Alteration of Piezo1 signaling in type 2 diabetic mice: focus on endothelium and BKCa channel.

Piezo1 mechanosensitive ion channel plays a important role in vascular physiology and disease. This study aimed to elucidate the altered signaling elicited by Piezo1 activation in the arteries of type 2 diabetes. Ten- to 12-week-old male C57BL/6 (control) and type 2 diabetic mice (db-/db-) were used. The second-order mesenteric arteries (~ 150 µm) were used for isometric tension experiments. Western blot analysis and immunofluorescence staining were performed to observe protein expression. Piezo1 was significantly decreased in mesenteric arteries of type 2 diabetic mice compared to control mice, as analyzed by western blot and immunofluorescence staining. Piezo1 agonist, Yoda1, concentration-dependently induced relaxation of mesenteric arteries in both groups. Interestingly, the relaxation response was significantly greater in control mice than in db-/db- mice. The removal of endothelium reduced relaxation responses induced by Yoda1, which was greater in control mice than db-/db- mice. Furthermore, the relaxation response was reduced by pre-treatment with various types of K+ channel blockers in endothelium-intact arteries in control mice. In endothelium-denuded arteries, pre-incubation with charybdotoxin, an Ca2+-activated K+ channel (BKCa channel) blocker, significantly attenuated Yoda1-induced relaxation in db-/db- mice, while there was no effect in control mice. Co-immunofluorescence staining showed co-localization of Piezo1 and BKCa channel was more pronounced in db-/db- mice than in control mice. These results indicate that the vascular responses induced by Piezo1 activation are different in the mesenteric resistance arteries in type 2 diabetic mice.

BKCa channels are involved in spontaneous and lipopolysaccharide-stimulated uterine contraction in late gestation mice†.

The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) is one of the most abundant potassium channels in the myometrium. Previous work conducted by our group has identified a link between inflammation, BKCa channels and excitability of myometrial smooth muscle cells. Here, we investigate the role of BKCa channels in spontaneous and lipopolysaccharide (LPS)-stimulated uterine contraction to gain a better understanding of the relationship between the BKCa channel and uterine contraction in basal and inflammatory states. Uteri of C57BL/6 J mice on gestational day 18.5 (GD18.5) were obtained and either fixed in formalin or used immediately for tension recording or isolation of primary myocytes for patch-clamp. Paraffin sections were used for immunofluorescenctdetection of BKCa and Toll-like receptor (TLR4). For tension recordings, LPS was administered to determine its effect on uterine contractions. Paxilline, a BKCa inhibitor, was used to dissect the role of BKCa in uterine contraction in basal and inflammatory states. Finally, patch-clamp recordings were performed to investigate the relationship between LPS, the BKCa channel and membrane currents in mouse myometrial smooth muscle cells (mMSMCs). We confirmed the expression of BKCa and TLR4 in the myometrium of GD18.5 mice and found that inhibiting BKCa channels with paxilline suppressed both spontaneous and LPS-stimulated uterine contractions. Furthermore, application of BKCa inhibitors (paxilline or iberiotoxin) after LPS inhibited BKCa channel activity in mMSMCs. Moreover, pretreatment with BKCa inhibitor or the TLR4 inhibitor suppressed LPS-activated BKCa currents. Our study demonstrates that BKCa channels are involved in both basal and LPS-stimulated uterine contraction in pregnant mice.

Enhanced Contraction of Arterial Smooth Muscle Cell in Skin Artery Is Sensitive to Hyperpolarization Mediated by BKCa Channel in Chronic Constriction Injury Model Rat.

