Membrane Potential Of Muscle Cells Research Articles

Pulmonary Fibrosis Augments CGRP-Dependent Hyperpolarization and Vasodilation of Mouse Pulmonary Arteries

Calcitonin gene related peptide (CGRP) hyperpolarizes membrane potential (Vm) of pulmonary arterial smooth muscle cells (SMCs) and endothelial cells (ECs) through PKA-dependent activation of KATP channels. CGRP can diminish the severity of pulmonary fibrosis (PF), however, the effects on vascular signaling were poorly defined. We hypothesized that hyperpolarization to CGRP would be augmented in a mouse model of PF. PF was induced by intratracheal delivery of bleomycin (3 wk), and saline was used as a control (sham). Pulmonary arteries (PAs; 100-150 μm diameter) from male C57BL/6J mice (age, ~5 mo), were cannulated, pressurized to 16 cmH2O, and studied at 37°C. Alternatively, intact endothelial tubes were studied at 32°C following dissociation of SMCs. Vm was recorded continuously using intracellular microelectrodes. At rest, Vm was similar for SMCs (bleomycin = -43±3 mV, sham = -44±3 mV; n = 5/group) and ECs (bleomycin = -45±4 mV, sham = -44±3 mV; n=5/group). Increasing concentrations of CGRP (10−10-10−6 M) evoked hyperpolarization of Vm with greater potency in SMCs (EC50: bleomycin = 2 nM, sham = 15 nM) and ECs (EC50: bleomycin = 3 nM, sham = 8 nM) from PF mice, without changing effcacy (SMCs: bleomycin ΔVm = -24±4 mV, sham ΔVm = -21±3 mV; ECs: bleomycin ΔVm = -26±2 mV, sham ΔVm=23±4 mV). Consistently, vasodilation to CGRP in PAs preconstricted with uridine triphosphate (UTP, 5 μM; EC50) was enhanced in PF (92±6% maximum dilation;) vs. sham (83±4% maximum dilation) mice (n=5/group). Greater vasodilation and hyperpolarization of SMCs to CGRP persisted in endothelium-disrupted PAs and during treatment with L-NAME (10−4 M) indicating that ECs are not required for greater responsiveness to CGRP in SMCs. With no effect on resting Vm, inhibition of KATP channels with 1 μM glibenclamide or PKA with protein kinase inhibitor [PKI-(14-22)-amide, myristoylated; 1 μM] significantly attenuated (p<0.05) hyperpolarization of SMCs and ECs (ΔVm ≤5 mV for all groups). Both glibenclamide and PKI attenuated vasodilation to CGRP in PAs and eliminated differences between groups. In a mouse model of PF, CGRP-dependent hyperpolarization of pulmonary arterial SMCs and ECs is augmented through increased PKA-dependent activation of KATP channels leading to increased vasodilator sensitivity. AHA CDA#931652. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Minimal model for the dependence of stresses in the wall of a cerebral vessel on the parameters of a smooth muscle cell

A minimal mathematical model of the wall of a small arterial vessel is described. This model is created based on the published results of experiments performed on rat cerebral vessels. It is assumed that the active stress has only a circumferential component and depends on the circumferential stretch, calcium concentration in the cytoplasm, and the membrane potential of smooth muscle cells. The presented model for a small artery can qualitatively reproduce the results of more sophisticated models for other vessels under normal physiological conditions. Unlike a similar model, that accounts for only one cellular parameter, the addition of membrane potential as one of the main parameters was crucial to reveal a qualitative change in the dependency of circumferential stress on stretch and the radial coordinate with an alteration in vascular tone. At fixed values of membrane potential and calcium concentration in the phase of a development of vascular tone, stress decreases as it approaches the outer wall of the vessel and increases as stretch increases, and after it is formed, the direction of changes in the circumferential stress reverses.

Physiological functions and pathological involvement of ion channel trafficking in the vasculature.

A variety of ion channels regulate membrane potential and calcium influx in arterial smooth muscle and endothelial cells to modify vascular functions, including contractility. The current (I) generated by a population of ion channels is equally dependent upon their number (N), open probability (Po) and single channel current (i), such that I=N.PO .i. A conventional view had been that ion channels traffic to the plasma membrane in a passive manner, resulting in a static surface population. It was also considered that channels assemble with auxiliary subunits prior to anterograde trafficking of the multimeric complex to the plasma membrane. Recent studies have demonstrated that physiological stimuli can regulate the surface abundance (N) of several different ion channels in arterial smooth muscle and endothelial cells to control arterial contractility. Physiological stimuli can also regulate the number of auxiliary subunits present in the plasma membrane to modify the biophysical properties, regulatory mechanisms and physiological functions of some ion channels. Furthermore, ion channel trafficking becomes dysfunctional in the vasculature during hypertension, which negatively impacts the regulation of contractility. The temporal kinetics of ion channel and auxiliary subunit trafficking can also vary depending on the signalling mechanisms and proteins involved. This review will summarize recent work that has uncovered the mechanisms, functions and pathological modifications of ion channel trafficking in arterial smooth muscle and endothelial cells.

