Ion Secretion Research Articles

Salinity and prolactin regulate anoctamin 1 in the model teleost, Fundulus heteroclitus.

To maintain internal ion balance in marine environments, teleost fishes leverage seawater (SW)-type ionocytes to actively secrete Na+ and Cl- into the environment. It is well established that SW-type ionocytes use apically expressed cystic fibrosis transmembrane conductance regulator 1 (Cftr1) as a conduit for Cl- to exit the gill. Here, we investigated whether the Ca2+-activated Cl- channel, anoctamin 1 (Ano1), provides an additional path for Cl--secretion in euryhaline mummichogs (Fundulus heteroclitus). Two ano1 gene isoforms, denoted ano1.1a and -1.1b, exhibited higher expression in the gill and opercular epithelium of mummichogs long-term acclimated to SW versus fresh water (FW). Branchial ano1.1b and cftr1 expression was increased in mummichogs sampled 24 h after transfer from FW to SW; ano1.1a and -1.1b were upregulated in the gill and opercular epithelium following transfer from SW to hypersaline SW. Alternatively, the expression of ano1.1a, -1.1b, and cftr1 in the gill and opercular epithelium was markedly decreased after transfer from SW to FW. Given its role in attenuating ion secretion, we probed whether prolactin downregulates ano1 isoforms. In addition to attenuating cftr1 expression, a prolactin injection reduced branchial ano1.1a and -1.1b levels. Given how Ano1 mediates Cl- secretion by mammalian epithelial cells, the salinity- and prolactin-sensitive nature of ano1 expression reported here indicates that Ano1 may constitute a novel Cl--secretion pathway in ionocytes. This study encourages a wider evaluation of this putative Cl--secretion pathway and its regulation by hormones in teleost fishes.NEW & NOTEWORTHY In this study, we provide evidence in a teleost fish that the Ca2+-activated Cl- channel, anoctamin 1 may provide an additional path for Cl- secretion by seawater-type ionocytes. Not only is this the first report of a Cftr-independent Cl--secreting pathway conferring survival in seawater but also the first description of its regulation by the pituitary hormone prolactin.

The Influence of Retinol Ointment on Rabbit Skin (Oryctolagus cuniculus) Ion Transport-An In Vitro Study.

Retinoids are known to improve the condition of the skin. Transepithelial transport of sodium and chloride ions is important for proper skin function. So far, the effect of applying vitamin A preparations to the skin on ion transport has not been evaluated. In the study, electrophysiological parameters, including transepithelial electric potential (PD) and transepithelial resistance (R), of rabbit skin specimens after 24 h exposure to retinol ointment (800 mass units/g) were measured in a modified Ussing chamber. The R of the fragments incubated with retinol was significantly different than that of the control skin samples incubated in iso-osmotic Ringer solution. For the controls, the PD values were negative, whereas the retinol-treated specimens revealed positive PD values. Mechanical-chemical stimulation with the use of inhibitors of the transport of sodium (amiloride) or chloride (bumetanide) ions revealed specific changes in the maximal and minimal PD values measured for the retinol-treated samples. Retinol was shown to slightly modify the transport pathways of sodium and chloride ions. In particular, an intensification of the chloride ion secretion from keratinocytes was observed. The proposed action may contribute to deep hydration and increase skin tightness, limiting the action of other substances on its surface.

Isolation, Characterization, and Genome Insight of Pseudomonas jordanii: A Novel Endophyte Enhancing Durum Wheat (Triticum turgidum ssp. durum) Growth under Salinity Stress

Pseudomonas jordanii strain G34 is a moderately halophilic endophytic bacterium isolated from the root tissue of durum wheat plants growing in the saline environment of the Jordan Valley’s Ghor Sweimeh region. Microscopic and biochemical analyses of P. jordanii strain G34 revealed that it is a Gram-negative, non-motile rod. It also exhibits capsule formation, catalase and oxidase positive reactions, indole positivity, citrate utilization, and non-glucose fermenting capability. Pseudomonas jordanii strain G34 showed growth-promoting effects on durum wheat seedlings grown under severe salinity stress conditions up to a 200 mM NaCl concentration. The draft genome of P. jordanii strain G34 comprises 5,142,528 base pairs (bp) and possesses a G + C content of 64.0%. It contains 57 RNA coding genes and is predicted to encode a total of 4675 protein-coding genes. Putative genes linked to various aspects of the bacterial endophyte lifestyle were identified including ion transport, motility, secretion, adhesion, delivery systems, and plant cell wall modification. Performing a comprehensive phylogenomic analysis identified P. jordanii as a new species, with its closest relative being P. argentinensis LMG 22563, sharing only around 40.2% digital DNA-DNA hybridization identity. Pseudomonas jordanii strain G34 holds great potential for future use as a biofertilizer in saline environments.

