H 2 S 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 a, f , Luan K.M. Souza a, f , Nayara A. Sousa a, f , Thiago S.L. Araújo a, f , Simone de Araújo b, f , Dvison M. Pacífico c , Irismara S. Silva d , Renan O. Silva e , Lucas A.D. Nicolau e , Fabiana M. Souza a , Marcelo C. Filgueiras a, f , Jefferson S. Oliveira a , Marcellus H.L.P. Souza e , Jand Venes R. Medeiros a, b, f, * a Biotechnology and Biodiversity Center Research, BIOTEC, Federal University of Piauí, Parnaíba, Piaui, Brazil b Medicinal Plant Research Center, NPPM, Federal University of Piaui, Teresina, Piauí, Brazil c Department of Morphology, Faculty of Medicine, Federal University Cear a, Fortaleza, Ceara, Brazil d Physiology and Pharmacology, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil e Department of Physiology and Pharmacology, Federal University of Cear a, Fortaleza, Ceara, Brazil f Laboratory of Experimental Physiopharmacology, Federal University of Piauí, Parnaíba, Piauí, Brazil article info Article history: Received 24 May 2017 Received in revised form 18 September 2017 Accepted 20 September 2017 Available online xxx Keywords: Gaseous mediators Diarrhea diseases Cholera AMPK abstract Hydrogen sulphide (H 2 S) is a gasotransmitter that participates in various physiological and pathophys- iological processes within the gastrointestinal tract. We studied the effects and possible mechanism of action of H 2 S in secretory diarrhoea caused by cholera toxin (CT). The possible mechanisms of action of H 2 S were investigated using an intestinal fluid secretion model in isolated intestinal loops on anaes- thetized mice treated with CT. NaHS and Lawesson's reagent and L-cysteine showed antisecretory activity through reduction of intestinal fluid secretion and loss of Cl  induced by CT. Pretreatment with an in- hibitor of cystathionine-g-lyase (CSE), DL-propargylglycine (PAG), reversed the effect of L-cysteine and caused severe intestinal secretion. Co-treatment with PAG and a submaximal dose of CT increased in- testinal fluid secretion, thus supporting the role of H 2 S in the pathophysiology of cholera. CT increased the expression of CSE and the production of H 2 S. Pretreatment with PAG did not reverse the effect of SQ 22536 (an AC inhibitor), bupivacaine (inhibitor of cAMP production), KT-5720 (a PKA inhibitor), and AICAR (an AMPK activator). The treatment with Forskolin does not reverse the effects of the H 2 S donors. Co-treatment with either NaHS or Lawesson's reagent and dorsomorphin (an AMPK inhibitor) did not reverse the effect of the H 2 S donors. H 2 S has antisecretory activity and is an essential molecule for protection against the intestinal secretion induced by CT. Thus, H 2 S donor drugs are promising candi- dates for cholera therapy. However, more studies are needed to elucidate the possible mechanism of action. © 2017 Elsevier Inc. All rights reserved. 1. Introduction Cholera, a disease characterised by acute secretory diarrhoea, is caused by intestinal infection from the gram-negative bacterium Vibrio cholerae serogroups O1 and O139 [1]. This disease causes large epidemics worldwide and is a 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 [3]. CT 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 GM1 ganglio- side receptor, which is then internalized through retrograde endocytosis [4]. Within the cell, the A subunit causes constitutive * Corresponding author. Av. S~ ao Sebasti~ ao, 2819, CEP: 64202-020, Parnaíba, PI, Brazil. E-mail address: jandvenes@ufpi.edu.br (J.V.R. Medeiros). Contents lists available at ScienceDirect Nitric Oxide journal homepage: www.elsevier.com/locate/yniox https://doi.org/10.1016/j.niox.2017.09.007 1089-8603/© 2017 Elsevier Inc. All rights reserved. Nitric Oxide xxx (2017) 1e12 Please cite this article in press as: F.B.M. Sousa, et al., H 2 S is a key antisecretory molecule against cholera toxin-induced diarrhoea in mice: Evidence for non-involvement of the AC/cAMP/PKA pathway and AMPK, Nitric Oxide (2017), https://doi.org/10.1016/j.niox.2017.09.007