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This study explores the therapeutic benefits of tannic acid (TnA) in an experimental protocol of chronic hypermethioninemia in rats. Rats were categorized into four groups: Group I - control, Group II - TnA 30 mg/kg, Group III - methionine (Met) 0.2-0.4 g/kg + methionine sulfoxide (MS) 0.05-0.1 g/kg, Group IV - TnA/Met + MS. Saline was administered by subcutaneous pathway into groups I and II twice daily from postnatal day 6 (P6) to P28, whereas those in groups III and IV received Met + MS. From P28 to P35, groups II and IV received TnA orally. Animals from group III presented cognitive and memory impairment assessed through object recognition and Y-maze tests (p < 0.05). Elevated levels of reactive species, lipid peroxidation, and nitrites followed by a decline in sulfhydryl content, catalase activity, and superoxide dismutase activity were observed in animals treated with Met + MS (p < 0.05). However, TnA treatment reversed all these effects (p < 0.05). In group III, there was an increase in acetylcholinesterase activity and IL-6 levels, coupled with a reduction in Na + /K + -ATPase activity (p < 0.05). TnA was able to protect against these effects (p < 0.05). The gene expression of catalase, brain-derived neurotrophic factor, and nuclear factor erythroid 2-related factor 2 was decreased in the hippocampus and striatum from group III (p < 0.05). TnA reversed almost all of these alterations (p < 0.05). These findings suggest that TnA is a therapeutic target for patients with hypermethioninemia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Na + /K + -ATPase (NKA) is a plasma membrane ion-transporting protein involved in the generation and maintenance of Na + and K + gradients across the cell membrane, which can produce a driving force for the secondary transport of metabolic substrates. NKA also regulates intracellular calcium that is responsible for modulating numerous cellular processes, while it interacts with many other proteins and functions as a signal transducer, with several signaling pathways being involved. Thus, NKA has become an important target for the treatment of human diseases. Cardiac glycosides are well-known NKA inhibitors, of which (+)-digoxin or digoxin has been long used for the treatment of congestive heart failure. Also, digoxin has exhibited potential antitumor activity, by targeting directly HIF-1α, NKA, and NF-κB. Thus, the function of NKA in human cardiovascular diseases and cancer and the therapeutic effects of digoxin on these diseases are summarized in the present review, with the correlations among digoxin, NKA, cardiovascular diseases, and cancer being discussed. Presented herein are also the antitumor potential of monosaccharide cardiac glycoside analogues of digoxin, including (-)-cryptanoside A, (-)-oleandrin, (-)-ouabain, and (+)-strebloside. It is hoped that this contribution will provide some helpful information for the design and discovery of new cardiac glycoside-type therapeutic agents for the treatment of cardiovascular diseases and cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study aimed to elucidate vincristine (VCR)-induced peripheral neuropathy in aged rats, a poorly understood neurotoxicity. Both young and old Wistar rats were administered VCR (0.1 mg/kg, intraperitoneally (i.p.)) and compared to age-matched controls (0.9% saline; 10 mg/mL, i.p.). Mechanical (MN) and thermal nociceptive (TN) responses were assessed on days 0, 6, 11, and 17. Locomotor response, cognitive ability, and anxious-like behavior were evaluated on days 14, 15, and 16. Results showed MN and TN responses in both young and old VCR-exposed rats. In old rats, VCR exacerbated MN (on days 6, 11, and 17) and TN (on days 6 and 17) responses. VCR also induced cognitive impairments and anxiety-like behavior. Histological analysis revealed Wallerian degeneration in the spinal cords of VCR-exposed rats accompanied by macrophage migration. Furthermore, VCR increased Ca 2+ -ATPase activity while inhibiting Na + , K + -ATPase activity in young and old rats. VCR altered the homeostasis of Mg 2+ -ATPase activity. Lipid peroxidation and nitrite and nitrate levels increased in young and old rats exposed to VCR. This study provides valuable insights into VCR's mechanistic pathways in aged rats, emphasizing the need for further research in this area.

Caloric restriction mimetics mimic the beneficial effects of dietary restriction approaches, such as caloric restriction approaches, without limiting the dietary intake. Chlorogenic acid (CGA) acts as a caloric restriction mimetic; however, its neuroprotective mechanisms remain ambiguous. Therefore, in this study, we aimed to elucidate the neuroprotective effects of CGA on SH-SY5Y cells treated with amyloid-beta (Aβ)1-42. Aβ1-42 (1.25 μM) induced oxidative stress by significantly increasing the pro-oxidant levels and decreasing the antioxidant levels in the cells. Additionally, it decreased the Na + /K + -ATPase and Ca 2+ -ATPase ion transporter activities and enhanced the acetylcholinesterase activity. However, CGA (100 μM) reversed these Aβ1-42-induced changes in SH-SY5Y cells. Overall, CGA exerted neuroprotective effects against Aβ1-42-induced oxidative stress and impaired ion transporter and acetylcholinesterase activities, serving as a promising therapeutic candidate for neurodegenerative diseases, such as Alzheimer's disease., (© 2024 Wiley Periodicals LLC.)

Theories have been widely proposed and tested for impacts of soil nitrogen (N) on phytochemical defenses. Among the hundreds of distinct cardenolide toxins produced by milkweeds (Asclepias spp.), few contain N, yet these appear to be the most toxic against specialist herbivores. Because N- and non-N-cardenolides coexist in milkweed leaves and likely have distinct biosynthesis, they present an opportunity to address hypotheses about drivers of toxin expression. We tested effects of soil N and herbivore-damage on cardenolide profiles of two milkweed species differing in life-history strategies (Asclepias syriaca and A. curassavica), and the toxicity of their leaves. In particular leaf extracts were tested against the target enzymes (Na + /K + -ATPase extracted from neural tissue) from both monarch butterflies (Danaus plexippus) as well as less cardenolide-resistant queen butterflies, D. gilippus. Increasing soil N enhanced biomass of Asclepias syriaca but had weak effects on cardenolides, including causing a significant reduction in the N-cardenolide labriformin; feeding by monarch caterpillars strongly induced N-cardenolides (labriformin), its precursors, and total cardenolides. Conversely, soil N had little impact on A. curassavica biomass, but was the primary driver of increasing N-cardenolides (voruscharin, uscharin and their precursors); caterpillar induction was weak. Butterfly enzyme assays revealed damage-induced cardenolides substantially increased toxicity of both milkweeds to both butterflies, swamping out effects of soil N on cardenolide concentration and composition. Although these two milkweed species differentially responded to soil N with allocation to growth and specific cardenolides, leaf toxicity to butterfly Na + /K + -ATPases was primarily driven by herbivore-induced defense. Thus, both biotic and abiotic factors shape the composition of phytochemical defense expression, and their relative importance may be dictated by plant life-history differences., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Spreading depolarization (SD) describes the near-complete depolarization of central nervous system (CNS) neural cells as a consequence of chemical, electrical, or metabolic perturbations. It is well established as the central mechanism underlying insect coma and various mammalian neurological dysfunctions. Despite significant progress in our understanding, the question remains: which cation channel, if any, generates SD in the CNS? Previously, we speculated that the sodium-potassium ATPase (NKA) might function as a large-conductance ion channel to initiate SD in insects, potentially mediated by a palytoxin (PLTX)-like endogenous activator. In the current study, we evaluate the effectiveness and properties of PLTX as an SD initiator in Locusta migratoria . Whereas bath-applied PLTX failed to ignite SD, direct injection into the neuropil triggered SD in 57% of the preparations. Notably, PLTX-induced SD onset was significantly more rapid compared with ouabain (OUA) injection and azide controls, though their electrophysiological features remained similar. Furthermore, PLTX-induced SD was recoverable and resulted in a greater frequency of repetitive SD events compared with ouabain. Surprisingly, prior PLTX treatment disrupted the onset and recovery of subsequent SD evoked by other means. PLTX injection could attenuate the amplitude and even completely inhibit the onset of azide-induced SD at higher doses. These results show that PLTX can trigger repetitive and reversible SD-like events in locusts and simultaneously interfere with anoxic SD occurrence. We suggest that the well-documented NKA pump conversion into an open nonselective cationic channel is a plausible mechanism of SD activation in the locust CNS, warranting additional investigations. NEW & NOTEWORTHY Spreading depolarization (SD) is a critical mechanism underlying central nervous system (CNS) shutdown and injury under stress, yet the initiating ion channel remains unknown. Here, we used the marine poison palytoxin (PLTX), which converts the sodium-potassium ATPase (NKA) into an open channel, to initiate SD in intact locust CNS. We show for the first time that PLTX-induced SD is rapid and recoverable in vivo, providing support that NKA conversion to a channel may be the SD-initiating mechanism.

