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Domestic effluent discharges change water quality and habitat conditions in urban watersheds, though less known about how these impact fish communities. This work assessed the impact of chronic wastewater pollution on biotic and abiotic factors in six urban streams in Patagonia. Stream hydrological features, water quality conditions and fish communities were analyzed during a one-year period. The oxygen saturation and water velocity showed significant differences between up- and downstream locations of wastewater treatment plants (WWTPs). Chemical parameters revealed an eutrophication process downstream of the WWTP input, with increased biological oxygen demanding (BOD), nitrogen, ammonium, soluble reactive phosphorus, and chlorophyll a concentrations, indicating nutrient enrichment that can lead to a potential for algal growth. The study highlighted significant differences in fish abundance, density, and biomass between reaches upstream (Control) and downstream (Impacted) of the WWTP discharges, suggesting a detrimental impact on fish communities. While juveniles, females and males of the native Catfish (Hatcheria macraei) preferred pristine zones, juveniles and males of the native Perch (Percichthys spp.) displayed preferences for areas with higher nutrient levels. Exotic species like Rainbow Trout (Oncorhynchus mykiss) (juveniles, females and males) preferred low-nutrient, high-quality habitats, while juveniles, females and males of Brown Trout (Salmo trutta) were found near the WWTP facilities. Although some previous studies have suggested that nutrient enrichment might benefit fish, our findings highlight the negative impacts on fish abundance and distribution due to WWTPs. Fish species appear to demonstrate certain degrees of tolerance to pollution, with larger individuals displaying greater tolerance. Although the pollution levels may did not result in an irreversible collapse of the system, the absence of fish in the stream with the highest pollution level would indicate an ongoing environmental deterioration. Anthropogenic activities, especially municipal effluent discharge, exacerbate environmental degradation and demand specific management actions to maintain ecosystem integrity.

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Human activities such as agriculture and urbanization generate a large number of substances like personal care products, pharmaceutical compounds, and pesticides, which often reach aquatic environments and represent a threat to biodiversity. Many organisms have developed different evolutionary strategies to remove pervasive substances from their bodies, allowing them to persist even in polluted environments, and one of these is the multixenobiotic resistance (MXR) mechanism associated with the expression of membrane proteins like P-glycoprotein (P-gp). Numerous chemical compounds with diverse functions and structures can modulate this mechanism, which can be employed as a pollution biomarker. We examined the MXR activity in two species of snails that inhabit Patagonian freshwaters. Functional assay measurements of MXR were conducted on the native Chilina dombeiana and the exotic Physella acuta in stream reaches affected by anthropogenic impacts. Results indicated that at agricultural sites, C. dombeiana snails had a more active MXR system than organisms sampled at reference and moderately disturbed urban sites, whereas P. acuta snails from agricultural and highly disturbed urban sites showed better detoxifying activity than organisms from reference sites. Only in exotic snails, part of this activity was due to the action of P-gp. The most important environmental variables explaining MXR activity were ammonium, nitrate and nitrite, phosphates, and electrical conductivity. These results show the promise of measuring MXR activity in native and exotic snails, as a biomarker in the environmental monitoring of Patagonian freshwaters.

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Constructed wetlands are environmental solutions that mitigate the impacts of urban effluents. It is unclear how the performance of these wastewater treatment plants (WWTP) is affected by climatic conditions. The dissipation of nutrients, suspended solids, and changes in dissolved oxygen were investigated on a monthly basis over two years (2018/2019) at six sampling points across a WWTP located in Esquel, Patagonia. It was predicted that climatic variables (rain pattern and air temperature) would affect the functioning and efficiency of the WWTP (i.e., via nutrient load mitigation and sediment retention). Rainfall and temporal patterns differed markedly between and throughout the two years, leading to a clear seasonality in the transformation of pollutants. Nitrate loads were significantly higher in 2018 than in 2019 suggesting some degree of operational failure, whereas ammonia levels in treated effluents were extremely high during both years, with marked peaks occurring during autumn 2018 and summer 2019. The WWTP was moderately successful (~36%) in reducing TSS contents during 2018 but was inefficient in 2019. Ammonia levels in receiving waters underwent dilution due to rains rather than due to adequate WWTP nutrient retention. In terms of nutrients, effluent values exceeded those established by governmental regulation during most months, but worsened during summer coinciding with low flows. This lack of predictability for the values of the treated effluent strongly jeopardizes the ecological integrity and biodiversity of the receiving stream.

