Current-clamp Mode Research Articles

Abstract 074: Empagliflozin Improves Impaired Sensitivity Of Afferent Neurons With Renal Axons

Afferent renal nerve pathways likely play a role in salt sensitive hypertension. We recently reported that high salt diet (HS) impairs these afferent renal pathways in rats. Now we tested the hypothesis that during HS a decrease in sensitivity of renal afferent neurons is prevented by the SGLT2 inhibitor empagiflozin.Respective groups of rats were put on HS containing 8% NaCl or a normal diet. Two groups (HS, controls) received empagiflozin 20 mg/kg BW/day orally. Renal neurons were retrogradely labeled with DiI. In culture, labeled dorsal root ganglion neurons (DRG Th11-L2) with renal afferents were investigated electrophysiologically using current clamp mode to assess action potential generation during current injection. Neurons were characterized as tonic highly active (> 5 action potentials, AP) and phasic less active neurons (≤ 5 AP upon stimulation. )In neurons from rats on HS, the relation of tonic highly active neurons to less active phasic neurons shifted consistently towards phasic units (63,8% tonic neurons in controls vs. 42%* on HS, *p<0.05, z-test). However, continuous treatment with empagiflozin preserved the proportion of tonic neurons as in controls (67,9% on HS with concomitant administration of empagiflozin). In controls, empagiflozin did not affect the proportion of tonic to phasic neurons (63,8% tonic neurons in controls vs. 67,9% on HS & empagliflozin, p=0.7, z-test). Blood pressure and heart rate were not altered by HS and/or treatment with any chosen dose of empagiflozin.In rats, chronically elevated sodium intake (8% NaCl) reduced the sensitivity and stimulability of renal afferent DRG neurons. Under these circumstances, concomitant treatment with the SGLT2 inhibitor empagiflozin preserved the function of renal afferent DRG neurons. SGLT 2 inhibitors may help to treat dysfunction of renal innervation in cardiovascular disease.

Afterhyperpolarization potential modulated by local [K+]o in K+ diffusion-restricted extracellular space in the central clock of suprachiasmatic nucleus

BackgroundIntercellular coupling is essential for the suprachiasmatic nucleus (SCN) to serve as a coherent central clock. Synaptic release of neurotransmitters and neuropeptides is critical for synchronizing SCN neurons. However, intercellular coupling via non-synaptic mechanisms has also been demonstrated. In particular, the abundant perikaryal appositions with morphological specializations in the narrow extracellular space (ECS) may hinder molecular diffusion to allow for ion accumulation or depletion. MethodsThe SCN neurons were recorded in the whole-cell current-clamp mode, with pipette filled with high (26 mM)-Na+ or low (6 mM)-Na+ solution. ResultsCells recorded with high-Na+ pipette solution could fire spontaneous action potentials (AP) with peak AHP more negative than the calculated value of K+ equilibrium potential (EK) and with peak AP more positive than calculated ENa. Cells recorded with low-Na+ pipette solution could also have peak AHP more negative than calculated EK. In contrast, the resting membrane potential (RMP) was always less negative to calculated EK. The distribution and the averaged amplitude of peak AHP, peak AP, or RMP was similar between cells recorded with high-Na+ and low-Na+ solution pipette. In a number of cells, the peak AHP could increase from more positive to become more negative than calculated EK spontaneously or after treatments to hyperpolarize the RMP. TTX blocked the Na+ -dependent APs and tetraethylammonium (TEA), but not Ba2+ or Cd2+, markedly reduced the peak AHP. Perforated-patch cells could also but rarely fire APs with peak AHP more negative than calculated EK. ConclusionThe result of peak AHP negative to calculated EK indicates that local [K+]o sensed by the TEA-sensitive AHP K+ channels must be lower than bulk [K+]o, most likely due to K+ clearance from K+ diffusion-restricted ECS by the Na+/K+-ATPase. The K+ diffusion-restricted ECS may allow for K+-mediated ionic interactions among neurons to regulate SCN excitability.

Pharmacological activation of exchange protein directly activated by cAMP 1 (EPAC1) increases action potential duration in mouse atrial myocytes

