Effects of cloperastine, a non-narcotic antitussive, on the expression of GIRK channels in the brain of methamphetamine-induced hyperactive mice

Subjects and housing

Forty-eight male ddY mice (SLC, Shizuoka, Japan) were used for this study. They were kept in standard cages (33.8 long × 22.5 wide × 14.0 cm high) with bedding of paper chips in the animal centre of the Graduate School of Pharmaceutical Sciences of Kumamoto University. A 12:12-h light-to-darkness cycle was continued throughout the experiments, with lights switched on at 08:00–20:00, and the temperature was maintained at 22°C ± 2°C. Standard mouse food and tap water were made freely available to the mice. The mice were processed based on recommendations made by the Japanese Pharmacological Society. All experiments were approved by the Animal Experiment Committee at Kumamoto University. Reference no of the ethics approval of our study was AA17-189, and its date of approval was December 21, 2005.

Drugs

Cloperastine hydrochloride was bought from Sigma-Aldrich Japan (Tokyo, Japan). Methamphetamine hydrochloride was purchased from Dainippon Sumitomo Pharma (Osaka, Japan). The drugs were administered to the animals as indicated in the figure legends, and the doses were estimated using the respective free base.

Methamphetamine-induced hyperactive mice

We purchased 4-week-old male mice and performed handling for 1–2 weeks. Methamphetamine was administered subcutaneously (s.c.) at 0.5- or 2.0-mg/kg body weight.

Semiquantitative reverse transcription-polymerase chain reaction

The brain tissues were excised and the frontal cortex (FC), dorsal hippocampus (DH), striatum (ST) and ventral midbrain (VM) were quickly dissected and immersed in liquid nitrogen and stored at −80°C for later use. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA). The amount and quality of RNA were evaluated using their UV absorbance values at 260 nm and agarose gel electrophoresis, respectively. Reverse transcription (RT)-polymerase chain reaction (PCR) was carried out using an RNA PCR Kit Version 3 (Takara Shuzo, Ohtsu, Japan) in accordance with the steps advised by the manufacturers. For RT, total RNA (0.5 mg) was mixed in a 10 µL reaction mixture consisting of 5 mM MgCl₂, 1 mM dNTP mixture, 1 U RNase inhibitor, 125 nM oligo dT-adaptor primer and 0.25 U avian myeloblastosis virus reverse transcriptase XL (Life Sciences, Hialeah, FL) in RNA PCR buffer (10 mM Tris-HCl and 50 mM KCl). After producing cDNA on the first strand at 42°C for 60 min, a denaturation step at 99°C for 5 min was carried out. For the PCR, the template (10 µL) underwent amplification in a 50 µL PCR reaction mixture consisting of 200 nM of each PCR-primers for GIRK1, GIRK2, GIRK3, tyrosine hydroxylase (TH), dopamine D₁ receptor (D₁R), dopamine D₂ receptor (D₂R), or b-actin (see Supplementary materials and methods) and 1.25 U Taq polymerase (Takara Shuzo) in PCR buffer. Denaturation was executed at 94°C for 120 s and amplification was performed (1) at 94°C for 60 s and (2) at 56°C (D₁R and D₂R), 58°C (GIRK2), 60°C (GIRK1, GIRK3 and b-actin), 68°C (TH) for 60 s and (3) at 72°C for 120 s in an automated DNA Thermal Cycler (Mastercycler gradient, Eppendorf, Tokyo, Japan). The quantity of template and the number of amplification cycles were preliminarily optimised during the PCR reaction to avoid saturation (GIRK1; FC, DH: 23 cycles; ST: 25 cycles; VM: 30 cycles; GIRK2; FC, DH: 23 cycles; ST, VM: 28 cycles; GIRK3; FC, DH, ST: 25 cycles; VM: 30 cycles; TH; ST: 26 cycles; VM: 28 cycles; D₁R; ST: 25 cycles; VM: 32 cycles; D₂R; ST, VM: 28 cycles; b-actin; FC, DH and ST and VM: 20 cycles). After amplification, the RT-PCR reaction mixture (10 µL) underwent electrophoresis on a 2% agarose gel and was subsequently visualised by ethidium bromide staining using an ultraviolet transilluminator (TCP-15C, M&S Instruments, Osaka, Japan), and quantified by Scion Image beta 4.0.2 (Scion Corporation, USA). The mRNA levels of GIRK1, GIRK2, GIRK3, TH, D₁R and D₂R were then normalised to mRNA b-actin levels and expressed as arbitrary units.

Statistical analysis

The findings are reported as means ± standard error of the mean (SEM). In the case of comparisons between the two groups, statistical significance was assessed using the unpaired two-tailed t-test. For all comparisons, P < 0.05 was used as the statistically significant criterion.

