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The potassium released in the extracellular space during neuronal activity is rapidly removed by glia and neurons to maintain tissue homeostasis. Oligodendrocyte-derived myelin axonal coating contributes to potassium buffering and is therefore crucial to control brain excitability. We studied activity-dependent extracellular potassium ([K+] o) changes in the piriform cortex (PC), a region that features highly segregated bundles of myelinated and unmyelinated fibers. Four-aminopyridine (4AP; 50 μM) treatment or patterned high-frequency stimulations (hfST) were utilized to generate [K+] o changes measured with potassium-sensitive electrodes in the myelinated lateral olfactory tract (LOT), in the unmyelinated PC layer I and in the myelinated deep PC layers in the ex vivo isolated guinea-pig brain. Seizure-like events induced by 4AP are initiated by the abrupt [K+] o rise in the layer I formed by unmyelinated fibers (Uva et al., 2017). Larger [K+] o shifts occurred in unmyelinated layers compared to the myelinated LOT. LOT hfST that mimicks pre-seizure discharges also generated higher [K+] o changes in unmyelinated PC layer I than in LOT and deep PC layers. The treatment with the Kir4.1 potassium channel blocker BaCl 2 (100 μM) enhanced the [K+] o changes generated by hfST in myelinated structures. Our data show that activity-dependent [K+] o changes are intrinsically different in myelinated vs unmyelinated cortical regions. The larger [K+] o shifts generated in unmyelinated structures may represent a vehicle for seizure generation. • [K+] o shifts were measured in myelinated and unmyelinated piriform cortex layers • Activity-dependent [K+] o changes are larger in unmyelinated structures • Blockade of Kir4.1 potassium channels enhanced [K+] o changes [ABSTRACT FROM AUTHOR]

Organic nitrates are widely used to restore endogenous nitric oxide (NO) levels reduced by endothelial nitric oxide synthase dysfunction. However, these drugs are associated with undesirable side effects, including tolerance. This study aims to investigate the cardiovascular effects of the new organic nitrate 1,3-diisobutoxypropan-2-yl nitrate (NDIBP). Specifically, we assessed its effects on blood pressure, vascular reactivity, acute toxicity, and the ability to induce tolerance. In vitro and ex vivo techniques showed that NDIBP released NO both in a cell-free system and in isolated mesenteric arteries preparations through a process catalyzed by xanthine oxidoreductase. NDIBP also evoked endothelium-independent vasorelaxation, which was significantly attenuated by 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, 300 μM), a nitric oxide scavenger; 1-H-[1,2,4] oxadiazolo–[4,3-a]quinoxalin-1-one (ODQ, 10 μM), a soluble guanylyl cyclase inhibitor; tetraethylammonium (TEA, 3 mM), a potassium channel blocker; febuxostat (500 nM), a xanthine oxidase inhibitor; and proadifen (10 μM), an inhibitor of cytochrome P450 enzyme. Furthermore, this organic nitrate did not induce tolerance in isolated vessels and presented low toxicity following acute oral administration. In vivo changes on cardiovascular parameters were assessed using normotensive and renovascular hypertensive rats. NDIBP evoked a reduction of blood pressure that was significantly higher in hypertensive animals. Our results suggest that NDIBP acts as a NO donor, inducing blood pressure reduction without having the undesirable effects of tolerance. Those effects seem to be mediated by activation of NO-sGC-cGMP pathway and positive modulation of K+ channels in vascular smooth muscle. • NDIBP releases NO through a metabolic process catalyzed by XOR and CYP450. • NDIBP induces an endothelium-independent vasorelaxant effect. • NDIBP presents a low oral acute preclinical toxicity. • NDIBP induces hypotension and a dual chronotropic effect in rats. [ABSTRACT FROM AUTHOR]

Barium (Ba2+) is a classic permeant blocker of potassium (K+) channels. The "external lock-in effect" in barium block experiments, whereby the binding of external K+ impedes the forward translocation of the blocker, provides a powerful avenue to investigate the selectivity of the binding sites along the pore of potassium channels. Barium block experiments show that the external lock-in site is highly selective for K+ over Na+. Wild-type KcsA was crystallized in low K+ conditions, and the crystals were soaked in solutions containing various concentrations of barium. Structural analysis reveals open and closed gate conformations of the KcsA channel. Anomalous diffraction experiments show that Ba2+ primarily binds to the innermost site S4 of the selectivity filter of the open-gate conformation and also the site S2, but no binding is detected with the closed-gate conformation. Alchemical free-energy perturbation calculations indicate that the presence of a Ba2+ ion in the selectivity filter boosts the specificity of K+ binding relative to Na+ in the external sites S0–S2. Unlabelled Image • Barium (Ba2+) is a classic permeant blocker of potassium (K+) channels. • The binding of external K+ gives rise to the "external lock-in effect" by impeding the forward translocation of the blocker. • X-ray crystallography analysis reveals open and closed gate conformations of the KcsA channel with bound Ba2+ ion • Anomalous diffraction experiments show that Ba2+ primarily binds to the innermost site S4 of the selectivity filter of the open-gate conformation and also the site S2, but no binding is detected with the closed-gate conformation. • Molecular dynamics simulations and alchemical free-energy perturbation calculations indicate that the presence of Ba2+ in the selectivity filter boosts the specificity of K+ binding relative to Na+. [ABSTRACT FROM AUTHOR]

Resveratrol is a plant-derived phytoalexin with antioxidant, anti-inflammatory and cardio-protective properties and may be a promising therapeutic intervention strategy in cardiovascular disease. Here, we investigated the acute direct effects of trans-resveratrol (RV), on acetylcholine (ACh)-induced and flow-mediated dilation (FMD) of isolated pressurized femoral arteries of young (4-month-old) and old (26-month-old) mice. Vessel exposure to RV enhanced ACh (0.01–1.0 mM)-induced dilation (p < 0.05), but not FMD (@ 5–10 μL⋅min−1) (p < 0.05) in both young and old mice. After RV incubation, acute nitric oxide (NO) production by cultured endothelial cells was increased in response to 0.01 mM ACh, but reduced by flow (5–10 μL⋅min−1; p < 0.05). In isolated femoral arteries from endothelial nitric oxide synthase knockout (eNOS−/-) mice, RV had no overall effect on FMD, but potentiated ACh induced dilation, that was completely abolished by potassium channel blockers, Apamin and Tram 34 (p < 0.01). We demonstrate that the non-metabolised form of RV stimulates ACh-induced dilation via the NO and EDHF pathways, but not FMD by interaction with the cyclo-oxygenase pathway. Our findings have important implications in the use of RV (for both young and aged) under 'normal' non-diseased physiological states. • Resveratrol may be a promising therapy in cardiovascular disease and ageing. • We examined direct effects of resveratrol on femoral artery dilation in mice. • Resveratrol enhances acetylcholine-induced, but not flow mediated dilation. • Resveratrol stimulates ACh-induced dilation via the NO and EDHF pathways. • Femoral dilator effects depend on nature of stimulus in young and aged mice. [ABSTRACT FROM AUTHOR]