Atrial Fibrillation (AF) is the most common sustained cardiac arrhythmia worldwide. Several mechanisms are known to be involved in AF development and sustainability, such as abnormal Ca2+ cycling and action potential (AP) duration. Molecules involved in these mechanisms are potential relevant therapeutic candidates. One of these candidates is the Exchange Protein directly Activated by cAMP 1 (EPAC1), which has been recently shown to increase arrhythmogenic activity in ventricular cardiomyocytes and to be related to AF in an EPAC1-/- mouse model. Although strong evidence suggests that EPAC1 is involved in cardiac arrhythmia in mice, its role in AF has not been fully investigated. To investigate the effect of EPAC1 pharmacological activation in the development of atrial arrhythmogenic activity. Atrial myocytes from EPAC1-/- and Wild Type (WT) mice were dissociated by enzymatic digestion. AP were recorded using the whole cell configuration of the patch clamp technique in the current clamp mode. To assess the selective role of EPAC1, EPAC proteins were acutely activated by superfusion of 8-CPT-AM, a preferential EPAC1 activator (10 μM) and inhibited by superfusion of AM-001 compound, an EPAC1 non-competitive selective inhibitor (20 μM). Activation of EPAC1 by 8-CPT-AM drastically increased AP duration at 90% of repolarization (APD90) by 106.6 ± 14.3% (vs control; p < 0.0001) in WT atrial myocytes. This effect was attenuated by AM-001 perfusion (APD90 decreased by 38.9 ± 8.3% vs 8-CPT-AM; P < 0.05). Interestingly, EPAC1 deleted cardiomyocytes displayed a lower APD90 increase (+42.3 ± 15.1% vs control; P < 0.01) after treatment by 8-CPT-AM than that of WT atrial myocytes ( P < 0.05). Concomitant treatment of EPAC-/- cells with 8-CPT-AM and AM-001 failed to influence the effect on APD90 compared to 8-CPT-AM alone ( P = 0.85). Our results show that EPAC1 activation is involved in the modulation of AP duration in mouse atrial myocytes. In addition, our data suggest that EPAC1 could participate in AF initiation and/or sustainability by promoting an arrhythmogenic ectopic activity. Therefore, EPAC1 may constitute a therapeutic target in AF management.

Systemic administration of lipopolysaccharide induces hyperexcitability of prelimbic neurons via modulation of sodium and potassium currents

In C57BL/6 J mice, systemic inflammation was induced by administering bacterial LPS (1 mg/kg) intraperitoneally. In response, animals exhibited hypokinesia, piloerection, and a slight decrease in body temperature accompanied by increased serum levels of the proinflammatory cytokine TNF-α. 24 h after the immunogenic challenge, acute cortical slices were prepared, and whole-cell patch-clamp recordings were performed in morphologically identified prelimbic neurons of the mice’s prefrontal cortex. Electrophysiologic alterations included changes in the kinetics parameters of the fast-inactivating sodium and slow-inactivating potassium currents. In current-clamp mode, our recordings revealed alterations in several conductances that shape the intrinsic excitability of prelimbic neurons. The action potential exhibited changes in latency, amplitude, and the rheobase current to elicit firing discharge. Likewise, phase plots of the action potentials uncovered alterations in the repetitive firing of prelimbic neurons. Consistent with these changes, the afterhyperpolarization conductance and the slowly decaying, calcium‐dependent after-hyperpolarization current that follows an action potential were decreased in response to systemic LPS. Our data demonstrate that immune activation alters the ionic currents that shape the intrinsic excitability and predicts dysregulation of non-synaptic forms of neuronal plasticity modulated by the intrinsic excitability of prefrontal cortex neurons.

Makatoxin-3, a thermostable Nav1.7 agonist from Buthus martensii Karsch (BmK) scorpion elicits non-narcotic analgesia in inflammatory pain models

Ethnopharmacological relevanceChronic pain management represents a serious healthcare problem worldwide. The use of opioid analgesics for pain has always been hampered by their side effects; in particular, the addictive liability associated with chronic use. Finding a morphine replacement has been a long-standing goal in the field of analgesia. In traditional Chinese medicine, processed Buthus martensii Karsch (BmK) scorpion has been used as a painkiller to treat chronic inflammatory arthritis and spondylitis, so called “Scorpio-analgesia”. However, the molecular basis and the underline mechanism for the Scorpio-analgesia are still unclear. Aim of the studyThe study aims to investigate the molecular basis of “Scorpio analgesia” and identify novel analgesics from BmK scorpion. Materials and methodsIn this study, the analgesic abilities were determined using formalin-, acetic acid- and complete Freund's adjuvant-induced pain models. The effect of BmK venom and processed BmK venom on Nav1.7 were detected by whole-cell voltage-clamp recordings on HEK293-hNav1.7 stable cell line. Action potentials in Dorsal root ganglion (DRG) neurons induced by Makatoxin-3-R58A were recorded in current-clamp mode. The content of Makatoxin-3 was detected using competitive enzyme-linked immunosorbent assay based on the Makatoxin-3 antibody. High performance liquid chromatography, western blot and circular dichroism spectroscopy were used to analysis the stability of Makatoxin-3. ResultsHere we demonstrate that Makatoxin-3, an α-like toxin in BmK scorpion venom targeting Nav1.7 is the critical component in Scorpio-analgesia. The analgesic effect of Makatoxin-3 could not be reversed by naloxone and is more potent than Nav1.7-selective inhibitors and non-steroidal anti-inflammatory drugs in inflammatory models. Moreover, a R58A mutant of Makatoxin-3 is capable of eliciting analgesia effect without inducing pain response. ConclusionsThis study advances ion channel biology and proposes Nav1.7 agonists, rather than the presumed Nav1.7-only blockers, for non-narcotic relief of chronic pain.

