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. 2018 Jul 24;24(12):1231–1240. doi: 10.1111/cns.13033

2‐(4‐methyl‐thiazol‐5‐yl) ethyl nitrate maleate‐potentiated GABAA receptor response in hippocampal neurons

Xiao‐Mei Jiang 1, Wei‐Ping Wang 1, Zhi‐Hui Liu 1, Hua‐Jing Yin 1, Hao Ma 1, Nan Feng 1, Ling Wang 1, Hai‐Hong Huang 1, Xiao‐Liang Wang 1,✉
PMCID: PMC6489840  PMID: 30039924

Summary

Aims

2‐(4‐methyl‐thiazol‐5‐yl) ethyl nitrate maleate (NMZM), a derivative of clomethiazole (CMZ), had been investigated for the treatment of Alzheimer's disease (AD). The beneficial effects of NMZM in AD included reversing cognitive deficit, improving learning and memory as well as neuroprotection. The pharmacological effects of NMZM on GABAA receptors were reported previously; however, the mechanisms were unclear and were explored therefore.

Results

In this study, we demonstrated that NMZM improved learning and memory by alleviating scopolamine‐induced long‐term potentiation (LTP) suppression in the dentate gyrus of rats, indicating that NMZM had protective effects against scopolamine‐induced depression of LTP. Next, we investigated the action of NMZM on GABAA receptors in hippocampal neurons and the binding site of NMZM on GABAA receptors. NMZM directly activated GABAA receptors in hippocampal neurons in a weak manner. However, NMZM could potentiate the response of GABAA receptors to GABA and NMZM positively modulated GABAA receptors with an EC50 value of 465 μmol/L at 3 μmol/L GABA while this potentiation at low concentration of GABA (1, 3 μmol/L) was more significant than that at high concentration (10, 30 μmol/L). In addition, NMZM could enhance GABA currents after using diazepam and pentobarbital, the positive modulators of GABAA receptors. NMZM could not affect the etomidate‐potentiated GABAA current. It suggested that the binding site of NMZM on GABAA receptors is the same as etomidate.

Conclusions

These results provided support for the neuroprotective effect of NMZM, which was partly dependent on the potentiation of GABAA receptors. The etomidate binding site might be a new target for neuronal protection and for drug development.

Keywords: 2‐(4‐methyl‐thiazol‐5‐yl) ethyl nitrate maleate, clomethiazole, GABAA receptors, long‐term potentiation, neuroprotection

1. INTRODUCTION

GABAA receptor agonists are used to protect neuronal injury and ameliorate or reverse age‐related cognitive deficits and are suggested as a new therapeutic strategy recently.1, 2 In recent decades, it has been proposed that imbalance between excitatory and inhibitory neurotransmission systems might be a major cause of neurodegenerative disease. Therapeutic strategies aim to regulate excitatory and/or inhibitory neurotransmission and thereby to reestablish the balance between both systems has been demonstrated to improve AD symptoms.3, 4 Glutamate excitotoxicity is one of the most extensively explored mechanisms of neuron loss or cognitive deficit and plays an important role in AD. Currently, one of FDA‐approved pharmacological options for treatment of severe AD is NMDA antagonist memantine. It might prevent excitotoxicity by acting as a non‐competitive antagonist for NMDA receptors activated by glutamate in vivo. 5 On the other hand, enhancement of inhibitory neurotransmission by GABAA receptor agonists seems to be a new approach for treating AD. Several previous studies have indicated that selective GABAA receptor agonists are capable of protecting neurons from Aβ‐induced neurotoxicity in various brain regions in rodents.6, 7 Since neuroprotective effects of GABA modulators could be blocked by GABAA receptor antagonists, revealing that these neuroprotective effects were mediated by GABAA receptor activation or positive modulation8 may provide a new strategy for AD treatment.

