Channel Opening Activity Research Articles

Magnolol alleviates pulmonary fibrosis inchronic obstructive pulmonary disease by targeting transient receptor potential vanilloid 4-ankyrin repeat domain.

Transient receptor potential vanilloid 4 (TRPV4) plays a role in regulating pulmonary fibrosis (PF). While several TRPV4 antagonists including magnolol (MAG), have been discovered, the mechanism of action is not fully understood. This study aimed to investigate the effect of MAG on alleviating fibrosis in chronic obstructive pulmonary disease (COPD) based on TRPV4, and to further analyze its mechanism of action on TRPV4. COPD was induced using cigarette smoke and LPS. The therapeutic effect of MAG on COPD-induced fibrosis was evaluated. TRPV4 was identified as the main target protein of MAG using target protein capture with MAG probe and drug affinity response target stability assay. The binding sites of MAG at TRPV4 were analyzed using molecular docking and small molecule interaction with TRPV4-ankyrin repeat domain (ARD). The effects of MAG on TRPV4 membrane distribution and channel activity were analyzed by co-immunoprecipitation, fluorescence co-localization, and living cell assay of calcium levels. By targeting TRPV4-ARD, MAG disrupted the binding between phosphatidylinositol 3 kinase γ and TRPV4, leading to hampered membrane distribution on fibroblasts. Additionally, MAG competitively impaired ATP binding to TRPV4-ARD, inhibiting TRPV4 channel opening activity. MAG effectively blocked the fibrotic process caused by mechanical or inflammatory signals, thus alleviating PF in COPD. Targeting TRPV4-ARD presents a novel treatment strategy for PF in COPD.

Carba Analogues of Flupirtine and Retigabine with Improved Oxidation Resistance and Reduced Risk of Quinoid Metabolite Formation.

The KV7 potassium channel openers flupirtine and retigabine have been valuable options in the therapy of pain and epilepsy. However, as a result of adverse reactions, both drugs are currently no longer in therapeutic use. The flupirtine‐induced liver injury and the retigabine linked tissue discolouration do not appear related at first glance; nevertheless, both events can be attributed to the triaminoaryl scaffold, which is affected by oxidation leading to elusive reactive quinone diimine or azaquinone diimine metabolites. Since the mechanism of action, i. e. KV7 channel opening, seems not to be involved in toxicity, this study aimed to further develop safer replacements for flupirtine and retigabine. In a ligand‐based design strategy, replacing amino substituents of the triaminoaryl core with alkyl substituents led to carba analogues with improved oxidation resistance and negligible risk of quinoid metabolite formation. In addition to these improved safety features, some of the novel analogues exhibited significantly improved KV7.2/3 channel opening activity, indicated by an up to 13‐fold increase in potency and an efficacy of up to 176 % compared to flupirtine, thus being attractive candidates for further development.

Ketamine administration ameliorates anesthesia and surgery-induced cognitive dysfunction via activation of TRPV4 channel opening.

Perioperative neurocognitive disorder (PND) is a common complication associated with anesthesia and surgery in the elderly. The dysfunction of transient receptor potential vanilloid 4 (TRPV4) has been associated with a number of diseases, including Alzheimer's disease. Given that ketamine can reportedly improve PNDs, the present study sought to determine whether ketamine-induced PND alleviation was mediated by activation of TRPV4 channel opening. A total of 120, 20-month-old male C57BL/6 mice were randomly divided into five groups: Vehicle, PND (tibial fracture surgery), PND + ketamine (Ket), PND + Ket + HC-067047 (HC), and PND + HC groups. Ketamine (0.5 mg/kg) was administered intraperitoneally once a day for 3 days after surgery and HC-067047 (1 µmol/2 µl), an antagonist of TRPV4, was administered via the left lateral ventricle 30 min before ketamine treatment. Superoxide dismutase (SOD), malondialdehyde (MDA), lipid peroxidation (LPO), IL-1β, IL-6, adenosine monophosphate-activated protein kinase (AMPK), NF-κB, TNF-α and IFN-β levels were determined 3 days after surgery. At 28 days after surgery, fear conditioning and novel object recognition were assessed, and Aβ1-42 levels were measured and ionized calcium binding adaptor molecule 1 (Iba1) staining was conducted on day 31 after surgery. The results revealed that ketamine administration upregulated total SOD activity, downregulated MDA and LPO content, mitigated phosphorylated (p)-NF-κB, TNF-α mRNA and IFN-β mRNA expression in the hippocampus, and promoted p-AMPK 3 days after surgery. Furthermore, it was found that ketamine increased both context- and tone-dependent fear conditioning, and the time spent exploring a novel object, and reduced Aβ peptide levels and microglial activation 30 days after surgery. Notably, these changes could be reversed by HC-067047 to a certain extent. In conclusion, ketamine improved PND in aged mice after tibial fracture surgery and the potential mechanism may involve activation of the TRPV4/AMPK/NF-κB signaling pathway.

