J Korean Med Sci.  2011 Jan;26(1):92-99. 10.3346/jkms.2011.26.1.92.

The Role of Endothelin Receptor A during Myelination of Developing Oligodendrocytes

• Jung KJ
• Kim DW
• Lee HN
• Lee YS
• Lee SJ
• Che JH
• Lee YH
• Kang BC

• KMID: 1777982
• DOI: http://doi.org/10.3346/jkms.2011.26.1.92

Abstract

Endothelin (ET)-1 and its receptors (ETA and ETB receptor) are present in the central nervous system. ET exerts biological effects on gliogenesis and glial cell functions. In order to define a possible mechanism of ETA receptor signaling, the distribution of the ETA receptor in developing oligodendrocytes and the effects of ET-1 on the myelination of oligodendrocytes were examined. ETA receptor immunoreactivity was confined to the perivascular elements of the blood vessels during early postnatal development. However later in development, ETA receptor immunoreactivity was no longer observed in the vessels but became localized to the myelinating oligodendrocytes of the primitive corpus callosum of the white matter, apart from the vessels. ET-1 induced myelin basic protein (MBP) in primary oligodendrocyte precursor cell culture though the ETA receptor and was blocked by an ETA receptor antagonist. In addition, ET-1 evoked the release of Ca2+ which is a central regulator of oligodendrocyte differentiation. Our results provide a link between ET-1 and its ETA receptor and myelination during oligodendrocyte differentiation.

Keyword

Endothelins; Receptor, Endothelin A; Oligodendroglia; Myelination; Calcium

MeSH Terms

Animals
Brain/pathology
Calcium/metabolism
Calcium Signaling
Cells, Cultured
Endothelin-1/metabolism/physiology
Mice
Mice, Inbred ICR
Myelin Basic Proteins/genetics/metabolism
Myelin Sheath/*physiology
Oligodendroglia/cytology/*metabolism
Rats
Rats, Sprague-Dawley
Receptor, Endothelin A/metabolism/*physiology

Figure

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  Fig. 1 ETA receptor and MBP immunoreactivity with cresyl violet counterstain in the developing mouse brain. Ages examined were P1d (A, E, I), P1w (B, F, J), P2w (C, G, K), and P4w (D, H, L). During early development (P1d), ETA receptor immunoreactivity was associated with blood vessels (A). However at a later point in time, ETA receptor immunoreactivity disappears from the vessels (arrowheads in B), and is instead localized within round oligodendrocyte-like cells in the corpus callosum (CC) (C, D). MBP immunoreactivity appears at P1w in the primitive corpus callosum (arrows in F) and is strongly expressed in the corpus callosum later (G, H). Higher magnification of the rectangular area in A shows the perivascular expression of the ETA receptor (white arrowhead in I) at P1d, but ETA receptor immunoreactivity appears in primitive corpus callosum (arrows in J) and increases with development in oligodendrocyte-like cells of the white matter (K, L). Scale bar = 100 µm in A-H, 20 µm in I-L.

• 

  Fig. 2 Fluorescent double labeling of the ETA receptor and CD31, MBP and APC in the developing mouse brain. Double labeling for the ETA receptor (A, D, G) and CD31 (B), MBP (E) and APC (H) was examined at P1d (A-C), P1w (D-F) and P4w (G-I). The ETA receptor is associated with blood vessels and the perivascular elements is co-localized with CD31, a marker of endothelial cells (arrow in C) during early development. At a later point in time, the ETA receptor is co-localized with MBP expression (arrowheads in F). In addition, ETA receptor immunoreactivity is found with another mature oligodendrocyte marker, APC (G-I). Scale bar = 20 µm.

• 

  Fig. 3 Western blots for ETA receptor and MBP expression in mice brains during development. This figure shows a representative immunoblot from 3 independent experiments. ETA receptor expression is found at P1d and increased with further development. MBP expression is weakly found at P2w and increased at P4w and P8w.

• 

  Fig. 4 Fluorescent double labeling for the ETA receptor and MBP in primary OPCs after ET-1 treatment. The ETA receptor was expressed in a few cells of OPC cultures (A-C). Myelination was assessed by immunostaining with an antibody against MBP, a marker of mature oligodendrocytes. OPCs were treated with ET-1 (100 nM). ETA receptor/MBP positive cells increase at 24 and 48 hr after treatment with ET-1 (D-I). Scale bar = 20 µm.

• 

  Fig. 5 Western blot for MBP in primary OPCs after ET-1 treatment alone, or with an ETA receptor blocker. OPCs were treated with ET-1 (100 nM) alone and then preincubated with BQ123 (2 µM) for 2 hr before exposure to ET-1. Transcripts (A) and protein (B) of MBP increase after 48 hr of treatment with ET-1 and are blocked by the specific ETA receptor antagonist, BQ123. Data are expressed as optical densities and represent means ± SEM of three independent experiments. *P < 0.05.

• 

  Fig. 6 Effect of ET-1 on intracellular Ca2+ release in primary OPCs. (A) OPCs were loaded with Fura-2/AM and stimulated with ET-1 (100 nM), then imaged by confocal microscopy at 1 min intervals. (B) Graphs show changes in mean fluorescence intensity from 15 cells per microscopic field over time. The figure is representative of three independent experiments. Fura-2/AM was excited with the 340 nm/380 nm, and its fluorescence emission was recorded at 510 nm. The actual number of cells in each case was approximately 100 single cells.

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