2‐(4‐methyl‐thiazol‐5‐yl) ethyl nitrate (NMZ) is one of the derivatives of chlormethiazole (CMZ), which showed neuroprotective activity in rodent models of cerebral ischemia9 and in non‐human primates after focal cerebral ischemia10; however, the clinical trial for stroke was halted in phase III due to the failure of attaining the primary end point of improvement in the general population.11, 12 Previous studies revealed that NMZ has neuroprotective effects on Aβ, glutamate, or oxygen‐glucose deprivation (OGD)‐induced neuron injury in vitro, which could be antagonized by GABAA receptor antagonist bicuculline,13 an allosteric inhibitor of channel opening of the GABAA receptor. This result indicated that the neuroprotective effect of NMZ was mediated by the GABAA receptor. In vivo, NMZ has been shown to reverse the cognitive impairment induced by many chemical lesions14 and to improve the learning and memory in rats with cognitive deficits in the cholinergic systems of the basal forebrain15 and in APP/PS1 mice.13 NMZ‐treated 3 × Tg mice exhibited long‐term potentiation (LTP) improvement via NO/cGMP, cognitive deficits reversing.16 NMZ belongs to a series of NO‐releasing hybrid agents and it is believed to be a novel therapeutic compound to treat Alzheimer's disease (AD). Though NO/sGC/cGMP/CREB pathway is expected to be one of the primary mechanisms for its cognitive‐enabling, learning and memory improving, and neuroprotective effects,17 GABAA receptors may also be involved in the neuroprotective action of NMZ as described above.

NMZM (Figure 1) is a maleate salt of NMZ. Recently, NMZM was synthesized to improve its drugability by improving its solubility and absorption. We supposed that NMZM might also have the ability to play its neuroprotective effect by acting on the GABAA receptor. However, the electrophysiological mechanisms of NMZ/NMZM to improve the capability of learning and memory through GABAA receptors remain to be elucidated. In addition, the binding site of NMZM to act (direct or indirect) on GABAA receptors is also needed to be clarified. In the present study, we used scopolamine‐injured rat model and in vivo observed the LTP changes after treatment of NMZM. To record GABAA receptor‐mediated currents in native hippocampal neurons, patch‐clamp whole cell recording techniques were used. Due to the existence of multi‐subunits in the GABAA receptor, it is important to elucidate the binding site of NMZM at the receptor. Diazepam, pentobarbital, and etomidate (an anesthetic agent) are the most important drugs that act on GABAA receptors. Therefore, the role of the three main binding sites in regulating GABAA receptor‐mediated ion channels and the interaction between the agonists/antagonists with NMZM were studied. The modulation of NMZM on activity of GABAA was also investigated.

Figure 1.

Figure 1

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Chemical structure of 2‐(4‐methyl‐thiazol‐5‐yl) ethyl nitrate maleate (NMZM)

2. MATERIALS AND METHODS

2.1. Animals

Postnatal 12 hours, Wistar rats were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animal care and experiments were carried out in accordance with international and institutional guidelines for animal care and approved by Animal Care Committee of Peking Union Medical College and the Chinese Academy of Medical Sciences. All efforts were made to minimize suffering.

2.2. Drugs and reagents

NMZM was kindly provided by Professor Haihong Huang from the Department of Medical Synthetic Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences (CAMS). All chemicals were purchased from Sigma‐Aldrich (St. Louis, MO), unless otherwise specified. DMEM/F‐12, fetal bovine serum (FBS), horse serum, Neurobasal medium, B27 supplement, GlutaMAX supplement, and trypsin were purchased from Invitrogen (Carlsbad, CA).

