Temporal expression profiles of organic anion transport proteins in placenta and fetal liver of the rat

Physiological cholestasis linked to immature hepatobiliary transport systems for organic anions occurs in rat and human neonates. In utero, the placenta facilitates vectorial transfer of certain fetal-derived solutes to the maternal circulation for elimination. We compared the ontogenesis of organic anion transporters in the placenta and the fetal liver of the rat to assess their relative abundance throughout gestation and to determine whether the placenta compensates for the late maturation of transporters in the developing liver. The mRNA of members of the organic anion transporting polypeptide (Oatp) superfamily, the multidrug resistance protein (Mrp) family, one organic anion transporter (OAT), and the bile acid carriers Na(+)-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) was quantified by real-time PCR. The most abundant placental transporters were Oatp4a1, whose mRNA increased 10-fold during gestation, and Mrp1. Mrp1 immunolocalized predominantly to epithelial cells of the endoplacental yolk sac, suggesting an excretory role that sequesters fetal-derived solutes in the yolk sac cavity, and faintly to the basal syncytiotrophoblast surface. The mRNA levels of Oatp2b1, Mrp3, and Bsep in the placenta exceeded those in the fetal liver until day 20 of gestation, suggesting that the fetus relies on placental clearance of substrates when expression in the developing liver is low. Mrp3 immunolocalized to the epithelium of the endoplacental yolk sac and less abundantly in the labyrinth zone and endothelium of the maternal arteries. The placental expression of Oatp1a1, Oatp1a4, Oatp1a5, Oatp1b2, Oat, Ntcp, Mrp2, and Mrp6 was low.     

Impact of forskolin and amino acid depletion upon System A activity and SNAT expression in BeWo cells

Amino acid transport System A (SysA) plays an important role in mediating the transplacental transfer of neutral amino acids from mother to fetus. Given that prior work has demonstrated that SysA activity is regulated, both over gestation and in response to dietary restriction during pregnancy, we examined the response of SysA activity and sodium-dependent neutral amino acid transporter (SNAT; responsible for SysA activity) expression to cAMP analogues and amino acid deprivation in BeWo cells, an accepted model of placental syncytia. SysA activity was unaffected by forskolin, a cAMP agonist, at 48 and 72 h. Amino acid depletion was associated with an up-regulation of SysA activity, largely mediated through an enhancement of SNAT2 (Slc38a2) expression at both the protein and mRNA level. SNAT1 (Slc38a1) expression did not change in response to amino acid depletion, while SNAT4 (Slc38a4) could not be detected. In summary, SysA activity in BeWo cells responds to amino acid depletion through the differential regulation of SNAT subtypes.     

Insulin receptor gene expression during development: developmental regulation of insulin receptor mRNA abundance in embryonic rat liver and yolk sac, developmental regulation of insulin receptor gene splicing, and comparison to abundance of insulin-like growth factor 1 receptor mRNA.

Insulin gene expression has been demonstrated in nonpancreatic tissues early in development, suggesting that this hormone might have actions significant for the differentiating embryo. Because such actions imply ligand-receptor binding, we quantified mRNAs encoding the two known forms of insulin receptor in rat liver and yolk sac, two endodermally derived tissues shown to express insulin genes, between gestation days (E) 13 and E21 (mid-organogenesis to parturition). Because of its presumed importance for fetal growth, we estimated the abundance of mRNA encoding insulin-like growth factor 1 (IGF 1) receptor in the same samples for comparison. The abundance of insulin receptor mRNA exceeded that for IGF 1 receptor mRNA in liver and yolk sac at all times studied. This difference was greater in liver, where insulin receptor mRNAs were three to more than 50 times more abundant than IGF 1 receptor mRNA on gestation days E13-E16, times which antedate the development of significant hepatic metabolic actions of insulin. The marked abundance of mRNAs encoding insulin receptors is consistent with the hypothesis that insulin has significant actions in specific tissues during the organogenic period.     

