Tight junctions between endothelial cells of brain capillaries are the most important structural elements of the blood-brain barrier. Cultured brain endothelial cells are known to loose tight junction-dependent blood-brain barrier characteristics such as macromolecular impermeability and high electrical resistance. We have directly analyzed the structure and function of tight junctions in primary cultures of bovine brain endothelial cells using quantitative freeze-fracture electron microscopy, and ion and inulin permeability. The complexity of tight junctions, defined as the number of branch points per unit length of tight junctional strands, decreased 5 hours after culture but thereafter remained almost constant. In contrast, the association of tight junction particles with the cytoplasmic leaflet of the endothelial membrane bilayer (P-face) decreased continuously with a major drop between 16 hours and 24 hours. The complexity of tight junctions could be increased by elevation of intracellular cAMP levels while phorbol esters had the opposite effect. On the other hand, the P-face association of tight junction particles was enhanced by elevation of cAMP levels and by coculture of endothelial cells with astrocytes or exposure to astrocyte-conditioned medium. The latter effect on P-face association was induced by astrocytes but not fibroblasts. Elevation of cAMP levels together with astrocyte-conditioned medium synergistically increased transendothelial electrical resistance and decreased inulin permeability of primary cultures, thus confirming the effects on tight junction structure and barrier function. P-face association of tight junction particles in brain endothelial cells may therefore be a critical feature of blood-brain barrier function that can be specifically modulated by astrocytes and cAMP levels. Our results suggest an important functional role for the cytoplasmic anchorage of tight junction particles for brain endothelial barrier function in particular and probably paracellular permeability in general.

Arthur
F. E.
,
Shivers
R. R.
,
Bowman
P. D.
(
1987
).
Astrocyte-mediated induction of tight junctions in brain capillary endothelium: an efficient in vitro model.
Dev. Brain Res
36
,
155
159
Brightman
M. W.
,
Reese
T. S.
(
1969
).
Junctions between intimately apposed cell membranes in the vertebrate brain.
J. Cell Biol
40
,
648
677
Bundgaard
M.
,
Cserr
H.
,
Murray
M.
(
1979
).
Impermeability of hagfish cerebral capillaries to horseradish peroxidase An ultrastructural study.
Cell Tiss. Res
198
,
65
77
Butt
A. M.
,
Jones
H. C.
,
Abbott
N. J.
(
1990
).
Electrical-resistance across the blood-brain barrier in anesthetized rats- a developmental study.
J. Physiol
429
,
47
62
Citi
S.
(
1993
).
The molecular-organization of tight junctions.
J. Cell Biol
121
,
485
489
Claude
P.
,
Goodenough
D. A.
(
1973
).
Fracture faces of zonulae occludentes from “tight” and “leaky” epithelia.
J. Cell Biol
58
,
390
400
Claude
P.
(
1978
).
Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens.
J. Membr. Biol
32
,
219
232
Dermietzel
R.
,
Krause
D.
(
1991
).
Molecular anatomy of the blood-brain-barrier as defined by immunocytochemistry.
Int. Rev. Cytol
127
,
57
109
Drenckhahn
D.
,
Dermietzel
R.
(
1988
).
Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study.
J. Cell Biol
107
,
1037
1048
Frey
A.
,
Meckelein
B.
,
Weilerguttler
H.
,
Mockel
B.
,
Flach
R.
,
Gassen
H. G.
(
1991
).
Pericytes of the brain microvasculature express gamma-glutamyl transpeptidase.
Eur. J. Biochem
202
,
421
429
Gumbiner
B.
,
Simons
K.
(
1986
).
A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of a uvomorulin-like polypeptide.
J. Cell Biol
102
,
457
468
Itoh
M.
,
Nagafuchi
A.
,
Yonemura
S.
,
Kitani-Yasuda
T.
,
Tsukita
S.
,
Tsukita
S.
(
1993
).
The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy.
J. Cell Biol
121
,
491
502
Janzer
R. C.
,
Raff
M. C.
(
1987
).
Astrocytes induce blood-brain barrier properties in endothelial cells.
Nature
325
,
253
257
Kintner
C.
