Regulation of transporters by nuclear hormone receptors: implications during inflammation

Membrane transporters play a critical role in the absorption, distribution, and elimination of both endogenous substrates and xenobiotics. Defects in transporter function can lead to altered drug disposition including toxicity or loss of efficacy. Inflammation is one condition during which variable drug response has been demonstrated, and this can be attributed, at least in part, to changes in the expression of transporter genes. Thus, knowledge of the mechanisms behind transporter regulation can significantly contribute to our ability to predict variations in drug disposition among individuals and during inflammatory disease. The discovery of several xenobiotic-activated nuclear hormone receptors during the past decade including the pregnane X receptor, constitutive androstane receptor, and farnesoid X receptor has contributed greatly toward this endeavor. These receptors regulate the expression of transporters such as P-glycoprotein, MRP2, MRP3, BCRP, and OATP2 (Oatp1a1/OATP1B1), all of which undergo altered expression during an inflammatory response. Nuclear receptors may therefore play an important role in mediating this effect. This review presents what is currently known about the role of nuclear receptors in transporter regulation during inflammation. The use of this knowledge toward understanding interindividual variation in drug response and drug interactions during inflammation as well toward the development of therapeutics to treat transporter-related diseases will also be discussed.     

All aglow about presynaptic receptor regulation of neurotransmitter transporters

Mounting evidence supports the idea that neurotransmitter transporters are subject to many forms of post-translational regulation typically associated with receptors and ion channels, including receptor and kinase-mediated changes in transporter phosphorylation, cell surface trafficking, and/or catalytic activation. Although hints of this regulation can be achieved with traditional radiolabeled substrate flux techniques, higher resolution methods are needed that can localize transporter function in situ as well as permit real-time monitoring of transport function without confounds associated with coincident receptor activation. The elegant study by Bolan et al. (p. 1222) capitalizes on the fluorescent properties of a recently introduced substrate for the dopamine (DA) transporter (DAT), termed 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+), to illuminate a pertussis toxin-sensitive, extracellular signal-regulated kinase (ERK1/2)-dependent pathway by which presynaptic DA D(2) receptors regulate DATs.     

Agonistic and antagonistic bivalent ligands for serotonin and dopamine receptors including their transporters

This review deals with the literature (1982-2006) concerning bivalent ligands for dopamine (D) and serotonin (5-HT) receptors, as well as for their respective transporters. The design, synthesis, and pharmacological evaluation of bivalent agonists and antagonists for dopamine and serotonin receptors have been successfully pursued. Increased potencies for 5-HT(1B/1D) receptor agonists were achieved as well as improved selectivities. At these receptors, selectivity seems to depend strongly on spacer length, whereas the improved affinities seem to be based on the presence of two pharmacophores within one molecule. Intrinsic activities and pharmacokinetic properties may differ from those of the respective monovalent ligands. Additionally, improved central nervous system penetration was achieved. Bivalent dopamine receptor agonists and antagonists can exhibit selectivity profiles different from their monomeric analogues with no loss in potency. For dopamine antagonists, affinities depend strongly on spacer length. For agonistic dimers different pharmacokinetic properties were observed. Bivalency was also applied to inhibitors of monoamine re-uptake transporters. Selectivity profiles and affinities depend strongly on the length of the alkylene-spacer: For some dimeric inhibitors the norepinephrine transporter (NET) and the dopamine transporter (DAT) affinities changed gradually, but for the serotonin transporter (SERT) a pentamethylene spacer showed the highest potency. Because the bivalent ligand approach has just begun to be applied to these versatile, therapeutically important targets, many advances in affinity enhancement, as well as the achievement of novel selectivity profiles and improved pharmacokinetics can be expected.     