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Environmental impairment resulted from urbanizations can produce damage on freshwater species including strong physiological effects at individual or population level. The multixenobiotic resistance (MXR) is a defence mechanism which has been demonstrated in several aquatic organisms. The key mediators of MXR activity are ATP-binding cassette (ABC) proteins like P-glycoprotein (P-gp). This system protects aquatic organisms against the accumulation of xenobiotics by extruding them from cells in an energy-dependent manner. MXR has been pointed out as relevant in the ecotoxicological context and has been proposed as a biomarker for pollution assessment. Since fish species are common target in freshwater biomonitoring programs, the purpose of the study was to evaluate the MXR mechanism in native Hatcheria macraei (Patagonian catfish) and exotics Salmo trutta (brown trout), Oncorhynchus mykiss (rainbow trout) and Oncorhynchus tshawytscha (Chinook salmon) freshwater fishes widespread in Argentine Patagonia. We characterized the MXR mechanism using a combination of functional assays and Western blot analysis. Our results in different tissues such as liver, gills, muscle and epidermis indicate that the fishes studied have different species-specific levels of MXR activity, being gills and liver the tissues with greater detoxifying activity. Induction of MXR transport activity was also identified in liver tissue from rainbow trout from urban stream suggesting their suitability in the biomonitoring of aquatic environments subjected to urban contaminants.

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Cristina N. Horak and Yanina A. Assef (2017) P-glycoprotein (P-gp) mediated multixenobiotic resistance (MXR) is a mechanism analogous to multidrug resistance, which has been extensively characterized in mammalian tumours. The expression and function of the MXR mechanism has been demonstrated in numerous aquatic organisms and has been proposed as a biomarker for pollution assessment. A close relationship between thermal stress and MXR response has been reported in some aquatic organisms. Seasonal studies in freshwater organisms are scarce and conducted mainly in zebra mussel (Dreissena polymorpha), whose presence has not been reported in South America. The general purpose of the present study was to evaluate seasonal variation of a biomarker, the MXR mechanism, in the worldwide distributed freshwater snail P. acuta. We analyzed the in situ influence of temperature on the biomarker response over an 18-month field study. MXR defence system was evaluated by a combination of functional assays (RB accumulation) and molecular approaches to analyse P-gp expression. The results demonstrated a linear correlation between MXR response, at activity and expression level, and water temperature at sample site, in P. acuta snails. The characterization of the MXR system in worldwide distributed species, including the study of their seasonal fluctuations, could contribute to the increasing interest to incorporate this biomarker to provide an integrated assessment of mussel health status. This work supports the possible use of P. acuta snails with this purpose and also highlights that the occurrence of variations in MXR response related to water temperature has to be taken into account in the interpretation of in situ monitoring studies.

