Involvement of organic anion transporters in the efflux of uremic toxins across the blood-brain barrier

Renal failure causes multiple physiological changes involving CNS dysfunction. In cases of uremia, there is close correlation between plasma levels of uremic toxins [e.g. 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippurate (HA) and indoleacetate (IA)] and the degree of uremic encephalopathy, suggesting that uremic toxins are involved in uremic encephalopathy. In order to evaluate the relevance of uremic toxins to CNS dysfunction, we investigated directional transport of uremic toxins across the blood-brain barrier (BBB) using in vivo integration plot analysis and the brain efflux index method. We observed saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA, which was inhibited by probenecid. For all uremic toxins evaluated, apparent efflux clearance across the BBB was greater than apparent influx clearance, suggesting that these toxins are predominantly transported from the brain to blood across the BBB. Saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA was completely inhibited by benzylpenicillin, which is a substrate of rat organic anion transporter 3 (rOat3). Taurocholate and digoxin, which are common substrates of rat organic anion transporting polypeptide (rOatp), partially inhibited the efflux of [(3)H]CMPF. Transport experiments using a Xenopus laevis oocyte expression system revealed that CMPF, HA and IA are substrates of rOat3, and that CMPF (but not HA or IA) is a substrate of rOap2. These results suggest that rOat3 mediates brain-to-blood transport of uremic toxins, and that rOatp2 is involved in efflux of CMPF. Thus, conditions typical of uremia can cause inhibition of brain-to-blood transport involving rOat3 and/or rOatp2, leading to accumulation of endogenous metabolites and drugs in the brain.     

Organic anion transporter 3 is involved in the brain-to-blood efflux transport of thiopurine nucleobase analogs

Thiopurines are used as antileukemic drugs. However, during chemotherapy CNS relapses occur due to the proliferation of leukemic cells in the CNS resulting from restricted drug distribution in the brain. The molecular mechanism for this limited cerebral distribution remains unclear. The purpose of this study was to identify the transporter responsible for the brain-to-blood transport of thiopurines across the blood-brain barrier (BBB) using the brain efflux index method. [14C]6-Mercaptopurine (6-MP) and [3H]6-thioguanine were eliminated from rat brain in a time-dependent manner. The elimination of [14C]6-MP was inhibited by substrates of rat organic anion transporters (rOATs), including indomethacin and benzylpenicillin. rOAT1 and rOAT3 exhibited 6-MP uptake, while benzylpenicillin inhibited rOAT3-mediated uptake, but not that by rOAT1. rOAT3-mediated [14C]6-MP uptake was also inhibited by other thiopurine derivatives. Although methotrexate inhibited rOAT3-mediated [14C]6-MP uptake, the Ki value was 17.5-fold greater than the estimated brain concentration of methotrexate in patients receiving chemotherapy. Accordingly, 6-MP would undergo efflux transport by OAT3 from the brain without any inhibitory effect from coadministered methotrexate in the chemotherapy. In conclusion, rOAT3 is involved in the brain-to-blood transport of thiopurines at the BBB and is one mechanism of limited cerebral distribution.     

Uremic toxins inhibit transport by breast cancer resistance protein and multidrug resistance protein 4 at clinically relevant concentrations

