Inhibition of suplatast tosilate on EPSC in the single paratracheal ganglion neurons attached with presynaptic boutons.

Using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with nystatin-perforated patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. EPSC frequency was higher sensitive to suplatast than EPSC amplitude. IC50 for EPSC frequency was 1.1 × 10-5 M, being similar to that for the effect on histamine release from mast cells and lower than that for the inhibitory effect on cytokine production. Suplatast also inhibited the EPSCs potentiated by bradykinin (BK), but it did not affect the potentiation itself by BK. Thus suplatast inhibited the EPSC of PTG neurons attached with presynaptic boutons at both the presynaptic and postsynaptic sites.NEW & NOTEWORTHY In this study, using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. Thus suplatast inhibited the function of PTG neurons at both of presynaptic and postsynaptic sites.

Effects of enriched environment on micturition activity in freely moving C57BL/6J mice.

OBJECTIVES: An enriched environment (EE) has been known to promote structural changes in the brain and enhance learning and emotional performance. However, little is known about the effect of an EE on brain stem functions, such as the micturition function. In this study, we examined whether an EE affects micturition activity in mice. METHODS: Male C57BL/6J mice were used. We assessed the micturition activity of freely moving mice using a novel system developed in-house. RESULTS: During the dark period, but not light, the EE significantly increased voiding frequency, total voided volume, mean voided volume, voiding duration, mean flow rate, and maximum flow rate compared with the control environment. This EE effect on micturition function was associated with habituation to novel environments in the open-field test, but not with amelioration of motor coordination in the rotarod test. Interestingly, even after the mice were withdrawn from the EE, the improvements in micturition function persisted, while other behavioral changes were abolished. The relative value of voiding frequency and total voided volume during the light period, expressed as a percentage of 24 hours, increased with age when mice were reared in a standard environment. However, this age-related change was not observed in mice reared in an EE. CONCLUSIONS: These results suggest that an EE may promote micturition activity during the active phase of C57BL/6J mice, and its effects persist even after withdrawal from the EE. Furthermore, an EE may mitigate dysfunctions in micturition activity, such as polyuria, during the resting phase in aged mice.

Effects of tipepidine on MK-801-induced cognitive impairment in mice.

We previously reported that centrally acting non-narcotic antitussives, including tipepidine, inhibit G-protein-coupled inwardly rectifying potassium (GIRK) channel-activated currents of neurons. In addition, when administered at a cough suppressant dose, the drugs ameliorated the symptoms of various models of intractable brain disease in rodents. In the current study, we investigated whether tipepidine causes recovery from schizophrenia-like cognitive dysfunction, which was induced by MK-801 (0.2 mg/kg, i.p.) in mice. We also examined the effect of tipepidine and clozapine co-administration on the dysfunction. Moreover, we studied whether clozapine inhibits GIRK channel activated currents in single brain neurons using the patch-clamp technique. Tipepidine elicited recovery from MK-801-induced cognitive impairment in the novel objective recognition test and Y-maze test. Further, co-administration of tipepidine and clozapine, at subthreshold doses of each drug, improved MK-801-induced cognitive impairment in the novel objective recognition test. Clozapine (3 × 10-5 M) had a minor effect on baclofen-induced currents in dopamine neurons of the ventral tegmental area.

Direct Potentiation of Capsaicin Current by Histamine and Its Effect by Suplatast on Rat Trigeminal Ganglia Neurons.

In this study, we investigated the effect of histamine on capsaicin-induced current and its influence by suplatast in rat trigeminal ganglia neurons using a patch-clamp technique. We found that histamine directly potentiated capsaicin-induced currents in rat sensory neurons, and suplatast had little effect on this potentiation. Since it has been known that suplatast suppresses histamine release from mast cells, it is possible that suplatast inhibits the activation of nociceptive fibers in the pathological condition via prevention of histamine-induced potentiation of the transient receptor potential vanilloid 1 receptor-mediated currents.

Deletion of GIRK2 subunit containing GIRK channels of neurons expressing dopamine transporter decrease immobility time on forced swimming in mice.

