Inhibition of the ATP-sensitive potassium channel from mouse pancreatic β-cells by surfactants

1. We have used patch-clamp methods to study the effects of the detergents, Cremophor, Tween 80 and Triton X100 on the K_ATP channel in the pancreatic β-cell from mouse.
2. All three detergents blocked K_ATP channel activity with the following order of potency: Tween 80 (K_i<∼83 nM)>Triton X100 (K_i=350 nM)>Cremophor. In all cases the block was poorly reversible.
3. Single-channel studies suggested that at low doses, the detergents act as slow blockers of the K_ATP channel.
4. Unlike the block produced by tolbutamide, that produced by detergent was not affected by intracellular Mg²⁺-nucleotide, diazoxide or trypsin treatment, nor did it involve an acceleration of rundown or increase in ATP sensitivity of the chanel.
5. The detergents could block the pore-forming subunit, Kir6.2ΔC26, which can be expressed independently of SUR₁ (the regulatory subunit of the K_ATP channel). These data suggest that the detergents act on Kir6.2 and not SUR₁.
6. The detergents had no effect on another member of the inward rectifier family: Kir1.1a (ROMK1).
7. Voltage-dependent K-currents in the β-cell were reversibly blocked by the detergents with a far lower potency than that found for the K_ATP channel.
8. Like other insulin secretagogues that act by blocking the K_ATP channel, Cremophor elevated intracellular Ca²⁺ in single β-cells to levels that would be expected to elicit insulin secretion.
9. Given the role of the K_ATP channel in many physiological processes, we conclude that plasma borne detergent may have pharmacological actions mediated through blockage of the K_ATP channel

     

Glucotoxic conditions induce endoplasmic reticulum stress to cause caspase 3 mediated lamin B degradation in pancreatic β-cells: Protection by nifedipine

Nuclear lamins form the lamina on the interior of the nuclear envelope, and are involved in the regulation of various cellular processes, including DNA replication and chromatin organization. Despite this evidence, little is known about potential alterations in nuclear metabolism, specifically lamin structure and integrity in isolated β-cells subjected to stress conditions, including chronic exposure to hyperglycemia (i.e., glucotoxicity). Herein, we investigated effects of glucotoxic conditions on the catalytic activation of caspase 3 and the associated degradation of one of its substrate proteins, namely lamin-B. We report that incubation of insulin-secreting INS-1 832/13 cells, normal rat islets or human islets under glucotoxic conditions (20 mM; 12–48 h) results in the degradation of native lamin B leading to accumulation of the degraded products in non-relevant cellular compartments, including cytosol. Moreover, the effects of high glucose on caspase 3 activation and lamin B degradation were mimicked by thapsigargin, a known inducer of endoplasmic reticulum stress (ER stress). Nifedipine, a known blocker of calcium channel activation, inhibited high glucose-induced caspase 3 activation and lamin B degradation in these cells. 4-Phenyl butyric acid, a known inhibitor of ER stress, markedly attenuated glucose-induced CHOP expression (ER stress marker), caspase 3 activation and lamin B degradation. We conclude that glucotoxic conditions promote caspase 3 activation and lamin B degradation, which may, in part, be due to increased ER stress under these conditions. We also provide further evidence to support beneficial effects of calcium channel blockers against metabolic dysfunction of the islet β-cell induced by hyperglycemic conditions.     

Arsenic induces pancreatic β-cell apoptosis via the oxidative stress-regulated mitochondria-dependent and endoplasmic reticulum stress-triggered signaling pathways

