Asunto(s)

Humanos , Glándulas Salivales/crecimiento & desarrollo , Acuaporinas/fisiología , Desarrollo Fetal/fisiología , Morfogénesis/fisiología , Inmunohistoquímica , Permeabilidad de la Membrana Celular/fisiología , Técnicas para Inmunoenzimas , Acuaporina 3/fisiología , Acuaporina 1/fisiología , Acuaporina 5/fisiología

RESUMEN

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.

Asunto(s)

Acuaporinas/fisiología , Beta vulgaris/fisiología , Transporte Biológico/fisiología , Fosforilación/fisiología , Raíces de Plantas/fisiología , Salinidad , Plantones/fisiología , Estrés Fisiológico/fisiología , Productos Agrícolas/fisiología

RESUMEN

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.

Asunto(s)

Acuaporinas/metabolismo , Transporte Biológico/fisiología , Plantas/metabolismo , Estrés Fisiológico/fisiología , Acuaporinas/fisiología , Expresión Génica

RESUMEN

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.

Asunto(s)

Plantas/metabolismo , Estrés Fisiológico/fisiología , Transporte Biológico/fisiología , Acuaporinas/metabolismo , Expresión Génica , Acuaporinas/fisiología

RESUMEN

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.

Asunto(s)

Apoptosis/fisiología , Acuaporinas/fisiología , Trofoblastos/citología , Apoptosis/efectos de los fármacos , Acuaporina 3/antagonistas & inhibidores , Acuaporina 3/biosíntesis , Acuaporina 3/fisiología , Caspasa 3/análisis , Hipoxia de la Célula , Sulfato de Cobre/farmacología , Fragmentación del ADN , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Cloruro de Mercurio/farmacología , Técnicas de Cultivo de Órganos , Estrés Oxidativo , Oxígeno/farmacología , Embarazo , Sustancias Reactivas al Ácido Tiobarbitúrico/análisis , Adulto Joven , Proteína X Asociada a bcl-2/biosíntesis , Proteína X Asociada a bcl-2/genética

RESUMEN

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.

Asunto(s)

Acuaporinas/genética , Evolución Biológica , Cetáceos/genética , Delfines/genética , Evolución Molecular , Selección Genética , Animales , Acuaporina 1/genética , Acuaporina 1/fisiología , Acuaporina 2/genética , Acuaporina 2/fisiología , Acuaporina 3/genética , Acuaporina 3/fisiología , Acuaporina 4/genética , Acuaporina 4/fisiología , Acuaporina 6/genética , Acuaporina 6/fisiología , Acuaporinas/fisiología , Cetáceos/fisiología , Delfines/fisiología , Filogenia , Selección Genética/genética , Selección Genética/fisiología , Alineación de Secuencia

RESUMEN

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.

Asunto(s)

Acuaporinas/fisiología , Animales , Acuaporinas/clasificación , Bacterias , Transporte Biológico , Eucariontes , Evolución Molecular , Humanos , Familia de Multigenes , Filogenia , Plantas , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas

RESUMEN

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.

Asunto(s)

Acuaporinas/fisiología , Criopreservación/métodos , Óvulo/fisiología , Espermatozoides/fisiología , Agua/metabolismo , Animales , Acuaporinas/genética , Acuaporinas/metabolismo , Transporte Biológico , Membrana Celular/fisiología , Crioprotectores , Femenino , Humanos , Masculino

RESUMEN

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.

Asunto(s)

Acuaporinas/genética , Acuaporinas/fisiología , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Potencial de la Membrana Mitocondrial/genética , Mitocondrias Hepáticas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Adenosina Trifosfato/metabolismo , Western Blotting , Ciclosporina/farmacología , Células Hep G2 , Humanos , Peróxido de Hidrógeno/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Microscopía Fluorescente , Oxidantes/metabolismo , Permeabilidad , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Sales de Tetrazolio , Tiazoles , Azul de Tripano

RESUMEN

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 (τ∼5 s).