We previously reported that non-narcotic antitussives possessing inhibitory actions on G protein-coupled inwardly rectifying potassium (GIRK) channels have antidepressant-like effects in the forced swimming test in normal and adrenocoticotropic hormone (ACTH) treated rats. Furthermore, the antidepressant-like effects of the antitussives such as tipepidine were blocked by dopamine D1 receptor antagonist, and inhibitory actions on GIRK channels of dopamine neurons may be involved in the antidepressant-like effects of tipepidine. In this study, we generated GIRK2DATKO mice with Girk2/Kcnj6 conditional deletion and assessed depression-related behavior of the mice. The Cre/loxP system was used to selectively delete GIRK2 subunit containing GIRK channels in the neurons expressing dopamine transporter. First, deletion of GIRK2 subunits in the ventral tegmental area (VTA) neurons expressing dopamine transporters was confirmed by hisitochemically and electrophysiologically. In the mice, a significant decrease in the immobility time of forced swimming test was observed. Locomotor activity of the mice was not changed compared to that of GIRK2floxed mice, when tested in the open field. These results suggest that the antidepressant-like effect of antitussives such as tipepidine may be caused partly through the inhibitory actions on GIRK channels in the dopamine neurons.

The effects of suplatast tosilate on acutely dissociated sensory and paratracheal ganglia neurons.

In this study, we investigated the effects of suplatast on acutely dissociated single neurons of sensory and paratracheal ganglia using a patch-clamp technique. Suplatast had little effect on various responses caused by capsaicin, acid, bradykinin, serotonin and adenosine 5'-triphosphate in rat sensory neurons. Suplatast, even at 10-3 M, also did not induce any current at various membrane potentials in rat and guinea pig paratracheal ganglia neurons. Further, acetylcholine- and bradykinin-induced depolarizations were not affected by suplatast. On the other hand, in rat paratracheal ganglia neurons, 10-5 M nicotine-induced current were inhibited by suplatast in a concentration-dependent manner with a 50% inhibitory concentration of 9.86x10-5 M. The effect was noncompetitive and voltage-dependent. Furthermore, the effect was use-independent and not affected by the pretreatment time of suplatast. The results suggested that suplatast may inhibit neurotransmission at the paratracheal ganglia via the inhibition of nicotinic current. Thus, suplatast may attenuate cough production through the improvement of pathological conditions of the lower airway via suppressed acetylcholine release from the postganglionic nerve terminal.

Centrally acting non-narcotic antitussives prevent hyperactivity in mice: Involvement of GIRK channels.

We have previously reported that centrally acting non-narcotic antitussives inhibited G protein-coupled inwardly rectifying potassium (GIRK) channel-activated currents, and that the antitussives had multiple pharmacological actions on various models of intractable brain diseases in rodents. In this study, the question of whether these antitussives inhibit drug-induced hyperactivity in mice was investigated. Antitussives, such as cloperastine and tipepidine, at cough suppressant doses, inhibited an increase in ambulation of mice neonatally treated with 6-hydroxydopamine. In addition, all antitussives studied inhibited an increase in methamphetamine-induced hyperactivity in mice. Methylphenidate, which is used for treatment of ADHD, inhibited 6-hydroxydopamine-lesion-induced, but not methamphetamine-induced, hyperactivity in mice. By the rota-rod test, the drugs had little effect on motor coordination of the hyperactive mice. Significant correlation was found between the ameliorating effects of antitussives on methamphetamine-induced hyperactivity and their inhibitory actions on GIRK channel currents (coefficient factor, 0.998). Furthermore, tertiapin, a GIRK channel blocker, prevented an increase in methamphetamine-induced hyperactivity of mice. These results demonstrated that antitussive drugs (cloperastine, tipepidine and caramiphen) possessing inhibitory action on GIRK channels inhibit drug-induced hyperactivity in mice, suggesting that such antitussives may potentially be therapeutic for patients with ADHD.

Tipepidine, a non-narcotic antitussive, exerts an antidepressant-like effect in the forced swimming test in adrenocorticotropic hormone-treated rats.