Effect of different microplastics on the mobilization of soil inorganic phosphorus by exomycorrhizal fungi

<p>Microplastics (MPs) can affect the phosphorus (P) cycle in soil by changing soil properties and the function of microbial communities, but their impact on soil P availability remains unclear. To explore the effects of different types of MPs on the mobilization of soil inorganic P by exomycorrhizal fungi, Lactarius delicious (Ld) was used as the test strain, and polystyrene microspheres (PS), polypropylene (PP) and poly(lactic acid) (PLA) were used as test materials. The soil was used as the only P source for the media. The effect of different types of MPs on the mobilization of soil inorganic P by exomycorrhizal fungi was studied using liquid culture method. The results showed that exposure to MPs at low to medium concentrations (10 or 100 mg L-1) activated inorganic P in soil by stimulating the secretion of additional organic acids and hydrogen ions by ectomycorrhizal fungi, improving the available P in soil. The effect of mobilizing inorganic P was more significant in the treatment groups exposed to low and medium concentrations of biodegradable PLA. This may be related to the fact that PLA stimulated the ectomycorrhizal fungi to secrete more organic acids, and the release of a large amount of lactic acid after PLA was biodegraded. However, when the exposure concentration of MPs further increased, the growth of ectomycorrhizal fungal mycelium may be inhibited, the secretion of organic acids and hydrogen ions may be reduced, and the mobilization ability of soil inorganic P may be limited. In addition, it should be noted that high concentrations of non biodegradable MPs (PS and PP) had significant limiting effects on the mobilization of soil inorganic P by exomycorrhizal fungi. Therefore, our study results provide evidence that the presence of MPs may reduce the availability of soil P. Further research should focus on maintaining or improving P availability in plant-microbe systems in MP-polluted soil.</p>

Intestinal barrier function in the naked mole-rat: an emergent model for gastrointestinal insights.

The intestinal barrier plays a crucial role in homeostasis by both facilitating the absorption of nutrients and fluids and providing a tight shield to prevent the invasion by either pathogen or commensal microorganisms. Intestinal barrier malfunction is associated with systemic inflammation, oxidative stress, and decreased insulin sensitivity, which may lead to the dysregulation of other tissues. Therefore, a deeper understanding of physiological aspects related to an enhanced barrier function is of significant scientific and clinical relevance. The naked mole-rat has many unusual biological features, including attenuated colonic neuron sensitivity to acid and bradykinin and resistance to chemical-induced intestinal damage. However, insight into their intestinal barrier physiology is scarce. Here, we observed notable macroscopic and microscopic differences in intestinal tissue structure between naked mole-rats and mice. Moreover, naked mole-rats showed increased number of larger goblet cells and elevated mucus content. In measuring gut permeability, naked mole-rats showed reduced permeability compared with mice, measured as transepithelial electrical resistance, especially in ileum. Furthermore, intestinal ion secretion induced by serotonin, bradykinin, histamine, and capsaicin was significantly reduced in naked mole-rats compared with mice, despite the expression of receptors for all these agonists. In addition, naked mole-rats exhibited reduced prosecretory responses to the nonselective adenylate cyclase activator forskolin. Collectively, these findings indicate that naked mole-rats possess a robust and hard-to-penetrate gastrointestinal barrier that is resistant to environmental and endogenous irritants. Naked mole-rats may therefore provide valuable insights into the physiology of the intestinal barrier and set the stage for the development of innovative and effective therapies.NEW & NOTEWORTHY This is the first study to characterize the intestinal function of naked mole-rats. We found that these animals show a robust gut tissue structure, displaying thicker intestinal layers, longer villi, and larger crypts. Naked mole-rats showed more and larger goblet cells, with increased mucus content. Intestinal permeability, especially in the ileum, was substantially lower than that of mice. Finally, naked mole-rats showed reduced intestinal anion secretion in response to serotonin, bradykinin, histamine, capsaicin, and forskolin.

Role of CRF1 and CRF2 Receptors in Stress-induced Increase of Intestinal Epithelial Permeability in the Mouse Colon

Background and Aims: Stress is known to increase intestinal permeability, which may contribute to the development of irritable bowel syndrome (IBS) and other intestinal disorders. Corticotropin releasing factor (CRF) has been proven to mediate stress-induced changes in gut function, such as increased colonic motility, ion secretion, and intestinal permeability. Actions of CRF are mediated by two G-protein coupled receptors, CRF1 and CRF2; both are expressed in the intestine. However, the roles of the CRF1 and CRF2 receptors in stress-induced increase of intestinal epithelial permeability remain unknown. Methods: Adult male and female C57BL/6 mice were restrained (1h/d) for five consecutive days. The selective CRF1 and CRF2 receptor antagonists were injected intraperitoneally 15 min prior to the restraint stress. Control mice were kept in their home cages without restraint. After the last restraint stress/control session, the proximal colon was removed, and the mucosa/submucosa preparations were dissected and mounted onto the Ussing Chamber System for the measurement of intestinal permeability. Transepithelial resistance (TER) and FITC-inulin (4kD) flux across the colonic epithelium were used as indicators of paracellular permeability. Horseradish peroxidase (HRP, 40kD) flux was used to measure transcellular permeability. Results: Restraint stress did not alter TER or FITC-inulin flux in male mice. Pretreatment with the CRF1 receptor antagonist NBI 27914 or the CRF2 receptor antagonist antisauvagine-30 had no effect on TER or FITC-inulin flux in male mice. In female mice, restraint stress significantly reduced TER and increased FITC-inulin flux in the proximal colon, which were blocked by NBI 27914 but not by antisauvagine-30. Restraint stress significantly increased HRP flux in both male and female mice. In male mice, pretreatment with NBI 27914 significantly reduced stress-evoked increase of HRP flux, while antisauvagine-30 had no effect. In female mice, both NBI 27914 and antisauvagine-30 significantly reduced stress-evoked increase of HRP flux. Conclusions: Stress evokes different changes in epithelial permeability in the proximal colon of male and female mice. In male mice, stress does not influence paracellular permeability but increases transcellular permeability, which is mediated by the CRF1 receptor. In female mice, stress increases both transcellular and paracellular permeability. Stress-induced increase of paracellular permeability was mediated by the CRF1 receptor, but the increased transcellular permeability was mediated by both CRF1 and CRF2 receptors. University of Wisconsin-La Crosse Undergraduate Research Grant (CS and LB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Contrary effects of the gut metabolites deoxycholate and butyrate on the acetylcholine-evoked calcium response in an enteroendocrine cell model

