Vasorelaxant effects of the potassium channel opener SR 47063 on the isolated human saphenous vein and rat aorta

The vasorelaxant effects of SR 47063 (4-(2-cyanimino-1,2-dihydropyrid-1-yl)-2,2-dimethyl-6-nitrochromene), a new K+-channel opener structurally related to levcromakalim, were examined in isolated human saphenous vein (HSV) and rat aorta (RA). HSV or RA rings were precontracted with either KCl or noradrenaline and cumulative relaxant concentration-response curves were obtained for SR 47063 (0.1 nM to 1 µM) in the presence or absence of 3 µM glibenclamide. SR 47063 potently relaxed HSV and RA precontracted with 20 mM (but not 60 mM) KCl or 10 µM noradrenaline in a concentration-dependent manner, showing slightly greater activity in the aorta. The potency of the effect of SR 47063 on HSV and RA was 12- and 58-fold greater, respectively, than that reported for the structurally related K+-channel opener levcromakalim. The vasorelaxant action of SR 47063 in both blood vessels was strongly inhibited by 3 µM glibenclamide, consistent with a mechanism of action involving ATP-dependent K+-channels.

The mechanism of gentisic acid-induced relaxation of the guinea pig isolated trachea: the role of potassium channels and vasoactive intestinal peptide receptors

We examined some of the mechanisms by which the aspirin metabolite and the naturally occurring metabolite gentisic acid induced relaxation of the guinea pig trachea in vitro. In preparations with or without epithelium and contracted by histamine, gentisic acid caused concentration-dependent and reproducible relaxation, with mean EC50 values of 18 µM and Emax of 100% (N = 10) or 20 µM and Emax of 92% (N = 10), respectively. The relaxation caused by gentisic acid was of slow onset in comparison to that caused by norepinephrine, theophylline or vasoactive intestinal peptide (VIP). The relative rank order of potency was: salbutamol 7.9 > VIP 7.0 > gentisic acid 4.7 > theophylline 3.7. Gentisic acid-induced relaxation was markedly reduced (24 ± 7.0, 43 ± 3.9 and 78 ± 5.6%) in preparations with elevated potassium concentration in the medium (20, 40 or 80 mM, respectively). Tetraethylammonium (100 µM), a nonselective blocker of the potassium channels, partially inhibited the relaxation response to gentisic acid, while 4-AP (10 µM), a blocker of the voltage potassium channel, inhibited gentisic acid-induced relaxation by 41 ± 12%. Glibenclamide (1 or 3 µM), at a concentration which markedly inhibited the relaxation induced by the opener of ATP-sensitive K+ channels, levcromakalim, had no effect on the relaxation induced by gentisic acid. Charybdotoxin (0.1 or 0.3 µM), a selective blocker of the large-conductance Ca2+-activated K+ channels, caused rightward shifts (6- and 7-fold) of the gentisic acid concentration-relaxation curve. L-N G-nitroarginine (100 µM), a NO synthase inhibitor, had no effect on the relaxant effect of gentisic acid, and caused a slight displacement to the right in the relaxant effect of the gentisic acid curve at 300 µM, while methylene blue (10 or 30 µM) or ODQ (1 µM), the inhibitors of soluble guanylate cyclase, all failed to affect gentisic acid-induced relaxation. D-P-Cl-Phe6,Leu17[VIP] (0.1 µM), a VIP receptor antagonist, significantly inhibited (37 ± 7%) relaxation induced by gentisic acid, whereas CGRP (8-37) (0.1 µM), a CGRP antagonist, only slightly enhanced the action of gentisic acid. Taken together, these results provide functional evidence for the direct activation of voltage and large-conductance Ca+2-activated K+ channels, or indirect modulation of potassium channels induced by VIP receptors and accounts for the predominant relaxation response caused by gentisic acid in the guinea pig trachea.

