Disruption of the Na+/K+-ATPase-purinergic P2X7 receptor complex in microglia promotes stress-induced anxiety.

Na+/K+-ATPase (NKA) plays an important role in the central nervous system. However, little is known about its function in the microglia. Here, we found that NKAα1 forms a complex with the purinergic P2X7 receptor (P2X7R), an adenosine 5'-triphosphate (ATP)-gated ion channel, under physiological conditions. Chronic stress or treatment with lipopolysaccharide plus ATP decreased the membrane expression of NKAα1 in microglia, facilitated P2X7R function, and promoted microglia inflammatory activation via activation of the NLRP3 inflammasome. Accordingly, global deletion or conditional deletion of NKAα1 in microglia under chronic stress-induced aggravated anxiety-like behavior and neuronal hyperexcitability. DR5-12D, a monoclonal antibody that stabilizes membrane NKAα1, improved stress-induced anxiety-like behavior and ameliorated neuronal hyperexcitability and neurogenesis deficits in the ventral hippocampus of mice. Our results reveal that NKAα1 limits microglia inflammation and may provide a target for the treatment of stress-related neuroinflammation and diseases.

High sodium, rather than high blood pressure, induces immune cell activation and renal infiltration in ovariectomized adult Wistar rats.

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.

Paralog switching facilitates diadromy: ontogenetic, microevolutionary and macroevolutionary evidence.

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.

Colorectal and bladder prokinetic activity of [Lys5, MeLeu9, Nle10]-NKA(4-10) after intranasal or sublingual delivery in dogs.

The feasibility of eliciting defecation and urination after intranasal (IN) or sublingual (SL) delivery of a small peptide NK2 receptor agonist, [Lys5, MeLeu9, Nle10]-NKA(4-10), was examined using prototype formulations in dogs. In anesthetized animals, administration of 100 or 300 µg/kg IN or 2.0-6.7 mg/kg SL increased colorectal peak pressure and area under the curve. Peak bladder pressure was also increased at the same doses, and this was accompanied by highly efficient voiding at normal physiological bladder pressure. The onset of these effects was rapid (≤2.5 min), and the primary contractions lasted ∼25 min, returning to baseline in <60 min. Slight hypotension lasting a few minutes and causing <10% change from baseline was detected after higher doses and was statistically significant after only 100 µg/kg IN. In conscious dogs, there was a dose-related increase in voiding responses and reduction in the latency to urinate and defecate after 300 and 1000 µg/kg IN; emesis was also observed at these doses. SL administration of 6.7 mg/kg induced urination within 10 min, but not defecation or emesis. These findings support the feasibility of developing a convenient dosage form of small peptide NK2 receptor agonists as on-demand defecation or urination therapies.

Effects of Osmotic Stress on the mRNA Expression of prl, prlr, gr, gh, and ghr in the Pituitary and Osmoregulatory Organs of Black Porgy, Acanthopagrus schlegelii.

In euryhaline teleost black porgy, Acanthopagrus schlegelii, the glucocorticoid receptor (gr), growth hormone receptor (ghr), prolactin (prl)-receptor (prlr), and sodium-potassium ATPase alpha subunit (α-nka) play essential physiological roles in the osmoregulatory organs, including the gill, kidney, and intestine, during osmotic stress. The present study aimed to investigate the impact of pituitary hormones and hormone receptors in the osmoregulatory organs during the transfer from freshwater (FW) to 4 ppt and seawater (SW) and vice versa in black porgy. Quantitative real-time PCR (Q-PCR) was carried out to analyze the transcript levels during salinity and osmoregulatory stress. Increased salinity resulted in decreased transcripts of prl in the pituitary, α-nka and prlr in the gill, and α-nka and prlr in the kidney. Increased salinity caused the increased transcripts of gr in the gill and α-nka in the intestine. Decreased salinity resulted in increased pituitary prl, and increases in α-nka and prlr in the gill, and α-nka, prlr, and ghr in the kidney. Taken together, the present results highlight the involvement of prl, prlr, gh, and ghr in the osmoregulation and osmotic stress in the osmoregulatory organs (gill, intestine, and kidney). Pituitary prl, and gill and intestine prlr are consistently downregulated during the increased salinity stress and vice versa. It is suggested that prl plays a more significant role in osmoregulation than gh in the euryhaline black porgy. Furthermore, the present results highlighted that the gill gr transcript's role was solely to balance the homeostasis in the black porgy during salinity stress.

