KATP Channel Opener Research Articles

Meningeal KATP channels contribute to behavioral responses in preclinical migraine models

Abstract Human experimental studies have shown that levcromakalim, an ATP-sensitive potassium (KATP) channel opener, induces migraine attacks in people with migraine but not in healthy volunteers. However, the exact site of action for KATP channels in migraine pathophysiology remains unclear. This study investigates the role of these channels in the meninges in eliciting behavioral hypersensitivity responses in mice. The effects of KATP channel signaling were assessed using preclinical migraine models induced by repetitive stress or dural stimulation. Prolactin, CGRP, sodium nitroprusside (SNP), and KATP channel openers or blockers were administered systemically or onto the dura of mice followed by behavioral testing using periorbital von Frey or facial grimace measurements. Repetitive stress sensitized mice to a normally subthreshold systemic dose of levcromakalim. The KATP blocker glibenclamide significantly reduced responses to systemic SNP following repetitive stress. In naive mice, direct dural application of levcromakalim or SNP elicited periorbital hypersensitivity. Responses to dural levcromakalim were inhibited by coinjection with glibenclamide or sumatriptan. By contrast, injection of levcromakalim in the periorbital skin did not induce hypersensitivity. Moreover, repetitive stress sensitized mice to dural injection of normally subthreshold doses of levcromakalim or SNP. Finally, dural coinjection of glibenclamide inhibited periorbital hypersensitivity induced by CGRP or prolactin in female mice. These studies demonstrate that the meninges can be one site of action for the migraine-triggering effects of KATP channel openers. They also show that NO donors, CGRP, and prolactin can produce behavioral hypersensitivity through opening of KATP channels in the meninges.

AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone.

Pancreatic KATP channel trafficking defects underlie congenital hyperinsulinism (CHI) cases unresponsive to the KATP channel opener diazoxide, the mainstay medical therapy for CHI. Current clinically used KATP channel inhibitors have been shown to act as pharmacochaperones and restore surface expression of trafficking mutants; however, their therapeutic utility for KATP trafficking impaired CHI is hindered by high-affinity binding, which limits functional recovery of rescued channels. Recent structural studies of KATP channels employing cryo-electron microscopy (cryoEM) have revealed a promiscuous pocket where several known KATP pharmacochaperones bind. The structural knowledge provides a framework for discovering KATP channel pharmacochaperones with desired reversible inhibitory effects to permit functional recovery of rescued channels. Using an AI-based virtual screening technology AtomNet® followed by functional validation, we identified a novel compound, termed Aekatperone, which exhibits chaperoning effects on KATP channel trafficking mutations. Aekatperone reversibly inhibits KATP channel activity with a half-maximal inhibitory concentration (IC50) ~ 9 μM. Mutant channels rescued to the cell surface by Aekatperone showed functional recovery upon washout of the compound. CryoEM structure of KATP bound to Aekatperone revealed distinct binding features compared to known high affinity inhibitor pharmacochaperones. Our findings unveil a KATP pharmacochaperone enabling functional recovery of rescued channels as a promising therapeutic for CHI caused by KATP trafficking defects.

Mechanisms and Functions of Sweet Reception in Oral and Extraoral Organs.

The oral detection of sugars relies on two types of receptor systems. The first is the G-protein-coupled receptor TAS1R2/TAS1R3. When activated, this receptor triggers a downstream signaling cascade involving gustducin, phospholipase Cβ2 (PLCβ2), and transient receptor potential channel M5 (TRPM5). The second type of receptor is the glucose transporter. When glucose enters the cell via this transporter, it is metabolized to produce ATP. This ATP inhibits the opening of KATP channels, leading to cell depolarization. Beside these receptor systems, sweet-sensitive taste cells have mechanisms to regulate their sensitivity to sweet substances based on internal and external states of the body. Sweet taste receptors are not limited to the oral cavity; they are also present in extraoral organs such as the gastrointestinal tract, pancreas, and brain. These extraoral sweet receptors are involved in various functions, including glucose absorption, insulin release, sugar preference, and food intake, contributing to the maintenance of energy homeostasis. Additionally, sweet receptors may have unique roles in certain organs like the trachea and bone. This review summarizes past and recent studies on sweet receptor systems, exploring the molecular mechanisms and physiological functions of sweet (sugar) detection in both oral and extraoral organs.