Arsenic (As), a ubiquitous toxic metal, is an important environmental and industrial pollutant throughout the world. Inorganic As (iAs) is usually more harmful than organic ones and with a high risk of diabetes incidence by exposure. However, the toxicological effects of iAs on growth and function of pancreatic β-cells still remain unclear. Here, we found that iAs significantly decreased insulin secretion and cell viability, and increased ROS and MDA formation in pancreatic β-cell-derived RIN-m5F cells. iAs also induced the increases in sub-G1 hypodiploids, annexin V-Cy3 binding, and caspase-3 activity in RIN-m5F cells, indicating that iAs could induce β-cell apoptosis. Moreover, iAs induced MAPKs activation, mitochondria dysfunction, p53 up-regulation, Bcl-2 and Mdm-2 down-regulation, PARP, and caspase cascades, which displayed features of mitochondria-dependent apoptotic signals. In addition, exposure of RIN-m5F cells to iAs, could trigger ER stress as indicated by the enhancement in ER stress-related molecules induction (such as GRP78, GRP94, CHOP, and XBP1), procaspase-12 cleavage, and calpain activation. The iAs-induced apoptosis and its-related signalings could be effectively reversed by antioxidant N-acetylcysteine. We next observed that exposure of mice to iAs in drinking water for 6 consecutive weeks significantly decreased decreased the plasma insulin, elevated glucose intolerance and plasma lipid peroxidation, and induced islet cells apoptosis, which accompanied with arsenic accumulation in the whole blood and pancreas. N-acetylcysteine effectively antagonized the iAs-induced responses in mice. Taken together, these results suggest that iAs-induced oxidative stress causes pancreatic β-cells apoptosis via the mitochondria-dependent and ER stress-triggered signaling pathways.     

Regulation by tolbutamide and diazoxide of the electrical activity in mouse pancreatic β-cells recorded in vivo

1. The glucose-dependence of β-cell electrical activity and the effects of tolbutamide and diazoxide were studied in anaesthetized mice.
2. In untreated animals there was a direct relationship between glycaemia and the burst pattern of electrical activity. Animals with high glucose concentration showed continuous electrical activity. The application of insulin led to a steady decrease in blood glucose concentration and a transition from continuous to oscillatory activity at 7.7±0.1 mM glucose (mean±s.d.) and a subsequent transition from oscillatory to silent at 4.7±0.6 mM glucose.
3. At physiological blood glucose concentrations the electrical activity was oscillatory. The injection of tolbutamide (1800 mg kg⁻¹) transformed this oscillatory pattern into one of continuous electrical activity. The increased electrical activity was associated with a decrease in blood glucose concentration from 7.1±0.9 (control) to 5.5±1.0 mM (10 min after tolbutamide injection). The effects of tolbutamide are consistent with a direct blocking effect on the K_ATP channel that leads to membrane depolarization.
4. The injection of diazoxide (6000 mg kg⁻¹) hyperpolarized the cells and transformed the oscillatory pattern into a silent one. This is consistent with a direct stimulant effect by diazoxide on the K_ATP channel. The use of tolbutamide or diazoxide correspondingly led to the lengthening or shortening of the active phase of electrical activity, respectively. This indicates that in vivo, such activity can be modulated by the relative degree of activation or inhibition of the K_ATP channel.
5. These results indicate that under physiological conditions, tolbutamide and diazoxide have direct and opposite effects on the electrical activity of pancreatic β-cells, most likely through their action on K_ATP channels. This is consistent with previous work carried out on in vitro models and explains the drugs hypo- and hyperglycaemic effects.

     

D-saccharic acid-1,4-lactone ameliorates alloxan-induced diabetes mellitus and oxidative stress in rats through inhibiting pancreatic β-cells from apoptosis via mitochondrial dependent pathway