We investigated whether tipepidine exerts an antidepressant-like effect in the forced swimming test in adrenocorticotropic hormone (ACTH)-treated rats, which is known as a treatment-resistant depression model, and we studied the pharmacological mechanisms of the effects of tipepidine. Male Wistar rats (5-7 weeks old) were used in this study. Tipepidine (20 and 40 mg/kg, i.p.) decreased the immobility time in the forced swimming test in ACTH-treated rats. The anti-immobility effect of tipepidine was blocked by a catecholamine-depleting agent, alpha-methyl-p-tyrosine (300 mg/kg, s.c.), but not by a serotonin-depleting agent, p-chlorophenylalanine. The anti-immobility effect of tipepidine was also blocked by a dopamine D1 receptor antagonist, SCH23390 (0.02 mg/kg, s.c.) and an adrenaline α2 receptor antagonist, yohimbine (2 mg/kg, i.p.). In microdialysis technique, tipepidine (40 mg/kg, i.p.) increased the extracellular dopamine level of the nucleus accumbens (NAc) in ACTH-treated rats. These results suggest that tipepidine exerts an antidepressant-like effect in the forced swimming test in ACTH-treated rats, and that the effect of tipepidine is mediated by the stimulation of dopamine D1 receptors and adrenaline α2 receptors. The results also suggest that an increase in the extracellular dopamine level in the NAc may be involved in the antidepressant-like effect of tipepidine in ACTH-treated rats.

Tipepidine increases dopamine level in the nucleus accumbens without methamphetamine-like behavioral sensitization.

We previously reported that the novel antidepressant-like effect of tipepidine may be produced at least partly through the activation of mesolimbic dopamine neurons via inhibition of G protein-coupled inwardly rectifying potassium channels. In this study, we investigated whether tipepidine increases dopamine levels in the nucleus accumbens (NAc) in rats using an in vivo microdialysis technique. We further assessed whether tipepidine at antidepressant-like effective doses induces behavioral- and cross-sensitization of locomotor activity in rats using the open field test. We found that acute administration of tipepidine increased dopamine levels in the NAc in freely moving rats without increasing locomotor activity. Tipepidine at antidepressant-like effective doses (20 and 40 mg/kg, i.p.) did not cause behavioral sensitization in rats. Furthermore, cross-sensitization between tipepidine and methamphetamine was not observed in rats. These results further support our working hypothesis that tipepidine may produce a novel antidepressant-like effect through activation of ventral tegmental area-NAc dopaminergic neurons whose mechanisms differ from those contributing to the reinforcing effects of addictive drugs.

Novel antitussive effect of suplatast tosilate in guinea pigs.

We studied the antitussive effects of suplatast, a Th2 cytokine inhibitor, and compared them with the effects of codeine using an experimental cough model in guinea pigs. Suplatast and codeine dose-dependently inhibited cough caused by mechanical stimulation of the larynx, but they did not inhibit cough caused by mechanical stimulation of the bifurcation of the trachea. In guinea pigs with bronchitis, suplatast had an antitussive effect on cough caused by stimulation of the larynx, whereas codeine did not inhibit such cough. In SO2-exposed guinea pigs, suplatast tended to inhibit cough caused by mechanical stimulation of the tracheal bifurcation. Further, suplatast inhibited citric acid-induced cough augmented by pretreatment with an angiotensin-converting enzyme inhibitor, whereas codeine did not inhibit such cough. Suplatast also inhibited bradykinin-induced discharges of airway vagal afferent nerves and significantly inhibited 4-aminopyridine-induced discharges of airway vagal afferent nerves. These findings indicate that the antitussive effects of suplatast are mediated by a novel mechanism involving the peripheral nervous system.

Cholinergic EPSCs and their potentiation by bradykinin in single paratracheal ganglion neurons attached with presynaptic boutons.

