ATP Channel Blocker Research Articles

Role of l -arginine/nitric oxide/cyclic GMP/K ATP channel signaling pathway and opioid receptors in the antinociceptive effect of rutin in mice.

The l -arginine ( l -Arg)/nitric oxide/cyclic GMP/potassium channel (K ATP ) pathway and opioid receptors are known to play critical roles in pain perception and the antinociceptive effects of various compounds. While there is evidence suggesting that the analgesic effects of rutin may involve nitric oxide modulation, the direct link between rutin and the l -Arg/nitric oxide/cyclic GMP/K ATP pathway in the context of pain modulation requires further investigation. The antinociceptive effect of rutin was studied in male NMRI mice using the formalin test. To investigate the role of the l -Arg/nitric oxide/cyclic GMP/K ATP pathway and opioid receptors, the mice were pretreated intraperitoneally with different substances. These substances included l -Arg (a precursor of nitric oxide), S-nitroso- N -acetylpenicillamine (SNAP, a nitric oxide donor), N(gamma)-nitro- l -arginine methyl ester (L-NAME, an inhibitor of nitric oxide synthase), sildenafil (an inhibitor of phosphodiesterase enzyme), glibenclamide (a K ATP channel blocker), and naloxone (an opioid receptor antagonist). All pretreatments were administered 20 min before the administration of the most effective dose of rutin. Based on our investigation, it was found that rutin exhibited a dose-dependent antinociceptive effect. The administration of SNAP enhanced the analgesic effects of rutin during both the initial and secondary phases. Moreover, L-NAME, naloxone, and glibenclamide reduced the analgesic effects of rutin in both the primary and secondary phases. In conclusion, rutin holds significant value as a flavonoid with analgesic properties, and its analgesic effect is directly mediated through the nitric oxide/cyclic GMP/K ATP channel pathway.

Citrus flavonoids effects on human umbilical vein

• Hesperidin is more potent than diosmin as vasorelaxant of Human Umbilical Vein (HUV). • K ATP channel activation is involved in hesperidin-dependent vasorelaxation only. • Endothelium-dependent vasorelaxation to hesperidin is gender-sensitive. • Hesperidin may be beneficial in maintaining vascular and endothelial functions in HUV. Flavonoids intake is associated to beneficial effects on the cardiovascular system. Aim of this study was to investigate the biological activities of hesperidin and diosmin on the vascular tone of human umbilical vein (HUV). Umbilical cords obtained at delivery from healthy women were set up for recording of isometric tension; vasorelaxing activity of flavonoids was assessed in serotonin-precontracted HUV rings. Hesperidin evoked a more profound vasorelaxation than diosmin, and inhibition of NO-synthase significantly impaired relaxant responses to hesperidin but completely abolished those to diosmin. The residual vasorelaxation to hesperidin was prevented by glybenclamide (a K ATP channel blocker). Endothelium-dependent responses to hesperidin were larger in female- when compared to male-derived HUV. β-estradiol and a direct NO-donor compound caused a marked vasorelaxation irrespective of gender of the HUV. Diosmin and, in particular, hesperidin may exert a beneficial effect on the vascular and endothelial function of HUV, and help preventing fetal vascular complications.

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.

P3483Diet governs metabolic and electrical properties of the atrial myocardium in mice

