Function of monoamine neurotransmitter transporters

Monoamine transporters include plasma membrane and vesicular monoamine transporters(VMAT). The former selectively and Na+/Cl(-)-dependently transport dopamine, noradrenaline and serotonin into the cytoplasma, and the latter non-selectively carries monoamine into the vesicle. These transporters are composed of amino acid groups containing 12 folds more transmembrane components. Cytoplasmic transporters are a target site of certain drugs. Antiepileptic drugs such as SSRI and tricyclic antidepressants bind with serotonin transporter(SERT), noradrenaline transporter(NET) and/or dopamine transporters(DAT) to inhibit transport of monoamines into the cytoplasma, thereby increasing monoamine levels within the synaptic cleft. However, amphetamine, known to induce drug dependence, is transported by DAT and inhibit VMAT to induce reverse-transport of monoamines into the synaptic area, thereby producing psychiatric and behavioral alterations. Thus, monoamine transporters are target sites of drugs, and functional changes in the transporters may be involved in the pathogenesis of affective diseases, schizophrenia and/or personality disorders including neurogenerative diseases such as Parkinson's disease.     

Molecular and behavioral interactions between central melanocortins and cocaine

Behavioral and molecular studies have established a link between drugs of abuse and the central melanocortin system, particularly the melanocortin 4 receptor (MC4-R). The present study expands this line of investigation to characterization of the neurochemical and behavioral interactions between MC4-R and the psychomotor stimulant, cocaine. The results demonstrate that repeated, but not acute, cocaine administration up-regulates MC4-R mRNA expression in the striatum and hippocampus, but not cerebral cortex. Pharmacological studies indicate that the up-regulation of MC4-R expression occurs via dopamine D1 and D2 receptor-dependent mechanisms. The D1/D2 antagonist haloperidol and the D2-selective antagonist eticlopride mimic the effect of cocaine on MC4-R expression. In addition, coadministration of a D1-selective antagonist, SCH 23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine], completely blocks the up-regulation of MC4 mRNA by cocaine, demonstrating that D1 receptor activation is necessary for this response. Moreover, the results demonstrate that cocaine treatment increases behavioral responses (grooming and locomotor activity) to infusions of a melanocortin agonist, indicating that up-regulation of MC4-R expression results in functional consequences. These data further support a role for the melanocortin-MC4-R neuropeptide system in the biochemical and behavioral effects of cocaine.     

Molecular physiology of glucose transporters

Molecular cloning of cDNA encoding the human erythrocyte facilitated-diffusion glucose transporter (GT) has elucidated its structure and has permitted a careful study of its tissue distribution and of its involvement in processes such as insulin-stimulated glucose uptake by adipose cells or transformation-induced increase in glucose metabolism. An important outcome of these studies was the discovery that additional isoforms of this transporter were expressed in a tissue-specific manner; these comprise a family of structurally and functionally related molecules. Their tissue distribution, differences in kinetic properties, and differential regulation by ambient glucose and insulin levels suggest that they play specific roles in the control of glucose homeostasis. Herein, we will discuss the structure of three members of the GT family: erythroid/brain GT, liver GT, and adipose cell/muscle GT. In the light of their tissue-specific expression, kinetic parameters, and susceptibility to insulin action, we discuss their possible specific functions.     