Eugenosedin-A improves obesity-related hyperglycemia by regulating ATP-sensitive K+ channels and insulin secretion in pancreatic β cells

Eugenosedin-A (Eu-A) has been shown to protect against hyperglycemia- and hyperlipidemia-induced metabolic syndrome. We investigated the relationship of KATP channel activities and insulin secretion by Eu-A in vitro in pancreatic β-cells, and examined the effect of Eu-A on streptozotocin (STZ)/nicotinamide (NA)-induced type 2 diabetes mellitus (T2DM) in vivo. We isolated pancreatic islets from adult male Wistar rats (250-350g) and identified pancreatic β-cells by the cell size, capacitance and membrane potential. Perforated patch-clamp and inside-out recordings were used to monitor the membrane potential (current-clamp mode) and channel activity (voltage-clamp mode) of β-cells. The membrane potential of β-cells was raised by Eu-A and reversed by the KATP channel activator diazoxide. Eu-A inhibited the KATP channel activity measured at -60mV and increased the intracellular calcium concentration ([Ca2+]i), resulting in enhanced insulin secretion. Eu-A also reduced Kir6.2 protein on the cell membrane and scattered in the cytosol under normal glucose conditions (5.6mM). In our animal study, rats were divided into normal and STZ/NA-induced T2DM groups. Normal rats fed with regular chow were divided into control and control+Eu-A (5mg/kg/day, i.p.) groups. The STZ/NA-induced diabetic rats fed with a high-fat diet (HFD) were divided into three groups: T2DM, T2DM+Eu-A (5mg/kg/day, i.p.), and T2DM+glibenclamide (0.5mg/kg/day, i.p.; a KATP channel inhibitor). Both Eu-A and glibenclamide decreased the rats' blood glucose, prevented weight gain, and enhanced insulin secretion. We found that Eu-A blocked pancreatic β-cell KATP channels, caused membrane potential depolarization, and stimulated Ca2+ influx, thus increasing insulin secretion. Furthermore, Eu-A decreased blood glucose and increased insulin levels in T2DM rats. These results suggested that Eu-A might have clinical benefits for the control of T2DM and its complications.

Melatonin receptor blockade slows ventricular conduction by IK1 downregulation

Abstract Background Melatonin has been reported to prevent life-threatening arrhythmias in myocardial ischemia and reperfusion. The recent study from our group demonstrated that the antiarrhythmic action of melatonin was associated with enhanced recovery of resting membrane potential (RMP) and shortened activation time delay in the affected myocardium. This effect was independent of melatonin antioxidative properties, which raises a question about the involvement of melatonin receptor-dependent signaling in the electrophysiological action of melatonin. The aim of this study was to evaluate a role of MT1/MT2 melatonin receptors in the regulation of RMP and ventricular conduction velocity (CV). Methods Anesthetized male rats underwent coronary occlusion (5 min) and reperfusion (5 min). 5 min before ischemia induction the animals were intravenously injected with saline (control, n=7), melatonin (4 mg/kg, n=8), luzindole (MT1/MT2 receptor blocker, 0.4 mg/kg, n=6), and melatonin/luzindole combination (4 and 0.4 mg/kg, respectively, n=7). CV was measured in baseline and reperfusion by epicardial 64-lead mapping under epicardial pacing. In the enzymatically isolated ventricular myocytes (n=20 cells), voltage- and current-clamp modes of whole cell patch-clamp were used for measurements of IK1 and INa currents, and RMP, respectively. Melatonin (10 uM), luzindole (1 uM) and their combination were added to the bath solution. Results Luzindole application decreased baseline longitudinal CV as compared to placebo (52±3 vs 42±2 cm/s, p=0.028, respectively). Paired comparisons demonstrated that longitudinal CV decreased at reperfusion vs baseline in the control group (35±2 vs 53±6 cm/s, p=0.018) and in the luzindole group (33±4 vs 38±3 cm/s, p=0.039), whereas no significant differences were observed in the melatonin and melatonin/luzindole groups (40±5 vs 51±3 cm/s, p=0.114 and 39±3 vs 43±3 cm/s, p=0.498, respectively). Neither melatonin nor luzindole changed INa current characteristics (Figure, panels a and b). The average amplitude of IK1 current was 2.08±0.92 at −50 mV and −16.11±4.70 pA/pF at −120 mV in the normal Tyrode solution. Luzindole significantly reduced IK1 amplitude to 0.82±0.48 (p=0.028) and −5.6±1.6 (p=0.008) pA/pF at −50 and −120 mV, respectively. Addition of melatonin to the luzindole-containing solution partly abolished the luzindole effect (Figure, panel c). Luzindole also reduced RMP from −63±10 mV to −37±12 mV (p=0.013) and caused an increase in APD100 duration from 35±6 ms to 170±43 ms (p=0.003) (Figure, panel d). Conclusion Thus, MT1/MT2 receptor block with luzindole did not affect INa but led to the down-regulation of IK1 and depolarization of RMP. These effects can reduce accessibility of sodium channels and be the basis for the observed luzindole-dependent CV slowing. The obtained data support the significance of melatonin receptor-dependent signaling in the realization of antiarrhythmic effects of melatonin. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Russian Science Foundation

The suitability of high throughput automated patch clamp for physiological applications.

