Assuntos
Humanos , Glândulas Salivares/crescimento & desenvolvimento , Aquaporinas/fisiologia , Desenvolvimento Fetal/fisiologia , Morfogênese/fisiologia , Imuno-Histoquímica , Permeabilidade da Membrana Celular/fisiologia , Técnicas Imunoenzimáticas , Aquaporina 3/fisiologia , Aquaporina 1/fisiologia , Aquaporina 5/fisiologiaRESUMO
Soil salinity reduces root hydraulic conductivity (Lpr) of several plant species. However, how cellular signaling and root hydraulic properties are linked in plants that can cope with water restriction remains unclear. In this work, we exposed the halotolerant species red beet (Beta vulgaris) to increasing concentrations of NaCl to determine the components that might be critical to sustaining the capacity to adjust root hydraulics. Our strategy was to use both hydraulic and cellular approaches in hydroponically grown seedlings during the first osmotic phase of salt stress. Interestingly, Lpr presented a bimodal profile response apart from the magnitude of the imposed salt stress. As well as Lpr, the PIP2-aquaporin profile follows an unphosphorylated/phosphorylated pattern when increasing NaCl concentration while PIP1 aquaporins remain constant. Lpr also shows high sensitivity to cycloheximide. In low NaCl concentrations, Lpr was high and 70 % of its capacity could be attributed to the CHX-inhibited cell-to-cell pathway. More interestingly, roots can maintain a constant spontaneous exudated flow that is independent of the applied NaCl concentration. In conclusion, Beta vulgaris root hydraulic adjustment completely lies in a dominant cell-to-cell pathway that contributes to satisfying plant water demands.
Assuntos
Aquaporinas/fisiologia , Beta vulgaris/fisiologia , Transporte Biológico/fisiologia , Fosforilação/fisiologia , Raízes de Plantas/fisiologia , Salinidade , Plântula/fisiologia , Estresse Fisiológico/fisiologia , Produtos Agrícolas/fisiologiaRESUMO
Aquaporins (AQP) are channel proteins belonging to the Major Intrinsic Protein (MIP) superfamily that play an important role in plant water relations. The main role of aquaporins in plants is transport of water and other small neutral molecules across cellular biological membranes. AQPs have remarkable features to provide an efficient and often, specific water flow and enable them to transport water into and out of the cells along the water potential gradient. Plant AQPs are classified into five main subfamilies including the plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26 like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and X intrinsic proteins (XIPs). AQPs are localized in the cell membranes and are found in all living cells. However, most of the AQPs that have been described in plants are localized to the tonoplast and plasma membranes. Regulation of AQP activity and gene expression, are also considered as a part of the adaptation mechanisms to stress conditions and rely on complex processes and signaling pathways as well as complex transcriptional, translational and posttranscriptional factors. Gating of AQPs through different mechanisms, such as phosphorylation, tetramerization, pH, cations, reactive oxygen species, phytohormones and other chemical agents, may play a key role in plant responses to environmental stresses by maintaining the uptake and movement of water in the plant body.
Assuntos
Aquaporinas/metabolismo , Transporte Biológico/fisiologia , Plantas/metabolismo , Estresse Fisiológico/fisiologia , Aquaporinas/fisiologia , Expressão GênicaRESUMO
Abstract Aquaporins (AQP) are channel proteins belonging to the Major Intrinsic Protein (MIP) superfamily that play an important role in plant water relations. The main role of aquaporins in plants is transport of water and other small neutral molecules across cellular biological membranes. AQPs have remarkable features to provide an efficient and often, specific water flow and enable them to transport water into and out of the cells along the water potential gradient. Plant AQPs are classified into five main subfamilies including the plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26 like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and X intrinsic proteins (XIPs). AQPs are localized in the cell membranes and are found in all living cells. However, most of the AQPs that have been described in plants are localized to the tonoplast and plasma membranes. Regulation of AQP activity and gene expression, are also considered as a part of the adaptation mechanisms to stress conditions and rely on complex processes and signaling pathways as well as complex transcriptional, translational and posttranscriptional factors. Gating of AQPs through different mechanisms, such as phosphorylation, tetramerization, pH, cations, reactive oxygen species, phytohormones and other chemical agents, may play a key role in plant responses to environmental stresses by maintaining the uptake and movement of water in the plant body.