The interaction of nutrients and bacterial-derived products and their enteroendocrine effects are still enigmatic. We used the human enteroendocrine tumour cell line P-STS isolated from the terminal ileum as cellular sentinel. In this model, P-STS cells react to acetylcholine or histamine with an increase in intracellular calcium mediated by T-type voltage-gated calcium channels followed by serotonin secretion. Incubation with the secondary bile acid deoxycholate inhibited the calcium response to acetylcholine but not to histamine. No response to other nutrients or bacterial products tested (i.e. palmitate, lactate, acetate, proprionate, butyrate, the OR51E1 ligand isovalerate and the HCA2 ligand β-hydroxybutyrate) could be observed. The bile acid receptor TGR5 agonist CCDC did not inhibit the acetylcholine-induced calcium response, and the free fatty acid receptor 2 agonist 4-CMTB had no enhancing effect on the calcium response to acetylcholine. Only butyrate had an enhancing effect on the calcium response in P-STS cells when added for short-time pre-incubation before challenge with acetylcholine. From our data we propose i) an inhibitory effect of deoxycholate at the muscarinic acetylcholine receptor M3 without involvement of TGR5, and that ii) high butyrate concentrations derived from bacterial metabolism of carbohydrates might increase intestinal release of serotonin and potentially also other gut hormones and satiety-inducing peptides independently of known butyrate receptors, thereby influencing intestinal motility and ion secretion.

Molecular mechanisms of dexamethasone actions in COVID-19: Ion channels and airway surface liquid dynamics

The COVID-19 pandemic has been a global health crisis of unprecedented magnitude. In the battle against the SARS-CoV-2 coronavirus, dexamethasone, a widely used corticosteroid with potent anti-inflammatory properties, has emerged as a promising therapy in the fight against severe COVID-19.Dexamethasone is a synthetic glucocorticoid that exerts its therapeutic effects by suppressing the immune system and reducing inflammation. In the context of COVID-19, the severe form of the disease is often characterized by a hyperactive immune response, known as a cytokine storm. Dexamethasone anti-inflammatory properties make it a potent tool in modulating this exaggerated immune response.Lung inflammation may lead to excessive fluid accumulation in the airways which can reduce gas exchange and mucociliary clearance. Pulmonary oedema and flooding of the airways are hallmarks of severe COVID-19 lung disease. The volume of airway surface liquid is determined by a delicate balance of salt and water secretion and absorption across the airway epithelium. In addition to its anti-inflammatory actions, dexamethasone modulates the activity of ion channels which regulate electrolyte and water transport across the airway epithelium. The observations of dexamethasone activation of sodium ion absorption via ENaC Na+ channels and inhibition of chloride ion secretion via CFTR Cl− channels to decrease airway surface liquid volume indicate a novel therapeutic action of the glucocorticoid to reverse airway flooding.This brief review delves into the early non-genomic and late genomic signaling mechanisms of dexamethasone regulation of ion channels and airway surface liquid dynamics, shedding light on the molecular mechanisms underpinning the action of the glucocorticoid in managing COVID-19.

Spontaneous hierarchical surface engineering of minerals through coupled dissolution‐precipitation chemistry

AbstractPeculiar hierarchical microstructures in creatures inspire modern material design with distinct functionalities. Creatures can effortlessly construct sophisticated yet long‐range ordered microstructure across bio‐membrane through ion secretion and precipitation. However, microstructure biomimicry in current technology generally requires elaborate, point‐by‐point fabrication. Herein, a spontaneous yet controllable strategy is developed to achieve surface microstructure engineering through a natural surface phenomenon similar to ion secretion‐precipitation, that is, coupled dissolution‐precipitation. A series of hierarchical microstructures on mineral surfaces in fluids with tunable morphology, orientation, dimension, and spatial distribution are achieved by simply controlling initial dissolution and fluid chemistry. In seawater, long‐range ordered film of vertically aligned brucite flakes forms through interfacial dissolution, nucleation, and confinement‐induced orientation of flakes with vertically grown {110} plane, on the edge of which, fusiform aragonite epitaxially precipitates. With negligible initial surface dissolution, prismatic aragonite epitaxially grows on a calcite polyhedron‐packed surface. By tuning fluid chemistry, closely packed calcite polyhedron and loosely packed calcite micro‐pillars are engineered through rapid and retarded precipitation, respectively. Surprisingly, the spontaneously grown microstructures resemble those deliberately created by human or found in nature, and tremendously modulate surface functionality. These findings open new possibilities for facile and customizable engineering of microstructural surfaces, hierarchical heterostructures, and biomimetic materials.