Cannabinoid receptor (CBR) agonist could act as a protective agent against seizure susceptibility in animal models of epilepsy. Studies have shown that potassium channels could play a key role in ameliorating neuronal excitability. In this study, we attempted to evaluate how CBRs and Adenosine Tri-Phosphate (ATP)-sensitive potassium channels collaborate to affect seizure susceptibility by changing the clonic seizure threshold (CST). We used male Naval Medical Research Institute (NMRI) mice and treated them with the following drugs: cromakalim (a potassium channel opener, 10 μg/kg), glibenclamide (a potassium channel blocker, 0.03 and 1 mg/kg), 0.5 mg/kg of AM-251 (a selective CB1 antagonist), AM-630 (a selective CB2 antagonist), and 0.5, 3, and 10 mg/kg of WIN 55,212-2 (a nonselective agonist of CBRs); and CST was appraised after each type of administration. Also, we evaluated the ATP level of the hippocampus in each treatment to clarify the interaction between the cannabinoid system and potassium channel. Our results showed that administration of WIN 55,212-2 at 10 mg/kg significantly increased CST (P < 0.001). This change could be reversed by using AM-251(P < 0.001) but not AM-630. Also, either cromakalim (10 μg/kg) or glibenclamide (0.03 and 1 mg/kg) could not significantly affect the CST. In addition, glibenclamide (1 mg/kg) could reverse the anticonvulsant effect of WIN 55,212-2 (10 mg/kg) on CST (P < 0.001). However, the anticonvulsant effect was observed when cromakalim (10 μg/kg) was added to WIN 55,212-2 at its subeffective dose (3 mg/kg) in comparison to single-treated animals. Interestingly, we observed that CB1 agonist could significantly decrease ATP level. In conclusion, CB1 agonist accomplishes at least a part of its anticonvulsant actions through ATP-sensitive potassium channels, probably by decreasing the mitochondrial ATP level to open the potassium channel to induce its anticonvulsant effect. Unlabelled Image • Cannabinoid system agonist could increase the seizure threshold. • Anticonvulsant-like effects of cannabinoid receptor agonist are depended on CBR1. • K-channel blocker decreases the anticonvulsant-like effects of cannabinoid receptor agonist. • Cannabinoid receptor agonist increases the anticonvulsant-like effects of K-channel activator. • CBR1 agonist could increase K ATP activator by decreasing mitochondrial ATP level. [ABSTRACT FROM AUTHOR]

We examined the effects of nobiletin, a polymethoxyflavonoid, on the retinal microvascular diameter to determine if they depend on the endothelium and/or smooth muscle to reveal the signaling mechanisms involved in this vasomotor activity. Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Video microscopic techniques recorded diametric responses to nobiletin. The retinal arterioles dilated in a nobiletin concentration-dependent (100 pM-10 μM) manner and decreased by 50% after endothelial removal. The nitric oxide (NO) synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME), reduced nobiletin-induced vasodilation comparable to denudation. Blockade of soluble guanylyl cyclase by 1H-[1,2,4] oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) produced a similar inhibitory effect as that by L-NAME. Nobiletin-induced vasodilation was also inhibited by the nonselective potassium channel inhibitor, tetraethylammonium (TEA), and the voltage-gated K (Kv) inhibitor, 4-aminopyridine. Co-administration of L-NAME and TEA almost eliminated nobiletin-induced vasodilation. Nobiletin elicits both endothelium-dependent and -independent dilation of retinal arterioles mediated by NO release and Kv channel activation, respectively. • Nobiletin, a polymethoxyflavonoid, elicits both endothelium-dependent and -independent dilation of porcine retinal arterioles. • Nobiletin-induced vasodilation is mediated by NO release from the vascular endothelium. • Nobiletin-induced vasodilation is also caused by the activation of Kv channel of the vascular smooth muscle. [ABSTRACT FROM AUTHOR]

SKF-96365 is a TRPC channel antagonist commonly used to characterize the potential functions of TRPC channels in different systems, which was recently reported to induce QTc prolongation on ECG by inhibiting TRPC channels. The present study investigates whether the blockade of cardiac repolarization currents would be involved in the increase of QTc interval. Cardiac repolarization currents were recorded in HEK 293 cells stably expressing human ether-à-go-go-related gene potassium (hERG or hKv11.1) channels, hKCNQ1/hKCNE1 channels ( I Ks ) or hKir2.1 channels and cardiac action potentials were recorded in guinea pig ventricular myocytes using a whole-cell patch technique. The potential effect of SKF-96365 on QT interval was evaluated in ex vivo guinea pig hearts. It was found that SKF-96365 inhibited hERG current in a concentration-dependent manner (IC 50 , 3.4 μM). The hERG mutants S631A in the pore helix and F656 V of the S6 region reduced the inhibitory sensitivity with IC 50 s of 27.4 μM and 11.0 μM, suggesting a channel pore blocker. In addition, this compound inhibited I Ks and hKir2.1currents with IC 50 s of 10.8 and 8.7 μM. SKF-96365 (10 μM) significantly prolonged ventricular APD 90 in guinea pig ventricular myocytes and QTc interval in ex vivo guinea pig hearts. These results indicate that the TRPC channel antagonist SKF-96365 exerts blocking effects on hERG, I Ks , and hKir2.1 channels. Prolongation of ventricular APD and QT interval is related to the inhibition of multiple repolarization potassium currents, especially hERG channels. [ABSTRACT FROM AUTHOR]

