1. Peripheral inflammatory pain (λ-carrageenan injection) promotes localized paw edema and hyperalgesia Peripheral inflammatory pain reduces morphine efficacy Peripheral inflammatory pain reduces brain uptake of morphine PgP trafficking within caveolin- enriched domains is modulated by peripheral inflammatory pain Disassembly of high molecular weight complexes containing PgP and caveolin-1 is associated with increases in PgP-associated ATPase activity Figure 1. A. Edema in the hind paw 3h after λ-carrageenan injection. B. Measurement of paw edema and IR sensitivity in the injected paw and contralateral paw 3h post injection. Values are the mean ± S.E.M. Figure 2. Time course of morphine uptake in the brain using in situ perfusion of [ 3 H] morphine 3h post injection of vehicle (saline) or λ- carrageenen. Values are the mean ± S.E.M. Figure 3. Measurement of the antinociceptive effects of morphine using the tail flick assay. Top panel shows latency over time and the bottom panel shows the AUC calculations using the trapezoidal method. Values are the mean ± S.E.M. Figure 4. Measurement of morphine uptake in the brain after a single dose of cyclosporine A (CsA0 in naïve rates using in situ perfusion of [ 3 H]morphine. Values are the mean ± S.E.M. Figure 5. Detergent-free stepwise 5-30% OptiPrep gradients of isolated microvessels showing a redistribution of PgP and caveolin-1 to higher density fractions 3h after injection of λ-carrageenan. The top left panel indicates the density of each gradient fraction and the protein concentration. Values are the mean + S.E.M. Figure 6. Left and center panels: Detergent-free OptiPrep gradient fractions containing the major portion of PgP show a high molecular weight complex that contains PgP and caveolin-1 in control-treated animals that disassembles 3h after injection of λ- carrageenan. Right panel: Verapamil-induced ATPase activity in the selected fractions. Values are mean + S.E.M. λ-carrageenan modulates the disulfide bonding in PgP-containing high molecular weight complexes Figure 7. Immunoblot of the lower density major PgP- containing fractions. NR = samples not treated with reducing agents; R = samples treated with aqueous reducing agents. Identification of proteins that co-localize with PgP Figure 8. Detergent-free 10-35% stepwise OptiPrep gradients of microvessels isolated from control animals showing proteins that co-localize with PgP. The top panel indicates the density of each fraction and the protein concentration. Values are the mean ± S.E.M. Abbreviations are: GLUT1 (glucose transporter 1), plasma membrane marker; nucleoporin, nuclear membrane marker; PDI (protein disulfide isomerase); QSOX1 (quiescin Q6 sulfhydryl oxidase); ATPB (ATP synthase beta subunit). Introduction Pain is a major public health problem that afflicts millions of individuals resulting in significant health care costs and loss of productivity. Currently, opioid analgesics are the most prominent drugs used for pain management. Although these drugs bind receptors in the periphery and in the central nervous system (CNS), access to the CNS is required for optimal pain relief. The Blood-Brain Barrier (BBB) is particularly effective at preventing xenobiotic access to the CNS. P-glycoprotein (PgP) in microvessel endothelial cell membranes is the major obstacle for delivery of opioid analgesics to the CNS. The physiological function of PgP is to prevent systemic xenobiotic toxicity. PgP utilizes ATP hydrolysis to efflux a wide variety of compounds against a concentration gradient, thus preventing these compounds from accumulating to toxic levels within the cells. Opioid analgesics are among the PgP substrates. Activation of PgP at the BBB provides an additional obstacle for drug delivery to the CNS. The clinical challenge for pain management is to design treatment protocols that allow analgesic access to the CNS while maintaining a barrier that prevents toxic xenobiotics and infectious agents from reaching the CNS. In the current study we have used a peripheral inflammatory pain (PIP) model to study the role of trafficking in the up regulation of PgP. Our goal is to identify potential drug targets in the trafficking pathways that could be modulated for clinical gain. Model for PIP-induced PgP trafficking Figure 9. PIP signaling induces dissociation of some PgP from caveolin1 and relocation to other membrane structures. This could occur through signaling within and/or remodeling of caveolae to release stored inactive PgP resulting in the observed increased drug efflux. Conclusions and implications PIP induces disassembly of high molecular weight PgP and caveolin-1-containing complexes concomitant with increased PgP activity. PIP alters PgP trafficking suggesting a change in subcellular location. Loss of disulfide-bonded complexes accompany PgP trafficking suggesting that redox processes play a role in the PIP induced PgP trafficking/activation. ATP ADP ATP ADP ATPADP To nucleus PIP Lumen Cytosol Inflammatory, neuronal, endocrine signals (ROS, cytokines and etc.) Basal Drug EffluxIncreased Drug Efflux Cavin-1 PgP Caveolin-1