Background & Aims
Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. While the effect of bile acids (BAs) on intestinal mucosal injury is known, we investigated the contribution of BAs during the development of NEC in neonatal rats.
Premature rats were fed with cow’s milk–based formula and subjected to asphyxia and cold stress to develop NEC. Jejunal and ileal luminal BAs, portal blood BAs, and messenger RNA and protein for the apical sodium-dependent bile acid transporter, the ileal bile acid binding protein, and the heteromeric organic solute transporter (Ostα/Ostβ)were evaluated.
Ileal luminal BAs levels were increased significantly during disease development and the removal of ileal BAs significantly decreased the incidence and severity of disease. Furthermore, when NEC was reduced via treatment with epidermal growth factor (EGF), BA levels were reduced significantly. Jejunal luminal BA levels were similar between animals with NEC and controls, but portal/ileal luminal BA ratios were decreased significantly in animals with NEC. The apical sodium-dependent bile acid transporter was up-regulated at the site of injury in animals with NEC and decreased after EGF treatment; however, the ileal bile acid binding protein was up-regulated only in the NEC and EGF group. Ostα/Ostβ expression was low in all groups, and only slightly increased in the NEC group.
These data strongly suggest that BAs play a role in the development of ileal damage in experimental NEC and that alterations in BA transport in the neonatal ileum may contribute to disease development.
The heteromeric organic solute transporter alpha-beta (Ostα-Ostβ) is expressed at relatively high levels on the basolateral membrane of enterocytes, where it plays a critical role in the intestinal absorption of bile acids and the enterohepatic circulation. However, this transporter is also expressed in nearly all human tissues, including those that are not normally thought to be involved in bile acid homeostasis, indicating that Ostα-Ostβ may have additional roles beyond bile acid transport in these other tissues, or that bile acids and their derivatives are more pervasive than currently envisioned. Emerging data from different laboratories provide support for both of these hypotheses. In particular, recent studies indicate that tissues such as brain and ovary have the capacity to synthesize bile acids or bile acid precursors. In addition, studies examining Ostα-Ostβ substrate specificity have revealed that this transporter can also accept conjugated steroids, including some neurosteroids, and that the transporter is selectively expressed in steroidogenic cells of the brain and adrenal gland, suggesting a novel function for Ostα-Ostβ. The broad tissue expression of Ostα-Ostβ is also consistent with the emerging concept that bile acids and their derivatives act as signaling molecules in diverse tissues. Bile acids activate nuclear receptors such as the farnesoid X receptor (FXR/NR1H4), the pregnane X receptor and the vitamin D receptor, are ligands for a G-protein-coupled bile acid receptor (GPBAR1/TGR5), and can also activate protein kinases A and C as well as mitogen-activated protein kinase pathways. These signaling pathways are present in many tissues and regulate processes such as triglyceride, glucose and energy homeostasis. Note that although FXR and TGR5 are thought to function primarily as bile acid receptors, they are modulated by some other sterols and select lipid metabolites, and are also widely expressed in tissues, indicating a complex interplay among diverse regulatory networks that impact critical cell and organ functions. The present report summarizes the evidence for a pleiotropic role of Ostα-Ostβ in different tissues.
Ostα-Ostβ; Bile acids; Neurosteroids; Fxr; Ostα–/– mice
A variety of steroids, including pregnenolone sulfate (PREGS) and dehydroepiandrosterone sulfate (DHEAS) are synthesized by specific brain cells, and are then delivered to their target sites, where they exert potent effects on neuronal excitability. The present results demonstrate that [3H]DHEAS and [3H]PREGS are relatively high affinity substrates for the organic solute transporter, OSTα–OSTβ, and that the two proteins that constitute this transporter are selectively localized to steroidogenic cells in the cerebellum and hippocampus, namely the Purkinje cells and cells in the CA region in both mouse and human brain. Analysis of Ostα and Ostβ mRNA levels in mouse Purkinje and hippocampal cells isolated via laser capture microdissection supported these findings. In addition, Ostα-deficient mice exhibited changes in serum dehydroepiandrosterone (DHEA) and DHEAS levels, and in tissue distribution of administered [3H]DHEAS. OSTα and OSTβ proteins were also localized to the zona reticularis of human adrenal gland, the major region for DHEAS production in the periphery. These results demonstrate that OSTα-OSTβ is localized to steroidogenic cells of the brain and adrenal gland, and that it modulates DHEA/DHEAS homeostasis, suggesting that it may contribute to neurosteroid action.