A Novel Role of Uricosuric Agent Benzbromarone in BK Channel Activation and Reduction of Airway Smooth Muscle Contraction.

The uricosuric drug benzbromarone, widely used for treatment of gout, hyperpolarizes the membrane potential of airway smooth muscle cells, but how it works remains unknown. Here we show a novel role of benzbromarone in activation of large conductance calcium-activated K+ channels. Benzbromarone results in dose-dependent activation of macroscopic big potassium (BK) currents about 1.7- to 14.5-fold with an EC50 of 111 μM and shifts the voltage-dependent channel activation to a more hyperpolarizing direction about 10 to 54 mV in whole-cell patch clamp recordings. In single-channel recordings, benzbromarone decreases single BKα channel closed dwell time and increases the channel open probability. Coexpressing β1 subunit also enhances BK activation by benzbromarone with an EC50 of 67 μM and a leftward shift of conductance-voltage (G-V) curve about 44 to 138 mV. Site-directed mutagenesis reveals that a motif of three amino acids 329RKK331 in the cytoplasmic linker between S6 and C-terminal regulator of potassium conductance (RCK) gating ring mediates the pharmacological activation of BK channels by benzbromarone. Further ex vivo assay shows that benzbromarone causes reduction of tracheal strip contraction. Taken together, our findings demonstrate that uricosuric benzbromarone activates BK channels through molecular mechanism of action involving the channel RKK motif of S6-RCK linker. Pharmacological activation of BK channel by benzbromarone causes reduction of tracheal strip contraction, holding a repurposing potential for asthma and pulmonary arterial hypertension or BK channelopathies. SIGNIFICANCE STATEMENT: We describe a novel role of uricosuric agent benzbromarone in big potassium (BK) channel activation and relaxation of airway smooth muscle contraction. In this study, we find that benzbromarone is an activator of the large-conductance Ca2+- and voltage-activated K+ channel (BK channel), which serves numerous cellular functions, including control of smooth muscle contraction. Pharmacological activation of BK channel by the FDA-approved drug benzbromarone may hold repurposing potential for treatment of asthma and pulmonary arterial hypertension or BK channelopathies.

Ca2+-Activated K+ Channels and the Regulation of the Uteroplacental Circulation.

Adequate uteroplacental blood supply is essential for the development and growth of the placenta and fetus during pregnancy. Aberrant uteroplacental perfusion is associated with pregnancy complications such as preeclampsia, fetal growth restriction (FGR), and gestational diabetes. The regulation of uteroplacental blood flow is thus vital to the well-being of the mother and fetus. Ca2+-activated K+ (KCa) channels of small, intermediate, and large conductance participate in setting and regulating the resting membrane potential of vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) and play a critical role in controlling vascular tone and blood pressure. KCa channels are important mediators of estrogen/pregnancy-induced adaptive changes in the uteroplacental circulation. Activation of the channels hyperpolarizes uteroplacental VSMCs/ECs, leading to attenuated vascular tone, blunted vasopressor responses, and increased uteroplacental blood flow. However, the regulation of uteroplacental vascular function by KCa channels is compromised in pregnancy complications. This review intends to provide a comprehensive overview of roles of KCa channels in the regulation of the uteroplacental circulation under physiological and pathophysiological conditions.

Deciphering the potential role of Maca compounds prescription influencing gut microbiota in the management of exercise-induced fatigue by integrative genomic analysis.

