Inhibition Of Na Research Articles

Sex differences in solute transport along the nephrons: effects of Na+ transport inhibition.

Each day, ~1.7 kg of NaCl and 180 liters of water are reabsorbed by nephron segments in humans, with urinary excretion fine tuned to meet homeostatic requirements. These tasks are coordinated by a spectrum of renal Na+ transporters and channels. The goal of the present study was to investigate the extent to which inhibitors of transepithelial Na+ transport (TNa) along the nephron alter urinary solute excretion and how those effects may vary between male and female subjects. To accomplish that goal, we developed sex-specific multinephron models that represent detailed transcellular and paracellular transport processes along the nephrons of male and female rat kidneys. We simulated inhibition of Na+/H+ exchanger 3 (NHE3), bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC2), Na+-Cl- cotransporter (NCC), and amiloride-sensitive epithelial Na+ channel (ENaC). NHE3 inhibition simulations predicted a substantially reduced proximal tubule TNa, and NKCC2 inhibition substantially reduced thick ascending limb TNa. Both gave rise to diuresis, natriuresis, and kaliuresis, with those effects stronger in female rats. While NCC inhibition was predicted to have only minor impact on renal TNa, it nonetheless had a notable effect of enhancing excretion of Na+, K+, and Cl-, particularly in female rats. Inhibition of ENaC was predicted to have opposite effects on the excretion of Na+ (increased) and K+ (decreased) and to have only a minor impact on whole kidney TNa. Unlike inhibition of other transporters, ENaC inhibition induced stronger natriuresis and diuresis in male rats than female rats. Overall, model predictions agreed well with measured changes in Na+ and K+ excretion in response to diuretics and Na+ transporter mutations.

Na+ controls hypoxic signalling by the mitochondrial respiratory chain.

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of theinner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.

Inhibition of Na+/K+ ATPase blocks Zika virus infection in mice

Zika virus (ZIKV) is an infectious disease that has become an important concern worldwide, it associates with neurological disorders and congenital malformations in adults, also leading to fetal intrauterine growth restriction and microcephaly during pregnancy. However, there are currently no approved vaccines or specific antiviral drugs for preventing or treating ZIKV infection. Here, we show that two FDA-approved Na+/K+-ATPase inhibitors, ouabain and digoxin, can block ZIKV infection at the replication stage by targeting Na+/K+-ATPase. Furthermore, ouabain reduced the viral burden of ZIKV in adult mice, penetrated the placental barrier to enter fetal tissues, and protected fetal mice from ZIKV infection-induced microcephaly in a pregnant mouse model. Thus, ouabain has therapeutic potential for ZIKV.

Consequences of surface coatings and soil ageing on the toxicity of cadmium telluride quantum dots to the earthworm Eisenia fetida.

The bioaccumulation potential and toxic effects of engineered nanomaterials (ENMs) to earthworms are poorly understood. Two studies were conducted following OECD TG 222 on Eisenia fetida to assess the effects of CdTe QDs with different coatings and soil ageing respectively. Earthworms were exposed to carboxylate (COOH), ammonium (NH4+), or polyethylene glycol (PEG) coated CdTe QDs, or a micron scale (bulk) CdTe material, at nominal concentrations of 50, 500 and 2000 mg CdTe QD kg-1 dry weight (dw) for 28 days in Lufa 2.2 soil. In the fresh soil study, earthworms accumulated similar amounts of Cd and Te in the CdTe-bulk exposures, while the accumulation of Cd was higher than Te during the exposures to CdTe QDs. However, neither the total Cd, nor Te concentrations in the earthworms, were easily explained by the extractable metal fractions in the soil or particle dissolution. There were no effects on survival, but some retardation of growth was observed at the higher doses. Inhibition of Na+/K+-ATPase activity with disturbances to tissue electrolytes, as well as tissue Cu and Mn were observed, but without depletion of total glutathione in the fresh soil experiment. Additionally, juvenile production was the most sensitive endpoint, with estimated nominal EC50 of values >2000, 108, 65, 96 mg CdTe kg-1 for bulk, PEG-, COOH- and NH4+-coated CdTe QDs, respectively. In the aged soil study, the accumulation of Cd and Te was higher than in the fresh soil study in all CdTe QD exposures. Survival of the adult worms was reduced in the top CdTe-COOH and -NH4+ QD exposures by 55 ± 5 and 60 ± 25%, respectively; and with decreases in growth. The nominal EC50 values for juvenile production in the aged soil were 165, 88, 78 and 63 mg CdTe kg-1 for bulk, PEG-, COOH- and NH4+-coated CdTe QDs, respectively. In conclusion, exposure to nanoscale CdTe QDs, regardless of coating, caused more severe toxic effects that the CdTe bulk material and the toxicity increased after soil ageing. There were some coating-mediated effects, likely due to differences in the metal content and behaviour of the materials.