Organic solute transporter; neurosteroid transport; pregnenolone sulfate; dehydroepiandrosterone sulfate; Purkinje cells; CA region of the hippocampus
Organic solute transporter alpha-beta (Ostα-Ostβ) is a heteromeric bile acid and sterol transporter that facilitates the entero- and renal-hepatic circulation of bile acids. Hepatic expression of this basolateral membrane protein is increased in cholestasis, presumably to facilitate removal of toxic bile acids from the liver. In this study we show that the cholestatic phenotype induced by common bile duct ligation (BDL) is reduced in mice genetically deficient in Ostα. Although Ostα−/− mice have a smaller bile acid pool size which could explain lower serum and hepatic levels of bile acids after BDL, gallbladder bilirubin and urinary bile acid concentrations were significantly greater in Ostα−/− BDL mice, suggesting additional alternative adaptive responses. Livers of Ostα−/− mice had higher mRNA levels of constitutive androstane receptor (Car) than wild-type BDL mice and increased expression of Phase I enzymes (Cyp7a1, Cyp2b10, Cyp3a11), Phase II enzymes (Sult2a1, Ugt1a1) and Phase III transporters (Mrp2, Mrp3). Following BDL, the bile acid pool size increased in Ostα−/− mice and protein levels for the hepatic basolateral membrane export transporters, Mrp3 and Mrp4, and for the apical bilirubin transporter, Mrp2, were all increased. In the kidney of Ostα−/− mice after BDL the apical bile acid uptake transporter, Asbt, is further reduced, while apical export transporters, Mrp2 and Mrp4, are increased, resulting in a significant increase in urinary bile acid excretion. Conclusions: These findings indicate that loss of Ostα provides protection from liver injury in obstructive cholestasis through adaptive responses in both the kidney and liver that enhance clearance of bile acids into urine and through detoxification pathways most likely mediated by the nuclear receptor, Car.
adaptive regulation; urinary bile acids; nuclear receptor; kidney, bile acid homeostasis
In an attempt to identify genes involved in glutathione (GSH) transport, a human mammary gland cDNA library was screened for clones capable of complementing a defect in GSH uptake in yeast cells that lack Hgt1p, the primary yeast GSH uptake transporter. Five genes capable of rescuing growth on sulfur-deficient GSH-containing medium were identified: prostate transmembrane protein, androgen induced 1 (PMEPA1); lysosomal-associated protein transmembrane 4 alpha (LAPTM4α); solute carrier family 25, member 1 (SLC25A1); lipopolysaccharide-induced TNF factor (LITAF); and cysteine/tyrosine-rich-1 (CYYR1). All of these genes encode small integral membrane proteins of unknown function, although none appear to encode prototypical GSH transporters. Nevertheless, they all increased both intracellular glutathione levels and [3H]GSH uptake rates. [3H]GSH uptake was uniformly inhibited by high concentrations of unlabeled GSH, GSSG, and ophthalmic acid. Interestingly, each protein is predicted to contain Pro-Pro-x-Tyr (PY) motifs, which are thought to be important for regulating protein cell surface expression. Uptake of the endocytotic markers lucifer yellow and FM4-64 was also enhanced by each of the five genes. Mutations of the PY motifs in LITAF largely abolished all of its effects. In summary, although the results do not reveal novel GSH transporters, they identify five PY-containing human gene products that may influence plasma membrane transport activity.
PY-motif; Yeast; Membrane; Transport; Endocytosis; Glutathione
The liver is a major organ involved in regulating whole body manganese (Mn) homeostasis; however, the mechanisms of Mn transport across the hepatocyte basolateral and canalicular membranes remain poorly defined. To gain insight into these transport steps, the present study measured hepatic uptake and biliary excretion of Mn in an evolutionarily primitive marine vertebrate, the elasmobranch Leucoraja erinacea, the little skate. Mn was rapidly removed from the recirculating perfusate of isolated perfused skate livers in a dose-dependent fashion; however, only a small fraction was released into bile (<2% in 7h). Mn was also rapidly taken up by freshly isolated skate hepatocytes in culture. Mn uptake was inhibited by a variety of divalent metals, but not by cesium. Analysis of the concentration-dependence of Mn uptake revealed of two components, with apparent Km values 1.1 ± 0.1 μM and 112 ± 29 μM. The Km value for the high-affinity component was similar to the measured skate blood Mn concentration, 1.9 ± 0.5 μM. Mn uptake was reduced by nearly half when bicarbonate was removed from the culture medium, but was unaffected by a change in pH from 6.5 to 8.5, or by substitution of Na with Li or K. Mn efflux from the hepatocytes was also rapid, and was inhibited when cells were treated with 0.5 mM 2,4-dinitrophenol to deplete ATP levels. These data indicate that skate liver has efficient mechanisms for removing Mn from the sinusoidal circulation, whereas overall biliary excretion is low and appears to be mediated in part by an ATP-sensitive mechanism.
