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Physiological Reports Sep 2023Hypertension is a pandemic nowadays. We aimed to investigate whether chronic undernutrition modifies the response to the antihypertensive drug rostafuroxin in juvenile...
Hypertension is a pandemic nowadays. We aimed to investigate whether chronic undernutrition modifies the response to the antihypertensive drug rostafuroxin in juvenile hypertensive rats. Chronic undernutrition was induced in male rats using a multideficient diet known as the Regional Basic Diet (RBD), mimicking alimentary habits in impoverished regions worldwide. Animals were given RBD-or a control/CTRL normal diet for rodents-from weaning to 90 days, and rostafuroxin (1 mg/kg body mass) was orally administered from day 60 onwards. For the last 2 days, the rats were hosted in metabolic cages to measure food/energy, water, Na ingestion, and urinary volume. Rostafuroxin increased food/energy/Na intake in CTRL and RBD rats but had opposite effects on Na balance (intake minus urinary excretion). The drug normalized the decreased plasma Na concentration in RBD rats, increased urinary volume in RBD but not in CTRL, and decreased and increased urinary Na concentration in the RBD and CTRL groups, respectively. Rostafuroxin decreased the ouabain-sensitive (Na +K )ATPase and increased the ouabain-resistant Na -ATPase from proximal tubule cells in both groups and normalized the systolic blood pressure in RBD without effect in CTRL rats. We conclude that chronic undernutrition modifies the response of blood pressure and metabolic responses to rostafuroxin.
Topics: Male; Rats; Animals; Antihypertensive Agents; Ouabain; Malnutrition; Hypertension; Adenosine Triphosphatases
PubMed: 37667414
DOI: 10.14814/phy2.15820 -
International Journal of Molecular... Aug 2023(PE), the most severe presentation of hypertensive disorders of pregnancy, is the major cause of morbidity and mortality linked to pregnancy, affecting both mother and... (Review)
Review
(PE), the most severe presentation of hypertensive disorders of pregnancy, is the major cause of morbidity and mortality linked to pregnancy, affecting both mother and fetus. Despite advances in prophylaxis and managing PE, delivery of the fetus remains the only causative treatment available. Focus on complex pathophysiology brought the potential for new treatment options, and more conservative options allowing reduction of feto-maternal complications and sequelae are being investigated. Endogenous digitalis-like factors, which have been linked to the pathogenesis of preeclampsia since the mid-1980s, have been shown to play a role in the pathogenesis of various cardiovascular diseases, including congestive heart failure and chronic renal disease. Elevated levels of EDLF have been described in pregnancy complicated by hypertensive disorders and are currently being investigated as a therapeutic target in the context of a possible breakthrough in managing preeclampsia. This review summarizes mechanisms implicating EDLFs in the pathogenesis of preeclampsia and evidence for their potential role in treating this doubly life-threatening disease.
Topics: Female; Pregnancy; Humans; Hypertension, Pregnancy-Induced; Pre-Eclampsia; Cardenolides; Saponins
PubMed: 37628922
DOI: 10.3390/ijms241612743 -
Biomedicines Aug 2023Na/K ATPase is a protein involved in the active transport of ions across the cellular membrane. Ouabain is a cardiotonic glycoside that, by inhibiting the Na/K pump,...
Na/K ATPase is a protein involved in the active transport of ions across the cellular membrane. Ouabain is a cardiotonic glycoside that, by inhibiting the Na/K pump, interferes with cell processes mediated directly by the pump, but also indirectly influences other cellular processes such as cell cycle and proliferation, growth, cell differentiation, angiogenesis, migration, adhesion, and invasion. We used the SK-BR-3 breast cancer cell line, mesenchymal stem cells (MSCs), and tumor-associated fibroblasts (TAFs) in vitro to determine the effects of ouabain exposure on these cellular types. The results showed a multi-level effect of ouabain mainly on tumor cells, in a dose-dependent manner, while the TAFs and their normal counterparts were not significantly influenced. Following exposure to ouabain, the SK-BR-3 cells changed their morphologic appearance, decreased the expression of immunophenotypic markers (CD29, Her2, VEGF), the proliferation rate was significantly decreased (Ki67 index), the cells were blocked in the G phase of the cell cycle and suffered necrosis. These data were correlated with the variable expression of α and β Na/K pump subunits in tumor cells, resulting in decreased ability to adhere to the VCAM-1 substrate in functional flow chamber studies. Being indicative of the pro-apoptotic and inhibitory effect of ouabain on tumor invasion and metastasis, the results support the addition of ouabain to the oncological therapeutic arsenal, trailing the "repurposing drugs" approach.