During chronic kidney disease (CKD), there is a progressive accumulation of toxic solutes due to inadequate renal clearance. Here, the interaction between uremic toxins and two important efflux pumps, viz. multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP) was investigated. Membrane vesicles isolated from MRP4- or BCRP-overexpressing human embryonic kidney cells were used to study the impact of uremic toxins on substrate specific uptake. Furthermore, the concentrations of various uremic toxins were determined in plasma of CKD patients using high performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Our results show that hippuric acid, indoxyl sulfate and kynurenic acid inhibit MRP4-mediated [(3)H]-methotrexate ([(3)H]-MTX) uptake (calculated Ki values: 2.5 mM, 1 mM, 25 μM, respectively) and BCRP-mediated [(3)H]-estrone sulfate ([(3)H]-E1S) uptake (Ki values: 4 mM, 500 μM and 50 μM, respectively), whereas indole-3-acetic acid and phenylacetic acid reduce [(3)H]-MTX uptake by MRP4 only (Ki value: 2 mM and IC(50) value: 7 mM, respectively). In contrast, p-cresol, p-toluenesulfonic acid, putrescine, oxalate and quinolinic acid did not alter transport mediated by MRP4 or BCRP. In addition, our results show that hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid and phenylacetic acid accumulate in plasma of end-stage CKD patients with mean concentrations of 160 μM, 4 μM, 129 μM, 1 μM and 18 μM, respectively. Moreover, calculated Ki values are below the maximal plasma concentrations of the tested toxins. In conclusion, this study shows that several uremic toxins inhibit active transport by MRP4 and BCRP at clinically relevant concentrations.     

In vivo delivery of small interfering RNA targeting brain capillary endothelial cells

Brain capillary endothelial cells (BCECs) play an important role in blood-brain barrier (BBB) functions and pathophysiologic mechanisms in brain ischemia and inflammation. We try to suppress gene expression in BCECs by intravenous application of small interfering RNA (siRNA). After injection of large dose siRNA with hydrodynamic technique to mouse, suppression of endogenous protein and the BBB function of BCECs was investigated. The brain-to-blood transport function of organic anion transporter 3 (OAT3) that expressed in BCECs was evaluated by Brain Efflux Index method in mouse. The siRNA could be delivered to BCECs and efficiently inhibited endogenously expressed protein of BCECs. The suppression effect of siRNA to OAT3 is enough to reduce the brain-to-blood transport of OAT3 substrate, benzylpenicillin at BBB. The in vivo siRNA-silencing method with hydrodynamic technique may be useful for the study of BBB function and gene therapy targeting BCECs.     

OAT2 catalyses efflux of glutamate and uptake of orotic acid

OAT (organic anion transporter) 2 [human gene symbol SLC22A7 (SLC is solute carrier)] is a member of the SLC22 family of transport proteins. In the rat, the principal site of expression of OAT2 is the sinusoidal membrane domain of hepatocytes. The particular physiological function of OAT2 in liver has been unresolved so far. In the present paper, we have used the strategy of LC (liquid chromatography)-MS difference shading to search for specific and cross-species substrates of OAT2. Heterologous expression of human and rat OAT2 in HEK (human embryonic kidney)-293 cells stimulated accumulation of the zwitterion trigonelline; subsequently, orotic acid was identified as an excellent and specific substrate of OAT2 from the rat (clearance=106 μl·min?1·mg of protein?1) and human (46 μl·min?1·mg of protein?1). The force driving uptake of orotic acid was identified as glutamate antiport. Efficient transport of glutamate by OAT2 was directly demonstrated by uptake of [3H]glutamate. However, because of high intracellular glutamate, OAT2 operates as glutamate efflux transporter. Thus expression of OAT2 markedly increased the release of glutamate (measured by LC-MS) from cells, even without extracellular exchange substrate. Orotic acid strongly trans-stimulated efflux of glutamate. We thus propose that OAT2 physiologically functions as glutamate efflux transporter. OAT2 mRNA was detected, after laser capture microdissection of rat liver slices, equally in periportal and pericentral regions; previous reports of hepatic release of glutamate into blood can now be explained by OAT2 activity. A specific OAT2 inhibitor could, by lowering plasma glutamate and thus promoting brain-to-blood efflux of glutamate, alleviate glutamate exotoxicity in acute brain conditions.     

