GrimAge is elevated in older adults with mild COVID-19 an exploratory analysis.

COVID-19 has been contained; however, the side effects associated with its infection continue to be a challenge for public health, particularly for older adults. On the other hand, epigenetic status contributes to the inter-individual health status and is associated with COVID-19 severity. Nevertheless, current studies focus only on severe COVID-19. Considering that most of the worldwide population developed mild COVID-19 infection. In the present exploratory study, we aim to analyze the association of mild COVID-19 with epigenetic ages (HorvathAge, HannumAge, GrimAge, PhenoAge, SkinAge, and DNAmTL) and clinical variables obtained from a Mexican cohort of older adults. We found that all epigenetic ages significantly differ from the chronological age, but only GrimAge is elevated. Additionally, both the intrinsic epigenetic age acceleration (IEAA) and the extrinsic epigenetic age acceleration (EEAA) are accelerated in all patients. Moreover, we found that immunological estimators and DNA damage were associated with PhenoAge, SkinBloodHorvathAge, and HorvathAge, suggesting that the effects of mild COVID-19 on the epigenetic clocks are mainly associated with inflammation and immunology changes. In conclusion, our results show that the effects of mild COVID-19 on the epigenetic clock are mainly associated with the immune system and an increase in GrimAge, IEAA, and EEAA.

KS-WNK1 is Required for the Renal Response to Extreme Changes in Potassium Intake.

KS-WNK1 is an isoform of WNK1 kinase that is predominantly found in the distal convoluted tubule of the kidney. The precise physiological function of KS-WNK1 remains unclear. Some studies suggest that it could play a role in regulating potassium renal excretion by modulating the activity of the Na+-Cl- cotransporter (NCC). However, changes in the potassium diet from normal to high failed to reveal a role for KS-WNK1, but under a normal potassium diet, the expression of KS-WNK1 is negligible. It is only detectable when mice are exposed to a low potassium diet. In this study, we investigated the role of KS-WNK1 in regulating potassium excretion under extreme changes in potassium intake. After following a zero-potassium diet (0KD) for 10 days, KS-WNK1-/- mice had lower plasma levels of K+ and Cl-, while exhibiting higher urinary excretion of Na+, Cl-, and K+ compared to KS-WNK1+/+ mice. After 10 days of 0KD or normal-potassium diet (NKD), all mice were challenged with a high-potassium diet (HKD). Plasma K+ levels markedly increased after the HKD challenge only in mice previously fed with 0KD, regardless of genotype. KSWNK1+/+ mice adapt better to HKD-challenge than KS-WNK1-/- mice after a potassium-retaining state. The difference in the pNCC/NCC ratio between KS-WNK1+/+ and KS-WNK1-/- mice after 0KD and HKD indicates a role for KS-WNK1 in both, NCC phosphorylation and dephosphorylation. These observations show that KS-WNK1 helps the DCT to respond to extreme changes in potassium intake, such as those occurring in wildlife.

Reactive oxygen species downregulate dystroglycans in the megakaryocytes of rats with arterial hypertension.

Hypertension is a multifactorial disease characterized by vascular and renal dysfunction, cardiovascular remodeling, inflammation, and fibrosis, all of which are associated with oxidative stress. We previously demonstrated cellular reactive oxygen species (ROS) imbalances may impact the structural and biochemical functions of blood cells and reported downregulation of ß-dystroglycan (ß-Dg) and overexpression of the epithelial sodium channel (ENaC) contribute to the pathophysiology of hypertension. In this study, we aimed to determine the expression of dystroglycans (Dg) and ENaC in platelet progenitors (megakaryocytes) and their surrounding niches. Thin sections of bone marrow from 5- and 28-week-old spontaneous hypertensive rats (SHR) were compared to age-matched normotensive rats (WKY). Cytometry and immunohistochemical assays demonstrated an oxidative environment in SHR bone marrow, characterized by high levels of myeloperoxidase and 3-nitrotyrosine and downregulation of peroxiredoxin II. In addition, transmission electron micrography and confocal microscopy revealed morphological changes in platelets and Mgks from SHR rats, including swollen mitochondria. Quantitative qRT-PCR assays confirmed downregulation of Dg mRNA and immunohistochemistry and western-blotting validated low expression of ß-Dg, mainly in the phosphorylated form, in Mgks from 28-week-old SHR rats. Moreover, we observed a progressive increase in ß-1 integrin expression in Mgks and extracellular matrix proteins in Mgk niches in SHR rats compared to WKY controls. These results indicate accumulation of ROS promotes oxidative stress within the bone marrow environment and detrimentally affects cellular homeostasis in hypertensive individuals.

