Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 35.360
Filter
1.
World J Microbiol Biotechnol ; 40(7): 231, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38833075

ABSTRACT

To investigate the mechanism of Triton X-100 (TX-100) reducing the Ag+-resistance of Enterococcus faecalis (E. faecalis), and evaluate the antibacterial effect of TX-100 + Ag+ against the induced Ag+-resistant E. faecalis (AREf). The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of AgNO3 against E. faecalis with/without TX-100 were determined to verify the enhanced antibacterial activity. Transmission electron microscopy (TEM) was used to observe the morphological changes of E. faecalis after treatment. The intra- and extracellular concentration of Ag+ in treated E. faecalis was evaluated using inductively coupled plasma mass spectrometer (ICP-MS). The changes in cell membrane potential and integrity of treated E. faecalis were also observed using the flow cytometer. Moreover, AREf was induced through continuous exposure to sub-MIC of Ag+ and the antibacterial effect of TX-100 + Ag+ on AREf was further evaluated. The addition of 0.04% TX-100 showed maximal enhanced antibacterial effect of Ag+ against E. faecalis. The TEM and ICP-MS results demonstrated that TX-100 could facilitate Ag+ to enter E. faecalis through changing the membrane structure and integrity. Flow cytometry further showed the effect of TX-100 on membrane potential and permeability of E. faecalis. In addition, the enhanced antibacterial effect of TX-100 + Ag+ was also confirmed on induced AREf. TX-100 can facilitate Ag+ to enter E. faecalis through disrupting the membrane structure and changing the membrane potential and permeability, thus reducing the Ag+-resistance of E. faecalis and enhancing the antibacterial effect against either normal E. faecalis or induced AREf.


Subject(s)
Anti-Bacterial Agents , Drug Resistance, Bacterial , Enterococcus faecalis , Microbial Sensitivity Tests , Octoxynol , Silver , Enterococcus faecalis/drug effects , Enterococcus faecalis/growth & development , Octoxynol/pharmacology , Anti-Bacterial Agents/pharmacology , Silver/pharmacology , Cell Membrane/drug effects , Membrane Potentials/drug effects , Microscopy, Electron, Transmission , Silver Nitrate/pharmacology
2.
ACS Infect Dis ; 10(6): 2196-2211, 2024 Jun 14.
Article in English | MEDLINE | ID: mdl-38836553

ABSTRACT

The metabolic environment is responsible for antibiotic resistance, which highlights the way in which the antibiotic resistance mechanism works. Here, GC-MS-based metabolomics with iTRAQ-based proteomics was used to characterize a metabolic state in tetracycline-resistant Escherichia coli K12 (E. coli-RTET) compared with tetracycline-sensitive E. coli K12. The repressed pyruvate cycle against the elevation of the proton motive force (PMF) and ATP constructed the most characteristic feature as a consequence of tetracycline resistance. To understand the role of the elevated PMF in tetracycline resistance, PMF inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the pH gradient were used to investigate how the elevation influences bacterial viability and intracellular antibiotic concentration. A strong synergy was detected between CCCP and tetracycline to the viability, which was consistent with increasing intracellular drug and decreasing external pH. Furthermore, E. coli-RTET and E. coli-RGEN with high and low PMF concentrations were susceptible to gentamicin and tetracycline, respectively. The elevated PMF in E. coli-RTET was attributed to the activation of other metabolic pathways, except for the pyruvate cycle, including a malate-oxaloacetate-phosphoenolpyruvate-pyruvate-malate cycle. These results not only revealed a PMF-dependent mechanism for tetracycline resistance but also provided a solution to tetracycline-resistant pathogens by aminoglycosides and aminoglycoside-resistant bacteria by tetracyclines.


Subject(s)
Anti-Bacterial Agents , Membrane Potentials , Tetracycline Resistance , Tetracycline , Anti-Bacterial Agents/pharmacology , Tetracycline/pharmacology , Membrane Potentials/drug effects , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Escherichia coli/drug effects , Escherichia coli K12/drug effects , Proton-Motive Force/drug effects , Microbial Sensitivity Tests , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , Metabolomics , Hydrogen-Ion Concentration , Proteomics
3.
Biochem Biophys Res Commun ; 720: 150105, 2024 Aug 06.
Article in English | MEDLINE | ID: mdl-38754163

ABSTRACT

BACKGROUND: Dexmedetomidine (DEX), a highly selective α2-adrenoceptor agonist, can decrease the incidence of arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the underlying mechanisms by which DEX affects cardiac electrophysiological function remain unclear. METHODS: Ryanodine receptor (RyR2) heterozygous R2474S mice were used as a model for CPVT. WT and RyR2R2474S/+ mice were treated with isoproterenol (ISO) and DEX, and electrocardiograms were continuously monitored during both in vivo and ex vivo experiments. Dual-dye optical mapping was used to explore the anti-arrhythmic mechanism of DEX. RESULTS: DEX significantly reduced the occurrence and duration of ISO-induced of VT/VF in RyR2R2474S/+ mice in vivo and ex vivo. DEX remarkably prolonged action potential duration (APD80) and calcium transient duration (CaTD80) in both RyR2R2474S/+ and WT hearts, whereas it reduced APD heterogeneity and CaT alternans in RyR2R2474S/+ hearts. DEX inhibited ectopy and reentry formation, and stabilized voltage-calcium latency. CONCLUSION: DEX exhibited an antiarrhythmic effect through stabilizing membrane voltage and intracellular Ca2+. DEX can be used as a beneficial perioperative anesthetic for patients with CPVT or other tachy-arrhythmias.


