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 ��M) in the presence or absence of 3 ��M glibenclamide. SR 47063 potently relaxed HSV and RA precontracted with 20 mM (but not 60 mM) KCl or 10 ��M 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 ��M glibenclamide, consistent with a mechanism of action involving ATP-dependent K+-channels.

Participation of ATP-sensitive K+ channels in the peripheral antinociceptive effect of fentanyl in rats

We examined the effect of several K+ channel blockers such as glibenclamide, tolbutamide, charybdotoxin (ChTX), apamin, tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP), and cesium on the ability of fentanyl, a clinically used selective ��-opioid receptor agonist, to promote peripheral antinociception. Antinociception was measured by the paw pressure test in male Wistar rats weighing 180-250 g (N = 5 animals per group). Carrageenan (250 ��g/paw) decreased the threshold of responsiveness to noxious pressure (delta = 188.1 �� 5.3 g). This mechanical hyperalgesia was reduced by fentanyl (0.5, 1.5 and 3 ��g/paw) in a peripherally mediated and dose-dependent fashion (17.3, 45.3 and 62.6%, respectively). The selective blockers of ATP-sensitive K+ channels glibenclamide (40, 80 and 160 ��g/paw) and tolbutamide (80, 160 and 240 ��g/paw) dose dependently antagonized the antinociception induced by fentanyl (1.5 ��g/paw). In contrast, the effect of fentanyl was unaffected by the large conductance Ca2+-activated K+ channel blocker ChTX (2 ��g/paw), the small conductance Ca2+-activated K+ channel blocker apamin (10 ��g/paw), or the non-specific K+ channel blocker TEA (150 ��g/paw), 4-AP (50 ��g/paw), and cesium (250 ��g/paw). These results extend previously reported data on the peripheral analgesic effect of morphine and fentanyl, suggesting for the first time that the peripheral ��-opioid receptor-mediated antinociceptive effect of fentanyl depends on activation of ATP-sensitive, but not other, K+ channels.

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 ��M) or indomethacin (10 ��M), 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 ��M, an Na+/K+-ATPase inhibitor), but not K+ channel antagonists [tetraethylammonium (100 ��M, a nonselective K+ channel blocker), Tram-34 (10 ��M, blocker of intermediate conductance K+ channels) or UCL-1684 (0.1 ��M, 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.

Participation of the NO/cGMP/K+ATP pathway in the antinociception induced by Walker tumor bearing in rats

Implantation of Walker 256 tumor decreases acute systemic inflammation in rats. Inflammatory hyperalgesia is one of the most important events of acute inflammation. The L-arginine/NO/cGMP/K+ATP pathway has been proposed as the mechanism of peripheral antinociception mediated by several drugs and physical exercise. The objective of this study was to investigate a possible involvement of the NO/cGMP/K+ATP pathway in antinociception induced in Walker 256 tumor-bearing male Wistar rats (180-220 g). The groups consisted of 5-6 animals. Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. Walker tumor (4th and 7th day post-implantation) reduced prostaglandin E2- (PGE2, 400 ng/paw; 50 ��L; intraplantar injection) and carrageenan-induced hypernociception (500 ��g/paw; 100 ��L; intraplantar injection). Walker tumor-induced analgesia was reversed (99.3% for carrageenan and 77.2% for PGE2) by a selective inhibitor of nitric oxide synthase (L-NAME; 90 mg/kg, ip) and L-arginine (200 mg/kg, ip), which prevented (80% for carrageenan and 65% for PGE2) the effect of L-NAME. Treatment with the soluble guanylyl cyclase inhibitor ODQ (100% for carrageenan and 95% for PGE2; 8 ��g/paw) and the ATP-sensitive K+ channel (KATP) blocker glibenclamide (87.5% for carrageenan and 100% for PGE2; 160 ��g/paw) reversed the antinociceptive effect of tumor bearing in a statistically significant manner (P < 0.05). The present study confirmed an intrinsic peripheral antinociceptive effect of Walker tumor bearing in rats. This antinociceptive effect seemed to be mediated by activation of the NO/cGMP pathway followed by the opening of KATP channels.

