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The effect of polyphenols (PPs) on the absorption of ochratoxin A (OTA), a food-borne mycotoxin, was investigated in an in vitro model of the human intestinal barrier based on Caco-2 cells cultivated in a bicameral system. Two intraluminal concentrations of OTA approaching physiological levels were chosen (0.75 nM and 7.5 nM) through calculations based on estimated daily intakes. The transport of OTA from the apical to the basolateral side of Caco-2 cells, i.e. absorption, was directly proportional to its initial apical concentration. Very significant increase in both OTA absorption and cellular accumulation was observed upon co-incubation with certain PPs, i.e. chrysin, quercetin, genistein, biochanin A, resveratrol, at concentrations that should be encountered in the gastrointestinal tract, as well as with MK571, a specific inhibitor of MRPs efflux pumps. As these PPs have been reported to be metabolized in Caco-2 cells into substrates of MRP-2, we hypothesize that PPs and/or metabolites could impair the OTA efflux, previously proposed to be mediated by the MRP-2, through competitive inhibition for the pump. These data imply that interactions between OTA and PPs may lead to a greater bioavailability of the mycotoxin in the bloodstream with possible adverse effects for human health.

Searching for potent and subtype selective parent structures of the murine gamma-aminobutyric acid (GABA) transporter subtypes mGAT3 and mGAT4 a series of amino acids was characterised in a uniform [3H]GABA uptake test system based on transiently expressed mGAT1-4. From several potent inhibitors showing IC50 values at mGAT3 and mGAT4 in the low microM range cis-4-aminocrotonic acid and (RS)-2,3-diaminopropionic acid turned out to be most subtype selective for these transporters. With (RS)-isoserine--a compound unknown as GAT inhibitor until now--one of the most potent amino acids selectively inhibiting mGAT3 and mGAT4 was found. Furthermore, (2-amino-1,3-thiazol-4-yl)acetic acid was identified as the first parent structure exhibiting a clear, though still moderate, selective inhibition of GABA uptake at mGAT3.

Human concentrative nucleoside transporters 1, 2, and 3 (hCNT1, hCNT2, and hCNT3) exhibit different functional characteristics, and a better understanding of their permeant selectivities is critical for development of nucleoside analog drugs with optimal pharmacokinetic properties. In this study, the sensitivity of a high-throughput yeast expression system used previously for hCNT1 and hCNT3 was improved and used to characterize determinants for interaction of uridine (Urd) with hCNT2. The observed changes of binding energy between hCNT2 and different Urd analogs suggested that it interacts with C3'-OH, C5'-OH, and N3-H of Urd. The C2' and C5 regions of Urd played minor but significant roles for Urd-hCNT2 binding, possibly through Van der Waals interactions. Because the yeast assay only provided information about potential transportability, the permeant selectivities of recombinant hCNT1, hCNT2, and hCNT3 produced in Xenopus laevis oocytes were investigated using a two-electrode voltage clamp assay. hCNT1-mediated transport was sensitive to modifications of the N3, C3', and C5' positions of Urd. hCNT2 showed some tolerance for transporting Urd analogs with C2' or C5 modifications, little tolerance for N3 modifications, and no tolerance for any modifications at C3' or C5' of Urd. Although hCNT3 was sensitive to C3' modifications, it transported a broad range of variously substituted Urd analogs. The transportability profiles identified in this study, which reflected the binding profiles well, should prove useful in the development of anticancer and antiviral therapies with nucleoside drugs that are permeants of members of the hCNT protein family.

LcrV of Yersinia pestis is a major protective antigen proposed for inclusion in subunit plague vaccines. One way that anti-LcrV antibody is thought to protect is by inhibiting the delivery of toxins called Yops to host cells. The present study characterizes the relation between this inhibition and the phagocytosis of the bacteria. J774A.1 cells were infected with Y. pestis KIM5 in the presence of a protective polyclonal anti-LcrV antibody or a nonprotective polyclonal anti-YopM antibody, and delivery of YopH and YopE into the cytoplasm was assayed by immunoblotting. The ability to inhibit the delivery of these Yops depended upon having antibody bound to the cell surface; blocking conditions that prevented the binding of antibody to Fc receptors prevented the inhibition of Yop delivery. Anti-LcrV antibody also promoted phagocytosis of the yersiniae, whereas F(ab')(2) fragments did not. Further, anti-LcrV antibody could not inhibit the delivery of Yops into cells that were unable to phagocytose due to the presence of cytochalasin D. However, Yops were produced only by extracellular yersiniae. We hypothesize that anti-LcrV antibody does not directly inhibit Yop delivery but instead causes phagocytosis, with consequent inhibition of Yop protein production in the intracellular yersiniae. The prophagocytic effect of anti-LcrV antibody extended to mouse polymorphonuclear neutrophils (PMNs) in vitro, and PMNs were shown to be critical for protection: when PMNs in mice were ablated, the mice lost all ability to be protected by anti-LcrV antibody.

GABAergic inhibition in the brain can be classified as either phasic or tonic. gamma-Aminobutyric acid (GABA) uptake by GABA transporters (GATs) can limit the time course of phasic currents arising from endogenous and exogenous GABA, as well as decrease a tonically active GABA current. GABA transporter subtypes 1 and 3 (GAT-1 and GAT-3) are the most heavily expressed of the four known GAT subtypes. The role of GATs in shaping GABA currents in the neocortex has not been explored. We obtained patch-clamp recordings from layer II/III pyramidal cells and layer I interneurons in rat sensorimotor cortex. We found that selective GAT-1 inhibition with NO711 decreased the amplitude and increased the decay time of evoked inhibitory postsynaptic currents (IPSCs) but had no effect on the tonic current or spontaneous IPSCs (sIPSCs). GAT-2/3 inhibition with SNAP-5114 had no effect on IPSCs or the tonic current. Coapplication of NO711 and SNAP-5114 substantially increased tonic currents and synergistically decreased IPSC amplitudes and increased IPSC decay times. sIPSCs were not resolvable with coapplication of NO711 and SNAP-5114. The effects of the nonselective GAT antagonist nipecotic acid were similar to those of NO711 and SNAP-5114 together. We conclude that synaptic GABA levels in neocortical neurons are controlled primarily by GAT-1, but that GAT-1 and GAT-2/3 work together extrasynaptically to limit tonic currents. Inhibition of any one GAT subtype does not increase the tonic current, presumably as a result of increased activity of the remaining transporters. Thus neocortical GAT-1 and GAT-2/3 have distinct but overlapping roles in modulating GABA conductances.

Neurons in the external and internal segment of the globus pallidus (GPe and GPi, respectively) receive substantial GABAergic inputs from the striatum and through axon collaterals of neighboring pallidal neurons. The effects of GABA on pallidal activity depend on the synaptic localization of GABA receptors and the distribution and activity of GABA transporters (GATs). To explore the contribution of GABA receptors and transporters to pallidal function, we recorded the activity of single neurons in GPe or GPi before, during, and after local microinjections of GABAergic compounds in awake rhesus monkeys. Activation of GABA(A) or GABA(B) receptors with muscimol or baclofen, respectively, inhibited pallidal activity. These effects were reversed by concomitant infusion of the respective GABA receptor antagonists, gabazine and CGP-55845. Given alone, the antagonists were without consistent effect. Application of the selective GAT-1 inhibitor, SKF-89976A, and the semiselective GAT-3 blocker, SNAP-5114, decreased pallidal activity. Both GAT inhibitors increased GABA levels in the pallidum, as measured by microdialysis. Electron microscopic observations revealed that these transporters are located on glial processes and unmyelinated axonal segments, but rarely on terminals. Our results indicate that activation of GABA(A) and GABA(B) receptors inhibits neuronal activity in both segments of the pallidum. GAT-1 and GAT-3 are involved in the modulation of endogenous GABA levels and may be important in regulating the extrasynaptic levels of GABA. Together with previous evidence that a considerable proportion of pallidal GABA receptors are located outside the synaptic cleft, our experiments strongly support the importance of extrasynaptic GABAergic transmission in the primate pallidum.

