Kv Currents Research Articles

Protriptyline, a tricyclic antidepressant, inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

In this study, we explore the inhibitory effects of protriptyline, a tricyclic antidepressant drug, on voltage-dependent K+ (Kv) channels of rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Protriptyline inhibited the vascular Kv current in a concentration-dependent manner, with an IC50 value of 5.05 ± 0.97μM and a Hill coefficient of 0.73 ± 0.04. Protriptyline did not affect the steady-state activation kinetics. However, the drug shifted the steady-state inactivation curve to the left, suggesting that protriptyline inhibited the Kv channels by changing their voltage sensitivity. Application of 20 repetitive train pulses (1 or 2Hz) progressively increased the protriptyline-induced inhibition of the Kv current, suggesting that protriptyline inhibited Kv channels in a use (state)-dependent manner. The extent of Kv current inhibition by protriptyline was similar during the first, second, and third step pulses. These results suggest that protriptyline-induced inhibition of the Kv current mainly occurs principally in the closed state. The increase in the inactivation recovery time constant in the presence of protriptyline also supported use (state)-dependent inhibition of Kv channels by the drug. In the presence of the Kv1.5 inhibitor, protriptyline did not induce further inhibition of the Kv channels. However, pretreatment with a Kv2.1 or Kv7 inhibitor induced further inhibition of Kv current to a similar extent to that observed with protriptyline alone. Thus, we conclude that protriptyline inhibits the vascular Kv channels in a concentration- and use-dependent manner by changing their gating properties. Furthermore, protriptyline-induced inhibition of Kv channels mainly involves the Kv1.5.

Micro-computed tomography images of lung adenocarcinoma: detection of lepidic growth patterns.

ABSTRACTMicro-computed tomography (µCT) provides extremely high-resolution images of samples and can be employed as a non-destructive inspection tool. Using µCT, we can obtain images comparable with microscopic images. In this work, we have attempted to take high-resolution images of the human lung using µCT. Compared to clinical high-resolution computed tomography (HRCT) images of living body (in-vivo imaging), we can obtain extremely high-resolution images by µCT of ex-vivo tissues (resected lungs) as three-dimensional data. The purpose of this study was to distinguish between areas of normal lung and lung cancer by µCT images in order to study the feasibility of cancer diagnosis using this novel radiological image modality. Ten resected human lungs containing primary cancer were fixed by Heitzman’s methods to obtain high-resolution µCT images. After fixation of the lung, images of the specimens were taken by µCT between January 2016 and November 2017. The imaging conditions were tube voltage: 90 kV and tube current: 110 µA. To compare details of images gained by conventional HRCT and µCT, we measured the thickness of the alveolar walls of the normal lung area and the cancer area of which alveoli might be replaced by tumor cells, and compared their appearance by means of histopathological images. All the nodules were diagnosed as adenocarcinoma. The median whole tumor size was 18 mm (9 mm–24 mm). Each specimen was clearly divided into areas of normal alveolar wall and of thickened alveolar wall on µCT ‘visually’. Median thickness of alveolar walls of the normal lung was 0.037 mm (0.034 mm–0.048 mm), and that of the cancer area was 0.084 mm (0.074 mm–0.094 mm); there was a statistically significant difference between both thicknesses by Student’s t-test (P < 0.01). The area of thickened alveolar walls on µCT corresponded well with the area of microscopically lepidic growth patterns of adenocarcinoma. We found that µCT images could be correctly divided by alveolar walls into normal lung area and lung cancer area. Further detailed investigations with regard to µCT are needed to make comparable histological diagnoses using µCT images with conventional microscopic methods of pathological diagnoses.

