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-Amino Butyric Acid Type B Receptors Stimulate Neutrophil Chemotaxis during Ischemia-Reperfusion¹

Madhavi J. Rane^2,*, David Gozal^3,,, Waseem Butt^*, Evelyne Gozal^,, William M. Pierce, Jr, Shang Z. Guo, Rui Wu^*, Aviv D. Goldbart, Visith Thongboonkerd^*, Kenneth R. McLeish^*,,|| and Jon B. Klein^3,*,,¶,||

Departments of^* Medicine, Pediatrics, Pharmacology and Toxicology, Biochemistry and Molecular Biology, and ^¶ Core Proteomics Laboratory, University of Louisville, and ^|| Veterans Affairs Medical Center, Louisville, KY 40202

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 -amino butyric acid (GABA) type B receptor 2 (GABA_BR2) as an Akt binding protein in human neutrophils. Neutrophil lysates subjected to Akt immunoprecipitation followed by immunoblotting with anti-GABA_BR2 demonstrated Akt association with the intact GABA_BR. Similar results were obtained when reciprocal immunoprecipitations were performed with anti-GABA_BR2 Ab. Additionally, GABA_BR2 and Akt colocalization was demonstrated by confocal microscopy. A GABA_BR agonist, baclofen, activated Akt and stimulated neutrophil-directed migration in a PI3K-dependent manner, whereas CGP52432 a GABA_BR antagonist blocked such effects. Baclofen, stimulated neutrophil chemotaxis and tubulin reorganization in a PI3K-dependent manner. Additionally, a GABA_BR agonist failed to stimulate neutrophil superoxide burst. We are unaware of the association of GABA_BR with Akt in any cell type. The present study shows for the first time that a brain-specific receptor, GABA_BR2 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_BR is functionally expressed in neutrophils, and acts as a chemoattractant receptor via an Akt-dependent pathway. The GABA_BR potentially plays a significant role in the inflammatory response and neutrophil-dependent ischemia-reperfusion injury such as stroke.