The Arctic is warming three times faster than the global average, imposing challenges to cold-adapted fish, such as Arctic char (Salvelinus alpinus). We evaluated stress and metabolic responses of Arctic char to different thermal acclimation scenarios to determine whether responses to thermal variation differed from those to stable exposures. Fish were exposed for 7 days to one of four treatments: (1) control (12°C); (2) mean (16°C), corresponding to the mean temperature of the diel thermal cycle; (3) constant high temperature (20°C); and (4) diel thermal cycling (12 to 20°C every 24 h). Exposure to 20°C causes increases plasma lactate and glucose, an imbalance in antioxidant systems, and oxidative stress in the liver. The 20°C treatment also elevated fractional rates of protein synthesis and caused oxidative stress in the heart. Stress responses were more pronounced in diel thermal cycling than in mean (16°C) fish, indicating that peak exposure temperatures or variation are physiologically important. Cortisol was highest in diel thermal cycling fish and oxidative stress was noted in the liver. Gill Na+/K+-ATPase activity was also significantly reduced in diel thermal cycling fish, suggesting gill remodeling in response to an osmoregulatory stress. Exposure to a constant 20°C was more challenging than a diel thermal cycle, demonstrating the importance of daily cooling to recovery. Arctic char inhabit a thermally variable environment and understanding how this impacts their physiology will be critical for informing conservation strategies in the context of a rapidly warming Arctic., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Bipolar Disorder (BD) is a psychiatric disorder marked by mood swings between manic and depressive episodes. The reduction in the Na,K-ATPase (NKA) enzyme activity and the inability of individuals with BD to produce endogenous ouabain (EO) at sufficient levels to stimulate this enzyme during stressful events are factors proposed for BD etiology. According to these hypotheses, reduction in NKA activity would result in altered neuronal resting potential, leading to BD symptoms. Recently, damage to the adrenals (EO synthesis site) in coronavirus disease (COVID-19) patients has been reported, however studies pointing to the pathophysiological mechanisms shared by these two diseases are scarce. Through a literature review, this study aims to correlate COVID-19 and BD, focusing on the role of NKA and EO to identify possible mechanisms for the worsening of BD due to COVID-19. The search in the PubMed database for the descriptors ("bipolar disorder" AND "Na,K-ATPase"), ("bipolar disorder" AND "endogenous ouabain"), ("covid-19" AND "bipolar disorder") and ("covid-19" AND "adrenal gland") resulted in 390 articles. The studies identified the adrenals as a vulnerable organ to SARS-CoV-2 infection. Cases of adrenal damage in patients with COVID-19 showing lower levels of adrenal hormones were reported. Cases of COVID-19 patients with symptoms of mania were reported worldwide. Given these results, we propose that adrenal cortical cell damage could lead to EO deficiency following neuronal NKA activity impairment, with small reductions in activity leading to mania and greater reductions leading to depression., Competing Interests: Declaration of competing interest Louise Castro de Jesus, Cassiano Felippe Gonçalves-de-Albuquerque and Patrícia Burth authors of a manuscript titled“Onset of bipolar disorder by Covid-19: the roles of endogenous ouabain and the Na,K-ATPase “agree to submit this manuscript. This material has not been published and is not under active consideration by another journal. The authors declare do not have any potential competing interests. The authors disclose that they have no financial interest or commercial sponsor for this work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aging is characterized by a functional decline in several physiological systems. α-Klotho-hypomorphic mice (Kl -/- ) exhibit accelerated aging and cognitive decline. We evaluated whether male and female α-Klotho-hypomorphic mice show changes in the expression of synaptic proteins, N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits, postsynaptic density protein 95 (PSD-95), synaptophysin and synapsin, and the activity of Na + , K + -ATPase (NaK) isoforms in the cerebellum and hippocampus. In this study, we demonstrated that in the cerebellum, Kl -/- male mice have reduced expression of GluA1 (AMPA) compared to wild-type (Kl +/+ ) males and Kl -/- females. Also, Kl-/- male and female mice show reduced ɑ2/ɑ3-NaK and Mg 2+ -ATPase activities in the cerebellum, respectively, and sex-based differences in NaK and Mg 2+ -ATPase activities in both the regions. Our findings suggest that α-Klotho could influence the expression of AMPAR and the activity of NaK isoforms in the cerebellum in a sex-dependent manner, and these changes may contribute, in part, to cognitive decline., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Elisa Mitiko Kawamoto reports financial support was provided by University of São Paulo. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Investigating blood-brain barrier (BBB) dysfunction has become a pre-clinical and clinical research focus as it accompanies many neurological disorders. Nevertheless, knowledge of how diagnostic BBB tracers cross the endothelium from blood-to-brain or vice versa often remains incomplete. In particular, brain-to-blood transport (efflux) may reduce tracer extravasation of intravascularly (i.v.) applied tracers. Conversely, impaired efflux could mimic phenotypic extravasation. Both processes would affect conclusions on BBB properties primarily attributed to blood-to-brain leakage. Here, we specifically investigated efflux of fluorescent BBB tracers, focusing on the most common non-toxic marker, sodium fluorescein, which is applicable in patients. We used acute neocortical slices from mice and applied fluorescein, sulforhodamine-B, rhodamine-123, FITC dextran to the artificial cerebrospinal fluid. Anionic low molecular weight (MW) fluorescein and sulforhodamine-B, but not ~ 10-fold larger FITC-dextran and cationic low MW rhodamine-123, showed efflux into the lumen of blood vessels. Our data suggest that fluorescein efflux depends on organic anion transporter polypeptides (Oatp) rather than P-glycoprotein. Furthermore, sodium-potassium ATPase inhibition and incomplete oxygen-glucose deprivation (OGD, 20% O 2 ) reduced fluorescein efflux, while complete OGD (0% O 2 ) abolished efflux. We provide evidence for active efflux of fluorescein in vitro. Impaired efflux of fluorescein could thus contribute to the frequently observed BBB dysfunction in neuropathologies in addition to blood-to-brain leakage., (© 2024. The Author(s).)

Objectives: To observe the effect of acupuncture on serum gastrin content and urinary sodium excretion in spontaneously hypertensive rat (SHR), so as to explore its potential mechanism in the treatment of hypertension., Methods: Thirty-two male SHRs were randomly divided into model, hydrochlorothiazide, acupuncture and sham acupuncture groups, with 8 rats in each group, and 8 male Wistar-kyoto rats were taken as the control group. Rats in the hydrochlorothiazide group received gavage of hydrochlorothiazide solution (10 mg·kg -1 ·d -1 ), once daily for 4 weeks. Acupuncture was applied to bilateral "Renying" (ST9) and "Zusanli" (ST36) or non-acupoint on both sides for rats in the acupuncture and sham acupuncture groups, with manual stimulation every 10 minutes for a total of 20 minutes, once a day for a total of 4 weeks. The systolic blood pressure of the tail-artery was measured before and 1, 2, 3, and 4 weeks after the intervention. HE staining was used to observe the pathological changes in renal tissue. Serum gastrin contents were detected using enzyme-linked immunosorbent assay (ELISA). Urinary sodium content was measured by atomic absorption spectrometry. The expression levels of cholecystokinin B receptor (CCKBR) and Na + /K + -ATPase proteins in renal tissue were detected by Western blot, and the mRNA expression levels of CCKBR and the α1 subunit of Na + /K + - ATPase (ATP1A1) were detected by fluorescence quantitative PCR., Results: Compared with the control group, the systolic blood pressure of the tail artery in the model group were increased significantly before intervention and at the 1 st , 2 nd , 3 rd and 4 th weeks of intervention ( P <0.05). Before intervention, the 24 h urine volume of the model, hydrochlorothiazide, acupuncture and sham acupuncture groups were decreased significantly ( P <0.05). After intervention, the 24 h urine volume and urinary sodium excretion in the model group were significantly decreased ( P <0.05)；the expression levels of CCKBR protein and mRNA in renal tissue were significantly decreased ( P <0.05)；the expression levels of Na + /K + -ATPase protein and ATP1A1 mRNA were significantly increased ( P <0.05)；the glomerulus was mildly congested with a small amount of lymphocyte infiltration. Compared with the model group, the systolic blood pressure of the tail artery in the hydrochlorothiazide and the acupuncture groups were decreased significantly at the 2 nd , 3 rd and 4 th weeks of intervention ( P <0.05)；the 24 h urine volume and urinary sodium excretion in the hydrochlorothiazide and the acupuncture groups were significantly increased ( P <0.05)；the serum gastrin content and the expression levels of CCKBR protein and mRNA in renal tissue were significantly increased ( P <0.05)；the expression levels of Na + /K + -ATPase protein and ATP1A1 mRNA were significantly decreased ( P <0.05)；there were no obvious pathological changes in renal tissue. A small number of lymphocyte focal infiltration around blood vessels was observed in the kidney tissue of the sham acupuncture group., Conclusions: Acupuncture can significantly reduce the tail artery systolic blood pressure of SHR, which may be related to its effect in increasing serum gastrin content and CCKBR expression, inhibiting sodium pump reabsorption, thus promoting urinary sodium excretion.

Beta vulgaris L. is a root vegetable that is consumed mainly as a food additive. This study aimed to describe the protective effect of B. vulgaris on Fe 2+ -mediated oxidative liver damage through in vitro, ex vivo, and in silico studies to establish a strong rationale for its protective effect. To induce oxidative damage, we incubated the livers of healthy male rats with 0.1 mM FeSO 4 to induce oxidative injury and coincubated them with an aqueous extract of B. vulgaris root (BVFE) (15-240 µg/mL). Induction of liver damage significantly (p < .05) decreased the levels of GSH, SOD, CAT, and ENTPDase activities, with a corresponding increase in MDA and NO levels and Na + /K + ATPase, G6 Pase, and F-1,6-BPase enzyme activities. BVFE treatment (p < .05) reduced these levels and activities to almost normal levels, with the most prominent effects observed at 240 µg/mL BVFE. An HPLC investigation revealed sixteen compounds in BVFE, with quercetin being the most abundant. Chlorogenic acid and iso-orientation showed the highest binding affinities for G6 Pase and Na+/K + ATPase, respectively. These findings suggest that B. vulgaris can protect against Fe 2+ -mediated liver damage by suppressing oxidative stress and cholinergic and purinergic activities while regulating gluconeogenesis. Overall, the hepatoprotective activity of this extract might be driven by the synergistic effect of the identified compounds and their probable interactions with target proteins., (© 2024. The Author(s).)