Structural and functional analysis of salivary intercalated duct cells reveals a secretory phenotype.

Currently, all salivary ducts (intercalated, striated and collecting) are assumed to function broadly in a similar manner, reclaiming ions that were secreted by the secretory acinar cells while preserving fluid volume and delivering saliva to the oral cavity. Nevertheless, there has been minimal investigation into the structural and functional differences between distinct types of salivary duct cells. Therefore, in this study, the expression profile of proteins involved in stimulus-secretion coupling, as well as the function of the intercalated duct (ID) and striated duct cells, was examined. Particular focus was placed on defining differences between distinct duct cell populations. To accomplish this, immunohistochemistry and in situ hybridization were utilized to examine the localization and expression of proteins involved in reabsorption and secretion of ions and fluid. Further, in vivo calcium imaging was employed to investigate cellular function. Based on the protein expression profile and functional data, marked differences between the IDs and striated ducts were observed. Specifically, the ID cells express proteins native to the secretory acinar cells while lacking proteins specifically expressed in the striated ducts. Further, the ID and striated duct cells display different calcium signalling characteristics, with the IDs responding to a neural stimulus in a manner similar to the acinar cells. Overall, our data suggest that the IDs have a distinct role in the secretory process, separate from the reabsorptive striated ducts. Instead, based on our evidence, the IDs express proteins found in secretory cells, generate calcium signals in a manner similar to acinar cells, and, therefore, are likely secretory cells. KEY POINTS: Current studies examining salivary intercalated duct cells are limited, with minimal documentation of the ion transport machinery and the overall role of the cells in fluid generation. Salivary intercalated duct cells are presumed to function in the same manner as other duct cells, reclaiming ions, maintaining fluid volume and delivering the final saliva to the oral cavity. Here we systematically examine the structure and function of the salivary intercalated duct cells using immunohistochemistry, in situ hybridization and by monitoring in vivo Ca2+ dynamics. Structural data revealed that the intercalated duct cells lack proteins vital for reabsorption and express proteins necessary for secretion. Ca2+ dynamics in the intercalated duct cells were consistent with those observed in secretory cells and resulted from GPCR-mediated IP3 production.

Chitosan-Glycolic Acid Gel Modification of Chloride Ion Transport in Mammalian Skin: An In Vitro Study.

Chitosan, a polyaminosaccharide with high medical and cosmetic potential, can be combined with the beneficial properties of glycolic acid to form a gel that not only moisturizes the skin, but also has a regenerative effect. Its involvement in the activation of biochemical processes may be associated with the activity of skin ion channels. Therefore, the aim of the research was to evaluate the immediate (15 s) and long-term (24 h) effect of chitosan-glycolic acid gel (CGG) on the transepithelial electric potential and the transepithelial electric resistance (R) of skin specimens tested in vitro. Stimulation during immediate and prolonged application of CGG to skin specimens resulted in a significant decrease in the measured minimal transepithelial electric potential (PDmin). The absence of any change in the R after the CGG application indicates that it does not affect the skin transmission, or cause distortion, microdamage or changes in ion permeability. However, the reduction in potential may be due to the increased transport of chloride ions, and thus water, from outside the cell into the cell interior. Increased secretion of chloride ions is achieved by stimulating the action of the CFTR (cystic fibrosis transmembrane conductance). It can be assumed that chitosan gently stimulates the secretion of chlorides, while maintaining a tendency to reduce the transport of sodium ions, without causing deformation or tissue damage.

Na+ and K+ homeostasis in different organs of contrasting Zoysia japonica accessions under salt stress