Distinct co-tolerance responses to combined salinity and cadmium exposure in metallicolous and non-metallicolous ecotypes of Silene vulgaris

This study compared co-tolerance to salinity and cadmium and investigated its mechanisms in a facultative metallophyte Silene vulgaris originating from distinct habitats. Shoots of calamine (Cal) and non-metallicolous (N-Cal) ecotypes grown in vitro were exposed to 10 and 100mM NaCl, 5μM CdCl2 and their combinations. Stress effects were evaluated based on growth, oxidative stress parameters, and DNA content and damage. Tolerance mechanisms were assessed by analyzing non-enzymatic antioxidants, osmolytes and ion accumulation. Irrespective of the ecotype, Cd stimulated shoot proliferation (micropropagation coefficients MC=15.2 and 12.1 for Cal and N-Cal, respectively, growth tolerance index GTI=148.1 and 156.7%). In Cal ecotype this was attributed to an increase in glutathione content and reorganization of cell membrane structures under Cd exposure, whereas in N-Cal to enhanced synthesis of other non-enzymatic antioxidants, mainly carotenoids and ascorbate. Low salinity stimulated growth of Cal ecotype due to optimizing Cl- content. High salinity inhibited growth, especially in Cal ecotype, where it enhanced DNA damage and disturbed ionic homeostasis. Species-specific reaction to combined salinity and Cd involved a mutual inhibition of Na+, Cl- and Cd2+ uptake. N-Cal ecotype responded to combined stresses by enhancing its antioxidant defense, presumably induced by Cd, whereas the metallicolous ecotype triggered osmotic adjustment. The study revealed that in S. vulgaris Cd application ameliorated metabolic responses to simultaneous salinity exposure. It also shed a light on distinct strategies of coping with combined abiotic stresses in two ecotypes of the species showing high plasticity in environmental conditions.

Partial Inhibition of Na+/K+-ATPase and Plasma Membrane Ca2+-ATPase from the Rat Cerebral Cortex by S-Nitrosoglutathione

S-nitrosoglutathione (GSNO) is an endogenous S-nitrosylated glutathione derivative (GSH) involved in NO signaling. GSNO acts as a carrier of nitrosyl groups to cysteine (both free amino acid and its residues in proteins), a glutathionylation agent, and an NO donor. We investigated the effects of GSNO, GSH, and oxidized glutathione (GSSG) on the activity of two main ion pumps of the plasma membrane, Na+/K+-ATPase (NKA) and plasma membrane Ca2+-ATPase (PMCA). Plasma membranes were obtained from the rat cerebral cortex by differential centrifugation. Preincubation of the saponinpermeabilized plasma membranes at 37°C (but not at 0°C) with various concentrations of GSNO led to partial inhibition of ATPase activities of NKA and PMCA (on average, by 19.4 ± 2.2 and 42.1 ± 3% at 10 mM GSNO, respectively). The addition of GSH instead of GSNO did not affect the enzyme activities, while GSSG inhibited these enzymes much weaker than GSNO. The inhibited enzyme activities after the action of GSNO were partially restored after incubation of the membranes with a thiol-reducing agent, 2-mercaptoethanol. GSNO-dependent inhibition of NKA was not observed in the presence of ouabain (>1.0 μM), when only isozymes having an α1-subunit were active. GSNO inhibited both high- and lowsensitive to Ca2+ (calmodulin-dependent and calmodulin-independent) PMCA activities by 51 and 33%, respectively, with no changes in Km for Ca2+. It is assumed that GSNO or its degradation product(s), by modifying the thiol groups in NKA and PMCA molecules, partly inhibit their activity. Only NKA isozymes with α2 and α3 subunits, which are highly sensitive to ouabain, are likely to manifest such sensitivity. Inhibition of PMCA is not associated with a change in its sensitivity to Ca2+.