The antitumor effect of cardiotonic steroids (CTS) has stimulated the search for new methods to evaluate both kinetic and thermodynamic aspects of their binding to Na + /K + -ATPase (IUBMB Enzyme Nomenclature). We propose a real-time assay based on a chromogenic substrate for phosphatase activity (pNPPase activity), using only two concentrations with an inhibitory progression curve, to obtain the association rate ( k on ), dissociation rate ( k off ), and equilibrium ( K i ) constants of CTS for the structure-kinetics relationship in drug screening. We show that changing conditions (from ATPase to pNPPase activity) resulted in an increase of K i of the cardenolides digitoxigenin, essentially due to a reduction of k on In contrast, the K i of the structurally related bufadienolide bufalin increased much less due to the reduction of its k off partially compensating the decrease of its k on When evaluating the kinetics of 15 natural and semisynthetic CTS, we observed that both k on and k off correlated with K i (Spearman test), suggesting that differences in potency depend on variations of both k on and k off A rhamnose in C3 of the steroidal nucleus enhanced the inhibitory potency by a reduction of k off rather than an increase of k on Raising the temperature did not alter the k off of digitoxin, generating a ΔH ‡ ( k off ) of -10.4 ± 4.3 kJ/mol, suggesting a complex dissociation mechanism. Based on a simple and inexpensive methodology, we determined the values of k on , k off , and K i of the CTS and provided original kinetics and thermodynamics differences between CTS that could help the design of new compounds. SIGNIFICANCE STATEMENT: This study describes a fast, simple, and cost-effective method for the measurement of phosphatase pNPPase activity enabling structure-kinetics relationships of Na + /K + -ATPase inhibitors, which are important compounds due to their antitumor effect and endogenous role. Using 15 compounds, some of them original, this study was able to delineate the kinetics and/or thermodynamics differences due to the type of sugar and lactone ring present in the steroid structure., Competing Interests: No author has an actual or perceived conflict of interest with the contents of this article., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Tissue-specific differences in the expression of paralog genes, which are not essential in most cell types due to the buffering effect of the partner pair, can make for highly selective gene dependencies. To identify selective paralogous targets for acute myeloid leukemia (AML), we integrated the Cancer Dependency Map with numerous datasets characterizing protein-protein interactions, paralog relationships, and gene expression in cancer models. In this study, we identified ATP1B3 as a context-specific, paralog-related dependency in AML. ATP1B3, the β-subunit of the sodium-potassium pump (Na/K-ATP pump), interacts with the α-subunit ATP1A1 to form an essential complex for maintaining cellular homeostasis and membrane potential in all eukaryotic cells. When ATP1B3's paralog ATP1B1 is poorly expressed, elimination of ATP1B3 leads to the destabilization of the Na/K-ATP pump. ATP1B1 expression is regulated through epigenetic silencing in hematopoietic lineage cells through histone and DNA methylation in the promoter region. Loss of ATP1B3 in AML cells induced cell death in vitro and reduced leukemia burden in vivo, which could be rescued by stabilizing ATP1A1 through overexpression of ATP1B1. Thus, ATP1B3 is a potential therapeutic target for AML and other hematologic malignancies with low expression of ATP1B1. Significance: ATP1B3 is a lethal selective paralog dependency in acute myeloid leukemia that can be eliminated to destabilize the sodium-potassium pump, inducing cell death., (©2024 American Association for Cancer Research.)

Bufalin is a promising active ingredient in traditional Chinese medicine but has shown limited anticancer applications due to its toxicity. Here, we report BCNPs@gel, a bufalin-containing CaCO 3 nanoparticle hydrogel, for enhancing cancer treatment through inducing cellular pyroptosis. Under the tumor microenvironment's low pH conditions, bufalin and Ca 2+ are released from the delivery system. Bufalin serves as a direct anticancer drug and a Na + /K + -ATPase inhibitor by forcing the Na + /Ca 2+ exchanger to reverse its function, which transfers Ca 2+ into cytoplasm and ultimately causes Ca 2+ overload-triggered pyroptosis. Meanwhile, we found that bufalin can upregulate PD-L1 in tumor cells. In combination with the PD-1 antibody, the delivery system showed a greater performance during the cancer treatment. BCNPs@gel enhances antitumor efficiency, reduces systemic side effects, extends antitumor mechanism of bufalin, and provides new strategies for inducing pyroptosis and calcium overload in cancer immunotherapy via Na + /K + -ATPase inhibitor. This work provides an application model for numerous other traditional Chinese medicine ingredients.

The effects of Gastrodin (GD) on cerebral ischemia stimulated researchers to investigate its possible role in the progression of arrhythmia associated with cardiac ischemia-reperfusion (IR) damage in rats. 40 Sprague-Dawley rats were divided into four groups: Sham, Model, GD 50 mg/kg, and GD 100 mg/kg. Myocardial ischemia (MI) was caused by the procedure of ligating the left coronary artery, followed by reperfusion. Heart rate (HR), mean arterial pressure (MAP), and rate pressure product (RPP) in rats were assessed before and after ischemia and reperfusion, as well as cardiac arrhythmia in experimental rats. The I/R damage was evaluated by measuring levels of Na + -K + ATPase and Ca 2+ -Mg 2+ ATPase, Creatine Kinase-MB (CK-MB), Cardiac Troponin I (cTnI), Gap Junction α-1 (GJα-1), Phospho-GJα-1/total-GJα-1, Kir2.1, Bax, Bcl-2, and oxidative indicators. MGL's Autodock and Vina programs were used for in silico docking studies to identify possible interactions between GJα-1 and Gastrodin. The animals in the model group expressed a substantial decrease in HR, MAP, and RPP compared to the Sham group. GD-treated rats revealed slightly higher values compared to the model group. Expression of CK-MB and cTnI was reduced, and Na + -K + ATPase and Ca 2+ -Mg 2+ ATPase expression was increased on GD pre-conditioning. Phospho-Cx43/total-Cx43 ratio and Bax expression were increased, whereas GD reduced Bcl-2 expression. In silico molecular docking studies suggested the potential binding of GD with the GJα-1 protein, thus confirming the in vivo results. GD corrected the arrhythmia in rats subjected to I/R injury by increasing Na + -K + ATPase and Ca 2+ -Mg 2+ ATPase expression, targeting GJα-1, and modulating the expression of Kir2.1.

Na + -K + ATPase is an integral component of cardiac sarcolemma and consists of three major subunits, namely the α-subunit with three isoforms (α 1 , α 2 , and α 3 ), β-subunit with two isoforms (β 1 and β 2 ) and γ-subunit (phospholemman). This enzyme has been demonstrated to transport three Na and two K ions to generate a trans-membrane gradient, maintain cation homeostasis in cardiomyocytes and participate in regulating contractile force development. Na + -K + ATPase serves as a receptor for both exogenous and endogenous cardiotonic glycosides and steroids, and a signal transducer for modifying myocardial metabolism as well as cellular survival and death. In addition, Na + -K + ATPase is regulated by different hormones through the phosphorylation/dephosphorylation of phospholemman, which is tightly bound to this enzyme. The activity of Na + -K + ATPase has been reported to be increased, unaltered and depressed in failing hearts depending upon the type and stage of heart failure as well as the association/disassociation of phospholemman and binding with endogenous cardiotonic steroids, namely endogenous ouabain and marinobufagenin. Increased Na + -K + ATPase activity in association with a depressed level of intracellular Na + in failing hearts is considered to decrease intracellular Ca 2+ and serve as an adaptive mechanism for maintaining cardiac function. The slight to moderate depression of Na + -K + ATPase by cardiac glycosides in association with an increased level of Na + in cardiomyocytes is known to produce beneficial effects in failing hearts. On the other hand, markedly reduced Na + -K + ATPase activity associated with an increased level of intracellular Na + in failing hearts has been demonstrated to result in an intracellular Ca 2+ overload, the occurrence of cardiac arrhythmias and depression in cardiac function during the development of heart failure. Furthermore, the status of Na + -K + ATPase activity in heart failure is determined by changes in isoform subunits of the enzyme, the development of oxidative stress, intracellular Ca 2+ -overload, protease activation, the activity of inflammatory cytokines and sarcolemmal lipid composition. Evidence has been presented to show that marked alterations in myocardial cations cannot be explained exclusively on the basis of sarcolemma alterations, as other Ca 2+ channels, cation transporters and exchangers may be involved in this event. A marked reduction in Na + -K + ATPase activity due to a shift in its isoform subunits in association with intracellular Ca 2+ -overload, cardiac energy depletion, increased membrane permeability, Ca 2+ -handling abnormalities and damage to myocardial ultrastructure appear to be involved in the progression of heart failure.

