and 50 mg, kg (0.054 «: 0.006 gem) (Fig. 1C) as compared to the f20up in which the loop was injected with cholera toxin (0.162 + 0.009 gem") in what was observed an excessive fluid accumulation (loop weightlength ratio), 38 expected. We also evaluated the eect of the 1% carboxymethyeelulose solution by gavage inthe loops without the administration of cholera toxin, fand was not observed any signifeant diference in intestinal secretion as compared to the negative control (PBS) (data. not shown), Dose rates for NaHS and Lawesson's reagent (both at 27 yamol kg") and weysteine (50 mg kg") were chosen for sub- Sequent studies. We choose the 27 umol kg"! concentration of [NalS and Lawesson’s reagent because this dos is generally used in 16 BM Soa a. i aie 76 (2008) 152-163 ana gg a NIMS (3.9 and 27 una kg 9~ 6 (A), Lawson's tegen (3,9 and 27 una e PBS Sal NaHS. Law Leyseie PAG iTeyaine ‘Giles on ne Ect froze uiphice on intestinal ui accumulation in cola toxin-reatd intestinal sed ops, Mice were prewated by gavage with saline (25 mL gn ~ 8, {6(8). stein (10 and 50g he" 6 (Cand PAG (100 mg kg") and este 50 meg sn = 6{C) fr 30 min Before he inucton a athaeay CT (1 yoo The conel groups were peetrested with alin, apd the zat conea group rected PSS 10 nthe slated lop and the postive contol roupeceed CL. (0) Et of nalumialadmistaton vers gavage administration oH donors, obse¥ed in prevons tial against CTinduced roe (enestnal loos of each study soup nice were presented by gavage wit PC (100 man 6)rsaine(23 my n= 6 30min before the ction of does of CT (05 yatenp Al eres hrs ite SEM"? «O05 versa the soup treed th PBSin the loop: “P< 00 vers pov con BoD {prerestd with saline and Crin the ops)" <.05 vs. group preteated with -ystene (30 me KE) *P O03 versus te group peated with PAG 10 mK *) and eated wit Pin the loops, Values were deterred by one-way ANOVA followed by a Newman-Keuls test, other models of gastrointestinal diseases. Likewise, this dose is not considered high for applications in pathological processes in the ‘gastrointestinal tract [21]. In Fig. 1C it was also observed that the administration of PAG, a CSE inhibitor, reversed the antisecretory effect of t-cysteine (0.148 + 0.016 gem '). In Fig. IE, we have shown an intestinal loop isolated from each study group. To confirm the antisecretory effect observed by gavage administration of the HS donors, we tested NaHS and Lawesson's reagent directly inoculated into the loops. NaHS (27 iM) and Lawesson’s reagent (27 iM) administered intraluminal also significantly reduced (p < 0.001, Fig. 1D) the i testinal secretion (0.072 + 0.010 gem”) and (0.062 + 0.011 gem” respectively, ‘We also evaluated the fluid secretion in the presence of CBS inhibitor (carboxymethylhydroxylamine hemihydrochloride (CHH ~ 20 mg/kg, ip.)). The inhibition of the CBS did not reverse the antisecretory effect of t-cysteine (data not shown). Likewise, the {treatment with NaHS and Lawesson's reagent both 27 jmol kg”! and PBS in the loops did not present significant difference in i testinal secretion as compared to the negative control (PBS) (data not shown). This date indicate that HS not show secretory effect on physiological secretion, in this model 3.2. PAG increases secretion of intestinal fluid in combination with 4 submaximal dose of cholera toxin ‘To determine the role of H,S in intestinal secretion induced by cholera toxin, mice were pretreated with PAG (100 mg kg") fol- owed by intraluminal administration of a submaximal dose of cholera toxin (0.5 y/loop)- This was necessary because PAG did not ‘worsen intestinal secretion in the loop when administered at a CT dose of 1 igjloop. This may have occurted because this dose of cholera toxin resulted in the maximum secretion that can occur in the tissue. As shown in Fig. IF, PAG in combination with CT(03 yg) Joop), promoted a significant increase (0.089 + 0.005 g cm~ intestinal fluid secretion as compared tothe toxin group (p< 0.01) and PAG alone (without the administration of the toxin) (0.055 + 0.004 g cm ' p < 0,001) that did not cause an increase in intestinal secretion. Cholera toxin at this dose (0.5 yg/loop) pro- moted a significant increase in intestinal secretion (0.069 + 0.009 g cm) as compared to PBS group (0.046 + 0.005 g em”, p < 001). FAM Sosa ea / Nv Cue 75 (201) 152-163 157 3.3, HS reduces chloride ion concentration in intestinal fluid there was also staining in the crypts and some staining in the secretion deeper layers of intestinal tissue (muscular and serous) (arrows; ‘ig. 3B). Fig. 3A shows the negative control, without primary anti- ‘To confirm the antisecretory effect of HzS observed in previous body, in the loops of mice treated with PBS. The data from the assays, we analysed the concentration of chloride fons in the in- quantitative analysis of patterns of immunostaining are summa- testinal uid secretion accumulated in each closed loop. The rized in Fig. 3D. An unpaired t-test revealed that immunostaining of intense secretion of chloride ions into the intestinal lumen caused villous mucosal cells was highly similar between the groups treated by cholera toxin is responsible for increased intestinal secretion.In__with CT (13.50 + 156) and PBS (10.75 + 1.75) and no statistical the present study, the concentration of the chloride ions was differences were observed between them. However, analysis of the significantly high (p < 0.001) in intestinal loops injected with number of immunostained cells in the mucosal intestinal crypts cholera toxin (1565 + 1451 mEq L)compared with oopsinjected and other layers found a significant increase of immunostained only with PBS (1565 + 1451 mEq L-'). Treatment with NaHS, cells in loops treated with CT (25.00 + 430) and PBS (15 + 0.64, Lawesson's reagent, and L-cysteine significantly decreased the p< 0001). Based on these data, we next examined the levels of the levels of chloride in the loops (4.47 + 15.07 mEq L-1,63.16 + 20.07 H,S in the loops by a sulphide ion microelectrode. lis. 3E shows a meq Land 5727 + 11.35 mEq Lp <0.001, Fig. 2A) respectively. significant increase (p < 0.001 occurred in the levels Of H2S on the oops inoculated with CT (15030 1404 ymol g' of tissue) 3A. Effect of HaS on intestinal fluid absorption ‘compared with the loops treated with PBS(71.65 + 8.94 umolg "of tissue). ‘The effect of H,S on intestinal Nuid absorption was studied to evaluate whether the protective effect of HS on intestinal fluid 36, Roles of AC, cAMP, and PKA in the HLS antisecretory effect secretion stimulated by CT was involved in increasing intestinal absorption. Fig. 2B shows that administration of glucose solution To evaluate the involvement of AC in the antisecretory effect of (10 mM), promotes a significant intestinal absorption _4,§ (riz 4A), SQ.22536 (0.01 M), a specific inhibitor of AC, was (78.25 + 806%, p < 0.01) as compared to the basal level of intes- administered ditectiy into the isolated loop. AS expected. this tinal absorption observed in the PBS group (4426 + 5.90%). A produced a significant reduction in intestinal fluid. secretion similar response occurred in loops. treated with NaHS induced by CT (0.086 + 0012 g cm’, p < 0.001), However, pre- (22.64 + 8.03%), Lawesson's reagent (41.33 + 7.08%), or L-cysteine treatment with PAG (100 mg kg~') did not reverse the protective (62.75 + 10.73%),inwhich twasnot observed a signficanteffecton effect of SQ 22536 (0.085 + O01 g cm’, p 0001), These data intestinal absorption when compared to the PBS group. suggest that the antisecretory effect of HS is independent of AC. To confirm the non-involvement of AC, we also tested a specific AC 35. Cholera toxin increased expression of CSE and levels of HS in. activator, forskolin. As shown in Fig. 4B and C.a significant increase ‘mouse small intestine in intestinal secretion was observed in the intestinal loops treated ‘with forskolin (0103 = 0.014 g cm”, p <0.01) when compared to Fig, 3B show the presence of immunostaining in the loops the PBS group. This elect was expected with forskolin as AC acti- tweated with PBS, especially on the lamina propria layer of the vation causes an increase in the secretion. We also observed a ‘mucosa of the intestinal villi (arrow: Fg. 3), although some im- significant increase inthe intestinal secretion in the loops treated munostaining was also observed inthe blood vessels arrowhead: only with CT (0.115 009 g em”, p< 0001) or forskolin and CT Fg. 38) However, when the intestinal loops were inoculated with (0120 «0.011 cm ',p <.01), when compared tothe PBS group. CF (Fig, 30, staining was observed in other layers. In addition to Inanother set of experiments, we tested the non-involvement of AC Intense staining for CSE in the mucosal layers of the intestinal vil by treating the loops directly with forskolin followed by NaHS or A B 200 120 100 10 5 + a ge g gE + ee is® * . ®% 40 5 sy # * 0 ° ° POs Sal Lcyeisie Law WaHS PES Glucose Leysteine Law NaHS (oma) Gomeigt Gem) arm) mao’) mai’) tumothe Chek toxin 19 Fig 2 ecto drogen sulphide on he eves of ori ns 2 intestinal absorption (A et of yaeoge sulphide onthe lewlgfclrie on resent in ops treated with CE Mice were peated by garage with sain (2 mL yO NaHS (27pm. ~ 6), Lawes rage (27 uml hy. |, 6) or pst (50 mg hn ~ 6) OF 50 min before the inaction a arhoea by CT geo) (8 ect of reo slide onthe ines absorption. Mie were treated diet ft the iat testinal loop th PBS (200 pi and ghcose scan (10 mM) o PBS (20) containing eyscne (30 me "NaS (27 mol Ky "or Lawesons reagent (27 amok") 30 min before emova tthe ops Aleror rs se te SEM. ? O05 eras the US rested group inthe ops "P< 05 vers the positive cone group petted wih saline and CT ‘the loos) and“ O05 vers the gcoseeeate grup Values were determined by one-way ANOVA allowed by 2 New BM Soa a. i aie 76 (2008) 152-163 Fe. 3. mmunohistochemia staining for ytathanne yas (CS) (magnification: 40) n mse smal nest. (A) Negative onl without primary ansbady in oops of nce ested wit PBS) unnostanad for SE lope of mice esed wit BS or (Ch Se bar 50m (D) Quantitative analjss of mmunstalned ees observed the Toop cated with PS and Tin elation the vk crypts and ters ayes testinal sve. hole xin signal lceasod te expression of CSE nthe cys and thes layers ofits ies () level in ines lope of mice eed eth PRS a CT. eroes bas indate the SEM P08 verse the group ted wth PRS. Values were

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