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Introducción: las plantas aromáticas y medicinales son una fuente potencial de componentes antioxidantes. La Patagonia Argentina presenta un ambiente diverso en especies nativas, las cuales deberían ser estudiadas en mayor profundidad debido a su potencial farmacéutico, así como para contribuir a fomentar su conservación. Objetivos: estudiar la actividad antioxidante de infusiones, tinturas y aceites esenciales de las siguientes especies nativas de la Patagonia Argentina: Acantholippia seriphioides (A. Gray) Moldenke, Adesmia boronioides Hook. f., Buddleja globosa Hope, Fabiana imbricata Ruiz & Pav., Solidago chilensis Meyen. Identificar los componentes volátiles presentes en los aceites esenciales. Métodos: se obtuvieron infusiones y tinturas por la guía de las normas de la Farmacopea Argentina VI edición. Los aceites esenciales se obtuvieron por hidrodestilación con un aparato tipo Clevenger. El análisis de los componentes volátiles se realizó mediante cromatografía de gases-espectrometría de masas. Los ensayos de actividad antioxidante se realizaron por el método del difenil-picrilhidrazilo. Resultados: las especies presentaron el siguiente orden de actividad antioxidante: B. globosa > S. chilensis ≥ F. imbricata ≥ A . seriphioides > A. boronioides. Las infusiones de B. globosa, S. chilensis y A. seriphioides, presentaron una actividad antioxidante similar a Camellia sinensis (L.) Kuntze ("té verde") y superior a Ginkgo biloba (L.) Mant (especies reconocidas por su alto contenido de antioxidantes). La actividad encontrada para el aceite esencial de A. seriphioides se deba a sus contenidos en timol y carvacrol. En cuanto a la actividad de S. chilensis podría adjudicarse a su alto porcentaje de limoneno. Conclusiones: este trabajo es el primero que estudia la actividad antioxidante de plantas medicinales y aromáticas en la región noroeste de la Patagonia Argentina; los resultados obtenidos demuestran que las especies estudiadas de dicha región son una fuente rica en compuestos antioxidantes y de potencial valor como suplemento dietario(AU)

Introduction: medicinal and aromatic plants have potential as sources of antioxidant compounds. There is a great diversity of native species in Patagonia Argentina. It is worthy to study them because of its pharmaceutical potential and to help promote conservation. Objectives: to analyze antioxidant activities of herbal teas, tinctures and essential oils of native species from Patagonia Argentina: Acantholippia seriphioides (A. Gray) Moldenke, Adesmia boronioides Hook. f., Buddleja globosa Hope, Fabiana imbricata Ruiz & Pav., Solidago chilensis Meyen. Identify essential oils compounds. Methods: infusions and tinctures were obtained according to Pharmacopoeia Argentina VIth edition. The essential oils were obtained by hydrodistillation in a Clevenger apparatus. Volatiles compounds were analyzed by gas chromatography-mass spectrometry. Antioxidant activity assays were performed by difenil-picrilhidrazilo method. Results: antioxidant activity order was: B. globosa > S. chilensis ≥ F. imbricata ≥ A. seriphioides > A. boronioides. Infusions of B. globosa, S. chilensis and A. seriphioides presented an antioxidant activity similar to Camellia sinensis (L.) Kuntze ("green tea") and higher than Ginkgo biloba (L.) Mant. The A. seriphioides essential oil activity was probably obeyed to thymol and carvacrol presence. S. chilensis activity could be owing to its high limonene content. Conclusions: this study is the first report about antioxidant activity of medicinal and aromatic plants in the northwest region of Patagonia Argentina. The results showed that analyzed species are a rich source of antioxidant compounds and have potential value as a dietary supplement(AU)

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Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with α, ß, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na(+) currents (INa((amil))). We observed a marked reduction in INa((amil)) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant ß-mENaC subunit (ß-S518K) with an open probability of 1 had enhanced INa((amil)) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa((amil)) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 µg/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing α, ß and γ mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane.