In Gram-negative bacteria, efflux complexes, consisting of an inner-membrane pump, a periplasmic adaptor protein and outer-membrane channel, provide an efficient means for the export of structurally unrelated drugs, causing the multidrug-resistance phenotype. Resistance due to this antibiotic efflux is an increasing problem worldwide. A new molecular challenge is to combat this transport by searching for new molecules to block efflux and thus restore drug susceptibility to resistant clinical strains. Recent data shed new light on the structure and activity of the archetypal efflux pumps AcrAB-TolC and MexAB-OprM. Here, we describe recent insights into the molecular mechanisms of bacterial efflux pumps and their inhibitors. Current progress for the clinical use of efflux-pump inhibitors and new strategies to combat the drug-efflux mechanisms will be discussed.

Serotonin (5-hydroxytryptamine [5-HT]), is a neurohormone found in various nonneural tissues, including the gonads of many invertebrates, in which it regulates spawning and oocyte meiotic maturation. The possibility that a local serotonergic network might also exist in the female gonads of vertebrate species, including mammals, remains poorly documented. To clarify this possibility, we investigated mouse cumulus cells, oocytes, and embryos for three key serotonergic components, namely, 5-HT itself; the rate-limiting enzyme for its production, tryptophan hydroxylase 1 (TPH1); and the 5-HT-specific transporter (SLC6A4) required for modulating its cellular effects. Using a combination of reverse transcription-polymerase chain reaction analysis and immunofluorescence confocal microscopy, we showed that mouse cumulus cells, oocytes, and embryos contain 5-HT and SLC6A4, while only cumulus cells possess the 5-HT-producing enzyme TPH1 and may thus be the local source of 5-HT observed in their neighboring cells. With a semiquantitative assay in single cells, we demonstrated that 5-HT can actively be taken up by isolated oocytes when it is supplied exogenously in vitro. This 5-HT transport in isolated oocytes is driven by a classical serotonin transporter, expressed up to the blastocyst stage, that is sensitive to the antidepressants fluoxetine and fluvoxamine, which belong to the selective serotonin reuptake inhibitor family. All together, our results show that 5-HT may be produced locally by cumulus cells and that it can be actively taken up by mammalian oocytes and embryos as part of a likely larger serotonergic network possibly regulating various developmental processes much earlier than previously thought.

The mechanism of uptake of dietary niacin (nicotinic acid) by intestinal epithelial cells is not well understood, and nothing is known about regulation of the uptake process. In this investigation, we used human-derived intestinal epithelial Caco-2 cells and purified intestinal brush-border membrane vesicles (BBMVs) isolated from human organ donors to assess niacin uptake. Our findings show niacin uptake by Caco-2 cells to be 1) temperature and energy dependent; 2) Na+ independent, but highly dependent on extracellular acidic pH; 3) saturable as a function of concentration, with an apparent K(m) of 0.53 +/- 0.08 microM; 4) severely inhibited by the membrane-impermeable sulfhydryl group of reagents; and 5) highly specific for niacin but not affected by monocarboxylic acids. A marked trans stimulation in [3H]niacin efflux from preloaded Caco-2 cells by unlabeled niacin in the incubation buffer was also observed. These findings suggest the involvement of a specialized, pH-dependent, carrier-mediated mechanism for human intestinal niacin uptake. This suggestion was confirmed in studies with native human intestinal BBMVs. We also examined possible regulation of niacin uptake by Caco-2 cells via specific intracellular regulatory pathways. The results show that while the PKA-, PKC-, and Ca2+/calmodulin-mediated regulatory pathways play no role in regulating niacin uptake, a role for a protein tyrosine kinase (PTK)-mediated pathway is apparent. The results of these studies show for the first time the existence of a specialized, acidic pH-dependent, carrier-mediated system of niacin uptake by human intestinal epithelial cells that operates at the micromolar (physiological) range of niacin. The results also suggest the possible involvement of a PTK-mediated pathway in the regulation of niacin uptake.

In an effort to produce compounds with longer durations of action, we attempted to synthesize ketone analogs of methylphenidate which, however, appear to be highly unstable due to a highly acidic proton alpha to the ketone and phenyl groups. Nevertheless, vinylogous amide by products have been synthesized and tested for activity at dopamine, norepinephrine, and serotonin transporters. The compounds were found to be weak inhibitors of monoamine reuptake despite rigid three dimensional structures that are quite similar to the global minimum of threo-(R,R)-methylphenidate. The structures were confirmed by X-ray crystallography.

Background: Postmortem studies have reported several alterations in serotonin transporter (SERT) binding parameters in patients with schizophrenia. The aim of this study was to compare SERT availability in vivo in patients with schizophrenia and matched control subjects., Methods: Ten medication-free patients with schizophrenia and 10 healthy subjects underwent positron emission tomography (PET) scans for 90 min after 11C-3-amino-4-(2-dimethylaminomethylphenylthio)benzonitrile ([11C]DASB) injection. Metabolite-corrected arterial input function was measured. Regional distribution volumes (mL/g) were derived with a two tissue compartment kinetic model. Outcome measures for SERT availability included binding potential (BP) and the specific-to-nonspecific equilibrium partition coefficient (V3''). Ten brain regions with high density of SERT and where SERT availability can be reliably quantified with [11C]DASB were included in the analysis., Results: No significant differences were observed in regional BP or V3'' between patients and control subjects. No significant relationships were observed between regional SERT availability and severity of positive, negative, and depressive symptoms., Conclusions: This study failed to detect alterations of SERT availability in patients with schizophrenia; however, this study does not rule out the possibility that schizophrenia might be associated with alterations of SERT density in the cortical regions, where the [11C]DASB-specific binding signal is too low for reliable quantification of SERT.

To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of alpha,beta-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. Alpha,beta-unsaturated carbonyl compounds react with glutathione, and therefore either their parent compound or their intracellularly formed glutathione metabolite(s) can modulate MRP-activity. Inhibition was studied in Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, and isolated Sf9-MRP1 or Sf9-MRP2 membrane vesicles. In the latter model system metabolism is not an issue. Of the series tested, three distinct groups could be discriminated based on differences in interplay of glutathione metabolism with MRP1 inhibition. Curcumin inhibited MRP1 transport only in the vesicle model pointing at inhibition by the parent compound. The glutathione conjugates of curcumin also inhibit MRP1 mediated transport, but to a much lesser extent than the parent compound curcumin. In the cellular model system, it was demonstrated that glutathione conjugation of curcumin leads to inactivation of its inhibitory potential. Demethoxycurcumin and bisdemethoxycurcumin inhibited MRP1 in both the vesicle and cellular model pointing at inhibitory potency of at least the parent compound and possibly their metabolites. A second group, including caffeic acid phenethyl ester inhibited MRP1-mediated calcein transport only in the MDCKII-MRP1 cells, and not in the vesicle model indicating that metabolism appeared a prerequisite to generate the active inhibitor. Finally cinnamaldehyde, crotonaldehyde, trans-2-hexanal, citral, and acrolein did not inhibit MRP1. For MRP2, inhibition was much less in both model systems, with the three curcuminoids being the most effective. The results of this study show the importance to study the complex interplay between MRP-inhibitors and their cellular metabolism, the latter affecting the ultimate potential of a compound for cellular MRP-inhibition.

A series of lobelane analogues has been synthesized and their structure-activity relationships at the vesicular monoamine transporter-2 (VMAT2) have been evaluated. The most potent analogues in this series were the cis-2,6-piperidino analogues, 25b, 27b, 28b, and 30b, with K(i) values ranging from 430 to 580 nM.