Subject(s)
Arrhythmias, Cardiac , Calcium , Dexmedetomidine , Ryanodine Receptor Calcium Release Channel , Animals , Dexmedetomidine/pharmacology , Ryanodine Receptor Calcium Release Channel/metabolism , Ryanodine Receptor Calcium Release Channel/genetics , Calcium/metabolism , Mice , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/drug therapy , Arrhythmias, Cardiac/genetics , Membrane Potentials/drug effects , Isoproterenol/pharmacology , Tachycardia, Ventricular/metabolism , Tachycardia, Ventricular/genetics , Tachycardia, Ventricular/drug therapy , Anti-Arrhythmia Agents/pharmacology , Male , Action Potentials/drug effects , Mice, Inbred C57BL
4.
J Dent ; 146: 105046, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38729285

ABSTRACT

OBJECTIVES: The high prevalence of antibiotic-resistant bacteria poses a threat to the global public health. The appropriate use of adjuvants to restore the antimicrobial activity of antibiotics against resistant bacteria could be an effective strategy for combating antibiotic resistance. In this study, we investigated the counteraction of Triton X-100 (TX-100) and the mechanisms underlying the antibiotic resistance of Enterococcus faecalis (E. faecalis). METHODS: Standard, wild-type (WT), and induced antibiotic-resistant E. faecalis strains were used in this study. In vitro antibacterial experiments were conducted to evaluate the antimicrobial activities of gentamicin sulfate and ciprofloxacin hydrochloride in the presence and absence of 0.02 % TX-100 against both planktonic and biofilm bacteria. Transcriptomic and untargeted metabolomic analyses were performed to explore the molecular mechanisms of TX-100 as an antibiotic adjuvant. Additionally, membrane permeability, membrane potential, glycolysis-related enzyme activity, intracellular adenosine triphosphate (ATP), and expression levels of virulence genes were assessed. The biocompatibility of different drug combinations was also evaluated. RESULTS: A substantially low TX-100 concentration improved the antimicrobial effects of gentamicin sulfate or ciprofloxacin hydrochloride against antibiotic-resistant E. faecalis. Mechanistic studies demonstrated that TX-100 increased cell membrane permeability and dissipated membrane potential. Moreover, antibiotic resistance and pathogenicity of E. faecalis were attenuated by TX-100 via downregulation of the ABC transporter, phosphotransferase system (PTS), and ATP supply. CONCLUSIONS: TX-100 enhanced the antimicrobial activity of gentamicin sulfate and ciprofloxacin hydrochloride at a low concentration by improving antibiotic susceptibility and attenuating antibiotic resistance and pathogenicity of E. faecalis. CLINICAL SIGNIFICANCE: These findings provide a theoretical basis for developing new root canal disinfectants that can reduce antibiotic resistance.


Subject(s)
Anti-Bacterial Agents , Biofilms , Ciprofloxacin , Drug Resistance, Bacterial , Enterococcus faecalis , Gentamicins , Microbial Sensitivity Tests , Octoxynol , Enterococcus faecalis/drug effects , Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Ciprofloxacin/pharmacology , Gentamicins/pharmacology , Octoxynol/pharmacology , Cell Membrane Permeability/drug effects , Humans , Adenosine Triphosphate/metabolism , Membrane Potentials/drug effects , Glycolysis/drug effects
5.
Brain Res ; 1839: 149008, 2024 Sep 15.
Article in English | MEDLINE | ID: mdl-38761846

ABSTRACT

A synthetic inhibitor of capsaicin-induced TRPV1 channel activation is called capsazepine (CPZ). In this study, we aimed to explore the effects of CPZ on hyperpolarization-activated cationic current (Ih) and voltage-gated Na + current (INa) in pituitary tumor (GH3) cells. Through patch-clamp recordings, we found that CPZ concentration-dependently inhibited Ih amplitude and slowed its activation time course. The IC50 and KD values were 3.1 and 3.16 µM, respectively. CPZ also shifted the steady-state activation curve of Ih towards a more hyperpolarized potential. However, there was no change in the gating charge of the curve. A modified Markovian model predicted the CPZ-induced decrease in the voltage-dependent hysteresis of Ih. CPZ suppressed INa in GH3 cells, without altering its activation or inactivation time course. Additionally, exposure to CPZ reduced spontaneous firing. These findings suggest that CPZ's inhibitory effects on Ih and INa are direct and not dependent on vanilloid receptor binding. This could provide light on an unidentified ionic mechanism influencing the membrane excitability of neurons and endocrine or neuroendocrine cells in vivo.