manganese; transport; bile; little skate; liver
Reduced glutathione (GSH) is critical for many cellular processes, and both its intracellular and extracellular concentrations are tightly regulated. Intracellular GSH levels are regulated by two main mechanisms: by adjusting the rates of synthesis and of export from cells. Some of the proteins responsible for GSH export from mammalian cells have recently been identified, and there is increasing evidence that these GSH exporters are multispecific and multifunctional, regulating a number of key biological processes. In particular, the multidrug resistance-associated proteins (Mrp/Abcc) appear to mediate GSH export and homeostasis. The Mrp proteins mediate not only GSH efflux, but they also export oxidized glutathione derivatives (e.g., glutathione disulfide (GSSG), S-nitrosoglutathione (GS-NO), and glutathione-metal complexes), as well as other glutathione S-conjugates. The ability to export both GSH and oxidized derivatives of GSH, endows these transporters with the capacity to directly regulate the cellular thiol-redox status, and therefore the ability to influence many key signaling and biochemical pathways. Among the many processes that are influenced by the GSH transporters are apoptosis, cell proliferation, and cell differentiation. This report summarizes the evidence that Mrps contribute to the regulation of cellular GSH levels and the thiol redox state, and thus to the many biochemical processes that are influenced by this tripeptide.
Glutathione; glutathione transporters; multidrug resistance-associated proteins; thiol-redox status; apoptosis; xenobiotic export; signaling; cell proliferation and differentiation
The organic solute and steroid transporter, Ost alpha-Ost beta, is an unusual heteromeric carrier that appears to play a central role in the transport of bile acids, conjugated steroids, and structurally-related molecules across the basolateral membrane of many epithelial cells. The transporter’s substrate specificity, transport mechanism, tissue distribution, subcellular localization, transcriptional regulation, as well as the phenotype of the recently characterized Ost alpha-deficient mice all strongly support this model. Ost alpha-Ost beta is composed of a predicted 340-amino acid, 7-transmembrane (TM) domain protein (Ost alpha) and a putative 128-amino acid, single-TM domain polypeptide (Ost beta). Heterodimerization of the two subunits increases the stability of the individual proteins, facilitates their post-translational modifications, and is required for delivery of the functional transport complex to the plasma membrane. Ost alpha and Ost beta are expressed in nearly all human tissues that have been examined, but are most abundant in the small intestine, kidney, liver, testis, adrenal gland and other steroidogenic tissues. Ost alpha-Ost beta substrates include bile acids, steroids (estrone 3-sulfate, dehydroepiandrosterone 3-sulfate, and digoxin), and prostaglandin E2, indicating a role of Ost alpha-Ost beta in the disposition of key cellular metabolites and signaling molecules. Transport occurs by a facilitated diffusion mechanism, and thus Ost alpha-Ost beta can mediate cellular efflux or uptake depending on that substrate’s electrochemical gradient. Additional strong evidence for a role of Ost alpha-Ost beta in sterol homeostasis was provided by recent studies in Ost alpha-deficient mice. These mice display a marked defect in intestinal bile acid and conjugated steroid absorption; a decrease in bile acid pool size and serum bile acid levels; altered intestinal, hepatic and renal disposition of known substrates of the transporter; and altered serum triglyceride, cholesterol, and glucose levels. Taken together, these observations indicate that Ost alpha-Ost beta is essential for bile acid and sterol disposition, and suggest that the carrier may be involved in human conditions related to imbalances in bile acid or lipid homeostasis.
