Potencies of haloperidol metabolites as inhibitors of the human noradrenaline, dopamine and serotonin transporters in transfected COS-7 cells

Extrapyramidal symptoms, such as tardive dyskinesia, often develop in patients on long-term treatment with haloperidol. It has been proposed that these symptoms could be caused by neurotoxic effects of haloperidol metabolites following uptake by monoamine transporters, in an analogous mechanism to the neurotoxic effect of MPP+ (1-methyl-4-phenylpyridinium) metabolised from MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). In this study, the hypothesis was partially investigated by determining the potencies of haloperidol and reduced haloperidol and the corresponding pyridinium and tetrahydropyridine metabolites, compared with MPP+ and MPTP, as inhibitors of the noradrenaline transporter (NAT), dopamine transporter (DAT) and 5-HT transporter (SERT). Two days after COS-7 cells were transiently transfected with the cDNA for the human NAT, DAT or SERT (Lipofectamine method), the cells were incubated with 10 nM [3H]noradrenaline, dopamine or 5-HT, respectively, for 2 min at 37 C, in the absence or presence of various concentrations of the eight compounds or a specific uptake inhibitor (NAT: nisoxetine 1 microM; DAT: GBR 12909 1 microM; SERT: citalopram 10 microM). Specific amine uptake (fmol/ mg protein) was calculated as the difference in uptake in the absence and presence of the specific uptake inhibitor. Ki values were calculated for the eight compounds for inhibition of NAT, DAT and SERT. Haloperidol, its five metabolites and MPP+ and MPTP all inhibited NAT, DAT and SERT. For the pyridinium and tetrahydropyridine metabolites of haloperidol, there were not marked differences between their potencies as inhibitors between each other for NAT or DAT or between NAT and DAT, with all of the Ki values in the range of 5.8-16 microM. However, there were more marked differences for SERT, with all but one of the metabolites showing selectivity for inhibition of SERT relative to NAT and DAT. Haloperidol and reduced haloperidol had similar inhibitory potencies for all three transporters, and were clearly less potent than the other haloperidol metabolites only for inhibition of SERT. The lack of correlation between the inhibitory potencies of the haloperidol metabolites and their structural analogues, MPTP and MPP+, suggests that they are not likely to cause neurotoxicity by a mechanism analogous to that of the latter neurotoxin.     

Expression of human organic anion transporters in the choroid plexus and their interactions with neurotransmitter metabolites

The purpose of the present study was to elucidate the expression of human organic anion transporter 1 (hOAT1) and hOAT3 in the choroid plexus of the human brain and their interactions with neurotransmitter metabolites using stable cell lines. Immunohistochemical analysis revealed that hOAT1 and hOAT3 are expressed in the cytoplasmic membrane and cytoplasm of human choroid plexus. Neurotransmitter metabolites, namely, 5-methoxyindole-3-acetic acid (5-MI-3-AA), homovanillic acid (HVA), vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HI-3-AA), N-acetyl-5-hydroxytryptamine (NA-5-HTT), melatonin, 5-methoxytryptamine (5-MTT), 3,4-dihidroxymandelic acid (DHMA), 5-hydroxytryptophol, and 5-methoxytryptophol (5-MTP), but not methanephrine (MN), normethanephrine (NMN), and 3-methyltyramine (3-MT), at 2 mM, inhibited para-aminohippuric acid uptake mediated by hOAT1. On the other hand, melatonin, 5-MI-3-AA, NA-5-HTT, 5-MTT, 5-MTP, HVA, 5-HI-3-AA, VMA, DOPAC, 5-hydroxytryptophol, and MN, but not 3-MT, DHMA, and NMN, at 2 mM, inhibited estrone sulfate uptake mediated by hOAT3. Differences in the IC(50) values between hOAT1 and hOAT3 were observed for DHMA, DOPAC, HVA, 5-HI-3-AA, melatonin, 5-MI-3-AA, 5-MTP, 5-MTT, and VMA. HOAT1 and hOAT3 mediated the transport of VMA but not HVA and melatonin. These results suggest that hOAT1 and hOAT3 are involved in the efflux of various neurotransmitter metabolites from the cerebrospinal fluid to the blood across the choroid plexus.     

