ABSTRACT
OBJECTIVE: To investigate the role of N-methyl-D-aspartate receptor(NMDAR)/cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA) signaling pathways in regulating 2-chloroethanol-induced aquaporin-4(AQP4) expression in astrocytes(AS). METHODS: i) AS in logarithmic growth phase were treated with 2-chloroethanol at the doses of 0.0, 7.5, 15.0 and 30.0 mmol/L for 12 hours, and the cells were collected for detection. ii) The AS in logarithmic growth phase were divided into blank control group, inhibitor control group, 2-chloroethanol group, and inhibitor intervention group. The inhibitor included dizocilpine(MK-801) and N-(2-[p-bromocinnamylamino-]ethyl)-5-isoquinolinesulfonamide(H89). The blank control group did not receive any treatment. The inhibitor control group was treated with a concentration of 10.0 μmmol/L MK-801 or 15.0 μmmol/L H89. The MK-801 intervention group was pretreated with MK-801 at a concentration of 10.0 μmmol/L for 30 minutes. The H89 intervention group was pretreated with H89 at a concentration of 15.0 μmmol/L for 1 hour. After the intervention, the AS in 2-chloroethanol group and MK-801, H89 intervention group were stimulated with 2-chloroethanol at a dose of 30.0 mmol/L for 12 hours. iii) The AS in each group were collected and used for Western blotting and real-time fluorescence quantitative polymerase chain reaction analysis to detect the protein and mRNA expression of AQP4, NMDAR receptor main subunit(NR1), NMDAR receptor 2 B subunit(NR2 B) and calmodulin dependent protein kinaseⅡ(CaMKⅡ). The Western blotting was adopted to detect the expression of phosphorylase-CaMKⅡ(p-CaMKⅡ) and PKA. Colorimetric method was used to detect the concentration of calcium(Ca~(2+)) in AS. The enzyme-linked adsorption test was used to measure adenylate cyclase(AC) activity and cAMP levels. RESULTS: i) The relative expression of protein and mRNA of AQP4, NR1 and NR2 B, PKA at protein level and CaMKⅡ at mRNA level, and the ratio of p-CaMKⅡ/CaMKⅡ protein, the concentration of Ca~(2+), AC activity and cAMP level in 30.0 mmol/L group were higher then those of 0.0 mmol/L group in AS(P<0.05). The relative protein expression of PKA and the concentration of Ca~(2+) increased with the increase of 2-chloroethanol(P<0.05). ii) The relative protein expression of AQP4 and the concentration of Ca~(2+) in the 2-chloroethanol group were higher than that of the blank control group and MK-801 control group(P<0.05). The relative protein expression of AQP4 and the concentration of Ca~(2+) in MK-801 intervention group were lower than that in 2-chloroethanol group(P<0.05). The relative protein expression of AQP4 and PKA in 2-chloroethanol group were higher than that of the blank control group and H89 control group(P<0.05). The relative protein expression of AQP4 and PKA in H89 intervention group was lower than that in 2-chloroethanol group(P<0.05). CONCLUSION: The 2-chloroethanol timulation induces the expression of AQP4 by activating NMDAR/cAMP/PKA signaling pathway in AS.