Dihydroouabain, a novel radiosensitizer for cervical cancer identified by automated high-throughput screening

Background and purposeRadiotherapy plays a crucial role in the treatment of cervical cancer, but existing radiosensitizers have limited efficacy in clinical applications. The aims of this study were to establish and verify an efficient method for identifying new radiosensitizers, to use this to identify candidate radiosensitizers for cervical cancer, and to investigate the specific mechanisms of these when used in combination with radiotherapy. Materials and methodsAn automated platform for identifying radiosensitizers for cervical cancer was created based on high-throughput screening technology. The radiosensitizing effects of candidate compounds from the LOPAC1280 chemical library were evaluated in radiosensitive and radioresistant cervical cancer cells using a clonogenic survival assay, with cell cycle analyses, and western blot analyses performed for both cell lines. ResultsThe automated high-throughput screening approach identified four hit compounds. One of the most potent candidates was dihydroouabain (DHO), an inhibitor of Na+/K+-ATPase that has not previously been classified as a radiosensitizer. DHO significantly enhanced radiosensitivity in cervical cancer cells. It also abrogated radiation-induced S phase arrest in cervical cancer cells. Combination treatment significantly caused the inhibition of Chk1 and increased DNA double-strand breaks (DSB). ConclusionsDHO is a novel radiosensitizer for the treatment of cervical cancer. The automated high-throughput screening platform developed in this study proved to be powerful and effective, with the potential to be widely used in the future identification of radiosensitizers.

Selenium and Salt Interactions in Black Gram (Vigna mungo L): Ion Uptake, Antioxidant Defense System, and Photochemistry Efficiency.

Salinity is a major abiotic stress which limits crop production, especially under rainfed conditions. Selenium (Se), as an important micronutrient, plays a vital role in mitigating detrimental effects of different abiotic stresses. The objective of this research was to examine the effect of Se fertilization on black gram (Vigna mungo) under salt stress. Our results showed that salt stress (100 mM NaCl) in leaves significantly induced oxidative damage and caused a decline in relative water content, chlorophyll (Chl), stomatal conductance (gs), photochemical efficiency (Fv/Fm), sucrose, and reducing sugars. A low dose of Se (1.5 ppm) significantly reduced hydrogen peroxide content, malondialdehyde formation, cell membrane damage, and also improved antioxidative enzyme activities, including superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and glutathione peroxidase under salt stress. Se-treated plants exhibited higher Chl, gs, Fv/Fm, sucrose, and reducing sugars than untreated plants in response to salt stress. In addition, Se application enhanced Se uptake and reduced Na+ uptake, but Cl− remained unaffected. Our results indicated that a low dose of Se effectively alleviated salt damage via inhibition of Na+ uptake and enhanced antioxidant defense resulting in a significant decrease in oxidative damage, and maintained gaseous exchange and PS II function for sucrose and reducing sugars accumulation in black gram.

Effect of Carvacrol, TRP Channels Modulator, on Cardiac Electrical Activity.

