Expression of GFRalpha -1, receptor for GDNF, in rat brain capillary during postnatal development of the BBB

Hiroyuki Utsumi, Hideki Chiba, Yasuhiro Kamimura, Makoto Osanai, Yo Igarashi, Hirotoshi Tobioka, Michio Mori, and Norimasa Sawada

Department of Pathology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo, 060, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is well known that the blood-brain barrier (BBB) matures at ~2 wk after birth in the rat. Recently, we showed that glial cell line-derived neurotrophic factor (GDNF) enhances the barrier function of porcine endothelial cells forming the BBB in culture. In the present study, we examined the relation between permeability of the BBB, using Evans blue as a tracer, and expression of the GDNF family receptor (GFRalpha -1) during postnatal development of the BBB. Morphometric analysis showed that exudation of Evans blue from capillaries of the cerebral cortex progressively decreased until postnatal day 21. Inversely, immunohistochemical examinations showed expression of GFRalpha -1 in the capillaries at postnatal day 3 and expression that reached the same levels as observed in adult rats by postnatal day 10. However, c-ret, which is thought to mediate a signal evoked by binding of GDNF to GFRalpha -1, was not expressed in the capillaries of the brain cortex in 3-mo-old rats. On the other hand, the tight junction proteins occludin and ZO-1 appeared to be fully expressed at birth. The reciprocal relation between GFRalpha -1 expression and the permeability of the BBB strongly suggests active participation of GDNF in postnatal development of the BBB, although the mechanism(s) involved is still veiled.

blood-brain barrier; maturation; glial cell line-derived neurotrophic factor; glial cell line-derived neurotrophic factor family receptor alpha -1; Evans blue


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CAPILLARY ENDOTHELIUM in the brain forms a highly impermeable structure between the blood and the central nervous system (CNS) brain interstitium, called the blood-brain barrier (BBB), and plays an essential role in maintaining homeostasis of the CNS. In the BBB, highly impermeable tight junctions between endothelial cells are the most important cellular apparatus for paracellular barrier function as well as limited transcytosis of endothelial cells (23). It is well known that the BBB of the rat fully matures in postnatal weeks 3-4 (2, 3) and that exudation of tracers from the BBB ceases at ~2 wk after birth (18, 32). However, none of the molecular mechanisms of development of the BBB have yet been fully clarified.

Several proteins associated with tight junctions have been disclosed (5). Of these proteins, occludin and the claudins are considered to be essential for tight junctions because they are integral membrane proteins comprising tight junction strands (29). Occludin is known to be much more highly expressed in brain endothelial cells than in endothelial cells of nonneural tissue (12), whereas the expression of claudins in various tissues and cell types has not been fully clarified (19). To date, the roles of these proteins in regulation of tight junctions are mostly unknown.

In the brain, astrocytes have been suggested to contribute to development of the BBB because the cells have vascular feet ensheathing the brain capillaries. In addition to this anatomic observation, it has been reported that astrocytes presumably secrete unknown factors differentiating capillaries to the BBB-type capillary, in terms of forming impermeable tight junctions (1, 6, 15, 20, 23, 26). In this context, it was reported that interleukin-6 secreted by astrocytes contributes to induction of BBB properties such as activities of alkaline phosphatase and the Na+-K+-Cl- cotransporter (24, 25).

The rat glial cell line B49 secretes glial cell-derived neurotrophic factor (GDNF) to maintain dopaminergic (17) and motor neurons (11) in vivo. GDNF is a member of the transforming growth factor-beta family and binds to receptor GFRalpha -1 on the cell surface of neurons. GFRalpha -1 then interacts with c-ret, a receptor kinase (7, 16, 27, 28). Recently, we demonstrated that GFRalpha -1 was expressed in endothelial cells of the cerebral cortex in adult rats and that GDNF activated the barrier function of endothelial cells isolated from the porcine brain in vitro (13), suggesting that GDNF is a potent differentiating factor of the BBB. Thus in the present study we examined the expression of GFRalpha -1 and tight junction-associated proteins during postnatal maturation of the BBB in the rat as compared with leakage of Evans blue from the BBB as a marker of permeability. We show here a reciprocal relationship between leakage of Evans blue and expression of GFRalpha -1, strongly suggesting that GDNF is a potent differentiating factor for the BBB.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. F344/Jcl rats were used on postnatal days 1, 3, 5, 7, 10, and 14 and as adults (postnatal month 6) for immunohistochemical detection. Moreover, postnatal day 4 (n = 3), 7 (n = 3), 10 (n = 3), 14 (n = 3), and 21 (n = 3) rats as well as adult rats (n = 3, postnatal month 3) were used for evaluation of Evans blue leakage. Three 4-wk-old Crj:CD-1 (ICR) mice were used for preparation of total RNAs from the brain, spinal cord, and thymus, because the complete sequence of c-ret is only available for the mouse and human.

