Piezo1 and BKCa channels in human atrial fibroblasts: Interplay and remodelling in atrial fibrillation.

AIMS: Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of 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 changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF. METHODS AND RESULTS: Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or 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 cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent '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 BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected. CONCLUSIONS: Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. 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 support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical links between the two channel proteins.

Macrovascular Protecting Effects of Berberine through Anti-inflammation and Intervention of BKCa in Type 2 Diabetes Mellitus Rats.

OBJECTIVE: The aim of this study was to examine the effect of berberine on diabetes mellitus in vivo and in vitro, and elucidate the underlying mechanisms. METHODS: Rat models of type 2 diabetes mellitus (T2DM) were established and were treated with berberine. Pathological changes in the thoracic aorta, and inflammatory factor and adiponectin levels were investigated. Vascular smooth muscle cells (VSMCs) of the thoracic aorta were cultured and treated with berberine. Cellular proliferation, migration, and inflammatory factor levels were investigated. Responses of vascular rings to phenylephrine (PE) and sodium nitroprusside (SNP) after berberine intervention and the changes of relaxation responses to SNP after adding Iberiotoxin (IbTX) were investigated. RESULTS: Berberine ameliorated the pathological status of the thoracic aorta in the T2DM rats. Berberine significantly inhibited the C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) production, and increased the adiponectin level compared with the model group. Compared with the model group, berberine inhibited the proliferation and migration of VSMCs in vitro, and reduced tumor growth factor-ß1 (TGF-ß1), IL-6, and TNF-α levels. Furthermore, the contraction of thoracic aorta to PE was reduced, while the relaxation response of thoracic aorta to SNP was increased, after the berberine intervention in the T2DM rats. The relaxation response of thoracic aorta to SNP in the model and berberine groups decreased after the IbTX treatment. CONCLUSION: Protective effects of berberine against macrovascular complications induced by diabetes mellitus may be attributed to inhibiting of the inflammation and intervening of the calcium- activated potassium (BKCa).

Inhibition of intracellular Ca2+ mobilization and potassium channels activation are involved in the vasorelaxation induced by 7-hydroxycoumarin.

Coumarins exhibit a wide variety of biological effects, including activities in the cardiovascular system and the aim of this study was to evaluate the vascular therapeutic potential of 7-Hydroxicoumarin (7-HC). The vascular effects induced by 7-HC (0.001 µM-300 µM), were investigated by in vitro approaches using isometric tension measurements in rat superior mesenteric arteries and by in silico assays using Ligand-based analysis. Our results suggest that the vasorelaxant effect of 7-HC seems to rely on potassium channels, notably through large conductance Ca2+-activated K+ (BKCa) channels activation. In fact, 7-HC (300 µM) significantly reduced CaCl2-induced contraction as well as the reduction of intracellular calcium mobilization. However, the relaxation induced by 7-HC was independent of store-operated calcium entry (SOCE). Moreover, in silico analysis suggests that potassium channels have a common binding pocket, where 7-HC may bind and hint that its binding profile is more similar to quinine's than verapamil's. These results are compatible with the inhibition of Ca2+ release from intracellular stores, which is prompted by phenylephrine and caffeine. Taken together, these results demonstrate a therapeutic potential of 7-HC on the cardiovascular system, making it a promising lead compound for the development of drugs useful in the treatment of cardiovascular diseases.

Opening large-conductance potassium channels selectively induced cell death of triple-negative breast cancer.