Previous our study found that improvement of skin blood flow associated with neuropathic pain using vasodilators is useful for alleviation of neuropathic pain. In this study, we aimed to elucidate the mechanism underlying enhanced vasorelaxation induced by vasodilators, which increase cAMP and cyclic guanosine monophosphate (cGMP), in chronic constriction injury model rat. We assessed vasorelaxation effect of vasodilators by measurement of isometric contraction in isolated plantar artery from chronic constriction injury of sciatic nerve model rats. Nifedipine, a voltage-dependent Ca2+ channel inhibitor, NS1619, Ca2+-activated K+ (BKCa) channel opener, and diazoxide, an ATP-sensitive potassium channel opener, -induced vasorelaxation in ipsilateral plantar artery was enhanced compared to the these in contralateral plantar artery. Sodium nitroprusside (SNP), a nitric oxide (NO) donor, and substance P, a NK1 receptor agonist, caused vasorelaxation in both ipsilateral and contralateral artery. The vasorelaxation induced by SNP and substance P in ipsilateral artery is enhanced compared to the these in contralateral artery. Isoprenaline, a ß adrenoceptor agonist, and salbutamol, a ß2 adrenoceptor agonist, caused strong vasorelaxation in ipsilateral artery but not in contralateral artery. Iberiotoxin, a BKCa channel inhibitor, prominently suppressed the enhanced vasorelaxation induced by SNP, substance P, isoprenaline and salbutamol. In summary, the enhanced contraction of arterial smooth muscle cell in skin artery is sensitive to hyperpolarization in chronic constriction injury model rat. Furthermore, ß adrenoceptor agonist would be a good drug to improve the decreased skin blood flow because it has selective vasorelaxation to ipsilateral plantar artery.

Loss of IP3R-BKCa Coupling Is Involved in Vascular Remodeling in Spontaneously Hypertensive Rats.

Mechanisms by which BKCa (large-conductance calcium-sensitive potassium) channels are involved in vascular remodeling in hypertension are not fully understood. Vascular smooth muscle cell (VSMC) proliferation and vascular morphology were compared between hypertensive and normotensive rats. BKCa channel activity, protein expression, and interaction with IP3R (inositol 1,4,5-trisphosphate receptor) were examined using patch clamp, Western blot analysis, and coimmunoprecipitation. On inside-out patches of VSMCs, the Ca2+-sensitivity and voltage-dependence of BKCa channels were similar between hypertensive and normotensive rats. In whole-cell patch clamp configuration, treatment of cells with the IP3R agonist, Adenophostin A (AdA), significantly increased BKCa channel currents in VSMCs of both strains of rats, suggesting IP3R-BKCa coupling; however, the AdA-induced increases in BKCa currents were attenuated in VSMCs of hypertensive rats, indicating possible IP3R-BKCa decoupling, causing BKCa dysfunction. Co-immunoprecipitation and Western blot analysis demonstrated that BKCa and IP3R proteins were associated together in VSMCs; however, the association of BKCa and IP3R proteins was dramatically reduced in VSMCs of hypertensive rats. Genetic disruption of IP3R-BKCa coupling using junctophilin-2 shRNA dramatically augmented Ang II-induced proliferation in VSMCs of normotensive rats. Subcutaneous infusion of NS1619, a BKCa opener, to reverse BKCa dysfunction caused by IP3R-BKCa decoupling significantly attenuated vascular hypertrophy in hypertensive rats. In summary, the data from this study demonstrate that loss of IP3R-BKCa coupling in VSMCs induces BKCa channel dysfunction, enhances VSMC proliferation, and thus, may contribute to vascular hypertrophy in hypertension.

Differential Regulation by Mechanical Stretch of the Expressions of Large-Conductance Ca2+-Activated K+ Channel and L-Type Voltage-Dependent Ca2+ Channel in Rat Uterine Smooth Muscle Cells.

Large-conductance Ca2+-activated K+ (BKCa) channel and L-type voltage-dependent Ca2+ channel (L-VDCC) play important roles in regulating uterine contractility. The uterus stretch, occurring during pregnancy, is a critical factor to trigger uterine contraction. However, how mechanical stimuli impact the two channels remains unknown. Here we investigated the effects of exposure to mechanical stretches with varying magnitudes and durations on expressions of the two channels in rat uterine smooth muscle cells. Our results show that stretch down-regulates the BKCa channel expression but upregulates the L-VDCC expression. These findings are helpful to better understand the roles of L-VDCC and BKCa channel in stretch-triggered uterine contraction.