2.3. LTP recordings in vivo

Long‐term potentiation recording was conducted as previously described with slight modification.18 The rats were anesthetized and then placed in a stereotaxic frame. A stainless bipolar stimulating electrode was inserted in the perforant path (7.5 mm posterior to bregma, 4.2 mm lateral to the midline, 2.8‐3.5 mm ventral). A recording electrode was placed in the dentate gyrus (3.8 mm posterior to bregma, 2.0 mm lateral to the midline, 3.0‐3.5 mm ventral). Silver wire was fixed and used as reference and ground. The population spike (PS) was achieved from the dentate gyrus in response to stimulation in the perforant path at a frequency of 0.033 Hz with single constant current pulse (100 seconds in duration). The PS was recorded and amplified by TDT RA16PA amplifier and digitized by TDT RX7‐5 processor (Tucker‐Davis Technologies, USA). The electrodes were adjusted until the maximal response was observed. The intensity of the test stimuli was adjusted until it evoked about 40% of the maximum response of PS amplitude. LTP was induced by high frequency stimuli (HFS) delivered at 100 Hz, 10 stimuli, repeated 10 times at an interval of 300 ms Stable baseline PS responses were recorded for at least 15‐20 minutes prior to drug applications. Scopolamine (1 mg/kg) or vehicle or drugs were injected intraperitoneally.

2.4. Isolation and culture of primary hippocampal neurons

Primary cultures of hippocampal neurons were prepared from postnatal rat brains as described with slight modification.19 Briefly, 12 hours Wistar rat pups were decapitated and the hipposcampal tissues were dissected. Hippocampal neuron dissociation was carried out by mincing and adding trypsin (0.125%) for digestion for 15 minutes at 37°C. After the digestion was terminated by adding FBS to the culture medium, a single hippocampal cell could be harvested by gentle passage through a flame‐polished Pasteur pipette in the culture medium. Cells were then passed through a strainer (40‐μm pore size) and rinsed once in culture medium (DMEM/F‐12 medium with 10% FBS, 10% HS, and penicillin/streptomycin). Neurons were plated at a density of 2‐3 × 105 on poly‐D‐lysine‐coated plates. After 4‐hours culture for adherence, culture medium was placed by the final culture medium (neurobasal medium with additions of B27 supplement, GlutaMAX supplement, and penicillin/streptomycin). Cytosine arabinoside was added to the medium at the final concentration of 10 μmol/L for another 24 hours after 48 hours.

2.5. Electrophysiological procedures

Cover slips were placed in a recording chamber on a Nikon TE2000‐U inverted microscope and superfused continuously with an external solution at a rate of 1 mL/min at room temperature (23‐25°C). The external solution consisted of (in mmol/L): NaCl 150, KCl 5, CaCl2 2, MgCl2 1, HEPES 10, glucose 10, pH 7.4 with NaOH. Patch‐pipettes of borosilicate glass (VitalSense Scientific Instruments Co., Ltd. Wuhan, China) were pulled by a puller (Flaming/Brown; P‐97; Sutter Instrument, Novato, CA) to a tip resistance of 4‐6 MΩ for whole cell recordings. The pipette solution was composed of (in mmol/L): NaCl 10, CaCl2 1, EGTA 10, K‐gluconate 140, HEPES 10, ATP‐Mg 5, GTP‐Na2 0.2, pH 7.2 adjusted with KOH. High ATP and EGTA were included in the intracellular solution to prevent current rundown during a prolonged period of whole cell recording.20 The liquid junction potential (V LJ) between the external solution and pipette solution was calculated according to previous reports,21 which was determined to be 8.8 mV. Standard whole cell recordings were performed using an EPC‐10 patch‐clamp amplifier (HEKA Electronik, Lambrecht, Germany) linked to an IBM‐compatible computer driven by HEKA software. The current signals were filtered at 3 kHz with a low pass‐filter and sampled at 20 kHz. For acquisition and analysis, pulse 8.64 software was used. The holding potential for cells was −70 mV unless otherwise stated. When GABAA receptor was activated directly, GABA or drug was applied at a position about 7‐8 cells away from the recording cell by pressure ejection (0‐4 psi) from modified patch pipettes (tip size 20 μmol/L) for 2 seconds to directly activate the GABAA receptor; the drug‐elicited current amplitude was measured as the GABAA receptor response. When potentiation action on the GABAA receptor was investigated, drugs were administrated by superfusion while specific concentration of GABA was applied by pressure ejection to activate the GABAA receptor; the potentiation action was expected to be an increment of the current amplitude. The ejection of GABA or drug was performed every 2 minutes to make sure both thorough washout of GABA and recovery of receptors from desensitization. Once the control GABAA receptor response was stably determined, the effect of drugs on the response was examined.

