NADPH‐oxidase and mitochondrial sources contribute to adenosine‐induced O2.‐ generation in cerebral arterial muscle cells.

Abstract

Adenosine generally serves as signaling molecule and its actions are mediated by surface adenosine receptors. In isolated cerebral arterial muscle cells (CAMCs) adenosine evokes generation of O2.‐mediated by functional A2a and A2b adenosine receptor subtypes. CAMCs express the protein for the key redox component of NADPH‐oxidase gp91phox. To determine the sources of O2.‐ generated by adenosine we examined contributions of NADPH‐oxidase gp91phox and the mitochondria using fluorescent microscopy and fluorescent HPLC measurement methods of O2.‐ production. Stimulation of CAMCs preloaded with the superoxide detecting fluorescent probe hydroethidine (HE) with adenosine or with the A2a receptor agonist CGS‐21680 induced increased generation of O2.‐. Pretreatment of CAMCs with the specific peptide inhibitor of NADPH‐oxidase gp91ds‐tat or with the mitochondria uncoupling agent 2,4‐dinitrophenol attenuated both adenosine or CGS‐21680 induced generation of O2.‐. Furthermore, prior inhibition of the PI3‐kinase/Akt pathway with either wortmannin or LY294002 abrogated the ability of adenosine and CGS‐21680 to induce generation of O2..‐ in CAMCs. These findings suggest that CAMCs adenosine A2a and A2b receptor stimulation generates O2.‐ from NADPH‐oxidase and mitochondrial sources that appears to involve activities of the PI3‐kinase/Akt signaling pathway.