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Phytomedicine : International Journal... Apr 2023Sophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR's electrophysiological properties and druggability research are relatively...
BACKGROUND
Sophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR's electrophysiological properties and druggability research are relatively inadequate, which limits the development of SR as an antiarrhythmic candidate.
PURPOSE
To facilitate the development process of SR as an antiarrhythmic candidate, we performed integrated studies on the electrophysiological properties of SR in vitro and ex vivo to gain more comprehensive insights into the multi-ion channel blocking effects of SR, which provided the foundation for the further drugability studies in antiarrhythmic and safety studies. Firstly, SR's electrophysiological properties and antiarrhythmic potentials were recorded and assessed at the cell and tissue levels by comprehensively integrating the patch clamp with the Electrical and Optical Mapping systems. Subsequently, the antiarrhythmic effects of SR were validated by aconitine and ouabain-induced arrhythmia in vivo. Finally, the safety of SR as an antiarrhythmic candidate compound was evaluated based on the guidelines of the Comprehensive in Vitro Proarrhythmia Assay (CiPA).
STUDY DESIGN
The antiarrhythmic effect of SR was evaluated at the in vitro, ex vivo, and in vivo levels.
METHODS
Isolated primary cardiomyocytes and stable cell lines were prepared to explore the electrophysiologic properties of being a multiple ion-channel blocker in vitro by whole-cell patch clamp. Using electrical and optical mapping, the negative chronotropic effect of SR was determined in langendorff-perfused rat or guinea-pig hearts.The antiarrhythmic activity of SR was assessed by the ex vivo tachyarrhythmia models induced by left coronary artery ligation (LCAL) and isoproterenol (ISO). Canonical models of aconitine and ouabain-induced arrhythmia were used to verify the antiarrhythmic effects in vivo. Finally, the pro-arrhythmic risk of SR was detected in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hSCCMs) using a Microelectrode array (MEA).
RESULTS
Single-cell patch assay validated the multiple ion-channel blockers of SR in transient outward current potassium currents (Ito), l-type calcium currents (ICa-l), and rapid activation delayed rectifier potassium currents (IKr). SR ex vivo depressed heart rates (HR) and ventricular conduction velocity (CV) and prolonged Q-T intervals in a concentration-dependent manner. Consistent with the changes in HRs, SR extended the active time of hearts and increased the action potential duration measured at 90% repolarization (APD90). SR could also significantly lengthen the onset time and curtail the duration of spontaneous ventricular tachycardia (VT) in the ex vivo arrhythmic model induced by LCAL. Meanwhile, SR could also significantly upregulate the programmed electrical stimulation (PES) frequency after the ISO challenge in forming electrical alternans and re-entrant excitation. Furthermore, SR exerted antiarrhythmic effects in the tachyarrhythmia models induced by aconitine and ouabain in vivo. Notably, the pro-arrhythmic risk of SR was shallow for a moderate inhibition of the human ether-à-go-go-related gene (hERG) channel. Moreover, SR prolonged field potential duration (FPDc) of hSCCMs in a concentration-dependent manner without early after depolarization (EAD) and arrhythmia occurrence.
CONCLUSION
Our results indicated that SR manifested as a multiple ion-channel blocker in the electrophysiological properties and exerts antiarrhythmic effects ex vivo and in vivo. Meanwhile, due to the low pro-arrhythmic risk in the hERG inhibition assay and the induction of EAD, SR has great potential as a leading candidate in the treatment of ventricular tachyarrhythmia.
Topics: Rats; Humans; Animals; Guinea Pigs; Anti-Arrhythmia Agents; Matrines; Ouabain; Aconitine; Arrhythmias, Cardiac; Ion Channels; Myocytes, Cardiac; Isoproterenol; Potassium; Action Potentials
PubMed: 36738478
DOI: 10.1016/j.phymed.2023.154688 -
Journal of Neurology May 2023Charcot-Marie-Tooth disease (CMT) is a genetically and clinically heterogeneous group of inherited neuropathies. Monoallelic pathogenic variants in ATP1A1 were...
BACKGROUND
Charcot-Marie-Tooth disease (CMT) is a genetically and clinically heterogeneous group of inherited neuropathies. Monoallelic pathogenic variants in ATP1A1 were associated with axonal and intermediate CMT. ATP1A1 encodes for the catalytic α1 subunit of the Na/ K ATPase. Besides neuropathy, other associated phenotypes are spastic paraplegia, intellectual disability, and renal hypomagnesemia. We hereby report the first demyelinating CMT case due to a novel ATP1A1 variant.