High-Intensity Training Represses FXYD5 and Glycosylates Na,K-ATPase in Type II Muscle Fibres, Which Are Linked with Improved Muscle K+ Handling and Performance.

Na+/K+ ATPase (NKA) comprises several subunits to provide isozyme heterogeneity in a tissue-specific manner. An abundance of NKA α, ß, and FXYD1 subunits is well-described in human skeletal muscle, but not much is known about FXYD5 (dysadherin), a regulator of NKA and ß1 subunit glycosylation, especially with regard to fibre-type specificity and influence of sex and exercise training. Here, we investigated muscle fibre-type specific adaptations in FXYD5 and glycosylated NKAß1 to high-intensity interval training (HIIT), as well as sex differences in FXYD5 abundance. In nine young males (23.8 ± 2.5 years of age) (mean ± SD), 3 weekly sessions of HIIT for 6 weeks enhanced muscle endurance (220 ± 102 vs. 119 ± 99 s, p < 0.01) and lowered leg K+ release during intense knee-extensor exercise (0.5 ± 0.8 vs. 1.0 ± 0.8 mmol·min-1, p < 0.01) while also increasing cumulated leg K+ reuptake 0-3 min into recovery (2.1 ± 1.5 vs. 0.3 ± 0.9 mmol, p < 0.01). In type IIa muscle fibres, HIIT lowered FXYD5 abundance (p < 0.01) and increased the relative distribution of glycosylated NKAß1 (p < 0.05). FXYD5 abundance in type IIa muscle fibres correlated inversely with the maximal oxygen consumption (r = -0.53, p < 0.05). NKAα2 and ß1 subunit abundances did not change with HIIT. In muscle fibres from 30 trained males and females, we observed no sex (p = 0.87) or fibre type differences (p = 0.44) in FXYD5 abundance. Thus, HIIT downregulates FXYD5 and increases the distribution of glycosylated NKAß1 in type IIa muscle fibres, which is likely independent of a change in the number of NKA complexes. These adaptations may contribute to counter exercise-related K+ shifts and enhance muscle performance during intense exercise.

The Role of Substance P, Neurokinin A, Neuropeptide Y, and Cortisol in Assessing Neonatal Pain.

Introduction: Our aim was to investigate biomarkers of neonatal pain and their association with two pain scales. Methods: This prospective study included 54 full-term neonates. Levels of substance P (SubP), neurokinin A (NKA), neuropeptide Y (NPY), and cortisol were recorded and two pain scales (Premature Infant Pain Profile [PIPP] and Neonatal Infant Pain Scale [NIPS]) were used. Results: A statistically significant decrease in the levels of NPY (p = 0.02) and NKA (p = 0.03) was detected. A significant increase in NIPS scale (p < 0.001) and PIPP scale (p < 0.001) postpainful intervention was also detected. There was a positive correlation between cortisol and SubP (p = 0.01), NKA and NPY (p < 0.001) and between NIPS and PIPP (p < 0.001). A negative correlation was found for NPY with SubP (p = 0.004), cortisol (p = 0.02), NIPS (p = 0.001) and PIPP (p = 0.002). Conclusions: Novel biomarkers and pain scales may help in designing an objective tool for the quantification of neonatal pain in the everyday practice.

Extracellular tau oligomers affect extracellular glutamate handling by astrocytes through downregulation of GLT-1 expression and impairment of NKA1A2 function.

