RESUMO
Human mesenchymal stem cells (MSCs) are good candidates for brain cell replacement strategies and have already been used as adjuvant treatments in neurological disorders. MSCs can be obtained from many different sources, and the present study compares the potential of neuronal transdifferentiation in MSCs from adult and neonatal sources (Wharton's jelly (WhJ), dental pulp (DP), periodontal ligament (PDL), gingival tissue (GT), dermis (SK), placenta (PLAC), and umbilical cord blood (UCB)) with a protocol previously tested in bone marrow- (BM-) MSCs consisting of a cocktail of six small molecules: I-BET151, CHIR99021, forskolin, RepSox, Y-27632, and dbcAMP (ICFRYA). Neuronal morphology and the presence of cells positive for neuronal markers (TUJ1 and MAP2) were considered attributes of neuronal induction. The ICFRYA cocktail did not induce neuronal features in WhJ-MSCs, and these features were only partial in the MSCs from dental tissues, SK-MSCs, and PLAC-MSCs. The best response was found in UCB-MSCs, which was comparable to the response of BM-MSCs. The addition of neurotrophic factors to the ICFRYA cocktail significantly increased the number of cells with complex neuron-like morphology and increased the number of cells positive for mature neuronal markers in BM- and UCB-MSCs. The neuronal cells generated from UCB-MSCs and BM-MSCs showed increased reactivity of the neuronal genes TUJ1, MAP2, NF-H, NCAM, ND1, TAU, ENO2, GABA, and NeuN as well as down- and upregulation of MSC and neuronal genes, respectively. The present study showed marked differences between the MSCs from different sources in response to the transdifferentiation protocol used here. These results may contribute to identifying the best source of MSCs for potential cell replacement therapies.
RESUMO
Neuronal activity in the retina generates osmotic gradients that lead to Müller cell swelling, followed by a regulatory volume decrease (RVD) response, partially due to the isoosmotic efflux of KCl and water. However, our previous studies in a human Müller cell line (MIO-M1) demonstrated that an important fraction of RVD may also involve the efflux of organic solutes. We also showed that RVD depends on the swelling-induced Ca2+ release from intracellular stores. Here we investigate the contribution of taurine (Tau) and glutamate (Glu), the most relevant amino acids in Müller cells, to RVD through the volume-regulated anion channel (VRAC), as well as their Ca2+ dependency in MIO-M1 cells. Swelling-induced [3H]Tau/[3H]Glu release was assessed by radiotracer assays and cell volume by fluorescence videomicroscopy. Results showed that cells exhibited an osmosensitive efflux of [3H]Tau and [3H]Glu (Tau > Glu) blunted by VRAC inhibitors 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)-oxybutyric acid and carbenoxolone reducing RVD. Only [3H]Tau efflux was mainly dependent on Ca2+ release from intracellular stores. RVD was unaffected in a Ca2+-free medium, probably due to Ca2+-independent Tau and Glu release, but was reduced by chelating intracellular Ca2+. The inhibition of phosphatidylinositol-3-kinase reduced [3H]Glu efflux but also the Ca2+-insensitive [3H]Tau fraction and decreased RVD, providing evidence of the relevance of this Ca2+-independent pathway. We propose that VRAC-mediated Tau and Glu release has a relevant role in RVD in Müller cells. The observed disparities in Ca2+ influence on amino acid release suggest the presence of VRAC isoforms that may differ in substrate selectivity and regulatory mechanisms, with important implications for retinal physiology. NEW & NOTEWORTHY The mechanisms for cell volume regulation in retinal Müller cells are still unknown. We show that swelling-induced taurine and glutamate release mediated by the volume-regulated anion channel (VRAC) largely contributes the to the regulatory volume decrease response in a human Müller cell line. Interestingly, the hypotonic-induced efflux of these amino acids exhibits disparities in Ca2+-dependent and -independent regulatory mechanisms, which strongly suggests that Müller cells may express different VRAC heteromers formed by the recently discovered leucine-rich repeat containing 8 (LRRC8) proteins.
