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AIM: Apigenin, a natural bioflavonoid, is reported as an anti-diabetic agent since it possesses the ability to inhibit α-glucosidase activity, cause stimulation of insulin action and secretion, manage ROS, and prevent diabetes complications. Apigenin was identified as a new insulin secretagogue that enhances glucose-stimulated insulin secretion and seems like a better antidiabetic drug candidate. Here we explored the insulinotropic mechanism(s) of apigenin in vitro in mice islets and in vivo in diabetic rats. METHODS: Size-matched pancreatic islets were divided into groups and incubated in the presence or absence of apigenin and agonists or antagonists of major insulin signaling pathways. The secreted insulin was measured by ELISA. The intracellular cAMP was estimated by cAMP acetylation assay. The acute and chronic effects of apigenin were evaluated in diabetic rats. RESULTS: apigenin dose-dependently enhanced insulin secretion in isolated mice islets, and its insulinotropic effect was exerted at high glucose concentrations distinctly different from glibenclamide. Furthermore, apigenin amplified glucose-induced insulin secretion in depolarized and glibenclamide-treated islets. Apigenin showed no effect on intracellular cAMP concentration; however, an additive effect was observed by apigenin in both forskolin and IBMX-induced insulin secretion. Interestingly, H89, a PKA inhibitor, and U0126, a MEK kinase inhibitor, significantly inhibited apigenin-induced insulin secretion; however, no significant effect was observed by using ESI-05, an epac2 inhibitor. Apigenin improved glucose tolerance and increased glucose-stimulated plasma insulin levels in diabetic rats. Apigenin also lowered blood glucose in diabetic rats upon chronic treatment. CONCLUSION: Apigenin exerts glucose-stimulated insulin secretion by modulating the PKA-MEK kinase signaling cascade independent of K-ATP channels.
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Apigenina , Proteínas Quinasas Dependientes de AMP Cíclico , Diabetes Mellitus Experimental , Glucosa , Secreción de Insulina , Insulina , Animales , Apigenina/farmacología , Secreción de Insulina/efectos de los fármacos , Masculino , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Insulina/sangre , Ratones , Ratas , Transducción de Señal/efectos de los fármacos , Canales KATP/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , AMP Cíclico/metabolismo , Hipoglucemiantes/farmacología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ratones Endogámicos C57BL , Ratas Wistar , Glucemia/metabolismo , Glucemia/efectos de los fármacosRESUMEN
OBJECTIVE: Mitochondrial ATP-sensitive K+ (mitoKATP) channels are involved in neuronal and cardiac protection from ischemia and oxidative stress. Penile erection is a neurovascular event mediated by relaxation of the erectile tissue via nitric oxide (NO) released from nerves and endothelium. In the present study, we investigated whether mitoKATP channels play a role in the control of penile vascular tone and mitochondrial dynamics, and the involvement of NO. METHODS: The effect of the selective mitoKATP activator BMS191095 was examined on vascular tone, on mitochondrial bioenergetics by real-time measurements with Agilent Seahorse and on ROS production by MitoSOX fluorescence in freshly isolated microarteries. RESULTS: BMS191095 and diazoxide relaxed penile arteries, BMS191095 being one order of magnitude more potent. BMS191095-induced relaxations were reduced by mechanical endothelium removal and by inhibitors of the nitric oxide synthase (NOS) and PI3K enzymes. The NO-dependent component of the relaxation to BMS191095 was impaired in penile arteries from insulin resistant obese rats. The blockers of mitoKATP channel 5-HD, sarcolemma KATP (sarcKATP) channel glibenclamide, and large conductance Ca2+-activated K+ (BKCa) channel iberiotoxin, inhibited relaxations to BMS191095 and to the NO donor SNAP. BMS191095 reduced the mitochondrial bioenergetic profile of penile arteries and attenuated mitochondrial ROS production. Blockade of endogenous NO impaired and exogenous NO mimicked, respectively, the inhibitory effects of BMS191095 on basal respiration and oxygen consumed for ATP synthesis. Exogenous NO exhibited dual inhibitory/stimulatory effects on mitochondrial respiration. CONCLUSIONS: These results demonstrate that selective activation of mitoKATP channels causes penile vasodilation, attenuates ROS production and inhibits mitochondrial respiration in part by releasing endothelial NO. These mechanisms couple blood flow and metabolism in penile arterial wall and suggest that activation of vascular mitoKATP channels may protect erectile tissue against ischemic injury.
