AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na(+) transport across human airway epithelial cells

BACKGROUND AND PURPOSE

Pulmonary transepithelial Na⁺ transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na⁺ channels and basolateral Na⁺K⁺ ATPase activity.

EXPERIMENTAL APPROACH

H441 human airway epithelial cells were used to examine the effects of hypoxia on Na⁺ transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS.

KEY RESULTS

AMPK was activated by exposure to 3% or 0.2% O₂ for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm⁻²) was added to the apical surface of cells grown at the air–liquid interface. Only 0.2% O₂ activated AMPK in cells grown at the air–liquid interface. AMPK activation was associated with elevation of cellular AMP : ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive I_sc (I_ouabain) and apical amiloride-sensitive Na⁺ conductance (G_Na+). Modification of AMPK activity prevented the effect of hypoxia on I_ouabain (Na⁺K⁺ ATPase) but not apical G_Na+. Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical G_Na+ (epithelial Na⁺ channels).

CONCLUSIONS AND IMPLICATIONS

Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na⁺ channels and basolateral Na⁺K⁺ ATPase activity to decrease transepithelial Na⁺ transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions.     

Benzamil sensitive ion channels contribute to volume regulation in canine chondrocytes

Background and Purpose

Chondrocytes exist within cartilage and serve to maintain the extracellular matrix. It has been postulated that osteoarthritic (OA) chondrocytes lose the ability to regulate their volume, affecting extracellular matrix production. In previous studies, we identified expression of epithelial sodium channels (ENaC) in human chondrocytes, but their function remained unknown. Although ENaC typically has Na⁺ transport roles, it is also involved in the cell volume regulation of rat hepatocytes. ENaC is a member of the degenerin (Deg) family, and ENaC/Deg-like channels have a low conductance and high sensitivity to benzamil. In this study, we investigated whether canine chondrocytes express functional ENaC/Deg-like ion channels and, if so, what their function may be.

Experimental Approach

Canine chondrocytes were harvested from dogs killed for unassociated welfare reasons. We used immunohistochemistry and patch-clamp electrophysiology to investigate ENaC expression and video microscopy to analyse the effects of pharmacological inhibition of ENaC/Deg on cell volume regulation.

Key Results

Immunofluorescence showed that canine chondrocytes expressed ENaC protein. Single-channel recordings demonstrated expression of a benzamil-sensitive Na⁺ conductance (9 pS), and whole-cell experiments show this to be approximately 1.5 nS per cell with high selectivity for Na⁺. Benzamil hyperpolarized chondrocytes by approximately 8 mV with a pD₂ 8.4. Chondrocyte regulatory volume decrease (RVI) was inhibited by benzamil (pD₂ 7.5) but persisted when extracellular Na⁺ ions were replaced by Li⁺.

Conclusion and Implications

Our data suggest that benzamil inhibits RVI by reducing the influx of Na⁺ ions through ENaC/Deg-like ion channels and present ENaC/Deg as a possible target for pharmacological modulation of chondrocyte volume.     

Loss of Ca2+-mediated ion transport during colitis correlates with reduced ion transport responses to a Ca2+-activated K+ channel opener

Background and purpose:

Epithelial surface hydration is critical for proper gut function. However, colonic tissues from individuals with inflammatory bowel disease or animals with colitis are hyporesponsive to Cl⁻ secretagogues. The Cl⁻ secretory responses to the muscarinic receptor agonist bethanechol are virtually absent in colons of mice with dextran sodium sulphate (DSS)-induced colitis. Our aim was to define the mechanism underlying this cholinergic hyporesponsiveness.

Experimental approach:

Colitis was induced by 4% DSS water, given orally. Epithelial ion transport was measured in Ussing chambers. Colonic crypts were isolated and processed for mRNA expression via RT-PCR and protein expression via immunoblotting and immunolocalization.