Assuntos
Cálcio/metabolismo , Tamanho Celular , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Ácido Glutâmico/metabolismo , Taurina/metabolismo , Análise de Variância , Ânions/metabolismo , Antiulcerosos/farmacologia , Carbenoxolona/farmacologia , Ciclopentanos/farmacologia , Humanos , Indanos/farmacologia , Canais Iônicos/antagonistas & inibidores , Microscopia de Vídeo , Osmorregulação/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Retina/fisiologiaRESUMO
Taurine content is high (mM) in mammalian brain. By its major role as an osmolyte, taurine contributes to the cell volume control, which is particularly critical in the brain. Taurine participates in osmotic adjustments required to maintain the organization and size of intracellular compartments. It counteracts volume fluctuations in unbalanced transmembrane fluxes of ions and neurotransmitters, preserving the functional synaptic contacts. Taurine has a key role in the long-term adaptation to chronic hyponatremia as well as in other pathologies leading to brain edema. Together with other osmolytes, taurine corrects cell shrinkage, preventing mysfunction of organelles and apoptosis. Swelling corrective taurine efflux occurs through a leak pathway, likely formed by LCRR8 protein isoforms. Shrinkage-activated influx comes largely by the increased activity of the Na+/Cl--dependent transporter. The brain taurine pool results from the equilibrium between (i) dietary intake and active transport into the cell, (ii) synthesis in the brain itself or import of that synthesized elsewhere, and (iii) leak and posterior excretion. The interplay between these elements preserves brain taurine homeostasis in physiological conditions and permits the proper adjustments upon deviations of normal in the internal/external environment.
Assuntos
Encéfalo/metabolismo , Tamanho Celular , Homeostase/fisiologia , Taurina/metabolismo , Animais , Humanos , Concentração OsmolarRESUMO
Neural progenitors (NP), found in fetal and adult brain, differentiate into neurons potentially able to be used in cell replacement therapies. This approach however, raises technical and ethical problems which limit their potential therapeutic use. Alternately, NPs can be obtained by transdifferentiation of non-neural somatic cells evading these difficulties. Human bone marrow mesenchymal stromal cells (MSCs) are suggested to transdifferentiate into NP-like cells, which however, have a low proliferation capacity. The present study demonstrates the requisite of cell adhesion for proliferation and survival of NP-like cells and re-evaluates some neuronal features after differentiation by standard procedures. Mature neuronal markers, though, were not detected by these procedures. A chemical differentiation approach was used in this study to convert MSCs-derived NP-like cells into neurons by using a cocktail of six molecules, CHIR99021, I-BET151, RepSox, DbcAMP, forskolin and Y-27632, defined after screening combinations of 22 small molecules. Direct transdifferentiation of MSCs into neuronal cells was obtained with the small molecule cocktail, without requiring the NP-like intermediate stage.
Assuntos
Proliferação de Células/fisiologia , Transdiferenciação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Adolescente , Adulto , Amidas/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Colforsina/administração & dosagem , Combinação de Medicamentos , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Piridinas/administração & dosagem , Adulto JovemRESUMO
Volume changes deviating from original cell volume represent a major challenge for cellular homeostasis. Cell volume may be altered either by variations in the external osmolarity or by disturbances in the transmembrane ion gradients that generate an osmotic imbalance. Cells respond to anisotonicity-induced volume changes by active regulatory mechanisms that modify the intracellular/extracellular concentrations of K(+), Cl(-), Na(+), and organic osmolytes in the direction necessary to reestablish the osmotic equilibrium. Corrective osmolyte fluxes permeate across channels that have a relevant role in cell volume regulation. Channels also participate as causal actors in necrotic swelling and apoptotic volume decrease. This is an overview of the types of channels involved in either corrective or pathologic changes in cell volume. The review also underlines the contribution of transient receptor potential (TRP) channels, notably TRPV4, in volume regulation after swelling and describes the role of other TRPs in volume changes linked to apoptosis and necrosis. Lastly we discuss findings showing that multimers derived from LRRC8A (leucine-rich repeat containing 8A) gene are structural components of the volume-regulated Cl(-) channel (VRAC), and we underline the intriguing possibility that different heteromer combinations comprise channels with different intrinsic properties that allow permeation of the heterogenous group of molecules acting as organic osmolytes.