KATP channels in cerebral hemodynamics: a systematic review of preclinical and clinical studies.

Cumulative evidence suggests that ATP-sensitive potassium (KATP) channels act as a key regulator of cerebral blood flow (CBF). This implication seems to be complicated, since KATP channels are expressed in several vascular-related structures such as smooth muscle cells, endothelial cells and pericytes. In this systematic review, we searched PubMed and EMBASE for preclinical and clinical studies addressing the involvement of KATP channels in CBF regulation. A total of 216 studies were screened by title and abstract. Of these, 45 preclinical and 6 clinical studies were included. Preclinical data showed that KATP channel openers (KCOs) caused dilation of several cerebral arteries including pial arteries, the middle cerebral artery and basilar artery, and KATP channel inhibitor (KCI) glibenclamide, reversed the dilation. Glibenclamide affected neither the baseline CBF nor the baseline vascular tone. Endothelium removal from cerebral arterioles resulted in an impaired response to KCO/KCI. Clinical studies showed that KCOs dilated cerebral arteries and increased CBF, however, glibenclamide failed to attenuate these vascular changes. Endothelial KATP channels played a major role in CBF regulation. More studies investigating the role of KATP channels in CBF-related structures are needed to further elucidate their actual role in cerebral hemodynamics in humans. Systematic review registration: Prospero: CRD42023339278 (preclinical data) and CRD42022339152 (clinical data).

L-valine is a powerful stimulator of GLP-1 secretion in rodents and stimulates secretion through ATP-sensitive potassium channels and voltage-gated calcium channels

BackgroundWe previously reported that, among all the naturally occurring amino acids, l-valine is the most powerful luminal stimulator of glucagon-like peptide 1 (GLP-1) release from the upper part of the rat small intestine. This makes l-valine an interesting target for nutritional-based modulation of GLP-1 secretion. However, the molecular mechanism of l-valine-induced secretion remains unknown.MethodsWe aimed to investigate the effect of orally given l-valine in mice and to identify the molecular details of l-valine stimulated GLP-1 release using the isolated perfused rat small intestine and GLUTag cells. In addition, the effect of l-valine on hormone secretion from the distal intestine was investigated using a perfused rat colon.ResultsOrally given l-valine (1 g/kg) increased plasma levels of active GLP-1 comparably to orally given glucose (2 g/kg) in male mice, supporting that l-valine is a powerful stimulator of GLP-1 release in vivo (P > 0.05). Luminal l-valine (50 mM) strongly stimulated GLP-1 release from the perfused rat small intestine (P < 0.0001), and inhibition of voltage-gated Ca2+-channels with nifedipine (10 μM) inhibited the GLP-1 response (P < 0.01). Depletion of luminal Na+ did not affect l-valine-induced GLP-1 secretion (P > 0.05), suggesting that co-transport of l-valine and Na+ is not important for the depolarization necessary to activate the voltage-gated Ca2+-channels. Administration of the KATP-channel opener diazoxide (250 μM) completely blocked the l-valine induced GLP-1 response (P < 0.05), suggesting that l-valine induced depolarization arises from metabolism and opening of KATP-channels. Similar to the perfused rat small intestine, l-valine tended to stimulate peptide tyrosine-tyrosine (PYY) and GLP-1 release from the perfused rat colon.Conclusionsl-valine is a powerful stimulator of GLP-1 release in rodents. We propose that intracellular metabolism of l-valine leading to closure of KATP-channels and opening of voltage-gated Ca2+-channels are involved in l-valine induced GLP-1 secretion.

Non-vascular ATP-sensitive potassium channel activation does not trigger migraine attacks: A randomized clinical trial.

To investigate the role of NN414, a selective KATP channel opener for the Kir6.2/SUR1 channel subtype found in neurons and β-pancreatic cells, in inducing migraine attacks in individuals with migraine without aura. Thirteen participants were randomly allocated to receive NN414 and placebo on two days separated by at least one week. The primary endpoint was the difference in the incidence of migraine attacks after NN414 compared with placebo. The secondary endpoints were the difference in the area under the curve for headache intensity scores, middle cerebral artery blood flow velocity (VMCA), superficial temporal artery diameter, heart rate and mean arterial pressure. Twelve participants completed the study, with two (16.6%) reporting migraine attacks after NN414 compared to one (8.3%) after placebo (p = 0.53). The area under the curve for headache intensity, VMCA, superficial temporal artery diameter, heart rate and mean arterial pressure did not differ between NN414 and placebo (p > 0.05, all comparisons). The lack of migraine induction upon activation of the Kir6.2/SUR1 channel subtype suggests it may not contribute to migraine pathogenesis. Our findings point to KATP channel blockers that target the Kir6.1/SUR2B subtype, found in cerebral vasculature, as potential candidates for innovative antimigraine treatments.Registration number: NCT04744129.