METHODS
Whole-exome sequencing on the patient's genomic DNA and Sanger sequencing to validate and confirm the segregation of the identified p.P600R ATP1A1 variation were performed. To evaluate functional effects, blood-derived mRNA and protein levels of ATP1A1 and the auxiliary β1 subunit encoded by ATP1B1 were investigated. The ouabain-survival assay was performed in transfected HEK cells to assess cell viability, and two-electrode voltage clamp studies were performed in Xenopus oocytes.
RESULTS
The variant was absent in the local and global control datasets, falls within a highly conserved protein position, and is in a missense-constrained region. The expression levels of ATP1A1 and ATP1B1 were significantly reduced in the patient compared to healthy controls. Electrophysiology indicated that ATP1A1 injected Xenopus oocytes have reduced Na/ K ATPase function. Moreover, HEK cells transfected with a construct encoding ATP1A1 harbouring variants that confers ouabain insensitivity displayed a significant decrease in cell viability after ouabain treatment compared to the wild type, further supporting the pathogenicity of this variant.
CONCLUSION
Our results further confirm the causative role of ATP1A1 in peripheral neuropathy and broaden the mutational and phenotypic spectrum of ATP1A1-associated CMT.
Topics: Humans; Adenosine Triphosphatases; Charcot-Marie-Tooth Disease; Mutation; Ouabain; Phenotype; Proteins; Sodium-Potassium-Exchanging ATPase
PubMed: 36738336
DOI: 10.1007/s00415-023-11581-w -
European Journal of Dentistry Oct 2023The purpose of the present study was to evaluate the efficacy of spiral ganglion neuron (SGN) regeneration after dental pulp stem cell (DPSC) transplantation in a rat...
OBJECTIVES
The purpose of the present study was to evaluate the efficacy of spiral ganglion neuron (SGN) regeneration after dental pulp stem cell (DPSC) transplantation in a rat sensorineural hearing loss (HL) model.
MATERIALS AND METHODS
Sham or experimental HL was induced in adult Sprague-Dawley rats by cochlear round window surgery. An HL rat model was established with a single 10 mM ouabain intratympanic injection. After 7 days, the rats received DPSCs, stem cells from human exfoliated deciduous teeth (SHED), or culture medium in the sutural area to establish four groups: sham, HL-DPSC, HL-SHED, and HL-medium. Histological analyses were performed at 4, 7, and 10 weeks after transplantation, and the number of SGNs, specific SGN protein expression, and the function of SGNs were evaluated.
STATISTICAL ANALYSIS
Data were statistically by MS Excel and SPSS v.15.0. Intergroup level of significance was determined via a one-way analysis of variance and Duncan's multiple range test with 95% confidence intervals.
RESULTS
New SGN formation was observed in the HL-DPSC and HL-SHED rat groups. The number of SGNs was significantly higher in the HL-DPSC and HL-SHED groups than in the HL-medium group over 4 to 10-week survival period. HL-DPSC rats exhibited higher SGN density compared with that in HL-SHED group, which was statistically significant at week 10. The regenerated SGNs expressed cochlear wiring regulator GATA-binding-protein 3. Moreover, the SGNs from the HL-DPSC group also exhibited a higher expression of synaptic vesicle protein and regulated action potential-dependent neurotransmitter release compared with SGNs from the HL-SHED group.
CONCLUSIONS
Our findings suggest that DPSCs and SHED repair and regenerate SGNs in rat HL model. Dental pulp stem cells represent a promising treatment strategy for restoring damage to the sensory circuits associated with deafness.
PubMed: 36716786
DOI: 10.1055/s-0043-1761190 -
Frontiers in Plant Science 2022In plants under hypersaline stress, the main transporter that extrudes sodium ions (Na ) is the Na /H antiporter SOS1. Different from land plants, the intertidal...