AIMS: Several studies reported that astrocytes support neuronal communication by the release of gliotransmitters, including ATP and glutamate. Astrocytes also play a fundamental role in buffering extracellular glutamate in the synaptic cleft, thus limiting the risk of excitotoxicity in neurons. We previously demonstrated that extracellular tau oligomers (ex-oTau), by specifically targeting astrocytes, affect glutamate-dependent synaptic transmission via a reduction in gliotransmitter release. The aim of this work was to determine if ex-oTau also impair the ability of astrocytes to uptake extracellular glutamate, thus further contributing to ex-oTau-dependent neuronal dysfunction. METHODS: Primary cultures of astrocytes and organotypic brain slices were exposed to ex-oTau (200 nM) for 1 h. Extracellular glutamate buffering by astrocytes was studied by: Na+ imaging; electrophysiological recordings; high-performance liquid chromatography; Western blot and immunofluorescence. Experimental paradigms avoiding ex-oTau internalisation (i.e. heparin pre-treatment and amyloid precursor protein knockout astrocytes) were used to dissect intracellular vs extracellular effects of oTau. RESULTS: Ex-oTau uploading in astrocytes significantly affected glutamate-transporter-1 expression and function, thus impinging on glutamate buffering activity. Ex-oTau also reduced Na-K-ATPase activity because of pump mislocalisation on the plasma membrane, with no significant changes in expression. This effect was independent of oTau internalisation and it caused Na+ overload and membrane depolarisation in ex-oTau-targeted astrocytes. CONCLUSIONS: Ex-oTau exerted a complex action on astrocytes, at both intracellular and extracellular levels. The net effect was dysregulated glutamate signalling in terms of both release and uptake that relied on reduced expression of glutamate-transporter-1, altered function and localisation of NKA1A1, and NKA1A2. Consequently, Na+ gradients and all Na+ -dependent transports were affected.

Inflammatory and contractile profile in LPS-challenged equine isolated bronchi: Evidence for IL-6 as a potential target against AHR in equine asthma.

BACKGROUND: Airway inflammation and airway hyperresponsiveness (AHR) are pivotal characteristics of equine asthma. Lipopolysaccharide (LPS) may have a central role in modulating airway inflammation and dysfunction. Therefore, the aim of this study was to match the inflammatory and contractile profile in LPS-challenged equine isolated bronchi to identify molecular targets potentially suitable to counteract AHR in asthmatic horses. METHODS: Equine isolated bronchi were incubated overnight with LPS (0.1-100 ng/ml). The contractile response to electrical field stimulation (EFS) and the levels of cytokines, chemokines, and neurokinin A (NKA) were quantified. The role of capsaicin sensitive-sensory nerves, neurokinin-2 (NK2) receptor, transient receptor potential vanilloid type 1 receptors (TRPV1), and epithelium were also investigated. RESULTS: LPS 1 ng/ml elicited AHR to EFS (+238.17 ± 25.20% P < 0.001 vs. control). LPS significantly (P < 0.05 vs. control) increased the levels of IL-4 (+36.08 ± 1.62%), IL-5 (+38.60 ± 3.58%), IL-6 (+33.79 ± 2.59%), IL-13 (+40.91 ± 1.93%), IL-1ß (+1650.16 ± 71.16%), IL-33 (+88.14 ± 8.93%), TGF-ß (22.29 ± 1.03%), TNF-α (+56.13 ± 4.61%), CXCL-8 (+98.49 ± 17.70%), EOTAXIN (+32.26 ± 2.27%), MCP-1 (+49.63 ± 4.59%), RANTES (+36.38 ± 2.24%), and NKA (+112.81 ± 6.42%). Capsaicin sensitive-sensory nerves, NK2 receptor, and TRPV1 were generally involved in the LPS-mediated inflammation. Epithelium removal modulated the release of IL-1ß, IL-33, and TGF-ß. Only the levels of IL-6 fitted with AHR to a wide range of EFS frequencies, an effect significantly (P < 0.05) inhibited by anti-IL-6 antibody; exogenous IL-6 induced significant (P < 0.05) AHR to EFS similar to that elicited by LPS. CONCLUSION: Targeting IL-6 with specific antibody may represent an effective strategy to treat equine asthma, especially in those animals suffering from severe forms of this disease.

