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HaiXia Ren,1,* FangTing Yuan,1,2,* Wei Tan,1 YiJuan Ding,1 Ping An,1 HeSheng Luo1 1Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, People’s Republic of China; 2Key Laboratory of Hubei Province for Digestive System Diseases, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: HeSheng Luo Email LHSxhnk@163.comBackground and Aim: EVO is a natural alkaloid that reportedly has potential value in regulating gastrointestinal motility, but this conclusion remains controversial, and the molecular mechanism is unclear. In this study, we aimed to explore the effect of short-chain fatty acids on rat colonic hypermotility induced by water avoidance stress and the underlying mechanism.Methods: We constructed a hypermotile rat model by chronic water avoidance stress, and Western blot was used to detect the protein level of nNOS in colon tissue. The organ bath and multichannel physiological signal acquisition systems were used to examine the spontaneous contractions of smooth muscle strips. The whole‐cell patch‐clamp technique was used to investigate L‐type voltage‐dependent calcium and BKCa channel currents in colonic smooth muscle cells.Results: EVO inhibited the spontaneous contractions of colonic smooth muscle strips in a dose-dependent manner. Moreover, EVO decreased the fecal output induced by chronic water avoidance stress. TTX did not block the inhibitory effect of EVO on spontaneous colon contractions, while L-NNA, a selective nNOS synthase inhibitor, did partially abolish this inhibitory effect. The protein expression of nNOS in the colon tissues of rats administered EVO was significantly increased compared to that in control rats. EVO reversibly inhibited the L-type calcium channel current without changing the steady-state activation or inactivation in colonic smooth muscle cells. EVO significantly inhibited the BKCa current but did not change the shape of the I‐V curves.Conclusion: EVO inhibits gastrointestinal motility by inhibiting L-type calcium and BKCa channels in colonic smooth muscle cells and indirectly interacting with nNOS.Keywords: evodiamine, gastrointestinal motility, nitric oxide, L‐type voltage‐dependent calcium channels, BKCa channel

Age-related changes in the contribution of BKCa channels and NO to acetylcholine-induced dilation of pial arteries were studied in normotensive (Wistar–Kyoto) and spontaneously hypertensive rats (SHRs). Using intravital microphotography (×470), responses of pial arteries to acetylcholine chloride (ACh, 10−7 M, 5 min) with and without BKCa channel blockade by tetraethylammonium chloride (TEA, 2 mМ) and nitric oxide (NO) synthesis by L-NAME (10−3 M) were comparatively evaluated in WKY rats and SHRs aged 4 and 20 months. The evaluation criteria were the number and degree of arterial dilations arising in response to ACh after application of inhibitors, with the latter criterion being evaluated, in turn, by measuring the erythrocyte flow width in three individual groups of arteries: small (40 µm). It was established that in WKY rats aging leads to diminish the role of BKCa channels in ACh-induced dilation of all pial arteries, no matter the caliber. Similar processes were observed in the pial vascular bed of young SHRs. It appears that changes in the role of BKCa channels in vascular dilatatory responses in aging WKY rats and young SHRs are largely associated with the impairment of NO synthesis and changes in the role of NO-mediated mechanisms of vasodilation. Aging in SHRs is accompanied by increasing contribution of the NO-dependent mechanism to the regulation of ACh-induced dilatatory responses of small-caliber arteries.

The review addresses the Ca2+-dependent mitochondrial mechanisms of adaptation of cardiomyocytes to hypoxia and ischemia and analyzes signaling mechanisms responsible for expression of “antioxidant” genes and the formation of tolerance to hypoxia and ischemia. A special attention is paid to the role of the transcription factor Nrf2, reactive oxygen species and large-conductance Ca2+-activated K+ channels (BKCa channels) in the regulation of adaptive responses of cardiomyocytes in myocardial ischemia.

Large (big)-conductance calcium-activated potassium (BKCa) channels are expressed in migraine-related structures such as the cranial arteries, trigeminal ganglion and trigeminal spinal nucleus, and they play a substantial role in vascular tonus and neuronal excitability. Using synthetic BKCa channels openers was associated with headache as a frequent adverse effect in healthy volunteers. Additionally, BKCa channels are downstream molecules in migraine signalling pathways that are activated by several compounds known to provoke migraine, including calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating polypeptide (PACAP) and glyceryl trinitrate (GTN). Also, there is a high affinity and a close coupling between BKCa channels and ATP-sensitive potassium (KATP) channels, the role of which has recently been established in migraine pathophysiology. These observations raise the question as to whether direct BKCa channel activation can provoke migraine in migraine patients, and whether the BKCa channel could be a potential novel anti-migraine target. Hence, randomized and placebo-controlled clinical studies on BKCa channel openers or blockers in migraine patients are needed.

Trigeminal neuralgia (TN) is a severe chronic neuropathic pain. Despite numerous available medical interventions, the therapeutic effects are not ideal. To control the pain attacks, the need for more contemporary drugs continues to be a real challenge. Our previous study reported that Ca2+-activated K+ channels (BKCa) channels modulated by mitogen-activated protein kinases (MAPKs) in the trigeminal ganglia (TG) neurons play crucial roles in regulating TN, and some research studies demonstrated that inflammatory cytokine tumor necrosis factor alpha (TNF-α) could promote neuropathic pain. Meanwhile, the trigeminal nucleus caudalis (TNC), the first central site of the trigeminal nociceptive pathway, is responsible for processing sensory and pain signals from the peripheral orofacial area. Thus, this study is aimed to further investigate whether TNF-α and MAPKs phosphorylation in the TNC could mediate the pathogenesis of TN by modulating BKCa channels. The results showed that TNF-α of the TNC region is upregulated significantly in the chronic constriction injury of infraorbital nerve (ION-CCI) rats model, which displayed persistent facial mechanical allodynia. The normal rats with target injection of exogenous TNF-α to the fourth brain ventricle behaved just like the ION-CCI model rats, the orofacial mechanical pain threshold decreased clearly. Meanwhile, the exogenous TNF-α increased the action potential frequency and reduced the BKCa currents of TNC neurons significantly, which could be reversed by U0126 and SB203580, the inhibitors of MAPK. In addition, U0126, SB203580, and another MAPK inhibitor SP600125 could relieve the facial mechanical allodynia by being injected into the fourth brain ventricle of ION-CCI model rats, respectively. Taken together, our work suggests that the upregulation of TNF-α in the TNC region would cause the increase of MAPKs phosphorylation and then the negative regulation of BKCa channels, resulting in the TN.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science, Research and Arts Baden-Württemberg (MWK-BW Sonderlinie Medizin) Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence. One of the important indicators for AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to such changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in their expression and activity associated with AF. Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or with sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC-signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel and its pharmacology indicated presence of ‘big potassium channels’, BKCa. In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of stretch-induced BKCa current. No co-immunoprecipitation of Piezo1 and BKCa was detected. Human atrial fibroblasts express functional Piezo1 and BKCa channels. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data show the presence and activity of Piezo1 and BKCa in human atrial fibroblasts and suggest an interplay between the two in the absence of direct physical interactions.