Assuntos
Tamanho Celular , Canais Iônicos/metabolismo , Animais , Morte Celular , Sobrevivência Celular , Humanos , Proteínas de Membrana/metabolismo , Ligação ProteicaRESUMO
Taurine was previously reported to increase the proliferation of neural precursor cells (NPCs) from subventricular zone of the mouse brain. The results of a study that aimed to understand the mechanisms of this effect are presented here. Because taurine was not found in NPC nuclei, direct interactions with nuclear elements seem unlikely. A gene expression profile analysis indicated that genes that are regulated by taurine have roles in i) proliferation, including the Shh and Wnt pathways; ii) cellular adhesion; iii) cell survival; and iv) mitochondrial functioning. Cell cycle analysis of propidium iodide and CFSE-labeled cells using flow cytometry revealed an increase in the number of cells in the S-phase and a decrease in those in the G0/G1 phase in taurine-treated cultures. No changes in the length of the cell cycle were observed. Quantification of the viable, apoptotic, and necrotic cells in cultures using flow cytometry and calcein-AM, annexin-V, and propidium iodide staining showed reductions in the number of apoptotic and necrotic cells (18% to 11% and 13% to 10%, respectively) and increases in the number of viable cells (61% to 69%) in the taurine-treated cultures. Examination of the relative mitochondrial potential values by flow cytometry and rhodamine123 or JC-1 staining showed a 44% increase in the number of cells with higher mitochondrial potential and a 38% increase in the mitochondrial membrane potential in taurine cultures compared with those of controls. Taken together, the results suggest that taurine provides more favorable conditions for cell proliferation by improving mitochondrial functioning.
Assuntos
Proliferação de Células , Ventrículos Laterais/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Taurina/metabolismo , Animais , Ciclo Celular , Sobrevivência Celular , Células Cultivadas , Feminino , Ventrículos Laterais/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismoRESUMO
K(+)-Cl(-) cotransporter (KCC) isoforms 3 (KCC3) and 4 (KCC4) are expressed at the basolateral membrane of proximal convoluted tubule cells, and KCC4 is present in the basolateral membrane of the thick ascending loop of Henle's limb and α-intercalated cells of the collecting duct. Little is known, however, about the physiological roles of these transporters in the kidney. We evaluated KCC3 and KCC4 mRNA and protein expression levels and intrarenal distribution in male Wistar rats or C57 mice under five experimental conditions: hyperglycemia after a single dose of streptozotocin, a low-salt diet, metabolic acidosis induced by ammonium chloride in drinking water, and low- or high-K(+) diets. Both KCC3 mRNA and protein expression were increased during hyperglycemia in the renal cortex and at the basolateral membrane of proximal tubule cells but not with a low-salt diet or acidosis. In contrast, KCC4 protein expression was increased by a low-sodium diet in the whole kidney and by metabolic acidosis in the renal outer medulla, specifically at the basolateral membrane of α-intercalated cells. The increased protein expression of KCC4 by a low-salt diet was also observed in WNK4 knockout mice, suggesting that upregulation of KCC4 in these circumstances is not WNK4 dependent. No change in KCC3 or KCC4 protein expression was observed under low- or high-K(+) diets. Our data are consistent with a role for KCC3 in the proximal tubule glucose reabsorption mechanism and for KCC4 in salt reabsorption of the thick ascending loop of Henle's loop and acid secretion of the collecting duct.