Fat augments leptin-induced uterine contractions by decreasing JAK2 and BKCa channel expressions in late pregnant rats.

Altered lipid metabolism in obesity causes pregnancy complications in humans and animals. Leptin levels increase in pregnancy, as well as obesity. However, the effect of obesity on uterine leptin receptors and its distal signaling is not clear. The present study aimed to understand the effect of increased fat on leptin signaling in rat uterus. Wistar female rats were fed with an HF diet (40% Fat, 17% Sucrose, 1.25% Cholesterol, 0.75% Cholic acid) for 6 weeks before the mating and during pregnancy. HF diet significantly increased the fat depots, liver weight, serum, and tissue cholesterol levels. It produced fatty degeneration in the liver and caused infiltration of inflammatory cells, cystic endometrial glands, and sub endometrial fibrosis of the uterus. In isometric tension experiments, leptin caused a significant increase in uterine contractions in high fat-fed animals compared to control animals. Analysis of receptor expressions revealed no significant difference between the groups. However, a significant decrease in the JAK2 and BKCaα mRNA expression was observed in the uterus of high fat-fed rats. No change in the BKCaß, eNOS, iNOS, MLCP, and MLCK mRNA expressions was noticed in the HF group compared to the control. The findings of the present study suggest that the contractile response to leptin in the uterus of high fat-fed rats may be attributed to reduced signaling through JAK2 and, lowered expressions of BKCa channel α subunits.

External Hemin as an Inhibitor of Mitochondrial Large-Conductance Calcium-Activated Potassium Channel Activity.

The mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) is located in the inner mitochondrial membrane and seems to play a crucial role in cytoprotection. The mitoBKCa channel is regulated by many modulators, including activators, such as calcium ions and inhibitors, such as heme and its oxidized form hemin. Heme/hemin binds to the heme-binding motif (CXXCH) located between two RCK domains present in the mitochondrial matrix. In the present study, we used the patch-clamp technique in the outside-out configuration to record the activity of mitoBKCa channels. This allowed for the application of channel modulators to the intermembrane-space side of the mitoBKCa. We found that hemin applied in this configuration inhibits the activity of mitoBKCa. In addition, we proved that the observed hemin effect is specific and it is not due to its interaction with the inner mitochondrial membrane. Our data suggest the existence of a new potential heme/hemin binding site in the structure of the mitoBKCa channel located on the mitochondrial intermembrane space side, which could constitute a new way for the regulation of mitoBKCa channel activity.

Potassium Channels in the Uterine Vasculature: Role in Healthy and Complicated Pregnancies.

A progressive increase in maternal uterine and placental blood flow must occur during pregnancy to sustain the development of the fetus. Changes in maternal vasculature enable an increased uterine blood flow, placental nutrient and oxygen exchange, and subsequent fetal development. K+ channels are important modulators of vascular function, promoting vasodilation, inducing cell proliferation, and regulating cell signaling. Different types of K+ channels, such as Ca2+-activated, ATP-sensitive, and voltage-gated, have been implicated in the adaptation of maternal vasculature during pregnancy. Conversely, K+ channel dysfunction has been associated with vascular-related complications of pregnancy, including intrauterine growth restriction and pre-eclampsia. In this article, we provide an updated and comprehensive literature review that highlights the relevance of K+ channels as regulators of uterine vascular reactivity and their potential as therapeutic targets.

Involvement of the BNP/NPR-A/BKCa pathway in rat trigeminal ganglia following chronic constriction injury.

