Extract of Glycyrrhiza glabra root attenuates nociception in experimental pain models: the role of BKCa channels / Extracto de raíz de Glycyrrhiza glabra atenúa la nocicepción en modelos experimentales de dolor: el rol de los canales BKCa

Abstract: The aim of this study was to evaluate the functional interaction of Glycyrrhiza glabra root extract (GGRE) on the large conductance Ca 2+ - activated K + (BKCa) channels expressed in the peripheral nervo us system by using nociception and inflammation models in rodents in vivo . Besides toxicity studies and open field tests, nociception and inflammation tests were performed on rodents. Different doses of GGRE were given orally to rats and mice. Naloxone, in domethacin, morphine, NS1619 and iberiotoxin (IbTX) were administered. GGRE had both anti - nociceptive and anti - inflammatory activity in rats and mice. GGRE exhibited an analgesic effect by decreasing the time - course of the pain threshold or reaction time i n some nociceptive tests. Furthermore, GGRE reduced level of pro - inflammatory cytokines, including TNF - α and IL - 1ß. As a conclusion, GGRE can alleviate the pain sensation of the afferent nerves and can reduce inflammation and associated pain by activating B KCa channels and reducing the levels of TNF - α, IL1ß

Resumen: El objetivo de este estudio fue evaluar la interacción funcional del extracto de raíz de Glycyrr hiza glabra (GGRE) en los canales de K + (BKCa) activados por Ca 2+ de gran conductancia expresados en el sistema nervioso periférico mediante el uso de modelos de nocicepción e inflamación en roedores in vivo . Además de los estudios de toxicidad y las prueb as de campo abierto, se realizaron pruebas de nocicepción e inflamación en roedores. Se administraron por vía oral diferentes dosis de GGRE a ratas y ratones. Se administraron naloxona, indometacina, morfina, NS1619 e iberiotoxina (IbTX). GGRE tenía activi dad tanto antinociceptiva como antiinflamatoria en ratas y ratones. GGRE mostró un efecto analgésico al disminuir la evolución temporal del umbral del dolor o el tiempo de reacción en algunas pruebas nociceptivas. Además, GGRE redujo el nivel de citocinas proinflamatorias, incluidas TNF - α e IL - 1ß. Como conclusión, GGRE puede aliviar la sensación de dolor de los nervios aferentes y puede reducir la inflamación y el dolor asociado activando los canales BKCa y reduciendo los niveles de TNF - α, IL1ß.

Heme is required for carbon monoxide activation of mitochondrial BKCa channel.

Carbon monoxide (CO) is an endogenously synthesized gaseous mediator and is involved in the regulation of numerous physiological processes. Mitochondria, in which hemoproteins are abundant, are among the targets for CO action. Large-conductance calcium-activated (mitoBKCa) channels in the inner mitochondrial membrane share multiple biophysical similarities with the BKCa channels of the plasma membrane and could be a potential target for CO. To test this hypothesis, the activity of the mitoBKCa channels in human astrocytoma U-87 MG cell mitochondria was assessed with the patch-clamp technique. The effects of CO-releasing molecules (CORMs), such as CORM-2, CORM-401, and CORM-A1, were compared to the application of a CO-saturated solution to the mitoBKCa channels in membrane patches. The applied CORMs showed pleiotropic effects including channel inhibition, while the CO-containing solution did not significantly modulate channel activity. Interestingly, CO applied to the mitoBKCa channels, which were inhibited by exogenously added heme, stimulated the channel. To summarize, our findings indicate a requirement of heme binding to the mitoBKCa channel for channel modulation by CO and suggest that CORMs might have complex unspecific effects on mitoBKCa channels.

Arecoline inhibits intermediate-conductance calcium-activated potassium channels in human glioblastoma cell lines.