In plants under hypersaline stress, the main transporter that extrudes sodium ions (Na ) is the Na /H antiporter SOS1. Different from land plants, the intertidal macroalgae, contains an animal-type Na /K -ATPase as well as the SOS1 system. However, the contribution of Na /K -ATPase to the K /Na homeostasis of intertidal macroalgae remains unclear. In this study, we analyzed the function of Na /K -ATPase in the response of to salt stress from the perspective of ion transport dynamics. Both the transcript level of and enzyme activity of Na /K -ATPase increased in the early response of thalli to hypersaline stress. Addition of ouabain, an inhibitor of Na /K -ATPase, resulted in Na accumulation in the cells, severe K leakage from the thalli, and then remarkably disturbed the K /Na homeostasis in thalli. This disruption might induce a significant decrease in photosynthesis and a severe oxidative damage in thalli. Accordingly, these results suggested that the important role of Na /K -ATPase in the resistance of intertidal macroalgae to hypersaline stress, and shed light on the diversity of K /Na homeostasis maintenance mechanisms in plants.
PubMed: 36684749
DOI: 10.3389/fpls.2022.1040142 -
Acta Cirurgica Brasileira 2023To investigate the role of renal denervation (RDN) on endogenous ouabain (EO) secretion in spontaneously hypertensive rats (SHR).
PURPOSE
To investigate the role of renal denervation (RDN) on endogenous ouabain (EO) secretion in spontaneously hypertensive rats (SHR).
METHODS
Sixteen 12-week-old male SHR were randomly separated into the renal denervation group (RDNX group) and sham operation group (sham group), and eight age-matched Wistar Kyoto rats (WKY) were served as control group. EO concentrations, the Na+- K+-ATPaseactivity, and the expression of Na+-K+-ATPase were assessed.
RESULTS
EO levels in serum, kidneys and hypothalamus of sham group were higher than in RDNX group (p < 0.05). Renal Na+-K+-ATPase activity subjected to denervation surgery showed significantly reduction when compared with the sham groups (p < 0.05). A positive correlation existed between norepinephrine (NE) content and Na+-K+-ATPase activity in the kidney (r2 = 0.579). Renal Na+-K+-ATPase α1 subunit mRNA expression was down-regulated in the RDNX group compared with the sham group (P < 0.05), while renal Na+-K+-ATPase α1 subunit mRNA expression was no statistical significance between the groups (P = 0.63). Immunohistochemical analysis showed that there were significant differences in the renal expression of Na+-K+-ATPasebetween the three groups (P < 0.05).
CONCLUSIONS
These experiments demonstrate that RDN exerted an anti-hypertensive effect with reduction of EO levels and Na+-K+-ATPase activity and Na+-K+-ATPase α1 subunit expression of kidney in SHR.
Topics: Rats; Animals; Male; Rats, Inbred SHR; Ouabain; Hypertension; Kidney; Rats, Inbred WKY; Sodium; Sodium-Potassium-Exchanging ATPase; Denervation; RNA, Messenger
PubMed: 36629529
DOI: 10.1590/acb371102 -
Scientific Reports Dec 2022Na/K-ATPases (NKA) in the basolateral membrane of the intestinal enterocytes create a Na-gradient that drives both ion-coupled fluid uptake and nutrient transport. Being...
Na/K-ATPases (NKA) in the basolateral membrane of the intestinal enterocytes create a Na-gradient that drives both ion-coupled fluid uptake and nutrient transport. Being dependent on the same gradient as well as on the environmental salinity, these processes have the potential to affect each other. In salmonids, L-lysine absorption has been shown to be higher in freshwater (FW) than in seawater (SW) acclimated fish. Using electrophysiology (Ussing chamber technique), the aim was to explore if the decrease in L-lysine transport was due to allocation of the Na-gradient towards ion-driven fluid uptake in SW, at the cost of amino acid transport. Intestinal NKA activity was higher in SW compared to FW fish. Exposure to ouabain, an inhibitor of NKA, decreased L-lysine transport. However, exposure to bumetanide and hydrochlorothiazide, inhibitors of Na, K, 2Cl-co-transporter (NKCC) and Na, Cl-co-transporter (NCC) respectively, did not affect the rate of intestinal L-lysine transport. In conclusion, L-lysine transport is Na-dependent in rainbow trout and the NKA activity and thus the available Na-gradient increases after SW acclimation. This increased Na-gradient is most likely directed towards osmoregulation, as amino acid transport is not compromised in SW acclimated fish.
Topics: Animals; Oncorhynchus mykiss; Salinity; Lysine; Intestines; Sodium; Acclimatization; Symporters; Seawater; Gills; Sodium-Potassium-Exchanging ATPase
PubMed: 36564520
DOI: 10.1038/s41598-022-26904-6 -
Pharmaceuticals (Basel, Switzerland) Dec 2022Inflammation and loss of cholinergic transmission are involved in neurodegenerative diseases, but possible interactions between them within neurons, astrocytes, and...