Evaluation of Evans blue leakage. Evans blue (4% wt/vol) was intraperitoneally injected into the rats, and systematic distribution of the dye was confirmed by a change in skin color 2 h after injection. After decapitation, the brains were frozen in nitrogen, sectioned 5 µm thick, and mounted on glass slides. The extent of the autofluorescence of Evans blue in or over the capillaries was noted with a Nikon FX epifluorescence photomicroscope with the use of Photoshop (Adobe Photoshop 4.0J) under the same luminance conditions. Five micrographs were randomly taken from one rat, and then each area stained by Evans blue was measured by using Photoshop under the same luminance conditions. The area ratios of Evans blue leakage were determined by dividing the mean area of the postnatal rats by the mean area of adult rats. The mean ± SD of one area stained with Evans blue was calculated from the values of three rats.

RNA extraction and RT-PCR. RNA preparation and RT-PCR were performed according to standard protocols (21). Briefly, total RNA was isolated from the cerebral cortices and spinal cords of two male Crj:CD-1 (ICR) mice with the use of the single-step thiocyanate-phenol-chloroform extraction method as modified by Xie and Rothblum (30). RT-PCR was performed using an RT-PCR kit supplied by PerkinElmer (Branchburg, NJ) according to the manufacturer's recommendations. To confirm the finding that no c-ret was present in the cerebral cortex, total RNA was extracted from the various tissues of 3- to 4-wk-old mice followed by RT-PCR using 1 µg of the total RNA as a template. The primers for mouse c-ret (14) were 5'-GGATGCCCCTGGAGAAGTGCC-3' [nucleotides (nt) 171 to 191] and 5'-CATTCCTCACACTCGGGGCGC-3' (nt 1,598 to 1,578). PCR was performed for 40 cycles. Aliquots of PCR products (10 µl) were loaded on a 1% agarose gel containing ethidium bromide.

Immunohistochemistry. About 1 mm3 of the brain cortex tissue was put between glass slides. The slides were pressed against each other, pulled apart, and immediately immersed in a cold mixture of acetone and ethanol (1:1) for 5 min. This procedure is suitable for observation of brain tissues, particularly vessels, because the cell shape and tissue are well preserved by avoiding freezing. Five 6-µm-thick frozen sections of the brain cortex and spinal cord, as well as cultured cells on polycarbonate filters, were also dipped in the mixture for 5 min. After the slides were rinsed with phosphate-buffered saline (PBS), they were incubated overnight with the primary antibody at 4°C. The tissues were then incubated with appropriate secondary antibodies (DAKO, Glostrup, Denmark) labeled with FITC for 1 h at room temperature. Primary antibodies used included goat polyclonal anti-rat GFRalpha -1 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti ZO-1 (Zymed, San Francisco, CA), rabbit polyclonal anti-c-ret (Santa Cruz Biotechnology), and rabbit polyclonal anti-occludin (Zymed). Actin was visualized by using rhodamine-phalloidin. All samples were examined with a Nikon FX epifluorescence photomicroscope (Nikon, Tokyo, Japan) and/or a confocal laser scanning fluorescence imaging system (MRC-500J, Bio-Rad).

