Efflux Of Homovanillic Acid Research Articles

Rat organic anion transporter 3 (rOAT3) is responsible for brain-to-blood efflux of homovanillic acid at the abluminal membrane of brain capillary endothelial cells.

The mechanism that removes homovanillic acid (HVA), an end metabolite of dopamine, from the brain is still poorly understood. The purpose of this study is to identify and characterize the brain-to-blood HVA efflux transporter at the rat blood-brain barrier (BBB). Using the Brain Efflux Index method, the apparent in vivo efflux rate constant of [3H]HVA from the brain, k(eff), was determined to be 1.69 x 10(-2) minute(-1). This elimination was significantly inhibited by para-aminohippuric acid (PAH), benzylpenicillin, indoxyl sulfate, and cimetidine, suggesting the involvement of rat organic anion transporter 3 (rOAT3). rOAT3-expressing oocytes exhibited [3H]HVA uptake (K(m) = 274 micromol/L), which was inhibited by several organic anions, such as PAH, indoxyl sulfate, octanoic acid, and metabolites of monoamine neurotransmitters. Neurotransmitters themselves did not affect the uptake. Furthermore, immunohistochemical analysis suggested that rOAT3 is localized at the abluminal membrane of brain capillary endothelial cells. These results provide the first evidence that rOAT3 is expressed at the abluminal membrane of the rat BBB and is involved in the brain-to-blood transport of HVA. This HVA efflux transport system is likely to play an important role in controlling the level of HVA in the CNS.

Rat Organic Anion Transporter 3 (rOAT3) Is Responsible for Brain-to-Blood Efflux of Homovanillic Acid at the Abluminal Membrane of Brain Capillary Endothelial Cells

Rat Organic Anion Transporter 3 (rOAT3) Is Responsible for Brain-to-Blood Efflux of Homovanillic Acid at the Abluminal Membrane of Brain Capillary Endothelial Cells

Effects of peripheral and central catechol- O-methyltransferase inhibition on striatal extracellular levels of dopamine: a microdialysis study in freely moving rats

Tolcapone is a mixed (peripheral and central) catechol- O-methyltransferase (COMT) inhibitor, whereas entacapone is a preferential peripheral COMT inhibitor. Both drugs are able to decrease the peripheral conversion of l-DOPA into 3- O-methyl-DOPA and thereby increase plasma and cerebral levels of l-DOPA, the precursor of dopamine (DA). Tolcapone may also impair the extraneuronal catabolism of DA by inhibiting COMT activity in the brain. To evaluate the role played by peripheral and central COMT inhibition, we compared the effects of tolcapone and entacapone on COMT activity in peripheral tissues, and on striatal extracellular levels of l-DOPA and DA in rats. Tolcapone and entacapone, at the dose of 15 mg/kg p.o., were almost equally effective in inhibiting COMT activity in duodenum and liver. Tolcapone decreased striatal extracellular levels of homovanillic acid (HVA), thus confirming its central COMT inhibitory effect, whereas entacapone did not alter HVA efflux. Following l-DOPA/benserazide administration (50/15 mg/kg p.o.), both COMT inhibitors significantly increased striatal levels of l-DOPA and DA compared with saline. The levels of l-DOPA were similar after treatment with either COMT inhibitors, whereas the increase in DA output was significantly greater in rats given tolcapone compared to those given entacapone. We conclude that the blockade of central DA catabolism by tolcapone contributes to the greater increase in striatal DA levels achieved with this drug.

Involvement of intrinsic cholinergic and GABAergic innervation in the effect of NMDA on striatal dopamine efflux and metabolism as assessed by microdialysis studies in freely moving rats.

