Occludin oligomeric assembly at tight junctions of the blood-brain barrier is disrupted by peripheral inflammatory hyperalgesia.

Tight junctions (TJs) at the blood-brain barrier (BBB) dynamically alter paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS in response to external stressors, such as pain, inflammation, and hypoxia. In this study, we investigated the effect of lambda-carrageenan-induced peripheral inflammatory pain (i.e., hyperalgesia) on the oligomeric assembly of the key TJ transmembrane protein, occludin. Oligomerization of integral membrane proteins is a critical step in TJ complex assembly that enables the generation of tightly packed, large multiprotein complexes capable of physically obliterating the interendothelial space to inhibit paracellular diffusion. Intact microvessels isolated from rat brains were fractionated by detergent-free density gradient centrifugation, and gradient fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/ Western blot. Injection of lambda-carrageenan into the rat hind paw produced after 3 h a marked change in the relative amounts of oligomeric, dimeric, and monomeric occludin isoforms associated with different plasma membrane lipid raft domains and intracellular compartments in endothelial cells at the BBB. Our findings suggest that increased BBB permeability (i.e., leak) associated with lambda-carrageenan-induced peripheral inflammatory pain is promoted by the disruption of disulfide-bonded occludin oligomeric assemblies, which renders them incapable of forming an impermeant physical barrier to paracellular transport.

Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo.

Tight junctions (TJs) are major components of the blood-brain barrier (BBB) that physically obstruct the interendothelial space and restrict paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS. TJs are dynamic structures whose intricate arrangement of oligomeric transmembrane and accessory proteins rapidly alters in response to external stressors to produce changes in BBB permeability. In this study, we investigate the constitutive trafficking of the TJ transmembrane proteins occludin and claudin-5 that are essential for forming the TJ seal between microvascular endothelial cells that inhibits paracellular diffusion. Using a novel, detergent-free OptiPrep density-gradient method to fractionate rat cerebral microvessels, we identify a plasma membrane lipid raft domain that contains oligomeric occludin and claudin-5. Our data suggest that oligomerization of occludin involves disulfide bond formation within transmembrane regions, and that assembly of the TJ oligomeric protein complex is facilitated by an oligomeric caveolin scaffold. This is the first time that distribution of oligomeric TJ transmembrane proteins within plasma membrane lipid rafts at the BBB has been examined in vivo. The findings reported in this study are critical to understand the mechanism of assembly of the TJ multiprotein complex that is essential for maintaining BBB integrity.

Conjugation of low molecular weight poly(ethylene glycol) to biphalin enhances antinociceptive profile.

The objectives of this study were to examine the effect of poly(ethylene glycol) (PEG) conjugation on the tyrosine residues of biphalin to determine the proper size PEG for optimal efficacy and investigate the antinociceptive profile of PEG-biphalin against biphalin via three routes of administration. All antinociception evaluations were made using a radiant-heat tail flick analgesia meter. (2 kDa)(2) PEG-biphalin was identified as the optimal size of PEG to enhance the antinociceptive profile following intravenous administration of 685 nmol kg(-1) of biphalin or PEG-biphalin [(1 kDa)(2), (2 kDa)(2), (5 kDa)(2), (12 kDa)(2), (20 kDa)(2)]. (2 kDa)(2) PEG-biphalin displayed an area under the curve (AUC) approximately 2.5 times that of biphalin with enhanced analgesia up to 300 min postinjection. (2 kDa)(2) PEG-biphalin was equipotent to biphalin following intracerebroventricular administration (0.4 nmol kg(-1)). Both biphalin and (2 kDa)(2) PEG-biphalin were effectively antagonized with naloxone (10 mg kg(-1)) and a partial antagonistic effect was seen following pretreatment with naltrindole (20 mg kg(-1)). (2 kDa)(2) PEG-biphalin showed significantly increased potency (A(50)) when administered intravenously and subcutaneously. Additionally, (2 kDa)(2) PEG-biphalin demonstrated a significantly enhanced antinociceptive profile (AUC) via all routes of administration tested. These findings indicate that PEG conjugation to biphalin retains opioid-mediated effects observed with biphalin and is a valuable tool for eliciting potent, sustained analgesia via parenteral routes of administration.

Viability of microvascular endothelial cells to direct exposure of formalin, lambda-carrageenan, and complete Freund's adjuvant.

We investigated three inflammatory agents to establish if these substances elicit a direct effect on the functional and structural integrity of the blood-brain barrier. Cellular cytotoxicity and paracellular permeability were assessed in vitro using primary bovine brain microvascular endothelial cells exposed to formalin, lambda-carrageenan, or complete Freund's adjuvant for 1, 3, or 72 h, respectively. Results showed that only the highest concentration (0.025%) of formalin produced a decrease in cell viability (approximately 34%) and a significant increase in cell permeability to [(14)C]sucrose at 120 min (approximately 137%). Brain perfusion using female Sprague-Dawley rats showed no difference in paracellular permeability to [(14)C]sucrose for any inflammatory agent. Western blot analyses were performed on isolated rat brain microvessels to assess the structural integrity of blood-brain barrier tight junctions. Results indicate that expression of zonula occludens-1, occludin, claudin-1, and actin remain unchanged following intravenous exposure to inflammatory agents. This study confirms that changes seen at the blood-brain barrier following a peripheral inflammation are due to physiological responses to the given inflammatory agent and not to any direct interaction between the inflammatory agent and the brain microvasculature.