A growing number of nutraceuticals and cosmeceuticals have been utilized for millennia as anti-fatigue supplements in folk medicine. However, the anti-fatigue mechanism underlying is still far from being clearly explained. The aim of the study is to explore the underlying mechanism of the Maca compound preparation (MCP), a prescription for management of exercise-induced fatigue. In this study, mice weight-loaded swimming test was used to evaluate the anti-fatigue effect of MCP. MCP significantly improved the forelimb grip strength and Rota-rod test in behavioral tests via regulating energy metabolism. 16S rDNA sequencing results showed MCP can regulate the intestinal flora at the genus level by increasing several beneficial bacteria (i.e., Lactobacillus, Akkermansia and etc.), and decreasing the harmful bacteria (i.e., Candidatus_Planktophila and Candidatus_Arthromitus), where notable high relevance was observed between the fatigue-related biomarkers and fecal microbiota. The results of microbial function analysis suggested that MCP might improve exercise-induced fatigue by enhancing energy metabolism, carbohydrate and lipid metabolism and metabolism of terpenoids and polyketides and breakdown of amino acid metabolism. In addition, and H2O2-induced oxidative stress model on C2C12 cells was employed to further validate the regulation of MCP on energy metabolisms. MCP pre-treatment significantly reduced intracellular ROS accumulation, and increased glycogen content, ATP generation capacity and mitochondrial membrane potential of skeletal muscle cells, as well as conferred anti-cell necrosis ability. In conclusion, MCP plays a key role in regulating fatigue occurrence in exercising and gut microbiota balance, which may be of particular importance in the case of manual workers or sub-healthy populations.

ProBDNF and Brain-Derived Neurotrophic Factor Prodomain Differently Modulate Acetylcholine Release in Regenerating and Mature Mouse Motor Synapses.

The effects of brain-derived neurotrophic factor (BDNF) processing by-products (proBDNF and BDNF prodomain) on the activity of mouse neuromuscular junctions (NMJs) were studied in synapses formed during the reinnervation of extensor digitorum longus muscle (m. EDL) and mature synapses of the diaphragm. The parameters of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were analyzed in presence of each of the BDNF maturation products (both – 1 nM). In newly formed NMJs, proBDNF caused an increase in the resting membrane potential of muscle fibers and a decrease in the frequency of MEPPs, which was prevented by tertiapin-Q, a G-protein-coupled inwardly rectifying potassium channels (GIRK) blocker but not by p75 receptor signaling inhibitor TAT-Pep5. proBDNF had no effect on the parameters of EPPs. BDNF prodomain in newly formed synapses had effects different from those of proBDNF: it increased the amplitude of MEPPs, which was prevented by vesamicol, an inhibitor of vesicular acetylcholine (ACh) transporter; and reduced the quantal content of EPPs. In mature NMJs, proBDNF did not influence MEPPs parameters, but BDNF prodomain suppressed both spontaneous and evoked ACh release: decreased the frequency and amplitude of MEPPs, and the amplitude and quantal content of EPPs. This effect of the BDNF prodomain was prevented by blocking GIRK channels, by TAT-Pep5 or by Rho-associated protein kinase (ROCK) inhibitor Y-27632. At the same time, the BDNF prodomain did not show any inhibitory effects in diaphragm motor synapses of pannexin 1 knockout mice, which have impaired purinergic regulation of neuromuscular transmission. The data obtained suggest that there is a previously unknown mechanism for the acute suppression of spontaneous and evoked ACh release in mature motor synapses, which involves the activation of p75 receptors, ROCK and GIRK channels by BDNF prodomain and requires interaction with metabotropic purinoreceptors. In general, our results show that both the precursor of BDNF and the product of its maturation have predominantly inhibitory effects on spontaneous and evoked ACh release in newly formed or functionally mature neuromuscular junctions, which are mainly opposite to the effects of BDNF. The inhibitory influences of both proteins related to brain neurotrophin are mediated via GIRK channels of mouse NMJs.

Peripheral Coupling Sites Formed by STIM1 Govern the Contractility of Vascular Smooth Muscle Cells

Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs). However, the mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal‐interaction molecule 1), a single transmembrane‐domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER‐PM junctions in response to depletion of ER Ca2+ stores and initiates store‐operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR‐PM interacting sites were decreased and SR‐dependent Ca2+ signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen‐inducible, SMC‐specific STIM1‐knockout (Stim1‐smKO) mice. VSMCs from Stim1‐smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+‐release sites on the SR and Ca2+‐activated ion channels on the PM, accompanied by diminished channel activity. Stim1‐smKO mice were hypotensive and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 – independent of SR Ca2+ store depletion – is critically important for stable peripheral coupling in contractile VSMCs.

Potassium Channels in the Transition from Fetal to the Neonatal Pulmonary Circulation.