Despite the wide application of carvacrol (CAR) in medicines, dietary supplements, and foods, there is still insufficient electrophysiological data on the mechanisms of action of CAR, particularly with regard to heart function. Therefore, in this study, we attempted to elucidate whether CAR, whose inhibitory effect on both cardiac and vascular TRPM7 and L-type Ca2+ currents has been demonstrated previously, could modify cardiac electrical activity. We used a combination of optical mapping and microelectrode techniques to track the action potentials (APs) and the spread of electrical activity in a Langendorff-perfused rabbit heart model during atrial/endo/epicardial pacing. Simultaneously, ECG recordings were acquired. Because human trials on CAR are still lacking, we tested the action of CAR on human ventricular preparations obtained from explanted hearts. Activation time (AT), AP duration (APD), and conduction velocity maps were constructed. We demonstrated that at a low concentration (10 μM) of CAR, only marginal changes in the AP parameters were observed. At higher concentrations (≥100 μM), a decrease in AP upstroke velocity (dV/dtmax), suggesting inhibition of Na+ current, and APD (at 50 and 90% repolarization) was detected; also slowing in the spread of electrical signals via the atrioventricular node was observed, suggesting impaired functioning of Ca2+ channels. In addition, a decrease in the T-wave amplitude was seen on the ECG, suggesting an impaired repolarization process. Nevertheless, those changes occurred without a significant impact on the resting membrane potential and were reversible. We suggest that CAR might play a role in modulating cardiac electrical activity at high concentrations.

Inhibitors of Na+/K+ ATPase exhibit antitumor effects on multicellular tumor spheroids of hepatocellular carcinoma

Hepatocellular carcinoma (HCC), one of the most common malignant cancers worldwide, is associated with substantial mortality. Because HCCs have strong resistance to conventional chemotherapeutic agents, novel therapeutic strategies are needed to improve survival in patients with HCC. The multicellular tumor spheroid (MCTS) model is a powerful method for anticancer research because of its ability to mimic the complexity and heterogeneity of tumor tissue, the three-dimensional cellular context of tumor tissue, and the pathophysiological gradients of in vivo tumors. However, it is difficult to obtain meaningful results from the MCTS model without considering the conditions of clinical tumors. We, therefore, provided a proof of concept to determine whether spheroid models simulate in vivo tumor microenvironments. Through a high-throughput screening for HCC therapy using the MCTS model, we selected inhibitors of Na+/K+-ATPase (ouabain and digoxin) that could suppress cell growth and migration via inhibition of the epithelial-mesenchymal transition of HCC in vivo and in vitro. The results showed that this model provides a new paradigm for high-throughput drug screening and will significantly improve the efficiency of identifying new drugs for HCC treatment. Through utilization of MCTS models, here we found that inhibitors of Na+/K+-ATPase may be feasible as a novel target to sensitize HCC cells.

Melatonin protects against membrane alterations affected by ‘Artificial Light at Night’ in a circadian-disrupted model of rat

ABSTRACT Artificial Light at Night (ALAN) causes alteration in the redox status and membrane-bound transporters. Melatonin has pleiotropic effects on normal physiology and acts as a potential antioxidant. This study was undertaken to evaluate the effects of ALAN-induced alterations in the membrane transporters, oxidative stress biomarkers and to evaluate the protective effects of melatonin supplementation prior to exposure of light in a circadian-disrupted model of rat. Young male Wistar rats 250 ± 20 g were randomly segregated into following groups; Group (I) control, Group (II) melatonin-treated group (10 mg/kg, orally), Group (III) ALAN (500 lux), Group (IV) ALAN with melatonin, for 10 days. Results show that melatonin is an effective mediator of membrane-bound transporters revealed by activation of Na+/K+-ATPase (NKA), plasma membrane Ca2+-ATPase (PMCA) and suppression of Na+/H+ exchanger (NHE) activity. Oxidative stress biomarkers such as total thiol (T-SH), sialic acid (SA), lipid hydroperoxides (LHs) and protein carbonylation (PC) are also restored to the normal level after supplementation of melatonin. These findings demonstrate the protective effect of melatonin on membrane-bound enzymes along with other oxidative stress biomarkers against light-mediated oxidative damage in erythrocyte membrane.

