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Membrane-transport systems in the fenestrated capillaries of the area postrema in rat and calf.

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THE ANATOMICAL RECORD PART A 279A:664 – 670 (2004)
Membrane-Transport Systems in the
Fenestrated Capillaries of the Area
Postrema in Rat and Calf
Department of Veterinary Morphophysiology and Animal Productions, Faculty of
Veterinary Medicine, University of Bologna, Ozzano dell’Emilia, Italy
The capillaries of the area postrema (AP) lack the morphological peculiarity of the blood-brain barrier (BBB), and the AP neurons are considered
located outside the BBB. Using the immunofluorescent method, we have
investigated the expression of membrane transport systems that are instrumental to the BBB function, such as caveolin-1, -2, P-glycoprotein, and
glut-4, in the capillary endothelium of the rat and calf AP. The expression
of these molecules was verified after fibronectin immunostaining of the
microvessels. Both in the rat and calf, caveolin-1, -2, and P-glycoprotein
were expressed in the AP capillaries. A quantitative analysis revealed that
the proportion of the capillary profiles expressing these transport systems
was very close to 100% of the fibronectin immunolabelled profiles. On the
contrary, none of the AP capillaries showed glut-4 immunoreactivity. The
present investigation demonstrates that the endothelial layer of the AP
capillaries, in spite of the paracellular passage of polar molecules through
the leaky tight junctions and fenestrations, could be an active interface
which is able to control the entry of a wide range of blood-borne compounds
into the brain by means of specific mechanisms, including an efflux pump.
2004 Wiley-Liss, Inc.
Key words: receptor-mediated transport; efflux pump; immunohistochemistry; fenestrated endothelium; circumventricular organs; area postrema
The entry and distribution of organic compounds and ions
into the central nervous system depends on the blood-brain
barrier (BBB), which is made up of brain microvessel endothelial cells. These cells form a continuos layer; they are
characterized by low pinocytosis activity (Reese and Karnovsky, 1967) and are connected to each other by extensive
and complex tight junctions that prevent the paracellular
passage of most polar molecules (Kniesel and Wolburg, 2000;
Petty and Lo, 2002). Moreover, they are equipped with specific enzymatic and membrane transport systems in both
blood to brain (influx) and brain to blood (efflux) directions
(Tamai and Tsuji, 2000). Therefore, the passage across the
BBB results biochemically and locally selective. However, in
some cerebral areas referred to as circumventricular organs
(CVOs), the endothelial capillary layer lacks the morphological peculiarity of the BBB, because of the fenestrated endothelial cells and the numerous pinocytic vesicles (Dempsey,
1973; Klara and Brizzee, 1975, 1977; Coomber and Stewart,
1986; Casali et al., 1989; Lucchi et al., 1989; Gross, 1992), as
well as the leaky tight junctions (Petrov et al., 1994). Therefore, the CVOs are traditionally considered outside the BBB
Grant sponsor: Ministero dell’Istruzione, dell’Università e della
Ricerca (M.I.U.R).
*Correspondence to: Professor Maria Luisa Lucchi, Facoltà di
Medicina Veterinaria, Università di Bologna, Dipartimento di
Morfofisiologia Veterinaria e Produzioni Animali, Via Tolara di
Sopra 50, 40064 Ozzano dell’Emilia, Italy. Fax: 0039 051-792956.
Received 15 August 2003; Accepted 15 February 2004
DOI 10.1002/ar.a.20041
Published online 3 June 2004 in Wiley InterScience
TABLE 2. Calf area postrema*
TABLE 1. Rat area postrema*
Fibronectin Caveolin-1
Fibronectin Caveolin-2
Fibronectin Glut-4
⫾ 53.76
⫾ 53.93
⫾ 47.17
⫾ 46.97
⫾ 45.23
⫾ 45.18
⫾ 46.00
Fibronectin Caveolin-1
Fibronectin Caveolin-2
Fibronectin Glut-4
*Quantitative values of capillary density in double immunolabelling.
Mean value of the number of AP immunoreactive capillary
profiles in six rats.
