P-glycoprotein (PgP) is the major drug efflux pump in human cerebral microvessels. PgP prevents pathogens, toxins and therapeutic drugs from entering the CNS. Understanding the molecular regulation of PgP activity will suggest novel mechanisms to improve CNS drug delivery. Previously, we found that during peripheral inflammatory pain (PIP) (3 h after λ carrageenan injection in the rat paw), PgP traffics to the cortical microvessel endothelial cell plasma membrane concomitant with increased PgP activity. In the current study, we measured the changes in composition of PgP-containing protein complexes after PIP in rat microvessel isolates. We found that a portion of the PgP is contained in a multi-protein complex that also contains the caveolar proteins CAV1, SDPR, PTRF and PRKCDBP. With PIP, total CAV1 bound to PgP was unchanged; however, phosphorylated CAV1 (Y14P-CAV1) in the complex increased. There were few PgP/CAV1 complexes relative to total PgP and CAV1 in the microvessels suggesting CAV1 bound to PgP is unlikely to affect total PgP activity. However, both PgP and CAV1 trafficked away from the nucleus in response to PIP. These data suggest that P-CAV1 bound to PgP potentially regulates PgP trafficking and contributes to the acute PgP activity increase after a PIP stimulus.
AbbottNJPatabendigeAADolmanDEet al.Structure and function of the blood-brain barrier. Neurobiol Dis2010; 37: 13–25.
2.
ObermeierBDanemanRRansohoffRM. Development, maintenance and disruption of the blood-brain barrier. Nat Med2013; 19: 1584–1596.
3.
BorstPElferinkRO. Mammalian ABC transporters in health and disease. Annu Rev Biochem2002; 71: 537–592.
4.
MillerDSCannonRE. Signaling pathways that regulate basal ABC transporter activity at the blood- brain barrier. Curr Pharm Des2014; 20: 1463–1471.
5.
SeelbachMJBrooksTAEgletonRDet al.Peripheral inflammatory hyperalgesia modulates morphine delivery to the brain: a role for P-glycoprotein. J Neurochem2007; 102: 1677–1690.
6.
MillerDS. Regulation of P-glycoprotein and other ABC drug transporters at the blood-brain barrier. Trends Pharmacol Sci2010; 31: 246–254.
7.
AmbudkarSVKimIWSaunaZE. The power of the pump: mechanisms of action of P-glycoprotein (ABCB1). Eur J Pharm Sci2006; 27: 392–400.
8.
LooTWBartlettMCClarkeDM. Permanent activation of the human P-glycoprotein by covalent modification of a residue in the drug-binding site. J Biol Chem2003; 278: 20449–20452.
9.
ThomasHColeyHM. Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting p-glycoprotein. Cancer Control2003; 10: 159–165.
10.
LiangXJAszalosA. Multidrug transporters as drug targets. Curr Drug Targets2006; 7: 911–921.
11.
HawkinsBTSykesDBMillerDS. Rapid, reversible modulation of blood-brain barrier P-glycoprotein transport activity by vascular endothelial growth factor. J Neurosci2010; 30: 1417–1425.
12.
CannonREPeartJCHawkinsBTet al.Targeting blood-brain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain. Proc Natl Acad Sci U S A2012; 109: 15930–15935.
13.
MesevEVMillerDSCannonRE. Ceramide 1-phosphate increases P-glycoprotein transport activity at the blood-brain barrier via prostaglandin E2 signaling. Mol Pharmacol2017; 91: 373–382.
14.
BanksDBChanGNEvansRAet al.Lysophosphatidic acid and amitriptyline signal through LPA1R to reduce P-glycoprotein transport at the blood-brain barrier. J Cereb Blood Flow Metab2017; 38: 857–868.
15.
HuberJDHauVSBorgLet al.Blood-brain barrier tight junctions are altered during a 72-h exposure to lambda-carrageenan-induced inflammatory pain. Am J Physiol Heart Circ Physiol2002; 283: H1531–H1537.
16.
McCaffreyGSeelbachMJStaatzWDet al.Occludin oligomeric assembly at tight junctions of the blood-brain barrier is disrupted by peripheral inflammatory hyperalgesia. J Neurochem2008; 106: 2395–2409.
17.
McCaffreyGStaatzWDSanchez-CovarrubiasLet al.P-glycoprotein trafficking at the blood-brain barrier altered by peripheral inflammatory hyperalgesia. J Neurochem2012; 122: 962–975.
18.
TomeMEHerndonJMSchaeferCPet al.P-glycoprotein traffics from the nucleus to the plasma membrane in rat brain endothelium during inflammatory pain. J Cereb Blood Flow Metab2016; 36: 1913–1928.
