title: Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells.
abstract: The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144-7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.
authors.affiliation: Cellular Physiology Laboratory, Biomedical Department, Faculty of Health Sciences, Universidad de Antofagasta, Antofagasta 1270300, Chile.|Department of Education, Faculty of Education, Universidad de Antofagasta, Antofagasta 1270300, Chile.
authors.forename: Ana R
authors.initials: AR
authors.lastname: Beltrán
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo 3550308-1009, Brazil.
authors.forename: Luciene R
authors.initials: LR
authors.lastname: Carraro-Lacroix
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo 3550308-1009, Brazil.
authors.forename: Camila N A
authors.initials: CN
authors.lastname: Bezerra
authors.affiliation: Cellular Physiology Laboratory, Biomedical Department, Faculty of Health Sciences, Universidad de Antofagasta, Antofagasta 1270300, Chile.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Cornejo
authors.affiliation: Cellular Physiology Laboratory, Biomedical Department, Faculty of Health Sciences, Universidad de Antofagasta, Antofagasta 1270300, Chile.
authors.forename: Katrina
authors.initials: K
authors.lastname: Norambuena
authors.affiliation: Department of Basic Sciences, Faculty of Sciences, Universidad del Bío-Bío, Chillán 3780000, Chile.
authors.forename: Fernando
authors.initials: F
authors.lastname: Toledo
authors.affiliation: Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynecology, Faculty of Medicine, School of Medicine, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile.
authors.forename: Joaquín
authors.initials: J
authors.lastname: Araos
authors.affiliation: Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynecology, Faculty of Medicine, School of Medicine, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile.
authors.forename: Fabián
authors.initials: F
authors.lastname: Pardo
authors.affiliation: Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynecology, Faculty of Medicine, School of Medicine, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile.
authors.forename: Andrea
authors.initials: A
authors.lastname: Leiva
authors.affiliation: Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynecology, Faculty of Medicine, School of Medicine, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile.
authors.forename: Carlos
authors.initials: C
authors.lastname: Sanhueza
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo 3550308-1009, Brazil.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Malnic
authors.affiliation: Department of Physiology, Faculty of Pharmacy, Universidad de Sevilla, Seville E-41012, Spain.|University of Queensland Centre for Clinical Research (UQCCR), Faculty of Medicine and Biomedical Sciences, University of Queensland, Herston, QLD 4029, Queensland, Australia.|Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynecology, Faculty of Medicine, School of Medicine, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile.
authors.forename: Luis
authors.initials: L
authors.lastname: Sobrevia
authors.affiliation: Cellular Physiology Laboratory, Biomedical Department, Faculty of Health Sciences, Universidad de Antofagasta, Antofagasta 1270300, Chile.|Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynecology, Faculty of Medicine, School of Medicine, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Ramírez
mesh_terms: D000328:Adult; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D004355:Drug Stability; D004768:Enterotoxins / Q000737:chemistry / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D004926:Escherichia coli; D005786:Gene Expression Regulation / Q000187:drug effects; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000166:cytology*; D007700:Kinetics; D008297:Male; D011522:Protons; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D004768:Enterotoxins; D011522:Protons; C570475:SLC9A4 protein, human; D017923:Sodium-Hydrogen Exchangers; D000242:Cyclic AMP; D006152:Cyclic GMP