Asunto(s)

Acuaporinas/fisiología , Cuerpo Ciliar/fisiología , Conjuntiva/fisiología , Endotelio Corneal/fisiología , Células Epiteliales/fisiología , Cristalino/fisiología , Epitelio Pigmentado de la Retina/fisiología , Animales , Transporte Biológico , Cuerpo Ciliar/citología , Conjuntiva/citología , Endotelio Corneal/citología , Células Epiteliales/citología , Humanos , Cristalino/citología , Especificidad de Órganos , Isoformas de Proteínas/fisiología , Conejos , Epitelio Pigmentado de la Retina/citología , Agua/metabolismo , Equilibrio Hidroelectrolítico

RESUMEN

Osmolarity not only plays a key role in cellular homeostasis but also challenges cell survival. The molecular understanding of osmosis has not yet been completely achieved, and the discovery of aquaporins as molecular entities involved in water transport has caused osmosis to again become a focus of research. The main questions that need to be answered are the mechanism underlying the osmotic permeability coefficients and the extent to which aquaporins change our understanding of osmosis. Here, attempts to answer these questions are discussed. Critical aspects of the state of the state of knowledge on osmosis, a topic that has been studied since 19th century, are reviewed and integrated with the available information provided by in vivo, in vitro and in silico approaches.

Asunto(s)

Acuaporinas/fisiología , Ósmosis/fisiología , Animales , Acuaporinas/historia , Fenómenos Biofísicos , Permeabilidad de la Membrana Celular/fisiología , Biología Computacional , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Modelos Biológicos , Concentración Osmolar , Proteínas de Plantas/fisiología , Agua/metabolismo

RESUMEN

Brain edema is a severe clinical complication in a number of pathologies and is a major cause of increased morbidity and death. The swelling of astrocytes caused by a disruption of water and ion homeostasis, is the primary event contributing to the cytotoxic form of brain edema. Astrocyte cytotoxic swelling ultimately leads to transcapillary fluxes of ions and water into the brain parenchyma. This review focuses on the implication of transporters and channels in cytotoxic astrocyte swelling in hyponatremia, ischemia, trauma and hepatic encephalopathy. Emphasis is put on some salient features of the astrocyte physiology, all related to cell swelling, i.e. predominance of aquaporins, control of K(+) homeostasis and ammonia accumulation during the brain ammonia-detoxifying process.

Asunto(s)

Astrocitos/fisiología , Canales Iónicos/fisiología , Proteínas de Transporte de Membrana/fisiología , Animales , Acuaporinas/fisiología , Canales de Cloruro/fisiología , Encefalopatía Hepática/fisiopatología , Humanos

RESUMEN

Since few data are availble on the genetic responses to low temperatures, we investigated if cold storage of hepatocytes (0 degree C, mUW or BGS solutions, 72 h) can affect gene expression and/or cellular localization of AQP8 and their correlation with water movements. Cold preserved hepatocytes showed a significant decrease in water content (P less than 0.05) but were able to regulate their volume when they returned to physiological conditions. These changes were not related to modulation in the expression and the pattern of distribution of AQP8 suggesting that other mechanisms are involved. The study of the quantitative changes in the expression of genes coding for liver specific proteins in cold preserved hepatic cells is of interest in order to develop new preservation methods or solutions that could contribute to maintain the utility of these cells when destined to be applied in clinical models.

Asunto(s)

Acuaporinas/fisiología , Criopreservación/métodos , Hepatocitos/metabolismo , Soluciones Preservantes de Órganos , Agua/fisiología , Ácidos Alcanesulfónicos , Animales , Tamaño de la Célula , Supervivencia Celular/fisiología , Frío , Expresión Génica , Gluconatos , Hepatocitos/citología , Inmunohistoquímica , Hígado/citología , Hígado/metabolismo , Masculino , Microscopía Confocal , Concentración Osmolar , Ratas , Ratas Wistar , Sacarosa

RESUMEN

It has been established that the permeability of the human placenta increases with advancing gestation. Indirect evidence has also proposed that aquaporins (AQPs) may be involved in the regulation of placental water flow but the mechanisms are poorly understood. Five AQPs have been found in the human placenta and fetal membranes [AQP1, 3, 4, 8 and 9]. However, the physiological function(s) and the regulation of these proteins remain unknown. Emerging evidence has shown that human fetal membrane AQPs may have a role in intramembranous amniotic fluid water regulation and that alterations in their expression are related to polyhydramnios and oligohydramnios. In addition, we have observed a high expression of AQP3 and AQP9 in the apical membrane of the syncytiotrophoblast. Moreover, AQP9 was found to be increased in preeclamptic placentas, but it could not be related to its functionality for the transport of water and mannitol. However, a significant urea flux was seen. Since preeclampsia is not known to be associated with an altered water flux to the fetus we propose that AQP9 might not have a key role in water transport in human placenta, but a function in the energy metabolism or the urea uptake and elimination across the placenta. However, the role of AQP9 in human placenta is still speculative and needs further studies. Insulin, hCG, cAMP and CFTR have been found to be involved in the regulation of the molecular and functional expression of AQPs. Further insights into these mechanisms may clarify how water moves between the mother and the fetus.

