ARTICLE |
Correspondence to: Daniela Virgintino, Dept. of Human Anatomy and Histology, University of Bari School of Medicine, Piazza Giulio Cesare, I-70124 Bari, Italy. E-mail: virgintino@histology.uniba.it
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
P-Glycoprotein (P-gp) is an ATP-dependent efflux transporter that extrudes non-polar molecules, including cytotoxic substances and drugs, from the cells. It was initially found in cancer cells and then was shown to be a normal component of complex transport systems working at the bloodbrain barrier (BBB). Previous studies have demonstrated that, in the brain, P-gp is localized on the luminal plasmalemma of BBB endothelial cells and that it may interact with the caveolar compartment of these cells. The aim of this study was to identify the site of cellular expression of P-gp in human brain in situ and to morphologically determine whether an association may exist between P-gp and caveolin-1, a structural and functional protein of the caveolar frame. The study was carried out on human cerebral cortex by immunoconfocal microscopy with antibodies to both P-gp and caveolin-1. The results show that P-gp marks the microvessels of the cortex and that the transporter is localized in the luminal endothelial compartment, where it co-localizes with caveolin-1. The demonstration of this co-localization of P-gp with caveolin-1 contributes a morphological backing to biochemical studies on P-gp/caveolin-1 relationships and leads us to suggest that interactions between these molecules may occur at the BBB endothelia.
(J Histochem Cytochem 50:16711676, 2002)
Key Words: P-glycoprotein, caveolin-1, human brain microvessels, bloodbrain barrier, immunoconfocal microscopy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the central nervous system (CNS), highly specialized transporters work at the bloodtissue interface, contributing to the activities of the bloodbrain barrier (BBB), a sophisticated multicellular and multifunctional control unit that regulates the neuronal microenvironment and CNS homeostasis. Among the BBB-specific transporters, the P-glycoprotein (P-gp), a member of the ABC (ATP-binding cassette) transporter superfamily, is an intrinsic membrane protein functioning as an energy-dependent efflux pump and involved in vectorial back-transport of a wide range of hydrophobic compounds (
The precise function of P-gp in normal tissues is not completely understood. In somatic microvessels, in which P-gp was found on both luminal and abluminal membranes of the endothelial cells, the transporter could actively regulate the efflux of lipophilic substances from the endothelial cytoplasm on both blood and tissue fronts. On the contrary, in the BBB microvessels, where the highest levels of P-gp have been detected, the transporter was described only at the luminal endothelial membrane. This selective one-front localization suggests that P-gp plays a barrier protective role by extruding cytotoxic substances and drugs from the endothelial cells back into the bloodstream (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study included 10 samples of brain tissues, measuring 46 mm in maximal diameter, obtained from 10 patients (six men and four women; age range 5065 years, median 58 years) undergoing tumor resection for high grade (IIIIV) gliomas with concomitant mapping of tumor-free margins. The latter procedure consisted of intraoperative examination of hematoxylineosin (H&E)-stained frozen tissue sections obtained from half of each of the above 10 samples that confirmed absence of tumor invasion. The other halves of the tumor-free samples were immersed in a fixative mixture consisting of 2% paraformaldehyde plus 0.2% glutaraldehyde in PBS for 2 hr at 4C. Sections (25-µm) were prepared using a vibrating microtome (Leica; Milton Keynes, UK). Fluorescence single and double immunolabeling was carried out by a second-layer indirect method on free-floating sections. The primary antibodies were a mouse monoclonal antibody (MAb) raised against the cytoplasmic epitope of the plasma membrane-associated 170180-kD glycoprotein (1:20 dilution; clone JSB-1, IgG1 subclass; Novocastra Laboratories, Newcastle upon Tyne, UK) and a rabbit polyclonal antibody raised against the N-terminus of caveolin-1 of human origin (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). In addition, to further check the normal nature of the non-neoplastic brain tissue samples, the sections were subjected to double labeling for the glucose transporter isoform 1 (GLUT-1) as a marker for BBB endothelial cells and for the glial fibrillary acidic protein (GFAP) as a marker for astroglial cells, utilizing a rabbit polyclonal antibody anti-GLUT-1 (1:500 dilution; Chemicon International, Temecula, CA) and a mouse MAb anti-GFAP (1:100 dilution; Novocastra Laboratories). The immunostains were performed in parallel with those for P-gp/caveolin-1.