Cysteine-modification of Kv7 Channels as Analgesic Mechanism of Action of Acetaminophen

N-acetyl-p-benzoquinone imine (NAPQI), an active metabolite of acetaminophen, decreases excitability in first- and second-order neurons of the nociceptive pathway, which involves an activation of Kv7 channels. NAPQI modifies cysteine residues and both alkylation and oxidative modification of cysteine residues in Kv7 channels were shown to increase M-currents. The aim of the study is to determine, if modification of cysteine residues in Kv7 channels by NAPQI is responsible for the reported increase of Kv7 currents. Heterologously expressed Kv7.1 to Kv7.5 channels were recorded in the perforated voltage clamp mode and clamped to −30 mV. Once every 15 s, the cells were hyperpolarized to −80 mV to elicit a deactivation current. Activation curves were recorded between −150 mV and +50 mV in 10 mV increments. Current levels at +60 mV were evaluated. Activation curves were recorded before, after three and ten minutes of NAPQI application. The effects of NAPQI were tested on wt and mutated versions of the respective Kv7 channel subtypes. While currents through Kv7.3 channels remained unchanged and Kv7.1-currents were reduced, deactivation currents through heterologously expressed Kv7.2, Kv7.4 and Kv7.5 channels increased about two-fold after ten minutes of NAPQI application. All currents remained unchanged during a subsequent five-minute wash out phase. Maximal current levels of Kv7.2, 7.4, and Kv7.5 homomeric channels were increased but only Kv7.2 and Kv7.4 also showed a left shift in half-maximal channel activation. A triple-cysteine stretch in the S2-S3 linker region was identified as responsible for the NAPQI-mediated increase of Kv7 currents.This module of three subsequent cysteine residues was previously shown to mediate the current increase induced by oxidative modification. We conclude that covalent modification of this stretch of three cysteine residues is responsible for the increase of Kv7 currents in response to NAPQI application.

The muscarinic receptor antagonist tolterodine inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

We explored the effect of the muscarinic receptor antagonist tolterodine on voltage-dependent K+ (Kv) channels using the patch-clamp technique in coronary arterial smooth muscle cells freshly isolated from rabbits. Tolterodine inhibited Kv channels in a concentration-dependent manner, with an IC50 of 1.71 ± 0.33 μM and Hill coefficient of 0.69 ± 0.03. Tolterodine accelerated the decay rate of Kv channel inactivation. The apparent rate constants of association and dissociation for tolterodine were 1.79 ± 0.13 μM−1s−1, and 3.13 ± 0.96 s−1, respectively. Although 3 μM tolterodine had no effect on the steady-state activation of the Kv current, it shifted the steady-state inactivation curve towards a negative potential. Application of consecutive train steps (1 or 2 Hz) progressively decreased the Kv current and promoted its inhibition. Furthermore, the recovery time constant was augmented in the presence of tolterodine, indicating that tolterodine-induced Kv channel blockade is use (state) dependent. Pretreatment with inhibitors of the Kv1.5, Kv2.1, and Kv7 subtypes (DPO-1, guangxitoxin, and linopirdine) partially reduced the inhibitory effect of tolterodine on Kv channels. The alternative muscarinic receptor antagonist atropine did not inhibit the Kv current nor influence tolterodine-induced inhibition of the Kv current. Tolterodine induced vasoconstriction and membrane depolarization. Based on these results, we conclude that tolterodine inhibits Kv channels in concentration-, time-, and use (state)-dependent manners, irrespective of its antagonism of muscarinic receptors.

A CO2/O2 Mixed Gas DC Circuit Breaker With Superconducting Fault Current-Limiting Technology

Researchers have investigated the interruption properties of CO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> gas, including CO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> and O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> mixed gas. However, there has been no research on the passive resonance DC interrupting characteristics of CO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> /O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> mixed gas. The objective of this paper is to learn the passive resonance DC interrupting properties of CO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> /O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> mixed gas with superconducting fault current-limiting technology. A proposed passive resonance DCCB that includes a superconducting current-limiting module (SCLM) and a DC interrupting module (DCIM) connected in series was tested for DC voltages of 1.3, 3 and 10 kV and DC currents from 50 A to 10 kA. The experimental results show that the DCCB could successfully interrupt a 10 kA current in a 10 kV DC system. The response time of the DCCB was only 0.3 ms. After interruption, there was no overvoltage. The arcing time and the arc voltage of the SF <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">6</sub> gas and the CO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> /O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> mixed gas were also compared experimentally. Experimental results show that CO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> /O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> mixed gas may be a good substitute for the SF <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">6</sub> gas in passive resonance interruption. The CO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> /O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> mixed gas passive resonance DCCB combined with the SCLM is an effective way to interrupt high short-circuit fault currents in medium-voltage and high-voltage DC systems.