Conjugated steroids; bile acid transport; FXR; Ost alpha-Ost beta; Ost alpha−/− mice
Glutathione (GSH) plays an important role in a multitude of cellular processes, including cell differentiation, proliferation, and apoptosis, and as a result, disturbances in GSH homeostasis are implicated in the etiology and/or progression of a number of human diseases, including cancer, diseases of aging, cystic fibrosis, and cardiovascular, inflammatory, immune, metabolic, and neurodegenerative diseases. Because of GSH’s pleiotropic effects on cell functions, it has been quite difficult to define the role of GSH in the onset and/or the expression of human diseases, although significant progress is being made. GSH levels, turnover rates and/or oxidation state can be compromised by inherited or aquired defects in the enzymes, transporters, signaling molecules, or transcription factors that are involved in its homeostasis, or from exposure to reactive chemicals or metabolic intermediates. GSH deficiency or a decrease in the GSH/glutathione disulfide (GSSG) ratio manifests itself largely through an increased susceptibility to oxidative stress, and the resulting damage is thought to be involved in diseases such as cancer, Parkinson’s disease, and Alzheimer’s disease. In addition, imbalances in GSH levels affect immune system function, and are thought to play a role in the aging process. Just as low intracellular GSH levels decrease cellular antioxidant capacity, elevated GSH levels generally increase antioxidant capacity and resistance to oxidative stress, and this is observed in many cancer cells. The higher GSH levels in some tumor cells are also typically associated with higher levels of GSH-related enzymes and transporters. Although neither the mechanism nor the implications of these changes are well defined, the high GSH content makes cancer cells chemoresistant, which is a major factor that limits drug treatment. The present report highlights and integrates the growing connections between imbalances in GSH homeostasis and a multitude of human diseases.
Glutathione; neurodegenerative diseases; aging; cancer; cardiovascular diseases; metabolic diseases
The proteins responsible for reduced glutathione (GSH) export under both basal conditions and in cells undergoing apoptosis have not yet been identified, although recent studies implicate some members of the multidrug resistance-associated protein family (MRP/ABCC) in this process. To examine the role of MRP1 in GSH release, the present study measured basal and apoptotic GSH efflux in HEK293 cells stably transfected with human MRP1. MRP1-overexpressing cells had lower intracellular GSH levels and higher levels of GSH release, under both basal conditions and after apoptosis was induced with either Fas antibody or staurosporine. Despite the enhanced GSH efflux in MRP1-overexpressing cells, intracellular GSH levels were not further depleted when cells were treated with Fas antibody or staurosporine, suggesting an increase in GSH synthesis. MRP1-overexpressing cells were also less susceptible to apoptosis, suggesting that the stable intracellular GSH levels may have protected cells from death. Overall, these results demonstrate that basal and apoptotic GSH release are markedly enhanced in cells overexpressing MRP1, suggesting that MRP1 plays a key role in these processes. The enhanced GSH release, with a concurrent decrease of intracellular GSH, appears to be necessary for the progression of apoptosis.
Glutathione; Multidrug resistance-associated proteins; MRP1; apoptosis
The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E2 across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 was identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eiconsanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules.
Ost; organic solute and steroid transporter; little skate; L. erinacea; LEE-1 cell line; arachidonic acid
Bile acids are transported across the ileal enterocyte brush border membrane by the well characterized apical sodium-dependent bile acid transporter (Asbt) Slc10a2; however, the carrier(s) responsible for transporting bile acids across the ileocyte basolateral membrane into the portal circulation have not been fully identified. Transcriptional profiling of wild type and Slc10a2 null mice was employed to identify a new candidate basolateral bile acid carrier, the heteromeric organic solute transporter (Ost)α-Ostβ. By Northern blot analysis, Ostα and Ostβ mRNA was detected only in mouse kidney and intestine, mirroring the horizontal gradient of expression of Asbt in the gastrointestinal tract. Analysis of Ostα and Ostβ protein expression by immunohistochemistry localized both subunits to the basolateral surface of the mouse ileal enterocyte. The transport properties of Ostα-Ostβ were analyzed in stably transfected Madin-Darby canine kidney cells. Co-expression of mouse Ostα-Ostβ, but not the individual subunits, stimulated Na+-independent bile acid uptake and the apical-to-basolateral transport of taurocholate. In contrast, basolateral-to-apical transport was not affected by Ostα-Ostβ expression. Co-expression of Ostα and Ostβ was required to convert the Ostα subunit to a mature glycosylated endoglycosidase H-resistant form, suggesting that co-expression facilitates the trafficking of Ostα through the Golgi apparatus. Immunolocalization studies showed that co-expression was necessary for plasma membrane expression of both Ostα and Ostβ. These results demonstrate that the mouse Ostα-Ostβ heteromeric transporter is a basolateral bile acid carrier and may be responsible for bile acid efflux in ileum and other ASBT-expressing tissues.
Many people, by means of consumption of seafood or other anthropogenic sources, are exposed to levels of methylmercury (MeHg) that are generally considered to be quite low, but that may nevertheless produce irreversible brain damage, particularly in unborn babies. The only way to prevent or ameliorate MeHg toxicity is to enhance its elimination from the body.