The sections were immersed and gently agitated at room temperature as follows: (a) 30 min in blocking buffer (BB: PBS, 1% BSA, 2% FCS); (b) 30 min in 0.5% Triton X-100 in PBS; (c) 1 hr in either one or a mixture of the two primary antibodies (double labeling) suitably diluted in BB; (d) 1 hr in the appropriate secondary antibody/ies, Alexa Fluor 488 goat anti-mouse IgG (Fab'2-conjugated fragment; Molecular Probes Europe, Leiden, The Netherlands; for P-gp and GFAP), Alexa Fluor 568 goat anti-rabbit IgG conjugate (Molecular Probes; for caveolin-1 and GLUT-1) both diluted 1:200 in BB. The sections were washed three times for 10 min in PBS between each step. Nuclear counterstaining was performed by incubations either in RNase diluted 5 µl/ml in PBS (30 min at 37C) and then propidium iodide (Molecular Probes) diluted 1 µl/ml in PBS, or by TO-PRO-3 iodide (Molecular Probes) diluted 1:10 in PBS and added to the third wash after the conjugate antibodies. The sections were then transferred onto subbed slides (1% gelatin plus 1% formalin in distilled water), carefully drained, coverslipped in Vectashield anti-fade mounting medium (Vector Laboratories; Burlingame, CA), and sealed with nail polish. To fully preserve the fluorescence signal, no pretreatments were applied to avoid autofluorescence of both lipofuscin pigments and lipofuscin-like substances in neurons and perivascular cells (
The control sections were prepared (a) by substituting the primary antibodies with BB, (b) by substituting anti-P-gp with an inappropriate MAb of identical subclass and anti-caveolin-1 with normal rabbit serum, both at the same working dilution, (c) preadsorbing the primary antibody anti-caveolin-1 with an excess of the pure antigen available from the supplier, or (d) mismatching the secondary antibodies. The results of these negative controls were consistent with the expected results.
The sections were viewed under the Leica TCS SP confocal laser scanning microscope using x40 and x63 oil-immersion objective lenses with either x1 or x2 zoom factors. On the double-immunolabeled sections, a sequential scan procedure was applied during image acquisition of the two fluorophores, Alexa Fluor 488 (excitation at 488 nm and detection range 500535 nm; green fluorescence) and Alexa Fluor 568 (excitation with 568 nm and detection range 580620 nm; red fluorescence). Confocal images were taken at 0.5-µm intervals through the z-axis of the section covering in total 1520 µm in depth. Images from individual optical sections and multiple serial optical sections were analyzed, recorded digitally, and stored as TIFF files in Adobe Photoshop software (Adobe Systems; San Jose, CA).
All the investigation procedures on human tissues were carried out in accordance with the local institutional ethical committee policies.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Examination of the sections double immunolabeled for GLUT-1 and GFAP confirms the normal morphological features of the tissue. GLUT-1 is expressed by the endothelial cells of the cortex microvessels and GFAP marks astrocyte processes scattered in the neuropil and regularly distributed on the microvessel wall (Fig 1A).
