Hypoxic (low
oxygen) and reperfusion (post-hypoxic reoxygenation) phases of
stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood-brain barrier (BBB) that may lead to
cerebral edema. To investigate the effect of
hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ
protein occludin, rats were subjected to either normoxia (Nx, 21% O(2), 60 min), Hx (6% O(2), 60 min), or
hypoxia/reoxygenation (H/R, 6% O(2), 60 min followed by 21% O(2), 10 min).
After treatment, cerebral microvessels were isolated, fractionated by
detergent-free density gradient centrifugation, and
occludin oligomeric assemblies associated with plasma membrane
lipid rafts were solubilized by perfluoro-
octanoic acid (PFO) exclusively as high molecular weight
protein complexes. Analysis by non-reducing and reducing
sodium dodecyl sulfate (SDS)-
polyacrylamide gel electrophoresis/western blot of PFO-solubilized
occludin revealed that
occludin oligomeric assemblies co-localizing with 'TJ-associated' raft domains contained a high molecular weight 'structural core' that was resistant to disassembly by either SDS or a hydrophilic
reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO-solubilized
occludin oligomeric assemblies to SDS ex vivo revealed the non-covalent association of a significant amount of dimeric and monomeric
occludin isoforms to the
disulfide-bonded inner core, and dispersal of these non-covalently attached
occludin subunits to
lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of
isoform interaction within
occludin oligomeric assemblies at the BBB that enables
occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric
occludin isoforms with a variety of regulatory molecules within different plasma membrane
lipid raft domains.