Regulation of NRF2 by Na+/K+-ATPase: implication of tyrosine phosphorylation of Src

Tumors adapt well to the imbalanced redox status created by rapid growth and limited nutrient availability because they highly express high levels of NRF2 to counteract oxidative stress. Therefore, inhibition of NRF2 is currently considered a feasible strategy for development of chemotherapeutic agents. In the present study, we identified that Na+/K+-ATPase regulates NRF2 in A549 cells. Suppression of Na+/K+-ATPase by convallatoxin or siRNAs downregulates NRF2 in A549 cells, and this event is mediated by Ca2+-dependent induction of CSK1 and subsequent phosphorylation of SRC at Tyr 527. Consistent with this finding, knocking down the α1 or β1 subunit of Na+/K+-ATPase promotes the generation of intracellular ROS by cisplatin and potentiates cisplatin-induced apoptosis and autophagy in A549 cells. Our study reveals that the signaling axis composed of Na+/K+-ATPase, CSK1, and tyrosine phosphorylation of Src could be a useful target for development of NRF2 inhibitors.

Protective effects of rimeporide on left ventricular function in golden retriever muscular dystrophy dogs

BackgroundAlterations in intracellular Na+ and Ca2+ have been observed in patients with Duchenne muscular dystrophy (DMD) and in animal models of DMD, and inhibition of Na+-H+ exchanger 1 (NHE1) by rimeporide has previously demonstrated cardioprotective effects in animal models of myocardial ischemia and heart failure. Since heart failure is becoming a predominant cause of death in DMD patients, this study aimed to demonstrate a cardioprotective effect of chronic administration of rimeporide in a canine model of DMD. MethodsGolden retriever muscular dystrophy (GRMD) dogs were randomized to orally receive rimeporide (10 mg/kg, twice a day) or placebo from 2 months to 1 year of age. Left ventricular (LV) function was assessed by conventional and advanced echocardiography. ResultsCompared with placebo-treated GRMD, LV function deterioration with age was limited in rimeporide-treated GRMD dogs as indicated by the preservation of LV ejection fraction as well as overall cardiac parameters different from placebo-treated dogs, as revealed by composite cardiac scores and principal component analysis. In addition, principal component analysis clustered rimeporide-treated GRMD dogs close to healthy control dogs. ConclusionsChronic administration of the NHE1 inhibitor rimeporide exerted a protective effect against LV function decline in GRMD dogs. This study provides proof of concept to explore the cardiac effects of rimeporide in DMD patients.

Anti-Neuraminidase Bioactives from Manggis Hutan (Garcinia celebica L.) Leaves: Partial Purification and Molecular Characterization.

The neuraminidase enzyme (NA) from the influenza virus is responsible for the proliferation and infections of the virus progeny, prompting several efforts to discover and optimize effective neuraminidase inhibitors. The main aim of this study is to discover a new potential neuraminidase inhibitor that comes from Garcinia celebica leaves (GCL). The bioassay-guided isolation method was performed to obtain lead compounds. The binding interaction of the isolated compounds was predicted by using molecular docking studies. Friedeline (GC1, logP > 5.0), two lanastone derivatives (methyl-3α,23-dihydroxy-17,14-friedolanstan-8,14,24-trien-26-oat (GC2) and 24E-3a,9,23-trihydroxy-17,14-friedolanostan-14,24-dien-26-oate (GC3) with LogP > 5.0) and catechin (GC4, LogP = 1.4) were identified. The inhibitory potency of these four compounds on NA from C. perfringens and H1N1 was found to be as follows: GC4 > GC2 > GC3 > GC1. All compounds exhibited higher inhibitory activity towards C. perfringens NA compared to H1N1 NA. From the molecular docking results, GC4 favorably docked and interacted with Arg118, Arg371, Arg292, Glu276 and Trp178 residues, whilst GC2 interacted with Arg118, Arg371, Arg292, Ile222, Arg224 and Ser246. GC3 interacted with Tyr406 only. GC4 had potent NA inhibition with free energy of binding of −12 kcal/mol. In the enzyme inhibition study, GC4 showed the highest activity with an IC50 of 60.3 µM and 91.0 µM for C. perfringens NA and H1N1 NA—respectively.