Subject(s)
Capsaicin , TRPV Cation Channels , TRPV Cation Channels/metabolism , TRPV Cation Channels/antagonists & inhibitors , TRPV Cation Channels/drug effects , Capsaicin/pharmacology , Capsaicin/analogs & derivatives , Animals , Rats , Cell Line, Tumor , Patch-Clamp Techniques , Membrane Potentials/drug effects , Neurons/drug effects , Neurons/metabolism , Action Potentials/drug effects
6.
Acta Physiol (Oxf) ; 240(6): e14151, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38676357

ABSTRACT

AIMS: Ischaemic heart disease remains a significant cause of mortality globally. A pharmacological agent that protects cardiac mitochondria against oxygen deprivation injuries is welcome in therapy against acute myocardial infarction. Here, we evaluate the effect of large-conductance Ca2+-activated K+ channels (BKCa) activator, Compound Z, in isolated mitochondria under hypoxia and reoxygenation. METHODS: Mitochondria from mice hearts were obtained by differential centrifugation. The isolated mitochondria were incubated with a BKCa channel activator, Compound Z, and subjected to normoxia or hypoxia/reoxygenation. Mitochondrial function was evaluated by measurement of O2 consumption in the complexes I, II, and IV in the respiratory states 1, 2, 3, and by maximal uncoupled O2 uptake, ATP production, ROS production, transmembrane potential, and calcium retention capacity. RESULTS: Incubation of isolated mitochondria with Compound Z under normoxia conditions reduced the mitochondrial functions and induced the production of a significant amount of ROS. However, under hypoxia/reoxygenation, the Compound Z prevented a profound reduction in mitochondrial functions, including reducing ROS production over the hypoxia/reoxygenation group. Furthermore, hypoxia/reoxygenation induced a large mitochondria depolarization, which Compound Z incubation prevented, but, even so, Compound Z created a small depolarization. The mitochondrial calcium uptake was prevented by the BKCa activator, extruding the mitochondrial calcium present before Compound Z incubation. CONCLUSION: The Compound Z acts as a mitochondrial BKCa channel activator and can protect mitochondria function against hypoxia/reoxygenation injury, by handling mitochondrial calcium and transmembrane potential.


Subject(s)
Calcium , Mitochondria, Heart , Animals , Mice , Calcium/metabolism , Mitochondria, Heart/metabolism , Mitochondria, Heart/drug effects , Male , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Membrane Potential, Mitochondrial/drug effects , Reactive Oxygen Species/metabolism , Mice, Inbred C57BL , Hypoxia/metabolism , Membrane Potentials/drug effects , Oxygen Consumption/drug effects , Oxygen/metabolism
7.
Cell Physiol Biochem ; 58(2): 172-181, 2024 Apr 20.
Article in English | MEDLINE | ID: mdl-38643508

ABSTRACT

BACKGROUND/AIMS: Extracellular acidic conditions impair cellular activities; however, some cancer cells drive cellular signaling to adapt to the acidic environment. It remains unclear how ovarian cancer cells sense changes in extracellular pH. This study was aimed at characterizing acid-inducible currents in an ovarian cancer cell line and evaluating the involvement of these currents in cell viability. METHODS: The biophysical and pharmacological properties of membrane currents in OV2944, a mouse ovarian cancer cell line, were studied using the whole-cell configuration of the patch-clamp technique. Viability of this cell type in acidic medium was evaluated using the MTT assay. RESULTS: OV2944 had significant acid-sensitive outwardly rectifying (ASOR) Cl- currents at a pH50 of 5.3. The ASOR current was blocked by pregnenolone sulfate (PS), a steroid ion channel modulator that blocks the ASOR channel as one of its targets. The viability of the cells was reduced after exposure to an acidic medium (pH 5.3) but was slightly restored upon PS administration. CONCLUSION: These results offer first evidence for the presence of ASOR Cl- channel in ovarian cancer cells and indicate its involvement in cell viability under acidic environment.