The efficacy of antitumor immunity is associated with the metabolic state of cytotoxic T cells, which is sensitive to the tumor microenvironment. Whether ionic signals affect adaptive antitumor immune responses is unclear. In the present study, we show that there is an enrichment of sodium in solid tumors from patients with breast cancer. Sodium chloride (NaCl) enhances the activation state and effector functions of human CD8 + T cells, which is associated with enhanced metabolic fitness. These NaCl-induced effects translate into increased tumor cell killing in vitro and in vivo. Mechanistically, NaCl-induced changes in CD8 + T cells are linked to sodium-induced upregulation of Na + /K + -ATPase activity, followed by membrane hyperpolarization, which magnifies the electromotive force for T cell receptor (TCR)-induced calcium influx and downstream TCR signaling. We therefore propose that NaCl is a positive regulator of acute antitumor immunity that might be modulated for ex vivo conditioning of therapeutic T cells, such as CAR T cells., (© 2024. The Author(s).)

The predicted global warming of surface waters can be challenging to aquatic ectotherms like freshwater mussels. Especially animals in northern temperate latitudes may face and physiologically acclimate to significant stress from seasonal temperature fluctuations. Na + /K + -ATPase enzyme is one of the key mechanisms that allow mussels to cope with changing water temperatures. This enzyme plays a major role in osmoregulation, energy control, ion balance, metabolite transport and electrical excitability. Here, we experimentally studied the effects of temperature on Na + /K + -ATPase activity of gills in two freshwater mussel species, Anodonta anatina and Unio tumidus. The study animals were acclimated to three ambient temperatures (+4, +14, +24 °C) and Na + /K + -ATPase activity was measured at those temperatures for each acclimation group. Both species had their highest gill Na + /K + -ATPase activity at the highest acclimation temperature. Na + /K + -ATPase activity of gills exhibited species-specific differences, and was higher in A. anatina than U. tumidus in all test groups at all test temperatures. Temperature dependence of Na + /K + -ATPase was confirmed in both species, being highest at temperatures between +4 and + 14 °C when Q 10 values in the acclimation groups varied between 5.06 and 6.71. Our results underline the importance of Na + /K + -ATPase of gills for the freshwater mussels in warming waters. Because Na + /K + -ATPase is the driving force behind ciliary motion, our results also suggest that in warming waters A. anatina may be more tolerant at sustaining vigorous ciliary action (associated with elevated respiration rates and filter-feeding) than U. tumidus. Overall, our results indicate great flexibility of the mussel's ecophysiological characteristics as response to changing conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Decapod Crustacea exhibit a marine origin, but many taxa have occupied environments ranging from brackish to fresh water and terrestrial habitats, overcoming their inherent osmotic challenges. Osmotic and ionic regulation is achieved by the gill epithelia, driven by two active ATP-hydrolyzing ion transporters, the basal (Na + , K + )-ATPase and the apical V(H + )-ATPase. The kinetic characteristic of gill (Na + , K + )-ATPase and the mRNA expression of its α subunit have been widely studied in various decapod species under different salinity challenges. However, the evolution of the primary structure has not been explored, especially considering the functional modifications associated with decapod phylogeny. Here, we proposed a model for the topology of the decapod α subunit, identifying the sites and motifs involved in its function and regulation, as well as the patterns of its evolution assuming a decapod phylogeny. We also examined both the amino acid substitutions and their functional implications within the context of biochemical and physiological adaptation. The α-subunit of decapod crustaceans shows greater conservation (∼94% identity) compared to the β-subunit (∼40%). While the binding sites for ATP and modulators are conserved in the decapod enzyme, the residues involved in the α-β interaction are only partially conserved. In the phylogenetic context of the complete sequence of (Na + , K + )-ATPase α-subunit, most substitutions appear to be characteristic of the entire group, with specific changes for different subgroups, especially among brachyuran crabs. Interestingly, there was no consistent separation of α-subunit partial sequences related to habitat, suggesting that the convergent evolution for freshwater or terrestrial modes of life is not correlated with similar changes in the enzyme's primary amino acid sequence., Competing Interests: Declaration of competing interest All authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Marine fishes excrete excess H + using basolateral Na + -K + -ATPase (NKA) and apical Na + /H + exchanger 3 (NHE3) in gill ionocytes. However, the mechanisms that regulate H + excretion during exposure to environmentally relevant hypercapnia (ERH) remain poorly understood. Here, we explored transcriptomic, proteomic, and cellular responses in gills of juvenile splitnose rockfish ( Sebastes diploproa ) exposed to 3 days of ERH conditions (pH ∼7.5, ∼1,600 μatm Pco 2 ). Blood pH was fully regulated at ∼7.75 despite a lack of significant changes in gill 1 ) mRNAs coding for proteins involved in blood acid-base regulation, 2 ) total NKA and NHE3 protein abundance, and 3 ) ionocyte density. However, ERH-exposed rockfish demonstrated increased NKA and NHE3 abundance on the ionocyte plasma membrane coupled with wider apical membranes and greater extension of apical microvilli. The observed gill ionocyte remodeling is consistent with enhanced H + excretion that maintains blood pH homeostasis during exposure to ERH and does not necessitate changes at the expression or translation levels. These mechanisms of phenotypic plasticity may allow fishes to regulate blood pH during environmentally relevant acid-base challenges and thus have important implications for both understanding how organisms respond to climate change and for selecting appropriate metrics to evaluate its impact on marine ecosystems. NEW & NOTEWORTHY Splitnose rockfish exposed to environmentally relevant hypercapnia utilize existing proteins (rather than generate additional machinery) to maintain homeostasis.

Lowering the levels of the cellular prion protein (PrPC) is widely considered a promising strategy for the treatment of prion diseases. Building on work that established immediate spatial proximity of PrPC and Na+, K+-ATPases (NKAs) in the brain, we recently showed that PrPC levels can be reduced by targeting NKAs with their natural cardiac glycoside (CG) inhibitors. We then introduced C4'-dehydro-oleandrin as a CG with improved pharmacological properties for this indication, showing that it reduced PrPC levels by 84% in immortalized human cells that had been differentiated to acquire neural or astrocytic characteristics. Here we report that our lead compound caused cell surface PrPC levels to drop also in other human cell models, even when the analyses of whole cell lysates suggested otherwise. Because mice are refractory to CGs, we explored guinea pigs as an alternative rodent model for the preclinical evaluation of C4'-dehydro-oleandrin. We found that guinea pig cell lines, primary cells, and brain slices were responsive to our lead compound, albeit it at 30-fold higher concentrations than human cells. Of potential significance for other PrPC lowering approaches, we observed that cells attempted to compensate for the loss of cell surface PrPC levels by increasing the expression of the prion gene, requiring daily administration of C4'-dehydro-oleandrin for a sustained PrPC lowering effect. Regrettably, when administered systemically in vivo, the levels of C4'-dehydro-oleandrin that reached the guinea pig brain remained insufficient for the PrPC lowering effect to manifest. A more suitable preclinical model is still needed to determine if C4'-dehydro-oleandrin can offer a cost-effective complementary strategy for pushing PrPC levels below a threshold required for long-term prion disease survival., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: GS and DW declare that they are co-inventors on patent applications named ‘Compounds for altering levels of one or more NKA alpha subunits and their use in treating prion diseases or brain diseases associated with cellular prion protein’ filed in the US and Canada as well as a European patent application named ‘Compounds and methods to treat prion and related diseases’ based on a PCT international filing (PCT/CA2022/050358). These patent applications describe the use of CGs for the purpose of reducing steady-state levels of PrPC. This potential conflict-of-interest does not affect the adherence of all authors to journal policies on the sharing of data and materials. This does not alter our adherence to PLoS One policies on sharing data and materials., (Copyright: © 2024 Eid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The corneal endothelium is responsible for pumping fluid out of the stroma in order to maintain corneal transparency, which depends in part on the expression and activity of sodium-potassium pumps. In this study, we evaluated how physiologic pressure and flow influence transcription, protein expression, and activity of Na+/K+-ATPase. Native and engineered corneal endothelia were cultured in a bioreactor in the presence of pressure and flow (hydrodynamic culture condition) or in a Petri dish (static culture condition). Transcription of ATP1A1 was assessed using qPCR, the expression of the α1 subunit of Na+/K+-ATPase was measured using Western blots and ELISA assays, and Na+/K+-ATPase activity was evaluated using an ATPase assay in the presence of ouabain. Results show that physiologic pressure and flow increase the transcription and the protein expression of Na+/K+-ATPase α1 in engineered corneal endothelia, while they remain stable in native corneal endothelia. Interestingly, the activity of Na+/K+-ATPase was increased in the presence of physiologic pressure and flow in both native and engineered corneal endothelia. These findings highlight the role of the in vivo environment on the functionality of the corneal endothelium.

Natural compounds constitute a major resource for the development of medicines for multiple diseases. While many natural compounds show strong biological activity, the mechanisms that confer clinical benefits are often elusive and have been attributed to multiple pathways. Periplogenin (PPG), a natural compound isolated from Cortex periplocae, exhibits strong anti-tumor activities in several human cancer cell lines. However, its molecular mode of action remained unclear. In this study, we leveraged a forward genetic screening approach in DU145 prostate cancer cells to uncover the molecular target of PPG using chemical mutagenesis. Next generation sequencing revealed that a single amino acid substitution at amino acid 804 in ATP1A1 (ATPase Na + /K + Transporting Subunit Alpha 1) confers resistance to the cytotoxic activity of PPG. Mechanistically, ATP1A1 T804 forms a hydrogen bond with PPG which is abolished by the T804A substitution in ATP1A1, resulting in resistance to PPG treatment in vitro. Importantly, in vivo, PPG strongly suppressed tumor development in a DU145 xenograft model whereas DU145 xenograft tumors carrying a ATP1A1-T804A mutation were largely unaffected by the treatment. These findings demonstrate that PPG suppresses the growth of DU145 prostate cancer cells in vitro and in vivo by directly binding to ATP1A1 and highlight the power of our unbiased forward genetic screening approach to uncover direct drug target structures at single amino acid resolution., (© 2024. The Author(s).)

