KATP Channel Blocker Glibenclamide Research Articles

Levosimendan Relaxes Thoracic Aortic Smooth Muscle in Mice by Inhibiting PKC and Activating Inwardly Rectifying Potassium Channels.

Studies have examined the therapeutic effect of levosimendan on cardiovascular diseases such as heart failure, perioperative cardiac surgery, and septic shock, but the specific mechanism in mice remains largely unknown. This study aimed to investigate the relaxation mechanism of levosimendan in the thoracic aorta smooth muscle of mice. Levosimendan-induced relaxation of isolated thoracic aortic rings that were precontracted with norepinephrine (NE) or KCl was recorded in an endothelium-independent manner. Vasodilatation by levosimendan was not associated with the production of the endothelial relaxation factors NO and PGI2. The voltage-dependent K+ channel (KV) blocker (4-aminopyridine) and selective KCa blocker (tetraethylammonium) had no effect on thoracic aortas treated with levosimendan, indicating that KV and KCa channels may not be involved in the levosimendan-induced relaxation mechanism. Although the inwardly rectifying K+ channel (Kir) blocker (barium chloride) and the KATP channel blocker (glibenclamide) significantly inhibited levosimendan-induced vasodilation in the isolated thoracic aorta, barium chloride had a much stronger inhibitory effect on levosimendan-induced vasodilation than glibenclamide, suggesting that levosimendan-induced vasodilation may be mediated by Kir channels. The vasodilation effect and expression of Kir 2.1 induced by levosimendan were further enhanced by the PKC inhibitor staurosporine. Extracellular calcium influx was inhibited by levosimendan without affecting intracellular Ca2+ levels in the isolated thoracic aorta. These results suggest that Kir channels play a more important role than KATP channels in regulating vascular tone in larger arteries and that the activity of the Kir channel is enhanced by the PKC pathway.

Evidence for the involvement of TRPV2 channels in the modulation of vascular tone in the mouse aorta

AimsTransient receptor potential vanilloid 2 (TRPV2) channels are expressed in both smooth muscle and endothelial cells and participate in vascular mechanotransduction and sensing of high temperatures and lipids. Nevertheless, the impact of TRPV2 channel activation by agonists on the coordinated and cell-type specific modulation of vasoreactivity is unknown. Main methodsAorta from 2- to 4-months-old male Oncins France 1 mice was dissected and mounted in tissue baths for isometric tension measurements. TRPV2 channel expression was assessed by immunofluorescence and western blot in mice aortas and in cultured A7r5 rat aortic smooth muscle cells. Key findingsTRPV2 channels were expressed in all three mouse aorta layers. Activation of TRPV2 channels with probenecid evoked endothelium-dependent relaxations through a mechanism that involved activation of smooth muscle Kir and Kv channels. In addition, TRPV2 channel inhibition with tranilast increased endothelium-independent relaxations to probenecid and this effect was abrogated by the KATP channel blocker glibenclamide, revealing that smooth muscle TRPV2 channels induce negative feedback on probenecid relaxations mediated via KATP channel inhibition. Exposure to the NO donor sodium nitroprusside increased TRPV2 channel translocation to the plasma membrane in cultured smooth muscle cells and enhanced negative feedback on probenecid relaxations. SignificanceIn conclusion, we present the first evidence that TRPV2 channels may modulate vascular tone through a balance of opposed inputs from the endothelium and the smooth muscle leading to net vasodilation. The fact that TRPV2 channel-induced activity can be amplified by NO emphasizes the pathophysiological relevance of these findings.

