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.

Live cell monitoring of glycine betaine by FRET-based genetically encoded nanosensor.

Glycine betaine (GB) is one of the key compatible solutes that accumulate in the cell at exceedingly high level under the conditions of high salinity. It plays a crucial role in the maintenance of osmolarity of the cell without affecting the physiological processes. Analysis of stress-induced physiological conditions in living cells, therefore, requires real-time monitoring of cellular GB level. Glycine Betaine Optical Sensor (GBOS), a genetically-encoded FRET-based nanosensor developed in this study, allows the real-time monitoring of GB levels inside living cells. This nanosensor has been developed by sandwiching GB binding protein (ProX) between the Förster resonance energy transfer (FRET) pair, the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Conformational change in ProX, which was used as sensory domain, reported the change in the level of this compatible solute in in vitro and in vivo conditions. Binding of the GB to the sensory domain fetches close to both the fluorescent moieties that result in the form of increased FRET ratio. So, any change in the concentration of GB is correlated with change in FRET ratio. This sensor also reported the GB cellular dynamics in real-time in Escherichia coli cells after the addition of its precursor, choline. The GBOS was also expressed in yeast and mammalian cells to monitor the intracellular GB. Therefore, the GBOS represents a unique FRET-based nanosensor which allows the non-invasive ratiometric analysis of the GB in living cells.

In vitro effects of standardized extract of Bacopa monniera and its five individual active constituents on human P-glycoprotein activity.

1. For centuries Bacopa monniera (BM) has been used as an herbal drug for the treatment of various mental ailments. A chemically standardized alcoholic extract of BM is clinically available over the counter herbal remedy for memory enhancement in children and adults. Consumption of herbal preparations has been reported to alter the function of membrane transporters, especially P-glycoprotein (P-gp), ATP-dependent drug efflux transporter responsible for the development of herb-drug interactions. 2. In the present study, we evaluated the in vitro effect of BM extract and its five individual active constituents (namely, bacopaside I, bacopaside II and bacopasaponin C, bacoside A and bacoside A3) on P-gp function using luminescent P-gp ATPase assay and Rh123 transport assay across human MDR1 gene transfected LLC-GA5-COL150 cell line. 3. It was observed that BM extract and its five individual constituents inhibited both basal activity as well as verapamil-stimulated ATPase activity, suggesting their affinity towards P-gp. Further, BM and its five active constituents inhibited the rhodamine 123 (Rh123) transport across LLC-GA5-COL150 cell monolayer with bacopaside II being the most potent inhibitor of P-gp, which decreased P-gp efflux ratio of Rh123 by fourfold in comparison to control. 4. Our finding may prove beneficial in predicting the potential herb-drug interactions of BM on concomitant medication with P-gp substrate drugs in clinical settings.

Sindromes de resistencia a las hormonas tiroideas / Thyroide hormone resistance syndromes

La resistencia a hormonas tiroideas es un grupo de sindromes de causa genetica caracterizados por la disminucion de la sensibilidad tisular a estas hormonas. En la actualidad se distinguen tres formas, en los que la resistencia a la accion hormonal se debe, respectivamente, a mutaciones del gen que codifica el receptor nuclear de T3 TRbeta, a alteraciones en el transporte celular de T4 y T3, y a defectos en la conversion de T4 en T3 mediada por desyodasas. En esta revision se hace una exposicion resumida y actualizada de cada una de estas tres formas de resistencia y se discuten los mecanismos patogonicos y aproximaciones clinicas (AU)

Thyroid hormone resistance syndromes are a group of genetic conditions characterized by decreased tissue sensitivity to thyroid hormones. Three syndromes, in which resistance to hormone action is respectively due to mutations in the gene encoding for thyroid hormone receptor TR¦Â, impaired T4 and T3 transport, and impaired conversion of T4 to T3 mediated by deiodinases. An updated review of each of these forms of resistance is provided, and their pathogenetic mechanisms and clinical approaches are discussed (AU)

Tapasin shapes immunodominance hierarchies according to the kinetic stability of peptide-MHC class I complexes.

