KATP Channel Blocker Research Articles

Roles of Endothelial Motilin Receptor and Its Signal Transduction Pathway in Motilin-Induced Left Gastric Artery Relaxation in Dogs.

Background: Motilin increases left gastric artery (LGA) blood flow in dogs via the endothelial motilin receptor (MLNR). This article investigates the signaling pathways of endothelial MLNR.Methods: Motilin-induced relaxation of LGA rings was assessed using wire myography. Nitric oxide (NO), and cyclic guanosine monophosphate (cGMP) levels were measured using an NO assay kit and cGMP ELISA kit, respectively.Results: Motilin concentration-dependently (EC50=9.1±1.2×10−8M) relaxed LGA rings precontracted with U46619 (thromboxane A2 receptor agonist). GM-109 (MLNR antagonist) significantly inhibited motilin-induced LGA relaxation and the production of NO and cGMP. N-ethylmaleimide (NEM; G-protein antagonist), U73122 [phospholipase C (PLC) inhibitor], and 2-aminoethyl diphenylborinate [2-APB; inositol trisphosphate (IP3) blocker] partially or completely blocked vasorelaxation. In contrast, chelerythrine [protein kinase C (PKC) inhibitor] and H89 [protein kinase A (PKA) inhibitor] had no such effect. Low-calcium or calcium-free Krebs solutions also reduced vasorelaxation. N-nitro-L-arginine methyl ester [L-NAME; nitric oxide synthase (NOS) inhibitor] and ODQ [soluble guanylyl cyclase (sGC) inhibitor] completely abolished vasodilation and synthesis of NO and cGMP. Indomethacin (cyclooxygenase inhibitor), 18α-glycyrrhetinic acid [18α-GA; myoendothelial gap junction (MEGJ) inhibitor], and K+ channel inhibition through high K+ concentrations or tetraethylammonium (TEA-Cl; KCa channel blocker) partially decreased vasorelaxation, whereas glibenclamide (KATP channel blocker) had no such effect.Conclusion: The current study suggests that motilin-induced LGA relaxation is dependent on endothelial MLNR through the G protein-PLC-IP3 pathway and Ca2+ influx. The NOS-NO-sGC-cGMP pathway, prostacyclin, MEGJ, and K+ channels (especially KCa) are involved in endothelial-dependent relaxation of vascular smooth muscle (VSM) cells.

The anti-nociceptive activity of naringenin passes through L-arginine/NO/cGMP/KATP channel pathway and opioid receptors.

As a promising flavonoid, naringenin has shown potential anti-inflammatory and antioxidant properties mainly in inflammatory pain models by oral administration. Therefore, we investigated the antinociceptive activity of this compound by intraperitoneally (i.p.) administration, as well as, associated mechanism of action considering the involvement of L-arginine/nitric oxide (NO)/cyclic GMP (cGMP)/potassium channel (KATP) pathway and opioid receptors. The antinociceptive effect of naringenin was evaluated in male NMRI mice using formalin test at early and late phases. To assess the involvement of L-arginine/NO/cGMP/KATP pathway and opioid receptors, mice were pretreated i.p. with L-arginine (NO precursor), S-nitroso-N-acetylpenicillamine (SNAP, NO donor), N(gamma)-nitro-L-arginine methyl ester (L-NAME, inhibitor of nitric oxide synthase), sildenafil (inhibitor of phosphodiesterase enzyme), glibenclamide (KATP channel blocker) and naloxone (an opioid receptor antagonist), respectively 20 min before administration of the most effective dose of naringenin. Naringenin showed a dose-dependent antinociceptive effect at both early and late phases of the formalin test. The dose of 100 mg/kg of naringenin was identified as the most effective dose and selected for further experiments. Our mechanistic evaluations showed that L-arginine, SNAP and sildenafil could enhance the antinociceptive effects of naringenin, revealing the critical role of NO and cGMP during its antinociceptive effect. On the other hand, glibenclamide and naloxone could mitigate the antinociceptive potential of naringenin at both phases of formalin test, which confirmed the associated role of KATP channels and opioid receptors. In conclusion, naringenin could be a promising antinociceptive agent acting through opioid receptors and L-arginine/NO/cGMP/KATP channel pathway.

Pituitary Adenylate Cyclase Activating Polypeptide Inhibits A10 Dopamine Neurons and Suppresses the Binge-like Consumption of Palatable Food

Pituitary adenylate cyclase-activating polypeptide (PACAP) binds to PACAP-specific (PAC1) receptors in multiple hypothalamic areas, especially those regulating energy balance. PACAP neurons in the ventromedial nucleus (VMN) exert anorexigenic effects within the homeostatic energy balance circuitry. Since PACAP can also reduce the consumption of palatable food, we tested the hypothesis that VMN PACAP neurons project to the ventral tegmental area (VTA) to inhibit A10 dopamine neurons via PAC1 receptors and KATP channels, and thereby suppress binge-like consumption. We performed electrophysiological recordings in mesencephalic slices from male PACAP-Cre and tyrosine hydroxylase (TH)-Cre mice. Initially, we injected PACAP (30 pmol) into the VTA, where it suppressed binge intake in wildtype male but not female mice. Subsequent tract tracing studies uncovered projections of VMN PACAP neurons to the VTA. Optogenetic stimulation of VMN PACAP neurons in voltage clamp induced an outward current and increase in conductance in VTA neurons, and a hyperpolarization and decrease in firing in current clamp. These effects were markedly attenuated by the KATP channel blocker tolbutamide (100 μM) and PAC1 receptor antagonist PACAP6–38 (200 nM). In recordings from A10 dopamine neurons in TH-Cre mice, we replicated the outward current by perfusing PACAP1–38 (100 nM). This response was again completely blocked by tolbutamide and PACAP6–38, and associated with a hyperpolarization and decrease in firing. These findings demonstrate that PACAP activates PAC1 receptors and KATP channels to inhibit A10 dopamine neurons and sex-dependently suppress binge-like consumption. Accordingly, they advance our understanding of how PACAP regulates energy homeostasis via the hedonic energy balance circuitry.