SD, standard deviation; SE, standard error of mean; %, percentage of capillaries profiles expressing the second antigen.
and no data are available on the presence of receptor mediated vesicular transport and of efflux/influx transporters in
the endothelial cells of these cerebral areas.
In order to verify a transporter mediated permeation in
the capillaries of the CVOs, we have investigated on the
fenestrated endothelium of the area postrema (AP) the expression of caveolin-1 and -2, P-glycoprotein, and glut-4. As it
is well known, these membrane-transport systems are able
to catalyze a selective transport in the BBB endothelium
(Dehouck et al., 1997; Ikezu et al., 1998; Ngarmukos et al.,
2001; Bendayan et al., 2002; Virgintino et al., 2002a,b).
The AP represents a chemosensitive CVO involved in
emetic response and many other functions, such as neurosecretion, cardiovascular and respiratory regulation,
blood osmoreception, control of renal function, and caloric
homeostasis (Leslie, 1986; Borison, 1989). Also, the AP is
considered an important center for the integration of metabolic and hormonal control of nutrient intake (Edwards
et al., 1981; Ritter et al., 2000; Riediger et al., 2002). The
AP neurons have afferent and efferent connections (Morest, 1967; Vigier and Portalier, 1979; Manni et al., 1982;
Leslie, 1986; Koga and Fukuda, 1992; Ferguson, 1992;
Yuan and Barber, 1993) and are capable of serving as
targets for specific endogenous blood-borne humoral messengers. In fact, prolactin (Mangurian et al., 1999), angiotensin (Consolim-Colombo et al., 1996), and vasopressin
(Jurzak and Schmid, 1998) receptors have been evidenced
on the AP neurons.
This study has been performed on rat and calf because
they exhibit different behaviors to circulating emetic
agents. In fact, the i.v. injection of apomorphine causes the
expulsion of acidic abomasal contents back to preabomasal compartments (“internal vomiting”) in ruminants
only, while in rat it causes “clinical vomiting” (Eiler et al.,
Tissue Preparation
The medulla oblongata was removed from six adult
Sprague Dawley rats (350 – 430 g), killed under ethical
approval of the Committee of Animal Experimentation of
Bologna University, and from three calves (3–12 months),
butchered at a public slaughter-house. It was dissected
rostrally and caudally to the obex to take out a sample at
the level of the area postrema. The samples were im-
⫾ 15.50
⫾ 16.01
⫾ 17.67
⫾ 16.46
⫾ 14.19
⫾ 15.18
⫾ 13.61
*Quantitative values of capillary density in double immunolabelling.
Mean value of the number immunoreactive capillary profiles
counted in the left (1 mm2) and the right (1 mm2) sides of the
AP in three calves.
SD, standard deviation; SE, standard error of mean; %, percentage of capillaries profiles expressing the second antigen.
mersed in 2-dimethylbutane frozen in liquid nitrogen.
Cryostatic transverse serial sections (7 ␮m in thickness)
were collected on glass slides coated with poli-L-lysine
(Sigma-Aldrich, St. Louis, MO) and briefly fixed in cool
acetone (–20°C).
The sections of each AP were subdivided into four
groups and submitted to fluorescent double immunolabelling for: fibronectin and caveolin-1 (first group); fibronectin and P-glycoprotein (second group); fibronectin and
glut-4 (third group); and fibronectin and caveolin-2 (fourth
group). For each animal, the immunohistochemistry for a
given second antigen was performed every fourth section
of the series.
Fibronectin immunostaining was used as a capillary
marker (Gobel et al., 1990; Theilen and Kuschinsky, 1992)
to visualize the existing AP capillary profiles and to verify
the expression of the second antigen in the same structures.