BACKGROUND: Unlike other breast cancer subtypes that may be treated with a variety of hormonal or targeted therapies, there is a need to identify new, effective targets for triple-negative breast cancer (TNBC). It has recently been recognized that membrane potential is depolarized in breast cancer cells. The primary objective of the study is to explore whether hyperpolarization induced by opening potassium channels may provide a new strategy for treatment of TNBC. METHODS: Breast cancer datasets in cBioPortal for cancer genomics was used to search for ion channel gene expression. Immunoblots and immunohistochemistry were used for protein expression in culture cells and in the patient tissues. Electrophysiological patch clamp techniques were used to study properties of BK channels in culture cells. Flow cytometry and fluorescence microscope were used for cell viability and cell cycle studies. Ultrasound imaging was used to study xenograft in female NSG mice. RESULTS: In large datasets of breast cancer patients, we identified a gene, KCNMA1 (encoding for a voltage- and calcium-dependent large-conductance potassium channel, called BK channel), overexpressed in triple-negative breast cancer patients. Although overexpressed, 99% of channels are closed in TNBC cells. Opening BK channels hyperpolarized membrane potential, which induced cell cycle arrest in G2 phase and apoptosis via caspase-3 activation. In a TNBC cell induced xenograft model, treatment with a BK channel opener significantly slowed tumor growth without cardiac toxicity. CONCLUSIONS: Our results support the idea that hyperpolarization induced by opening BK channel in TNBC cells can become a new strategy for development of a targeted therapy in TNBC.

Total flavones of Rhododendron simsii Planch flower protect rat hippocampal neuron from hypoxia-reoxygenation injury via activation of BKCa channel.

OBJECTIVES: To study the effects of total flavones of Rhododendra simsii Planch flower (TFR) on hypoxia/reoxygenation (H/R) injury in rat hippocampal neurons and its underlying mechanism. METHOD: Model of H/R was established in newborn rat primary cultured hippocampal neuron. Lactate dehydrogenase (LDH) and neuron-specific enolase (NSE) activity as well as malondialdehyde (MDA) content in cultured supernatants of the neurons were examined. Methyl thiazolyl tetrazolium assay and Hoechst33258 staining were, respectively, used to detect cell viability and apoptosis of neurons. Protein expression and current of BKCa channel were assessed by using Western blotting and whole-cell patch-clamp methods, respectively. KEY FINDINGS: In the ranges of 3.7-300 mg/l, TFR significantly inhibited H/R-induced decrease of neuronal viability and increases of LDH, NSE and MDA in the supernatants as well as apoptosis; TFR 33.3, 100 and 300 mg/l markedly increased current of BKCa channel rather than the BKCa channel protein expression in the neurons. CONCLUSIONS: Total flavones of R. simsii Planch flower had a protective effect against H/R injury in rat hippocampal neuron, and activation of BKCa channel may contribute to the neuroprotection.

Propofol Relaxes Isolated Rat Aorta through BKCa Activation.

BACKGROUND: Propofol is an intravenous anesthetic that can be used for the induction and maintenance of anesthesia. In the present study, it was aimed to investigate the mechanism of vasodilator action of propofol in the rat aorta (RA). METHODS: The RA rings were suspended in isolated organ baths and tension was recorded isometrically. First, potassium chloride (KCl) and phenylephrine (PE) were added to organ baths to form precontraction. When the precontractions were stable, propofol (1, 10, and 100 µM) was added cumulatively to the baths. The antagonistic effect of propofol on KCl (45 mM), PE (1 µM), 5-hydroxytryptamine (5-HT) (30 µM), and calcium chloride (CaCl2) (10 µM to 10 mM) induced contractions in the vascular rings were investigated. Propofol-induced relaxations were also tested in the presence of the K+ channel inhibitors tetraethylammonium (TEA, 1 mM), glibenclamide (GLI, 10 µM), 4-aminopyridine (4-AP, 1 mM), and barium chloride (BaCl2, 30 µM). RESULTS: Preincubation with propofol (1, 10, and 100 µM) did not affect the basal tone but inhibited the contraction induced by KCl, PE, 5-HT, and CaCl2-induced contractions. Propofol-induced relaxation was not effected by 4-AP, GLI, and BaCl2. However, TEA inhibited propofol-induced relaxations significantly. CONCLUSIONS: The propofol induces relaxation in contracted RA and inhibits KCl, PE, 5-HT, and CaCl2-induced contractions. The results demonstrate that the mechanism of action of propofol-induced vasodilation in the RA may be related to large conductance Ca2+-activated K+ channel activation.