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Cell migration/proliferation processes associated with wound healing were measured in BeWo cells at 6 h, when mitosis is still scarce. Cells were cultured in medium with 1% fetal bovine serum to minimize proliferation. BeWo cell migration covered 20.6 +/- 7.0%, 38.0 +/- 5.4%, 16.6 +/- 4.8% and 13.7 +/- 3.6% of the wound when cultivated under control, aldosterone (100 nM, 12 h), aldosterone plus amiloride (10 muM) and amiloride treatments, respectively. When BeWo cells were treated with aldosterone, there was an increase in wound healing (P < 0.05), which was prevented by adding the ENaC blocker amiloride (P < 0.05, n = 16). Immunocytochemistry studies showed that the three ENaC subunits showed greater expression at the leading edge of the wound 3 h after injury, supporting the notion that these proteins participate in a postinjury signal. Antisense oligonucleotides directed against the alpha-ENaC subunit decreased the migratory response of the cells compared to the sense treated cells or the cells without oligonucleotides (P < 0.001, n = 16): 30.2 +/- 3.7%, 17.6 +/- 1.3%, 27.5 +/- 1.5% and 20.2 +/- 1.5% reinvasion of the wound with aldosterone, aldosterone plus antisense, aldosterone plus sense treatments and control conditions, respectively. Aldosterone and amiloride influence wound healing in BeWo cells, probably by their effects upon ENaCs, transmitting a signal to the cell cytoplasm for the release of several agents that promote cell migration.

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The multidrug resistance phenotype (MDR) is one of the major causes of failure in cancer chemotherapy and it is associated with the over-expression of P-glycoprotein (P-gp or MDR1) in tumor cell membranes. A constitutive NF-kappaB activity has been observed in several haematological malignancies and this is associated with its anti-apoptotic role. In the present work, the relationship between NF-kappaB and MDR phenotype was evaluated in wild type K562 human leukemic cells (K562-WT) and in its vincristine-resistant counterpart, K562-Vinc cells. These data showed that K562-Vinc cells, which express an active P-gp, exhibited MDR phenotype. The resistant indexes (IC(50)(K562-Vinc)/IC(50)(K562-WT)) for structurally unrelated drugs like imatinib, doxorubicin and colchicine were 8.0+/-0.3, 2.8+/-0.4 and 44.8+/-8.8, respectively. The imatinib resistance was reversed by P-gp blockade suggesting the involvement of P-gp in imatinib transport. We observed that NF-kappaB was constitutively activated in both cell lines but in a lesser extent in K562-Vinc. The inhibition of NF-kappaB with BAY 11-7082 increased the cytotoxicity of imatinib in K562-Vinc cells but not in K562-WT. Further, the co-administration of imatinib and BAY 11-7082 sensitized multidrug-resistant K562 cells to cell death as detected by increased percentage of annexin V positive cells. The induced cell death in K562-Vinc cells was associated with activation of caspases 9 and 3. Finally, we provide data showing that BAY 11-7082 down-regulates the expression of P-gp suggesting that the activity of NF-kappaB could be functionally associated to this protein in K562 cells. Our results indicate that the vincristine-resistant K562 cells which developed MDR phenotype, exhibited resistance to imatinib associated with a functional P-gp over-expression. This resistance could be partially overcome by the inhibition of NF-kappaB pathway.

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The syncytiotrophoblast acts in human placenta as a transporting barrier regulating the transference of nutrients, solutes and water between maternal and fetal blood. This transepithelial transport involves movement of Na+ and its contribution to the osmotic pressure is an important determinant of the extracellular fluid volume. ENaC is a channel that mediates entry of Na+ from the luminal fluid into the cells in many reabsorbing epithelia; it is aldosterone, vasopressin, insulin and catecholamine-inducible, modulated by estrogens and progesterone and blocked by amiloride and its analogs. Multiple proteases are involved in the proteolytic processing and activation of ENaC subunits and aldosterone alters the protease-protease inhibitors balance. ENaC is also expressed in human placenta; although its function is not well known, the Na+ conductive properties may participate in electrolyte and extracellular volume homeostasis. The activity of ENaC channels and other ion channels and transporters is regulated by the state of actin filaments; on the other hand, changes in volume influence the actin cytoskeleton. Thus, there is an interaction between ENaC and components of the apical membrane cytoskeleton. In addition to their role in cellular homeostasis and electrical properties, Na+ currents through ENaC and other sodium channels are involved in cell migration, well documented in normal and cancer cells. In this work we presented evidences supporting the hypothesis that ENaC channels are required for the migration of BeWo cells, a human hormone-synthesizing trophoblastic cell line that express the three subunits of the ENaC channels. BeWo cell line has also been used as a model to investigate the placental transport mechanisms.