After washing in phosphate-buffered solution (0.01 M
PBS), the sections were incubated for 30 min in 3% normal
donkey serum (S30; Chemicon, Temecula, CA) in PBS and
treated with 0.5% Triton X-100 in PBS for 30 min. Thereafter, the sections of the first, second, and third groups
were incubated in a mixture of mouse anti-fibronectin
(diluted 1:100 in PBS; EP5: Santa Cruz-8422, Santa Cruz,
CA), and then rabbit anti-caveolin-1 (diluted 1:100 in
PBS; N-20: Santa Cruz-894), rabbit anti-P-glycoprotein
(diluted 1:200 in PBS; H-241: Santa Cruz-8313), and rabbit anti-glut-4 (diluted 1:200 in PBS; H-61: Santa Cruz7938) antibodies for 1 hr, respectively. After washing in
PBS, the sections were incubated for 45 min with a mixture of FITC-conjugated donkey anti-mouse (diluted 1:200
in PBS; Jackson Immuno Research 715-095-150, West
Grove, PA) and TRITC-conjugated donkey anti-rabbit (diluted 1:200 in PBS; Jackson Immuno Research 711-025152), as secondary antibodies.
The sections of the fourth group were incubated in a
mixture of rabbit anti-fibronectin (diluted 1:1000 in PBS;
DAKO 0245, Glostrup, Denmark) and goat anti-caveolin-2
(diluted 1:200 in PBS; N-20: Santa Cruz-1858) antibodies
for 1 hr. After washing in PBS (3 ⫻ 10 min), the sections
were overlaid with a mixture of AMCA-conjugated donkey
Fig. 1. Area postrema of the rat. Paired images of capillaries immunostained for: a) fibronectin-FITC and
a1) caveolin-1-TRITC, bar ⫽ 20 ␮m; b) fibronectin-AMCA and b1) caveolin-2-FITC, bar ⫽ 20 ␮m; c)
fibronectin-FITC and c1) P-glycoprotein-TRITC, bar ⫽ 20 ␮m.
anti-rabbit (diluted 1:200 in PBS; Jackson Immuno Research 705-155-147) and FITC-conjugated donkey antigoat (diluted 1:200 in PBS; Santa Cruz-2024) as secondary
antibodies and incubated for 45 min.
The incubations were performed at room temperature in a
humid chamber. Control sections were prepared by omitting
the primary antibodies. After washing in PBS (3 ⫻10 min),
the slides were mounted with buffered glycerol, pH 8.6.
Fluorescent Microscopy
The sections were examined on an epifluorescent microscope (Axiophot, Carl Zeiss, Oberkochen, Germany)
Fig. 2. Area postrema of the calf. Paired images of capillaries immunostained for: a) fibronectin-FITC and
a1) caveolin-1-TRITC, bar ⫽ 50 ␮m; b) fibronectin-AMCA and b1) caveolin-2-FITC, bar ⫽ 20 ␮m; c)
fibronectin-FITC and c1) P-glycoprotein-TRITC, bar ⫽ 50 ␮m
equipped with fluorescent filter combinations for the detection of FITC, TRITC, and AMCA. The images were
recorded by using a Polaroid DMC digital camera (Polaroid Corp., Cambridge, MA) and DMC2 software. The
images were further processed using Adobe Photoshop
software (Adobe Systems, San Jose, CA).
The AP microvessels were first located by the presence
of the fluorophore that labels fibronectin; then the filter
was switched to visualize the fluorescence that labels the
second antigen. In this way, paired images of the same
field were recorded with the X 25 objective. Random sampling of areas within a given section was considered; 32
paired images, derived from no less than 16 different
sections of each group, were recorded.
Quantitative Analysis
For each double immunolabelling, an overall area of 1
mm2 was analyzed in each rat However, since the AP is a
bilateral organ in ruminants, an area of 1 mm2 was considered in each calf in each side (for a total of 2 mm2). In
this reference space, the capillary profiles immunolabelled
for fibronectin and for the second antigen, respectively,
were counted to have the density of both (N/reference
space). The counts were accomplished by two laboratory
members using a blind approach. Data were pooled by
summing the values from each animal, and the percentage
of the capillaries expressing the second antigen (caveolin-1, -2, P-glycoprotein, and glut-4) was determined (Tables 1 and 2). In addition, a linear regression analysis was
performed for each second antigen.
In the first, second, and fourth groups of sections, the
AP capillary profiles were double-immunolabelled (Figs. 1
and 2). In the third group of sections, none of the fibronectin immunolabelled AP capillary profiles showed glut-4
immunoreactivity that, on the contrary, was present in
the capillaries of the adjacent cerebral areas.