|
The cortex microvessels are heavily stained by P-gp immunolabeling, showing a distribution that appears to be independent of the lamination of the cortex. P-gp preferentially stains the cortex microvessels, whereas it appears weaker or undetectable on the larger vessels. It does not stain neurons and glial cells in the neuropil. In neurons, only the autofluorescence of lipofuscin is detectable and is always easily distinguishable from the bright-green P-gp-specific staining (Fig 1B). On reconstructed microvessels obtained with multiple optical planes, the P-gp labeling appears finely punctate and evenly distributed all along the vascular walls, with many scattered, more evident fluorescent puncta (Fig 1B). On longitudinal- and cross-sectioned microvessels, the P-gp labeling reveals the entire vessel profile and is seen to correspond to the thin endothelial cells that line the vessel walls (Fig 1C). Perivascular phagocytic cells containing autofluorescent lipofuscin-like granules are a constant feature of these vascular fields (Fig 1C). On the double P-gp/caveolin-1-immunolabeled sections (Fig 2A2L), observation at higher magnification on serial optical planes of cross-sectioned microvessels confirms that the expression of P-gp corresponds to the endothelial cells and also reveals that the transporter is localized in their luminal compartment (Fig 2A and Fig 2F). In the vascular wall, weaker reactivity is also detectable in cells in close contact with the endothelium and recognizable as pericytes owing to their position and nuclear shape (Fig 2F). Unlike P-gp, caveolin-1 stains the entire thickness of the endothelium from the luminal to the abluminal side, with a finely punctate pattern in the endothelial luminal compartment and larger fluorescent puncta in the abluminal one (Fig 2B and Fig 2G). Comparing the single channel signal of P-gp and caveolin-1, green and red fluorescence, respectively, it is evident that both proteins are present in the luminal compartment of the endothelial cells (Fig 2A, Fig 2B, Fig 2F, and Fig 2G). Nevertheless, only on the merged images, obtained by superimposing the two fluorescent signals, is the exact extent of P-gp/caveolin-1 co-localization revealed (Fig 2C2E and Fig 2H2L; yellow fluorescence). More frequently, P-gp and caveolin-1 appear to largely co-localize in the luminal compartment of the endothelial cells (Fig 2C2E), although elsewhere the fluorescent signals do not appear to overlap completely and co-localization is detectable only at the boundary between the luminal and abluminal endothelial cell compartments (Fig 2H2L).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A large body of work has analyzed P-gp expression, substrates, and activities in the CNS, obtaining significant information thanks to many different experimental approaches, such as cultures of cerebral endothelial cells, isolated brain microvessels, and the P-gp knockout mouse (
As to the mechanisms of action and regulation of the P-gp efflux pump in resistant and normal cells, previous biochemical studies demonstrated that drugs are sequestered in P-gp-containing vesicles and that the caveolar number and caveolin-1 levels parallel the expression of P-gp (
![]() |
Acknowledgments |
---|
Supported by grants from the Consiglio Nazionale delle Ricerche (to L.R.) and the Ministero dell'Istruzione, dell'Università e della Ricerca (to D.V.).
We thank Ms M.V.C. Pragnell, BA, for linguistic help and Ms M. Ambrosi for excellent technical assistance.
Received for publication March 27, 2002; accepted July 10, 2002.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beaulieu E, Demeule M, Ghitescu L, Beliveau R (1997) P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain. Biochem J 326:539-544[Medline]
Begley DJ, Lechardeur D, Chen Z-D, Rollinson C, Bardoul M, Roux F, Scherman D et al. (1996) Functional expression of P-glycoprotein in an immortalised cell line of rat brain endothelial cells, RBE4. J Neurochem 67:988-995[Medline]
CordonCardo C, O'Brien JP, Boccia J, Casals D, Bertino JR, Melamed MR (1990) Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J Histochem Cytochem 38:1277-1287[Abstract]