Microdialysis perfusion was used to study the participation of striatal cholinergic and gamma-aminobutyric acid-ergic (GABAergic) neurotransmission in basal and N-methyl-D-aspartate (NMDA) receptor-modulated dopamine release and metabolism in the striatum of the freely moving rat. Reverse dialysis of atropine (1-50 microM) induced a concentration-related increase in dopamine efflux and decrease in 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) efflux. (+)-Bicuculline (10-100 microM) similarly increased dopamine efflux, but was without consistent effect on metabolite efflux. Reverse dialysis of NMDA (1 mM) evoked an approximately twofold increase in dopamine efflux and decreased DOPAC and HVA efflux to 30-40% of basal levels. The effect of NMDA on dopamine efflux was completely abolished by coadministration of tetrodotoxin (TTX; 1 microM) or atropine (10 microM), and markedly potentiated (approximately fourfold) by coadministration of (+)-bicuculline (50 microM). The NMDA-induced decrease in dopamine metabolite efflux was inhibited by coadministration of TTX or (+)-bicuculline, but was unaffected by atropine. Our data suggest that dopamine release in the striatum is subject to both cholinergic and GABAergic tonic inhibitory mechanisms mediated through muscarinic and GABAA receptors, respectively. Furthermore, NMDA-stimulated dopamine release also involves obligatory cholinergic facilitation and an inhibitory GABAergic component mediated through these respective receptors.

L-arginine produces NO-independent increases in dopamine efflux in rat striatum.

The effect of L-arginine (L-ARG; 10-100 mM) on dopamine efflux from rat striatum was investigated using in vivo microdialysis. L-ARG (50 mM-100 mM), but not D-arginine (100 mM) nor L-citrulline (100 mM), produced a biphasic effect on dopamine efflux with an initial small reduction, followed by a large sustained increase. The effect of L-ARG was not prevented by nitric oxide synthase inhibition with NG-nitro-L-arginine methyl ester or 7-nitroindazole monosodium salt. Efflux of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was reduced by L-ARG (10-100 mM), D-arginine (100 mM) and L-citrulline (100 mM). These data suggest that changes in dopamine, DOPAC and HVA efflux produced by high concentrations of L-ARG occur independently of NO, and that the use of high L-ARG concentrations are inappropriate when investigating the role of NO in striatum.

Effects of COMT inhibitors on striatal dopamine metabolism: a microdialysis study

In vivo microdialysis was used to examine the effect of two new catechol- O-methyltransferase (COMT) inhibitors, Ro 40-7592 and OR-611, on extracellular levels of dopamine, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in rat striatum. The interactions of the COMT inhibitors with nomifensine, clorgyline and deprenyl were also studied. Ro 40-7592 (3–30 mg/kg. i.p.) decreased dose-dependently the efflux of HVA, increased that of DOPAC, and tended to increase that of dopamine. Higher doses of OR-611 (30–100mg/kg, i.p.) also decreased the dialysate level of HVA, increased that of DOPAC, and tended to increase that of dopamine. Ro 40-7592 was about ten-fold as potent as OR-611. Neither of the COMT inhibitors changed dialysate levels of 5-HIAA. An OR-611 dose of 10 mg/kg i.p. had no significant effect, in contrast to Ro 40-7592, on any of the parameters studied; this dose was thus used to differentiate between the effects of central and peripheral COMT inhibition. Both nomifensine (15 mg/kg. i.p.) and clorgyline (4/mg/kg, i.p.) alone elevated extracellular dopamine levels, and lowered those of DOPAC and HVA, though there were quantitative and temporal differences between the drugs. l-Deprenyl (1 mg/kg, i.p.) alone had no significant effect on any of the compounds measured. Ro 40-7592 (10 mg/kg, i.p.) potentiated the effect of nomifensine on dopamine efflux, and it tended to increase clorgy line-induced dopamine efflux, DOPAC levels in dialysates were significantly increased by combinations of Ro 40-7592 and nomifensine or clorgyline, whereas HVA levels remained about as low as they were after Ro 40-7592 had no significant interactions with l-deprenyl. OR-611 (10 mg/kg, i.p.) did not modify the effects on dopamine metabolism of nomifensine, clorgyline or l-deprenyl. These data show that Ro 40-7592 is a potent centrally active COMT inhibitor, whereas OR-611 is principally a peripherally active inhibitor. Use of drugs which inhibit brain COMT can considerable modify dopamine metabolism. COMT inhibitors may be of clinical significance in treating Parkinson's disease.

