Difference In Efflux Rate Research Articles

Mechanisms of glutathione-conjugate efflux from the brain into blood: Involvement of multiple transporters in the course

Accumulation of detrimental glutathione-conjugated metabolites in the brain potentially causes neurological disorders, and must therefore be exported from the brain. However, in vivo mechanisms of glutathione-conjugates efflux from the brain remain unknown. We investigated the involvement of transporters in glutathione-conjugates efflux using 6-bromo-7-[11C]methylpurine ([11C]1), which enters the brain and is converted into its glutathione conjugate, S-(7-[11C]methylpurin-6-yl)glutathione ([11C]2). In mice of control and knockout of P-glycoprotein/breast cancer resistance protein and multidrug resistance-associated protein 2 ([Mrp2] −/− ), [11C]2 formed in the brain was rapidly cleared, with no significant difference in efflux rate. In contrast, [11C]2 formed in the brain of Mrp1 −/− mice was slowly cleared, whereas [11C]2 microinjected into the brain of control and Mrp1 −/− mice was 75% cleared within 60 min, with no significant difference in efflux rate. These suggest that Mrp1 contributes to [11C]2 efflux across cell membranes, but not BBB. Efflux rate of [11C]2 formed in the brain was significantly lower in Mrp4 −/− and organic anion transporter 3 (Oat3) −/− mice compared with control mice. In conclusion, Mrp1, Oat3, and Mrp4 mediate [11C]2 efflux from the brain. Mrp1 may contribute to [11C]2 efflux from brain parenchymal cells, while extracellular [11C]2 is likely cleared across the BBB, partly by Oat3 and Mrp4.

Effects of toxicologically relevant xenobiotics and the lipid-derived electrophile 4-hydroxynonenal on macrophage cholesterol efflux: silencing carboxylesterase 1 has paradoxical effects on cholesterol uptake and efflux.

Cholesterol cycles between free cholesterol (unesterified) found predominantly in membranes and cholesteryl esters (CEs) stored in cytoplasmic lipid droplets. Only free cholesterol is effluxed from macrophages via ATP-binding cassette (ABC) transporters to extracellular acceptors. Carboxylesterase 1 (CES1), proposed to hydrolyze CEs, is inactivated by oxon metabolites of organophosphorus pesticides and by the lipid electrophile 4-hydroxynonenal (HNE). We assessed the ability of these compounds to reduce cholesterol efflux from foam cells. Human THP-1 macrophages were loaded with [3H]-cholesterol/acetylated LDL and then allowed to equilibrate to enable [3H]-cholesterol to distribute into its various cellular pools. The cholesterol-engorged cells were then treated with toxicants in the absence of cholesterol acceptors for 24 h, followed by a 24 h efflux period in the presence of toxicant. A concentration-dependent reduction in [3H]-cholesterol efflux via ABCA1 (up to 50%) was found for paraoxon (0.1–10 μM), whereas treatment with HNE had no effect. A modest reduction in [3H]-cholesterol efflux via ABCG1 (25%) was found after treatment with either paraoxon or chlorpyrifos oxon (10 μM each) but not HNE. No difference in efflux rates was found after treatments with either paraoxon or HNE when the universal cholesterol acceptor 10% (v/v) fetal bovine serum was used. When the re-esterification arm of the CE cycle was disabled in foam cells, paraoxon treatment increased CE levels, suggesting the neutral CE hydrolysis arm of the cycle had been inhibited by the toxicant. However, paraoxon also partially inhibited lysosomal acid lipase, which generates cholesterol for efflux, and reduced the expression of ABCA1 protein. Paradoxically, silencing CES1 expression in macrophages did not affect the percent of [3H]-cholesterol efflux. However, CES1 mRNA knockdown markedly reduced cholesterol uptake by macrophages, with SR-A and CD36 mRNA reduced 3- and 4-fold, respectively. Immunoblots confirmed SR-A and CD36 protein downregulation. Together, these results suggest that toxicants, e.g., oxons, may interfere with macrophage cholesterol homeostasis/metabolism.

Prediction of In Vivo Rat Biliary Drug Clearance from an In Vitro Hepatocyte Efflux Model

Well-established techniques are available to predict in vivo hepatic uptake and metabolism from in vitro data, but predictive models for biliary clearance remain elusive. Several studies have verified the expression and activity of ATP-binding cassette (ABC) efflux transporters central to biliary clearance in freshly isolated rat hepatocytes, raising the possibility of predicting biliary clearance from in vitro efflux measurements. In the present study, short-term plated rat hepatocytes were evaluated as a model to predict biliary clearance from in vitro efflux measurements before major changes in transporter expression known to take place in long-term hepatocyte cultures. The short-term cultures were carefully characterized for their uptake and metabolic properties using a set of model compounds. In vitro efflux was studied using digoxin, fexofenadine, napsagatran, and rosuvastatin, representing compounds with over 100-fold differences in efflux rates in vitro and 60-fold difference in measured in vivo biliary clearance. The predicted biliary clearances from short-term plated rat hepatocytes were within 2-fold of measured in vivo values. As in vitro efflux includes both basolateral and canalicular effluxes, pronounced basolateral efflux may introduce errors in predictions for some compounds. In addition, in vitro rat hepatocyte uptake rates corrected for simultaneous efflux predicted rat in vivo hepatic clearance of the biliary cleared compounds with less than 2-fold error. Short-term plated hepatocytes could thus be used to quantify hepatocyte uptake, metabolism, and efflux of compounds and considerably improve the prediction of hepatic clearance, especially for compounds with a large biliary clearance component.