Accumulating evidence indicates that the brain natriuretic peptide (BNP) and its receptor (natriuretic peptide receptor, NPR) are widely distributed in a variety of tissues including trigeminal ganglion (TG). Furthermore, recent studies support the involvement of the BNP-NPR-A pathway in acute and chronic pain. To investigate the role of this pathway in chronic pain, an infraorbital nerve-chronic constriction injury (ION-CCI) model of trigeminal neuralgia (TN) was produced in the rat. The time course of changes in mechanical pain threshold was examined. We observed an upregulation of BNP and NPR-A and a downregulation of large-conductance Ca2+-activated K+ (BKCa) mRNA and protein in rats subjected to ION-CCI. Patch clamping experiments in vitro found that BKCa currents were significantly reduced in rats subjected to ION-CCI. BNP increased BKCa currents in ION-CCI rats. These results suggest that BNP and NPR-A might serve as endogenous pain relievers in ION-CCI rats. Modulation of the BNP/NPR-A/BKCa channel pathway in TG may be a viable strategy for the treatment of TN.NEW & NOTEWORTHY BNP has been known to activate its receptor, NPR-A, to modulate inflammatory pain. However, the potential modulatory roles of BNP in TN have not been investigated in detail. We established an ION-CCI model of TN in the rat and observed an upregulation of BNP and NPR-A and a downregulation of BKCa in rats subjected to ION-CCI. Moreover, BNP can increase BKCa currents in ION-CCI rats. Thus, BNP and NPR-A might have inhibitory effects on trigeminal neuralgia through activating the BNP/NPR-A/BKCa channel pathway.

BKCa Channel Activation Attenuates the Pathophysiological Progression of Monocrotaline-Induced Pulmonary Arterial Hypertension in Wistar Rats.

PURPOSE: In the present study, the therapeutic efficacy of a selective BKCa channel opener (compound X) in the treatment of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) was investigated. METHODS: PAH was induced in male Wistar rats by a single injection of MCT. After two weeks, the MCT-treated group was divided into two groups that were either treated with compound X or vehicle. Compound X was administered daily at 28 mg/kg. Electrocardiographic, echocardiographic, and haemodynamic analyses were performed; ex vivo evaluations of pulmonary artery reactivity, right ventricle (RV) and lung histology as well as expression levels of α and ß myosin heavy chain, brain natriuretic peptide, and cytokines (TNFα and IL10) in heart tissue were performed. RESULTS: Pulmonary artery rings of the PAH group showed a lower vasodilatation response to acetylcholine, suggesting endothelial dysfunction. Compound X promoted strong vasodilation in pulmonary artery rings of both control and MCT-induced PAH rats. The untreated hypertensive rats presented remodelling of pulmonary arterioles associated with increased resistance to pulmonary flow; increased systolic pressure, hypertrophy and fibrosis of the RV; prolongation of the QT and Tpeak-Tend intervals (evaluated during electrocardiogram); increased lung and liver weights; and autonomic imbalance with predominance of sympathetic activity. On the other hand, treatment with compound X reduced pulmonary vascular remodelling, pulmonary flow resistance and RV hypertrophy and afterload. CONCLUSION: The use of a selective and potent opener to activate the BKCa channels promoted improvement of haemodynamic parameters and consequent prevention of RV maladaptive remodelling in rats with MCT-induced PAH.

Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells.

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated K+ channel (BKCa) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.

Isoliquiritigenin-induced vasodilation by activating large-conductance Ca2+ -activated K+ channels in mouse mesenteric arteries.