From the quantitative analysis, it was found that in
both the rat and calf AP, the proportion of the capillaries
immunostained for caveolin -1, -2, and P-glycoprotein,
respectively, was always very close to 100% of the fibronectin immunostained profiles. In fact, no significant
differences were found among the counts of the fibronectin
immunostained AP capillary profiles and that of the capillaries also expressing the second antigen (Tables 1 and
2). Moreover, the linear regression analysis showed that
the AP capillary profiles expressing the second antigen
had a significant positive association with the fibronectin
labelled ones both in the rat and calf (Figs. 3 and 4).
No immunofluorescence was observed in the control sections processed after omission of the primary antibodies.
This study demonstrates the presence of caveolin-1, -2,
and P-glycoprotein, and the absence of glut-4 in the fenestrated capillary endothelial layer of the rat and calf AP.
Since fibronectin is a reliable marker of the brain microvessels (Gobel et al., 1990; Theilen and Kuschinsky,
1992), the quantitative analysis shows that caveolin-1, -2,
and P-glycoprotein are expressed in a proportion very
close to 100% of the AP capillaries.
As it is well known, caveolin-1 and -2 are the principal
components of the caveolae. They may regulate specific
biological actions mediated by receptors (Schlegel and
Lisanti, 2001) and are implicated in the receptor-mediated
uptake and transcytotic vesicular transport of molecules,
such as albumin (Pelkmans and Helenius, 2002), LDL
(Dehouck et al., 1997), and insulin (Ikezu et al., 1998).
Therefore, their expression can account for the large number of shuttle vesicles (50 –100 nm in diameter) described
in the endothelial cells of the AP capillaries (Coomber and
Stewart, 1986).
In addition, the expression of P-glycoprotein provides
evidence of a carrier-mediated efflux transport system in
the AP fenestrated endothelial cells. P-glycoprotein is a
Fig. 3. Area postrema of the rat. Graphs showing density of fibronectin (FIBRO) immunolabelled capillaries in relation to the number of capillaries expressing: a) caveolin-1 (CAV-1); b) caveolin-2 (CAV-2); c) Pglycoprotein (P-GLY). The level of significance was set at P ⬍ 0.05. A
solid triangle shows duplicate points.
and/or prevent nonessential compounds from entering the
cells (Borst et al., 1993).
Recent studies on different cell types demonstrate that
caveolin-1 and -2 can form a stable hetero-oligomeric complex (Scherer et al., 1997). Moreover, an interaction between caveolin-1 and P-glycoprotein has been demonstrated in the BBB endothelial layer (Demeule et al., 2000;
Bendayan et al., 2002; Virgintino et al., 2002a,b). Since
the statistical analysis shows that virtually all the rat and
calf AP capillary profiles are immunostained for caveolin-1, -2, and P-glycoprotein, it seems reasonable to assume that these molecules can also interact in the fenestrated endothelial cells of the AP.
The absence of glut-4, as well as of glut-1 (Young and
Wang, 1990), i.e., of glucose transporters, in a glucoreceptive cerebral area, such as the AP (Ritter et al., 2000;
Riediger et al., 2002), can be explained with the transendothelial passage of glucose across the leaky tight junctions and fenestrations of the AP endothelial cells.
On the basis of this study, it is possible to conclude that
the AP capillary endothelial layer, in spite of its paracellular permeability, could represent a dynamic interface
that is able to regulate the movement of a wide range of
blood-borne compounds by means of different mechanisms, including an active efflux process.
Fig. 4. Area postrema of the calf. Graphs showing density of fibronectin (FIBRO) immunolabelled capillaries in relation to the number of
capillaries expressing: a) caveolin-1 (CAV-1); b) caveolin-2 (CAV-2); c)
P-glycoprotein (P-GLY). The level of significance was set at P ⬍ 0.05.
membrane-associated energy-dependent efflux transporter with a huge variety of lipophilic large molecules
(Shapiro and Ling, 1998; Schinkel, 1999; Tsuji and Tamai,
1999; Tamai and Tsuji, 2000), and it functions to detoxify
Bendayan R, Lee G, Bendayan M. 2002. Functional expression and
localization of p-glycoprotein at the blood brain barrier. Microsc Res
Tech 57:365–380.
