Deciphering the function of the fifth class of Gα proteins: regulation of ionic homeostasis as unifying hypothesis.

Trimeric G proteins transduce signals from a superfamily of receptors and each G protein controls a wide range of cellular and systemic functions. Their highly conserved alpha subunits fall in five classes, four of which have been well investigated (Gs, Gi, G12, Gq). In contrast, the function of the fifth class, Gv is completely unknown, despite its broad occurrence and evolutionary ancient origin (older than metazoans). Here we show a dynamic presence of Gv mRNA in several organs during early development of zebrafish, including the hatching gland, the pronephros and several cartilage anlagen, employing in situ hybridisation. Next, we generated a Gv frameshift mutation in zebrafish and observed distinct phenotypes such as reduced oviposition, premature hatching and craniofacial abnormalities in bone and cartilage of larval zebrafish. These phenotypes could suggest a disturbance in ionic homeostasis as a common denominator. Indeed, we find reduced levels of calcium, magnesium and potassium in the larvae and changes in expression levels of the sodium potassium pump atp1a1a.5 and the sodium/calcium exchanger ncx1b in larvae and in the adult kidney, a major osmoregulatory organ. Additionally, expression of sodium chloride cotransporter slc12a3 and the anion exchanger slc26a4 is altered in complementary ways in adult kidney. It appears that Gv may modulate ionic homeostasis in zebrafish during development and in adults. Our results constitute the first insight into the function of the fifth class of G alpha proteins.

Antibacterial Activity and Mechanism of Action of Osthole against Listeria monocytogenes.

The purpose of this study was to investigate the antibacterial activity and mechanism of action of osthole against Listeria monocytogenes. The antibacterial activity of osthole was evaluated by determining the minimum inhibitory concentration (MIC) and growth curve. Cell morphology, membrane permeability, membrane integrity, bacterial physiology, and metabolism were explored using different methods to elucidate the mechanism of action of osthole. It was shown that the MIC of osthole against L. monocytogenes was 62.5 µg/mL and it inhibited the growth of L. monocytogenes effectively in a concentration-dependent manner. Scanning electron microscopy (SEM) images demonstrated morphology changes of L. monocytogenes, including rough surface, cell shrinkage, and rupture. It was found that extracellular conductivity and macromolecule content were increased significantly in the presence of osthole, indicating the disruption of cell membrane integrity and permeability. Laser confocal microscopy results supported the conclusion that osthole caused severe damage to the cell membrane. It was also noticed that osthole depleted intracellular adenosine triphosphate (ATP), inhibited Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity, and promoted the accumulation of intracellular reactive oxygen species (ROS), leading to cell death. This study suggests that osthole is a promising antibacterial agent candidate against L. monocytogenes, and it shows potential in the prevention and control of foodborne pathogens.

[Effect and mechanism of Linggui Zhugan Decoction in regulating Sig1R on Angâ ¡-induced cardiomyocyte hypertrophy].

This study aims to explore the mechanism of Linggui Zhugan Decoction(LGZGD) in inhibiting Angiotensin Ⅱ(AngⅡ)-induced cardiomyocyte hypertrophy by regulating sigma-1 receptor(Sig1R). The model of H9c2 cardiomyocyte hypertrophy induced by AngⅡ in vitro was established by preparing LGZGD-containing serum and blank serum. H9c2 cells were divided into normal group, AngⅡ model group, 20% normal rat serum group(20% NSC), and 20% LGZGD-containing serum group. After the cells were incubated with AngⅡ(1 µmol·L~(-1)) or AngⅡ with serum for 72 h, the surface area of cardiomyocytes was detected by phalloidine staining, and the activities of Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase were detected by micromethod. The mitochondrial Ca~(2+) levels were detected by flow cytometry, and the expression levels of atrial natriuretic peptide(ANP), brain natriuretic peptide(BNP), Sig1R, and inositol 1,4,5-triphosphate receptor type 2(IP_3R_2) were detected by Western blot. The expression of Sig1R was down-regulated by transfecting specific siRNA for investigating the efficacy of LGZGD-containing serum on cardiomyocyte surface area, Na~+-K~+-ATPase activity, Ca~(2+)-Mg~(2+)-ATPase activity, mitochondrial Ca~(2+), as well as ANP, BNP, and IP_3R_2 protein expressions. The results showed that compared with the normal group, AngⅡ could significantly increase the surface area of cardiomyocytes and the expression of ANP and BNP(P<0.01), and it could decrease the activities of Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase, the concentration of mitochondrial Ca~(2+), and the expression of Sig1R(P<0.01). In addition, IP_3R_2 protein expression was significantly increased(P<0.01). LGZGD-containing serum could significantly decrease the surface area of cardiomyocytes and the expression of ANP and BNP(P<0.05, P<0.01), and it could increase the activities of Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase, the concentration of mitochondrial Ca~(2+ )(P<0.01), and the expression of Sig1R(P<0.05). In addition, IP_3R_2 protein expression was significantly decreased(P<0.05). However, after Sig1R was down-regulated, the effects of LGZGD-containing serum were reversed(P<0.01). These results indicated that the LGZGD-containing serum could inhibit cardiomyocyte hypertrophy induced by AngⅡ, and its pharmacological effect was related to regulating Sig1R, promoting mitochondrial Ca~(2+ )inflow, restoring ATP synthesis, and protecting mitochondrial function.