Abstract Background Obesity is associated with an increased risk of atrial fibrillation. This epidemiologic observation may not only be due to shared co-morbidities but also from direct impacts of metabolic disorders on myocardium Here, we examined the impact of high fat diet (HFD) induced obesity on atrial properties in mice. Methods Eight weeks old C57Bl/6J mice were subjected to 2 or 4 months of HFD (60% fat) or normal diet (ND, 4% fat). Rapid burst atrial pacing were delivered using a trans-esophageal probe to induced AF in anesthetized animals. Action potential (AP) were recorded in left atrial trabeculae using glass microelectrode technique. Potassium currents were recorded in isolated atrial myocytes using the whole cell or perforated patch clamp configurations. Metabolomic and lipidomic analysis were performed on whole left atria. Mitochondrial respiration was studied in situ in saponin-permeabilized atrial muscle fibres using a Clarke electrode and oxygen consumption was measured after successive addition of ADP (2 mM), malate (4 mM), palmitoyl-CoA and carnitine (pCoA-Ca) (100 μM and 2 mM), pyruvate (1 mM), glutamate (10 mM), succinate (15 mM), amytal (1 mM) and tetramethyl-paraphenylenediamine (TMPD)/ascorbate (0.5/0.5 mM). Results HFD mice showed a higher atrial vulnerability to AF as indicated by longer AF episodes compared to ND mice. APs were shorter in HFD versus ND mice (AP duration measured at 90% of repolarization: 47.9±2.4 msec in HFD vs 58.2±1.4 msec in ND, P<0.001 after 2 months of HFD) and more sensitive to the K ATP channel blocker, glibenclamide. In perforated but not whole cell, patch clamped atrial myocytes, the K-ATP component of the potassium current was enhanced in HFD mice (at +70 mV: 0.41±0.12 pA/pF in HFD vs 0.13±0.08 pA/pF in ND, P<0.05). Metabolomic analysis indicated an increased consumption of free fatty acid by the beta-oxidation and an accumulation of long chain fatty acid. Although the mitochondrial respiration measurements showed a trend towards a lower complex IV-driven respiration in HFD mice, no significant difference between ND and HFD was noted regarding complex I and complex II-driven respiration. However, the ability of fatty acids (pCoA-Car) to support mitochondrial respiration was higher in HFD Conclusions High fat diet induces a shift from carbon hydrate to a beta oxidation of the atrial myocardium metabolism together with an enhanced use of fatty acids by mitochondria. These metabolic changes could result in an enhanced activity of K-ATP channels and AP shortening that might contribute to AF vulnerability in this clinical setting. Acknowledgement/Funding ANR-10-IAHU-05

Amino acid sensing in hypothalamic tanycytes via umami taste receptors

ObjectiveHypothalamic tanycytes are glial cells that line the wall of the third ventricle and contact the cerebrospinal fluid (CSF). While they are known to detect glucose in the CSF we now show that tanycytes also detect amino acids, important nutrients that signal satiety. MethodsCa2+ imaging and ATP biosensing were used to detect tanycyte responses to l-amino acids. The downstream pathway of the responses was determined using ATP receptor antagonists and channel blockers. The receptors were characterized using mice lacking the Tas1r1 gene, as well as an mGluR4 receptor antagonist. ResultsAmino acids such as Arg, Lys, and Ala evoke Ca2+ signals in tanycytes and evoke the release of ATP via pannexin 1 and CalHM1, which amplifies the signal via a P2 receptor dependent mechanism. Tanycytes from mice lacking the Tas1r1 gene had diminished responses to lysine and arginine but not alanine. Antagonists of mGluR4 greatly reduced the responses to alanine and lysine. ConclusionTwo receptors previously implicated in taste cells, the Tas1r1/Tas1r3 heterodimer and mGluR4, contribute to the detection of a range of amino acids by tanycytes in CSF.

Remote Mechanisms of Myocardial Protection

Background: Remote ischemic preconditioning (rIPC) has been suggested to reduce infarct size through the activation of a parasym- pathetic neural pathway. However, the intracellular mechanisms responsible for this protection remain unclear. Objective: The aim of this study was to describe some of the intracellular protective signals activated at the cardiac level by rIPC prior to myocardial ischemia. Methods: Isolated rat hearts were subjected to 30 minutes of global ischemia and 120 minutes of reperfusion (I/R). In a second group, before the isolation of the heart, a rIPC protocol (three cycles of left femoral artery ischemia/reperfusion) was performed, followed by the I/R protocol. Additionally, four experimental groups were studied, in which prior to the rIPC protocol a bilateral cervical vagotomy (VS (vagal section)) was performed or atropine (muscarinic receptor blocker), L-NAME (NO synthesis inhibitor), and 5-HD (mK + ATP channel blocker) was administered, respectively. Infarct size and eNOS phosphorylation were measured in I/R, rIPC, and VS groups. Finally, mitochondrial H2O2 production was assessed. Results: Remote ischemic preconditioning significantly decreased infarct size and this effect was abolished by VS and atropine, L-NAME, and 5-HD treatments. Furthermore, rIPC increased eNOS phosphorylation and this effect was abolished by VS. Finally, rIPC increased the mitochondrial H2O2 production, and this effect was also abolished by VS. Conclusions: Remote ischemic preconditioning activates a muscarinic vagal pathway involving eNOS phosphorylation, opening of mitochondrial mK + ATP channels, and the production of mitochondrial H2O2.