Borison HL. 1989. Area postrema: chemoreceptor circumventricular
organ of the medulla oblongata. Prog Neurobiol 32:351–390.
Borst P, Schinker AH, Smit JJ, Wagenaar E, Van Deemter L, Smith
AJ, Eijdems EW, Baas F, Zaman GJ. 1993. Classical and novel
forms of multidrug resistence and the physiological functions of
P-glycoproteins in mammals. Pharmacol Ther 60:289 –299.
Casali AM, Lucchi ML, Millo R, Florian FM, Ferreri Santi L, Re G,
Cavalli G. 1989. L’organo subfornicale oggi: aspetti morfologici e
ruolo funzionale. Arch Ital Anat Embriol 94:1–53.
Consolim-Colombo FM, Hay M, Smith TC, Elizondo-Fournier M,
Bishop VS. 1996. Subcellular mechanisms of angiotensin II and
arginine vasopressin activation of area postrema neurons. Am J
Physiol 271:34 – 41.
Coomber BL, Stewart PA. 1986. Three-dimensional reconstruction of
vesicles in endothelium of blood-brain barrier versus highly permeable microvessels. Anat Rec 215:256 –261.
Dehouck B, Fenart L, Dehouck MP, Pierce A, Torpier G, Cecchelli R.
1997. A new function for the LDL receptor: transcytosis of LDL
across the blood-brain barrier. J Cell Biol 138:877– 889.
Demeule M, Jodoin J, Gingras D, Béliveau R. 2000. P-glycoprotein is
localized in caveolae in resistant cells and in brain capillaries.
FEBS Lett 446:219 –224.
Dempsey EW. 1973. Neural and vascular ultrastructure of the area
postrema in the rat. J Comp Neurol 150:177–200.
Edwards GL, Ritter RC. 1981. Ablation of the area postrema causes
exaggerated consumption of preferred foods in the rat. Brain Res
Eiler H, Lyke WA, Johnson R. 1981. Internal vomiting in the
ruminant: effect of apomorphine on ruminal pH in sheep. Amer J
Vet Res 42:202–204.
Ferguson AV. 1992. Neurophysiological analysis of mechanisms for
subfornical organ and area postrema involvement in autonomic
control. Prog Brain Res 91:413– 421.
Gobel U, Theilen H, Kuschinsky W. 1990. Congruence of total and
perfused capillary network in rat brains. Circ Res 66:271–281.
Gross PM. 1992. Circumventricular organ capillaries. Prog Brain Res
91:219 –233.
Jurzak M, Schmid HA. 1998. Vasopressin and sensory circumventricular organs. Prog Brain Res 119:221–245.
Klara PM, Brizzee KR. 1975. The ultrastructural morphology of the
squirrel monkey area postrema. Cell Tiss Res 160:315–326.
Klara PM, Brizzee KR. 1977. Ultrastructure of the feline area postrema. J Comp Neurol 171:409 – 432.
Kniesel U, Wolburg H. 2000. Tight junctions of the blood-brain barrier. Cell Mol Neurobiol 20:57–76.
Koga T, Fukuda H. 1992. Neurons in the nucleus of the solitary tract
mediating inputs from ematic vagal afferents and the area postrema to the pattern generator for the emetic act in dogs. Neurosci
Res 14:166 –179.
Ikezu T, Ueda H, Trapp BD, Nishiyama K, Sha JF, Volonte D, Galbiati F, Byrd AL, Bassell G, Serizawa H, Lane WS, Lisanti MP,
Okamoto T. 1998. Affinity-purification and characterization of
caveolins from the brain: differential expression of caveolin-1, -2,
and -3 in brain endothelial and astroglial cell types. Brain Res
Leslie RA. 1986. Comparative aspects of the area postrema: finestructural considerations help to determine its function. Cell Mol
Neurobiol 6:95–120.