Glutaric aciduria type I and methylmalonic aciduria: Simulation of cerebral import and export of accumulating neurotoxic dicarboxylic acids in in vitro models of the blood–brain barrier and the choroid plexus

Intracerebral accumulation of neurotoxic dicarboxylic acids (DCAs) plays an important pathophysiological role in glutaric aciduria type I and methylmalonic aciduria. Therefore, we investigated the transport characteristics of accumulating DCAs – glutaric (GA), 3-hydroxyglutaric (3-OH-GA) and methylmalonic acid (MMA) – across porcine brain capillary endothelial cells (pBCEC) and human choroid plexus epithelial cells (hCPEC) representing in vitro models of the blood–brain barrier (BBB) and the choroid plexus respectively. We identified expression of organic acid transporters 1 (OAT1) and 3 (OAT3) in pBCEC on mRNA and protein level. For DCAs tested, transport from the basolateral to the apical site (i.e. efflux) was higher than influx. Efflux transport of GA, 3-OH-GA, and MMA across pBCEC was Na⁺-dependent, ATP-independent, and was inhibited by the OAT substrates para-aminohippuric acid (PAH), estrone sulfate, and taurocholate, and the OAT inhibitor probenecid. Members of the ATP-binding cassette transporter family or the organic anion transporting polypeptide family, namely MRP2, P-gp, BCRP, and OATP1B3, did not mediate transport of GA, 3-OH-GA or MMA confirming the specificity of efflux transport via OATs. In hCPEC, cellular import of GA was dependent on Na⁺-gradient, inhibited by NaCN, and unaffected by probenecid suggesting a Na⁺-dependent DCA transporter. Specific transport of GA across hCPEC, however, was not found. In conclusion, our results indicate a low but specific efflux transport for GA, 3-OH-GA, and MMA across pBCEC, an in vitro model of the BBB, via OAT1 and OAT3 but not across hCPEC, an in vitro model of the choroid plexus.     

Metabolomic search for uremic toxins as indicators of the effect of an oral sorbent AST-120 by liquid chromatography/tandem mass spectrometry

An oral sorbent AST-120 composed of spherical porous carbon particles has superior adsorption ability for certain small-molecular-weight organic compounds known to accumulate in patients with chronic renal failure (CRF). A metabolomic approach was applied to search for uremic toxins as possible indicators of the effect of AST-120. Serum metabolites in normal and CRF rats before and after administration of AST-120 for 3 days were analyzed by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) and principal component analysis. Further, serum and urine levels of the indicators were quantified by selected reaction monitoring of LC/ESI-MS/MS. Indoxyl sulfate was the first principal serum metabolite, which could differentiate CRF from both normal and AST-120-administered CRF rats, followed by hippuric acid, phenyl sulfate and 4-ethylphenyl sulfate. CRF rats showed increased serum levels of indoxyl sulfate, hippuric acid, phenyl sulfate, 4-ethylphenyl sulfate and p-cresyl sulfate. Administration of AST-120 for 3 days to the CRF rats reduced the serum and urine levels of these metabolites. In conclusion, indoxyl sulfate is the best indicator of the effect of AST-120 in CRF rats. Hippuric acid, phenyl sulfate and 4-ethylphenyl sulfate are suggested as the additional indicators. 4-Ethylphenyl sulfate is a newly identified uremic substance.     

Delivery of peptides to the brain: emphasis on therapeutic development

Peptides and regulatory proteins hold great promise as therapeutic agents for the central nervous system (CNS). However, the blood-brain barrier (BBB) is a major obstacle to the delivery of these potential therapeutics to their site of action. We concentrate here on the vascular BBB, which is comprised of the capillary bed of the brain specially modified to prevent the production of a plasma ultrafiltrate. For many peptides and proteins, this physical barrier is reinforced by enzymatic activities at the BBB, CNS, and peripheral tissues, short half-lives and large volumes of distribution in the blood, binding proteins in blood, and brain-to-blood efflux systems. Nevertheless, there are pathways through which substances can cross. Small, lipid soluble substances cross by the nonsaturable mechanism of transmembrane diffusion, but even water-soluble peptides can cross to some degree. Many endogenous peptides and regulatory proteins cross the BBB by way of selective, saturable transport systems. For enzymatically resistant substances with long circulating half-lives and small volumes of distribution, such as antibodies, erythropoietin, and enzymes, substances can enter the CNS in therapeutic amounts through the residual leak of the BBB, termed the extracellular pathways. Recent examples show that the BBB transporters for peptides and regulatory substances are modifiable. This provides both a therapeutic opportunity and the potential for disease to arise from BBB dysfunctions. In the last case, the BBB itself is a therapeutic target.     

