Myrna Virreira

Congenital Chagas disease involves Trypanosoma cruzi sub-lineage IId in the northwestern province of Salta, Argentina

Trypanosoma cruzi is genetically classified into six discrete phylogenetic lineages on the basis of different genetic markers. Identifying lineages circulating among humans in different areas is essential to understand the molecular epidemiology of Chagas disease. In the present study, 18 T. cruzi isolates from congenitally infected newborns in the northwestern province of Salta-Argentina were studied by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD). All isolates were typed by MLEE and RAPD as belonging to T. cruzi IId. Analysis of minor variants of TcIId using probes hybridizing with hypervariable domains of kDNA minicircles, detected three variants with a similar distribution among the isolates. Our findings confirm the presence of T. cruzi IId among congenitally infected newborns in northwestern Argentina and support the assumption that human infection by T. cruzi in the Southern Cone countries of Latin America is due principally to T. cruzi II.

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.

Contrasting effects of glycerol and urea transport on rat pancreatic beta-cell function.

Background/aims: Pancreatic β-cell function is influenced by changes in cell volume. Such volume changes depend on water permeability of the plasma membrane, conferred in part by aquaporins. Islet cells express aquaporin 7 (AQP7), which is permeable to urea and glycerol in addition to water. We therefore investigated the effects of glycerol and urea on rat pancreatic β-cell function. Methods: Electrical activity and whole-cell current were studied using the perforated patch technique. Cell volume was measured by video-imaging and insulin release by radioimmunoassay. Aquaporin 7 expression was studied by RT-PCR, Western blot and double fluorescent immunolabelling. Results: The isosmotic addition of glycerol and urea resulted in depolarization of the plasma membrane and electrical activity, accompanied by β-cell swelling, activation of the volume-regulated anion channel (VRAC) and insulin release. However, the effects of glycerol, in contrast to urea, persisted throughout exposure to the osmolyte. Glycerol also caused β-cell activation when added hyperosmotically. A non-metabolizable glycerol analogue had comparable effects to urea on β-cells. The expression of AQP7 was demonstrated in rat β-cells. Conclusion: Glycerol and urea can activate β-cells via their rapid uptake across the β-cell plasma membrane, possibly via AQP7. This results in cell swelling, VRAC activation, electrical activity and insulin release. Glycerol appears to exert an additional effect, possibly related to its intracellular metabolism.

A new role for aquaporin 7 in insulin secretion.

Bacgrouns/Aims: Several insulinotropic agents were recently reported to cause β-cell swelling. The possible participation of AQP7 to water transport was investigated in AQP7+/ + or AQP7-/- mice. Methods: Aquaporin expression, insulin secretion, cell swelling and electrical activity were investigated in pancreatic islets. Results: RT-PCR revealed the expression of AQP5 and AQP8 mRNA. Double immunofluorescent labeling indicated their presence in β-cells. Whilst basal insulin release from isolated pancreatic islets incubated at 2.8 mM D-glucose did not differ between AQP7+/ + or AQP7-/- mice, the secretion of insulin evoked by the omission of 50 mM NaCl, the substitution of 50 mM NaCl by 100 mM glycerol or a rise in D-glucose concentration to 8.3 mM and 16.7 mM was severely impaired in the islets from AQP7-/- mice. Yet, exposure of β-cells to either the hypotonic medium or a rise in D-glucose concentration caused a similar degree of swelling and comparable pattern of electrical activity in cells from AQP7+/ + and AQP7-/- mice. Both the cell swelling and change in membrane potential were only impaired in AQP7-/- cells when exposed to 50 mM glycerol. Conclusion: It is proposed, therefore, that AQP7 may, directly or indirectly, play a role at a distal site in the exocytotic pathway.

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

Both mouse and rat pancreatic islet β‐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 [2‐3H]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 [2‐3H]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, [2‐3H]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. J. Cell. Physiol. 221: 424–429, 2009.

Analysis of aquaporin expression in liver with a focus on hepatocytes

A deeper understanding of aquaporins (AQPs) expression and transcriptional regulation will provide useful information for liver pathophysiology. We established a complete AQPs mRNA expression profile in human and mouse liver, as well as protein localization of expressed AQPs. Additionally, the modulation of AQPs mRNA levels in response to various agents was determined in human HuH7 cells and in primary culture of mouse hepatocytes. AQP1, AQP3, AQP7, AQP8, and AQP9 mRNA and protein expressions were detected in human liver, while only AQP6 and AQP11 mRNAs were detected. We reported for the first time the localization of AQP3 in Kupffer cells, AQP7 in hepatocytes and endothelial cells, and AQP9 in cholangiocytes. In addition, we confirmed the localization of AQP1 in endothelial cells, and of AQP8 and AQP9 in hepatocytes. On HuH7 cells, we reported the presence of AQP4 mRNA, confirmed the presence of AQP3, AQP7, and AQP11 mRNAs, but not of AQP8 mRNA. On primary culture of murine hepatocytes, AQP1 and AQP7 mRNAs were identified, while the presence of AQP3, AQP8, AQP9, and AQP11 mRNAs was confirmed. At the protein level, murine endothelial liver cells expressed AQP1 and AQP9, while hepatocytes expressed AQP3, AQP7, AQP8, and AQP9, and macrophages expressed AQP3. Dexamethasone, forskolin, AICAR, rosiglitazone, octanoylated, and non-octanoylated ghrelin regulated some AQP expression in primary culture of murine hepatocytes and human HuH7 cells. Additional studies will be required to further assess the role of AQPs expression in human and murine liver and understand the transcriptional regulation of AQPs in hepatocytes under pathophysiological conditions.

