• Acute Regulation of P-glycoprotein at the Blood-Brain Barrier by Peripheral Inflammatory Pain

      Davis, Thomas P.; Seelbach, Melissa Jessica; Davis, Thomas P.; French, Edward D.; Lynch, Ronald M.; Stamer, W. Daniel; Vanderah, Todd W. (The University of Arizona., 2007)
      P-glycoprotein (Pgp; ABCB1) is a well known transporter involved in energy-dependent-drug efflux activity. At the brain capillary endothelium, its luminal membrane location is ideal for its ascribed role in the physiological efflux of a wide array of structurally and functionally diverse compounds from the brain. This is a critical issue in regards to the delivery of central nervous system (CNS)-acting therapeutics. Moreover, a dysregulation of Pgp has been implicated in specific CNS disease states, including Alzheimer's disease, epilepsy, and brain cancer where an upregulation of Pgp has been well established as a mediator of multi-drug resistance. Inflammation is a common component in all of these conditions. Previously our laboratory has reported changes in BBB molecular and functional properties during inflammatory pain (Huber et al. 2001). This has led us to investigate the effects of peripheral inflammatory pain on Pgp efflux transport properties at the BBB, in vivo. In the present study we examined the effects of lambda-carrageenan-induced inflammatory pain (i.e. hyperalgesia; CIP) on the molecular and functional properties of Pgp at the BBB. Western blots using enriched fractions of isolated rat brain microvessels revealed that Pgp expression at the BBB was increased by CIP and that this increase occurred predominantly within the membrane region of the cell. Additionally, both in situ brain perfusions and whole body antinociceptive profiling of the Pgp substrate and opioid analgesic, [3H] morphine, indicate that changes in Pgp at the BBB, mediated by peripheral inflammation, can impact brain uptake of morphine. To further elucidate the mechanism(s) behind the rapid upregulation (3 h) of Pgp at this region, we explored regulation of Pgp at the plasma membrane. Our findings show that CIP induces a movement of Pgp within these domains and that Pgp co-localizes with caveolin-1 and clathrin, key structural proteins associated with caveolae and clathrin-pit lipid rafts, respectively. Our data indicate for the first time that peripheral inflammatory pain induces functional and molecular changes in Pgp, a critical efflux transporter, at the BBB in vivo and that these alterations may be mediated in part via a proteolipidic re-organization mechanism.
    • NSAIDs Modulate Morphine Transport at the Blood-Brain Barrier: A Role for P-glycoprotein

      Davis, Thomas P.; Sanchez Covarrubias, Lucy; Ronaldson, Patrick T.; French, Edward; Vanderah, Todd; Palmer, John D.; Davis, Thomas P. (The University of Arizona., 2013)
      Our laboratory has previously demonstrated that experimental peripheral inflammatory pain (PIP), induced by subcutaneous plantar injection of λ-carrageenan in Sprague Dawley rats, results in increased expression and activity of the ATP-dependent efflux transporter P-glycoprotein (P-gp) that is endogenously expressed at the blood-brain barrier (BBB). Increased P-gp functional expression was associated with a significant reduction in CNS uptake of morphine and, subsequently, reduced morphine analgesic efficacy. The present study examined whether the PIP-induced increase in P-gp functional expression was due to changes in intracellular trafficking (i.e., localization of P-gp), mediated by changes in the association of P-gp and caveolin-1, a key trafficking protein. These studies also determined if the drug diclofenac, a non-steroidal antiinflammatory (NSAID) that is commonly administered in conjunction with opioids during peripheral inflammatory pain (PIP), altered or modulated P-gp functional expression providing evidence of a drug-drug interaction. Confocal microscopy and subcellular fractionation revealed that under conditions of PIP, the disassembly of high-molecular weight P-gp-containing structures result in an increase in P-gp ATPase activity and changes in the localization of P-gp. Western blot analysis demonstrated further an increase in P-gp expression in rat brain microvessels following PIP induction and also after diclofenac treatment alone in the absence of PIP. Additionally, in situ brain perfusion studies showed that both PIP and diclofenac treatment alone increased P-gp efflux activity resulting in decreased radiolabeled- morphine uptake into the brain. This concurrent administration of NSAIDs and opioids in the presence of a pathophysiological stressor (i.e., pain/inflammation) may result in clinically significant drug-drug interactions that may impair the desired pharmacologic response and analgesic effects of opioids. Such interactions can lead to significant modifications to pain management in clinical settings. Therefore: The central hypothesis of this work is that the pathophysiological stressor peripheral inflammatory pain (PIP) and the pharmacological agent diclofenac modulate P-glycoprotein functional expression at the BBB. This hypothesis may be broken down further into two parts: 1) PIP induced changes in P-gp functional expression are mediated via changes in Pgp intracellular trafficking. 2) The non-steroidal anti-inflammatory drug Diclofenac, a drug commonly used to treat pain, modulates P-gp functional expression at the BBB thus decreasing morphine uptake into the CNS.