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.     

Dominant expression of androgen receptors and their functional regulation of organic anion transporter 3 in rat brain capillary endothelial cells; comparison of gene expression between the blood-brain and -retinal barriers

Brain and retinal capillary endothelial cells (BCECs and RCECs, respectively) exhibit a barrier structure and function. Comparison of gene expression in these cells could clarify the selective function of each barrier. The purpose of this study was to identify the genes selectively expressed at the blood-brain barrier (BBB) and to clarify the function of the selective gene, androgen receptor (AR). Gene expression was compared by a differential display using conditionally immortalized rat BCECs and RCECs (TR-BBB and TR-iBRB, respectively). A total of 12 gene fragments were identified as the selective genes dominantly expressed in TR-BBB cells. The most selective fragment in TR-BBB cells had the highest homology with the 3'-UTR of human and mouse AR. Rat AR mRNA was detected in TR-BBB cells and the brain capillary rich fraction, but not in TR-iBRB cells. Expression of organic anion transporter 3 (OAT3) mRNA in TR-BBB cells was induced by treatment with dihydrotestosterone (DHT), an AR ligand, and this induction was suppressed by flutamide. Moreover, uptake of benzylpenicillin by TR-BBB cells was also induced by DHT treatment. In contrast, OAT3 mRNA expression in TR-iBRB cells was not affected by DHT treatment. The brain-to-blood efflux rate of benzylpenicillin was not affected by gender. These results suggest that AR is involved in the functional regulation of OAT3 at the BBB, but not at the inner blood-retinal barrier (iBRB), and this regulation is not affected by gender. The BBB function will be affected by the androgen levels in the brain and/or plasma via AR.     

Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta

A cDNA encoding a novel multispecific organic anion transporter, OAT4, was isolated from a human kidney cDNA library. The OAT4 cDNA consisted of 2210 base pairs that encoded a 550-amino acid residue protein with 12 putative membrane-spanning domains. The amino acid sequence of OAT4 showed 38 to 44% identity to those of other members of the OAT family. Northern blot analysis revealed that OAT4 mRNA is abundantly expressed in the placenta as well as in the kidney. When expressed in Xenopus oocytes, OAT4 mediated the high affinity transport of estrone sulfate (K(m) = 1.01 microM) and dehydroepiandrosterone sulfate (K(m) = 0.63 microM) in a sodium-independent manner. OAT4 also mediated the transport of ochratoxin A. OAT4-mediated transport of estrone sulfate was inhibited by several sulfate conjugates, such as p-nitrophenyl sulfate, alpha-naphthyl sulfate, beta-estradiol sulfate, and 4-methylumbelliferyl sulfate. By contrast, glucuronide conjugates showed little or no inhibitory effect on the OAT4-mediated transport of estrone sulfate. OAT4 interacted with chemically heterogeneous anionic compounds, such as nonsteroidal anti-inflammatory drugs, diuretics, sulfobromophthalein, penicillin G, and bile salts, whereas tetraethylammonium, an organic cation, did not. OAT4 is the first member of the multispecific organic anion transporter family, which is expressed abundantly in the placenta. OAT4 might be responsible for the elimination and detoxification of harmful anionic substances from the fetus.     