Implication des canaux K+ dans les processus de r��paration de l�����pith��lium respiratoire sain et fibrose kystique

La pathologie de la fibrose kystique (FK) est caus��e par des mutations du g��ne codant pour le canal Cl- CFTR. Au niveau respiratoire, cette dysfonction du transport trans��pith��lial de Cl- occasionne une alt��ration de la composition et du volume du liquide de surface des voies a��riennes. Une accumulation de mucus d��shydrat�� favorise alors la colonisation bact��rienne et une r��ponse inflammatoire chronique, entra��nant des l��sions ��pith��liales s��v��res au niveau des voies a��riennes et des alv��oles pouvant culminer en d��faillance respiratoire. Le principal objectif de mon projet de ma��trise ��tait d�����tudier les processus de r��paration de l�����pith��lium alv��olaire sain, l�����pith��lium bronchique sain et FK �� l���aide d���un mod��le in vitro de plaies m��caniques. Nos r��sultats d��montrent la pr��sence d���une boucle autocrine EGF/EGFR contr��lant les processus de migration cellulaire et de r��paration des l��sions m��caniques. D���autre part, nos exp��riences montrent que l���EGF stimule l���activit�� et l���expression des canaux K+ KATP, KvLQT1 et KCa3.1 des cellules ��pith��liales respiratoires. L���activation de ces canaux est cruciale pour les processus de r��paration puisque la majeure partie de la r��paration stimul��e �� l���EGF est abolie en pr��sence d���inhibiteurs de ces canaux. Nous avons ��galement observ�� que les cellules FK pr��sentent un d��lai de r��paration, probablement caus�� par un d��faut de la r��ponse EGF/EGFR et une activit��/expression r��duite des canaux K+. Nos r��sultats permettent de mieux comprendre les m��canismes de r��gulation des processus de r��paration de l�����pith��lium sain et FK. De plus, ils ouvrent de nouvelles options th��rapeutiques visant �� promouvoir, �� l���aide d���activateurs de canaux K+ et de facteurs de croissance, la r��g��n��ration de l�����pith��lium respiratoire chez les patients atteints de FK.

Implications des canaux K+ sur la r��gulation g��nique du canal ENaC, et impact de l'hyperglyc��mie sur le transport ionique et la r��paration de l'��pith��lium respiratoire

Dans mon projet de doctorat, j���ai ��tudi�� des fonctions primordiales de l�����pith��lium respiratoire telles que la r��gulation du transport ionique, la clairance liquidienne et la r��paration ��pith��liale. J���ai particuli��rement mis l���emphase sur le r��le des canaux potassiques qui interviennent dans ces trois fonctions de l�����pith��lium respiratoire. J���ai tout d���abord prouv�� que la modulation des canaux potassiques r��gulait l���activit�� du promoteur de ��ENaC, en partie via la voie de signalisation ERK1/2, dans des cellules alv��olaires. Cette r��gulation entra��ne une variation de l���expression g��nique et prot��ique du canal ENaC. Physiologiquement, il en r��sulte une augmentation du ph��nom��ne de clairance liquidienne suite �� l���activation des canaux K+, tandis que l���inhibition de ces canaux la diminue s��v��rement. J���ai aussi pu d��montrer que l���absence de canal KvLQT1 entra��nait une diminution du courant (ENaC) sensible �� l���amiloride, dans les cellules de trach��e en culture primaire, isol��es de souris KO pour kcnq1. Dans la seconde partie de mon ��tude, j���ai ��valu�� l���impact de l���hyperglyc��mie sur la capacit�� de transport ionique et de r��paration de cellules ��pith��liales bronchiques saines ou Fibrose Kystique. Mes r��sultats montrent que l���hyperglyc��mie diminue le transport trans��pith��lial de chlore et le transport basolat��ral de potassium. Des ��tudes pr��alables du laboratoire ayant montr�� que les canaux K+ et Cl- contr��lent les processus de r��paration, j���ai donc ��valu�� si ceux-ci ��taient modifi��s par l���hyperglyc��mie. Et en effet, l���hyperglyc��mie ralentit la vitesse de r��paration des cellules issues des voies a��riennes (CFBE-wt et CFBE-��F508). J���ai donc d��montr�� que le transport de potassium intervenait dans des fonctions cl��s de l�����pith��lium respiratoire, comme dans la r��gulation g��nique de canaux ioniques, le contr��le de la clairance liquidienne alv��olaire, et que l���hyperglyc��mie diminuait le transport ionique (K+ et Cl-) et la r��paration ��pith��liale.