Zoysia japonica Steud., a typical halophyte, can secrete salt through salt glands, which is an important salt stress tolerance mechanism. However, the Na+ and K+ homeostasis in different organs under salt stress has not been reported. In this study, two salt-tolerant (Z011 and Z015) and two salt-sensitive (Z004 and Z103) Zoysia japonica accessions were investigated. We analyzed their Na+ and K+ absorption, transport, distribution and secretion in different organs under salt stress. Under 200 mM NaCl treatment, the Na+ content in the roots of the salt-tolerant accessions was significantly lower but K+ levels and K+/Na+ ratio were both prominently higher than those in the salt-sensitive accessions. Na+ and K+ flux assays in different root zones suggested that, under salt stress, Na+ efflux in the root maturation zone of the salt-tolerant accessions increased significantly compared with the control. The K+ efflux in the root maturation zone of the salt-sensitive accessions showed a prominent increase, but no significant differences were seen in the root meristematic or elongation zones, implying that the root maturation zone is the crucial salt tolerance regulation area of Zoysia japonica for Na+ and K+ absorption and efflux. Studies on cross-sectional tissues of different organs indicated that the roots of the salt-tolerant accessions had a stronger compartmentalization ability aiming to inhibit the entry of Na+ into the stele and prevent the loss of K+ than those of the salt-sensitive accessions. Furthermore, the salt-tolerant accessions could secrete more Na+ and K+ through the salt glands of leaves, while Na+ secretion was much more than K+ under salt stress. Therefore, the efflux and compartmentalization of ions by roots, selective transport of ions by stolons and ion secretion by leaves coordinately regulate the Na+ and K+ homeostasis of Zoysia japonica under salt stress.

Divergent molecular responses of greater amberjack (Seriola dumerili) to acute salinity stress revealed by comparative transcriptome analysis

Greater amberjack (Seriola dumerili) is an important commercial fish for its high growth rate and excellent flesh quality. However, its sensitivity to variations of water salinity poses challenges to the cage culture. In this study, the greater amberjack were reared in the optimum salinity (30 ppt, CK) and undesired regimes (10 and 40 ppt) for 72 hours. The molecular adaptive mechanisms to salinity stress were revealed by the comparative transcriptome analysis for the gills and kidneys. In gills, a total of 445 and 423 differentially expressed genes (DEGs) were identified in 10 and 40 ppt salinity stress groups, respectively. Those DEGs were involved in cartilage and skeletal development, ions transport, and immune response. The major ion secretion and osmoregulation transport proteins gene slc12a2/nkcc1 and cftr expression levels were significantly down-regulated at 10 ppt, but slightly activated at 40 ppt, compared with the control group. The expression changes in response to the Na+, K+ movement, and Cl- ion secretion reduced under the hypo-osmotic exposure and ion excretion boost upon hyper-salinity stress. Meanwhile, the cartilage and skeletal development were enhanced in the gills by hypo- or hyper-salinity stimuli, which is critical for maintaining gill structures and improving respiration and osmoregulation under salinity stress. In kidneys, 600 and 539 DEGs were identified in 10 and 40 ppt groups, respectively. Those DEGs were enriched in oxygen transport, pronephros development, regulation of growth, blood coagulation, ion transmembrane transport, and immune response. While the known renal Na+/Cl– co-transporter gene slc12a3/ncc expression level was significantly down-regulated at 10 ppt, the organic cation transporter 2 gene slc22a2, ammonium transmembrane transport gene rhd and rhag expression levels were overexpressed under the hyper-salinity condition at 40 ppt, contributing to the salts secretion and ammonium transport regulation, to combat the osmotic influx of salts following the drink of seawater and elevated ammonia production upon high salinity stress. These findings advance our knowledge of adaptative mechanisms to the salinity stress and provide theoretical guidance for the optimal breeding mode for the aquaculture of greater amberjack.

Human pancreatic ductal organoids with controlled polarity provide a novel ex vivo tool to study epithelial cell physiology

Epithelial ion and fluid secretion determine the physiological functions of a broad range of organs, such as the lung, liver, or pancreas. The molecular mechanism of pancreatic ion secretion is challenging to investigate due to the limited access to functional human ductal epithelia. Patient-derived organoids may overcome these limitations, however direct accessibility of the apical membrane is not solved. In addition, due to the vectorial transport of ions and fluid the intraluminal pressure in the organoids is elevated, which may hinder the study of physiological processes. To overcome these, we developed an advanced culturing method for human pancreatic organoids based on the removal of the extracellular matrix that induced an apical-to-basal polarity switch also leading to reversed localization of proteins with polarized expression. The cells in the apical-out organoids had a cuboidal shape, whereas their resting intracellular Ca2+ concentration was more consistent compared to the cells in the apical-in organoids. Using this advanced model, we demonstrated the expression and function of two novel ion channels, the Ca2+ activated Cl− channel Anoctamin 1 (ANO1) and the epithelial Na+ channel (ENaC), which were not considered in ductal cells yet. Finally, we showed that the available functional assays, such as forskolin-induced swelling, or intracellular Cl− measurement have improved dynamic range when performed with apical-out organoids. Taken together our data suggest that polarity-switched human pancreatic ductal organoids are suitable models to expand our toolset in basic and translational research.Graphical abstract

Evaluation of Selenomethionine Entrapped in Nanoparticles for Oral Supplementation Using In Vitro, Ex Vivo and In Vivo Models