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En la placenta humana, el sinciciotrofoblasto es la barrera que regula el transporte de nutrientes, solutos y agua entre la sangre materna y fetal. Dentro de este movimiento transepitelial se encuentra el del Na+, su contribución a la presión osmótica es fundamental en la regulación del volumen de líquido extracelular. El canal epitelial de sodio sensible al amiloride (ENaC) media el transporte de Na+ desde el lumen hacia el interior celular en numerosos epitelios absortivos. Está regulado por la aldosterona, vasopresina, catecolaminas, estrógenos y progesterona. Es bloqueado por el amiloride y sus análogos. Para su activación, diversas proteasas lo escinden en la membrana plasmática y esto a su vez es regulado por la aldosterona. El ENaC está expresado también en la placenta humana y aunque su función no es conocida, podría participar en la homeostasis de agua y electrolitos. El ENaC también es influenciado por el estado de las proteínas del citoesqueleto y los cambios en el volumen celular alteran a su vez a éste. De esta manera existe una relación entre el ENaC y el citoesqueleto. Además, las corrientes de Na+ por el ENaC y otros canales de sodio participan en la migración celular en células normales y cancerosas. Aquí presentamos evidencias que avalan la hipótesis que el ENaC es necesario para la migración celular en células BeWo, derivadas del trofoblasto humano, que sintetizan hormonas y expresan el ENaC. Las células BeWO han sido utilizadas como modelo experimental para estudiar el transporte en células de placenta.

The syncytiotrophoblast acts in human placenta as a transporting barrier regulating the transference of nutrients, solutes and water between maternal and fetal blood. This transepithelial transport involves movement of Na+ and its contribution to the osmotic pressure is an important determinant of the extracellular fluid volume. ENaC is a channel that mediates entry of Na+ from the luminal fluid into the cells in many reabsorbing epithelia; it is aldosterone, vasopressin, insulin and catecholamine-inducible, modulated by estrogens and progesterone and blocked by amiloride and its analogs. Multiple proteases are involved in the proteolytic processing and activation of ENaC subunits and aldosterone alters the protease-protease inhibitors balance. ENaC is also expressed in human placenta; although its function is not well known, the Na+ conductive properties may participate in electrolyte and extracellular volume homeostasis. The activity of ENaC channels and other ion channels and transporters is regulated by the state of actin filaments; on the other hand, changes in volume influence the actin cytoskeleton. Thus, there is an interaction between ENaC and components of the apical membrane cytoskeleton. In addition to their role in cellular homeostasis and electrical properties, Na+ currents through ENaC and other sodium channels are involved in cell migration, well documented in normal and cancer cells. In this work we presented evidences supporting the hypothesis that ENaC channels are required for the migration of BeWo cells, a human hormone-synthesizing trophoblastic cell line that express the three subunits of the ENaC channels. BeWo cell line has also been used as a model to investigate the placental transport mechanisms.