Cocaine esterase: interactions with cocaine and immune responses in mice

Cocaine esterase (CocE) is the most efficient protein catalyst for the hydrolysis of cocaine characterized to date. The aim of this study was to investigate the in vivo potency of CocE in blocking cocaine-induced toxicity in the mouse and to assess CocE's potential immunogenicity. Cocaine toxicity was quantified by measuring the occurrence of convulsions and lethality. Intravenous administration of CocE (0.1-1 mg) 1 min before cocaine administration produced dose-dependent rightward shifts of the dose-response curve for cocaine toxicity. More important, i.v. CocE (0.1-1 mg), given 1 min after the occurrence of cocaine-induced convulsions, shortened the recovery time after the convulsions and saved the mice from subsequent death. Effects of repeated exposures to CocE were evaluated by measuring anti-CocE antibody titers and the protective effects of i.v. CocE (0.32 mg) against toxicity elicited by i.p. cocaine (320 mg/kg) (i.e., 0-17% occurrence of convulsions and lethality). CocE retained its potency against cocaine toxicity in mice after a single prior CocE exposure (0.1-1 mg), and these mice did not show an immune response. CocE retained similar effectiveness in mice after three prior CocE exposures (0.1-1 mg/week for 3 weeks), although these mice displayed 10-fold higher antibody titers. CocE partially lost effectiveness (i.e., 33-50% occurrence of convulsions and lethality) in mice with four prior exposures to CocE (0.1-1 mg/2 week for four times), and these mice displayed approximately 100-fold higher antibody titers. These results suggest that CocE produces robust protection and reversal of cocaine toxicity, indicating CocE's therapeutic potential for acute cocaine toxicity. Repeated CocE exposures may increase its immunogenicity and partially reduce its protective ability.     

Molecular biology of mammalian glucose transporters

The oxidation of glucose represents a major source of metabolic energy for mammalian cells. However, because the plasma membrane is impermeable to polar molecules such as glucose, the cellular uptake of this important nutrient is accomplished by membrane-associated carrier proteins that bind and transfer it across the lipid bilayer. Two classes of glucose carriers have been described in mammalian cells: the Na(+)-glucose cotransporter and the facilitative glucose transporter. The Na(+)-glucose cotransporter transports glucose against its concentration gradient by coupling its uptake with the uptake of Na+ that is being transported down its concentration gradient. Facilitative glucose carriers accelerate the transport of glucose down its concentration gradient by facilitative diffusion, a form of passive transport. cDNAs have been isolated from human tissues encoding a Na(+)-glucose-cotransporter protein and five functional facilitative glucose-transporter isoforms. The Na(+)-glucose cotransporter is expressed by absorptive epithelial cells of the small intestine and is involved in the dietary uptake of glucose. The same or a related protein may be responsible for the reabsorption of glucose by the kidney. Facilitative glucose carriers are expressed by most if not all cells. The facilitative glucose-transporter isoforms have distinct tissue distributions and biochemical properties and contribute to the precise disposal of glucose under varying physiological conditions. The GLUT1 (erythrocyte) and GLUT3 (brain) facilitative glucose-transporter isoforms may be responsible for basal or constitutive glucose uptake. The GLUT2 (liver) isoform mediates the bidirectional transport of glucose by the hepatocyte and is responsible, at least in part, for the movement of glucose out of absorptive epithelial cells into the circulation in the small intestine and kidney. This isoform may also comprise part of the glucose-sensing mechanism of the insulin-producing beta-cell. The subcellular localization of the GLUT4 (muscle/fat) isoform changes in response to insulin, and this isoform is responsible for most of the insulin-stimulated uptake of glucose that occurs in muscle and adipose tissue. The GLUT5 (small intestine) facilitative glucose-transporter isoform is expressed at highest levels in the small intestine and may be involved in the transcellular transport of glucose by absorptive epithelial cells. The exon-intron organizations of the human GLUT1, GLUT2, and GLUT4 genes have been determined. In addition, the chromosomal locations of the genes encoding the Na(+)-dependent and facilitative glucose carriers have been determined. Restriction-fragment-length polymorphisms have also been identified at several of these loci.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of methotrexate transport and its drug interactions with human organic anion transporters