Mitochondrial K+ transport and cardiac protection during ischemia/reperfusion

Mitochondrial ion transport, oxidative phosphorylation, redox balance, and physical integrity are key factors in tissue survival following potentially damaging conditions such as ischemia/reperfusion. Recent research has demonstrated that pharmacologically activated inner mitochondrial membrane ATP-sensitive K+ channels (mitoK ATP) are strongly cardioprotective under these conditions. Furthermore, mitoK ATP are physiologically activated during ischemic preconditioning, a procedure which protects against ischemic damage. In this review, we discuss mechanisms by which mitoK ATP may be activated during preconditioning and the mitochondrial and cellular consequences of this activation, focusing on end-effects which may promote ischemic protection. These effects include decreased loss of tissue ATP through reverse activity of ATP synthase due to increased mitochondrial matrix volumes and lower transport of adenine nucleotides into the matrix. MitoK ATP also decreases the release of mitochondrial reactive oxygen species by promoting mild uncoupling in concert with K+/H+ exchange. Finally, mitoK ATP activity may inhibit mitochondrial Ca2+ uptake during ischemia, which, together with decreased reactive oxygen release, can prevent mitochondrial permeability transition, loss of organelle function, and loss of physical integrity. We discuss how mitochondrial redox status, K+ transport, Ca2+ transport, and permeability transitions are interrelated during ischemia/reperfusion and are determinant factors regarding the extent of tissue damage.

A key role for Na+/K+-ATPase in the endothelium-dependent oscillatory activity of mouse small mesenteric arteries

Oscillatory contractile activity is an inherent property of blood vessels. Various cellular mechanisms have been proposed to contribute to oscillatory activity. Mouse small mesenteric arteries display a unique low frequency contractile oscillatory activity (1 cycle every 10-12 min) upon phenylephrine stimulation. Our objective was to identify mechanisms involved in this peculiar oscillatory activity. First-order mesenteric arteries were mounted in tissue baths for isometric force measurement. The oscillatory activity was observed only in vessels with endothelium, but it was not blocked by L-NAME (100 µM) or indomethacin (10 µM), ruling out the participation of nitric oxide and prostacyclin, respectively, in this phenomenon. Oscillatory activity was not observed in vessels contracted with K+ (90 mM) or after stimulation with phenylephrine plus 10 mM K+. Ouabain (1 to 10 µM, an Na+/K+-ATPase inhibitor), but not K+ channel antagonists [tetraethylammonium (100 µM, a nonselective K+ channel blocker), Tram-34 (10 µM, blocker of intermediate conductance K+ channels) or UCL-1684 (0.1 µM, a small conductance K+ channel blocker)], inhibited the oscillatory activity. The contractile activity was also abolished when experiments were performed at 20°C or in K+-free medium. Taken together, these results demonstrate that Na+/K+-ATPase is a potential source of these oscillations. The presence of α-1 and α-2 Na+/K+-ATPase isoforms was confirmed in murine mesenteric arteries by Western blot. Chronic infusion of mice with ouabain did not abolish oscillatory contraction, but up-regulated vascular Na+/K+-ATPase expression and increased blood pressure. Together, these observations suggest that the Na+/K+ pump plays a major role in the oscillatory activity of murine small mesenteric arteries.

Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle

The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F1α (6-keto-PGF1α; a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM22-52, a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP8-37, a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with NG-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K+ channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K+ channels), and apamin (Ca2+-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K+ channels.

Implication des canaux K+ dans les processus de réparation de l’épithélium respiratoire sain et fibrose kystique

La pathologie de la fibrose kystique (FK) est causée par des mutations du gène codant pour le canal Cl- CFTR. Au niveau respiratoire, cette dysfonction du transport transépithélial de Cl- occasionne une altération de la composition et du volume du liquide de surface des voies aériennes. Une accumulation de mucus déshydraté favorise alors la colonisation bactérienne et une réponse inflammatoire chronique, entraînant des lésions épithéliales sévères au niveau des voies aériennes et des alvéoles pouvant culminer en défaillance respiratoire. Le principal objectif de mon projet de maîtrise était d’étudier les processus de réparation de l’épithélium alvéolaire sain, l’épithélium bronchique sain et FK à l’aide d’un modèle in vitro de plaies mécaniques. Nos résultats démontrent la présence d’une boucle autocrine EGF/EGFR contrôlant les processus de migration cellulaire et de réparation des lésions mécaniques. D’autre part, nos expériences montrent que l’EGF stimule l’activité et l’expression des canaux K+ KATP, KvLQT1 et KCa3.1 des cellules épithéliales respiratoires. L’activation de ces canaux est cruciale pour les processus de réparation puisque la majeure partie de la réparation stimulée à l’EGF est abolie en présence d’inhibiteurs de ces canaux. Nous avons également observé que les cellules FK présentent un délai de réparation, probablement causé par un défaut de la réponse EGF/EGFR et une activité/expression réduite des canaux K+. Nos résultats permettent de mieux comprendre les mécanismes de régulation des processus de réparation de l’épithélium sain et FK. De plus, ils ouvrent de nouvelles options thérapeutiques visant à promouvoir, à l’aide d’activateurs de canaux K+ et de facteurs de croissance, la régénération de l’épithélium respiratoire chez les patients atteints de FK.