Oxidative stress plays a vital role in diabetic complications. To suppress the oxidative stress mediated damage in diabetic pathophysiology, a special focus has been given on naturally occurring antioxidants present in normal diet. d-saccharic acid 1,4-lactone (DSL), a derivative of d-glucaric acid, is present in many dietary plants and is known for its detoxifying and antioxidant properties. The aim of the present study was to evaluate the beneficial role of DSL against alloxan (ALX) induced diabetes in the pancreas tissue of Swiss albino rats. A dose-dependent study for DSL (20-120 mg/kg body weight) was carried out to find the effective dose of the compound in ALX-induced diabetic rats. ALX exposure elevated the blood glucose, glycosylated Hb, decreased the plasma insulin and disturbed the intra-cellular antioxidant machineries whereas oral administration of DSL at a dose of 80 mg/kg body weight restored these alterations close to normal. Investigating the mechanism of the protective activity of DSL we observed that it prevented the pancreatic β-cell apoptosis via mitochondria-dependent pathway. Results showed decreased mitochondrial membrane potential, enhanced cytochrome c release in the cytosol and reciprocal regulation of Bcl-2 family proteins in the diabetic rats. These events were also found to be associated with increased level of Apaf-1, caspase 9, and caspase 3 that ultimately led to pancreatic β-cell apoptosis. DSL treatment, however, counteracted these changes. In conclusion, DSL possesses the capability of ameliorating the oxidative stress in ALX-induced diabetes and thus could be a promising approach in lessening diabetic complications.     

Effect of β-glucan originated from Aureobasidium pullulans on asthma induced by ovalbumin in mouse

The objective of this study is to detect the effect of beta-glucan derived from Aureobasidium pullulans SM-2001, a UV induced mutant of A. pullulans on the ovalbumin (OVA) induced allergic asthma. The test articles were orally administered to OVA-inducing asthmatic mice 4 days after sensitization for 13 days at 31.25, 62.5 or 125 mg/kg levels. Three days after the OVA sensitization, ten mice were selected per group based on body weight and were sacrificed three days after the OVA aerosol challenge. The changes on the body weight, lung weight, total leukocytes in peripheral blood and total cells in bronchoalveolar lavage fluid (BALF) were observed with changes on the lung histopathology and histomorphometry. The results were compared with dexamethasone (DEXA) 3 mg/kg intraperitoneally treated mice. The results showed increases of body weight after the OVA aerosol challenge, lung weight, total leukocytes and eosinophils in peripheral blood, total cell numbers, neutrophil and eosinophils in BALF were detected in the OVA control compared to sham control (non-OVA). However, these changes from asthmatic responses were significantly or dose-dependently decreased in the beta-glucan-dosing groups compared to those of the OVA control. Therefore, it is concluded that beta-glucan has favorable effects on asthmatic response induced by OVA. It was found that beta-glucan 125 mg/kg showed similar or slightly lower efficacy compared with DEXA 3 mg/kg.     

The β-carboline alkaloid harmol induces cell death via autophagy but not apoptosis in human non-small cell lung cancer A549 cells

β-Carboline alkaloids are naturally occurring plant substances that have a wide spectrum of neuropharmacological, psychopharmacological, and antitumor effects. Recently, we have demonstrated that harmol, a β-carboline alkaloid, induces apoptosis by caspase-8 activation independently from Fas/Fas ligand interaction in human non-small cell lung cancer (NSCLC) H596 cells. Here, we found that harmol induces autophagy and cell death in human NSCLC A549 cells. Although harmol induced cell death in A549 cells in a significant dose- and time-dependent manner, it did not induce caspase-3, caspase-8, or caspase-9 activity. Furthermore, cleavage of poly-(ADP-ribose)-polymerase was not induced in A549 cells by harmol treatment. Autophagy, but not apoptosis, was detected by electron microscopy in A549 cells treated with 70 μM harmol. Pretreatment of A549 cells with 3-methyladenine, an autophagy inhibitor, as well as small interfering RNA (siRNA)-mediated knockdown of LC3, both suppressed harmol-induced cell death. These suggest that the induction of autophagy by harmol precedes cell death. The cytotoxicity of some anticancer agents is reportedly linked to autophagy induction. The 2 major autophagy regulatory pathways are the Akt/mammalian target of rapamycin (mTOR) pathway and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Although harmol treatment showed no effect on the Akt/mTOR pathway, it transiently activated the ERK1/2 pathway. However, inhibition of the ERK1/2 pathway using the mitogen-activated protein kinase (MEK)/ERK inhibitor U0126 partially suppressed autophagy. Therefore, although activation of the ERK1/2 pathway might be related to harmol-induced autophagy, another major pathway may also be involved in A549 cells.     