Role of orphan nuclear receptors in the regulation of drug-metabolising enzymes

During the past several years, important advances have been made in our understanding of the mechanisms that regulate the expression of genes that determine drug clearance, including phase I and phase II drug-metabolising enzymes and drug transporters. Orphan nuclear receptors have been recognised as key mediators of drug-induced changes in both metabolism and efflux mechanisms. In this review, we summarise recent findings regarding the function of nuclear receptors in regulating drug-metabolising and transport systems, and the relevance of these receptors to clinical drug-drug interactions and the development of new drugs. Emphasis is given to two newly recognised 'orphan' receptors (the pregnane X receptor [PXR] and the constitutive androstane receptor [CAR]) and their regulation of cytochrome P450 enzymes, such as CYP3A4, CYP2Cs and CYP2B6; and transporters, such as P-glycoprotein (MDR1), multidrug resistance-associated proteins (MRPs) and organic anion transporter peptide 2 (OATP2). Although 'cross-talk' occurs between these two receptors and their target sequences, significant species differences exist between ligand-binding and activation profiles for both receptors, and PXR appears to be the predominant or 'master' regulator of hepatic drug disposition in humans. Several important physiological processes, such as cholesterol synthesis and bile acid metabolism, are also tightly controlled by certain ligand-activated orphan nuclear receptors (farnesoid X receptor [FXR] and liver X receptor [LXR]). In general, their ability to bind a broad range of ligands and regulate an extensive array of genes that are involved in drug clearance and disposition makes these orphan receptors attractive targets for drug development. Drugs have the capacity to alter nuclear receptor expression (modulators) and/or serve as ligands for the receptors (agonists or antagonists), and thus can have synergistic or antagonistic effects on the expression of drug-metabolising enzymes and transporters. Coadministration of drugs that are nuclear receptor agonists or antagonists can lead to severe toxicity, a loss of therapeutic efficacy or an imbalance in physiological substrates, providing a novel molecular mechanism for drug-drug interactions.     

Serotonin receptor and transporter ligands - current status

The serotonin (5-HT) receptor system has 14 different subtypes classified by pharmacology and function. Many ligands are widely used for therapeutic and diagnostic purposes in some severe human diseases. Most of the ligands that are specific for each 5-HT receptor have distinctive chemical structures with regard to pharmacophore elements including 4-arylpiperazine, benzimidazole, benzamide, chroman, aminopyridazine, tetralin, and polycycles. However, their affinity and selectivity for 5-HT, dopamine and alpha1 receptors depend on their substituents and linker spacers. 5-HT transporter inhibitors have also been developed as potential antidepressants. In contrast to classical tricyclic compounds, newly developed secondary amine derivatives such as paroxetine and tetralin show high binding affinity and selectivity. Radioisotope-labeled ligands have also been developed, including [carbonyl-(11)C]WAY 100635 for 5-HT1A receptor, [(11C) or (18)F]ketanserine derivatives for 5-HT(2) receptor, [(125)I]DAIZAC for 5-HT(3) receptor, and [123I]IDAM for 5-HT transporter, and these are accumulated in brain regions that are rich in the respective receptors. This review summarizes the recent development of 5-HT receptor- and transporter-specific ligands and their pharmacological properties on the basis of their chemical structures.     

Review of the pharmacological and clinical profile of rimcazole

Rimcazole is a carbazole derivative that acts in part as a sigma receptor antagonist. Wellcome Research Laboratories introduced this compound during the 1980s when it was hypothesized to be a novel antipsychotic with an improved side effect profile. However, subsequent clinical trials demonstrated that rimcazole lacked efficacy in schizophrenic patients and it is now primarily used as an experimental tool. In addition to its actions as a sigma receptor antagonist, rimcazole also has high affinity for dopamine transporters, and in recent years it has served as a lead compound for the development of novel dopamine transporter ligands. Although rimcazole cannot be considered a selective ligand for sigma receptors, the recent development of other selective agonists and antagonists for sigma receptors have aided in clarifying the involvement of these receptors in the actions of rimcazole. Many of the physiological and behavioral effects of rimcazole can in fact be ascribed to its action as a sigma receptor antagonist, although there are exceptions. Rimcazole is likely to have a continued role in elucidating sigma receptor function in either in vitro or in vivo systems where sigma receptor-mediated effects can be studied independently of the influence of dopamine and serotonin transporters.     