We have found that bradykinin (BK) potentiates the nicotine-induced currents in airway paratracheal/parabronchial ganglia (PTG) neurons. In this study, we investigated if BK affects the cholinergic synaptic transmission in rat PTG neurons attached with synaptic buttons. Excitatory postsynaptic currents (EPSCs) were recorded in acutely dissociated PTG neurons attached with presynaptic boutons. EPSC frequency was increased in the high-K(+) external solution without affecting their amplitude. Activation and deactivation kinetics also did not change in the high-K(+) solution. Cd(2+) inhibited the EPSC frequency at 10(-7) M and also amplitude at higher concentrations without changing the kinetics. Mecamylamine inhibited both the amplitude and frequency of EPSCs and reduced the activation and deactivation kinetics. 10(-8) M BK potentiated the EPSC amplitude to 1.37 ± 0.19 times of preapplication control. In addition, its frequency was increased to 2.04 ± 0.41 times. BK did not affect the activation and deactivation kinetics. The effects of BK were mimicked by [Hyp(3)]-BK, a B2 kinin receptor agonist, whereas HOE 140, a B2 kinin receptor antagonist, abolished the effects of BK. In conclusion, BK potentiates the cholinergic synaptic transmission via B2 kinin receptors in the PTG. Since predominant control of airway function is thought to be exerted by cholinergic nerves arising from the PTG, the present findings might underlie at least partly the inflammatory pathological conditions of the lower airway.

Potent cough suppression by physiologically active substance in human plasma.

Human plasma contains wide variety of bioactive proteins that have proved essential in therapeutic discovery. However many human plasma proteins remain orphans with unknown biological functions. Evidences suggest that some plasma components target the respiratory system. In the present study we adapted heparin affinity chromatography to fractionate human plasma for functional bioassay. Fractions from pooled human plasma yielded particular plasma fractions with strong cough suppressing effects. Purification yielded a fraction that was finally identified as an activated blood coagulation factor fXIa using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS). The fraction almost completely suppressed coughs induced by either chemical or mechanical stimulation applied to larynx or bifurcation of guinea-pig trachea. Cough suppressing effect of the fraction and commercially available fXIa were one million times stronger than codeine and codeine only partially suppressed the mechanically triggered coughing in animal model. Recent reviews highlighted prominent shortcomings of current available antitussives, including narcotic opioids such as codeine and their unpleasant or intolerable side effects. Therefore, safer and more effective cough suppressants would be welcome, and present findings indicate that fXIa in human plasma as a very promising, new therapeutic candidate for effective antitussive action.

Cloperastine rescues impairment of passive avoidance response in mice prenatally exposed to diethylstilbestrol.

We previously reported that prenatal exposure to diethylstilbestrol (DES) impaired passive avoidance responses in mice. Apart from the above, we also found that cloperastine, a centrally acting antitussive, ameliorated depression-like and anxiety-like behaviors in rodents at antitussive-effective doses. In this study, we investigated whether or not cloperastine rescues impairment of passive avoidance responses in mice prenatally exposed to DES. Male DES-exposed mice were subcutaneously administered cloperastine at 10 or 30 mg/kg twice a day from 32 to 41 days after birth and subjected to behavioral testing 42 to 46 days after birth. Cloperastine at 10 and 30 mg/kg ameliorated DES-induced impairment of passive avoidance responses. In addition, cloperastine affected the levels of 5-HT1A receptors, GIRK and BDNF in the hippocampus of DES-exposed mice. However, the number of BrdU-positive cells in the hippocampus of DES-exposed mice was not changed by chronic administration of cloperastine. These findings suggest that the action of endocrine disruptors in the brain may not always be irreversible, and that the symptoms caused by endocrine disruptors might be curable with drugs such as cloperastine.

Effect of tipepidine with novel antidepressant-like action on c-fos-like protein expression in rat brain.

We previously reported that tipepidine, a centrally acting non-narcotic antitussive, has an antidepressant-like effect in normal and imipramine treatment-resistant depression model rats. Recently, mapping the induction of c-fos-like immunoreactivity (FLI) in the rat brain showed FLI-positive neurons in several brain areas after acute administration of different classes of antidepressants. Here, the effect of a single injection of an antidepressive dose of tipepidine on FLI was studied in seven areas of the rat brain including the central nucleus of the amygdala (CeA) and the nucleus accumbens (NAc). Desipramine was also used for comparison. Rats were anesthetized and perfused 2h after injection with tipepidine (20 and 40mg/kg, i.p.), desipramine (10mg/kg, i.p.), or saline. Then, immunostaining of FLI-positive neurons in brain slices was performed with conventional methods. A single injection of tipepidine increased FLI-positive neurons in the CeA, similar to preexisting antidepressants, and induced the characteristic pattern of an increase in FLI-positive neurons in six other brain areas including the NAc, an effect that was different from other antidepressants. In addition, a single injection of desipramine (10mg/kg) or tipepidine (20mg/kg) decreased the immobility time in the forced swimming test to a similar extent. The results obtained from the previous behavioral study and the current immunohistochemical study suggest that tipepidine may be a novel antidepressant.