Subject(s)
Cell Survival , Ovarian Neoplasms , Pregnenolone , Animals , Female , Mice , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Cell Line, Tumor , Pregnenolone/pharmacology , Hydrogen-Ion Concentration , Cell Survival/drug effects , Chloride Channels/metabolism , Chloride Channels/antagonists & inhibitors , Patch-Clamp Techniques , Membrane Potentials/drug effects
8.
Nat Commun ; 15(1): 3521, 2024 Apr 25.
Article in English | MEDLINE | ID: mdl-38664456

ABSTRACT

Recently, a novel cyclo-heptapeptide composed of alternating D,L-amino acids and a unique thiazolidine heterocycle, called lugdunin, was discovered, which is produced by the nasal and skin commensal Staphylococcus lugdunensis. Lugdunin displays potent antimicrobial activity against a broad spectrum of Gram-positive bacteria, including challenging-to-treat methicillin-resistant Staphylococcus aureus (MRSA). Lugdunin specifically inhibits target bacteria by dissipating their membrane potential. However, the precise mode of action of this new class of fibupeptides remains largely elusive. Here, we disclose the mechanism by which lugdunin rapidly destabilizes the bacterial membrane potential using an in vitro approach. The peptide strongly partitions into lipid compositions resembling Gram-positive bacterial membranes but less in those harboring the eukaryotic membrane component cholesterol. Upon insertion, lugdunin forms hydrogen-bonded antiparallel ß-sheets by the formation of peptide nanotubes, as demonstrated by ATR-FTIR spectroscopy and molecular dynamics simulations. These hydrophilic nanotubes filled with a water wire facilitate not only the translocation of protons but also of monovalent cations as demonstrated by voltage-clamp experiments on black lipid membranes. Collectively, our results provide evidence that the natural fibupeptide lugdunin acts as a peptidic channel that is spontaneously formed by an intricate stacking mechanism, leading to the dissipation of a bacterial cell's membrane potential.


Subject(s)
Methicillin-Resistant Staphylococcus aureus , Methicillin-Resistant Staphylococcus aureus/drug effects , Molecular Dynamics Simulation , Water/chemistry , Membrane Potentials/drug effects , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Staphylococcus lugdunensis/drug effects , Staphylococcus lugdunensis/chemistry , Staphylococcus lugdunensis/metabolism , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Spectroscopy, Fourier Transform Infrared , Microbial Sensitivity Tests , Nanotubes/chemistry , Antimicrobial Peptides/chemistry , Antimicrobial Peptides/pharmacology
9.
Int J Biol Macromol ; 267(Pt 2): 131581, 2024 May.
Article in English | MEDLINE | ID: mdl-38615866

ABSTRACT

Using Escherichia coli as a model, this manuscript delves into the intricate interactions between dimethyl sulfoxide (DMSO) and membranes, cellular macromolecules, and the effects on various aspects of bacterial physiology. Given DMSO's wide-ranging use as a solvent in microbiology, we investigate the impacts of both non-growth inhibitory (1.0 % and 2.5 % v/v) and slightly growth-inhibitory (5.0 % v/v) concentrations of DMSO. The results demonstrate that DMSO causes alterations in bacterial membrane potential, influences the electrochemical characteristics of the cell surface, and exerts substantial effects on the composition and structure of cellular biomolecules. Genome-wide gene expression data from DMSO-treated E. coli was used to further investigate and bolster the results. The findings of this study provide valuable insights into the complex relationship between DMSO and biological systems, with potential implications in drug delivery and cellular manipulation. However, it is essential to exercise caution when utilizing DMSO to enhance the solubility and delivery of bioactive compounds, as even at low concentrations, DMSO exerts non-inert effects on cellular macromolecules and processes.


Subject(s)
Cell Membrane , Dimethyl Sulfoxide , Escherichia coli , Dimethyl Sulfoxide/pharmacology , Dimethyl Sulfoxide/chemistry , Escherichia coli/drug effects , Cell Membrane/metabolism , Cell Membrane/drug effects , Dose-Response Relationship, Drug , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Macromolecular Substances/pharmacology , Membrane Potentials/drug effects
10.
Pflugers Arch ; 476(5): 809-820, 2024 May.
Article in English | MEDLINE | ID: mdl-38421408

ABSTRACT

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520 AM -loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10 nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30 mV but not - 40 mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20 mV in a manner sensitive to iberiotoxin (100 nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10 µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3 µM) or adenylyl cyclase (SQ22536 100 µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100 nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.


Subject(s)
Large-Conductance Calcium-Activated Potassium Channels , Muscle Contraction , Muscle, Smooth , Parathyroid Hormone-Related Protein , Urinary Bladder , Animals , Rats , Urinary Bladder/metabolism , Urinary Bladder/physiology , Urinary Bladder/drug effects , Parathyroid Hormone-Related Protein/pharmacology , Parathyroid Hormone-Related Protein/metabolism , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle, Smooth/metabolism , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Rats, Sprague-Dawley , Male , Calcium/metabolism , Membrane Potentials/drug effects , Membrane Potentials/physiology
11.
Cell Stress Chaperones ; 28(2): 151-165, 2023 03.
Article in English | MEDLINE | ID: mdl-36653727