This study explored whether KMUP-1 improved chronic constriction injury (CCI)-induced BKCa current inhibition in dorsal root ganglion (DRG) neurons. Rats were randomly assigned to four groups: sham, sham + KMUP-1, CCI, and CCI + KMUP-1 (5 mg/kg/day, i.p.). DRG neuronal cells (L4–L6) were isolated on day 7 after CCI surgery. Perforated patch-clamp and inside-out recordings were used to monitor BKCa currents and channel activities, respectively, in the DRG neurons. Additionally, DRG neurons were immunostained with anti-NeuN, anti-NF200 and anti-BKCa. Real-time PCR was used to measure BKCa mRNA levels. In perforated patch-clamp recordings, CCI-mediated nerve injury inhibited BKCa currents in DRG neurons compared with the sham group, whereas KMUP-1 prevented this effect. CCI also decreased BKCa channel activity, which was recovered by KMUP-1 administration. Immunofluorescent staining further demonstrated that CCI reduced BKCa-channel proteins, and KMUP-1 reversed this. KMUP-1 also changed CCI-reduced BKCa mRNA levels. KMUP-1 prevented CCI-induced neuropathic pain and BKCa current inhibition in a peripheral nerve injury model, suggesting that KMUP-1 could be a potential agent for controlling neuropathic pain.

Background: The current study presents the novel angiotensin II receptor blocker fluorophenyl benzimidazole (FPD) as an antihypertensive agent in the SHR model of hypertension. We investigated the role of cGMP, voltage-dependent L-type calcium channels, and BKCa channels in the vasorelaxant mechanisms of FPD in the rat superior mesenteric artery.Methods: The antihypertensive effect of FPD was examined using an invasive technique measuring blood pressure in SHR animals. Using a myograph, tension measurement was completed in the superior mesenteric artery to elucidate the mechanisms of vasorelaxation involving AT1 receptors, the NO/cGMP pathway, L-type calcium channels, and BKCa channels. Ion flux (Ca2+, K+) studies were conducted in aortic smooth muscle cells. Putative targets proteins were determined by in silico docking studies. A safety evaluation of FPD was carried out using Swiss albino mice.Results: FPD significantly decreased blood pressure in SHR. It relaxed superior mesenteric arteries in a concentration-dependent manner and significantly inhibited angiotensin II-induced contraction. The relaxation response was also mediated by an increase in tissue cGMP levels, inhibition of L-type calcium channels, and the opening of BKCa channels. FPD further enhanced efflux of K+ and inhibited Bay K8644-stimulated Ca2+ influx in aortic smooth muscle cells and docked well in an in silico study with the targets. It was well tolerated in the toxicity study.Conclusion: The present study reports the antihypertensive activity of novel AT-1 receptor blocker FPD at 50 and 100 mg kg−1 with cGMP, L-type calcium channels, and BKCa channels as putative targets of vasorelaxation, and was found safe in oral toxicity.

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.

Epilepsy is a neurological disorder characterized by unpredictable seizures, which are bursts of electrical activity that temporarily affect the brain. Cereblon (CRBN), a DCAFs (DDB1 and CUL4-associated factors), is a well-established protein associated with human mental retardation. Being a substrate receptor of the cullin-RING E3 ubiquitin ligase (CRL) 4 complex, CRBN mediates ubiquitination of several substrates and conducts multiple biological processes. In the central nervous system, the largeconductance Ca2+-activated K+ (BKCa) channel, which is the substrate of CRBN, is an important regulator of epilepsy. Despite the functional role and importance of CRBN in the brain, direct injection of pentylenetetrazole (PTZ) to induce seizures in CRBN knock-out mice has not been challenged. In this study, we investigated the effect of PTZ in CRBN knock-out mice. Here, we demonstrate that, compared with WT mice, CRBN knock-out mice do not show the intensification of seizures by PTZ induction. Moreover, electroencephalography recordings were also performed in the brains of both WT and CRBN knockout mice to identify the absence of significant differences in the pattern of seizure activities. Consistently, immunoblot analysis for validating the protein level of the CRL4 complex containing CRBN (CRL4Crbn) in the mouse brain was carried out. Taken together, we found that the deficiency of CRBN does not affect PTZ-induced seizure. [BMB Reports 2020; 53(9): 484-489].

H2S is an endogenous gasotransmitter that increases cerebral blood flow. In the cerebral vascular endothelium, H2S is produced by cystathionine δ-lyase (CSE). Endothelin-1 (ET-1) has constrictor and dilator influences on the cerebral circulation. The mechanism of the vasodilation caused by ET-1 may involve endothelium-derived factors. We hypothesize that ET-1-elicited dilation of pial arterioles requires an elevation of H2S production in the cerebral vascular endothelium. We investigated the effects of ET-1 on CSE-catalyzed brain H2S production and pial arteriolar diameter using cranial windows in newborn pigs in vivo. H2S was measured in periarachnoid cerebrospinal fluid. ET-1 (10−12–10−8 M) caused an elevation of H2S that was reduced by the CSE inhibitors propargylglycine (PPG) and β-cyano-l-alanine (BCA). Low doses of ET-1 (10−12–10−11 M) produced vasodilation of pial arterioles that was blocked PPG and BCA, suggesting the importance of H2S influences. The vasodilator effects of H2S may require activation of smooth muscle cell membrane ATP-sensitive K+ (KATP) channels and large-conductance Ca2+-activated K+ (BK) channels. The KATP inhibitor glibenclamide and the BK inhibitor paxilline blocked CSE/H2S-dependent dilation of pial arterioles to ET-1. In contrast, the vasoconstrictor response of pial arterioles to 10−8 M ET-1 was not modulated by PPG, BCA, glibenclamide, or paxilline and, therefore, was independent of CSE/H2S influences. Pial arteriolar constriction response to higher levels of ET-1 was independent of CSE/H2S and KATP/BKCa channel activation. These data suggest that H2S is an endothelium-derived factor that mediates the vasodilator effects of ET-1 in the cerebral circulation via a mechanism that involves activation of KATP and BK channels in vascular smooth muscle. NEW & NOTEWORTHY Disorders of the cerebral circulation in newborn infants may lead to lifelong neurological disabilities. We report that vasoactive peptide endothelin-1 exhibits vasodilator properties in the neonatal cerebral circulation by stimulating production of H2S, an endothelium-derived messenger with vasodilator properties. The ability of endothelin-1 to stimulate brain production of H2S may counteract the reduction in cerebral blood flow and prevent the cerebral vascular dysfunction caused by stroke, asphyxia, cerebral hypoxia, ischemia, and vasospasm.