(-)-Carveol Prevents Gastric Ulcers via Cytoprotective, Antioxidant, Antisecretory and Immunoregulatory Mechanisms in Animal Models.

Background: (-)-Carveol (p-Mentha-6,8-dien-2-ol) is a monocyclic monoterpenic alcohol, present in essential oils of plant species such as Cymbopogon giganteus, Illicium pachyphyllum and in spices such as Carum carvi (cumin). Pharmacological studies report its antitumor, antimicrobial, neuroprotective, vasorelaxant, antioxidant and anti-inflammatory activity.Hypothesis/Purpose: The objective of this study was to evaluate the acute non-clinical oral toxicity, gastroprotective activity of monoterpene (-)-Carveol in animal models and the related mechanisms of action.Methods: Acute toxicity was assessed according to OECD guide 423 in mice. Ethanol, stress, NSAIDs and pylorus ligation-induced gastric ulcer models were used to investigate antiulcer properties. The related mechanisms of action were using the ethanol-gastric lesions protocol.Results: (-)-Carveol has low toxicity, with a lethal dose 50% (LD50) equal to or greater than 2,500 mg/kg according to OECD guide nº 423. In all gastric ulcer induction methods evaluated, (-)-Carveol (25, 50, 100 and 200 mg/kg, p.o.) significantly reduced the ulcerative lesion in comparison with the respective control groups. To investigate the mechanisms involved in the gastroprotective activity, the antisecretory or neutralizing of gastric secretion, cytoprotective, antioxidant and immunoregulatory effects were evaluated. In the experimental protocol of pylorus ligation-induced gastric ulcer, (-)-Carveol (100 mg/kg) reduced (p < 0.001) the volume of gastric secretion in both routes (oral and intraduodenal). The previous administration of blockers NEM (sulfhydryl groups blocker), L-NAME (nitric oxide synthesis inhibitor), glibenclamide (KATP channel blocker) and indomethacin (cyclo-oxygenase inhibitor), significantly reduced the gastroprotection exercised by (-)-Carveol, suggesting the participation of these pathways in its gastroprotective activity. In addition, treatment with (-)-Carveol (100 mg/kg) increased (p < 0.001) mucus adhered to the gastric wall. Treatment also increased (p < 0.001) levels of reduced glutathione (GSH), superoxide dismutase (SOD) and interleukin-10 (IL-10). It also reduced (p < 0.001) malondialdehyde (MDA), myeloperoxidase (MPO), interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels.Conclusion: Thus, it is possible to infer that (-)-Carveol presents gastroprotective activity related to antisecretory, cytoprotective, antioxidant and immunomodulatory mechanisms.

The Effect of K ATP Channel Blocker Glibenclamide on CGRP-Induced Headache and Hemodynamic in Healthy Volunteers.

BackgroundCalcitonin gene-related peptide (CGRP) dilates cranial arteries and triggers headache. The CGRP signaling pathway is partly dependent on activation of ATP-sensitive potassium (KATP) channels. Here, we investigated the effect of the KATP channel blocker glibenclamide on CGRP-induced headache and vascular changes in healthy volunteers.MethodsIn a randomized, double-blind, placebo-controlled, cross-over study, 20 healthy volunteers aged 18–27 years were randomly allocated to receive an intravenous infusion of 1.5 μg/min CGRP after oral pretreatment with glibenclamide (glibenclamide-CGRP day) or placebo (placebo-CGRP day). The primary endpoints were the difference in incidence of headache and the difference in area under the curve (AUC) for headache intensity scores (0–14 h) between glibenclamide and placebo. The secondary endpoints were the difference in AUC for middle cerebral artery blood flow velocity (VMCA), superficial temporal artery (STA) and radial artery (RA) diameter, facial flushing, heart rate (HR) and mean arterial blood pressure (MAP) (0–4 h) between glibenclamide and placebo.ResultsWe found no significant difference in the incidence of headache between glibenclamide-CGRP day (14/20, 70%) and placebo-CGRP day (19/20, 95%) (P = 0.06). The AUC for headache intensity, VMCA, STA, RA, facial skin blood flow, HR, and MAP did not differ between glibenclamide-CGRP day compared to placebo-CGRP day (P > 0.05).ConclusionPretreatment with a non-selective KATP channel inhibitor glibenclamide did not attenuate CGRP-induced headache and hemodynamic changes in healthy volunteers. We suggest that CGRP-induced responses could be mediated via activation of specific isoforms of sulfonylurea receptor subunits of KATP channel.