Glibenclamide promotes FGF21 secretion in interscapular BAT and attenuates depression-like behaviors in male mice with HFD-induced obesity

AimsEpidemiological evidence suggests that comorbidity of obesity and depression is extremely common and continues to grow in prevalence. However, the mechanisms connecting these two conditions are unknown. In this study, we explored how treatment with KATP channel blocker glibenclamide (GB) or the well-known metabolic regulator FGF21 impact male mice with high-fat diet (HFD)-induced obesity and depressive-like behaviors. Materials and methodsMice were fed with HFD for 12 weeks and then treated with recombinant FGF21 protein by infusion for 2 weeks, followed by intraperitoneal injection of 3 mg/kg recombinant FGF21 once per day for 4 days. Measurements were made of catecholamine levels, energy expenditure, biochemical endpoints and behavior tests, including sucrose preference and forced swim tests were. Alternatively, animals were infused with GB into brown adipose tissue (BAT). The WT-1 brown adipocyte cell line was used for molecular studies. Key findingsCompared to HFD controls, HFD + FGF21 mice exhibited less severe metabolic disorder symptoms, improved depressive-like behaviors, and more extensive mesolimbic dopamine projections. FGF21 treatment also rescued HFD-induced dysregulation of FGF21 receptors (FGFR1 and co-receptor β-klotho) in the ventral tegmental area (VTA), and it altered dopaminergic neuron activity and morphology in HFD-fed mice. Importantly, we also found that FGF21 mRNA level and FGF21 release were increased in BAT after administration of GB, and GB treatment to BAT reversed HFD-induced dysregulation of FGF21 receptors in the VTA. SignificanceGB administration to BAT stimulates FGF21 production in BAT, corrects HFD-induced dysregulation of FGF21 receptor dimers in VTA dopaminergic neurons, and attenuates depression-like symptoms.

Recovery of the Functional Activity of K<sub>ATP</sub>-Channels of Pial Arteries after Ischemia/Reperfusion Using Cell Therapy

This study aims to examine the efficiency of intravenous transplantation of human mesenchymal stem cells (hMSCs) performed 7 days after cerebral ischemia/reperfusion (I/R) for recovery of the functional activity of KATP-channels of cerebral arteries. Using a device for intravital visualization of pial vessels, the reaction of arteries to the KATP-channel blocker glibenclamide (GB), the activator of the same channels of pinacidil (PI), acetylcholine (ACh), and ACh against a background of GB action (ACh/GB) 14 and 21 days after I/R and intravenous hMSC transplantation performed 7 days after ischemic exposure. On exposure to GB 14 days after I/R, 1.5–1.8 times fewer arteries narrowed than in the sham–operated (SO) rats. By day 21 after I/R, the constriction reaction was completely restored, except for arteries with a diameter more 40 μm. In the cell–therapy group, the constrictor response to GB was completely recovered to the level of SO animals in arteries with a diameter less than 40 μm by 14 day after I/R exposure; in arteries with a diameter of more than 40 μm, the constriction reaction did not recover until 21 days. The number of dilations per ACh/GB compared to a clear ACh in SO rats was reduced in 1.6–1.8 times on 14 day after I/R and in 1.6–6.6 after 21 days. In I/R animals on 14 day, the number of dilatations per ACh/GB compared to clear ACh was significantly increased in arteries with a diameter of more than 20 μm by 1.5–1.7 times, and after 21 days in arteries with a diameter of more than 40 μm by 1.2 times. After the introduction of hMSC, GB blocked ACh–mediated dilation in arteries less than 40 μm in diameter both on days 14 and 21 after I/R. In arteries with a diameter of more than 40 μm the functional activity of KATP-channels did not recover until 21 days. Conclusion. I/R of the rat cerebral cortex reduces the contribution of KATP-channels to maintaining the basal tone of the pial arteries and almost completely excludes these channels from the formation of ACh–mediated dilation during 21 days of the postischemic period. Practically did not participate in the dilatory response. Intravenous transplantation of hMSC, performed 7 days after I/R, results in restoration of participation of SMC KATP-channels in maintaining the basal tone and ACh–mediated dilatation of pial arteries with a diameter less than 40 μm already 14 days after I/R.

