Miriam Echevarría

Detailed localization of aquaporin-4 messenger RNA in the CNS : Preferential expression in periventricular organs

We have performed a detailed in situ hybridization study of the distribution of aquaporin-4 messenger RNA in the CNS. Contrary to expectation, we demonstrate that aquaporin-4 is ubiquitously expressed in the CNS. Strong hybridization labeling was detected in multiple olfactory areas, cortical cells, medial habenular nucleus, bed nucleus of the stria terminalis, tenia tecta, pial surface, pontine nucleus, hippocampal formation and multiple thalamic and hypothalamic areas. A low but significant hybridization signal was found, among others, in the choroid plexus of the lateral ventricles, ependymal cells, dorsal raphe and cerebellum. Overall, a preferential distribution of aquaporin-4 messenger RNA-expressing cells was evident in numerous periventricular organs. From the distribution study, the presence of aquaporin-4 messenger RNA-expressing cells in neuronal layers was evident in neuronal layers including the CA1 -CA3 hippocampal pyramidal cells, granular dentate cells and cortical cells. Further evidence of neuronal expression comes from the semicircular arrangement of aquaporin-4 messenger RNA-expressing cells in the bed nucleus of the stria terminalis and medial habenular nucleus exhibiting Nissl-stained morphological features typical of neurons. Combined glial fibrillary acidic protein immunohistochemistry and aquaporin-4 messenger RNA in situ hybridization demonstrated that aquaporin-4 messenger RNA is expressed by glial fibrillary acidic protein-lacking cells. We conclude that aquaporin-4 messenger RNA is present in a collection of structures typically involved in the regulation of water and sodium intake and that aquaporin-4 water channels could be the osmosensor mechanism responsible for detecting changes in cell volume by these cells.

Development of Cytosolic Hypoxia and Hypoxia-inducible Factor Stabilization Are Facilitated by Aquaporin-1 Expression

O2 is essential for aerobic life, and the classic view is that it diffuses freely across the plasma membrane. However, measurements of O2 permeability of lipid bilayers have indicated that it is much lower than previously thought, and therefore, the existence of membrane O2 channels has been suggested. We hypothesized that, besides its role as a water channel, aquaporin-1 (AQP-1) could also work as an O2 transporter, because this transmembrane protein appears to be CO2-permeable and is highly expressed in cells with rapid O2 turnover (erythrocytes and microvessel endothelium). Here we show that in mammalian cells overexpressing AQP-1 and exposed to hypoxia, the loss of cytosolic O2, as well as stabilization of the O2-dependent hypoxia-inducible transcription factor and expression of its target genes, is accelerated. In normoxic endothelial cells, knocking down AQP-1 produces induction of hypoxia-inducible genes. Moreover, lung AQP-1 is markedly up-regulated in animals exposed to hypoxia. These data suggest that AQP-1 has O2 permeability and thus could facilitate O2 diffusion across the cell membrane.

Localization of Aquaporin-3 mRNA and protein along the gastrointestinal tract of Wistar rats

Abstract Since specific proteins responsible for water transport (aquaporins, AQPs) have been identified in a great variety of tissues, we decided to study the presence of AQP3 in the gastrointestinal tract (GIT) of Wistar rats. Poly(A+) RNA was purified from the mucosa of the stomach, jejunum, ileum and colon, and gross detection of AQP3 mRNA was done by Northern blot analysis. In situ hybridization studies were carried out to precisely localize the distribution of this transcript. Sections of the different tissues were hybridized with @400-bp [35S]riboprobes. The results presented here demonstrate that AQP3 is expressed throughout the GIT, with its expression in the colon and ileum greater than that in the stomach. Immunohistochemistry experiments, using a polyclonal antibody against AQP3, revealed that AQP3 protein is present at the basolateral membrane of the epithelial cells lining the villus tip of the small intestine and colon. The finding of AQP3 in the intestinal epithelia strongly suggests that this protein functions as a pathway for water transport in this epithelium.

Targeting aquaporin function: potent inhibition of aquaglyceroporin-3 by a gold-based compound

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC50 = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.

Induction of the glucose-6-phosphate dehydrogenase gene expression by chronic hypoxia in PC12 cells

We studied the regulation of glucose‐6‐phosphate dehydrogenase (G6PD) gene expression by chronic hypoxia. G6PD mRNA level and activity were increased in PC12 cells by hypoxia in a dose‐ and time‐dependent manner. Cobalt chloride and dimethyloxalylglycine, which can mimic hypoxia, also activated G6PD gene expression. Interestingly, hypoxia‐induced G6PD expression followed a time course much slower than that of phosphoglycerate kinase 1 (PGK1), a hypoxia‐inducible factor (HIF)‐dependent glycolytic enzyme. Hypoxic‐G6PD induction was almost negligible in non‐excitable Buffalo rat liver cells, although in these cells PGK1 was strongly upregulated by low PO2. Furthermore, G6PD but not PGK1 induction was blocked by the antioxidants glutathione and N‐acetylcysteine. These results suggest the dependence of G6PD gene expression on HIF and intracellular redox status and the differential hypoxic regulation of glucose‐metabolizing enzymes.