Although automated patch clamp (APC) devices have been around for many years and have become an integral part of many aspects of drug discovery, high throughput instruments with gigaohm seal data quality are relatively new. Experiments where a large number of compounds are screened against ion channels are ideally suited to high throughput APC, particularly when the amount of compound available is low. Here we evaluate different APC approaches using a variety of ion channels and screening settings. We have performed a screen of 1920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPR. Additionally, we tested the effect of 36 arthropod venoms on NaV 1.9 using a single 384-well plate on the SyncroPatch 384. As an example for mutant screening, a range of acid-sensing ion channel variants were tested and the success rate increased through fluorescence-activated cell sorting (FACS) prior to APC experiments. Gigaohm seal data quality makes the 384-format accessible to recording of primary and stem cell-derived cells on the SyncroPatch 384. We show recordings in voltage and current clamp modes of stem cell-derived cardiomyocytes. In addition, the option of intracellular solution exchange enabled investigations into the effects of intracellular Ca2+ and cAMP on TRPC5 and HCN2 currents, respectively. Together, these data highlight the broad applicability and versatility of APC platforms and also outlines some limitations of the approach. KEY POINTS: High throughput automated patch clamp (APC) can be used for a variety of applications involving ion channels. Lower false positive rates were achieved using automated patch clamp versus a fluorometric imaging plate reader (FLIPR) in a high throughput compound screen against NMDA receptors. Genetic variants and mutations can be screened on a single 384-well plate to reduce variability of experimental parameters. Intracellular solution can be perfused to investigate effects of ions and second messenger systems without the need for excised patches. Primary cells and stem cell-derived cells can be used on high throughput APC with reasonable success rates for cell capture, voltage clamp measurements and action potential recordings in current clamp mode.

Вплив тромбіну у плазматичному іонному середовищі на розвиток епілептиформної активності культивованих нейронів гіпокампа щурів

The mechanisms of epileptiform neuronal activity develop- ment under blood-brain barrier (BBB) dysfunction remains relevant in modern psychoneurology. In the present work we mimic some effects of BBB disruption in the culture of hip- pocampal neurons to examined the effect of serum-adapted ionic environment on the impulse activity of hippocampal neurons and the role of serum protein thrombin in induction of epileptiform neuronal activity. Using the whole-cell patch- clamp method under current-clamp mode we analyzed the spontaneous action potentials (AP) in the single hippocampal neurons. The changing of ionic extracellular neuronal environ- ment to such serum-adapted contributed to the development of epileptiform tonic activity of cultured hippocampal neurons and led to increase the average APs frequency by 65.1 ± 17.9% (n = 5) in neurons with spontaneous firing activity (FA) and to occurrence of tonic electrical activity (1.65 ± 0.4 s-1) in neurons without firing activity. Glutamate NMDA receptors significantly contribute to epileptiform tonic activity formation in neurons with FA, while their role in tonic activity providing in neurons without FA was insignificant. Thrombin (5 U/ml) in the serum-adapted ionic solution significantly enhanced of epileptiform activity in neurons with and without spontaneous FA: APs frequency increased in these neuronal groups by 117.3 ± 25.6% (n = 3) and by 61.8 ± 11.5% (n = 3), respective- ly, compared with that in the serum-adapted ionic solution only. Blockade of thrombin protease activated receptor 1 (PAR-1) by application of SCH 79797 (10 μm) canceled the thrombin’s effect in neurons without spontaneous FA, and significantly reduced such in neurons with FA. Therefore, the change of ionic extracellular neuronal environment to serum-adapted stimulates the occurrence of epileptiform activity in hippo- campal neurons, that is apparently associated with NMDA- receptors activation in neurons with FA. The proepileptiform action of thrombin was mostly mediated by PAR-1 activation. Thrombin-dependent regulation of the hippocampal single neurons firing activity involves the mechanisms different from the modulation of glutamate NMDA receptors in these cells.

A method of marking neurons on brain slices using patch clamp technique

Objective: To introduce a method of marking neurons using patch clamp technique. Methods: The brain slices of the target area was cut with a vibrating microtome. The glass microelectrode was perfused with the electrode liquid containing NeurobiotinTM Tracer, and the whole-cell patch-clamp recording was performed. After recording, the brain slices were fixed and rinsed with 4% paraformaldehyde. After stained in phosphate buffer with Streptavidin-Texas Red and Triton X-100 for at least 2 hours, the neurons can be observed under a fluorescence microscope. Results: The cell membrane voltage was clamped at -70 mV, and the neuron showed a gradually increasing membrane current after step stimulation. When recording in the current clamp mode, the step stimulus caused the neuron to depolarize to the threshold potential and then burst into action potentials. The morphology of intact neurons with clear cell body and protrusions of a neuron could be observed under a fluorescence microscope. Conclusion: This method is suitable for observing the morphological features of the recorded neuron after patch clamp experiments, which is easy to operate, and the image is intuitive and clear.