Effects of haloperidol on neurotransmitter activity in the rat vas deferens

1. The effects of haloperidol on the responses of the isolated rat vasa deferentia to catecholamines and ACh were studied. 2. Haloperidol produced a competitive antagonism to responses elicited by NA and DA in the vas deferens. 3. The M1 and M2 muscarinic responses to ACh of the vas deferens were potentiated by this neuroleptic. 4. The AChE activity of the vas deferens was significantly depressed by pretreatment with haloperidol. 5. The ability of haloperidol to lower AChE activity was compared with that of neostigmine and it may be due to a similar molecular mechanism. 6. The present results suggest that haloperidol has anti-AChE properties that may be responsible for the potentiation of the responses to ACh. 7. The study indicates that haloperidol has a wider range of pharmacological actions than previously reported.     

The functional evaluation of human peptide/histidine transporter 1 (hPHT1) in transiently transfected COS-7 cells

Recently, the expression of the human peptide/histidine transporter (hPHT1, SLC15A4) mRNA was observed in the GI tract and in Caco-2 cells, suggesting that it may participate in the intestinal absorption of peptide-based agents. This study aims to elucidate the: (i) protein expression pattern of hPHT1 (SLC15A4) in human small intestine; (ii) cloning of the hPHT1 full-length sequence; (iii) functional characterization of hPHT1 in transiently transfected COS-7 cells. The expression of hPHT1 was measured using Western blot and immunohistochemical analysis. The hPHT1 full-sequence was amplified from BeWo cells, inserted into the pcDNA3.1–V5/His TOPO^® plasmid and transiently transfected into COS-7 cells to investigate the uptake kinetics of [³H]histidine and [³H]carnosine. Time, pH and sodium-dependent uptake studies were performed in mock (empty vector) and hPHT1–COS-7 cells. Results demonstrated hPHT1 protein expression in different intestinal regions. Histidine and carnosine uptake was linear in hPHT1–COS-7 cells over 15 min and was found to be pH-dependent. These substrates and valacyclovir showed significantly higher uptake at pH 5.0 in the hPHT1 transients when contrasted to the mock COS-7 cells, whereas glycylsarcosine uptake was significantly lower and unaffected by pH. Other di- and tripeptides also showed affinity for hPHT1. This study presents the initial functional characterization, the protein expression of the hPHT1 transporter and provides insight into a potentially different route for increasing peptide and peptide-based drug transport.     

Behavioral and pharmacokinetic interaction between morphine and haloperidol in the rat

Tail-flick latencies and morphine concentrations in the blood serum, brain (without striatum) and spinal cord were measured in rats receiving 10 mg/kg ip morphine with or without haloperidol pretreatment (0.1 or 1 mg/kg sc). Haloperidol pretreatment dose-dependently potentiated the analgesic action of morphine and interfered with tissue morphine levels. Morphine levels in the spinal cord were similar to those in the blood serum and were dose-dependently increased by haloperidol pretreatment; in the brain the appearance of morphine was delayed, the levels lower than in the blood serum, and the effect of both doses of haloperidol (augmentation of morphine level) was similar. The results indicate that the analgesic effect of morphine in the tail-flick test is correlated better with the spinal than cerebral morphine levels and that potentiation of morphine analgesia by haloperidol is due, at least in part, to pharmacokinetic interaction.     

Effects of the dithiocarbamate fungicide propineb in primary neuronal cell cultures and skeletal muscle cells of the rat