Propofol inhibits stromatoxin-1-sensitive voltage-dependent K+ channels in pancreatic β-cells and enhances insulin secretion.

BackgroundProper glycemic control is an important goal of critical care medicine, including perioperative patient care that can influence patients’ prognosis. Insulin secretion from pancreatic β-cells is generally assumed to play a critical role in glycemic control in response to an elevated blood glucose concentration. Many animal and human studies have demonstrated that perioperative drugs, including volatile anesthetics, have an impact on glucose-stimulated insulin secretion (GSIS). However, the effects of the intravenous anesthetic propofol on glucose metabolism and insulin sensitivity are largely unknown at present.MethodsThe effect of propofol on insulin secretion under low glucose or high glucose was examined in mouse MIN6 cells, rat INS-1 cells, and mouse pancreatic β-cells/islets. Cellular oxygen or energy metabolism was measured by Extracellular Flux Analyzer. Expression of glucose transporter 2 (GLUT2), potassium channels, and insulin mRNA was assessed by qRT-PCR. Protein expression of voltage-dependent potassium channels (Kv2) was also assessed by immunoblot. Propofol’s effects on potassium channels including stromatoxin-1-sensitive Kv channels and cellular oxygen and energy metabolisms were also examined.ResultsWe showed that propofol, at clinically relevant doses, facilitates insulin secretion under low glucose conditions and GSIS in MIN6, INS-1 cells, and pancreatic β-cells/islets. Propofol did not affect intracellular ATP or ADP concentrations and cellular oxygen or energy metabolism. The mRNA expression of GLUT2 and channels including the voltage-dependent calcium channels Cav1.2, Kir6.2, and SUR1 subunit of KATP, and Kv2 were not affected by glucose or propofol. Finally, we demonstrated that propofol specifically blocks Kv currents in β-cells, resulting in insulin secretion in the presence of glucose.ConclusionsOur data support the hypothesis that glucose induces membrane depolarization at the distal site, leading to KATP channel closure, and that the closure of Kv channels by propofol depolarization in β-cells enhances Ca2+ entry, leading to insulin secretion. Because its activity is dependent on GSIS, propofol and its derivatives are potential compounds that enhance and initiate β-cell electrical activity.

GLP-1 Relaxes Rat Coronary Arteries by Enhancing ATP-Sensitive Potassium Channel Currents.

GLP-1 is a new type of antidiabetic agent that possesses many beneficial effects. Although its cardiovascular actions have been widely examined, little is known about GLP-1's effects on the rat coronary artery (RCA) or about the mechanisms underpinning these effects. Here, we report that GLP-1 inhibits depolarization- or thromboxane receptor agonist (U46619)-induced RCA contraction in a dosage-dependent manner. Vasorelaxation was attenuated by denuding the endothelium, L-NAME (nitric oxide synthase inhibitor), and glyburide (KATP channel blocker) but was not affected by indomethacin (cyclooxygenase inhibitor), iberiotoxin [Ca2+-activated K+ channel (KCa) blocker], or 4-aminopyridine (KV channel blocker). Furthermore, GLP-1 increased outward K+ currents by enhancing the KATP channel in rat coronary arterial smooth muscle cells (RCASMCs). These results show that GLP-1 is an endothelial-dependent vasospasmolytic agent in the RCA and imply that the relaxant effect is regulated by enhancing KATP rather than KV or KCa currents in RCASMCs.

