Anoctamin-1/TMEM16A is the major apical iodide channel of the thyrocyte.

Iodide is captured by thyrocytes through the Na(+)/I(-) symporter (NIS) before being released into the follicular lumen, where it is oxidized and incorporated into thyroglobulin for the production of thyroid hormones. Several reports point to pendrin as a candidate protein for iodide export from thyroid cells into the follicular lumen. Here, we show that a recently discovered Ca(2+)-activated anion channel, TMEM16A or anoctamin-1 (ANO1), also exports iodide from rat thyroid cell lines and from HEK 293T cells expressing human NIS and ANO1. The Ano1 mRNA is expressed in PCCl3 and FRTL-5 rat thyroid cell lines, and this expression is stimulated by thyrotropin (TSH) in rat in vivo, leading to the accumulation of the ANO1 protein at the apical membrane of thyroid follicles. Moreover, ANO1 properties, i.e., activation by intracellular calcium (i.e., by ionomycin or by ATP), low but positive affinity for pertechnetate, and nonrequirement for chloride, better fit with the iodide release characteristics of PCCl3 and FRTL-5 rat thyroid cell lines than the dissimilar properties of pendrin. Most importantly, iodide release by PCCl3 and FRTL-5 cells is efficiently blocked by T16Ainh-A01, an ANO1-specific inhibitor, and upon ANO1 knockdown by RNA interference. Finally, we show that the T16Ainh-A01 inhibitor efficiently blocks ATP-induced iodide efflux from in vitro-cultured human thyrocytes. In conclusion, our data strongly suggest that ANO1 is responsible for most of the iodide efflux across the apical membrane of thyroid cells.

Does NAD(P)H oxidase-derived H2O2 participate in hypotonicity-induced insulin release by activating VRAC in ß-cells?

NAD(P)H oxidase (NOX)-derived H(2)O(2) was recently proposed to act, in several cells, as the signal mediating the activation of volume-regulated anion channels (VRAC) under a variety of physiological conditions. The present study aims at investigating whether a similar situation prevails in insulin-secreting BRIN-BD11 and rat ß-cells. Exogenous H(2)O(2) (100 to 200 µM) at basal glucose concentration (1.1 to 2.8 mM) stimulated insulin secretion. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous H(2)O(2). In patch clamp experiments, exogenous H(2)O(2) was observed to stimulate NPPB-sensitive anion channel activity, which induced cell membrane depolarization. Exposure of the BRIN-BD11 cells to a hypotonic medium caused a detectable increase in intracellular level of reactive oxygen species (ROS) that was abolished by diphenyleneiodonium chloride (DPI), a universal NOX inhibitor. NOX inhibitors such as DPI and plumbagin nearly totally inhibited insulin release provoked by exposure of the BRIN-BD11 cells to a hypotonic medium. Preincubation with two other drugs also abolished hypotonicity-induced insulin release and reduced basal insulin output: 1) N-acetyl-L-cysteine (NAC), a glutathione precursor that serves as general antioxidant and 2) betulinic acid a compound that almost totally abolished NOX4 expression. As NPPB, each of these inhibitors (DPI, plumbagin, preincubation with NAC or betulinic acid) strongly reduced the volume regulatory decrease observed following a hypotonic shock, providing an independent proof that VRAC activation is mediated by H(2)O(2). Taken together, these data suggest that NOX-derived H(2)O(2) plays a key role in the insulin secretory response of BRIN-BD11 and native ß-cells to extracellular hypotonicity.

Functional role of aquaglyceroporin 7 expression in the pancreatic beta-cell line BRIN-BD11.

Both mouse and rat pancreatic islet beta-cells were recently found to express aquaglyceroporin 7 (AQP7). In the present study, the expression and role of AQP7 in the function of BRIN-BD11 cells were investigated. AQP7 mRNA and protein were detected by RT-PCR and Western blot analysis, respectively. In an isoosmolar medium, the net uptake of [2-(3)H]glycerol displayed an exponential time course reaching an equilibrium plateau value close to its extracellular concentration. Within 2 min of incubation in a hypotonic medium (caused by a 50 mM decrease in NaCl concentration), the [2-(3)H]glycerol uptake averaged 143.2 +/- 3.8% (n = 24; P < 0.001) of its control value in isotonic medium, declining thereafter consistently with previously demonstrated volume regulatory decrease. When isoosmolarity was restored by the addition of 100 mM urea to the hypotonic medium, [2-(3)H]glycerol uptake remained higher (112.1 +/- 2.8%, n = 24; P < 0.001) than its matched control under isotonic conditions, indicating rapid entry of urea and water. Insulin release by BRIN-BD11 cells was 3 times higher in hypotonic than in isotonic medium. When glycerol (100 mM) or urea (100 mM) were incorporated in the hypotonic medium, the insulin release remained significantly higher than that found in the control isotonic medium, averaging respectively 120.2 +/- 4.2 and 107.0 +/- 3.8% of the paired value recorded in the hypotonic medium. These findings document the rapid entry of glycerol and urea in BRIN-BD11 cells, likely mediated by AQP7.

Detección de la heterogeneidad molecular de Trypanosoma cruzi / Detection of molecular heterogeneity of Trypanosoma cruzi

Congenital transmission of T. cruzi in Cochabamba affects 6% of newborns from infected mothers. Only limited information is available on the type of transmitted parasites. However, it is well established that T. cruzi isolated from various vectors as well from host animals are highly heterogeneous. In our presentation we analyse aspects of molecular heterogeneity of T. cruzi and we review methods used for the molecular typing of T. cruzi lineages. Experimentally, we performed the PCR amplification of [quot ]Sequence-characterised region Markers[quot ] for typing T. cruzi isolated from umbilical blood of newborns in Cochabamba. We compared these results with those we obtained from general infected population. All 16 analysed, congenitally infected samples were of lineage IId. Our data also indicated that this lineage was found in about 80% of samples originated from general infected population in Cochabamba.

Comparación de métodos de PCR para el diagnóstico de la infección congenita por Trypanosoma cruzi / Comparison of PCR methods for the diagnosis of congenital Trypanosoma cruzi infection

PCR is a potentially interesting diagnostic tool to detect congenital T. cruzi infection. We have compared the sensitivity and capacity of a battery of T. cruzi PCR primers to detect the complete spectrum of known T. cruzi lineages, in order to improve and simplify the detection of infection in neonatal blood. We found that the primers Tcz1/Tcz2, targeting the 195 bp satellite repeat, detected all the parasitic lineages with the same sensitivity For all other tested primers (nDNA primers: BP1/BP2, 01/02, Pon1/ Pon2 and Tca1/Tca2; kDNA primers: S35VS36, 121/122), either, the intensity of amplicons varied according to T. cruzi lineages, or the assess were less sensitive. In order to better assess such PCR protocol, we assayed 311 samples of neonatal blood previously tested with parasitological methods. Reliability of our PCR test was demonstrated since all the 18 blood samples from newborns with congenital T. cruzi infection were positive, whereas the remaining samples (30 from control newborns of uninfected mothers and 262 out of 263 from babies, parasitologically negative, born from infected mothers) were negative. As our PCR method is simple, reliable, robust and cheap, it appears suitable for the detection of T. cruzi infection in neonatal blood.

Anencefalia en embarazo de 16 semanas / Anencephalia in pregnancy of 16 weeks

Paciente de 21 años de edad, primigrávida, vista en consulta a las 15 semanas de gestación. Realizados los estudios de rutina, mediante la ultrasonografía, se diagnostica anecefalia, razón por la que luego de evaluar la situación se decide interrumpir el embarazo