Capsaicin-Sensitive Sensory Nerves Mediate the Cellular and Microvascular Effects of H2S via TRPA1 Receptor Activation and Neuropeptide Release.

It is supposed that TRPA1 receptor can be activated by hydrogen sulphide (H2S). Here, we have investigated the role of TRPA1 receptor in H2S-induced [Ca(2+)]i increase in trigeminal ganglia (TRG) neurons, and the involvement of capsaicin-sensitive sensory nerves in H2S-evoked cutaneous vasodilatation. [Ca(2+)]i was measured with ratiometric technique on TRG neurons of TRPA1(+/+) and TRPA1(-/-) mice after NaHS, Na2S, allylisothiocyanate (AITC) or KCl treatment. Microcirculatory changes in the ear were detected by laser Doppler imaging in response to topical NaHS, AITC, NaOH, NaSO3 or NaCl. Mice were either treated with resiniferatoxin (RTX), or CGRP antagonist BIBN4096, or NK1 receptor antagonist CP99994, or K(+) ATP channel blocker glibenclamide. Alpha-CGRP(-/-) and NK1 (-/-) mice were also investigated. NaHS and Na2S increased [Ca(2+)]i in TRG neurons derived from TRPA(+/+) but not from TRPA1(-/-) mice. NaHS increased cutaneous blood flow, while NaOH, NaSO3 and NaCl did not cause significant changes. NaHS-induced vasodilatation was reduced in RTX-treated animals, as well as by pre-treatment with BIBN4096 or CP99994 alone or in combination. NaHS-induced vasodilatation was significantly smaller in alpha-CGRP(-/-) or NK1 (-/-) mice compared to wild-types. H2S activates capsaicin-sensitive sensory nerves through TRPA1 receptors and the resultant vasodilatation is mediated by the release of vasoactive sensory neuropeptides CGRP and substance P.

Hypothermia improves oral and gastric mucosal oxygenation during hypoxic challenges

Therapeutic hypothermia, used primarily for protective effects after hypoxia, improves oral and gastric mucosal microvascular oxygenation (μHbO₂) during additional haemorrhage. Therefore, we questioned whether hypothermia likewise improves μHbO₂ during hypoxic challenges. Since both hypothermia and hypoxia reduce cardiac output (e.g. by myofilament Ca(2+) desensitization), and modulate vasomotor tone via K(+) ATP channels, we hypothesized that the Ca(2+) sensitizer levosimendan and K(+) ATP channel blocker glibenclamide would support the cardiovascular system. The effects of mild hypothermia (34°C) on μHbO₂ during hypoxia [Formula: see text] were analysed in a cross-over study on five anaesthetized dogs and compared with normothermia (37.5°C) and hypoxia. During hypothermia, but before hypoxia, glibenclamide (0.2 mg kg(-1)) or levosimendan (20 µg kg(-1)+0.25 µg kg(-1) min(-1)) was administered. Systemic haemodynamic variables, gastric and oral mucosal microvascular oxygenation (reflectance spectrophotometry), and perfusion (laser Doppler flowmetry) were recorded continuously. Data are presented as mean (sem), P<0.05. Hypoxia during normothermia reduced gastric μHbO₂ by 27 (3)% and oral μHbO₂ by 28 (3)% (absolute change). During hypothermia, this reduction was attenuated to 16 (3)% and 13 (1)% (absolute change). This effect was independent of microvascular flow that did not change during hypoxia and hypothermia. Additional administration of levosimendan during hypothermia restored reduced cardiac output but did not change flow or μHbO₂ compared with hypothermia alone. Glibenclamide did not exert any additional effects during hypothermia. Hypothermia attenuates the decrease in μHbO₂ during additional hypoxic challenges independent of systemic or regional flow changes. A reduction in cardiac output during hypothermia is prevented by Ca(2+) sensitization with levosimendan but not by K(+) ATP channel blockade with glibenclamide.