Isoliquiritigenin (ISL) is a flavonoid substance with a chalcone structure, which exerts anti-tumour, anti-oxidation and anti-inflammatory activity. The large-conductance calcium-activated potassium channel (BKCa ) is an important potassium channel with negative feedback regulation on the vascular smooth muscle cells (VSMCs) membrane. The activation of BKCa channel causes the hyperpolarization of VSMCs. It plays an important role in relaxation of blood vessels. Previous studies have shown that ISL causes the relaxation of the aorta and the basilar artery of the rat. However, there have not been studies on regulation of ISL in mesenteric arteries. To examine whether ISL causes the relaxation of the mesenteric artery of mice, we recorded vasodilation of mouse mesenteric arterial rings with a myograph. After contraction of arterial rings with phenylephrine, we added ISL to the arterial rings and measured its relaxation effect. To further examine which channel was involved in this relaxation effect, we tested the effects of ISL on endothelium-dependent and endothelium-independent vasodilation. Then we used BKCa channel blockers tetraethylammonium and iberiotoxin, to detect whether the BKCa channel is involved in ISL-induced vasodilation. Mesenteric arterial smooth muscle cells were isolated by enzyme digestion. Bis-(1, 3-dibutylbarbituric acid) trimethine oxonol staining was used to measure membrane potential of mesenteric arterial smooth muscle cells. We identified a vasodilation effect caused by ISL on mouse mesenteric arterial rings pre-contracted by phenylephrine in a concentration-dependent manner, with an EC50 of 13.71 ± 1.1 µmol/L. The vasodilation effect of ISL is endothelium-independent. K+ channel inhibitors tetraethylammonium and iberiotoxin reduced the vasodilation induced by ISL which suggested the involvement of BKCa channel.

The large-conductance voltage- and Ca2+ -activated K+ channel and its γ1-subunit modulate mouse uterine artery function during pregnancy.

KEY POINTS: The uterine artery (UA) markedly vasodilates during pregnancy to direct blood flow to the developing fetus. Inadequate UA vasodilatation leads to intrauterine growth restriction and fetal death. The large-conductance voltage- and Ca2+ -activated K+ (BKCa ) channel promotes UA vasodilatation during pregnancy. We report that BKCa channel activation increases the UA diameter at late pregnancy stages in mice. Additionally, a BKCa channel auxiliary subunit, γ1, participates in this process by increasing channel activation and inducing UA vasodilatation at late pregnancy stages. Our results highlight the importance of the BKCa channel and its γ1-subunit for UA functional changes during pregnancy. ABSTRACT: Insufficient vasodilatation of the uterine artery (UA) during pregnancy leads to poor utero-placental perfusion, contributing to intrauterine growth restriction and fetal loss. Activity of the large-conductance Ca2+ -activated K+ (BKCa ) channel increases in the UA during pregnancy, and its inhibition reduces uterine blood flow, highlighting a role of this channel in UA adaptation to pregnancy. The auxiliary γ1-subunit increases BKCa activation in vascular smooth muscle, but its role in pregnancy-associated UA remodelling is unknown. We explored whether the BKCa and its Î³1-subunit contribute to UA remodelling during pregnancy. Doppler imaging revealed that, compared to UAs from wild-type (WT) mice, UAs from BKCa knockout (BKCa-/- ) mice had lower resistance at pregnancy day 14 (P14) but not at P18. Lumen diameters were twofold larger in pressurized UAs from P18 WT mice than in those from non-pregnant mice, but this difference was not seen in UAs from BKCa-/- mice. UAs from pregnant WT mice constricted 20-50% in response to the BKCa blocker iberiotoxin (IbTX), whereas UAs from non-pregnant WT mice only constricted 15%. Patch-clamp analysis of WT UA smooth muscle cells confirmed that BKCa activity increased over pregnancy, showing three distinct voltage sensitivities. The γ1-subunit transcript increased 7- to 10-fold during pregnancy. Furthermore, γ1-subunit knockdown reduced IbTX sensitivity in UAs from pregnant mice, whereas γ1-subunit overexpression increased IbTX sensitivity in UAs from non-pregnant mice. Finally, at P18, γ1-knockout (γ1-/- ) mice had smaller UA diameters than WT mice, and IbTX-mediated vasoconstriction was prevented in UAs from γ1-/- mice. Our results suggest that the γ1-subunit increases BKCa activation in UAs during pregnancy.