Key results:

Expression of muscarinic M₃ receptors in colonic epithelium was not decreased during colitis. Short-circuit current (I_SC) responses to other Ca²⁺-dependent secretagogues (histamine, thapsigargin, cyclopiazonic acid and calcium ionophore) were either absent or severely attenuated in colonic tissue from DSS-treated mice. mRNA levels of several ion transport molecules (a Ca²⁺-regulated Cl⁻ channel, the intermediate-conductance Ca²⁺-activated K⁺ channel, the cystic fibrosis transmembrane conductance regulator, the Na⁺/K⁺-ATPase pump or the Na⁺/K⁺/2Cl⁻ co-transporter) were not reduced in colonic crypts from DSS-treated mice. However, protein expression of Na⁺/K⁺-ATPase α1 subunits was decreased twofold during colitis. Activation of Ca²⁺-activated K⁺ channels increased I_SC significantly less in DSS colons compared with control, as did the protein kinase C activator, phorbol 12-myristate 13-acetate.

Conclusions and implications:

Decreased Na⁺/K⁺-ATPase expression probably contributes to overall epithelial hyporesponsiveness during colitis, while dysfunctional K⁺ channels may account, at least partially, for lack of epithelial secretory responses to Ca²⁺-mediated secretagogues.     

Epithelial Na⁺ channel activity in human airway epithelial cells: the role of serum and glucocorticoid-inducible kinase 1

BACKGROUND AND PURPOSE

Glucocorticoids appear to control Na⁺ absorption in pulmonary epithelial cells via a mechanism dependent upon serum and glucocorticoid-inducible kinase 1 (SGK1), a kinase that allows control over the surface abundance of epithelial Na⁺ channel subunits (α-, β- and γ-ENaC). However, not all data support this model and the present study re-evaluates this hypothesis in order to clarify the mechanism that allows glucocorticoids to control ENaC activity.

EXPERIMENTAL APPROACH

Electrophysiological studies explored the effects of agents that suppress SGK1 activity upon glucocorticoid-induced ENaC activity in H441 human airway epithelial cells, whilst analyses of extracted proteins explored the associated changes to the activities of endogenous protein kinase substrates and the overall/surface expression of ENaC subunits.

KEY RESULTS

Although dexamethasone-induced (24 h) ENaC activity was dependent upon SGK1, prolonged exposure to this glucocorticoid did not cause sustained activation of this kinase and neither did it induce a coordinated increase in the surface abundance of α-, β- and γ-ENaC. Brief (3 h) exposure to dexamethasone, on the other hand, did not evoke Na⁺ current but did activate SGK1 and cause SGK1-dependent increases in the surface abundance of α-, β- and γ-ENaC.

CONCLUSIONS AND IMPLICATIONS

Although glucocorticoids activated SGK1 and increased the surface abundance of α-, β- and γ-ENaC, these responses were transient and could not account for the sustained activation of ENaC. The maintenance of ENaC activity did, however, depend upon SGK1 and this protein kinase must therefore play an important but permissive role in glucocorticoid-induced ENaC activation.     

The guinea-pig tracheal potential difference as an in vivo model for the study of epithelial sodium channel function in the airways

Background and purpose:

The epithelial sodium channel (ENaC) is a key regulator of airway mucosal hydration and mucus clearance. Negative regulation of airway ENaC function is predicted to be of clinical benefit in the cystic fibrosis lung. The aim of this study was to develop a small animal model to enable the direct assessment of airway ENaC function in vivo.

Experimental approach:

Tracheal potential difference (TPD) was utilized as a measure of airway epithelial ion transport in the guinea-pig. ENaC activity in the trachea was established with a dose–response assessment to a panel of well-characterized direct and indirect pharmacological modulators of ENaC function, delivered by intra-tracheal (i.t.) instillation.

Key results:

The TPD in anaesthetized guinea-pigs was attenuated by the direct ENaC blockers: amiloride, benzamil and CF552 with ED₅₀ values of 16, 14 and 0.2 μg kg⁻¹ (i.t.), respectively. 5-(N-Ethyl-N-isopropyl) amiloride, a structurally related compound but devoid of activity on ENaC, was without effect on the TPD. Intra-tracheal dosing of the Kunitz-type serine protease inhibitors aprotinin and placental bikunin, which have previously been demonstrated to inhibit proteolytic activation of ENaC, likewise potently attenuated TPD in guinea-pigs, whereas α₁-antitrypsin and soya bean trypsin inhibitor were without effect.