ABSTRACT

Endoplasmic reticulum (ER) stress and associated oxidative stress are involved in the genesis and progression of skeletal muscle diseases such as myositis and atrophy or muscle wasting. Targeting the ER stress and associated downstream pathways can aid in the development of better treatment strategies for these diseases with limited therapeutic approaches. There is a growing interest in identifying natural products against ER stress due to the lower toxicity and cost effectiveness. In the present study, we investigated the protective effect of Tangeretin, a citrus methoxyflavone found in citrus peels against Tunicamycin (pharmacological ER stress inducer)-induced ER stress and associated complications in rat skeletal muscle L6 cell lines. Treatment with Tunicamycin for a period of 24 h resulted in the upregulation of ER stress marker proteins, ER resident oxidoreductases and cellular reactive oxygen species (ROS). Co-treatment with Tangeretin was effective in alleviating Tunicamycin-induced ER stress and associated redox-related complications by significantly downregulating the unfolded protein response (UPR), ER resident oxidoreductase proteins, cellular ROS and improving the antioxidant enzyme activity. Tunicamycin also induced upregulation of phosphorylated p38 MAP Kinase and loss of mitochondrial membrane potential. Tangeretin significantly reduced the levels of phosphorylated p38 MAP Kinase and improved the mitochondrial membrane potential. From the results, it is evident that Tangeretin can be explored further as a potential candidate for skeletal muscle diseases involving protein misfolding and ER stress.


Subject(s)
Flavones , Myoblasts, Skeletal , Animals , Rats , Endoplasmic Reticulum Stress/drug effects , Cell Line , Flavones/pharmacology , Dose-Response Relationship, Drug , Oxidative Stress/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism , Mitochondrial Membranes/metabolism , Membrane Potentials/drug effects , Myoblasts, Skeletal/drug effects
12.
Biol Bull ; 245(1): 45-56, 2023 Aug.
Article in English | MEDLINE | ID: mdl-38820288

ABSTRACT

AbstractExtracellular calcium has been known to be required for in situ nematocyst discharge for more than 60 years, yet calcium's role in nematocyst discharge is poorly understood. Currently, we know that extracellular calcium plays at least two distinct roles in in situ nematocyst discharge. First, calcium plays a role in the triggering of discharge by physical contact, most likely involving transient receptor potential channels. Second, activated L-type calcium channels desensitize nematocyst discharge predisposed to discharge by stimulated chemoreceptors for N-acetylated sugars, such as N-acetylneuraminic acid (NANA). It is not known whether the stimulated NANA signaling pathway activates L-type channels electrogenically through membrane depolarization or directly by phosphorylation of the channel. We hypothesize that the activated NANA signaling pathway initiates desensitization by depolarizing cell membrane potentials to activate voltage-gated L-type calcium channels. Consistent with our hypothesis, we show that depolarization induced by blocking voltage-gated potassium channels with 4-aminopyridine selectively activates Ca2+ influx into tentacle ectodermal cells via L-type channels and inhibits in situ nematocyst discharge from chemosensitized anemones. Furthermore, preventing membrane depolarization with valinomycin or hyperpolarizing resting membrane potentials with low-potassium seawater suppresses NANA-induced Ca2+ influx, prevents desensitization of in situ nematocyst discharge, and enhances NANA sensitivity. Thus, changing resting membrane potentials modulates NANA sensitivity, and NANA-induced depolarization drives desensitization. We suggest that desensitization of the NANA signaling pathway occurs by a feedback pathway involving calcium channels that are activated by NANA-induced depolarization. Elucidating the desensitization pathway may suggest methods to protect or prevent public health cases of nematocyst stinging.


Subject(s)
Chemoreceptor Cells , Membrane Potentials , Nematocyst , Sea Anemones , Animals , Sea Anemones/physiology , Membrane Potentials/physiology , Membrane Potentials/drug effects , Nematocyst/physiology , Chemoreceptor Cells/physiology , Chemoreceptor Cells/metabolism , Calcium/metabolism , Calcium Channels, L-Type/metabolism , Signal Transduction
13.
Int J Mol Sci ; 23(4)2022 Feb 15.
Article in English | MEDLINE | ID: mdl-35216258

ABSTRACT

Neuropathic pain is a form of chronic pain arising from damage of the neural cells that sense, transmit or process sensory information. Given its growing prevalence and common refractoriness to conventional analgesics, the development of new drugs with pain relief effects constitutes a prominent clinical need. In this respect, drugs that reduce activity of sensory neurons by modulating ion channels hold the promise to become effective analgesics. Here, we evaluated the mechanical antinociceptive effect of IQM-PC332, a novel ligand of the multifunctional protein downstream regulatory element antagonist modulator (DREAM) in rats subjected to chronic constriction injury of the sciatic nerve as a model of neuropathic pain. IQM-PC332 administered by intraplantar (0.01-10 µg) or intraperitoneal (0.02-1 µg/kg) injection reduced mechanical sensitivity by ≈100% of the maximum possible effect, with ED50 of 0.27 ± 0.05 µg and 0.09 ± 0.01 µg/kg, respectively. Perforated-patch whole-cell recordings in isolated dorsal root ganglion (DRG) neurons showed that IQM-PC332 (1 and 10 µM) reduced ionic currents through voltage-gated K+ channels responsible for A-type potassium currents, low, T-type, and high voltage-activated Ca2+ channels, and transient receptor potential vanilloid-1 (TRPV1) channels. Furthermore, IQM-PC332 (1 µM) reduced electrically evoked action potentials in DRG neurons from neuropathic animals. It is suggested that by modulating multiple DREAM-ion channel signaling complexes, IQM-PC332 may serve a lead compound of novel multimodal analgesics.