A subgroup of light-driven sodium pumps with an additional Schiff base counterion.

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

The involvement of Akt, mTOR, and S6K in the in vivo effect of IGF-1 on the regulation of rat cardiac Na+/K+-ATPase.

BACKGROUND: We previously demonstrated that insulin-like growth factor-1 (IGF-1) regulates sodium/potassium adenosine triphosphatase (Na+/K+-ATPase) in vascular smooth muscle cells (VSMC) via phosphatidylinositol-3 kinase (PI3K). Taking into account that others' work show that IGF-1 activates the PI3K/protein kinase B (Akt) signaling pathway in many different cells, we here further questioned if the Akt/mammalian target of rapamycin (mTOR)/ribosomal protein p70 S6 kinase (S6K) pathway stimulates Na+/K+-ATPase, an essential protein for maintaining normal heart function. METHODS AND RESULTS: There were 14 adult male Wistar rats, half of whom received bolus injections of IGF-1 (50 µg/kg) for 24 h. We evaluated cardiac Na+/K+-ATPase expression, activity, and serum IGF-1 levels. Additionally, we examined the phosphorylated forms of the following proteins: insulin receptor substrate (IRS), phosphoinositide-dependent kinase-1 (PDK-1), Akt, mTOR, S6K, and α subunit of Na+/K+-ATPase. Additionally, the mRNA expression of the Na+/K+-ATPase α1 subunit was evaluated. Treatment with IGF-1 increases levels of serum IGF-1 and stimulates Na+/K+-ATPase activity, phosphorylation of α subunit of Na+/K+-ATPase on Ser23, and protein expression of α2 subunit. Furthermore, IGF-1 treatment increased phosphorylation of IRS-1 on Tyr1222, Akt on Ser473, PDK-1 on Ser241, mTOR on Ser2481 and Ser2448, and S6K on Thr421/Ser424. The concentration of IGF-1 in serum positively correlates with Na+/K+-ATPase activity and the phosphorylated form of mTOR (Ser2448), while Na+/K+-ATPase activity positively correlates with the phosphorylated form of IRS-1 (Tyr1222) and mTOR (Ser2448). CONCLUSION: These results indicate that the Akt/mTOR/S6K signalling pathway may be involved in the IGF-1 regulating cardiac Na+/K+-ATPase expression and activity.

Negative regulation of thyroid adenoma-associated protein (THADA) in the cardiac glycoside-induced anti-cancer effect.

Cardiac glycosides, known as inhibitors of Na+,K+-ATPase, have anti-cancer effects such as suppression of cancer cell proliferation and induction of cancer cell death. Here, we examined the signaling pathway elicited by cardiac glycosides in the human hepatocellular carcinoma HepG2 cells and human epidermoid carcinoma KB cells. Three kinds of cardiac glycosides (ouabain, oleandrin, and digoxin) inhibited the cancer cell proliferation and decreased the expression level of thyroid adenoma-associated protein (THADA). Interestingly, the knockdown of THADA inhibited cancer cell proliferation, and the proliferation was significantly rescued by re-expression of THADA in the THADA-knockdown cells. In addition, the THADA-knockdown markedly decreased the expression level of L-type amino acid transporter LAT1. Cardiac glycosides also reduced the LAT1 expression. The LAT1 inhibitor, JPH203, significantly weakened the cancer cell proliferation. These results suggest that the binding of cardiac glycosides to Na+,K+-ATPase negatively regulates the THADA-LAT1 pathway, exerting the anti-proliferative effect in cancer cells.