Large-conductance, calcium-activated potassium channels (BK channels) and the Na/K-ATPase are expressed universally in vascular smooth muscle. The Na/K-ATPase may act via changes in the intracellular Ca 2+ concentration mediated by the Na/Ca exchanger (NCX) and via Src kinase. Both pathways are known to regulate BK channels. Whether BK channels functionally interact in vascular smooth muscle cells with the Na/K-ATPase remains to be elucidated. Thus, this study addressed the hypothesis that BK channels limit ouabain-induced vasocontraction. Rat mesenteric arteries were studied using isometric myography, FURA-2 fluorimetry and proximity ligation assay. The BK channel blocker iberiotoxin potentiated methoxamine-induced contractions. The cardiotonic steroid, ouabain (10 -5  M), induced a contractile effect of IBTX at basal tension prior to methoxamine administration and enhanced the pro-contractile effect of IBTX on methoxamine-induced contractions. These facilitating effects of ouabain were prevented by the inhibition of either NCX or Src kinase. Furthermore, inhibition of NCX or Src kinase reduced the BK channel-mediated negative feedback regulation of arterial contraction. The effects of NCX and Src kinase inhibition were independent of each other. Co-localization of the Na/K-ATPase and the BK channel was evident. Our data suggest that BK channels limit ouabain-induced vasocontraction by a dual mechanism involving the NCX and Src kinase signaling. The data propose that the NCX and the Src kinase pathways, mediating the ouabain-induced activation of the BK channel, act in an independent manner., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Rapid eye movement sleep (REMS) maintains brain excitability at least by regulating Na-K ATPase activity. Although REMS deprivation (REMSD)-associated elevated noradrenaline (NA) increases Na-K ATPase protein expression, its mRNA transcription did not increase. We hypothesized and confirmed both in vivo as well as in vitro that elevated mRNA stability explains the apparent puzzle. The mRNA stability was measured in control and REMSD rat brain with or without in vivo treatment with α 1 -adrenoceptor (AR) antagonist, prazosin (PRZ). Upon REMSD, Na-K ATPase α1-, and α2-mRNA stability increased significantly, which was prevented by PRZ. To decipher the molecular mechanism of action, we estimated NA-induced Na-K ATPase mRNA stability in Neuro-2a cells under controlled conditions and by transcription blockage using Actinomycin D (Act-D). NA increased Na-K ATPase mRNA stability, which was prevented by PRZ and propranolol (PRP, β-AR antagonist). The knockdown assay confirmed that the increased mRNA stabilization was induced by elevated cytoplasmic abundance of Human antigen R (HuR) and involving (Phospholipase C) PLC-mediated activation of Protein Kinase C (PKC). Additionally, using cell-impermeable Enz-link sulfo NHS-SS-Biotin, we observed that NA increased Na-K ATPase α1-subunits on the Neuro-2a cell surface. We conclude that REMSD-associated elevated NA, acting on α1- and β-AR, increases nucleocytoplasmic translocation of HuR and increases Na-K ATPase mRNA stability, resulting in increased Na-K ATPase protein expression. The latter then gets translocated to the neuronal membrane surface involving both PKC and (Protein Kinase A) PKA-mediated pathways. These findings may be exploited for the amelioration of REMSD-associated chronic disorders and symptoms., (© 2024 International Society for Neurochemistry.)

The treatment of non-small cell lung cancer (NSCLC) is known as a significant level of unmet medical need in spite of the progress in targeted therapy and personalized therapy. Overexpression of the Na + /K + -ATPase contributes to NSCLC progression, suggesting its potentiality in antineoplastic approaches. Epi-reevesioside F, purified from Reevesia formosana, showed potent anti-NSCLC activity through inhibiting the Na + /K + -ATPase, leading to internalization of α1- and α3-subunits in Na + /K + -ATPase and suppression of Akt-independent mTOR-p70S6K-4EBP1 axis. Epi-reevesioside F caused a synergistic amplification of apoptosis induced by gefitinib but not cisplatin, docetaxel, etoposide, paclitaxel, or vinorelbine in both NCI-H460 and A549 cells. The synergism was validated by enhanced activation of the caspase cascade. Bax cleavage, tBid formation, and downregulation of Bcl-xL and Bcl-2 contributed to the synergistic apoptosis induced by the combination treatment of epi-reevesioside F and gefitinib. The increase of membrane DR4 and DR5 levels, intracellular Ca 2+ concentrations, and active m-calpain expression were responsible for the caspase-8 activation and Bax cleavage. The increased α-tubulin acetylation and activation of MAPK (i.e., p38 MAPK, Erk, and JNK) depending on cell types contributed to the synergistic mechanism under combination treatment. These signaling pathways that converged on profound c-Myc downregulation led to synergistic apoptosis in NSCLC. In conclusion, the data suggest that epi-reevesioside F inhibits the Na + /K + -ATPase and displays potent anti-NSCLC activity. Epi-reevesioside F sensitizes gefitinib-induced apoptosis through multiple pathways that converge on c-Myc downregulation. The data support the inhibition of Na + /K + -ATPase as a switch-on mechanism to sensitize gefitinib-induced anti-NSCLC activity., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In this study, marine medaka (Oryzias melastigma) embryos were exposed to different concentrations of water-accommodated fractions (WAFs) and chemically enhanced water-accommodated fractions (CEWAFs) of Oman crude oil for 14 d by semi-static exposure methods. The effects on growth and development and energy metabolism process were evaluated. Results showed that embryo survival and hatchability were decreased in a dose-dependent manner with an increase in the concentration of petroleum hydrocarbon compounds, whereas the malformation exhibited a dose-dependent increase. Compared to the control, the adenosine triphosphate (ATP) content and Na + -K + -ATPase (NKA) activities of embryos exposed to both WAFs and CEWAFs were reduced, while intracellular reactive oxygen species (ROS) levels and NADH oxidase (NOX) activities were increased. Our study demonstrated that exposure to crude oil dispersed by chemical dispersant affected the growth and development of marine medaka embryos, caused oxidative stress while produced a series of malformations in the body and dysregulation in energy metabolism. In comparison, the toxic effects of chemically dispersed crude oil might be more severe than the oil itself in the equivalent diluted concentration treatment solution. These would provide more valuable and reliable reference data for the use of chemical dispersants in oil spills., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pesticides are widely used to control weeds and pests in agricultural settings but harm non-target aquatic organisms. In this study, our objective was to evaluate the effect of short-term exposure (one week) to environmentally relevant concentrations of pesticides mixture (low concentration: 0.4 μg/l atrazine, 0.5 μg/l Roundup®, and 0.5 μg/l 2,4-D; high concentration: 0.8 μg/l atrazine, 1 μg/l Roundup®, and 1 μg/l 2,4-D) on tissue architecture, body fluid conditions, and 3-nitrotyrosine protein (NTP) and Na + /K + -ATPase, expressions in tissues of American oyster (Crassostrea virginica) under controlled laboratory conditions. Histological analysis demonstrated the atrophy in the gills and digestive glands of oysters exposed to pesticides mixture. Periodic acid-Schiff (PAS) staining showed the number of hemocytes in connective tissue increased in low- and high-concentration pesticides exposure groups. However, pesticides treatment significantly (P < 0.05) decreased the amount of mucous secretion in the gills and digestive glands of oysters. The extrapallial fluid (i.e., body fluid) protein concentrations and glucose levels were dropped significantly (P < 0.05) in oysters exposed to high-concentration pesticides exposure groups. Moreover, immunohistochemical analysis showed significant upregulations of NTP and Na + /K + -ATPase expressions in the gills and digestive glands in pesticides exposure groups. Our results suggest that exposure to environmentally relevant pesticides mixture causes morphological changes in tissues and alters body fluid conditions and NTP and Na + /K + -ATPase expressions in tissues, which may lead to impaired physiological functions in oysters., Competing Interests: Declaration of competing interest There are no competing interests in this research work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Epilepsy is a chronic neurological disease. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient diagnosed as epilepsy caused by ATP1A2 gene mutation. Induced pluripotent stem cells (iPSCs) were developed using non-integrating episomal vectors containing OCT4, SOX2, KLF4, BCL-XL and C-MYC. The established iPSC line (SDCHi007-A) displayed pluripotent cell morphology, high expression levels of pluripotency markers, differentiation potential in vitro, normal karyotype, and remaining the original ATP1A2 gene mutation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Evidence suggests that increasing salt intake in pregnancy lowers blood pressure, protecting against preeclampsia. We hypothesized that sodium (Na + ) evokes beneficial placental signals that are disrupted in preeclampsia., Methods: Blood and urine were collected from nonpregnant women of reproductive age (n=26) and pregnant women with (n=50) and without (n=55) preeclampsia, along with placental biopsies. Human trophoblast cell lines and primary human trophoblasts were cultured with varying Na + concentrations., Results: Women with preeclampsia had reduced placental and urinary Na + concentrations, yet increased urinary angiotensinogen and reduced active renin, aldosterone concentrations, and osmotic response signal TonEBP (tonicity-responsive enhancer binding protein) expression. In trophoblast cell cultures, TonEBP was consistently increased upon augmented Na + exposure. Mechanistically, inhibiting Na + /K + -ATPase or adding mannitol evoked the TonEBP response, whereas inhibition of cytoskeletal signaling abolished it., Conclusions: Enhanced Na + availability induced osmotic gradient-dependent cytoskeletal signals in trophoblasts, resulting in proangiogenic responses. As placental salt availability is compromised in preeclampsia, adverse systemic responses are thus conceivable., Competing Interests: None.

