Inhibition of Gap Junction Elevates Glutamate Uptake in Cultured Astrocytes
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ORIGINAL PAPER
Inhibition of Gap Junction Elevates Glutamate Uptake in Cultured Astrocytes Katsura Takano1 · Masato Ogawa1 · Kenji Kawabe1 · Mitsuaki Moriyama1 · Yoichi Nakamura1
Received: 12 April 2017 / Revised: 26 May 2017 / Accepted: 27 May 2017 © Springer Science+Business Media New York 2017
Abstract Glutamate uptake is a main function of astrocytes to keep extracellular glutamate levels low and protect neurons against glutamate-induced excitotoxicity. On the other hand, astrocyte networks formed by gap junctions, which are consisted with connexins and connecting neighboring cells, are reported to play a critical role in maintaining the homeostasis in the brain. In the present study, we examined the effects of gap junction inhibitors on the glutamate uptake activity in cultured rat cortical astrocytes. At first, we confirmed the effects of gap junction inhibitors, 1-octanol and carbenoxolone, on cell–cell communication by the scrape-loading assay using a fluorescent dye Lucifer yellow. Both of 1-octanol and carbenoxolone treatments for 20 min in cultured astrocytes significantly suppressed the cell–cell communication assessed as the distance of dyespreading. 1-octanol and carbenoxolone increased the glutamate uptake by astrocytes and glutamate aspartate transporter (GLAST) expression on the cell membrane. These results suggest that gap junction inhibitors increase the glutamate uptake activity through the increase of GLAST proteins located on the cell membrane. The regulation of gap junction in astrocytes might protect neurons against glutamate-induced excitotoxicity. Keywords Astrocyte · Gap junction · Carbenoxolone · 1-Octanol · Glutamate uptake
* Katsura Takano [email protected]‑u.ac.jp 1
Laboratory of Integrative Physiology in Veterinary Sciences, Osaka Prefecture University, 1‑58, Rinku‑Ourai Kita, Izumisano, Osaka 598‑8531, Japan
Abbreviations CNS Central nervous system Cx Connexin DMEM Dulbecco’s modified eagle medium FBS Fetal bovine serum HHBSS HEPES-buffered Hank’s balanced salt solution HKR HEPES-buffered Krebs Ringer solution HRP Horseradish peroxidase PBS Phosphate-buffered saline
Introduction Astrocytes play various important roles in central nervous system (CNS), such as maintenance of blood brain barrier, control of ionic balance in brain parenchyma, cerebrovascular regulation, and scavenging some neurotransmitters including glutamate [1–4]. These functions of astrocytes serve the maintenance of brain homeostasis. Glutamate is the main excitatory neurotransmitter in the CNS [5]. High concentrations of extracellular glutamate represent a potent neurotoxin which leads to neuronal over-stimulation and subsequent excitotoxic cell death [6]. Astrocytes are known to play complex roles in the control of extracellular glutamate homeostasis [7]. On one hand, astrocytes modulate the duration of glutamatergic neurotransmission and synaptic strength by clearing extracellular glutamate through the high affinity sodium-dependent glutamate transporters, excitatory amino acid transporter
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