Localization of the Neuropeptide Arginine Vasotocin and Its Receptor in the Osmoregulatory Organs of Black Porgy, Acanthopagrus schlegelii: Gills, Kidneys, and Intestines.

The neurohypophysial hormone arginine vasotocin (avt) and its receptor (avtr) regulates ions in the osmoregulatory organs of euryhaline black porgy (Acanthopagrus schlegelii). The localization of avt and avtr transcripts in the osmoregulatory organs has yet to be demonstrated. Thus, in the present study, we performed an in situ hybridization analysis to determine the localization of avt and avtr in the gills, kidneys, and intestines of the black porgy. The avt and avtr transcripts were identified in the filament and lamellae region of the gills in the black porgy. However, the basal membrane of the filament contained more avt and avtr transcripts. Fluorescence double tagging analysis revealed that avt and avtr mRNAs were partially co-localized with α-Nka-ir cells in the gill filament. The proximal tubules, distal tubules, and collecting duct of the kidney all had positive hybridization signals for the avt and avtr transcripts. Unlike the α-Nka immunoreactive cells, the avt and avtr transcripts were found on the basolateral surface of the distal convoluted tubule and in the entire cells of the proximal convoluted tubules of the black porgy kidney. In the intestine, the avt and avtr transcripts were found in the basolateral membrane of the enterocytes. Collectively, this study provides a summary of evidence suggesting that the neuropeptides avt and avtr with α-Nka-ir cells may have functions in the gills, kidneys, and intestines via ionocytes.

Ovariectomy and high salt increase blood pressure and alter sodium transport proteins in peripheral blood mononuclear cells of adult Wistar rats.

NEW FINDINGS: What is the central question of this study? In a model of salt-sensitive hypertension in ovariectomized (oVx) adult Wistar rats, what is the expression of proteins related to sodium transport in peripheral blood mononuclear cells (PBMCs), and how does the response of proteins to high sodium intake compare with changes in blood pressure in intact female rats? What is the main finding and its importance? Sodium transport proteins in PBMCs react to high sodium and blood pressure markedly differently in oVx versus intact female rats. Protein expression shows sodium and pressure sensitivity. Renal immune cells increase in oVx under high salt. ABSTRACT: Hypertension is a worldwide public health problem. High sodium consumption is associated with hypertension, and hypertensive mechanisms involve immunity cells. Peripheral blood mononuclear cells (PBMCs) are endowed with proteins related to sodium transport. We studied their abundance in PBMCs from intact (IF) or ovariectomized (oVx) adult Wistar rats under normal (NS) or high (HS) salt intake. Ovariectomy was performed at 60 days of life. At 145 days, one group of IF and oVx rats received NS or HS intake for 5 days. Another group of IF HS and oVx HS rats received hydralazine (HDZ) to reduce blood pressure (BP). Sodium balance and BP were recorded. Expression of Na+ ,K+ -ATPase (NKA), Na+ -K+ -2Cl- cotransporter 1 (NKCC1), serum/glucocorticoid-regulated kinase 1 (SGK1), dopamine D1 like receptor (D1DR), CD4+ and CD8+ were determined in PBMCs and CD45+ leukocytes in renal tissue. IF HS rats showed increased natriuresis and normal BP. NKA and CD4+ expression diminished in IF HS. Instead, oVx HS rats had sodium retention and high BP and increased the expression of NKA, NKCC1, D1DR, CD4+ and CD8+ in PBMCs. Renal CD45+ leukocytes increased in oVx HS rats. HDZ decreased BP in all rats. Upon HDZ treatment, NKA did not change, NKCC1 decreased in oVx HS rats, while SGK1 increased in both IF HS and oVx HS rats. Hormonal background determines BP response and the expression of proteins related to sodium transport in PBMCs and renal immune cells at HS intake. The analysis of NKA, NKCC1 and SGK1 expression in PBMCs differentiated salt-sensitivity from BP variations.