A digoxin derivative that potently reduces intraocular pressure: efficacy and mechanism of action in different animal models.

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) (Macaca fascicularis). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow.NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model (Macaca fascicularis), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.

Targeting NKAα1 to treat Parkinson's disease through inhibition of mitophagy-dependent ferroptosis.

Dysfunction of the Na+/K+-ATPase (NKA) has been documented in various neurodegenerative diseases, yet the specific role of NKAα1 in Parkinson's disease (PD) remains incompletely understood. In this investigation, we utilized NKAα1 haploinsufficiency (NKAα1+/-) mice to probe the influence of NKAα1 on dopaminergic (DA) neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our findings reveal that NKAα1+/- mice displayed a heightened loss of DA neurons and more pronounced motor dysfunction compared to the control group when exposed to MPTP. Intriguingly, this phenomenon coincided with the activation of ferroptosis and impaired mitophagy both in vivo and in vitro. To scrutinize the role and underlying mechanism of NKAα1 in PD, we employed DR-Ab, an antibody targeting the DR-region of the NKA α subunit. Our study demonstrates that the administration of DR-Ab effectively reinstated the membrane abundance of NKAα1, thereby mitigating MPTP-induced DA neuron loss and subsequent improvement in behavioral deficit. Mechanistically, DR-Ab heightened the formation of the surface NKAα1/SLC7A11 complex, inhibiting SLC7A11-dependent ferroptosis. Moreover, DR-Ab disrupted the cytosolic interaction between NKAα1 and Parkin, facilitating the translocation of Parkin to mitochondria and enhancing the process of mitophagy. In conclusion, this study establishes NKAα1 as a key regulator of ferroptosis and mitophagy, identifying its DR-region as a promising therapeutic target for PD.

Analysis of the sodium pump subunit ATP1A3 in glioma patients: Potential value in prognostic prediction and immunotherapy.

The ATP1A3 gene is associated with the development and progression of neurological diseases. However, the pathological function and therapeutic value of ATP1A3 in glioblastoma (GBM) remains unknown. In this study, we tried to explore the correlation between the ATP1A3 gene expression and immune features in GBM samples. We found that ATP1A3 gene expression levels showed significant negative correlation with immune checkpoints such as PD-L1, CTLA-4 and IDO1. Next, ATP1A3 gene expression levels showed significant negative correlation with the anti-cancer immune cell process, the immune score and stromal score. By grouping ATP1A3 expression levels, we found that that immunomodulator-related genes and tumor-associated immune cell effector gene expression levels were associated with lower ATP1A3 expression. In addition, immunotherapy prediction pathway activity and a majority of the anti-cancer immune cell process activity levels were also showed to be correlated with lower ATP1A3 gene expression. Further, nine prognostic factors were identified by prognostic analysis, and a GBM prognostic model (risk score) was established. We applied the model to the TCGA GBM training set sample and the GSE4412 validation set sample and found that patients in the high risk score subgroup had significantly shorter survival time, demonstrating the prognostic value and prognostic efficacy of the risk score. Furthermore, ATP1A3 overexpression has also been found to sensitize cancer cells to anti-PD-1 therapy. In conclusion, we showed that ATP1A3 is a highly promising treatment target in GBM and the risk score is an independent prognostic factor for cancer and can be used to help guide the prediction of survival time in patients with GBM.

Inhibition the ubiquitination of ENaC and Na,K-ATPase with erythropoietin promotes alveolar fluid clearance in sepsis-induced acute respiratory distress syndrome.