Gas Signaling Molecules and Mitochondrial Potassium Channels.

Recently, gaseous signaling molecules, such as carbon monoxide (CO), nitric oxide (NO), and hydrogen sulfide (H2S), which were previously considered to be highly toxic, have been of increasing interest due to their beneficial effects at low concentrations. These so-called gasotransmitters affect many cellular processes, such as apoptosis, proliferation, cytoprotection, oxygen sensing, ATP synthesis, and cellular respiration. It is thought that mitochondria, specifically their respiratory complexes, constitute an important target for these gases. On the other hand, increasing evidence of a cytoprotective role for mitochondrial potassium channels provides motivation for the analysis of the role of gasotransmitters in the regulation of channel function. A number of potassium channels have been shown to exhibit activity within the inner mitochondrial membrane, including ATP-sensitive potassium channels, Ca2+-activated potassium channels, voltage-gated Kv potassium channels, and TWIK-related acid-sensitive K⁺ channel 3 (TASK-3). The effects of these channels include the regulation of mitochondrial respiration and membrane potential. Additionally, they may modulate the synthesis of reactive oxygen species within mitochondria. The opening of mitochondrial potassium channels is believed to induce cytoprotection, while channel inhibition may facilitate cell death. The molecular mechanisms underlying the action of gasotransmitters are complex. In this review, we focus on the molecular mechanisms underlying the action of H2S, NO, and CO on potassium channels present within mitochondria.

Chronic hypoxia upregulates DNA methyltransferase and represses large conductance Ca2+-activated K+ channel function in ovine uterine arteries.

Chronic hypoxia during gestation suppresses large-conductance Ca2+-activated K+ (BKCa) channel function and impedes uterine arterial adaptation to pregnancy. This study tested the hypothesis that chronic hypoxia has a direct effect in upregulating DNA methyltransferase (DNMT) and epigenetically repressing BKCa channel beta-1 subunit (KCNMB1) expression in uterine arteries. Resistance-sized uterine arteries were isolated from near-term pregnant sheep maintained at ∼300 m above sea level or animals acclimatized to high-altitude (3,801 m) hypoxia for 110 days during gestation. For ex vivo hypoxia treatment, uterine arteries from normoxic animals were treated with 21.0% O2 or 10.5% O2 for 48 h. High-altitude hypoxia significantly upregulated DNMT3b expression and enzyme activity in uterine arteries. Similarly, ex vivo hypoxia treatment upregulated DNMT3b expression and enzyme activity that was blocked by a DNMT inhibitor 5-aza-2'-deoxycytidine (5-Aza). Of importance, 5-Aza inhibited hypoxia-induced hypermethylation of specificity protein (SP) 1 binding site at the KCNMB1 promoter and restored transcription factor binding to the KCNMB1 promoter, resulting in the recovery of KCNMB1 gene expression in uterine arteries. Furthermore, 5-Aza blocked the effect of hypoxia and rescued BKCa channel activity and reversed hypoxia-induced decrease in BKCa channel-mediated relaxations and increase in myogenic tone of uterine arteries. Collectively, these results suggest that chronic hypoxia during gestation upregulates DNMT expression and activity, resulting in hypermethylation and repression of KCNMB1 gene and BKCa channel function, impeding uterine arterial adaptation to pregnancy.

Resveratrol attenuates cortical neuron activity: roles of large conductance calcium-activated potassium channels and voltage-gated sodium channels.