Glibenclamide Posttreatment Does Not Inhibit Levcromakalim Induced Headache in Healthy Participants: A Randomized Clinical Trial

Intravenous infusion of ATP-sensitive potassium channel (KATP) opener levcromakalim causes headache in humans and implicates KATP channels in headache pathophysiology. Whether KATP channel blocker glibenclamide inhibits levcromakalim-induced headache has not yet been elucidated. We aimed to investigate the effect of posttreatment with glibenclamide on levcromakalim-induced headache in healthy participants. In a double blind, randomized, three-arm, placebo-controlled, crossover study, 20 healthy participants were randomized to receive 20 mL of levcromakalim (0.05 mg/min (50 mg/mL)) or 20 mL placebo (isotonic saline) intravenously over 20 min followed by oral administration of 10 mg glibenclamide or placebo. Fifteen participants completed all three study days. The primary endpoint was the difference in incidence of headache (0–12 h) between glibenclamide and placebo. More participants developed headache on levcromakalim-placebo day (15/15, 100%) (P = 0.013) and levcromakalim-glibenclamide day (13/15, 86%) compared to placebo-placebo day (7/15, 46%) (P = 0.041). We found no difference in headache incidence between levcromakalim-placebo day and levcromakalim-glibenclamide day (P = 0.479). The AUC0–12 h for headache intensity was significantly larger in levcromakalim-placebo day and levcromakalim-glibenclamide day compared to placebo-placebo day (106.3 ± 215.8) (P < 0.01). There was no difference in the AUC0–12 h for headache intensity between the levcromakalim-placebo day (494 ± 336.6) and the levcromakalim-glibenclamide day (417 ± 371.6) (P = 0.836). We conclude that non-specific KATP channel inhibitor glibenclamide did not attenuate levcromakalim-induced headache in healthy volunteers. Future studies should clarify the involvement of the distinct isoforms of sulfonylurea receptor subunits of KATP channels in the pathogenesis of headache and migraine.

Protective Effect of Foxtail Millet Protein Hydrolysate on Ethanol and Pyloric Ligation-Induced Gastric Ulcers in Mice.

Foxtail millet has been traditionally considered to possess gastroprotective effects, but studies evaluating its use as a treatment for gastric ulcers are lacking. Here, we assessed the antiulcer effects of foxtail millet protein hydrolysate (FPH) and explored its mechanism by using blocking agents. In a mouse model of ethanol-induced gastric ulcers, pretreatment with FPH reduced the ulcerative lesion index, downregulated the expression of inflammatory cytokines in the gastric tissue, increased the activity of antioxidant enzymes, and improved the oxidative status. FPH increased constitutive the activity of nitric oxide synthase (cNOS), NO levels, and mucin expression in gastric mucosa, and inhibited the activation of the ET-1/PI3K/Akt pathway. In a mouse model of pyloric ligation-induced gastric ulcers, FPH inhibited gastric acid secretion and decreased the activity of gastric protease. Pretreatment of mice with the sulfhydryl blocker NEM and the NO synthesis inhibitor L-NAME abolished the gastroprotective effect of FPH, but not the KATP channel blocker glibenclamide and the PGE2 synthesis blocker indomethacin. Among the peptides identified in FPH, 10 peptides were predicted to have regulatory effects on the gastric mucosa, and the key sequences were GP and PG. The results confirmed the gastroprotective effect of FPH and revealed that its mechanism was through the regulation of gastric mucosal mucus and NO synthesis. This study supports the health effects of a millet-enriched diet and provides a basis for millet protein as a functional food to improve gastric ulcers and its related oxidative stress.

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.

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.

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.

The role of KATP channel blockade and activation in the protection against neurodegeneration in the rotenone model of Parkinson's disease

AimsSeveral studies suggested that ATP-sensitive potassium channels (KATP) are potential therapeutic targets for protection against various neurodegenerative disorders, yet, there is an ongoing controversy regarding their role in Parkinson's disease (PD). Thus, the aim of the current study is to investigate the protective effect of KATP blockade and activation in the mice rotenone model of PD. Main methodsPD has been induced by 9 subcutaneous injections of rotenone (1.5 mg/kg; 3 times/week) in adult male Swiss albino mice. For 3 consecutive weeks, parkinsonian mice were either untreated or treated with L-dopa (25 mg/kg), the KATP channel blocker glibenclamide (3 mg/kg) or the KATP channel opener nicorandil (6 mg/kg). Key findingsGlibenclamide significantly improved motor performance in the wire hanging and stair tests and halted the decline in striatal dopamine content as well as dopaminergic neurons' density. In addition, it reduced the rotenone-induced apoptosis as portrayed in the immunohistopathological examination via increasing Bcl-2 and decreasing caspases-3, -8, -9 contents. Furthermore, through its anti-inflammatory potential, glibenclamide reduced tumor necrosis factor-alpha level. On the other hand, nicorandil failed to mitigate the rotenone-induced neurodegenerative consequences. SignificanceKATP channel blockade by glibenclamide has neuroprotective effect against rotenone-induced neurotoxicity, that was mediated by its anti-inflammatory effect along with hindering apoptosis through extrinsic and intrinsic pathways.

