title: Short- and long-term regulation of intestinal Na+/H+ exchange by Toll-like receptors TLR4 and TLR5.
abstract: Inappropriate activation of pattern recognition receptors has been described as a potential trigger in the development of inflammatory bowel disease (IBD). In this study, we evaluated the activity and expression of Na(+)/H(+) exchanger (NHE) subtypes in T84 intestinal epithelial cells during Toll-like receptor 4 (TLR4) activation by monophosphoryl lipid A and TLR5 by flagellin. NHE activity and intracellular pH were evaluated by spectrofluorescence. Additionally, kinase activities were evaluated by ELISA, and siRNA was used to specifically inhibit adenylyl cyclase (AC). Monophosphoryl lipid A (MPLA) (0.01-50.00 μg/ml) and flagellin (10-500 ng/ml) inhibited NHE1 activity in a concentration-dependent manner (MPLA short term -25.2 ± 5.0%, long term -31.9 ± 4.0%; flagellin short term -14.9 ± 2.0%, long term -19.1 ± 2.0%). Both ligands triggered AC3, PKA, PLC, and PKC signal molecules. Long-term exposure to flagellin and MPLA induced opposite changes on NHE3 activity; flagellin increased NHE3 activity (∼10%) with overexpression of membrane protein, whereas MPLA decreased NHE3 activity (-17.3 ± 3.0%). MPLA and flagellin simultaneously had synergistic effects on NHE activity. MPLA and flagellin impaired pHi recovery after intracellular acidification. The simultaneous exposure to MPLA and flagellin induced a substantial pHi reduction (-0.55 ± 0.03 pH units). Activation of TLR4 and TLR5 exerts marked inhibition of NHE1 activity in intestinal epithelial cells. Transduction mechanisms set into motion during TLR4-mediated and long-term TLR5-mediated inhibition of NHE1 activity involve AC3, PKA, PLC, and PKC. However, short- and long-term TLR4 activation and TLR5 activation might use different signaling pathways. The physiological alterations on intestinal epithelial cells described here may be useful in the development of better IBD therapeutics.
authors: Department of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal;
authors: José Miguel
authors: http://orcid.org/0000-0002-3485-1298
authors: JM
authors: Cabral
authors: Department of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal;
authors: Daniela
authors: D
authors: Grácio
authors: Department of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal; MedInUP, Centre for Drug Discovery and Innovative Medicines, University of Porto, Porto, Portugal;
authors: Patrício
authors: P
authors: Soares-da-Silva
authors: Department of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal; MedInUP, Centre for Drug Discovery and Innovative Medicines, University of Porto, Porto, Portugal; Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal fm@med.up.pt.
authors: Fernando
authors: F
authors: Magro
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival; D006224:Cricetinae; D005408:Flagellin / Q000494:pharmacology; D020868:Gene Silencing; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology; D008050:Lipid A / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D020033:Protein Isoforms; D051381:Rats; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D051198:Toll-Like Receptor 5 / Q000235:genetics / Q000378:metabolism*
chemical_list: D008050:Lipid A; D020033:Protein Isoforms; D017923:Sodium-Hydrogen Exchangers; D051197:Toll-Like Receptor 4; D051198:Toll-Like Receptor 5; D005408:Flagellin; C048436:monophosphoryl lipid A
keywords: Na+/H+ exchanger; Toll-like receptor; inflammation; inflammatory bowel disease; intestine