The favorable kinetics and balance of nebivolol-stimulated nitric oxide and peroxynitrite release in human endothelial cells

BackgroundNebivolol is a third-generation beta-blocker used to treat hypertension. The vasodilation properties of nebivolol have been attributed to nitric oxide (NO) release. However, the kinetics and mechanism of nebivolol-stimulated bioavailable NO are not fully understood.MethodsUsing amperometric NO and peroxynitrite (ONOO-) nanosensors, β3-receptor (agonist: L-755,507; antagonists: SR59230A and L-748,337), ATP efflux (the mechanosensitive ATP channel blocker, gadolinium) and P2Y-receptor (agonists: ATP and 2-MeSATP; antagonist: suramin) modulators, superoxide dismutase and a NADPH oxidase inhibitor (VAS2870), we evaluated the kinetics and balance of NO and ONOO- stimulated by nebivolol in human umbilical vein endothelial cells (HUVECs). NO and ONOO- were measured with nanosensors (diameter ~ 300 nm) placed 5 ± 2 μm from the cell membrane and ATP levels were determined with a bioluminescent method. The kinetics and balance of nebivolol-stimulated NO and ONOO- were compared with those of ATP, 2-MeSATP, and L-755,507.ResultsNebivolol stimulates endothelial NO release through β3-receptor and ATP-dependent, P2Y-receptor activation with relatively slow kinetics (75 ± 5 nM/s) as compared to the kinetics of ATP (194 ± 10 nM/s), L-755,507 (108 ± 6 nM/s), and 2-MeSATP (105 ± 5 nM/s). The balance between cytoprotective NO and cytotoxic ONOO- was expressed as the ratio of [NO]/[ONOO-] concentrations. This ratio for nebivolol was 1.80 ± 0.10 and significantly higher than that for ATP (0.80 ± 0.08), L-755,507 (1.08 ± 0.08), and 2-MeSATP (1.09 ± 0.09). Nebivolol induced ATP release in a concentration-dependent manner.ConclusionThe two major pathways (ATP efflux/P2Y receptors and β3 receptors) and several steps of nebivolol-induced NO and ONOO- stimulation are mainly responsible for the slow kinetics of NO release and low ONOO-. The net effect of this slow kinetics of NO is reflected by a favorable high ratio of [NO]/[ONOO-] which may explain the beneficial effects of nebivolol in the treatment of endothelial dysfunction, hypertension, heart failure, and angiogenesis.

KATP channels of parafacial respiratory group (pFRG) neurons are involved in H2S-mediated central inhibition of respiratory rhythm in medullary slices of neonatal rats

We recently found that hydrogen sulfide (H2S) participates in inhibitory regulation of rhythmic respiration by acting on the parafacial respiratory group (pFRG) in medullary slices of neonatal rats. The present study investigated whether ATP-sensitive potassium (KATP) channels are expressed in neurons of the pFRG, and, if so, whether they play a role in central regulation of respiratory activity, in particular the H2S-mediated central inhibition of respiratory rhythm in medullary slices of neonatal rats. Immunohistochemical techniques revealed that KATP channels are expressed in neurons of the pFRG region. Micro-injection of the KATP channel activator, pinacidil, into the pFRG region inhibited the discharge rhythm of hypoglossal rootlets, whereas injection of the KATP channel blocker, glibenclamide (Gl), had no effect. Micro-injection of the H2S donor sodium hydrosulfide (NaHS) into the pFRG region produced identical inhibitory responses to those induced by pinacidil. However, combined micro-injection of Gl and NaHS eliminated inhibitory effects of NaHS and converted to minor excitatory effects on the respiratory rhythm. It can be concluded that KATP channels of pFRG neurons are involved in the central regulation of respiratory rhythm and H2S-mediated inhibitory actions on respiratory rhythm in medullary slices of neonatal rats.