Testing of NKA expression by mobile real time PCR is an efficient indicator of smoltification status of farmed Atlantic salmon.

Assessment of seawater readiness of freshwater salmon smolts is a crucial husbandry step with economic implications in salmon aquaculture but current methods rely on delayed centralised enzymic activity measurement. The efficiency of a qRT-PCR assay for sodium potassium ATPase (NKA) α1a mRNA was tested in a 3-year study on 19 hatcheries across Scotland incorporating environmental factors such as temperature and metal contamination. The NKA qRT-PCR assay was transferred to a mobile laboratory and on-site testing was carried out at 3 hatchery sites. For the first two years standard enzymatic and gene expression assays had similar success rates in detecting smoltification (NKA activity 60%, qRT-PCR 57%). In the third year, all but one site were determined as sea water ready by qRT-PCR but only at 4 by enzymatic testing. On site testing with mobile qRT-PCR was successfully performed on four farm sites. Altogether, high sensitivity was shown for the in lab (98.9%, SE 0.24) and mobile (93.43%, SE 0.119) assays when tested using a quantitative RNA standard. Some indication for obscured smoltification assay results due to environmental increased heavy metal contamination was observed. Our results prove it is possible to test a smoltification marker on site and provide results on the day of testing during the smolt period allowing for informed decisions on seawater transfer.

Distribution of Na+/K+-ATPase-immunoreactive ionocytes varies between two superorders of ray-finned fish: Ostariophysi and Acanthopterygii.

Ray-finned fishes of the superorder Ostariophysi are primarily freshwater (FW), and normally stenohaline. Differently, fishes of the superorder Acanthopterygii are essentially marine, and frequently euryhaline, with some secondary FW. Na+/K+-ATPase-immunoreactive ionocytes were localized in the branchial epithelia of 4 species of Ostariophysi and 3 of Acanthopterygii. The Ostariophysi grass carp (Ctenopharyngodon idella, Cypriniformes), twospot Astyanax (Astyanax bimaculatus) and piracanjuba (Brycon orbignyanus), Characiformes, and the jundiá (Rhamdia quelen, Siluriformes), all from FW, displayed ionocytes in the filament plus secondary lamellae (F + SL). In their turn, all the three species of Acanthopterygii showed immunoreactive ionocytes in the filaments only (F). They were the Nile tilapia (Oreochromis niloticus, Cichliformes) in FW, the dog snapper (Lutjanus jocu, Perciformes) in seawater (SW), and the green puffer (Sphoeroides greeleyi, Tetraodontiformes) in SW. Ionocytes normally extend their distribution to the secondary lamellae (F + SL) in Ostariophysi. In Acanthopterygii, we find more plasticity: ionocytes are more frequently restricted to the filament in SW, but also spread to SL in FW. It may be that the occurrence of ionocytes in SL is the ancestral condition, but some euryhaline acanthopterygians rely on the space of the SL for placement of additional ionocytes when in FW absorbing salt. Our study contributed to the identification of the pattern of ionocyte distribution in gills of Ostariophysi in respect to that of Acanthopterygii.

Salt-water acclimation of the estuarine crocodile Crocodylus porosus involves enhanced ion transport properties of the urodaeum and rectum.