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Óxido Nítrico , Canales de Potasio , Vasodilatación , Masculino , Ratas , Animales , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Adenosina Trifosfato , RespiraciónRESUMEN
Dopaminergic neurons in the substantia nigra (SN) expressing SUR1/Kir6.2 type ATP-sensitive potassium channels (K-ATP) are more vulnerable to rotenone or metabolic stress, which may be an important reason for the selective degeneration of neurons in Parkinson's disease (PD). Baicalein has shown neuroprotective effects in PD animal models. In this study, we investigated the effect of baicalein on K-ATP channels and the underlying mechanisms in rotenone-induced apoptosis of SH-SY5Y cells. K-ATP currents were recorded from SH-SY5Y cells using whole-cell voltage-clamp recording. Drugs dissolved in the external solution at the final concentration were directly pipetted onto the cells. We showed that rotenone and baicalein opened K-ATP channels and increased the current amplitudes with EC50 values of 0.438 µM and 6.159 µM, respectively. K-ATP channel blockers glibenclamide (50 µM) or 5-hydroxydecanoate (5-HD, 250 µM) attenuated the protective effects of baicalein in reducing reactive oxygen species (ROS) content and increasing mitochondrial membrane potential and ATP levels in rotenone-injured SH-SY5Y cells, suggesting that baicalein protected against the apoptosis of SH-SY5Y cells by regulating the effect of rotenone on opening K-ATP channels. Administration of baicalein (150, 300 mg·kg-1·d-1, i.g.) significantly inhibited rotenone-induced overexpression of SUR1 in SN and striatum of rats. We conducted surface plasmon resonance assay and molecular docking, and found that baicalein had a higher affinity with SUR1 protein (KD = 10.39 µM) than glibenclamide (KD = 24.32 µM), thus reducing the sensitivity of K-ATP channels to rotenone. Knockdown of SUR1 subunit reduced rotenone-induced apoptosis and damage of SH-SY5Y cells, confirming that SUR1 was an important target for slowing dopaminergic neuronal degeneration in PD. Taken together, we demonstrate for the first time that baicalein attenuates rotenone-induced SH-SY5Y cell apoptosis through binding to SUR1 and activating K-ATP channels.
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Flavanonas , Neuroblastoma , Canales de Potasio de Rectificación Interna , Humanos , Ratas , Animales , Canales KATP , Rotenona/farmacología , Receptores de Sulfonilureas , Canales de Potasio de Rectificación Interna/metabolismo , Gliburida/farmacología , Simulación del Acoplamiento Molecular , Apoptosis , Neuronas Dopaminérgicas/metabolismo , Adenosina Trifosfato/farmacologíaRESUMEN
AIMS: Transient 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 METHODS: Aorta 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 FINDINGS: TRPV2 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. SIGNIFICANCE: In 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.
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Células Endoteliales , Probenecid , Ratones , Ratas , Masculino , Animales , Probenecid/farmacología , Mecanotransducción Celular , Aorta/metabolismo , Vasodilatación , Adenosina Trifosfato/metabolismo , Endotelio Vascular/fisiologíaRESUMEN
OBJECTIVES: The goal of the research is to investigate the protocatechuic acid (PCA) potential action, a phenolic acid derivative, on pain induced by neuropathy and to determine its efficacy on activation of KATP type channels and A1 receptors. MATERIALS AND METHODS: Neuropathic pain by cause of sciatic nerve damage was induced in Sprague-Dawley rats. Anti-allodynic and anti-hyperalgesic effects were evaluated with von Frey apparatus and Hargreave's plantar test apparatus, respectively. The effects of PCA at the doses of 75, 150 and 300 mg/kg, carbamazepine at the doses of 50 and 100 mg/kg, combination of low effective doses of PCA and carbamazepine were tested. Pretreatments 3 µg/kg DPCPX as adenosine A1 receptor antagonist and 60.7 nmol glibenclamide as KATP channel blocker were applied for mechanistic studies. RESULTS: PCA showed anti-allodynic and anti-hyperalgesic effects without impairing locomotor activity. In addition, the combination treatment was found to be more effective than the separate individual treatments of drugs. KATP channel activation related with A1 receptor stimulation makes a significant contribution to the anti-allodynia and anti-hyperalgesia induced by PCA. CONCLUSIONS: It can be said that PCA has similar effects with carbamazepine, which is used in clinical practice, and that PCA can take place as an adjuvant drug in neuropathic pain with the combination group. In addition, it is seen that the undesirable effects that drugs can cause alone can be avoided and a more effective treatment potential can be created with multiple mechanisms.
