Inhibition by lead of production and secretion of transthyretin in the choroid plexus: its relation to thyroxine transport at blood-CSF barrier.
(1/601)
Long-term, low-dose Pb exposure in rats is associated with a significant decrease in transthyretin (TTR) concentrations in the CSF. Since CSF TTR, a primary carrier of thyroxine in brain, is produced and secreted by the choroid plexus, in vitro studies were conducted to test whether Pb exposure interferes with TTR production and/or secretion by the choroid plexus, leading to an impaired thyroxine transport at the blood-CSF barrier. Newly synthesized TTR molecules in the cultured choroidal epithelial cells were pulse-labeled with [35S]methionine. [35S]TTR in the cell lysates and culture media was immunoprecipitated and separated by SDS-PAGE, and quantitated by autoradiography and liquid scintillation counting. Pb treatment did not significantly alter the protein concentrations in the culture, but inhibited the synthesis of total [35S]TTR (cells + media), particularly during the later chase phase. Two-way ANOVA of the chase phase revealed that Pb exposure (30 microM) significantly suppressed the rate of secretion of [35S]TTR compared to the controls (p < 0.05). Accordingly, Pb treatment caused a retention of [35S]TTR by the cells. In a two-chamber transport system with a monolayer of epithelial barrier, Pb exposure (30 microM) reduced the initial release rate constant (kr) of [125I]T4 from the cell monolayer to the culture media and impeded the transepithelial transport of [125I]T4 from the basal to apical side of epithelial cells by 27%. Taken together, these in vitro data suggest that sequestration of Pb in the choroid plexus hinders the production and secretion of TTR by this tissue. Consequently, this may alter the transport of thyroxine across this blood-CSF barrier. (+info)
Atm is dispensable for p53 apoptosis and tumor suppression triggered by cell cycle dysfunction.
(2/601)
Both p53 and ATM are checkpoint regulators with roles in genetic stabilization and cancer susceptibility. ATM appears to function in the same DNA damage checkpoint pathway as p53. However, ATM's role in p53-dependent apoptosis and tumor suppression in response to cell cycle dysregulation is unknown. In this study, we tested the role of murine ataxia telangiectasia protein (Atm) in a transgenic mouse brain tumor model in which p53-mediated apoptosis results in tumor suppression. These p53-mediated activities are induced by tissue-specific inactivation of pRb family proteins by a truncated simian virus 40 large T antigen in brain epithelium. We show that p53-dependent apoptosis, transactivation, and tumor suppression are unaffected by Atm deficiency, suggesting that signaling in the DNA damage pathway is distinct from that in the oncogene-induced pathway. In addition, we show that Atm deficiency has no overall effect on tumor growth and progression in this model. (+info)
Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood-cerebrospinal-fluid drug-permeability barrier.
(3/601)
The blood-brain barrier and a blood-cerebrospinal-fluid (CSF) barrier function together to isolate the brain from circulating drugs, toxins, and xenobiotics. The blood-CSF drug-permeability barrier is localized to the epithelium of the choroid plexus (CP). However, the molecular mechanisms regulating drug permeability across the CP epithelium are defined poorly. Herein, we describe a drug-permeability barrier in human and rodent CP mediated by epithelial-specific expression of the MDR1 (multidrug resistance) P glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP). Noninvasive single-photon-emission computed tomography with 99mTc-sestamibi, a membrane-permeant radiopharmaceutical whose transport is mediated by both Pgp and MRP, shows a large blood-to-CSF concentration gradient across intact CP epithelium in humans in vivo. In rats, pharmacokinetic analysis with 99mTc-sestamibi determined the concentration gradient to be greater than 100-fold. In membrane fractions of isolated native CP from rat, mouse, and human, the 170-kDa Pgp and 190-kDa MRP are identified readily. Furthermore, the murine proteins are absent in CP isolated from their respective mdr1a/1b(-/-) and mrp(-/-) gene knockout littermates. As determined by immunohistochemical and drug-transport analysis of native CP and polarized epithelial cell cultures derived from neonatal rat CP, Pgp localizes subapically, conferring an apical-to-basal transepithelial permeation barrier to radiolabeled drugs. Conversely, MRP localizes basolaterally, conferring an opposing basal-to-apical drug-permeation barrier. Together, these transporters may coordinate secretion and reabsorption of natural product substrates and therapeutic drugs, including chemotherapeutic agents, antipsychotics, and HIV protease inhibitors, into and out of the central nervous system. (+info)
Organic cation transport in rat choroid plexus cells studied by fluorescence microscopy.
(4/601)
Quinacrine uptake and distribution were studied in a primary culture of rat choroid plexus epithelial cells using conventional and confocal fluorescence microscopy and image analysis. Quinacrine rapidly accumulated in cells, with steady-state levels being achieved after 10-20 min. Uptake was reduced by other organic cations, e.g., tetraethylammonium (TEA), and by KCN. Quinacrine fluorescence was distributed in two cytoplasmic compartments, one diffuse and the other punctate. TEA efflux experiments indicated that more than one-half of intracellular organic cation was in a slowly emptying compartment. The protonophore monensin both emptied that TEA compartment and abolished punctate quinacrine fluorescence, suggesting that a large fraction of total intracellular organic cation was sequestered in acidic vesicles, e.g., endosomes. Finally, quinacrine-loaded vesicles were seen to move within the cytoplasm and to abruptly release their contents at the blood side of the cell; the rate of release was greatly reduced by the microtubule disrupter nocodazole. (+info)
Choline uptake across the ventricular membrane of neonate rat choroid plexus.
