γ-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 γ-amino butyric acid (GABA) type B receptor 2 (GABABR2) as an Akt binding protein in human neutrophils. Neutrophil lysates subjected to Akt immunoprecipitation followed by immunoblotting with anti-GABABR2 demonstrated Akt association with the intact GABABR. Similar results were obtained when reciprocal immunoprecipitations were performed with anti-GABABR2 Ab. Additionally, GABABR2 and Akt colocalization was demonstrated by confocal microscopy. A GABABR agonist, baclofen, activated Akt and stimulated neutrophil-directed migration in a PI3K-dependent manner, whereas CGP52432, a GABABR antagonist blocked such effects. Baclofen, stimulated neutrophil chemotaxis and tubulin reorganization in a PI3K-dependent manner. Additionally, a GABABR agonist failed to stimulate neutrophil superoxide burst. We are unaware of the association of GABABR with Akt in any cell type. The present study shows for the first time that a brain-specific receptor, GABABR2 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 GABABR is functionally expressed in neutrophils, and acts as a chemoattractant receptor via an Akt-dependent pathway. The GABABR potentially plays a significant role in the inflammatory response and neutrophil-dependent ischemia-reperfusion injury such as stroke.