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Neuralgia , Ratas , Animales , Ratas Sprague-Dawley , Neuralgia/tratamiento farmacológico , Hiperalgesia/tratamiento farmacológico , Carbamazepina/uso terapéutico , Adenosina Trifosfato/uso terapéuticoRESUMEN
Naringin (NRG) is a flavonoid with recognized cardioprotective effects. Then, it was investigated the cardioprotective mechanisms of NRG against ischemia-reperfusion (I/R) injury. The rats were pretreated for 7 days (v.o.) with NRG (25 mg/kg) or n-acetylcysteine (NAC, 100 mg/kg) and their isolated hearts were subjected to global ischemia (30 min) and reperfusion (60 min). Furthermore, isolated hearts were perfused with 5 µM NRG in the presence of 10 µM glibenclamide (GLI) and subjected to I/R protocol. In healthy ventricular cardiomyocyte, it was evaluated the acute effect of 5 µM NRG on the GLI sensitive current. The results showed that NRG pretreatment restored the cardiac function and electrocardiogram (ECG) alterations induced by I/R injury, decreasing arrhythmia scores and the occurrence of severe arrhythmias. Lactate dehydrogenase and infarct area were decreased while superoxide dismutase (SOD), catalase and citrate synthase activities increased. Expression of SOD CuZn and SOD Mn not was altered. NRG treatment decreased reactive oxygen species (ROS) generation and lipid peroxidation without alter sulfhydryl groups and protein carbonylation. Also, NRG (5 µM) increased the glibenclamide sensitive current in isolated cardiomyocytes. In isolated heart, the cardioprotection of NRG was significantly reduced by GLI. Furthermore, NRG promoted downregulation of Bax expression and Bax/Bcl-2. Histopathological analysis showed that NRG decreased cell edema, cardiomyocytes and nucleus diameter. Thus, NRG has a cardioprotective effect against cardiac I/R injury which is mediated by its antioxidant and antiapoptotic actions and KATP channels activation.
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Previous report indicated that nicorandil potentiated morphine antinociception and attenuated hepatic injury in liver fibrotic rats. Herein, the underlying mechanisms of nicorandil/morphine interaction were investigated using pharmacological, biochemical, histopathological, and molecular docking studies. Male Wistar rats were injected intraperitoneally (i.p.) with carbon tetrachloride (CCl4, 40%, 2 ml/kg) twice weekly for 5 weeks to induce hepatic fibrosis. Nicorandil (15 mg/kg/day) was administered per os (p.o.) for 14 days in presence of the blockers; glibenclamide (KATP channel blocker, 5 mg/kg, p.o.), L-NG-nitro-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor, 15 mg/kg, p.o.), methylene blue (MB, guanylyl cyclase inhibitor, 2 mg/kg, i.p.) and naltrexone (opioid antagonist, 20 mg/kg, i.p.). At the end of the 5th week, analgesia was evaluated using tail flick and formalin tests along with biochemical determinations of liver function tests, oxidative stress markers and histopathological examination of liver tissues. Naltrexone and MB inhibited the antinociceptive activity of the combination. Furthermore, combined nicorandil/morphine regimen attenuated the release of endogenous peptides. Docking studies revealed a possible interaction of nicorandil on µ, κ and δ opioid receptors. Nicorandil/morphine combination protected against liver damage as evident by decreased liver enzymes, liver index, hyaluronic acid, lipid peroxidation, fibrotic insults, and increased superoxide dismutase activity. Nicorandil/morphine hepatoprotection and antioxidant activity were inhibited by glibenclamide and L-NAME but not by naltrexone or MB. These findings implicate opioid activation/cGMP versus NO/KATP channels in the augmented antinociception, and hepatoprotection, respectively, of the combined therapy and implicate provoked cross talk by nicorandil and morphine on opioid receptors and cGMP signaling pathway. That said, nicorandil/morphine combination provides a potential multitargeted therapy to alleviate pain and preserve liver function.
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Analgésicos Opioides , Morfina , Ratas , Masculino , Animales , Morfina/farmacología , Morfina/uso terapéutico , Analgésicos Opioides/farmacología , Nicorandil/farmacología , Nicorandil/uso terapéutico , NG-Nitroarginina Metil Éster/farmacología , Ratas Wistar , Naltrexona , Gliburida/farmacología , Gliburida/uso terapéutico , Simulación del Acoplamiento Molecular , Dolor/tratamiento farmacológico , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Adenosina Trifosfato , Óxido Nítrico/metabolismo , GMP Cíclico/metabolismo , Analgésicos/farmacologíaRESUMEN
Over the last decades, lactate has emerged as important energy substrate for the brain fueling of neurons. A growing body of evidence now indicates that it is also a signaling molecule modulating neuronal excitability and activity as well as brain functions. In this review, we will briefly summarize how different cell types produce and release lactate. We will further describe different signaling mechanisms allowing lactate to fine-tune neuronal excitability and activity, and will finally discuss how these mechanisms could cooperate to modulate neuroenergetics and higher order brain functions both in physiological and pathological conditions.