In skeletal muscle, Na + ,K + -ATPase (NKA), a heterodimeric (α/β) P-type ATPase, has an essential role in maintenance of Na + and K + homeostasis, excitability, and contractility. AMP-activated protein kinase (AMPK), an energy sensor, increases the membrane abundance and activity of NKA in L6 myotubes, but its potential role in regulation of NKA content in skeletal muscle, which determines maximum capacity for Na + and K + transport, has not been clearly delineated. We examined whether energy stress and/or AMPK affect expression of NKA subunits in rat L6 and primary human myotubes. Energy stress, induced by glucose deprivation, increased protein content of NKAα1 and NKAα2 in L6 myotubes, while decreasing the content of NKAα1 in human myotubes. Pharmacological AMPK activators (AICAR, A-769662, and diflunisal) modulated expression of NKA subunits, but their effects only partially mimicked those that occurred in response to glucose deprivation, indicating that AMPK does not mediate all effects of energy stress on NKA expression. Gene silencing of AMPKα1/α2 increased protein levels of NKAα1 in L6 myotubes and NKAα1 mRNA levels in human myotubes, while decreasing NKAα2 protein levels in L6 myotubes. Collectively, our results suggest a role for energy stress and AMPK in modulation of NKA expression in skeletal muscle. However, their modulatory effects were not conserved between L6 myotubes and primary human myotubes, which suggests that coupling between energy stress, AMPK, and regulation of NKA expression in vitro depends on skeletal muscle cell model., (© 2024. The Author(s).)

Anadromous salmon populations are declining in the Pacific Northwest, with high mortality during the transition from fresh- to seawater as smolts, a stage particularly vulnerable to adverse environmental conditions. This study seeks to explore the impacts of warming and ocean acidification on the transition of life in freshwater to life at sea in Chinook salmon smolts. In a fully factorial experiment, we transitioned Chinook salmon from fresh- to seawater at current and future conditions of temperature (13 °C and 16 °C, respectively) and ocean acidification (400 and 1400 atm CO 2 ), including a fluctuating CO 2 treatment (between control and high CO 2 ) that may be more representative of natural environmental conditions associated with upwelling and tidal cycling. We hypothesized that constant elevated CO 2 levels would impair smoltification success immediately following seawater transfer, but that fluctuating conditions would be even more physiologically challenging. We predicted that elevated temperatures would exacerbate these effects. To test this, we measured plasma ion concentrations, gill Na + /K + -ATPase (NKA) isoform mRNA and protein expression, as well as condition indices in freshwater and following 1, 3, 6, and 18 days in seawater at the respective treatments. We confirmed the existence of gill freshwater and seawater isoforms of NKA (α1a and α1b, respectively) in Chinook salmon for the first time, and found an upregulation of both isoforms in the fluctuating CO 2 treatment but a reduction of the number of NKA α1b cells 3-days post seawater transfer at 13 °C. At 16 °C, NKA α1b was upregulated in high CO 2 levels, with an elevated hematocrit indicating fish were likely stressed. Taken together, plasma ions, gill NKA and condition indices revealed a complex response to interacting warming and acidification during the first few days in seawater, however there were no longer-term adverse physiological responses. Thus, Chinook salmon appear to be relatively resilient to near-future climate change., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Pufferfish of the genus Takifugu possess tetrodotoxin (TTX), known as "pufferfish toxin" and it is believed that pufferfish eggs and newly hatched larvae utilize TTX as a defensive substance against predators. However, the mechanism for the placement of TTX to specific cells on the larval body surface during the developmental process remains unknown. In this study, we clarify the distribution and characteristics of TTX-rich cells. We performed whole-mount immunohistochemistry (IHC) using anti-TTX monoclonal antibody on larvae of two pufferfish species, Takifugu rubripes and Takifugu alboplumbeus, just after hatching. This allowed observation of the TTX location and compared it with those of wheat germ agglutinin (WGA)-positive (periodic acid-Schiff (PAS)-positive) cells for mucous cells and IHC using anti-Na + /K + -ATPase (NKA) monoclonal antibody for ionocytes. As a result, uniformly scattered localization of TTX-rich cells was commonly observed in the epidermis of the larvae of the two Takifugu species. TTX-rich cells were WGA-negative (PAS-negative) and structurally distinct from NKA-positive cells, suggesting that TTX-rich cells are unreported small cells unique to pufferfish skin, but not mucous cells nor ionocytes., (© 2024. The Author(s).)

Global warming significantly impacts aquatic ecosystems, with changes in the salt environment negatively affecting the physiological responses of fish. We investigated the impact of hyposalinity on the physiological responses and intestinal microbiota of Sebastes schlegelii under the context of increased freshwater influx due to climate change. We focused on the osmoregulatory capacity, oxidative stress responses, and alterations in the intestinal microbiome of S. schlegelii under low-salinity conditions. Our findings revealed compromised osmoregulatory capacity in S. schlegelii under low-salinity conditions, accompanied by the activation of oxidative stress responses, indicating physiological adaptations to cope with environmental stress. Specifically, changes in Na + /K + -ATPase (NKA) activity in gill tissues were associated with decreased osmoregulatory capacity. Furthermore, the analysis of the intestinal microbiome led to significant changes in microbial diversity. Exposure to low-salinity environments led to dysbiosis, with notable decreases in the relative abundance of Gammaproteobacteria at the class level and specific genera such as Enterovibrio, and Photobacterium. Conversely, Bacilli classes, along with genera like Mycoplasma, exhibited increased proportions in fish exposed to low-salinity conditions. These findings underscore the potential impact of environmental salinity changes on the adaptive capacity of fish species, particularly in the context of aquaculture. Moreover, they highlight the importance of considering both physiological and microbial responses in understanding the resilience of aquatic organisms to environmental stress. Additionally, they highlight the importance of intestinal microbiota analyses in understanding the immune system and disease management in fish., Competing Interests: Declarations Approval for animal research was obtained from the Institutional Animal Care and Use Committee of Korea Maritime and Ocean University (approved no # KMOU IACUC 2023–05). Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

We used an animal model of salt-sensitive hypertension (SSH) in which ovariectomized (oVx) rats developed hypertension with high salt (HS) intake. Hypertension is accompanied by changes in the percentage of CD4 + T lymphocytes, immune CD45 + cell infiltration into renal tissue, and changes in Na + , K + - ATPase (NKA) expression in both renal tissue and peripheral blood mononuclear cells (PBMCs). To determine whether the observed changes resulted from HS intake, high blood pressure, or both, hydralazine (HDZ) was used to lower blood pressure. The oVx HS rats received two HDZ schedules either to prevent or to treat hypertension. NKA was overexpressed in the kidneys of all oVx groups and in PBMCs of oVx HS rats. This pattern was not altered with HDZ treatment. Changes in CD4 + T lymphocytes and renal infiltration of CD45 + cells were not reversed either. High salt, but not high blood pressure, induces immune cell activation and renal infiltration. Overexpressed NKA is the primary event, and HS is the perturbation to the system in this model of SSH, which resembles the postmenopausal state., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In this study, we assessed the impact of hepatocyte growth factor (HGF) on corneal endothelial cells (CECs), finding that HGF concentrations of 100-250 ng/mL significantly increased CEC proliferation by 30%, migration by 32% and improved survival under oxidative stress by 28% compared to untreated controls ( p < 0.05). The primary objective was to identify non-fibrotic pharmacological strategies to enhance corneal endothelial regeneration, addressing a critical need in conditions like Fuchs' endothelial dystrophy (FED), where donor tissue is scarce. To confirm the endothelial nature of the cultured CECs, Na + /K + -ATPase immunohistochemistry was performed. Proliferation rates were determined through BrdU incorporation assays, while cell migration was assessed via scratch assays. Cell viability was evaluated under normal and oxidative stress conditions using WST-1 assays. To ensure that HGF treatment did not trigger epithelial-mesenchymal transition, which could lead to undesirable fibrotic changes, α-SMA staining was conducted. These comprehensive methodologies provided robust data on the effects of HGF, confirming its potential as a therapeutic agent for corneal endothelial repair without inducing harmful EMT, as indicated by the absence of α-SMA expression. These findings suggest that HGF holds therapeutic promise for enhancing corneal endothelial repair, warranting further investigation in in vivo models to confirm its clinical applicability.