Inhibition of Na(+),K(+)-ATPase and activation of myosin ATPase by calix[4]arene C-107 cause stimulation of isolated smooth muscle contractile activity

Inhibition of Na(+),K(+)-ATPase and activation of myosin ATPase by calix[4]arene C-107 cause stimulation of isolated smooth muscle contractile activity

Biochemical Implications of Biotransformation of Some Toxic Floras Using Natural Local Enzyme Sources.

Recently, it has been established that simultaneous saccharification and fermentation is a potent technique for the detoxification of harmful plant materials. Following encouraging simultaneous medicinal applications of snail slime and yeast, we exploited their hydrolytic and fermentation potentials to prevent toxicities of the selected floras; Erythrodontium barteri (EB), bracken fern (BF), and crustose lichens (CL). The applicability of the saccharification process has been described in a patent (WO2005010193A2). The plants were bioprocessed using snail digestive juice and yeast slurry and their health effects were evaluated. Seventy rats were divided equally into groups, treated with single doses of aqueous extracts of the plants and their bioprocessed forms, and compared with control rats. The plants showed very high antinutrients levels, which significantly reduced after SSF with enhanced flavonoids, alkaloids and phenols. Potential alterations of WBC differentials, RBC, liver and renal function markers indices were mitigated by bioprocessed extracts. MDA, SOD, GRase, XO and XDH levels in rats administered the bEB and CL were equivalent to the levels found for the control rats. Some bioprocessed plants produced unaltered insulin, ghrelin, and leptin levels. The bioprocessed extracts, when compared to the effects of unprocessed extracts, produced lower TNF-α, Caspase-3, and adiponectin levels and mitigated the potential suppression of Na+/K+-ATPase levels. Potential depletion of inhibin-B, testosterone, estrogen, and prolactin was mitigated after bioprocessing. This study, thus, validates the application of bioprocessing using snail digestive juice and yeast as an effective approach to reduce the potential toxicities of harmful plants.

Interaction between the autophagy protein Beclin 1 and Na+,K+-ATPase during starvation, exercise, and ischemia.

Autosis is a distinct form of cell death that requires both autophagy genes and the Na+,K+-ATPase pump. However, the relationship between the autophagy machinery and Na+,K+-ATPase is unknown. We explored the hypothesis that Na+,K+-ATPase interacts with the autophagy protein Beclin 1 during stress and autosis-inducing conditions. Starvation increased the Beclin 1/Na+,K+-ATPase interaction in cultured cells, and this was blocked by cardiac glycosides, inhibitors of Na+,K+-ATPase. Increases in Beclin 1/Na+,K+-ATPase interaction were also observed in tissues from starved mice, livers of patients with anorexia nervosa, brains of neonatal rats subjected to cerebral hypoxia-ischemia (HI), and kidneys of mice subjected to renal ischemia/reperfusion injury (IRI). Cardiac glycosides blocked the increased Beclin 1/Na+,K+-ATPase interaction during cerebral HI injury and renal IRI. In the mouse renal IRI model, cardiac glycosides reduced numbers of autotic cells in the kidney and improved clinical outcome. Moreover, blockade of endogenous cardiac glycosides increased Beclin 1/Na+,K+-ATPase interaction and autotic cell death in mouse hearts during exercise. Thus, Beclin 1/Na+,K+-ATPase interaction is increased in stress conditions, and cardiac glycosides decrease this interaction and autosis in both pathophysiological and physiological settings. This crosstalk between cellular machinery that generates and consumes energy during stress may represent a fundamental homeostatic mechanism.

A neural circadian signal essential for ovulation is generated in the suprachiasmatic nucleus during each stage of the oestrous cycle.