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Ácido Láctico , Neuronas , Ácido Láctico/metabolismo , Neuronas/metabolismo , Transducción de Señal , Encéfalo/metabolismo , Astrocitos/metabolismoRESUMEN
Kir6.2/SUR1 is an ATP-regulated potassium channel that acts as an intracellular metabolic sensor, controlling insulin and appetite-stimulatory neuropeptides secretion. In this Letter, we present the SAR around a novel Kir6.2/SUR1 channel opener scaffold derived from an HTS screening campaign. New series of compounds with tractable SAR trends and favorable potencies are reported.
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Receptores de Sulfonilureas , Receptores de Sulfonilureas/metabolismoRESUMEN
BACKGROUND: Early post-stroke seizure frequently occurs in stroke survivors within the first few days and is associated with poor functional outcomes. Therefore, efficient treatments of such complications with less adverse effects are pivotal. In this study, we investigated the possible beneficial effects of lasmiditan and sumatriptan against post-stroke seizures in mice and explored underlying mechanisms in their effects. METHODS: Stroke was induced by double ligation of the right common carotid artery in mice. Immediately after the ligation, lasmiditan (0.1 mg/kg, intraperitoneally [i.p.]) or sumatriptan (0.03 mg/kg, i.p.) were administered. Twenty-four hours after the stroke induction, seizure susceptibility was evaluated using the pentylenetetrazole (PTZ)-induced clonic seizure model. In separate experiments, naltrexone (a non-specific opioid receptor antagonist) and glibenclamide (a KATP channel blocker) were administered 15 min before lasmiditan or sumatriptan injection. To evaluate the underlying signaling pathways, ELISA analysis of inflammatory cytokines (TNF-α and IL-1ß) and western blot analysis of anti- and pro-apoptotic markers (Bcl-2 and Bax) were performed on mice isolated brain tissues. RESULTS: Lasmiditan (0.1 mg/kg, i.p.) and sumatriptan (0.03 mg/kg, i.p.) remarkably decreased seizure susceptibility in stroke animals by reducing inflammatory cytokines and neuronal apoptosis. Concurrent administration of naltrexone (10 mg/kg, i.p.) or glibenclamide (0.3 mg/kg, i.p.) with lasmiditan or sumatriptan resulted in a higher neuroprotection against clonic seizures and efficiently reduced the inflammatory and apoptotic markers. CONCLUSION: Lasmiditan and sumatriptan significantly increased post-stroke seizure thresholds in mice by suppressing inflammatory cytokines and neuronal apoptosis. Lasmiditan and sumatriptan seem to exert higher effects on seizure threshold with concurrent administration of the opioid receptors or KATP channels modulators.
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Fármacos Neuroprotectores , Accidente Cerebrovascular , Adenosina Trifosfato , Animales , Anticonvulsivantes/farmacología , Benzamidas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Gliburida/farmacología , Gliburida/uso terapéutico , Ratones , Modelos Teóricos , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Pentilenotetrazol , Piperidinas , Bloqueadores de los Canales de Potasio , Canales de Potasio/metabolismo , Piridinas , Receptores Opioides , Convulsiones/tratamiento farmacológico , Convulsiones/etiología , Convulsiones/prevención & control , Accidente Cerebrovascular/tratamiento farmacológico , Sumatriptán , Factor de Necrosis Tumoral alfa , Proteína X Asociada a bcl-2RESUMEN
ATP-sensitive potassium channels (KATP channels), a group of vital channels that link the electrical activity of the cell membrane with cell metabolism, were discovered on the ventricular myocytes of guinea pigs by Noma using the patch-clamp technique in 1983. Subsequently, KATP channels have been found to be expressed in pancreatic ß cells, cardiomyocytes, skeletal muscle cells, and nerve cells in the substantia nigra (SN), hippocampus, cortex, and basal ganglia. KATP channel openers (KCOs) diazoxide, nicorandil, minoxidil, and the KATP channel inhibitor glibenclamide have been shown to have anti-hypertensive, anti-myocardial ischemia, and insulin-releasing regulatory effects. Increasing evidence has suggested that KATP channels also play roles in Alzheimer's disease (AD), Parkinson's disease (PD), vascular dementia (VD), Huntington's disease (HD) and other neurodegenerative diseases. KCOs and KATP channel inhibitors protect neurons from injury by regulating neuronal excitability and neurotransmitter release, inhibiting abnormal protein aggregation and Ca2+ overload, reducing reactive oxygen species (ROS) production and microglia activation. However, KATP channels have dual effects in some cases. In this review, we focus on the roles of KATP channels and their related openers and inhibitors in neurodegenerative diseases. This will enable us to precisely take advantage of the KATP channels and provide new ideas for the treatment of neurodegenerative diseases.