After repeated-dose toxicity studies with the fungicide propineb, reversible effects on muscle functions were found. Therefore, mechanistic investigations should contribute to clarification of its mode of action in relation to disulfiram and diethyldithiocarbamate neurotoxicity or direct effects on muscle cells. In principle, besides the dithiocarbamate effects, two different mechanisms have been discussed for this fungicide. One mechanism is the degradation to carbon disulfide (CS(2)) and propylenthiourea (PTU) and the other are direct effects of zinc. Primary neuronal cell cultures of the rat are a well established model to identify neurotoxic compounds like n-hexane or acrylamide. In this cell culture model, endpoints such as viability, energy supply, glucose consumption and cytoskeleton elements were determined. Additionally, skeletal muscle cells were used for comparison. Propineb and its metabolite PTU were investigated in comparison to CS(2), disulfiram and diethyldithiocarbamate. The toxicity of zinc was tested using zinc chloride (ZnCl(2)). It was clearly shown that propineb exerted strong effects on the cytoskeleton of neuronal and non-neuronal cell cultures (astrocytes, muscle cells). This was similar to ZnCl(2,) but not to CS(2). With CS(2) and disulfiram effects on the energy supply were more prominent. In conclusion, the toxicity of propineb is not comparable to disulfiram, diethyldithiocarbamate or CS(2) neurotoxicity. In regard to these findings, a direct reversible effect of propineb on skeletal muscle cells seems to be more likely.     

Further structural analysis of urinary metabolites of suprofen in the rat

The urinary metabolites of 2-(4-(2-thienylcarbonyl)phenyl)propionic acid (suprofen, S) in rats were analyzed by radio-GC, GC/MS, or 1H NMR technique. Radio-GC analysis of trimethylsilylated materials after TLC separation of intact urine showed the presence of three radioactive peaks with the retention times corresponding to the authentic S, 2-(4-(2-thienylhydroxymethyl)phenyl)propionic acid, and 2-(4-carboxyphenyl)propionic acid. About 40% of the total radioactivity appearing in the 0-24-hr urine was accounted for by the three metabolites and their conjugates. The identification of these metabolites was confirmed by comparison of the MS spectra of urine, in which rats were administered an equimolar mixture of S and S[phenyl-d4], with those of synthetic standards. The labile metabolites of S, corresponding to about 32% of the total radioactivity appearing in the 0-24-hr urine, were isolated and purified by ether extraction from the fresh urine and GC/MS or HPLC. GC/MS of the methylated metabolite revealed the consistent presence of the ion peaks at m/z 304, 245, 217, and 141, indicative of a dimethylated product with monohydroxy group on the thiophene ring. Analysis of the 1H NMR spectrum demonstrated the metabolite to be 2-(4-(5-hydroxy-2-thienylcarbonyl)phenyl)propionic acid.     

Efflux studies allow further characterisation of the noradrenaline and 5hydroxytryptamine transporters in rat lungs

The aim of the present study was to further characterise the noradrenaline and 5hydroxytryptamine [5HT] transporters in rat lungs by examining the efflux of noradrenaline and 5HT, respectively. Lungs from rats were isolated and perfused via the pulmonary artery. After loading the tissue with ³H5HT or ³Hnoradrenaline the efflux of the relevant amine from the lungs was examined for 1525min. The rate constant for efflux of ³H5HT increased by 81% when Na⁺ ions were removed from the perfusion solution; increased gradually when a selective 5HT transporter inhibitor, 200nM citalopram, was added to the perfusion solution for the final 6min of efflux; and increased markedly and rapidly when substrates of the 5HT transporter, tryptamine (18μM) and 7methyltryptamine (12μM), were added for the final 6min of efflux. These effects of the substrates were abolished by 1μM citalopram, but were not significantly affected by 1μM desipramine, a selective uptake₁ inhibitor. On the other hand, the previously described substrateinduced increase in the rate of efflux of noradrenaline was significantly reduced by desipramine but was unaffected by citalopram. The results show that efflux of 5HT is mediated only by the 5HT transporter, with no significant contribution of uptake₁, and efflux of noradrenaline from rat lungs is mediated only by uptake₁ and not by the 5HT transporter. The effects of dopamine on the efflux of noradrenaline over a concentration range of 100-600nM were investigated and the results showed that 50% of the maximal increase in the rate of efflux occurred at a concentration of 275nM. This value did not differ from the K_m for uptake of dopamine. This result implies that the only factor affecting the substrate-induced increase in noradrenaline efflux is the affinity of the substrate for uptake₁. The efflux of noradrenaline was also examined in the absence and presence of two concentrations of desipramine (0.35and 1.5μM). Analysis of these results showed that uptake₁ contributed approximately 81% and diffusion 19% to the total efflux of noradrenaline and that 90% of the total noradrenaline efflux was subject to reuptake by uptake₁ into the pulmonary endothelial cells. Received: 23 January 1997 / Accepted: 26 March 1997     