Peptides derived from retroviral envelope proteins have been shown to possess a wide range of immunosuppressive and anti-inflammatory activities. We have previously reported identification of such a peptide derived from the envelope protein coded by a human endogenous retrovirus (HERV). In this study, we identify that in vitro the peptide inhibits the K Ca 3.1 potassium channel, a potential target for therapy of immune diseases. We describe in vitro ENV59-GP3 effects with respect to potency of inhibition on K Ca 3.1 channels and calcium influx. Furthermore, we asses in vivo the effect of blocking K Ca 3.1 with ENV59-GP3 peptide or K Ca 3.1-blocker NS6180 on protection against DSS-induced acute colitis. ENV59-GP3 peptide treatment showed reduction of the disease score in the DSS-induced acute colitis mice model, which was comparable to effects of the K Ca 3.1 channel blocker NS6180. Analysis of cytokine production from DSS-mice model treated animals revealed equipotent inhibitory effects of the ENV59-GP3 and NS6180 compounds on the production of IL-6, TNF-α, IL-1β. These findings altogether suggest that ENV59-GP3 functions as a K Ca 3.1 channel inhibitor and underline the implications of using virus derived channel blockers for treatment of autoimmune diseases. Additionally, they open the possibilities whether K Ca 3.1 inhibition is efficacious in patients with inflammatory bowel diseases. • Potassium channels might be regarded as a new group of therapeutic targets for IBD. • Immunosuppressive peptide from human endogenous retroviral envelope protein, ENV59 inhibits K Ca 3.1 potassium channel. • Inhibition of K Ca 3.1 with ENV59-GP3 peptide or K Ca 3.1-blocker NS6180 protects against DSS-induced acute colitis. • Virus derived channel blockers might be effective for ameliorating of autoimmune diseases. [ABSTRACT FROM AUTHOR]

Accurate diagnosis of predisposition to long QT syndrome is crucial for reducing the risk of cardiac arrhythmias. In recent years, drug-induced provocative tests have proved useful to unmask some latent mutations linked to cardiac arrhythmias. In this study we expanded this concept by developing a prototype for a computational provocative screening test to reveal genetic predisposition to acquired long-QT syndrome (aLQTS). We developed a computational approach to reveal the pharmacological properties of I Kr blocking drugs that are most likely to cause aLQTS in the setting of subtle alterations in I Kr channel gating that would be expected to result from benign genetic variants. We used the model to predict the most potentially lethal combinations of kinetic anomalies and drug properties. In doing so, we also implicitly predicted ideal inverse therapeutic properties of K channel openers that would be expected to remedy a specific defect. We systematically performed “ in silico mutagenesis” by altering discrete kinetic transition rates of the Fink et al. Markov model of human I Kr channels, corresponding to activation, inactivation, deactivation and recovery from inactivation of I Kr channels. We then screened and identified the properties of I Kr blockers that caused acquired long QT and therefore unmasked mutant phenotypes for mild, moderate and severe variants. Mutant I Kr channels were incorporated into the O'Hara et al. human ventricular action potential (AP) model and subjected to simulated application of a wide variety of I Kr –drug interactions in order to identify the characteristics that selectively exacerbate the AP duration (APD) differences between wild-type and I Kr mutated cells. Our results show that drugs with disparate affinities to conformation states of the I Kr channel are key to amplify variants underlying susceptibility to acquired long QT syndrome, an effect that is especially pronounced at slow frequencies. Finally, we developed a mathematical formulation of the M54T MiRP1 latent mutation and simulated a provocative test. In this setting, application of dofetilide dramatically amplified the predicted QT interval duration in the M54T hMiRP1 mutation compared to wild-type. [ABSTRACT FROM AUTHOR]

Abstract: Field potential duration (FPD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which can express QT interval in an electrocardiogram, is reported to be a useful tool to predict K+ channel and Ca2+ channel blocker effects on QT interval. However, there is no report showing that this technique can be used to predict multichannel blocker potential for QT prolongation. The aim of this study is to show that FPD from MEA (Multielectrode array) of hiPS-CMs can detect QT prolongation induced by multichannel blockers. hiPS-CMs were seeded onto MEA and FPD was measured for 2min every 10min for 30min after drug exposure for the vehicle and each drug concentration. IKr and IKs blockers concentration-dependently prolonged corrected FPD (FPDc), whereas Ca2+ channel blockers concentration-dependently shortened FPDc. Also, the multichannel blockers Amiodarone, Paroxetine, Terfenadine and Citalopram prolonged FPDc in a concentration dependent manner. Finally, the IKr blockers, Terfenadine and Citalopram, which are reported to cause Torsade de Pointes (TdP) in clinical practice, produced early afterdepolarization (EAD). hiPS-CMs using MEA system and FPDc can predict the effects of drug candidates on QT interval. This study also shows that this assay can help detect EAD for drugs with TdP potential. [Copyright &y& Elsevier]

Highlights: [•] Ctri9577 toxin effectively inhibited Kv4.3 channel currents with IC50 value of 1.34±0.03μM. [•] Ctri9577 toxin was a novel gating modifier affecting gating kinetics of Kv4.3 channels. [•] Ctri9577 toxin interacted with the linker between the S3 and S4 helical domains. [•] Ctri9577 toxin less inhibited the currents of Kv4.1 and Kv4.2 channels. [ABSTRACT FROM AUTHOR]

Highlights: [•] DREAM increases apoptosis in neural cells. [•] DREAM binds hexokinase I in a calcium-dependent manner. [•] DREAM reduces mitochondrial HK I concentrations. [•] DREAM-dependent apoptosis is reduced by overexpression of hexokinase I. [•] DREAM and hexokinase I interact to regulate apoptosis in cells. [ABSTRACT FROM AUTHOR]