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Canales KATP , Enfermedades Neurodegenerativas , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Cobayas , Humanos , Canales KATP/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Nicorandil/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
High doses of salicylate induce tinnitus in humans and experimental animals. The Dorsal Cochlear Nucleus is implicated with the genesis of tinnitus, and increased activity in this nucleus is seen in animal models of tinnitus. Incubation of brainstem slices containing the DCN with millimolar salicylate reduces the spontaneous firing of glycinergic cartwheel neurons and glycinergic neurotransmission on fusiform neurons, the principal neuron of this nucleus. However, the mechanism of salicylate mediating this effect is not known. Recently, we have shown that KATP channels strongly modulate the spontaneous firing of cartwheel neurons. We tested if KATP channels could mediate the effects of salicylate on cartwheel neurons. Perfusion of 1.4 mM salicylate hyperpolarizes the membrane of cartwheel neurons and stops firing. Salicylate produces an outward current similar to the KATP current seen in quiet cartwheel neurons. Activation of this current is occluded by the KATP agonist diazoxide, which is produced by the opening of KATP channels. The antagonist of AMP-kinase (AMPK), dorsomorphim, inhibited salicylate effects, suggesting that they could be mediated by activation of this kinase. Still, the AMPK agonist, AICAR, did not reproduce salicylate effects but occluded them. Additionally, inhibiting mitochondrial ATP synthesis with the protonophore CCCP reproduced, albeit with less efficacy, and inhibited the effects of salicylate. We concluded that salicylate in millimolar concentrations opens KATP channels in DCN cartwheel neurons, inhibiting spontaneous firing of these neurons, probably by activating AMPK and reducing mitochondrial ATP synthesis.
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Núcleo Coclear , Acúfeno , Proteínas Quinasas Activadas por AMP , Adenosina Trifosfato/farmacología , Animales , Núcleo Coclear/fisiología , Canales KATP/farmacología , Neuronas , Ratas , Salicilatos/farmacologíaRESUMEN
OBJECTIVE: The mechanisms by which glucose stimulates insulin secretion from ß-cells are well established and involve inhibition of ATP-sensitive K+ (KATP) channels, followed by a rise in [Ca2+]c that triggers exocytosis. However, the mechanisms by which glucose controls glucagon release from α-cells are much less known. In particular, it is debated whether the sugar controls glucagon secretion by changing α-cell [Ca2+]c, and whether KATP channels or paracrine factors are involved. The present study addresses these issues. METHODS: We tested the effect of a decrease or an increase of glucose concentration (Gx, with x = concentration in mM) on α-cell [Ca2+]c and glucagon secretion. α-cell [Ca2+]c was monitored using GluCreGCaMP6f mice expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells. [Ca2+]c was compared between dispersed α-cells and α-cells within islets to evaluate the potential contribution of an indirect effect of glucose. The same protocols were used for experiments of glucagon secretion from whole islets and [Ca2+]c measurements to test if changes in glucagon release mirror those in α-cell [Ca2+]c. RESULTS: Blockade of KATP channels by sulfonylureas (tolbutamide 100 µM or gliclazide 25 µM) strongly increased [Ca2+]c in both dispersed α-cells and α-cells within islets. By contrast, glucose had no effect on [Ca2+]c in dispersed α-cells, whereas it affected it in α-cells within islets. The effect of glucose was however different in islets expressing (Sst+/+) or not somatostatin (SST) (Sst-/-). Decreasing glucose concentration from G7 to G1 modestly increased α-cell [Ca2+]c, but to a slightly larger extent in Sst+/+ islets than in Sst-/- islets. This G1-induced [Ca2+]c rise was also observed in the continuous presence of sulfonylureas in both Sst+/+ and Sst-/- islets. Increasing glucose concentration from G7 to G20 did not affect α-cell [Ca2+]c in Sst+/+ islets which remained low, whereas it strongly increased it in Sst-/- islets. The observations that this increase was seen only in α-cells within islets but never in dispersed α-cells and that it was abrogated by the gap junction inhibitor, carbenoxolone, point to an indirect effect of G20 and suggest that, in Sst-/- islets, G20-stimulated ß-cells entrain α-cells whereas, in Sst+/+ islets, the concomitant release of SST keeps α-cell [Ca2+]c at low levels. The [Ca2+]c lowering effect of endogenous SST is also supported by the observation that SST receptor antagonists (SSTR2/3) increased [Ca2+]c in α-cells from Sst+/+ islets. All these [Ca2+]c changes induced parallel changes in glucagon release. To test if glucose also controls glucagon release independently of [Ca2+]c changes, additional experiments were performed in the continuous presence of 30 mM K+ and the KATP channel opener diazoxide (250 µM). In these conditions, α-cell [Ca2+]c within islets was elevated and its steady-state level was unaffected by glucose. However, decreasing the glucose concentration from G7 to G1 stimulated glucagon release whereas increasing it from G1 to G15 inhibited it. These effects were also evident in Sst-/- islets, and opposite to those on insulin secretion. CONCLUSIONS: We propose a model according to which glucose controls α-cell [Ca2+]c and glucagon secretion through multiple mechanisms. Increasing the glucose concentration modestly decreases [Ca2+]c in α-cells independently of their KATP channels and partly via SST. The involvement of SST increases with the glucose concentration, and one major effect of SST is to keep α-cell [Ca2+]c at low levels by counteracting the effect of an entrainment of α-cells by ß-cells when ß-cells become stimulated by glucose. All these [Ca2+]c changes induce parallel changes in glucagon release. Glucose also decreases the efficacy of Ca2+ on exocytosis by an attenuating pathway that is opposite to the well-established amplifying pathway controlling insulin release in ß-cells.