Increased sarcolemma chloride conductance as one of the mechanisms of action of carbonic anhydrase inhibitors in muscle excitability disorders

To get insight into the mechanism of action of carbonic anhydrase inhibitors (CAI) in neuromuscular disorders, we investigated effects of dichlorphenamide (DCP) and acetazolamide (ACTZ) on ClC-1 chloride channels and skeletal muscle excitability. We performed patch-clamp experiments to test drugs on chloride currents in HEK293T cells transfected with hClC-1. Using the two-intracellular microelectrode technique in current-clamp mode, we measured the effects of drugs on the resting chloride conductance and action potential properties of sarcolemma in rat and mouse skeletal muscle fibers. Using BCECF dye fluorometry, we measured the effects of ACTZ on intracellular pH in single rat muscle fibers. Similarly to ACTZ, DCP (100 μM) increased hClC-1 chloride currents in HEK cells, because of the negative shift of the open probability voltage dependence and the slowing of deactivation kinetics. Bendroflumethiazide (BFT, 100 μM), structurally related to DCP but lacking activity on carbonic anhydrase, had little effects on chloride currents. In isolated rat muscle fibers, 50–100 μM of ACTZ or DCP, but not BFT, induced a ~ 20% increase of the resting chloride conductance. ACTZ reduced action potential firing in mouse muscle fibers. ACTZ (100 μM) reduced intracellular pH to 6.8 in rat muscle fibers. These results suggest that carbonic anhydrase inhibitors can reduce muscle excitability by increasing ClC-1 channel activity, probably through intracellular acidification. Such a mechanism may contribute in part to the clinical effects of these drugs in myotonia and other muscle excitability disorders.

Accelerated Production and Maturation of Human Induced Pluripotent Stem Cell Derived Nociceptors

The scarce availability and prohibitive costs of primary human DRG tissues limits the scalability of in vitro models for pain drug discovery and disease modeling. Human induced pluripotent stem cell (hiPSC)-derived sensory neurons provide a near-limitless amount of tissue for these applications. Using an accelerated, step-wise, directed differentiation protocol that proceeds through a novel ‘primal ectoderm’ population, we are able to manufacture nociceptors rapidly and at scale by relying on a simplified chemically-defined basal medium, recombinant growth factors and small molecules. Following production and only one week of maturation, these nociceptors have a consistent molecular phenotype and express key therapeutic pain targets such as human voltage-gated sodium channels Nav1.7, 1.8, and 1.9, which are primarily responsible for eliciting nociceptor action potentials, as well as the human capsaicin receptor TRPV1. Using whole-cell patch clamp electrophysiology, we demonstrate that these neurons fire action potentials and possess burst-firing ability by day 9 in vitro as assessed by performing stepwise depolarizing current injections in current clamp mode. We also show these neurons exhibit inward and outward currents in response to voltage steps in voltage clamp mode. We have also demonstrated that these neurons are responsive to ligands such as capsaicin using calcium imaging region-of-interest analysis. Greater than 30% of neuronal cell bodies respond to 100 nM capsaicin following an additional 2 to 3 weeks of maturation. Importantly, this response was ablated by capsazepine, thus confirming response specificity. These data demonstrate that hiPSC nociceptors have the potential to screen and identify novel analgesic or anti-nociceptive compounds for use in humans.

K V 7 Channel Subtype Expression and Physiological Roles in Rat Urinary Bladder Smooth Muscle Excitability and Contractility