Perillyl alcohol protects against ethanol induced acute liver injury in Wistar rats by inhibiting oxidative stress, NFκ-B activation and proinflammatory cytokine production

Oxidative stress and inflammation are two major etiological factors that are suggested to play key roles in the development of ethanol induced liver injury. Release of proinflammatory cytokine like tumor necrosis factor alpha (TNF-α) and activation of nuclear factor kappa-B (NFκ-B) may strongly intensify inflammation and cell damage. Additionally, reactive oxygen species (ROS) also exerts significant effect in this whole cell signaling machinery. The present study was designed to investigate the protective effects of perillyl alcohol (POH) on ethanol-induced acute liver injury in Wistar rats and its probable mechanism. We have successfully demonstrated that pre-treatment with POH, besides exerting antioxidant activity might be able to modulate TNF-α release and NFκ-B activation. Rats were divided into five groups and treated with ethanol or POH via an intragastric tube for one week. Control group was treated with vehicle, and ethanol treated group was given ethanol (5 g/kg body wt). Animal of treatment groups were pretreated with POH (50 & 100 mg/kg body wt) and have been given ethanol. Serum aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase and hepatic malondialdehyde were increased significantly by ethanol treatment. Ethanol administration decreased hepatic reduced glutathione content and various antioxidant enzymes activity. TNF-α production and NFκ-B activation was also found to be increased after ethanol administration. POH pre-treatment significantly ameliorates ethanol induced acute liver injury possibly by inhibition of lipid peroxidation, replenishment of endogenous enzymatic and non-enzymatic defense system, downregulation of TNF-α as well as NFκ-B.     

The role of endoplasmic reticulum Ca2+-independent phospholipase a2γ in oxidant-induced lipid peroxidation, Ca2+ release, and renal cell death

Oxidant-induced lipid peroxidation and cell death are major components of ischemia/reperfusion and toxicant injury. Our previous studies showed that renal proximal tubular cells (RPTCs) express Ca(2+)-independent phospholipase A(2)γ (iPLA(2)γ) in endoplasmic reticulum (ER) and mitochondria and that iPLA(2)γ is cytoprotective. Our present studies reveal the role of ER-iPLA(2)γ in oxidant-induced ER lipid peroxidation, Ca(2+) release, and cell death. Oxidant tert-butyl hydroperoxide (TBHP) caused ER lipid peroxidation and Ca(2+) release in isolated rabbit kidney cortex microsomes. ER-iPLA(2)γ inhibition, using bromoenol lactone (BEL), potentiated both oxidant-induced ER lipid peroxidation and Ca(2+) release. Assessment of fatty acids using electrospray ionization-mass spectrometry revealed that ER-iPLA(2)γ mediates the TBHP-induced release of arachidonic acid (20:4), linoleic acid (18:2), and their oxidized forms (18:2-OH, 18:2-OOH, 20:4-OH, 20:4-OOH, 20:4-(OH)(3). iPLA(2)γ inhibition also accelerated oxidant-induced ER Ca(2+) release in RPTC. Depletion of ER Ca(2+) stores in RPTC with thapsigargin, an ER Ca(2+) pump inhibitor, prior to TBHP exposure reduced necrotic cell death and blocked the potentiation of TBHP-induced necrotic cell death by BEL. Together, these data provide strong evidence that ER-iPLA(2)γ protects renal cells from oxidant-induced necrotic cell death by releasing unsaturated and/or oxidized fatty acids from ER membranes, thereby preserving ER membrane integrity and preventing ER Ca(2+) release.     