Primary cultures of porcine brain capillary endothelial cells. Porcine brain capillary endothelial cells were purified as described previously (13). Briefly, cortical gray matter of brains obtained from miniature pigs weighing ~20 kg was minced with scissors into small pieces and digested in 0.25% dispase (Godo Shusei, Tokyo, Japan) and 0.12% collagenase (Yakult, Tokyo) in Ca2+-, Mg2+-free Hanks' balanced saline solution (HBSS) at 39°C for 60 min. During the enzyme digestion, the solution containing the tissues was bubbled with a mixture of 95% O2-5% CO2. After extensive pipetting, the capillaries were separated from the remaining slurry by centrifugation at 1,000 g for 15 min in PBS containing 25% bovine serum albumin. After several rinses by centrifugation, fragments of capillaries were seeded onto 12-well tissue culture plates coated with type IV collagen (Nitta Gelatin, Osaka, Japan) in Dulbecco's modified Eagle's medium 1:1 with Ham's F-12 nutrient (D/F12) mixture (Kyokuto), supplemented with 15% heat-inactivated fetal bovine serum (Moregate), 75 µg/ml endothelial growth supplement (Sigma, St. Louis, MO), 80 µg/ml heparin (Sigma), 5 µg/ml insulin (Collaborative Biomedical, Bedford, MA), 5 µg/ml transferrin (Collaborative Biomedical), 5 ng/ml selenous acid (Collaborative Biomedical), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. At day 1 after plating, 60 nM vincristin (Sigma) was added to the cultures to eliminate undesirable cells until the cell density reached subconfluence (4). The medium was renewed every other day. When the endothelial cells reached subconfluence, they were released by 0.25% trypsin-EDTA (GIBCO, Grand Island, NY) and seeded at 25, 000 cells/filter on 0.33-cm2 rat-tail collagen-coated polycarbonate Costar Transwell filters (0.4-µm pore size; Costar, Cambridge, MA) for transcellular electrical resistance (TER) measurements and immunocytochemical examinations. After 4 days of cultivation without vincristin after passage, the medium of the endothelial cells was changed to medium containing 0.1 ng GDNF/ml, 125 µM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP; Sigma), and 17.5 µM phosphodiesterase inhibitor (PDE-I) RO20-1724 (RBI, Natick, MA). The cells were then cultured for 8 h.

Measurement of TER. The TER of the endothelial cells on the filters was measured using an Epithelial Voltohmmeter (World Precision Instruments, Sarasota, FL) equipped with STX-2 Ag-AgCl electrodes (Endohm, World Precision Instruments). The measurements of TER were performed at 37°C on a thermal plate (Fine, Tokyo, Japan). TER was expressed in standard units of ohm · cm2. For calculation of the resistance of endothelial cell monolayers, resistance of blank filters was subtracted from that of filters covered with cells. Each value was calculated from five or six cultures.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Evaluation of Evans blue leakage. First, we evaluated the function of the BBB by using leakage of Evans blue from capillaries of the cerebral cortex. Extensive exudation of Evans blue in the surrounding capillaries was observed widely at postnatal day 4. With time, the leakage of this dye decreased (Fig. 1). Five micrographs randomly taken under the same luminance conditions per rat were analyzed to calculate the mean value of the Evans blue-stained area, and then the mean value and SD were calculated by using three rats per age group. Thus area ratios of Evans blue leakage (postnatal rats/adult rats) were determined (Fig. 2). The ratios at postnatal days 4, 7, and 14 were ~4.5, 2.3, and 1.9 times those of adults, respectively. At postnatal day 21, the degree of dye leakage was not significantly different from that observed in adult rats. The diameter of the capillaries did not significantly change during the observation period (data not shown).


View larger version (92K):
[in this window]
[in a new window]
 
Fig. 1.   Photomicrographs of Evans blue-stained areas in the cerebral cortex. A: postnatal day 4. B: postnatal day 7. C: postnatal day 14. D: postnatal month 3 (adult rat). Original magnification, ×120.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Morphometric evaluation of Evans blue leakage under the same luminance conditions using Photoshop. P4, P7, P14, and P21, postnatal days 4, 7, 14, and 21; 3M, 3-mo-old adult rats. Values at each time point are means ± SD for 3 rats. *P < 0.05; **P < 0.01, significant difference from 3M.

Expression of GFRalpha -1 and c-ret protein in the cerebral cortex capillaries during postnatal development of the BBB. We immunohistochemically investigated expression of GFRalpha -1 during postnatal development of the BBB to clarify the relationship between GDNF and the postnatal development. At birth, expression of GFRalpha -1 was not detected. GFRalpha -1 was initially expressed in the capillaries at postnatal day 3. Its signals became stronger with time. By postnatal day 10, GFRalpha -1 appeared to be fully expressed as detected in adult rats.

Figure 3 is representative of 10 separate experiments for staining of GFRalpha -1 with the use of adult tissues as a positive control. Figure 3H shows a frozen section confirming that GFRalpha -1 was exclusively detected on the capillaries in the brain cortex. However, capillaries of the hypophysis, which do not form a BBB, never expressed GFRalpha -1 (data not shown).