Airway smooth muscle expresses abundant BKCachannels, but their role in regulating contractions remains controversial. This study examines the effects of two potent BKCachannel openers on agonist-induced phasic contractions in rabbit and mouse bronchi. First, we demonstrated the ability of 10 μM GoSlo-SR5-130 to activate BKCachannels in inside-out patches from rabbit bronchial myocytes, where it shifted the activation V1/2by −88 ± 11 mV (100 nM Ca2+, n = 7). In mouse airway smooth muscle cells, GoSlo-SR5-130 dose dependently shifted V1/2by 12–83 mV over a concentration range of 1–30 μM. Compound X, a racemic mixture of two enantiomers, reported to be potent BKCachannel openers, shifted V1/2by 20–79 mV over a concentration range of 0.3–3 μM. In rabbit bronchial rings, exposure to histamine (1 μM) induced phasic contractions after a delay of ~35 min. These were abolished by GoSlo-SR5-130 (30 μM). Nifedipine (100 nM) and CaCCinhA01 (10 μM), a TMEM16A blocker, also abolished histamine-induced phasic contractions. In mouse bronchi, similar phasic contractions were evoked by exposure to U46619 (100 nM) and carbachol (100 nM). In each case, these were inhibited by concentrations of GoSlo-SR5-130 and compound X that shifted the activation V1/2of BKCachannels in the order of −80 mV. In conclusion, membrane potential-dependent regulation of L-type Ca2+channels appears to be important for histamine-, U46619-, and carbachol-induced phasic contractions in airway smooth muscle. Contractions can be abolished by BKCachannel openers, suggesting that these channels are potential targets for treating some causes of airway obstruction.

Conventional treatments have failed to improve the prognosis of heart failure with preserved ejection fraction (HFpEF) patients. Thus, the purpose of this study was to determine the therapeutic efficacy of chronic interval exercise training (IT) on large-conductance Ca2+-activated K+ (BKCa) channel-mediated coronary vascular function in heart failure. We hypothesized that chronic interval exercise training would attenuate pressure overload-induced impairments to coronary BKCa channel-mediated function. A translational large-animal model with cardiac features of HFpEF was used to test this hypothesis. Specifically, male Yucatan miniswine were divided into three groups ( n = 7/group): control (CON), aortic banded (AB)-heart failure (HF), and AB-interval trained (HF-IT). Coronary blood flow, vascular conductance, and vasodilatory capacity were measured after administration of the BKCa channel agonist NS-1619 both in vivo and in vitro in the left anterior descending coronary artery and isolated coronary arterioles, respectively. Skeletal muscle citrate synthase activity was decreased and left ventricular brain natriuretic peptide levels increased in HF vs. CON and HF-IT animals. A parallel decrease in NS-1619-dependent coronary vasodilatory reserve in vivo and isolated coronary arteriole vasodilatory responsiveness in vitro were observed in HF animals compared with CON, which was prevented in the HF-IT group. Although exercise training prevented BKCa channel-mediated coronary vascular dysfunction, it did not change BKCa channel α-subunit mRNA, protein, or cellular location (i.e., membrane vs. cytoplasm). In conclusion, these results demonstrate the viability of chronic interval exercise training as a therapy for central and peripheral adaptations of experimental heart failure, including BKCa channel-mediated coronary vascular dysfunction. NEW & NOTEWORTHY Conventional treatments have failed to improve the prognosis of heart failure with preserved ejection fraction (HFpEF) patients. Our findings show that chronic interval exercise training can prevent BKCa channel-mediated coronary vascular dysfunction in a translational swine model of chronic pressure overload-induced heart failure with relevance to human HFpEF.

Activation of large-conductance Ca2+-activated K+ (BKCa) channels evokes cell survival programs that mitigate intestinal ischemia and reperfusion (I/R) inflammation and injury 24 h later. The goal of the present study was to determine the roles of reactive oxygen species (ROS) and heme oxygenase (HO)-1 in delayed acquisition of tolerance to I/R induced by pretreatment with the BKCa channel opener NS-1619. Superior mesentery arteries were occluded for 45 min followed by reperfusion for 70 min in wild-type (WT) or HO-1-null (HO-1−/−) mice that were pretreated with NS-1619 or saline vehicle 24 h earlier. Intravital microscopy was used to quantify the numbers of rolling and adherent leukocytes. Mucosal permeability, tumor necrosis factor-α (TNF-α) levels, and HO-1 activity and expression in jejunum were also determined. I/R induced leukocyte rolling and adhesion, increased intestinal TNF-α levels, and enhanced mucosal permeability in WT mice, effects that were largely abolished by pretreatment with NS-1619. The anti-inflammatory and mucosal permeability-sparing effects of NS-1619 were prevented by coincident treatment with the HO-1 inhibitor tin protoporphyrin-IX or a cell-permeant SOD mimetic, Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), in WT mice. NS-1619 also increased jejunal HO-1 activity in WT animals, an effect that was attenuated by treatment with the BKCa channel antagonist paxilline or MnTBAP. I/R also increased postischemic leukocyte rolling and adhesion and intestinal TNF-α levels in HO-1−/− mice to levels comparable to those noted in WT animals. However, NS-1619 was ineffective in preventing these effects in HO-1-deficient mice. In summary, our data indicate that NS-1619 induces the development of an anti-inflammatory phenotype and mitigates postischemic mucosal barrier disruption in the small intestine by a mechanism that may involve ROS-dependent HO-1 activity. NEW & NOTEWORTHY Antecedent treatment with the large-conductance Ca2+-activated K+ channel opener NS-1619 24 h before ischemia-reperfusion limits postischemic tissue injury by an oxidant-dependent mechanism. The present study shows that NS-1619-induced oxidant production prevents ischemia-reperfusion-induced inflammation and mucosal barrier disruption in the small intestine by provoking increases in heme oxygenase-1 activity.

In fragile X syndrome (FXS), sensory hypersensitivity and impaired habituation is thought to result in attention overload and various behavioral abnormalities in reaction to the excessive and remanent salience of environment features that would normally be ignored. This phenomenon, termed sensory defensiveness, has been proposed as the potential cause of hyperactivity, hyperarousal, and negative reactions to changes in routine that are often deleterious for FXS patients. However, the lack of tools for manipulating sensory hypersensitivity has not allowed the experimental testing required to evaluate the relevance of this hypothesis. Recent work has shown that BMS-204352, a BKCa channel agonist, was efficient to reverse cortical hyperexcitability and related sensory hypersensitivity in the Fmr1-KO mouse model of FXS. In the present study, we report that exposing Fmr1-KO mice to novel or unfamiliar environments resulted in multiple behavioral perturbations, such as hyperactivity, impaired nest building and excessive grooming of the back. Reversing sensory hypersensitivity with the BKCa channel agonist BMS-204352 prevented these behavioral abnormalities in Fmr1-KO mice. These results are in support of the sensory defensiveness hypothesis, and confirm BKCa as a potentially relevant molecular target for the development of drug medication against FXS/ASD.