Subject(s)
Analgesics/pharmacology , Kv Channel-Interacting Proteins/metabolism , Neuralgia/drug therapy , Neuralgia/etiology , Peripheral Nerve Injuries/complications , Animals , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Hyperalgesia/drug therapy , Hyperalgesia/metabolism , Ligands , Male , Membrane Potentials/drug effects , Neuralgia/metabolism , Peripheral Nerve Injuries/metabolism , Rats , Rats, Sprague-Dawley , Sciatic Nerve/drug effects , Sciatic Nerve/metabolism , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/metabolism
14.
Molecules ; 27(4)2022 Feb 16.
Article in English | MEDLINE | ID: mdl-35209129

ABSTRACT

Excess synaptic glutamate release has pathological consequences, and the inhibition of glutamate release is crucial for neuroprotection. Kaempferol 3-rhamnoside (KR) is a flavonoid isolated from Schima superba with neuroprotective properties, and its effecton the release of glutamate from rat cerebrocortical nerve terminals was investigated. KR produced a concentration-dependent inhibition of 4-aminopyridine (4-AP)-evoked glutamate release with half-maximal inhibitory concentration value of 17 µM. The inhibition of glutamate release by KR was completely abolished by the omission of external Ca2+ or the depletion of glutamate in synaptic vesicles, and it was unaffected by blocking carrier-mediated release. In addition, KR reduced the 4-AP-evoked increase in Ca2+ concentration, while it did not affect 4-AP-evoked membrane potential depolarization. The application of selective antagonists of voltage-dependent Ca2+ channels revealed that the KR-mediated inhibition of glutamate release involved the suppression of P/Q-type Ca2+ channel activity. Furthermore, the inhibition of release was abolished by the calmodulin antagonist, W7, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN62, but not by the protein kinase A (PKA) inhibitor, H89, or the protein kinase C (PKC) inhibitor, GF109203X. We also found that KR reduced the 4-AP-induced increase in phosphorylation of CaMKII and its substrate synapsin I. Thus, the effect of KR on evoked glutamate release is likely linked to a decrease in P/Q-type Ca2+ channel activity, as well as to the consequent reduction in the CaMKII/synapsin I pathway.


Subject(s)
Calcium Channels/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cerebral Cortex/metabolism , Glutamic Acid/metabolism , Kaempferols/pharmacology , Synapses/drug effects , Synapses/metabolism , Animals , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Cerebral Cortex/cytology , Dose-Response Relationship, Drug , Kaempferols/chemistry , Membrane Potentials/drug effects , Molecular Structure , Phosphorylation , Rats , Signal Transduction/drug effects , Synapsins/metabolism
15.
Cells ; 11(2)2022 01 11.
Article in English | MEDLINE | ID: mdl-35053351

ABSTRACT

Freshly isolated primary cardiomyocytes (CM) are indispensable for cardiac research. Experimental CM research is generally incompatible with life of the donor animal, while human heart samples are usually small and scarce. CM isolation from animal hearts, traditionally performed by coronary artery perfusion of enzymes, liberates millions of cells from the heart. However, due to progressive cell remodeling following isolation, freshly isolated primary CM need to be used within 4-8 h post-isolation for most functional assays, meaning that the majority of cells is essentially wasted. In addition, coronary perfusion-based isolation cannot easily be applied to human tissue biopsies, and it does not straightforwardly allow for assessment of regional differences in CM function within the same heart. Here, we provide a method of multi-day CM isolation from one animal heart, yielding calcium-tolerant ventricular and atrial CM. This is based on cell isolation from cardiac tissue slices following repeated (usually overnight) storage of the tissue under conditions that prolong CM viability beyond the day of organ excision by two additional days. The maintenance of cells in their near-native microenvironment slows the otherwise rapid structural and functional decline seen in isolated CM during attempts for prolonged storage or culture. Multi-day slice-based CM isolation increases the amount of useful information gained per animal heart, improving reproducibility and reducing the number of experimental animals required in basic cardiac research. It also opens the doors to novel experimental designs, including exploring same-heart regional differences.