Synthesis and BK channel-opening activity of 2-amino-1,3-thiazole derivatives

A series of 2-amino-5-arylmethyl- or 5-heteroarylmethyl-1,3-thiazole derivatives were synthesized and evaluated for BK channel-opening activities in cell-based fluorescence assay and electrophysiological recording. The assay results indicated that the activities of the investigated compounds were influenced by the physicochemical properties of the substituent at benzene ring.

Fast desensitization of acetylcholine receptors induced by a spider toxin

ABSTRACT Nicotinic acetylcholine receptors (nAChRs) are members of the “cys-loop” ligand-gated ion channel superfamily that play important roles in both the peripheral and central system. At the neuromuscular junction, the endplate current is induced by ACh binding and nAChR activation, and then, the current declines to a small steady state, even though ACh is still bound to the receptors. The kinetics of nAChRs with high affinity for ACh but no measurable ion conductance is called desensitization. This adopted desensitization of nAChR channel currents might be an important mechanism for protecting cells against uncontrolled excitation. This study aimed to show that Grammostola spatulata toxin (GsMTx4), which was first purified and characterized from the venom of the tarantula Grammostola spatulata (now genus Phixotricus), can facilitate the desensitization of nAChRs in murine C2C12 myotubes. To examine the details, muscle-type nAChRs, which are expressed heterologously in HEK293T cells, were studied. A single channel current was recorded under the cell-attached configuration, and the channel activity (NPo) decayed much faster after the addition of GsMTx-4 to the pipette solution. The channel kinetics were further analyzed, and GsMTx-4 affected the channel activity of nAChRs by prolonging the closing time without affecting channel conductance or opening activity. The interaction between nAChRs embedded in the lipid membrane and toxin inserted into the membrane may contribute to the conformational change in the receptor and thus change the channel activity. This new property of GsMTx-4 may lead to a better understanding of the desensitization of ligand-gated channels and disease therapy.

Sulfide Analogues of Flupirtine and Retigabine with Nanomolar KV 7.2/KV 7.3 Channel Opening Activity.

The potassium channel openers flupirtine and retigabine have proven to be valuable analgesics or antiepileptics. Their recent withdrawal due to occasional hepatotoxicity and tissue discoloration, respectively, leaves a therapeutic niche unfilled. Metabolic oxidation of both drugs gives rise to the formation of electrophilic quinones. These elusive, highly reactive metabolites may induce liver injury in the case of flupirtine and blue tissue discoloration after prolonged intake of retigabine. We examined which structural features can be altered to avoid the detrimental oxidation of the aromatic ring and shift oxidation toward the formation of more benign metabolites. Structure-activity relationship studies were performed to evaluate the KV 7.2/3 channel opening activity of 45 derivatives. Sulfide analogues were identified that are devoid of the risk of quinone formation, but possess potent KV 7.2/3 opening activity. For example, flupirtine analogue 3-(3,5-difluorophenyl)-N-(6-(isobutylthio)-2-(pyrrolidin-1-yl)pyridin-3-yl)propanamide (48) has 100-fold enhanced activity (EC50 =1.4 nm), a vastly improved toxicity/activity ratio, and the same efficacy as retigabine in vitro.

Flupirtine Analogues: Explorative Synthesis and Influence of Chemical Structure on KV7.2/KV7.3 Channel Opening Activity.

Neuronal voltage‐gated potassium channels KV7.2/KV7.3 are sensitive to small‐molecule drugs such as flupirtine, even though physiological response occurs in the absence of ligands. Clinically, prolonged use of flupirtine as a pain medication is associated with rare cases of drug‐induced liver injury. Thus, safety concerns prevent a broader use of this non‐opioid and non‐steroidal analgesic in therapeutic areas with unmet medical needs such as hyperactive bladder or neonatal seizures. With the goal of studying influences of chemical structure on activity and toxicity of flupirtine, we explored modifications of the benzylamino bridge and the substitution pattern in both rings of flupirtine. Among twelve derivatives, four novel thioether derivatives showed the desired activity in cellular assays and may serve as leads for safer KV channel openers.