The dopamine transporter: a vigilant border control for psychostimulant action

Neurotransmission within the mesocorticolimbic dopamine system has remained the central focus of investigation into the molecular, cellular and behavioral properties of psychostimulants for nearly three decades. The primary means by which dopamine transmission in the synapse is terminated is via the dopamine transporter (DAT), the presynaptic plasmalemmal protein that is responsible for the reuptake of released dopamine. Numerous abused as well as clinically important drugs have important pharmacological interactions with DAT. In general, these compounds fall into two categories: those that block dopamine transport (e.g., cocaine, methylphenidate) and those that serve as substrates for transport [e.g., dopamine, amphetamine and 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy")]. Recent data from in vitro and in vivo studies have suggested that DAT, like other biogenic amine transporters, share several characteristics with classical ligand-gated ion channels. In addition, substrates for transport promote redistribution of DAT away from the plasma membrane, while transport inhibitors such as cocaine disrupt this process. In addition, presynaptic autoreceptors for dopamine have been implicated in the modulation of DAT surface expression and function. The present chapter summarizes some of the recent discoveries pertaining to the electrogenic properties of DAT and their potential relevance to the effects of amphetamine-like stimulants on DAT function. Although there are a number of intracellular and extracellular modulatory influences on dopamine clearance that may play particular roles in psychostimulant action, we specifically focus on the differential direct modulation of DAT function by transport substrates and inhibitors, and we also discusses the role of presynaptic D2 receptors in transport regulation.     

Structure and function of the dopamine transporter

The dopamine transporter mediates uptake of dopamine into neurons and is a major target for various pharmacologically active drugs and environmental toxins. Since its cloning, much information has been obtained regarding its structure and function. Binding domains for dopamine and various blocking drugs including cocaine are likely formed by interactions with multiple amino acid residues, some of which are separate in the primary structure but lie close together in the still unknown tertiary structure. Chimera and site-directed mutagenesis studies suggest the involvement of both overlapping and separate domains in the interaction with substrates and blockers, whereas recent findings with sulfhydryl reagents selectively targeting cysteine residues support a role for conformational changes in the binding of blockers such as cocaine. The dopamine transporter can also operate in reverse, i.e. in an efflux mode, and recent mutagenesis experiments show different structural requirements for inward and outward transport. Strong evidence for dopamine transporter domains selectively influencing binding of dopamine or cocaine analogs has not yet emerged, although the development of a cocaine antagonist at the level of the transporter remains a possibility.     

Cannabinoid receptors as therapeutic targets

The cannabinoid receptors CB1 and CB2 are family A, G-protein Coupled Receptors that mediate the effects of cannabinoids, a class of compounds that are so named because the first members were isolates of the cannabis plant. In recent history, there has been much anecdotal evidence that the potent and diverse physiological responses produced by these compounds can be turned to therapeutic benefit for a wide variety of maladies. The remarkable abundance of cannabinoid receptors and the discovery of several endogenous ligands along with enzyme and transporter proteins for which they are substrates, suggests that an endogenous cannabinoid neuromodulatory system is an important mediator of biological function. For these reasons CB1 and CB2 receptors are attractive targets for the design of therapeutic ligands. The action of these receptors, however, may also be modulated by manipulating the enzymes and membrane transporters that regulate the endogenous ligands. Despite the range of physiological processes and activities that are mediated by cannabinoid receptors, it is clear that it is possible to produce ligands that result in differential responses. In this paper, we review the pharmacophoric elements that lead to these differential responses and in order to discuss them in context we present an overview of structural aspects governing cannabinoid receptor function, the cannabinergic system and its physiological functions.     