Vasorelaxing Activity of Stilbenoid and Phenanthrene Derivatives from Brasiliorchis porphyrostele: Involvement of Smooth Muscle CaV1.2 Channels.

Five compounds, 3,4'-dihydroxy-3',5,5'-trimethoxydihydrostilbene, 1: ; 3,4'-ihydroxy-3',5'-dimethoxydihydrostilbene, 2: ; 3,4'-dihydroxy-5,5'-dimethoxydihydrostilbene, 3: ; 9,10-dihydro-2,7-dihydroxy-4,6-dimethoxyphenanthrene, 4: ; and the previously unreported 1,2,6,7-tetrahydroxy-4-methoxyphenanthrene, 5: were isolated from the South American orchid, Brasiliorchis porphyrostele. An in-depth analysis of their vascular effects was performed on in vitro rat aorta rings and tail main artery myocytes. Compounds 1: - 4: were shown to possess vasorelaxant activity on rings pre-contracted by the α 1 receptor agonist phenylephrine, the CaV1.2 stimulator (S)-(-)-Bay K 8644, or depolarized with high K+ concentrations. However, compound 5: was active solely on rings stimulated by 25 mM but not 60 mM K+. The spasmolytic activity of compounds 1: and 4: was significantly affected by the presence of an intact endothelium. The KATP channel blocker glibenclamide and the KV channel blocker 4-aminopyridine significantly antagonized the vasorelaxant activity of compounds 4: and 1: , respectively. In patch-clamp experiments, compounds 1: - 4: inhibited Ba2+ current through CaV1.2 channels in a concentration-dependent manner, whereas neither compound 4: nor compound 1: affected K+ currents through KATP and KV channels, respectively. The present in vitro, comprehensive study demonstrates that Brasiliorchis porphyrostele may represent a source of vasoactive agents potentially useful for the development of novel antihypertensive agents that has now to be validated in vivo in animal models of hypertension.

The surprising complexity of KATP channel biology and of genetic diseases.

The ATP-sensitive K+ channel (KATP) is formed by the association of four inwardly rectifying K+ channel (Kir6.x) pore subunits with four sulphonylurea receptor (SUR) regulatory subunits. Kir6.x or SUR mutations result in KATP channelopathies, which reflect the physiological roles of these channels, including but not limited to insulin secretion, cardiac protection, and blood flow regulation. In this issue of the JCI, McClenaghan et al. explored one of the channelopathies, namely Cantu syndrome (CS), which is a result of one kind of KATP channel mutation. Using a knockin mouse model, the authors demonstrated that gain-of-function KATP mutations in vascular smooth muscle resulted in cardiac remodeling. Moreover, they were able to reverse the cardiovascular phenotypes by administering the KATP channel blocker glibenclamide. These results exemplify how genetic mutations can have an impact on developmental trajectories, and provide a therapeutic approach to mitigate cardiac hypertrophy in cases of CS.

KATP Channel Blocker Glibenclamide Prevents Radiation-Induced Lung Injury and Inhibits Radiation-Induced Apoptosis of Vascular Endothelial Cells by Increased Ca2+ Influx and Subsequent PKC Activation.