Abstract: Scorpion venom contains a group of two-domain peptides that function to block potassium channels or have cytolytic activities. These peptides, whose functions are poorly studied, are named β-KTx or scorpine-like peptides. Ev37, the first identified gene in the Euscorpiidae family, which encoded a novel scorpine-like peptide, was cloned from the venom cDNA library of scorpion Euscorpiops validus. Sequence analysis showed that the mature Ev37 peptide contained 78 amino acid residues, which formed two structural domains: a putative α-helical N-terminus and a C-terminus with the cysteine-stabilized α/β motif. The peptide rEv37 and two truncated peptides representing the individual structural domains (Ev37-N and Ev37-C) were expressed in Escherichia coli and purified for functional study. Unlike classic scorpine-like peptides, rEv37 and truncated peptides showed no cytolytic activity against bacteria or eukaryotic cells. Interestingly, rEv37 selectively inhibited Kv1.3 channel without effectively blocking Kv1.1 and Kv1.2 channels. Neither Ev37-N nor Ev37-C blocked Kv1.3 channel, suggesting that both the N-terminal and C-terminal domain of Ev37 are likely involved in the interaction with Kv1.3 channel. These results not only enrich our knowledge of scorpion toxins from scorpine-like subfamily but also provide a novel template with unique structure for designing new types of selective Kv1.3 blockers. [Copyright &y& Elsevier]

Abstract: Potassium channels are involved in the maintenance of resting membrane potential, control of cardiac and neuronal excitability, neurotransmitters release, muscle contractility and hormone secretion. The Tityus stigmurus scorpion is widely distributed in Northeastern Brazil and known to cause severe human envenomations, inducing pain, hypoesthesia, edema, erythema, paresthesia, headaches and vomiting. Most potassium channel blocking peptides that have been purified from scorpion venoms contain 30–40 amino acids with three or four disulfide bridges. These peptides belong to α-KTx subfamily. On the other hand, the β-KTx subfamily is poorly characterized, though it is very representative in some scorpion venoms. A transcriptomic approach of T. stigmurus scorpions developed by our group revealed the repertoire of possible molecules present in the venom, including many toxins of the β-KTx subfamily. One of the ESTs found, named TSTI0003C has a cDNA sequence of 538bp codifying a mature protein with 47 amino acid residues, corresponding to 5299Da. This β-KTx peptide is a new member of the BmTXKβ-related toxins, and was here named TstKMK. The three-dimensional structure of this potassium channel toxin of the T. stigmurus scorpion was obtained by computational modeling and refined by molecular dynamic simulations. Furthermore, we have made docking simulations using a Shaker kV-1.2 potassium channel from rats as receptor model and proposed which amino acid residues and interactions could be involved in its blockade. [Copyright &y& Elsevier]

Abstract: Stroke causes CNS injury associated with strong fast microglial activation as part of the inflammatory response. In rat models of stroke, sulphonylurea receptor blockade with glibenclamide reduced cerebral edema and infarct volume. We postulated that glibenclamide administered during the early stages of stroke might foster neuroprotective microglial activity through ATP-sensitive potassium (KATP) channel blockade. We found in vitro that BV2 cell line showed upregulated expression of KATP channel subunits in response to pro-inflammatory signals and that glibenclamide increases the reactive morphology of microglia, phagocytic capacity and TNFα release. Moreover, glibenclamide administered to rats 6, 12 and 24h after transient Middle Cerebral Artery occlusion improved neurological outcome and preserved neurons in the lesioned core three days after reperfusion. Immunohistochemistry with specific markers to neuron, astroglia, microglia and lymphocytes showed that resident amoeboid microglia are the main cell population in that necrotic zone. These reactive microglial cells express SUR1, SUR2B and Kir6.2 proteins that assemble in functional KATP channels. These findings provide that evidence for the key role of KATP channels in the control of microglial reactivity are consistent with a microglial effect of glibenclamide into the ischemic brain and suggest a neuroprotective role of microglia in the early stages of stroke. [Copyright &y& Elsevier]

Abstract: We have demonstrated that the natural flavone acacetin selectively inhibits ultra-rapid delayed rectifier potassium current (I Kur) in human atria. However, molecular determinants of this ion channel blocker are unknown. The present study was designed to investigate the molecular determinants underlying the ability of acacetin to block hKv1.5 channels (coding IKur) in human atrial myocytes using the whole-cell patch voltage-clamp technique to record membrane current in HEK 293 cells stably expressing the hKv1.5 gene or transiently expressing mutant hKv1.5 genes generated by site-directed mutagenesis. It was found that acacetin blocked hKv1.5 channels by binding to both closed and open channels. The blockade of hKv1.5 channels by acacetin was use- and frequency-dependent, and the IC50 of acacetin for inhibiting hKv1.5 was 3.5, 3.1, 2.9, 2.1, and 1.7μM, respectively, at 0.2, 0.5, 1, 3, and 4Hz. The mutagenesis study showed that the hKv1.5 mutants V505A, I508A, and V512A in the S6-segment remarkably reduced the channel blocking properties by acacetin (IC50, 29.5μM for V505A, 19.1μM for I508A, and 6.9μM for V512A). These results demonstrate the novel information that acacetin mainly blocks open hKv1.5 channels by binding to their S6 domain. The use- and rate-dependent blocking of hKv1.5 by acacetin is beneficial for anti-atrial fibrillation. [Copyright &y& Elsevier]

Abstract: The human ether-a-go-go-related gene (hERG) channel, a member of a family of voltage-gated potassium (K+) channels, plays a critical role in the repolarization of the cardiac action potential. The reduction of hERG channel activity as a result of adverse drug effects or genetic mutations may cause QT interval prolongation and potentially leads to acquired long QT syndrome. Thus, screening for hERG channel activity is important in drug development. Cardiotoxicity associated with the inhibition of hERG channels by environmental chemicals is also a public health concern. To assess the inhibitory effects of environmental chemicals on hERG channel function, we screened the National Toxicology Program (NTP) collection of 1408 compounds by measuring thallium influx into cells through hERG channels. Seventeen compounds with hERG channel inhibition were identified with IC50 potencies ranging from 0.26 to 22μM. Twelve of these compounds were confirmed as hERG channel blockers in an automated whole cell patch clamp experiment. In addition, we investigated the structure–activity relationship of seven compounds belonging to the quaternary ammonium compound (QAC) series on hERG channel inhibition. Among four active QAC compounds, tetra-n-octylammonium bromide was the most potent with an IC50 value of 260nM in the thallium influx assay and 80nM in the patch clamp assay. The potency of this class of hERG channel inhibitors appears to depend on the number and length of their aliphatic side-chains surrounding the charged nitrogen. Profiling environmental compound libraries for hERG channel inhibition provides information useful in prioritizing these compounds for cardiotoxicity assessment in vivo. [Copyright &y& Elsevier]