Voltage-dependent block of N-methyl-D-aspartate receptors by dopamine D1 receptor ligands

Accumulating evidence indicates that dopamine and D1 receptor ligands modulate N-methyl D-aspartate (NMDA) receptors through a variety of D1 receptor-dependent mechanisms. In this study, we reveal a distinct D1 receptor-independent mechanism by which NMDA receptors are modulated. Using the human embryonic kidney (HEK) cell recombinant system and dissociated neurons, we have discovered that dopamine and several D1 ligands act as voltage-dependent, open-channel blockers for NMDA receptors, regardless of whether they are agonists or antagonists for D1 receptors. Analysis of structural and functional relationships of D1 ligands revealed the elements that are critical for their binding to NMDA receptors. Furthermore, using D1 receptor knockout mice, we verified that this channel-blocking effect was independent of D1 receptors. Finally, we demonstrated that D1 ligands functionally interact with Mg(2+) block through multiple sites, implying a possible role of the direct channel block under physiological conditions. Our results suggest that the direct inhibition of NMDA receptors by dopamine D1 receptor ligands is due to the channel pore block rather than receptor-receptor interactions.     

Pharmacology of monoamine neurotransmitter transporters

Following exocytotic release, the biogenic amine neurotransmitters, norepinephrine, dopamine, and serotonin are removed from the synaptic cleft by the respective transporter, NET, DAT, and SERT, located on the plasma membrane and then re-stored into synaptic vesicles by vesicular monoamine transporter, VMAT. The molecular cloning of these transporters revealed that NET, DAT, and SERT are members of a sodium-dependent neurotransmitter transporter gene family, while VMATs arise from proton-dependent transporter gene family. Structural features common to NET, DAT, and SERT reveal a putative 12 transmembrane-spanning domain structure with cytosolic N- and C-terminal regions. Recent evidence suggest the regulation of the functional expression of these transporters via phosphorylation, which include direct phosphorylation of transporter proteins and/or of associated proteins that may control transporter function/expression. In addition, the substrates and inhibitors for these transporters appear capable of regulating transporter cell surface expression, thereby suggesting both activity-dependent and pharmacological regulatory mechanisms for transporter expression. Analyses of the genes provide new insight into their relation to neuronal diseases since NET, DAT and SERT are the molecular targets for many antidepressants as well as drugs of abuse such as cocaine and amphetamine. The availability of cDNAs of these and vesicular transporters has permitted detailed pharmacological studies in heterologous expression systems, and thus would promise the development of novel drugs with diverse chemical structures.     

Peptide transporters and their roles in physiological processes and drug disposition

1. The peptide transporters belong to the peptide transporter (PTR) family and serve as integral membrane proteins for the cellular uptake of di- and tripeptides in the organism. By their ability also to transport peptidomimetics and other substrates with therapeutic activities or precursors of pharmacologically active agents, they are of considerable importance in pharmacology. 2. PEPT1 is the low-affinity, high-capacity transporter and is mainly expressed in the small intestine, whereas PEPT2 is the high-affinity, low-capacity transporter and has a broader distribution in the organism. 3. Targeted mouse models have revealed PEPT2 to be the dominant transporter for the reabsorption of di- and tripeptides and its pharmacological substrates in the organism, and for the removal of these substrates from the cerebrospinal fluid. Moreover, the peptide transporters undergo physiological and pharmacological regulation and, of great interest, are present in disease states where PEPT1 exhibits ectopic expression in colonic inflammation. 4. The paper reviews the structural characteristics of the peptide transporters, the structural requirements for substrates, the distribution of the peptide transporters in the organism, and finally their regulation in the organism in healthy and pathological situations.     

Dopamine transporter in vitro binding and in vivo imaging in the brain.

Much research interest has lately been focused on the dopamine transporter function in brain. Recent findings indicate that dopamine reuptake is more like a highly regulated than a constitutive determinant of dopamine clearance. Positron emission tomography (PET) and single-photon emission tomography (SPET) offer unique methods to study dopamine transporter function. Results from in vivo PET and SPET studies correspond well with in vitro studies performed on post mortem human brain tissue. Considering some of the variances between in vitro and in vivo receptor binding phenomena it may be that the role of a compound to alter binding to monoamine uptake sites in vitro does not indicate its potential to affect monoamine transporters after administration in vivo. This discrepancy may be better understood taking into account recent studies indicating the possibility of a rapid regulation of transporter function and surface expression. Furthermore, the dopamine transporter is a fruitful target for CNS drug discovery. Fundamental nature of drug actions in vivo may be studied using demonstrated in vitro and in vivo imaging methods.     