(
1992
).
Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain.
Cell
69
,
225
236
Kniesel
U.
,
Wolburg
H.
(
1993
).
Tight junction complexity in the retinal-pigment epithelium of the chicken during development.
Neurosci. Lett
149
,
71
74
Krause
D.
,
Mischeck
U.
,
Galla
H. J.
,
Dermietzel
R.
(
1991
).
Correlation of zonula-occludens ZO-1 antigen expression and transendothelial resistance in porcine and rat cultured cerebral endothelial-cells.
Neurosci. Lett
128
,
301
304
Lampugnani
M. G.
,
Resnati
M.
,
Raiteri
M.
,
Pigott
R.
,
Pisacane
A.
,
Houen
G.
,
Ruco
L. P.
,
Dejana
E.
(
1992
).
A novel endothelial-specific membrane-protein is a marker of cell-cell contacts.
J. Cell Biol
118
,
1511
1522
Madara
J. L.
,
Dharmsathaphorn
K.
(
1985
).
Occluding junction structure-function relationships in a cultured epithelial monlayer.
J. Cell Biol
101
,
2124
2133
Madara
J. L.
(
1987
).
Intestinal absorptive cell tight junctions are linked to cytoskeleton.
Amer. J. Physiol
253
,
171
–.
Madara
J. L.
,
Pappenheimer
J. R.
(
1987
).
Structural basis for physiological regulation of paracellular pathways in intestinal epithelia.
J. Membr. Biol
100
,
149
164
Mandel
L. J.
,
Bacallao
R.
,
Zampighi
G.
(
1993
).
Uncoupling of the molecular fence and paracellular gate functions in epithelial tight junctions.
Nature
361
,
552
555
Marcial
M. A.
,
Carlson
S. L.
,
Madar
J. L.
(
1984
).
Partitioning of paracellular conductance along the ileal crypt-villus axis: a hypothesis based on structural analysis with detailed considerations of tight junction structure-function relationship.
J. Membr. Biol
80
,
59
80
Meyer
J.
,
Rauh
J.
,
Galla
H. J.
(
1991
).
The susceptibility of cerebral endothelial-cells to astroglial induction of blood-brain-barrier enzymes depends on their proliferative state.
J. Neurochem
57
,
1971
1977
Muhleisen
H.
,
Wolburg
H.
,
Betz
E.
(
1989
).
Freeze-fracture analysis of endothelial cell membranes in rabbit carotid arteries subjected to short-term atherogenic stimuli.
Virch. Arch. B Cell Pathol
56
,
413
417
Nagy
Z.
,
Peters
H.
,
Huttner
I.
(
1984
).
Fracture faces of cell junctions in cerebral endothelium during normal and hyperosmotic conditions.
Lab. Invest
50
,
313
322
Nigam
S. K.
,
Denisenko
N.
,
Rodriguez-Boulan
E.
,
Citi
S.
(
1991
).
The role of phosphorylation in development of tight junctions in cultured renal epithelial (mdck) cells.
Biochem. Biophys. Res. Commun
181
,
548
553
Noble
M.
,
Fok-Seang
J.
,
Cohen
J.
(
1984
).
Glia are a unique substrate for the in vitro growth of central nervous system neurons.
J. Neurosci
4
,
1892
1903
Ozawa
M.
,
Kemler
R.
(
1992
).
Molecular-organization of the uvomorulin-catenin complex.
J. Cell Biol
116
,
989
996
Reese
T. S.
,
Karnovsky
M. J.
(
1967
).
Fine structural localization of a blood-brain barrier to exogenous peroxidase.
J. Cell Biol
34
,
207
217
Risau
W.
,
Hallmann
R.
,
Albrecht
U.
(
1986
).
Differentiation-dependent expression of protein in brain endothelium during development of the blood-brain barrier.
Dev. Biol
117
,
537
545
Risau
W.
,
Hallmann
R.
,
Albrecht
U.
,
Henke-Fahle
S.
(
1986
).
Brain induces the expression of an early cell surface marker for blood-brain barrier specific endothelium.
EMBO J
5
,
3179
3183
Risau
W.
,
Engelhardt
B.