PubMed: 37626702
DOI: 10.3390/biomedicines11082205 -
Cells Aug 2023Na/K-ATPase maintains transmembrane ionic gradients and acts as a signal transducer when bound to endogenous cardiotonic steroids. At subnanomolar concentrations,...
Na/K-ATPase maintains transmembrane ionic gradients and acts as a signal transducer when bound to endogenous cardiotonic steroids. At subnanomolar concentrations, ouabain induces neuroprotection against calcium overload and apoptosis of neurons during excitotoxic stress. Here, the role of lipid rafts in interactions between Na/K-ATPase, sodium-calcium exchanger (NCX), and N-methy-D-aspartate receptors (NMDARs) was investigated. We analyzed 0.5-1-nanometer ouabain's effects on calcium responses and miniature post-synaptic current (mEPSCs) frequencies of cortical neurons during the action of NMDA in rat primary culture and brain slices. In both objects, ouabain attenuated NMDA-evoked calcium responses and prevented an increase in mEPSC frequency, while the cholesterol extraction by methyl-β-cyclodextrin prevented the effects. The data support the conclusions that (i) ouabain-induced inhibition of NMDA-elicited calcium response involves both pre- and post-synapse, (ii) the presence of astrocytes in the tripartite synapse is not critical for the ouabain effects, which are found to be similar in cell cultures and brain slices, and (iii) ouabain action requires the integrity of cholesterol-rich membrane microdomains in which the colocalization and functional interaction of NMDAR-transferred calcium influx, calcium extrusion by NCX, and Na/K-ATPase modulation of the exchanger occur. This regulation of the molecules by cardiotonic steroids may influence synaptic transmission, prevent excitotoxic neuronal death, and interfere with the pharmacological actions of neurological medicines.
Topics: Rats; Animals; Ouabain; Calcium; N-Methylaspartate; Neurons; Cholesterol; Adenosine Triphosphatases
PubMed: 37566090
DOI: 10.3390/cells12152011 -
Cellular Physiology and Biochemistry :... Jul 2023The functional significance of the Na/Ca exchanger (NCX) in basolateral membranes in the proximal tubule remains controversial. The key factor in crosstalk between the...
BACKGROUND/AIMS
The functional significance of the Na/Ca exchanger (NCX) in basolateral membranes in the proximal tubule remains controversial. The key factor in crosstalk between the apical and basolateral sides is not known.
METHODS
We investigated the basolateral membranes, using double-barreled Ca or pH ion-selective microelectrodes. We used doubly perfused bullfrog kidneys in vivo, and switched the basolateral solution (renal portal vein) to experimental solutions.
RESULTS
In the control, cellular pH (pH) was 7.33 ± 0.032 (mean ± SE, n = 7) and in separate experiments, cellular Ca activity (aCa) was 249.6 ± 35.54 nM (n = 28). Changing to respiratory acidosis, pH was significantly acidified by 0.123 pH units on average and the change of aCa was +53.1 nM (n = 9 ns). In metabolic acidosis, pH was reduced by 0.151 while aCa was reduced by 143.4. Using the 30 mM K solution, pH was increased by 0.233 while aCa was reduced by 203.9, with depolarization. Both ionomycin and ouabain caused aCa to increase. In the 0.5 mM Na solution (replaced with BIDAC Cl), pH was reduced by 0.177. No changes in aCa (+49.8 ns) were observed although we recorded depolarization of 15.2 mV. In the 0.5 mM Na solution, replaced with raffinose, no changes in aCa (-126.4 ns) were observed with depolarization (6.5 ns).