Estuarine crocodiles, Crocodylus porosus, inhabit freshwater, estuarine and marine environments. Despite being known to undertake extensive movements throughout and between hypo-osmotic and hyperosmotic environments, little is known about the role of the cloaca in coping with changes in salinity. We report here that, in addition to the well-documented functional plasticity of the lingual salt glands, the middle of the three cloacal segments (i.e. the urodaeum) responds to increased ambient salinity to enhance solute-coupled water absorption. This post-renal modification of urine serves to conserve water when exposed to hyperosmotic environments and, in conjunction with lingual salt gland secretions, enables C. porosus to maintain salt and water balance and thereby thrive in hyperosmotic environments. Isolated epithelia from the urodaeum of 70% seawater-acclimated C. porosus had a strongly enhanced short-circuit current (an indicator of active ion transport) compared with freshwater-acclimated crocodiles. This enhanced active ion absorption was driven by increased Na+/K+-ATPase activity, and possibly enhanced proton pump activity, and was facilitated by the apical epithelial Na+ channel (ENaC) and/or the apical Na+/H+ exchanger (NHE2), both of which are expressed in the urodaeum. NHE3 was expressed at very low levels in the urodaeum and probably does not contribute to solute-coupled water absorption in this cloacal segment. As C. porosus does not appear to drink water of salinities above 18 ppt, observations of elevated short-circuit current in the rectum as well as a trend for increased NHE2 expression in the oesophagus, the anterior intestine and the rectum suggest that dietary salt intake may stimulate salt and possibly water absorption by the gastrointestinal tract of C. porosus living in hyperosmotic environments.

Tambaqui (Colossoma macropomum) acclimated to different tropical waters from the Amazon basin shows specific acute-stress responses.

Stress responses in teleosts include the release of hormones into the bloodstream. Their effects depend on the species and on the environmental conditions. The Amazon basin collects waters of diverse chemical composition, and some fish are able to inhabit several of them. However, the effects of these waters on the stress axis are still unknown. Here we show how acute air-exposure differently affects stress biomarkers in tambaqui (Colossoma macropomum), a tropical model species, when acclimated to two Amazonian waters (Rio Negro -RN- water rich in humic acids and poor in ions, and groundwater -IG- with no humic acids and higher concentration of ions). This study described primary and secondary stress responses after air exposure including plasma cortisol, energy metabolites, pH and ions, skin mucus energy metabolites, as well as gills and kidney Na+/K+-ATPase and Na+/H+-exchanger (NHE) activities. Several differences were described in these stress biomarkers due to the acclimation water. The most remarkable ones include increased mucus glucose only in RN-fish, and mucus lactate only in IG-fish after air exposure. Moreover, an inverse relationship between plasma cortisol and Na+ concentrations as well as a direct relationship between plasma ammonia and branchial NHE activity were observed only in RN-fish. Our results demonstrate how important is to study stress responses in fish acclimated to different environments, as physiological differences can be magnified during episodes of high energy expenditure. In addition to having a direct application in aquaculture, this study will improve the management of critical ecosystems such as the Amazon.

Ion regulation in the antennal glands differs among Ocypodoidea and Grapsoidea crab species.

Gills and the antennal gland are ion-regulatory organs in crabs. Previous studies have suggested that the differences in the morphology and ion regulation of gills and accessory respiratory organs between ocypodid and grapsid species are related to their distinct evolutionary transition to land habitats. In addition, Na+, K+-ATPase (NKA) activity and Na+ and NH4+ regulation in the antennal gland differ between ocypodid and grapsid species, which had different terrestrial adaptation trajectories. This study used five Ocypodoidea species and three Grapsoidea species from the intertidal and supratidal zones to further investigate the differences in ion regulation and NKA activity in the antennal gland between these crab families in different habitats. Crabs were transferred to 5 practical salinity unit (PSU) water, and osmolality, Na+ and Cl- concentrations in the urine and hemolymph, and NKA activity in the antennal gland were examined. Phylogenetic ANOVA results showed that the NKA activity in the antennal gland was higher in the ocypodid than grapsid groups, and Moran's I autocorrelation analysis also indicated that NKA activity in the antennal gland was phylogenetically correlated among crabs. K-means clustering showed a difference among the crabs in the crabs' Na+ and Cl- concentrations in the urine/hemolymph, NKA activities in the antennal gland and gill 6, and number of pairs of gills. Crabs with relatively high antennal gland NKA activity were found not only in the Ocypode species, which are better adapted to terrestrial environments, but also in two intertidal species of Gelasiminae. In conclusion, part of the Ocypodidae lineage may have a) the ability to reabsorb Na+ and b) higher NKA activity in the antennal gland than other families, and this phenomenon is phylogenetically correlated in Ocypodoidea and Grapsoidea. The physiological diversity in osmoregulation among intertidal and costal species provides a base to further investigate their ecological niches and guilds.