Lysophosphatidic acid receptor LPA1 trafficking and interaction with Rab proteins, as evidenced by Förster resonance energy transfer.

LPA1 internalization to endosomes was studied employing Förster Resonance Energy Transfer (FRET) in cells coexpressing the mCherry-lysophosphatidic acid LPA1 receptors and distinct eGFP-tagged Rab proteins. Lysophosphatidic acid (LPA)-induced internalization was rapid and decreased afterward: phorbol myristate acetate (PMA) action was slower and sustained. LPA stimulated LPA1-Rab5 interaction rapidly but transiently, whereas PMA action was rapid but sustained. Expression of a Rab5 dominant-negative mutant blocked LPA1-Rab5 interaction and receptor internalization. LPA-induced LPA1-Rab9 interaction was only observed at 60 min, and LPA1-Rab7 interaction after 5 min with LPA and after 60 min with PMA. LPA triggered immediate but transient rapid recycling (i.e., LPA1-Rab4 interaction), whereas PMA action was slower but sustained. Agonist-induced slow recycling (LPA1-Rab11 interaction) increased at 15 min and remained at this level, whereas PMA action showed early and late peaks. Our results indicate that LPA1 receptor internalization varies with the stimuli.

Expression of voltage-gated Ca2+ channels, Insp3Rs, and RyRs in the immature mouse ovary.

BACKGROUND: The postnatal mammalian ovary undergoes a series of changes to ensure the maturation of sufficient follicles to support ovulation and fecundation over the reproductive life. It is well known that intracellular [Ca2+]i signals are necessary for ovulation, fertilization, and egg activation. However, we lack detailed knowledge of the molecular identity, cellular distribution, and functional role of Ca2+ channels expressed during folliculogenesis. In the neonatal period, ovarian maturation is controlled by protein growth factors released from the oocyte and granulosa cells. Conversely, during the early infantile period, maturation becomes gonadotropin-dependent and is controlled by granulosa and theca cells. The significance of intracellular Ca2+ signaling in folliculogenesis is supported by the observation that mice lacking the expression of Ca2+/calmodulin-dependent kinase IV in granulosa cells suffer abnormal follicular development and impaired fertility. RESULTS: Using immunofluorescence in frozen ovarian sections and confocal microscopy, we assessed the expression of high-voltage activated Ca2+ channel alpha subunits and InsP3 and ryanodine receptors in the postnatal period from 3 to 16 days. During the neonatal stage, oocytes from primordial and primary follicles show high expression of various Ca2+-selective channels, with granulosa and stroma cells expressing significantly less. These channels are likely involved in supporting Ca2+-dependent secretion of peptide growth factors. In contrast, during the early and late infantile periods, Ca2+ channel expression in the oocyte diminishes, increasing significantly in the granulosa and particularly in immature theca cells surrounding secondary follicles. CONCLUSIONS: The developmental switch of Ca2+ channel expression from the oocytes to the perifollicular cells likely reflects the vanishing role of the oocytes once granulosa and theca cells take control of folliculogenesis in response to gonadotropins acting on their receptors.

Positively charged amino acids at the N terminus of select mitochondrial proteins mediate early recognition by import proteins αß'-NAC and Sam37.