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Calcio , Exocitosis , Células Secretoras de Glucagón , Glucagón , Glucosa , Somatostatina , Adenosina Trifosfato , Animales , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Exocitosis/fisiología , Glucagón/biosíntesis , Glucagón/metabolismo , Células Secretoras de Glucagón/metabolismo , Glucosa/análisis , Glucosa/metabolismo , Canales KATP/metabolismo , Ratones , Somatostatina/metabolismoRESUMEN
Eugenosedin-A (Eu-A) has been shown to protect against hyperglycemia- and hyperlipidemia-induced metabolic syndrome. We investigated the relationship of KATP channel activities and insulin secretion by Eu-A in vitro in pancreatic ß-cells, and examined the effect of Eu-A on streptozotocin (STZ)/nicotinamide (NA)-induced type 2 diabetes mellitus (T2DM) in vivo. We isolated pancreatic islets from adult male Wistar rats (250-350 g) and identified pancreatic ß-cells by the cell size, capacitance and membrane potential. Perforated patch-clamp and inside-out recordings were used to monitor the membrane potential (current-clamp mode) and channel activity (voltage-clamp mode) of ß-cells. The membrane potential of ß-cells was raised by Eu-A and reversed by the KATP channel activator diazoxide. Eu-A inhibited the KATP channel activity measured at - 60 mV and increased the intracellular calcium concentration ([Ca2+]i), resulting in enhanced insulin secretion. Eu-A also reduced Kir6.2 protein on the cell membrane and scattered in the cytosol under normal glucose conditions (5.6 mM). In our animal study, rats were divided into normal and STZ/NA-induced T2DM groups. Normal rats fed with regular chow were divided into control and control+Eu-A (5 mg/kg/day, i.p.) groups. The STZ/NA-induced diabetic rats fed with a high-fat diet (HFD) were divided into three groups: T2DM, T2DM+Eu-A (5 mg/kg/day, i.p.), and T2DM+glibenclamide (0.5 mg/kg/day, i.p.; a KATP channel inhibitor). Both Eu-A and glibenclamide decreased the rats' blood glucose, prevented weight gain, and enhanced insulin secretion. We found that Eu-A blocked pancreatic ß-cell KATP channels, caused membrane potential depolarization, and stimulated Ca2+ influx, thus increasing insulin secretion. Furthermore, Eu-A decreased blood glucose and increased insulin levels in T2DM rats. These results suggested that Eu-A might have clinical benefits for the control of T2DM and its complications.
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Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hiperglucemia/tratamiento farmacológico , Piperazinas/farmacología , Animales , Glucemia/efectos de los fármacos , Dieta Alta en Grasa , Gliburida/farmacología , Hiperglucemia/etiología , Hipoglucemiantes/farmacología , Secreción de Insulina/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Canales KATP/metabolismo , Masculino , Obesidad/complicaciones , Ratas , Ratas WistarRESUMEN
PURPOSE: Studies argue in favor of hydrogen sulfide (H2S) as the next potent therapeutic agent for neurodegenerative diseases. In present study, we investigated the effect of long term treatment with NaHS (as donor of H2S) on induction and progress of the 6-hydroxydopamine (6-OHDA) -induced Parkinsonism in rat. METHODS: The 6-OHDA was injected into medial forebrain bundle of right hemisphere by stereotaxic surgery. Behavioral tests and treatments were carried out to eight weeks after the toxin. Immunohistochemistry and western blotting were carried out to evaluate the survival of tyrosine hydroxylase (TH) -positive neurons in substantia nigra (SN) and also expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP), the markers of endoplasmic reticulum (ER) stress, in striatum and SN. RESULTS: Eight weeks assessment of the behavioral symptoms showed that NaHS especially at dose of 100 µmol/kg attenuates remarkably induction of the Parkinsonism and prevents its progress. NaHS also increased the survival of TH- positive neurons and suppressed 6-OHDA- induced overexpression of GRP78 and CHOP. Blockade of ATP-sensitive potassium (K-ATP) channels with glibenclamide (Glib) prevented markedly the effect of NaHS on both the induction phase and survival of TH- positive neurons. But Glib did not affect the preventing effect of NaHS on the progress phase and its suppressing effect on the overexpression of ER stress markers. CONCLUSION: H2S attenuates induction of the 6-OHDA- induced Parkinsonism and also increases the survival of dopaminergic neurons through activation of K-ATP channels. H2S also prevents progress of the Parkinsonism probably through suppression of ER stress.