Imaging and characterization of stretch‐induced ATP release from alveolar A549 cells

Abstract Mechano-transduction at cellular and tissue levels often involves ATP release and activation of the purinergic signalling cascade. In the lungs, stretch is an important physical stimulus but its impact on ATP release, the underlying release mechanisms and transduction pathways are poorly understood. Here, we investigated the effect of unidirectional stretch on ATP release from human alveolar A549 cells by real-time luciferin-luciferase bioluminescence imaging coupled with simultaneous infrared imaging, to monitor the extent of cell stretch and to identify ATP releasing cells. In subconfluent (<90%) cell cultures, single 1 s stretch (10-40%)-induced transient ATP release from a small fraction (1.5%) of cells that grew in number dose-dependently with increasing extent of stretch. ATP concentration in the proximity (150 μm) of releasing cells often exceeded 10 μm, sufficient for autocrine/paracrine purinoreceptor stimulation of neighbouring cells. ATP release responses were insensitive to the putative ATP channel blockers carbenoxolone and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid, but were inhibited by N-ethylmaleimide and bafilomycin. In confluent cell cultures, the maximal fraction of responding cells dropped to <0.2%, but was enhanced several-fold in the wound/scratch area after it was repopulated by new cells during the healing process. Fluo8 fluorescence experiments revealed two types of stretch-induced intracellular Ca(2+) responses, rapid sustained Ca(2+) elevations in a limited number of cells and delayed secondary responses in neighbouring cells, seen as Ca(2+) waves whose propagation was consistent with extracellular diffusion of released ATP. Our experiments revealed that a single >10% stretch was sufficient to initiate intercellular purinergic signalling in alveolar cells, which may contribute to the regulation of surfactant secretion and wound healing.

The effect of hypoxia and reoxygenation in the response of mesangial cells to angiotensin II in vitro

Mesangial cells (MC) may be involved in the glomerular alterations induced by ischemia/reperfusion injury. To evaluate the response of immortalized MC (IMC) to 30 minutes of hypoxia followed by reoxygenation periods of 30 minutes (H/R30) or 24 hours (H/R24). The intracellular calcium concentration ([Ca+2]i) was measured before (baseline) and after adding angiotensin II (AII, 10-5 M) in the presence and absence of glybenclamide (K ATP channel blocker). We estimated the level of intracellular ATP, nitric oxide (NO) and PGE2. ATP concentration decreased after hypoxia and increased after reoxygenation. Hypoxia and H/R induced increases in basal [Ca+2]i. AII induced increases in [Ca+2]i in normoxia (97 ± 9%), hypoxia (72 ± 10%) or HR30 (85 ± 17%) groups, but there was a decrease in the response to AII in group H/R24 since the elevation in [Ca+2]i was significantly lower than in control (61 ± 10%, p < 0.05). Glybenclamide did not modify this response. It was observed a significant increase in NO generation after 24 hours of reoxygenation, but no difference in PGE2 production was observed. Data suggest that H/R injury is characterized by increased basal [Ca+2]i and by an impairment in the response of cells to AII. Results suggest that the relative insensibility to AII may be at least in part mediated by NO but not by prostaglandins or vasodilator K ATP channels. H/R caused dysfunction in IMC characterized by increases in basal [Ca+2]i during hypoxia and reduction in the functional response to AII during reoxygenation.

Bacterial lipopolysaccharide protects the retina from light‐induced damage

Light-induced damage is a widely used model to study retinal degeneration. We examined whether bacterial lipopolysaccharide (LPS) protects the retina against light-induced injury. One day before intense light exposure for 24 h, rats were intravitreally injected with LPS in one eye and vehicle in the contralateral eye. At several time points after light exposure, rats were subjected to electroretinography and histological analysis. Bax, Bcl-xL, p-Akt, and p-Stat3 levels were assessed by Western blotting, and retinal thiobarbituric acid reactive substances levels were measured as an index of lipid peroxidation. One group of animals received injections of dexamethasone, aminoguanidine (an inducible NOS inhibitor), 5-hydroxydecanoic acid (a mitochondrial K(+) /ATP channel blocker), or wortmannin [a phosphoinositide-3-kinase (PI3K) inhibitor] in order to analyze their effect on the protection induced by LPS. LPS afforded significant morphologic and functional protection in eyes exposed to intense light. Light damage induced an increase in mitochondrial Bax/cytoplasmic Bax ratio, and lipid peroxidation which were prevented by LPS. Dexamethasone and wortmannin (but not aminoguanidine or 5-hydroxydecanoic acid) prevented the effect of LPS. Moreover, wortmannin prevented the effect of LPS on p-Akt levels. These results indicate that LPS provides retinal protection against light-induced stress, probably through a PI3K/Akt-dependent mechanism.