Peptides Are Cardioprotective Drugs of the Future: The Receptor and Signaling Mechanisms of the Cardioprotective Effect of Glucagon-like Peptide-1 Receptor Agonists.

The high mortality rate among patients with acute myocardial infarction (AMI) is one of the main problems of modern cardiology. It is quite obvious that there is an urgent need to create more effective drugs for the treatment of AMI than those currently used in the clinic. Such drugs could be enzyme-resistant peptide analogs of glucagon-like peptide-1 (GLP-1). GLP-1 receptor (GLP1R) agonists can prevent ischemia/reperfusion (I/R) cardiac injury. In addition, chronic administration of GLP1R agonists can alleviate the development of adverse cardiac remodeling in myocardial infarction, hypertension, and diabetes mellitus. GLP1R agonists can protect the heart against oxidative stress and reduce proinflammatory cytokine (IL-1β, TNF-α, IL-6, and MCP-1) expression in the myocardium. GLP1R stimulation inhibits apoptosis, necroptosis, pyroptosis, and ferroptosis of cardiomyocytes. The activation of the GLP1R augments autophagy and mitophagy in the myocardium. GLP1R agonists downregulate reactive species generation through the activation of Epac and the GLP1R/PI3K/Akt/survivin pathway. The GLP1R, kinases (PKCε, PKA, Akt, AMPK, PI3K, ERK1/2, mTOR, GSK-3β, PKG, MEK1/2, and MKK3), enzymes (HO-1 and eNOS), transcription factors (STAT3, CREB, Nrf2, and FoxO3), KATP channel opening, and MPT pore closing are involved in the cardioprotective effect of GLP1R agonists.

The impact of ATP-sensitive potassium channel modulation on mitochondria in a Parkinson’s disease model using SH-SY5Y cells depends on their differentiation state

Inward rectifying potassium channels sensitive to ATP levels (KATP) have been the subject of investigation for several decades. Modulators of KATP channels are well-established treatments for metabolic as well as cardiovascular diseases. Experimental studies have also shown the potential of KATP modulation in neurodegenerative disorders. However, to date, data regarding the effects of KATP antagonists/agonists in experiments related to neurodegeneration remain inconsistent. The main source of confusion in evaluating available data seems to be the choice of experimental models. The present study aims to provide a comprehensive understanding of the effects of both opening and blocking KATP channels in two forms of SH-SY5Y cells. Our results offer valuable insights into the significance of metabolic differences between differentiated and non-differentiated SH-SY5Y cells, particularly in the context of glibenclamide and diazoxide effects under normal conditions and during the initiation of pathological events simulating Parkinson’s disease in vitro. We emphasize the analysis of mitochondrial functions and changes in mitochondrial network morphology. The heightened protein expression of KATP channels identified in non-differentiated SH-SY5Y cells seems to be a platform for a more significant impact of KATP modulators in this cell type. The efficiency of rotenone treatment in inducing morphological changes in the mitochondrial network depends on the differentiation status of SH-SY5Y cells.

Purinergic inhibitory regulation of esophageal smooth muscle is mediated by P2Y receptors and ATP-dependent potassium channels in rats

Purines such as ATP are regulatory transmitters in motility of the gastrointestinal tract. The aims of this study were to propose functional roles of purinergic regulation of esophageal motility. An isolated segment of the rat esophagus was placed in an organ bath, and mechanical responses were recorded using a force transducer. Exogenous application of ATP (10–100 μM) evoked relaxation of the esophageal smooth muscle in a longitudinal direction under the condition of carbachol (1 μM) -induced precontraction. Pretreatment with a non-selective P2 receptor antagonist, suramin (500 μM), and a P2Y receptor antagonist, cibacron blue F3GA (200 μM), inhibited the ATP (100 μM) -induced relaxation, but a P2X receptor antagonist, pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (50 μM), did not affect it. A blocker of ATP-dependent potassium channels (KATP channels), glibenclamide (200 μM), inhibited the ATP-induced relaxation and application of an opener of KATP channels, nicorandil (50 μM), produced relaxation. The findings suggest that ATP is involved in inhibitory regulation of the longitudinal smooth muscle in the muscularis mucosae of the rat esophagus via activation of P2Y receptors and then opening of KATP channels.