KATP channel blockers control glucagon secretion by distinct mechanisms: A direct stimulation of α-cells involving a [Ca2+]c rise and an indirect inhibition mediated by somatostatin

ObjectiveGlucagon is secreted by pancreatic α-cells in response to hypoglycemia and its hyperglycemic effect helps to restore normal blood glucose. Insulin and somatostatin (SST) secretions from β- and δ-cells, respectively, are stimulated by glucose by mechanisms involving an inhibition of their ATP-sensitive K+ (KATP) channels, leading to an increase in [Ca2+]c that triggers exocytosis. Drugs that close KATP channels, such as sulfonylureas, are used to stimulate insulin release in type 2 diabetic patients. α-cells also express KATP channels. However, the mechanisms by which sulfonylureas control glucagon secretion are still largely debated and were addressed in the present study. In particular, we studied the effects of KATP channel blockers on α-cell [Ca2+]c and glucagon secretion in the presence of a low (1 mM) or a high (15 mM) glucose concentration and evaluated the role of SST in these effects. MethodsUsing a transgenic mouse model expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells, we measured [Ca2+]c in α-cells either dispersed or within whole islets (by confocal microscopy). By measuring [Ca2+]c in α-cells within islets and glucagon secretion using the same perifusion protocols, we tested whether glucagon secretion correlated with changes in [Ca2+]c in response to sulfonylureas. We studied the role of SST in the effects of sulfonylureas using multiple approaches including genetic ablation of SST, or application of SST-14 and SST receptor antagonists. ResultsApplication of the sulfonylureas, tolbutamide, or gliclazide, to a medium containing 1 mM or 15 mM glucose increased [Ca2+]c in α-cells by a direct effect as in β-cells. At low glucose, sulfonylureas inhibited glucagon secretion of islets despite the rise in α-cell [Ca2+]c that they triggered. This glucagonostatic effect was indirect and attributed to SST because, in the islets of SST-knockout mice, sulfonylureas induced a stimulation of glucagon secretion which correlated with an increase in α-cell [Ca2+]c. Experiments with exogenous SST-14 and SST receptor antagonists indicated that the glucagonostatic effect of sulfonylureas mainly resulted from an inhibition of the efficacy of cytosolic Ca2+ on exocytosis. Although SST-14 was also able to inhibit glucagon secretion by decreasing α-cell [Ca2+]c, no decrease in [Ca2+]c occurred during sulfonylurea application because it was largely counterbalanced by the direct stimulatory effect of these drugs on α-cell [Ca2+]c. At high glucose, i.e., in conditions where glucagon release was already low, sulfonylureas stimulated glucagon secretion because their direct stimulatory effect on α-cells exceeded the indirect effect by SST. Our results also indicated that, unexpectedly, SST-14 poorly decreased the efficacy of Ca2+ on exocytosis in β-cells. ConclusionsSulfonylureas exert two opposite actions on α-cells: a direct stimulation as in β-cells and an indirect inhibition by SST. This suggests that any alteration of SST paracrine influence, as described in diabetes, will modify the effect of sulfonylureas on glucagon release. In addition, we suggest that δ-cells inhibit α-cells more efficiently than β-cells.

Optimizing Nicorandil for Spinal Cord Protection in a Murine Model of Complex Aortic Intervention

There are currently no clinically utilized pharmacological agents for the induction of metabolic tolerance to spinal cord ischemia-reperfusion injury in the setting of complex aortic intervention. Nicorandil, a nitric oxide donor and ATP-sensitive potassium (KATP) channel opener, has shown promise in neuroprotection. However, the optimized clinical application of the drug and its mechanism of neuroprotection remains unclear. We hypothesized that 3-days pretreatment would confer the most effective neuroprotection, mediated by mitochondrial KATP channel activation. Spinal cord injury was induced by 7 minutes of thoracic aortic cross-clamping in adult male C57BL/6 mice. Time course: mice received 0.1 mg/kg nicorandil for 10 min, 4 hours, and 3 consecutive days prior to ischemia compared with control. Dose challenge: mice received 3-days nicorandil pretreatment comparing 0.1 mg/kg, 1.0 mg/kg, 5.0 mg/kg, and saline administration. Mitochondrial KATP channel blocker 5-hydroxy-decanoate (5HD) was co-administered to elucidate mechanism. Limb motor function was evaluated, and viable anterior horn neurons quantified. Nicorandil pretreatment at 4 hours and 3 days before ischemia demonstrated significant motor function preservation; administration 10 minutes before ischemia showed no neuroprotection. All nicorandil doses showed significant motor function preservation. Three days administration of Nicorandil 1.0 mg/kg was most potent. Neuroprotection was completely abolished by 5HD co-administration. Histological analysis showed significant neuron preservation with nicorandil pretreatment, which was attenuated by 5HD co-administration. Three days administration of Nicorandil 1.0 mg/kg showed near-total motor function preservation in a murine spinal cord ischemia-reperfusion model, mediated by the mitochondrial KATP channel.