Decomposition kinetics of soil carbon of different age from a forest exposed to 8 years of elevated atmospheric CO 2 concentration

Ecosystem exposure to elevated atmospheric CO 2 concentration can often leads to increased ecosystem carbon (C) fluxes, as well as greater net primary production. Changes in the soil C pool with elevated [CO 2] are more difficult to measure and therefore remain poorly understood. In this study, we carried out a series of laboratory soil incubations, in order to determine whether 8 years of ecosystem exposure to elevated [CO 2] altered decomposition dynamics of two age classes of soil C in a temperate coniferous forest. Our objectives were to determine whether there were differences in the decomposition kinetics of soil C up to 8 years old (C post-tr) and soil C older than 8 years (C pre-tr), in the absence of concurrent plant activity. We collected soil from the Duke Forest Free Air CO 2 Enrichment site in North Carolina and incubated whole and crushed (all macroaggregates dispersed) soil from two depth increments (0–5 cm and 5–15 cm) for 102–127 days. We found that mineral soil from the treatment plots had higher respiration rates in the absence of concurrent plant activity than mineral soil from plots under ambient CO 2 conditions. These differences in respiration rate were only significant in 0–5 cm soil and could be largely explained by higher initial respiration rates of soil collected from the CO 2-treated plots. Disruption of soil macroaggregates did not result in a difference in efflux rate in soil from this forest under ambient or elevated CO 2 conditions at either depth. The specific respiration rate of C post-tr was higher than that of C pre-tr in the top 5 cm of soil, while the opposite was true for 5–15 cm of soil. Even though C post-tr was assimilated by the ecosystem more recently than C pre-tr, their decay constants were similar at both depths. These results suggest that, in the absence of plant activity, the mineralization of soil C of different ages in this forest may be under similar biological and/or biochemical control. Therefore, if the higher initial rates of decomposition of C post-tr seen in these experiments are sustained in the field, greater labile pool size of recently added C, and potentially faster cycling of this pool, may in part explain higher soil respiration rates and limited soil C accumulation under elevated [CO 2] in this forest.

Online Fluorescent Method to Assess BCRP/ABCG2 Activity in Suspension Cells

An online method was developed to monitor BCRP mediated efflux of fluorescent substrates in suspension cells. To this end, a 2-compartment system consisting of a transwell cup and a cuvette was used. In this system we were able to observe differences in efflux kinetics between BCRP overexpressing RPMI 8226/MR cells and parental myeloid RPMI 8226(s) cells using only 50,000 cells per experiment. 8226/MR cells displayed a larger cellular efflux rate of the BCRP substrate Hoechst 33342, as compared to the wildtype cells. This difference in efflux rate was completely decreased in the presence of the BCRP inhibitor Ko143.

Online Fluorescent Method to Assess BCRP/ABCG2 Activity in Suspension Cells

An online method was developed to monitor BCRP mediated efflux of fluorescent substrates in suspension cells. To this end, a 2‐compartment system consisting of a transwell cup and a cuvette was used. In this system we were able to observe differences in efflux kinetics between BCRP overexpressing RPMI 8226/MR cells and parental myeloid RPMI 8226(s) cells using only 50,000 cells per experiment. 8226/MR cells displayed a larger cellular efflux rate of the BCRP substrate Hoechst 33342, as compared to the wildtype cells. This difference in efflux rate was completely decreased in the presence of the BCRP inhibitor Ko143.

Methotrexate and γ- tert-butyl methotrexate transport in CEM and CEM/MTX human leukemic lymphoblasts

In a continuing investigation of determinants of their 200-fold methotrexate resistance and their collateral sensitivity to γ- tert-butyl methotrexate, the ability of CEM/MTX cells to transport the two drugs was analyzed and compared with that of CEM cells. The K m and V max values for the influx of methotrexate into CEM cells did not differ significantly from those of CEM/MTX cells, and this was the case for γ- tert-butyl methotrexate as well. Surface binding and influx rates were proportional to cell surface area, but differences in efflux rates and methotrexate uptake were too large to be explained on this basis. Neither methotrexate nor trimetrexate competed with γ- tert-butyl methotrexate influx in CEM cells. However, both drugs perturbed the γ- tert-butyl methotrexate steady state in CEM cells, resulting in slightly less uptake than with γ- tert-butyl methotrexate alone. However, the major difference between the two cell types was in the methotrexate uptake plateau, which was much greater in the case of the parental cell line. A related observation was the more rapid efflux of methotrexate from CEM/MTX cells than from CEM cells. The poor uptake, the associated meager capacity to polyglutamylate methotrexate and the enhanced methotrexate efflux appear to be responsible for its decreased activity against CEM/MTX cells. Half-lives for γ- tert-butyl methotrexate efflux were the same in both cell lines, allowing the drug to accumulate to cytotoxic levels despite its inability to form polyglutamates.