Sepsis-induced acute respiratory distress syndrome (ARDS) causes significant fatalities worldwide and lacks pharmacological intervention. Alveolar fluid clearance (AFC) plays a pivotal role in the remission of ARDS and is markedly impaired in the pathogenesis of ARDS. Here, we demonstrated that erythropoietin could effectively ameliorate lung injury manifestations and lethality, restore lung function and promote AFC in a rat model of lipopolysaccharide (LPS)-induced ARDS. Moreover, it was proven that EPO-induced restoration of AFC occurs through triggering the total protein expression of ENaC and Na,K-ATPase channels, enhancing their protein abundance in the membrane, and suppressing their ubiquitination for degeneration. Mechanistically, the data indicated the possible involvement of EPOR/JAK2/STAT3/SGK1/Nedd4-2 signaling in this process, and the pharmacological inhibition of the pathway markedly eliminated the stimulating effects of EPO on ENaC and Na,K-ATPase, and subsequently reversed the augmentation of AFC by EPO. Consistently, in vitro studies of alveolar epithelial cells paralleled with that EPO upregulated the expression of ENaC and Na,K-ATPase, and patch-clamp studies further demonstrated that EPO substantially strengthened sodium ion currents. Collectively, EPO could effectively promote AFC by improving ENaC and Na,K-ATPase protein expression and abundance in the membrane, dependent on inhibition of ENaC and Na,K-ATPase ubiquitination, and resulting in diminishing LPS-associated lung injuries.

The role of the antennal glands and gills in acid-base regulation and ammonia excretion of a marine osmoconforming brachyuran.

The excretory mechanisms of stenohaline marine osmoconforming crabs are often compared to those of the more extensively characterized euryhaline osmoregulating crabs. These comparisons may have limitations, given that unlike euryhaline brachyurans the gills of stenohaline marine osmoconformers possess ion-leaky paracellular pathways and lack the capacity to undergo ultrastructural changes that can promote ion-transport processes in dilute media. Furthermore, the antennal glands of stenohaline marine osmoconformers are poorly characterized making it difficult to determine what role urinary processes play in excretion. In the presented study, ammonia excretory processes as well as related acid-base equivalent transport rates and mechanisms were investigated in the Dungeness crab, Metacarcinus magister - an economically valuable stenohaline marine osmoconforming crab. Isolated and perfused gills were found to predominantly eliminate ammonia through a microtubule network-dependent active NH4+ transport mechanism that is likely performed by cells lining the arterial pockets of the gill lamella where critical Na+/K+-ATPase detection was observed. The V-type H+-ATPase - a vital component to transbranchial ammonia excretion mechanisms of euryhaline crabs - was not found to contribute significantly to ammonia excretion; however, this may be due to the transporter's unexpected apical localization. Although unconnected to ammonia excretion rates, a membrane-bound isoform of carbonic anhydrase was localized to the apical and basolateral membranes of lamella suited for respiration. Urine was found to contain significantly less ammonia as well as carbonate species than the hemolymph, indicating that unlike those of some euryhaline crabs the antennal glands of the Dungeness crab reabsorb these molecules rather than eliminate them for excretion.

Bioinformatic Study of Possible Acute Regulation of Acid Secretion in the Stomach.

The gastric H+,K+-ATPase is an integral membrane protein which derives energy from the hydrolysis of ATP to transport H+ ions from the parietal cells of the gastric mucosa into the stomach in exchange for K+ ions. It is responsible for the acidic environment of the stomach, which is essential for digestion. Acid secretion is regulated by the recruitment of the H+,K+-ATPase from intracellular stores into the plasma membrane on the ingestion of food. The similar amino acid sequences of the lysine-rich N-termini α-subunits of the H+,K+- and Na+,K+-ATPases, suggests similar acute regulation mechanisms, specifically, an electrostatic switch mechanism involving an interaction of the N-terminal tail with the surface of the surrounding membrane and a modulation of the interaction via regulatory phosphorylation by protein kinases. From a consideration of sequence alignment of the H+,K+-ATPase and an analysis of its coevolution with protein kinase C and kinases of the Src family, the evidence points towards a phosphorylation of tyrosine-7 of the N-terminus by either Lck or Yes in all vertebrates except cartilaginous fish. The results obtained will guide and focus future experimental research.

PEBP4 deficiency aggravates LPS-induced acute lung injury and alveolar fluid clearance impairment via modulating PI3K/AKT signaling pathway.