The serotonin transporter (SERT) regulates 5-hydroxytryptamine (serotonin) (5-HT) neurotransmission and is a high-affinity target for antidepressants and psychostimulants. In the present study, we investigated the mechanisms that contribute to a previously unidentified biphasic regulation of endogenous SERTs expressed in the platelets. Treatment of rat platelets with beta-phorbol 12-myristate 13-acetate (PMA) for 5 min or less resulted in a rapid inhibition of SERT involving changes in intrinsic activity of the transporter (increased K(m) and decreased V(max)). beta-PMA treatment for 30 min or more produced a sustained inhibition of SERT with a decrease only in the V(max). Whereas inhibition of SERT activity was detected from 1 to 45 min after phorbol ester addition, the decrease in surface SERT required at least 30 min of phorbol ester incubation. Increased endocytosis of SERT accounted for the decrease in surface SERT at the later point. Protein kinase C (PKC)-mediated phosphorylation of SERT occurs on the plasma membrane during the initial phase of rapid transporter inhibition, and later, the phosphorylated SERT enters the intracellular pool. beta-PMA-induced phosphorylation of SERT occurs initially on serine residues(s) and then on threonine residue(s). The initial serine phosphorylation corresponded to the first phase of rapid inhibition mediated by changes in intrinsic activity and/or silencing of SERT. The later phosphorylation on threonine residue(s) corresponded to the later phase of sustained inhibition mediated by an enhanced endocytosis of SERT. Together, these data reveal that in platelets, SERT function is regulated by PKC in a biphasic manner involving both trafficking-dependent and independent mechanisms and that these two events occur at distinct phases of transporter phosphorylation.

Protein reserves in the cereal endosperm are sequentially degraded to small peptides and amino acids during germination and these are translocated across the scutellum to support growth of the embryo. Peptide transport in the germinating barley grain is mediated by specific carriers localized to the plasma membrane of the scutellar epithelium. In isolated barley embryos peptide transport is rapidly inhibited by amino acid concentrations comparable with those found in the post-germination barley grain. However, this inhibition of HvPTR1 activity is not effected at either the transcriptional or translational level. The protein phosphatase inhibitor okadaic acid repressed transport of Ala-[14C]Phe, but not [14C]Ala, into the barley scutellar epithelium. In vivo [32P]orthophosphate labelling studies of barley scutellar tissue in combination with immunoprecipitation studies using antiserum raised to HvPTR1 showed that HvPTR1 (66 kDa) is phosphorylated in the presence of amino acids. Immunopurified HvPTR1 was further demonstrated to be phosphorylated on serine residues. Digestion with the N-glycosidase enzyme PNGase F results in a shift in the molecular mass of the protein by 10 kDa, indicating that HvPTR1 is an N-linked glycoprotein. These results provide strong circumstantial evidence that HvPTR1 peptide transport activity in the germinating barley grain is regulated at the post-translational level by phosphorylation in response to rising levels of amino acids emanating from the endosperm as a result of storage protein breakdown and mobilization. This is potentially an important element in balancing the flux of organic nitrogen and carbon from the endosperm to embryo during germination and seedling establishment.

Although many studies have revealed alterations in neurotransmission during ischaemia, few works have been devoted to the neurochemical effects of mild hypoxia, a situation encountered during life in altitude or in several pathologies. In that context, the present work was undertaken to determine the in vivo mechanisms underlying the striatal dopamine efflux induced by mild hypoxaemic hypoxia. For that purpose, the extracellular concentrations of dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid were simultaneously measured using brain microdialysis during acute hypoxic exposure (10% O(2), 1h) in awake rats. Hypoxia induced a +80% increase in dopamine. Application of the dopamine transporters inhibitor, nomifensine (10 microM), just before the hypoxia prevented the rise in dopamine during the early part of hypoxia; in contrast the application of nomifensine after the beginning of hypoxia, failed to alter the increase in dopamine. Application of the voltage-dependent Na(+) channel blocker tetrodotoxin abolished the increase in dopamine, whether administered just before or after the beginning of hypoxia. These data show that the neurochemical mechanisms of the dopamine efflux may change over the course of the hypoxic exposure, dopamine transporters being involved only at the beginning of hypoxia.

In the present study, the effects of myricetin on either MRP1 or MRP2 mediated vincristine resistance in transfected MDCKII cells were examined. The results obtained show that myricetin can inhibit both MRP1 and MRP2 mediated vincristine efflux in a concentration dependent manner. The IC50 values for cellular vincristine transport inhibition by myricetin were 30.5+/-1.7 microM for MRP1 and 24.6+/-1.3 microM for MRP2 containing MDCKII cells. Cell proliferation analysis showed that the MDCKII control cells are very sensitive towards vincristine toxicity with an IC50 value of 1.1+/-0.1 microM. The MDCKII MRP1 and MRP2 cells are less sensitive towards vincristine toxicity with IC50 values of 33.1+/-1.9 and 22.2+/-1.4 microM, respectively. In both the MRP1 and MRP2 cells, exposure to 25 microM myricetin enhances the sensitivity of the cells towards vincristine toxicity to IC50 values of 7.6+/-0.5 and 5.8+/-0.5 microM, respectively. The increase of sensitivity represents a reversal of the resistance towards vincristine as a result of MRP1 and MRP2 inhibition. Thus, the present study demonstrates the ability of the flavonoid myricetin to modulate MRP1 and MRP2 mediated resistance to the anticancer drug vincristine in transfected cells, indicating that flavonoids might be a valuable adjunct to chemotherapy to block MRP mediated resistance.

A key to obtaining an X-ray structure of the lactose permease of Escherichia coli (LacY) (Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R., and Iwata, S. (2003) Science 301, 549-716) was the use of a mutant in which Cys154 (helix V) is replaced with Gly. LacY containing this mutation strongly favors an inward-facing conformation, which binds ligand with high affinity, but catalyzes little transport and exhibits few if any of the ligand-dependent conformational changes observed with wild-type LacY. The X-ray structure demonstrates that helix V crosses helix I in the approximate middle of the membrane in such a manner that Cys154 lies close to Gly24 (helix I). Therefore, it seems likely that replacing Cys154 with Gly may lead to tighter packing between helices I and V, thereby resulting in the phenotype observed. Consistently, replacement of Gly24 with Cys in the C154G mutant rescues significant transport activity, and the mutant exhibits properties similar to wild-type LacY with respect to substrate binding and thermostability. However, the only other replacements that rescue transport to any extent whatsoever are Val and Asp, both of which are much less effective than Cys. The results suggest that, although helix packing probably plays an important role with respect to the properties of the C154G mutant, the ability of Cys at position 24 to rescue transport activity of C154G is more complicated than simple replacement of bulk between positions 24 and 154. Rather, activity is dependent on more subtle interactions between the helices, and mutations that disrupt interactions between helix IV and loop 6-7 or between helices II and IV also rescue transport in the C154G mutant.

Serotonin (5-hydroxytryptamine, 5-HT) is an amine neurotransmitter derived from tryptophan and is important in brain systems regulating mood, emotional behavior, and sleep. Selective serotonin reuptake inhibitor (SSRI) drugs are used to treat disorders such as depression, stress, eating disorders, autism, and schizophrenia. It is thought that these drugs act to prolong the action of 5-HT by blocking reuptake. This may lead to decreased 5-HT content in the nerve fibers themselves; however, this has not previously been directly demonstrated. We have studied the effects of administration of two drugs, imipramine and citalopram, on levels of 5-HT in nerve fibers in the murine brain. Quantitative analysis of the areal density of 5-HT fibers throughout the brain was performed using ImageJ software. While a high density of fibers was observed in mid- and hind-brain regions and areas such as thalamus and hypothalamus, densities were far lower in areas such as cortex, where SSRIs might be thought to exert their actions. As anticipated, imipramine and citalopram produced a decline in 5-HT levels in nerve fibers, but the result was not uniform. Areas such as inferior colliculus showed significant reduction whereas little, if any, change was observed in the adjacent superior colliculus. The reason for, and significance of, this regionality is unclear. It has been proposed that serotonin effects in the brain might be linked to changes in glutamatergic transmission. Extracellular glutamate levels are regulated primarily by glial glutamate transporters. Qualitative evaluation of glutamate transporter immunolabeling in cortex of control and drug-treated mice revealed no discernable difference in intensity of glutamate transporter immunoreactivity. These data suggest that changes in intracellular and extracellular levels of serotonin do not cause concomitant changes in astroglial glutamate transporter expression, and thus cannot represent a mechanism for the delayed efficacy of antidepressants when administered clinically.