Inflammation and loss of cholinergic transmission are involved in neurodegenerative diseases, but possible interactions between them within neurons, astrocytes, and microglia have not yet been investigated. We aimed to compare store-operated Ca entry (SOCE) in neurons, astrocytes, and microglia following cholinergic dysfunction in combination with (or without) an inflammatory stimulus and to investigate the effects of linalyl acetate (LA) on this process. We used the SH-SY5Y, U373, and BV2 cell lines related to neurons, astrocytes, and microglia, respectively. Scopolamine or lipopolysaccharide (LPS) was used to antagonize the muscarinic receptors or induce inflammatory responses, respectively. The concentration of intracellular Ca was measured using Fura-2 AM. Treatment with scopolamine and LPS significantly increased SOCE in the neuron-like cells and microglia but not in the scopolamine-pretreated astrocytes. LA significantly reduced SOCE in the scopolamine-pretreated neuron-like cells and microglia exposed to LPS, which was partially inhibited by the Na-K ATPase inhibitor ouabain and the Na/Ca exchanger (NCX) inhibitor Ni. Notably, SOCE was significantly reduced in the LPS plus scopolamine-pretreated cells mixed with astrocytes and microglia, with a two-fold increase in the applied number of astrocytes. LA may be useful in protecting neurons and microglia by reducing elevated SOCE that is induced by inflammatory responses and inhibiting the muscarinic receptors via Na-K ATPase and the forward mode of NCX. Astrocytes may protect microglia by reducing increased SOCE under the conditions of inflammation and a muscarinic receptor blockade.
PubMed: 36558972
DOI: 10.3390/ph15121521 -
Cancers Dec 2022Anaplastic thyroid carcinoma (ATC) is a rare, but aggressive, carcinoma derived from follicular cells. While conventional treatments may improve patients' survival, the...
Anaplastic thyroid carcinoma (ATC) is a rare, but aggressive, carcinoma derived from follicular cells. While conventional treatments may improve patients' survival, the lethality remains high. Therefore, there is an urgent need for more effective ATC treatments. Cardiotonic steroids, such as ouabain, have been shown to have therapeutic potential in cancer treatment. Thus, we aimed to evaluate ouabain's effects in human anaplastic thyroid cells. For this, 8505C cells were cultured in the presence or absence of ouabain. Viability, cell death, cell cycle, colony formation and migratory ability were evaluated in ouabain-treated and control 8505C cells. The expression of differentiation and epithelial-to-mesenchymal transition (EMT) markers, as well as IL-6, TGFb1 and their respective receptors were also quantified in these same cells. Our results showed that ouabain in vitro decreased the number of viable 8505C cells, possibly due to an inhibition of proliferation. A reduction in migration was also observed in ouabain-treated 8505C cells. In contrast, decreased mRNA levels of PAX8 and TTF1 differentiation markers and increased levels of the N-cadherin EMT marker, as well as IL-6 and TGFb1, were found in ouabain-treated 8505C cells. In short, ouabain may have anti-proliferative and anti-migratory effect on 8505C cells, but maintains an aggressive and undifferentiated profile.
PubMed: 36551653
DOI: 10.3390/cancers14246168 -
Frontiers in Molecular Neuroscience 2022Zebrafish regenerate their retinas following damage, resulting in restoration of visual function. Here we evaluate recovery of retinal function through qualitative and...
INTRODUCTION
Zebrafish regenerate their retinas following damage, resulting in restoration of visual function. Here we evaluate recovery of retinal function through qualitative and quantitative analysis of the electroretinogram (ERG) over time following retinal damage, in correlation to histological features of regenerated retinal tissue.
METHODS
Retinas of adult zebrafish were lesioned by intravitreal injection of 10 μM (extensive lesion; destroys all neurons) or 2 μM (selective lesion; spares photoreceptors) ouabain. Unlesioned contralateral retinas served as controls. Function of retinal circuitry was analyzed at selected timepoints using ERG recordings from live zebrafish, and whole eyes were processed for histological analyses immediately thereafter.