Conclusions and implications:

The pharmacological sensitivity of the TPD to amiloride analogues and also to serine protease inhibitors are both consistent with that of ENaC activity in the guinea-pig trachea. The guinea-pig TPD therefore represents a suitable in vivo model of human airway epithelial ion transport.     

Bepridil up-regulates cardiac Na+ channels as a long-term effect by blunting proteasome signals through inhibition of calmodulin activity

Background and purpose:

Bepridil is an anti-arrhythmic agent with anti-electrical remodelling effects that target many cardiac ion channels, including the voltage-gated Na⁺ channel. However, long-term effects of bepridil on the Na⁺ channel remain unclear. We explored the long-term effect of bepridil on the Na⁺ channel in isolated neonatal rat cardiomyocytes and in a heterologous expression system of human Na_v1.5 channel.

Experimental approach:

Na⁺ currents were recorded by whole-cell voltage-clamp technique. Na⁺ channel message and protein were evaluated by real-time RT-PCR and Western blot analysis.

Key results:

Treatment of cardiomyocytes with 10 µmol·L⁻¹ bepridil for 24 h augmented Na⁺ channel current (I_Na) in a dose- and time-dependent manner. This long-term effect of bepridil was mimicked or masked by application of W-7, a calmodulin inhibitor, but not KN93 [2-[N-(2-hydroxyethyl)-N-(4-methoxy benzenesulphonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], a Ca²⁺/calmodulin-dependent kinase inhibitor. During inhibition of protein synthesis by cycloheximide, the I_Na increase due to bepridil was larger than the increase without cycloheximide. Bepridil and W-7 significantly slowed the time course of Na_v1.5 protein degradation in neonatal cardiomyocytes, although the mRNA levels of Na_v1.5 were not modified. Bepridil and W-7 did not increase I_Na any further in the presence of the proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide]. Bepridil, W-7 and MG132 but not KN93 significantly decreased 20S proteasome activity in a concentration-dependent manner.

Conclusions and implications:

We conclude that long-term exposure of cardiomyocytes to bepridil at therapeutic concentrations inhibits calmodulin action, which decreased degradation of the Na_v1.5 α-subunit, which in turn increased Na⁺ current.     

Hypericin prolongs action potential duration in hippocampal neurons by acting on K+ channels

Background and purpose:

Synaptic deficiency is generally accepted to be involved in major depression, and accordingly classic antidepressants exert their effects through enhancing synaptic efficiency. Hypericin is one of the major active constituents of extracts of St. John''s Wort (Hypericum perforatum L.) with antidepressive actions, but little is known about its therapeutic mechanisms. Our aim was to explore whether hypericin has a modulatory effect on neuronal action potential (AP) duration by acting on voltage-gated ion channels.

Experimental approach:

We used voltage-clamp and current-clamp techniques in a whole-cell configuration to study primary cultures of neonatal rat hippocampal neurones. We measured the effects of extracellularly applied hypericin on AP duration as well as on voltage-gated Na⁺, I_A and I_K currents.

Key results:

Extracellularly applied hypericin dose-dependently increased AP duration but barely affected its amplitude. Further analysis revealed that hypericin inhibited both transient I_A and delayed rectifier I_K potassium currents. In contrast, hypericin exerted no significant effect on both Na⁺ peak current and its decay kinetics.

Conclusions and implications:

Extracellularly applied hypericin increased AP duration, which might be ascribed to its effect on I_A and I_K currents. As a small increase in AP duration could lead to a dramatic increase in synaptic efficiency, our results imply that hypericin might exert its antidepressant effects by enhancing presynaptic efficiency.     