Determination of rat urinary metabolites of icariin in vivo and estrogenic activities of its metabolites on MCF-7 cells

To confirm that the estrogenic activity of icariin is based on the close relationship between the structures of its metabolites and the effects of their binding to target hormone receptors, the metabolism of icariin in rat urine was analyzed in vivo, and the estrogenic activity of its metabolites was measured in cultured MCF-7 human breast cancer cells, respectively. By CZE analysis, peaks corresponding to the relative positions of desmethylicaritin and icaritin were observed in the urine sample. Structural analysis following LC-ESI-MS revealed molecular ions [M-H]- of 512.8, 353.3, and 367.0 for metabolites consistent with those of icariside II, desmethylicaritin, and icaritin. Icariin, icaritin, and desmethylicaritin were analyzed for their estrogenicity using MCF7-cell proliferation (E-screen test). MCF-7 cells were cultured in an estradiol free medium and then exposed to 10(-8) to 10(-5) mol/L icariin and its metabolites, icaritin and desmethylicaritin, for 6 days. Icaritin and desmethylicaritin significantly increased cell proliferation, and the cell number increased from 1.61 to 4.14 fold compared with the untreated control, but the parent compound icariin failed to exhibit this effect. These results indicate that icariin is converted to icariside II, desmethylicaritin, and icaritin in vivo, and that the latter two act as a weak xenoestrogen on MCF-7 cells.     

Studies on the interaction of five triazole fungicides with human renal transporters in cells

The widespread use of triazole fungicides in agricultural production poses a potential risk to human health. This study investigates the interaction of five triazole fungicides, i.e., tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole with human renal transporters, including OAT1, OAT3, OCT2, OCTN1, OCTN2, MATE1, MATE2-K, MRP2, MDR1, and BCRP, using transgenic cell models. For uptake transporters, triticonazole was the substrate of OAT1 and OAT3 and the inhibitor of OCT2. Tebuconazole and penconazole inhibited OCTN2 (100 μM), while tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole inhibited MATE1 (100 μM). Tebuconazole and hexaconazole inhibited MATE2-K (100 μM). All five triazole fungicides were not substrates or strong inhibitors of MRP2, MDR1, and BCRP efflux transporters. Penconazole inhibited OCT2 with IC₅₀ = 1.12 μM. Penconazole and uniconazole inhibited MATE1 with IC₅₀ = 0.94 μM and 0.87 μM. Tebuconazole and hexaconazole inhibited MATE2-K with IC₅₀ = 0.96 μM and 1.04 μM, indicating that triazole fungicides may inhibit renal drug transporter activity at low concentrations. Triticonazole was transported by OAT1 and OAT3, and the K_m values of triticonazole were 5.81 ± 1.75 and 47.35 ± 14.27, respectively. Tebuconazole and uniconazole were transported by OAT3, and the K_m values of tebuconazole and uniconazole were 30.28 ± 7.18 and 87.61 ± 31.70, respectively, which may induce nephrotoxicity.     

Regulated expression and function of the somatodendritic catecholamine neurotransmitter transporters

Termination of neurotransmission at catecholaminergic synapses is well documented by the transporters for dopamine and norepinephrine, members of the Na(+)/Cl(-)-dependent neurotransmitter transporter family, which accumulates released transmitters within their nerve endings, respectively. Although somatodendritic expression of the transporters and the effects of cocaine and amphetamine on those have been reported, their role is still obscure. Recent findings of the transporter function as an ion channel and/or its reverse transport property provide a clue to identify the role of these transporters in the somatodendrites and their consequential interaction with uptake inhibitors. Differences in ionic environment and maturity of the release machinery in the somatodendrites at developmental stages influence the transporter functions, resulting in the formation of both positive and negative feedback loop of catecholaminergic neurons.     

