title: Gamma-amino butyric acid type B receptors stimulate neutrophil chemotaxis during ischemia-reperfusion.
abstract: Serine/threonine kinase Akt, or protein kinase B, has been shown to regulate a number of neutrophil functions. We sought to identify Akt binding proteins in neutrophils to provide further insights into understanding the mechanism by which Akt regulates various neutrophil functions. Proteomic and immunoprecipitation studies identified gamma-amino butyric acid (GABA) type B receptor 2 (GABA(B)R2) as an Akt binding protein in human neutrophils. Neutrophil lysates subjected to Akt immunoprecipitation followed by immunoblotting with anti-GABA(B)R2 demonstrated Akt association with the intact GABA(B)R. Similar results were obtained when reciprocal immunoprecipitations were performed with anti-GABA(B)R2 Ab. Additionally, GABA(B)R2 and Akt colocalization was demonstrated by confocal microscopy. A GABA(B)R agonist, baclofen, activated Akt and stimulated neutrophil-directed migration in a PI3K-dependent manner, whereas CGP52432, a GABA(B)R antagonist blocked such effects. Baclofen, stimulated neutrophil chemotaxis and tubulin reorganization in a PI3K-dependent manner. Additionally, a GABA(B)R agonist failed to stimulate neutrophil superoxide burst. We are unaware of the association of GABA(B)R with Akt in any cell type. The present study shows for the first time that a brain-specific receptor, GABA(B)R2 is present in human neutrophils and that it is functionally associated with Akt. Intraventricular baclofen pretreatment in rats subjected to a stroke model showed increased migration of neutrophils to the ischemic lesion. Thus, the GABA(B)R is functionally expressed in neutrophils, and acts as a chemoattractant receptor via an Akt-dependent pathway. The GABA(B)R potentially plays a significant role in the inflammatory response and neutrophil-dependent ischemia-reperfusion injury such as stroke.
authors.affiliation: Department of Medicine, University of Louisville, Louisville, KY 40202, USA. mrane@louisville.edu
authors.forename: Madhavi J
authors.initials: MJ
authors.lastname: Rane
authors.forename: David
authors.initials: D
authors.lastname: Gozal
authors.forename: Waseem
authors.initials: W
authors.lastname: Butt
authors.forename: Evelyne
authors.initials: E
authors.lastname: Gozal
authors.forename: William M
authors.initials: WM
authors.lastname: Pierce
authors.forename: Shang Z
authors.initials: SZ
authors.lastname: Guo
authors.forename: Rui
authors.initials: R
authors.lastname: Wu
authors.forename: Aviv D
authors.initials: AD
authors.lastname: Goldbart
authors.forename: Visith
authors.initials: V
authors.lastname: Thongboonkerd
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: McLeish
authors.forename: Jon B
authors.initials: JB
authors.lastname: Klein
mesh_terms: D000818:Animals; D001418:Baclofen / Q000494:pharmacology; D002478:Cells, Cultured; D002544:Cerebral Infarction / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D018613:Microscopy, Confocal; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D020033:Protein Isoforms / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology; D040901:Proteomics / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D018080:Receptors, GABA-B / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D015427:Reperfusion Injury / Q000378:metabolism* / Q000473:pathology* / Q000503:physiopathology; D014404:Tubulin / Q000378:metabolism
chemical_list: D001418:Baclofen; D019869:Phosphatidylinositol 3-Kinases; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D018080:Receptors, GABA-B; D014404:Tubulin; C494918:AKT1 protein, human; C494920:Akt1 protein, rat