AGEs impair Kv channel-mediated vasodilation of coronary arteries by activating the NF-κB signaling pathway in ZDF rats

Excessive formation of advanced glycation end products (AGEs) impairs voltage-gated potassium (Kv) channels in rat coronary artery smooth muscle cells (CSMCs), resulting in weakened Kv-mediated coronary vasodilation. We hypothesized that induction of the nuclear factor-κB (NF-κB) signaling pathway by AGEs plays a significant role in the regulation of Kv channel-mediated vasodilation in Zucker diabetic fatty (ZDF) rats. Assays of mRNA transcripts, protein expression, and intracellular localization as well as patch-clamp experiments in cultured CSMCs revealed that AGEs significantly induced activation of the NF-κB signaling pathway, reduced Kv1.2/1.5 expression, and inhibited Kv currents. In addition, silencing of the receptor for AGEs (RAGE) or p65 with siRNA and treatment with alagrebrium (ALA) or pyrrolidine dithiocarbamate (PDTC) alleviated the AGE-induced impairment of Kv channels in CSMCs. Compared with Zucker lean (ZL) rats, the amount of AGEs, RAGE protein expression, and NF-κB activity in coronary arteries were higher in ZDF rats; whereas Kv1.2/1.5 expression was significantly lower in ZDF rats. Reduced Kv1.2/1.5 expression in coronary arteries and impaired Kv-mediated coronary relaxation tested by wire myography in ZDF rats were markedly improved by treatment with aminoguanidine (AG), ALA, or PDTC. These effects were accompanied by diminished NF-κB activity, inflammation, and oxidative stress. Taken together, these results indicate that an increased interaction between AGEs and RAGE in diabetic rats leads to impaired Kv channel-mediated coronary vasodilation. Moreover, activation of the NF-κB signaling pathway and a subsequent increase of inflammation and oxidative stress may play an important role in AGE-induced impairment of coronary vasodilation in diabetes.

Crosstalk in the non-classical signal transduction of testosterone and retinol in immature rat testes

This study aimed to investigate the effects of the interaction between testosterone and retinol on the rapid responses of cultured Sertoli cells obtained from 10-day-old immature rat testes. Non-classical actions of testosterone and retinol were investigated, and the activities of L-type voltage-dependent calcium channels (L-VDCC) and voltage-dependent potassium channels (Kv) were determined by measuring 45Ca2+ influx in whole testis. Additionally, the effects of testosterone and retinol on these channels were studied in primary culture of Sertoli cells using the patch-clamp technique. 45Ca2+ influx was used to observe a dose–response curve on tissues treated with retinol and/or testosterone for 2 min (10−12, 10−9 and 10−6 M and 10−9 and 10−6 M), and a concentration of 10−6 M was selected to investigate the mechanism of action of testosterone and retinol on rapid responses. Participation of the L-VDCC and Kv channels was investigated using nifedipine and tetraethylammonium chloride (TEA) inhibitors, respectively. Both, testosterone and retinol act through non-classical mechanisms, stimulating 45Ca2+ influx in immature rat testes. The response to testosterone was abolished by nifedipine and TEA, whereas the effects of retinol were partially blocked by nifedipine and completely inhibited by TEA. Retinol amplified the testosterone-induced effect on 45Ca2+ influx in the testes, suggesting a crosstalk between rapid responses (calcium influx) and cell repolarization via activation of Kv channels. Whole-cell electrophysiology data demonstrated that testosterone and retinol increased voltage-dependent potassium currents (Kv) in Sertoli cells; inhibition of these responses by TEA confirmed the involvement of TEA-sensitive K+ channels in these effects. Taken together, we demonstrate, for the first time, crosstalk between testosterone and retinol that is mediated by a non-classical mechanism involving the L-VDCC-triggered cell depolarization and activation of repolarization by Kv currents in Sertoli cells. These ionic modulations play a physiological role in Sertoli cells and male fertility via stimulation of secretory activities.