Regulation of the sodium/sulfate co-transporter by farnesoid X receptor alpha

Fxralpha is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxralpha is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxralpha target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxralpha response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxralpha agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxralpha-/- mice, after treatment with the hepatotoxin alpha-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxralpha-/- mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxralpha target gene expressed in the kidney and intestine and that in the absence of Fxralpha, mice waste sulfate into the urine. Thus, Fxralpha is necessary for normal sulfate homeostasis in vivo.     

SNAT4 isoform of system A amino acid transporter is expressed in human placenta

The system A amino acid transporter is encoded by three members of the Slc38 gene family, giving rise to three subtypes: Na+-coupled neutral amino acid transporter (SNAT)1, SNAT2, and SNAT4. SNAT2 is expressed ubiquitously in mammalian tissues; SNAT1 is predominantly expressed in heart, brain, and placenta; and SNAT4 is reported to be expressed solely by the liver. In the placenta, system A has an essential role in the supply of neutral amino acids needed for fetal growth. In the present study, we examined expression and localization of SNAT1, SNAT2, and SNAT4 in human placenta during gestation. Real-time quantitative PCR was used to examine steady-state levels of system A subtype mRNA in early (6-10 wk) and late (10-13 wk) first-trimester and full-term (38-40 wk) placentas. We detected mRNA for all three isoforms from early gestation onward. There were no differences in SNAT1 and SNAT2 mRNA expression with gestation. However, SNAT4 mRNA expression was significantly higher early in the first trimester compared with the full-term placenta (P < 0.01). We next investigated SNAT4 protein expression in human placenta. In contrast to the observation for gene expression, Western blot analysis revealed that SNAT4 protein expression was significantly higher at term compared with the first trimester (P < 0.05). Immunohistochemistry and Western blot analysis showed that SNAT4 is localized to the microvillous and basal plasma membranes of the syncytiotrophoblast, suggesting a role for this isoform of system A in amino acid transport across the placenta. This study therefore provides the first evidence of SNAT4 mRNA and protein expression in the human placenta, both at the first trimester and at full term.     

Quantitation of milk proteins and their mRNAs in rat mammary gland at various stages of gestation and lactation.

The content of alpha-lactalbumin and three species of caseins, 42K, 29K, and 25K, have been measured along with the levels and activities of their mRNAs in the rat mammary gland. Changes in these values were followed during gestation and lactation. An increment of 3- to 4-fold over the virgin level was observed for both alpha-lactalbumin and 42K casein during the 1st day of gestation. From this point on, the level of 42K remained unchanged during the 1st week of gestation and increased thereafter. After the increment of the 1st day, the alpha-lactalbumin content decreased rapidly during the 2nd day of gestation, continued to decrease more slowly until the 12th day, and then started to increase thereafter. During the 2nd and 3rd week of gestation. the amounts of alpha-lactalbumin within the gland increased continuously but not uniformly and caseins accumulated rapidly with a tendency to plateau around the 13th to 16th day of gestation. The relative proportions remained, respectively, 42K greater than 29K greater than 25K greater than alpha-lactalbumin until parturition. At the onset of lactation, both alpha-lactalbumin and casein content increased sharply, the relative proportion for caseins changed to 42K greater than 25K greater than 29K greater than alpha-lactalbumin and remained so throughout the lactation period. alpha-Lactalbumin and casein mRNA activity, as judged by the wheat germ translational system, remained unchanged during the 1st week of gestation, then showed a steady but not uniform increase from the 7th day of gestation until parturition. These activities increased sequentially during lactation, alpha-lactalbumin reaching a plateau by the 1st week, caseins between the 1st and 2nd week, and other mRNAs by the end of the 2nd week of lactation. By the 21st day of lactation, the activity of all mRNA had declined. The levels of alpha-lactalbumin mRNA and 16 S doublet casein mRNA sequences measured with the cDNA probes increased by about 8-fold for alpha-lactalbumin mRNA and 6-fold for casein mRNA during the 1st week of gestation. These levels declined slightly early in the 2nd week and then continued to increase until parturition with a shoulder in the levels around the 13th to 16th day. During lactation, these levels increased until the 8th to 12th day and from then on declined. The content of alpha-lactalbumin and caseins, as well as the measurement of sequences and activities of their mRNAs, showed that in the rat mammary gland these differentiated functions are already expressed at the onset of gestation. Both concentration and activity of mRNA are out of phase with protein levels during the 1st week of gestation but they remain in phase thereafter.     

