Institute of Neuroscience, The Fourth Military Medical University, Xi’an 710032, China

Abstract 

Objective 
To investigate whether hypertonic saline (HS) can induce the synthesis and release of glutamate in cultured hypothalamic astrocytes or C6 cell line. 
Methods 
Astrocytes were isolated, cultured, purified and identified from the hypothalamus of newborn rat (1 day). The astrocytes were randomly divided into five groups: isotonic (IS) and HS groups, astrocytes were incubated by IS and HS (320 mOsm NaCl) medium, respectively, for 1, 3, 5, 10 or 15 min; carbenoxolone (CBX) +IS and CBX+HS groups, astrocytes were pre-treated with CBX (100 mmol/L) for 1 h at 37 °C in a 5% CO2 / 95% atmosphere, then removed to IS and HS medium, respectively, for 1, 3, 5, 10 or 15 min; Ca2++HS group, astrocytes were pre-incubated with Ca2+ (1 000 μmol/L) for 1 h at 37 °C in a 5% CO2 / 95% atmosphere, followed by a wash with isotonic FBS/DMEM, and then removed to hypertonic saline for 1, 3, 5, 10 or 15 min. The media of five groups were collected to analyze the medium glutamate concentration with high performance liquid chromatography. The astrocytes were fixed and double immunofluorescent stained with anti-glial fibrillary acidic protein (GFAP) and anti-glutamate. The C6 cells were divided into four groups: IS, HS, CBX+IS and CBX+HS groups, and used for quantitative measurement of glutamate in cells by flow cytometry (FCM). 
Results 
(1) Anti-GFAP immunofluorescent signal revealed no significant difference among various time points in each group, or among the five groups. (2) The anti-glutamate immunofluorescent signal was increased in HS group and peaked at 5 min, and decreased and returned to the level of IS group at 15 min (P < 0.01 vs the 5 min of HS group). In CBX+HS group, the glutamate intensity was higher than that in CBX+IS and HS groups. (3) The medium glutamate concentration had no change after treatment with HS for 1 and 3 min, while increased markedly after treatment for 5 min to 15 min (P < 0.01 vs 1 min and 3 min). On the contrary, the medium glutamate concentrations in the CBX+HS or Ca2++HS group were significant lower than that in the HS group (P < 0.01). (4) FCM showed HS and CBX+HS induced glutamate increase in C6 cells. 
Conclusion 
HS induced cultured rat hypothalamic astrocytes or C6 cells to synthesize and release glutamate; CBX could block glutamate release, but could not disrupt glutamate synthesis.

Keywords

astrocytes; hypertonic stimulation; carbenoxolone; connexin 43; high performance liquid chromatography; immunofluorescent

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