First report on BaltCRP, a cysteine-rich secretory protein (CRISP) from Bothrops alternatus venom: Effects on potassium channels and inflammatory processes

CRISPs represent a family of cysteine-rich secretory proteins with molecular mass between 20 and 30 kDa and a highly conserved specific pattern of 16 cysteine residues. In this work, we isolated and characterized a novel CRISP from Bothrops alternatus venom, named BaltCRP, also evaluating its effects on different isoforms of potassium channels (Kv1.1; Kv1.2; Kv1.3; Kv1.4; Kv1.5; Kv2.1; Kv10.1 and Shaker) and on inflammatory processes in vivo. This toxin has a molecular mass of 24.4 kDa and pI around 7.8. Electrophysiological experiments using voltage clamp techniques showed that BaltCRP can affect the currents of Kv1.1; Kv1.3; Kv2.1 and Shaker channels. In addition, BaltCRP induced inflammatory responses characterized by an increase of leukocytes in the peritoneal cavity of mice, also stimulating the production of mediators such IL-6, IL-1β, IL-10, PGE2, PGD2, LTB4 and CysLTs. Altogether, these results demonstrated that BaltCRP can help understand the biological effects evoked by snake venom CRISPs, which could eventually lead to the development of new molecules with therapeutic potential.

The anticholinergic drug oxybutynin inhibits voltage-dependent K+ channels in coronary arterial smooth muscle cells.

This study demonstrates the inhibitory effect of anticholinergic drug oxybutynin on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Oxybutynin inhibited vascular Kv channels in a concentration-dependent manner, with an IC50 value of 11.51±0.38μmol/L and a Hill coefficient (n) of 2.25±0.12. Application of oxybutynin shifted the activation curve to the right and the inactivation curve to the left. Pretreatment with the Kv1.5 subtype inhibitor DPO-1 and the Kv2.1 subtype inhibitor guangxitoxin suppressed the oxybutynin-induced inhibition of the Kv current. However, application of the Kv7 subtype inhibitor linopirdine did not affect the inhibition by oxybutynin of the Kv current. The anticholinergic drug atropine did not inhibit the Kv current nor influence oxybutynin-induced inhibition of the Kv current. From these results, we concluded that oxybutynin inhibited the vascular Kv current in a concentration-dependent manner by influencing the steady-state activation and inactivation curves independent of its anticholinergic effect.

Inhibition of Kv2.1 Potassium Channels by MiDCA1, A Pre-Synaptically Active PLA2-Type Toxin from Micrurus dumerilii carinicauda Coral Snake Venom.

MiDCA1, a phospholipase A2 (PLA2) neurotoxin isolated from Micrurus dumerilii carinicauda coral snake venom, inhibited a major component of voltage-activated potassium (Kv) currents (41 ± 3% inhibition with 1 μM toxin) in mouse cultured dorsal root ganglion (DRG) neurons. In addition, the selective Kv2.1 channel blocker guangxitoxin (GxTx-1E) and MiDCA1 competitively inhibited the outward potassium current in DRG neurons. MiDCA1 (1 µM) reversibly inhibited the Kv2.1 current by 55 ± 8.9% in a Xenopus oocyte heterologous system. The toxin showed selectivity for Kv2.1 channels over all the other Kv channels tested in this study. We propose that Kv2.1 channel blockade by MiDCA1 underlies the toxin’s action on acetylcholine release at mammalian neuromuscular junctions.

Contribution of DNMT1 to Neuropathic Pain Genesis Partially through Epigenetically Repressing Kcna2 in Primary Afferent Neurons.

Expressional changes of pain-associated genes in primary sensory neurons of DRG are critical for neuropathic pain genesis. DNA methyltransferase (DNMT)-triggered DNA methylation silences gene expression. We show here that DNMT1, a canonical maintenance methyltransferase, acts as the de novo DNMT and is required for neuropathic pain genesis likely through repressing at least DRG Kcna2 gene expression in male mice. Peripheral nerve injury upregulated DNMT1 expression in the injured DRG through the transcription factor cAMP response element binding protein-triggered transcriptional activation of Dnmt1 gene. Blocking this upregulation prevented nerve injury-induced DNA methylation within the promoter and 5'-untranslated region of Kcna2 gene, rescued Kcna2 expression and total Kv current, attenuated hyperexcitability in the injured DRG neurons, and alleviated nerve injury-induced pain hypersensitivities. Given that Kcna2 is a key player in neuropathic pain, our findings suggest that DRG DNMT1 may be a potential target for neuropathic pain management.SIGNIFICANCE STATEMENT In the present study, we reported that DNMT1, a canonical DNA maintenance methyltransferase, is upregulated via the activation of the transcription factor CREB in the injured DRG after peripheral nerve injury. This upregulation was responsible for nerve injury-induced de novo DNA methylation within the promoter and 5'-untranslated region of the Kcna2 gene, reductions in Kcna2 expression and Kv current and increases in neuronal excitability in the injured DRG. Since pharmacological inhibition or genetic knockdown of DRG DNMT1 alleviated nerve injury-induced pain hypersensitivities, DRG DNMT1 contributes to neuropathic pain genesis partially through repression of DRG Kcna2 gene expression.