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The present study was performed to assay sodium currents in BeWo cells. These cells comprise a human trophoblast cell line which displays many of the biochemical and morphological properties similar to those reported for the in uterus proliferative cytotrophoblast. For whole-cell patch-clamp experiments, BeWo cells treated for 12 h with 100 nM aldosterone were exposed to 8Br-cAMP, a membrane-permeable cAMP analogue, to induce channel activity. Cells showed an amiloride-sensitive ion current (IC50 of 5.77 microM). Ion substitution experiments showed that the amiloride-sensitive current carried cations with a permeability rank order of Li+ > Na+ > K+ > NMDG (PLi/PNa = 1.3, PK/PNa = 0.6, PNMDG/PNa = 0.2). In cells pretreated with aldosterone, we observed that nearly half of successful patches had sodium channels with a linear conductance of 6.4 +/- 1.8 pS, a low voltage-independent Po and a PK/PNa of 0.19. Using RT-PCR, we determined that control cells express the alpha-, but not beta- and gamma-, epithelial sodium channel (ENaC) mRNA. When cells were treated with aldosterone (100 nM, 12 h), all alpha-, beta- and gamma-ENaC mRNAs were detected. The presence of ENaC subunit proteins in these cells was confirmed by Western blot analysis and immunolocalization with specific ENaC primary antibodies. In summary, our results suggest that BeWo cells express ENaC subunits and that aldosterone was able to modulate a selective response by generating amiloride-sensitive sodium currents similar to those observed in other human tissues.

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The human ether-a-go-go related gene (HERG1) K+ channel is expressed in neoplastic cells, in which it was proposed to play a role in proliferation, differentiation and/or apoptosis. K562 cells (a chronic myeloid leukemic human cell line) express both the full-length (herg1a) and the N-terminally truncated (herg1b) isoforms of the gene, and this was confirmed with Western blots and coimmunoprecipitation experiments. Whole-cell currents were studied with a tail protocol. Seventy-eight percent of cells showed a HERG1-like current: repolarization to voltages negative to -40 mV produced a transient peak inward tail current, characteristic of HERG1 channels. Cells were exposed to a HERG-specific channel blocker, E4031. Half-maximal inhibitory concentration (IC50) of the blocker was 4.69 nM: The kinetics of the HERG1 current in K562 cells resembled the rapid component of the native cardiac delayed rectifier current, known to be conducted by heterotetrameric HERG1 channels. Fast and slow deactivation time constants at -120 mV were 27.5 and 239.5 ms, respectively. Our results in K562 cells suggest the assembling of heterotetrameric channels, with some parameters being dominated by one of the isoforms and other parameters being intermediate. Hydrogen peroxide was shown to increase HERG1a K+ current in heterologous expression systems, which constitutes an apoptotic signal. However, we found that K562 HERG1 whole-cell currents were not activated by H2O2.

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The syncytiotrophoblast (SCT), a multinucleated epithelium forming the outer layer of chorionic villi, acts in human placenta as a transporting barrier regulating the transference of nutrients, solutes and water between maternal and fetal blood. Electrolyte homeostasis and extracellular fluid volume are maintained primarily by regulated Na+ transport. The present study was conducted to analyze the presence of the epithelial Na channel (ENaC) in placental tissue from normal and pre-eclamptic women and in BeWo cell, a model of a human SCT. Changes in the expression of these proteins during sodium transport across the placenta may be related to the pathogeny of pre-eclampsia. The role that ENaC and Na+ transport deregulation play on human placental tissues still remains unknown although in aldosterone-responsive epithelial cells (kidney, colon), abnormalities upregulating its activity lead to increased Na+ uptake and hypertension (i.e. Liddle's syndrome) whereas a diminished channel activity can result in the pseudohypoaldosteronisn syndrome with salt loss and hypotension. Our results show that ENaC is expressed in the apical membrane of normal syncytiotrophoblast. The amplified fragment of alpha-ENaC was cloned and sequenced having a 100% identity with the sequence of (alpha-ENaC obtained from GenBank (SCNN1A, accession number Z92981). We found that the transcription of the alpha-ENaC mRNA was not detectable in preeclamptic placentas and the protein was not observed with immunohistochemistry staining, probably indicating a low protein expression level. In BeWo cells ENac was found and its expression is regulated by aldosterone, vasopressin, progesterone and estradiol. With patch clamp techniques we studied the currents trough ENaO channels in Bewo cells. We observed currents that were blocked by 10 microM amiloride in cells incubated in 100 nM aldosterone for 12 hs. The amplitude of this current was 20-fold the basal current, a reversal potential of 3 mV and a conductance of 127 +/- 26 pS/pF with pulses between -60 and -140 mV. These characteristics are similar to those reported in ENaC channels in several tissues. Although their roles in placenta are still poorly understood, the differences in the expression of ENaC in pre-eclamptic placentas may have consequences for ion transport and these data could lead to future studies concerning the mechanism involved in the pathophysiology of pre-eclampsia.