Differential Effects of Angiotensin II on Intra-Renal Hemodynamics in Rats; Contribution of Prostanoids, NO and K&amp;lt;sup&amp;gt;+&amp;lt;/sup&amp;gt; Channels

Many agents are known to cause qualitative and quantitative differences in intrarenal blood flow. This study tested the hypothesis that angiotensin II (AII) evokes a differential effect on cortical (CBF) and medullary blood flow (MBF) and that AT2 receptor mediates AII-induced increase in renal MBF by mechanisms related to nitric oxide (NO) and prostanoids. AII (100, 300 and 1000 ng/kg/min) increased mean arterial blood pressure (MABP) by 24% ± 7% (p G nitro-L-arginine (L-NNA), an inhibitor of NO synthase (100 mg/L in drinking water) enhanced AII effects on MABP (169 ± 75, p ATP channel blocker, did not affect intra-renal hemodynamics elicited by AII. Blockade of AT2 receptors with PD123319 (50 μg/kg/min) did not change basal or AII-induced changes MABP or CBF but blunted AII-induced increase in MBF by 60% ± 11 % (p 2 receptor agonist, elicited a reduction in MABP and increases in CBF and MBF that were abolished or attenuated by PD123319. These findings demonstrate that AII elicited differential changes in intrarenal blood flow; an AT1-mediated reduction in CBF but an AT2-mediated increase in MBF. The AT2 receptor-mediated increase in MBF involves guanylase cyclase, NO and dilator prostanoids but not KATP channels.

Morphologic and pharmacological investigations in the epicatechin gastroprotective effect.

Previous studies of the gastroprotective activity of plants have highlighted the importance of the polyphenolic compound epicatechin (EC) in the treatment of gastric ulcers. This paper aimed to evaluate and characterize the gastroprotective mechanism of action of EC using male rats. The gastroprotective action of EC was analyzed in gastric ulcers induced by ethanol or indomethacin. The involvement of sulfhydryl (SH) groups, K+ ATP channels, α 2 adrenoceptors, gastric antisecretory activity, and the amount of mucus in the development of gastric ulcers were investigated. The lowest effective dose of EC providing gastroprotective effects was 50 mg/kg in the ethanol-induced gastric ulcers and 25 mg/kg in the indomethacin-induced gastric ulcers. The gastroprotection seen upon treatment with EC was significantly decreased in rats pretreated with a SH compound reagent or an α 2-receptor antagonist, but not with a K+ ATP channel blocker. Furthermore, oral treatment with EC increased mucus production and decreased H+ secretion. Immunohistochemistry demonstrated the involvement of superoxide dismutase (SOD), nitric oxide (NO), and heat shock protein-70 (HSP-70) in the gastroprotection. These results demonstrate that EC provides gastroprotection through reinforcement of the mucus barrier and neutralization of gastric juice and this protection occurs through the involvement of SH compounds, α 2-adrenoceptors, NO, SOD, and HSP-70.