Radiation-induced lung injury (RILI) is a common and severe side effect of thoracic radiotherapy, which compromises patients' quality of life. Recent studies revealed that early vascular injury, especially microvascular damage, played a central role in the development of RILI. For this reason, early vascular protection is essential for RILI therapy. The ATP-sensitive K+ (KATP) channel is an ATP-dependent K+ channel with multiple subunits. The protective role of the KATP channel in vascular injury has been demonstrated in some published studies. In this work, we investigated the effect of KATP channel on RILI. Our findings confirmed that the KATP channel blocker glibenclamide, rather than the KATP channel opener pinacidil, remitted RILI, and in particular, provided protection against radiation-induced vascular injury. Cytology experiments verified that glibenclamide enhanced cell viability, increased the potential of proliferation after irradiation and attenuated radiation-induced apoptosis. Involved mechanisms included increased Ca2+ influx and PKC activation, which were induced by glibenclamide pretreatment. In conclusion, the KATP channel blocker glibenclamide remitted RILI and inhibited the radiation-induced apoptosis of vascular endothelial cells by increased Ca2+ influx and subsequent PKC activation.

Effect of gestational diabetes mellitus and pregnancy-induced hypertension on human umbilical vein smooth muscle KATP channels

Gestational diabetes mellitus (GDM) and pregnancy-induced hypertension (PIH) can jeopardize mother and/or fetus. Vascular ATP-sensitive potassium (KATP) channels most likely participate in the processes of diabetes and hypertension. The aim of this research was to examine whether GDM and PIH cause changes in the expression and function of KATP channels in vascular smooth muscle of human umbilical vein (HUV). Western blot and immunohistochemistry detected significantly decreased expression of Kir6.1 subunit of KATP channels in GDM and PIH, while the expression of SUR2B was unchanged. In GDM, a K+ channel opener, pinacidil caused reduced relaxation of the endothelium-denuded HUVs compared to normal pregnancy. However, its effects in HUVs from PIH subjects were similar to normal pregnancy. In all groups KATP channel blocker glibenclamide antagonized the relaxation of HUV induced by pinacidil without change in the maximal relaxations indicating additional KATP channel-independent mechanisms of pinacidil action. Iberiotoxin, a selective antagonist of large-conductance calcium-activated potassium channels, inhibited the relaxant effect of pinacidil in PIH, but not in normal pregnancy and GDM. Experiments performed in K+-rich solution confirmed the existence of K+-independent effects of pinacidil, which also appear to be impaired in GDM and PIH. Thus, the expression of KATP channels is decreased in GDM and PIH. In GDM, vasorelaxant response of HUV to pinacidil is reduced, while in PIH it remains unchanged. It is very likely that KATP channels modulation and more detailed insight in KATP channel-independent actions of pinacidil may be precious in the therapy of pathological pregnancies.

Hypoxia augments NaHS-induced ANP secretion via KATP channel, HIF-1α and PPAR-γ pathway

It has been reported that sodium hydrosulfide (NaHS) stimulated high stretch induced-atrial natriuretic peptide (ANP) secretion via ATP sensitive potassium (KATP) channel. KATP channel is activated during hypoxic condition as a compensatory mechanism. However, whether NaHS affects ANP secretion during hypoxia remains obscure. The purpose of the present study is to discover the impact of NaHS on ANP secretion during hypoxia and to unravel its signaling pathway. Isolated beating rat atria were perfused with buffer exposed to different O2 tension (to 100% O2, normoxia; to 20% O2, hypoxia). The ANP secretion increased negatively correlated with O2 tension. NaHS (50 μM) did not show any significant effect on low stretch induced-ANP secretion in normoxic condition but augmented low stretch induced-ANP secretion in hypoxic condition. The augmentation of NaHS-induced ANP secretion during hypoxia was blocked by the pretreatment with KATP channel blocker (glibenclamide) and was enhanced by the pretreatment with KATP channel activator (pinacidil). Hypoxia increased the expression of PPAR-γ protein but did not change the expression of HIF-1α protein and eNOS phosphorylation. The NaHS-induced ANP secretion during hypoxia was also blocked by the pretreatment with HIF-1α inhibitor (2-methoxy- estradiol), PPAR−γ inhibitor (GW9662) but not by NOS inhibitor (L-NAME) and endothelin receptor inhibitor (bosentan). The intravenous infusion of NaHS increased plasma ANP level in monocrotaline-treated rats but not in sham rats. These results suggest that hypoxia augmented NaHS-induced ANP secretion partly through KATP channel, HIF-1α, and PPAR−γ pathway.