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Progresión de la Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Sulfuro de Hidrógeno/uso terapéutico , Canales KATP/metabolismo , Oxidopamina/toxicidad , Trastornos Parkinsonianos/metabolismo , Animales , Estrés del Retículo Endoplásmico/fisiología , Gasotransmisores/farmacología , Gasotransmisores/uso terapéutico , Sulfuro de Hidrógeno/farmacología , Masculino , Trastornos Parkinsonianos/inducido químicamente , Ratas , Ratas WistarRESUMEN
BACKGROUND: Openers of mitochondrial adenosine triphosphate-dependent potassium (mKATP) channels like diazoxide increase reactive oxygen species (ROS) production in cardiac cells and reduce Ca2+ elevations produced by ischemia-reperfusion, protecting the heart from damage. In this study we tested the hypothesis that opening mKATP channels regulates expression of the major components of store-operated Ca2+ entry (SOCE) STIM1 and Orai1. RESULTS: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot experiments showed that diazoxide increased expression of STIM1 and Orai1 at the mRNA and protein levels, respectively, in adult rat cardiomyocytes. Immunofluorescence analyses revealed that diazoxide also disrupted the striated distribution pattern of STIM1. These effects were prevented by the ROS scavenger N-acetyl cysteine (NAC), the mKATP channel antagonist 5-hydroxydecanoate (5-HD), or the protein synthesis inhibitor cycloheximide (CHX). Confocal microscopy revealed that diazoxide also led to nuclear translocation of the transcription factors c-Fos and NFκB, which was also blocked by NAC or 5-HD. Finally, the MAPK pathway inhibitor UO126 attenuated diazoxide-induced upregulation of STIM1 and Orai1 expression. CONCLUSIONS: Our results suggest that opening mitochondrial potassium ATP channels with diazoxide upregulates the expression of STIM1 and Orai1 by de novo synthesis by a mechanism that involves NFkB, c-Fos, and ROS via MAPK/ERK signaling.
RESUMEN
OBJECTIVE: Elevations in pancreatic α-cell intracellular Ca2+ ([Ca2+]i) lead to glucagon (GCG) secretion. Although glucose inhibits GCG secretion, how lactate and pyruvate control α-cell Ca2+ handling is unknown. Lactate enters cells through monocarboxylate transporters (MCTs) and is also produced during glycolysis by lactate dehydrogenase A (LDHA), an enzyme expressed in α-cells. As lactate activates ATP-sensitive K+ (KATP) channels in cardiomyocytes, lactate may also modulate α-cell KATP. Therefore, this study investigated how lactate signaling controls α-cell Ca2+ handling and GCG secretion. METHODS: Mouse and human islets were used in combination with confocal microscopy, electrophysiology, GCG immunoassays, and fluorescent thallium flux assays to assess α-cell Ca2+ handling, Vm, KATP currents, and GCG secretion. RESULTS: Lactate-inhibited mouse (75 ± 25%) and human (47 ± 9%) α-cell [Ca2+]i fluctuations only under low-glucose conditions (1 mM) but had no effect on ß- or δ-cells [Ca2+]i. Glyburide inhibition of KATP channels restored α-cell [Ca2+]i fluctuations in the presence of lactate. Lactate transport into α-cells via MCTs hyperpolarized mouse (14 ± 1 mV) and human (12 ± 1 mV) α-cell Vm and activated KATP channels. Interestingly, pyruvate showed a similar KATP activation profile and α-cell [Ca2+]i inhibition as lactate. Lactate-induced inhibition of α-cell [Ca2+]i influx resulted in reduced GCG secretion in mouse (62 ± 6%) and human (43 ± 13%) islets. CONCLUSIONS: These data demonstrate for the first time that lactate entry into α-cells through MCTs results in KATP activation, Vm hyperpolarization, reduced [Ca2+]i, and inhibition of GCG secretion. Thus, taken together, these data indicate that lactate either within α-cells and/or elevated in serum could serve as important modulators of α-cell function.