The transition from the fetal to the neonatal circulation includes dilatation of the pulmonary arteries (PA) and closure of the Ductus Arteriosus Botalli (DAB). The resting membrane potential and various potassium channel activities in smooth muscle cells (SMC) from fetal and neonatal PA and DAB obtained from the same species has not been systematically analyzed. The key issue addressed in this paper is how the resting membrane potential and the whole-cell potassium current (IK) change when PASMC or DABSMC are transitioned from hypoxia, reflecting the fetal state, to normoxia, reflecting the post-partal state. Patch-clamp measurements were employed to characterize whole-cell K+ channel activity in fetal and post-partal (newborn) PASMC and DABSMC. The main finding of this paper is that the SMC from both tissues use a similar set of K+ channels (voltage-dependent (Kv), calcium-sensitive (KCa), TASK-1 and probably also TASK-2 channels); however, their activity level depends on the cell type and the oxygen level. Furthermore, we provide the first evidence for pH-sensitive non-inactivating K+ current in newborn DABSMC and PASMC, suggesting physiologically relevant TASK-1 and TASK-2 channel activity, the latter particularly in the Ductus Arteriosus Botalli.

STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells.

Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs). The mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal interaction molecule 1), a single-transmembrane-domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER-PM junctions in response to depletion of ER Ca2+ stores and initiates store-operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR-PM interacting sites were decreased, and SR-dependent Ca2+-signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen-inducible, SMC-specific STIM1-knockout (Stim1-smKO) mice. VSMCs from Stim1-smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+-release sites on the SR and Ca2+-activated ion channels on the PM, accompanied by diminished channel activity. Stim1-smKO mice were hypotensive, and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 - independent of SR Ca2+ store depletion - is critically important for stable peripheral coupling in contractile VSMCs.

Tokishakuyakusan ameliorates lowered body temperature after immersion in cold water through the early recovery of blood flow in rats

Ethnopharmacological relevance‘Cold feeling’ is a subjective feeling of unusual coldness that aggravates fatigue, stiffness, and other symptoms, thereby reducing quality of life. Tokishakuyakusan (TSS) is a Kampo medicine reported to improve cold feeling and is used to treat symptoms aggravated by cold feeling. However, the mechanism of action of TSS is unclear. Cold feeling may involve reduced blood flow and subsequent inhibition of heat transport. Therefore, elucidating the effects of TSS on blood flow is one of the most important research topics for clarifying the mechanism of action of TSS. Aim of the studyWe aimed to evaluate the effect of TSS on recovery from lowered body temperature by the immersion of rats in cold water and to clarify the involvement of blood flow in the action of TSS. Materials and methodsAfter female Wistar rats underwent 9 days of low room temperature stress loading (i.e. room temperature of 18 °C), they were subjected to immersion in cold water (15 °C) for 15 min. Body surface temperature, rectal temperature, and plantar temperature were measured before and after immersion in cold water. Blood flow was measured before and after immersion in cold water without low room temperature stress loading. TSS (0.5 g/kg or 1 g/kg) or the vehicle (i.e. distilled water) was orally administered once daily for 10 days for the measurement of body temperature or once 30 min before immersion in cold water for the measurement of blood flow. In addition, we examined the effect of TSS on calcitonin gene-related peptide (CGRP) release from dorsal root ganglion (DRG) cells, the effect of TSS ingredients on transient receptor potential (TRP) channels, and the effect of TSS ingredients on the membrane potential of vascular smooth muscle cells and evaluated the mechanism of the effects of TSS on blood flow. ResultsBody temperature and blood flow decreased after immersion in cold water and then recovered over time. A comparison of body temperature at each timepoint or area under the curve showed that TSS (1 g/kg) accelerated the recovery of body surface temperature, rectal temperature, and blood flow. TSS significantly increased CGRP release from DRG cells, which disappeared after pretreatment with HC-030031 (a transient receptor potential ankyrin 1 [TRPA1] antagonist). The effects of seven TSS ingredients on TRP channels were examined. The agonistic effect on TRPA1 was observed for atractylodin, atractylodin carboxylic acid and levistolide A. Among the TSS ingredients, atractylodin carboxylic acid had significant hyperpolarising effects. ConclusionsThe mechanism by which TSS accelerates the recovery of lowered body temperature in rats after immersion in cold water may involve the acceleration of the recovery of lowered blood flow. Increased CGRP release from DRG cells by TSS, TRPA1 activation by TSS ingredients, and membrane potential changes in vascular smooth muscle cells caused by TSS ingredients are part of the mechanism of action of TSS. These findings may partly contribute to the interpretation of the beneficial effects of TSS on cold feeling.