Life-threatening drug interactions are known to occur between methotrexate and nonsteroidal anti-inflammatory drugs (NSAIDs), probenecid, and penicillin G. The purpose of this study was to characterize methotrexate transport, as well as to determine the site and the mechanism of drug interactions in the proximal tubule. Mouse proximal tubule cells stably expressing basolateral human organic anion transporters (hOAT1 and hOAT3) and apical hOAT (hOAT4) were established. The K(m) values for hOAT1-, hOAT3-, and hOAT4-mediated methotrexate uptake were 553.8 microM, 21.1 microM, and 17.8 microM, respectively. NSAIDs (salicylate, ibuprofen, ketoprofen, phenylbutazone, piroxicam, and indomethacin), probenecid, and penicillin G dose dependently inhibited methotrexate uptake mediated by hOAT1, hOAT3, and hOAT4. Kinetic analysis of inhibitory effects of these drugs on hOAT3-mediated methotrexate uptake revealed that these inhibitions were competitive. The K(i) values for the effects of salicylate, phenylbutazone, indomethacin, and probenecid on hOAT3-mediated methotrexate uptake were comparable with therapeutically relevant plasma concentrations of unbound drugs. In addition, in the presence of human serum albumin, the K(i) values were comparable with therapeutically relevant total plasma concentrations of drugs. In conclusion, these results suggest that methotrexate is taken up via hOAT3 and hOAT1 at the basolateral side of the proximal tubule and effluxed or taken up at the apical side via hOAT4. In addition, hOAT1, hOAT3, and hOAT4 are the sites of drug interactions between methotrexate and NSAIDs, probenecid, and penicillin G. Furthermore, it was predicted that hOAT3 is the site of drug interactions between methotrexate and salicylate, phenylbutazone, indomethacin, and probenecid in vivo.     

Behavioral and neurochemical interactions between cocaine and buprenorphine: implications for the pharmacotherapy of cocaine abuse

Intravenous self-administration studies in nonhuman primates suggest that the opioid receptor agonist-antagonist buprenorphine may be useful in the pharmacotherapy of cocaine abuse. In the present studies, behavioral and neurochemical interactions between cocaine and buprenorphine were examined using a conditioned place preference (CPP) procedure and in vivo microdialysis. Cocaine-induced CPP was linearly related to the dose administered (0-5.0 mg/kg). Buprenorphine (0-0.9 mg/kg) also elicited CPP in a dose-related manner; an inverted U-shaped function was obtained. Subthreshold doses of cocaine (1.5 mg/kg) and buprenorphine (0.01 mg/kg), themselves incapable of eliciting CPP, produced a significant CPP when given together. Moderate doses of cocaine (5.0 mg/kg) and buprenorphine (0.075 mg/kg), which were individually capable of eliciting CPP, produced a significantly larger CPP when given in combination. In the in vivo microdialysis studies, a low dose of buprenorphine (0.01 mg/kg) produced a progressive increase in extracellular dopamine in the nucleus accumbens, reaching approximately 200% of basal levels after 5 hr. Cocaine (5.0 mg/kg) rapidly increased extracellular dopamine concentrations (180% of basal values within 20 min), which returned to baseline in 2 to 3 hr. This effect of cocaine was significantly potentiated by coadministering buprenorphine (0.01 mg/kg); under this condition the peak increase in extracellular dopamine reached 260% of baseline values. These neurochemical findings are consistent with the CPP results and indicate that buprenorphine can interact with cocaine in a synergistic manner. In contrast to previous speculations, these results suggest that buprenorphine may enhance rather than attenuate the rewarding properties of cocaine.     

Nucleoside phosphonate interactions with multiple organic anion transporters in renal proximal tubule

The interactions of two antiviral, acyclic nucleoside phosphonates, adefovir and cidofovir, with xenobiotic transporters was studied in intact killifish (Fundulus heteroclitus) renal proximal tubules by using fluorescent substrates, confocal microscopy, and quantitative image analysis. Both drugs reduced in a concentration-dependent manner the transport of fluorescein on the classical organic anion system and transport of fluorescein-methotrexate on multidrug resistance-associated protein 2 (Mrp2). Neither drug inhibited transport of a fluorescent cyclosporin A derivative on P-glycoprotein. Inhibition of Mrp2-mediated transport was abolished by 50 microM p-aminohippurate, indicating that adefovir and cidofovir entered the cells at the basolateral membrane on the classical organic anion transport system (OAT1). Comparison of the inhibitory potencies of the nucleoside phosphonates with other substrates and inhibitors showed them to be moderate inhibitors of OAT1- and Mrp2-mediated transport.     