Asunto(s)
Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Membrana Celular/fisiología , Glucagón/fisiología , Células Secretoras de Glucagón/fisiología , Glucosa/farmacología , Humanos , Islotes Pancreáticos/metabolismo , Canales KATP/metabolismo , Ácido Láctico/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Páncreas/metabolismo , Cultivo Primario de Células , Ácido Pirúvico/farmacologíaRESUMEN
AIMS: Several 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 METHODS: PD 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 FINDINGS: Glibenclamide 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. SIGNIFICANCE: KATP 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.
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Gliburida/farmacología , Canales KATP/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Trastornos Parkinsonianos/tratamiento farmacológico , Bloqueadores de los Canales de Potasio/farmacología , Animales , Apoptosis/efectos de los fármacos , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Canales KATP/metabolismo , Levodopa/farmacología , Masculino , Ratones , Nicorandil/farmacología , Trastornos Parkinsonianos/fisiopatología , Rotenona/toxicidadRESUMEN
BACKGROUND: Obesity, a critical feature in metabolic disorders, is associated with medical depression. Recent evidence reveals that brown adipose tissue (BAT) activity may contribute to mood disorders, Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels regulate BAT sympathetic nerve activity. However, the mechanism through which BAT activity affects mood control remains unknown. We hypothesized the BAT is involved in depressive-like symptoms regulation by trafficking KATP channels. METHODS: Eight-week-old male B6 mice fed with a high-fat diet (HFD) for 12 weeks exhibited characteristics of metabolic disorders, including hyperglycemia, hyperinsulinemia, and hyperlipidemia, as well as depressive symptoms. In this study, we surgically removed interscapular BAT in mice, and these mice exhibited immobility in the forced swim test and less preference for sugar water compared with other mice. To delineate the role of KATP channels in BAT activity regulation, we implanted a miniosmotic pump containing glibenclamide (GB), a KATP channel blocker, into the interscapular BAT of HFD-fed mice. RESULTS: GB infusion improved glucose homeostasis, insulin sensitivity, and depressive-like symptoms. KATP channel expression was lower in HFD-fed mice than in chow-fed mice. Notably, GB infusion in HFD-fed mice restored KATP channel expression. CONCLUSION: KATP channels are functionally expressed in BAT, and inhibiting BAT-KATP channels improves metabolic syndromes and reduces depressive symptoms through beta-3-adrenergic receptor-mediated protein kinase A signaling.
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Tejido Adiposo Pardo/efectos de los fármacos , Neuronas Dopaminérgicas/efectos de los fármacos , Gliburida/farmacología , Red Nerviosa/efectos de los fármacos , Obesidad , Recompensa , Tejido Adiposo Pardo/inervación , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/fisiopatología , Animales , Células Cultivadas , Citoprotección/efectos de los fármacos , Dieta Alta en Grasa , Neuronas Dopaminérgicas/fisiología , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Canales KATP/antagonistas & inhibidores , Canales KATP/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Red Nerviosa/fisiología , Obesidad/metabolismo , Obesidad/fisiopatología , Obesidad/psicología , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/metabolismo , Termogénesis/efectos de los fármacosRESUMEN
AMP-activated protein kinase (AMPK) is a cellular energy sensor activated during energy stress to stimulate ATP production pathways and restore homeostasis. AMPK is widely expressed in the kidney and involved in mitochondrial protection and biogenesis upon acute renal ischemia, AMPK activity being blunted in metabolic disease-associated kidney disease. Since little is known about AMPK in the regulation of renal blood flow, the present study aimed to assess the role of AMPK in renal vascular function. Functional responses to the selective AMPK activator A769662 were assessed in intrarenal small arteries isolated from the kidney of renal tumour patients and Wistar rats and mounted in microvascular myographs to perform simultaneous measurements of intracellular calcium [Ca2+]i and tension. Superoxide (O2.-) and hydrogen peroxide (H2O2) production were measured by chemiluminescence and fluorescence and protein expression by Western blot. Activation of AMPK with A769662 increased AMPKα phosphorylation at Thr-172 and induced potent relaxations compared to AICAR in isolated human and rat intrarenal arteries, through both endothelium-dependent mechanisms involving nitric oxide (NO) and intermediate-conductance calcium-activated potassium (IKCa) channels, as well as activation of ATP-sensitive (KATP) channels and sarcoplasmic reticulum Ca2+-ATPase (SERCA) in vascular smooth muscle (VSM). Furthermore, AMPK activator reduced NADPH oxidase 4 (Nox4) and Nox2-derived reactive oxygen species (ROS) production. These results demonstrate that A769662 has potent vasodilator and antioxidant effects in intrarenal arteries. The benefits of AMPK activation in rat kidney are reproduced in human arteries and therefore vascular AMPK activation might be a therapeutic target in the treatment of metabolic disease-associated kidney injury.