Introduction Voltage-gated K+ (KV) channels, specifically type 7 (KV7), are emerging key regulators of urinary bladder smooth muscle (UBSM) excitability and contractility. The KV7 channel family consists of 5 pore-forming α-subunits (KV7.1–KV7.5) capable of assembling as homotetrameric (e.g. KV7.4) or heterotetrameric (e.g. KV7.4/KV7.5) channels; thus, further increasing species/tissue expression diversity and regulatory potential. However, much remains unknown about KV7 channel properties in UBSM and the translational value of the common experimental animal model - the rat. Hypothesis KV7 channels, especially homomeric or heteromeric KV7.4 and KV7.5 subtypes, regulate rat UBSM excitability and contractility. Methods UBSM tissue without mucosa was obtained from adult male Sprague Dawley rats. UBSM homogenates were used for Western blots to detect KV7 channel subtypes with or without surface biotinylation. UBSM isometric tension recordings were carried out in the presence of tetrodotoxin for spontaneous, 20 mM K+-induced and 1 µM carbachol-induced contractions or in its absence for the electrical field stimulated (EFS) contractions. For electrophysiological amphotericin-B perforated patch-clamp studies, single UBSM cells were prepared using enzymatic dissociation. UBSM cell membrane potential was recorded in current-clamp (I=0) mode at room temperature. The following KV7 channel modulators were tested: XE991 (KV7.1-KV7.5 subtype blocker), retigabine (KV7.2-KV7.5 activator), and ICA-069673 (selective activator for KV7.2/KV7.3, which at higher concentrations preferentially enhances KV7.4 and KV7.4/KV7.5 but not KV7.5 channels). Results Western blot experiments detected KV7.4 and KV7.5 channels in UBSM under both non- and surface-biotinylation assay conditions. The latter condition revealed that for both KV7.4 and KV7.5 subtypes surface localization predominated (>75 %). In isometric tension experiments, retigabine attenuated contractility in a concentration-dependent manner for all four contraction protocols (EC50 values: 2.6-58.7 µM for the amplitude and force, with complete inhibition of contractility at 100 µM, n=3-10). XE991 (10 µM) depolarized the membrane potential of isolated UBSM cells (Δ10.5±3.6 mV, n=5, p<0.05). In contrast, retigabine (10 µM, Δ-6.5±1.8 mV, n=4, p<0.04) and ICA-069673 (10 µM, Δ-11.5±2.4 mV, n=4, p<0.02), when applied separately, induced UBSM cell hyperpolarization. In the presence of XE991, ICA-069673 (10 µM) did not significantly change the membrane potential of isolated UBSM cells (Δ0.4±1.2 mV, n=4, p>0.05). Conclusions Our data show that both KV7.4 and KV7.5 channel subtypes are expressed in rat UBSM and display preferential cell membrane localization. Pharmacological modulation of KV7 channels can control UBSM cell excitability and tissue contractility. The hyperpolarizing effect of ICA-069673, which is blocked by XE991, revealed a critical role of KV7.4 homomeric and/or KV7.4/KV7.5 heteromeric channels in UBSM function.

AFFERENT RENAL NERVE PATHWAYS IN RATS – RESPONSIVENESS TO HIGH SALT INTAKE

Objective: Afferent renal nerve pathways are likely involved in the development of salt sensitive hypertension. We recently reported that intrarenal NaCl elicited a long-lasting sympatho-depression via a neuro-humoral TRPV1 dependent and tachykinin mediated renal afferent nerve pathway. We now wanted to test the hypothesis that high sodium intake impairs this afferent sympatho-depressory mechanism. Design and method: Respective groups were put on tap water, 0.9 % saline for drinking or chow containing 8% NaCl. Cultured dorsal root ganglion neurons (DRG Th11-L2) of rats with renal afferents were investigated in current clamp mode to assess action potential generation during current injection. Rats were equipped with femoral catheters for blood pressure (BP) &amp; heart rate (HR) assessment, drug application, a renal arterial catheter for intrarenal administration (IRA) of NaCl boli (10 % NaCl, 10 microl) or Capsaicin (CAP 10 ∗10-7 M, 10 microl) and a bipolar electrode for renal sympathetic nerve activity (RSNA) recordings; eventually an intravenous (iv) bolus of the NK1-receptor blocker RP67580 (10∗10-3 M, 15 microl) was administered. Results are mean ± SEM. Results: In neurons from rats on high salt diet, but not on 0.9 % saline or controls the relation of tonic highly active neurons to less active neurons shifted towards less active units. (62% tonic neurons in the control group and 63% tonic neurons in the saline group vs. 40%∗ tonic neurons in the high salt group, ∗ p &lt; 0.05, z-test, mean +/-SEM). However, cultured renal neurons from rats on 0,9% saline or on high salt diet exhibited increased action potential production upon stimulation (controls 13,3+/11,03 APs/600ms vs. 0.9% saline 19,8+/-2,33∗ APs/600ms vs. high salt diet 22,2+/-4,54 APs/600ms, ∗ p &lt; 0.05, t-test, mean+/-SEM). 10% NaCl boli IRA induced decreases of RSNA from baseline 4.1 ± 0.6 microV∗sec to 2.2 ± 0.8 microV∗sec impaired in rats on 8% NaCl. Conclusions: In rats on high salt diet highly active tonic neurons with renal afferents in vitro decreased at the expense of less active phasic neurons in spite of tonic neurons producing more action potentials upon stimulation. The sodium inducible long-lasting sympatho-depression via a neuro-humoral tachykinin mediated afferent renal nerve pathway was eventually impaired.

Neurogenic substance P\u2014influences on action potential production in afferent neurons of the kidney?

