BackgroundAn array of environmental compounds is known to possess endocrine disruption (ED) potentials. Bisphenol A (BPA) and bisphenol A dimethacrylate (BPA-DM) are monomers used to a high extent in the plastic industry and as dental sealants. Alkylphenols such as 4-n-nonylphenol (nNP) and 4-n-octylphenol (nOP) are widely used as surfactants.ObjectivesWe investigated the effect in vitro of these four compounds on four key cell mechanisms including transactivation of a) the human estrogen receptor (ER), b) the human androgen receptor (AR), c) the aryl hydrocarbon receptor (AhR), and d) aromatase activity.ResultsAll four compounds inhibited aromatase activity and were agonists and antagonists of ER and AR, respectively. nNP increased AhR activity concentration-dependently and further increased the 2,3,7,8-tetrachlorodibenzo-p-dioxin AhR action. nOP caused dual responses with a weak increased and a decreased AhR activity at lower (10−8 M) and higher concentrations (10−5–10−4 M), respectively. AhR activity was inhibited with BPA (10−5–10−4 M) and weakly increased with BPA-DM (10−5 M), respectively. nNP showed the highest relative potency (REP) compared with the respective controls in the ER, AhR, and aromatase assays, whereas similar REP was observed for the four chemicals in the AR assay.ConclusionOur in vitro data clearly indicate that the four industrial compounds have ED potentials and that the effects can be mediated via several cellular pathways, including the two sex steroid hormone receptors (ER and AR), aromatase activity converting testosterone to estrogen, and AhR; AhR is involved in syntheses of steroids and metabolism of steroids and xenobiotic compounds.
The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for renal electrolyte balance and its phosphorylation causes an increase in its transport activity and cellular localization. Here, we generated phospho-specific antibodies against two conserved N-terminal phosphorylation sites (Thr53, Thr58 and Thr53/Thr58) to assess the role of arginine vasopressin (AVP) in regulating NCC in rodent kidney in vivo. Immunohistochemistry showed distinct staining of phosphorylated NCC (pNCC) at the apical plasma membrane domain of distal convoluted tubule (DCT) cells. Unlike total NCC, pNCC was localized only to the apical plasma membrane as determined by immunogold electron microscopy. In AVP-deficient Brattleboro rats, acute deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) exposure significantly increased pNCC abundance at the apical plasma membrane by about threefold, whereas total NCC and its cellular distribution were not affected. dDAVP significantly increased the abundance of phosphorylated STE20/SPS1-related proline-alanine-rich kinase and oxidative stress-response kinase (SPAK and OSR1), kinases implicated in NCC phosphorylation. Intracellular calcium levels in early and late DCTs were increased in response to 1 min superfusion of dDAVP, confirming that these segments are AVP responsive. In rats fed a high-salt diet with angiotensin (ANG) type 1-receptor blockade, similar increases in pNCC and active SPAK and OSR1 were detected following chronic or acute dDAVP, thus indicating the effects of AVP are independent of ANGII. Our results show that AVP is a potent regulator of NCC activity.
The renal late distal convoluted tubules and connecting tubules are sites for the fine regulation of Na(+) and Ca(2+) reabsorption. The role of these segments in Na(+) and K(+) homeostasis is possibly underestimated, as the tubules are technically difficult to isolate in sufficient numbers and purity for functional analysis. To overcome these difficulties, we have developed a transgenic mouse model expressing enhanced green fluorescent protein in late distal convoluted tubules and connecting tubules. Enhanced green fluorescent protein expression was driven by the promoter for the transient receptor potential subfamily V, member 5. Confocal fluorescence microscopy allowed detection of enhanced green fluorescent protein in living, isolated late distal convoluted tubules and connecting tubules and in the initial cortical collecting ducts. Enhanced green fluorescent protein expression was validated by double- and triple-fluorescence immunolabeling with specific tubule markers. Freshly isolated late distal convoluted tubules and connecting tubules increased their intracellular Ca(2+) levels in response to the V(2) receptor-specific agonist deamino-Cys,d-Arg(8)-vasopressin (2 x 10(-10) M) after 1 min of superfusion. In addition, both late distal convoluted tubules and connecting tubules displayed a concentration-dependent intracellular Ca(2+) response to 1alpha,25-dihydroxyvitamin D(3) (range 10(-10) to 10(-8) M). This suggests that 1alpha,25-dihydroxyvitamin D(3) can act through a nongenomic signaling pathway in these tubules. In conclusion, the transgenic mouse model, expressing enhanced green fluorescent protein, is suitable for rapid isolation of viable late distal convoluted tubules, connecting tubules, and initial cortical collecting ducts and provides an ideal tool for a more exhaustive functional characterization of these segments.
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