Dopamine and serotonin metabolites in rat cerebroventricular fluid following withdrawal of haloperidol or electroshock treatment

Levels of the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and of the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) were determined in the CSF of rats at various times after repeated electroshock treatment (EST) or chronic administration of haloperidol. The acidic metabolites were analyzed in 25 l CSF using HPLC with an electrochemical detector. A significant decrease in the CSF levels of DOPAC and HVA was found 4 days after the last administration of chronic haloperidol, EST, or both. The decrease in the level of the dopamine metabolites indicated a slower dopamine turnover, which might have resulted from hypersensitivity of presynaptic dopamine receptors after these treatments. Rats treated with haloperidol also showed an increase in 5-HIAA levels, possibly due to enhanced serotonin turnover. The 5-HIAA increase following haloperidol was prevented by a concurrent administration of EST, suggesting attenuation by EST of the haloperidol-induced enhancement of serotonin turnover.     

Clozapine, but not haloperidol, increases neuropeptide Y neuronal expression in the rat hypothalamus

Many atypical antipsychotic drugs, such as clozapine, can induce significant weight gain which can have serious implications for drug compliance and morbidity. Food intake and weight gain are regulated primarily by the hypothalamus; the arcuate nucleus (ARC) of the hypothalamus is the region initially mediating the effects of circulating hormones on food intake. Neuropeptide Y (NPY) is an important hypothalamic peptide involved in body weight regulation. Immunohistochemical staining of NPY in the ARC was carried out in male Sprague-Dawley rats treated with haloperidol (1.5 mg/kg, i.p.) or clozapine (25 mg/kg, i.p.) for 3 weeks. Clozapine, but not haloperidol, produced an increase in NPY immunoreactivity in the ARC, suggesting that effects on NPY may be involved in increases in body weight following clozapine treatment.     

Interaction of nucleoside analogues with nucleoside transporters in rat brain endothelial cells

A number of nucleoside analogues, consisting of antiviral compounds and agents designed as adenosine A1 receptor agonists, were examined for nucleoside transporter affinity using an in vitro model of the blood-brain barrier (BBB), the rat brain endothelial cell line, RBE4. Structure-activity relationships (SAR) were also performed to identify the key structural requirements for transporter recognition and the suitability of these systems for carrier-mediated strategies to deliver therapeutics across the BBB. Adenosine receptor agonists did not show transport affinity for concentrative nucleoside carriers, but exhibited affinity for equilibrative systems (Ki=10.8-97.9 microM) within the range of Kms for natural substrates. However, none of the antiviral compounds tested in this study showed affinity for either class of nucleoside transporter. SAR studies suggest that the hydroxyl group located at the 3'-position of the ribose moiety is an essential requirement for transporter recognition. This may explain the inability of nucleoside derived anti-viral compounds to use these systems despite the significant structural homology with naturally occurring nucleosides. Sites have also been identified which accommodate structural additions with retention of carrier affinity, suggesting that compounds which fail to penetrate the BBB could be attached to these sites for carrier-mediated delivery using a prodrug strategy.     

Dissociation of nitric oxide generation and kainate-mediated neuronal degeneration in primary cultures of rat cerebellar granule cells.

In the presence of physiological concentrations of Mg2+ and in glycine-free buffer, the relationship between KA-mediated generation of NO and neurotoxicity in cultures of cerebellar granule cells of the rat was examined. The neuronal damage elicited by KA was not dependent on the presence of L-arginine, a precursor of NO, since neither the potency nor magnitude of KA-mediated cell death was altered in either the absence or presence of exogenously applied L-arginine. Similarly, with the exception of 4-hydroxy-azobenzene-4'-sulfonic acid, disodium salt dihydrate (HBS), the salt associated with NG-monomethyl-L-arginine (di-(p-hydroxyazobenzene-p'-sulfonate) (MA(HBS)), treatment with several different competitive NO synthetase inhibitors did not provide protection against the toxicity of KA. However, the ability of KA to induce neuronal damage was significantly decreased in cerebellar granule cells treated with either HBS or alpha-tocopherol (VE). On the basis of these results, it is concluded that the generation of free radicals may be involved in the process of KA-elicited neuronal death in cultures of cerebellar granule cells but that this is unrelated to the synthesis of NO. This conclusion agrees with both in vivo and in vitro studies, implicating the involvement of free radicals in non-NMDA mediated neuronal damage.