Morphological and physiological traits of Mediterranean sticklebacks living in the Camargue wetland (Rhone river delta).

Three-spined sticklebacks (Gasterosteus aculeatus L.) living at the southern limit of the species distribution range could possess specific morphological and physiological traits that enable these fish to live at the threshold of their physiological capacities. Morphological analysis was carried out on samples of sticklebacks living in different saline habitats of the Camargue area (Rhone delta, northern Mediterranean coast) obtained from 1993 to 2017. Salinity acclimation capacities were also investigated using individuals from freshwater-low salinity drainage canals and from mesohaline-euryhaline lagoons. Fish were maintained in laboratory conditions at salinity values close to those of their respective habitats: low salinity (LS, 5‰) or seawater (SW, 30‰). Fish obtained from a mesohaline brackish water lagoon (BW, 15‰) were acclimated to SW or LS. Oxygen consumption rates and branchial Na+ /K+ -ATPase (NKA) activity (indicator of fish osmoregulatory capacity) were measured in these LS or SW control fish and in individuals subjected to abrupt SW or LS transfers. At all the studied locations, only the low-plated "leiurus" morphotype showed no spatial or temporal variations in their body morphology. Gill rakers were only longer and denser in fish sampled from the LS-freshwater (FW) drainage canals. All fish presented similar physiological capacities. Oxygen consumption rates were not influenced by salinity challenge except in SW fish transferred to LS immediately and 1 h after transfer. However, and as expected, gill NKA activity was salinity dependent. Sticklebacks of the Camargue area sampled from habitats with contrasted saline conditions are homogenously euryhaline, have low oxygen consumption rates and do not appear to experience significantly greater metabolic costs when challenged with salinity. However, an observed difference in gill raker length and density is most probably related to the nutritional condition of their habitat, indicating that individuals can rapidly acclimatize to different diets.

Evidence for heterogeneous subsarcolemmal Na+ levels in rat ventricular myocytes.

The intracellular Na+ concentration ([Na+]) regulates cardiac contractility. Previous studies have suggested that subsarcolemmal [Na+] is higher than cytosolic [Na+] in cardiac myocytes, but this concept remains controversial. Here, we used electrophysiological experiments and mathematical modeling to test whether there are subsarcolemmal pools with different [Na+] and dynamics compared with the bulk cytosol in rat ventricular myocytes. A Na+ dependency curve for Na+-K+-ATPase (NKA) current was recorded with symmetrical Na+ solutions, i.e., the same [Na+] in the superfusate and internal solution. This curve was used to estimate [Na+] sensed by NKA in other experiments. Three experimental observations suggested that [Na+] is higher near NKA than in the bulk cytosol: 1) when extracellular [Na+] was high, [Na+] sensed by NKA was ~6 mM higher than the internal solution in quiescent cells; 2) long trains of Na+ channel activation almost doubled this gradient; compared with an even intracellular distribution of Na+, the increase of [Na+] sensed by NKA was 10 times higher than expected, suggesting a local Na+ domain; and 3) accumulation of Na+ near NKA after trains of Na+ channel activation dissipated very slowly. Finally, mathematical models assuming heterogeneity of [Na+] between NKA and the Na+ channel better reproduced experimental data than the homogeneous model. In conclusion, our data suggest that NKA-sensed [Na+] is higher than [Na+] in the bulk cytosol and that there are differential Na+ pools in the subsarcolemmal space, which could be important for cardiac contractility and arrhythmogenesis. NEW & NOTEWORTHY Our data suggest that the Na+-K+-ATPase-sensed Na+ concentration is higher than the Na+ concentration in the bulk cytosol and that there are differential Na+ pools in the subsarcolemmal space, which could be important for cardiac contractility and arrhythmogenesis. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/heterogeneous-sodium-in-ventricular-myocytes/ .