BACKGROUND: Resveratrol, a phytoalexin found in grapes and red wine, exhibits diverse pharmacological activities. However, relatively little is known about whether resveratrol modulates the ion channels in cortical neurons. The large-conductance calcium-activated potassium channels (BKCa) and voltage-gated sodium channels were expressed in cortical neurons and play important roles in regulation of neuronal excitability. The present study aimed to determine the effects of resveratrol on BKCa currents and voltage-gated sodium currents in cortical neurons. RESULTS: Resveratrol concentration-dependently increased the current amplitude and the opening activity of BKCa channels, but suppressed the amplitude of voltage-gated sodium currents. Similar to the BKCa channel opener NS1619, resveratrol decreased the firing rate of action potentials. In addition, the enhancing effects of BKCa channel blockers tetraethylammonium (TEA) and paxilline on action potential firing were sensitive to resveratrol. Our results indicated that the attenuation of action potential firing rate by resveratrol might be mediated through opening the BKCa channels and closing the voltage-gated sodium channels. CONCLUSIONS: As BKCa channels and sodium channels are critical molecular determinants for seizure generation, our findings suggest that regulation of these two channels in cortical neurons probably makes a considerable contribution to the antiseizure activity of resveratrol.

N-terminal isoforms of the large-conductance Ca²âº-activated K⁺ channel are differentially modulated by the auxiliary ß1-subunit.

The large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BK(Ca) α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary ß1-subunits, we examined whether ß1 differentially modulates the N-terminal BK(Ca) α-subunit isoforms. In the absence of ß1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas ß1 did not modulate the voltage-activation curve of MSSN, ß1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 µM and 100 µM Ca(2+). The ß1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the ß1-subunit may be due to the differential effect of ß1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related ß2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to ß1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BK(Ca) channel activity in cells, highlighting a novel mechanism of BK(Ca) channel regulation.

The role of large-conductance, calcium-activated potassium channels in a rat model of trigeminal neuropathic pain.

BACKGROUND: Trigeminal neuralgia is a disorder of paroxysmal and severely disabling facial pain and continues to be a real therapeutic challenge. At present there are few effective drugs. Here the aim of this study was to investigate the role of BKCa channels in trigeminal neuropathic pain. METHODS: Rats were divided into two groups: a sham and a chronic constriction injury of infraorbital branch of trigeminal nerve (ION-CCI) group. Nociceptive behavior testing, immunohistochemistry, RT-PCR, Western blotting and whole-cell patch clamp recording were used. RESULTS: Relative to the sham group, rats with ION-CCI consistently displayed lower mechanical pain thresholds in the vibrissal pad region from day 6 to 42 after ION-CCI operation. ION-CCI induced a significant down-regulation of BKCa channels both in mRNA and protein levels in the ipsilateral trigeminal ganglion (TG), a lower threshold intensity of action potential, and decreased total BKCa currents in cultured TG neurons. TG target injection of NS1619 (20-100 µg), an opener of BKCa channels, dose-dependently increased the mechanical pain threshold, which was blocked by the BKCa channel inhibitor iberiotoxin (IbTX, 20 µg). NS1619 (10 µM) significantly increased the mean threshold intensities of action potentials in ION-CCI rats, while failing to affect those in the sham rats. The levels of phosphorylated extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinases (JNK) in TG were significantly increased after ION-CCI operation. The ERK1/2 antagonist U0126, p38 antagonist SB203580 and JNK antagonist SP600125 significantly reversed the facial mechanical allodynia in ION-CCI rats. However, the ERK1/2 antagonist U0126, p38 antagonist SB203580 but not JNK antagonist SP600125 significantly increased BKCa currents in ION-CCI TG neurons. CONCLUSIONS: Our results indicate the important involvement of mainly ERK and p38 MAPK pathways in modulating BKCa channels in ION-CCI TG neurons. BKCa channels represent a new therapeutic target for the clinical treatment of trigeminal neuropathic pain.

Effect of the novel BKCa channel opener LDD175 on the modulation of corporal smooth muscle tone.