Effects of methamphetamine on the expression levels of GIRK channels, TH and dopamine receptors in the brain of normal mice

To ascertain if the ameliorative effects of antitussives on hyperactivity were related to the GIRK channels, we determined the expression levels of the GIRK channels by semiquantitative RT-PCR analysis in some brain regions of methamphetamine-induced hyperactive mice. We then examined the levels of (1) TH, the rate-determining enzyme in dopamine synthesis and (2) GIRK channels in the ST and the VM involved in the nigrostriatal dopamine system. TH mRNA levels were significantly increased in both the ST and the VM by acute injection of methamphetamine at 0.5 and 2.0 mg/kg [Table 1]. In the brain, three subunits of GIRK channel (GIRK1, GIRK2 and GIRK3) have broad and overlapping distributions, while the fourth subunit (GIRK4) occurs mainly in the heart.^[7-12] Therefore, we have examined GIRK1, GIRK2 and GIRK3 expression levels, but not GIRK4. Methamphetamine at 0.5 and 2.0 mg/kg significantly increased GIRK2, but not GIRK1 and GIRK3 mRNA in both the ST and the VM [Table 1]. Apart from the above, the levels of mRNA of GIRK1 and GIRK3 were significantly increased in the DH, whereas methamphetamine at 0.5 and 2.0 mg/kg significantly reduced GIRK1 mRNA levels in the FC [Table 1]. In addition, we performed immunohistochemical staining for GIRK2 at 180 min after injection of methamphetamine. In the hippocampus and VM, including the substantia nigra, GIRK2 immunoreactivity was increased in mice injected with methamphetamine compared to controls [Supplementary resources].

In this study, our findings suggest that methamphetamine activated the nigrostriatal system and GIRK2. It was known that GIRK channels were coupled to the dopamine D₂ receptors, but not the dopamine D₁ receptors. Therefore, we also examined dopamine receptors mRNA levels to determine, whether the action of methamphetamine was associated with the GIRK channels coupled with dopamine receptors. Dopamine D₂ receptor mRNA levels were increased by methamphetamine in both the ST and the VM. In contrast, methamphetamine at 0.5 and 2.0 mg/kg significantly reduced the dopamine D₁ receptor mRNA levels in the ST [Table 1].

Moreover, we examined the time-course of the expression levels of the mRNA of both GIRK2 subunits and dopamine receptors after methamphetamine injection. GIRK2 mRNA levels had increased significantly at 40 min but decreased at 180 min after methamphetamine injection. On the other hand, dopamine D₂ receptors mRNA levels were both significantly increased at 40 and 180 min after methamphetamine injection compared to the control [Figure 1].

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Figure 1:

Relative mRNA expression of G protein-coupled inwardly rectifying potassium channel 2 (GIRK2) subunits and dopamine receptors in the ventral midbrain after methamphetamine injection. (a) Semiquantitative reverse transcription-polymerase chain reaction analysis of GIRK2 subunits, dopamine D₁ receptors and dopamine D₂ receptors in the ventral midbrain. Samples were obtained from mice s.c. injected with saline (Ctrl) or 0.5-mg/kg methamphetamine. Expression of β-actin was also analysed in the same sample, as a housekeeping gene. The brain was removed at 40 min (left) or 180 min (right) after injection of methamphetamine. (b) Relative expression levels of each mRNA in the ventral midbrain. The optical density of each mRNA was normalised to that of corresponding β-actin. Mean ± SEM of 5 to 6 mice. **P < 0.01 versus 0 min group. Ctrl: Control, METH: Methamphetamine, D₁R: Dopamine D₁ receptors, D₂R: Dopamine D₂ receptors.

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Effects of centrally-acting non-narcotic antitussive cloperastine on the expression levels of GIRK2 subunits and dopamine D₂ receptors in the VM of methamphetamine-induced hyperactive mice

Finally, we evaluated the effects of cloperastine on the concentration of GIRK2 subunits and dopamine D₂ receptors in the VM, which is involved in the origin of the dopaminergic cell bodies, of methamphetamine-induced hyperactive mice. First, cloperastine at a dose of 20 mg/kg was injected and 10 min thereafter, methamphetamine was injected. Subsequently, 40 min after methamphetamine injection, GIRK2 mRNA levels were significantly reduced in the VM by subcutaneous injection of cloperastine administered at 20 mg/kg dose. However, dopamine D₂ receptors mRNA levels did not change [Figure 2]. In addition, 180 min after methamphetamine injection, the levels of the mRNA of GIRK2 subunits and dopamine D₂ receptors did not change by subcutaneous injection of cloperastine using a 20 mg/kg dose (data not shown).

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Figure 2:

Effects of cloperastine on the expression of G protein-coupled inwardly rectifying potassium channel 2 (GIRK2) subunits and dopamine D₂ receptors in the ventral midbrain after methamphetamine injection. The brain was removed 40 min after the injection of 0.5-mg/kg methamphetamine, s.c. Relative mRNA expression of GIRK2 subunits was significantly decreased by cloperastine administered at a 20-mg/kg dose, s.c. injection 10 min before the injection of methamphetamine. However, relative mRNA expression of the dopamine D₂ receptors did not change. Mean ± SEM of 3 to 4 mice. *P < 0.05 versus methamphetamine control (vehicle + methamphetamine 0.5 mg/kg) group. METH: Methamphetamine; CP: Cloperastine.

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