Muscarinic suppression of ATP-sensitive K+ channels mediated by the M3/Gq/11/phospholipase C pathway contributes to mouse ileal smooth muscle contractions.

ATP-sensitive K+ (KATP) channels are expressed in gastrointestinal smooth muscles, and their activity is regulated by muscarinic receptor stimulation. However, the physiological significance and mechanisms of muscarinic regulation of KATP channels are not fully understood. We examined the effects of the KATP channel opener cromakalim and the KATP channel blocker glibenclamide on electrical activity of single mouse ileal myocytes and on mechanical activity in ileal segment preparations. To explore muscarinic regulation of KATP channel activity and its underlying mechanisms, the effect of carbachol (CCh) on cromakalim-induced KATP channel currents ( IKATP) was studied in myocytes of M2 or M3 muscarinic receptor-knockout (KO) and wild-type (WT) mice. Cromakalim (10 µM) induced membrane hyperpolarization in single myocytes and relaxation in segment preparations from WT mice, whereas glibenclamide (10 µM) caused membrane depolarization and contraction. CCh (100 µM) induced sustained suppression of IKATP in cells from both WT and M2KO mice. However, CCh had a minimal effect on IKATP in M3KO and M2/M3 double-KO cells. The Gq/11 inhibitor YM-254890 (10 μM) and PLC inhibitor U73122 (1 μM), but not the PKC inhibitor calphostin C (1 μM), markedly decreased CCh-induced suppression of IKATP in WT cells. These results indicated that KATP channels are constitutively active and contribute to the setting of resting membrane potential in mouse ileal smooth muscles. M3 receptors inhibit the activity of these channels via a Gq/11/PLC-dependent but PKC-independent pathways, thereby contributing to membrane depolarization and contraction of smooth muscles. NEW & NOTEWORTHY We systematically investigated the regulation of ATP-sensitive K+ channels by muscarinic receptors expressed on mouse ileal smooth muscles. We found that M3 receptors inhibit the activity of ATP-sensitive K+ channels via a Gq/11/PLC-dependent, but PKC-independent, pathway. This muscarinic suppression of ATP-sensitive K+ channels contributes to membrane depolarization and contraction of smooth muscles.

Role of cGMP and EDHF Pathways in Hydrogen Sulfide‐induced Vasorelaxation in the Human Artery

BackgroundEndogenous H2S generated by mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT) in endothelium as a physical vasodilator and blood pressure regulator may play an important protective role in the pathogenesis of coronary heart disease. The present study aimed to investigate the role of cGMP and endothelium‐derived hyperpolarizing factor (EDHF) in H2S‐induced vasorelaxation in the human internal mammary artery (IMA).MethodsIn the human IMA segments from 54 patients undergoing coronary artery bypass grafting, the acetylcholine (ACh)‐induced relaxation and the effect of CAT inhibitor (aminooxyacetic acid [AOAA] and L‐aspartate) and KCa channel blocker or KATP channel blocker were studied in a myograph. Enzyme‐linked Immunosorbent Assay for PDE5A and western blotting analysis for eNOS and p‐eNOSser1177 were performed. Methylene blue assay was used to quantify the contents of H2S.ResultsThe maximal relaxation induced by ACh was significantly attenuated by AOAA or L‐aspartate, Iberiotixin (BKCa channel blocker) (P&lt;0.01), or IKCa channel blocker plus SKCa channel blocker (TRAM‐34+Apamin) (P&lt;0.01), or KATP channel blocker (Glibenclamide) (P&lt;0.01). The H2S donor NaHS increased eNOS phosphorylation at its activating site S1177 and down‐regulated PDE5A activity. Incubation with ACh for 4 hours significantly increased the H2S concentration.ConclusionsWe for the first time demonstrated in the human IMA that endogenous H2S could be produced under the stimulation of ACh and that CAT is one of the key enzymes in production of H2S in the endothelial cell of the IMA. H2S may activate eNOS to produce NO that activates PKG and may also inhibit PDE5A activity; both enhance the cGMP pathway‐mediated relaxation. Further, H2S as a potential EDHF directly stimulates KCa channels to induce vasodilatation. These findings may provide new therapeutical targets for the treatment of vasospasm of coronary artery bypass grafting vessels and provide a pharmacological basis for the development of new vasodilator drugs.Support or Funding InformationSupported by grants from the National Natural Science Foundation of China (81170148 &amp; 81641017), Zhejiang Provincial Natural Science Foundation (LY15H020008), &amp; Tianjin Binhai Key Platform for Creative Research Program (2012‐BK110004), Binhai New Area Health Bureau (2012BWKZ008, 2016BWKY007, 2016BWKZ003).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of KATP channels in human cervical cancer: Potential tools for diagnosis and therapy.