Pulmonary arterial 5-hydroxytryptamine (serotonin) (5-HT) transporter (SERT)-, 5-HT receptor expression, and 5-HT-induced vasoconstriction can be increased in pulmonary hypertension. These variables were studied in normoxic and hypoxic Fawn-Hooded (FH) and Sprague-Dawley (SD) rats. Furthermore, we compared the functional effects of SERT inhibitors and 5-HT receptor antagonists against 5-HT-induced vasoconstriction of pulmonary arteries. SERT and 5-HT(1B) expression was greater in FH rat lungs than in SD rats, as was 5-HT-mediated vasoconstriction. The 5-HT(2A) receptor antagonist ketanserin and the 5-HT(1B) receptor antagonist SB224289 (1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydro-spiro-[furo] 2, 3-f]indole-3,4'-piperidine]) inhibited responses to 5-HT in all vessels. The combined 5-HT(1B) receptor/SERT antagonist LY393558 (1-[2-[4-(6-fluoro-1H-indol-3-yl)-3,6-dihydro-1(2H)-pyridinyl]ethyl]-3-isopropyl-6-(methylsulfonyl)-3,4-dihydro-1H-2,1,3-benzothiadiazine-2,2-dioxide) was the most potent inhibitor of constriction in all vessels. SERT inhibitors citalopram and fluoxetine inhibited responses to 5-HT in SD vessels. However, these inhibitors potentiated responses to 5-HT in FH vessels. After exposure of rats to 2 weeks of hypoxia, there was increased 5-HT-mediated vasoconstriction and a profound decrease in SERT expression in both the FH and SD rat lung. Accordingly, citalopram had no effect on 5-HT-induced constriction in SD rat vessels and markedly less effect in FH rat vessels. Ketanserin, SB224289, and LY393558 inhibited responses to 5-HT in all hypoxic rat vessels. LY393558 was the most potent antagonist, and there was synergy between the effects of fluoxetine and SB224289 when given simultaneously. The results suggest that, in FH rats, SERT inhibitors may increase pulmonary vasoconstriction, but this can be inhibited by simultaneous 5-HT(1B) receptor antagonism. There is synergy between the inhibitory effects of 5-HT(1B) receptor antagonists and SERT inhibitors on 5-HT-induced pulmonary vasoconstriction.

In mycophenolate mofetil (MMF)-treated organ transplant recipients, lower mycophenolic acid (MPA) plasma concentrations have been found in cyclosporine (CsA) compared with tacrolimus (Tac)-based immunosuppressive regimens. We previously demonstrated that CsA decreases exposure to MPA and increases exposure to its metabolite MPA-glucuronide (MPAG), possibly by interfering with the biliary excretion of MPAG. To elucidate the role of the multidrug resistance-associated protein (Mrp)-2 in the interaction between MMF and CsA, we treated three groups of 10 Mrp2-deficient rats (TR- rat) for 6 days with either vehicle, CsA (8 mg/kg) or Tac (4 mg/kg) by oral gavage. Hereafter, co-administration with MMF (20 mg/kg) was started in all groups and continued through day 14. The 24-h MPA/MPAG area under the concentration-time curve (AUC) was determined after single (day 7) and multiple MMF doses (day 14). On both study days, there were no significant differences in the mean MPA and MPAG AUC between CsA and Tac-treated animals. We conclude that the pharmacokinetics of MMF are comparable in Mrp2-deficient rats receiving either CsA or Tac as co-medication. This finding suggests that CsA-mediated inhibition of the biliary excretion of MPAG by the Mrp2 transporter is the mechanism responsible for the interaction between CsA and MMF.

The potent tropane analog, WF-23 [2beta-propanoyl-3beta-(2-naphthyl) tropane], blocks dopamine, serotonin, and norepinephrine transporters with high affinity in vitro and blocks transporters for at least 2 days following a single in vivo administration. Previous studies demonstrated desensitization of monoamine receptor-coupled G-proteins in brain following chronic treatment of rats with WF-23. The current study sought to determine the time course of this desensitization and the behavioral effects of receptor desensitization. Rats were treated with 1 mg/kg WF-23 and injected i.p. every 48 h for 1 to 21 days. Receptor activation of G-proteins was determined by guanosine 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding in brain sections for monoamine receptors, as well as mu opioid receptors as a nonmonoamine receptor control. Chronic treatment with WF-23 produced significant reductions in D(2), 5-hydroxytryptamine 1A, and alpha(2)-adrenergic receptor-stimulated [(35)S]GTPgammaS binding; however, the time course of desensitization varied with different receptors. There was no effect of WF-23 treatment on mu opioid-stimulated [(35)S]GTPgammaS binding at any time point. Consistent with previous studies, there was no effect of WF-23 treatment on D(2) receptor binding, as determined by [(3)H]spiperone autoradiography. Locomotor activity was significantly increased for up to 48 h following acute administration of WF-23, demonstrated by increased photocell beam interruptions. WF-23-induced increases in locomotor activity occurred following repeated administration, as above, for up to 7 days. Following 7 days of treatment, there was a significant decrease in WF-23-increased locomotor activity. This reduction occurred at the same time point as the decrease in D(2) receptor/G-protein coupling, suggesting a role of D(2) desensitization in producing tolerance to WF-23-mediated behavior.

Objective: Sibutramine, a serotonin and norepinephrine transporter inhibitor, is widely used as an adjunctive obesity treatment. There have been concerns that norepinephrine reuptake inhibition with sibutramine could exacerbate arterial hypertension., Design: Combined analysis of two placebo-controlled trials., Subjects: The combined data set consisted of 1336 patients. Of these patients, 966 were randomized to sibutramine and 370 were randomized to placebo., Measurements: Body weight, blood pressure, heart rate (HR)., Results: Sibutramine reduced body weight regardless of basal blood pressure. In the complete set of patients, systolic blood pressure did not change with either intervention over the 48-week period (-0.1+/-15.5 mmHg with sibutramine, -0.2+/-15.2 mmHg with placebo, P=0.9). The change in diastolic blood pressure over the 48 week period was 0.3+/-9.5 mmHg with sibutramine and -0.8+/-9.2 mmHg with placebo (P=0.049). The blood pressure response was not exacerbated in patients with grade 1 or 2 hypertension or in patients with isolated systolic hypertension. Sibutramine treatment caused a slight increase in supine HR that was sustained throughout the studies., Conclusions: Sibutramine treatment is unlikely to elicit a critical increase in blood pressure even in hypertensive patients. However, blood pressure and HR should be monitored closely. In patients who experience a clinically significant and sustained increase in blood pressure, the drug should probably be discontinued.