Arecoline (ARE) is an alkaloid-type natural product from areca nut. This compound has numerous pharmacological and toxicological effects. Whether this agent interacts with ion channels to perturb functional activity of cells remains unknown. The effects of ARE on ionic currents were studied in glioma cell lines (U373 and U87MG) using patch-clamp technique. Like TRAM-34(1-[(2-chlorophenyl)-diphenylmethyl]pyrazole), ARE suppressed the amplitude of whole-cell voltage-gated K(+) currents in U373 cells elicited by a ramp voltage clamp. In cell-attached configuration, ARE did not modify the single-channel conductance of intermediate-conductance Ca(2+)-activated K(+) (IKCa) channels; however, it did reduce channel activity. Its inhibition of IKCa channels was accompanied by a significant lengthening in the slow component of mean closed time of IKCa channels. Based on minimal kinetic scheme, the dissociation constant (KD) required for ARE-mediated prolongation of mean closed time was 11.2µM. ARE-induced inhibition of IKCa channels was voltage-dependent. Inability of ARE to perturb the activity of large-conductance Ca(2+)-activated K(+) (BKCa) channels was seen. Under current-clamp recordings, ARE depolarized the membrane of U373 cells and DCEBIO reversed ARE-induced depolarization. Similarly, ARE suppressed IKCa-channel activities in oral keratinocytes. This study provides the evidence that ARE block IKCa channels in a concentration, voltage and state-dependent manner. ARE-induced block of IKCa channels is unrelated to the binding of muscarinic receptors. The effects of ARE on these channels may partially be responsible for the underlying cellular mechanisms by which it influences the functional activities of glioma cells or oral keratinocytes, if similar findings occur in vivo.

Rottlerin-induced BKCa channel activation impairs specific contractile responses and promotes vasodilation.

BACKGROUND: Activation of large conductance calcium-activated potassium (BKCa) channels is cardioprotective for ischemic injury and can enhance vasorelaxation. Rottlerin has recently been identified as a potent BKCa activator. We demonstrated that rottlerin improves cardiac function and increases coronary flow when used as a cardioplegia additive in rat and mouse models of cardioplegic arrest and reperfusion. In this study we examined the effectiveness and specificity of the putative BKCa activator rottlerin on vascular reactivity in response to specific contractile and dilatory agonists. METHODS: Aortic rings from wild-type (wt) and BKCa knock-out (KO) mice were mounted in a tissue bath with force transducers. The vasodilatory effect of rottlerin was evaluated after pre-constriction with U46619. Dose responses to the contractile agonists U46619 and phenylephrine (PE), and vasodilation responses to rottlerin, hydrogen sulfide (H2S), and sodium nitroprusside (SNP) were performed after pretreatment with rottlerin. Similar studies were performed in pig coronary vessels. RESULTS: The BKCa KO mouse aortic rings exhibited spontaneous contraction and had greater contractile responses to U46619 and reduced vasodilation to SNP compared with wt mice. The wt and KO responses to phenylephrine were similar. Rottlerin dose dependently dilated wild-type vessels, but not in BKCa KO animals. Pretreatment with rottlerin caused depressed U46619 responses, but had no effect on PE, SNP, or H2S-mediated responses. However, pig coronary vessels pretreated with rottlerin exhibited reduced contractile responses and enhanced nitric oxide-dependent dilation. CONCLUSIONS: Rottlerin directly causes vasodilation through BKCa channel dependent mechanisms. The BKCa channel activator pretreatment enhances vasodilatory responses and impairs specific vasoconstrictive agonists.

Slo1 is the principal potassium channel of human spermatozoa.

Mammalian spermatozoa gain competence to fertilize an oocyte as they travel through the female reproductive tract. This process is accompanied by an elevation of sperm intracellular calcium and a membrane hyperpolarization. The latter is evoked by K(+) efflux; however, the molecular identity of the potassium channel of human spermatozoa (hKSper) is unknown. Here, we characterize hKSper, reporting that it is regulated by intracellular calcium but is insensitive to intracellular alkalinization. We also show that human KSper is inhibited by charybdotoxin, iberiotoxin, and paxilline, while mouse KSper is insensitive to these compounds. Such unique properties suggest that the Slo1 ion channel is the molecular determinant for hKSper. We show that Slo1 is localized to the sperm flagellum and is inhibited by progesterone. Inhibition of hKSper by progesterone may depolarize the spermatozoon to open the calcium channel CatSper, thus raising [Ca(2+)] to produce hyperactivation and allowing sperm to fertilize an oocyte. DOI:http://dx.doi.org/10.7554/eLife.01009.001.

Blockade of ATP-sensitive potassium channels prevents the attenuation of the exercise pressor reflex by tempol in rats with ligated femoral arteries.