ATP1A3 is a Na,K-ATPase gene expressed specifically in neurons in the brain. Human mutations are dominant and produce an unusually wide spectrum of neurological phenotypes, most notably rapid-onset dystonia parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). Here we compared heterozygotes of two mouse lines, a line with little or no expression ( Atp1a 3 tm1Ling/+ ) and a knock-in expressing p.Asp801Tyr (D801Y, Atp1a3 +/D801Y ). Both mouse lines had normal lifespans, but Atp1a3 +/D801Y had mild perinatal mortality contrasting with D801N mice ( Atp1a3 +/D801N ), which had high mortality. The phenotypes of Atp1a 3 tm1Ling/+ and Atp1a3 +/D801Y were different, and testing of each strain was tailored to its symptom range. Atp1a 3 tm1Ling/+ mice displayed little at baseline, but repeated ethanol intoxication produced hyperkinetic motor abnormalities not seen in littermate controls. Atp1a3 +/D801Y mice displayed robust phenotypes: hyperactivity, diminished posture consistent with hypotonia, and deficiencies in beam walk and wire hang tests. Symptoms also included qualitative motor abnormalities that are not well quantified by conventional tests. Paradoxically, Atp1a3 +/D801Y showed sustained better performance than wild type on the accelerating rotarod. Atp1a3 +/D801Y mice were overactive in forced swimming and afterward had intense shivering, transient dystonic postures, and delayed recovery. Remarkably, Atp1a3 +/D801Y mice were refractory to ketamine anesthesia, which elicited hyperactivity and dyskinesia even at higher dose. Neither mouse line exhibited fixed dystonia (typical of RDP patients), spontaneous paroxysmal weakness (typical of AHC patients), or seizures but had consistent, measurable neurological abnormalities. A gradient of variation supports the importance of studying multiple Atp1a3 mutations in animal models to understand the roles of this gene in human disease., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Liu et al.)

FGF12 belongs to a subfamily of FGF proteins called FGF homologous factors (FHFs), which until recently were thought to be non-signaling intracellular proteins. Our recent studies have shown that although they lack a conventional signal peptide for secretion, they can reach the extracellular space, especially under stress conditions. Here, we unraveled that the long "a" isoform of FGF12 is secreted in a pathway involving the A1 subunit of Na(+)/K(+) ATPase (ATP1A1), Tec kinase and lipids such as phosphatidylinositol and phosphatidylserine. Further, we showed that the short "b" isoform of FGF12, which binds ATP1A1 and phosphatidylserine less efficiently, is not secreted from cells. We also indicated regions in the FGF12a protein sequence that are crucial for its secretion, including N-terminal fragment and specific residues, and proposed that liquid-liquid phase separation may be important in this process. Our results strongly suggest that the mechanism of this process is very similar for all unconventionally secreted FGF proteins., (© 2024. The Author(s).)

Objective: To evaluate the effects of fine particle matter (PM 2.5 ) and ozone (O 3 ) combined exposure on adenosine triphosphate (ATP) amount and ATPase activities in nasal mucosa of Sprague Dawley (SD) rats., Methods: Twenty male SD rats were divided into control group ( n =10) and exposure group ( n =10) by random number table method. The rats were fed in the conventional clean environment and the air pollutant exposure system established by our team, respectively, and exposed for 208 d. During the exposure period, the concentrations of PM 2.5 and O 3 in the exposure system were monitored, and a comprehensive assessment of PM 2.5 and O 3 in the exposure system was conducted by combining self-measurement and site data. On the 208 d of exposure, the core, liver, spleen, kidney, testis and other major organs and nasal mucosal tissues of the rats were harvested. Each organ was weighed and the organ coefficient calculated. The total amount of ATP was measured by bioluminescence, and the activities of Na + -K + -ATPase and Ca 2+ -ATPase were detected by spectrophotometry. The t test of two independent samples was used to compare the differences among the indicator groups., Results: From the 3rd week to the end of exposure duration, the body weight of the rats in the exposure group was higher than that in the control group ( P < 0.05), and there was no significant difference in organ coefficients between the two groups. The average daily PM 2.5 concentration in the exposure group was (30.68±19.23) μg/m 3 , and the maximum 8 h ozone concentration (O 3 -8 h) was (82.45±35.81) μg/m 3 . The chemiluminescence value (792.4±274.1) IU/L of ATP in nasal mucosa of the rats in the exposure group was lower than that in the control group (1 126.8±218.1) IU/L. The Na + -K + -ATPase activity (1.53±0.85) U/mg in nasal mucosa of the rats in the exposure group was lower than that in the control group (4.31±1.60) U/mg ( P < 0.05). The protein content of nasal mucosa in the control group and the exposure group were (302.14±52.51) mg/L and (234.58±53.49) mg/L, respectively, and the activity of Ca 2+ -ATPase was (0.81±0.27) U/mg and (0.99±0.73) U/mg, respectively. There was no significant difference between the groups., Conclusion: The ability of power capacity decreased in the rat nasal mucossa under the sub-chronic low-concentration exposure of PM 2.5 and O 3 .

Background: The renal proximal tubule (RPT) plays a pivotal role in regulating sodium reabsorption and thus blood pressure (BP). Transient receptor potential ankyrin 1 (TRPA1) has been reported to protect against renal injury by modulating mitochondrial function. We hypothesize that the activation of TRPA1 by its agonist cinnamaldehyde may mitigate high-salt intake-induced hypertension by inhibiting urinary sodium reabsorption through restoration of renal tubular epithelial mitochondrial function., Methods: Trpa1-deficient (Trpa1-/-) mice and wild-type (WT) mice were fed standard laboratory chow [normal diet (ND) group, 0.4% salt], standard laboratory chow with 8% salt [high-salt diet (HS) group], or standard laboratory chow with 8% salt plus 0.015% cinnamaldehyde [high-salt plus cinnamaldehyde diet (HSC) group] for 6 months. Urinary sodium excretion, reactive oxygen species (ROS) production, mitochondrial function, and the expression of sodium hydrogen exchanger isoform 3 (NHE3) and Na+/K+-ATPase of RPTs were determined., Results: Chronic dietary cinnamaldehyde supplementation reduced tail systolic BP and 24-hour ambulatory arterial pressure in HS-fed WT mice. Compared with the mice fed HS, cinnamaldehyde supplementation significantly increased urinary sodium excretion, inhibited excess ROS production, and alleviated mitochondrial dysfunction of RPTs in WT mice. However, these effects of cinnamaldehyde were absent in Trpa1-/- mice. Furthermore, chronic dietary cinnamaldehyde supplementation blunted HS-induced upregulation of NHE3 and Na+/K+-ATPase in WT mice but not Trpa1-/- mice., Conclusions: The present study demonstrated that chronic activation of Trpa1 attenuates HS-induced hypertension by inhibiting urinary sodium reabsorption through restoring renal tubular epithelial mitochondrial function. Renal TRPA1 may be a potential target for the management of excessive dietary salt intake-associated hypertension., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Journal of Hypertension, Ltd. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Slow brain rhythms, for example during slow-wave sleep or pathological conditions like seizures and spreading depolarization, can be accompanied by oscillations in extracellular potassium concentration. Such slow brain rhythms typically have a lower frequency than tonic action-potential firing. They are assumed to arise from network-level mechanisms, involving synaptic interactions and delays, or from intrinsically bursting neurons. Neuronal burst generation is commonly attributed to ion channels with slow kinetics. Here, we explore an alternative mechanism generically available to all neurons with class I excitability. It is based on the interplay of fast-spiking voltage dynamics with a one-dimensional slow dynamics of the extracellular potassium concentration, mediated by the activity of the Na+/K+-ATPase. We use bifurcation analysis of the complete system as well as the slow-fast method to reveal that this coupling suffices to generate a hysteresis loop organized around a bistable region that emerges from a saddle-node loop bifurcation-a common feature of class I excitable neurons. Depending on the strength of the Na+/K+-ATPase, bursts are generated from pump-induced shearing the bifurcation structure, spiking is tonic, or cells are silenced via depolarization block. We suggest that transitions between these dynamics can result from disturbances in extracellular potassium regulation, such as glial malfunction or hypoxia affecting the Na+/K+-ATPase activity. The identified minimal mechanistic model outlining the sodium-potassium pump's generic contribution to burst dynamics can, therefore, contribute to a better mechanistic understanding of pathologies such as epilepsy syndromes and, potentially, inform therapeutic strategies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Behbood et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Status epilepticus (SE) is a medical emergency associated with high mortality and morbidity. Na + , K + -ATPase, is a promising therapeutic target for SE, given its critical role in regulation of neuron excitability and cellular homeostasis. We investigated the effects of a Na + , K + -ATPase-activating antibody (DRRSAb) on short-term electrophysiological and behavioral consequences of pilocarpine-induced SE. Rats were submitted to pilocarpine-induced SE, followed by intranasal administration (2 μg/nostril). The antibody increased EEG activity following SE, namely, EEG power in theta, beta, and gamma frequency bands, assessed by quantitative analysis of EEG power spectra. One week later, DRRSAb-treated animals displayed less behavioral hyperreactivity in pick-up tests and better performance in novel object recognition tests, indicating that the intranasal administration of this Na + , K + -ATPase activator immediately after SE improves behavioral outcomes at a later time point. These results suggest that Na + , K + -ATPase activation warrants further investigation as an adjunctive therapeutic strategy for SE.