Inhibitory effects of openers of large-conductance Ca2+-activated K+ channels on agonist-induced phasic contractions in rabbit and mouse bronchial smooth muscle.

Airway smooth muscle expresses abundant BKCa channels, but their role in regulating contractions remains controversial. This study examines the effects of two potent BKCa channel openers on agonist-induced phasic contractions in rabbit and mouse bronchi. First, we demonstrated the ability of 10 µM GoSlo-SR5-130 to activate BKCa channels in inside-out patches from rabbit bronchial myocytes, where it shifted the activation V1/2 by -88 ± 11 mV (100 nM Ca2+, n = 7). In mouse airway smooth muscle cells, GoSlo-SR5-130 dose dependently shifted V1/2 by 12-83 mV over a concentration range of 1-30 µM. Compound X, a racemic mixture of two enantiomers, reported to be potent BKCa channel openers, shifted V1/2 by 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 V1/2 of BKCa channels 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 BKCa channel openers, suggesting that these channels are potential targets for treating some causes of airway obstruction.

Lipopolysaccharide stimulates BK channel activity in bladder umbrella cells.

Bladder urothelium plays an active role in response to bacterial infection. There is little known about the electrophysiological activity in urothelial cells in this process. We used a nonenzymatic method to isolate bladder urothelial tissue and to patch clamp umbrella cells in situ. A 200 pS conductance potassium (K+) channel was detected from female C57BL6 mice. Of 58 total patches, 17.2% patches displayed the 200 pS K+ conductance channel. This K+ conductance channel showed Ca2+ sensitivity and voltage dependence. Specific big-conductance potassium channel (BK) inhibitors (paxilline, iberiotoxin) blocked the 200 pS K+ conductance channel activity. RT-PCR and immunoblot confirmed BK channel pore-forming α-subunit (BK-α) mRNA and protein in urothelium. Immunohistochemistry also showed the BK-α located in urothelium. The above data provided evidence that the 200 pS K+ conductance channel was a BK channel. Lipopolysaccharide (LPS), a component of uropathogenic Escherichia coli, was used to investigate the role of BK channel in the pathogenesis of urinary tract infection. BK channel activity as NPo increased threefold within 30 min of exposure to LPS. mRNAs for LPS receptors (TLR4, CD14, MD-2) were expressed in the urothelium but not in lamina propria or detrusor. Blockade of the receptors by an antagonist (polymyxin B) abrogated LPS's effect on BK channel. The involvement of protein kinase A (PKA) on BK channel activity was demonstrated by applying PKA blockers (H89 and PKI). Both PKA inhibitors abolished the BK channel activity induced by LPS. In conclusion, BK channel was identified in bladder umbrella cells, and its activity was significantly increased by LPS.

Methylglyoxal Impairs ß2-Adrenoceptor-Mediated Vasodilatory Mechanisms in Rat Retinal Arterioles.

Methylglyoxal, a highly reactive dicarbonyl compound, is formed as a by-product of glycolysis and plays an important role in the pathogenesis of diabetic complications, including diabetic retinopathy. However, it remains to be determined how methylglyoxal affects the regulatory mechanisms of retinal blood flow. In this study, we examined the effects of methylglyoxal on ß2-adrenoceptor-mediated vasodilatory mechanisms in rat retinal arterioles. The retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles in the fundus images. Intravitreal injection of methylglyoxal significantly diminished the vasodilation of retinal arterioles induced by the ß2-adrenoceptor agonist salbutamol. The vasodilator effect of BMS-191011, a large-conductance Ca2+-activated K+ (BKCa) channel opener, on retinal arterioles was also attenuated by methylglyoxal. In contrast, methylglyoxal had no significant effect on retinal vasodilator response to forskolin. Methylglyoxal attenuated retinal vasodilator response to salbutamol under blockade of BKCa channels with iberiotoxin, an inhibitor of the channels. These results suggest that methylglyoxal attenuates ß2-adrenoceptor-mediated retinal vasodilation by impairing the coupling of the ß2-adrenoceptor to the guanine nucleotide-binding protein (Gs protein) and the function of the BKCa channel. Increased methylglyoxal in the eyes may contribute to the impairment of regulatory mechanisms of retinal blood flow in patients with diabetic retinopathy.