Design, synthesis and evaluation of substituted piperidine based KCNQ openers as novel antiepileptic agents

Epilepsy is a kind of disease with complicated pathogenesis. KCNQ (Kv7) is a voltage dependent potassium channel that is mostly associated with epilepsy and thus becomes an important target in the treatment of epilepsy. In this paper, a series of substituted piperidine derivatives targeting KCNQ were designed and synthesized by using scaffold hopping and active substructure hybridization. Compounds were evaluated by fluorescence-based thallium influx assay, Rb+ flow assay and electrophysiological patch-clamp assay. Results showed that some compounds possessed more potent potassium channel opening activity than Retigabine. More significantly, compound 11 was found to have good pharmacokinetic profiles in vivo.

Synthesis and potassium KV7 channel opening activity of thioether analogues of the analgesic flupirtine.

Flupirtine, an opener of neuronal voltage gated potassium channels (KV7.2/3), has been used as a therapeutic alternative for pain treatment in patients refractory to NSAIDs and opioids. Because flupirtine is associated with rare but fatal drug-induced liver injury that may result from the formation of toxic metabolites upon metabolic oxidation, we synthesized novel derivatives with the goal of identifying equally active and ultimately safer KV7.2/3 channel openers. Four thioether analogues were designed to lack a nitrogen atom that would be a prerequisite for the formation of toxic para-quinone diimines, and form sulfoxide and sulfone metabolites instead. KV7.2/3 channel opening activity and hepatotoxicity data of twelve novel flupirtine analogues, four thioethers and their respective sulfoxide and sulfone metabolites are reported.

Nicorandil inhibits osteoclast differentiation in vitro

Nicorandil is a hybrid angina therapeutic agent that has nitric oxide (NO) action and the ability to open ATP-sensitive K+ channels (KATP channels). A transient increase in NO and intracellular Ca2+ has been demonstrated to be highly involved in the differentiation and activation of osteoclasts. The objective of this study was to verify that the pharmacological effect of nicorandil suppresses the differentiation process of osteoclasts in vitro.Although little authentic NO production was detected in the culture medium in osteoclast formation assays, NO production increased only in the presence of nicorandil. The number of osteoclasts decreased markedly at late time-points after nicorandil addition compared with the number at early time-points. Both the number of TRAP-positive multinucleated cells and the number of cells that obtained F-actin rings decreased in the presence of nicorandil in a concentration-dependent manner. The osteo assay showed that the bone resorption area was also reduced with nicorandil in a concentration-dependent manner. An inhibition recovery experiment was conducted by adding a soluble guanylyl cyclase (sGC) inhibitor (ODQ) and a KATP channel-opening inhibitor (glibenclamide) during the osteoclast formation process. In the inhibition recovery experiment, the inhibitory effect of nicorandil on osteoclastogenesis was blocked by the addition of ODQ and glibenclamide. These results suggest that both the NO and KATP channel-opening activity of nicorandil inhibit osteoclast differentiation. Further study of nicorandil may lead to the development of drugs for osteoporosis treatment.

The synthesis and BK channel-opening activity of N-acylaminoalkyloxime derivatives of dehydroabietic acid.

A series of N-acylaminoalkyloxime derivatives of dehydroabietic acid were synthesized and evaluated for BK channel-opening activities in an assay system of CHO-K1 cells expressing hBKα channels. The structure-activity relationship study revealed that a non-covalent interaction between the S atom of the 2-thiophene and the carbonyl O atom may contribute to conformation restriction for interaction with the ion channel. This research could guide the design and synthesis of novel abietane-based BK channel opener.

Actions of KMUP-1, a xanthine and piperazine derivative, on voltage-gated Na(+) and Ca(2+) -activated K(+) currents in GH3 pituitary tumour cells.