Peripherally injected linalool and bergamot essential oil attenuate mechanical allodynia via inhibiting spinal ERK phosphorylation.

Bergamot essential oil (BEO) is one of the most common essential oil containing linalool and linalyl acetate as major volatile components. This study investigated the effect of intraplantar (i.pl.) bergamot essential oil (BEO) or linalool on neuropathic hypersensitivity induced by partial sciatic nerve ligation (PSNL) in mice. The i.pl. injection of BEO or linalool into the ipsilateral hindpaw to PSNL reduced PSNL-induced mechanical allodynia in a dose-dependent manner. Peripheral (i.pl.) injection of BEO or linalool into the contralateral hindpaw did not yield anti-allodynic effects, suggesting a local anti-mechanical allodynic effect of BEO or linalool in PSNL mice. Anti-mechanical hypersensitivity of morphine was enhanced by the combined injection of BEO or linalool at an ineffective dose when injected alone. We also examined the possible involvement of spinal extracellular signal-regulated protein kinase (ERK) in BEO or linalool-induced anti-mechanical allodynia. In western blotting analysis, i.pl. injection of BEO or linalool resulted in a significant blockade of spinal ERK activation induced by PSNL. These results suggest that i.pl. injection of BEO or linalool may reduce PSNL-induced mechanical allodynia followed by decreasing spinal ERK activation.

Aminophylline, administered at usual doses for rodents in pharmacological studies, induces hippocampal neuronal cell injury under low tidal volume hypoxic conditions in guinea-pigs.

OBJECTIVES: To establish whether aminophylline, administered at usual doses for rodents in pharmacological studies, induces brain injury in systemic hypoxaemia in guinea-pigs. METHODS: A hypoxaemia (partial oxygen tension of arterial blood (PaO2) = 40-60 mmHg) model was developed by low tidal volume mechanical ventilation in guinea-pigs. KEY FINDINGS: Under hypoxic conditions, aminophylline significantly increased the concentration of brain-specific creatine kinase in the serum in a dose- and time-dependent manner. A reduced number of hippocampal neuronal cells in the CA1 region, an increase in the concentration of neuron-specific enolase (NSE) in cerebrospinal fluid (CSF), an increase in lipid hydroperoxides and a decrease in the ratio of glutathione to glutathione disulfide in the brain tissues were also observed. These effects were not observed when aminophylline at the same doses was administered under normoxic conditions (PaO2 = 80-100 mmHg). There was no difference in either serum or CSF concentrations of theophylline between normoxic and hypoxic conditions. Another methylxanthine, caffeine, did not increase the concentration of NSE in CSF. CONCLUSIONS: Aminophylline potentially induces brain damage under hypoxic conditions. We suggest that aminophylline treatment has adverse effects in patients with hypoxaemia subsequent to respiratory disorders such as asthma.

Multiple pharmacological actions of centrally acting antitussives--Do they target G protein-coupled inwardly rectifying K⁺ (GIRK) channels?

Antitussive drugs have been used for decades and their modes of action are well elucidated. However, recent studies on the mechanism of their antitussive action seem to be opening a new way for discovery or development of novel drugs for intractable brain diseases including psychiatric disorders. Antitussives inhibit the currents caused by activation of G protein-coupled inwardly rectifying K⁺ (GIRK) channels in neurons. In our own studies carried out so far, we found that antitussives possessing an inhibitory action on GIRK channels, similar to the effects of an enriched environment, ameliorate symptoms of intractable brain diseases in various animal models. In this review, the multiple pharmacological actions of the antitussives are described, and their mechanisms are discussed addressing GIRK channels as a possible molecular target.