The quest for endothelial atypical cannabinoid receptor: BKCa channels act as cellular sensors for cannabinoids in in vitro and in situ endothelial cells.

Endothelium-dependent component of cannabinoid-induced vasodilation has been postulated to require G-protein-coupled non-CB1/CB2 endothelial cannabinoid (eCB) receptor. GPR18 was proposed as a candidate for eCBR. To address the hypothesis that the effects attributed to eCBR are mediated by G-protein-coupled receptor (GPCR)-independent targets, we studied the electrical responses in endothelial cells, focusing on BKCa channels. In patches excised from endothelial-derived EA.hy926 cells, N-arachidonoyl glycine (NAGly) and abnormal cannabidiol (abn-cbd), prototypical agonists for eCB receptor, stimulate single BKCa activity in a concentration- and Ca2+-dependent manner. The postulated eCB receptor inhibitors rimonabant and AM251 were found to inhibit basal and stimulated by NAGly- and abn-cbd BKCa activity in cell-free patches. In isolated mice aortas, abn-cbd and NAGly produced endothelial cell hyperpolarization that was sensitive to paxilline, a selective BKCa inhibitor, but not to GPR18 antibody, and mimicked by NS1619, a direct BKCa opener. In excised patches from mice aortic endothelium, single channel activity with characteristics similar to BKCa was established by the addition of abn-cbd and NAGly. We conclude that the two cannabinoids abn-cbd and NAGly initiate a GPR18-independent activation of BKCa channels in mice aortic endothelial cells that might contribute to vasodilation to cannabinoids.

Potential Involvement of Impaired BKCa Channel Function in Sensory Defensiveness and Some Behavioral Disturbances Induced by Unfamiliar Environment in a Mouse Model of Fragile X Syndrome.

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.

Melatonin mediates vasodilation through both direct and indirect activation of BKCa channels.

Melatonin, synthesized primarily by the pineal gland, is a neuroendocrine hormone with high membrane permeability. The vascular effects of melatonin, including vasoconstriction and vasodilation, have been demonstrated in numerous studies. However, the mechanisms underlying these effects are not fully understood. Large-conductance Ca2+-activated K+ (BKCa) channels are expressed broadly on smooth muscle cells and play an important role in vascular tone regulation. This study explored the mechanisms of myocyte BKCa channels and endothelial factors underlying the action of melatonin on the mesenteric arteries (MAs). Vascular contractility and patch-clamp studies were performed on myocytes of MAs from Wistar rats. Melatonin induced significant vasodilation on MAs. In the presence of Nω-nitro-l-arginine methyl ester (l-NAME), a potent endothelial oxide synthase (eNOS) inhibitor, melatonin elicited concentration-dependent relaxation, with lowered pIC50 The effect of melatonin was significantly attenuated in the presence of BKCa channel blocker iberiotoxin or MT1/MT2 receptor antagonist luzindole in both (+) l-NAME and (-) l-NAME groups. In the (+) l-NAME group, iberiotoxin caused a parallel rightward shift of the melatonin concentration-relaxation curve, with pIC50 lower than that of luzindole. Both inside-out and cell-attached patch-clamp recordings showed that melatonin significantly increased the open probability, mean open time and voltage sensitivity of BKCa channels. In a cell-attached patch-clamp configuration, the melatonin-induced enhancement of BKCa channel activity was significantly suppressed by luzindole. These findings indicate that in addition to the activation of eNOS, melatonin-induced vasorelaxation of MAs is partially attributable to its direct (passing through the cell membrane) and indirect (via MT1/MT2 receptors) activation of the BKCa channels on mesenteric arterial myocytes.