CONCLUSION
Our results suggest that thermodynamic calculations of cellular Na concentration led to the conclusion that either a Na/HCO exchanger (NBC) or NCX could be present in the same basolateral membrane. H ions are the most plausible key factor in the crosstalk.
Topics: Animals; Rana catesbeiana; Sodium-Calcium Exchanger; Cell Membrane; Ions; Hydrogen-Ion Concentration
PubMed: 37522756
DOI: 10.33594/000000641 -
Life (Basel, Switzerland) Jul 2023The signaling or non-pumping Na,K-ATPase function was first observed by us in the nociceptive neuron; Na,K-ATPase transduced the signals from the opioid-like receptors...
The signaling or non-pumping Na,K-ATPase function was first observed by us in the nociceptive neuron; Na,K-ATPase transduced the signals from the opioid-like receptors to Na1.8 channels. This study elucidates the role of the rhamnosyl residue of ouabain in the activation of the Na,K-ATPase signaling function. The effects resulting from activation of Na,K-ATPase signaling by the Ca chelate complex of ouabain (EO) are not manifested upon removal of the rhamnosyl residue, as demonstrated in viable cells by the highly sensitive patch-clamp and organotypic cell culture methods. Docking calculations show that the rhamnosyl residue is involved in five intermolecular hydrogen bonds with the Na,K-ATPase α1-subunit, which are fundamentally important for activation of the Na,K-ATPase signaling function upon EO binding. The main contribution to the energy of EO binding is provided by its steroid core, which forms a number of hydrogen bonds and hydrophobic interactions with Na,K-ATPase that stabilize the ligand-receptor complex. Another critically important role in EO binding is expected to be played by the chelated Ca cation, which should switch on strong intermolecular ionic interactions between the EO molecule and two α1-Na,K-ATPase amino acid residues, Glu116 and Glu117.
PubMed: 37511875
DOI: 10.3390/life13071500 -
Molecular Medicine Reports Sep 2023The Na/K‑ATPase/Src complex is reportedly able to affect reactive oxygen species (ROS) amplification. However, it has remained elusive whether NADPH oxidases (NOXs)...
The Na/K‑ATPase/Src complex is reportedly able to affect reactive oxygen species (ROS) amplification. However, it has remained elusive whether NADPH oxidases (NOXs) are involved in this oxidant amplification loop in renal fibrosis. To test this hypothesis, interactions between oxidative features and Na/K‑ATPase/Src activation were examined in a mouse model of unilateral urethral obstruction (UUO)‑induced experimental renal fibrosis. Both 1‑tert‑butyl‑3‑(4‑chlorophenyl)‑1H‑pyrazolo[3,4‑d]pyrimidin‑4‑amine (PP2) and apocynin significantly attenuated the development of UUO‑induced renal fibrosis. Apocynin administration attenuated the expression of NOXs and oxidative markers (e.g., nuclear factor erythroid 2‑related factor 2, heme oxygenase‑1,4‑hydroxynonenal and 3‑nitrotyrosine); it also partially restored Na/K‑ATPase expression and inhibited the activation of the Src/ERK cascade. Furthermore, administration of PP2 after UUO induction partially reversed the upregulation of NOX2, NOX4 and oxidative markers, while inhibiting the activation of the Src/ERK cascade. Complementary experiments in LLC‑PK1 cells corroborated the in vivo observations. Inhibition of NOX2 by RNA interference attenuated ouabain‑induced oxidative stress, ERK activation and E‑cadherin downregulation. Thus, it is indicated that NOXs are major contributors to ROS production in the Na/K‑ATPase/Src/ROS oxidative amplification loop, which is involved in renal fibrosis. The disruption of this vicious feed‑forward loop between NOXs/ROS and redox‑regulated Na/K‑ATPase/Src may have therapeutic applicability for renal fibrosis disorders.
Topics: Mice; Animals; NADPH Oxidases; Reactive Oxygen Species; Oxidants; Kidney Diseases; Oxidative Stress; Fibrosis; Adenosine Triphosphatases; NADPH Oxidase 4
PubMed: 37417374
DOI: 10.3892/mmr.2023.13048 -
PloS One 2023Biliary tract cancer is a deadly disease with limited therapeutic options. Ouabain is a well-known inhibitor of the pumping function of Na+/K+-ATPase, though there is...