Acute lung injury (ALI) is a common clinical syndrome, which often results in pulmonary edema and respiratory distress. It has been recently reported that phosphatidylethanolamine binding protein 4 (PEBP4), a basic cytoplasmic protein, has anti-inflammatory and hepatoprotective effects, but its relationship with ALI remains undefined so far. In this study, we generated PEBP4 knockout (KO) mice to investigate the potential function of PEBP4, as well as to evaluate the capacity of alveolar fluid clearance (AFC) and the activity of phosphatidylinositide 3-kinases (PI3K)/serine-theronine protein kinase B (PKB, also known as AKT) signaling pathway in lipopolysaccharide (LPS)-induced ALI mice models. We found that PEBP4 deficiency exacerbated lung pathological damage and edema, and increased the wet/dry weight ratio and total protein concentration of bronchoalveolar lavage fluid (BALF) in LPS-treated mice. Meanwhile, PEBP4 KO promoted an LPS-induced rise in the pulmonary myeloperoxidase (MPO) activity, serum interleuin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α levels, and pulmonary cyclooxygenase-2 (COX-2) expression. Mechanically, PEBP4 deletion further reduced the protein expression of Na+ transport markers, including epithelial sodium channel (ENaC)-α, ENaC-γ, Na,K-ATPase α1, and Na,K-ATPase ß1, and strengthened the inhibition of PI3K/AKT signaling in LPS-challenged mice. Furthermore, we demonstrated that selective activation of PI3K/AKT with 740YP or SC79 partially reversed all of the above effects caused by PEBP4 KO in LPS-treated mice. Altogether, our results indicated the PEBP4 deletion has a deterioration effect on LPS-induced ALI by impairing the capacity of AFC, which may be achieved through modulating the PI3K/AKT pathway.

Effect of sulfate on the osmoregulatory and physio-biochemical responses of GIFT (Oreochromis niloticus) juveniles reared in potassium-deficient medium saline waters.

The inland saline waters were continuously observed to have low potassium concentrations compared to their seawater counterpart of the same salinity. We hypothesize that the toxic effect of sulfate may manifest in low potassium saline (LPSW) waters compared to brackish water of the same salinity. Thus, LC50 trials were performed in GIFT (genetically improved farmed tilapia) fry (0.5 ± 0.02 g) to determine the acute sulfate toxicity in freshwater (FW, 0.5 g L-1), artificial seawater (ASW, 10 g L-1), and LPSW (10 g L-1). The median lethal concentrations (96h LC50) of sulfate ion in FW, LPSW, and ASW for the GIFT were 5.30 g L-1, 2.56 g L-1, and 2.98 g L-1, respectively. A second experiment was conducted for 21 days, exposing fish to a sub-lethal level of sulfate ion (SO42-) concentration (1000 mg L-1, one-fifth of FW LC50) with different types of waters (FW, freshwater, 0.5 g L-1; ASW, artificial seawater, 10 g L-1; LPSW, low potassium saline water, 10 g L-1) with and without sulfate inclusion to constitute the treatments as follows, (FW, FW + SO4, ASW, ASW + SO4, LPSW, LPSW + SO4). The effect of sulfate on GIFT reared in sulfate-rich potassium-deficient medium saline water was evaluated by focusing on the hematological adjustments, stress-induced oxidative damage, and osmoregulatory imbalances. The survival was not altered due to the sulfate concentration and K+ deficiency; however, there were significant changes in branchial NKA (Na+/K+-ATPase) activity and osmolality. The increase in NKA was highest in LPSW treatment, suggesting that internal ionic imbalance was triggered due to an interactive effect of sulfate and K+ deficiency. The cortisol levels showed a pronounced increase due to sulfate inclusion irrespective of K+ deficiency. The antioxidant enzymes, i.e., SOD (superoxide dismutase), catalase, GST (glutathione-S-transferase), and GPX (glutathione peroxidase), reflected a similar pattern of increment in the gills and liver of the LPSW + SO4 groups, suggesting a poor antioxidant status of the exposed group. The hepatic peroxidation status, i.e. TBARS (thiobarbituric acid reactive substances), and the peroxide values were enhanced due to both K+ deficiency and sulfate inclusion, suggesting a possible lipid peroxidation in the liver due to handling the excess sulfate anion concentration. The hematological parameters, including haemoglobin, total erythrocyte count, and hematocrit level, reduced significantly in the LPSW + SO4 group, indicating a reduced blood oxygen capacity due to the sulfate exposure and water potassium deficiency. The hepatic acetylcholine esterase activity was suppressed in all the treatments with sulfate inclusion, while the highest suppression was observed in the LPSW + SO4 group. Thus, it is concluded that sulfate-induced physiological imbalances manifest more in potassium-deficient water, indicating that environmental sulfate is more detrimental to inland saline water than freshwater or brackish water of the same salinity.