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El sinciciotrofoblasto (SCT) de placenta humana regula la transferencia de solutos y agua entre la sangre fetal y materna. En el presente trabajo observamos que el canal de sodio ENaC (asociado a cuadros como el síndrome de Liddle y pseudohipoaldosteronismo) está presente en la membrana apical del SCT y que la subunidad del canal tiene una expresión reducida en placentas con hipertensión gestacional (preeclampsia). Realizamos estudios a nivel de expresión de ARN (RT-PCR) y a nivel proteico (western blot e inmunohistoquímica). En la línea celular BeWo (modelo de SCT humano) el canal se encuentra presente y la expresión del mismo es regulada por las hormonas aldosterona, vasopresina, estradiol y progesterona. Analizamos la actividad del ENaC por electrofisiología y observamos corrientes sensibles a amiloride (10 μM) cuando las células BeWo se cultivaron 12 horas con aldosterona (100 nM). Esta corriente presentó una magnitud 20 veces mayor que las corrientes basales, un potencial de reversión cercano a 3 mV y una conductancia de 127 ± 26 pS/ pF entre los pulsos de –60 y –140 mV aplicados. Las características de esta corriente son similares a las producidas por ENaC en otros tejidos y evidencian la presencia de un canal funcional. El papel del ENaC en el SCT es poco comprendido, aunque la diferencia de expresión en la preeclampsia podría tener consecuencias para el transporteplacentario de agua y iones. Nuestros datos son un aporte para futuros estudios de los mecanismos involucrados en la patofisiología de la preeclampsia.

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El sinciciotrofoblasto (SCT) de placenta humana regula la transferencia de solutos y agua entre la sangre fetal y materna. En el presente trabajo observamos que el canal de sodio ENaC (asociado a cuadros como el síndrome de Liddle y pseudohipoaldosteronismo) está presente en la membrana apical del SCT y que la subunidad del canal tiene una expresión reducida en placentas con hipertensión gestacional (preeclampsia). Realizamos estudios a nivel de expresión de ARN (RT-PCR) y a nivel proteico (western blot e inmunohistoquímica). En la línea celular BeWo (modelo de SCT humano) el canal se encuentra presente y la expresión del mismo es regulada por las hormonas aldosterona, vasopresina, estradiol y progesterona. Analizamos la actividad del ENaC por electrofisiología y observamos corrientes sensibles a amiloride (10 μM) cuando las células BeWo se cultivaron 12 horas con aldosterona (100 nM). Esta corriente presentó una magnitud 20 veces mayor que las corrientes basales, un potencial de reversión cercano a 3 mV y una conductancia de 127 ± 26 pS/ pF entre los pulsos de ¹60 y ¹140 mV aplicados. Las características de esta corriente son similares a las producidas por ENaC en otros tejidos y evidencian la presencia de un canal funcional. El papel del ENaC en el SCT es poco comprendido, aunque la diferencia de expresión en la preeclampsia podría tener consecuencias para el transporteplacentario de agua y iones. Nuestros datos son un aporte para futuros estudios de los mecanismos involucrados en la patofisiología de la preeclampsia. (AU)