Pharmacological mechanisms of antidepressant-like effect of tipepidine in the forced swimming test.

We previously reported that the centrally acting non-narcotic antitussive, tipepidine, produces a novel antidepressant-like effect in the forced swimming test in rats, but the mechanism of the antidepressant-like effect of tipepidine is not clear. We investigated the pharmacological mechanism of the antidepressant-like effect of tipepidine in the forced swimming test in rats. A catecholamine-depleting agent, alpha-methyl-p-tyrosine (AMPT; 300 mg/kg, s.c.), was given 6h before the first injection and with the last injection of tipepidine (40 mg/kg, i.p.). A serotonin (5-HT)-depleting agent, p-chlorophenylalanine (PCPA; 350 mg/kg, i.p.), was given 72 h and 48 h before the pretest session. The dopamine D(1) receptor antagonist, SCH23390 (0.02 mg/kg, s.c.) was given 15min before each of the three injections of tipepidine. The dopamine D(2) receptor antagonist raclopride (0.2mg/kg, s.c.), the alpha 1 adrenoceptor antagonist prazosin (1mg/kg, i.p.), the alpha 2 adrenoceptor antagonist yohimbine (2mg/kg, i.p.) and the beta adrenoceptor antagonist propranolol (2mg/kg, i.p.) were given 30 min before each of the three injections of tipepidine. AMPT, but not PCPA, significantly inhibited the immobility time-reducing effect of tipepidine in the forced swimming test. Furthermore, the effect of tipepidine was significantly inhibited by SCH23390 and yohimbine. However, raclopride, prazosin, and propranolol failed to block the effect of tipepidine. The results suggest that the antidepressant-like effect of tipepidine in the forced swimming test may be due at least in part to the effects of dopamine and noradrenaline released at the dopamine D(1) receptor and alpha 2 adrenoceptor, respectively.

The effect of aging and an ovariectomy operation on the level of phosphorylated CaM kinase II in the hippocampus of female mice prenatally exposed to diethylstilbestrol.

The effects of aging and an ovariectomy operation on the brain-disrupting actions caused by prenatal exposure to diethylstilbestrol (DES) were studied in mice. In the young DES-exposed female mice, the level of hippocampal phosphorylated CaM kinase II (pCaMKII) was not changed. However, at 8 months, the level of hippocampal pCaMKII in the DES-exposed female mice significantly increased compared to control. Moreover, the ovariectomy significantly increased the level of pCaMKII in the hippocampus but not the cortex of DES-exposed female mice. These findings suggest that the influence of prenatally-exposed DES on the hippocampal pCaMKII may be affected by the endogenous female sex hormones such as estrogen.

[Is the GIRK channel a possible target in the development of a novel therapeutic drug of urinary disturbance?].

Clinically, both overactive bladder (OAB) and dysuria are known to occur in patients with cerebral infarction (CI). A few anticholinergic drugs are used to treat OAB in such patients, although the effect is not satisfactory. On the other hand, little or no therapeutic drug is available for dysuria after CI. We previously reported that dextromethorphan (DM) and cloperastine (CP), centrally acting antitussives, reduce the frequency of micturition reflex and increase the threshold pressure in anesthetized rats. In this article, we describe the effects of DM and CP on urinary disturbances at 24 h after CI, induced by occlusion of the left middle cerebral artery in conscious rats. We also briefly review the structure, function, and distribution of G-protein-coupled inwardly rectifying K(+) (GIRK) channels in the brain, since both drugs have potent inhibitory effect on GIRK channel-activated currents in brain neurons. Of the two drugs, CP at antitussive-effective doses ameliorated both OAB and dysuria 24 h after CI in rats. On the other hand, DM aggravated the dysuria, although it significantly ameliorated the OAB. These results suggest that CP may have some therapeutic value for the treatment of OAB and dysuria after CI. At the present time, mechanisms of the effect of CP are unknown. However, several lines of evidence including pharmacological findings support the idea that the effects of CP may be produced at least partly by an increase in the level of 5-HT in the brain through an inhibitory effect on GIRK channel-activating currents.