Neuroprotection by Transgenic Expression of Glucose-6-Phosphate Dehydrogenase in Dopaminergic Nigrostriatal Neurons of Mice

Oxidative damage to dopaminergic nigrostriatal (DNS) neurons plays a central role in the pathogenesis of Parkinsons disease (PD). Glucose-6-phosphate dehydrogenase (G6PD) is a key cytoprotective enzyme that provides NADPH, the major source of the reducing equivalents of a cell. Mutations of this enzyme are the most common enzymopathies worldwide. We have studied in vivo the role of G6PD overexpressed specifically in the DNS pathway and show that the increase of G6PD activity in the soma and axon terminals of DNS neurons, separately from other neurons or glial cells, protects them from parkinsonism. Analysis of DNS neurons by histological, neurochemical, and functional methods showed that even a moderate increase of G6PD activity rendered transgenic mice more resistant than control littermates to the toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The neuroprotective action of G6PD was also observed in aged animals despite that they had a greater susceptibility to MPTP. Therefore, overexpression of G6PD in dopaminergic neurons or pharmacological activation of the native enzyme should be considered as potential therapeutic strategies to PD.

Rat Adrenal Chromaffin Cells Are Neonatal CO2 Sensors

We studied the participation of adrenal medulla (AM) chromaffin cells in hypercapnic chemotransduction. Using amperometric recordings, we measured catecholamine (CAT) secretion from cells in AM slices of neonatal and adult rats perfused with solutions bubbled with different concentrations of CO2. The secretory activity augmented from 1.74 ± 0.19 pC/min at 5% CO2 to 6.36 ± 0.77 pC/min at 10% CO2. This response to CO2 was dose dependent and appeared without changes in extracellular pH, although it was paralleled by a drop in intracellular pH. Responsiveness to hypercapnia was higher in neonatal than in adult slices. The secretory response to hypercapnia required extracellular Ca2+ influx. Both the CO2-induced internal pH drop and increase in CAT secretion were markedly diminished by methazolamide (2 μm), a membrane-permeant carbonic anhydrase (CA) inhibitor. We detected the presence of two CA isoforms (CAI and CAII) in neonatal AM slices by in situ hybridization and real-time PCR. The expression of these enzymes decreased in adult AM together with the disappearance of responsiveness to CO2. In patch-clamped chromaffin cells, hypercapnia elicited a depolarizing receptor potential, which led to action potential firing, extracellular Ca2+ influx, and CAT secretion. This receptor potential (inhibited by methazolamide) was primarily attributable to activation of a resting cationic conductance. In addition, voltage-gated K+ current amplitude was also decreased by high CO2. The CO2-sensing properties of chromaffin cells may be of physiologic relevance, particularly for the adaptation of neonates to extrauterine life, before complete maturation of peripheral and central chemoreceptors.

Functional and Transcriptional Induction of Aquaporin-1 Gene by Hypoxia; Analysis of Promoter and Role of Hif-1α

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O2 transport. It has been reported that this protein contributes to gas permeation (CO2, NO and O2) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5′ proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl2) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.

Functional Inhibition of Aquaporin-3 With a Gold-Based Compound Induces Blockage of Cell Proliferation

AQP3 has been correlated with higher transport of glycerol, increment of ATP content, and larger proliferation capacity. Recently, we described the gold(III) complex Auphen as a very selective and potent inhibitor of AQP3s glycerol permeability (Pgly). Here we evaluated Auphen effect on the proliferation of various mammalian cell lines differing in AQP3 expression level: no expression (PC12), moderate (NIH/3T3) or high (A431) endogenous expression, cells stably expressing AQP3 (PC12‐AQP3), and human HEK293T cells transiently transfected (HEK‐AQP3) for AQP3 expression. Proliferation was evaluated in the absence or presence of Auphen (5 μM) by counting number of viable cells and analyzing 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation. Auphen reduced ≈50% the proliferation in A431 and PC12‐AQP3, ≈15% in HEK‐AQP3 and had no effect in PC12‐wt and NIH/3T3. Strong arrest in the S‐G2/M phases of the cell cycle, supported by analysis of cyclins (A, B1, D1, E) levels, was observed in AQP3‐expressing cells treated with Auphen. Flow‐cytometry of propidium iodide incorporation and measurements of mitochondrial dehydrogenases activity confirmed absence of cytotoxic effect of the drug. Functional studies evidenced ≈50% inhibition of A431 Pgly by Auphen, showing that the compound’s antiproliferative effect correlates with its ability to inhibit AQP3 Pgly. Role of Cys‐40 on AQP3 permeability blockage by Auphen was confirmed by analyzing the mutated protein (AQP3‐Ser‐40). Accordingly, cells transfected with mutated AQP3 gained resistance to the antiproliferative effect of Auphen. These results highlight an Auphen inhibitory effect on proliferation of cells expressing AQP3 and suggest a targeted therapeutic effect on carcinomas with large AQP3 expression. J. Cell. Physiol. 229: 1787–1801, 2014.

Chronic and progressive Parkinson's disease MPTP model in adult and aged mice.

Despite the different animal models of Parkinsons disease developed during the last years, they still present limitations modelling the slow and progressive process of neurodegeneration. Here, we undertook a histological, neurochemical and behavioural analysis of a new chronic parkinsonian mouse model generated by the subcutaneous administration of low doses of MPTP (20 mg/kg, 3 times per week) for 3 months, using both young adult and aged mice. The MPTP‐induced nigrostriatal neurodegeneration was progressive and was accompanied by a decrease in striatal dopamine levels and motor impairment. We also demonstrated the characteristic neuroinflammatory changes (microglial activation and astrogliosis) associated with the neurodegenerative process. Aged animals showed both a faster time course of neurodegeneration and an altered neuroinflammatory response. The long‐term systemic application of low MPTP doses did not induce any increase in mortality in either young adult or aged mice and better resembles the slow evolution of the neurodegenerative process. This treatment could be useful to model different stages of Parkinsons disease, providing a better understanding of the pathophysiology of the disease and facilitating the testing of both protective and restorative treatments.