Identification of proton-translocating adenosine triphosphatases in rat cerebral microvessels

To determine if proton translocating adenosine triphosphatases (ATPases) can be localized at the blood-brain barrier, isolated rat cerebral microvessels and cerebral synaptosomal preparations were assayed for ATPase activity in the presence of various inhibitors. N-ethylmaleimide-sensitivity could be consistently found in both cerebral microvessel and synaptosomal preparations. There was no vanadate sensitive component in the presence of ouabain, oligomycin and EGTA. Immunoblotting of cerebral microvessels and synaptosomes with a monoclonal antibody (E11) against the 31 kDa subunit of the vacuolar type H-ATPase pump identified a discrete 31 kDa band. Diffuse immunocytochemical staining of cerebral cortical tissue, predominantly in choroid plexus, could be found with E11 but not with HK alpha N2, an H,K-ATPase specific antibody, nor with a non-specific mouse monoclonal antibody (MOPC-21). Immunoblotting with HK alpha N2 showed an immunoreactive 76 kDa band, not present with the preimmune serum or the antibody preabsorbed with the immunizing synthetic peptide. It is concluded that the vascular type H-ATPase and not the gastric H,K-ATPase is present in cerebral tissue including cerebral microvessels and choroid plexus. Non-specific immunoreactivity may account for the 76 kDa band observed in the immunoblots using the HK alpha N2 antibody although presence of a degradation product of H,K-ATPase can not be ruled out. The functional role of the vacuolar H-ATPase in the blood-brain barrier remains to be determined.