Relative contribution of OAT1 and OAT3 transport activities in isolated perfused rabbit renal proximal tubules

The expression of both OAT1 and OAT3 along the isolated rabbit renal proximal tubule (RPT) was determined using RT-PCR. They were found to be very strong in S2 segment and weak in S1 and S3 segments. We further examined the relative transport activity of these transporters in isolated perfused rabbit RPT using [(3)H]para-aminohippurate ([(3)H]PAH), and estrone sulfate ([(3)H]ES) as specific substrates for rbOAT1 and rbOAT3, respectively. The transport activity of OAT1 was in the order S2>S1=S3 segments and that of OAT3 was in the order S1=S2>S3 segments. The addition of alpha-ketoglutarate (100 muM) in the bathing medium increased both OAT1 and OAT3 transport activities in all segments of proximal tubule. The kinetics of [(3)H]succinic acid transport, used to measure the activity of sodium dicarboxylate transporter 3 (NaDC3), were examined. The J(max) for succinic acid was in the order S2>S3 and unmeasurable in the S1 segment. Our data indicate that both OAT1 and OAT3 play quantitatively significant roles in the renal transport of organic anions along the proximal tubule but predominately in S2 segment. The relative contribution of both transporters depends on their relative expression levels and may possibly be affected by the activity of NaDC3 in RPT.     

Nature and consequences of mammalian brain and CSF efflux transporters: four decades of progress

In the last 40 years, especially with the application of new neurochemical and molecular biological techniques, there has been explosive progress in understanding how certain ligands and drugs are transported across the blood-brain barrier and choroid plexus out of brain and CSF. In the CNS, there are several separate efflux transporters with very broad specificity that are responsible for much of the efflux transport. This review focuses on three such transporters: organic acid transporter-3, peptide transporter-2 and P-glycoprotein for which there is substantial new information including 'knockout' models in mice and, in one case, dogs. Moreover, the structural biology and transport mechanism of P-glycoprotein at 3.8 angstroms is described. The overall objective is to show how this new knowledge provides a more thorough understanding (e.g., of molecular mechanisms) of efflux transport and in several cases leads to clinically relevant information that allows better treatment of certain CNS disorders (e.g., meningitis and brain cancer).     

Homeostatic role of the active transport in elimination of [3H]benzylpenicillin out of the cerebrospinal fluid system

Cerebral acidic metabolites and penicillin are organic anions which can be carried by active transport into capillaries of the central nervous system (CNS). However, it is generally believed that these metabolites are mainly delivered from CNS to cerebrospinal fluid (CSF) and eliminated by CSF circulation over cortex and its absorption into dural venous sinuses. To test this hypothesis we studied fate of penicillin ([3H]benzylpenicillin) in the CSF under control conditions and when its active transport was blocked by probenecid. After application of penicillin into cisterna magna of control dogs, it is distributed only in traces to lumbar, ventricular and cortical CSF. However, when active transport of penicillin across capillary wall is blocked by probenecid, its disappearance from cisterna is slowed down and its distribution is greatly enhanced so that at 300 min penicillin concentrations in cisternal, lumbar and cortical CSF approach or equal each other. Disappearance of penicillin from cisternal CSF shows a single exponential course (half-time 30 min) in control, while in probenecid pretreated dogs this is a slow multiexponential process. The results indicate that the active transport across capillary wall in CNS, but not generally postulated unidirectional CSF circulation over cortex and its absorption into dural venous sinuses, is instrumental in elimination of cerebral acidic metabolites and in such a way homeostasis in brain and cerebrospinal fluid is maintained.     