Regional effects of ceruletide, a cholecystokinin-8 analog, on the striatal monoaminergic systems in food-deprived mice

To clarify the pharmacological mechanism by which ceruletide affects involuntary movements, we used 20-h food-deprived mice to examine the acute effects of ceruletide (600 μg/kg, i.p.) on the histochemistry of striatal dopaminergic and serotonergic systems. The latter was unchanged but a reduction in catecholamine fluorescence was seen which was restricted to the ventrolateral (VL) portion of the striatum. Biochemical assays also indicated decreased levels of dopamine (DA) and its metabolites in this restricted region of the striatum with little or no change in noradrenaline, serotonin or their metabolites. In all regions examined, except the dorsomedial part of the mid-striatum, the ration of dopamine metabolites to dopamine was higher in the ceruletide-treated group than in controls, suggesting increased DA release. Further pharmacological experiments showed that, compared to results in mice receiving only ceruletide: the ceruletide-induced decreases in DA and dihydroxyphenylacetic acid (DOPAC) in VL were less after α-methyl- p-tyrosine treatment; ceruletide caused no significant decrease in either DA or DOPAC after pargyline pretreatment, although the low levels of homovanillic acid (HVA) were still further significantly reduced; and the ceruletide-induced decrease of DA was reduced, that of DOPAC was abolished and that of HVA enhanced by nomifensine pretreatment. These results suggested that ceruletide might induce a more rapid degradation of DA in VL and increased efflux of HVA through the blood-brain barrier. This evidence suggests that ceruletide has a regionally specific effect on the striatal dopaminergic system which may relate to the amelioration of involuntary movements.

Amphetamine‐Induced Dopamine Release in the Rat Striatum: An In Vivo Microdialysis Study

The effects of a number of biochemical and pharmacological manipulations on amphetamine (AMPH)-induced alterations in dopamine (DA) release and metabolism were examined in the rat striatum using the in vivo brain microdialysis method. Basal striatal dialysate concentrations were: DA, 7 nM; dihydroxyphenylacetic acid (DOPAC), 850 nM; homovanillic acid (HVA), 500 nM; 5-hydroxyindoleacetic acid (5-HIAA), 300 nM; and 3-methoxytyramine (3-MT), 3 nM. Intraperitoneal injection of AMPH (4 mg/kg) induced a substantial increase in DA efflux, which attained its maximum response 20-40 min after drug injection. On the other hand, DOPAC and HVA efflux declined following AMPH. The DA response, but not those of DOPAC and HVA, was dose dependent within the range of AMPH tested (2-16 mg/kg). High doses of AMPH (greater than 8 mg/kg) also decreased 5-HIAA and increased 3-MT efflux. Depletion of vesicular stores of DA using reserpine did not affect significantly AMPH-induced dopamine efflux. In contrast, prior inhibition of catecholamine synthesis, using alpha-methyl-p-tyrosine, proved to be an effective inhibitor of AMPH-evoked DA release (less than 35% of control). Moreover, the DA releasing action of AMPH was facilitated in pargyline-pretreated animals (220% of control). These data suggest that AMPH releases preferentially a newly synthesised pool of DA. Nomifensine, a DA uptake inhibitor, was an effective inhibitor of AMPH-induced DA efflux (18% of control). On the other hand, this action of AMPH was facilitated by veratrine and ouabain (200-210% of control). These results suggest that the membrane DA carrier may be involved in the actions of AMPH on DA efflux.

Acute effects of nomifensine on in vivo uptake and metabolism of dopamine, noradrenaline and serotonin in the rat brain.