Diazoxide and moderate-intensity exercise improve skeletal muscle function by decreasing oxidants and enhancing antioxidant defenses in hypertensive male rats.

High sodium intake is decisive in the incidence increase and prevalence of hypertension, which has an impact on skeletal muscle functionality. Diazoxide is an antihypertensive agent that inhibits insulin secretion and is an opener of KATP channels (adosine triphosphate sensitive potasium channels). For this reason, it is hypothesized that moderate-intensity exercise and diazoxide improve skeletal muscle function by reducing the oxidants in hypertensive rats. Male Wistar rats were assigned into eight groups: control (CTRL), diazoxide (DZX), exercise (EX), exercise + diazoxide (EX + DZX), hypertension (HTN), hypertension + diazoxide (HTN + DZX), hypertension + exercise (HTN + EX), and hypertension + exercise + diazoxide (HTN + EX + DZX). To induce hypertension, the rats received 8% NaCl dissolved in water orally for 30 days; in the following 8 weeks, 4% NaCl was supplied to maintain the pathology. The treatment with physical exercise of moderate intensity lasted 8 weeks. The administration dose of diazoxide was 35 mg/kg intraperitoneally for 14 days. Tension recording was performed on the extensor digitorum longus and the soleus muscle. Muscle homogenates were used to measure oxidants using fluorescent probe and the activity of antioxidant systems. Diazoxide and moderate-intensity exercise reduced oxidants and increased antioxidant defenses.

Structure of an open KATP channel reveals tandem PIP2 binding sites mediating the Kir6.2 and SUR1 regulatory interface

ATP-sensitive potassium (KATP) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic β-cells. KATP channel opening is stimulated by PIP2 and inhibited by ATP. Mutations that increase channel opening by PIP2 reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has implicated a role for PIP2 in KATP channel function, previously solved open-channel structures have lacked bound PIP2, and mechanisms by which PIP2 regulates KATP channels remain unresolved. Here, we report the cryoEM structure of a KATP channel harboring the neonatal diabetes mutation Kir6.2-Q52R, in the open conformation, bound to amphipathic molecules consistent with natural C18:0/C20:4 long-chain PI(4,5)P2 at two adjacent binding sites between SUR1 and Kir6.2. The canonical PIP2 binding site is conserved among PIP2-gated Kir channels. The non-canonical PIP2 binding site forms at the interface of Kir6.2 and SUR1. Functional studies demonstrate both binding sites determine channel activity. Kir6.2 pore opening is associated with a twist of the Kir6.2 cytoplasmic domain and a rotation of the N-terminal transmembrane domain of SUR1, which widens the inhibitory ATP binding pocket to disfavor ATP binding. The open conformation is particularly stabilized by the Kir6.2-Q52R residue through cation-π bonding with SUR1-W51. Together, these results uncover the cooperation between SUR1 and Kir6.2 in PIP2 binding and gating, explain the antagonistic regulation of KATP channels by PIP2 and ATP, and provide a putative mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.

Activation of ATP-sensitive potassium channels prevents doxorubicin-induced mitochondrial dysfunction in the heart and impaired vascular responses in rats