Subject(s)
Biomedical Research , Heart Atria/cytology , Heart Ventricles/cytology , Myocytes, Cardiac/cytology , Animals , Calcium/pharmacology , Cell Separation , Cell Shape/drug effects , Cells, Cultured , Gene Expression Regulation/drug effects , Membrane Potentials/drug effects , Rabbits , Vasoconstriction/drug effects , Vasodilation/drug effects
16.
Biochem Pharmacol ; 197: 114928, 2022 03.
Article in English | MEDLINE | ID: mdl-35063442

ABSTRACT

Na+ channels undergo multiple inactivated states with different kinetics, which set the refractory period of neuronal discharges, but isolating the intermediate inactivated state has been challenging. Most classical Na+channel-inhibiting anticonvulsants bind to the fast inactivated state to reduce Na+currents and cellular excitability. These anticonvulsants have the slow binding kinetics and thus necessitate long depolarization for drug action, a "use-dependent" effect sparing most normal activities. Rufinamide is a new anticonvulsant targeting Na+channels, and has a therapeutic effect on Lennox-Gastaut syndrome (LGS) which is refractory to classicalNa+channel inhibitors. The efficacy on LGS, whose epileptiform discharges largely involve short depolarization or bursts, is primarily due to the very fast binding kinetics of rufinamide. Could the very fast kinetics of rufinamide lead to indiscriminate inhibition of neuronal activities ? Onhippocampal neurons from male and female mice, wefound that rufinamide most effectively shifts the Na+channel inactivation curve if the inactivating pulse is 1 s, rather than 0.1 or 18 s, in duration. Rufinamide also shows a maximal slowing effect on the recovery kinetics from the inactivation driven by modest depolarization (e.g. -60 mV) of intermediate length (e.g. 50-300 ms). Consistently, rufinamide selectively inhibits the burst discharges at 50-300 ms on a plateau of ∼-60 mV. This is mechanistically ascribable to selective binding of rufinamide to an intermediate inactivated state withan apparent dissociation constantof ∼40 µM. Being the first molecule embodying the evasive transitional gating state, rufinamide could have a unique anti-seizure profile with a novel form of use-dependent action.


Subject(s)
Anticonvulsants/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Triazoles/pharmacology , Voltage-Gated Sodium Channel Blockers/pharmacology , Voltage-Gated Sodium Channels/physiology , Animals , Dose-Response Relationship, Drug , Female , Male , Mice , Mice, Inbred C57BL , Organ Culture Techniques , Protein Stability/drug effects , Voltage-Gated Sodium Channels/chemistry
17.
Molecules ; 27(1)2022 Jan 05.
Article in English | MEDLINE | ID: mdl-35011544

ABSTRACT

Natural plant compounds, such as betaine, are described to have nematocidal properties. Betaine also acts as a neurotransmitter in the free-living model nematode Caenorhabditis elegans, where it is required for normal motility. Worm motility is mediated by nicotinic acetylcholine receptors (nAChRs), including subunits from the nematode-specific DEG-3 group. Not all types of nAChRs in this group are associated with motility, and one of these is the DEG-3/DES-2 channel from C. elegans, which is involved in nociception and possibly chemotaxis. Interestingly, the activity of DEG-3/DES-2 channel from the parasitic nematode of ruminants, Haemonchus contortus, is modulated by monepantel and its sulfone metabolite, which belong to the amino-acetonitrile derivative anthelmintic drug class. Here, our aim was to advance the pharmacological knowledge of the DEG-3/DES-2 channel from C. elegans by functionally expressing the DEG-3/DES-2 channel in Xenopus laevis oocytes and using two-electrode voltage-clamp electrophysiology. We found that the DEG-3/DES-2 channel was more sensitive to betaine than ACh and choline, but insensitive to monepantel and monepantel sulfone when used as direct agonists and as allosteric modulators in co-application with betaine. These findings provide important insight into the pharmacology of DEG-3/DES-2 from C. elegans and highlight the pharmacological differences between non-parasitic and parasitic nematode species.


Subject(s)
Aminoacetonitrile/analogs & derivatives , Caenorhabditis elegans Proteins/metabolism , GABA Plasma Membrane Transport Proteins/metabolism , Ion Channel Gating/drug effects , Receptors, Nicotinic/metabolism , Aminoacetonitrile/pharmacology , Animals , Caenorhabditis elegans , Membrane Potentials/drug effects , Sulfones/pharmacology , Xenopus laevis
18.
Biochem Biophys Res Commun ; 592: 44-50, 2022 02 12.
Article in English | MEDLINE | ID: mdl-35026604

ABSTRACT

The purpose of this study was to investigate the antimicrobial effect and mechanism of slightly acidic electrolyzed water (SAEW) against Shewanella putrefaciens (S. putrefaciens) and Staphylococcus saprophytic (S. saprophyticus). The results showed that SAEW exhibited strong antimicrobial activity against tested bacteria, which was positively correlated to the available chlorine concentration (ACC) of SAEW. The mortality rate of S. putrefaciens and S. saprophyticus was up to 96% and 85%, respectively, when the ACC of SAEW was 60.0 mg/L. The results of scanning electron microscopy (SEM) showed that the cell morphology and structure were destroyed by SAEW. Besides, the results of confocal laser scanning microscopy (CLSM), leakage of DNA and protein provided evidence that SAEW induced membrane permeabilization in cells. Compared with the control, the intracellular reactive oxygen species (ROS) generated by SAEW was strengthened significantly with the increase of ACC, and the cells were injured to death accordingly.