Using N-acetylcysteine (NAC), we aimed to devise a monitoring protocol for early detection of acute exposure or relatively low MeHg levels in a rodent model, and to test whether NAC reduces MeHg levels in the developing embryo.
NAC produced a transient, dose-dependent acceleration of urinary MeHg excretion in rats of both sexes. Approximately 5% of various MeHg doses was excreted in urine 2 hr after injection of 1 mmol/kg NAC. In pregnant rats, NAC markedly reduced the body burden of MeHg, particularly in target tissues such as brain, placenta, and fetus. In contrast, NAC had no significant effect on urinary MeHg excretion in preweanling rats.
Because NAC causes a transient increase in urinary excretion of MeHg that is proportional to the body burden, it is promising as a biomonitoring agent for MeHg in adult animals. In view of this and because NAC is effective at enhancing MeHg excretion when given either orally or intravenously, can decrease brain and fetal levels of MeHg, has minimal side effects, and is widely available in clinical settings, NAC should be evaluated as a potential antidote and biomonitoring agent in humans.
N-acetylcysteine; antidote; biomarker; biomonitoring; embryotoxicity; methylmercury; toxicity
Intracellular concentrations of essential metals are normally maintained within a narrow range, whereas the nonessential metals generally lack homeostatic controls. Some of the factors that contribute to metal homeostasis have recently been identified at the molecular level and include proteins that mediate import of essential metals from the extracellular environment, those that regulate delivery to specific intracellular proteins or compartments, and those that mediate metal export from the cell. Some of these proteins appear highly selective for a given essential metal; however, others are less specific and interact with multiple metals, including toxic metals. For example, DCT1 (divalent cation transporter-1; also known as NRAMP2 or DMT1) is considered to be a major cellular uptake mechanism for Fe(2+) and other essential divalent metals, but this protein also mediates uptake of Cd(2+), Pb(2+), and possibly of other toxic divalent metals. The ability of nonessential metals to interact with binding sites for essential metals is critical for their ability to gain access to specific cellular compartments and for their ability to disrupt normal biochemical or physiological functions. Another major mechanism by which metals traverse cell membranes and produce cell injury is by forming complexes whose overall structures mimic those of endogenous molecules. For example, it has long been known that arsenate and vanadate can compete with phosphate for transport and metabolism, thereby disrupting normal cellular functions. Similarly, cromate and molybdate can mimic sulfate in biological systems. Studies in our laboratory have focused on the transport and toxicity of methylmercury (MeHg) and inorganic mercury. Mercury has a high affinity for reduced sulfhydryl groups, including those of cysteine and glutathione (GSH). MeHg-l-cysteine is structurally similar to the amino acid methionine, and this complex is a substrate for transport systems that carry methionine across cell membranes. Once MeHg has entered the cell, some of it binds to GSH, and the resulting MeHg-glutathione complex appears to be a substrate for proteins that mediate cellular export of glutathione S-conjugates, including the apically located MRP2 (multidrug resistance-associated protein 2) transporter, a member of the adenosine triphosphate-binding cassette protein superfamily. Because other toxic metals also form complexes with endogenous molecules, comparable mechanisms may be involved in their membrane transport and disposition.
To better define the role of glutathione (GSH) in cell differentiation, the present study measured GSH concentrations during terminal HL-60 cell differentiation, in the presence and absence of differentiation-inducing agents, and in the presence and absence of GSH altering agents. Interestingly, there was a small transient increase in intracellular GSH levels during dimethyl sulfoxide (DMSO) or 1α,25-dihydroxyvitamin D3 (VD3) induced differentiation. This increase coincided with an increase in nitroblue tetrazolium (NBT) reduction capacity, a measure of superoxide anion production, but there was no apparent change in the GSH/glutathione disulfide (GSSG) ratio. Surprisingly, treatment of cells with low doses of 1-chloro-2,4-dinitrobenzene (CDNB; 5 µM) or diethylmaleate (DEM; 0.5 mM), which transiently deplete GSH levels to about 40% of control levels, resulted in enhanced differentiation of HL-60 cells exposed to VD3 or all-trans-retinoic acid (ATRA), as well as under un-induced conditions (i.e., spontaneous differentiation). Enhanced differentiation occurred when cells were treated with the GSH-depleting agents 4 hours after treatment with differentiation inducers. These findings indicate that intracellular GSH levels are regulated in a complex fashion during HL-60 cell differentiation, and that transient GSH depletion using low doses of CDNB and DEM enhances the differentiation process.
glutathione; cell differentiation; thiol levels; vitamin D3; all-trans retinoic acid; NADPH oxidase; N-acetylcysteine