Selenium methionine (SeMet) is an essential micronutrient required for normal body function and is associated with additional health benefits. However, oral administration of SeMet can be challenging due to its purported narrow therapeutic index, low oral bioavailability, and high susceptibility to oxidation. To address these issues, SeMet was entrapped in zein-coated nanoparticles made from chitosan using an ionic gelation formulation. The high stability of both the SeMet and selenomethionine nanoparticles (SeMet-NPs) was established using cultured human intestinal and liver epithelial cells, rat liver homogenates, and rat intestinal homogenates and lumen washes. Minimal cytotoxicity to Caco-2 and HepG2 cells was observed for SeMet and SeMet-NPs. Antioxidant properties of SeMet were revealed using a Reactive Oxygen Species (ROS) assay, based on the observation of a concentration-dependent reduction in the build-up of peroxides, hydroxides and hydroxyl radicals in Caco-2 cells exposed to SeMet (6.25-100 μM). The basal apparent permeability coefficient (Papp) of SeMet across isolated rat jejunal mucosae mounted in Ussing chambers was low, but the Papp was increased when presented in NP. SeMet had minimal effects on the electrogenic ion secretion of rat jejunal and colonic mucosae in Ussing chambers. Intra-jejunal injections of SeMet-NPs to rats yielded increased plasma levels of SeMet after 3 h for the SeMet-NPs compared to free SeMet. Overall, there is potential to further develop SeMet-NPs for oral supplementation due to the increased intestinal permeability, versus free SeMet, and the low potential for toxicity.

Electrically-evoked oscillating calcium transients in mono- and co-cultures of iPSC glia and sensory neurons.

Activated glia are known to exhibit either neuroprotective or neurodegenerative effects, depending on their phenotype, while participating in chronic pain regulation. Until recently, it has been believed that satellite glial cells and astrocytes are electrically slight and process stimuli only through intracellular calcium flux that triggers downstream signaling mechanisms. Though glia do not exhibit action potentials, they do express both voltage- and ligand-gated ion channels that facilitate measurable calcium transients, a measure of their own phenotypic excitability, and support and modulate sensory neuron excitability through ion buffering and secretion of excitatory or inhibitory neuropeptides (i.e., paracrine signaling). We recently developed a model of acute and chronic nociception using co-cultures of iPSC sensory neurons (SN) and spinal astrocytes on microelectrode arrays (MEAs). Until recently, only neuronal extracellular activity has been recorded using MEAs with a high signal-to-noise ratio and in a non-invasive manner. Unfortunately, this method has limited compatibility with simultaneous calcium transient imaging techniques, which is the most common method for monitoring the phenotypic activity of astrocytes. Moreover, both dye-based and genetically encoded calcium indicator imaging rely on calcium chelation, affecting the culture's long-term physiology. Therefore, it would be ideal to allow continuous and simultaneous direct phenotypic monitoring of both SNs and astrocytes in a high-to-moderate throughput non-invasive manner and would significantly advance the field of electrophysiology. Here, we characterize astrocytic oscillating calcium transients (OCa2+Ts) in mono- and co-cultures of iPSC astrocytes as well as iPSC SN-astrocyte co-cultures on 48 well plate MEAs. We demonstrate that astrocytes exhibit OCa2+Ts in an electrical stimulus amplitude- and duration-dependent manner. We show that OCa2+Ts can be pharmacologically inhibited with the gap junction antagonist, carbenoxolone (100 μM). Most importantly, we demonstrate that both neurons and glia can be phenotypically characterized in real time, repeatedly, over the duration of the culture. In total, our findings suggest that calcium transients in glial populations may serve as a stand-alone or supplemental screening technique for identifying potential analgesics or compounds targeting other glia-mediated pathologies.

Abstract OT3-30-01: A randomized double-blind placebo controlled phase 3 trial on the effect of Salovum™ and SPC-Flakes™ on abemaciclib-induced gastrointestinal toxicity in early breast cancer – the ASF-BC study