Abstract: The goal of this study was to investigate the efflux of K+ from human corneal limbal epithelial cells (HCLE) exposed to ambient levels of UVB, which is known to cause apoptosis, and to examine the effect of K+ channel blockers on loss of potassium induced by UVB. HCLE cells were exposed to 100–200 mJ/cm2 UVB, followed by incubation in culture media with 5.5–100 mM K+, BDS-1, Ba2+ or ouabain. To measure intracellular cations, cells were washed in 280 mM sucrose and lysed in DI water. K+ and Na+ levels in lysates were measured by ion chromatography. HCLE cells showed maximal loss of K+ i 10 min after exposure to UVB and 5.5 mM K+ media, with recovery of normal K+ levels after 90 min. Treatment with 1 μM BDS-1 following UVB exposure reduced the loss of K+ by HCLE cells. Exposure to 0.1–5 mM Ba2+ inhibited UVB-induced K+ loss in a time and dose-dependent manner. These results confirm that blocking K+ channels in HCLE cells exposed to UVB prevents efflux of K+, confirming that UVB activates K+ channels in these cells. Electrophysiology data show that K+ channels remain highly active at least 90 min after UVB exposure. HCLE cells exposed to UVB and incubated in 0.01–1 μM ouabain did not recover from UVB-induced K+ loss. These data suggest that the Na/K pump may act to restore [K+]i to control levels in HCLE cells following UVB exposure and that the pump is not damaged by exposure to UVB. Incubation of HCLE cells exposed to UVB in medium with 25–100 mM K+ media prevented K+ efflux at extracellular concentrations as low as 25 mM (the concentration in tear fluid), maintaining control levels of K+ i. In all experiments inward fluxes and intracellular Na+ levels mirrored K+ changes, albeit at the expected lower concentrations. The prevention of UVB-induced Ki + loss by 25 mM Ko + is consistent with the possible contribution of the relatively high K+ concentration in tears to protection of the corneal epithelium from ambient UVB. [Copyright &y& Elsevier]

Abstract: Multiple sclerosis (MS) is a severely debilitating neurodegenerative diseases marked by progressive demyelination and axonal degeneration in the CNS. Although inflammation is the major pathology of MS, the mechanism by which it occurs is not completely clear. The primary symptoms of MS are movement difficulties caused by conduction block resulting from the demyelination of axons. The possible mechanism of functional loss is believed to be the exposure of potassium channels and increase of outward current leading to conduction failure. 4-Aminopyridine (4-AP), a well-known potassium channel blocker, has been shown to enhance conduction in injured and demyelinated axons. However, 4-AP has a narrow therapeutic range in clinical application. Recently, we developed a new fast potassium channel blocker, 4-aminopyridine-3-methanol (4-AP-3-MeOH). This novel 4-AP derivative is capable of restoring impulse conduction in ex vivo injured spinal cord without compromising the ability of axons to follow multiple stimuli. In the current study, we investigated whether 4-AP-3-MeOH can enhance impulse conduction in an animal model of MS. Our results showed that 4-AP-3-MeOH can significantly increase axonal conduction in ex vivo experimental autoimmune encephalomyelitis mouse spinal cord. [ABSTRACT FROM AUTHOR]

Abstract: Raloxifene is widely used in the treatment of postmenopausal osteoporosis and also has been shown to be cardioprotective. The effect of raloxifene on cardiac ion channels is not fully understood. The present study investigated whether raloxifene could affect the cloned hERG channel (I hERG) and recombinant human cardiac KCNQ1/KCNE1 channel (I Ks) stably expressed in HEK 293 cells using a patch-clamp technique. Raloxifene blocked I hERG with an IC50 of 1.1μM and decreased I Ks (IC50: 4.8μM) without affecting activation kinetics. In addition, raloxifene significantly decreased I Na (IC50: 2.8μM) in guinea pig ventricular myocytes. However, this drug (1μM) did not increase QRS and QTc interval in isolated guinea pig hearts. These results demonstrate that raloxifene, despite its inhibitory action on delayed rectifier potassium currents, does not prolong ECG QTc interval, suggesting that raloxifene is likely a safe selective estrogen receptor modulator with less cardiac toxicity. [ABSTRACT FROM AUTHOR]

Abstract: Besides their role in the generation of action potentials, voltage-gated potassium channels are implicated in cellular processes ranging from cell division to cell death. The K+ channel regulator protein (KCNRG), identified as a putative tumor suppressor, reduces K+ currents through human K+ channels hKv1.1 and hKv1.4 expressed in Xenopus oocytes. Current attenuation requires the presence of the N-terminal T1 Domain and immunoprecipitation experiments suggest association of KCNRG with the N-terminus of the channel. Our data indicates that KCNRG is an ER-associated protein, which we propose regulates Kv1 family channel proteins by retaining a fraction of channels in endomembranes. [Copyright &y& Elsevier]

Inflammation and oxidative stress contribute to the pathophysiology of diabetic neuropathy. According to recent evidence, the modulation of macrophage polarization in peripheral nerves represents a potential therapeutic target for diabetic neuropathy. Xanthine oxidase, which is a form of xanthin oxidoreductase, is the rate-limiting enzyme that catalyzes the degradation of hypoxanthine and xanthine into uric acid. Activation of xanthine oxidase promotes oxidative stress and macrophage activation. A preclinical study reported the beneficial effects of xanthine oxidase inhibitors on peripheral nerve dysfunction in experimental models of diabetes. However, the detailed mechanisms remain unknown. In this study, we examined the effect of the xanthine oxidase inhibitor topiroxostat on macrophage polarization and peripheral neuropathy in an obese diabetic model, db/db mice. First, the effects of xanthine oxidase inhibitors on cultured macrophages and dorsal root ganglion neurons exposed to xanthine oxidase were assessed. Furthermore, five-week-old db/db mice were administered the xanthine oxidase inhibitors topiroxostat [1 mg/kg/day (dbT1) or 2 mg/kg/day (dbT2)] or febuxostat [1 mg/kg (dbF)]. Glucose metabolism and body weight were evaluated during the experimental period. At 4 and 8 weeks of treatment, peripheral nerve functions such as nerve conduction velocities, thermal thresholds and pathology of skin and sciatic nerves were evaluated. The mRNA expression of molecules related to inflammation and oxidative stress was also measured in sciatic nerves. Untreated db/db mice and the nondiabetic db strain (db/m) were studied for comparison. An in vitro study showed that topiroxostat suppressed macrophage activation and proinflammatory but not anti-inflammatory polarization, and prevented the reduction in neurite outgrowth from neurons exposed to xanthine oxidase. Neuropathic changes exemplified by delayed nerve conduction and reduced intraepidermal nerve fiber density developed in db/db mice. These deficits were significantly prevented in the treated group, most potently in dbT2. Protective effects were associated with the suppression of macrophage infiltration, cytokine expression, and oxidative stress in the sciatic nerve and decreased plasma xanthine oxidoreductase activity. Our results revealed the beneficial effects of the xanthine oxidase inhibitor topiroxostat on neuropathy development in a mouse model of type 2 diabetes. The suppression of proinflammatory macrophage activation and oxidative stress-induced damage were suggested to be involved in this process. • Activation of xanthine oxidase evokes reactive oxygen species and inflammation. • Pathogenesis of diabetic neuropathy may include activation of xanthine oxidase. • Inhibition of xanthine oxidase depleted nerve oxidative stress and inflammation. • Extracellular xanthine oxidase inhibited neurite outgrowth in primary neuron culture. • Plasma xanthine oxidase may be a new therapeutic target of diabetic neuropathy. [ABSTRACT FROM AUTHOR]