2.6. Data analysis

The drug‐elicited current amplitude was measured as the GABAA receptor response. The potentiation effect was determined by normalizing the GABAA receptor response in the presence of drugs to the GABAA receptor response in the absence of drugs. Normalization was calculated by the following equation: % potentiation = (peak current in the presence of drugs/peak current in the absence of drugs) × 100. Data are expressed as mean ± SEM. Concentration‐response/potentiation data were fitted to a logistic equation (Origin pro7.5). EC50 is the concentration of drug inducing a half‐maximal response/potentiation unless otherwise specified; statistical analysis was performed using Student's t test.

3. RESULTS

3.1. NMZM enhanced LTP in scopolamine rat model

Long‐term potentiation (LTP) recording is a widely documented method for investigating the synaptic basis of learning and memory and it is induced by high frequency stimuli (HFS, described before). NMZM were given ip 40 minutes before HFS. NMZM had no significant effect on the LTP of normal control rats (1 mg/kg: 188% ± 14% vs control group: 189% ± 21%, P > 0.05, n = 4; 3 mg/kg: 207% ± 21% vs control group: 189% ± 21%, P > 0.05, n = 4; Figure 2A,B). Scopolamine‐mediated suppression of LTP was significantly alleviated by NMZM 1 mg/kg and 3 mg/kg (183% ± 10% vs scop. group 153% ± 6%, P < 0.05, and 211% ± 14% vs scop. group 153% ± 6%, P < 0.01, respectively). These results indicated that NMZM had protective effects against scopolamine‐induced damage of LTP (Figure 2C,D).

Figure 2.

Figure 2

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Effects of NMZM on the suppression of LTP induced by scopolamine (1 mg/kg, scop.) in the dentate gyrus of rats. A and B, After baseline recording for 15 min, rats were injected intraperitoneally (ip) with NMZM (1, 3 mg/kg) or vehicle. A series of high frequency stimulation (HFS) was given 40 min later. NMZM had no significant effect on the induction of LTP (1 mg/kg: 188% ± 14% vs control group 189% ± 21%, P > 0.05, n = 4; 3 mg/kg: 207% ± 21% vs control group 189% ± 21%, P > 0.05, n = 4). C and D, After baseline recording for 15 min, rats were injected intraperitoneally (ip) with NMZM (1, 3 mg/kg) or vehicle followed by injection of scopolamine (1 mg/kg) or vehicle after 10 min; the control group was given vehicle intraperitoneally only. A series of high frequency stimulation (HFS) was given 30 min later. Scopolamine‐mediated suppression of LTP was significantly alleviated by NMZM (1 mg/kg: 183% ± 10% vs scop. group 153% ± 6%, P < 0.05, n = 7. 3 mg/kg: 211% ± 14% vs scop. group 153% ± 6%, P < 0.01, n = 6.). The difference of PS amplitude at 55‐60 min between groups was detected by one‐way ANOVA, # P < 0.05 vs control group, * P < 0.05 vs scop. group

3.2. Activation of GABAA receptor by NMZM in hippocampal neurons

GABA at concentrations ranging from 1 to 100 μmol/L was applied to hippocampal neurons; the inward chloride currents were measured as response of GABAA receptor to GABA (Figure 3A). The inward currents were inhibited by GABAA receptor antagonist bicuculline (10 μmol/L, Figure 3B). To examine if NMZM could directly activate GABAA receptor, NMZM was administrated in the absence and presence of bicuculline. We found that NMZM had a very weak action on the direct activation of GABAA receptor till the concentration reached 1 mmol/L (Figure 3C). The response of GABAA current to NMZM was about 59 ± 7 pA (n = 4) and it was almost completely inhibited by bicuculline 10 μmol/L, indicating a week activation of NMZM on GABAA receptors.