Aim: To investigate the effect of 5-hydroxytryptamine transporter (5-HTT) inhibitor fluoxetine and antisense oligodeoxynucleotide (ODN) to extracelluar signal-regulated kinases (ERKs) on pulmonary arterial smooth muscle cells (PASMCs) proliferation induced by 5-HT., Methods: Liposomal transfection was used to introduce ODNs to ERK1/2 into cultured rat PASMCs and the transfection efficiency was measured by observing the uptake of the fluorecein isothiocynate (FITC)-labeled antisense ODN in PASMCs. The effects of 5-HTT selective inhibitor fluoxetine and ODNs on the proliferation of PASMCs were evaluated by cell number counting and cell cycle analysis, and measured by microculture tetrazolium (MTT) assay and flow cytometry (FCM), respectively., Results: Liposomes mediated the transfection of ODNs into PASMCs with high efficiency. MTT assay showed fluoxetine (10 micromol/L, 1 micromol/L, and 100 nmol/L) concentration dependently inhibited the proliferation of PASMCs induced by 5-HT (1 micromol/L) in vitro. The proliferation rate of PASMCs by 5-HT was significantly inhibited by pretreatment with ERK1/2 antisense ODN (0.2 micromol/L) from 251%+/-18% to 86%+/-5% (P<0.01). Flow cytometric analysis of cell cycle distribution showed that the increase of 5-HT induced S phase fraction (SPF) and proliferation index (PI) were significantly inhibited by fluoxetine (1 micromol/L) or antisense ODN with SPF from 36%+/-4% to 26%+/-3% and 24%+/-4%, and PI from 34%+/-2% to 29%+/-2% and 24%+/-2%, respectively., Conclusion: 5-HTT mediates the mitogenic effect of 5-HT on PASMCs and the proliferation of PASMCs induced by 5-HT is dependent on ERKs signal pathway.

gamma-Aminobutyric acid (GABA) transporters (GATs) play an important role in inhibitory neurotransmission by clearing synaptically released GABA and by maintaining low resting levels of GABA in synaptic and extrasynaptic regions. In certain brain regions, vesicular zinc is colocalized and coreleased with glutamate and modulates the behavior of a number of channels, receptors, and transporters. We examined the effect of zinc on expressed GATs (GAT1, GAT2, GAT3, and GAT4) in Xenopus laevis oocytes by using tracer flux and electrophysiological methods. We show that zinc is a potent inhibitor of GAT4 (K(i) of 3 muM). Immunolocalization of GAT4 in the hippocampus revealed dense localization in the CA1 and CA3 regions of the hippocampus, regions which are known to be heavily populated by zinc-containing glutamatergic neurons. The results suggest a physiological role of synaptically released zinc in the hippocampus, because zinc released from hyperactive glutamatergic neurons may simultaneously bring about elevated GABAergic inhibition. Therefore, this mode of zinc function signifies a link between excitatory and inhibitory neurotransmission and may play a neuroprotective role against glutamate-induced excitotoxicity.

Cocaine, methylphenidate and other drugs that block dopamine transport indirectly promote immediate early gene expression, via dopamine-mediated activation of D1 dopamine receptors. Increased expression of the immediate early gene (IEG) c-fos, initiates a cascade of intracellular events that may underlie neuroadaptive changes following repeated exposure to the drugs. We investigated whether substrates (dopamine, norepinephrine) of the human dopamine (hDAT) and norepinephrine (hNET) transporters can directly induce c-Fos protein in HEK-293 (HEK) cells transfected with the hDAT and hNET and whether PKC modulators affect this process. Dopamine and norepinephrine robustly induced c-Fos immunofluorescence in both hDAT and hNET cells, but not in untransfected HEK-293 cells, demonstrating that catecholamine-induced c-Fos induction was DAT- and NET-dependent. The PKC activator PMA induced c-Fos in hDAT, hNET and HEK cell lines indicating that PKC stimulated c-Fos independent of transporters. The PKC inhibitor bisindolylmaleimide I (BIS) significantly increased c-Fos expression in hDAT cells, but not in hNET or HEK-293 cells, suggesting that inhibition of DAT-mediated phosphorylation results in c-Fos induction. BIS pretreatment abolished norepinephrine-induced c-Fos expression hNET but not dopamine-induced c-Fos induction in hDAT cells. In conclusion, induction of c-Fos by dopamine and norepinephrine requires the presence of hDAT and hNET but the contributions of hDAT and hNET to c-Fos induction is distinguishable on the basis of differing responses to a PKC inhibitor. These findings present a cell system and methodology for investigating the potential contribution of monoamine transporters to pre-synaptic neuroadaptation.

Pre-synaptic norepinephrine (NE) and dopamine (DA) transporters (NET and DAT) terminate catecholamine synaptic transmission through reuptake of released neurotransmitter. Recent studies reveal that NET and DAT are tightly regulated by receptor and second messenger-linked signaling pathways. Common approaches for studying these transporters involve use of radiolabeled substrates or antagonists, methods possessing limited spatial resolution and that bear limited opportunities for repeated monitoring of living preparations. To circumvent these issues, we have explored two novel assay platforms that permit temporally resolved quantitation of transport activity and transporter protein localization. To monitor the binding and transport function of NET and DAT in real-time, we have investigated the uptake of the fluorescent organic compound 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (ASP+). We have extended our previous single cell level application of this substrate to monitor transport activity via high-throughput assay platforms. Compared to radiotracer uptake methods, acquisition of ASP+ fluorescence is non-isotopic and allows for continuous, repeated transport measurements on both transfected and native preparations. Secondly, we have extended our application of small-molecule-conjugated fluorescent CdSe/ZnS nanocrystals, or quantum dots (Qdots), to utilize antibody and peptide ligands that can identify surface expressed transporters, receptors and other membrane proteins in living cell systems. Unlike typical organic fluorophores, Qdots are highly resistant to bleaching and can be conjugated to multiple ligands. They can also be illuminated by conventional light sources, yet produce narrow, gaussian emission spectra compatible with multiple target visualization (multiplexing). Together, these approaches offer novel opportunities to investigate changes in transporter function and distribution in real-time with superior spatial and temporal resolution.

The structural requirements for high affinity at the serotonin transporter (5-HTT) have been investigated through the preparation of rigid paroxetine analogs. Tropane-derived analogs (4a-i) of paroxetine (2) were designed and synthesized as potential inhibitors of serotonin reuptake based on the structural and biological similarity between the two compound classes. Overall, the affinity of tropane-derived analogs at the 5-HTT was found to be at least an order of magnitude lower than that of paroxetine and ranged from 2-400nM. The reduced affinity at the 5-HTT may be attributed to the inability of the rigid tropane-derived analogs to adopt conformations favored by the 5-HTT. Within the series of tropane analogs, the 2beta,3beta- and 2beta,3alpha-isomers, 4a and 4d, were the most potent at the DAT and NET and are also significantly more potent than paroxetine (2) suggesting that their reduced conformational flexibility maximizes residence time in conformations favored by these transporters. Examination of the previously published preparation and structural assignment of 4a by additional NMR and X-ray crystallographic data has established that nucleophilic addition to the intermediate 2beta-methanesulfonyloxymethyl-3beta-(4-fluorophenyl)tropane unexpectedly provided the aza-bicyclo[3.2.2]nonane derivative 10a.

A "multitarget multiribozyme" (MTMR) was constructed. It consists of three trans-acting hammerhead ribozymes directed against the transcripts of the ABC transporters MDR1/P-gp, BCRP, and MRP2; three cis-acting MDR1/P-gp-specific ribozymes; and three MDR1/P-gp-homologous spacer sequences. The trans-acting hammerhead ribozymes are liberated from the MTMR through autocatalytic self-cleavage by the cis-acting ribozymes. The MTMR was characterized with regard to its kinetic parameters. Comparison of the MTMR-specific kinetic values with those of the corresponding monoribozymes demonstrated that MTMR fragments could cleave their specific substrates without loss of efficiency. The MTMR was applied to three cell models, each overexpressing another ABC transporter, i.e., the gastric carcinoma cell line EPG85-257RDB expresses MDR1/P-gp, the cell variant EPG85-257RNOV synthesizes BCRP, and the ovarian carcinoma line A2780RCIS produces MRP2. In all cellular systems, the MTMR could specifically decrease the expression of the respective ABC transporter at the mRNA level (97% decrease in the MDR1/P-gp mRNA, 80% decrease in the BCRP mRNA, 96% decrease in the MRP2 mRNA) and the protein level. Resistance against the selection drug was reversed completely (100% in EPG85-257RDB) or by 94 (EPG85-257RNOV) or 63% (A2780RCIS). Thus, the MTMR technology provides a novel tool for gene therapeutic applications to reverse different ABC-transporter-dependent drug-resistant phenotypes.