To examine the contributions of calcium-activated K+ (K Ca) channels and nitric oxide synthase (NOS) to adenosine triphosphate (ATP)-induced cutaneous vasodilation in healthy older adults. In eleven older adults (69 ± 2 years, 5 females), cutaneous vascular conductance, normalized to maximum vasodilation (%CVC max) was assessed at four dorsal forearm skin sites that were continuously perfused with either 1) lactated Ringer solution (Control), 2) 50 mM tetraethylammonium (TEA, K Ca channel blocker), 3) 10 mM N ω-nitro-L-arginine (L-NNA, NOS inhibitor), or 4) combined 50 mM TEA +10 mM L-NNA, via microdialysis. Local skin temperature was fixed at 33 °C at all sites with local heaters throughout the protocol while the cutaneous vasodilator response was assessed during coadministration of ATP (0.03, 0.3, 3, 30, 300 mM; 20 min per dose), followed by 50 mM sodium nitroprusside and local skin heating to 43 °C to achieve maximum vasodilation (20–30 min). Blockade of K Ca channels blunted %CVC max relative to Control from 0.3 to 300 mM ATP (All P < 0.05). A similar response was observed for the combined K Ca channel blockade and NOS inhibition site from 3 to 300 mM ATP (All P < 0.05). Conversely, NOS inhibition alone did not influence %CVC max across all ATP doses (All P > 0.05). In healthy older adults, K Ca channels play an important role in modulating ATP-induced cutaneous vasodilation, while the NOS contribution to this response is negligible. • K Ca channels modulate ATP-mediated cutaneous vasodilation in older adults. • Nitric oxide synthase does not modulate ATP-mediated vasodilation in older adults. • K Ca channel modulation of ATP-mediated vasodilation is NOS-independent. [ABSTRACT FROM AUTHOR]

The voltage-gated potassium channel Kv4.2 is a critical regulator of dendritic excitability in the hippocampus and is crucial for dendritic signal integration. Kv4.2 mRNA and protein expression as well as function are reduced in several genetic and pharmacologically induced rodent models of epilepsy and autism. It is not known, however, whether reduced Kv4.2 is just an epiphenomenon or a disease-contributing cause of neuronal hyperexcitability and behavioral impairments in these neurological disorders. To address this question, we used male and female mice heterozygous for a Kv.2 deletion and adult-onset manipulation of hippocampal Kv4.2 expression in male mice to assess the role of Kv4.2 in regulating neuronal network excitability, morphology and anxiety-related behaviors. We observed a reduction in dendritic spine density and reduced proportions of thin and stubby spines but no changes in anxiety, overall activity, or retention of conditioned freezing memory in Kv4.2 heterozygous mice compared with wildtype littermates. Using EEG analyses, we showed elevated theta power and increased spike frequency in Kv4.2 heterozygous mice under basal conditions. In addition, the latency to onset of kainic acid-induced seizures was significantly shortened in Kv4.2 heterozygous mice compared with wildtype littermates, which was accompanied by a significant increase in theta power. By contrast, overexpressing Kv4.2 in wildtype mice through intrahippocampal injection of Kv4.2-expressing lentivirus delayed seizure onset and reduced EEG power. These results suggest that Kv4.2 is an important regulator of neuronal network excitability and dendritic spine morphology, but not anxiety-related behaviors. In the future, manipulation of Kv4.2 expression could be used to alter seizure susceptibility in epilepsy. Unlabelled Image • Kv4.2 heterozygous mice have reduced dendritic spine density and altered morphology. • Kv4.2 heterozygous mice do not display autistic-like or anxiety-related phenotypes. • EEG analyses of Kv4.2 heterozygous mice suggest neuronal network hyperexcitability. • Reduced Kv4.2 decreases latency to kainic acid-induced seizure onset. • Lentiviral Kv4.2 overexpression increases latency to seizure onset. [ABSTRACT FROM AUTHOR]

Environmental toxicants have the potential to contribute to the pathophysiology of multiple complex diseases, but the underlying mechanisms remain obscure. One such toxicant is the widely used fungicide ziram, a dithiocarbamate known to have neurotoxic effects and to increase the risk of Parkinson's disease. We have used Drosophila melanogaster as an unbiased discovery tool to identify novel molecular pathways by which ziram may disrupt neuronal function. Consistent with previous results in mammalian cells, we find that ziram increases the probability of synaptic vesicle release by dysregulation of the ubiquitin signaling system. In addition, we find that ziram increases neuronal excitability. Using a combination of live imaging and electrophysiology, we find that ziram increases excitability in both aminergic and glutamatergic neurons. This increased excitability is phenocopied and occluded by null mutant animals of the ether a-go-go (eag) potassium channel. A pharmacological inhibitor of the temperature sensitive hERG (human ether-a-go-go related gene) phenocopies the excitability effects of ziram but only at elevated temperatures. s eizure (sei), a fly ortholog of hERG, is thus another candidate target of ziram. Taken together, the eag family of potassium channels emerges as a candidate for mediating some of the toxic effects of ziram. We propose that ziram may contribute to the risk of complex human diseases by blockade of human eag and sei orthologs, such as hERG. Unlabelled Image • The neurotoxicant ziram disrupts neuronal activity via two distinct mechanisms. • Mechanism 1: increased synaptic release by inhibition of the ubiquitin signaling system. • Mechanism 2: increased excitability by inhibition of the eag family of K channels. • Both pathways may contribute to human disease. • Environmental toxicants can identify novel disease pathways. [ABSTRACT FROM AUTHOR]

