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A label-free impedance-based whole cell assay revealed a new G protein-coupled receptor ligand for mouse microglial cell migration

Author:
Fukano, Yasufumi, Okino, Nozomu, Furuya, Shigeki, Ito, Makoto
Source:
Biochemical and biophysical research communications 2016 v.478 no.2 pp. 624-630
ISSN:
0006-291X
Subject:
G-protein coupled receptors, adenosine diphosphate, cell movement, chemoattractants, complement, enzyme inhibitors, ligands, mice, neuroglia, pertussis toxin, protein kinases, uridine, uridine triphosphate
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.
Agid:
6056379