Attenuation of mitochondrial, but not cytosolic, Ca^2＋ overload reduces myocardial injury induced by ischemia and reperfusion

AIM: Attenuation of mitochondrial Ca2+ ([Ca2+]m), but not cytosolic Ca2+ ([Ca2+]c), overload improves contractile recovery. We hypothesized that attenuation of [Ca2+]m, but not [Ca2+]c, overload confers cardioprotection against ischemia/reperfusion-induced injury. METHODS: Infarct size from isolated perfused rat heart, cell viability, and electrically-induced Ca2+ transient in isolated rat ventricular myocytes were measured. We determined the effects of BAPTA-AM, a Ca2+ chelator, at concentrations that abolish the overload of both [Ca2+]c and [Ca2+]m, and ruthenium red, an inhibitor of mitochondrial uniporter of Ca2+ transport, at concentrations that abolish the overload of [Ca2+]m, but not [Ca2+]c, on cardiac injury induced by ischemia/reperfusion. RESULTS: Attenuation of both [Ca2+]m and [Ca2+]c by BAPTA-AM, and attenuation of [Ca2+]m, but not [Ca2+]c, overload by ruthenium red, reduced the cardiac injury observations, indicating the importance of [Ca2+]m in cardioprotection and contractile recovery in response to ischemia/reperfusion. CONCLUSION: The study has provided unequivocal evidence using a cause-effect approach that attenuation of [Ca2+]m, but not [Ca2+]c, overload is responsible for cardioprotection against ischemia/reperfusion-induced injury. We also confirmed the previous observation that attenuation of [Ca2+]m, but not [Ca2+]c, by ruthenium red improves contractile recovery following ischemia/reperfusion.     

Cassia tora prevents Aβ1-42 aggregation,inhibits acetylcholinesterase activity and protects against Aβ1-42-induced cell death and oxidative stress in human neuroblastoma cells

BackgroundAlzheimer’s is a complex neurodegenerative disease and is characterized by extraneuronal accumulation of β-amyloid peptide. Because of its complex nature, multi-target directed ligands (MTDLs) are increasingly being considered as promising anti-Alzheimer therapeutic agents. This study is aimed at determining the effects of Cassia tora ethyl acetate fraction on several Alzheimer-associated deleterious events in test tubes as well as in human neuroblastoma SK-N-SH and SH-SY5Y cell lines.MethodEthyl acetate fraction of C. tora was purified by chromatography, characterized by ¹H and ¹³C NMR, and tested for its ability to prevent Aβ _1-42 aggregation by thioflavin-T fluorescence and transmission electron microscopy. We also analyzed the intracellular ROS level and cytotoxicity in SK-N-SH and SH-SY5Y cell lines.ResultsThe extract inhibits the formation of Aβ _1-42 aggregation from monomers and oligomers, as also acetylcholinesterase activity, Aβ _1-42 -induced cell death, and Aβ _1-42 -dependent intracellular ROS production in both SK-N-SH and SH-SY5Y cells. In-depth chromatographic and spectroscopic analysis of the extract revealed that the active molecules are most likely triglycerides of oleic acid (C₁₈H₃₄O₂).ConclusionWe demonstrate for the first time that Cassia tora fraction prevents Aβ _1-42 aggregation, inhibits acetylcholinesterase and alleviates Aβ _1-42 -induced oxidative stress in human neuroblastoma cells. We further suggest the possible use of triglycerides of oleic acid as efficient anti-Alzheimer agents.     

Central antinociception induced by μ-opioid receptor agonist morphine,but not δ- or κ-, is mediated by cannabinoid CB1 receptor

Background and purpose:

It has been demonstrated that cannabinoids evoke the release of endogenous opioids to produce antinociception; however, no information exists regarding the participation of cannabinoids in the antinociceptive mechanisms of opioids. The aim of the present study was to determine whether endocannabinoids are involved in central antinociception induced by activation of µ-, δ- and κ-opioid receptors.

Experimental approach:

Nociceptive threshold to thermal stimulation was measured according to the tail-flick test in Swiss mice. Morphine (5 µg), SNC80 (4 µg), bremazocine (4 µg), AM251 (2 and 4 µg), AM630 (2 and 4 µg) and MAFP (0.1 and 0.4 µg) were administered by the intracerebroventricular route.

