Membrane-transport systems in the fenestrated capillaries of the area postrema in rat and calf.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 279A:664 – 670 (2004) Membrane-Transport Systems in the Fenestrated Capillaries of the Area Postrema in Rat and Calf CRISTIANO BOMBARDI, ANNAMARIA GRANDIS, ROBERTO CHIOCCHETTI, MARIA LUISA LUCCHI,* EMILIO CALLEGARI, AND RUGGERO BORTOLAMI Department of Veterinary Morphophysiology and Animal Productions, Faculty of Veterinary Medicine, University of Bologna, Ozzano dell’Emilia, Italy ABSTRACT 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, © 2004 WILEY-LISS, INC. 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. E-mail: firstname.lastname@example.org Received 15 August 2003; Accepted 15 February 2004 DOI 10.1002/ar.a.20041 Published online 3 June 2004 in Wiley InterScience (www.interscience.wiley.com). 665 TRANSPORT SYSTEMS IN AREA POSTREMA CAPILLARIES TABLE 2. Calf area postrema* TABLE 1. Rat area postrema* Double immunolabellings Fibronectin Caveolin-1 Fibronectin Caveolin-2 Fibronectin P-glycoprotein Fibronectin Glut-4 N/mm2a SD 347.83 347.17 346.67 344.83 351.17 349.83 346.17 0 ⫾ 53.76 ⫾ 53.93 ⫾ 47.17 ⫾ 46.97 ⫾ 45.23 ⫾ 45.18 ⫾ 46.00 ⫾0 % Double immunolabellings N/2mm2a Fibronectin Caveolin-1 99.81 Fibronectin Caveolin-2 99.47 99.62 Fibronectin P-glycoprotein Fibronectin Glut-4 0 *Quantitative values of capillary density in double immunolabelling. a 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., 1981). MATERIAL AND METHODS 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- 608.33 606.67 609.67 607.00 604.67 602.67 619.33 0 SD ⫾ 15.50 ⫾ 16.01 ⫾ 17.67 ⫾ 16.46 ⫾ 14.19 ⫾ 15.18 ⫾ 13.61 ⫾0 % 99.73 99.56 99.67 0 *Quantitative values of capillary density in double immunolabelling. a 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). Immunohistochemistry 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 666 BOMBARDI ET AL. 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) TRANSPORT SYSTEMS IN AREA POSTREMA CAPILLARIES 667 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; 32 668 BOMBARDI ET AL. 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. RESULTS 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. DISCUSSION 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. TRANSPORT SYSTEMS IN AREA POSTREMA CAPILLARIES 669 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. 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