Previous studies indicate that sex-related differences exist in the regulation of cutaneous vasodilation, however, the mechanisms remain unresolved. We assessed if sex-differences in young adults exist for cholinergic, nicotinic, and β-adrenergic cutaneous vasodilation with a focus on nitric oxide synthase (NOS), cyclooxygenase (COX), and K+ channel mechanisms. In twelve young men and thirteen young women, four intradermal forearm skin sites were perfused with the following: 1) lactated Ringer's solution (control), 2) 10 mM N ω-nitro- l -arginine, a non-selective NOS inhibitor, 3) 10 mM ketorolac, a non-selective COX inhibitor, or 4) 50 mM BaCl 2 , a nonspecific K+ channel blocker. At all four sites, cutaneous vasodilation was induced by 1) 10 mM nicotine, a nicotinic receptor agonist, 2) 100 μM isoproterenol, a nonselective β-adrenergic receptor agonist, and 3) 2 mM and 2000 mM acetylcholine, an acetylcholine receptor agonist. Nicotine and isoproterenol were administered for 3 min, whereas each acetylcholine dose was administered for 25 min. Regardless of treatment site, cutaneous vasodilation in response to nicotine and a high dose of acetylcholine (2000 mM) were lower in women than men. By contrast, isoproterenol induced cutaneous vasodilation was greater in women vs. men. Irrespective of sex, NOS inhibition or K+ channel blockade attenuated isoproterenol-mediated cutaneous vasodilation, whereas K+ channel blockade decreased nicotine-induced cutaneous vasodilation. Taken together, our findings indicate that while the mechanisms underlying cutaneous vasodilation are comparable between young men and women, sex-related differences in the magnitude of cutaneous vasodilation do exist and this response differs as a function of the receptor agonist. • We assessed if sex-differences in young adults exist for cholinergic, nicotinic, and β-adrenergic cutaneous vasodilation • Mechanisms underlying cutaneous vasodilation are not different between young women and young men • Sex modulates the magnitude of cutaneous vasodilation and this response differs as a function of the receptor agonist. [ABSTRACT FROM AUTHOR]

• Multifunctional compounds are smart strategy for the treatment of multifactorial diseases. • Benzothiazole derivatives were evaluated for their UV-filtering, antioxidant, antifungal and antiproliferative activities. • Compounds 4g and 4k were potential multifunctional compounds. Oxidative stress is the product or aetiology of various multifactorial diseases; on the other hand, the development of multifunctional compounds is a recognized strategy for the control of complex diseases. To this end, a series of benzothiazole derivatives was synthesized and evaluated for their multifunctional effectiveness as antioxidant, sunscreen (filter), antifungal and antiproliferative agents. Compounds were easily synthesized via condensation reaction between 2-aminothiophenols and different benzaldehydes. SAR study, particularly in position 2 and 6 of benzothiazoles, led to the identification of 4g and 4k as very interesting potential compounds for the design of multifunctional drugs. In particular, compound 4g is the best blocker of hERG potassium channels expressed in HEK 293 cells exhibiting 60.32% inhibition with IC 50 = 4.79 μM. [ABSTRACT FROM AUTHOR]

On two rat cell lines, pheochromocytoma PC12 and ascites hepatoma AS-30D, and on rat liver mitochondria we studied action of paxilline (lipophilic mycotoxin from fungus Penicillium paxilli which is blocker of large-conductance potassium channels) against harmful effects of Cd(II) - one of the most dangerous toxic metals and environmental pollutants. We investigated an influence of paxilline on cell viability and mitochondrial function in the presence and in the absence of Cd2+. As found, paxilline protected partially from the Cd2+-induced cytotoxicity, namely taken in concentration of 1 μM it decreased the Cd2+-induced cell necrosis in average by 10–14 or 13–23% for AS-30D and PC12 cells, respectively. Nevertheless, paxilline did not affect the Cd2+-induced apoptosis of AS-30D cells. The alleviating concentration of paxilline reduced an intracellular production of reactive oxygen species (ROS) in PC12 cells intoxicated by Cd2+ and enhanced the ROS production in control AS-30D cells; however, it weakly affected mitochondrial membrane potential of the cells in the absence and in the presence of Cd2+. The ameliorative concentration of paxilline decreased the maximal respiration rates of control cells of both types after short-term (3–5 h) treatment with it while the rates reached their control levels after long-term (24–48 h) incubation with the drug. Paxilline was not protective against the Cd2+-induced membrane permeability and respiration rate changes in isolated rat liver mitochondria. As result, the mitochondrial electron transport chain was concluded to contribute in the mitigating effect of paxilline against the Cd2+-produced cell injury. • Paxilline attenuated Cd2+-produced toxic effects. • Paxilline decreased Cd2+-induced necrosis of both AS-30D and PC12 cells. • Paxilline reduced ROS formation in PC12 cells treated by Cd2+ and enhanced it in untreated AS-30D cells. • Paxilline produced reversible decline of mitoETC activity that was the early cellular effect of the drug. • mitoETC was proposed to contribute in the protective action of paxilline. [ABSTRACT FROM AUTHOR]