7-[2-[4-(2-Chlorophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) is a xanthine-based derivative. It has soluble GC activation and K(+) -channel opening activity. Effects of this compound on ion currents in pituitary GH3 cells were investigated in this study. The aim of this study was to evaluate effects of KMUP-1 on the amplitude and gating of voltage-gated Na(+) current (INa ) in pituitary GH3 cells and in HEKT293T cells expressing SCN5A. Both the amplitude of Ca(2+) -activated K(+) current and the activity of large-conductance Ca(2+) -activated K(+) (BKCa ) channels were also studied. KMUP-1 depressed the transient and late components of INa with different potencies. The IC50 values required for its inhibitory effect on transient and late INa were 22.5 and 1.8 μM respectively. KMUP-1 (3 μM) shifted the steady-state inactivation of INa to a hyperpolarized potential by -10 mV, despite inability to alter the recovery of INa from inactivation. In cell-attached configuration, KMUP-1 applied to bath increased BKCa -channel activity; however, in inside-out patches, this compound applied to the intracellular surface had no effect on it. It prolonged the latency in the generation of action currents elicited by triangular voltage ramps. Additionally, KMUP-1 decreased the peak INa with a concomitant increase of current inactivation in HEKT293T cells expressing SCN5A. Apart from activating BKCa channels, KMUP-1 preferentially suppresses late INa . The effects of KUMP-1 on ion currents presented here constitute an underlying ionic mechanism of its actions.

WS06.3 Evaluation of novel corrector–potentiator combinations for treating cystic fibrosis in various assays

Defects in CFTR that result in cystic fibrosis can be broadly categorized into three processes: translational, folding/maturation, and functional. To address the underlying causes affecting the folding/maturation and functional properties of CFTR, two biomolecular activities are required, namely correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective opening of the CFTR channel. Combined, these activities allow chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. Previously, we reported the discovery of new corrector molecules via a HTS screen on CFBe41o-cells harboring HRP-tagged F508del CFTR and on U2OS cells ProLink-tagged F508del CFTR (NACFC 2014). Corrector compounds from two distinct series exhibited channel opening activity in primary cells derived from F508del CFTR homozygous patients as measured by trans-epithelial clamp circuit (TECC). Here, we report a series of assays developed to better understand the mechanism of action of the corrector molecules. More specifically, assays using differently tagged F508del CFTR were employed to evaluate the effect of correctors alone and in various combinations with a potentiator on the correction time-dependency and on the cell surface residence time of F508del CFTR. Other characterization endpoints will also be discussed. In summary, we developed several assays to evaluate the impact of corrector/potentiator combinations on the rescue of F508del CFTR, and to understand the contribution of each component in combination cocktails.

Oxidation potentials of N-modified derivatives of the analgesic flupirtine linked to potassium KV 7 channel opening activity but not hepatocyte toxicity.

Openers of neuronal voltage-gated potassium channels (KV ) are of interest as therapeutic agents for treating pain (flupirtine) and epilepsy (retigabine). In an effort to better understand the mechanisms of action and toxicity of flupirtine, we synthesized nine novel analogues with varying redox behavior. Flupirtine can be oxidatively metabolized into azaquinone diimines; thus, the oxidation potentials of flupirtine and its analogues were measured by cyclic voltammetry. KV 7.2/3 (KCNQ2/3) opening activity was determined by an established assay with HEK293 cells overexpressing these channels. A link was found between the oxidation potentials of the compounds and their EC50 values for potassium channel opening activity. On the other hand, no correlation was observed between oxidation potentials and cytotoxicity in cultures of transgenic mouse hepatocytes (TAMH). These results support the idea that oxidative metabolites of flupirtine contribute to the mechanism of action, similar to what was recently proposed for acetaminophen (paracetamol), but not to hepatotoxicity.

Intestinal formation of trans-crocetin from saffron extract (Crocus sativus L.) and in vitro permeation through intestinal and blood brain barrier