Figure 3.

Figure 3

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NMZM directly activated GABAA receptor in hippocampal neurons in a weak manner. A, Currents induced by application of GABA (1‐100 μmol/L); The holding potential was −70 mV. B, Current induced by application of GABA (10 μmol/L). C, NMZM (1 mmol/L) induced current was inhibited by bicuculline (10 μmol/L). Data were obtained from 4 neurons

3.3. NMZM enhanced the currents activated by GABA in hippocampal neurons

To observe the action of NMZM in modulating GABAA receptor‐mediated chloride currents, NMZM was applied to hippocampal neurons in the presence of GABA. NMZM showed a concentration‐dependent enhancing effect on the current induced by 3 μmol/L GABA. NMZM potentiated this current by 166 + 10% and 291 + 22% at concentrations of 0.3 and 1 mmol/L, respectively (Figure 4A). Figure 4B indicated GABAA receptor‐mediated chloride current amplitude in the presence of NMZM (0.3 and 1 mmol/L), suggesting a direct potentiation of NMZM on GABAA receptor‐mediated chloride currents. Figure 4C exhibited that NMZM had a more significant potentiation on the current evoked by low concentrations of GABA (1 and 3 μmol/L) compared to that by higher concentrations of GABA (10 and 30 μmol/L). The EC50 for the enhancing effect of NMZM on 3 μmol/L GABA‐evoked current was determined to be 465 μmol/L (Figure 4C). These results suggested that NMZM potentiated the currents activated by GABA in hippocampal neurons.

Figure 4.

Figure 4

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NMZM positively modulated GABAA receptor‐mediated current in hippocampal neurons. A, Representative traces of the responses to the application of 3 μmol/L GABA in the absence or presence of NMZM 300 and 1000 μmol/L. The holding potential was −70 mV. B, GABAA receptor‐mediated current amplitude in the presence of NMZM (300 and 1000 μmol/L; n = 4‐8). C, GABA‐evoked current potentiation by NMZM was normalized to the corresponding concentration of GABA‐evoked response. GABA‐evoked current potentiation by NMZM showed that low concentration (1 μmol/L in hollow circle from 6 to 8 neurons and 3 μmol/L in solid circle from 5 to 7 neurons) GABA‐evoked current potentiation by NMZM was more significant than high concentration (10 μmol/L in hollow triangle from four neurons and 30 μmol/L in solid triangle from 4 to 6 neurons) GABA‐evoked current potentiation by NMZM using t test analysis. D, Dose‐response curve for 3 μmol/L GABA‐evoked current potentiation by NMZM. Data normalized to the 3 μmol/L GABA response. The EC50 for the enhancing effect of NMZM on 3 μmol/L GABA‐evoked current is determined to be 465 μmol/L. Data were obtained from 5 to 9 neurons

3.4. Flumazenil, a diazepam binding site antagonist, affected NMZM‐potentiated GABAA currents

The above study confirmed that the GABAA‐mediated current was potentiated by NMZM. However, it remains to be studied if a classic antagonist of GABAA receptor inhibits the effects of NMZM. Flumazenil (flu) is a well‐known antagonist of diazepam (DZP) which is one of the classical agents of benzodiazepines and it has a specific binding site on the GABAA receptor. Flumazenil 10 μmol/L inhibited diazepam (100 nmol/L)‐potentiated GABAA currents (3 μmol/L GABA‐evoked current, Figure 5A,B), but it had no effect on NMZM (500 μmol/L)‐potentiated GABAA current. These results suggested that the binding site of NMZM on GABA receptor was not the same as the binding site of benzodiazepines. Therefore, the binding site of NMZM on GABAA receptor needs to be further studied.

Figure 5.