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El sinciciotrofoblasto (SCT) de placenta humana regula la transferencia de solutos y agua entre la sangre fetal y materna. En el presente trabajo observamos que el canal de sodio ENaC (asociado a cuadros como el síndrome de Liddle y pseudohipoaldosteronismo) está presente en la membrana apical del SCT y que la subunidad del canal tiene una expresión reducida en placentas con hipertensión gestacional (preeclampsia). Realizamos estudios a nivel de expresión de ARN (RT-PCR) y a nivel proteico (western blot e inmunohistoquímica). En la línea celular BeWo (modelo de SCT humano) el canal se encuentra presente y la expresión del mismo es regulada por las hormonas aldosterona, vasopresina, estradiol y progesterona. Analizamos la actividad del ENaC por electrofisiología y observamos corrientes sensibles a amiloride (10 μM) cuando las células BeWo se cultivaron 12 horas con aldosterona (100 nM). Esta corriente presentó una magnitud 20 veces mayor que las corrientes basales, un potencial de reversión cercano a 3 mV y una conductancia de 127 ± 26 pS/ pF entre los pulsos de ¹60 y ¹140 mV aplicados. Las características de esta corriente son similares a las producidas por ENaC en otros tejidos y evidencian la presencia de un canal funcional. El papel del ENaC en el SCT es poco comprendido, aunque la diferencia de expresión en la preeclampsia podría tener consecuencias para el transporteplacentario de agua y iones. Nuestros datos son un aporte para futuros estudios de los mecanismos involucrados en la patofisiología de la preeclampsia. (AU)

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In this study, the expression and functional characterization of currents through the CFTR (cystic fibrosis transmembrane regulator) and ORCC (outwardly rectifying chloride channels) were determined in wild-type K562 chronic human leukemia cells (K562-WT) and in its resistant counterpart, the vincristine resistant cell line (K562-Vinc). Expression of the CFTR and MDR1 (multidrug resistant) gene products was determined by a semi-quantitative RT-PCR protocol. The amplified products in K562-WT and K562-Vinc showed two bands corresponding to CFTR and MDR1. MDR1 mRNA increased by 20-fold in K562-Vinc whereas no change in CFTR mRNA levels was observed. CFTR and ORCC channel activity were measured with a whole cell configuration of the patch clamp technique. Forskolin (40 microM n activator of adenylate cyclase, added to the extracellular side increased the current in both cell lines. A fraction of the activated whole cell currents was inhibited by 500 microM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and subsequent addition of 500 microM diphenylamine-2-carboxylate (DPC plus DIDS) further inhibited the remaining currents. The levels of forskolin-activated currents and subsequent blockade were similar in both cell lines. The effect of forskolin was prevented in cells previously exposed to 500 microM DPC. The effects of DIDS and DPC on the forskolin-activated whole cell currents support the idea that both CFTR and ORCC are generating a significant fraction of these currents with DIDS inhibiting ORCC currents and DPC inhibiting CFTR currents when the blockers are added one after another to the extracellular side. Finally, we show that exposure of K562 cells to vincristine which results in the over expression of MDR1 is not accompanied by a significant down regulation of CFTR as in other cells.

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In this study, two electrode voltage clamp technique was used to assess the ionic current of oocytes of the South American toad Bufo arenarum and to study the dependence of these currents on the extracellular and intracellular Ca2+ concentrations. Ca2+ chelators, ionomycin -a calcium ionophore- and thapsigargin, a blocker of the Ca2+ pump of the sarcoplasmic reticulum, were used. The main results were the following: Most oocytes showed a voltage activated rectifying conductance. Ionomycin (1 microM) increased inward and outward currents in control solution. The effect of ionomycin was blocked partially at negative potentials and was blocked completely at positive potentials in absence of extracellular Ca2+. When the oocytes were treated with thapsigargin (2 microM) or BAPTA-am, a membrane-permeant intracellular chelator in control solution (10 microM), ionomycin did not increased either inward nor outward currents. The conclusion of our experiments is that there are two sources of Ca2+ for activation of the current induced by ionomycin, the cytoplasmic stores and the extracellular space. We believe ionomycin directly translocates Ca2+ from the SER into the cytoplasm but not from the extracellular medium. Ca2+ entry probably occurs through store-operated-Ca-channels.

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In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 microM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.

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