2180-P: HERG2 Channel Plays a Key Role in Insulin Secretion

Background: Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is mainly mediated by KATP channels. A problem commonly associated with drugs like the sulfonylureas, KATP channel inhibitor, is that KATP current inhibition is independent of the basal glucose levels and hypoglycemia is frequently observed. Nevertheless, the role of non-KATP K+ channels in insulin secretion is largely unknown. Here, we show that a voltage-gated K+ (Kv) channel, HERG2, plays a key role in insulin secretion. Aims: To investigate the effects of HERG2 channel in β-cells. Methods: Using TALEN-based method, we created Herg2 gene knock out (KO) mice. Patch-clamp experiments were performed to record Kv currents and action potential in primary cultured β-cells from wild type (WT) and KO mice. INS-1 cells, rat β-cell line, were transfected with shRNA of Herg2 gene or scramble shRNA to generated Herg2 transiently knock down (KD) or WT cells. Glucose-stimulated calcium levels were measured and GSIS experiments were performed in INS-1WT and INS-1KD cells. Results: The Kv currents of β-cells from the KO mice decreased approximately 37.6% compared with that of the WT mice, indicating HERG2 channel constitutes 37.6% of all Kv currents. The action potential duration was significantly prolonged in KO β-cells, whereas the action potential magnitude and resting potential remained unchanged compared with WT β-cells. INS-1KD showed enhanced glucose-induced elevation of the intracellular Ca2+ concentrations, the most important ion that triggers insulin secretion in β-cell. Consistently, Herg2 knockdown significantly enhanced insulin secretion at a high glucose concentration, but did not potentiate insulin secretion at basal glucose concentration, suggesting the glucose-dependent insulin secretion of HERG2 channel. Conclusions HERG2, as a non-KATP K+ channel, regulates the action potential duration of β-cells and plays a key role in insulin secretion. Inhibition of HERG2 currents leads to enhanced insulin secretion only in high glucose levels. Disclosure M. Zhao: None. J. Yang: None.

Activation of Kv 7 channels as a novel mechanism for NO/cGMP-induced pulmonary vasodilation.

The NO/cGMP pathway represents a major physiological signalling controlling tone in pulmonary arteries (PA), and drugs activating this pathway are used to treat pulmonary arterial hypertension. Kv channels expressed in PA smooth muscle cells (PASMCs) are key determinants of vascular tone. We aimed to analyse the contribution of Kv 1.5 and Kv 7 channels in the electrophysiological and vasodilating effects evoked by NO donors and the GC stimulator riociguat in PA. Kv currents were recorded in isolated rat PASMCs using the patch-clamp technique. Vascular reactivity was assessed in a wire myograph. The NO donors diethylamine NONOate diethylammonium (DEA-NO) and sodium nitroprusside hyperpolarized the membrane potential and induced a bimodal effect on Kv currents (augmenting the current between -40 and -10mV and decreasing it at more depolarized potentials). The hyperpolarization and the enhancement of the current were suppressed by Kv 7 channel inhibitors and by the GC inhibitor ODQ but preserved when Kv 1.5 channels were inhibited. Additionally, DEA-NO enhanced Kv 7.5 currents in COS7 cells expressing the KCNQ5 gene. Riociguat increased Kv currents at all potentials ≥-40mV and induced membrane hyperpolarization. Both effects were prevented by Kv 7 inhibition. Likewise, PA relaxation induced by NO donors and riociguat was attenuated by Kv 7 inhibitors. NO donors and riociguat enhance Kv 7 currents, leading to PASMC hyperpolarization. This mechanism contributes to NO/cGMP-induced PA vasodilation. Our study identifies Kv 7 channels as a novel mechanism of action of vasodilator drugs used in the treatment of pulmonary arterial hypertension.