Abstract Background: In patients with high-risk luminal breast cancer, the addition of CDK 4/6-inhibitor abemaciclib to adjuvant endocrine therapy for two years has been associated with improved disease-free survival and is now recommended as the preferred treatment strategy for this patient group. However, patients treated with abemaciclib frequently (82%) experience diarrhea which primarily occurs during the first three months from treatment initiation and seems to impact patients’ quality of life. No proactive strategy to reduce the occurrence of abemaciclib-induced diarrhea is proposed but patients are recommended to start with loperamide upon the occurrence of diarrhea to be combined with treatment interruption and dose adjustment as needed. Cholera induced diarrhea, as well as other forms of diarrhea-inducing agents, has been shown to elicit a stimulated, endogenous production of a protein, named ”antisecretory factor”, ASF, which acts by modulating secretion of water and ions but also counteracts inflammatory processes. ASF is commercialized as Salovum® and registered by the EU authorities as ”Food for specific medical purposes”. Another way to increase ASF and, thus, to achieve benefit, is to induce its production/conversion by ingestion of malted oat flakes (SPC-flakes®) which has been recommended or considered for several secretory pathological conditions. Salovum has been shown to rapidly, ie within hours to a few days, antagonize diarrheal diseases of various etiologies. It has also been used against high fluid passages and inflammation in Crohn disease, Colitis ulcerosa and carcinoids in adults. SPC-flakes have similar effects but need weeks of administration to emerge. Importantly, to raise body ASF, by Salovum or SPC-flakes, for the above indications has not been associated with adverse effects. Methods: This is a randomized double-blind multicenter phase III study aiming to investigate a proactive strategy including Salovum and SPC-flakes to prevent the occurrence of abemaciclib-induced diarrhea in patients with early breast cancer treated with abemaciclib. A total of 100 patients will be randomized, in a 1:1 manner, between 13 weeks with Salovum/SPC flakes (A) or placebo (B). The study will be conducted in up to ten different oncology departments in Sweden starting in Q3 2022. Primary objective: Occurrence of any-grade (mild, moderate, severe) diarrhea according to the Systemic Treatment-Induced Diarrhea Assessment Tool (STIDAT; patient-reported outcome). Secondary objectives: Occurrence of any-grade diarrhea according to CTCAE v. 5.0, health-related quality of life (QoL), other adverse events related to abemaciclib, adherence to planned abemaciclib treatment, adherence to and pharmacodynamic effect from study products, safety of investigational products, sick leave duration, breast cancer recurrence. Investigational product, dosage and mode of administration: Salovum/placebo egg powder high in antisecretory factor, 4 g. Four sachets, ie 16 g q 8 h for 5 - 7 days before start of abemaciclib. The appropriate amount of Salovum is mixed with 100 – 200 ml of suitable liquid, e.g., fruit juice, and ingested orally. SPC-flakes/placebo flat dose of 75 g/d divided in 2 – 4 doses started in parallel with Salovum/placebo to be continued during the first 12 weeks of treatment with abemaciclib. End of study: All included patients will be followed using questionnaires up to 12 weeks from initiation of abemaciclib. After 12 weeks, all the patients will be followed through electronic medical records until the end of abemaciclib treatment (up to two years) for collecting potential adverse events and information on sick leave. Data from electronical medical records regarding recurrence and subsequent therapy will be collected until breast cancer recurrence or up to 5 years from initiation of abemaciclib. Citation Format: Henrik Lindman, Peter Nygren, Antonis Valachis. A randomized double-blind placebo controlled phase 3 trial on the effect of Salovum™ and SPC-Flakes™ on abemaciclib-induced gastrointestinal toxicity in early breast cancer – the ASF-BC study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-30-01.

Abstract P2-26-19: Slc26a9 cooperates with HER2 to regulate the progression and development of HER2-positive breast cancer

Abstract Goals: Ion transporters play an important regulatory role in the progression and development of breast cancer (BC). Slc26a9 is a member of the Slc26a anion transporter family, which is mainly involved in regulating the secretion of chloride ions and bicarbonate, but the role of Slc26a9 in HER2-positive BC is still unclear. Methods: Tissue microarray and BC cell line were used to detect the expression level of Slc26a9 and its clinical relevance. By changing the expression level of Slc26a9 gene in BC cells, the effect of Slc26a9 gene on the biological behavior of BC cells and its related molecular mechanism were discussed. Results: We found that the expression of Slc26a9 was significantly upregulated in BC compared with adjacent tissues, and the upregulated Slc26a9 was associated with TNM staging and poor prognosis in BC patients. In addition, the expression of Slc26a9 was significantly upregulated in HER2-positive BC compared with HER2-negative BC, and similar results were obtained in BC cell lines, with Slc26a9 was the highest expression in HER2-enriched SKBR3 cells. Functionally, the proliferation, migration, invasion and anti-apoptotic abilities of SKBR3 cells were significantly inhibited after silencing Slc26a9, and tumorigenesis and metastasis were significantly inhibited in vivo. On the contrary, overexpression of Slc26a9 resulted in the opposite result. Mechanistically, overexpression of Slc26a9 activated the PI3K/AKT/mTOR signaling pathway, the key signaling pathway implicated in HER2-positive breast carcinogenesis, and promoted the expression of downstream proliferation related genes CCND1 (Cyclin D1) and c-Myc, and downregulated the expression of apoptosis related genes Caspase9, apoptosis-inducing factor (AIF) and endonuclease G (Endo G), indicating the simultaneous inhibition of caspase dependent and independent apoptosis pathway. At the same time, accompanied by changes in markers of epithelial-mesenchymal transition (EMT), including downregulation of E-cadherin and ZO-1, and upregulation of N-cadherin and Fibronectin, and SKBR3 cells changed from epithelioid morphology to mesenchymal morphology. In addition, immunofluorescence and protein nucleoplasm separation experiments showed that Slc26a9 upregulated the expression of HER2 and co-localized with HER2 in the nucleus. Co-immunoprecipitation experiments proved that Slc26a9 interacted with HER2. Furthermore, trastuzumab downregulated the expression of Slc26a9 by targeting HER2 in SKBR3 cells. Moreover, when Slc26a9 was overexpressed, the inhibitory effect of trastuzumab on HER2 was partially reversed, and we also verified the PI3K/AKT/mTOR signaling pathway. Not only that, we found that Slc26a9 was significantly upregulated in drug-resistant cell lines relative to parental cells by constructing SKBR3 drug-resistant cell lines, indicating that Slc26a9 expression was significantly correlated with chemotherapy resistance in HER2-positive BC. Conclusion(s): Slc26a9 may interact with HER2 in the form of molecular chaperones to activate PI3K/AKT/mTOR signaling pathway to promote the progression and development of HER2-positive BC and be associated with chemotherapy resistance, but the precise molecular mechanism needs further exploration. Conflict of Interest: No significant relationships. Citation Format: Zhengxing Zhou, Zhiyuan Ma, Xuemei Liu, Chengmin Zhang, Hu Wang, Renmin Mu, Xiaoming Cheng, Biguang Tuo, Taolang Li. Slc26a9 cooperates with HER2 to regulate the progression and development of HER2-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-19.