SNPs for Genes Encoding the Mitochondrial Proteins Sirtuin3 and Uncoupling Protein 2 Are Associated With Disease Severity, Type 2 Diabetes, and Outcomes in Patients With Pulmonary Arterial Hypertension and This Is Recapitulated in a New Mouse Model Lacking Both Genes.

BackgroundIsolated loss‐of‐function single nucleotide polymorphisms (SNPs) for SIRT3 (a mitochondrial deacetylase) and UCP2 (an atypical uncoupling protein enabling mitochondrial calcium entry) have been associated with both pulmonary arterial hypertension (PAH) and insulin resistance, but their collective role in animal models and patients is unknown.Methods and ResultsIn a prospective cohort of patients with PAH (n=60), we measured SNPs for both SIRT3 and UCP2, along with several clinical features (including invasive hemodynamic data) and outcomes. We found SIRT3 and UCP2 SNPs often both in the same patient in a homozygous or heterozygous manner, correlating positively with PAH severity and associated with the presence of type 2 diabetes and 10‐year outcomes (death and transplantation). To explore this mechanistically, we generated double knockout mice for Sirt3 and Ucp2 and found increasing severity of PAH (mean pulmonary artery pressure, right ventricular hypertrophy/dilatation and extensive vascular remodeling, including inflammatory plexogenic lesions, in a gene dose‐dependent manner), along with insulin resistance, compared with wild‐type mice. The suppressed mitochondrial function (decreased respiration, increased mitochondrial membrane potential) in the double knockout pulmonary artery smooth muscle cells was associated with apoptosis resistance and increased proliferation, compared with wild‐type mice.ConclusionsOur work supports the metabolic theory of PAH and shows that these mice exhibit spontaneous severe PAH (without environmental or chemical triggers) that mimics human PAH and may explain the findings in our patient cohort. Our study offers a new mouse model of PAH, with several features of human disease that are typically absent in other PAH mouse models.

Acid-sensing ion channel 1 contributes to pulmonary arterial smooth muscle cell depolarization following hypoxic pulmonary hypertension.

Pulmonary hypertension is characterized by sustained vasoconstriction and remodelling of the small pulmonary arteries, which is associated with persistent depolarization of the resting membrane potential (Em ) of pulmonary arterial smooth muscle cells (PASMCs). It is well-known that the underlying mechanism of this depolarization includes inhibition of K+ channels; however, whether other ion channels contribute to this depolarization is unknown. We previously reported that acid-sensing ion channel 1 (ASIC1), a non-selective cation channel (NSCC) that conducts both Na+ and Ca2+ , is present in PASMCs and contributes to the development of chronic hypoxia (CH)-induced pulmonary hypertension. Therefore, we tested the hypothesis that ASIC1-mediated Na+ influx contributes to PASMC Em regulation following CH-induced pulmonary hypertension. Using sharp electrode intracellular recordings in isolated, pressurized small pulmonary arteries from rats and mice, we show that exposure to CH leads to PASMC membrane depolarization compared with control animals, and this is independent of intraluminal pressure-induced depolarization. In addition to a decrease in PASMC whole-cell K+ currents following CH, we demonstrate that whole-cell NSCC currents are increased and essential to the persistent CH-induced Em depolarization in PASMCs. Both the specific inhibitor of ASIC1, psalmotoxin 1, and global knockout of ASIC1 (Asic1-/- ) prevents CH-induced Em depolarization and largely inhibits whole-cell NSCC currents, without affecting whole-cell K+ currents. Our results show a combination of factors, including inhibition of K+ efflux and augmented Na+ influx, mediate CH-induced PASMC depolarization. Furthermore, this study demonstrates a novel role for ASIC1 in the regulation of Em in PASMCs during CH-induced pulmonary hypertension. KEY POINTS: In pulmonary hypertensive patients and animal models of pulmonary hypertension, the resting membrane potential (Em ) of pulmonary arterial smooth muscle cells (PASMCs) is persistently depolarized. In addition to the well-established reduction of K+ conductance, we show that non-selective cation channel currents are increased and essential to the persistent Em depolarization in PASMCs following chronic hypoxia (CH)-induced pulmonary hypertension. The current study provides novel evidence that acid-sensing ion channel 1 (ASIC1)-mediated Na+ influx induces membrane depolarization and regulates Em in PASMCs following CH exposure. Although fairly quiescent under control conditions, our findings demonstrate a pathological function of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension.

Network Pharmacology/Metabolomics-Based Validation of AMPK and PI3K/AKT Signaling Pathway as a Central Role of Shengqi Fuzheng Injection Regulation of Mitochondrial Dysfunction in Cancer-Related Fatigue.