19.
JodoinJDemeuleMFenartLet al.P-glycoprotein in blood-brain barrier endothelial cells: interaction and oligomerization with caveolins. J Neurochem2003; 87: 1010–1023.
20.
BarakatSDemeuleMPilorgetAet al.Modulation of p-glycoprotein function by caveolin-1 phosphorylation. J Neurochem2007; 101: 1–8.
21.
Vilas-BoasVSilvaRNunesCet al.Mechanisms of P-gp inhibition and effects on membrane fluidity of a new rifampicin derivative, 1,8-dibenzoyl-rifampicin. Toxicol Lett2013; 220: 259–266.
22.
TroostJLindenmaierHHaefeliWEet al.Modulation of cellular cholesterol alters P-glycoprotein activity in multidrug-resistant cells. Mol Pharmacol2004; 66: 1332–1339.
23.
GreenwoodJPryceGDevineLet al.SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics. J Neuroimmunol1996; 71: 51–63.
24.
DemeusePFragnerPLeroy-NouryCet al.Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines. J Neurochem2004; 88: 23–31.
25.
TomeMESchaeferCPJacobsLMet al.Identification of P-glycoprotein co-fractionating proteins and specific binding partners in rat brain microvessels. J Neurochem2015; 134: 200–210.
26.
McCaffreyGStaatzWDQuigleyCAet al.Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo. J Neurochem2007; 103: 2540–2555.
27.
SchindelinJArganda-CarrerasIFriseEet al.Fiji: an open-source platform for biological-image analysis. Nat Methods2012; 9: 676–682.
28.
BendayanRRonaldsonPTGingrasDet al.In situ localization of P-glycoprotein (ABCB1) in human and rat brain. J Histochem Cytochem2006; 54: 1159–1167.
29.
WangXLiuTBaiYet al.Polymerase I and transcript release factor acts as an essential modulator of glioblastoma chemoresistance. PLoS One2014; 9: e93439.
30.
SannaEMiottiSMazziMet al.Binding of nuclear caveolin-1 to promoter elements of growth-associated genes in ovarian carcinoma cells. Exp Cell Res2007; 313: 1307–1317.
31.
LudwigAHowardGMendoza-TopazCet al.Molecular composition and ultrastructure of the caveolar coat complex. PLoS Biol2013; 11: e1001640.
32.
HillMMBastianiMLuetterforstRet al.PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function. Cell2008; 132: 113–124.
33.
ZimnickaAMHusainYSShajahanANet al.Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae. Mol Biol Cell2016; 27: 2090–2106.
34.
ZhangYQuXTengYet al.Cbl-b inhibits P-gp transporter function by preventing its translocation into caveolae in multiple drug-resistant gastric and breast cancers. Oncotarget2015; 6: 6737–6748.
35.
MinshallRDTiruppathiCVogelSMet al.Vesicle formation and trafficking in endothelial cells and regulation of endothelial barrier function. Histochem Cell Biol2002; 117: 105–112.
36.
de LA, DonovanLArcaroA. Lipid rafts and caveolae in signaling by growth factor receptors. Open Biochem J2007; 1: 12–32.
37.
MohanJMorenBLarssonEet al.Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae. J Cell Sci2015; 128: 979–991.
38.
RadevaGPeraboJSharomFJ. P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains. FEBS J2005; 272: 4924–4937.
39.
HawkinsBTRigorRRMillerDS. Rapid loss of blood-brain barrier P-glycoprotein activity through transporter internalization demonstrated using a novel in situ proteolysis protection assay. J Cereb Blood Flow Metab2010; 30: 1593–1597.
40.
Schmidt-GlenewinkelHReinzEBulashevskaSet al.Multiparametric image analysis reveals role of Caveolin1 in endosomal progression rather than internalization of EGFR. FEBS Lett2012; 586: 1179–1189.
41.
NetheMHordijkPL. A model for phospho-caveolin-1-driven turnover of focal adhesions. Cell Adh Migr2011; 5: 59–64.
42.
Gottlieb-AbrahamEShvartsmanDEDonaldsonJCet al.Src-mediated caveolin-1 phosphorylation affects the targeting of active Src to specific membrane sites. Mol Biol Cell2013; 24: 3881–3895.
43.
CaiTWangHChenYet al.Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase. J Cell Biol2008; 182: 1153–1169.
44.
GobeilFJr.BernierSGVazquez-TelloAet al.Modulation of pro-inflammatory gene expression by nuclear lysophosphatidic acid receptor type-1. J Biol Chem2003; 278: 38875–38883.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