RESULTS
Qualitative and quantitative assessment of waveforms during retinal regeneration revealed dynamic changes that were heterogeneous on an individual level within each sampling time, but still followed common waveform recovery patterns on a per-fish and population-level basis. Early in the regeneration period (13-30 days post injury; DPI), for both lesion types, b-waves were essentially not detected, and unmasked increased apparent amplitudes, implicit times, and half-widths of a-waves (vs. controls). In control recordings, d-waves were not obviously detected, but apparent d-waves (OFF-bipolar responses) from regenerating retinas of several fish became prominent by 30DPI and dominated the post-photoreceptor response (PPR). Beyond 45DPI, b-waves became detectable, and the ratio of apparent d- to b-wave contributions progressively shifted with most, but not all, fish displaying a b-wave dominated PPR. At the latest timepoints (extensive, 90DPI; selective, 80DPI), recordings with measurable b-waves approached a normal waveform (implicit times and half-widths), but amplitudes were not restored to control levels. Histological analyses of the retinas from which ERGs were recorded showed that as regeneration progressed, PKCa + ON-bipolar terminals and parvalbumin + amacrine cell processes became more stereotypically positioned within the deep sublaminae of the INL over recovery time after each lesion type, consistent with the shift in PPR seen in the ERG recordings.
DISCUSSION
Taken together, these data suggest that photoreceptor-OFF-bipolar component/connectivity may functionally recover and mature earlier during regeneration compared to the photoreceptor-ON-bipolar component, though the timeframe in which such recovery happens is heterogeneous on a per-fish basis. Collectively our studies suggest gradual restoration of ON-bipolar functional circuitry during retinal regeneration.
PubMed: 36533135
DOI: 10.3389/fnmol.2022.1070509 -
International Journal of Molecular... Dec 2022Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline...
Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (P) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos. Their average mean diameters were determined by Dynamic Light Scattering (DLS) (212 ± 19 nm) and by Atomic Force Microcopy (AFM) (180 ± 85 nm). The MVs had a specific activity for TNAP of 9.2 ± 4.6 U·mg confirming that the MVs were mineralization competent. The ability to hydrolyze ATP was assayed by a colorimetric method and by P NMR with and without Levamisole and SBI-425 (two TNAP inhibitors), ouabain (an NKA inhibitor), and ARL-67156 (an NTPDase1, NTPDase3 and Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) competitive inhibitor). The mineralization profile served to monitor the formation of precipitated calcium phosphate complexes, while IR spectroscopy allowed the identification of apatite. Proteoliposomes containing NKA with either dipalmitoylphosphatidylcholine (DPPC) or a mixture of 1:1 of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) served to verify if the proteoliposomes were able to initiate mineral formation. Around 69-72% of the total ATP hydrolysis by MVs was inhibited by 5 mM Levamisole, which indicated that TNAP was the main enzyme hydrolyzing ATP. The addition of 0.1 mM of ARL-67156 inhibited 8-13.7% of the total ATP hydrolysis in MVs, suggesting that NTPDase1, NTPDase3, and/or NPP1 could also participate in ATP hydrolysis. Ouabain (3 mM) inhibited 3-8% of the total ATP hydrolysis by MVs, suggesting that NKA contributed only a small percentage of the total ATP hydrolysis. MVs induced mineralization via ATP hydrolysis that was significantly inhibited by Levamisole and also by cleaving TNAP from MVs, confirming that TNAP is the main enzyme hydrolyzing this substrate, while the addition of either ARL-6715 or ouabain had a lesser effect on mineralization. DPPC:DPPE (1:1)-NKA liposome in the presence of a nucleator (PS-CPLX) was more efficient in mineralizing compared with a DPPC-NKA liposome due to a better orientation of the NKA active site. Both types of proteoliposomes were able to induce apatite formation, as evidenced by the presence of the 1040 cm band. Taken together, the findings indicated that the hydrolysis of ATP was dominated by TNAP and other phosphatases present in MVs, while only 3-8% of the total hydrolysis of ATP could be attributed to NKA. It was hypothesized that the loss of Na/K asymmetry in MVs could be caused by a complete depletion of ATP inside MVs, impairing the maintenance of symmetry by NKA. Our study carried out on NKA-liposomes confirmed that NKA could contribute to mineral formation inside MVs, which might complement the known action of PHOSPHO1 in the MV lumen.
Topics: Animals; Chick Embryo; Phosphoric Monoester Hydrolases; Sodium-Potassium-Exchanging ATPase; Calcification, Physiologic; Alkaline Phosphatase; Hydrolysis; Adenosine Triphosphate; Liposomes; Minerals; Calcinosis
PubMed: 36499456
DOI: 10.3390/ijms232315072