The Slo(w) path to identifying the mitochondrial channels responsible for ischemic protection.

Mitochondria play an important role in tissue ischemia and reperfusion (IR) injury, with energetic failure and the opening of the mitochondrial permeability transition pore being the major causes of IR-induced cell death. Thus, mitochondria are an appropriate focus for strategies to protect against IR injury. Two widely studied paradigms of IR protection, particularly in the field of cardiac IR, are ischemic preconditioning (IPC) and volatile anesthetic preconditioning (APC). While the molecular mechanisms recruited by these protective paradigms are not fully elucidated, a commonality is the involvement of mitochondrial K+ channel opening. In the case of IPC, research has focused on a mitochondrial ATP-sensitive K+ channel (mitoKATP), but, despite recent progress, the molecular identity of this channel remains a subject of contention. In the case of APC, early research suggested the existence of a mitochondrial large-conductance K+ (BK, big conductance of potassium) channel encoded by the Kcnma1 gene, although more recent work has shown that the channel that underlies APC is in fact encoded by Kcnt2 In this review, we discuss both the pharmacologic and genetic evidence for the existence and identity of mitochondrial K+ channels, and the role of these channels both in IR protection and in regulating normal mitochondrial function.

The Cardioprotective Effect of Dexmedetomidine in Rats Is Dose-Dependent and Mediated by BKCa Channels.

The alpha-2 receptor agonist Dexmedetomidine (Dex) protects the heart against ischemia-reperfusion injury. We investigated the signaling cascade underlying Dex-induced acute cardioprotection, with special emphasis on large-conductance Ca2+-sensitive potassium (BKCa) channels. Rats were anesthetized with pentobarbital. Hearts were isolated, mounted on a Langendorff system and perfused with Krebs-Henseleit buffer. Hearts underwent 33 minutes of ischemia followed by 60 minutes of reperfusion. Before the beginning of ischemia, Dex was administered at different doses (0.1-30 nM) for characterization of a dose-effect relationship. In another set of experiments, Dex (3 nM) was administered together with the BKCa channel inhibitor paxilline and the connexin-43 inhibitor peptide Gap27. Also, the BKCa channel opener NS1619 was administered. In control animals, infarct size was 49% ± 5%. Dex at 3-30 nM reduced infarct size to ∼22%, whereas lower (0.1-1 nM) doses reduced infarct size to ∼38%. Paxilline (1 µM) and GAP27 (6 µM) blocked the Dex-induced cardioprotection. NS1619 (10 µM) reduced infarct size to about the same magnitude as did the higher doses of Dex. Functional heart parameters and coronary flow were not different between the study groups. In male rats, the Dex-induced protection against ischemia-reperfusion injury involves connexin-43 and activation of BKCa channels.

Stimulatory actions of a novel thiourea derivative on large-conductance, calcium-activated potassium channels.

In this study, we examine whether an anti-inflammatory thiourea derivative, compound #326, actions on ion channels. The effects of compound #326 on Ca2+ -activated K+ channels were evaluated by patch-clamp recordings obtained in cell-attached, inside-out or whole-cell configuration. In pituitary GH3 cells, compound #326 increased the amplitude of Ca2+ -activated K+ currents (IK(Ca) ) with an EC50 value of 11.6 µM, which was reversed by verruculogen, but not tolbutamide or TRAM-34. Under inside-out configuration, a bath application of compound #326 raised the probability of large-conductance Ca2+ -activated K+ (BKCa ) channels. The activation curve of BKCa channels was shifted to less depolarised potential with no modification of the gating charge of the curve; consequently, the difference of free energy was reduced in the presence of this compound. Compound #326-stimulated activity of BKCa channels is explained by a shortening of mean closed time, despite its inability to alter single-channel conductance. Neither delayed-rectifier nor erg-mediated K+ currents was modified. Compound #326 decreased the peak amplitude of voltage-gated Na+ current with no clear change in the overall current-voltage relationship of this current. In HEK293T cells expressing α-hSlo, compound #326 enhanced BKCa channels effectively. Intriguingly, the inhibitory actions of compound #326 on interleukin 1ß in lipopolysaccharide-activated microglia were significantly reversed by verruculogen, whereas BKCa channel inhibitors suppressed the expressions of inducible nitric oxide synthase. The BKCa channels could be an important target for compound #326 if similar in vivo results occur, and the multi-functionality of BKCa channels in modulating microglial immunity merit further investigation.