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 3.   Immunohistochemistry of glial cell-derived neurotrophic factor family (GDNF) receptor alpha -1 (GFRalpha -1) in the cerebral cortex of the rat during postnatal maturation of the blood-brain barrier (BBB). A: postnatal day 1. B: postnatal day 3. C: postnatal day 5. D: postnatal day 7. E: postnatal day 10. F: postnatal day 14. G and H: postnatal month 6. A-G: pressed brain cortex. H: frozen section. GFRalpha -1 preferentially localized on the cell membranes of capillary endothelial cells. Original magnification, ×150.

Although the actions of GDNF are considered to be mediated by a multicomponent receptor complex composed of c-ret and GFRalpha -1 (7, 16, 27, 28), c-ret was not detected immunohistochemically in the cerebral cortex of the rat, though the cells of the ventral horn and vestibulospinal tract were positive for c-ret (Fig. 4). Figure 4 is representative of three separate experiments for staining of c-ret using triplicate sections of each tissue. This was confirmed by RT-PCR of 40 cycles performed using various mouse tissues, because the complete sequence of c-ret is available only for the human and mouse. The results of the RT-PCR showed that c-ret was expressed in the thymus and spinal cord, but not in the cerebral cortex, of three 3- to 4-wk-old mice (Fig. 5). Figure 5 is representative of three separate experiments.


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 4.   Immunohistochemistry of c-ret in the adult rat cerebral cortex. A: brain cortex. B: spinal cord. Original magnification, ×80.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5.   Detection of c-ret mRNA in the spinal cord, brain cortex, and thymus of 3- to 4-wk-old mice by RT-PCR. Expression of c-ret was clearly detected in the spinal cord and thymus but not in the brain cortex.

Expression of tight junction-associated proteins in the capillaries during postnatal development of the BBB. Tight junctions are an important cellular apparatus for regulating the paracellular pathway of the BBB. Thus, as an initial approach to investigate the involvement of tight junction-associated proteins in development of the BBB, two well-known proteins, occludin and ZO-1, were studied by using an immunohistochemical technique. It was reported that ZO-1 is expressed at postnatal days 8 and 70, whereas occludin is expressed at postnatal day 70 but hardly detectable at postnatal day 8 (12). In the present experiments, even at birth, these proteins appeared to be expressed at cell-cell contacts of endothelial cells of the cerebral capillaries. The intensity of the junctional staining of the proteins in the brain capillaries at birth was comparable to that in adult rats (Fig. 6). Figure 6 is representative of 10 separate experiments for staining of ZO-1 and occludin.


View larger version (83K):
[in this window]
[in a new window]
 
Fig. 6.   Immunohistochemical expression of tight junction-associated proteins ZO-1 (A, C, and E) and occludin (B, D, and F) in the cerebral capillaries of the rat. A and B: postnatal day 1. C and D: postnatal day 7. E and F: postnatal month 6. These proteins localized at cell-cell contacts of the cerebral capillaries. Original magnification, ×300.

Effects of GDNF on occludin of cultured porcine endothelial cells forming the BBB. To examine the relationship between the expression of occludin and ZO-1 and the permeability of tight junctions, endothelial cells of the porcine cerebral cortex were cultured and treated with GDNF or agents elevating the intracellular cAMP level (CPT-cAMP and PDE-I) for 8 h. The results are summarized in Fig. 7A. The TER of the cells treated with GDNF and the cAMP-elevating agents was significantly higher than those of the cells treated with the agents alone. The TER of the cells treated with GDNF alone, however, did not increase. These results are consistent with our previous report that GDNF enhances barrier function (13). Under these conditions, we immunocytochemically examined the localization and staining intensity of occludin, showing no marked alteration of occludin by GDNF or the cAMP-elevating agents. On the other hand, the distribution of actin was altered from a stress-fiber dominant to a circumferential pattern by addition of the cAMP-elevating agents, whereas GDNF had no effect on actin organization (Fig. 7B). Figure 7B is representative of five separate experiments for staining of occludin and actin.