We recently showed that a substance P (SP)–dependent sympatho-inhibitory mechanism via afferent renal nerves is impaired in mesangioproliferative nephritis. Therefore, we tested the hypothesis that SP released from renal afferents inhibits the action potential (AP) production in their dorsal root ganglion (DRG) neurons. Cultured DRG neurons (Th11-L2) were investigated in current clamp mode to assess AP generation during both TRPV1 stimulation by protons (pH 6) and current injections with and without exposure to SP (0.5 µmol) or CGRP (0.5 µmol). Neurons were classified as tonic (sustained AP generation) or phasic (≤ 4 APs) upon current injection; voltage clamp experiments were performed for the investigation of TRPV1-mediated inward currents due to proton stimulation. Superfusion of renal neurons with protons and SP increased the number of action potentials in tonic neurons (9.6 ± 5 APs/10 s vs. 16.9 ± 6.1 APs/10 s, P < 0.05, mean ± SD, n = 7), while current injections with SP decreased it (15.2 ± 6 APs/600 ms vs. 10.2 ± 8 APs/600 ms, P < 0.05, mean ± SD, n = 29). Addition of SP significantly reduced acid-induced TRPV1-mediated currents in renal tonic neurons (− 518 ± 743 pA due to pH 6 superfusion vs. − 82 ± 50 pA due to pH 6 with SP superfusion). In conclusion, SP increased action potential production via a TRPV1-dependent mechanism in acid-sensitive renal neurons. On the other hand, current injection in the presence of SP led to decreased action potential production. Thus, the peptide SP modulates signaling pathways in renal neurons in an unexpected manner leading to both stimulation and inhibition of renal neuronal activity in different (e.g., acidic) environmental contexts.

Effect of etomidate on the reuniens neuronal activity.

To explore the effect of etomidate on the neuronal activity of ventral thalamic reuniens nucleus and the underlying mechanisms. Whole-cell patch clamp method was used to explore the effect of etomidate on the activity of ventral thalamic reuniens neurons in the acute brain slices obtained from 4-5 weeks old C57BL/6J mice. The electrophysiological characteristics of ventral thalamic reuniens neurons were recorded in the current clamp mode, and then the effects of etomidate (0.5, 2.0, 8.0 μmol/L etomidate groups) and intralipid (intralipid group) on the discharge frequency and membrane potential of ventral thalamic reuniens neurons were recorded. During the experiment, the ventral thalamic reuniens neuron firing rates (RNFRs) were recorded as FB, FD and Fw before, after administration, and after elution; and the membrane potential was recorded as MPB and MPD before, after administration. The chlorine channel of gamma-amino butyric acid Type A (GABAA) receptor was blocked with 100 μmol/L picrotoxin (PTX). The RNFRs were recorded as FBS, FETO and FETO+PTX before, after perfusing etomidate with sub-anesthesia concentration (0.5 μmol/L) and after perfusing both PTX and etomidate. In the intralipid group, there was no significant difference among the FB, FD and Fw (P>0.05). But in the etomidate groups (0.5, 2.0, 8.0 μmol/L), the FD was less than the FB, there was significant difference (all P<0.01); the Fw was higher than the FD, there was significant difference (all P<0.05). Moreover, there was significant difference in the inhibitory degree of the RNFRs between the 0.5 μmol/L etomidate group and the 8.0 μmol/L etomidate group (P<0.05). In the experiment to explore the mechanism of etomidate (0.5 μmol/L), the FETO was compared with the FBS, there was significant difference (P<0.01); but when the FETO+PTX was compared with the FBS, there was no significant difference (P>0.05). Etomidate can inhibit the activity of ventral thalamic reuniens neurons in concentration-dependent manner, and which is reversible. Etomidate with sub-anesthetic concentration inhibits the activity of ventral thalamic reuniens neurons via targeting the GABAA receptor.

Isoliquiritigenin, an active ingredient of Glycyrrhiza, elicits antinociceptive effects via inhibition of Nav channels.

Glycyrrhiza extract has been used for the treatment of oral and gastric ulcers, but the analgesic mechanism remains unknown. In the present study, we investigated the effects of isoliquiritigenin, an active ingredient of Glycyrrhiza, on Nav channels in vitro and nociceptive behaviors in vivo. In an autopatch-clamp study, isoliquiritigenin inhibited the currents of Nav1.1, Nav1.3, Nav1.6, Nav1.7, and Nav1.8 in a channel expression system. In small- and medium-sized cultured trigeminal ganglion neurons, the compound suppressed Nav currents in many neurons (78%) and Kv currents in all neurons, dose-dependently. In current-clamp mode, isoliquiritigenin blocked action potential generation in many neurons (64%), but it conversely accelerated action potential generation in the remaining neurons. The opposing effects on action potentials were reproduced in a computational simulation of a modified Hodgkin-Huxley-based model, based on the electrophysiological data. In behavioral experiments, local treatment with isoliquiritigenin suppressed nociceptive behaviors in response to oral ulcer development or nociceptive TRP channel agonists in the oral mucosa and hind paw. These results suggest that isoliquiritigenin exerts an analgesic effect predominantly via inhibitory action on Nav channels on sensory nociceptive fibers. This pharmacological mechanism indicates that isoliquiritigenin is useful for pain relief and provides scientific evidence for Glycyrrhiza at the ingredient level.

Pathological activation of CaMKII induces arrhythmogenicity through TRPM4 overactivation.