Key results:

The CB₁-selective cannabinoid receptor antagonist AM251 completely reversed the central antinociception induced by morphine in a dose-dependent manner. In contrast, the CB₂-selective cannabinoid receptor antagonist AM630 did not antagonize this effect. Additionally, the administration of the anandamide amidase inhibitor, MAFP, significantly enhanced the antinociception induced by morphine. In contrast, the antinociceptive effects of δ- and κ-opioid receptor agonists were not affected by the cannabinoid antagonists. The antagonists alone caused no hyperalgesic or antinociceptive effects.

Conclusions and implications:

The results provide evidence for the involvement of cannabinoid CB₁ receptors in the central antinociception induced by activation of µ-opioid receptors by the agonist morphine. The release of endocannabinoids appears not to be involved in central antinociception induced by activation of κ- and δ-opioid receptors.     

Lithospermic acid B protects β-cells from cytokine-induced apoptosis by alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1

Lithospermic acid B (LAB) has been reported to protect OLETF rats, an established type 2 diabetic animal model, from the development of diabetes-related vascular complications. We investigated whether magnesium lithospermate B (LAB) has a protective role under cytokine-induced apoptosis in INS-1 cells in vitro and whether it slows the development of diabetes in OLETF rats in vivo.Pretreatment with 50 μM LAB significantly reduced the 1000 U/mL INF-γ and 100 U/mL IL-1β-induced INS-1 cell death. LAB significantly alleviated cytokine-induced phosphorylations of p38 and JNK in accordance with a decrease in cleaved caspase-3 activity in beta-cells. LAB also protected against the cytokine-induced caspase-3 apoptotic pathway via significant activation of Nrf2-HO (heme-oxigenase)-1 and Sirt1 expression. OLETF rats treated with 40 mg/kg/day LAB showed a significant improvement in glucose tolerance compared to untreated OLETF control rats in vivo.Our results suggest that the cytoprotective effects of LAB on pancreatic β-cells are related with both alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1.     

Long-term in vitro treatment of INS-1 rat pancreatic β-cells by unsaturated free fatty acids protects cells against gluco- and lipotoxicities via activation of GPR40 receptors

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Peripheral antinociception induced by δ-opioid receptors activation, but not μ- or κ-, is mediated by Ca²⁺-activated Cl⁻ channels

Studies have demonstrated that the L-arginine/NO/cGMP pathway and the potassium and calcium channels are involved in the mechanisms underlying opioid receptor activation. As additional pathways may participate in the observed antinociceptive effects following opioid exposure, the aim of our study was to determine whether Ca(2+)-activated Cl(-) channels (CaCCs) are involved in peripheral antinociception induced by μ-, δ- and κ-opioid receptor activation. Hyperalgesia was induced by intraplantar injection of prostaglandin E(2) (PGE(2), 2 μg). Nociceptive thresholds to pressure (grams) were measured using an algesimetric apparatus 3h following injection. The μ-opioid receptor agonist morphine (200 μg), δ-opioid receptor agonist (+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80, 80 μg), κ-opioid receptor agonist bremazocine (50 μg), CaCCs blocker niflumic acid (8-64 μg), CaCCs blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 32-128 μg), nitric oxide donor sodium nitroprusside (SNP, 500 μg) and cGMP exogenous analogs dibutyryl cGMP (db-cGMP, 100 μg) were also administered into the paw. The CaCCs blocker niflumic acid and NPPB partially reversed the peripheral antinociception induced by exposure to the SNC80 in a dose-dependent manner. In contrast, niflumic acid did not modify the antinociceptive effect observed following exposure to morphine or bremazocine. Additionally, the peripheral antinociception induced by the NO donor SNP or by db-cGMP was not inhibited by niflumic acid. These results provide evidence for the involvement of CaCCs in the peripheral antinociception induced by SNC80. CaCCs activation does not appear to be involved when μ- and κ-opioid receptors are activated. In addition, we did not observe a link between CaCCs and the L-arginine/NO/GMPc pathway.