Two novel thiazolidine compounds, GW604714X and GW450863X, were found to be potent inhibitors of mitochondrial respiration supported by pyruvate but not other substrates. Direct measurement of pyruvate transport into rat liver and yeast mitochondria confirmed that these agents inhibited the mitochondrial pyruvate carrier (MPC) with K(i) values <0.1 muM. Inhibitor titrations of pyruvate-dependent respiration by heart mitochondria gave values (+/-S.E.) for the concentration of inhibitor binding sites (pmol per mg protein) and their K(i) (nM) of 56.0+/-0.9 and 0.057+/-0.010 nM for the more hydrophobic GW604714X; for GW450863X the values were 59.9+/-4.6 and 0.60+/-0.12 nM. [(3)H]-methoxy-GW450863X binding was also used to determine the MPC content of the heart, kidney, liver and brain mitochondria giving values of 56, 40, 26 and 20 pmol per mg protein respectively. Binding to yeast mitochondria was <10% of that in rat liver mitochondria, consistent with the slow rate of pyruvate transport into yeast mitochondria. [(3)H]-methoxy-GW450863X binding was inhibited by GW604714X and by the established MPC inhibitor, UK5099. The absorbance spectra of GW450863X and GW604714X were markedly changed by the addition of beta-mercaptoethanol suggesting that the novel inhibitors, like alpha-cyanocinnamate, possess an activated double bond that attacks a critical cysteine residue on the MPC. However, no labelled protein was detected following SDS-PAGE suggesting that the covalent modification is reversible. GW604714X and GW450863X inhibited l-lactate transport by the plasma membrane monocarboxylate transporter MCT1, but at concentrations more than four orders of magnitude greater than the MPC.

Multidrug resistance protein 2 (MRP2, ABCC2) is a drug efflux pump belonging to the ATP-binding cassette (ABC) transporter superfamily. MRP2 is present predominantly at the biliary pole of hepatocytes and is also expressed in the kidney and intestine. It plays a major role in hepato-biliary elimination of many structurally diverse xenobiotics, including organic anions and drug conjugates, and therefore most likely contributes to pharmacokinetic parameters of these compounds. MRP2 also handles endogenous molecules such as bilirubin, and its overexpression has been shown to confer a multidrug resistance phenotype to tumoral cells. MRP2 expression can be regulated by endogenous substances such as inflammatory cytokines and biliary acids. The MRP2 levels and activity can also be affected by a large panel of xenobiotics, including chemopreventive agents and ligands of the pregnane X receptor, which may be a potential source of drug-drug interactions and drug adverse effects. MRP2 appears therefore as one of the major drug efflux pumps of the organism, whose functional and regulatory features are important to consider, notably for drug disposition.

Neurotransmitter transporters play a major role in achieving low concentrations of their respective transmitter in the synaptic cleft. The GABA transporter GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins which possess 12 putative transmembrane domains and three N-glycosylation sites in the extracellular loop between transmembrane domain 3 and 4. To study the significance of N-glycosylation, green fluorescence protein (GFP)-tagged wild type GAT1 (NNN) and N-glycosylation defective mutants (DDQ, DGN, DDN and DDG) were expressed in CHO cells. Compared with the wild type, all N-glycosylation mutants showed strongly reduced protein stability and trafficking to the plasma membrane, which however were not affected by 1-deoxymannojirimycin (dMM). This indicates that N-glycosylation, but not terminal trimming of the N-glycans is involved in the attainment of a correctly folded and stable conformation of GAT1. All N-glycosylation mutants were expressed on the plasma membrane, but they displayed markedly reduced GABA-uptake activity. Also, inhibition of oligosaccharide processing by dMM led to reduction of this activity. Further experiments showed that both N-glycosylation mutations and dMM reduced the V(max) value, while not increasing the K(m) value for GABA uptake. Electrical measurements revealed that the reduced transport activity can be partially attributed to a reduced apparent affinity for extracellular Na+ and slowed kinetics of the transport cycle. This indicates that N-glycans, in particular their terminal trimming, are important for the GABA-uptake activity of GAT1. They play a regulatory role in the GABA translocation by affecting the affinity and the reaction steps associated with the sodium ion binding.

Background: (2S,3S)-2-(3-Chlorophenyl)-3,5,5,-trimethyl-2-morpholinol hydrochloride (radafaxine) is a new antidepressant that blocks dopamine transporters (DAT). A concern with drugs that block (DAT) is their potential reinforcing effects and abuse liability. Using positron emission tomography (PET) we have shown that for DAT-blocking drugs to produce reinforcing effects they must induce >50% DAT blockade and the blockade has to be fast (within 15 minutes). This study measures the potency and kinetics for DAT blockade by radafaxine in human brain., Methods: PET and [11C]cocaine were used to estimate DAT blockade at 1, 4, 8, and 24 hours after radafaxine (40 mg p.o.) in 8 controls. Plasma pharmacokinetics and behavioral and cardiovascular effects were measured in parallel., Results: DAT blockade by radafaxine was slow, and at 1 hour, it was 11%. Peak blockade occurred at about 4 hours and was 22%. Blockade was long lasting: at 8 hours 17%, and at 24 hours 15%. Peak plasma concentration occurred about 4 to 8 hours. No behavioral or cardiovascular effects were observed., Conclusions: The relatively low potency of radafaxine in blocking DAT and its slow blockade suggests that it is unlikely to have reinforcing effects. This is consistent with preclinical studies showing no self-administration. This is the first utilization of PET to predict abuse liability of a new antidepressant in humans based on DAT occupancy and pharmacokinetics.

Parkinson's disease is a neurodegenerative disorder associated with progressive loss of dopaminergic cells in the substantia nigra. Oxidative stress has been implicated in the pathogenesis of the disease, and dopamine has been suggested as a contributing factor that generates reactive oxygen species due to its unstable catechol moiety. We have previously shown that tetrahydrobiopterin (BH4), an obligatory cofactor for dopamine synthesis, also contributes to the vulnerability of dopamine-producing cells by generating oxidative stress. This study shows that the presence of dopamine in the cytosol enhances the cell's vulnerability to BH4. Upon exposure to ketanserin, a vesicular monoamine transporter inhibitor, BH4-induced dopaminergic cell death is exacerbated, accompanied by increased lipid peroxidation and protein bound quinone. While intracellular amount of DOPAC is elevated by ketanserin, the monoamine oxidase inhibitor pargyline showed no significant protection. Instead, the thiol agent N-acetylcysteine and quinone reductase inducer dimethyl fumarate abolish BH4/ketanserin-induced cell death, suggesting that quinone production plays an important role. Therefore, it can be concluded that the presence of dopamine in the cytosol seems to contribute to the cells' vulnerability to BH4 and that vesicular monoamine transporter plays a protective role in dopaminergic cells by sequestering dopamine not only from monoamine oxidase but also from BH4-induced oxidative stress.

Drugs that target the dopamine transporter (DAT) have been proposed as pharmacotherapies to treat cocaine abuse. Accordingly, it is paramount to understand pharmacological interactions between cocaine and DAT inhibitors. The present study characterized acute interactions between cocaine and several DAT inhibitors (RTI-177, FECNT, RTI-112) that differed in selectivity for monoamine transporters on operant behavior and in vivo neurochemistry in squirrel monkeys. RTI-177 and FECNT, two DAT inhibitors with low affinity at norepinephrine transporters (NET), produced dose-dependent stimulant effects on behavior maintained by a fixed-interval schedule of stimulus termination. Compared to cocaine, RTI-177 and FECNT had a slower onset and longer duration of action. In vivo microdialysis in the caudate nucleus of awake monkeys confirmed dose-dependent increases in extracellular dopamine that corresponded to behavioral effects. Among the drugs characterized, RTI-112 is reportedly the least selective for binding to DAT, NET, and serotonin transporters (SERT). Interestingly, RTI-112 failed to produce significant behavioral-stimulant effects, and its effects on extracellular dopamine were highly variable across subjects. The results indicate that the pharmacological profile of DAT inhibitors may be influenced by actions at multiple monoamine transporters. Importantly, there was little evidence of additivity on behavioral or neurochemical measures when cocaine was administered in combination with behavioral-stimulant doses of the DAT inhibitors.