The presence of impermeant molecules within a cell can lead to an increase in cell volume through the influx of water driven by osmosis. This phenomenon is known as the Donnan (or Gibbs-Donnan) effect. Animal cells actively transport ions to counteract the Donnan effect and regulate their volume, actively pumping Na+ out and K+ into their cytosol using the Na+/K+ ATPase (NKA) pump. The pump-leak equations (PLEs) are a system of algebraic-differential equations to model the membrane potential, ion (Na+, K+, and Cl-), and water flux across the cell membrane, which provide insight into how the combination of passive ions fluxes and active transport contribute to stabilizing cell volume. Our broad objective is to provide analytical insight into the PLEs through three lines of investigation: (1) we show that the provision of impermeant extracellular molecules can stabilize the volume of a passive cell; (2) we demonstrate that the mathematical form of the NKA pump is not as important as the stoichiometry for cell stabilization; and (3) we investigate the interaction between the NKA pump and cation-chloride co-transporters (CCCs) on cell stabilization, showing that NCC can destabilize a cell while NKCC and KCC can stabilize it. We incorporate extracellular impermeant molecules, NKA pump, and CCCs into the PLEs and derive the exact formula for the steady states in terms of all the parameters. This analytical expression enables us to easily explore the effect of each of the system parameters on the existence and stability of the steady states., (© 2024 Aminzare and Kay.)

We studied the effect of intramuscular injection of physostigmine and neostigmine on Na + ,K + -ATPase activity in erythrocytes of rats subjected to intense physical exercise. Both anticholinesterase drugs had a significant effect on the development of the stress response, which was expressed in a decrease in the manifestation of its individual components such as the concentration of ascorbic acid in the adrenal glands, stress-related erythrocyte polycythemia, and LPO indicators. Anticholinesterase drugs reverse the stress-induced decrease in Na + ,K + -ATPase activity, as well as changes in its magnesium-dependent properties. There were no changes in the activity of the studied enzyme in the erythrocyte ghosts. We associate the observed differences with the correction of the functions of the cholinergic components of the hypothalamic-pituitary-adrenal axis leading to the development of a hypoergic type stress reaction., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Identifying how the demands of migration are met at the level of gene expression is critical for understanding migratory physiology and can potentially reveal how migratory forms evolve from nonmigratory forms and vice versa. Among fishes, migration between freshwater and seawater (diadromy) requires considerable osmoregulatory adjustments, powered by the ion pump Na + , K + -ATPase (NKA) in the gills. Paralogs of the catalytic α-subunit of the pump (NKA α1a and α1b) are reciprocally upregulated in fresh- and seawater, a response known as paralog-switching, in gills of some diadromous species. We tested ontogenetic changes in NKA α-subunit paralog expression patterns, comparing pre-migrant and migrant alewife (Alosa pseudoharengus) sampled in their natal freshwater environment and after 24 h in seawater. In comparison to pre-migrants, juvenile out-migrants exhibited stronger paralog switching via greater downregulation of NKA α1a in seawater. We also tested microevolutionary changes in the response, exposing juvenile diadromous and landlocked alewife to freshwater (0 ppt) and seawater (30 ppt) for 2, 5, and 15 days. Diadromous and landlocked alewife exhibited salinity-dependent paralog switching, but levels of NKA α1b transcription were higher and the decrease in NKA α1a was greater after seawater exposure in diadromous alewife. Finally, we placed alewife α-subunit NKA paralogs in a macroevolutionary context. Molecular phylogenies show alewife paralogs originated independently of paralogs in salmonids and other teleosts. This study demonstrated that NKA paralog switching is tied to halohabitat profile and that duplications of the NKA gene provided the substrate for multiple, independent molecular solutions that support a diadromous life history., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The Macrobrachium amazonicum complex is composed of at least the Macrobrachium amazonicum and Macrobrachium pantanalense species, with the latter described from specimens originally identified as part of an endemic M. amazonicum population in the Brazilian Pantanal region. While there may be a reproductive barrier between these two Macrobrachium species, both are phylogenetically close, with small genetic distance. However, there is currently no available biochemical information of Macrobrachium pantanalense (Na + , K + )-ATPase. Here, we report the kinetic characteristics of the gill (Na + , K + )-ATPase in two populations of M. pantanalense from Baiazinha Lagoon (Miranda, MS, Brazil) and Araguari River (Uberlândia, MG, Brazil), and compare them with Macrobrachium amazonicum populations from the Paraná-Paraguay River Basin. (Na + , K + )-ATPase activities were 67.9 ± 3.4 and 93.3 ± 4.1 nmol Pi min -1  mg -1 protein for the Baiazinha Lagoon and Araguari River populations, respectively. Two ATP hydrolyzing sites were observed for the Araguari River population while a single ATP site was observed for the Baiazinha Lagoon shrimps. Compared to the Araguari River population, a 3-fold greater apparent affinity for Mg 2+ and Na + was estimated for the Baiazinha Lagoon population, but no difference in K + affinity and ouabain inhibition was seen. The kinetic differences observed in the gill (Na + , K + )-ATPase between the two populations of M. pantanalense, compared with those of various M. amazonicum populations, highlight interspecific divergence within the Macrobrachium genus, now examined from a biochemical perspective., Competing Interests: Declaration of competing interest All authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sodium p-perfluorous nonenoxybenzene sulfonate (OBS)-one of the main alternatives to perfluorooctane sulfonate-has been increasingly detected in both aquatic environments and human bodies. Therefore, the pathogenic risks of OBS exposure warrant attention, especially its central nervous system toxicity mechanism under long-term exposure. In this study, the effects and mechanisms of OBS on the zebrafish brain at 40 days post exposure were examined. The results demonstrated that at 3.2 μg/L, OBS had no significant effect on the zebrafish brain, but 32 μg/L OBS caused depression or poor social behavior in zebrafish and reduced both their memory and survival ability. These changes were accompanied by histological damage and cell apoptosis. Furthermore, OBS caused the accumulation of excessive reactive oxygen species in the fish brain, leading to oxidative stress and subsequently cell apoptosis. Moreover, an imbalance of both inflammatory factors (IL-6, IL-1β, IL-10, TNF-α, and NF-κB) and neurotransmitters (GABA and Glu) led to neuroinflammation. Additionally, 32 μg/L OBS induced decreases in mitochondrial membrane potential and Na + -K + -ATPase activity, leading to both mitochondrial structural damage and the emergence of mitochondrial autophagosomes, partly explaining the neurotoxicity of OBS. These results help to analyze the target sites and molecular mechanisms of OBS neurotoxicity and provide a basis for the scientific evaluation of its health risks to humans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The aim of the study was to investigate the effect of ripasudil on corneal endothelial cell survival and migration after two types of descemetorhexis on a human ex vivo model. Eleven human corneoscleral buttons were incubated in either 50 ml organ culture medium containing 10 μM ripasudil or 50 μl dimethyl sulfoxide (DMSO), the vehicle in ripasudil for 2 days prior to wound creation then for 14 days after. The wound was created with either full trephination scoring or by shallow trephination plus manual peeling. At day 14, immunohistochemistry with vimentin and Na+/K+/ATPase markers was conducted. Tissues were assessed at day 3, 7 and 14 for morphology, cell migration, cell viability and cell density. Full trephination scoring created more damage on tissues compared to shallow trephination with full Descemet membrane peeling. In the full trephination scoring group, no differences in cell viability were noted when ripasudil and DMSO were compared. With the peeling method, Ripasudil could protect the endothelial cell death and maintain the morphology compared to the control. At day 14, no differences in the peripheral cell viability and density were found between ripasudil and DMSO, although the ripasudil group presented significantly increased central cell count and cell viability. Increased cell migration was noted with ripasudil and the initial cell morphology of those migrated cells was similar to that of fibroblasts. In conclusion, ex vivo modelling suggested that peeling resulted in less cell damage than scoring and ripasudil maintained better morphology and promoted migration. These effects might be via transformation of endothelial cells into a more motile spindle-like phenotype., Competing Interests: Declaration of competing interest Gregory Moloney – Kowa Industry (consultant). The remaining authors have no conflicts of interest to disclose., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In teleost fish, branchial ionocytes are important sites for osmoregulation and acid-base regulation by maintaining ionic balance in the body fluid. During the early developmental stages before the formation of the gills, teleost ionocytes are localized in the yolk-sac membrane and body skin. By comparing with teleost fish, much less is known about ionocytes in developing embryos of elasmobranch fish. The present study investigated the development of ionocytes in the embryo and larva of cloudy catshark, Scyliorhinus torazame. We first observed ionocyte distribution by immunohistochemical staining with anti-Na + /K + -ATPase (NKA) and anti-vacuolar-type H + -ATPase (V-ATPase) antibodies. The NKA- and V-ATPase-rich ionocytes appeared as single cells in the gill filaments from stage 31, the stage of pre-hatching, while the ionocytes on the body skin and yolk-sac membrane were also observed. From stage 32, in addition to single ionocytes on the gill filaments, some outstanding follicular structures of NKA-immunoreactive cells were developed to fill the inter-filament region of the gill septa. The follicular ionocytes possess NKA in the basolateral membrane and Na + /H + exchanger 3 in the apical membrane, indicating that they are involved in acid-base regulation like single NKA-rich ionocytes. Three-dimensional analysis and whole-mount immunohistochemistry revealed that the distribution of follicular ionocytes was limited to the rostral side of gill septum. The rostral sides of gill septum might be exposed to faster water flow than caudal side because the gills of sharks gently curved backward. This dissymmetric distribution of follicular ionocytes is considered to facilitate efficient body-fluid homeostasis of catshark embryo., (© 2024. The Author(s).)