Characteristics of drug interactions with recombinant biogenic amine transporters expressed in the same cell type

We characterized the effects of drugs on the uptake of [3H]neurotransmitter by and the binding of [125I](3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester ([125I]RTI-55) to the recombinant human dopamine (hDAT), serotonin (hSERT), or norepinephrine (hNET) transporters stably expressed in human embryonic kidney 293 cells. RTI-55 had similar affinity for the hDAT and hSERT and lower affinity for hNET (Kd = 1. 83, 0.98, and 12.1 nM, respectively). Kinetic analysis of [125I]RTI-55 binding indicated that the dissociation rate (k-1) was significantly lower for hSERT and the association rate (k+1) was significantly lower for hNET compared with the hDAT. The potency of drugs at blocking [3H]neurotransmitter uptake was highly correlated with potency at blocking radioligand binding for hDAT and hSERT. Substrates were more potent at the inhibition of [3H]neurotransmitter uptake than radioligand binding. The potency of drugs was highly correlated between displacement of [3H]nisoxetine (Kd = 6.0 nM) and [125I]RTI-55 from the hNET, suggesting that these radioligands recognize similar sites on the transporter protein. The correlation observed between inhibitory potency for uptake and binding of either ligand at the hNET was lower than correlations between uptake and binding for hDAT and hSERT. The present results indicate that the cocaine analog [125I]RTI-55 has unique binding properties at each of the transporters and that the use of recombinant transporters expressed by a single cell type can provide a powerful screening tool for drugs interacting with biogenic amine transporters, such as possible cocaine antagonists.     

Molecular pharmacological characterization of two multidrug transporters in Lactococcus lactis

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in Lactococcus lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.     

Pharmacological characterization of nicotine's interaction with cocaine and cocaine analogs.

Cocaine and a number of 3beta-phenyltropane cocaine analogs were investigated for their potential to block various pharmacological effects of nicotine in animals. They blocked the antinociceptive effect of nicotine in the tail-flick test after systemic administration in a dose-dependent manner. Similarly, cocaine was also able to block nicotine-induced motor impairment in mice. Furthermore, cocaine blocked nicotine-induced seizures at a lower potency than for antinociception, but failed to block nicotine's effect on body temperature and drug discrimination. The antagonistic potencies of the 3beta-phenyltropane cocaine analogs were not correlated with their affinity for monoamines transporters. Additionally, bupropion, nomifensin, GBR 12909, and nisoxetine, but not methylphenidate and fluoxetine, blocked nicotine-induced antinociception; however, their antagonistic potencies were unrelated to their affinities for the transporters. Taken together, these results suggest that the mechanism of cocaine's antagonistic activity is not related to its binding and uptake of inhibition on monoamine neurotransporters. The failure of lidocaine and procaine to antagonize nicotine's effects in the tail-flick assay rules out local anesthetic effects. In addition, cocaine blocked differentially the response of nicotine in the oocyte receptor expression system for the alpha4beta2 and alpha3beta2 subtypes in a dose-dependent manner. Our results suggest that cocaine is a noncompetitive nicotinic antagonist with some selectivity for neuronal nicotinic receptor subtypes. Our studies also demonstrate that 3beta-phenyltropane analogs constitute a new class of nicotinic antagonists. Elucidation of the mechanism of action of this new class of antagonists may provide an explanation for the effectiveness of agents such as bupropion for the treatment of smoking cessation.     

Cardiotoxicity and late onset seizures with citalopram overdose

A 31-year-old man ingested 400 mg of citalopram (Celexa) after an argument with his parents and girlfriend 13 h before presentation. Paramedics witnessed the patient having a generalized clonic seizure. The electrocardiogram (EKG) revealed a wide QRS complex, prolongation of the QTc interval, and left bundle branch pattern. He was treated with sodium bicarbonate with resolution of these changes. The patient was continued on a sodium bicarbonate infusion and demonstrated no further EKG abnormalities. Sodium bicarbonate should be considered as a treatment modality in patients with EKG abnormalities of prolongation of QRS or QTc interval after citalopram overdose.     