This study aimed to investigate the expression and function of BKCa channels in the Sphincter of Oddi (SO) in a rabbit model of hypercholesterolemia (HC). New Zealand white rabbits were randomly divided into 2 groups: the control group was fed standard chow (n = 18) whereas the high-cholesterol group was fed cholesterol-enriched chow containing 1.5% cholesterol (n = 18). The serum cholesterol level was significantly greater in the HC groups than in the control group, but there was no significant difference in body weight between the control and HC groups. Although the total protein expression of BKCa α- and β1-subunit was not significantly different between the control and HC groups, the Tyr-phosphorylation of BKCa α-subunit was significantly decreased in the HC group than in the control group. In addition, hypercholesterolemia significantly increased Acetylcholine (ACh)-induced contraction of the SO rings. Pretreatment with 30 μM NS1619, a BKCa channel agonist, significantly reduced ACh-induced c...

Verteporfin (VP), a benzoprophyrin derivative, has been clinically tailored as a photosensitizer and recently known to suppress YAP-TEAD complex accompanied by suppression of the growth in an array of neoplastic cells. However, the detailed information is little available regarding possible modifications of it and its related compounds on transmembrane ionic currents, despite its growing use in clinical settings. In this study, from whole cell recordings, VP (0.3-100 M) increased the amplitude of Ca2+-activated K+ currents (IK(Ca)) in pituitary tumor (GH3) cells in a concentration-dependent manner with an EC50 value of 2.4 M. VP-stimulated IK(Ca) in these cells was suppressed by further addition of either paxilline, iberiotoxin or dithiothreitol, but not by that of tobultamide or TRAM-39. VP at a concentration of 10 M mildly but significantly suppressed the amplitude of delayed-rectifier K+ current; however, it had minimal effects on M-type K+ current. In cell-attached current recordings, addition of VP to the recording medium enhanced the activity of large-conductance Ca2+-activated K+(BKCa) channels. In the presence of VP, additional illumination with light intensity of 5.5 mW/cm2raised the probability of BKCa-channel openings further. Addition of VP decreased the peak amplitude of L-type Ca2+ current together with slowed inactivation time course of the current; however, it failed to modify voltage-gated Na+ current. Illumination of GH3 cells in continued presence of VP also induced a non-selective cation current. Additionally, VP increased the activity of BKCa channels in human 13-06-MG glioma cells with an EC50 value of 1.9 M. Therefore, the effects of VPon ionic currents described herein tend to be upstream of its inhibition of YAP-TEAD complex and they are conceivably likely to contribute to the underlying mechanisms through which it and its structurally similar compounds effect the modifications in functional activities of pituitary or glial neoplastic cells, if the in vivo findings occur.

Flavonoids have shown beneficial effects in various disease conditions as reported by various previous studies. Biochanin-A is a flavonoid present in various plants in nature. Present investigation was done to assess the vasorelaxant potential of biochanin-A on isolated coronary artery of goat and its possible mechanism of action. Vascular reactivity experiments were done on circumflex coronary artery of goats using the tension experiments. Goat coronary arterial rings were relaxed with biochanin-A in concentration (0.1–100 μM)-dependent manner. Endothelium had no effect on biochanin-A-induced relaxation. Maximum relaxation induced by biochanin-A was 116.54 ± 12.21% in endothelium-intact artery and it was not significantly different with maximal relaxation (108.22 ± 1.88%) of endothelium-denuded vessel. L-NAME (100 μM) did not show any effect on biochanin-A-induced relaxation. TEA (BKCa channel blocker), and BaCl2 (KIR blocker) had no effect on biochanin-A-induced relaxation. However, biochanin-A-induced maximal relaxation (71.72 ± 4.50%) was reduced significantly (P

As a novel gasotransmitter, hydrogen sulfide (H2S) elicits various physiological actions including smooth muscle relaxation and promotion of transepithelial ion transport. However, the pro-secretory function of H2S in the male reproductive system remains largely unclear. The aim of this study is to elucidate the possible roles of H2S in modulating rat epididymal intraluminal ionic microenvironment essential for sperm storage. The results revealed that endogenous H2S-generating enzymes cystathionine β-synthetase (CBS) and cystathionine γ-lyase (CSE) were both expressed in rat epididymis. CBS located predominantly in epithelial cells whilst CSE expressed primarily in smooth muscle cells. The relative expression level of CBS and CSE escalated from caput to cauda regions of epididymis, which was paralleled to the progressively increasing production of endogenous H2S. The effect of H2S on epididymal epithelial ion transportation was investigated using short-circuit current (I SC), measurement of intracellular ion concentration and in vivo rat epididymal microperfusion. Our data showed that H2S induced transepithelial K+ secretion via adenosine triphosphate-sensitive K+ (KATP) channel and large conductance Ca2+-activated K+ (BKCa) channel. Transient receptor potential vanilloid 4 (TRPV4) channel-mediated Ca2+ influx was implicated in the activation of BKCa channel. In vivo studies further demonstrated that H2S promoted K+ secretion in rat epididymal epithelium. Inhibition of endogenous H2S synthesis caused a significant decrease in K+ concentration of cauda epididymal intraluminal fluid. Moreover, our data demonstrated that high extracellular K+ concentration actively depressed the motility of cauda epididymal sperm in a pH-independent manner. Collectively, the present study demonstrated that H2S was vital to the formation of high K+ concentration in epididymal intraluminal fluid by promoting the transepithelial K+ secretion, which might contribute to the maintenance of the cauda epididymal sperm in quiescent dormant state before ejaculation.

Aims Regulation of vascular tone by 3′,5′-cyclic adenosine monophosphate (cAMP) involves many effectors including the large conductance, Ca2+-activated, K+ (BKCa) channels. In arteries, cAMP is mainly hydrolyzed by type 3 and 4 phosphodiesterases (PDE3, PDE4). Here, we examined the specific contribution of BKCa channels to tone regulation by these PDEs in rat coronary arteries, and how this is altered in heart failure (HF). Methods and results Concomitant application of PDE3 (cilostamide) and PDE4 (Ro-20-1724) inhibitors increased BKCa unitary channel activity in isolated myocytes from rat coronary arteries. Myography was conducted in isolated, U46619-contracted coronary arteries. Cilostamide (Cil) or Ro-20-1724 induced a vasorelaxation that was greatly reduced by iberiotoxin (IBTX), a BKCa channel blocker. Ro-20-1724 and Cil potentiated the relaxation induced by the β-adrenergic agonist isoprenaline (ISO) or the adenylyl cyclase activator L-858051 (L85). IBTX abolished the effect of PDE inhibitors on ISO but did not on L85. In coronary arteries from rats with HF induced by aortic stenosis, contractility and response to acetylcholine were dramatically reduced compared with arteries from sham rats, but relaxation to PDE inhibitors was retained. Interestingly, however, IBTX had no effect on Ro-20-1724- and Cil-induced vasorelaxations in HF. Expression of the BKCa channel α-subunit, of a 98 kDa PDE3A and of a 80 kDa PDE4D were lower in HF compared with sham coronary arteries, while that of a 70 kDa PDE4B was increased. Proximity ligation assays demonstrated that PDE3 and PDE4 were localized in the vicinity of the channel. Conclusion BKCa channels mediate the relaxation of coronary artery induced by PDE3 and PDE4 inhibition. This is achieved by co-localization of both PDEs with BKCa channels, enabling tight control of cAMP available for channel opening. Contribution of the channel is prominent at rest and on β-adrenergic stimulation. This coupling is lost in HF.