Assuntos
Plantas/metabolismo , Estresse Fisiológico/fisiologia , Transporte Biológico/fisiologia , Aquaporinas/metabolismo , Expressão Gênica , Aquaporinas/fisiologiaRESUMO
STUDY HYPOTHESIS: Are the placental aquaporins (AQPs) involved in the apoptosis of human trophoblast? STUDY FINDING: The general blocking of placental AQPs with HgCl2 and, in particular, the blocking of AQP3 activity with CuSO4 abrogated the apoptotic events of human trophoblast cells. WHAT IS KNOWN ALREADY: Although apoptosis of trophoblast cells is a natural event involved in the normal development of the placenta, it is exacerbated in pathological processes, such as pre-eclampsia, where an abnormal expression and functionality of placental AQPs occur without alterations in the feto-maternal water flux. Furthermore, fluctuations in O2 tension are proposed to be a potent inducer of placental apoptotic changes and, in explants exposed to hypoxia/reoxygenation (H/R), transcellular water transport mediated by AQPs was undetectable. This suggests that AQPs might be involved in processes other than water transport, such as apoptosis. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Explants from normal term placentas were maintained in culture under conditions of normoxia, hypoxia and H/R. Cell viability was determined by assessing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide incorporation. For the general or specific inhibition of AQPs, 0.3 mM HgCl2, 5 mM CuSO4, 0.3 mM tetraethylammonium chloride (TEA) or 0.5 mM phloretin were added to the culture medium before explants were exposed to each treatment. Oxidative stress parameters and apoptotic indexes were evaluated in the presence or absence of AQPs blockers. AQP3 expression was confirmed by western blot and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: First, we observed that in H/R treatments cell viability decreased by 20.16 ± 5.73% compared with those explants cultured in normoxia (P = 0.009; n = 7). Hypoxia did not modify cell viability significantly. Both hypoxia and H/R conditions induced oxidative stress. Spontaneous chemiluminescence and thiobarbituric acid reactive substance levels were significantly increased in explants exposed to hypoxia (n = 6 per group, P = 0.0316 and P = 0.0009, respectively) and H/R conditions (n = 6 per group, P = 0.0281 and P = 0.0001, respectively) compared with those cultured in normoxia. Regarding apoptosis, H/R was a more potent inducer of trophoblast apoptosis than hypoxia alone. Bax expression and the number of apoptotic nuclei were significantly higher in explants cultured in H/R compared with normoxia and hypoxia conditions (n = 12, P = 0.0135 and P = 0.001, respectively). DNA fragmentation was only observed in H/R and, compared with normoxia and hypoxia, the activity of caspase-3 was highest in explants cultured in H/R (n = 12, P = 0.0001). In explants exposed to H/R, steric blocking of AQP activity with HgCl2 showed that DNA degradation was undetectable (n = 12, P = 0.001). Bax expression and caspase-3 activity were drastically reduced (n = 12, P = 0.0146 and P = 0.0001, respectively) compared with explants cultured in H/R but not treated with HgCl2. Similar results were observed in explants exposed to H/R when we blocked AQP3 activity with CuSO4. DNA degradation was undetectable and the number of apoptotic nuclei and caspase-3 activity were significantly decreased compared with explants cultured in H/R but not treated with CuSO4 (n = 12, P = 0.001 and P = 0.0001, respectively). However, TEA and phloretin treatments, to block AQP1/4 or AQP9, respectively, failed in abrogate apoptosis. In addition, we confirmed the expression and localization of AQP3 in explants exposed to H/R. LIMITATIONS, REASONS FOR CAUTION: Our studies are limited by the number of experimental conditions tested, which do not fully capture the variability in oxygen levels, duration of exposure and alternating patterns of oxygen seen in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that any alteration in placental AQP expression might disturb the equilibrium of the normal apoptotic events and may be an underlying cause in the pathophysiology of placental gestational disorders such as pre-eclampsia. Furthermore, the dysregulation of placental AQPs may be one of the crucial factors in triggering the clinical manifestations of pre-eclampsia. LARGE SCALE DATA: n/a. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by UBACyT 20020090200025 and 20020110200207 grants and PIP-CONICET 11220110100561 grant, and the authors have no conflict of interest to declare.