Assuntos
Rim/metabolismo , Simportadores/metabolismo , Acidose/induzido quimicamente , Acidose/metabolismo , Cloreto de Amônio , Animais , Transporte Biológico , Glicemia/metabolismo , Dieta Hipossódica , Modelos Animais de Doenças , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Hiperglicemia/induzido quimicamente , Hiperglicemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Potássio na Dieta/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Cloreto de Sódio na Dieta/metabolismo , Estreptozocina , Simportadores/genéticaRESUMO
The K(+):Cl(-) cotransporter (KCC) activity is modulated by phosphorylation/dephosphorylation processes. In isotonic conditions, KCCs are inactive and phosphorylated, whereas hypotonicity promotes their dephosphorylation and activation. Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation. However, here we show that the double mutant KCC3-T991A/T1048A could be further activated by hypotonicity, suggesting that additional phosphorylation site(s) are involved. We observed that in vitro activated STE20/SPS1-related proline/alanine-rich kinase (SPAK) complexed to its regulatory MO25 subunit phosphorylated KCC3 at Ser-96 and that in Xenopus laevis oocytes Ser-96 of human KCC3 is phosphorylated in isotonic conditions and becomes dephosphorylated during incubation in hypotonicity, leading to a dramatic increase in KCC3 function. Additionally, WNK3, which inhibits the activity of KCC3, promoted phosphorylation of Ser-96 as well as Thr-991 and Thr-1048. These observations were corroborated in HEK293 cells stably transfected with WNK3. Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3. However, when compared with the double mutant KCC3-T991A/T1048A, the triple mutant KCC3-S96A/T991A/T1048A activity in isotonic conditions was significantly higher, and it was not further increased by hypotonicity or inhibited by WNK3. We conclude that serine residue 96 of human KCC3 is a third site that has to be dephosphorylated for full activation of the cotransporter during hypotonicity.
Assuntos
Pressão Osmótica/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Oócitos/citologia , Oócitos/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Serina/genética , Serina/metabolismo , Simportadores/genética , Xenopus laevisRESUMO
Taurine is present at high concentrations in the fetal brain and is required for optimal brain development. Recent studies have reported that taurine causes increased proliferation of neural stem/progenitor neural cells (neural precursor cells, NPCs) obtained from embryonic and adult rodent brain. The present study is the first to show that taurine markedly increases cell numbers in cultures and neuronal generation from human NPCs (hNPCs). hNPCs obtained from 3 fetal brains (14-15 weeks of gestation) were cultured and expanded as neurospheres, which contained 76.3% nestin-positive cells. Taurine (5-20 mM) increased the number of hNPCs in culture, with maximal effect found at 10 mM and 4 days of culture. The taurine-induced increase ranged from 57 to 188% in the 3 brains examined. Taurine significantly enhanced the percentage of neurons formed from hNPCs under differentiating conditions, with increases ranging from 172 to 480% over controls without taurine. Taurine also increased the cell number and neuronal generation in cultures of the immortalized human cell line ReNcell VM. These results suggest that taurine has a positive influence on hNPC growth and neuronal formation.
Assuntos
Encéfalo/citologia , Células-Tronco Neurais/citologia , Neurogênese , Taurina/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/embriologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feto , Humanos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurônios/citologiaRESUMO
The involvement of WNK3 (with no lysine [K] kinase) in cell volume regulation evoked by anisotonic conditions was investigated in two modified stable lines of HEK293 cells: WNK3+, overexpressing WNK3 and WNK3-KD expressing a kinase inactive by a punctual mutation (D294A) at the catalytic site. This different WNK3 functional expression modified intracellular Cl(-) concentration with the following profile: WNK3+ > control > WNK3-KD cells. Stimulated with 15% hypotonic solutions, WNK3+ cells showed less efficient RVD (13.1%), lower Cl(-) efflux and decreased (94.5%) KCC activity. WNK3-KD cells showed 30.1% more efficient RVD, larger Cl(-) efflux and 5-fold higher KCC activity, increased since the isotonic condition. Volume-sensitive Cl(-) currents were similar in controls, WNK3+ cells, and WNK3-KD cells. Taurine efflux was not evoked at H15%. These results show a WNK3 influence on RVD in HEK293 cells via increasing KCC activity. Hypertonic medium induced cell shrinkage and RVI. In both WNK3+ and WNK3-KD cells, RVI and NKCC activity were increased, in WNK3+ cells presumably by enhanced NKCC phosphorylation, and in WNK3-KD cells via the [Cl(-)](i) reduction induced by the higher KCC activity in characteristic of these cells. These results support the role of WNK3 in modulation of intracellular Cl(-) concentration, in RVD, and indirectly on RVI, via its effects on KCC and NKCC activity. WNK3 in HEK293 cells is expressed as puncta at the intercellular junctions and diffusely at the cytosol, while the inactive kinase was found concentrated at the Golgi area. Cells with inactive WNK3 exhibited a marked change of cell phenotype.