One of the side effects of the anticancer drug doxorubicin is its mitotoxicity. At the same time, a sufficient number of functioning mitochondria is required for normal energy supply to the heart. The system of ATP-sensitive potassium channels (KATP -channels) of cytoplasmic and mitochondrial membranes is considered to be the central metabolic sensor of energy supply, and their opening triggers mechanisms of protection against cell damage and death under the influence of pathological factors. The aim of our study was to investigate the effect of KATP-channel opener flocalin on doxorubicin-induced mitochondrial dysfunction in the heart, impaired vascular contraction-relaxation function, and oxidative stress. Acute cardiotoxicity was modelled by short-term intraperitoneal injection of doxorubicin in a total dose of 15 mg/kg. To prevent damage, animals were administered flocalin at a dose of 2.5 mg/kg for 5 consecutive days. It was found that the rate of formation of superoxide anion (•O2 - ) and hydroxyl radical (•OH) in the heart mitochondria significantly increased after administration of doxorubicin by 10.5 and 3.4 times, respectively, and the level of H2O2 increased by 5.3 times. When rats were administered flocalin against the background of doxorubicin, oxidative stress indicators were significantly reduced, namely, the rate of •O2 - and •ON generation was 4 and 1.6 times lower, respectively, and the H2O2 levels were 4.6 times lower. Under conditions of impaired redox status in the rat heart after doxorubicin administration, mitochondrial permeability transition pore opening was activated: the amplitude of spontaneous swelling doubled, and Ca2+-induced swelling increased 1.5-fold. The use of flocalin reduced the amplitude of mitochondrial swelling in calcium-free medium by 84.6%, and under the conditions of action inducer of mPTP opening calcium, this index was restored to control values. Endothelium-dependent relaxation of aorta preparations of doxorubicin-treated animals to acetylcholine (0.1 μmol/l) was 47% less than in the control group. Contractions of aortic rings in these animals under the influence of norepinephrine (10 μmol/l) were reduced by 59% compared to control rats. When flocalin, a KATP-channel opener, was injected into rats, the contractile responses of isolated rat aortic rings were restored almost to the values of control animals, while the endothelium-dependent vasodilator effects of acetylcholine (0.1 μmol/l) under the influence of flocalin were restored by 69% compared with animals injected with doxorubicin. Thus, the opening of KATP-channels by flocalin prevents doxorubicininduced mitochondrial dysfunction in the heart, reduces oxidative stress and prevents vascular contractionrelaxation disorders.

Nicorandil and carvedilol mitigates motor deficits in experimental autoimmune encephalomyelitis-induced multiple sclerosis: Role of TLR4/TRAF6/MAPK/NF-κB signalling cascade

Multiple sclerosis (MS) is an inflammatory demyelinating neurodegenerative disease that negatively affects neurotransmission. It can be pathologically mimicked by experimental autoimmune encephalomyelitis (EAE) animal model. ATP-sensitive potassium channels (KATP) plays a crucial role in the control of neuronal damage, however their role in MS are still obscure. Additionally, Carvedilol showed a promising neuroprotective activity against several neurological disorders. Therefore, the present study aimed to investigate the potential neuroprotective effect of KATP channel opener (nicorandil) as well as α and β adrenoceptor antagonist (Carvedilol) against EAE induced neurodegeneration in mice. Mice was treated with nicorandil (6 mg/kg/day; p.o.) and carvedilol (10 mg/kg/day; p.o.) for 14 days. Nicorandil and carvedilol showed improvement in clinical scoring, behaviour and motor coordination as established by histopathological investigation and immunohistochemical detection of MBP. Furthermore, both treatments downregulated the protein expression of TLR4/ MYD88/TRAF6 signalling cascade with downstream inhibition of (pT183/Y185)-JNK/p38 (pT180/Y182)-MAPK axis leading to reduction of neuroinflammatory status, as witnessed by reduction of NF-κB, TNF-α, IL-1β and IL-6 contents. Moreover, nicorandil and carvedilol attenuated oxidative damage by increasing Nrf2 content and SOD activity together with reduction of MDA content. In addition, an immunomodulating effect via inhibiting the gene expression of CD4, TGF-β, and IL-17 as well as TGF-β, IL-17, and IL-23 contents along with anti-apoptotic effect by decreasing Bax protein expression and Caspase-3 content and increasing Bcl-2 protein expression was observed with nicorandil and carvedilol treatments. In conclusion, nicorandil and carvedilol exerted a neuroprotective activity against EAE induced neuronal loss via inhibition of TLR4/MYD88/TRAF6/JNK/p38-MAPK axis besides antioxidant and anti-apoptotic effects.

Roles of endothelial prostaglandin I2 in maintaining synchronous spontaneous Ca2+ transients in rectal capillary pericytes.