Apigenin relaxes rat intrarenal arteries, depresses Ca2+-activated Cl− currents and augments voltage-dependent K+ currents of the arterial smooth muscle cells

The vasorelaxant effect of apigenin (API) has been demonstrated in a number of vascular beds. We aimed to study the possible involvement of Cl− channels and K+ channels in API-induced vasorelaxation in intrarenal arteries. The vascular tone of isolated rat intrarenal arteries (RIRAs) was measured with a myograph. The myocyte transmembrane Cl− currents through Ca2+ activated Cl− channels (CaCCs) and the K+ currents through voltage-dependent (Kv) K+ channels were recorded using a patch clamp in the single arterial smooth muscle cells (ASMCs) isolated freshly from RIRAs. Preincubation with API (10–100 μM) concentration-dependently depressed the contractions induced by KCl, thromboxane A2 analog U46619, phenylephrine and vasopressin. The IC50 values were 13.27–26.26 μM. Instant application of API elicited immediate relaxations in RIRAs precontracted with these vasoconstrictors. The RC50 values were 5.80–24.33 μM. Chloride deprivation, Cl− channel blockers, Kv blocker and nitric oxide synthase inhibitor attenuated API-induced RIRA relaxation. At 10–100 μM, API depressed CaCC currents, but augmented Kv currents. Taken together, the present study demonstrated that API depresses contractions induced by various vasoconstrictors in RIRAs, suppresses CaCC currents and augments Kv currents in RIRA ASMCs.

Inhibitory Effect of Tricyclic Antidepressant Doxepin on Voltage-Dependent K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells.

Doxepin, tricyclic antidepressant, is widely used for the treatment of depressive disorders. Our present study determined the inhibitory effect of doxepin on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Vascular Kv currents were inhibited by doxepin in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC50) value of 6.52 ± 1.35μM and a Hill coefficient of 0.72 ± 0.03. Doxepin did not change the steady-state activation curve or inactivation curve, suggesting that doxepin does not alter the gating properties of Kv channels. Application of train pulses (1 or 2Hz) slightly reduced the amplitude of Kv currents. However, the inhibition of Kv channels by train pulses were not changed in the presence of doxepin. Pretreatment with Kv1.5 inhibitor, DPO-1, effectively reduced the doxepin-induced inhibition of the Kv current. However, pretreatment with Kv2.1 inhibitor (guangxitoxin) or Kv7 inhibitor (linopirdine) did not change the inhibitory effect of doxepin on Kv currents. Inhibition of Kv channels by doxepin caused vasoconstriction and membrane depolarization. Therefore, our present study suggests that doxepin inhibits Kv channels in a concentration-dependent, but not use-, and state-dependent manners, irrespective of its own function.

Luteolin-induced coronary arterial relaxation involves activation of the myocyte voltage-gated K+ channels and inward rectifier K+ channels