CordonCardo C, O'Brien JP, Casals D, RittmanGrauer L, Biedler JL, Melamed MR, Bertino JR (1989) Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proc Natl Acad Sci USA 86:695-698[Abstract]
Demeule M, Jodoin J, Gingras D, Beliveau R (2000) P-glycoprotein is localized in caveolae in resistant cells and in brain capillaries. FEBS Lett 466:219-224[Medline]
Demeule M, Labelle M, Régina A, Berthelet F, Beliveau R (2001) Isolation of endothelial cell from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms. Biochem Biophys Res Commun 281:827-834[Medline]
Feng Y, Venema VJ, Venema RC, Tsai N, Behzadian MA, Caldwell RB (1999) VEGF-induced permeability increase is mediated by caveolae. Invest Ophthalmol Vis Sci 40:157-167[Abstract]
Ferté J (2000) Analysis of the tangled relationships between P-glycoprotein-mediated multidrug resistance and the lipid phase of the cell membrane. Eur J Biochem 267:277-294
Golden PL, Pardridge WM (1999) P-glycoprotein on astrocyte foot processes of unfixed isolated human brain capillaries. Brain Res 819:143-146[Medline]
Greenwood J (1992) Characterization of a rat retinal endothelial cell culture and the expression of P-glycoprotein in brain and retinal endothelium in vitro. J Neuroimmunol 39:123-132[Medline]
Hegmann EJ, Bauer HC, Kerbel RS (1992) Expression and functional activity of P-glycoprotein in cultured cerebral capillary endothelial cells. Cancer Res 52:6969-6975[Abstract]
Jetté L, Têtu B, Béliveau R (1993) High levels of P-glycoprotein detected in isolated brain capillaries. Biochim Biophys Acta 1150:147-154[Medline]
Lavie Y, Fiucci G, Czarny M, Liscovitch M (1999) Changes in membrane microdomains and caveolae constituents in multidrug-resistant cancer cells. Lipids 34:57-63
Megard I, Garrigues A, Orlowski S, Jorajuria S, Clayette P, Ezan E, Mabondzo A (2002) A co-culture-based model of human blood-brain barrier: application to active transport of indinavir and in vivo-in vitro correlation. Brain Res 927:153-167[Medline]
Miller DS, Nobmann SN, Gutmann H, Toeroek M, Drewe J, Fricker G (2000) Xenobiotic transport across isolated brain microvessels studied by confocal microscopy. Mol Pharmacol 58:1357-1367
Pardridge WM, Golden PL, Kang YS, Bickel U (1997) Brain microvascular and astrocyte localization of P-glycoprotein. J Neurochem 68:1278-1285[Medline]
Parton RG (1996) Caveolae and caveolins. Curr Opin Cell Biol 8:542-548[Medline]
Schinkel AH (1999) P-glycoprotein, a gatekeeper in the blood brain barrier. Adv Drug Deliv Rev 36:179-194[Medline]
Schinkel AH, Smith JJM, van Tellingen O, Brijnen JH, Wagenaar E, van Deemter L, Mol CAAM et al. (1994) Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77:491-502[Medline]
Schlegel A, Lisanti MP (2001) Caveolae and their coat proteins, the caveolins: from electron microscopic novelty to biological launching pad. J Cell Physiol 186:329-337[Medline]
Schnell SA, Staines WA, Wessendorf MW (1999) Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem 47:719-730
Shapiro AB, Fox K, Lee P, Yang YD, Ling V (1998) Functional intracellular P-glycoprotein. Int J Cancer 76:857-864[Medline]
Stewart PA, Beliveau R, Rogers KA (1996) Cellular localization of P-glycoprotein in brain versus gonadal capillaries. J Histochem Cytochem 44:679-685
Tani E, Yamagata S, Ito Y (1977) Freeze-fracture of capillary endothelium in rat brain. Cell Tissue Res 176:157-165[Medline]
Tatsuta T, Naito M, Oh-haras T, Sugawara I, Tsuruo T (1992) Functional involvement of P-glycoprotein in blood-brain barrier. J Biol Chem 267:20383-20391
Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC (1987) Cellular localization of the multidrug-resistence gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci USA 84:7735-7738[Abstract]
Tsuji A, Tamai I (1999) Carrier-mediated or specialized transport of drugs across the blood brain barrier. Adv Drug Deliv Rev 36:277-290[Medline]
Xu HL, Galea E, Santizo RA, Baughman VL, Pelligrino DA (2001) The key role of caveolin-1 in estrogen-mediated regulation of endothelial nitric oxide synthase function in cerebral arterioles in vivo. J Cereb Blood Flow Metab 21:907-913[Medline]