Mechanotransduction in A549 Alveolar Cells via Cell Stretch-Induced ATP Release

Mechanotransduction at the cellular/tissue level often involves release of signaling molecules. Among them, purines appear to be the most primitive and widespread chemical messengers in the animal and plant kingdoms. Their release is highly mechano-sensitive, but the release pathways and regulatory mechanisms are not well understood. Here, we investigated the effect of unidirectional stretch on ATP release from human lung A549 alveolar cells grown on a flexible substrate. We used real-time luciferin-luciferase bioluminescence imaging combined with IR imaging to simultaneously monitor cellular ATP release and extend of cell stretch. Single 1-s stretch of 15-30% induced transient ATP release that ceased in 2-3 min and was restricted to a limited number of cells. The number of responding cells increased dose-dependently with the extent of stretch but did not involve cell damage. Calibration of the ATP response showed that local ATP concentration in the close proximity (≤150 μm) to stretch-activated cells may exceed 1 μM or even 10 μM. These concentrations are sufficient for autocrine/paracrine stimulation of cell surface purinergic receptors on the neighboring cells. ATP responses were insensitive to putative ATP channel blockers carbenoxolone or NPPB (100 μM), inhibitors of pannexin or anion channels respectively, but were abolished by N-ethylmaleimide. Fluo8 fluorescence measurement of stretch-induced intracellular Ca2+ responses revealed that limited number of cells displayed rapid responses, which peaked in <1-s and ceased in 1-3 min. This is similar to stretch-induced ATP responses and suggests functional connection between the two signals. Experiments show that cell stretch induces ATP release via cell-regulated process, likely exocytosis. Mechano-sensitive ATP release, via autocrine/paracrine effects, initiates purinergic signaling cascade in other cells and may function as a general intercellular mechanotransduction paradigm in the lung and other tissues.

Weak Magnetic Field at 16 Hz Affects Cardiac Myocyte Ca2+ Transients and Reduces Cells Damage Caused by Hypoxia

Effects of extremely low intensity and frequencies of Weak Electromagnetic Fields (WMF) on the cardiovascular system have been reported. However, little is known about the physical mechanism of interactions between magnetic fields and physiology. The aim of this study was to explore the effect of WMF at Extremely Low Frequencies (ELF) on Ca 2+ transients, and to investigate the influence of magnetic field following hypoxic stress, in cardiac cell cultures. Indo-1 loaded cells, which exposed to WMF at 16 Hz, demonstrated a faster reduction in cytosolic Ca 2+ transients compared to control cells (75±4% vs. 25±8%). This effect was not observed in other low frequencies. BDM (2,3-butanedione 2-monoxime), which inhibits contraction but not Ca 2+ transient, did not inhibit the effect of WMF on the reduction of Ca 2+ transients at 16 Hz, suggesting that the effect probably involves ion regulation. However, treatment with the K ATP channel blocker, glibenclamide, followed by 16 Hz exposure, blocked the reduction in cytosolic Ca 2+ transients, while treatment with chromanol 293B, a selective blocker of the delayed rectifier K + current channels (I ks ), or the potassium channel opener, pinacidil, did not inhibit the effect. In addition, cardiomyocytes exposure to WMF-ELF at 16 Hz 30 min prior to hypoxia, exhibited a significant reduction in lactate dehydrogenate level and low percentages of death cells vs. hypoxic treatment. In conclusion, WMF at low frequency of 16 Hz induced changes in cardiomyocyte contractions by reducing Ca 2+ transients, most probably through activation of K ATP ion channels. Cells subjected to WMF frequency prior to hypoxic stress, exhibited significantly reduced cardiomyocytes damage.

Glibenclamide reduces proinflammatory cytokines in an ex vivo model of human endotoxinaemia under hypoxaemic conditions

Aims In vivo application of the K ATP-channel blocker glibenclamide can reverse endotoxin-induced hypotension, vascular hyporeactivity and shock in experimental animals. The hypothesis of the present study is, that the drug effects might not only be based on direct inhibition of K ATP-channels of vascular smooth muscle cells, but might also reflect reduction of shock-induced excess proinflammatory cytokines and procoagulatory molecules produced in the blood monocytes. Main methods Human whole blood (normoxaemic or hypoxaemic) supplemented ex vivo with 100 ng/ml LPS was used to assess glibenclamide (3–100 μM) effects on IL-1beta, IL-6, TNF-alpha, tissue factor, and plasminogen-activator-inhibitor-2 (PAI-2). Co-incubations with monocytes and erythrocytes and cytosolic calcium measurements were performed to reveal their purinergic intercellular interaction. Key findings In heparinized blood, glibenclamide reduced LPS-induced release of IL-1beta and TNF-alpha, tissue factor and PAI-2 mRNA in a concentration-dependent manner. When samples were subjected to strong hypoxemia using 95% N 2/5% CO 2, these parameters became even more sensitive to the drug. No drug effect was observable in citrated blood or in isolated monocytes. IL-1beta mRNA inhibition by glibenclamide appeared to be dependent on P2X7-receptor activation of monocytes by ATP-releasing erythrocytes during hypoxia. Cytosolic calcium values as well as the duration of calcium transients elicited by P2X7-receptor stimulation in isolated monocytes were strongly increased during hypoxia, both of which could be abolished by glibenclamide. Significance We conclude that the anti-inflammatory effect of glibenclamide is mainly based on the reduction of calcium entry by drug-induced depolarization of hypoxic monocytes. Thus, glibenclamide possesses a potentially beneficial shock-specific anti-inflammatory action.