Actions of veratridine on tetrodotoxin-sensitive voltage-gated Na+ currents,NaV1.6, in murine vas deferens myocytes

Background and purpose:

The effects of veratridine, an alkaloid found in Liliaceae plants, on tetrodotoxin (TTX)-sensitive voltage-gated Na⁺ channels were investigated in mouse vas deferens.

Experimental approach:

Effects of veratridine on TTX-sensitive Na⁺ currents (I_Na) in vas deferens myocytes dispersed from BALB/c mice, homozygous mice with a null allele of Na_V1.6 (Na_V1.6^−/−) and wild-type mice (Na_V1.6^+/+) were studied using patch-clamp techniques. Tension measurements were also performed to compare the effects of veratridine on phasic contractions in intact tissues.

Key results:

In whole-cell configuration, veratridine had a concentration-dependent dual action on the peak amplitude of I_Na: I_Na was enhanced by veratridine (1–10 µM), while higher concentrations (≥30 µM) inhibited I_Na. Additionally, two membrane current components were evoked by veratridine, namely a sustained inward current during the duration of the depolarizing rectangular pulse and a tail current at the repolarization. Although veratridine caused little shift of the voltage dependence of the steady-state inactivation curve and the activation curve for I_Na, veratridine enhanced a non-inactivating component of I_Na. Veratridine caused no detectable contractions in vas deferens from Na_V1.6^−/− mice, although in tissues from Na_V1.6^+/+ mice, veratridine (≥3 µM) induced TTX-sensitive contractions. Similarly, no detectable inward currents were evoked by veratridine in Na_V1.6^−/− vas deferens myocytes, while veratridine elicited both the sustained and tail currents in cells taken from Na_V1.6^+/+ mice.

Conclusions and implications:

These results suggest that veratridine possesses a dual action on I_Na and that the veratridine-induced activation of contraction is induced by the activation of Na_V1.6 channels.     

Electrophysiological mechanisms of sophocarpine as a potential antiarrhythmic agent

Aim:

To examine the electrophysiological effects of sophocarpine on action potentials (AP) and ionic currents of cardiac myocytes and to compare some of these effects with those of amiodarone.

Methods:

Langendorff perfusion set-up was used in isolated guinea pig heart, and responses to sophocarpine were monitored using electrocardiograph. Conventional microelectrode, voltage clamp technique and perforated patch were employed to record fast response AP (fAP), slow response AP (sAP) and ionic currents in guinea pig papillary muscle or rabbit sinus node cells.

Results:

Tachyarrhythmia produced by isoprenaline (15 μmol/L) could be reversed by sophocarpine (300 μmol/L). Sophocarpine (10 μmol/L) decreased the amplitude by 4.0%, maximal depolarization velocity (V_max) of the fAP by 24.4%, and Na⁺ current (I_Na) by 18.0%, while it prolonged the effective refractory period (ERP) by 21.1%. The same concentration of sophocarpine could also decrease the amplitude and V_max of the sAP, by 26.8% and 25.7%, respectively, and attenuated the Ca²⁺ current (I_CaL) and the K⁺ tail current substantially. Comparison of sophocarpine with amiodarone demonstrated that both prolonged the duration and the ERP of fAP and sAP, both decreased the amplitude and V_max of the fAP and sAP, and both slowed the automatic heart rate.

Conclusion:

Sophocarpine could reverse isoprenaline-induced arrhythmia and inhibit I_Na, I_CaL, and I_Kr currents. The electrophysiological effects of sophocarpine are similar to those of amiodarone, which might be regarded as a prospective antiarrhythmic agent.     

Inhibitory effects of tetrandrine on the Na+ channel of human atrial fibrillation myocardium

Aim:

Tetrandrine (Tet) is a Ca²⁺ channel blocker and has antiarrhythmic effects. Less information exists with regard to the mechanisms underlying its antiarrhythmic action other than blocking Ca²⁺ channels. In this study, the effects of Tet on the Na⁺ current (I_Na) in the atrial myocardium of patients in atrial fibrillation (AF) and sinus rhythm (SR) were investigated, and the characteristics of the Na⁺ current were synchronously compared between the AF and SR patients.