The brain vesicular monoamine transporter (VMAT2) is part of the re-uptake mechanism which regulates monoaminergic neurotransmission. We demonstrated previously a high degree of similarity between the pharmacodynamic characteristics of platelet and brain VMAT2. Nicotine induced increase of dopamine and serotonin neurotransmission in limbic structures may alter the expression of VMAT2 in brains of smokers. In this study we measured the VMAT2 pharmacodynamic characteristics using high-affinity [3H]dihydrotetrabenazine (TBZOH) binding to platelets of smokers (n=15) compared to sex and age matched healthy nonsmokers controls (n=14). A significant decrease (17%, P=0.02) in VMAT2 density (Bmax) was observed in platelets of smokers compared to nonsmokers. There was no significant difference in the affinity of [3H]TBZOH to its platelet binding site and the VMAT2 density did not correlate with the heaviness of smoking. The decreased density of the VMAT2 in the platelets of smokers may reflect nicotine induced desensitization of VMAT2, a phenomenon that may be relevant to the addictive properties of nicotine.

The interaction of the S- and R-enantiomers (escitalopram and R-citalopram) of citalopram, with high- and low-affinity binding sites in COS-1 cell membranes expressing human SERT (hSERT) were investigated. Escitalopram affinity for hSERT and its 5-HT uptake inhibitory potency was in the nanomolar range and approximately 40-fold more potent than R-citalopram. Escitalopram considerably stabilised the [3H]-escitalopram/SERT complex via an allosteric effect at a low-affinity binding site. The stereoselectivity between escitalopram and R-citalopram was approximately 3:1 for the [3H]-escitalopram/hSERT complex. The combined effect of escitalopram and R-citalopram was additive. Paroxetine and sertraline mainly stabilised the [3H]-paroxetine/hSERT complex. Fluoxetine, duloxetine and venlafaxine have only minor effects. 5-HT stabilised the [125I]-RTI-55, [3H]-MADAM, [3H]-paroxetine, [3H]-fluoxetine and [3H]-venlafaxine/SERT complex to some extent. Thus, escitalopram shows a unique interaction with the hSERT compared with other 5-HT reuptake inhibitors (SSRIs) and, in addition to its 5-HT reuptake inhibitory properties, displays a pronounced effect via an affinity-modulating allosteric site.

BMS-505130 is a potent and selective serotonin transport inhibitor; K(i) for binding to the serotonin transporter = 0.18 nM (K(i) values for binding to the norepinephrine and dopamine transporters = 4.6 and 2.1 microM, respectively). In platelet serotonin uptake studies BMS-505130 (5 mg/kg, p.o.) produced a robust inhibition of serotonin uptake. In microdialysis studies oral dosing with BMS-505130 produced a dose-dependent increase in cortical serotonin levels that reached a maximal effect of 200% above baseline at a dose of 1 mg/kg, p.o.; the peak serotonin response was transient in nature. Following oral administration, peak plasma concentrations of BMS-505130 reached Tmax at 1.6 +/- 0.7 h and then declined to concentrations <10% of Cmax within the following 6 h; plasma half-life following i.v. dosing was 0.46 +/- 0.02 h. Parallel microdialysis and pharmacokinetic studies revealed that changes in serotonin levels in the cortex mirrored changes in the brain concentration of BMS-505130. In a behavioral assay known to be sensitive to selective serotonin reuptake inhibitors (SSRIs), mouse tail suspension, BMS-505130 produced a robust response after either oral or intraperitoneal dosing. BMS-505130 exhibits a pharmacological, neurochemical and behavioral profile consistent with a potent SSRI. Moreover, BMS-505130's short half-life may be advantageous for the treatment of premature ejaculation where an acute effect to delay ejaculation followed by a relatively rapid fall in SSRI plasma concentrations might be desirable.

Several synthetic approaches to N-alkylated derivatives of 4-hydroxypyrrolidine-2-carboxylic acid and 4-hydroxypyrrolidine-2-acetic acid are described. The final compounds have been evaluated as potential inhibitors of the GABA transport proteins GAT-1 and GAT-3. The biological assays used were based on bovine material or porcine brain. As compared to the corresponding 4-unsubstituted compounds, the 4-hydroxypyrrolidine-2-carboxylic acid and 4-hydroxypyrrolidine-2-acetic acid derivatives showed a significant decrease in the inhibitory potency at both GAT-1 and GAT-3 with only four compounds having reasonable affinity to GAT-1 (IC(50): 5.1, 6.6 and 9.4 microM) or GAT-3 (IC(50): 19.9 microM), respectively. The biological data of the 4-hydroxypyrrolidine-2-acetic acid derivatives indicates that (2S)-configuration at the C-2 position for potent inhibition of GAT-1 and (4R)-configuration at the C-4 position for potent inhibition of GAT-3 may be crucial.

Purpose: The aim of this study was to investigate the feasibility of assessing dopamine transporter binding after treatment with methylphenidate in the rat using a recently developed high-resolution small animal single-photon emission computed tomograph (TierSPECT) and [123I]FP-CIT., Methods: [123I]FP-CIT was administered intravenously 1 h after intraperitoneal injection of methylphenidate (10 mg/kg) or vehicle. Animals underwent scanning 2 h after radioligand administration. The striatum was identified by superimposition of [123I]FP-CIT scans with bone metabolism and perfusion scans obtained with 99mTc-DPD and 99mTc-tetrofosmin, respectively. As these tracers do not pass the blood-brain barrier, their distribution permits the identification of extracerebral anatomical landmarks such as the orbitae and the harderian glands. The cerebellum was identified by superimposing [123I]FP-CIT scans with images of brain perfusion obtained with 99mTc-HMPAO., Results: Methylphenidate-treated animals and vehicle-treated animals yielded striatal equilibrium ratios (V''3) of 0.24+/-0.26 (mean +/- SD) and 1.09+/-0.42, respectively (t test, two-tailed, p<0.0001). Cortical V''3 values amounted to 0.05+/-0.28 (methylphenidate) and 0.3+/-0.39 (saline, p=0.176). This first in vivo study of rat dopamine transporter binding after pre-treatment with methylphenidate showed a mean reduction of 78% in striatal [123I]FP-CIT accumulation., Conclusion: The results can be interpreted in terms of a pharmacological blockade in the rat striatum and show that in vivo quantitation of dopamine transporter binding is feasible with [123I]FP-CIT and the TierSPECT. This may be of future relevance for in vivo investigations on rat models of attention deficit/hyperactivity disorder. Furthermore, our findings suggest that investigations in other animal models, e.g. of Parkinson's and Huntington's disease, may be feasible using SPECT radioligands and small animal imaging systems.

The role of dopamine in iron uptake into catecholaminergic neurons, and dopamine oxidation to aminochrome and its one-electron reduction in iron-mediated neurotoxicity, was studied in RCSN-3 cells, which express both tyrosine hydroxylase and monoamine transporters. The mean +/- SD uptake of 100 microm 59FeCl3 in RCSN-3 cells was 25 +/- 4 pmol per min per mg, which increased to 28 +/- 8 pmol per min per mg when complexed with dopamine (Fe(III)-dopamine). This uptake was inhibited by 2 microm nomifensine (43%p < 0.05), 100 microm imipramine (62%p < 0.01), 30 microm reboxetine (71%p < 0.01) and 2 mm dopamine (84%p < 0.01). The uptake of 59Fe-dopamine complex was Na+, Cl- and temperature dependent. No toxic effects in RCSN-3 cells were observed when the cells were incubated with 100 microm FeCl3 alone or complexed with dopamine. However, 100 microm Fe(III)-dopamine in the presence of 100 microm dicoumarol, an inhibitor of DT-diaphorase, induced toxicity (44% cell death; p < 0.001), which was inhibited by 2 microm nomifensine, 30 microm reboxetine and 2 mm norepinephrine. The neuroprotective action of norepinephrine can be explained by (1) its ability to form complexes with Fe3+, (2) the uptake of Fe-norepinephrine complex via the norepinephrine transporter and (3) lack of toxicity of the Fe-norepinephrine complex even when DT-diaphorase is inhibited. These results support the proposed neuroprotective role of DT-diaphorase and norepinephrine.