In hollow visceral organs, capillary pericytes appear to drive spontaneous Ca2+ transients in the upstream arterioles. Here, mechanisms underlying the intercellular synchrony of pericyte Ca2+ transients were explored. Ca2+ dynamics in NG2 chondroitin sulphate proteoglycan (NG2)-expressing capillary pericytes were examined using rectal mucosa-submucosa preparations of NG2-GCaMP6mice. Spontaneous Ca2+ transients arising from endoplasmic reticulum Ca2+ release were synchronously developed amongst capillary pericytes in a gap junction blocker (3μM carbenoxolone)-sensitive manner and could spread into upstream vascular segments. Spontaneous Ca2+ transients were suppressed by the Ca2+ -activated Cl- channel (CaCC) blocker niflumic acid and their synchrony was diminished by a TMEM16A inhibitor (3μM Ani9) in accordance with TMEM16A immunoreactivity in pericytes. In capillaries where cyclooxygenase (COX)-2 immunoreactivity was expressed in endothelium but not pericytes, non-selective COX inhibitors (1μM indomethacin or 10μM diclofenac) or COX-2 inhibitor (10μM NS 398) disrupted the synchrony of spontaneous Ca2+ transients and raised the basal Ca2+ level. Subsequent prostaglandin I2 (PGI2 ; 100nM) or the KATP channel opener levcromakalim restored the synchrony with a reduction in the Ca2+ level. PGI2 receptor antagonist (1μM RO1138452) also disrupted the synchrony of spontaneous Ca2+ transients and increased the basal Ca2+ level. Subsequent levcromakalim restored the synchrony and reversed the Ca2+ rise. Thus, the synchrony of spontaneous Ca2+ transients in pericytes appears to be developed by the spread of spontaneous transient depolarisations arising from the opening of TMEM16A CaCCs. Endothelial PGI2 may play a role in maintaining the synchrony, presumably by stabilising the resting membrane potential in pericytes. KEY POINTS: Capillary pericytes in the rectal mucosa generate synchronous spontaneous Ca2+ transients that could spread into the upstream vascular segment. Spontaneous Ca2+ release from the endoplasmic reticulum (ER) triggers the opening of Ca2+ -activated Cl- channel TMEM16A and resultant depolarisations that spread amongst pericytes via gap junctions, establishing the synchrony of spontaneous Ca2+ transients in pericytes. Prostaglandin I2 (PGI2 ), which is constitutively produced by the endothelium depending on cyclooxygenase-2, appears to prevent premature ER Ca2+ releases in the pericytes allowing periodic, regenerative Ca2+ releases. Endothelial PGI2 may maintain the synchrony of pericyte activity by stabilising pericyte resting membrane potential by opening of KATP channels.

Effects of nicorandil on QT prolongation and myocardial damage caused by citalopram in rats

ABSTRACT Citalopram is a selective serotonin re-uptake inhibitor (SSRI) antidepressant; it exhibits the greatest cardiotoxic effect among SSRIs. Citalopram can cause drug-induced long QT syndrome (LQTS) and ventricular arrhythmias. We investigated the protective effect of nicorandil, a selective mitochondrial KATP (mito-KATP) channel opener, on LQTS and myocardial damage caused by citalopram in male rats. In a preliminary study, we determined that the minimum citalopram dose that prolonged the QT interval was 102 mg/kg injected intraperitoneally. For the main study, rats were divided randomly into five experimental groups: untreated control, normal saline + citalopram, nicorandil + citalopram, 5-hydroxydecanoate (5-HD) + citalopram, 5-HD + nicorandil + citalopram. Biochemical and histologic data from blood and heart tissue samples from six untreated control rats were evaluated. Electrocardiographic parameters including QRS duration, QT interval, corrected QT interval (QTc) and heart rate (HR) were assessed, and biochemical parameters including malondialdehyde, reduced glutathione, glutathione peroxidase, superoxide dismutase were measured. We also performed histomorphologic and immunohistochemical examination of heart tissue. Citalopram prolonged QT-QTc intervals significantly and increased significantly the histomorphologic score and proportion of apoptotic cells, but produced no differences in the oxidant and antioxidant parameters. Nicorandil did not prevent citalopram induced QT-QTc interval prolongation and produced no significant changes in oxidant and antioxidant parameters; however, it did reduce histologic damage and apoptosis caused by citalopram.

KATP channels are regulators of programmed cell death and targets for the creation of novel drugs against ischemia/reperfusion cardiac injury.