Anesthetic Activation of Central Respiratory Chemoreceptor Neurons Involves Inhibition of a THIK-1-Like Background K(+) Current

At surgical depths of anesthesia, inhalational anesthetics cause a loss of motor response to painful stimuli (i.e., immobilization) that is characterized by profound inhibition of spinal motor circuits. Yet, although clearly depressed, the respiratory motor system continues to provide adequate ventilation under these same conditions. Here, we show that isoflurane causes robust activation of CO(2)/pH-sensitive, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) of the rodent brainstem, in vitro and in vivo. In brainstem slices from Phox2b-eGFP mice, the firing of pH-sensitive RTN neurons was strongly increased by isoflurane, independent of prevailing pH conditions. At least two ionic mechanisms contributed to anesthetic activation of RTN neurons: activation of an Na(+)-dependent cationic current and inhibition of a background K(+) current. Single-cell reverse transcription-PCR analysis of dissociated green fluorescent protein-labeled RTN neurons revealed expression of THIK-1 (TWIK-related halothane-inhibited K(+) channel, K(2P)13.1), a channel that shares key properties with the native RTN current (i.e., suppression by inhalational anesthetics, weak rectification, inhibition by extracellular Na(+), and pH-insensitivity). Isoflurane also increased firing rate of RTN chemosensitive neurons in urethane-anesthetized rats, again independent of CO(2) levels. In these animals, isoflurane transiently enhanced activity of the respiratory system, an effect that was most prominent at low levels of respiratory drive and mediated primarily by an increase in respiratory frequency. These data indicate that inhalational anesthetics cause activation of RTN neurons, which serve an important integrative role in respiratory control; the increased drive provided by enhanced RTN neuronal activity may contribute, in part, to maintaining respiratory motor activity under immobilizing anesthetic conditions.

K-ATP(+) Channels-Independent Analgesic Action of Crotalus durissus cumanensis venom

The effect was investigated of the K+ channel blocker, glibenclamide, on the ability of Crotalus durissus cumanensis venom (CDCM) to promote peripheral antinociception. This was measured by formalin-induced nociception in male Swiss mice. CDCM (200 and 300 mu g/kg) produced an antinociceptive effect during phase 2 in the formalin test. The effect of CDCM (200 mu g/kg) was unaffected by the ATP-sensitive K+ channel blocker glibenclamide (2 mg/kg). These results suggest that CDCM is effective against acute pain. However, the ATP-sensitive K+ channels pathway is not contributable to the antinoeiceptive mechanism of CDCM.

Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of Type II Bartter's syndrome and in adaptation to a high-K diet

Type II Bartter's syndrome is a hereditary hypokalemic renal salt-wasting disorder caused by mutations in the ROMK channel (Kir1.1; Kcnj1), mediating potassium recycling in the thick ascending limb of Henle's loop (TAL) and potassium secretion in the distal tubule and cortical collecting duct (CCT). Newborns with Type II Bartter are transiently hyperkalemic, consistent with loss of ROMK channel function in potassium secretion in distal convoluted tubule and CCT. Yet, these infants rapidly develop persistent hypokalemia owing to increased renal potassium excretion mediated by unknown mechanisms. Here, we used free-flow micropuncture and stationary microperfusion of the late distal tubule to explore the mechanism of renal potassium wasting in the Romk-deficient, Type II Bartter's mouse. We show that potassium absorption in the loop of Henle is reduced in Romk-deficient mice and can account for a significant fraction of renal potassium loss. In addition, we show that iberiotoxin (IBTX)-sensitive, flow-stimulated maxi-K channels account for sustained potassium secretion in the late distal tubule, despite loss of ROMK function. IBTX-sensitive potassium secretion is also increased in high-potassium-adapted wild-type mice. Thus, renal potassium wasting in Type II Bartter is due to both reduced reabsorption in the TAL and K secretion by max-K channels in the late distal tubule. �� 2006 International Society of Nephrology.

The peripheral L-arginine-nitric oxide-cyclic GMP pathway and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine on neuropathic pain in rats

Crotalphine, a 14 amino acid peptide first isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces a peripheral long-lasting and opioid receptor-mediated antinociceptive effect in a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. In the present study, we further characterized the molecular mechanisms involved in this effect, determining the type of opioid receptor responsible for this effect and the involvement of the nitric oxide-cyclic GMP pathway and of K+ channels. Crotalphine (0.2 or 5 mu g/kg, orally; 0.0006 mu g/paw), administered on day 14 after nerve constriction, inhibited mechanical hyperalgesia and low-threshold mechanical allodynia. The effect of the peptide was antagonized by intraplantar administration of naltrindole, an antagonist of delta-opioid receptors, and partially reversed by norbinaltorphimine, an antagonist of kappa-opioid receptors. The effect of crotalphine was also blocked by 7-nitroindazole, an inhibitor of the neuronal nitric oxide synthase; by 1H-(1,2,4) oxadiazolo[4,3-a]quinoxaline-1-one, an inhibitor of guanylate cyclase activation; and by glibenclamide, an ATP-sensitive K+ channel blocker. The results suggest that peripheral delta-opioid and kappa-opioid receptors, the nitric oxide-cyclic GMP pathway, and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine. The present data point to the therapeutic potential of this peptide for the treatment of chronic neuropathic pain. Behavioural Pharmacology 23:14-24 (C) 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

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.

Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History

Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories���hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.

Ceramide-induced inhibition of T lymphocyte voltage-gated potassium channel is mediated by���tyrosine���kinases

The n-type K+ channel (n-K+, Kv1.3) in lymphocytes has been recently implicated in the regulation of Fas-induced programmed cell death. Here, we demonstrate that ceramide, a lipid metabolite synthesized upon Fas receptor ligation, inhibits n-K+ channel activity and induces a tyrosine phosphorylation of the Kv1.3 protein in Jurkat T lymphocytes. Tyrosine phosphorylation of the n-K+ channel correlated with an activation of the Src-like tyrosine kinase p56lck upon cellular treatment with the ceramide analog C6-ceramide. Because genetic deficiency of p56lck or inhibition of Src-like tyrosine kinases by herbimycin A prevented ceramide-mediated n-K+ channel inhibition and tyrosine phosphorylation, we propose a ceramide-initiated activation of p56lck resulting in tyrosine phosphorylation and inhibition of the n-K+ channel protein.

ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces���cerevisiae

A K+ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K+ uptake. Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K+ transport capacity on yeast cells when heterologously expressed. In Xenopus laevis oocytes, expression yields an ungated K+-selective current whose attributes resemble the ���leak��� conductance thought to mediate the resting potential of vertebrate myelinated neurons but whose molecular nature has long remained elusive. The predicted protein has two pore (P) domains and four membrane-spanning helices and is a member of a newly recognized K+ channel family. Expression of the channel in flies and yeast cells makes feasible studies of structure and in vivo function using genetic approaches that are not possible in higher animals.