Astrocytic Na+, K+ ATPases in physiology and pathophysiology.

The Na+, K+ ATPases play a fundamental role in the homeostatic functions of astrocytes. After a brief historic prologue and discussion of the subunit composition and localization of the astrocytic Na+, K+ ATPases, the review focuses on the role of the astrocytic Na+, K+ pumps in extracellular K+ and glutamate homeostasis, intracellular Na+ and Ca2+ homeostasis and signaling, regulation of synaptic transmission and neurometabolic coupling between astrocytes and neurons. Loss-of-function mutations in the gene encoding the astrocytic α2 Na+, K+ ATPase cause a rare monogenic form of migraine with aura (familial hemiplegic migraine type 2). On the other hand, the α2 Na+, K+ ATPase is upregulated in spinal cord and brain samples from amyotrophic lateral sclerosis and Alzheimer disease patients, respectively. In the last part, the review focuses on i) the migraine relevant phenotypes shown by familial hemiplegic migraine type 2 knock-in mice with 50 % reduced expression of the astrocytic α2 Na+, K+ ATPase and the insights into the pathophysiology of migraine obtained from these genetic mouse models, and ii) the evidence that upregulation of the astrocytic α2 Na+, K+ ATPase in mouse models of amyotrophic lateral sclerosis and Alzheimer disease promotes neuroinflammation and contributes to progressive neurodegeneration.

Female tilapia, Oreochromis sp. mobilised energy differently for growth and reproduction according to living environment.

This study was conducted to investigate the energy mobilisation preference and ionoregulation pattern of female tilapia, Oreochromis sp. living in different environments. Three different treatments of tilapia as physiology compromising model were compared; tilapia cultured in recirculating aquaculture system (RAS as Treatment I-RAS), tilapia cultured in open water cage (Treatment II-Cage) and tilapia transferred from cage and cultured in RAS (Treatment III-Compensation). Results revealed that tilapia from Treatment I and III mobilised lipid to support gonadogenesis, whilst Treatment II tilapia mobilised glycogen as primary energy for daily exercise activity and reserved protein for growth. The gills and kidney Na+/K+ ATPase (NKA) activities remained relatively stable to maintain homeostasis with a stable Na+ and K+ levels. As a remark, this study revealed that tilapia strategized their energy mobilisation preference in accessing glycogen as an easy energy to support exercise metabolism and protein somatogenesis in cage culture condition, while tilapia cultured in RAS mobilised lipid for gonadagenesis purposes.

Involvement of the Na+, K+-ATPase α1 Isoform and Endogenous Cardiac Steroids in Depression- and Manic-like Behaviors.

Bipolar disorder (BD) is a severe and common chronic mental illness characterized by recurrent mood swings between depression and mania. The biological basis of the disease is poorly understood, and its treatment is unsatisfactory. Na+, K+-ATPase is a major plasma membrane transporter and signal transducer. The catalytic α subunit of this enzyme is the binding site for cardiac steroids. Three α isoforms of the Na+, K+-ATPase are present in the brain. Previous studies have supported the involvement of the Na+, K+-ATPase and endogenous cardiac steroids (ECS) in the etiology of BD. Decreased brain ECS has been found to elicit anti-manic and anti-depressive-like behaviors in mice and rats. However, the identity of the specific α isoform involved in these behavioral effects is unknown. Here, we demonstrated that decreasing ECS through intracerebroventricular (i.c.v.) administration of anti-ouabain antibodies (anti-Ou-Ab) decreased the activity of α1+/- mice in forced swimming tests but did not change the activity in wild type (wt) mice. This treatment also affected exploratory and anxiety behaviors in α1+/- but not wt mice, as measured in open field tests. The i.c.v. administration of anti-Ou-Ab decreased brain ECS and increased brain Na+, K+-ATPase activity in wt and α1+/- mice. The serum ECS was lower in α1+/- than wt mice. In addition, a study in human participants demonstrated that serum ECS significantly decreased after treatment. These results suggest that the Na+, K+-ATPase α1 isoform is involved in depressive- and manic-like behaviors and support that the Na+, K+-ATPase/ECS system participates in the etiology of BD.