Aims: Extracts of saffron (Crocus sativus L.) have traditionally been used against depressions. Recent preclinical and clinical investigations have rationalized this traditional use. Trans-crocetin, a saffron metabolite originating from the crocin apocarotenoids, has been shown to exert strong NMDA receptor affinity and is thought to be responsible for the CNS activity of saffron. Pharmacokinetic properties of the main constituents from saffron have only been described to a limited extent. Therefore the present in vitro study aimed to determine if crocin-1 and trans-crocetin are able to pass the intestinal barrier and to penetrate the blood brain barrier (BBB). Additionally, the intestinal conversion of glycosylated crocins to the lipophilic crocetin had to be investigated. Experiments with Caco-2 cells and two different porcine BBB systems were conducted. Further on, potential intestinal metabolism of saffron extract was investigated by ex vivo experiments with murine intestine.Methodology: In vitro Caco-2 monolayer cell culture was used for investigation of intestinal permeation of crocin-1 and trans-crocetin. In vitro models of porcine brain capillary endothelial cells (BCEC) and blood cerebrospinal fluid barrier (BCSFB) were used for monitoring permeation characteristics of trans-crocetin through the blood brain barrier (BBB). Intestine tissue and feces homogenates from mice served for metabolism experiments.Results: Crocin-1, even at high concentrations (1000 µM) does not penetrate Caco-2 monolayers in relevant amounts. In contrast, trans-crocetin permeates in a concentration-independent manner (10–114 µM) the intestinal barrier by transcellular passage with about 32% of the substrate being transported within 2 h and a permeation coefficient of Papp 25.7 × 10−6 ± 6.23 × 10−6 cm/s. Trans-crocetin serves as substrate for pGP efflux pump. Trans-crocetin permeates BBB with a slow but constant velocity over a 29 h period (BCEC system: Papp 1.48 × 10−6 ± 0.12 × 10−6 cm/s; BCSFB system Papp 3.85 × 10−6 ± 0.21 × 10−6 cm/s). Conversion of glycosylated crocins from saffron extract to trans-crocetin occurs mainly by intestinal cells, rather than by microbiological fermentation in the colon.Conclusion: The here described in vitro studies have shown that crocins from saffron are probably not bioavailable in the systemic compartment after oral application. On the other side the investigations clearly have pointed out that crocins get hydrolyzed in the intestine to the deglycosylated trans-crocetin, which subsequently is absorbed by passive transcellular diffusion to a high extend and within a short time interval over the intestinal barrier. Crocetin will penetrate in a quite slow process the blood brain barrier to reach the CNS. The intestinal deglycosylation of different crocins in the intestine is mainly due to enzymatic processes in the epithelial cells and only to a very minor extent due to deglycosylation by the fecal microbiome. On the other side the fecal bacteria degrade the apocarotenoid backbone to smaller alkyl units, which do not show any more the typical UV absorbance of crocins. As previous studies have shown strong NMDA receptor affinity and channel opening activity of trans-crocetin the use of saffron for CNS disorders seems to be justified from the pharmacokinetic and pharmacodynamic background.

構造解明を指向したポリオール天然物シンビオジノライドの合成研究

Symbiodinolide is a polyol marine natural product isolated from dinoflagellate Symbiodinium sp. This natural product exhibits voltage-dependent N-type Ca2+ channel-opening activity at 7 nM and COX-1 inhibition activity at 2 µM. The planar structure of symbiodinolide was elucidated by the detailed 2D NMR analysis. However, the huge and complex molecular structure of this molecule featuring a molecular weight of 2,860 and the presence of 61 chiral centers has hampered the complete stereochemical determination. Herein, we report our synthetic study of symbiodinolide toward the structural elucidation.

Anti-hyperalgesic effect of a benzilidine-cyclohexanone analogue on a mouse model of chronic constriction injury-induced neuropathic pain: Participation of the κ-opioid receptor and KATP

The present study investigated the analgesic effect of a novel synthetic cyclohexanone derivative, 2,6-bis-4-(hydroxyl-3-methoxybenzilidine)-cyclohexanone or BHMC in a mouse model of chronic constriction injury-induced neuropathic pain. It was demonstrated that intraperitoneal administration of BHMC (0.03, 0.1, 0.3 and 1.0mg/kg) exhibited dose-dependent inhibition of chronic constriction injury-induced neuropathic pain in mice, when evaluated using Randall–Selitto mechanical analgesiometer. It was also demonstrated that pretreatment of naloxone (non-selective opioid receptor blocker), nor-binaltorphimine (nor-BNI, selective κ-opioid receptor blocker), but not β-funaltrexamine (β-FN, selective μ-opioid receptor blocker) and naltrindole hydrochloride (NTI, selective δ-opioid receptor blocker), reversed the anti-nociceptive effect of BHMC. In addition, the analgesic effect of BHMC was also reverted by pretreatment of 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ, soluble guanosyl cyclase blocker) and glibenclamide (ATP-sensitive potassium channel blocker) but not Nω-nitro-l-arginine (l-NAME, a nitric oxide synthase blocker). Taken together, the present study demonstrated that the systemic administration of BHMC attenuated chronic constriction, injury-induced neuropathic pain. We also suggested that the possible mechanisms include κ-opioid receptor activation and nitric oxide-independent cyclic guanosine monophosphate activation of ATP-sensitive potassium channel opening.