Various ion channels, including ATP-sensitive potassium (KATP) channels, are expressed in cancer and have been suggested as potential tumor markers and therapeutic targets. KATP channels are composed of at least two types of subunit, an inwardly rectifying K+ channel (Kir6.x) and a sulfonylurea receptor (SUR). However, the association between KATP channels and cervical cancer remains elusive. The present study determined that the Kir6.2, SUR1 and SUR2 subunits are expressed in cervical cancer cell lines and/or human biopsies. The potential association of subunit expression with tumor differentiation and invasion was analyzed. The effect of the KATP channel blocker glibenclamide on the proliferation of cervical cancer cell lines was also studied. Five cervical cancer cell lines, two primary cultures of cervical cancer cells, one normal keratinocyte cell line and 74 human biopsies were used in the experiments. The mRNA and protein levels of the Kir6.2 subunit were assessed by reverse transcription-polymerase chain reaction and immunochemistry, respectively. Cell proliferation was evaluated by MTT assay. Kir6.2 subunit overexpression compared with control, was observed in some cervical cancer cell lines and cervical tumor tissues. Additionally, increased KATP channel expression was observed in high-grade, poorly differentiated and invasive human cervical cancer biopsies. Kir6.2 subunit expression was not observed in the majority of the non-cancerous cervical tissues. The effect of the KATP channel blocker glibenclamide on the proliferation of five different cervical cancer cell lines was studied, revealing that as Kir6.2 mRNA expression increased, the inhibitory effect of glibenclamide also increased. The results of the present study suggest, for the first time to the best of our knowledge, that the KATP channel subunits, Kir6.2 and SUR2, could potentially represent tools for diagnosing and treating cervical cancer.

Role of l-arginine/SNAP/NO/cGMP/KATP channel signalling pathway in antinociceptive effect of α-terpineol in mice.

The main purpose of this study was to assess the role of l-arginine/SNAP/NO/cGMP/KATP channel pathway in analgesic effects of α-terpineol in mice. Male NMRI mice were pretreated intraperitoneally with NO precursor (l-arginine, 100mg/kg), NO synthase inhibitor (l-NAME, 30mg/kg), NO donor (SNAP, 1mg/kg), guanylyl cyclase inhibitor (methylene blue, 20mg/kg), PDE inhibitor (sildenafil, 0.5mg/kg), KATP channel blocker (glibenclamide, 10mg/kg) and naloxone (2mg/kg) 20min before the administration of α-terpineol. The formalin test was performed 20min after the administration of α-terpineol, and nociceptive responses of mice were recorded during 30min. A significant and dose-dependent antinociception was produced by α-terpineol (40 and 80mg/kg) in both the phases of formalin test. The antinociceptive effect of α-terpineol was significantly potentiated by l-arginine in the second phase while significantly antagonized by l-NAME in both phases of formalin test. Also, SNAP and sildenafil non-significantly enhanced-while methylene blue significantly diminished-the antinociceptive effect of α-terpineol in both phases of formalin test. Glibenclamide significantly reversed the α-terpineol-induced antinociception, indicating the involvement of KATP channels in antinociceptive effect of α-terpineol. These results indicate that the antinociceptive effect of α-terpineol is mediated through l-arginine/SNAP/NO/cGMP/KATP channel pathway.