Figure 5

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Potentiation of the GABA response to GABAA receptor in hippocampal neurons by NMZM was not inhibited by flumazenil(flu). A and B, Potentiation of the GABA response to GABAA receptor by diazepam(DZP) was inhibited by flu. Data were obtained from 4 neurons. # P < 0.05 compared with 3 μmol/L GABA response to GABAA receptor. * P < 0.05 compared with potentiation of 3 μmol/L GABA response to GABAA receptor by DZP. C and D, Potentiation of the GABA response to GABAA receptor by NMZM was not inhibited by flu in the case that flu was added after potentiation occurred. Data were obtained from 6 neurons. # P < 0.05 compared with 3 μmol/L GABA response to GABAA receptor. E and F, Potentiation of the GABA response to GABAA receptor by NMZM was not inhibited by flu in the case that flu was added before potentiation occurred. Data were obtained from 6 neurons. *P < 0.05 compared with 3 μmol/L GABA response to GABAA receptor in the presence of flu

3.5. Interaction of NMZM with well‐known positive allosteric modulators on binding site of GABAA receptor

3.5.1. Diazepam (benzodiazepine site)

As a positive modulator of the benzodiazepine site, diazepam was used to further investigate the interaction between NMZM and diazepam in potentiation of GABAA current through the diazepam binding site. In spite of flumazenil having no effect on the potentiation of the GABAA receptor, we explored whether NMZM and diazepam acted via a common site on the GABAA receptor. Firstly, we examined the potentiation of GABA response to GABAA receptor by diazepam. NMZM exhibited a concentration‐dependent enhancing effect on membrane current induced by 3 μmol/L GABA (Figure 6A). The EC50 for the enhancing effect of diazepam on 3 μmol/L GABA‐evoked current was determined to be 97 nmol/L (Figure 6B). Our results demonstrated that the modest positive modulation of GABAA receptor by diazepam (100 nmol/L) was further potentiated by NMZM (1 mmol/L, Figure 6C,D). Therefore, we suggested that the binding site of NMZM was not the same as diazepam on GABAA receptors.

Figure 6.

Figure 6

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Diazepam (DZP) did not block the modulatory effect of NMZM on GABAA receptor. A, Representative traces illustrated the enhancing effect of DZP on 3 μmol/L GABA‐evoked current; the holding potential was −70 mV. B, dose‐response curve for 3 μmol/L GABA‐evoked current potentiation by diazepam. Data were normalized to the 3 μmol/L GABA response. The EC50 for the enhancing effect of diazepam on 3 μmol/L GABA‐evoked current is determined to be 97 nmol/L. Data were obtained from 4 to 6 neurons. C, Representative traces illustrated the enhancing effect of DZP on 3 μmol/L GABA‐evoked current in the absence or presence of NMZM. D, Potentiation of GABA response to GABAA receptor by DZP was further enhanced by NMZM. Data were obtained from 9 neurons. # P < 0.05 compared with 3 μmol/L GABA response to GABAA receptor. *P < 0.05 compared with potentiation of 3 μmol/L GABA response to GABAA receptor by DZP

3.5.2. Etomidate (etomidate site)

Since NMZM did not act through the same binding site as diazepam on GABAA receptors, next we explored other binding sites, in which one of them was the etomidate site. Potentiation of the GABAA receptor by etomidate is influenced by the transformation of an amino acid residue located within the TM2 domain of the β‐subunit.22 By exploring whether the potentiation of GABAA receptor by NMZM is influenced by etomidate, we estimated that the binding site on the GABAA receptor of NMZM is similar to that of etomidate. Firstly, we examined the potentiation of GABA response to GABAA receptor by etomidate. Etomidate exhibited a concentration‐dependent enhancing effect on membrane current induced by 3 μmol/L GABA (Figure 7A). The EC50 for the enhancing effect of etomidate on 3 μmol/L GABA‐evoked current was determined to be 1.2 μmol/L (Figure 7B). Our results demonstrated that the modest positive modulation of GABAA receptor by etomidate (1 μmol/L) was not further potentiated when combined with NMZM (1000 μmol/L; Figure 7C,D); it suggested that etomidate had no effect on NMZM‐induced modulation of the GABAA receptor. In this case, we suggested that the binding site of NMZM was the same as etomidate on GABAA receptors.