Regulation of the dopamine transporter by phosphorylation

The dopamine transporter (DAT) is a neuronal phosphoprotein and target for psychoactive drugs that plays a critical role in terminating dopaminergic transmission by reuptake of dopamine from the synaptic space. Control of DAT activity and plasma membrane expression are therefore central to drug actions and the spatial and temporal regulation of synaptic dopamine levels. DATs rapidly traffic between the plasma membrane and endosomal compartments in both constitutive and protein kinase C-dependent manners. Kinase activators, phosphatase inhibitors, and transported substrates modulate DAT phosphorylation and activity, but the underlying mechanisms and role of phosphorylation in these processes are poorly understood. Complex adaptive changes in DAT function potentially related to these processes are also induced by psychostimulant and therapeutic transport blockers such as cocaine and methylphenidate. This chapter provides an overview of the current state of knowledge regarding DAT phosphorylation and its relationship to transporter activity and trafficking. A better understanding of how dopaminergic neurons regulate DAT function and the role of phosphorylation may lead to the identification of novel therapeutic targets for the treatment and prevention of dopaminergic disorders.     

Peptide transporters and their roles in physiological processes and drug disposition

1.?The peptide transporters belong to the peptide transporter (PTR) family and serve as integral membrane proteins for the cellular uptake of di- and tripeptides in the organism. By their ability also to transport peptidomimetics and other substrates with therapeutic activities or precursors of pharmacologically active agents, they are of considerable importance in pharmacology.

2.?PEPT1 is the low-affinity, high-capacity transporter and is mainly expressed in the small intestine, whereas PEPT2 is the high-affinity, low-capacity transporter and has a broader distribution in the organism.

3.?Targeted mouse models have revealed PEPT2 to be the dominant transporter for the reabsorption of di- and tripeptides and its pharmacological substrates in the organism, and for the removal of these substrates from the cerebrospinal fluid. Moreover, the peptide transporters undergo physiological and pharmacological regulation and, of great interest, are present in disease states where PEPT1 exhibits ectopic expression in colonic inflammation.

4.?The paper reviews the structural characteristics of the peptide transporters, the structural requirements for substrates, the distribution of the peptide transporters in the organism, and finally their regulation in the organism in healthy and pathological situations.     

Cannabinoids inhibit the synaptic uptake of adenosine and dopamine in the rat and mouse striatum

Adenosine, dopamine and endocannabinoids strictly modulate the release of one another in the dorsolateral striatum thereby controlling synaptic plasticity. As a second level of interaction, they regulate the action of one another via receptor heteromer formation. Here we investigated a putative third level of interaction, i.e. the possible control by cannabinoids of synaptic dopamine and adenosine reuptake. We found that a large number of endo- and exogenous cannabinoid ligands inhibit the uptake of [(3)H]adenosine and [(3)H]dopamine in rat sriatal nerve terminals. Maximal effects were often comparable to those of the dopamine transporter inhibitor, GBR12783 and the equilibrative nucleoside transporter inhibitor, dipyridamole. Cannabinoid ligands were generally more potent to inhibit the uptake of adenosine than that of dopamine. The inhibitory effect was: (1) unrelated to the pharmacological profile(s) of the ligands at the cannabinoid CB(1), CB(2), GPR55 and at the vanilloid TRPV(1) receptors; (2) not prevented by the cannabinoid CB(1) receptor antagonist/inverse agonist, LY320135; and (3) maintained in the cannabinoid CB(1) receptor knockout mice. In the same experiments, only O-2050, cannabidiol, and WIN55212-3 inhibited the simultaneously measured DL-TBOA-sensitive uptake of [(14)C]glutamate. In summary, many cannabinoid ligands are able to inhibit the synaptic uptake of adenosine and dopamine. These effects are not mediated by cannabinoid CB(1) receptors, and should be an additional mechanism to consider when interpreting synaptic effects of cannabinoids.     