Asunto(s)

Acuaporinas/fisiología , Membranas Extraembrionarias/metabolismo , Placenta/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Preeclampsia/metabolismo , Embarazo

RESUMEN

The mechanism of epithelial fluid transport remains unsolved, which is partly due to inherent experimental difficulties. However, a preparation with which our laboratory works, the corneal endothelium, is a simple leaky secretory epithelium in which we have made some experimental and theoretical headway. As we have reported, transendothelial fluid movements can be generated by electrical currents as long as there is tight junction integrity. The direction of the fluid movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Residual endothelial fluid transport persists even when no anions (hence no salt) are being transported by the tissue and is only eliminated when all local recirculating electrical currents are. Aquaporin (AQP) 1 is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability (by ∼40%) but fluid transport much less (∼20%), which militates against the presence of sizable water movements across the cell. In contrast, AQP1 null mice cells have reduced regulatory volume decrease (only 60% of control), which suggests a possible involvement of AQP1 in either the function or the expression of volume-sensitive membrane channels/transporters. A mathematical model of corneal endothelium we have developed correctly predicts experimental results only when paracellular electro-osmosis is assumed rather than transcellular local osmosis. Our evidence therefore suggests that the fluid is transported across this layer via the paracellular route by a mechanism that we attribute to electro-osmotic coupling at the junctions. From our findings we have developed a novel paradigm for this preparation that includes 1) paracellular fluid flow; 2) a crucial role for the junctions; 3) hypotonicity of the primary secretion; and 4) an AQP role in regulation rather than as a significant water pathway. These elements are remarkably similar to those proposed by the laboratory of Adrian Hill for fluid transport across other leaky epithelia.

Asunto(s)

Acuaporinas/fisiología , Líquidos Corporales/metabolismo , Epitelio/fisiología , Uniones Estrechas/fisiología , Animales , Acuaporinas/genética , Transporte Biológico/fisiología

RESUMEN

This review focuses on current knowledge on hepatocyte aquaporins (AQPs) and their significance in bile formation and cholestasis. Canalicular bile secretion results from a combined interaction of several solute transporters and AQP water channels that facilitate water flow in response to the osmotic gradients created. During choleresis, hepatocytes rapidly increase their canalicular membrane water permeability by modulating the abundance of AQP8. The question was raised as to whether the opposite process, i.e. a decreased canalicular AQP8 expression would contribute to the development of cholestasis. Studies in several experimental models of cholestasis, such as extrahepatic obstructive cholestasis, estrogen-induced cholestasis, and sepsis-induced cholestasis demonstrated that the protein expression of hepatocyte AQP8 was impaired. In addition, biophysical studies in canalicular plasma membranes revealed decreased water permeability associated with AQP8 protein downregulation. The combined alteration in hepatocyte solute transporters and AQP8 would hamper the efficient coupling of osmotic gradients and canalicular water flow. Thus cholestasis may result from a mutual occurrence of impaired solute transport and decreased water permeability.

Asunto(s)

Acuaporinas/fisiología , Colestasis Intrahepática/fisiopatología , Acuaporinas/metabolismo , Bilis/metabolismo , Colestasis Intrahepática/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Ósmosis/fisiología