Subject(s)
Anti-Bacterial Agents/pharmacology , Electrolysis , Shewanella putrefaciens/drug effects , Staphylococcus/drug effects , Water/pharmacology , Antioxidants/pharmacology , Bacterial Proteins/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , DNA, Bacterial/metabolism , Membrane Potentials/drug effects , Microbial Sensitivity Tests , Models, Biological , Reactive Oxygen Species/metabolism , Shewanella putrefaciens/ultrastructure , Staphylococcus/ultrastructure
19.
Food Chem Toxicol ; 160: 112804, 2022 Feb.
Article in English | MEDLINE | ID: mdl-34990786

ABSTRACT

A significant rise in the incidence of obesity and type 2 diabetes has occurred worldwide in the last two decades. Concurrently, a growing body of evidence suggests a connection between exposure to environmental pollutants, particularly insecticides, and the development of obesity and type 2 diabetes. This review summarizes key evidence of (1) the presence of different types of neuronal receptors - target sites for neurotoxic insecticides - in non-neuronal cells, (2) the activation of these receptors in non-neuronal cells by membrane-depolarizing insecticides, and (3) changes in metabolic functions, including lipid and glucose accumulation, associated with changes in membrane potential. Based on these findings, we propose that changes in membrane potential (Vmem) by certain insecticides serve as a novel regulator of lipid and glucose metabolism in non-excitable cells associated with obesity and type 2 diabetes.


Subject(s)
Cell Membrane/drug effects , Diabetes Mellitus, Type 2/etiology , Environmental Pollutants/toxicity , Insecticides/toxicity , Obesity/etiology , Animals , Cell Membrane/genetics , Cell Membrane/metabolism , Diabetes Mellitus, Type 2/epidemiology , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Environmental Exposure/adverse effects , Humans , Membrane Potentials/drug effects , Obesity/epidemiology , Obesity/genetics , Obesity/metabolism
20.
Mol Pharmacol ; 101(3): 132-143, 2022 03.
Article in English | MEDLINE | ID: mdl-34969832

ABSTRACT

Calcium- and voltage-gated K+ channels of large conductance (BKs) are expressed in the cell membranes of all excitable tissues. Currents mediated by BK channel-forming slo1 homotetramers are consistently inhibited by increases in membrane cholesterol (CLR). The molecular mechanisms leading to this CLR action, however, remain unknown. Slo1 channels are activated by increases in calcium (Ca2+) nearby Ca2+-recognition sites in the slo1 cytosolic tail: one high-affinity and one low-affinity site locate to the regulator of conductance for K+ (RCK) 1 domain, whereas another high-affinity site locates within the RCK2 domain. Here, we first evaluated the crosstalking between Ca2+ and CLR on the function of slo1 (cbv1 isoform) channels reconstituted into planar lipid bilayers. CLR robustly reduced channel open probability while barely decreasing unitary current amplitude, with CLR maximal effects being observed at 10-30 µM internal Ca2+ CLR actions were not only modulated by internal Ca2+ levels but also disappeared in absence of this divalent. Moreover, in absence of Ca2+, BK channel-activating concentrations of magnesium (10 mM) did not support CLR action. Next, we evaluated CLR actions on channels where the different Ca2+-sensing sites present in the slo1 cytosolic domain became nonfunctional via mutagenesis. CLR still reduced the activity of low-affinity Ca2+ (RCK1:E379A, E404A) mutants. In contrast, CLR became inefficacious when both high-affinity Ca2+ sites were mutated (RCK1:D367A,D372A and RCK2:D899N,D900N,D901N,D902N,D903N), yet still was able to decrease the activity of each high-affinity site mutant. Therefore, BK channel inhibition by CLR selectively requires optimal levels of Ca2+ being recognized by either of the slo1 high-affinity Ca2+-sensing sites. SIGNIFICANCE STATEMENT: Results reveal that inhibition of calcium/voltage-gated K+ channel of large conductance (BK) (slo1) channels by membrane cholesterol requires a physiologically range of internal calcium (Ca2+) and is selectively linked to the two high-affinity Ca2+-sensing sites located in the cytosolic tail domain, which underscores that Ca2+ and cholesterol actions are allosterically coupled to the channel gate. Cholesterol modification of BK channel activity likely contributes to disruption of normal physiology by common health conditions that are triggered by disruption of cholesterol homeostasis.


Subject(s)
Calcium/metabolism , Cholesterol/metabolism , Cytosol/metabolism , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/antagonists & inhibitors , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/metabolism , Animals , Calcium Channel Blockers/pharmacology , Cytosol/drug effects , HEK293 Cells , Humans , Large-Conductance Calcium-Activated Potassium Channels/antagonists & inhibitors , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Membrane Potentials/drug effects , Membrane Potentials/physiology , Protein Structure, Secondary , Rats
SELECTION OF CITATIONS
SEARCH DETAIL
...