title: Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism.
abstract: Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintains the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt.
authors: Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile. Electronic address: capuebla@uc.cl.
authors: Carlos
authors: C
authors: Puebla
authors: Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile; Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valparaíso, Universidad de Valparaíso, Valparaíso, Chile.
authors: Bruno A
authors: BA
authors: Cisterna
authors: Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.
authors: Daniela P
authors: DP
authors: Salas
authors: Laboratorios de Biomedicina, Departamento de Ciencias Preclínicas, Facultad de Medicina, Universidad Católica del Maule, Talca, Chile.
authors: Fernando
authors: F
authors: Delgado-López
authors: Translational Research Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States.
authors: Paul D
authors: PD
authors: Lampe
authors: Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile; Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valparaíso, Universidad de Valparaíso, Valparaíso, Chile. Electronic address: jsaez@bio.puc.cl.
authors: Juan C
authors: JC
authors: Sáez
mesh_terms: D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects* / Q000201:enzymology; D002463:Cell Membrane Permeability / Q000187:drug effects*; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects* / Q000201:enzymology; D005753:Gastric Mucosa / Q000187:drug effects* / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D019787:Linoleic Acid / Q000494:pharmacology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D034622:RNA Interference; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D012694:Serine; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
chemical_list: D018031:Connexin 43; C473034:FFAR1 protein, human; C500216:GJA1 protein, human; C543076:GJA1 protein, mouse; C580592:Gja1 protein, rat; D047428:Protein Kinase Inhibitors; D043562:Receptors, G-Protein-Coupled; D012694:Serine; D019787:Linoleic Acid; D051057:Proto-Oncogene Proteins c-akt; D002118:Calcium
keywords: Akt kinase, G protein-coupled receptor (GPCR); Connexon (hemichannel); FFAR1; Polyunsaturated fatty acid (PUFA); Protein phosphorylation; Transmembrane permeability