Peptide loading of MHC class I molecules involves multiple cofactors including tapasin. We showed previously in vitro that tapasin edits the peptide repertoire by favoring the binding of peptides with slow dissociation rates. Here, using tapasin-deficient mice and a DNA vaccine that primes directly, we confirm that tapasin establishes hierarchical responses in vivo according to peptide-MHC stability. In contrast, this hierarchy is lost when the peptides are cross-presented via an alternative DNA vaccine. By regulating transgene expression, we found that the dominant response modifier was antigen persistence. Our findings reveal strategies for activating T cells against low-affinity peptides, of potential importance for patients with repertoires narrowed by deletional tolerance.

Stereoselectivity of the reduced folate carrier in Caco-2 cells.

Stereoselectivity of the human reduced folate carrier (RFC1) was examined in Caco-2 cells using methotrexate (l-amethopterin or l-MTX) and its antipode (d-amethopterin or d-MTX) as model substrates. The initial uptake rate of folic acid (FA) was concentration dependent, with a K(m) value of approximately 0.6 microM. The Eadie-Hofstee plot of the RFC1-mediated FA uptake revealed a single component for FA uptake into Caco-2 cells, demonstrating that only RFC1 is involved in FA uptake. l-MTX inhibited FA uptake in a competitive manner with a K(i) value of approximately 2 microM, similar to the K(m) value of l-MTX. d-MTX also competitively inhibited FA uptake with a K(i) value being approximately 120 microM, indicating that the affinity of d-MTX is ca. 60-fold less than that of l-MTX. The stereoselectivity of human RFC1 observed in the present study was consistent not only with the stereoselectivity of rabbit RFC1 observed in rabbit intestinal brush border membrane vesicles but also with the reported differences in oral absorption of amethopterin enantiomers in humans.

Antifolate resistance in a HeLa cell line associated with impaired transport independent of the reduced folate carrier.

Prior studies from this laboratory documented the prevalence of methotrexate (MTX) transport activity with a low pH optimum in human solid tumor cell lines. In HeLa cells, this low pH activity has high affinity for pemetrexed [PMX (Alimta)] and is reduced folate carrier (RFC)-independent because it is not diminished in a RFC-null subline (R5). R5 cells also have residual transport activity, with high specificity for PMX, at neutral pH. In the current study, a R5 subline, R1, was selected under MTX selective pressure at a modest reduction in pH. There was markedly decreased MTX and PMX transport at both pH 5.5 and pH 7.4. When MTX was removed, there was a slow return of transport activity, and when MTX was added back, there was loss of transport at both pH values within 8 weeks. In R1 cells, there was a marked decrease in accumulation of PMX, MTX, and folic acid along with a decrease in growth inhibition by these and other antifolates that require a facilitative process to gain entry into cells. These data demonstrate that (i) RFC-independent transport in HeLa cells at low and neutral pH contributes to antifolate activity (in particular, to PMX activity) and can be diminished by antifolate selective pressure and (ii) the loss of these activities results in marked resistance to PMX, an agent for which there is little or no loss of activity when transport mediated by RFC is abolished. These observations suggest that transport activity in RFC-null HeLa R5 cells at neutral and low pH may reflect the same carrier-mediated process.

Taurine uptake by human retinal pigment epithelium: implications for the transport of small solutes between the choroid and the outer retina.

PURPOSE: To characterize the Michaelis-Menten kinetics of the taurine transporter (TT) in retinal pigment epithelium (RPE) freshly isolated from human donor eyes. To identify the rate limiting compartment in the pathway of taurine delivery from the choroidal blood supply to the outer retina composed by Bruch's-choroid (BC) and the RPE in the human older age group. METHODS: In human donor samples (4 melanoma-affected eyes, and 14 control eyes; age range, 62-93 years), radiochemical techniques were used to determine the RPE taurine accumulation at various exogenous concentrations. The transport capability of human RPE was obtained from a kinetic analysis of the high-affinity carrier over a substrate concentration of 1 to 60 microM taurine. RESULTS: Uptake of taurine into human RPE at a taurine concentration of 1 microM was independent of donor age (P > 0.05) and averaged at 2.83 +/- 0.27 (SEM) pmol/10 minutes per 6-mm trephine. Taurine transport by human RPE was mediated by a high-affinity carrier of K(m) 50 microM and V(max) of 267 pmol/10 minutes per 5-mm disc. CONCLUSIONS: In human donor RPE, uptake of taurine remained viable in the age range 62 to 93 years. Taurine transport rates in the RPE were lower than across the isolated BC complex, and thus the data suggest that the former compartment houses the rate-limiting step in the delivery of taurine to the outer retina.