Diverse mechanisms underlying the regulation of ion channels by carbon monoxide

Carbon monoxide is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, nitric oxide and cGMP levels, as well as regulate MAP kinase signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca2+ -sensitive K+ channels. More recent studies have revealed the ability of CO to inhibit T-type Ca2+ channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

Diverse mechanisms underlying the regulation of ion channels by carbon monoxide

Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca2+ -sensitive K+ channels. More recent studies have revealed the ability of CO to inhibit T-type Ca2+ channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

Morphine peripheral analgesia depends on activation of the PI3K gamma/AKT/nNOS/NO/K(ATP) signaling pathway

Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3K gamma/AKT protein kinase B (AKT) and culminated in increasedactivation of K(ATP) channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.

Nicorandil protects cardiac mitochondria against permeability transition induced by ischemia-reperfusion

Ischemia followed by reperfusion is known to negatively affect mitochondrial function by inducing a deleterious condition termed mitochondrial permeability transition. Mitochondrial permeability transition is triggered by oxidative stress, which occurs in mitochondria during ischemia-reperfusion as a result of lower antioxidant defenses and increased oxidant production. Permeability transition causes mitochondrial dysfunction and can ultimately lead to cell death. A drug able to minimize mitochondrial damage induced by ischemia-reperfusion may prove to be clinically effective. We aimed to analyze the effects of nicorandil, an ATP-sensitive potassium channel agonist and vasodilator, on mitochondrial function of rat hearts and cardiac HL-1 cells submitted to ischemia-reperfusion. Nicorandil decreased mitochondrial swelling and calcium uptake. It also decreased reactive oxygen species formation and thiobarbituric acid reactive substances levels, a lipid peroxidation biomarker. We thus confirm previous reports that nicorandil inhibits mitochondrial permeability transition and demonstrate that nicorandil inhibits this process by preventing oxidative damage and mitochondrial calcium overload induced by ischemia-reperfusion, resulting in improved cardiomyocyte viability. These results may explain the good clinical results obtained when using nicorandil in the treatment of ischemic heart disease.

The locus coeruleus and central chemosensitivity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K + (K ATP) channels, increasing intracellular Ca 2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K ATP channel opener and 10 μM nifedipine, a Ca 2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K ATP and L-type Ca 2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. © 2012 Elsevier Ltd.

Ethanol induces vascular relaxation via redox-sensitive and nitric oxide-dependent pathways

We investigated the role of reactive oxygen species (ROS) and nitric oxide (NO) in ethanol-induced relaxation. Vascular reactivity experiments showed that ethanol (0.03-200 mmol/L) induced relaxation in endothelium-intact and denuded rat aortic rings isolated from male Wistar rats. Pre-incubation of intact or denuded rings with L-NAME (non selective NOS inhibitor, 100 mu mol/L), 7-nitroindazole (selective nNOS inhibitor, 100 mu mol/L), ODQ (selective inhibitor of guanylyl cyclase enzyme, I mu mol/L), glibenclamide (selective blocker of ATP-sensitive K+ channels, 3 mu mol/L) and 4-aminopyridine (selective blocker of voltage-dependent K+ channels, 4-AP, 1 mmol/L) reduced ethanol-induced relaxation. Similarly, tiron (superoxide anion (O-2(-)) scavenger, 1 mmol/L) and catalase (hydrogen peroxide (H2O2) scavenger, 300 U/mL) reduced ethanol-induced relaxation to a similar extent in both endothelium-intact and denuded rings. Finally, prodifen (non-selective cytochrome P450 enzymes inhibitor, 10 mu mol/L) and 4-methylpyrazole (selective alcohol dehydrogenase inhibitor, 10 mu mol/L) reduced ethanol-induced relaxation. In cultured aortic vascular smooth muscle cells (VSMCs), ethanol stimulated generation of NO, which was significantly inhibited by L-NAME. In endothelial cells, flow cytometry studies showed that ethanol increased cytosolic Ca2+ concentration ([Ca2+]c), O-2(-) and cytosolic NO concentration ([NO]c). Tiron inhibited ethanol-induced increase in [Ca-2]c and [NO]c. The major new finding of this work is that ethanol induces relaxation via redox-sensitive and NO-cGMP-dependent pathways through direct effects on ROS production and NO signaling. These findings identify putative molecular mechanisms whereby ethanol, at pharmacological concentrations, influences vascular reactivity. (C) 2011 Elsevier Inc. All rights reserved.