Biliary tract cancer is a deadly disease with limited therapeutic options. Ouabain is a well-known inhibitor of the pumping function of Na+/K+-ATPase, though there is evidence that low concentrations of ouabain lead to a reduction of cell viability of cancer cells independent of its inhibition of the pumping function of the Na+/K+-ATPase. Regarding the impact of ouabain on biliary tract cancer, no data is currently available. Therefore, we aimed for a first-time investigation of ouabain as a potential anti-neoplastic biliary tract cancer agent using comprehensive human biliary tract cancer in vitro models. We found that ouabain has a strong cell line-dependent cytotoxic effect with IC50 levels in the (low) nanomolar-range and that this effect was not associated with the mRNA expression levels of the Na+/K+-ATPase α, β and fxyd-subunits. Regarding the mode of cytotoxicity, we observed induction of apoptosis in biliary tract cancer cells upon treatment with ouabain. Interestingly, cytotoxic effects of ouabain at sub-saturating (< μM) levels were independent of cellular membrane depolarization and changes in intracellular sodium levels. Furthermore, using a 3D cell culture model, we found that ouabain disturbs spheroid growth and reduces the viability of biliary tract cancer cells within the tumor spheroids. In summary, our data suggest that ouabain possesses anti-biliary tract cancer potential at low μM-concentration in 2D and 3D in vitro biliary tract cancer models and encourage further detailed investigation.
Topics: Humans; Ouabain; Biliary Tract Neoplasms; Antineoplastic Agents; Apoptosis; Sodium-Potassium-Exchanging ATPase
PubMed: 37390071
DOI: 10.1371/journal.pone.0287769 -
Journal of Ethnopharmacology Oct 2023Herb-induced liver injury is poorly described for African herbal remedies, such as Acokanthera oppositifolia. Although a commonly used treatment for pain, snake bites...
ETHNOPHARMACOLOGICAL RELEVANCE
Herb-induced liver injury is poorly described for African herbal remedies, such as Acokanthera oppositifolia. Although a commonly used treatment for pain, snake bites and anthrax, it is also a well-known arrow poison, thus toxicity is to be expected.
AIM OF THE STUDY
The cytotoxicity and preliminary mechanisms of toxicity in HepG2 hepatocarcinoma cells were assessed.
MATERIALS AND METHODS
The effect of hot water and methanol extracts were on cell density, oxidative status, mitochondrial membrane potential, fatty acids, caspase-3/7 activity, adenosine triphosphate levels, cell cycling and viability was assessed. Phytochemicals were tentatively identified using ultra-performance liquid chromatography.
RESULTS
The hot water extract displayed an IC of 24.26 μg/mL, and reduced proliferation (S- and G2/M-phase arrest) and viability (by 30.71%) as early as 24 h after incubation. The methanol extract had a comparable IC of 26.16 μg/mL, and arrested cells in the G2/M-phase (by 18.87%) and induced necrosis (by 13.21%). The hot water and methanol extracts depolarised the mitochondrial membrane (up to 0.84- and 0.74-fold), though did not generate reactive oxygen species. The hot water and methanol extracts decreased glutathione (0.42- and 0.62-fold) and adenosine triphosphate (0.08- and 0.26-fold) levels, while fatty acids (2.00- and 4.61-fold) and caspase-3/7 activity (1.98- and 5.82-fold) were increased.
CONCLUSION
Extracts were both cytostatic and cytotoxic in HepG2 cells. Mitochondrial toxicity was evident and contributed to reducing adenosine triphosphate production and fatty acid accumulation. Altered redox status perturbed proliferation and promoted necrosis. Extracts of A. oppositifolia may thus promote necrotic cell death, which poses a risk for inflammatory hepatotoxicity with associated steatosis.
Topics: Humans; Hep G2 Cells; Methanol; Cytostatic Agents; Caspase 3; Plant Extracts; Carcinoma, Hepatocellular; Antineoplastic Agents; Necrosis; Liver Neoplasms; Water; Apocynaceae; Adenosine Triphosphate; Apoptosis
PubMed: 37182674
DOI: 10.1016/j.jep.2023.116617