Na+-K+-ATPase and nka genes in spotted sea bass (Lateolabrax maculatus) and their involvement in salinity adaptation.

Euryhaline teleosts can survive in a wide salinity range via alteration of the molecular mechanisms to maintain internal ionic and osmotic balance in osmoregulatory organs such as gill，kidney and intestine. Na+/K+-ATPase (NKA), plays a crucial role in sustaining intracellular homeostasis and is characterized by association of multiple isoforms of α- and ß-subunits. To gain insight into the potential function of nka genes in salinity adaptation, 5 nkaα genes (nkaα1a, nkaα1b, nkaα2, nkaα3a, nkaα3b) and 7 nkaß genes (nkaß1a, nkaß1b, nkaß2a, nkaß2b, nkaß3a, nkaß3b and nkaß4) were identified from transcriptomic and genomic databases of Lateolabrax maculatus. The annotation and evolutionary footprint of these nka genes was revealed via the analysis of phylogenetic tree, gene synteny, copy numbers, exon-intron structures and motif compositions. The expressions of 12 nka genes in spotted sea bass was tested in ten tissues (kidney, gonad, stomach, intestine, gill, muscle, heart, spleen, liver and brain) and 6 genes (nkaα1a, nkaα1b, nkaα3a, nkaα3b, nkaß1b and nkaß2a) showed high expression in osmoregulatory organs. Furthermore, the responses of NKA and potential salinity-sensitive nka genes were examined under different salinity treatment (0 ppt, 12 ppt, 30 ppt, 45 ppt). Results showed that the enzyme activity of NKA was highest in gill and exhibited salinity dependent variation, with the highest activity identified in 45 ppt. Different nkaα/ß-isoforms showed their diverse responses to salinity changes and the expression of nka genes including nkaα1a, nkaα3b, nkaß1b in gill, nkaα3a in kidney and nkaß2a in intestine were transcriptionally regulated by altered salinity. Notably, the expression patterns of nkaα1a and nkaß1b in gill showed similar variation trend with NKA activity, suggesting that nkaα1a/ß1b could be the major function isoforms involved in primary ion transport during salinity adaptation. Our results provided insights into the roles of nkas in osmotic regulation and a theoretical basis for future studies that focus on detailed molecular mechanisms in salinity adaptation of euryhaline teleosts.

Effect of combined stress (salinity and temperature) in European sea bass Dicentrarchus labrax osmoregulatory processes.

European sea bass Dicentrarchus labrax undertake seasonal migrations to estuaries and lagoons that are characterized by fluctuations in environmental conditions. Their ability to cope with these unstable habitats is undeniable, but it is still not clear how and to what extent salinity acclimation mechanisms are affected at temperatures higher than in the sea. In this study, juvenile sea bass were pre-acclimated to seawater (SW) at 18°C (temperate) or 24°C (warm) for 2weeks and then transferred to fresh water (FW) or SW at the respective temperature. Transfer to FW for two weeks resulted in decreased blood osmolalities and plasma Cl- at both temperatures. In FW warm conditions, plasma Na+ was ~15% lower and Cl- was ~32% higher than in the temperate-water group. Branchial Na+/K+-ATPase (NKA) activity measured at the acclimation temperature (Vapparent) did not change according to the conditions. Branchial Na+/K+-ATPase activity measured at 37°C (Vmax) was lower in warm conditions and increased in FW compared to SW conditions whatever the considered temperature. Mitochondrion-rich cell (MRC) density increased in FW, notably due to the appearance of lamellar MRCs, but this increase was less pronounced in warm conditions where MRC's size was lower. In SW warm conditions, pavement cell apical microridges are less developed than in other conditions. Overall gill morphometrical parameters (filament thickness, lamellar length and width) differ between fish that have been pre-acclimated to different temperatures. This study shows that a thermal change affects gill plasticity affecting whole-organism ion balance two weeks after salinity transfer.