GPR55 agonist lysophosphatidylinositol and lysophosphatidylcholine inhibit endothelial cell hyperpolarization via GPR-independent suppression of Na+-Ca2+ exchanger and endoplasmic reticulum Ca2+ refilling.

Lysophosphatidylinositol (LPI) and lysophosphatidylcholine (LPC) are lipid signaling molecules that induce endothelium-dependent vasodilation. In addition, LPC suppresses acetylcholine (Ach)-induced responses. We aimed to determine the influence of LPC and LPI on hyperpolarizing responses in vitro and in situ endothelial cells (EC) and identify the underlying mechanisms. Using patch-clamp method, we show that LPI and LPC inhibit EC hyperpolarization to histamine and suppress Na+/Ca2+ exchanged (NCX) currents in a concentration-dependent manner. The inhibition is non-mode-specific and unaffected by intracellular GDPßS infusion and tempol, a superoxide dismutase mimetic. In excised mouse aorta, LPI strongly inhibits the sustained and the peak endothelial hyperpolarization induced by Ach, but not by SKA-31, an opener of Ca2+-dependent K+ channels of intermediate and small conductance. The hyperpolarizing responses to consecutive histamine applications are strongly reduced by NCX inhibition. In a Ca2+-re-addition protocol, bepridil, a NCX inhibitor, and KB-R7943, a blocker of reversed NCX, inhibit the hyperpolarizing responses to Ca2+-re-addition following Ca2+ stores depletion. These finding indicate that LPC and LPI inhibit endothelial hyperpolarization to Ach and histamine independently of G-protein coupled receptors and superoxide anions. Reversed NCX is critical for ER Ca2+ refilling in EC. The inhibition of NCX by LPI and LPC underlies diminished endothelium-dependent responses and endothelial dysfunction accompanied by increased levels of these lipids in the blood.

Coronary arterial BK channel dysfunction exacerbates ischemia/reperfusion-induced myocardial injury in diabetic mice.

The large conductance Ca(2+)-activated K(+) (BK) channels, abundantly expressed in coronary artery smooth muscle cells (SMCs), play a pivotal role in regulating coronary circulation. A large body of evidence indicates that coronary arterial BK channel function is diminished in both type 1 and type 2 diabetes. However, the consequence of coronary BK channel dysfunction in diabetes is not clear. We hypothesized that impaired coronary BK channel function exacerbates myocardial ischemia/reperfusion (I/R) injury in streptozotocin-induced diabetic mice. Combining patch-clamp techniques and cellular biological approaches, we found that diabetes facilitated the colocalization of angiotensin II (Ang II) type 1 receptors and BK channel α-subunits (BK-α), but not BK channel ß1-subunits (BK-ß1), in the caveolae of coronary SMCs. This caveolar compartmentation in vascular SMCs not only enhanced Ang II-mediated inhibition of BK-α but also produced a physical disassociation between BK-α and BK-ß1, leading to increased infarct size in diabetic hearts. Most importantly, genetic ablation of caveolae integrity or pharmacological activation of coronary BK channels protected the cardiac function of diabetic mice from experimental I/R injury in both in vivo and ex vivo preparations. Our results demonstrate a vascular ionic mechanism underlying the poor outcome of myocardial injury in diabetes. Hence, activation of coronary BK channels may serve as a therapeutic target for cardiovascular complications of diabetes.