Chinese herbal medicines have multiple targets and properties, and their use in multidisciplinary cancer therapies has consequently received increasing attention. Here, we have investigated the possible active ingredients associated with cancer-related fatigue (CRF) in the Shengqi Fuzheng Injection (SFI). In vitro cell models were used to measure the regulation effects of SFI on CRF. Metabolomic analysis was used to identify the potential genes and pathways in C2C12 mouse myoblasts treated with SFI, and the interaction of compounds and CRF targets was predicted using network pharmacology and molecular docking analyses. The putative pathways were further verified using immuno-blotting assays. The results showed that SFI significantly inhibited muscle cell apoptosis and increased the mitochondrial membrane potential of muscle cells. The network pharmacology analysis results identified 36 candidate compounds, and 244 potential targets were yielded by SFI, and they shared 10 key targets associated with cancer-related fatigue. According to the enrichment analysis and experimental validation, SFI might ameliorate muscle cell mitochondrial function by activating AMPK and inhibiting the PI3K/Akt signaling pathways, and the expression changes of mitochondrial metabolic enzymes MnSOD and apoptosis-associated proteins Bax and Bcl-2 were also triggered. The functions and mechanisms of SFI in anticancer-related fatigue were found here to be at least partly due to the targeting of the AMPK and PI3K/Akt signaling pathways, and this has highlighted new potential applications for network pharmacology when researching Chinese Medicines.

Mechanisms of K ATP channel Activation by Metabolic Stress in Mediating Lymphatic Contractile Dysfunction

Lymphatic contractile dysfunction has been implicated in the sub-clinical edema observed in multiple disease states, including obesity, metabolic syndrome and inflammatory disease. The strong positive correlation between the upregulation or hyperactivation of KATP channels in lymphatic muscle and lymphatic contractile dysfunction suggests that KATP channel activation be a common cause of the contractile dysfunction. We investigated the role of KATP channels in lymphatic muscle in response to decreases in the intracellular ATP/ADP ratio and the increased generation of reactive oxygen species (ROS), both of which occur in metabolic diseases. We analyzed spontaneous lymphatic contractions and smooth muscle cell membrane potentials in isolated, pressurized popliteal lymphatic vessels from mice. Exposure of lymphatic vessels from WT mice to the mitochondrial complex inhibitor (antimycin, 30 nM) or the oxidative phosphorylation inhibitor (CCCP, 1 µM) led to an attenuated or complete block of lymphatic pumping, which was rescued by the KATP channel inhibitor glibenclamide (GLIB, 1 µM). In contrast, lymphatic vessels from Kir6.1-/- mice were resistant to antimycin and CCCP. Antimycin inhibited spontaneous action potentials in lymphatic muscle from WT vessels and treatment of GLIB rescued action potential generation. Antimycin increased DHR fluorescence in WT lymphatic vessels, confirming the production of ROS. The ROS scavengers, tiron (1 mM) and catalase (250 U/ml) blocked the increase in DHR fluorescence to antimycin. After pretreatment with tiron (1 mM), WT lymphatic vessels were resistant to the effects of antimycin, and there were no further changes in response to GLIB, suggesting that KATP channels were activated by ROS production. Similar results were found for catalase (250 U/ml) treatment. However, neither tiron nor catalase blocked the inhibitory effect of CCCP on lymphatic pumping. Based on these results, we propose that KATP channels in lymphatic muscle are activated by directly by metabolic stress or indirectly by ROS generation, leading to the inhibition of spontaneous lymphatic contractions and lymphatic contractile dysfunction.

Hypoxia and metabolic inhibitors alter the intracellular ATP:ADP ratio and membrane potential in human coronary artery smooth muscle cells.

ATP-sensitive potassium (KATP) channels couple cellular metabolism to excitability, making them ideal candidate sensors for hypoxic vasodilation. However, it is still unknown whether cellular nucleotide levels are affected sufficiently to activate vascular KATP channels during hypoxia. To address this fundamental issue, we measured changes in the intracellular ATP:ADP ratio using the biosensors Perceval/PercevalHR, and membrane potential using the fluorescent probe DiBAC4(3) in human coronary artery smooth muscle cells (HCASMCs). ATP:ADP ratio was significantly reduced by exposure to hypoxia. Application of metabolic inhibitors for oxidative phosphorylation also reduced ATP:ADP ratio. Hyperpolarization caused by inhibiting oxidative phosphorylation was blocked by either 10 µM glibenclamide or 60 mM K+. Hyperpolarization caused by hypoxia was abolished by 60 mM K+ but not by individual K+ channel inhibitors. Taken together, these results suggest hypoxia causes hyperpolarization in part by modulating K+ channels in SMCs.

Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug

In addition to antibacterial and antitumor effects, synthetic ruthenium complexes have been reported to inhibit several medicinally important enzymes, including acetylcholinesterase (AChE). They may also interact with muscle-type nicotinic acetylcholine receptors (nAChRs) and thus affect the neuromuscular transmission and muscle function. In the present study, the effects of the organometallic ruthenium complex of 5-nitro-1,10-phenanthroline (nitrophen) were evaluated on these systems. The organoruthenium-nitrophen complex [(η6-p-cymene)Ru(nitrophen)Cl]Cl; C22H21Cl2N3O2Ru (C1-Cl) was synthesized, structurally characterized and evaluated in vitro for its inhibitory activity against electric eel acetylcholinesterase (eeAChE), human recombinant acetylcholinesterase (hrAChE), horse serum butyrylcholinesterase (hsBChE) and horse liver glutathione-S-transferase. The physiological effects of C1-Cl were then studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations, by means of single twitch measurements and electrophysiological recordings. The compound C1-Cl acted as a competitive inhibitor of eeAChE, hrAChE and hsBChE with concentrations producing 50 % inhibition (IC50) of enzyme activity ranging from 16 to 26 μM. Moreover, C1-Cl inhibited the nerve-evoked isometric muscle contraction (IC50 = 19.44 μM), without affecting the directly-evoked muscle single twitch up to 40 μM. The blocking effect of C1-Cl was rapid and almost completely reversed by neostigmine, a reversible cholinesterase inhibitor. The endplate potentials were also inhibited by C1-Cl in a concentration-dependent manner (IC50 = 7.6 μM) without any significant change in the resting membrane potential of muscle fibers up to 40 μM. Finally, C1-Cl (5–40 μM) decreased (i) the amplitude of miniature endplate potentials until a complete block by concentrations higher than 25 μM and (ii) their frequency at 10 μM or higher concentrations. The compound C1-Cl reversibly blocked the neuromuscular transmission in vitro by a non-depolarizing mechanism and mainly through an action on postsynaptic nAChRs. The compound C1-Cl may be therefore interesting for further preclinical testing as a new competitive neuromuscular blocking, and thus myorelaxant, drug.

A sex-specific, COX-derived/thromboxane receptor activator causes depolarization and vasoconstriction in male mice mesenteric resistance arteries.

We investigated whether sex differences exist in cyclooxygenase-dependent effects on membrane potential and relaxation in mice mesenteric resistance arteries. Mesenteric small arteries of 9- to 12-week-old, male and female, wild-type mice, db/+ mice and diabetic db/db mice were mounted in myographs for measurements of isobaric diameter and smooth muscle cell membrane potential. Acetylcholine caused smaller dilation of arteries from male db/+ mice compared to arteries from female db/+ mice. In the presence of the NO synthase inhibitor Nω -nitro-L-arginine methyl ester (L-NAME), acetylcholine-induced dilation of arteries from males increased in the presence of indomethacin and the COX-1-specific inhibitor FR122047. The presence of indomethacin was also associated with a more negative membrane potential in arteries from males. In arteries from db/db mice, no sex differences were seen. In arteries from male but not female wild-type mice, the thromboxane receptor blocker SQ29,548 increased relaxation to acetylcholine. In contrast to arteries from female mice, COX (most likely COX-1)-derived prostanoids and activation of thromboxane receptors counteract acetylcholine vasodilatation probably through increased smooth muscle depolarization in arteries from male mice. In mice with diabetes and pronounced endothelial dysfunction, inhibition of COX did not enhance acetylcholine vasodilatation.

Efficacy, potency and kinetics of activation process of Kv7.4 and Kv7.5 channel with novel pharmacological activators targeted for Lower Urinary Tract Syndrome (LUTS)