View larger version (63K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of GDNF on the transcellular electrical resistance (TER) and localization of occludin and actin of cultured porcine endothelial cells forming the BBB. A: effects of GDNF on TER. Values are means ± SD calculated from 5 or 6 cultures. *Significant difference (P < 0.05) from cultures before treatment. **Significant difference (P < 0.01) from cultures treated with cAMP + phosphodiesterase inhibitor (PDE-I). B: effects of GDNF on the localization of occludin (a-d) and actin (e-h). a and e: control; b and f: cAMP + PDE-I; c and g: GDNF; d and h: GDNF + cAMP + PDE-I. Original magnification, ×400.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Many studies have shown that the BBB of immature animals is more permeable than that of adults (2, 3, 18, 32). In newborn mice, the BBB is immature and some amino acids penetrate more freely into the brain than in adults (22). Xu et al. (31, 32) have demonstrated the extravasation of tracers, such as rhodamine isothiocyanate and ferritin, into the corpus callosum in early postnatal rats but not in rats older than 13-14 days. In the present experiment, we consistently observed extensive exudation of Evans blue at postnatal day 4, and the BBB became much less permeable against Evans blue by postnatal day 21, as in adult rats. Expression of GFRalpha -1 progressively increased from postnatal days 3-5 to postnatal day 14 with the postnatal development of the BBB, strongly suggesting the participation of GDNF in the postnatal maturation of the BBB.

We immunohistochemically detected expression of GFRalpha -1 in the capillaries of the cerebrum but not in the endothelial cells of lung, tongue, hypophysis, and other tissues (data not shown). These findings are consistent with results obtained by in situ hybridization showing that GDNF and GFRalpha -1mRNAs are weakly detected in the cerebral cortex (10, 33). Thus GFRalpha -1 was confirmed to be expressed in the brain capillaries forming the BBB as well as in neurons in the brain. Our previous study revealed that GDNF induced the barrier function of endothelial cells isolated from the porcine brain. The present finding of a reciprocal relation between GFRalpha -1 expression and leakage of Evans blue during postnatal development of the BBB provide further support for a role of GDNF in development of the BBB.

It has been reported that the actions of GDNF are mediated by a multicomponent receptor complex composed of c-ret and GFRalpha -1 (7, 16, 27, 28). Immunohistochemically, however, we failed to detect c-ret expression in capillaries of the cerebral cortex in the present study. Our observation is consistent with a previous report using an in situ hybridization technique in which no signal of c-ret was detected in brain cortex (10, 33). Thus these observations clearly indicate that c-ret is not always necessary for the signal transduction of GDNF, particularly in the endothelial cells of the BBB.

In endothelial cells forming the BBB, the P-face-associated particles of tight junctions observed in freeze fractures are suggested to play an important role in regulating the paracellular pathway (34). Thus the elucidation of which integral membrane proteins are crucial for tight junction formation is of importance. Of the tight junction-associated proteins (5), the integral membrane proteins occludin and the claudins are capable of forming tight junction strands, although occludin forms much shorter strands than the claudins (9). Regarding occludin expression during postnatal development of the BBB, it has been reported that occludin is expressed in the brain capillaries on postnatal day 70 but not on postnatal day 8 (12). It has also been reported that occludin expression in endothelial cells forming the BBB is not altered by treatment with astrocyte-conditioned medium and cAMP, which significantly increase TER (12). This prompted us to examine the cellular distribution of occludin in the development of the BBB. In the present experiments, however, occludin was clearly detected at cell junction areas of endothelial cells in the brain cortex, even at birth. We also observed that the localization and staining intensity of occludin were not changed by treatment with cAMP and GDNF. Thus it is suggested that the presence of occludin alone cannot account for formation of tight junctions in endothelial cells forming the BBB.

The claudin family, a newly disclosed membrane protein family of tight junctions (19), has been suggested to be a prime candidate for tight junction strands (8, 9, 29). Of the proteins of this family, it is reported that claudin 5 is highly expressed in capillaries of the brain and lung (4, 19). The tissue distribution of occludin is well correlated to the distribution of tight junctions compared with that of the claudin family; however, the role of occludin in the functioning of tight junctions remains to be clarified.

In conclusion, we demonstrated that GFRalpha -1 was detected in rat brain capillaries and that its expression began on postnatal days 3-5 and eventually reached the level observed in adult rats with a progressive decrease in the leakage of Evans blue. Thus it is strongly suggested that expression of GDNF is deeply involved in the postnatal development of the BBB.


    ACKNOWLEDGEMENTS

We thank Kim Barrymore for help with the manuscript.


    FOOTNOTES

This study was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sports and Science and the Ministry of Welfare of Japan, as well as the Akiyama Foundation, the Naitou Foundation, and the Hokkaido Geriatrics Research Institute.