(5/601)
The uptake of [3H]choline from the cerebrospinal fluid (CSF) side of the rat neonatal choroid plexus was characterized in primary cultures of the choroidal epithelium grown on solid supports. Cell-to-medium concentration ratios were approximately 5 at 1 min and as high as 70 at 30 min. Apical choline uptake was facilitated; the Km was approximately 50 microM. Several organic cations (e.g., hemicholinium-3 and N1-methylnicotinamide) inhibited uptake. The reduction or removal of external Na+ or the addition of 5 mM LiCl had no effect on uptake. However, increasing external K+ concentration from 3 to 30 mM depolarized ventricular membrane potential (-70 to -15 mV) and reduced uptake to 45% of that for the control. Treatment with 1 mM ouabain or 2 mM BaCl2 reduced uptake 45%, and intracellular acidification reduced uptake to approximately 90% of that for controls. These data indicate that the uptake of choline from CSF across the ventricular membrane of the neonatal choroidal epithelium is not directly coupled to Na+ influx but is sensitive to plasma membrane electrical potential. (+info)
Transepithelial transport of organic anions across the choroid plexus: possible involvement of organic anion transporter and multidrug resistance-associated protein.
(6/601)
Transport characteristics of 17beta-estradiol 17beta-D-glucuronide (E217betaG), a dual substrate of the transporters for cellular uptake (organic anion-transporting polypeptide 1 or oatp1) and cellular excretion (multidrug resistance-associated protein 1or MRP1), in the rat choroid plexus were studied in vivo and in vitro. The uptake of E217betaG into isolated choroid plexus was mediated by an energy-dependent system with a Km of 3.4 microM. Together with the previous finding that oatp1 is localized on the apical membrane of choroid plexus, these results suggest that oatp1 is responsible for the uptake of this ligand. After intracerebroventricular administration, elimination of E217betaG from cerebrospinal fluid was probenecid sensitive and much more rapid than that of inulin; less than 2% of the administered E217betaG and 40 to 50% of inulin remained in the cerebrospinal fluid 20 min after intracerebroventricular administration. In addition, the amount of E217betaG associated with choroid plexus at 20 min was negligible, suggesting the presence of an efficient excretion system on the basolateral membrane of choroid plexus. Expression of MRP1 was detected in choroid plexus. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analyses indicated that the expression level of MRP1 in choroid plexus is about four or five times higher than that in the lung, one of the tissues exhibiting high expression of MRP1. Together with the in vivo vectorial transport of E217betaG, these results can be accounted for by assuming that there is basolateral localization of MRP1 in choroid plexus. Combined, oatp1 and MRP1 may synergistically mediate the efficient transcellular transport of E217betaG across choroid plexus. (+info)
Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in Bruch's membrane of the aging eye.
(7/601)
PURPOSE: To develop an antibody that recognizes a variety of advanced glycation endproduct (AGE) epitopes. METHODS: Glycolaldehyde was used to modify bovine serum albumin and HPLC analysis was used to measure pentosidine formation as an indicator of AGE formation. A polyclonal anti-AGE antibody was synthesized by injecting glycolaldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin column, and characterized by immunoblot analysis. RESULTS: HPLC analysis of glycolaldehyde treated bovine serum albumin detected high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody recognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evidence of AGEs in Bruch's membrane (including basal laminar deposits and drusen), choroidal extracellular matrix, and vessel walls in an 82 year old nondiabetic globe. A similar staining pattern was observed in an age-matched diabetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe. CONCLUSIONS: A unique anti-AGE antibody was synthesized that recognizes a variety of AGE epitopes including carboxymethyllysine and pentosidine. Its best use might be in broad surveys of the age-dependent accumulation of a large number of AGE epitopes that might not be revealed by antibodies to pentosidine or CML. (+info)
Gli3 is required for Emx gene expression during dorsal telencephalon development.
(8/601)
Dentate gyrus and hippocampus as centers for spatial learning, memory and emotional behaviour have been the focus of much interest in recent years. The molecular information on its development, however, has been relatively poor. To date, only Emx genes were known to be required for dorsal telencephalon development. Here, we report on forebrain development in the extra toes (Xt(J)) mouse mutant which carries a null mutation of the Gli3 gene. This defect leads to a failure to establish the dorsal di-telencephalic junction and finally results in a severe size reduction of the neocortex. In addition, Xt(J)/Xt(J) mice show absence of the hippocampus (Ammon's horn plus dentate gyrus) and the choroid plexus in the lateral ventricle. The medial wall of the telencephalon, which gives rise to these structures, fails to invaginate during embryonic development. On a molecular level, disruption of dorsal telencephalon development in Xt(J)/Xt(J) embryos correlates with a loss of Emx1 and Emx2 expression. Furthermore, the expression of Fgf8 and Bmp4 in the dorsal midline of the telencephalon is altered. However, expression of Shh, which is negatively regulated by Gli3 in the spinal cord, is not affected in the Xt(J)/Xt(J) forebrain. This study therefore implicates Gli3 as a key regulator for the development of the dorsal telencephalon and implies Gli3 to be upstream of Emx genes in a genetic cascade controlling dorsal telencephalic development. (+info)