Lucchi ML, Lalatta Costerbosa G, Barazzoni AM, Faccioli G,
Petrosino G, Bortolami R. 1989. The fine structure of the area
postrema of the sheep. Arch Ital Biol 127:37– 61.
Mangurian LP, Jurjus AR, Walsh RJ. 1999. Prolactin receptors localization to the area postrema. Brain Res 836:218 –220.
Manni E, Lucchi ML, Filippi GM, Bortolami R. 1982. Area postrema
and the mesencephalic trigeminal nucleus. Exp Neurol 77:39 –55.
Morest DK. 1967. Experimental study of the projections of the nucleus
of the tractus solitarius and the area postrema in the cat. J Comp
Neurol 130:277–300.
Ngarmukos C, Baur EL, Kumagai AK. 2001. Co-localization of
GLUT-1 and GLUT-4 in the blood brain barrier of the rat ventromedial hypothalamus. Brain Res 900:1– 8.
Pelkmans L, Helenius A. 2002. Endocytosis via caveolae. Traffic
Petrov T, Howarth AG, Krukoff TL, Stevenson BR. 1994. Distribution
of the tight junction-associated protein ZO-1 in circumventricular
organs of the CNS. Mol Brain Res 21:235–246.
Petty MA, Lo E. 2002. Junctional complexes of the blood-brain
barrier: permeability changes in neuroinflammation. Prog Neurobiol 68:311–323.
Reese TS, Karnovsky MJ. 1967. Fine structural localization of a
blood-brain barrier to exogenous peroxidase. J Cell Biol 34:207–217.
Riediger T, Schmid HA, Lutz TA, Simon E. 2002. Amylin and glucose
co-activate area postrema neurons of the rat. Neurosci Lett 328:
Ritter S, Dinh TT, Zhang Y. 2000. Localization of hindbrain glucoreceptive sites controlling food intake and blood glucose. Brain Res
856:37– 47.
Scherer PE, Lewis RY, Volonté D, Engelman JA, Galbiati F, Cuet J,
Kohth DS, van Donselaar E, Peters P, Lisanti MP. 1997. Cell-type
and tissue-specific expression of caveolin-2. Caveolins 1 and 2 colocalize and form a stable hetero-oligomeric complex in vivo. J Biol
Chem 272:29337–29346.
Schinkel AH. 1999. P-glycoprotein, a gatekeeper in the blood-brain
barrier. Adv Drug Deliv Rev 36:179 –194.
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.
Shapiro AB, Ling W. 1998. The mechanism of ATP-dependent multidrug transport by P-glycoprotein. Acta Physiol Scand 643(suppl):
Tamai I, Tsuji A. 2000. Transporter-mediated permeation of drugs
across the blood-brain barrier. J Pharm Sci 89:1371–1388.
Theilen H, Kuschinsky W. 1992. Fluorescence labeling of the capillary
network in the rat brains. J Cereb Blood Flow 12:347–350.
Tsuji A, Tamai I. 1999. Carrier-mediated or specialized transport of
drugs across the blood-brain barrier. Adv Drug Deliv Rev 36:277–
Vigier D, Portalier P. 1979. Efferent projections of the area postrema
demonstrated by autoradiography. Arch Ital Biol 117:308 –324.
Virgintino D, Robertson D, Errede M, Benagiano V, Girolamo F,
Maiorano E, Roncali L, Bertossi M. 2002a. Expression of p-glycoprotein in human cerebral cortex microvessels. J Histochem Cytochem 50:1671–1676.
Virgintino D, Robertson D, Errede M, Benagiano V, Tauer U, Roncali
L, Bertossi M. 2002b. Expression of caveolin-1 in human brain
microvessels. Neurosci 115:145–152.
Young JK, Wang C. 1990. Glucose transporter immunoreactivity in
the hypothalamus and area postrema. Brain Res Bull 24:525-528.
Yuan CS, Barber WD. 1993. Area postrema: gastric vagal input from
proximal stomach and interactions with nucleus tractus solitarius
in cat. Brain Res Bull 30:119-125.
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capillaries, transport, area, postrema, rat, system, membranes, calf, fenestrated
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