Methods:

Na⁺ currents were recorded using the whole-cell patch clamp technique in single atrial myocyte of the AF and the normal SR groups. The effects of Tet (40–120 μmol/L) on the Na⁺ current in the two groups were then observed.

Results:

Tet (60–120 μmol/L) decreased I_Na density in a concentration-dependent manner and made the voltage-dependent activation curve shift to more positive voltages in the SR and AF groups. After exposure to Tet, the voltage-dependent inactivation curve of I_Na was shifted to more negative voltages in the two groups. Tet delayed the time-dependent recovery of I_Na in a concentration dependent manner in both AF and SR cells; however, there were no differences in the effects of Tet on I_Na density and properties in the two groups. The I_Na density of AF patients did not differ from that of the SR patients.

Conclusion:

Tet can block sodium channels with slow recovery kinetics, which may explain the mechanisms underlying the antiarrhythmic action of Tet. The decreased conduction velocity (CV) in AF patients is not caused by the Na⁺ current.     

Piceatannol, a derivative of resveratrol, moderately slows INa inactivation and exerts antiarrhythmic action in ischaemia-reperfused rat hearts

Background and purpose:

Piceatannol is more potent than resveratrol in free radical scavenging in association with antiarrhythmic and cardioprotective activities in ischaemic-reperfused rat hearts. The present study aimed to investigate the antiarrhythmic efficacy and the underlying ionic mechanisms of piceatannol in rat hearts.

Experimental approach:

Action potentials and membrane currents were recorded by the whole-cell patch clamp techniques. Fluo-3 fluorimetry was used to measure cellular Ca²⁺ transients. Antiarrhythmic activity was examined from isolated Langendorff-perfused rat hearts.

Key results:

In rat ventricular cells, piceatannol (3–30 µmol·L⁻¹) prolonged the action potential durations (APDs) and decreased the maximal rate of upstroke (V_max) without altering Ca²⁺ transients. Piceatannol decreased peak I_Na and slowed I_Na inactivation, rather than induced a persistent non-inactivating current, which could be reverted by lidocaine. Resveratrol (100 µmol·L⁻¹) decreased peak I_Na without slowing I_Na inactivation. The inhibition of peak I_Na or V_max was associated with a negative shift of the voltage-dependent steady-state I_Na inactivation curve without altering the activation threshold. At the concentrations more than 30 µmol·L⁻¹, piceatannol could inhibit I_Ca,L, I_to, I_Kr, Ca²⁺ transients and Na⁺-Ca²⁺ exchange except I_K1. Piceatannol (1–10 µmol·L⁻¹) exerted antiarrhythmic activity in isolated rat hearts subjected to ischaemia-reperfusion injury.

Conclusions and implications:

The additional hydroxyl group on resveratrol makes piceatannol possessing more potent in I_Na inhibition and uniquely slowing I_Na inactivation, which may contribute to its antiarrhythmic actions at low concentrations less than 10 µmol·L⁻¹.     

Dysregulation of epithelial Na+ absorption induced by inhibition of the kinases TORC1 and TORC2

BACKGROUND AND PURPOSE

Although the serum and glucocorticoid-inducible protein kinase 1 (SGK1) appears to be involved in controlling epithelial Na⁺ absorption, its role in this physiologically important ion transport process is undefined. As SGK1 activity is dependent upon target of rapamycin complex 2 (TORC2)-catalysed phosphorylation of SGK1-Ser⁴²², we have explored the effects of inhibiting TORC2 and/or TORC1 upon the hormonal control of Na⁺ absorption.

EXPERIMENTAL APPROACH

Na⁺ absorption was quantified electrometrically in mouse cortical collecting duct cells (mpkCCD) grown to confluence on permeable membranes. Kinase activities were assessed by monitoring endogenous protein phosphorylation, with or without TORC1/2 inhibitors (TORIN1 and PP242) and the TORC1 inhibitor: rapamycin.

