Effects of cloperastine, a non-narcotic antitussive, on the expression of GIRK channels in the brain of methamphetamine-induced hyperactive mice
How to cite this article: Soeda F, Kinoshita M, Fujieda Y, Takahama K. Effects of cloperastine, a non-narcotic antitussive, on the expression of GIRK channels in the brain of methamphetamine-induced hyperactive mice. Indian J Physiol Pharmacol 2023;67:57-63.
Centrally-acting antitussives with inhibitory effects on G protein-coupled inwardly rectifying potassium (GIRK) channels have been shown to also inhibit methamphetamine-induced hyperactivity in mice. In this study, we examined if cloperastine, which is the most potent inhibitor of the GIRK channels among antitussives, is sensitive to the expression levels of GIRK channels in the brain of methamphetamine-treated mice.
Materials and Methods:
The brain tissues have been removed and the total RNA has been extracted from tissues. The mRNA levels were evaluated using semiquantitative reverse transcription-polymerase chain reaction.
The concentration levels of the mRNA of GIRK channels within the ventral midbrain of methamphetamine-treated mice increased as compared with that in control and cloperastine reduced an upregulation in GIRK2, one of the subunits of the GIRK channels, by the injection of methamphetamine.
These findings suggest that cloperastine might ameliorate hyperactivity by inhibiting the GIRK channels in the brain.
G protein-coupled inwardly rectifying potassium channels
Previously, we have reported that centrally-acting antitussives (CAAs), like cloperastine, exhibit inhibitory effects on G protein-coupled inwardly rectifying potassium (GIRK) channel-activated currents and alleviate not only persistent cough but also intractable brain disease symptoms in rats and mice. For example, CAAs have been reported to improve cerebral infarction-associated urinary disorders among rodents. Moreover, the drug markedly lowers the immobility time in the forced swimming test among both adrenocorticotropic hormone-treated and normal rodents,[2,3] alluding to an antidepressant effect of the medication. In addition, mice who received the antitussives tend to have lower anxiety levels, based on their reduced performance (using number of marbles buried) in a marble-burying test, pointing out that the medication may exhibit anti-anxiety properties. Furthermore, a chronic administration of cloperastine has been reported to improve learning and memory disorders caused by endocrine disruptors such as diethylstilbestrol. Overall, these observations imply that antitussives, such as cloperastine, may improve disrupted brain conditions induced by several diseases or environmental toxins.
Hyperactive behaviours are symptoms of a severe developmental illness. For instance, attention deficit hyperactivity disorder is currently defined as a developmental disorder, in which all clinical criteria manifest as behavioural changes. Hyperactivity, impulsivity and inattention are currently considered the primary clinical symptoms (Diagnostic and statistical manual of mental disorders: DSM-5; American Psychiatric Association; 2013). Our previous study suggested that CAAs, such as cloperastine, ameliorated methamphetamine-induced hyperactivity (MIH). In addition, there was a significant correlation between the ameliorative effects of antitussives on MIH and the blockade of GIRK channel currents.
This study investigated whether the blockade of brain GIRK channels was involved in the effects of CAAs on MIH in mice, as the first step toward the understanding of action mechanisms of CAAs on hyperactivity in mice.
MATERIALS AND METHODS
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.
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.
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].
|METH 0.5 (vehicle±METH 0.5 mg/kg)||METH 2.0 (vehicle±METH 2.0 mg/kg)|
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].
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).