Figure 7.

Figure 7

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Etomidate (eto) influenced the GABAA receptor modulatory effect of NMZM. A, Representative traces illustrated the enhancing effect of eto on 3 μmol/L GABA‐evoked current. The holding potential was −70 mV. B, Dose‐response curve for 3 μmol/L GABA‐evoked current potentiation by eto. Data were normalized to the 3 μmol/L GABA response. The EC50 for the enhancing effect of eto on 3 μmol/L GABA‐evoked current is determined to be 1.2 μmol/L. Data were obtained from 4 to 6 neurons. C, Representative traces illustrated the enhancing effect of eto on 3 μmol/L GABA‐evoked current in the absence or presence of NMZM. D, Potentiation of GABA response to GABAA receptor by eto was not further enhanced by NMZM. Data were obtained from 6 neurons. # P < 0.05 compared with 3 μmol/L GABA response to GABAA receptor

3.5.3. Barbiturate (Pentobarbital site)

Previous studies have identified that β‐subunit is a key subunit in the potentiation of GABAA receptor by pentobarbital.23, 24, 25 Similarly, we estimated the possible binding site of NMZM on GABAA receptor and whether potentiation of GABAA receptor by NMZM was influenced by pentobarbital. It was observed that pentobarbital exhibited a concentration‐dependent enhancing effect on membrane current induced by 3 μmol/L GABA (Figure 8A). The EC50 for the enhancing effect of pentobarbital on 3 μmol/L GABA‐evoked current is determined to be 25 μmol/L (Figure 8B). Our results demonstrated that the modest positive modulation of GABAA receptor by pentobarbital (30 μmol/L) was further potentiated by NMZM (1000 μmol/L; Figure 8C,D). Therefore, pentobarbital did not block the effect of NMZM on GABA‐induced GABAA receptor activation. According to this result, we suggested that the binding site of NMZM was not the same as pentobarbital, which is at the barbiturate site.

Figure 8.

Figure 8

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Pentobarbital (PB) did not block the GABAA receptor modulatory effect of NMZM. A, Representative traces illustrated the enhancing effect of PB on 3 μmol/L GABA‐evoked current. The holding potential was −70 mV. B, Dose‐response curve for 3 μmol/L GABA‐evoked current potentiation by PB. Data were normalized to the 3 μmol/L GABA response. The EC50 for the enhancing effect of PB on 3 μmol/L GABA‐evoked current was determined to be 25 μmol/L. Data were obtained from 3 to 4 neurons. C, Representative traces illustrated the enhancing effect of PB on 3 μmol/L GABA‐evoked current in the absence or presence of NMZM. D, Potentiation of GABA response to GABAA receptor by PB was further enhanced by NMZM. # P < 0.05 compared with 3 μmol/L GABA response to GABAA receptor. *P < 0.05 compared with potentiation of 3 μmol/L GABA response to GABAA receptor by PB

4. DISCUSSION

Since glutamate‐induced excitotoxicity in AD is well acknowledged, GABAA receptors' dysfunction is also shown in AD; activating or potentiating GABAA receptors seems to be more and more important as a potential therapy for AD. CMZ ameliorated glutamate‐induced excitotoxicity by enhancing the function of GABAA receptor mediated by the inhibitory neurotransmitter, GABA, in the brain.26, 27, 28 It indicated that acting through GABAA receptor potentiation could alleviate or reverse neuronal death by regulating excitatory and/or inhibitory neurotransmission. Excitotoxicity was a cause of neuronal dysfunction and neuron death, with features of over‐activation of glutamate receptors, excessive intracellular calcium, and mitochondrial damage. CMZ as an neuroprotective agent showed a potential therapeutic effect on neuronal injury29 by regulating excitatory and/or inhibitory neurotransmission balance in neurodegenerative diseases, which acts as the GABAA receptor positive allosteric modulator.30, 31, 32 As one of the CMZ derivatives, NMZ also exhibited neuroprotective effect,33 and it was reported to modify the symptoms and condition of AD in several animal models.16