The use of percutaneous coronary intervention (PCI) in patients with ST-segment elevation myocardial infarction (STEMI) is associated with a mortality rate of 5%-7%. It is clear that there is an urgent need to develop new drugs that can effectively prevent cardiac reperfusion injury. ATP-sensitive K+ (KATP ) channel openers (KCOs) can be classified as such drugs. KCOs prevent irreversible ischemia and reperfusion injury of the heart. KATP channel opening promotes inhibition of apoptosis, necroptosis, pyroptosis, and stimulation of autophagy. KCOs prevent the development of cardiac adverse remodeling and improve cardiac contractility in reperfusion. KCOs exhibit antiarrhythmic properties and prevent the appearance of the no-reflow phenomenon in animals with coronary artery occlusion and reperfusion. Diabetes mellitus and a cholesterol-enriched diet abolish the cardioprotective effect of KCOs. Nicorandil, a KCO, attenuates major adverse cardiovascular event and the no-reflow phenomenon, reduces infarct size, and decreases the incidence of ventricular arrhythmias in patients with acute myocardial infarction. The cardioprotective effect of KCOs is mediated by the opening of mitochondrial KATP (mitoKATP ) and sarcolemmal KATP (sarcKATP ) channels, triggered free radicals' production, and kinase activation.

CL-705G: a novel chemical Kir6.2-specific KATP channel opener.

Background: KATP channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of KATP channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various KATP channel subclasses. Methods and Results: We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked "hits", which were tested for activity against KATP channels using patch clamping and thallium (Tl+) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl+ fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC50 of 9µM and 11μM, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na+ currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Δ36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP2 depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism. Conclusion: CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener.

UPLC-QTOF-MS/MS-based metabolomic approach and gastroprotective effect of two chemotypes of Egletes viscosa (L.) less. against ethanol-induced gastric ulcer in mice.

Egletes viscosa (L.) (macela) is a native wild herb that can be found in different states of northeastern Brazil. The infusions of its flower buds are traditionally used for the treatment of gastrointestinal disorders. E. viscosa possesses two chemotypes (named A and B), distinguishable by the composition of the essential oil from the flower buds. Although there are previous studies of the gastroprotective effect of the isolated constituents of E. viscosa, its infusions have not been investigated yet. The present study aimed to evaluate and compare the chemical composition and the gastroprotective effect of flower bud infusions of E. viscosa from chemotype A (EVCA) and chemotype B (EVCB). Sixteen infusions were brewed with flower buds according to the traditional preparation mode and were analyzed through a UPLC-QTOF-MS/MS based metabolomic approach for determination of their metabolic fingerprints and quantification of bioactive compounds. Afterward, these data were analyzed by chemometric methods (OPLS-DA) for discrimination of the two chemotypes. Additionally, infusions of EVCA and EVCB (50, 100 and 200mg/kg, p.o.) were evaluated on gastric ulcers induced by absolute ethanol (96%, 0.2mL, p.o.) in mice. To elucidate the gastroprotective mechanisms, the effect of EVCA and EVCB on gastric acid secretion and gastric wall mucus was determined and the role of TRPV1 channels, prostaglandins, nitric oxide and KATP channels were assessed. Moreover, the oxidative stress-related parameters and the histological aspects of the stomach tissue were analyzed. The chemotypes can be discriminated from each other using UPLC-QTOF-MS/MS chemical fingerprints. Both chemotypes presented similar chemical compositions, consisting basically of caffeic acid derivatives, flavonoids and diterpenes. The quantification of bioactive compounds demonstrated that chemotype A possesses more ternatin, tanabalin and centipedic than chemotype B. EVCA and EVCB (50, 100 and 200mg/kg, p.o.) significantly decreased the severity of ethanol-induced gastric lesions, as shown by a reduction in histological alterations and leucocyte infiltration in gastric tissue. The gastroprotective mechanism of both infusions involves an antioxidant effect, maintenance of gastric mucus and reduction gastric secretion. Stimulation of endogenous prostaglandins and nitric oxide release, activation of TRPV1 channels, and KATP channels are also involved in the gastroprotection of the infusions. The gastroprotective effect of EVCA and EVCB was equivalent and mediated through antioxidant and antisecretory actions, including the activation of TRPV1 receptors, stimulation of endogenous prostaglandins and nitric oxide, and opening of KATP channels. The presence of caffeic acid derivatives, flavonoids and diterpenes in both infusions is involved in mediating this protective effect. Our findings support the traditional use of infusions of E. viscosa for gastric disorders regardless of the chemotype.

