Background-We recently identified agonistic autoantibodies directed against the angiotensin AT1 receptor (AT 1 -AA) in the plasma of preeclamptic women. To elucidate their role further, we studied the effects of AT 1 -AA on reactive oxygen species (ROS), NADPH oxidase expression, and nuclear factor-B (NF-B) activation. Methods and Results-We investigated human vascular smooth muscle cells (VSMC) and trophoblasts, as well as placentas. AT 1 -AA were isolated from sera of preeclamptic women. Angiotensin II (Ang II) and AT 1 -AA increased ROS production and the NADPH oxidase components, p22, p47, and p67 phox in Western blotting. We next tested if AT 1 -AA lead to NF-B activation in VSMC and trophoblasts. AT 1 -AA activated NF-B. Inhibitor-B␣ (I-B␣) expression was reduced in response to AT 1 -AA. AT1 receptor blockade with losartan, diphenylene iodonium, tiron, and antisense against p22 phox all reduced ROS production and NF-B activation. VSMC from p47phoxϪ/Ϫ mice showed markedly reduced ROS generation and NF-B activation in response to Ang II and AT1-AA. The p22, p47, and p67 phox expression in placentas from preeclamptic patients was increased, compared with normal placentas. Furthermore, NF-B was activated and I-B␣ reduced in placentas from preeclamptic women. Conclusions-NADPH oxidase is potentially an important source of ROS that may upregulate NF-B in preeclampsia. We suggest that AT 1 -AA through activation of NADPH oxidase could contribute to ROS production and inflammatory responses in preeclampsia.

Human organic anion transporter 4 (hOAT4) is located at the apical membrane of proximal tubule cells and involved in renal secretion and reabsorption of endogenous substances as well as many drugs and xenobiotics. This study reevaluated the physiologic role, transport mode, and driving forces of hOAT4. 6-Carboxyfluorescein (6-CF) uptake into HEK293 cells that stably expressed hOAT4 was saturable, resulting in a K m of 108 M.

We have cloned human sodium-dependent organic anion transporter (SOAT) cDNA, which consists of 1502 bp and encodes a 377-amino acid protein. SOAT shows 42% sequence identity to the ileal apical sodium-dependent bile acid transporter ASBT and 33% sequence identity to the hepatic Na ؉ / taurocholate-cotransporting polypeptide NTCP. Immunoprecipitation of a SOAT-FLAG-tagged protein revealed a glycosylated form at 46 kDa that decreased to 42 kDa after PNGase F treatment. SOAT exhibits a seven-transmembrane domain topology with an outside-to-inside orientation of the N-terminal and C-terminal ends. SOAT mRNA is most highly expressed in testis. Relatively high SOAT expression was also detected in placenta and pancreas. We established a stable SOAT-HEK293 cell line that showed sodium-dependent transport of dehydroepiandrosterone sulfate, estrone-3-sulfate, and pregnenolone sulfate with apparent K m values of 28.7, 12.0, and 11.3 M, respectively. Although bile acids, such as taurocholic acid, cholic acid, and chenodeoxycholic acid, were not substrates of SOAT, the sulfoconjugated bile acid taurolithocholic acid-3-sulfate was transported by SOAT-HEK293 cells in a sodium-dependent manner and showed competitive inhibition of SOAT transport with an apparent K i value of 0.24 M. Several nonsteroidal organosulfates also strongly inhibited SOAT, including 1-(-sulfooxyethyl)pyrene, bromosulfophthalein, 2-and 4-sulfooxymethylpyrene, and ␣-naphthylsulfate. Among these inhibitors, 2-and 4-sulfooxymethylpyrene were competitive inhibitors of SOAT, with apparent K i values of 4.3 and 5.5 M, respectively, and they were also transported by SOAT-HEK293 cells.

Human trophoblasts depend on the supply of external precursors, such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16 alpha-OH-DHEA-S, for synthesis of estrogens. The aim of the present study was to characterize the uptake of DHEA-S by isolated mononucleated trophoblasts (MT) and to identify the involved transporter polypeptides. The kinetic analysis of DHEA-(35)S uptake by MT revealed a saturable uptake mechanism (K(m) = 26 microM, V(max) = 428 pmol x mg protein(-1) x min(-1)), which was superimposed by a nonsaturable uptake mechanism (diffusion constant = 1.2 microl x mg protein(-1) x min(-1)). Uptake of [(3)H]DHEA-S by MT was Na(+) dependent and inhibited by sulfobromophthalein (BSP), steroid sulfates, and probenecid, but not by steroid glucuronides, unconjugated steroids, conjugated bile acids, ouabain, p-aminohippurate (PAH), and bumetanide. MT took up [(35)S]BSP, [(3)H]estrone-sulfate, but not (3)H-labeled ouabain, estradiol-17beta-glucuronide, taurocholate, and PAH. RT-PCR revealed that the organic anion-transporting polypeptides OATP-B, -D, -E, and the organic anion transporter OAT-4 are highly expressed, and that OATP-A, -C, -8, OAT-3, and Na(+)-taurocholate cotransporting polypeptide (NTCP) are not or are only lowly expressed in term placental tissue and freshly isolated and cultured trophoblasts. Immunohistochemistry of first- and third-trimester placenta detected OAT-4 on cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast. Our results indicate that uptake of steroid sulfates by isolated MT is mediated by OATP-B and OAT-4 and suggest a physiological role of both carrier proteins in placental uptake of fetal-derived steroid sulfates.