title: A label-free impedance-based whole cell assay revealed a new G protein-coupled receptor ligand for mouse microglial cell migration.
abstract: We report the usefulness of an impedance-based label-free whole cell assay to identify new ligands for G protein-coupled receptors (GPCRs) involved in microglial cell migration. Authentic GPCR ligands were subjected to the impedance-based cell assay in order to examine the responses of ligands for MG5 mouse microglial cells. Complement component 5 (C5a), adenosine 5'-diphosphate (ADP), uridine 5'-triphosphate (UTP), lysophosphatidic acid (LPA), and lysophosphatidylserine (LysoPS) were found to elicit different cellular impedance patterns, i.e. C5a, ADP, and UTP caused a transient increase in cellular impedance, while LPA and LysoPS decreased it. The responses for C5a and ADP were abolished by pertussis toxin (PTX), but not rho-associated protein kinase inhibitor, Y-27632, indicating that C5a and ADP elicited responses through the Gαi pathway. On the other hand, the response for UTP, LPA or LysoPS was not cancelled by PTX or Y-27632. In a modified Boyden chamber assay, C5a and ADP, but not UTP, LPA, or LysoPS, induced the migration of MG5 cells. These results suggest that PTX-sensitive increase in cellular impedance with the assay is characteristic for ligands of GPCRs involved in microglial cell migration. We found using this assay that 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a new chemoattractant inducing microglial cell migration through the activation of Gαi.
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; Project Facilitation Department, Sohyaku. Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Aoba-ku, Yokohama-shi, Kanagawa 227-0033, Japan. Electronic address: Fukano.yasufumi@mc.mt-pharma.co.jp.
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Fukano
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Nozomu
authors.initials: N
authors.lastname: Okino
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Furuya
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: makotoi@agr.kyushu-u.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D017097:Electric Impedance; D008024:Ligands; D051379:Mice; D017628:Microglia / Q000166:cytology* / Q000187:drug effects; D037342:Pertussis Toxin / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
chemical_list: D001095:Arachidonic Acids; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; C080828:5-oxo-6,8,11,14-eicosatetraenoic acid; D037342:Pertussis Toxin
keywords: 5-Oxo-ETE; G protein-coupled receptor (GPCR); Impedance-based cell assay; Microglia; Migration