Pancreatic beta-cells: role of glycerol and aquaglyceroporin 7

Pancreatic β-cells originate from gut endoderm during development. Pancreatic endocrine cells represent about 10% of the mature pancreatic cells, and β-cells represent the majority of endocrine cells. β-cells secrete insulin in response to elevation of nutrient concentrations. Insulin maintains glucose homeostasis by stimulating glucose uptake into muscle and adipose tissue. Aquaglyceroporin 7, permeable to water, glycerol and urea, is expressed in pancreatic β-cells and was recently described as being involved in the control of insulin secretion.

Expression of the electrogenic Na+-HCO3-- cotransporter NBCe1 in tumoral insulin-producing BRIN-BD11 cells

Background/aims: The expression of the electrogenic Na+-HCO3--cotransporter NBCe1 was recently documented in rat pancreatic islet B-cells, it being speculated that such a protein participates in the extrusion of bicarbonate generated by the oxidative catabolism of nutrients from insulin-producing cells. Considering the prevalence of a Crabtree effect in tumoral insulin-producing cells, the possible presence of NBCe1 was now investigated in BRIN-BD11 cells, an insulin-producing cell line established by electrofusion of normal pancreatic B-cells with immortalized RINm5F cells. Methods: The possible presence of NBCe1 in BRIN-BD11 cells was investigated by RT-PCR, western blot analysis and immunocytochemistry. The release of insulin and net uptake of 22Na+ were also measured in the BRIN-BD11 cells. Results: RT-PCR, western blot analysis and immunocytochemistry documented the presence of NBCe1 in BRIN-BD11 cells. A reported inhibitor of NBCe1, i.e. tenidap, (50-100 μM), inhibited basal and hypotonicity-induced insulin release from the BRIN-BD11 cells, whilst increasing the net uptake of 22Na+ by the same cells. The latter effect was, in relative terms, more pronounced in the presence than absence of ouabain. Conclusion: BRIN-BD11 cells, like normal pancreatic islet B-cells, express NBCe1, with predominance of the B variant of this electrogenic Na+-HCO3--cotransporter.

Immunocytochemistry of GLUT2, uptake of fluorescent desnitroso-streptozotocin analogs and phosphorylation of D-glucose in INS-1E cells.

The non-invasive imaging of GLUT2-expressing cells remains a challenge. As streptozotocin, and similarly alloxan, may be transported into cells by GLUT2, the major aim of the present study was to assess the possible use of fluorescent desnitroso-streptozotocin analogs for in vitro labeling of GLUT2-expressing cells. INS-1E cells, human embryonic kidney (HEK) cells, rat isolated pancreatic islets, rat hepatic cells, rat exocrine pancreatic cells and tumoral insulin-producing BRIN-BD11 cells were incubated in the presence of two distinct fluorescent desnitroso-streptozotocin analogs, probes A and B. The immunocytochemistry of GLUT2 in INS-1E cells and the phosphorylation of D-glucose by INS-1E cell homogenates were also examined. The uptake of probes A and B (12.0 µM) by INS-1E cells yielded apparent intracellular concentrations approximately one order of magnitude higher than the extracellular concentration. The two probes differed from one another by the absolute values for their respective uptake and time course, but not so by the pattern of their concentration dependency. Comparable results were recorded in HEK cells, rat isolated pancreatic islets and hepatocytes. Vastly different findings were recorded, however, in rat exocrine pancreatic cells, which do not express GLUT2. Moreover, an unusual concentration dependency for the uptake of each probe was observed in tumoral BRIN-BD11 cells. It is proposed that suitable fluorescent desnitroso-streptozotocin analogs may be used to label GLUT2-expressing cells.

Glucose activation of the glucagon receptor gene: Functional dissimilarity with several other glucose response elements

Abstract:  The glucagon receptor (GLR) expression is positively regulated by glucose. This regulation is allowed by the presence, in the promotor of the rat GLR gene, of a sequence feature similar to the two E‐boxes motifs constituting the carbohydrate response elements (ChoRE) described for several glycolytic and lipogenic enzyme genes. Using reporter gene assays, we demonstrated here that, despite structural homologies with these ChoREs, the GLR gene glucose response element presents various functional dissimilarities.Testing glucose analogs, we demonstrated that, as for other genes, the glucose must be first phosphorylated. However, at variance with others homologue genes, our data showed the implication of the nonoxidative branch of the pentose phosphate pathway in the transmission of the glucose signal and lack of inhibition by adenosine monophosphate (AMP)‐kinase . Furthermore, the activity of our reporter gene was strongly stimulated by butyrate, propionate, and acetate. This observation contrasts with fatty‐acid‐induced inhibition of the glucose activation, observed for all other genes containing homolog ChoREs. We also showed that glucose and butyrate influence the reporter gene expression via different features.