Relaxation of Goat Detrusor Muscle by L-arginine Involves NO-dependent but Guanylyl Cyclase Independent Activation of KCa Channels

Background: Urinary incontinence is a major problem both in man and animals particularly dogs. L-arginine, the precursor of NO, relaxes coronary artery smooth muscle by opening of KATP channels. L-arginine has beneficial circulatory effects in patients with essential and secondary hypertension. However, not much is known about the role of L-arginine on bladder physiology. In view of this, the present work investigated the functional role of L- arginine on detrusor smooth muscle of goat. Methods: Detrusor strips of goat, collected from local abattoir were mounted in a thermostatically controlled (37° ± 0.5°C) organ bath (20 ml capacity) containing physiological solution. After 1 hr of equilibrium, carbachol (CCh) (10-5 M) was used to induce sub-maximal contraction. L-arginine (10-3 M) was added at the plateau of contraction to see any observable effect in absence and presence of modulators of NO and ion channels. Result: L- arginine (10-3 M) reversed the contractions induced by CCh (10-5 M) on detrusor tissues. Methylene blue (MB) (10-5 M), the non-specific guanylyl cyclase inhibitor, failed to attenuate the relaxant response of L-arginine but, the NO synthase inhibitor L-NAME (3 x 10-6 M) inhibited the relaxant response of L-arginine. The KATP channel blocker glibenclamide (10-6 M) failed to inhibit the relaxation induced by L-arginine while KCa channel blocker tetraethylammonium (TEA) (10-3 M) inhibited the relaxant response of L-arginine. The results of the present study suggest that L-arginine produces relaxation of goat detrusor muscle and the L-arginine-elicited relaxation is NO-dependent but guanylyl cyclase independent which activates KCa channels.

Изменение вклада К АТФ -каналов в поддержание базального тонуса и дилатацию пиальных артериальных сосудов крыс после ишемии

The changes in the contribution of ATP-sensitive potassium channels (KATP) in basal tone and acetylcholine (ACh) mediated dilation of pial arterial vessels after global cerebral ischemia (2VO + hypo model) were studied in Wistar rats. Vascular examination was performed 7, 14 and 21 days after ischemia. False- operated rats served as a control. The change in the contribution of KATP channels in basal tone was assessed using intravital microphotography. The number of vessels that constricted in response to the effect of the KATP channel blocker (glibenclamide, 10 μM) was assessed. The change in the contribution of channels in vascular dilatation was assessed by comparing the number and degree of arterial dilatation in response to ACh (10-7 M, 5 min) before and after the use of a KATP channel blocker. It was found that ischemia decreases the role of ATP-sensitive potassium channels in basal tone of the pial arterial vessels for 14 days after ischemic exposure. At the same time, the contribution of KATP - channels in the ACh mediated dilator reactions of pial vessels decreases. And by 21 days after ischemia, KATP channels do not participate in the dilator response to ACh.

Hydrogen sulfide as a mediator of endothelium-dependent relaxation evoked by Moringa oleifera leaf extract in mesenteric arterial beds isolated from L-NAME hypertensive rats.

Aqueous extract of Moringa oleifera leaves (MOE) is a potent inducer of endothelium-dependent relaxation of mesenteric resistance arteries of rats induced to be hypertensive using Nω-nitro-L-arginine methyl ester (L-NAME). Hydrogen sulfide (H2S) has been shown to participate in endothelium-dependent relaxation of small resistance arteries. Therefore, this study aimed to investigate whether endothelial H2S-dependent signaling plays a role in the vasorelaxation in response to MOE. Mesenteric arterial beds isolated from L-NAME hypertensive rats were set up in an exvivo perfusion system for measurement of vasoreactivity. All experiments were performed in the presence of the nitric oxide synthase inhibitor, L-NAME (100µM) and the cyclooxygenase inhibitor, indomethacin (10µM) to prevent the formation of nitric oxide and prostanoids, respectively. In the presence of the nitric oxide synthase inhibitor, L-NAME and the cyclooxygenase inhibitor, indomethacin, the endothelium-dependent vasorelaxation induced by MOE (0.001-3mg) was completely inhibited by DL-propargylglycine (100µM), which inhibits the H2Sgenerating enzyme, cystathionine γ-lyase. This H2Sdependent response was reduced by the KATP channel blocker; glibenclamide (10µM), the KCa channel blocker; tetraethylammonium (1µM), and the myo-endothelial gap-junctional uncoupler; 18α-glycyrrhetinic acid (10µM). In contrast, the muscarinic receptor antagonist, atropine (100µM), did not affect the response to MOE. The results may suggest that H2S is the likely mediator of endothelium-dependent relaxation in response to MOE in mesenteric arterial beds of L-NAME-induced hypertensive rats. MOE-induced H2S-dependent vasorelaxation involves activation of KATP and KCa channels and requires myo-endothelial gap-junctional communication.

Pathophysiological Consequences of KATP Channel Overactivity and Pharmacological Response to Glibenclamide in Skeletal Muscle of a Murine Model of Cantù Syndrome.