Spatial and temporal patterns of expression of 11beta-hydroxysteroid dehydrogenase types 1 and 2 messenger RNA and glucocorticoid receptor protein in the murine placenta and uterus during late pregnancy

To gain insight into the role of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes and actions of glucocorticoids in the murine placenta and uterus, the expression pattern of the mRNA for 11beta-HSD1 and 11beta-HSD2 and the glucocorticoid receptor (GR) protein were determined from Embryonic Day 12.5 (E12.5, term = E19) to E18.5 by in situ hybridization and immunohistochemistry, respectively. Consistent with its putative role in regulating the transplacental passage of maternal glucocorticoid to the fetus, 11beta-HSD2 mRNA was highly expressed in the labyrinthine zone (the major site of maternal/fetal exchange) at E12.5, and its level decreased dramatically at E16.5, when it became barely detectable. Remarkably, the silencing of 11beta-HSD2 gene expression coincided with the onset of 11beta-HSD1 gene expression in the labyrinth at E16.5 when moderate levels of 11beta-HSD1 mRNA were detected and maintained to E18.5. By contrast, neither 11beta-HSD1 mRNA nor 11beta-HSD2 mRNA were detected in any cell types within the basal zone from E12.5 to E18.5. Moreover, the expression of 11beta-HSD1 and 11beta-HSD2 in the decidua exhibited a high degree of cell specificity in that the mRNA for both 11beta-HSD1 and 11beta-HSD2 was detected in the decidua-stroma but not in the compact decidua. A distinct pattern was also observed within the endometrium where the mRNA for 11beta-HSD1 was expressed in the epithelium, whereas that for 11beta-HSD2 was confined strictly to the stroma. By comparison, the expression of GR in the placenta and uterus was ubiquitous and unremarkable throughout late pregnancy. In conclusion, the present study demonstrates for the first time remarkable spatial and temporal patterns of expression of 11beta-HSD1 and 11beta-HSD2 and GR in the murine placenta and uterus and highlights the intricate control of not only transplacental passage of maternal glucocorticoid to the fetus but also local glucocorticoid action during late pregnancy.     

A decrease in DKK1, a WNT inhibitor, contributes to placental lipid accumulation in an obesity-prone rat model

Placenta, as the sole transport mechanism between mother and fetus, links the maternal physical state and the immediate as well as lifelong outcomes of the offspring. The present study examined the consequences of maternal obesity on placental lipid accumulation and metabolism. Pregnant obesity-prone (OP) and obesity-resistant (OR) rat strains were fed a control diet throughout gestation. Placentas were collected on Gestational Day 21 for mRNA and oxidative stress analysis, and frozen placental sections were analyzed for fat accumulation as well as beta-catenin and Dickkopf homolog 1 (Xenopus laevis) (DKK1) localization. JEG3 trophoblast cells were cultured in vitro to determine the relationship between DKK1 and lipid accumulation. Maternal plasma and placental nonesterified fatty acids and triglycerides (TG) were elevated in OP dams. Placental Dkk1 mRNA content was 4-fold lower in OP placentas, and a significant increase was noted in beta-catenin accumulation as well as in mRNA content of fat transport and TG synthesis genes, including Ppard (peroxisome proliferator-activated receptor delta), Slc27a1 (fatty acid transport protein 1; also known as Fatp1), Cd36 (cluster of differentiation 36; also known as fatty acid translocation [Fat]), Lipin1, and Lipin3. Significant lipid accumulation was found within the decidual zones in OP, but not OR, placentas, and thickness of the decidual and junctional zones was significantly smaller in OP than in OR placentas. Overexpression of DKK1 in JEG3 cells decreased lipid accumulation and mRNA content of PPARD, SLC27A1, CD36, LIPIN1, and LIPIN3. Our results demonstrate that DKK1 is regulating certain aspects of placental lipid metabolism through the WNT signaling pathway.     

