Role of the purinergic system in controlling blood-brain barrier functions upon ischemic conditions: focus on ATP-metabolizing enzymes

The blood-brain barrier (BBB) is composed of endothelial cells, pericytes and astrocytes, and serves as interface between the blood flow and the central nervous system (CNS). Since BBB deregulation plays important role in the pathogenesis of several CNS diseases spanning from brain tumors to stroke, understanding the molecular mechanisms controlling BBB functions might help unveiling new therapeutic targets to brain pathologies. Extracellular nucleotides are important signaling molecules both in physiological and pathological conditions. Their actions are mediated by 7 ionotropic P2X and 8 metabotropic P2Y purinergic receptors, and terminated by metabolizing enzymes, namely ectonucleotidases (NTPDases) and 5\u2019-nucleotidase. To date the role of purinergic transmission in controlling BBB functions is not fully understood. Therefore, we used a new in vitro cell culture model of BBB to investigate the expression and distribution of NTPDases and P2Y receptors either in control conditions or following exposure to ischemia. RT-PCR analysis showed that astrocytes and pericytes expressed all the cloned P2Y receptors, that endothelial cells showed only the P2Y1,2,4 subtypes, and that NTPDase1 and 2 were expressed by all the three types of cell. Application of oxygen-glucose deprivation (OGD), which mimics cytotoxicity induced by ischemia in vivo, showed that endothelial cells were extremely susceptible to cell death, whereas astrocytes and pericytes were more resistant. A semi-quantitative assay highlighted increased ecto-ATPase activity following exposure to OGD in the three types of cell, both when grown separately and in triple co-culture. Our data show the usefulness of this new in vitro model to demonstrate a role for extracellular nucleotides in modulating BBB responses to ischemic events, and to determine if the purinergic system could represent a new target for the development of effective therapies to brain pathologies