Cantù syndrome (CS) arises from mutations in ABCC9 and KCNJ8 genes that lead to gain of function (GOF) of ATP-sensitive potassium (KATP) channels containing SUR2A and Kir6.1 subunits, respectively, of KATP channels. Pathological consequences of CS have been reported for cardiac and smooth muscle cells but consequences in skeletal muscle are unknown. Children with CS show muscle hypotonia and adult manifest fatigability. We analyzed muscle properties of Kir6.1[V65M] CS mice, by measurements of forelimb strength and ultrasonography of hind-limb muscles, as well as assessing KATP channel properties in native Flexor digitorum brevis (FDB) and Soleus (SOL) fibers by the patch-clamp technique in parallel with histopathological, immunohistochemical and Polymerase Chain Reaction (PCR) analysis. Forelimb strength was lower in Kir6.1wt/VM mice than in WT mice. Also, a significant enhancement of echodensity was observed in hind-limb muscles of Kir6.1wt/VM mice relative to WT, suggesting the presence of fibrous tissue. There was a higher KATP channel current amplitude in Kir6.1wt/VM FDB fibers relative to WT and a reduced response to glibenclamide. The IC50 of glibenclamide to block KATP channels in FDB fibers was 1.3 ± 0.2 × 10−7 M in WT and 1.2 ± 0.1 × 10−6 M in Kir6.1wt/VM mice, respectively; and it was 1.2 ± 0.4 × 10−7 M in SOL WT fibers but not measurable in Kir6.1wt/VM fibers. The sensitivity of the KATP channel to MgATP was not modified in Kir6.1wt/VM fibers. Histopathological/immunohistochemical analysis of SOL revealed degeneration plus regressive-necrotic lesions with regeneration, and up-regulation of Atrogin-1, MuRF1, and BNIP3 mRNA/proteins in Kir6.1wt/VM mice. Kir6.1wt/VM mutation in skeletal muscle leads to changes of the KATP channel response to glibenclamide in FDB and SOL fibers, and it is associated with histopathological and gene expression changes in slow-twitch muscle, suggesting marked atrophy and autophagy.

Abstract 13107: Nitroglycerin Pretreatment Induces Metabolic Tolerance to Spinal Cord Ischemia-reperfusion Injury Through Nitric Oxide-mediated Mitochondrial Katp Channel Activation

Introduction: Pharmacologic induction of metabolic tolerance to spinal cord ischemia-reperfusion injury (SCI) after thoracoabdominal aortic intervention is not well established. We previously demonstrated that nicorandil pretreatment preserved motor function in a murine SCI model via direct mitochondrial ATP-sensitive potassium (KATP) channel activation and nitric oxide (NO) donation. However, the independent role of NO-mediated neuroprotection has not been elucidated. Hypothesis: Nitroglycerin pretreatment will induce neuroprotection through NO-mediated KATP channel activation. Methods: SCI was induced by 7 minutes of thoracic aortic cross-clamping in adult male C57BL/6 mice. Pretreatment constituted intraperitoneal injection 3 consecutive days before injury. Experimental groups: sham (no pretreatment or ischemia, n=10), SCI control (normal saline, n=20), nitroglycerin 1 mg/kg (n=18), nitroglycerin 1 mg/kg + 5-hydroxydecanonate 5 mg/kg (5HD, mitochondrial KATP channel blocker, n=13), 5HD 5 mg/kg (n=10), nitroglycerin 1 mg/kg + carboxy-PTIO (cPTIO) 1 mg/kg (NO scavenger, n=16), and cPTIO 1 mg/kg (n=10). Limb motor function and the number of viable neurons within the anterior horn of the spinal cord were evaluated. Results: Compared to SCI control, motor function was significantly preserved in the nitroglycerin pretreatment group at every time point after ischemia. In the nitroglycerin+5HD and nitroglycerin+cPTIO groups, motor preservation was significantly attenuated compared to nitroglycerin pretreatment (p&lt;0.001). Histological analysis showed significant neuron preservation in the nitroglycerin pretreatment group compared with SCI control (p=0.011). This preservation was completely attenuated with 5HD or cPTIO co-administration (p=0.001). Conclusions: Nitroglycerin pretreatment significantly preserved motor function in a murine SCI model through NO-mediated KATP channel activation.

Protective effect of modafinil on skin flap survival in the experimental random-pattern skin flap model in rats: The role of ATP-sensitive potassium channels and nitric oxide pathway

Background The brain-stimulating agent modafinil acts through nitric oxide (NO) and adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, involved in the skin flap survival (SFS). The main aim of this study was to investigate the efficacy of modafinil on SFS in rats through the involvement of NO pathway and KATP channels. Methods Using controlled experiment study design, we enrolled a sample of Wistar male rats. Different doses of modafinil (10, 25, 50, and 100 mg/kg) were injected intraperitoneally (i.p.) before the surgery. L-NAME (non-selective nitric oxide synthase [NOS] inhibitor), aminoguanidine (inducible NOS inhibitor), and 7-nitroindazole (neuronal NOS inhibitor) were administered prior to modafinil. The role of KATP channels was determined by coadministering glibenclamide (KATP channel blocker) or cromakalim (KATP channel opener) with modafinil. The predictor variables were administration of different doses of modafinil, and the coadministration of modafinil with L-NAME, aminoguanidine, 7-nitroindazole, glibenclamide, and cromakalim. The main outcome variables included the percentage of necrotic area (PNA) in flap tissues, histopathological results, vascular endothelial growth factor (VEGF) immunohistochemical (IHC) staining, and nitrite concentrations. Appropriate statistics were computed considering p-value ≤ 0.05 significant. Results Modafinil 25 mg/kg was the most effective dose (PNA: 26 [95% CI: 19–33]) vs. control (PNA: 81 [95% CI: 71–92]) (p< 0.001). All NOS inhibitors significantly reversed the protective effect of modafinil (p< 0.001). Non-effective dose of cromakalim had a synergistic effect with the sub-effective dose of modafinil (10 mg/kg), while glibenclamide reversed the effect of modafinil 25 mg/kg (p< 0.001). Conclusions Modafinil increases SFS mediated by NO pathway and KATP channels, which could therefore be a target to improve SFS. The brain-stimulating agent modafinil acts through nitric oxide (NO) and adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, involved in the skin flap survival (SFS). The main aim of this study was to investigate the efficacy of modafinil on SFS in rats through the involvement of NO pathway and KATP channels. Using controlled experiment study design, we enrolled a sample of Wistar male rats. Different doses of modafinil (10, 25, 50, and 100 mg/kg) were injected intraperitoneally (i.p.) before the surgery. L-NAME (non-selective nitric oxide synthase [NOS] inhibitor), aminoguanidine (inducible NOS inhibitor), and 7-nitroindazole (neuronal NOS inhibitor) were administered prior to modafinil. The role of KATP channels was determined by coadministering glibenclamide (KATP channel blocker) or cromakalim (KATP channel opener) with modafinil. The predictor variables were administration of different doses of modafinil, and the coadministration of modafinil with L-NAME, aminoguanidine, 7-nitroindazole, glibenclamide, and cromakalim. The main outcome variables included the percentage of necrotic area (PNA) in flap tissues, histopathological results, vascular endothelial growth factor (VEGF) immunohistochemical (IHC) staining, and nitrite concentrations. Appropriate statistics were computed considering p-value ≤ 0.05 significant. Modafinil 25 mg/kg was the most effective dose (PNA: 26 [95% CI: 19–33]) vs. control (PNA: 81 [95% CI: 71–92]) (p< 0.001). All NOS inhibitors significantly reversed the protective effect of modafinil (p< 0.001). Non-effective dose of cromakalim had a synergistic effect with the sub-effective dose of modafinil (10 mg/kg), while glibenclamide reversed the effect of modafinil 25 mg/kg (p< 0.001). Modafinil increases SFS mediated by NO pathway and KATP channels, which could therefore be a target to improve SFS.