Role of BK(Ca) Potassium Channels in the Mechanisms of Modulatory Effects of IL-10 on Hypoxia-Induced Changes in Activity of Hippocampal Neurons.

We studied the contribution of large conductance Ca(2+)-activated potassium channels (BKCa) in the mechanisms of neuromodulatory effects of anti-inflammatory cytokine IL-10 on hypoxiainduced changes in activity of CA1 pyramidal neurons in rat hippocampus. We used the method of registration of population spikes from CA1 pyramidal neurons in hippocampal slices before, during, and after exposure to short-term episodes of hypoxia. Selective blocker (iberiotoxin) and selective activator of BKCa (BMS-191011) were used to evaluate the contribution of these channels in the mechanisms of suppressive effects of IL-10 on changes in neuronal activity during hypoxia and development of post-hypoxic hyperexcitability. It was shown that BKCa are involved in the modulatory effects of IL-10 on hypoxia-induced suppression of activity of CA1 pyramidal neurons in the hippocampus and development of post-hypoxic hyperexcitability in these neurons.

The synthesis and BK channel-opening activity of N-acylaminoalkyloxime derivatives of dehydroabietic acid.

A series of N-acylaminoalkyloxime derivatives of dehydroabietic acid were synthesized and evaluated for BK channel-opening activities in an assay system of CHO-K1 cells expressing hBKα channels. The structure-activity relationship study revealed that a non-covalent interaction between the S atom of the 2-thiophene and the carbonyl O atom may contribute to conformation restriction for interaction with the ion channel. This research could guide the design and synthesis of novel abietane-based BK channel opener.

Echinacoside induces rat pulmonary artery vasorelaxation by opening the NO-cGMP-PKG-BKCa channels and reducing intracellular Ca2+ levels.

AIM: Sustained pulmonary vasoconstriction as experienced at high altitude can lead to pulmonary hypertension (PH). The main purpose of this study is to investigate the vasorelaxant effect of echinacoside (ECH), a phenylethanoid glycoside from the Tibetan herb Lagotis brevituba Maxim and Cistanche tubulosa, on the pulmonary artery and its potential mechanism. METHODS: Pulmonary arterial rings obtained from male Wistar rats were suspended in organ chambers filled with Krebs-Henseleit solution, and isometric tension was measured using a force transducer. Intracellular Ca(2+) levels were measured in cultured rat pulmonary arterial smooth muscle cells (PASMCs) using Fluo 4-AM. RESULTS: ECH (30-300 µmol/L) relaxed rat pulmonary arteries precontracted by noradrenaline (NE) in a concentration-dependent manner, and this effect could be observed in both intact endothelium and endothelium-denuded rings, but with a significantly lower maximum response and a higher EC50 in endothelium-denuded rings. This effect was significantly blocked by L-NAME, TEA, and BaCl2. However, IMT, 4-AP, and Gli did not inhibit ECH-induced relaxation. Under extracellular Ca(2+)-free conditions, the maximum contraction was reduced to 24.54%±2.97% and 10.60%±2.07% in rings treated with 100 and 300 µmol/L of ECH, respectively. Under extracellular calcium influx conditions, the maximum contraction was reduced to 112.42%±7.30%, 100.29%±8.66%, and 74.74%±4.95% in rings treated with 30, 100, and 300 µmol/L of ECH, respectively. After cells were loaded with Fluo 4-AM, the mean fluorescence intensity was lower in cells treated with ECH (100 µmol/L) than with NE. CONCLUSION: ECH suppresses NE-induced contraction of rat pulmonary artery via reducing intracellular Ca(2+) levels, and induces its relaxation through the NO-cGMP pathway and opening of K(+) channels (BKCa and KIR).