Cardioprotective effect of polydatin against ischemia/reperfusion injury: Roles of protein kinase C and mito K ATP activation

Polydatin preconditioning (PPC) has been reported to be protective against brain and intestine ischemia/reperfusion injury (I/R injury), but whether polydatin exerts cardioprotective effect against myocardial ischemia/reperfusion and the underlying mechanisms remain unclear. Previous studies have demonstrated that oxidative stress plays an important role in the process of I/R. Elevation of oxidative agents and decline in anti-oxidant substance would promote I/R. Meanwhile, the activation of PKC signaling seems to mediate the cardioprotective effects of many drugs by alleviating Ca 2+ influx. In the present study, we reported for the first time that intravenous administration of polydatin before I/R significantly limited the infarct size, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) leakage from the damaged myocardium after I/R. The activity of SOD and the content of MDA remarkably changed in the presence of polydatin as well. However, the cardiac function-preserving and myocardial enzymes leakage-limiting effects of polydatin vanished in the presence of PKC inhibitors and mito K ATP channel blockers. But there was not a significant change in the activity of SOD and MDA content. We therefore conclude that PPC exerts cardioprotective effect by the activation of PKC-K ATP-dependent signaling and the direct anti-oxidative stress mechanisms.

Hydrogen sulphide inhibits carbachol-induced contractile responses in β-escin permeabilized guinea-pig taenia caecum

Hydrogen sulphide (H 2S) is an endogenous mediator producing a potent relaxation response in vascular and non-vascular smooth muscles. While ATP-sensitive potassium channels are mainly involved in this relaxant effect in vascular smooth muscle, the mechanism in other smooth muscles has not been revealed yet. In the present study, we investigated how H 2S relaxes non-vascular smooth muscle by using intact and β-escin permeabilized guinea-pig taenia caecum. In intact tissues, concentration-dependent relaxation response to H 2S donor NaHS in carbachol-precontracted preparations did not change in the presence of a K ATP channel blocker glibenclamide, adenylate cyclase inhibitor SQ-22536, guanylate cyclase inhibitor ODQ, protein kinase A inhibitor KT-5720, protein kinase C inhibitor H-7, tetrodotoxin, apamin/charybdotoxin, NOS inhibitor L-NAME and cyclooxygenase inhibitor indomethacin. We then studied how H 2S affected carbachol- or Ca 2+-induced contractions in permeabilized tissues. When Ca 2+ was clamped to a constant value ( pCa6), a further contraction could be elicited by carbachol that was decreased by NaHS. This decrease in contraction was reversed by catalase but not by superoxide dismutase or N-acetyl cysteine. The sarcoplasmic reticulum Ca 2+-ATPase pump inhibitor, cyclopiazonic acid, also decreased the carbachol-induced contraction that was further inhibited by NaHS. Mitochondrial proton pump inhibitor carbonyl cyanide p-trifluromethoxyphenylhydrazone also decreased the carbachol-induced contraction but this was not additionally changed by NaHS. The carbachol-induced Ca 2+ sensitization, calcium concentration–response curves, IP 3- and caffeine-induced contractions were not affected by NaHS. In conclusion, we propose that hydrogen peroxide and mitochondria may have a role in H 2S-induced relaxation response in taenia caecum.