• Gap junction blocker carbenoxolone strongly inhibited medaka ovulation. • Carbenoxolone specifically inhibited mmp15/Mmp15 expression in ovulating follicles. • Carbenoxolone inhibited the follicular expression of both Cdk9 and Ccni mRNA. • Carbenoxolone inhibited Pgr phosphorylation. • Connexins 34.5 and 35.4 were dominantly expressed in the ovulating follicles. Using medaka, we found that in vitro follicle ovulation, but not germinal vesicle breakdown, was inhibited by three gap junction blockers, carbenoxolone, mefloquine, and flufenamic acid. The blockers specifically inhibited follicular expression of matrix metalloproteinase-15 mRNA and the protein (mmp15 /Mmp15), a protease indispensable for medaka ovulation, indicating that gap junctional communication may be required for successful ovulation and mmp15 /Mmp15 expression. Further experiments using carbenoxolone as the representative of the gap junction blockers showed that expression of nuclear progestin receptor (Pgr), a transcription factor required for mmp15 expression, was not affected by carbenoxolone treatment, but the formation of phosphorylated Pgr was considerably suppressed. Carbenoxolone treatment caused a decrease in the Pgr binding to the promoter region of mmp15. mRNA expression of cyclin-dependent protein kinase-9 (cdk9) and cyclin I (ccni) , whose translation products are demonstrated to be involved in Pgr phosphorylation in the medaka ovulating follicles, was suppressed by carbenoxolone treatment. Transcripts of connexin 34.5 (cx34.5) and connexin 35.4 (cx35.4) were dominantly expressed in the follicle cells of ovulating follicles. The results indicate that gap junctional communication plays an important role in medaka ovulation. [ABSTRACT FROM AUTHOR]

The voltage-gated potassium (Kv) channel blockers tetraethylammonium (TEA) and 4-aminopyridine (4-AP) have shown beneficial effects on some neurological disorders. But their involvements in diabetes-associated cognitive dysfunction are still unknown. The present study aims to investigate whether the blockade of Kv channels by TEA and 4-AP alleviate cognitive decline in diabetes. In vivo, the effects of TEA and 4-AP (5 mg/kg body weight per day, 1 mg/kg body weight per day intraperitoneal injected for 4 weeks, respectively) were investigated in streptozotocin-induced C57BL/6 diabetic mice. In vitro study, we investigated the effects of TEA and 4-AP on the high glucose (HG) -stimulated primary cortical neurons. The results showed that TEA and 4-AP ameliorated the cognitive decline of diabetic mice in the Morris water maze test, improved the ultrastructure of pancreatic β cells, hippocampal neurons and synapses, decreased oxidative stress, modulated apoptosis-related proteins, and activated phosphatidylinositol 3-kinase (PI3K)/ Protein kinase-B (PKB or Akt) signaling pathway. In the HG-stimulated primary cultured cortical neurons, TEA and 4-AP increased the cell viability, decreased oxidative stress; prevented apoptosis and activated PI3K/Akt signaling pathway. Additionally, the PI3K inhibitor LY294002 partially abolished the effects of TEA and 4-AP. These findings indicate that the blockade of Kv channels by TEA and 4-AP ameliorates the diabetes-associated cognitive dysfunction via PI3K/Akt pathway, suggesting that targeting Kv channels could be a promising strategy for the treatments of cognitive impairments in diabetes. • TEA and 4-AP attenuated cognitive decline in STZ-induced diabetic mice. • TEA and 4-AP exerted neuroprotective effects in vivo and in vitro. • Blocking Kv channel alleviates diabetic cognitive dysfunction via PI3K/Akt pathway. • Kv channel blockers are promising therapeutics for diabetic cognitive impairment. [ABSTRACT FROM AUTHOR]

Objective: To observe the cardiovascular functions of hydrogen sulfide (H2S) within the caudal ventrolateral medulla (CVLM) and to explore its mechanisms in rats. Methods: Sixty-seven male SD rats were employed in present study. Among them, 34 rats were randomized into control group (aCSF, 100 nL, n =6), NaHS group (sodium hydrosulfide, NaHS, 2, 20 or 200pmol, n =7 for each dose), S-ademetionine (SAM, 10nmol, n =7). In 33 rats, hydroxylamine (HA, n =7), kynurenic acid (KYN, n =7) or glibenclamide (GLI, n =7) were prior applied before NaHS (20pmol) was microinjected into the CVLM. Results: Unilateral microinjection of NaHS (2 to 200pmol), a H2S donor, into the CVLM caused the transient and dose-dependent hypotension and bradycardia (P <0.05). Microinjection of CBS allosteric activator S-ademetionine (SAM) into the CVLM also produced significant decreases in BP and HR, which was very similar to those of NaHS. Pretreatment with hydroxylamine, a CBS inhibitor, failed to affect the cardiovascular functions of intra-NTS NaHS. However, pretreatment with kynurenic acid (KYN, 5nmol) or glibenclamide (Gli, 10nmol), a K ATP channel blocker, eliminated the on BP responses by 40% and 69.5%, HR responses 33.3% and 63.6%, respectively, of intra-CVLM NaHS (20pmol). Conclusion: These data support the hypothesis that endogenous H2S produces cardiovascular inhibition functions in the CVLM, mainly mediated by K ATP channels opening mechanism. [Copyright &y& Elsevier]

Background: Ischemia-reperfusion injury (IRI) is responsible for primary liver dysfunction and failure after transplantation. The mitochondrial pathway appears to be involved in liver ischemia-reperfusion injury. Mitochondrial ATP-sensitive K (mitoKATP) channels play a central role in protecting the heart from injury in ischemic preconditioning. The selective mitoKATP channel agonist diazoxide potently reduced mitochondrial injury by preventing cytochrome c loss from the intermembrane space. Therefore, this study sought to determine whether diazoxide can attenuate ischemia-reperfusion injury induced by orthotopic liver transplantation (OLT) in mice. Furthermore, it was found that up-regulation of the Bcl-2 gene is a mechanism of diazoxide cytoprotection. Materials and Methods: Donors were treated with diazoxide, Bcl-2 siRNA, or diazoxide + Bcl-2 siRNA and vehicle 10 min or 24 h before liver harvesting. Liver grafts were then orthotopically transplanted into their corresponding recipients. Results: Liver injury, as judged by transaminase level and histologic examination, was significantly lower in the diazoxide group compared with vehicle controls. The percentage of apoptotic cells and the amount of cytochrome c in the cytosol 6 h after transplant were also markedly reduced in diazoxide-treated grafts compared with vehicle-treated controls. Diazoxide notably up-regulated expression of Bcl-2, while siRNA knockdown of Bcl-2 abolished the cytoprotective effects of diazoxide. Conclusions: Diazoxide attenuated graft injury after mouse liver transplantation. One mechanism of diazoxide protection involves the induction of Bcl-2, an anti-apoptotic protein. Diazoxide might be useful clinically in hepatic surgery and transplantation. [ABSTRACT FROM AUTHOR]