Glucagon-Like Peptide-1 Analog Liraglutide Attenuates Pressure-Overload Induced Cardiac Hypertrophy and Apoptosis through Activating ATP Sensitive Potassium Channels.

This study aimed to investigate whether inhibition of glucagon-like peptide-1 (GLP-1) on pressure overload induced cardiac hypertrophy and apoptosis is related to activation of ATP sensitive potassium (KATP) channels. Male SD rats were randomly divided into five groups: sham, control (abdominal aortic constriction), GLP-1 analog liraglutide (0.3mg/kg/twice day), KATP channel blocker glibenclamide (5mg/kg/day), and liraglutide plus glibenclamide. Relative to the control on week 16, liraglutide upregulated protein and mRNA levels of KATP channel subunits Kir6.2/SUR2 and their expression in the myocardium, vascular smooth muscle, aortic endothelium, and cardiac microvasculature. Consistent with a reduction in aortic wall thickness (61.4 ± 7.6 vs. 75.0 ± 7.6μm, p < 0.05), liraglutide enhanced maximal aortic endothelium-dependent relaxation in response to acetylcholine (71.9 ± 8.7 vs. 38.6 ± 4.8%, p < 0.05). Along with a reduction in heart to body weight ratio (2.6 ± 0.1 vs. 3.4 ± 0.4, mg/g, p < 0.05) by liraglutide, hypertrophied cardiomyocytes (371.0 ± 34.4 vs. 933.6 ± 156.6μm2, p < 0.05) and apoptotic cells (17.5 ± 8.2 vs. 44.7 ± 7.9%, p < 0.05) were reduced. Expression of anti-apoptotic protein BCL-2 and contents of myocardial ATP were augmented, and expression of cleaved-caspase 3 and levels of serum Tn-I/-T were reduced. Echocardiography and hemodynamic measurement showed that cardiac systolic function was enhanced as evidenced by increased ejection fraction (88.4 ± 4.8 vs. 73.8 ± 5.1%, p < 0.05) and left ventricular systolic pressure (105.2 ± 10.8 vs. 82.7 ± 7.9mmHg, p < 0.05), and diastolic function was preserved as shown by a reduction of ventricular end-diastolic pressure (-3.1 ± 2.9 vs. 6.7 ± 2.8mmHg, p < 0.05). Furthermore, left ventricular internal diameter at end-diastole (5.8 ± 0.5 vs. 7.7 ± 0.6mm, p < 0.05) and left ventricular internal diameter at end-systole (3.0 ± 0.6 vs. 4.7 ± 0.4mm, p < 0.05) were improved. Dietary administration of glibenclamide alone did not alter all the parameters measured but significantly blocked liraglutide-exerted cardioprotection. Liraglutide ameliorates cardiac hypertrophy and apoptosis, potentially via activating KATP channel-mediated signaling pathway. These data suggest that liraglutide might be considered as an adjuvant therapy to treat patients with heart failure.

High fat diet induces change in mitochondria respiration, activation of K-ATP channel and action potential shortening in mice