Large-conductance Ca2+-activated K+ (BKCa) channels are thought to play a key role in the regulation of corpus cavernosum smooth muscle (CCSM) excitability. Few BKCa channel openers have been accepted for clinical development. The effect of the novel BKCa channel opener GoSlo-SR5-130 on electrical activity in isolated rabbit CCSM cells and mechanical activity in strips of rabbit CCSM was examined. Single-channel currents were observed in inside-out patches. These channels were sensitive to Ca2+, blocked by penitrem A, and had a conductance of 291 ± 20 pS ( n = 7). In the presence of GoSlo-SR5-130, the number of open BKCa channels increased. Using voltage-ramp protocols, GoSlo-SR5-130 caused currents to activate at more negative potentials in a concentration-dependent manner, shifting the half-maximal activation voltage potential to the left on the voltage axis. Therefore, BKCa channels were open within the physiological range of membrane potentials in the presence of GoSlo-SR5-130. GoSlo-SR5-130 also resulted in an increase in the activity of spontaneous transient outward currents in myocytes isolated from CCSM, and this effect was reversed by iberiotoxin. In current-clamp mode, GoSlo-SR5-130 hyperpolarized the cell membrane. Isometric tension recording of strips of rabbit corpus cavernosum showed that GoSlo-SR5-130 inhibited spontaneous contractions in a concentration-dependent manner. This effect was reversed in the presence of iberiotoxin, suggesting that GoSlo-SR5-130 exerts its effect through BKCa channels. These findings suggest that GoSlo-SR5-130 is an effective tool for the study of BKCa channels and that these channels can modulate CCSM activity and are possible targets for the treatment of erectile dysfunction.

Previous studies suggest that the large-conductance Ca2+-activated K+ (BKCa) channel and Rho-kinase play major roles in the control of urinary bladder tone. Here, we investigated their involvement in β-adrenoceptor (AR)-mediated relaxation of rat and human bladder. Concentration-response curves of isoprenaline and mirabegron-induced bladder relaxation were generated against passive tension and KCl- and carbachol-induced tone, in the absence or presence of the BKCa channel inhibitor iberiotoxin (100 nM) or the Rho-kinase inhibitor Y27,632 (1 μM). Myosin light chain (MLC) phosphorylation was studied by Western blot. In rat, iberiotoxin only slightly altered isoprenaline- and mirabegron-induced relaxation against KCl-induced tone but attenuated relaxation by both agonists against carbachol-induced tone. Y27,632 enhanced isoprenaline- or mirabegron-induced relaxation only against carbachol-induced tone. In humans, iberiotoxin slightly enhanced relaxation by both agonists against carbachol-induced pre-contraction. Y27,632 did not change isoprenaline-induced relaxation but enhanced that by mirabegron. Under passive tension, MLC phosphorylation was markedly reduced by both β-AR agonists, an effect insensitive to Y27,632. In the presence of carbachol, both β-AR agonists increased MLC phosphorylation, an effect reduced by Y27,632 only in the presence of 1 μM carbachol. These results indicate that the extent of BKCa channel and Rho-kinase involvement in relaxation induced by β-AR agonists depends on pre contractile stimulus and species. Electronic supplementary material The online version of this article (doi:10.1007/s00210-015-1128-z) contains supplementary material, which is available to authorized users.

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.

Formaldehyde (FA) has been linked to the detrimental cardiovascular effects. Here, we explored the effects and mechanisms of FA on rat aortas both in vivo and in vitro. The results presented that FA evidently lowered the blood pressures of rats. The expression levels of BKCa subunits α and β1 and iNOS of the aortas were up-regulated by FA in vivo. However, FA markedly reduced the levels of Cav1.2 and Cav1.3, which are the subunits of L-Ca2+ channel. Furthermore, the contents of NO, cGMP and iNOS in the aortas were augmented by FA. To further confirm these findings, the mechanisms accredited to these effects were examined in vitro. The data showed that FA contracted the isolated aortic rings at low concentrations ( 500 μM). The FA-induced vasoconstriction at low concentrations was blocked partly by an inhibitor of ACE. The relaxation caused by FA at high concentrations was attenuated by the inhibitors of NO-cGMP pathway, L-Ca2+ channel and BKCa channel, respectively. Similarly, the expression of iNOS was strongly enhanced by FA in vitro. The effects of FA on the aortic rings with endothelium were significantly greater than those on the rings without endothelium. Our results indicate that the vasoconstriction of FA at low concentrations might be partially pertinent to endothelin, and the FA-caused vasorelaxation at high concentrations is possibly associated with the NO-cGMP pathway, L-Ca2+ channel and BKCa channel. This study will improve our understanding of the pathogenic mechanisms for FA-related cardiovascular diseases.

Various types of mechanosensitive ion channels, including cationic stretch-activated channels (SACNS) and stretch-activated BKca (SAKca) channels, modulate heart rhythm. Bepridil has been used as an antiarrhythmic drug with multiple pharmacological effects; however, whether it is effective for mechanically induced arrhythmia has not been well investigated. To test the effects of Bepridil on SAKca channels activity, cultured chick embryonic ventricular myocytes were used for single-channel recordings. Bepridil significantly reduced the open probability of the SAKca channel (PO). Next, to test the effects of bepridil on stretch-induced extrasystoles (SIE), we used an isolated 2-week-old Langendorff-perfused chick heart. The left ventricle (LV) volume was rapidly changed, and the probability of SIE was calculated in the presence and absence of bepridil, and the effect of the drug was compared with that of Gadolinium (Gd3+). Bepridil decreased the probability of SIE despite its suppressive effects on SAKca channel activity. The effects of Gd3+, which blocks both SAKca and SACNS, on the probability of SIE were the same as those of bepridil. Our results suggest that bepridil blocks not only SAKca channels but possibly also blocks SACNS, and thus decreases the stretch-induced cation influx (stabilizing membrane potential) to compensate and override the effects of the decrease in outward SAKca current (destabilizing membrane potential).