KEY RESULTS

Inhibition of TORC1/2 (TORIN1, PP242) suppressed basal SGK1 activity, prevented insulin- and dexamethasone-induced SGK1 activation, and caused modest (10–20%) inhibition of basal Na⁺ absorption and substantial (∼80%) inhibition of insulin/dexamethasone-induced Na⁺ transport. Inhibition of TORC1 did not impair SGK1 activation or insulin-induced Na⁺ transport, but did inhibit (∼80%) dexamethasone-induced Na⁺ absorption. Arginine vasopressin stimulated Na⁺ absorption via a TORC1/2-independent mechanism.

CONCLUSION AND IMPLICATIONS

Target of rapamycin complex 2, but not TORC1, is important to SGK1 activation. Signalling via phosphoinositide-3-kinase/TORC2/SGK1 can explain insulin-induced Na⁺ absorption. TORC2, but not TORC1, is also involved in glucocorticoid-induced SGK1 activation but its role is permissive. Glucocorticoid-induced Na⁺ transport displayed a requirement for TORC1 activity. Therefore, TORC1 and TORC2 contribute to the regulation of Na⁺ absorption. Pharmacological manipulation of TORC1/2 signalling may provide novel therapies for Na⁺-sensitive hypertension.     

Effects of peroxisome proliferator-activated receptor γ agonists on Na+ transport and activity of the kinase SGK1 in epithelial cells from lung and kidney

Background and purpose:

Peroxisome proliferator-activated receptor γ (PPARγ) agonists, such as rosiglitazone and pioglitazone, sensitize cells to insulin, and are therefore used to treat type 2 diabetes. However, in some patients, these drugs induce oedema, and the present study tests the hypothesis that this side effect reflects serum and glucocorticoid-inducible kinase 1 (SGK1)-dependent enhancement of epithelia Na⁺ absorption.

Experimental approach:

Na⁺ absorbing epithelial cells (H441 cells, mpkCCD cells) on permeable membranes were mounted in Ussing chambers, and the effects of rosiglitazone (2 µM) and pioglitazone (10 µM) on transepithelial Na⁺ absorption were quantified electrometrically. Changes in SGK1 activity were assessed by monitoring phosphorylation of residues within an endogenous protein.

Key results:

Both cell types absorbed Na⁺ via an electrogenic process that was enhanced by insulin. In mpkCCD cells, this stimulation of Na⁺ transport was associated with increased activity of SGK1, whereas insulin regulated Na⁺ transport in H441 cells through a mechanism that did not involve activation of this kinase. Rosiglitazone and pioglitazone had no discernible effect on transepithelial Na⁺ absorption in unstimulated or insulin-stimulated cells and failed to alter cellular SGK1 activity.

Conclusions and implications:

Our results do not support the view that PPARγ agonists stimulate epithelial Na⁺ absorption or alter the control of cellular SGK1 activity. It is therefore likely that other mechanisms are involved in PPARγ-mediated fluid retention, and a better understanding of these mechanisms may help with the identification of patients likely to develop oedema or heart failure when treated with these drugs.     

Ketamine attenuates the Na+-dependent Ca2+ overload in rabbit ventricular myocytes in vitro by inhibiting late Na+ and L-type Ca2+ currents

Aim:

Intracellular Ca²⁺ ([Ca²⁺]_i) overload occurs in myocardial ischemia. An increase in the late sodium current (I_NaL) causes intracellular Na⁺ overload and subsequently [Ca²⁺]_i overload via the reverse-mode sodium-calcium exchanger (NCX). Thus, inhibition of INaL is a potential therapeutic target for cardiac diseases associated with [Ca²⁺]_i overload. The aim of this study was to investigate the effects of ketamine on Na⁺-dependent Ca²⁺ overload in ventricular myocytes in vitro.

Methods:

Ventricular myocytes were enzymatically isolated from hearts of rabbits. I_NaL, NCX current (I_NCX) and L-type Ca²⁺ current (I_CaL) were recorded using whole-cell patch-clamp technique. Myocyte shortening and [Ca²⁺]_i transients were measured simultaneously using a video-based edge detection and dual excitation fluorescence photomultiplier system.