We reported previously that tempol attenuated the exercise pressor and muscle mechanoreceptor reflexes in rats whose femoral arteries were ligated, whereas tempol did not attenuate these reflexes in rats whose femoral arteries were freely perfused. Although the mechanism whereby tempol attenuated these reflexes in rats whose femoral artery was ligated was independent of its ability to scavenge reactive oxygen species, its nature remains unclear. An alternative explanation for the tempol-induced attenuation of these reflexes involves ATP-sensitive potassium channels (K(ATP)) and calcium-activated potassium channels (BK(Ca)), both of which are opened by tempol. We tested the likelihood of this explanation by measuring the effects of either glibenclamide (0.1 mg/kg), which blocks K(ATP) channels, or iberiotoxin (20 or 40 µg/kg), which blocks BK(Ca) channels, on the tempol-induced attenuation of the exercise pressor and muscle mechanoreceptor reflexes in decerebrated rats whose femoral arteries were ligated. We found that glibenclamide prevented the tempol-induced attenuation of both reflexes, whereas iberiotoxin did not. We also found that the amount of protein comprising the pore of the K(ATP) channel in the dorsal root ganglia innervating hindlimbs whose femoral artery was ligated was significantly greater than that in the dorsal root ganglia innervating hindlimbs whose femoral arteries were freely perfused. In contrast, the amounts of protein comprising the BK(Ca) channel in the dorsal root ganglia innervating the ligated and freely perfused hindlimbs were not different. We conclude that tempol attenuated both reflexes by opening K(ATP) channels, an effect that hyperpolarized muscle afferents stimulated by static contraction or tendon stretch.

Cysteine residue 911 in C-terminal tail of human BK(Ca)α channel subunit is crucial for its activation by carbon monoxide.

The large conductance, voltage- and calcium-activated potassium channel, BK(Ca), is a known target for the gasotransmitter, carbon monoxide (CO). Activation of BK(Ca) by CO modulates cellular excitability and contributes to the physiology of a diverse array of processes, including vascular tone and oxygen-sensing. Currently, there is no consensus regarding the molecular mechanisms underpinning reception of CO by the BK(Ca). Here, employing voltage-clamped, inside-out patches from HEK293 cells expressing single, double and triple cysteine mutations in the BK(Ca) α-subunit, we test the hypothesis that CO regulation is conferred upon the channel by interactions with cysteine residues within the RCK2 domain. In physiological [Ca(2+)](i), all mutants carrying a cysteine substitution at position 911 (C911G) demonstrated significantly reduced CO sensitivity; the C911G mutant did not express altered Ca(2+)-sensitivity. In contrast, histidine residues in RCK1 domain, previously shown to ablate CO activation in low [Ca(2+)](i), actually increased CO sensitivity when [Ca(2+)](i) was in the physiological range. Importantly, cyanide, employed here as a substituent for CO at potential metal centres, occluded activation by CO; this effect was freely reversible. Taken together, these data suggest that a specific cysteine residue in the C-terminal domain, which is close to the Ca(2+) bowl but which is not involved in Ca(2+) activation, confers significant CO sensitivity to BK(Ca) channels. The rapid reversibility of CO and cyanide binding, coupled to information garnered from other CO-binding proteins, suggests that C911 may be involved in formation of a transition metal cluster which can bind and, thereafter, activate BK(Ca).

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells.

Plasma membrane large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK(Ca) channel regulation by IP(3) and IP(3)Rs in rat and mouse cerebral artery smooth muscle cells. IP(3) activated BK(Ca) channels both in intact cells and in excised inside-out membrane patches. IP(3) caused concentration-dependent BK(Ca) channel activation with an apparent dissociation constant (K(d)) of approximately 4 microM at physiological voltage (-40 mV) and intracellular Ca(2+) concentration ([Ca(2+)](i); 10 microM). IP(3) also caused a leftward-shift in BK(Ca) channel apparent Ca(2+) sensitivity and reduced the K(d) for free [Ca(2+)](i) from approximately 20 to 12 microM, but did not alter the slope or maximal P(o). BAPTA, a fast Ca(2+) buffer, or an elevation in extracellular Ca(2+) concentration did not alter IP(3)-induced BK(Ca) channel activation. Heparin, an IP(3)R inhibitor, and a monoclonal type 1 IP(3)R (IP(3)R1) antibody blocked IP(3)-induced BK(Ca) channel activation. Adenophostin A, an IP(3)R agonist, also activated BK(Ca) channels. IP(3) activated BK(Ca) channels in inside-out patches from wild-type (IP(3)R1(+/+)) mouse arterial smooth muscle cells, but had no effect on BK(Ca) channels of IP(3)R1-deficient (IP(3)R1(-/-)) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP(3)R1 is located in close spatial proximity to BK(Ca) alpha subunits. The IP(3)R1 monoclonal antibody coimmunoprecipitated IP(3)R1 and BK(Ca) channel alpha and beta1 subunits from cerebral arteries. In summary, data indicate that IP(3)R1 activation elevates BK(Ca) channel apparent Ca(2+) sensitivity through local molecular coupling in arterial smooth muscle cells.