Nicorandil Exerts Anticonvulsant Effects in Pentylenetetrazol-Induced Seizures and Maximal-Electroshock-Induced Seizures by Downregulating Excitability in Hippocampal Pyramidal Neurons.

N-(2-hydroxyethyl) nicotinamide nitrate (nicorandil), a nitrate that activates adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, is generally used in the treatment of angina and offers long-term cardioprotective effects. It has been reported that several KATP channel openers can effectively alleviate the symptoms of seizure. The purpose of this study was to investigate the improvement in seizures induced by nicorandil. In this study, seizure tests were used to evaluate the effect of different doses of nicorandil by analysing seizure incidence, including minimal clonic seizure and generalised tonic-clonic seizure. We used a maximal electroshock seizure (MES) model, a metrazol maximal seizure (MMS) model and a chronic pentylenetetrazol (PTZ)-induced seizure model to evaluate the effect of nicorandil in improving seizures. Each mouse in the MES model was given an electric shock, while those in the nicorandil group received 0.5, 1, 2, 3 and 6mg/kg of nicorandil by intraperitoneal injection, respectively. In the MMS model, the mice in the PTZ group and the nicorandil group were injected subcutaneously with PTZ (90mg/kg), and the mice in the nicorandil group were injected intraperitoneally with 1, 3 and 5mg/kg nicorandil, respectively. In the chronic PTZ-induced seizure model, the mice in the PTZ group and the nicorandil group were injected intraperitoneally with PTZ (40mg/kg), and the mice in the nicorandil group were each given 1 and 3mg/kg of PTZ at a volume of 200nL. Brain slices containing the hippocampus were prepared, and cell-attached recording was used to record the spontaneous firing of pyramidal neurons in the hippocampal CA1 region. Nicorandil (i.p.) significantly increased both the maximum electroconvulsive protection rate in the MES model and the seizure latency in the MMS model. Nicorandil infused directly onto the hippocampal CA1 region via an implanted cannula relieved symptoms in chronic PTZ-induced seizures. The excitability of pyramidal neurons in the hippocampal CA1 region of the mice was significantly increased after both the acute and chronic administration of PTZ. To a certain extent, nicorandil reversed the increase in both firing frequency and proportion of burst spikes caused by PTZ (P < 0.05). Our results suggest that nicorandil functions by downregulating the excitability of pyramidal neurons in the hippocampal CA1 region of mice and is a potential candidate for the treatment of seizures.

Rapamycin treatment unmasks a sex-specific pattern of scar expansion of the infarcted rat heart: The relationship between mTOR and KATP channel.

Inhibition of the mammalian target of rapamycin (mTOR) with the macrolide rapamycin or pharmacological suppression of KATP channel opening translated to scar expansion of the myocardial infarcted (MI) adult female rodent heart. The present study tested the hypotheses that rapamycin-mediated scar expansion was sex-specific and that mTOR signaling directly influenced KATP channel subunit expression/activity. Scar size was significantly larger in post-MI male rats as compared to the previous data reported in post-MI female rats. The reported scar expansion of rapamycin-treated post-MI female rats was not observed following the administration of the macrolide to post-MI male rats. Protein levels of the KATP channel subunits Kir6.2 and SUR2A and phosphorylation of the serine2448 residue of mTOR were similar in the normal heart of adult male and female rats. By contrast, greater tuberin inactivation characterized by the increased phosphorylation of the threonine1462 residue and reduced raptor protein levels were identified in the normal heart of adult female rats. Rapamycin pretreatment of phorbol 12,13-dibutyrate (PDBu)-treated neonatal rat ventricular cardiomyocytes (NNVMs) suppressed hypertrophy, inhibited p70S6K phosphorylation, and attenuated SUR2A protein upregulation. In the presence of low ATP levels, KATP channel activity detected in untreated NNVMs was significantly attenuated in PDBu-induced hypertrophied NNVMs via a rapamycin-independent pathway. Thus, rapamycin administration to post-MI rats unmasked a sex-specific pattern of scar expansion and mTOR signaling in PDBu-induced hypertrophied NNVMs significantly increased SUR2A protein levels. However, the biological advantage associated with SUR2A protein upregulation was partially offset by an mTOR-independent pathway that attenuated KATP channel activity in PDBu-induced hypertrophied NNVMs.