Obesity is associated with an increased risk of atrial fibrillation (AF). This epidemiologic observation may not be only due to shared co-morbidities but also from direct impacts of metabolic disorders on myocardium. To examine the impact of high fat diet (HFD) induced obesity on electrical properties in mice atria. Eight weeks old C57Bl/6 J mice were subjected to 4 months of HFD (60% fat) or normal diet (ND, 4% fat). Rapid burst atrial pacing were delivered using a trans-esophageal probe to induced AF in anesthetized animals. Action potential (AP) were recorded in left atrium using glass microelectrode technique. Potassium currents were recorded in isolated atrial myocytes using whole cell or perforated patch-clamp configurations. Mitochondrial respiration was studied in saponin-permeabilized atrial muscle fibers using a Clarke electrode. HFD mice showed higher atrial vulnerability to AF as indicated by longer AF episodes. APs were shorter in HFD versus ND mice (AP duration measured at 90% of repolarization: 49.3 ± 1.6 msec in HFD vs. 56.7 ± 2.3 msec in ND, P < 0.01). Glibenclamide, a KATP channel blocker, reversed AP shortening whereas cromakalim, a KATP channel activator, had no effect. Only in perforated patch-clamp configuration, without intracellular dialysis, KATP component of potassium current was enhanced in HFD mice (at +70 mV: 0.41 ± 0.12 pA/pF in HFD vs. 0.13 ± 0.08 pA/pF in ND, P < 0.05). There was no change in mitochondria respiration capacity between ND and HFD, however the ability of palmitoyl-CoA to support mitochondrial respiration was higher in HFD (25.99 ± 4.11% vs. 46.9 ± 3.96% in ND, P = 0.001). In addition, HADHA activity was enhanced in HFD mice. Obesity in mice is associated with atrial AP shortening which appears to result from increased activity of KATP-regulated currents and be associated with a metabolic shift. Shortening of AP duration could contribute to higher vulnerability to AF during obesity.

The antinociceptive mechanisms of melatonin: role of L-arginine/nitric oxide/cyclic GMP/KATP channel signaling pathway.

Pain is one of the most common medical challenges, reducing life quality. Despite the progression in pain management, it has remained a clinical challenge, which raises the need for investigating novel antinociceptive drugs with correspondence signaling pathways. Besides, the precise antinociceptive mechanisms of melatonin are not revealed. Accordingly, owing to the critical role of L-arginine/nitric oxide (NO)/cyclic GMP (cGMP)/KATP in the antinociceptive responses of various analgesics, the role of this signaling pathway is evaluated in the antinociceptive effects of melatonin. Male NMRI mice were intraperitoneally pretreated with the injection of L-arginine (NO precursor, 100 mg/kg), N(gamma)-nitro-L-arginine methyl ester [L-NAME, NO synthase (NOS) inhibitor, 30 mg/kg], S-nitroso-N-acetylpenicillamine (SNAP, NO donor, 1 mg/kg), sildenafil (phosphodiesterase inhibitor, 0.5 mg/kg), and glibenclamide (KATP channel blocker, 10 mg/kg) alone and before the administration of the most effective dose of melatonin amongst the intraperitoneal doses of 50, 100, and 150 mg/kg. The formalin test (2%, 25 µL, intra-plantarly) was done following the melatonin administration, then the nociceptive responses of mice were evaluated during the early phase for 5 min and the late phase for 15 min. The results showed that 100 mg/kg dose of melatonin carried out the most antinociceptive effects. While the antinociceptive effect of melatonin was increased by L-arginine, SNAP, and sildenafil, it was significantly reduced by L-NAME and glibenclamide in both phases of the formalin test, with no relation to the sedative effects of melatonin evaluated by the inclined plane test. In conclusion, the antinociceptive effect of melatonin is mediated through the L-arginine/NO/cGMP/KATP pathway.

The Involvement of ATP-Sensitive Potassium Channels in the Nebivolol-Induced Relaxation of Endothelium-Intact Aorta Isolated from Rats

Objective: Nebivolol is a highly selective beta-1 adrenergic receptor blocker with additional vasorelaxant properties. The vasorelaxant effect of nebivolol has been mainly attributed to endothelium-dependent mechanisms including beta-adrenergic receptors. However, the involvement of ATP-sensitive potassium (KATP) channels, another potential mechanism for vasorelaxant effect, in the vasorelaxant response to nebivolol remains unclear. Therefore, this study was aimed to investigate the role of KATP channels in the nebivolol-induced vasorelaxation in the isolated rat aorta Methods: The rat thoracic aortic rings isolated from Sprague-Dawley rats were mounted in organ bath chambers containing Krebs-Henseleit solution at 37 oC continuously bubbled with 95% O2 and 5% CO2. After an equilibration period, the presence of endothelium was confirmed by the response (more than 50%) to acetylcholine (10 μM) in aortic rings precontracted with phenylephrine (1 μM). After washout, in control group, the endothelium-intact aortic rings were contracted by potassium chloride (30 mM) before the cumulative addition of nebivolol (0.0001-100 μM). In some experiments, the relaxant response to nebivolol (0.0001-100 μM) was also obtained in the presence of glibenclamide (KATP channel blocker, 10 μM) or Nω-Nitro-L-arginine methyl ester (L-NAME: eNOS inhibitor, 100 μM) in the endothelium-intact aortic rings precontracted with potassium chloride (30 mM). Data were presented as means±SEM. Multiple comparisons of groups were performed by using ANOVA followed by post-hoc Bonferroni test. Results: Nebivolol elicited a concentration dependent vasorelaxant effect in the endothelium-intact aortic rings. Relaxant response to nebivolol was significantly inhibited by the presence of glibenclamide or L-NAME (p&amp;lt; 0.05). Although Emax values were not found significantly different among groups, pD2 values of nebivolol were reduced in the endothelium-intact aortic rings incubated with glibenclamide or L-NAME. Conclusion: These results demonstrate for the first time the involvement of KATP channels in the nebivolol-induced vasorelaxation in the endothelium-intact aorta precontracted with potassium chloride.