Molecular mechanisms on drug transporters in the drug absorption and disposition]

The membrane transport processes of drugs are critical issues to determine their absorption, distribution and elimination. Recently, various drug transporters have been identified and characterized. The enterocyte peptide transporter PEPT1 mediates the absorption of peptide-like drugs including beta-lactam antibiotics as well as valacyclovir lacking peptide bond. In the kidney, the basolateral organic anion transporters (OAT1, OAT3) and cation transporters (OCT1, OCT2) mediate renal distribution of hydrophilic anionic and cationic drugs, respectively. The brush-border type OAT-K1/K2 were suggested to be a target transporter for methotrexate-leucovorine rescue therapy. The ATP-driven efflux pump P-glycoprotein appeared to be an interaction site between digoxin and clarithromycin or itraconazole in the kidney. In addition, the intestinal P-glycoprotein was suggested to act as an absorptive barrier for tacrolimus in recipients of liver and small bowel transplantation.     

Behavioral effects of cocaine: interactions with D1 dopaminergic antagonists and agonists in mice and squirrel monkeys.

The present study compared interactions among dopamine D1-like agonists and partial agonists with cocaine on the locomotor stimulant effects of cocaine, as well as the discriminative-stimulus effects of cocaine, and effects of cocaine on rates of responding. Cocaine alone produced a dose-related stimulation of locomotor activity in Swiss-Webster mice and a dose-related increase in the proportion of responses on the cocaine-appropriate response key in squirrel monkeys (Saimiri sciureus) trained to discriminate cocaine (0.3 mg/kg i.m.) from saline. None of the D1 dopaminergic agents fully reproduced these effects, with SKF 77434 producing marginal stimulation of locomotor activity and SCH 23390, SCH 39166, and SKF 77434 producing some, although incomplete substitution for cocaine in monkeys discriminating cocaine. The D1 dopamine antagonists SCH 23390, SCH 39166, and A-69024 dose-dependently shifted the cocaine dose-effect curve for locomotor activity to the right and decreased the efficacy of cocaine. The same compounds shifted the discriminative-stimulus effects of cocaine to the right without altering efficacy of cocaine. In contrast to the effects on locomotor activity, the maximal shift to the right in the discriminative-stimulus effects of cocaine was approximately 3-fold, with higher doses of the antagonists producing no greater shifts in the cocaine dose-effect curve than with intermediate doses. The partial D1 agonists (+/-)-SKF 38393, (+)-SKF 38393, and SKF 77434 also dose-dependently shifted the dose-effect curve for locomotor stimulant effects to the right and decreased the maximal effect of cocaine. These compounds only shifted the discriminative-stimulus effects of cocaine to a 2-fold maximum. In general, cocaine effects on rates of responding in the subjects discriminating cocaine from saline were only minimally antagonized by coadministration of the D1 dopaminergic agents. Both potency for producing behavioral effects alone and in antagonizing the effects of cocaine were related to binding affinities assessed by displacement of [(3)H]SCH 23390 from rat striatum. These results suggest that actions mediated by D1-like receptors contribute to the behavioral effects of cocaine. However, the various limitations to the degree of antagonism accomplished indicate that D1-like dopaminergic actions appear to be more involved in the effects of cocaine on locomotor activity, relatively less involved in the discriminative-stimulus effects of cocaine, and least involved in the effects of cocaine on operant response rates. This differential involvement of D1 dopamine receptors in these various behavioral effects of cocaine suggests problems in predicting clinical efficacy of at least D1 receptor antagonists as potential treatments for cocaine abuse. Additional studies are necessary to determine whether the antagonism of cocaine can predict therapeutic efficacy at all, and, if so, which effects when antagonized are the best predictors.