TRPM4 is a Ca2+-activated nonselective cation channel involved in cardiovascular physiology and pathophysiology. Based on cellular experiments and numerical simulations, the present study aimed to explore the potential arrhythmogenicity of CaMKII-mediated TRPM4 channel overactivation linked to Ca2+ dysregulation in the heart. The confocal immunofluorescence microscopy, western blot, and proximity ligation assay (PLA) in HL-1 atrial cardiomyocytes and/or TRPM4-expressing TSA201 cells suggested that TRPM4 and CaMKII proteins are closely localized. Co-expression of TRPM4 and CaMKIIδ or a FRET-based sensor Camui in HEK293 cells showed that the extent of TRPM4 channel activation was correlated with that of CaMKII activity, suggesting their functional interaction. Both expressions and interaction of the two proteins were greatly enhanced by angiotensin II treatment, which induced early afterdepolarizations (EADs) at the repolarization phase of action potentials (APs) recorded from HL-1 cells by the current clamp mode of patch clamp technique. This arrhythmic change disappeared after treatment with the TRPM4 channel blocker 9-phenanthrol or CaMKII inhibitor KN-62. In order to quantitatively assess how CaMKII modulates the gating behavior of TRPM4 channel, the ionomycin-permeabilized cell-attached recording was employed to obtain the voltage-dependent parameters such as steady-state open probability and time constants for activation/deactivation at different [Ca2+]i. Numerical simulations incorporating these kinetic data into a modified HL-1 model indicated that > 3-fold increase in TRPM4 current density induces EADs at the late repolarization phase and CaMKII inhibition (by KN-62) completely eliminates them. These results collectively suggest a novel arrhythmogenic mechanism involving excessive CaMKII activity that causes TRPM4 overactivation in the stressed heart.

Ginsenoside Rg3 Alleviates Antithyroid Cancer Drug Vandetanib-Induced QT Interval Prolongation.

Inhibition of human ether-a-go-go-related gene (hERG) potassium channel is responsible for acquired long QT syndromes, which leads to life-threatening cardiac arrhythmia. A multikinase inhibitor, vandetanib, prolongs the progression-free survival time in advanced medullary thyroid cancer. However, vandetanib has been reported to induce significant QT interval prolongation, which limits its clinical application. Some studies have showed that ginsenoside Rg3 decelerated hERG K(+) channel tail current deactivation. Therefore, in this study, we aim to confirm whether ginsenoside Rg3 targeting hERG K(+) channel could be used to reverse the vandetanib-induced QT interval prolongation. Electrocardiogram (ECG) and monophasic action potential (MAP) were recorded using electrophysiology signal sampling and analysis system in Langendorff-perfused rabbit hearts. The current clamp mode of the patch-clamp technique was used to record transmembrane action potential. The whole-cell patch-clamp technique was used to record the hERG K+ current. In Langendorff-perfused hearts, vandetanib prolonged the QT interval in a concentration-dependent manner with an IC50 of 1.96 μmol/L. In human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), vandetanib significantly prolonged the action potential duration at 50%, 70%, and 90% repolarization (APD50, APD70, and APD90). In stable transfected human hERG gene HEK293 cells, vandetanib caused concentrate-dependent inhibition in the step and tail currents of hERG. As expected, ginsenoside Rg3 relieved vandetanib-induced QT interval prolongation in Langendorff-perfused heart and reversed vandetanib-induced APD prolongation in hiPSC-CMs. Furthermore, ginsenoside Rg3 alleviated vandetanib-induced hERG current inhibition and accelerated the process of the channel activation. Ginsenoside Rg3 may be a promising cardioprotective agent against vandetanib-induced QT interval prolongation through targeting hERG channel. These novel findings highlight the therapeutic potential of ginsenoside to prevent vandetanib-induced cardiac arrhythmia.

Antidiabetic compounds 8a, 8b, 8k, and 9h enhance insulin secretion: activity and mechanism.

This study primarily investigated the effects of hypoglycemic compounds (Imeglimin derivatives) on insulin secretion in type 2 diabetes mellitus (T2DM), and further explored the possible mechanism underlying these effects. Firstly, Metformin was used as the initiating compound to synthesize three sets of derivatives which contained Imeglimin structure core. At the cellular level, we screened compounds with better effect on the activity of insulin receptor tyrosine protein kinase (IFcTPK) after the islet β cells were treated with the compounds of different concentrations. The insulin secretion was assessed using radioimmunoassay and the cytotoxicity to islet β cells was evaluated by means of MTT assay following treatment with the compounds. The Ca2+-related mechanism by which these compounds promote insulin secretion was elucidated with whole cell recordings from current-clamp mode. Totally, 48 synthesized compounds were generated, wherein 10 compounds could increase the activity of IFcTPK in HIT-T15 cells better among these compounds. The modified Imeglimin, especially in the structure of hydrophilic hydroxyl or piperidine rings, could improve the activity of the compound to promote insulin secretion. Furthermore, the compounds 8a, 8b, 8k, and 9h revealed high insulin secretion-promoting activity. These compounds enhanced insulin secretion in islet β cells by repressing the ATP-sensitive K(+) and voltage-gated K+ pathway. Our findings indicate that the hypoglycemic compounds 8a, 8b, 8k, and 9h confer better promotive effect on insulin secretion, which provides a reference for the development of drugs with better hypoglycemic activity.