Evolutionary trade-offs in osmotic and ionic regulation and expression of gill ion transporter genes in high latitude, cold clime Neotropical crabs from the 'end of the world'.

Osmoregulatory findings on crabs from high Neotropical latitudes are entirely lacking. Seeking to identify the consequences of evolution at low temperature, we examined hyperosmotic/hypo-osmotic and ionic regulation and gill ion transporter gene expression in two sub-Antarctic Eubrachyura from the Beagle Channel, Tierra del Fuego. Despite sharing the same osmotic niche, Acanthocyclus albatrossis tolerates a wider salinity range (2-65‰ S) than Halicarcinus planatus (5-60‰ S); their respective lower and upper critical salinities are 4‰ and 12‰ S, and 63‰ and 50‰ S. Acanthocyclus albatrossis is a weak hyperosmotic regulator, while H. planatus hyperosmoconforms; isosmotic points are 1380 and ∼1340 mOsm kg-1 H2O, respectively. Both crabs hyper/hypo-regulate [Cl-] well with iso-chloride points at 452 and 316 mmol l-1 Cl-, respectively. [Na+] is hyper-regulated at all salinities. mRNA expression of gill Na+/K+-ATPase is salinity sensitive in A. albatrossis, increasing ∼1.9-fold at 5‰ compared with 30‰ S, decreasing at 40-60‰ S. Expression in H. planatus is very low salinity sensitive, increasing ∼4.7-fold over 30‰ S, but decreasing at 50‰ S. V-ATPase expression decreases in A. albatrossis at low and high salinities as in H. planatus. Na+/K+/2Cl- symporter expression in A. albatrossis increases 2.6-fold at 5‰ S, but decreases at 60‰ S versus 30‰ S. Chloride uptake may be mediated by increased Na+/K+/2Cl- expression but Cl- secretion is independent of symporter expression. These unrelated eubrachyurans exhibit similar systemic osmoregulatory characteristics and are better adapted to dilute media; however, the expression of genes underlying ion uptake and secretion shows marked interspecific divergence. Cold clime crabs may limit osmoregulatory energy expenditure by hyper/hypo-regulating hemolymph [Cl-] alone, apportioning resources for other energy-demanding processes.

Enhanced Na+ and Cl− sequestration and secretion selectivity contribute to high salt tolerance in the tetraploid recretohalophyte Plumbago auriculata Lam.

Enhanced secretion of Na+ and Cl- in leaf glands and leaf vacuolar sequestration of Na+ or root retention of Cl-, combined with K+ retention, contribute to the improved salt tolerance of tetraploid recretohalophyte P. auriculata. Salt stress is one of the major abiotic factors threatening plant growth and development, and polyploids generally exhibit higher salt stress resistance than diploids. In recretohalophytes, which secrete ions from the salt gland in leaf epidermal cells, the effects of polyploidization on ion homeostasis and secretion remain unknown. In this study, we compared the morphology, physiology, and ion homeostasis regulation of diploid and autotetraploid accessions of the recretohalophyte Plumbago auriculata Lam. after treatment with 300mM NaCl for 0, 2, 4, 6, and 8 days. The results showed that salt stress altered the morphology, photosynthetic efficiency, and chloroplast structure of diploid P. auriculata to a greater extent than those of its tetraploid counterpart. Moreover, the contents of organic osmoregulatory substances (proline and soluble sugars) were significantly higher in the tetraploid than in the diploid, while those of H2O2 and malondialdehyde (MDA) were significantly lower. Analysis of ion homeostasis revealed that the tetraploid cytotype accumulated more Na+ in stems and leaves and more Cl- in roots but less K+ loss in roots compared with diploid P. auriculata. Additionally, the rate of Na+ and Cl- secretion from the leaf surface was higher, while that of K+, Mg2+, and Ca2+ secretion was lower in tetraploid plants. X-ray microanalysis of mesophyll cells revealed that Na+ mainly accumulated in different cellular compartments in the tetraploid (vacuole) and diploid (cytoplasm) plants. Our results suggest that polyploid recretohalophytes require the ability to sequester Na+ and Cl-(via accumulation in leaf cell vacuoles or unloading by roots) and selectively secrete these ions (through salt glands) together with the ability to prevent K+ loss (by roots). This mechanism required to maintain K+/Na+ homeostasis in polyploid recretohalophytes under high salinity provides new insights in the improved maintenance of ion homeostasis in polyploids under salt stress.