Synthesis and BK channel-opening activity of novel N-acylhydrazone derivatives from dehydroabietic acid

Abstract A series of hydrazone and N -acylhydrazone derivatives of dehydroabietic acid were synthesized and evaluated for BK channel-opening activities in an assay system of CHO-K1 cells expressing hBK α channels. The assay results indicated that the activities of the investigated compounds were influenced by the physicochemical properties of the substituent at hydrazone moiety.

4-Chloro-7-Trifluoromethyl-10H- Benzo[4,5]furo[3,2-b]Indole-1-Carboxylic Acid (TBIC), a Putative BKCa Channel Opener with Uterine Relaxant Activities

In the present study, we examined the uterine relaxant activity of 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid (TBIC), a putative opener of the large conductance Ca²⁺-activated K⁺ (BK_Ca) channel. TBIC concentration-dependently inhibited spontaneous uterine contractions (EC₅₀ = 4.63 µmol/l; E_max = 94.85 ± 1.85%; 100 µmol/l, n = 6). It also reduced contractions induced by oxytocin (EC₅₀ = 4.10 µmol/l; E_max = 84.3 ± 3.83%; 100 µmol/l, n = 6), prostaglandin F₂_α (EC₅₀ = 2.14 µmol/l; E_max = 73.70 ± 5.21%; 100 µmol/l, n = 6) and acetylcholine (EC₅₀ = 4.37 µmol/l; E_max = 83.67 ± 4.82; 100 µmol/l, n = 6). TBIC decreased KCl (20 mmol/l) -induced contractions (EC₅₀ = 3.04 µmol/l; E_max = 94.0 ± 3.12%; 100 µmol/l, n = 6) indicating its K⁺ channel opening activity. BK_Ca channel blockers penitrem A (100 nmol/l) and tetraethylammonium chloride (1 mmol/l) attenuated the inhibitory activities of TBIC (p < 0.001) but not other K⁺ channel blockers such as barium chloride and glibenclamide (K_IR and K_ATP channel blockers, respectively). These results demonstrate the uterine relaxant effects of TBIC in a mechanism of action largely referable to the potentiation of the BK_Ca channels. We have provided evidence for the potential use of TBIC as a tocolytic agent and support for the utility of BK_Ca channel openers in pathophysiologic conditions involving smooth muscle hyperactivity.

Effect of YM934, a Novel K+‐channel Opener, on Non‐adrenergic Non‐cholinergic Neural Bronchoconstriction in Guinea‐pigs

The effect of YM934 (2-(3,4-dihydro-2,2-dimethyl-6-nitro-2H-1,4-benzoxazin-4-yl)pyridine N-oxide) on non-adrenergic non-cholinergic (NANC) neural bronchoconstriction was investigated in anaesthetized guinea-pigs, and compared with that of levcromakalim. After pre-treatment with atropine (1 mg kg−1, i.v.) and propranolol (1 mg kg−1, i.v.), bilateral vagal stimulation evoked a NANC neural bronchoconstriction. YM934 (10–100 μg kg−1, i.v.) and levcromakalim (30–300 μg kg−1, i.v.) dose-dependently inhibited the increase in airway resistance evoked by vagal stimulation with ED50 values of 55·7 and 113·4 μg kg−1, i.v., respectively. The effect of YM934 was about twice as potent as that of levcromakalim. The inhibitory effect of YM934 was antagonized by glibenclamide (25 mg kg−1, i.v.), an ATP-sensitive K+-channel blocker. YM934 (100 μg kg−1, i.v.) had no inhibitory effect on exogenous substance P- (20 μg kg−1, i.v.) or neurokinin A- (1–1·5 μg kg−1, i.v.) induced bronchoconstriction. These results suggest that YM934 has a potent inhibitory effect on NANC neural bronchoconstriction by reducing the release of neuropeptides from sensory nerve terminals, and that the action of YM934 is mainly attributable to its ATP-sensitive K+-channel opening activity.