Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress
| Autor: | Colin L. Willis, Jeffrey J. Lochhead, Carolyn A. Quigley, William D. Staatz, Gwen McCaffrey, Thomas P. Davis, Nicole Nametz, Sharon Hom |
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| Rok vydání: | 2009 |
| Předmět: |
Models
Molecular Protein subunit Blotting Western Brain Edema Biology Blood–brain barrier Occludin Biochemistry Cell junction Article Tight Junctions Diffusion Rats Sprague-Dawley Cellular and Molecular Neuroscience Membrane Microdomains Western blot Stress Physiological medicine Animals Hypoxia Brain Lipid raft medicine.diagnostic_test Tight junction Membrane Proteins Cerebral Arteries Rats Protein Subunits medicine.anatomical_structure Blood-Brain Barrier Reperfusion Injury Paracellular transport cardiovascular system Biophysics Electrophoresis Polyacrylamide Gel Female Protein Multimerization Subcellular Fractions |
| Zdroj: | Journal of Neurochemistry. 110:58-71 |
| ISSN: | 1471-4159 0022-3042 |
| DOI: | 10.1111/j.1471-4159.2009.06113.x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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