A major challenge in eukaryotic cells is the proper distribution of nuclear-encoded proteins to the correct organelles. For a subset of mitochondrial proteins, a signal sequence at the N terminus (matrix-targeting sequence [MTS]) is recognized by protein complexes to ensure their proper translocation into the organelle. However, the early steps of mitochondrial protein targeting remain undeciphered. The cytosolic chaperone nascent polypeptide-associated complex (NAC), which in yeast is represented as the two different heterodimers αß-NAC and αß'-NAC, has been proposed to be involved during the early steps of mitochondrial protein targeting. We have previously described that the mitochondrial outer membrane protein Sam37 interacts with αß'-NAC and together promote the import of specific mitochondrial precursor proteins. In this work, we aimed to detect the region in the MTS of mitochondrial precursors relevant for their recognition by αß'-NAC during their sorting to the mitochondria. We used targeting signals of different mitochondrial proteins (αß'-NAC-dependent Oxa1 and αß'-NAC-independent Mdm38) and fused them to GFP to study their intracellular localization by biochemical and microscopy methods, and in addition followed their import kinetics in vivo. Our results reveal the presence of a positively charged amino acid cluster in the MTS of select mitochondrial precursors, such as Oxa1 and Fum1, which are crucial for their recognition by αß'-NAC. Furthermore, we explored the presence of this cluster at the N terminus of the mitochondrial proteome and propose a set of precursors whose proper localization depends on both αß'-NAC and Sam37.

Improved post-stroke spontaneous recovery by astrocytic extracellular vesicles.

Spontaneous recovery after a stroke accounts for a significant part of the neurological recovery in patients. However limited, the spontaneous recovery is mechanistically driven by axonal restorative processes for which several molecular cues have been previously described. We report the acceleration of spontaneous recovery in a preclinical model of ischemia/reperfusion in rats via a single intracerebroventricular administration of extracellular vesicles released from primary cortical astrocytes. We used magnetic resonance imaging and confocal and multiphoton microscopy to correlate the structural remodeling of the corpus callosum and striatocortical circuits with neurological performance during 21 days. We also evaluated the functionality of the corpus callosum by repetitive recordings of compound action potentials to show that the recovery facilitated by astrocytic extracellular vesicles was both anatomical and functional. Our data provide compelling evidence that astrocytes can hasten the basal recovery that naturally occurs post-stroke through the release of cellular mediators contained in extracellular vesicles.

Senescence in Primary Rat Astrocytes Induces Loss of the Mitochondrial Membrane Potential and Alters Mitochondrial Dynamics in Cortical Neurons.

The decline in brain function during aging is one of the most critical health problems nowadays. Although senescent astrocytes have been found in old-age brains and neurodegenerative diseases, their impact on the function of other cerebral cell types is unknown. The aim of this study was to evaluate the effect of senescent astrocytes on the mitochondrial function of a neuron. In order to evaluate neuronal susceptibility to a long and constant senescence-associated secretory phenotype (SASP) exposure, we developed a model by using cellular cocultures in transwell plates. Rat primary cortical astrocytes were seeded in transwell inserts and induced to premature senescence with hydrogen peroxide [stress-induced premature senescence (SIPS)]. Independently, primary rat cortical neurons were seeded at the bottom of transwells. After neuronal 6 days in vitro (DIV), the inserts with SIPS-astrocytes were placed in the chamber and cocultured with neurons for 6 more days. The neuronal viability, the redox state [reduced glutathione/oxidized glutathione (GSH/GSSG)], the mitochondrial morphology, and the proteins and membrane potential were determined. Our results showed that the neuronal mitochondria functionality was altered after being cocultured with senescent astrocytes. In vivo, we found that old animals had diminished mitochondrial oxidative phosphorylation (OXPHOS) proteins, redox state, and senescence markers as compared to young rats, suggesting effects of the senescent astrocytes similar to the ones we observed in vitro. Overall, these results indicate that the microenvironment generated by senescent astrocytes can affect neuronal mitochondria and physiology.

Development of the hypersecretory phenotype in the population of adrenal chromaffin cells from prehypertensive SHRs.