Role of Adenosine Triphosphate Sensitive Potassium Channels in Glucocorticoid Induced Myopathy of a Rat Model

Objective: To clarify adenosine triphosphate sensitive potassium (KATP ) channels' role in glucocorticoid induced myopathy induction. Materials and methods: 48 adult male rats divided into two experiments. Experiment Ⅰ: 32 rats divided equally into four group; Group Ⅰ: control group, Group Ⅱ: (Dexa group) (Dexamethasone 500 μg/kg once daily i.p), Group ΙΙΙ (Dexa+ Nicorandil) (Nicorandil (3 mg/kg) per oral once daily), Group ΙV (Dexa+Glibenclamide) (Glibenclamide (10 mg/kg) per oral once daily) all for 10 days. By the end of the experiment, blood samples were collected for blood glucose, serum insulin, potassium and creatine kinase-MM. Tibialis anterior muscles were isolated for contractility recording, oxidative stress measurement, histopathological examination and immunohistochemistry for insulin like growth factor-1(IGF-1). Experiment Ⅱ:16 rats (8 normal and 8 glucocorticoid induced myopathy rats). From each rat, the tibialis anterior was isolated and incubated with nicorandil (100 μmol /L for 10 minutes) and glibenclamide (1μmol /L for 5 minutes) for contractility recording. Results: Contractile properties, histopathological changes and IGF-1 muscle expression are improved with glibenclamide (KATP channel blocker) and worsened with nicorandil (KATP channel opener). CK-MM level significantly increased with nicorandil, while normal with glibenclamide. Serum potassium and blood glucose significantly increased with nicorandil and decreased with glibenclamide. Serum insulin significantly decreased with nicorandil and increased with glibenclamide. The oxidative stress state insignificantly changed with both drugs (p value ≤0.05 was considered to be statistically significant). Conclusion: Opening of KATP channels seems to have a role in the induction of glucocorticoid-induced myopathy probably by decreasing IGF-1 expression.

Glucagonotropic and Glucagonostatic Effects of KATP Channel Closure and Potassium Depolarization.

The role of depolarization in the inverse glucose-dependence of glucagon secretion was investigated by comparing the effects of KATP channel block and of high potassium. The secretion of glucagon and insulin by perifused mouse islets was simultaneously measured. Lowering glucose raised glucagon secretion before it decreased insulin secretion, suggesting an alpha cell-intrinsic signal recognition. Raising glucose affected glucagon and insulin secretion at the same time. However, depolarization by tolbutamide, gliclazide, or 15 mM KCl increased insulin secretion before the glucagon secretion receded. In contrast to the robust depolarizing effect of arginine and KCl (15 and 40 mM) on single alpha cells, tolbutamide was of variable efficacy. Only when applied before other depolarizing agents had tolbutamide a consistent depolarizing effect and regularly increased the cytosolic Ca2+ concentration. When tested on inside-out patches tolbutamide was as effective on alpha cells as on beta cells. In the presence of 1 µM clonidine, to separate insulinotropic from glucagonotropic effects, both 500 µM tolbutamide and 30 µM gliclazide increased glucagon secretion significantly, but transiently. The additional presence of 15 or 40 mM KCl in contrast led to a marked and lasting increase of the glucagon secretion. The glucagon secretion by SUR1 knockout islets was not increased by tolbutamide, whereas 40 mM KCl was of unchanged efficiency. In conclusion a strong and sustained depolarization is compatible with a marked and lasting glucagon secretion. KATP channel closure in alpha cells is less readily achieved than in beta cells, which may explain the moderate and transient glucagonotropic effect.

Dextromethorphan and Dextrorphan Influence Insulin Secretion by Interacting with KATP and L-type Ca2+ Channels in Pancreatic β-Cells.

The NMDA receptor antagonist dextromethorphan (DXM) and its metabolite dextrorphan (DXO) have been recommended for treatment of type 2 diabetes mellitus because of their beneficial effects on insulin secretion. This study investigates how different key points of the stimulus-secretion coupling in mouse islets and β-cells are influenced by DXM or DXO. Both compounds elevated insulin secretion, electrical activity, and [Ca2+]c in islets at a concentration of 100 µM along with a stimulating glucose concentration. DXO and DXM increased insulin secretion approximately 30-fold at a substimulatory glucose concentration (3 mM). Patch-clamp experiments revealed that 100 µM DXM directly inhibited KATP channels by about 70%. Of note, DXM decreased the current through L-type Ca2+ channels about 25%, leading to a transient reduction in Ca2+ action potentials. This interaction might explain why elevating DXM to 500 µM drastically decreased insulin release. DXO inhibited KATP channels almost equally. In islets of KATP channel-deficient sulfonylurea receptor 1 knockout mice, the elevating effects of 100 µM DXM on [Ca2+]c and insulin release were completely lost. By contrast, 100 µM DXO still increased glucose-stimulated insulin release around 60%. In summary, DXM-induced alterations in stimulus-secretion coupling of wild-type islets result from a direct block of KATP channels and are partly counteracted by inhibition of L-type Ca2+ channels. The stimulatory effect of DXO seems to be based on a combined antagonism on KATP channels and NMDA receptors and already occurs under resting conditions. Consequently, both compounds seem not to be suitable candidates for treatment of type 2 diabetes mellitus. SIGNIFICANCE STATEMENT: This study shows that the use of dextromethorphan as an antidiabetic drug can cause unpredictable alterations in insulin secretion by direct interaction with KATP and L-type Ca2+ channels besides its actual target, the NMDA receptor.