Substituted methcathinones differ in transporter and receptor interactions

The use of synthetic methcathinones, components of “bath salts,” is a world-wide health concern. These compounds, structurally similar to methamphetamine (METH) and 3,4-methylendioxymethamphetamine (MDMA), cause tachycardia, hallucinations and psychosis. We hypothesized that these potentially neurotoxic and abused compounds display differences in their transporter and receptor interactions as compared to amphetamine counterparts. 3,4-Methylenedioxypyrovalerone and naphyrone had high affinity for radioligand binding sites on recombinant human dopamine (hDAT), serotonin (hSERT) and norepinephrine (hNET) transporters, potently inhibited [³H]neurotransmitter uptake, and, like cocaine, did not induce transporter-mediated release. Butylone was a lower affinity uptake inhibitor. In contrast, 4-fluoromethcathinone, mephedrone and methylone had higher inhibitory potency at uptake compared to binding and generally induced release of preloaded [³H]neurotransmitter from hDAT, hSERT and hNET (highest potency at hNET), and thus are transporter substrates, similar to METH and MDMA. At hNET, 4-fluoromethcathinone was a more efficacious releaser than METH. These substituted methcathinones had low uptake inhibitory potency and low efficacy at inducing release via human vesicular monoamine transporters (hVMAT2). These compounds were low potency (1) h5-HT_1A receptor partial agonists, (2) h5-HT_2A receptor antagonists, (3) weak h5-HT_2C receptor antagonists. This is the first report on aspects of substituted methcathinone efficacies at serotonin (5-HT) receptors and in superfusion release assays. Additionally, the drugs had no affinity for dopamine receptors, and high-nanomolar to mid-micromolar affinity for hSigma1 receptors. Thus, direct interactions with hVMAT2 and serotonin, dopamine, and hSigma1 receptors may not explain psychoactive effects. The primary mechanisms of action may be as inhibitors or substrates of DAT, SERT and NET.     

Regulation of dopamine and MPP+ transport by catecholamine transporters.

Following exocytotic release of the biogenic amine neurotransmitters, norepinephrine and dopamine, are removed from the synaptic cleft by the respective transporter, norepinephrine transporter (NET) and dopamine transporter (DAT) located on the plasma membrane. The catecholamine transporters are the molecular targets for psychoactive drugs as well as drugs of abuse such as cocaine and amphetamine and the Parkinsonism-inducing neurotoxin, MPP+. Nicotine regulates the transport of catecholamines and MPP+ and may exert self-medicating effects for depression, schizophrenia and attention deficit hyperactivity disorder, and neuroprotective effects against MPP+ through the regulation of the transporters. The availability of cDNAs of these transporters has permitted detailed pharmacological studies in heterologous expression systems for determining the mechanisms of action of nicotine on transporters. Moreover, functional analysis of the effect of single amino acid substitution suggests that specific residues in DAT molecules may play a significant role in interaction with MPP+ and cocaine, and thus would promise a development of novel drugs with diverse chemical structures.     

2-Carbomethoxy-3-aryl-8-bicyclo[3.2.1]octanes: potent non-nitrogen inhibitors of monoamine transporters

Cocaine is a potent central nervous system stimulant with severe addiction liability. Its reinforcing and stimulant properties derive from inhibition of monoamine transport systems, in particular the dopamine transporter (DAT). This inhibition results in an increase in synaptic dopamine with subsequent stimulation of postsynaptic dopamine receptors. A wide variety of ligands manifest potent inhibition of the DAT, and these ligands include 3-aryltropane as well as 8-oxa-3-aryltropane analogues of cocaine. There has been considerable effort to determine structure-activity relationships of cocaine and congeners, and it is becoming clear that these inhibitors do not all interact with the DAT in the same manner. The functional role of the 8-heteroatom is the focus of this study. We describe the preparation and biology of a series of 2-carbomethoxy-3-arylbicyclo[3.2.1]octane analogues. Results show that methylene substitution of the amine or ether function of the 8-hetero-2-carbomethoxy-3-arylbicyclo[3.2.1]octanes yields potent inhibitors of monoamine transport. Therefore neither nitrogen nor oxygen are prerequisites for binding of tropane-like ligands to monoamine transporters.