The Cyst Epithelium in Polycystic Kidney Disease Patients Displays Normal Apical-Basolateral Cell Polarity.

The main characteristic of polycystic kidney disease is the development of multiple fluid-filled renal cysts. The discovery of mislocalized sodium-potassium pump (Na,K-ATPase) in the apical membrane of cyst-lining epithelia alluded to reversal of polarity as a possible explanation for the fluid secretion. The topic of apical Na,K-ATPase in cysts remains controversial. We investigated the localization of the Na,K-ATPase and assessed the apical-basolateral polarization of cyst-lining epithelia by means of immunohistochemistry in kidney tissue from six polycystic kidney disease patients undergoing nephrectomy. The Na,K-ATPase α1 subunit was conventionally situated in the basolateral membrane of all immunoreactive cysts. Proteins of the Crumbs and partitioning defective (Par) complexes were localized to the apical membrane domain in cyst epithelial cells. The apical targeting protein Syntaxin-3 also immunolocalized to the apical domain of cyst-lining epithelial cells. Proteins of the basolateral Scribble complex immunolocalized to the basolateral domain of cysts. Thus, no deviations from the typical epithelial distribution of basic cell polarity proteins were observed in the cysts from the six patients. Furthermore, we confirmed that cysts can originate from virtually any tubular segment with preserved polarity. In conclusion, we find no evidence of a reversal in apical-basolateral polarity in cyst-lining epithelia in polycystic kidney disease.

Sensational site: the sodium pump ouabain-binding site and its ligands.

Cardiotonic steroids (CTS), used by certain insects, toads, and rats for protection from predators, became, thanks to Withering's trailblazing 1785 monograph, the mainstay of heart failure (HF) therapy. In the 1950s and 1960s, we learned that the CTS receptor was part of the sodium pump (NKA) and that the Na+/Ca2+ exchanger was critical for the acute cardiotonic effect of digoxin- and ouabain-related CTS. This "settled" view was upended by seven revolutionary observations. First, subnanomolar ouabain sometimes stimulates NKA while higher concentrations are invariably inhibitory. Second, endogenous ouabain (EO) was discovered in the human circulation. Third, in the DIG clinical trial, digoxin only marginally improved outcomes in patients with HF. Fourth, cloning of NKA in 1985 revealed multiple NKA α and ß subunit isoforms that, in the rodent, differ in their sensitivities to CTS. Fifth, the NKA is a cation pump and a hormone receptor/signal transducer. EO binding to NKA activates, in a ligand- and cell-specific manner, several protein kinase and Ca2+-dependent signaling cascades that have widespread physiological effects and can contribute to hypertension and HF pathogenesis. Sixth, all CTS are not equivalent, e.g., ouabain induces hypertension in rodents while digoxin is antihypertensinogenic ("biased signaling"). Seventh, most common rodent hypertension models require a highly ouabain-sensitive α2 NKA and the elevated blood pressure is alleviated by EO immunoneutralization. These numerous phenomena are enabled by NKA's intricate structure. We have just begun to understand the endocrine role of the endogenous ligands and the broad impact of the ouabain-binding site on physiology and pathophysiology.

Impacts of temperature and turbidity on the gill physiology of darter species.

Fish gills are complex organs that have direct contact with the environment and perform numerous functions including gas exchange and ion regulation. Determining if gill morphometry can change under different environmental conditions to maintain and/or improve gas exchange and ion regulation is important for understanding if gill plasticity can improve survival with increasing environmental change. We assessed gill morphology (gas exchange and ion regulation metrics), hematocrit and gill Na+/K+ ATPase activity of wild-captured blackside darter (Percina maculata), greenside darter (Etheostoma blennioides), and johnny darter (Etheostoma nigrum) at two temperatures (10 and 25 °C) and turbidity levels (8 and 94 NTU). Samples were collected August and October 2020 in the Grand River to assess temperature differences, and August 2020 in the Thames River to assess turbidity differences. Significant effects of temperature and/or turbidity only impacted ionocyte number, lamellae width, and hematocrit. An increase in temperature decreased ionocyte number while an increase in turbidity increased lamellae width. Hematocrit had a species-specific response for both temperature and turbidity. Findings suggest that the three darter species have limited plasticity in gill morphology, with no observed compensatory changes in hematocrit or Na+/K+ ATPase activity to maintain homeostasis under the different environmental conditions.