Transport of organic anions across the basolateral membrane of proximal tubule cells

Renal proximal tubules secrete diverse organic anions (OA) including widely prescribed anionic drugs. Here, we review the molecular properties of cloned transporters involved in uptake of OA from blood into proximal tubule cells and provide extensive lists of substrates handled by these transport systems. Where tested, transporters have been immunolocalized to the basolateral cell membrane. The sulfate anion transporter 1 (sat-1) cloned from human, rat and mouse, transported oxalate and sulfate. Drugs found earlier to interact with sulfate transport in vivo have not yet been tested with sat-1. The Na⁺-dicarboxylate cotransporter 3 (NaDC-3) was cloned from human, rat, mouse and flounder, and transported three Na⁺ with one divalent di- or tricarboxylate, such as citric acid cycle intermediates and the heavy metal chelator 2,3-dimercaptosuccinate (succimer). The organic anion transporter 1 (OAT1) cloned from several species was shown to exchange extracellular OA against intracellular α-ketoglutarate. OAT1 translocated, e.g., anti-inflammatory drugs, antiviral drugs, β-lactam antibiotics, loop diuretics, ochratoxin A, and p-aminohippurate. Several OA, including probenecid, inhibited OAT1. Human, rat and mouse OAT2 transported selected anti-inflammatory and antiviral drugs, methotrexate, ochratoxin A, and, with high affinities, prostaglandins E₂ and F_2α. OAT3 cloned from human, rat and mouse showed a substrate specificity overlapping with that of OAT1. In addition, OAT3 interacted with sulfated steroid hormones such as estrone-3-sulfate. The driving forces for OAT2 and OAT3, the relative contributions of all OA transporters to, and the impact of transporter regulation by protein kinases on renal drug excretion in vivo must be determined in future experiments. Electronic Publication     

Interactions of racemic mefloquine and its enantiomers with P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT

The brain distribution of the enantiomers of the antimalarial drug mefloquine is stereoselective according to the species. This stereoselectivity may be related to species-specific differences in the properties of some membrane-bound transport proteins, such as P-glycoprotein (P-gp). The interactions of racemic mefloquine and its individual enantiomers with the P-glycoprotein efflux transport system have been analysed in immortalised rat brain capillary endothelial GPNT cells. Parallel studies were carried out for comparison in human colon carcinoma Caco-2 cells. The cellular accumulation of the P-glycoprotein substrate, [(3)H]vinblastine, was significantly increased both in GPNT cells and in Caco-2 cells when treated with racemic mefloquine and the individual enantiomers. In GPNT cells, the (+)-stereoisomer of mefloquine was up to 8-fold more effective than its antipode in increasing cellular accumulation of [(3)H]vinblastine, while in Caco-2 cells, both enantiomers were equally effective. These results suggest that racemic mefloquine and its enantiomers are effective inhibitors of P-gp. Furthermore, a stereoselective P-glycoprotein inhibition is observed in rat cells but not in human cells. The efflux of [(14)C]mefloquine from GPNT cells was decreased when the cells were incubated with the P-gp modulators, verapamil, cyclosporin A or chlorpromazine, suggesting that MQ could be a P-gp substrate.     

Effect of Various Centrally Acting Drugs on the Efflux of Dopamine Metabolites from the Rat Brain

The influence of chlorpromazine, haloperidol, morphine, chloral hydrate, gamma-butyrolactone, probenecid, kainic acid, oxotremorine, pargyline, yohimbine, (+)-amphetamine, and cocaine on the efflux rate of 3,4-dihydroxyphenylacetic acid (DOPAC) from four brain areas was studied. All drugs studied except pargyline and morphine had an effect on the transport of DOPAC and homovanillic acid (HVA) from the brain. Nine drugs inhibited the efflux of DOPAC and HVA, whereas (+)-amphetamine stimulated this transport. These data suggest that most centrally acting drugs can interfere with the elimination of 3,4-dihydroxyphenylethylamine (DA or dopamine) metabolites from the brain. These effects are heterogeneously distributed throughout the brain and are probably related to indirect nonspecific drug effects. This implies that drug-induced changes in DA metabolite concentrations, especially when these changes are slight to moderate, cannot directly be translated to changes in the production rate of these metabolites. By studying five control groups, we concluded that formation and transport of DOPAC are not synchronized in the various brain areas.