Results:

Ketamine (20, 40, 80 μmol/L) inhibited I_NaL in a concentration-dependent manner. In the presence of sea anemone toxin II (ATX, 30 nmol/L), I_NaL was augmented by more than 3-fold, while ketamine concentration-dependently suppressed the ATX-augmented I_NaL. Ketamine (40 μmol/L) also significantly suppressed hypoxia or H₂O₂-induced enhancement of I_NaL. Furthermore, ketamine concentration-dependently attenuated ATX-induced enhancement of reverse-mode I_NCX. In addition, ketamine (40 μmol/L) inhibited I_CaL by 33.4%. In the presence of ATX (3 nmol/L), the rate and amplitude of cell shortening and relaxation, the diastolic [Ca²⁺]_i, and the rate and amplitude of [Ca²⁺]_i rise and decay were significantly increased, which were reverted to control levels by tetrodotoxin (TTX, 2 μmol/L) or by ketamine (40 μmol/L).

Conclusion:

Ketamine protects isolated rabbit ventricular myocytes against [Ca²⁺]_i overload by inhibiting I_NaL and I_CaL.     

Inhibition of vascular calcium-gated chloride currents by blockers of KCa1.1, but not by modulators of KCa2.1 or KCa2.3 channels

Background and purpose:

Recent pharmacological studies have proposed there is a high degree of similarity between calcium-activated Cl⁻ channels (CaCCs) and large conductance, calcium-gated K⁺ channels (K_Ca1.1). The goal of the present study was to ascertain whether blockers of K_Ca1.1 inhibited calcium-activated Cl⁻ currents (I_ClCa) and if the pharmacological overlap between K_Ca1.1 and CaCCs extends to intermediate and small conductance, calcium-activated K⁺ channels.

Experimental approaches:

Whole-cell Cl⁻ and K⁺ currents were recorded from murine portal vein myocytes using the whole-cell variant of the patch clamp technique. CaCC currents were evoked by pipette solutions containing 500 nM free [Ca²⁺].

Key results:

The selective K_Ca1.1 blocker paxilline (1 µM) inhibited I_ClCa by ∼90%, whereas penitrem A (1 µM) and iberiotoxin (100 and 300 nM) reduced the amplitude of I_ClCa by ∼20%, as well as slowing channel deactivation. Paxilline also abolished the stimulatory effect of niflumic acid on the CaCC. In contrast, an antibody against the Ca²⁺-binding domain of murine K_Ca1.1 had no effect on I_ClCa while inhibiting spontaneous K_Ca1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K⁺ channels (K_Ca2.1 and K_Ca2.3), namely 1-EBIO, (100 µM); NS309, (1 µM); TRAM-34, (10 µM); UCL 1684, (1 µM) had no effect on I_ClCa.

Conclusions and implications:

These data show that the selective K_Ca1.1 blockers also reduce I_ClCa considerably. However, the pharmacological overlap that exists between CaCCs and K_Ca1.1 does not extend to the calcium-binding domain or to other calcium-gated K⁺ channels.     

Blocking effect of methylflavonolamine on human NaV1.5 channels expressed in Xenopus laevis oocytes and on sodium currents in rabbit ventricular myocytes

Aim:

To investigate the blocking effects of methylflavonolamine (MFA) on human Na_V1.5 channels expressed in Xenopus laevis oocytes and on sodium currents (I_Na) in rabbit ventricular myocytes.

Methods:

Human Na_V1.5 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. I_Na and action potentials in rabbit ventricular myocytes were studied using the whole-cell recording.