We assessed the impact of chronic swim stress in rats (daily for 3 weeks) on vesicular monoamine transporter 2 (VMAT2) in the nucleus accumbens and striatum. Exposure to repeated swim stress resulted in significant reduction in VMAT2 density in nucleus accumbens (20%, p<0.01) and striatum subregions (21-38%, p<0.001). The down-regulation of VMAT2 in this dopaminergic regions may serve as an adaptatory mechanism in the response to prolonged stress, and may be relevant to chronic stress-induced depression.

In the present study, the effects of a large series of flavonoids on multidrug resistance proteins (MRPs) were studied in MRP1 and MRP2 transfected MDCKII cells. The results were used to define the structural requirements of flavonoids necessary for potent inhibition of MRP1- and MRP2-mediated calcein transport in a cellular model. Several of the methoxylated flavonoids are among the best MRP1 inhibitors (IC(50) values, ranging between 2.7 and 14.3 microM) followed by robinetin, myricetin and quercetin (IC(50) values ranging between 13.6 and 21.8 microM). Regarding inhibition of MRP2 activity especially robinetin and myricetin appeared to be good inhibitors (IC(50) values of 15.0 and 22.2 microM, respectively). Kinetic characterization revealed that the two transporters differ marginally in the apparent K(m) for the substrate calcein. For one flavonoid, robinetin, the kinetics of inhibition were studied in more detail and revealed competitive inhibition with respect to calcein, with apparent inhibition constants of 5.0 microM for MRP1 and 8.5 microM for MRP2. For inhibition of MRP1, a quantitative structure activity relationship (QSAR) was obtained that indicates three structural characteristics to be of major importance for MRP1 inhibition by flavonoids: the total number of methoxylated moieties, the total number of hydroxyl groups and the dihedral angle between the B- and C-ring. Regarding MRP2 mediated calcein efflux inhibition, only the presence of a flavonol B-ring pyrogallol group seems to be an important structural characteristic. Overall, this study provides insight in the structural characteristics involved in MRP inhibition and explores the differences between inhibitors of these two transporters, MRP1 and MRP2. Ultimately, MRP2 displays higher selectivity for flavonoid type inhibition than MRP1.

Two stilbenes (trans-piceid and its aglycone trans-resveratrol) were investigated in the uptake across the apical membrane of the human intestinal cell line Caco-2 in order to determine their mechanisms of transport. The uptake was quantified using a reverse phase high-performance liquid chromatography method with fluorescence detection. The rate of cellular accumulation in the cells was found to be higher for trans-resveratrol than for trans-piceid. In addition, trans-resveratrol uses passive transport to cross the apical membrane of the cells, whereas the transport of trans-piceid is likely active. With regard to the mechanisms of transport, the involvement of the active transporter SGLT1 in the absorption of trans-piceid was deduced using various inhibitors directly or indirectly exploiting the activity of this transporter (glucose, phlorizin, and ouabain). Moreover, we investigated the involvement of the multidrug-related protein 2 (MRP2), an efflux pump present on the apical membrane, in stilbene efflux by Caco-2 cells. The effect of MK-571 (an MRP inhibitor) seems to implicate MRP2 as responsible for apical efflux of trans-piceid and trans-resveratrol.

Adaptations in neurons of the midbrain periaqueductal gray (PAG) induced by chronic morphine treatment mediate expression of many signs of opioid withdrawal. The abnormally elevated action potential rate of opioid-sensitive PAG neurons is a likely cellular mechanism for withdrawal expression. We report here that opioid withdrawal in vitro induced an opioid-sensitive cation current that was mediated by the GABA transporter-1 (GAT-1) and required activation of protein kinase A (PKA) for its expression. Inhibition of GAT-1 or PKA also prevented withdrawal-induced hyperexcitation of PAG neurons. Our findings indicate that GAT-1 currents can directly increase the action potential rates of neurons and that GAT-1 may be a target for therapy to alleviate opioid-withdrawal symptoms.

Monoamine transporters were originally associated simply with the termination of synaptic monoamine function. In addition to amine reuptake, however, the transporters can act as ion channels that affect exocytotic neurotransmitter release and can operate in reverse mode, mediating non-exocytotic amine release. Activity at the plasma membrane is controlled by trafficking, which is modulated by interaction with both substrates and inhibitors and by cytosolic kinases and phosphatases. Monoamine transporters also constitute the principal sites of action of many psychoactive drugs, including amphetamines and cocaine, as well as therapeutic drugs for the treatment of depression, addiction and attention deficit hyperactivity disorder, each modifying the balance of presynaptic neurotransmitter function.

Adenosine, as the brain's endogenous anticonvulsant, is considered to be responsible for seizure arrest and postictal refractoriness. On the other hand, deficiencies within the adenosine-based neuromodulatory system may contribute to epileptogenesis. Based on these natural mechanisms and on findings that adenosine and its analogs can suppress pharmacoresistant seizures, a new field of adenosine-based therapies has emerged, including the use of adenosine receptor agonists and adenosine transport inhibitors, or the inhibition of adenosine kinase, which is thought to be the key enzyme for the regulation of intra- and extracellular adenosine levels. However, most of these pharmacological approaches are limited by strong systemic side effects ranging from a decrease of heart rate, blood pressure, and body temperature to sedation. Recently, new strategies have been developed aimed at the local reconstitution of the inhibitory adenosinergic tone by intracerebral implantation of cells engineered to release adenosine. Adenosine-releasing cells or devices implanted into or near a seizure focus offer new hopes for a side effect-free therapy for pharmacoresistant epilepsy.

Modulators and inhibitors of multidrug efflux transporters, like P-glycoprotein, are used to reduce or inhibit multidrug resistance, MDR, which leads to a failure of the chemotherapy of e.g. cancers, epilepsy, bacterial, parasitic, and fungal diseases. Binding and transport of first-, second-, and third-generation modulators and inhibitors of P-glycoprotein are discussed, taking into account the properties of the drug (H-bonding potential, dimensions, and pK(a) values) as well as the properties of the membrane.

Green tea extracts (GTE) might modulate ABC transporter gene expression or function. This may be relevant in the treatment of cancer or in influencing intestinal drug permeability. To gain more insight on the influence of a GTE on secretory transport proteins we investigated the influence of GTE and several green tea components on the mRNA expression level of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) in human gastrointestinal epithelial LS-180 cells. Furthermore, the functional activity of MRP2, using glutathione methylfluorescein (GS-MF) or [3H]methotrexate (MTX) as substrate, was investigated in canine kidney cells stably overexpressing human MRP2 (MDCK-MRP2). GTE, at a concentration of 0.01 mg/mL, did not increase mRNA expression of P-gp or MRP2 in LS-180 cells. Functional assays in MDCK-MRP2 cells using GS-MF did not show any effect of 0.01 mg/mL GTE on MRP2 activity. In the same cell line the cellular accumulation of MTX (a specific substrate of MRP2) was significantly increased with the MRP-specific inhibitor MK-571 or with 1 mg/mL GTE, but not with 0.1 mg/mL. The green tea components (-)-epigallocatechin gallate, (-)-epigallocatechin, theanine, or caffeine, each in corresponding concentrations to the respective concentration of GTE, did not show any effect on MRP2 function. These data demonstrate that the mRNA expression patterns of P-gp and MRP2 in LS-180 cells are not altered by 0.01 mg/mL of GTE. However, MRP2 function was inhibited by 1 mg/mL GTE, whereas none of the green tea components tested were responsible for this effect.