Present address of H. Utsumi: Toxicology Laboratories, Yoshitomi Pharmaceutical Industries, Ltd, 214-1, Yamazaki, Fukusaki-cho, Kanzaki-gun, 679-2200, Japan.

Address for reprint requests and other correspondence: N. Sawada, Dept. of Pathology, Sapporo Medical Univ. School of Medicine, South-1, West-17, Chuo-ku, Sapporo, 060, Japan (E-mail: sawadan{at}sapmed.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 13 July 1999; accepted in final form 18 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arthur, FE, Shivers RR, and Bowman PD. Astrocyte-mediated induction of tight junctions in brain capillary endothelium. Dev Brain Res 433: 155-159, 1987.

2.   Butt, M, Jones H, and Abbott NJ. Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study. J Physiol (Lond) 429: 47-62, 1990[Abstract].

3.   Caley, DW, and Maxwell DS. Development of the blood vessels and extracellular spaces during postnatal maturation of rat cerebral cortex. J Comp Neurol 138: 31-47, 1970[ISI][Medline].

4.   Chen, Z, Zandonatti M, Jakubowski D, and Fox HS. Brain capillary endothelial cells express MBEC1, a protein that is related to the Clostridium perfringens enterotoxin receptors. Lab Invest 78: 353-363, 1998[ISI][Medline].

5.   Citi, S, and Cordenonsi M. Tight junction proteins. Biochim Biophys Acta 1448: 1-11, 1998[ISI][Medline].

6.   Dehouck, MP, Meresse S, Delorme P, Fruchart JC, and Cecchelli R. An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro. J Neurochem 54: 1798-1801, 1990[ISI][Medline].

7.   Durbec, P, Marcos-Gutierrez CV, Kilkenny C, Grigoriou M, Wartiowaara K, Suvanto P, Smith D, Ponder B, Costanini F, Saarma M, Sariola H, and Pachnis V. GDNF signaling through the ret receptor tyrosine kinase. Nature 381: 789-793, 1996[ISI][Medline].

8.   Furuse, M, Fujita K, Hiiragi T, Fujimoto K, and Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141: 1539-1550, 1998[Abstract/Free Full Text].

9.   Furuse, M, Sasaki H, Fujimoto K, and Tsukita S. A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin. J Cell Biol 143: 391-401, 1998[Abstract/Free Full Text].

10.   Glazner, GW, Mu X, and Springer JE. Localization of glial cell line-derived neurotrophic factor receptor alpha and c-ret mRNA in rat central nervous system. J Comp Neurol 391: 42-49, 1998[ISI][Medline].

11.   Henderson, CE, Phillips HS, Pollock RA, Davies AM, Lemeulle C, Armanini M, Simpson LC, Moffet B, Vandlen RA, Koliatsos VE, and Rosenthal A. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science 266: 1162-1164, 1994[ISI][Medline].

12.   Hirase, T, Staddon JM, Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Fujimoto K, Tsukita S, and Rubin LL. Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci 110: 1603-1613, 1997[Abstract/Free Full Text].

13.   Igarashi, Y, Utsumi H, Chiba H, Yamada-Sasamori Y, Tobioka H, Furuuchi K, Kokai Y, Nakagawa T, Mori M, and Sawada N. Glial cell line-derived neurotrophic factor (GDNF) enhances barrier function of endothelial cells forming the blood-brain barrier. Biochem Biophys Res Commun 261: 108-112, 1999[ISI][Medline].

14.   Iwamoto, T, Taniguchi M, Asai N, Ohkusu K, Nakashima I, and Takahashi M. cDNA cloning of mouse ret proto-oncogene and its sequence similarity to the cadherin superfamily. Oncogene 8: 1087-1091, 1993[ISI][Medline].

15.   Janzer, RC, and Raff MC. Astrocytes induce blood-brain barrier properties in endothelial cells. Nature 325: 253-257, 1987[ISI][Medline].

16.   Jing, S, Wen D, Yu Y, Holst PL, Luo Y, Fang M, Tamir R, Antonio L, Hu Z, Cuppels R, Louis JC, Hu S, Altrock BW, and Fox GM. GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha , a novel receptor for GDNF. Cell 85: 1113-1124, 1996[ISI][Medline].

17.   Lin, LFH, Doherty DH, Lile JD, Bektesh S, and Collins F. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260: 1130-1132, 1993[ISI][Medline].

