Gabriela I. Marino

High levels of human chorionic gonadotropin (hCG) correlate with increased aquaporin-9 (AQP9) expression in explants from human preeclamptic placenta.

Trophoblastic abnormalities have a central role in the pathophysiology of preeclampsia, and some placental hormones, such as human chorionic gonadotropin (hCG), could affect the placental function. Here, we hypothesized that the elevated serum levels of hCG may be involved in the increased aquaporin-9 (AQP9) protein expression in preeclamptic placentas via adenosine 3′,5′-cyclic phosphate (cAMP) pathways. Normal placental explants were cultured with different concentrations of recombinant hCG or 8-Br-cAMP, a potent analogue of cAMP. We evaluated AQP9 protein expression and localization. After both treatments, we localized AQP9 in the apical membrane of syncytiotrophoblast and in the cytoplasm. We also observed a concentration-dependent effect on AQP9 protein expression. In addition, water uptake increased 1.6-fold in explants treated with hCG. Our results suggest that hCG may increase AQP9 protein expression and functionality via cAMP pathways. Although, in preeclamptic placentas high levels of hCG may upregulate AQP9 protein expression, AQP9 functionality was reduced possibly by other factors.

ENaC Channels in Oocytes from Xenopus laevis and their Regulation by xShroom1 Protein

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, β, 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 β-mENaC subunit (β-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, β 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.

Cystic fibrosis transmembrane regulator (CFTR) in human trophoblast BeWo cells and its relation to cell migration

INTRODUCTION ENaC and CFTR are coexpressed in epithelia and have positive or negative functional interactions. In addition, ENaC and CFTR promote migration in placental trophoblastic cells and human airway cells, respectively. Here we tested the idea if CFTR is functionally expressed in BeWo cells, a trophoblastic cell line, and if it is involved in their migratory behavior. METHODS CFTR expression was studied in BeWo cells with RT-PCR, biotinylation and Western blot. Ion currents were analyzed with patch clamp, and cell migration with the wound healing method. RESULTS The mature CFTR 160-kDa band was present, and its localization at the surface membrane was confirmed. Forskolin (20 μM), an adenylate cyclase activator, was used for channel activation, and subsequently CFTR(inh)-172 (2 μM) for its inhibition. The conductances in the presence of CFTR(inh)-172 plus forskolin (16.0 ± 0.7 pS/pF and 32.6 ± 1.5 pS/pF) were significantly lower than in presence of only forskolin (29.7 ± 0.9 and 47.0 ± 2.0 pS/pF). The conductance of CFTR(inh)-172 inhibited currents was 14.9 ± 0.7 pS/pF with a linear I-V relationship illustrating the nonrectifying properties of the CFTR. Cell migration was measured and covered 11.2 ± 0.4, 24.0 ± 1.7 and 13.9 ± 1.0% of the wound when cells were cultivated under control, forskolin, and forskolin plus CFTR(inh)-172, respectively. Proliferation was not changed by any of the treatments. CONCLUSIONS Our results shows that BeWo cells functionally express the CFTR which plays a role in the wound healing increasing the cell migration process.

CFTR channel in oocytes from Xenopus laevis and its regulation by xShroom1 protein.

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in Xenopus laevis oocytes, and it is required for the expression of the epithelial sodium channel (ENaC). As there is a close relationship between ENaC and the cystic fibrosis transmembrane regulator (CFTR), we examined the action of xShroom1 on CFTR expression and activity. Biotinylation was used to measure CFTR surface expression, and currents were registered with voltage clamp when stimulated with forskolin and 3-isobutyl-1-methylxanthine. Oocytes were coinjected with CFTR complementary RNAs (cRNAs) and xShroom1 sense or antisense oligonucleotides. We observed an increment in CFTR currents and CFTR surface expression in oocytes coinjected with CFTR and xShroom1 antisense oligonucleotides. MG-132, a proteasome inhibitor, did not prevent the increment in currents when xShroom1 was suppressed by antisense oligonucleotides. In addition, we inhibited the delivery of newly synthesized proteins to the plasma membrane with BFA and we found that the half-life of plasma membrane CFTR was prolonged when coinjected with the xShroom1 antisense oligonucleotides. Chloroquine, an inhibitor of the late endosome/lysosome, did not significantly increase CFTR currents when xShroom1 expression was inhibited. The higher expression of CFTR when xShroom1 is suppressed is in concordance with the functional studies suggesting that the suppression of the xShroom1 protein resulted in an increment in CFTR currents by promoting the increase of the half-life of CFTR in the plasma membrane. The role of xShroom1 in regulating CFTR expression could be relevant in the understanding of the channel malfunction in several diseases.

xshroom1 Regulates the Number of ENaC Channels Inserted in the Membrane of Oocytes From XENOPUS Laevis

Shroom is a family of proteins linked to the actin cytoskeleton. We studied its effect upon the currents through ENaC channels (INa amil) in oocytes (X. laevis) injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We observed a strong reduction in INa amil with the xShroom1 antisense: inward conductances (Ginward) (−160 to 0 mV) were 36 ± 12 µS and 1.80 ±.50 µS with xShroom1 sense and antisense. Similar results were obtained in oocytes expressing a mutant β-mENaC subunit (β-S518K) with a open probability of 1 (Ginward 65 ± 10 µS and 1.80 ± 2.0 µS for oocytes with xShroom1 sense or xShroom1 antisense. The negative effects of xShroom1 antisense can not be reversed with forskolin which reduced the rate of ENaC retrieval: Ginward : 124 ± 27 µS and 7.0 ± 1.9 µS with xShroom1 sense or xShroom1 antisense. Trypsin in the range of ng/ml activates the membrane-resident ENaC channels (Bengrine et al.2007), being this effect dependent on activation of G-proteins. Addition of 20 ng/ml of trypsin led to a slow increase in INa amil with xShroom1 sense and it had no effect in most of the oocytes coinjected with ENaC and xShroom1 antisense (2 out of 20). Trypsin were without effects on the endogenous conductances. These data are consistent with the idea that the reduced INa amil when xShroom1 is blocked is most probably due to a lack of functional ENaC channels in the plasma membrane.Acknowledgements: ENaC cDNAs were provided by Dr M. Carattino (Pittsburgh, Pa). and the set for oocytes was a gift of Dr C. Peracchia (Rochester, NY). Supported by grants MO35 (UBA) and PICT 38181.