P2Y1 receptors mediate an activation of neuronal calcium-dependent K+ channels.

Molecularly defined P2Y receptor subtypes are known to regulate the functions of neurons through an inhibition of K(V)7 K(+) and Ca(V)2 Ca(2+) channels and via an activation or inhibition of Kir3 channels. Here, we searched for additional neuronal ion channels as targets for P2Y receptors. Rat P2Y(1) receptors were expressed in PC12 cells via an inducible expression system, and the effects of nucleotides on membrane currents and intracellular Ca(2+) were investigated. At a membrane potential of 30 mV, ADP induced transient outward currents in a concentration-dependent manner with half-maximal effects at 4 µm. These currents had reversal potentials close to the K(+) equilibrium potential and changed direction when extracellular Na(+) was largely replaced by K(+), but remained unaltered when extracellular Cl() was changed. Currents were abolished by P2Y(1) antagonists and by blockade of phospholipase C. ADP also caused rises in intracellular Ca(2+), and ADP-evoked currents were abolished when inositol trisphosphate-sensitive Ca(2+) stores were depleted. Blockers of K(Ca)2, but not those of K(Ca)1.1 or K(Ca)3.1, channels largely reduced ADP-evoked currents. In hippocampal neurons, ADP also triggered outward currents at 30 mV which were attenuated by P2Y(1) antagonists, depletion of Ca(2+) stores, or a blocker of K(Ca)2 channels. These results demonstrate that activation of neuronal P2Y(1) receptors may gate Ca(2+)-dependent K(+) (K(Ca)2) channels via phospholipase C-dependent increases in intracellular Ca(2+) and thereby define an additional class of neuronal ion channels as novel effectors for P2Y receptors. This mechanism may form the basis for the control of synaptic plasticity via P2Y(1) receptors.

Adrenaline-induced colonic K+ secretion is mediated by KCa1.1 (BK) channels.

Colonic epithelial K(+) secretion is a two-step transport process with initial K(+) uptake over the basolateral membrane followed by K(+) channel-dependent exit into the lumen. In this process the large-conductance, Ca(2+)-activated K(Ca)1.1 (BK) channel has been identified as the only apparent secretory K(+) channel in the apical membrane of the murine distal colon. The BK channel is responsible for both resting and Ca(2+)-activated colonic K(+) secretion and is up-regulated by aldosterone. Agonists (e.g. adrenaline) that elevate cAMP are potent activators of distal colonic K(+) secretion. However, the secretory K(+) channel responsible for cAMP-induced K(+) secretion remains to be defined. In this study we used the Ussing chamber to identify adrenaline-induced electrogenic K(+) secretion. We found that the adrenaline-induced electrogenic ion secretion is a compound effect dominated by anion secretion and a smaller electrically opposing K(+) secretion. Using tissue from (i) BK wildtype (BK(+/+)) and knockout (BK(/)) and (ii) cystic fibrosis transmembrane regulator (CFTR) wildtype (CFTR(+/+)) and knockout (CFTR(/)) mice we were able to isolate the adrenaline-induced K(+) secretion. We found that adrenaline-induced K(+) secretion: (1) is absent in colonic epithelia from BK(/) mice, (2) is greatly up-regulated in mice on a high K(+) diet and (3) is present as sustained positive current in colonic epithelia from CFTR(/) mice. We identified two known C-terminal BK alpha-subunit splice variants in colonic enterocytes (STREX and ZERO). Importantly, the ZERO variant known to be activated by cAMP is differentially up-regulated in enterocytes from animals on a high K(+) diet. In summary, these results strongly suggest that the adrenaline-induced distal colonic K(+) secretion is mediated by the BK channel and probably involves aldosterone-induced ZERO splice variant up-regulation.

The molecular mechanism of dexamethasone-mediated effect on the blood-brain tumor barrier permeability in a rat brain tumor model.