INTRODUCTION: The BKCa channel has been reported to play an important role in erectile function. Recently, novel BKCa channel activator, LDD175, was introduced. AIM: This study aims to investigate whether LDD175 relaxes corporal smooth muscle (CSM) via BKCa channel activation. METHODS: After isolation of CSM strip from a male rabbit model, contraction studies using organ bath was performed. Isolating human tissue and cell cultures, electrophysiological studies were done via whole-cell patch-clamp recording. MAIN OUTCOME MEASURES: Vasodilatory effects of LDD175 were evaluated by cumulative addition ranging from 10(-7) to 10(-4) M in corpus cavernosal strips after precontraction with 10(-5) M phenylephrine via organ bath system. Using cultured human CSM cells, patch-clamp recording was performed. Erectile function was measured by in vivo rat cavernous nerve stimulation. RESULTS: LDD175 caused an endothelium-independent relaxation of corporal tissues, and this effect was abolished by pretreatment with iberiotoxin. The relaxation effect of 10(-4) M LDD175 was greater than that of 10(-6) M udenafil (54.0 ± 3.1% vs. 34.5 ± 3.9%, P < 0.05); 10(-5) M LDD175 with 10(-6) M udenafil caused a greater relaxation effect on strips than 10(-5) M LDD175 or 10(-6) M udenafil alone (50.7%, 34.1%, vs. 20.7%, respectively, P < 0.001). In patch-clamp recordings, LDD175 increased K(+) currents in a dose-dependent manner, and washout of LDD175 or the addition of iberiotoxin fully reversed the increase. Intravenous LDD175 improved erectile function measured by area under the curve (AUC) of the intracavernosal pressure (ICP)/arterial blood pressure (ABP) ratio (1,612.1 ± 135.6 vs. 1,093.7 ± 123.1, P < 0.05). There was no difference between 10 mg/kg LDD175 and 1 mg/kg udenafil regarding maximal ICP, maximal ICP/ABP ratio, and the AUC of the ICP/ABP ratio (P > 0.05). CONCLUSIONS: LDD175 leads to an endothelium-independent relaxation of erectile tissue, primarily through the opening of BKCa channels. The results suggest that LDD175 might be a new candidate treatment for erectile dysfunction.

GAL-021, a new intravenous BKCa-channel blocker, is well tolerated and stimulates ventilation in healthy volunteers.

BACKGROUND: Potassium-channels in the carotid body and the brainstem are important regulators of ventilation. The BKCa-channel contains response elements for CO, O2, and CO2. Its block increases carotid body signalling, phrenic nerve activity, and respiratory drive. GAL-021, a new BKCa-channel blocker, increases minute ventilation in rats and non-human primates. This study assessed the single-dose safety, tolerability, pharmacokinetics (PKs), and pharmacodynamics (PDs) of GAL-021 in healthy volunteers. METHODS: Thirty subjects participated in a nine-period, randomized, double-blinded, placebo-controlled, crossover, ascending dose, first-in-human study with i.v. infusions of 0.1-0.96 mg kg(-1) h(-1) for 1 h and intermediate doses up to 4 h. RESULTS: Adverse event rates were generally similar among dose levels and between placebo- and GAL-021-treated subjects. At higher GAL-021 doses, a mild/moderate burning sensation at the infusion site occurred during the infusion. No clinically significant changes in vital signs or clinical chemistries were noted. Minute ventilation increased (AUE0-1 h ≈ 16%, P<0.05) and end-tidal carbon dioxide ([Formula: see text]) decreased (AUE0-1 h ≈ 6%, P<0.05) during the first hour at 0.96 mg kg(-1) h(-1) with 1/2-maximal [Formula: see text] and [Formula: see text]-change occurring by 7.5 min. Drug concentration rose rapidly during the infusion and decreased rapidly initially (distribution t1/2 of 30 min) and then more slowly (terminal t1/2 of 5.6 h). CONCLUSIONS: GAL-021 was safe and generally well tolerated with adverse events comparable with placebo except for an infusion site burning sensation. GAL-021 stimulated ventilation at the highest doses suggesting that greater infusion rates may be required for maximum PD effects. GAL-021 had PK characteristics consistent with an acute care medication.