The hypersecretory phenotype of adrenal chromaffin cells (CCs) from early spontaneously hypertensive rats (SHRs) mainly results from enhanced Ca2+-induced Ca2+-release (CICR). A key question is if these abnormalities can be traced to the prehypertensive stage. Spontaneous and stimulus-induced catecholamine exocytosis, intracellular Ca2+ signals, and dense-core granule size and density were examined in CCs from prehypertensive and hypertensive SHRs and compared with age-matched Wistar-Kyoto rats (WKY). During the prehypertensive stage, the depolarization-elicited catecholamine exocytosis was ~ 2.9-fold greater in SHR than in WKY CCs. Interestingly, in half of CCs the exocytosis was indistinguishable from WKY CCs, while it was between 3- and sixfold larger in the other half. Likewise, caffeine-induced exocytosis was ~ twofold larger in prehypertensive SHR. Accordingly, depolarization and caffeine application elicited [Ca2+]i rises ~ 1.5-fold larger in prehypertensive SHR than in WKY CCs. Ryanodine reduced the depolarization-induced secretion in prehypertensive SHR by 57%, compared to 14% in WKY CCs, suggesting a greater contribution of intracellular Ca2+ release to exocytosis. In SHR CCs, the mean spike amplitude and charge per spike were significantly larger than in WKY CCs, regardless of age and stimulus type. This difference in granule content could explain in part the enhanced exocytosis in SHR CCs. However, electron microscopy did not reveal significant differences in granule size between SHRs and WKY rats' adrenal medulla. Nonetheless, preSHR and hypSHR display 63% and 82% more granules than WKY, which could explain in part the enhanced catecholamine secretion. The mechanism responsible for the heterogeneous population of prehypertensive SHR CCs and the bias towards secreting more medium and large granules remains unexplained.

Early Post-stroke Activation of Vascular Endothelial Growth Factor Receptor 2 Hinders the Receptor 1-Dependent Neuroprotection Afforded by the Endogenous Ligand.

Vascular endothelial growth factor (VEGF) has long been connected to the development of tissue lesion following ischemic stroke. Contradictory findings either situate VEGF as a promoter of large infarct volumes or as a potential attenuator of damage due to its well documented neuroprotective capability. The core of this discrepancy mostly lies on the substantial number of pleiotropic functions driven by VEGF. Mechanistically, these effects are activated through several VEGF receptors for which various closely related ligands exist. Here, we tested in an experimental model of stroke how the differential activation of VEGF receptors 1 and 2 would modify functional and histological outcomes in the acute phase post-ischemia. We also assessed whether VEGF-mediated responses would involve the modulation of inflammatory mechanisms and how this trophic factor acted specifically on neuronal receptors. We produced ischemic infarcts in adult rats by transiently occluding the middle cerebral artery and induced the pharmacological inhibition of VEGF receptors by i.c.v. administration of the specific VEGFR2 inhibitor SU1498 and the pan-VEGFR blocker Axitinib. We evaluated the neurological performance of animals at 24 h following stroke and the occurrence of brain infarctions analyzed at the gross metabolic and neuronal viability levels. We also assessed the induction of peripheral pro- and anti-inflammatory cytokines in the cerebrospinal fluid and blood and assessed the polarization of activated microglia. Finally, we studied the direct involvement of cortical neuronal receptors for VEGF with in vitro assays of excitotoxic damage. Preferential VEGFR1 activation by the endogenous ligand promotes neuronal protection and prevents the presentation of large volume infarcts that highly correlate with neurological performance, while the concomitant activation of VEGFR2 reduces this effect, even in the presence of exogenous ligand. This process partially involves the polarization of microglia to the state M2. At the cellular level, neurons also responded better to the preferential activation of VEGFR1 when challenged to N-methyl-D-aspartate-induced excitotoxicity. Endogenous activation of VEGFR2 hinders the neuroprotective mechanisms mediated by the activation of VEGFR1. The selective modulation of these concurrent processes might enable the development of therapeutic approaches that target specific VEGFR1-mediated signaling during the acute phase post-stroke.

Network Pharmacology Uncovers Anticancer Activity of Mammea-Type Coumarins from Calophyllum brasiliense.

Mammea-type coumarins are a particular type of secondary metabolites biosynthesized by the tropical rainforest tree Calophyllum Brasiliense, which is distributed from South America to Mexico. Particularly, mammea A/BA and A/BB (alone or as a mixture) possess biological properties such as cytotoxic and antitumoral activities, however, most of its molecular targets remain unknown. In this context, novel bioinformatic approaches, such as network pharmacology analysis, have been successfully used in herbal medicine to accelerate research in this field, and the support of experimental validations has been shown to be quite robust. In the present study, we performed a network pharmacology analysis to assess the possible molecular biological networks that interact with mammea A/BA and A/BB. Moreover, we validated the most relevant networks experimentally in vitro on K562 cancer cells. The results of the network pharmacology analysis indicate that mammea A/BA and A/BB interacts with cell death, PI3K/AKT, MAPK, Ras, and cancer pathways. The in vitro model shows that mammea A/BA and A/BB induce apoptosis through the overexpression of the proapoptotic proteins Bax and Bak, disrupt the autophagic flux as seen by the cytosolic accumulation of LC3-II and p62, disrupting the mitochondria ultrastructure and concomitantly increase the intracellular calcium concentration. Additionally, docking analysis predicted a possible interaction with a rapamycin-binding domain of mTOR. In conclusion, we validated network pharmacology analysis and report, for the first time, that mammea A/BA and A/BB coumarins induce apoptosis through the inhibition of the autophagic flux, possibly interacting with mTOR.