Epidemiological investigations have revealed that sulfur dioxide (SO2) exposure is linked to cardiovascular diseases. Our previous study indicated that the vasorelaxant effect of SO2 might be partly related to ATP-sensitive K(+) (KATP), big-conductance Ca(2+)-activated K(+) (BKCa) and L-type calcium (L-Ca(2+)) channels. The present study was designed to further investigate the effects of gaseous SO2 and its derivatives on the gene and protein expression of these channels in the rat aortas in vitro. The results showed that the mRNA and protein levels of the KATP channel subunits Kir6.1, Kir6.2 and SUR2B of the rat aortas in SO2 and its derivatives groups were higher than those in control group. Similarly, the expression of the BKCa channel subunits α and β1 was increased by SO2 and its derivatives. However, SO2 and its derivatives at 1500μM significantly decreased the expression of the L-Ca(2+) channel subunits Cav1.2 and Cav1.3. Histological examination of the rat aorta tissues showed moderate injury of tunica media induced by SO2 and its derivatives at 1500μM. These results suggest that SO2 and its derivatives can activate the KATP and BKCa channels by increasing the expression of Kir6.1, Kir6.2, SUR2B and α, β1, respectively, while also inhibiting the L-Ca(2+) channels by decreasing the expression of Cav1.2 and Cav1.3 of the rat aortas. The molecular mechanism of the vasorelaxant effect of SO2 and its derivatives might be related to the expression changes of KATP, BKCa and L-Ca(2+) channel subunits, which may play a role in the pathogenesis of SO2-associated cardiovascular diseases.

In the present study, we report that neolignan1 (Diethyl-4,4ʹ-dihydroxy-8,3ʹ-neolign-7,7ʹ-dien-9,9ʹ-dionate) relaxes the superior mesenteric artery in a concentration dependent manner (pD2 value 5.392 ± 0.04; n = 8 for endothelium intact and 5.204 ± 0.03; n = 8 for endothelium denuded mesenteric rings, respectively). The relaxation response of neolignan1 was found to be endothelium independent and sensitive to 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-on (ODQ; 1 μM) and tetraethyl ammonium (TEA; 1 mM). In-silico studies showed good LibDock score (92.66) of neolignan1 with BKCa channel and are in well corroboration with ex-vivo study. Further, neolignan1 significantly decreased the systolic blood pressure, diastolic blood pressure and mean arterial pressure in the Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; 50 mg/kg) treated Wistar rats at the dose of 30 and 100 mg/kg given once orally for 15 days. In addition, neolignan1 is well tolerated up to 100 mg/kg when given as a repeated dose, once orally for 28 days in Swiss albino mice. Neolignan1 was well absorbed from oral route, reached peak at 4 h and eliminated below detection level by 12 h after administration. Our present study concludes that neolignan1 produced relaxation in superior mesenteric artery by opening of BKCa channel and produced significant antihypertensive activity in L-NAME treated Wistar rats and was well tolerated by the experimental animal.

s Background Cold is an important factor contributing to the high incidence of hypertension. Large conductance calcium-activated potassium (BKCa) channels modulate vascular smooth muscle tone. The objective of this study is to determine whether BKCa channels in vascular smooth muscle cells (VSMC) play a role in the development of hypertension during chronic exposure to cold. Methods Forty-eight rats were divided into cold-treated group and control group randomly, and then, rats of cold-treated group were exposed to 4±1 °C (4 h/day) and rats of another group were kept at 25±1 °C. Results Compared to control group, systolic blood pressure of the rats exposed to cold for 2, 4, 6, 8 weeks were increased by 7.14%, 20.42%, 40.54%, 28.74%, respectively. The BKCa current and BKCa peak current density in aortic VSMC increased significantly as the time of exposure to cold went until the 6th week exposure to cold. At the 6th weeks, the BKCa peak current density in the cold exposed group compared to the control group were increased 345.21% (n=6, P Conclusion Our data showed that BKCa channels have apparent change with the development of hypertension during repeated cold exposure, suggesting that BKCa channel could be thought as a new target for prevention and treating cold-induced hypertension.

Expression and activation of the large conductance calcium and voltage-gated potassium (BK Ca ) channels encoded by Kcnma1 gene is shown to be vital in cardioprotection from ischemia-reperfusion injury. BK Ca channels present in SA node cells regulate the heart rate, and in blood vessels play an active role in vascular relaxation. However, the role of BK Ca in regulation of structure and function of the heart is not fully-established. Using Kcnma1 -/- mice, we have observed structural changes in cardiomyocytes and compromised cardiac function as compared to wild type mice. Absence of BK Ca resulted in significant increase in size of adult cardiomyocytes (from 7.95 + 0.1 um 2 to 9.68 + 0.1 um 2 , p < 0.01, n=480 cells each) and also increased cardiac fibrosis. Further to determine underlying signaling mechanisms in cardiac hypertrophy, we performed microarray analysis of RNAs isolated from wild type and Kcnma1 -/- mice (n=3) hearts. We found up regulation of a class of cardiac hypertrophy markers (myosin variants) and changes in the expression of several mitochondrial genes (such as ND4) directly associated with heart diseases in Kcnma1 -/- mice. To evaluate the functional consequence of absence of BK Ca , we performed high-resolution echocardiography on wild type and Kcnma1 -/- mice. Under anesthesia (1.5% isoflurane), left ventricle of Kcnma1 -/- mice showed significant reduction (p < 0.05) in ejection fraction (56 + 2 %, n=7) as compared to wild type (74 + 3 %, n=6) as well as fractional shortening (23 + 3 %, n=7, and 39 + 3 %, n=6, respectively). Similarly, right ventricle had a lower ejection fraction (35.7 + 4% vs 56.9 + 5 %, n > 5) in Kcnma1 -/- as compared to wild type mice. In agreement with our histopathology and microarray data, Kcnma1 -/- mice showed increased posterior wall thickness (0.75 + 0.3 mm vs 0.62 + 0.1 mm) and interventricular septum thickness (0.83 + 0.4 mm, n=7 vs 0.68 + 0.3 mm, n=6) . Together, these data imply that BK Ca plays a direct role in cardiac hypertrophy and cardiac function.

Large-conductance Ca2+-activated potassium channels (BKCa) are highly expressed in the lateral amygdala (LA), which is closely involved in assigning stress disorders, but data on their role in the neuronal circuits of stress disorders are limited. In the present study, a significant reduction in BKCa channel expression in the amygdala of mice accompanied anxiety-like behaviour induced by acute stress. Whole-cell patch-clamp recordings from LA neurons of the anxious animals revealed a pronounced reduction in the fast after-hyperpolarization (fAHP) of action potentials mediated by BKCa channels that led to hyperexcitability of the LA neurons. Activation of BKCa channels in the LA reversed stress-induced anxiety-like behaviour after stress. Furthermore, down-regulated BKCa channels notably increased the evoked NMDA receptor-mediated excitatory postsynaptic potentials at the thalamo-LA synapses. These data demonstrate, for the first time, that restraint stress-induced anxiety-like behaviour could at least partly be explained by alterations in the functional BKCa channels in the LA.