Ether-A -go-go 1 (Eag1) Potassium Channel Expression in Dopaminergic Neurons of Basal Ganglia is Modulated by 6-Hydroxydopamine Lesion

The ether A go-go (Eag) gene encodes the voltage-gated potassium (K+) ion channel Kv10.1, whose function still remains unknown. As dopamine may directly affect K+ channels, we evaluated whether a nigrostriatal dopaminergic lesion induced by the neurotoxin 6-hydroxydopamine (6-OHDA) would alter Eag1-K+ channel expression in the rat basal ganglia and related brain regions. Male Wistar rats received a microinjection of either saline or 6-OHDA (unilaterally) into the medial forebrain bundle. The extent of the dopaminergic lesion induced by 6-OHDA was evaluated by apomorphine-induced rotational behavior and by tyrosine hydroxylase (TH) immunoreactivity. The 6-OHDA microinjection caused a partial or complete lesion of dopaminergic cells, as well as a reduction of Eag1+ cells in a manner proportional to the extent of the lesion. In addition, we observed a decrease in TH immunoreactivity in the ipsilateral striatum. In conclusion, the expression of the Eag1-K+-channel throughout the nigrostriatal pathway in the rat brain, its co-localization with dopaminergic cells and its reduction mirroring the extent of the lesion highlight a physiological circuitry where the functional role of this channel can be investigated. The Eag1-K+ channel expression in dopaminergic cells suggests that these channels are part of the diversified group of ion channels that generate and maintain the electrophysiological activity pattern of dopaminergic midbrain neurons.

Nerve excitability studies characterize Kv1.1 fast potassium channel dysfunction in patients with episodic ataxia type 1

Episodic ataxia type 1 is a neuronal channelopathy caused by mutations in the KCNA1 gene encoding the fast K(+) channel subunit K(v)1.1. Episodic ataxia type 1 presents with brief episodes of cerebellar dysfunction and persistent neuromyotonia and is associated with an increased incidence of epilepsy. In myelinated peripheral nerve, K(v)1.1 is highly expressed in the juxtaparanodal axon, where potassium channels limit the depolarizing afterpotential and the effects of depolarizing currents. Axonal excitability studies were performed on patients with genetically confirmed episodic ataxia type 1 to characterize the effects of K(v)1.1 dysfunction on motor axons in vivo. The median nerve was stimulated at the wrist and compound muscle action potentials were recorded from abductor pollicis brevis. Threshold tracking techniques were used to record strength-duration time constant, threshold electrotonus, current/threshold relationship and the recovery cycle. Recordings from 20 patients from eight kindreds with different KCNA1 point mutations were compared with those from 30 normal controls. All 20 patients had a history of episodic ataxia and 19 had neuromyotonia. All patients had similar, distinctive abnormalities: superexcitability was on average 100% higher in the patients than in controls (P < 0.00001) and, in threshold electrotonus, the increase in excitability due to a depolarizing current (20% of threshold) was 31% higher (P < 0.00001). Using these two parameters, the patients with episodic ataxia type 1 and controls could be clearly separated into two non-overlapping groups. Differences between the different KCNA1 mutations were not statistically significant. Studies of nerve excitability can identify K(v)1.1 dysfunction in patients with episodic ataxia type 1. The simple 15 min test may be useful in diagnosis, since it can differentiate patients with episodic ataxia type 1 from normal controls with high sensitivity and specificity.