Astroglial cell-induced expression of Na-K-Cl cotransporter in brain microvascular endothelial cells

D. Sun, C. Lytle, Martha E O'Donnell

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

Endothelial cells of the blood-brain barrier (BBB) are characterized by extensive tight junctions and asymmetric distribution of specific enzymes and transport systems. Maintenance of the BBB endothelial phenotype depends on astrocyte-endothelial interactions. We showed previously that cultured cerebral microvascular endothelial cells (CMEC) exhibit robust Na-K-Cl cotransport activity. In the present study, we evaluated the expression of Na-K-Cl cotransport protein in CMEC by quantitative Western blot analysis and found that a protein of ~170 kDa was recognized by a monoclonal antibody against the cotransporter. Exposure of CMEC to astroglial cells or their conditioned media increased the expression of the CMEC cotransport protein by ~55%. Using a monoclonal antibody against the α-subunit of chicken Na-K- ATPase, we found that these treatments also increased expression of Na-K- ATPase protein by a similar amount. By comparing bumetanide-sensitive K influx and [3H]bumetanide binding of apical vs. basolateral surfaces of CMEC, we found both cotransporter activity and [3H]bumetanide binding to be ~90% epical and 10% basolateral. Coculture of the CMEC with astroglial cells increased cotransport activity and [3H]bumetanide binding at both surfaces, with the asymmetric distribution maintained. These results indicate that the cotransporter is regulated by astroglial cells and that an apically distributed CMEC cotransporter may function in tandem with the basolateral Na-K-ATPase to mediate vectorial transport of Na and Cl across the BBB.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume269
Issue number6 38-6
StatePublished - 1995

Fingerprint

Sodium-Potassium-Chloride Symporters
Endothelial cells
endothelial cells
Brain
Endothelial Cells
Bumetanide
brain
blood-brain barrier
sodium-potassium-exchanging ATPase
Blood-Brain Barrier
cells
Adenosine Triphosphatases
monoclonal antibodies
Proteins
proteins
Monoclonal Antibodies
tight junctions
Tight Junctions
astrocytes
coculture

Keywords

  • blood-brain barrier
  • bumetanide
  • C6 glial cells
  • cultured cerebral microvascular endothelial cells
  • primary astrocytes
  • sodium-potassium- adenosinetriphosphatase

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Agricultural and Biological Sciences(all)

Cite this

@article{8c24a08078094d71af245a6658096fcf,
title = "Astroglial cell-induced expression of Na-K-Cl cotransporter in brain microvascular endothelial cells",
abstract = "Endothelial cells of the blood-brain barrier (BBB) are characterized by extensive tight junctions and asymmetric distribution of specific enzymes and transport systems. Maintenance of the BBB endothelial phenotype depends on astrocyte-endothelial interactions. We showed previously that cultured cerebral microvascular endothelial cells (CMEC) exhibit robust Na-K-Cl cotransport activity. In the present study, we evaluated the expression of Na-K-Cl cotransport protein in CMEC by quantitative Western blot analysis and found that a protein of ~170 kDa was recognized by a monoclonal antibody against the cotransporter. Exposure of CMEC to astroglial cells or their conditioned media increased the expression of the CMEC cotransport protein by ~55{\%}. Using a monoclonal antibody against the α-subunit of chicken Na-K- ATPase, we found that these treatments also increased expression of Na-K- ATPase protein by a similar amount. By comparing bumetanide-sensitive K influx and [3H]bumetanide binding of apical vs. basolateral surfaces of CMEC, we found both cotransporter activity and [3H]bumetanide binding to be ~90{\%} epical and 10{\%} basolateral. Coculture of the CMEC with astroglial cells increased cotransport activity and [3H]bumetanide binding at both surfaces, with the asymmetric distribution maintained. These results indicate that the cotransporter is regulated by astroglial cells and that an apically distributed CMEC cotransporter may function in tandem with the basolateral Na-K-ATPase to mediate vectorial transport of Na and Cl across the BBB.",
keywords = "blood-brain barrier, bumetanide, C6 glial cells, cultured cerebral microvascular endothelial cells, primary astrocytes, sodium-potassium- adenosinetriphosphatase",
author = "D. Sun and C. Lytle and O'Donnell, {Martha E}",
year = "1995",
language = "English (US)",
volume = "269",
journal = "American Journal of Physiology - Renal Fluid and Electrolyte Physiology",
issn = "1931-857X",
publisher = "American Physiological Society",
number = "6 38-6",

}

TY - JOUR

T1 - Astroglial cell-induced expression of Na-K-Cl cotransporter in brain microvascular endothelial cells

AU - Sun, D.