Exposure to perinatal hypoxia results in alteration of the adult pulmonary circulation, which is linked among others to alterations in K(+) channels in pulmonary artery (PA) smooth muscle cells. In particular, large conductance Ca(2+)-activated K(+) (BK(Ca)) channels protein expression and activity were increased in adult PA from mice born in hypoxia compared with controls. We evaluated long-term effects of perinatal hypoxia on the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway-mediated activation of BK(Ca) channels, using isoproterenol, forskolin, and dibutyryl-cAMP. Whole-cell outward current was higher in pulmonary artery smooth muscle cells from mice born in hypoxia compared with controls. Spontaneous transient outward currents, representative of BK(Ca) activity, were present in a greater proportion in pulmonary artery smooth muscle cells of mice born in hypoxia than in controls. Agonists induced a greater relaxation in PA of mice born in hypoxia compared with controls, and BK(Ca) channels contributed more to the cAMP/PKA-mediated relaxation in case of perinatal hypoxia. In summary, perinatal hypoxia enhanced cAMP-mediated BK(Ca) channels activation in adult murine PA, suggesting that this pathway could be a potential target for modulating adult pulmonary vascular tone after perinatal hypoxia.

Background: It remains unclear whether sarcolemmal BKCa channels in post-hatch chick ventricular myocytes contribute to stretch-induced extrasystoles (SIE), and whether they are stretch-activated BKCa (SAKCa) channels or a non-stretch-sensitive BKCa variant. Methods and Results: To determine the role of sarcolemmal BKCa channels in SIE and their stretch sensitivity, an isolated 2-week-old Langendorff-perfused chick heart and mathematical simulation were used. The ventricular wall was rapidly stretched by application of a volume change pulse. As the speed of the stretch increased, the probability of SIE also significantly increased, significantly shortening the delay between SIE and the initiation of the stretch. Application of 100nmol/L of Grammostola spatulata mechanotoxin 4, a cation-selective stretch-activated channel (SAC) blocker, significantly decreased the probability of SIE. The application of Iberiotoxin, however, a BKCa channel blocker, significantly increased the probability of SIE, suggesting that a K+ efflux via a sarcolemmal BKCa channel reduces SIE by balancing out stretch-induced cation influx via SACs. The simulation using a cardiomyocyte model combined with a new stretch sensitivity model that considers viscoelastic intracellular force transmission showed that stretch sensitivity in BKCa channels is required to reproduce the present wet experimental results. Conclusions: Sarcolemmal BKCa channels in post-hatch chick ventricular myocytes are SAKCa channels, and they have a suppressive effect on SIE. (Circ J 2011; 75: 2552-2558)

We have previously reported the electrophysiological properties of sarcolemmal stretch-activated BKCa (SAKCA) channels cloned from cultured chick embryonic ventricular myocytes. However, the role of BKCa channels in the electrophysiology of the more mature heart is not clear. We have investigated the effects on the BKCa current of axial stretch in post-hatch ventricular myocytes. Whole-cell currents of ventricular myocytes isolated from 2-week-old chicks were recorded using the patch-clamp technique, while the cells were either held at resting length or stretched to cause a 10% increase in sarcomere length using a pair of carbon fibres attached to opposite ends of the cell. Stretch did not affect whole-cell currents immediately after the stretch was applied. However, sustained stretch for 3 min significantly increased outward currents. This stretch-induced change was reversed by applying 10 nm iberiotoxin, a specific BKCa channel blocker, or a Na+–Ca2+-free environment. These results were reproduced in a computer simulation study. The present study is the first report about the sarcolemmal BKCa current from post-hatch ventricular myocytes. The present results suggest that axial stretch activates BKCa channels via a stretch-induced increase in the cytosolic Na+ concentration followed by an increased Ca2+ influx.

This investigation was designed to examine the hypothesis that impaired function of coronary microvascular large-conductance Ca2+-activated K+ (BKCa) channels in metabolic syndrome (MetS) significantly attenuates the balance between myocardial oxygen delivery and metabolism at rest and during exercise-induced increases in myocardial oxygen consumption (MV̇o2). Studies were conducted in conscious, chronically instrumented Ossabaw swine fed a normal maintenance diet (11% kcal from fat) or an excess calorie atherogenic diet (43% kcal from fat, 2% cholesterol, 20% kcal from fructose) that induces many common features of MetS. Data were collected under baseline/resting conditions and during graded treadmill exercise before and after selective blockade of BKCa channels with penitrem A (10 μg/kg iv). We found that the exercise-induced increases in blood pressure were significantly elevated in MetS swine. No differences in baseline cardiac function or heart rate were noted. Induction of MetS produced a parallel downward shift in the relationship between coronary venous Po2 and MV̇o2 ( P < 0.001) that was accompanied by a marked release of lactate (negative lactate uptake) as MV̇o2 was increased with exercise ( P < 0.005). Inhibition of BKCa channels with penitrem A did not significantly affect blood pressure, heart rate, or the relationship between coronary venous Po2 and MV̇o2 in lean or MetS swine. These data indicate that BKCa channels are not required for local metabolic control of coronary blood flow under physiological (lean) or pathophysiological (MetS) conditions. Therefore, diminished function of BKCa channels does not contribute to the impairment of myocardial oxygen-supply demand balance in MetS.

Large-conductance calcium and voltage-dependent potassium (BK(Ca)) channel is an important determinant of vascular tone. It is activated by hydrogen peroxide (H(2)O(2)) which occurs in various physiological and pathological processes. However, the regulation mechanism is not fully understood. In the present study, the mSlo in the presence or absence of hbeta1 were cotransfected with the PTEN(wt), PTEN(C124S), PTEN(G129E) in HEK 293 cells. Typical BK(Ca) channel currents could be recorded in cell-attached configurations. We found that PTEN(wt) reduced the H(2)O(2)-induced BK(Ca) channel activation during the initial 10 min treatment. In contrast, coexpression with catalytically inactive PTEN(C124S)/PTEN(G129E) mutants that lack lipid phosphatase activity produced no regulation on the H(2)O(2)-induced BK(Ca) channel activation. These results demonstrated that PTEN regulated the H(2)O(2)-induced BK(Ca) channel activation through phosphatidylinositol 3-phosphatse. However, the inhibitory effect of PTEN on the H(2)O(2)-induced BK(Ca) channel activation was attenuated when cells were treated with H(2)O(2) at concentrations higher than 100 microM or at 100 microM for long-term treatment. In addition, the p-AKT expression level in PTEN(wt) overexpressing cells was lower than that in control cells, and the increase of cytoplasmic free calcium concentration ([Ca(2+)](i)) induced by H(2)O(2) was also inhibited. These findings may elucidate a new mechanism for H(2)O(2)-induced BK(Ca) channel activation and provide some evidences for the role of PTEN on vasodilation induced by H(2)O(2).