What is the central question of this study? Is the suprachiasmatic nucleus the structure that generates the neural circadian signals that occur during every stage of the oestrous cycle, not only pro-oestrus, and are these signals essential for proper regulation of ovulation? What is the main finding and its importance? Transient inhibition of Na+ -dependent action potentials in the suprachiasmatic nucleus by tetrodotoxin microinjection at 14.00h inhibits ovulation irrespective of the stage of the oestrous cycle at which the procedure is performed. Microinjection of saline solution into the suprachiasmatic nucleus has a disruptive effect on ovulation that depends on the stage of the cycle at which it is administered. Reproduction is a highly timed process that depends on both the reproductive and circadian systems. The core oscillator of the latter resides at the suprachiasmatic nuclei (SCN) and it is pivotal for the regulation of the pro-oestrus pre-ovulatory surge of gonadotropins in females. There is evidence to suggest that this system may be involved in the regulation of neuroendocrine events that are essential for ovulation and that occur prior to pro-oestrus. We explored this possibility by transiently inactivating the SCN. Female rats were implanted with guide cannulas aimed at the SCN. After recovery of the oestrous cycle, animals were injected with tetrodotoxin (TTX), artificial cerebrospinal fluid (ACSF) or saline solution while freely moving. Injections were performed at 14.00h of each stage of the oestrous cycle. Animals were killed on the next predicted oestrus day, the number of ova shed was counted and intact rats at oestrus stage were used as absolute control. ACSF did not modify ovulation. Saline solution blocked ovulation in oestrus- and dioestrus-injected rats. Irrespectively of the stage of the oestrous cycle, TTX blocked ovulation. These results lead us to suggest that a neural circadian signal, pivotal for triggering the gonadotropin pre-ovulatory surge, arises from the SCN during the critical window of pro-oestrus. We also suggest that a similar signal, needed for the regulation of other events that are indispensable for proper regulation of ovulation, is also generated in this nucleus during the other stages of the cycle at a similar time.

Ascleposide, a natural cardenolide, induces anticancer signaling in human castration-resistant prostatic cancer through Na+ /K+ -ATPase internalization and tubulin acetylation.

Cardiac glycosides, which inhibit Na+ /K+ -ATPase, display inotropic effects for the treatment of congestive heart failure and cardiac arrhythmia. Recent studies have suggested signaling downstream of Na+ /K+ -ATPase action in the regulation of cell proliferation and apoptosis and have revealed the anticancer activity of cardiac glycosides. The study aims to characterize the anticancer potential of ascleposide, a natural cardenolide, and to uncover its primary target and underlying mechanism against human castration-resistant prostate cancer (CRPC). Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay, carboxyfluorescein succinimidyl ester staining assay and clonogenic examination. Flow cytometric analysis was used to detect the distribution of cell cycle phase, mitochondrial membrane potential, intracellular Na+ and Ca2+ levels, and reactive oxygen species production. Protein expression was examined using Western blot analysis. Endocytosis of Na+ /K+ -ATPase was determined using confocal immunofluorescence microscopic examination. Ascleposide induced an increase of intracellular Na+ and a potent antiproliferative effect. It also induced a decrease of G1 phase distribution while an increase in both G2/M and apoptotic sub-G1 phases, and downregulated several cell cycle regulator proteins, including cyclins, Cdk, p21, and p27 Cip/Kip proteins, Rb and c-Myc. Ascleposide decreased the expression of antiapoptotic Bcl-2 members (eg, Bcl-2 and Mcl-1) but upregulated proapoptotic member (eg, Bak), leading to a significant loss of mitochondrial membrane potential and activation of both caspase-9 and caspase-3. Ascleposide also dramatically induced tubulin acetylation, leading to inhibition of the catalytic activity of Na+ /K+ -ATPase. Notably, extracellular high K+ (16 mM) significantly blunted ascleposide-mediated effects. Furthermore, ascleposide induced a p38 MAPK-dependent endocytosis of Na+ /K+ -ATPase and downregulated the protein expression of Na+ /K+ -ATPase α1 subunit. Ascleposide displays antiproliferative and apoptotic activities dependent on the inhibition of Na+ /K+ -ATPase pumping activity through p38 MAPK-mediated endocytosis of Na+ /K+ -ATPase and downregulation of α1 subunit, which in turn cause tubulin acetylation and cell cycle arrest. Cell apoptosis is ultimately triggered by the activation of caspase cascade attributed to mitochondrial damage through the downregulation of Bcl-2 and Mcl-1 protein expressions while upregulation of Bak protein levels. The data also suggest the potential of ascleposide in anti-CRPC development.

Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts.

Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.