Select nutrients in the ovine uterine lumen. ii. glucose transporters in the uterus and peri-implantation conceptuses

Total glucose in ovine uterine lumenal fluid increases 6-fold between Days 10 and 15 of gestation, but not the estrous cycle; however, mechanisms for glucose transport into the uterine lumen and uptake by conceptuses (embryo/fetus and associated membranes) are not established. This study determined the effects of the estrous cycle, pregnancy, progesterone (P4), and interferon tau (IFNT) on expression of both facilitative (SLC2A1, SLC2A3, and SLC2A4) and sodium-dependent (SLC5A1 and SLC5A11) glucose transporters in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy, as well as in conceptuses from Days 10 to 20 of pregnancy. The SLC2A1 and SLC5A1 mRNAs and proteins were most abundant in uterine luminal epithelia and superficial glandular epithelia (LE/sGE), whereas SLC2A4 was present in stromal cells and glandular epithelia (GE). SLC5A11 mRNA was most abundant in endometrial GE, whereas SLC2A3 mRNA was not detectable in endometria. SLC2A1, SLC2A3, SLC2A4, SLC5A1, and SLC5A11 were expressed in the trophectoderm and endoderm of conceptuses. Steady-state levels of SLC2A1, SLC5A1, and SLC5A11 mRNAs, but not SLC2A4 mRNA, were greater in endometria from pregnant than from cyclic ewes. Progesterone increased SLC2A1, SLC5A11, and SLC2A4 mRNAs in the LE/sGE and SLC5A1 in the GE of ovariectomized ewes. Expression of SLC5A1 was inhibited by ZK136,317 (progesterone receptor antagonist), and the combination of ZK136,317 and IFNT further decreased expression in GE. In constrast, P4 induced and IFNT stimulated expression of SLC2A1 and SLC5A11, and these effects were blocked by ZK136,317. Results of this study indicate differential expression of facilitative and sodium-dependent glucose transporters in ovine uteri and conceptuses for transport and uptake of glucose, and that P4 or P4 and IFNT regulate their expression during the peri-implantation period of pregnancy.     

Ion and solute transport by Prestin in Drosophila and Anopheles

The gut and Malpighian tubules of insects are the primary sites of active solute and water transport for controlling hemolymph and urine composition, pH, and osmolarity. These processes depend on ATPase (pumps), channels and solute carriers (Slc proteins). Maturation of genomic databases enables us to identify the putative molecular players for these processes. Anion transporters of the Slc4 family, AE1 and NDAE1, have been reported as HCO(3)(-) transporters, but are only part of the story. Here we report Dipteran (Drosophila melanogaster (d) and Anopheles gambiae (Ag)) anion exchangers, belonging to the Slc26 family, which are multi-functional anion exchangers. One Drosophila and two Ag homologues of mammalian Slc26a5 (Prestin) and Slc26a6 (aka, PAT1, CFEX) were identified and designated dPrestin, AgPrestinA and AgPrestinB. dPrestin and AgPrestinB show electrogenic anion exchange (Cl(-)/nHCO(3)(-), Cl(-)/SO(4)(2-) and Cl(-)/oxalate(2-)) in an oocyte expression system. Since these transporters are the only Dipteran Slc26 proteins whose transport is similar to mammalian Slc26a6, we submit that Dipteran Prestin are functional and even molecular orthologues of mammalian Slc26a6. OSR1 kinase increases dPrestin ion transport, implying another set of physiological processes controlled by WNK/SPAK signaling in epithelia. All of these mRNAs are highly expressed in the gut and Malpighian tubules. Dipteran Prestin proteins appear suited for central roles in bicarbonate, sulfate and oxalate metabolism including generating the high pH conditions measured in the Dipteran midgut lumen. Finally, we present and discuss Drosophila genetic models that integrate these processes.     