AU - Lytle, C.

AU - O'Donnell, Martha E

PY - 1995

Y1 - 1995

N2 - Endothelial cells of the blood-brain barrier (BBB) are characterized by extensive tight junctions and asymmetric distribution of specific enzymes and transport systems. Maintenance of the BBB endothelial phenotype depends on astrocyte-endothelial interactions. We showed previously that cultured cerebral microvascular endothelial cells (CMEC) exhibit robust Na-K-Cl cotransport activity. In the present study, we evaluated the expression of Na-K-Cl cotransport protein in CMEC by quantitative Western blot analysis and found that a protein of ~170 kDa was recognized by a monoclonal antibody against the cotransporter. Exposure of CMEC to astroglial cells or their conditioned media increased the expression of the CMEC cotransport protein by ~55%. Using a monoclonal antibody against the α-subunit of chicken Na-K- ATPase, we found that these treatments also increased expression of Na-K- ATPase protein by a similar amount. By comparing bumetanide-sensitive K influx and [3H]bumetanide binding of apical vs. basolateral surfaces of CMEC, we found both cotransporter activity and [3H]bumetanide binding to be ~90% epical and 10% basolateral. Coculture of the CMEC with astroglial cells increased cotransport activity and [3H]bumetanide binding at both surfaces, with the asymmetric distribution maintained. These results indicate that the cotransporter is regulated by astroglial cells and that an apically distributed CMEC cotransporter may function in tandem with the basolateral Na-K-ATPase to mediate vectorial transport of Na and Cl across the BBB.

AB - Endothelial cells of the blood-brain barrier (BBB) are characterized by extensive tight junctions and asymmetric distribution of specific enzymes and transport systems. Maintenance of the BBB endothelial phenotype depends on astrocyte-endothelial interactions. We showed previously that cultured cerebral microvascular endothelial cells (CMEC) exhibit robust Na-K-Cl cotransport activity. In the present study, we evaluated the expression of Na-K-Cl cotransport protein in CMEC by quantitative Western blot analysis and found that a protein of ~170 kDa was recognized by a monoclonal antibody against the cotransporter. Exposure of CMEC to astroglial cells or their conditioned media increased the expression of the CMEC cotransport protein by ~55%. Using a monoclonal antibody against the α-subunit of chicken Na-K- ATPase, we found that these treatments also increased expression of Na-K- ATPase protein by a similar amount. By comparing bumetanide-sensitive K influx and [3H]bumetanide binding of apical vs. basolateral surfaces of CMEC, we found both cotransporter activity and [3H]bumetanide binding to be ~90% epical and 10% basolateral. Coculture of the CMEC with astroglial cells increased cotransport activity and [3H]bumetanide binding at both surfaces, with the asymmetric distribution maintained. These results indicate that the cotransporter is regulated by astroglial cells and that an apically distributed CMEC cotransporter may function in tandem with the basolateral Na-K-ATPase to mediate vectorial transport of Na and Cl across the BBB.

KW - blood-brain barrier

KW - bumetanide

KW - C6 glial cells

KW - cultured cerebral microvascular endothelial cells

KW - primary astrocytes

KW - sodium-potassium- adenosinetriphosphatase

UR - http://www.scopus.com/inward/record.url?scp=0029202141&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029202141&partnerID=8YFLogxK

M3 - Article

C2 - 8572180

AN - SCOPUS:0029202141

VL - 269

JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

SN - 1931-857X

IS - 6 38-6

ER -