18.   Lossinsky, AS, Vorbrodt AW, and Wisniewski HM. Characterization of endothelial cell transport in the developing mouse blood-brain barrier. Dev Neurosci 8: 61-75, 1986[ISI][Medline].

19.   Morita, K, Furuse M, Fujimoto K, and Tsukita S. Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc Natl Acad Sci USA 96: 511-516, 1999[Abstract/Free Full Text].

20.   Rubin, LL, Hall DE, Portes S, Barbu K, Cannon C, Horner HC, Janatpour M, Liaw CW, Manning K, Morales J, Tanner LI, Tomaselli KJ, and Bard F. A cell culture model of the blood-brain barrier. J Cell Biol 115: 1725-1735, 1991[Abstract].

21.   Sambrook, J, Fritch EF, and Maniatis T. Molecular Cloning. A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

22.   Seta, K, Sershen H, and Lajtha A. Cerebral amino acid uptake in vivo in newborn mice. Brain Res 47: 415-425, 1972[ISI][Medline].

23.   Staddon, JM, and Rubin LL. Cell adhesion, cell junctions and the blood-brain barrier. Curr Opin Neurobiol 6: 622-627, 1996[ISI][Medline].

24.   Sun, D, Lytle C, and O'Donnell IL-6 secreted by astroglial cells regulates Na-K-Cl cotransport in brain microvessel endothelial cells. Am J Physiol Cell Physiol 272: C1829-C1835, 1997[Abstract/Free Full Text].

25.   Takemoto, H, Kaneda K, Hosokawa M, Ide M, and Fukushima H. Conditioned media of glial cell lines induce alkaline phosphatase activity in cultured artery endothelial cells. Identification of interleukin-6 as an induction factor. FEBS Lett 350: 99-103, 1994[ISI][Medline].

26.   Tao-Cheng, JH, Nagy Z, and Brightman MW. Tight junctions of brain endothelium in vivo are enhanced by astroglia. J Neurosci 7: 3293-3299, 1987[Abstract].

27.   Treanor, JJS, Goodman L, Sauvage F, Stone DM, Poulsen KT, Beck CD, Gray C, Armanini MP, Pollock RA, Hefti F, Buj-Bello A, Davies AM, Asai N, Takahashi M, Vandlen R, Henderson CE, and Rosenthal A. Characterization of a multicomponent receptor for GDNF. Nature 382: 80-83, 1996[ISI][Medline].

28.   Trupp, M, Arenas E, Fainzilber M, Nilsson AS, Sieber BA, Grigoriou M, Kilkenny C, Salazer-Grueso E, Pachnis V, Arumäe U, Sariola H, Saarma M, and Ibáñez CF. Functional receptor for GDNF encoded by the c-ret proto-oncogene. Nature 381: 785-789, 1996[ISI][Medline].

29.   Tsukita, S, and Furuse M. Occludin and claudins in tight-junction strands: leading or supporting players? Trends Cell Biol 9: 268-273, 1999[ISI][Medline].

30.   Xie, W, and Rothblum LI. Rapid, small-scale RNA isolation from tissue culture cells. Biotechniques 11: 325-327, 1991.

31.   Xu, J, Kaur C, and Ling EA. Variation with age in the labelling of amoeboid microglial cells in rats following intraperitoneal or intravenous injection of a fluorescent dye. J Anat 182: 55-63, 1993[ISI][Medline].

32.   Xu, J, and Ling EA. Studies of the ultrastructure and permeability of the blood-brain barrier in the developing corpus callosum in postnatal rat brain using electron dense tracers. J Anat 184: 227-237, 1994[ISI][Medline].

33.   Widenfalk, J, Nosrat C, Tomac A, Westphal H, Hoffer B, and Olson L. Neurturin and glial cell line-derived neurotrophic factor receptor-beta (GDNFR-beta ), novel proteins related to GDNF and GDNFR-alpha with specific cellular patterns of expression suggesting roles in the developing and adult nervous system and in peripheral organs. J Neurosci 17: 8506-8519, 1997[Abstract/Free Full Text].

34.   Wolburg, H, Neuhaus J, Kniesel U, Krauss B, Schmid EM, Öcalan M, Farrell C, and Risau W. Modulation of tight junction structure in blood-brain barrier endothelial cells. Effects of tissue culture, second messengers and cocultured astrocytes. J Cell Sci 107: 1347-1357, 1994[Abstract/Free Full Text].


Am J Physiol Cell Physiol 279(2):C361-C368
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society