Amiloride suppresses pilocarpine-induced seizures via ASICs other than NHE in rats

Abstract

Background and purpose: Although recent studies have indicated that acid-sensing ion channels (ASICs) may play an important role in suppressing status epilepticus (SE) in rats, the precise mechanism is unclear. We attempted to investigate the antiepileptic effect of amiloride in SE rats and its mechanism.

Methods: Rats with seizures induced by Li-pilocarpine were randomly divided into four groups, phosphate buffer saline (PBS) group, amiloride group, levetiracetam group and acidic liquid group, respectively. The electroencephalogram (EEG) of each group was recorded. Then rats treated with different drugs (2 h after amiloride or PBS injection or 1 h after PBS injection) and a normal control group was selected for reverse transcription-polymerase chain reaction (RT-PCR). The expression of ASIC1a, ASIC3 and sodium-hydrogen exchanger (NHE) in each group was detected.

Results: Amiloride reduced the frequency of discharge in 60~90 min after injection significantly. In acidic liquid group, the epileptic discharge was increased in 0~30 min. Moreover, the expression of ASIC1a, ASIC3 and NHE was obviously increased in the SE groups. Compared with SE groups, the expression of ASIC1a and ASIC3 mRNA in amiloride group decreased significantly. While NHE mRNA expression in the SE groups showed no significant difference.

Conclusion: Amiloride inhibited pilocarpine-induced SE and the anti-epileptic mechanism was associated with deactivation of the ASIC1a and ASIC3 instead of NHE in rats.

Keywords: ASICs; Amiloride; EEG; NHE; epilepsy; pilocarpine.

Figure 1

Effect of amiloride on the frequency of EEG discharge in rats. The difference of the frequency of rats EEG discharge in 30~90 min among the four groups after abdominal injection was statistically significant (P _30~60<0.05, P _60~90<0.01). Moreover, in both of amiloride group and levetiraceta group, the frequency of EGG discharge was significantly declined in 60~90 min after injection (P _60~90<0.05), compared with PBS group, respectively. There was no statistical difference between amiloride group and levetiraceta group. On the contrary, in acidic liquid group, the frequency of EGG discharge in 0~30 min (P _{0~30 min}<0.05) was moderately increased, while there was no statistically significant difference in 30~90 min between acidic liquid group and PBS group. *Compared with PBS group, P<0.05.

Figure 2

Expression of ASIC1a mRNA was increased by seizures. A: Photograph of an agarose gel showed ASIC1a mRNA expression. The expression of ASIC1a mRNA was decreased in amiloride group measured by RT-PCR. B: Compared with normal control group, the expression of ASIC1a mRNA was increased significantly in both SE 1 h group and SE 2 h group (P<0.01). Compared with other SE groups, the expression of ASIC1a mRNA in amiloride group was decreased significantly (P<0.01). **P<0.01, amiloride group vs. SE 1 h group, #P<0.01, amiloride group vs. SE 2 h group.

Figure 3

Expression of ASIC3 mRNA was increased by seizures. A: Photograph of an agarose gel showed ASIC3 mRNA expression. The expression of ASIC3 mRNA was decreased in the amiloride group measured by RT-PCR. B: Compared with normal control group, the expression of ASIC3 mRNA was increased significantly in SE 1 h group, SE 2 h group and amiloride group (P<0.01). Compared with other SE groups, the expression of ASIC3 mRNA in the amiloride group was decreased significantly (P<0.01). **P<0.01, amiloride group vs. SE 1 h group, #P<0.01, amiloride group vs. SE 2 h group.

Figure 4

Expression of NHE mRNA was not affected by amiloride. A: Photograph of an agarose gel showed NHE mRNA expression. The decline of NHE mRNA was not evident in amiloride group measured by RT-PCR. B: Compared with the normal control group, the expression of NHE mRNA was increased significantly in SE 1 h group, SE 2 h group and amiloride group (P<0.01). On the contrary, compared with 1 h seizure group and 2 h seizure group, the expression of NHE mRNA in the amiloride-treated group was not statistically different.