Nomifensine, in contrast to all other antidepressants, inhibits the neuronal uptake of dopamine. The effect of this drug (10 mg/kg intraperitoneally) on the metabolism of dopamine, serotonin and noradrenaline was studied in the rat brain. After 1.5 hours, nomifensine increased the concentrations of homovanillic acid (HVA) in corpus striatum and total (free and conjugated) 3-methoxy, 4-hydroxyphenylglycol (MOPEG) in mesencephalon, but had no effect on the 5-hydroxyindoleacetic acid (5-HIAA) in pons/medulla oblongata and mesencephalon. The effect was identical with that of desipramine (25 mg/kg) on MOPEG and of imipramine (25 mg/kg) on 5-HIAA. Two methods ordinarily used for estimating turnover rates of monoamines were compared: accumulation of acid metabolites after probenecid (measuring efflux of metabolites from the brain) and accumulation of monoamine precursors after decarboxylase inhibition (measuring amine synthesis). The efflux was reduced for 5-HIAA and MOPEG but increased for HVA after nomifensine. Imipramine had the same effect on 5-HIAA and desipramine on MOPEG. Desipramine decreased the efflux of HVA from corpus striatum. In contrast, nomifensine did not change the synthesis of noradrenaline and serotonin significantly. Imipramine reduced the synthesis of serotonin in pons/medulla oblongata. In corpus striatum nomifensine, unlike imipramine, increased the concentration of 5-HIAA and synthesis of serotonin in spite of a decrease in efflux, probably because of a secondary effect from the dopaminergic action. The conclusion was made that there were more than one compartment of monoamine metabolites. The antidepressants could to some extent lead to a shift in the metabolism to sites more distant to the transport mechanism.

Aspects of influx and efflux of homovanillic acid of rat cerebrospinal fluid

In rats, different methods of perfusion (lumbar-cisternal or ventricular-cisternal) with artificial cerebrospinal fluid were performed at velocities of about 30 and 180 μl/min. Levels of homovanillic acid (HVA) in the outflow were assayed with a semi-automated fluorimetric technique. Intravenous administration of 20 μg HVA did not substantially enhance the outflow of this acid, indicating that in our preparations HVA found in the perfusate is of central origin. In the lumbar-cisternal preparation probenecid (200 mg/kg, i.p.) was found to inhibit the efflux of a significant fraction of HVA added to the medium, at both perfusion rates. The proportion of HVA eliminated by a probenecid sensitive transport was much higher at the lower rate of perfusion. Following probenecid the increase of endogenous HVA in the ventricular-cisternal perfusate was higher at a lower rate of perfusion. We determined the turnover rate of HVA in the whole brain and compared this value with the HVA outflow in the various preparations. The highest efflux of HVA was found in the ventricular-cisternal preparation during probenecid treatment and did not appear to be dependent upon the rate of perfusion. A maximal value of 3.5% of HVA formed in the central nervous system was found to be released into the cerebrospinal fluid.

Carrier-mediated blood-brain barrier transport of short-chain monocarboxylic organic acids

Carrier-mediated blood-brain barrier transport of short-chain monocarboxylic organic acids

Amyotrophic lateral sclerosis. A study of central monoamine metabolism and therapeutic trial of levodopa.

Cerebrospinal fluid (CSF) levels of homovanillic acid (HVA), a major catabolite of dopamine, were substantially less in 21 patients with amyotrophic lateral sclerosis (ALS) than in 19 controls. There was also a significant reduction in the probenecid-induced accumulation of HVA in the CSF of ALS patients as compared with control subjects. Since probenecid inhibits the efflux of HVA from the central nervous system, the results suggest that central dopamine synthesis may be diminished in ALS. A therapeutic trial of levodopa, the immediate precursor of dopamine, in ten ALS patients failed to improve motor function. Defects in central dopamine metabolism, inferred from these tests applicable to the living patient, evidently may attend neurologic disorders which are unresponsive to precursor loading with levodopa.

Parkinsonism-dementia and amyotrophic lateral sclerosis of Guam. Effect of probenecid on monoamine catabolite levels in cerebrospinal fluid.

Changes in cerebrospinal fluid (CSF) levels of homovanillic acid (HVA), a major catabolite of dopamine and of 5-hydroxyindoleacetic acid (5-HIAA), the principle product of serotonin degradation, were compared in Guamanian parkinsonism-dementia (PD) and amyotrophic lateral sclerosis (ALS) patients with Guamanian and North American control subjects during the administration of probenecid. The rise of both HVA and 5-HIAA was significantly less in PD patients than in control subjects. Values for both these monoamine catabolites in patients with ALS consistently fell between those of PD patients and controls. Since probenecid inhibits the efflux of HVA and 5-HIAA from CSF, these results suggest that a substantial reduction in the synthesis of both dopamine and serotonin may attend PD and ALS of Guam.