RESUMEN

Although bacterial lipopolysaccharides (LPS) are known to cause cholestasis in sepsis, the molecular mechanisms accounting for this effect are only partially known. Because aquaporin-8 (AQP8) seems to facilitate the canalicular osmotic water movement during hepatocyte bile formation, we studied its gene and functional expression in LPS-induced cholestasis. By subcellular fractionation and immunoblotting analysis, we found that 34-kDa AQP8 was significantly decreased by 70% in plasma (canalicular) and intracellular (vesicular) liver membranes. However, expression and subcellular localization of hepatocyte sinusoidal AQP9 were unaffected. Immunohistochemistry for liver AQPs confirmed these observations. Osmotic water permeability (P(f)) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (65 +/- 1 vs. 49 +/- 1 microm/s) by LPS, consistent with defective canalicular AQP8 functional expression. By Northern blot analysis, we found that 1.5-kb AQP8 mRNA expression was increased by 80%, suggesting a posttranscriptional mechanism of protein reduction. The tumor necrosis factor-alpha (TNF-alpha) receptor fusion protein TNFp75:Fc prevented the LPS-induced impairment of AQP8 expression and bile flow, suggesting the cytokine TNF-alpha as a major mediator of LPS effect. Accordingly, studies in hepatocyte primary cultures indicated that recombinant TNF-alpha downregulated AQP8. The effect of TNF-alpha was prevented by the lysosomal protease inhibitors leupeptin or chloroquine or by the proteasome inhibitors MG132 or lactacystin, suggesting a cytokine-induced AQP8 proteolysis. In conclusion, our data suggest that LPS induces the TNF-alpha-mediated posttranscriptional downregulation of AQP8 functional expression in hepatocytes, a mechanism potentially relevant to the molecular pathogenesis of sepsis-associated cholestasis.

Asunto(s)

Acuaporinas/fisiología , Colestasis/etiología , Colestasis/fisiopatología , Lipopolisacáridos/farmacología , Hígado/metabolismo , Sepsis/complicaciones , Sepsis/fisiopatología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Northern Blotting , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Citocinas/sangre , Regulación hacia Abajo/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Inmunohistoquímica , Luz , Hígado/efectos de los fármacos , Masculino , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Dispersión de Radiación , Fracciones Subcelulares/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/sangre

RESUMEN

Las acuaporinas constituyen una familia de canales que regulan el trasporte del agua en la membrana de todos los órganos. Desde la descripción de la primer acuaporina por Peter Agre y col., por lo que recibió el Premio Nobel de Química en el 2003, mucha información ha sido obtenida sobre el significado de estos canales proteicos. Están involucradas en múltiples desórdenes como la diabetes insípida nefrogénica, pérdida de la visión, el edema cerebral, la respuesta al ayuno, la intoxicación por arsénico, etc. Su descubrimiento enriqueció la comprensión de la regulación de los volúmenes corporales. En este artículo se hace una breve revisión de los conocimientos actuales sobre las mismas.

Asunto(s)

Humanos , Animales , Acuaporinas/clasificación , Acuaporinas/fisiología , Equilibrio Hidroelectrolítico/fisiología , Arginina Vasopresina , Enfermedades Renales

Asunto(s)

Humanos , Acuaporinas/fisiología , Movimientos del Agua

RESUMEN

BACKGROUND INFORMATION: Water is crucial for plant development and growth, and its transport pathways inside a plant are an ongoing topic for study. Plants express a large number of membrane intrinsic proteins whose role is now being re-evaluated by considering not only the control of the overall plant water balance but also in adaptation to environmental challenges that may affect their physiology. In particular, we focused our work on water movements across the root cell TP (tonoplast), the delimiting membrane of the vacuole. This major organelle plays a central role in osmoregulation. RESULTS: An enriched fraction of TP vesicles from Beta vulgaris (red beet) storage roots obtained by a conventional method was used to characterize its water permeability properties by means of the stopped-flow technique. The preparation showed high water permeability (485 microm x s(-1)), consistent with values reported in the literature. The water permeability was strongly blocked by HgCl(2) (reduced to 16%) and its energy activation was low. These observations allow us to postulate the presence of functional water channels in this preparation. Moreover, Western-blot analysis demonstrated the presence of a tonoplast intrinsic protein. With the purpose of studying the regulation of water channels, TP vesicles were exposed to different acidic pH media. When the pH of a medium was low (pH 5.6), the water permeability exhibited a 42% inhibition. CONCLUSIONS: Our findings prove that although almost all water channels present in the TP vesicles of B. vulgaris root are sensitive to HgCl(2), not all are inhibited by pH. This interesting selectivity to acidification of the medium could play a role in adapting the water balance in the cell-to-cell pathway.

Asunto(s)

Acuaporinas/fisiología , Beta vulgaris/metabolismo , Raíces de Plantas/metabolismo , Agua/metabolismo , Concentración de Iones de Hidrógeno , Cloruro de Mercurio/farmacología , Permeabilidad/efectos de los fármacos , Raíces de Plantas/citología , Vesículas Transportadoras/efectos de los fármacos , Vesículas Transportadoras/fisiología