This study was performed to determine whether dexamethasone (DEX) had an effect on calcium-activated potassium channels (K(Ca) channels) and Occludin protein in blood-brain tumor barrier (BTB). Using a rat brain glioma model, we found that the expression of K(Ca) channels protein and Occludin protein was significantly increased in brain tumor tissue after DEX treatment for 3 days. Compared with non-DEX-treated animals, Evans Blue levels were greatly attenuated in DEX-treated animals. These effects were significantly reversed by the glucocorticoid receptor antagonist RU38486. In addition, DEX treatment enhanced the density of I(KCa) in the rat brain microvascular endothelial cells (RBMECs) in vitro BTB. All of these results strongly suggest that DEX could be involved in the regulation of both transcellular and paracellular pathway.

Analysing steroid modulation of BK(Ca) channels reconstituted into planar lipid bilayers.

A number of recent studies have described the activation of BK(Ca) channels by steroid hormones such as estrogen. The proposed mechanisms are diverse and include both the direct interaction with the ion channel subunits and the stimulation via receptor activation and cell signalling pathways. To investigate the activation of BK(Ca) channels by estrogen we devised a cell-free system by incorporating recombinant channels of known subunit composition into artificial bilayers and recorded single channel currents. This chapter describes the methods used to prepare purified membrane fractions from cultured cells and the construction of artificial phospholipids bilayers for the incorporation and recording of ion channels.

C-type natriuretic peptide hyperpolarizes and relaxes human penile resistance arteries.

INTRODUCTION: In addition to nitric oxide (NO), it is thought that an endothelium-derived hyperpolarizing factor (EDHF) plays an important role in the relaxation of penile arteries. Recently, it has been shown that C-type natriuretic peptide (CNP) shows the characteristics of EDHF in systemic small arteries. AIM: To investigate the mechanism involved in CNP-evoked vasodilatation and to address whether CNP is an EDHF in human penile resistance arteries. METHODS: Erectile tissue was obtained in connection with transsexual operations. Intracavernous penile resistance arteries were isolated and mounted in microvascular myographs for recording of isometric tension. Membrane potential was recorded by the use of a small glass electrode inserted in the smooth muscle layer. MAIN OUTCOME MEASURE: In vitro evidence for hyperpolarization and vasorelaxation induced by CNP. RESULTS: Acetylcholine (ACh) and CNP hyperpolarized smooth muscle membrane potential in resting penile resistance arteries. In penile small arteries incubated with inhibitors of NO synthase and cyclooxygenase and contracted with phenylephrine, ACh and CNP evoked concentration-dependent relaxations with maximum of 56 +/- 6% and 71 +/- 6%, respectively. Addition of a combination of blockers of small- and intermediate-conductance calcium-activated K(+) channels, apamin plus charybdotoxin, respectively, and a combination thought to block the smooth muscle response of EDHF-type relaxation, barium plus ouabain, markedly reduced ACh- and CNP-evoked relaxation. Iberiotoxin, a blocker of big-conductance calcium-activated K(+) channels inhibited the vasorelaxant responses evoked by ACh and CNP. A selective natriuretic peptide receptor type C (NPR-C) agonist, C-atrial natriuretic factor(4-23) (cANF(4-23)), induced relaxations with less maximum response compared to CNP. CONCLUSION: The present findings suggest that CNP possesses the characteristics of an EDHF in human penile resistance arteries. By activation of natriuretic peptide receptor type B and NPR-C receptors, CNP causes relaxation by activation, respectively, of large-conductance calcium-activated K(+) channels and Na(+)/K(+)-adenosine triphosphatase (ATPase), and barium-sensitive inward rectifier K(+) channels. Modulation of the CNP pathway opens for new treatment modalities of erectile dysfunction.

Modulation of the BK channel by estrogens: examination at single channel level.

BK channels regulate vascular tone by hyperpolarizing smooth muscle in response to fluctuating calcium concentrations. Oestrogen has been reported to lower blood pressure by increasing BK channel open probability through direct binding to the regulatory beta1-subunit(s) associated with the channel. The present investigation demonstrates that 17beta-oestradiol activates the BK channel complex by increasing the burst duration of channel openings. A subconductance state was observed in 25% of recordings following the addition of 17beta-oestradiol and could reflect uncoupling between the pore forming alpha1-subunit and the regulatory beta1-subunit. We also present evidence that more than one beta1-subunit is required to facilitate binding of 17beta-oestradiol to the channel complex.