In the present study, we demonstrated that NMZM, the derivative of CMZ, acted as a GABAA receptor positive allosteric modulator, as well as the possible binding site of NMZM on GABAA receptors. We firstly showed that NMZM could prevent scopolamine‐induced LTP deficits. LTP is a commonly used electrophysiological model for evaluating the functions of learning and memory. It indicated that NMZM might improve learning and memory under the condition of cholinergic system dysfunction. Secondly, we found that in the absence of GABA, NMZM might directly activate the GABAA receptor though a very weak effect. This effect of NMZM was blocked by bicuculline, a GABAA receptor antagonist. However, NMZM might potentiate the response of GABAA receptors activated by GABA. The potentiation at low concentrations of GABA (1, 3 μmol/L) was more significant than that at higher concentrations of GABA (10, 30 μmol/L). The enhancing effect of NMZM was probably due to an allosteric action on the GABAA receptor; it is similar to some other positive allosteric modulators such as pentobarbital, diazepam, etomidate, and neurosteroids. 34, 35 As the above drugs were used frequently in clinic, we compared the effects of NMZM on GABAA receptor with these drugs in order to elucidate the binding site of NMZM on GABAA receptors. We found that the possible binding site of NMZM might be the same as etomidate. NMZM could not enhance etomidate‐induced potentiation of GABA‐activated response of the GABAA receptor, but it could potentiate GABAA currents significantly after diazepam‐ or pentobarbital‐induced enhancement of the current.

Several studies had also revealed the neuroprotective effects of etomidate in different animal models: for example, in experimental spinal cord injury rats36 or in ischemia‐reperfusion model.37, 38 However, these studies did not indicate that the neuroprotective effects were mediated by the GABAA receptor and the relevant ion channel currents, but focused on other research areas, such as antioxidation and so on. Accordingly, positive allosteric modulators which bind to the etomidate binding site may entrust GABAA receptor the ability of neuroprotection. Therefore, it is suggested that etomidate binding sites might be used as a new drug target for the treatment of AD via neuroprotective action.

The GABAA receptors are made up of subunits which form a receptor complex. Humans have 19 receptor subunits including α (1‐6), β (1‐3), and γ (1‐3). The most common complex that includes around 40% of the GABAA receptors is the α1β2γ2 combination.39, 40 Barbiturates bind at a β‐subunit that is distinct from the benzodiazepine binding site.41 Etomidate binds to the β‐subunit.42 Figure 9 illustrated the structure of the GABAA receptor and its associated binding sites, which bind to ligand or allosteric modulators such as benzodiazepines, barbiturates, and etomidate.

Figure 9.

Figure 9

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The structure of five combined subunits of the GABAA receptor and its related binding sites. The binding site of GABA is at the interface between α and β subunits. The benzodiazepine binding site is positioned at the interface between α and γ2 subunits. Etomidate binds to the β‐subunits. Barbiturates, ethanol, and neurosteroids bind to sites in the membrane‐spanning transmembrane regions of the subunits

In summary, NMZM could improve LTP deficits induced by scopolamine, which demonstrated that NMZM had a capability of improving learning and memory. NMZM potentiated GABAA receptor as a positive allosteric modulator, but it is not a direct activator. The binding site of NMZM on GABAA receptor may be the same as etomidate. Our results indicated that the modulation of GABAA receptor is an important mechanism of NMZM in neuroprotection and it might benefit for dementia.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

ACKNOWLEDGMENT

This work was supported by National Major Special Project on New Drug Innovation of China (2018ZX09711001‐004‐004), Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study (BZ0150), and CAMS Initiative for Innovative Medicine (CAMS‐12M‐1‐010).

Jiang X‐M, Wang W‐P, Liu Z‐H, et al. 2‐(4‐methyl‐thiazol‐5‐yl) ethyl nitrate maleate‐potentiated GABAA receptor response in hippocampal neurons. CNS Neurosci Ther. 2018;24:1231–1240. 10.1111/cns.13033

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