Assuntos
Apoptose/fisiologia , Aquaporinas/fisiologia , Trofoblastos/citologia , Apoptose/efeitos dos fármacos , Aquaporina 3/antagonistas & inibidores , Aquaporina 3/biossíntese , Aquaporina 3/fisiologia , Caspase 3/análise , Hipóxia Celular , Sulfato de Cobre/farmacologia , Fragmentação do DNA , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Cloreto de Mercúrio/farmacologia , Técnicas de Cultura de Órgãos , Estresse Oxidativo , Oxigênio/farmacologia , Gravidez , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Adulto Jovem , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
BACKGROUND: Marine mammals are well adapted to their hyperosmotic environment. Several morphological and physiological adaptations for water conservation and salt excretion are known to be present in cetaceans, being responsible for regulating salt balance. However, most previous studies have focused on the unique renal physiology of marine mammals, but the molecular bases of these mechanisms remain poorly explored. Many genes have been identified to be involved in osmotic regulation, including the aquaporins. Considering that aquaporin genes were potentially subject to strong selective pressure, the aim of this study was to analyze the molecular evolution of seven aquaporin genes (AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP9) comparing the lineages of cetaceans and terrestrial mammals. RESULTS: Our results demonstrated strong positive selection in cetacean-specific lineages acting only in the gene for AQP2 (amino acids 23, 83, 107,179, 180, 181, 182), whereas no selection was observed in terrestrial mammalian lineages. We also analyzed the changes in the 3D structure of the aquaporin 2 protein. Signs of strong positive selection in AQP2 sites 179, 180, 181, and 182 were unexpectedly identified only in the baiji lineage, which was the only river dolphin examined in this study. Positive selection in aquaporins AQP1 (45), AQP4 (74), AQP7 (342, 343, 356) was detected in cetaceans and artiodactyls, suggesting that these events are not related to maintaining water and electrolyte homeostasis in seawater. CONCLUSIONS: Our results suggest that the AQP2 gene might reflect different selective pressures in maintaining water balance in cetaceans, contributing to the passage from the terrestrial environment to the aquatic. Further studies are necessary, especially those including other freshwater dolphins, who exhibit osmoregulatory mechanisms different from those of marine cetaceans for the same essential task of maintaining serum electrolyte balance.
Assuntos
Aquaporinas/genética , Evolução Biológica , Cetáceos/genética , Golfinhos/genética , Evolução Molecular , Seleção Genética , Animais , Aquaporina 1/genética , Aquaporina 1/fisiologia , Aquaporina 2/genética , Aquaporina 2/fisiologia , Aquaporina 3/genética , Aquaporina 3/fisiologia , Aquaporina 4/genética , Aquaporina 4/fisiologia , Aquaporina 6/genética , Aquaporina 6/fisiologia , Aquaporinas/fisiologia , Cetáceos/fisiologia , Golfinhos/fisiologia , Filogenia , Seleção Genética/genética , Seleção Genética/fisiologia , Alinhamento de SequênciaRESUMO
Aquaporins (AQPs) are a family of channel proteins, which transport water and/or small solutes across cell membranes. AQPs are present in Bacteria, Eukarya, and Archaea. The classical AQP evolution paradigm explains the inconsistent phylogenetic trees by multiple transfer events and emphasizes that the assignment of orthologous AQPs is not possible, making it difficult to integrate functional information. Recently, a novel phylogenetic framework of eukaryotic AQP evolution showed congruence between eukaryotic AQPs and organismal trees identifying 32 orthologous clusters in plants and animals (Soto et al. Gene 503:165-176, 2012). In this article, we discuss in depth the methodological strength, the ability to predict functionality and the AQP community perception about the different paradigms of AQP evolution. Moreover, we show an updated review of AQPs transport functions in association with phylogenetic analyses. Finally, we discuss the possible effect of AQP data integration in the understanding of water and solute transport in eukaryotic cells.