,
Wekerle
H.
(
1990
).
Immune function of the blood-brain barrier: Incomplete presentation of protein (auto-)antigens by rat brain microvascular endothelium in vitro.
J. Cell Biol
110
,
1757
1766
Risau
W.
,
Wolburg
H.
(
1990
).
Development of the blood-brain barrier.
Trends Neurosci
13
,
174
178
Risau
W.
,
Wolburg
H.
(
1991
).
The importance of the blood-brain barrier in fetuses and embryos- reply.
Trends Neurosci
14
,
15
15
Risau
W.
,
Dingler
A.
,
Albrecht
U.
,
Dehouck
M.-P.
,
Cecchelli
R.
) (
1992
).
Blood-brain barrier pericytes are the main source of gamma-glutamyltranspeptidase activity in brain capillaries.
J. Neurochem
58
,
667
672
Rubin
L. L.
,
Hall
D. E.
,
Porter
S.
,
Barbu
K.
,
Cannon
C.
,
Horner
H. C.
,
Janatpour
M.
,
Liaw
C. W.
,
Manning
K.
,
Morales
J.
,
Tanner
L. I.
,
Tomaselli
K. J.
, et al. 
(
1991
).
A cell-culture model of the blood-brain barrier.
J. Cell Biol
115
,
1725
1735
Saunders
N. R.
,
Dziegielewska
K. M.
,
Mollgard
K.
(
1991
).
The importance of the blood-brain barrier in fetuses and embryos.
Trends Neurosci
14
,
14
14
Schulze
C.
,
Firth
J. A.
(
1992
).
Interendothelial junctions during blood-brain barrier development in the rat, morphological changes at the level of individual tight junctional contacts.
Dev. Brain Res
69
,
85
95
Seulberger
H.
,
Lottspeich
F.
,
Risau
W.
(
1990
).
The inducible blood-brain barrier specific molecule HT7 is a novel immunoglobulin-like cell surface glycoprotein.
EMBO J
9
,
2151
2158
Simionescu
M.
,
Ghinea
N.
,
Fixman
A.
,
Lasser
M.
,
Kukes
L.
,
Simionescu
N.
,
Palade
G. E.
(
1988
).
The cerebral microvasculature of the rat: structure and luminal surface properties during early development.
J. Submicrosc. Cytol
20
,
243
261
Stevenson
B. R.
,
Anderson
J. M.
,
Godenough
D. A.
,
Mooseker
M. S.
(
1988
).
Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance.
J. Cell Biol
107
,
2401
2408
Stevenson
B. R.
,
Anderson
J. M.
,
Braun
D.
,
Mooseker
M. S.
(
1989
).
Phosphorylation of the tight-junction protein ZO-1 in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance.
Biochem. J
263
,
597
599
Stewart
P. A.
,
Wiley
M. J.
(
1981
).
Developing nervous tissue induces formation of blood-brain barrier characteristics in invading endothelial cells: A study using quail-chick transplantation chimeras.
Dev. Biol
84
,
183
192
Stewart
P. A.
,
Hayakawa
E. M.
(
1987
).
Interendothelial junctional changes underlie the developmental ‘tightening’ of the blood-brain barrier.
Dev. Brain Res
32
,
271
281
Tao-Cheng
J.
,
Nagy
Z.
,
Brightman
M. W.
(
1987
).
Tight junctions of brain endothelium in vitro are enhanced by astroglia.
J. Neurosci
7
,
3293
3299
van Deurs
B.
,
Luft
J. H.
(
1979
).
Effects of glutaraldehyde fixation on the structure of tight junctions. A quantitative freeze-fracture analysis.
J. Ultrastruct. Res
68
,
160
172
Wakai
S.
,
Hirokawa
N.
(
1978
).
Development of the blood-brain barrier to horseradish peroxidase in the chick embryo.
Cell Tiss. Res
195
,
195
203
Zampighi
G.
,
Bacallao
R.
,
Mandel
L.
,
Cereijido
M.
(
1991
).
Structural differences of the tight junction between low and high resistance MDCK monolayers.
J. Cell Biol
115
,
479
–.
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