The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability.

Many orally administered drugs must overcome several barriers before reaching their target site. The first major obstacle to cross is the intestinal epithelium. Although lipophilic compounds may readily diffuse across the apical plasma membrane, their subsequent passage across the basolateral membrane and into blood is by no means guaranteed. Efflux proteins located at the apical membrane, which include P-glycoprotein (Pgp; MDR1) and MRP2, may drive compounds from inside the cell back into the intestinal lumen, preventing their absorption into blood. Drugs may also be modified by intracellular phase I and phase II metabolising enzymes. This process may not only render the drug ineffective, but it may also produce metabolites that are themselves substrates for Pgp and/or MRP2. Drugs that reach the blood are then passed to the liver, where they are subject to further metabolism and biliary excretion, often by a similar system of ATP-binding cassette (ABC) transporters and enzymes to that present in the intestine. Thus a synergistic relationship exists between intestinal drug metabolising enzymes and apical efflux transporters, a partnership that proves to be a critical determinant of oral bioavailability. The effectiveness of this system is optimised through dynamic regulation of transporter and enzyme expression; tissues have a remarkable capacity to regulate the amounts of protein both at transcriptional and post-transcriptional levels in order to maintain homeostasis. This review addresses the progress to date on what is known about the role and regulation of drug efflux mechanisms in the intestine and liver.

Effect of excipients on the stability and transport of recombinant human epidermal growth factor (rhEGF) across Caco-2 cell monolayers.

The effect of sixteen excipients on the transport of recombinant human epidermal growth factor (rhEGF) across Caco-2 cell monolayers was examined at 37 degrees C. The apparent apical to basolateral (A-B) permeability (Papp) of 30 microM rhEGF was 8.15 x 10(-7) cm/sec, indicative of a poor level of absorption in the GI tract. The Papp was 1.7- and 6.3-fold greater than the Papp in the basolateral to apical (B-A) direction and the A-B permeability of mannitol, respectively, and decreased dramatically to a negligible level at 4 degrees C, consistent with a receptor mediated transcytosis of rhEGF. The stability of rhEGF was very poor, undergoing more than 85% degradation in 2 h in the transport medium at 37 degrees C. A significant increase in the Papp could be achieved by the addition of certain excipients, as exemplified by 23, 21, 20 and 16-fold increases, in the presence of sodium taurochenodeoxycholate (NaTCDC), sodium taurodeoxycholate (NaTDC), sodium glycodeoxycholate (NaGDC) and sodium laurylsulfate (SLS) (all at a concentration of 1% w/v), respectively. A significant increase in stability could also be achieved by the addition of some of the excipients, as represented by 1% SLS, which nearly completely stabilized the rhEGF. Unfortunately, however, an increase in the Papp of rhEGF could not be achieved without a simultaneous and extensive decrease in the integrity of the cell membranes. Thus, more efficient excipients, that specifically enhance the permeation of rhEGF and do not alter the membrane integrity, should be pursued in order to safely enhance the permeation of rhEGF.

Kinetic analysis about the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) by isolated rat hepatocytes.