Assuntos
Aquaporinas/fisiologia , Animais , Aquaporinas/classificação , Bactérias , Transporte Biológico , Eucariotos , Evolução Molecular , Humanos , Família Multigênica , Filogenia , Plantas , Conformação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
The discovery of water channels (aquaporins, AQPs) was a landmark event for the clarification of water transport through the plasma membrane. AQPs belong to a family of intrinsic membrane proteins that act as selective channels for water and for solutes such as glycerol and urea. AQPs were found in different tissues and organs, including male and female reproductive systems. In the swine female reproductive system, the AQPs were localized in the uterus, oviduct, and ovary, as well as in the granulosa cells from primordial follicles. Knowing the involvement of AQPs with the male and female germ cells, as well as their acknowledged role in transporting water through the plasma membrane, the research of these proteins in cryopreservation processes becomes essential. Thus, this review aims to describe the structure and function of AQPs in membranes, highlighting their role in the reproductive system (male and female). We also discuss the involvement of AQPs in cryopreservation, focusing on the effect and importance of these proteins on the rates of vitrification protocols for preantral follicles present in the ovarian tissue of domestic mammals.
Assuntos
Aquaporinas/fisiologia , Criopreservação/métodos , Óvulo/fisiologia , Espermatozoides/fisiologia , Água/metabolismo , Animais , Aquaporinas/genética , Aquaporinas/metabolismo , Transporte Biológico , Membrana Celular/fisiologia , Crioprotetores , Feminino , Humanos , MasculinoRESUMO
Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H(2)O(2) across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H(2)O(2) release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p<0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H(2)O(2) release, assessed by Amplex Red, was reduced by about 45% (p<0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+120%, p<0.05) and loss of mitochondrial membrane potential (-80%, p<0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H(2)O(2) release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death.
Assuntos
Aquaporinas/genética , Aquaporinas/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Potencial da Membrana Mitocondrial/genética , Mitocôndrias Hepáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Western Blotting , Ciclosporina/farmacologia , Células Hep G2 , Humanos , Peróxido de Hidrogênio/metabolismo , L-Lactato Desidrogenase/metabolismo , Microscopia de Fluorescência , Oxidantes/metabolismo , Permeabilidade , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Sais de Tetrazólio , Tiazóis , Azul TripanoRESUMO
Water is a major component of the eye, and water channels (aquaporins) are ubiquitous in ocular tissues, and quite abundant at their different locations. AQP1 is expressed in corneal endothelium, lens epithelium, ciliary epithelium, and retinal pigment epithelium. AQP3 is expressed in corneal epithelium, and in conjunctival epithelium. AQP4 is expressed in ciliary epithelium and retinal Muller cells. AQP5 is expressed in corneal epithelium, and conjunctival epithelium. AQP0 is expressed in lens fiber cells. It is known that five ocular tissues transport fluid, namely: (1) Corneal endothelium; (2) Conjunctival epithelium; (3) Lens epithelium; (4) Ciliary epithelium; (5) Retinal pigment epithelium. For the corneal endothelium, aquaporins are not the main route for trans-tissue water movement, which is paracellular. Instead, we propose that aquaporins allow fast osmotic equilibration of the cell, which is necessary to maintain optimal rates of fluid movement since the cyclic paracellular water transfer mechanism operates separately and tends to create periodic osmotic imbalances (τâ¼5 s).