Calcium signaling and expression of voltage-gated calcium channels in the mouse ovary throughout the estrous cycle†.

The estrous cycle is an iterative change in the anatomy, endocrinology, physiology, and behavior to provide maximum fecundity. Ovarian steroid production involves gonadotropin-induced [Ca2+]i raises due in part to voltage-gated Ca2+ channels (VGCCs) whose identity and tissue distribution in situ is largely unknown. Using fluorescence Ca2+ imaging and confocal microscopy, we recorded both spontaneous and depolarization-induced Ca2+ signals in living mouse ovarian slices. They were most prominent in theca cells (TCs) and oocytes. The presence of Ca2+ channel subunits CaV 1.2, CaV 1.3, CaV 2.1, CaV 2.2, and CaV 3 was examined with immunofluorescence of ovarian sections. CaV 1.2 and CaV 1.3 (L-type Ca2+ channels) are present in the stroma, granulosa cells (GCs), and corpora lutea (CL). Intriguingly subunits that are characteristic of nerve cells are also expressed: P/Q-type (CaV 2.1; α1A) in the stroma and CL cells and N-type (CaV 2.2; α1B) in perifollicular smooth muscle cells. The expression of α1 subunits fluctuates along the estrous cycle: in metestrus-diestrus (the quiescent stage of the cycle), CL and GCs are similarly stained, while in proestrus (stage of maximal ovarian stimulation) CL staining increases relatively to GCs. Also in proestrus, CaV 3 Ca2+ channel subunits are expressed more in CL compared to GC suggesting a more significant role of Ca2+ channels. In estrus, CaV 3 subunits from mesenchymal and interfollicular stromal cells become intensely stained. Our study represents an important step in understanding the role of VGCCs in ovarian physiology and possibly in ovarian cancer and other reproductive pathologies.

Methylprednisolone Administration Following Spinal Cord Injury Reduces Aquaporin 4 Expression and Exacerbates Edema.

Spinal cord injury (SCI) is an incapacitating condition that affects motor, sensory, and autonomic functions. Since 1990, the only treatment administered in the acute phase of SCI has been methylprednisolone (MP), a synthetic corticosteroid that has anti-inflammatory effects; however, its efficacy remains controversial. Although MP has been thought to help in the resolution of edema, there are no scientific grounds to support this assertion. Aquaporin 4 (AQP4), the most abundant component of water channels in the CNS, participates in the formation and elimination of edema, but it is not clear whether the modulation of AQP4 expression by MP plays any role in the physiopathology of SCI. We studied the functional expression of AQP4 modulated by MP following SCI in an experimental model in rats along with the associated changes in the permeability of the blood-spinal cord barrier. We analyzed these effects in male and female rats and found that SCI increased AQP4 expression in the spinal cord white matter and that MP diminished such increase to baseline levels. Moreover, MP increased the extravasation of plasma components after SCI and enhanced tissue swelling and edema. Our results lend scientific support to the increasing motion to avoid MP treatment after SCI.

Autophagy fails to prevent glucose deprivation/glucose reintroduction-induced neuronal death due to calpain-mediated lysosomal dysfunction in cortical neurons.

Autophagy is triggered during nutrient and energy deprivation in a variety of cells as a homeostatic response to metabolic stress. In the CNS, deficient autophagy has been implicated in neurodegenerative diseases and ischemic brain injury. However, its role in hypoglycemic damage is poorly understood and the dynamics of autophagy during the hypoglycemic and the glucose reperfusion periods, has not been fully described. In the present study, we analyzed the changes in the content of the autophagy proteins BECN1, LC3-II and p62/SQSTM1 by western blot, and autophagosome formation was followed through time-lapse experiments, during glucose deprivation (GD) and glucose reintroduction (GR) in cortical cultures. According to the results, autophagosome formation rapidly increased during GD, and was followed by an active autophagic flux early after glucose replenishment. However, cells progressively died during GR and autophagy inhibition reduced neuronal death. Neurons undergoing apoptosis during GR did not form autophagosomes, while those surviving up to late GR showed autophagosomes. Calpain activity strongly increased during GR and remained elevated during progressive neuronal death. Its activation led to the cleavage of LAMP2 resulting in lysosome membrane permeabilization (LMP) and release of cathepsin B to the cytosol. Calpain inhibition prevented LMP and increased the number of neurons containing lysosomes and autophagosomes increasing cell viability. Taken together, the present results suggest that calpain-mediated lysosome dysfunction during GR turns an adaptive autophagy response to energy stress into a defective autophagy pathway, which contributes to neuronal death. In these conditions, autophagy inhibition results in the improvement of cell survival.

The Ketone Body, ß-Hydroxybutyrate Stimulates the Autophagic Flux and Prevents Neuronal Death Induced by Glucose Deprivation in Cortical Cultured Neurons.

Glucose is the major energy substrate in brain, however, during ketogenesis induced by starvation or prolonged hypoglycemia, the ketone bodies (KB), acetoacetate and ß-hydroxybutyrate (BHB) can substitute for glucose. KB improve neuronal survival in diverse injury models, but the mechanisms by which KB prevent neuronal damage are still not well understood. In the present study we have investigated whether protection by the D isomer of BHB (D-BHB) against neuronal death induced by glucose deprivation (GD), is related to autophagy. Autophagy is a lysosomal-dependent degradation process activated during nutritional stress, which leads to the digestion of damaged proteins and organelles providing energy for cell survival. Results show that autophagy is activated in cortical cultured neurons during GD, as indicated by the increase in the levels of the lipidated form of the microtubule associated protein light chain 3 (LC3-II), and the number of autophagic vesicles. At early phases of glucose reintroduction (GR), the levels of p62 declined suggesting that the degradation of the autophagolysosomal content takes place at this time. In cultures exposed to GD and GR in the presence of D-BHB, the levels of LC3-II and p62 rapidly declined and remained low during GR, suggesting that the KB stimulates the autophagic flux preventing autophagosome accumulation and improving neuronal survival.

EGF regulates claudin-2 and -4 expression through Src and STAT3 in MDCK cells.

Epidermal Growth Factor (EGF) is a key regulator of epithelial paracellular permeability, a property that depends on tight junctions (TJ) and can be evaluated through the measurement of the transepithelial electrical resistance (TER). EGF increases the TER of MDCK monolayers by inducing ERK1/2-dependent downregulation of claudin-2 (CLDN-2) and upregulation of claudin-4 (CLDN-4). Because either increments or decrements in TER often involve Src activation and epithelial cell differentiation occasionally depends on STAT3, here we investigated whether EGF might control CLDN-2 downregulation and CLDN-4 upregulation through those proteins. We found that EGF induces Src activation necessary for the reduction of CLDN-2 at the TJ, the degradation of this CLDN, the reduction of the cellular levels of its mRNA and the resulting increase of TER. EGF-induced changes on CLDN-2 protein and mRNA also depend on STAT3 activity. This growth factor increases the levels of STAT3 phosphorylated at Y705 in the nucleus, a process that depends on Src activation. Interestingly, Src and STAT3 activation do not exclusively mediate the EGF-induced downregulation of CLDN-2, but they are also implicated in the EGF-induced CLDN-4 transcription, translation, and exocytic fusion into TJ. Our results indicate that EGF controls the levels of CLDN-2 and -4 proteins and mRNAs through Src and STAT3 activity.

21-Benzylidene digoxin: a proapoptotic cardenolide of cancer cells that up-regulates Na,K-ATPase and epithelial tight junctions.

Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na+ and K+ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD), and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α1, but not α2 and α3 isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1) up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2) cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3) changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions.

Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways.

In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.

Ouabain modulates epithelial cell tight junction.

Epithelial cells treated with high concentrations of ouabain (e.g., 1 microM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell-cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K(+) pumping nor disturb the K(+) balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (J(DEX)). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, J(DEX), and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell-cell contacts.