The purpose of the present study was to investigate the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) using isolated rat hepatocytes. The initial uptake rate of ANS by isolated hepatocytes was determined. The uptake process of ANS was saturable, with a Km of 29.1 +/- 3.2 microM and Vmax of 2.9 +/- 0.1 mmol/min/mg protein. Subsequently, the initial efflux rate of ANS from isolated hepatocytes was determined by resuspending preloaded cells to 3.0% (w/v) BSA buffer. The efflux process for total ANS revealed a little saturability. The mean value of the efflux clearance was 2.2 +/- 0.1 microL/min/mg protein. The efflux rate of ANS from hepatocytes was markedly decreased at 4 degrees C, indicating that the apparent efflux of ANS might not be attributed to the release of ANS bound to the cell surface, but to the efflux of ANS from intracellular space. The efflux clearance was furthermore corrected for the unbound intracellular ANS concentration on the basis of its binding parameters to cytosol. The relation between efflux rate and unbound ANS concentration was fitted well to the Michaelis-Menten equation with a saturable and a nonsaturable components. The Vmax and Km values were 0.54 mmol/min/mg protein, and 10.0 microM, respectively. Based on the comparison of the ratios of Vmax to Km (Vmax/Km) corresponding to the transport clearance, the influx clearance was two times higher than the efflux clearance. Together with our preliminary studies that ATP suppression in hepatocytes substantially inhibited ANS influx rate, we concluded that the hepatic uptake of ANS is actively taken up into hepatocytes via the carrier mediated transport system.

The impact of efflux transporters in the brain on the development of drugs for CNS disorders.

The development of drugs to treat disorders of the CNS requires consideration of achievable brain concentrations. Factors that influence the brain concentrations of drugs include the rate of transport into the brain across the blood-brain barrier (BBB), metabolic stability of the drug, and active transport out of the brain by efflux mechanisms. To date, three classes of transporter have been implicated in the efflux of drugs from the brain: multidrug resistance transporters, monocarboxylic acid transporters, and organic ion transporters. Each of the three classes comprises multiple transporters, each of which has multiple substrates, and the combined substrate profile of these transporters includes a large number of commonly used drugs. This system of transporters may therefore provide a mechanism through which the penetration of CNS-targeted drugs into the brain is effectively minimised. The action of these efflux transporters at the BBB may be reflected in the clinic as the minimal effectiveness of drugs targeted at CNS disorders, including HIV dementia, epilepsy, CNS-based pain, meningitis and brain cancers. Therefore, modulation of these efflux transporters by design of inhibitors and/or design of compounds that have minimal affinity for these transporters may well enhance the treatment of intractable CNS disorders.

Cocaine increases dopamine uptake and cell surface expression of dopamine transporters.

In HEK 293 cells expressing the human dopamine transporter (DAT), a 10-min incubation with 10 microM cocaine followed by extensive washing resulted in a 30% increase in [3H]dopamine (DA) uptake as well as an increase in cell surface DAT in biotinylation experiments. Consistent with this novel regulation, [3H]DA uptake into synaptosomes prepared from the nucleus accumbens of rats sacrificed 30 min after a single cocaine injection (30 mg/kg) was significantly increased compared to controls (56% increase in V(max), no change in K(m)). In addition, DA clearance in the striatum of anesthetized rats was increased after local application of a low (3 pmol) but not high (65 pmol) dose of cocaine, presumably as a result of mobilization of DAT to the cell surface. Cocaine-induced increases in cell surface expression of DAT and associated changes in DA clearance represent a novel mechanism that may play a role in its addictive properties.

Selection and neutrality in lactose operons of Escherichia coli.

The kinetics of the permeases and beta-galactosidases of six lactose operons which had been transduced into a common genetic background from natural isolates of Escherichia coli were investigated. The fitnesses conferred by the operons were determined using chemostat competition experiments in which lactose was the sole growth-limiting factor. The cell wall is demonstrated to impose a resistance to the diffusion of galactosides at low substrate concentrations. A steady state model of the flux of lactose through the metabolic pathway (diffusion, uptake and hydrolysis) is shown to be proportional to fitness. This metabolic model is used to explain why an approximately twofold range in activity among the permease alleles confers a 13% range in fitness, whereas a similar range in activity among alleles of the beta-galactosidase confers a 0.5% range in fitness. This metabolic model implies that selection need not be maximized when a resource is scarce.