Our previous research reported that non-narcotic antitussives blocked GIRK channel currents coupled to various receptors, such as 5-HT1A, dopamine D2, noradrenaline α2, GABAB and delta-opioid.[13-15] Furthermore, we have shown that non-narcotic antitussives decreased MIH at effective antitussive doses and found that there is a correlation between the suppression of hyperactivity and the inhibition of GIRK channel currents. Activation of GPCR-mediated GIRK channels stabilises the expression of neurons through the hyperpolarisation induced by outward currents of K+. Therefore, some investigators including us have postulated that the inhibition of GIRK channels should stimulate neurons, facilitating the expression of neurotransmitters.[15-18] In fact, non-narcotic antitussives such as cloperastine and tipepidine have been reported to increase levels of neurotransmitters and neurotrophic factors such as dopamine and BDNF.[5,19] In this study, it was suggested that cloperastine inhibited the expression levels of GIRK channels in the region containing the nucleus of origin in dopamine. It was found that the administration of methamphetamine upregulated the expression levels of GIRK2 subunits and dopamine D2 receptors. However, the administration of cloperastine inhibited the expression levels of GIRK2 subunits, but not those of dopamine D2 receptors, 40 min after methamphetamine injection. Therefore, it was suggested that cloperastine may selectively affect GIRK channels in the brain. Although not shown in the data, the expression level of GIRK2 subunits and dopamine D2 receptors did not change at 180 min after methamphetamine injection, suggesting that the action of cloperastine may be transient. We have previously reported that cloperastine inhibited MIH, and the results were indicated at 40 min after methamphetamine injection. Therefore, it is possible that the effects of cloperastine, as observed in this study, could be the mechanism of inhibitory action in MIH.
Furthermore, the present study evaluated the expression levels of GIRK channels in each part of the brain after an acute injection of methamphetamine. As far as we know, there was no previous related study; therefore, we examined reports of changes in mRNA and protein molecules due to the acute injection of methamphetamine. As obtained in the results, there were many reports that mRNA was examined about 1–4 h after treatment with methamphetamine,[20-26] and proteins were examined about 1.5–24 h after treatment with methamphetamine.[27,28] In addition, it was reported that the amount of extracellular dopamine in the ST was maximum at 40 min after an acute injection of 0.5-mg/kg methamphetamine. Therefore, in this study, samples at 40 min and 180 min after the injection of methamphetamine were used for the measurement of the levels of mRNA of GIRK channels by semiquantitative RT-PCR. For the measurement of GIRK channel proteins by immunohistochemistry, samples taken 180 min after methamphetamine injection were used. The results of this study suggest that the acute injection of methamphetamine alters the mRNA expression levels of GIRK channels in the brain. Moreover, the pattern of fluctuation differed by region of the brain. At present, the mechanism is unknown; however, it is generally believed that acute injection of methamphetamine involves the activation of the substantia nigra-striatal pathway that projects from dopamine cells present in the substantia nigra to the ST. In the present study, the expression levels of TH, a rate-limiting enzyme in the dopamine synthesis pathway, and dopamine D2 receptors coupled to GIRK channels in the ST and the VM were increased by the acute injection of methamphetamine, indicating the pathogenic mechanism of MIH. Furthermore, by comparing changes between GIRK2 subunits and dopamine D2 receptors after methamphetamine injection, the change in GIRK2 expression levels was transient. Therefore, in this animal model, it is possible that acute injection of methamphetamine increased the expression levels of the GIRK channels in the substantia nigrastriatal pathway, and as a result, the expression levels of the dopamine D2 receptors coupled to the GIRK channels correspondingly increased.
Obviously, methamphetamine injection has resulted in differences in the expression profiles for striatal dopamine receptors. Reports supporting this finding indicate that mice with genetically altered dopamine D1 receptor deficiency enhanced their locomotor activity, whereas mice with genetically engineered dopamine D2 receptor deficiency decreased their locomotor activity. Although the detailed mechanism is not known, it is assumed that the sensitivity of methamphetamine to dopamine receptors may differ in the ST.
These findings indicate that MIH in mice involves an increase in GIRK channels coupled to dopamine D2 receptors. Empirically, it was also suggested that the CAAs, such as cloperastine, may decrease the expression levels of GIRK channels in the brain with the attendant effect of affecting the release of neurotransmitters, such as dopamine, and thus suppress hyperactivity.
This study was funded by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology.
Declaration of patient consent
Patient’s consent not required as there are no patients in this study.
Conflicts of interest
There are no conflicts of interest.
Supplementary resources available on
Financial support and sponsorship
Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology.