RESUMO
Kidney-specific with-no-lysine kinase 1 (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 have suggested 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 with 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 phosphorylated NCC-to-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 distal convoluted tubule to respond to extreme changes in potassium intake, such as those occurring in wildlife.NEW & NOTEWORTHY The findings of this study demonstrate that kidney-specific with-no-lysine kinase 1 plays a role in regulating urinary electrolyte excretion during extreme changes in potassium intake, such as those occurring in wildlife. .
Assuntos
Camundongos Knockout , Potássio na Dieta , Proteína Quinase 1 Deficiente de Lisina WNK , Animais , Masculino , Camundongos , Rim/metabolismo , Túbulos Renais Distais/metabolismo , Camundongos Endogâmicos C57BL , Fosforilação , Potássio/urina , Potássio/metabolismo , Potássio/sangue , Potássio na Dieta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Eliminação Renal , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/genética , FemininoRESUMO
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.
Assuntos
Distroglicanas , Hipertensão , Ratos , Animais , Espécies Reativas de Oxigênio , Ratos Endogâmicos SHR , Megacariócitos/metabolismo , Ratos Endogâmicos WKY , Hipertensão/metabolismoRESUMO
LPA3 receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA3-mediated calcium signaling, the agonists, and the phorbol ester-induced LPA3 internalization. Pitstop 2 (clathrin heavy chain inhibitor) markedly reduced LPA-induced receptor internalization; in contrast, phorbol ester-induced internalization was only delayed. LPA induced rapid ß-arrestin-LPA3 receptor association. The agonist and the phorbol ester-induced marked LPA3 receptor phosphorylation, and phosphorylation sites were detected using mass spectrometry. Phosphorylated residues were detected in the intracellular loop 3 (S221, T224, S225, and S229) and in the carboxyl terminus (S321, S325, S331, T333, S335, Y337, and S343). Interestingly, phosphorylation sites are within sequences predicted to constitute ß-arrestin binding sites. These data provide insight into LPA3 receptor signaling and regulation.
Assuntos
Lisofosfolipídeos , Receptores de Ácidos Lisofosfatídicos , Transdução de Sinais , Humanos , beta-Arrestinas/metabolismo , Sítios de Ligação , Sinalização do Cálcio , Células HEK293 , Lisofosfolipídeos/metabolismo , Fosforilação , Receptores de Ácidos Lisofosfatídicos/metabolismoRESUMO
Lysophosphatidic acid (LPA) type 3 (LPA3) receptor mutants were generated in which the sites detected phosphorylated were substituted by non-phosphorylatable amino acids. Substitutions were made in the intracellular loop 3 (IL3 mutant), the carboxyl terminus (Ctail), and both domains (IL3/Ctail). The wild-type (WT) receptor and the mutants were expressed in T-REx HEK293 cells, and the consequences of the substitutions were analyzed employing different functional parameters. Agonist- and LPA-mediated receptor phosphorylation was diminished in the IL3 and Ctail mutants and essentially abolished in the IL3/Ctail mutant, confirming that the main phosphorylation sites are present in both domains and their role in receptor phosphorylation eliminated by substitution and distributed in both domains. The WT and mutant receptors increased intracellular calcium and ERK 1/2 phosphorylation in response to LPA and PMA. The agonist, Ki16425, diminished baseline intracellular calcium, which suggests some receptor endogenous activity. Similarly, baseline ERK1/2 phosphorylation was diminished by Ki16425. An increase in baseline ERK phosphorylation was detected in the IL3/Ctail mutant. LPA and PMA-induced receptor interaction with ß-arrestin 2 and LPA3 internalization were severely diminished in cells expressing the mutants. Mutant-expressing cells also exhibit increased baseline proliferation and response to different stimuli, which were inhibited by the antagonist Ki16425, suggesting a role of LPA receptors in this process. Migration in response to different attractants was markedly increased in the Ctail mutant, which the Ki16425 antagonist also attenuated. Our data experimentally show that receptor phosphorylation in the distinct domains is relevant for LPA3 receptor function.
Assuntos
Lisofosfolipídeos , Receptores de Ácidos Lisofosfatídicos , Transdução de Sinais , Humanos , Fosforilação , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Células HEK293 , Lisofosfolipídeos/metabolismo , Cálcio/metabolismo , Endocitose , MutaçãoRESUMO
Biological membranes are composed of a lipid bilayer with embedded proteins, including ion channels like the epithelial sodium channel (ENaC), which are critical for sodium homeostasis and implicated in arterial hypertension (HTN). Changes in the lipid composition of the plasma membrane can significantly impact cellular processes related to physiological functions. We hypothesized that the observed overexpression of ENaC in neutrophils from HTN patients might result from alterations in the structuring domains within the plasma membrane, disrupting the endocytic processes responsible for ENaC retrieval. This study assessed the structural lipid composition of neutrophil plasma membranes from HTN patients along with the expression patterns of key elements regulating ENaC at the plasma membrane. Our findings suggest alterations in microdomain structure and SGK1 kinase activity, which could prolong ENaC presence on the plasma membrane. Additionally, we propose that the proteasomal and lysosomal degradation pathways are insufficient to diminish ENaC presence at the plasma membrane in HTN. These results highlight the importance of understanding ENaC retrieval mechanisms and suggest that targeting these mechanisms could provide insights for developing drugs to prevent and treat HTN.
Assuntos
Membrana Celular , Endocitose , Canais Epiteliais de Sódio , Hipertensão , Neutrófilos , Canais Epiteliais de Sódio/metabolismo , Humanos , Neutrófilos/metabolismo , Hipertensão/metabolismo , Hipertensão/patologia , Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Masculino , Feminino , Proteínas Imediatamente Precoces/metabolismo , Pessoa de Meia-Idade , Microdomínios da Membrana/metabolismoRESUMO
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.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Mitocondriais , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Aminoácidos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Vesículas Extracelulares , Acidente Vascular Cerebral , Animais , Astrócitos , Axônios , Modelos Animais de Doenças , Humanos , Imageamento por Ressonância Magnética , Ratos , Recuperação de Função Fisiológica/fisiologia , Acidente Vascular Cerebral/patologiaRESUMO
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.
Assuntos
Células Cromafins/fisiologia , Hipertensão/fisiopatologia , Animais , Cálcio/metabolismo , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Exocitose/fisiologia , Hipertensão/metabolismo , Masculino , Fenótipo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Rianodina/metabolismoRESUMO
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.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Ciclo Estral/fisiologia , Ovário/metabolismo , Animais , Cálcio/metabolismo , Feminino , Imunofluorescência , Camundongos , Folículo Ovariano/metabolismoRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Calophyllum/química , Cumarínicos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Sítios de Ligação , Biologia Computacional , Cumarínicos/química , Cumarínicos/isolamento & purificação , Humanos , Células K562 , Transdução de Sinais , Biologia de Sistemas/métodos , Serina-Treonina Quinases TOR/químicaRESUMO
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.
Assuntos
Aquaporina 4/metabolismo , Edema/induzido quimicamente , Edema/metabolismo , Metilprednisolona/administração & dosagem , Traumatismos da Medula Espinal/tratamento farmacológico , Corticosteroides/administração & dosagem , Animais , Modelos Animais de Doenças , Edema/complicações , Feminino , Regulação da Expressão Gênica , Hemorragia , Masculino , Microscopia Confocal , Ratos , Ratos Long-Evans , Medula Espinal/patologia , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/metabolismoRESUMO
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.
Assuntos
Ácido 3-Hidroxibutírico/metabolismo , Autofagia , Córtex Cerebral/citologia , Glucose/metabolismo , Neurônios/citologia , Ácido 3-Hidroxibutírico/química , Ácido 3-Hidroxibutírico/farmacologia , Animais , Sobrevivência Celular , Células Cultivadas , Córtex Cerebral/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos Wistar , Proteína Sequestossoma-1 , EstereoisomerismoRESUMO
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.
Assuntos
Claudina-2/biossíntese , Claudina-4/biossíntese , Fator de Crescimento Epidérmico/fisiologia , Fator de Transcrição STAT3/metabolismo , Quinases da Família src/metabolismo , Animais , Butadienos/farmacologia , Claudina-2/genética , Claudina-4/genética , Cães , Regulação para Baixo , Impedância Elétrica , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Indóis/farmacologia , Células Madin Darby de Rim Canino , Maleimidas/farmacologia , Nitrilas/farmacologia , Fosforilação , Biossíntese de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/biossíntese , Fator de Transcrição STAT3/biossíntese , Junções Íntimas/fisiologia , Transcrição Gênica , Regulação para Cima , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/biossínteseRESUMO
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.
Assuntos
Endocitose/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ouabaína/farmacologia , Proteólise/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Animais , Células Cultivadas , Cães , Células Madin Darby de Rim CaninoRESUMO
Aging is characterized by the decline in many of the individual's capabilities. It has been recognized that the brain undergoes structural and functional changes during aging that are occasionally associated with the development of neurodegenerative diseases. In this sense, altered glutamatergic neurotransmission, which involves the release, binding, reuptake, and degradation of glutamate (Glu) in the brain, has been widely studied in physiological and pathophysiological aging. In particular, changes in glutamatergic neurotransmission are exacerbated during neurodegenerative diseases and are associated with cognitive impairment, characterized by difficulties in memory, learning, concentration, and decision-making. Thus, in the present manuscript, we aim to highlight the relevance of glutamatergic neurotransmission during cognitive impairment to develop novel strategies to prevent, ameliorate, or delay cognitive decline. To achieve this goal, we provide a comprehensive review of the changes reported in glutamatergic neurotransmission components, such as Glu transporters and receptors during physiological aging and in the most studied neurodegenerative diseases. Finally, we describe the current therapeutic strategies developed to target glutamatergic neurotransmission.
RESUMO
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.
Assuntos
COVID-19 , Humanos , Idoso , Masculino , Feminino , México/epidemiologia , Epigênese Genética , Idoso de 80 Anos ou mais , Índice de Gravidade de Doença , SARS-CoV-2 , Envelhecimento/genética , Envelhecimento/fisiologia , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Células Epiteliais/efeitos dos fármacos , Ouabaína/farmacologia , Junções Íntimas/efeitos dos fármacos , Animais , Proteína Tirosina Quinase CSK , Dextranos/química , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Íons , Modelos Biológicos , Potássio/química , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/metabolismo , Quinases da Família srcRESUMO
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.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Receptores de Ácidos Lisofosfatídicos , Receptores de Ácidos Lisofosfatídicos/metabolismo , Fosforilação , Acetato de Tetradecanoilforbol/farmacologia , Endossomos/metabolismo , Lisofosfolipídeos/farmacologia , Lisofosfolipídeos/metabolismoRESUMO
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.
Assuntos
Folículo Ovariano , Ovário , Animais , Feminino , Gonadotropinas , Células da Granulosa/metabolismo , Mamíferos , Camundongos , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , Células Tecais/metabolismoRESUMO
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.