Among several types of Kv7 (KCNQ) voltage gated potassium channel family, Kv7.4 (KCNQ4) and Kv7.5 (KCNQ5) channels show prominent expression in smooth muscle, cardiac tissues and vasculature. In general, activation of the channel would mediate strong efflux of potassium and lead membrane potential to hyperpolarization, though the extent may depend on established potassium concentration gradient and membrane potential. Once membrane potential is hyperpolarized, it would decrease the excitability of excitable cells. Decreased excitability may reduce calcium spikes and Ca‐CaM mediated activation of myosin light chain kinase (MLCK) in smooth muscle cells. Recently, it was reported that Kv7.4 channels are highly expressed in corpus cavernosum smooth muscle cells and Kv7.5 channels in prostate smooth muscle cells, respectively. Since smooth muscle relaxation of corpus cavernosum and smooth muscle tension in prostate are in the center of pathophysiology of erectile dysfunction and LUTS, we examined the effect of novel Kv7.4 and Kv7.5 activators to the channel activation process.In HEK293 cells expressing human Kv7.4 or Kv7.5 channels, we performed whole‐cell patch clamp technique with two different voltage protocols in order to measure specific activity of the channel. For kinetic analysis of the activation process, voltage steps from –100 mV to +100 mV with 10 mV of step interval was applied. For each step, pulse duration was 3 seconds for activation process of Kv7.4 and Kv7.5 channels is rather slow. Intersweep duration was 10 seconds and holding potential was –80 mV. For measurement of time‐dependent action of the drug, 10 seconds single step with +50 mV was applied in every 30s with holding potential of –80 mV. The composition of internal solution is as following; 120 KCl, 5.37 CaCl2, 1.75 MgCl2, 10 EGTA, 10 HEPES, 4 ATP, 0.2 GTP. pH was adjusted to 7.3 using KOH and [Ca2+]free = 100 nM. Normal tyrode solution was used for external solution and drugs stocked with DMSO was diluted to corresponding concentrations with the Normal tyrode solution.We have found that URO‐K10, a novel activator, increases not only maximum conductance of the channel but also time constant of activation gate. In comparison to positive control, ML‐213, a well‐known Kv7.4 channel activator, URO‐K10 showed significant supremacy. EC50 of URO‐K10 was 0.304 ± 0.058 μM for Kv7.4 channel which is smaller than EC50 of ML‐213 (0.8 ± 0.3 μM). URO‐K10 not only increased the maximum conductance of the channel but also left‐shifted activation curve in terms of Boltzmann distribution. Half‐maximal voltage (V1/2) of Kv7.4 channel without any of the drugs was −43.03 mV whereas V1/2 of URO‐K10 activated channel was −67.65 mV, which is near the resting membrane potential of smooth muscle cells. ML‐213 activated channels showed V1/2 of −51.91 mV.Support or Funding InformationThis work was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information & Communication Technology), and Future Planning (MSIP) of the Korean Government (2018R1A41023822 to I. So) and by the Education and Research Encouragement Fund of Seoul National University Hospital (I. So). Jung Eun Lee was supported by the BK plus program from the MSIP.

The Participation of Endocannabinoid Receptors in the Regulation of Spontaneous Synaptic Activity at Neuromuscular Junctions of Mice

The mechanisms underlying changes in spontaneous release of acetylcholine (ACh) induced by exogenous agonists and antagonists of endocannabinoid receptors were investigated by recording miniature endplate potentials (MEPPs) in motor synapses of mouse diaphragm preparations with intracellular microelectrodes. There have not been observed any statistically significant changes either in the membrane potential of muscle fibers or in the frequency, amplitude, and time parameters of the MEPPs under the action of AM-251 (1 μM), antagonist/inverse agonist of CB1 receptors, for 60 min. Activation of CB receptors by their exogenous agonist WIN 55,212-2 (WIN) (20 μM) for 1 h did not cause statistically significant changes in the amplitude of the MEPPs. However, MEPP frequency increased by more than 50% as compared to the control under the action of WIN for 30–60 min. Blocking of CB1 receptors by AM-251 (1 μM) prevented the WIN-induced increase in MEPP frequency. A protein kinase A inhibitor H-89 (1 μM) did not prevent the enhancement of the ACh secretion upon activation of CB receptors by WIN. WIN lost the ability to increase MEPP frequency in the presence of a phospholipase C inhibitor U73122 (5 μM), which did not affect by itself the amplitude and temporal characteristics or MEPP frequency. A protein kinase C inhibitor, chelerythrin (4 μM) or ryanodine, which blocks the ryanodine receptors (RyRs) at a concentration of 5 μM, also prevented the WIN-induced increase in MEPP frequency. Thus, a signaling cascade triggered by stimulation of presynaptic CB1 receptors with subsequent activation of phospholipase C, protein kinase C, and the release of stored calcium, leading to an increase in the frequency of spontaneous quantal release of ACh, has been detected for the first time in mouse motor synapses. This suggests the presence of CB1 receptors on the presynaptic membrane in mouse motor nerve terminals and the possibility of activation of these receptors by endocannabinoids for potentiating the spontaneous secretion of ACh.