The normal physiological contraction of the urinary bladder, which is required for voiding, is predominantly mediated by muscarinic receptors, primarily the M-3 subtype, with the M-2 subtype providing a secondary backup role. Bladder relaxation, which is required for urine storage, is mediated by beta-adrenoceptors, in most species involving a strong beta(3)-component. An excessive stimulation of contraction or a reduced relaxation of the detrusor smooth muscle during the storage phase of the micturition cycle may contribute to bladder dysfunction known as the overactive bladder. Therefore, interference with the signal transduction of these receptors may be a viable approach to develop drugs for the treatment of overactive bladder. The prototypical signaling pathway of M-3 receptors is activation of phospholipase C (PLC), and this pathway is also activated in the bladder. Nevertheless, PLC apparently contributes only in a very minor way to bladder contraction. Rather, muscarinic-receptor-mediated bladder contraction involves voltage-operated Ca2+ channels and Rho kinase. The prototypical signaling pathway of beta-adrenoceptors is an activation of adenylyl cyclase with the subsequent formation of cAMP. Nevertheless, cAMP apparently contributes in a minor way only to beta-adrenoceptor-mediated bladder relaxation. BKCa channels may play a greater role in beta-adrenoceptor-mediated bladder relaxation. We conclude that apart from muscarinic receptor antagonists and beta-adrenoceptor agonists, inhibitors of Rho kinase and activators of BKCa channels may have potential to treat an overactive bladder

Mitochondrial Ca2+-activated large-conductance K+ (BKCa) channels are thought to provide protection during ischemic insults in the heart. Rottlerin (mallotoxin) has been implicated as a potent BKCa activator. The purpose of this study was twofold: 1) to investigate the efficacy of BKCa channel activation as a cardioprotective strategy during ischemic cardioplegic arrest and reperfusion (CP/R) and 2) to assess the specificity of rottlerin for BKCa channels. Wild-type (WT) and BKCa knockout (KO) mice were subjected to an isolated heart model of ischemic CP/R. A mechanism of rottlerin-induced cardioprotection was also investigated using H9c2 cells subjected to in vitro CP/reoxygenation and assessed for mitochondrial membrane potential and reactive oxygen species (ROS) production. CP/R decreased left ventricular developed pressure, positive and negative first derivatives of left ventricular pressure, and coronary flow (CF) in WT mice. Rottlerin dose dependently increased the recovery of left ventricular function and CF to near baseline levels. BKCa KO hearts treated with or without 500 nM rottlerin were similar to WT CP hearts. H9c2 cells subjected to in vitro CP/R displayed reduced mitochondrial membrane potential and increased ROS generation, both of which were significantly normalized by rottlerin. We conclude that activation of BKCa channels rescues ischemic damage associated with CP/R, likely via effects on improved mitochondrial membrane potential and reduced ROS generation.

We studied the properties and roles of large-conductance calcium-sensitive potassium channels (BK"C"a) in cultured rat cerebellar granule neurons. In intact cells these channels had chord conductances of 130-200pS. The conductance, threshold for channel activation and the mean open time and open probability depend on the age of cells. In computer simulations the elimination of BK"C"a channel decreases the maximum and minimum peak values of an action potential, causes reduced hyperpolarization and increases firing frequency. The observed alterations may be linked to the complexity of granule neuron excitability during maturation and provide new insight into modeling the developmental changes in small CNS neurons.

Epoxyeicosatrienoic acids (EETs) are metabolized by soluble epoxide hydrolase (sEH) to form dihydroxyeicosatrienoic acids (DHETs) and are putative endothelium-derived hyperpolarizing factors (EDHFs). EDHFs modulate microvascular tone; however, the chemical identity of EDHF in the human coronary microcirculation is not known. We examined the capacity of EETs, DHETs, and sEH inhibition to affect vasomotor tone in isolated human coronary arterioles (HCAs). HCAs from right atrial appendages were prepared for videomicroscopy and immunohistochemistry. In vessels preconstricted with endothelin-1, three EET regioisomers (8,9-, 11,12-, and 14,15-EET) each induced a concentration-dependent dilation that was sensitive to blockade of large-conductance Ca2+-activated K+(BKCa) channels by iberiotoxin. EET-induced dilation was not altered by endothelial denudation. 8,9-, 11,12-, and 14,15-DHET also dilated HCA via activation of BKCachannels. Dilation was less with 8,9- and 14,15-DHET but was similar with 11,12-DHET, compared with the corresponding EETs. Immunohistochemistry revealed prominent expression of cytochrome P-450 (CYP450) 2C8, 2C9, and 2J2, enzymes that may produce EETs, as well as sEH, in HCA. Inhibition of sEH by 1-cyclohexyl-3-dodecylurea (CDU) enhanced dilation caused by 14,15-EET but reduced dilation observed with 11,12-EET. DHET production from exogenous EETs was reduced in vessels pretreated with CDU compared with control, as measured by liquid chromatography electrospray-ionization mass spectrometry. In conclusion, EETs and DHETs dilate HCA by activating BKCachannels, supporting a role for EETs/DHETs as EDHFs in the human heart. CYP450s and sEH may be endogenous sources of these compounds, and sEH inhibition has the potential to alter myocardial perfusion, depending on which EETs are produced endogenously.

Potassium (K+) channels play a pivotal role in the CNS, controlling cell excitability thereby raising their therapeutic application. In realisation of the utility of K+channels, many pharmaceutical companies have developed a plethora of antagonists and openers for a range of disorders, including stroke, epilepsy, pain and cognition. The most promising targets, including BKCa, SKCa and KCNQ channels, will be reviewed in this article. The focus will be upon the most recent K+ channel modulator patents for CNS disorders and future developments of drugs for the treatment of CNS disorders.

We report here a combination of site-directed mutations that eliminate the high-affinity Ca(2+) response of the large-conductance Ca(2+)-activated K(+) channel (BK(Ca)), leaving only a low-affinity response blocked by high concentrations of Mg(2+). Mutations at two sites are required, the "Ca(2+) bowl," which has been implicated previously in Ca(2+) binding, and M513, at the end of the channel's seventh hydrophobic segment. Energetic analyses of mutations at these positions, alone and in combination, argue that the BK(Ca) channel contains three types of Ca(2+) binding sites, one of low affinity that is Mg(2+) sensitive (as has been suggested previously) and two of higher affinity that have similar binding characteristics and contribute approximately equally to the power of Ca(2+) to influence channel opening. Estimates of the binding characteristics of the BK(Ca) channel's high-affinity Ca(2+)-binding sites are provided.