Results:

MFA and lidocaine inhibited human Na_V1.5 channels expressed in Xenopus oocytes in a positive rate-dependent and concentration-dependent manner, with IC₅₀ values of 72.61 μmol/L and 145.62 μmol/L, respectively. Both of them markedly shifted the steady-state activation curve of I_Na toward more positive potentials, shifted the steady-state inactivation curve of I_Na toward more negative potentials and postponed the recovery of the I_Na inactivation state. In rabbit ventricular myocytes, MFA inhibited I_Na with a shift in the steady-state inactivation curve toward more negative potentials, thereby postponing the recovery of the I_Na inactivation state. This shift was in a positive rate-dependent manner. Under current-clamp mode, MAF significantly decreased action potential amplitude (APA) and maximal depolarization velocity (V_max) and shortened action potential duration (APD), but did not alter the resting membrane potential (RMP). The demonstrated that the kinetics of sodium channel blockage by MFA resemble those of class I antiarrhythmic agents such as lidocaine.

Conclusion:

MFA protects the heart against arrhythmias by its blocking effect on sodium channels.     

The endocannabinoid system in renal cells: regulation of Na+ transport by CB1 receptors through distinct cell signalling pathways

Background and Purpose

The function of the endocannabinoid system (ECS) in renal tissue is not completely understood. Kidney function is closely related to ion reabsorption in the proximal tubule, the nephron segment responsible for the re-absorption of 70–80% of the filtrate. We studied the effect of compounds modulating the activity of cannabinoid (CB) receptors on the active re-absorption of Na⁺ in LLC-PK1 cells.

Experimental Approach

Changes in Na⁺/K⁺-ATPase activity were assessed after treatment with WIN55,212-2 (WIN), a non-selective lipid agonist, and haemopressin (HP), an inverse peptide agonist at CB₁ receptors. Pharmacological tools were used to investigate the signalling pathways involved in the modulation of Na⁺ transport.

Key Results

In addition to CB₁ and CB₂ receptors and TRPV1 channels, the mRNAs encoding for enzymes of the ECS were also expressed in LLC-PK1. WIN (10⁻⁷ M) and HP (10⁻⁶ M) altered Na⁺ re-absorption in LLC-PK1 in a dual manner. They both acutely (after 1 min) increased Na⁺/K⁺-ATPase activity in a TRPV1 antagonist-sensitive way. WIN''s stimulating effect persisted for 30 min, and this effect was partially blocked by a CB₁ antagonist or a PKC inhibitor. In contrast, HP inhibited Na⁺/K⁺-ATPase after 30 min incubation, and this effect was attenuated by a CB₁ antagonist or a PKA inhibitor.

Conclusion and Implications

The ECS is expressed in LLC-PK1 cells. Both CB₁ receptors and TRPV1 channels regulate Na⁺/K⁺-ATPase activity in these cells, and are modulated by lipid and peptide CB₁ receptor ligands, which act via different signalling pathways.     

Metergoline inhibits the neuronal Navl.2 voltage- dependent Na^＋ channels expressed in Xenopus oocytes

Aim： Metergoline is an ergot-derived psychoactive drug that acts as a ligand for serotonin and dopamine receptors. The aim of this study was to investigate the regulatory effects of metergoline on the neuronal Nav1.2 voltage-dependent Na^＋ channels in vitro. Methods： Xenopus oocytes were injected with cRNAs encoding rat brain Nav1.2 α and β1 subunits. Voltage-activated Na^＋ currents were recorded using two-electrode voltage clamp technique. Drugs were applied though perfusion. Results： Both metergoline and lidocaine reversibly and concentration-dependently inhibited the peak of Na^＋ currents with IC50 values of 3.6±4.2 and 916.9±98.8 μmol/L, respectively. Metergoline （3 pmol/L） caused a 6.8±1.2 mV depolarizing shift of the steady-state activation curve of the Na^＋ currents, and did not alter the inactivation curve. In contrast, lidocaine （3 μmol/L） caused a 12.7±1.2 mV hyperpolarizing shift of the inactivation curve of the Na^＋ currents without changing the steady-state activation curve. Both metergoline and lidocaine produced tonic and use-dependent inhibition on the peak of Na^＋ currents. Conclusion： Metergoline exerts potent inhibition on the activity of neuronal Nav1.2 channels, which may contribute to its actions on the central nervous system.