Effect of phospholipid fatty acid composition of endothelial cells on cholesterol efflux rates.

Human endothelial cells (EA.hy 926 line) were loaded with cationized low density lipoprotein (LDL) and subsequently incubated with fatty acid/bovine serum albumin complexes. The fatty acids were palmitic, oleic, linoleic, arachidonic, and eicosapentaenoic acids. The preincubations resulted in extensively modified fatty acid profiles in cell membrane phospholipids and in cellular cholesteryl esters. The cholesterol efflux from these fatty acid-modified cells was measured using 0.2 mg high density lipoprotein3 (HDL3)/ml medium. The efflux was significantly higher for the palmitic acid-treated cells, compared to all other fatty acid treatments. These differences in efflux rates were not caused by changes in the binding of HDL3 to high affinity receptors on the EA.hy 926 cells. Efflux mediated by dimethyl suberimidate-treated HDL3, which does not interact with high affinity HDL receptors, was similar to efflux induced by native HDL3 after all fatty acid treatments. Our results indicate that high affinity HDL receptors are not important for HDL-mediated efflux of cell cholesterol. The fatty acid composition of the cell membrane phospholipids may be an important determinant.

Effects of TSH on Iodide Transport by MouseThyroid Lobesin Vitro

Thyroidal iodide concentration by in vitro mouse thyroid lobes was studied by determining the equilibrium tissue/medium iodide, concentration ratio (T/M[I-]), THYROIDAL I- influx, and thyroidal I-efflux in the presence and absence of ClO4-. Chronic thyroid stimulation by low dietary iodine increased the T/M[I-], and the increase was linearly related to I- influx. There was no difference in efflux rate when animals fed low and high iodine diets were compared, although the efflux with C104- added to block influx was increased by low iodine diet. Addition of TSH in vitro caused a delayed fall in T/M[I-] with a similar TSH concentration-dependence as colloid droplet formation. The TSH effect was mimicked by exogenous cyclic AMP but could be dissociated from stimulation of hormone release by colchicine. Thyroidal I- efflux was increased up to 20-fold by C104-. In the presence of C104- short-term and equilibrium-labeled I- exited at the same rate, but in the absence of C104- short-term-labeled I- efflux was consistently higher. The TSH-induced fall in T/M I- could be accounted for by increased iodide efflux. As TSH also increased efflux when influx was blocked by C104-, the TSH effect would seem due to an increased intrinsic follicular leakiness.

Determination of the relative potency of two excitant amino acids

Recent reports have suggested that some cells in the CNS differ in their relative sensitivity to iontophoretically applied aspartate and glutamate. Attempts to investigate this phenomenon present difficulties since transport numbers for the same substance show considerable variation among different micropipette barrels. A method is described which allows corrections to be made for differences in efflux rates from different barrels. Efflux of tritiated glutamate and aspartate into saline for each pipette is measured by liquid scintillation counting before and after its use in vivo. Calibration curves of efflux (pmol min −1) against current (nA min −1) are then constructed for each barrel containing amino acid. Using these calibrations, log dose-response curves for aspartate and glutamate can be plotted for each neurone by derivation from the apparent dose-response curves in which firing rate is plotted against log current (nA). When the method was tested on brainstem neurones it was found that the apparent relative sensitivity of most cells was changed following correction, and in some cases reversed.

A comparison of the effux rates of AIB from kidney cortex slices of mature and newborn rats.

Earlier observations of the pattern of uptake of amino acids by rat kidney cortex slices taken from mature and from newborn animals indicated that although the initial rate of entry was invariably more rapid in the mature tissue, the concentration gradient eventually achieved was consistently higher in the newborn tissues. Assuming that the rate of entry was relatively constant, the final concentration differences could only be explained if there was a significantly more rapid efflux of amino acid from the mature tissues. Studies were therefore carried out to measure the rate of efflux of 14C-labelled α-aminoisobutyric acid (AIB) from newborn and mature tissues previously loaded with this nonmetabolized amino acid. It was observed that the rate of efflux was markedly greater in the case of the mature tissues. At 1 h the proportion of AIB which had effluxed was about twice as great in the mature as compared with the newborn tissue. A comparatively small fraction of the efflux from both types of tissue was shown to be dependent on the concentration of external AIB and may have resulted from an exchange diffusion process. This fraction did not account for any significant part of the difference in efflux rates, which probably was the result of different rates of passive diffusion.