AimsLuteolin has been shown to be beneficial to cardiovascular tissues and organs. We aimed to study its vasospasmolytic effects against various vasoconstrictors in the isolated rat coronary arteries (RCAs) and its electrophysiological effects on K+ currents via voltage-gated potassium (Kv) channels and inward rectifier potassium (Kir) channels in freshly isolated rat coronary arterial smooth muscle cells (RCASMCs). Main methodsThe vascular tone of the endothelium-denuded RCAs was recorded by a wire myograph. Kv currents and Kir currents in RCASMCs were assessed using whole-cell patch clamp. Key findingsPreincubation with luteolin depressed the contractions elicited by KCl, thromboxane A2 analog U46619, vasopressin, Kir blocker BaCl2, Kv blocker 4-aminopyridine and elevation of extracellular calcium ([Ca2+]o) in high K+ depolarizing solution. Instant application of luteolin produced concentration-dependent relaxations in the endothelium-denuded RCAs precontracted with KCl or U46619. Both 4-aminopyridine and BaCl2 attenuated luteolin-induced relaxation in U46619-precontracted RCAs, while neither nitric oxide synthetase inhibitor NG-nitro-L-arginine methyl ester nor cyclooxygenase inhibitor indomethacin affected the relaxation. Luteolin augmented both Kv currents and Kir currents in RCASMCs and the augmentations were antagonized by 4-aminopyridine and BaCl2, respectively. SignificanceThe present results demonstrated that luteolin antagonizes various vasoconstrictors in RCAs and augments both Kv currents and Kir currents in RCASMCs, suggesting that the direct action of luteolin on Kv channels and Kir channels is contributory to its vasospasmolytic effect. These findings indicate that luteolin may be a promising food additive with the aim of preventing coronary arterial spasm.

Inhibitory effect of immunosuppressive drug tacrolimus on voltage-gated K+ current in rabbit coronary arterial smooth muscle cells

In the present study, we investigated the inhibitory effect of tacrolimus, a macrolide immunosuppressive drug that acts through calcineurin inhibition, on voltage-gated K+ (Kv) channels expressed in native smooth muscle cells isolated from the coronary arteries of rabbits. Tacrolimus reduced the amplitude of Kv currents in a dose-dependent manner with an IC50 value and Hill coefficient of 7.80 ± 3.01 μM and 1.07 ± 0.25, respectively. Tacrolimus caused a shift in the activation curve toward a more positive potential and in the inactivation curve toward a more negative potential. Tacrolimus-induced inhibition of Kv current was increased by the application of train pulses (1 or 2 Hz). Furthermore, the recovery time constant of inactivation was extended in the presence of tacrolimus, suggesting that tacrolimus inhibited Kv channels in a use-dependent manner. Two kinds of Kv subtype inhibitors, DPO-1 and guangxitoxin did not affect the degree of tacrolimus-induced inhibition of Kv current. Furthermore, pretreatment with another calcineurin inhibitor, cyclosporine A, did not affect the Kv current, and did not alter the inhibitory effect of tacrolimus. Using perforated-patch clamp experiments, inhibition of Kv channels by tacrolimus caused membrane depolarization. From these results, we concluded that tacrolimus inhibited the vascular Kv currents in a dose, state (open and closed)-dependent manner.

Re-Education of Tumor Associated Macrophages by Trabectedin

Immune cells have an important role in the tumor-microenvironment. Macrophages may tune the immune response toward inflammatory or tolerance pathways. Tumor associated macrophages (TAM) have immunosuppressive functions and they are considered a therapeutic target in cancer. The aim of this study was to evaluate the effects of trabectedin, a new class of antitumor agent, on the tumor-microenvironment through the study of electrophysiological and molecular phenotype of macrophages. Experiments were performed using the whole-cell configuration of the patch-clamp technique in resident peritoneal mouse macrophages under different types of polarization. Trabectedin decreased macrophage viability and increased ROS production. Trabectedin does not directly interact with KV1.5 and KV1.3 channels, but treatment (16 h) of macrophages with sub-cytotoxic concentrations (0.1-5 nM) increased their KV current in a concentration-dependent manner due to an upregulation of KV1.3 channels. In vitro generated TAM (TAMiv), by a co-culture of ID8 cells and macrophages, exhibited a M2 phenotype. TAMiv generated a small KV current, similarly to M2 polarized macrophages, and expressed high levels of M2 markers. In this study, we demonstrated that TAMiv polarization could be re-educated by using sub-cytotoxic concentration of trabectedin. TAMiv treated with sub-cytotoxic concentration of trabectedin exhibited an upregulation of KV1.3 channels and their M2 phenotype changed towards M1 pro-inflammatory one. Funded by PharmaMar, CSIC 201820E104, CIBERCV and SAF2016-75021-R.