Different tissue distribution and hormonal regulation of messenger RNAs encoding rat insulin-like growth factor-binding proteins-1 and -2.

The insulin-like growth factor-binding proteins IGFBP-1 and IGFBP-2 are low mol wt IGFBPs that are similar in structure. They are not glycosylated and have a homologous amino acid sequence, including the number and position of 18 cysteine residues and a carboxyl-terminal Arg-Gly-Asp sequence that can be recognized by cell adhesion receptors. The present study demonstrates that expression of mRNAs encoding the two BPs differs in some fetal rat tissues and in the livers of adult rats after hypophysectomy, fasting, or streptozotocin-induced diabetes. As determined by Northern blot hybridization using cDNA probes for rat IGFBP-2 or human IGFBP-1, both mRNAs are expressed at high levels in liver of 21-day gestation and 1-day-old rats and at lower levels in 21- and 65-day-old rat liver. Levels of both mRNAs are higher in liver than in other fetal rat tissues. The relative abundance of the two mRNAs in most fetal tissues is similar to that in liver, except that kidney and brain have 8-fold and more than 25-fold higher relative levels of IGFBP-2 mRNA, respectively. IGFBP-2 mRNA is about 10- to 20-fold increased after hypophysectomy or fasting, whereas IGFBP-1 mRNA is relatively unchanged. IGFBP-2 mRNA levels are decreased completely by refeeding fasted rats for 3 days, but only partially decreased by treatment of hypophysectomized rats with GH, cortisone acetate, T4, and testosterone for 4 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological roles of renal anion transporters NaS1 and Sat1

This review will briefly summarize current knowledge on the renal anion transporters sodium-sulfate cotransporter-1 (NaS1; Slc13a1) and sulfate-anion transporter-1 (Sat1; Slc26a1). NaS1 and Sat1 mediate renal proximal tubular sulfate reabsorption and thereby regulate blood sulfate levels. Sat1 also mediates renal oxalate transport and controls blood oxalate levels. Targeted disruption of murine NaS1 and Sat1 leads to hyposulfatemia and hypersulfaturia. Sat1 null mice also exhibit hyperoxalemia, hyperoxaluria, and calcium oxalate urolithiasis. NaS1 and Sat1 null mice also have other phenotypes that result due to changes in blood sulfate and oxalate levels. Experimental data indicate that NaS1 is essential for maintaining sulfate homeostasis, whereas Sat1 controls both sulfate and oxalate homeostasis in vivo.     

Aminoaciduria, but normal thyroid hormone levels and signalling, in mice lacking the amino acid and thyroid hormone transporter Slc7a8

LAT2 (system L amino acid transporter 2) is composed of the subunits Slc7a8/Lat2 and Slc3a2/4F2hc. This transporter is highly expressed along the basolateral membranes of absorptive epithelia in kidney and small intestine, but is also abundant in the brain. Lat2 is an energy-independent exchanger of neutral amino acids, and was shown to transport thyroid hormones. We report in the present paper that targeted inactivation of Slc7a8 leads to increased urinary loss of small neutral amino acids. Development and growth of Slc7a8(-/-) mice appears normal, suggesting functional compensation of neutral amino acid transport by alternative transporters in kidney, intestine and placenta. Movement co-ordination is slightly impaired in mutant mice, although cerebellar development and structure remained inconspicuous. Circulating thyroid hormones, thyrotropin and thyroid hormone-responsive genes remained unchanged in Slc7a8(-/-) mice, possibly because of functional compensation by the thyroid hormone transporter Mct8 (monocarboxylate transporter 8), which is co-expressed in many cell types. The reason for the mild neurological phenotype remains unresolved.     

Epithelial cell stretching and luminal acidification lead to a retarded development of stria vascularis and deafness in mice lacking pendrin

Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4(-/-), are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO(3) (-) transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E) 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4(-/-) mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4(+/-) and Slc26a4(-/-) mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4(-/-) mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4(-/-) mice, and possibly in humans, lacking functional pendrin expression.