cDNA array reveals increased expression of glucose-dependent insulinotropic polypeptide following chronic clozapine treatment: role in atypical antipsychotic drug-induced adverse metabolic effects.

Clozapine is an atypical antipsychotic drug with unique pharmacological and therapeutic properties. Unlike the typical antipsychotic drug, haloperidol, clozapine does not cause extrapyramidal side effects; however, weight gain, dyslipidemia, and type II diabetes are commonly associated with the use of this drug in subjects with schizophrenia. The aim of this study was to profile gene expression in the rat striatum following clozapine treatment. Chronic treatment with clozapine revealed upregulation of several genes including the glucose-dependent insulinotropic polypeptide (GIP) gene by over 200% in the rat striatum. The cDNA array results for the GIP gene were confirmed by real-time RT-PCR as well as by radioimmunoassay. Expression of the GIP gene in the central nervous system is consistent with the results of retinal GIP gene expression as reported by other investigators. Taken together, these findings implicate the possible role of GIP as a neuromodulator in the central nervous system. GIP is an insulinotropic agent with stimulatory effects on insulin synthesis and release from the pancreas. However, changes in brain GIP levels are most likely unrelated to the metabolic adverse effects (dyslipidemia, type II diabetes, weight gain) associated with clozapine treatment. Therefore, we also measured GIP gene expression in the K-cell-rich regions, duodenum and jejunum (small intestine), and plasma GIP levels using radioimmunoassay following chronic treatment with clozapine. GIP mRNA levels in the small intestine and the plasma GIP at the protein level were significantly elevated in clozapine-treated subjects. Furthermore, as observed in humans, chronic clozapine treatment also caused weight gain, and increased levels of insulin, triglycerides and leptin in the plasma. These results suggest that adverse metabolic effects associated with clozapine treatment may be related to its ability to increase intestinal gene expression for GIP.

Direct binding and functional coupling of alpha-synuclein to the dopamine transporters accelerate dopamine-induced apoptosis.

Mutations in alpha-synuclein, a protein highly enriched in presynaptic terminals, have been implicated in the expression of familial forms of Parkinson's disease (PD) whereas native alpha-synuclein is a major component of intraneuronal inclusion bodies characteristic of PD and other neurodegenerative disorders. Although overexpression of human alpha-synuclein induces dopaminergic nerve terminal degeneration, the molecular mechanism by which alpha-synuclein contributes to the degeneration of these pathways remains enigmatic. We report here that alpha-synuclein complexes with the presynaptic human dopamine transporter (hDAT) in both neurons and cotransfected cells through the direct binding of the non-A beta amyloid component of alpha-synuclein to the carboxyl-terminal tail of the hDAT. alpha-Synuclein--hDAT complex formation facilitates the membrane clustering of the DAT, thereby accelerating cellular dopamine uptake and dopamine-induced cellular apoptosis. Since the selective vulnerability of dopaminergic neurons in PD has been ascribed in part to oxidative stress as a result of the cellular overaccumulation of dopamine or dopamine-like molecules by the presynaptic DAT, these data provide mechanistic insight into the mode by which the activity of these two proteins may give rise to this process.

Direct protein-protein coupling enables cross-talk between dopamine D5 and gamma-aminobutyric acid A receptors.

GABA(A) (gamma-aminobutyric-acid A) and dopamine D1 and D5 receptors represent two structurally and functionally divergent families of neurotransmitter receptors. The former comprises a class of multi-subunit ligand-gated channels mediating fast interneuronal synaptic transmission, whereas the latter belongs to the seven-transmembrane-domain single-polypeptide receptor superfamily that exerts its biological effects, including the modulation of GABA(A) receptor function, through the activation of second-messenger signalling cascades by G proteins. Here we show that GABA(A)-ligand-gated channels complex selectively with D5 receptors through the direct binding of the D5 carboxy-terminal domain with the second intracellular loop of the GABA(A) gamma2(short) receptor subunit. This physical association enables mutually inhibitory functional interactions between these receptor systems. The data highlight a previously unknown signal transduction mechanism whereby subtype-selective G-protein-coupled receptors dynamically regulate synaptic strength independently of classically defined second-messenger systems, and provide a heuristic framework in which to view these receptor systems in the maintenance of psychomotor disease states.

A cognate dopamine transporter-like activity endogenously expressed in a COS-7 kidney-derived cell line.

The activity of the dopamine transporter is an important mechanism for the maintenance of normal dopaminergic homeostasis by rapidly removing dopamine from the synaptic cleft. In kidney-derived COS-7, COS-1 and HEK-293 but not in other mammalian cell lines (CHO, Y1, Ltk-), we have characterized a putative functional dopamine transporter displaying a high affinity (Km approximately 250 nM) and a low capacity (approximately 0.1 pmol/10(5) cells/min) for [3H]dopamine uptake. Uptake displayed a pharmacological profile clearly indicative of the neuronal dopamine transporter. Estimated Ki values of numerous substrates and inhibitors for the COS-dopamine transporter and the cloned human neuronal transporter (human dopamine transporter) correlate well with the exception of a few notable compounds, including the endogenous neurotransmitter dopamine, the dopamine transporter inhibitor GBR 12,909 and the dopaminergic agonist apomorphine. As with native neuronal and cloned dopamine transporters, the uptake velocity was sodium-sensitive and reduced by phorbol ester pre-treatment. Two mRNA species of 3.8 and 4.0 kb in COS-7 cells were revealed by Northern blot analysis similar in size to that seen in native neuronal tissue. A reverse-transcribed PCR analysis confirmed the existence of a processed dopamine transporter. However, no immunoreactive proteins of expected dopamine transporter molecular size or [3H]WIN 35,428 binding activity were detected. A partial cDNA of 1.3 kb, isolated from a COS-1 cDNA library and encoding transmembrane domains 1-6, displayed a deduced amino acid sequence homology of approximately 96% to the human dopamine transporter. Taken together, the data suggest the existence of a non-neuronal endogenous high affinity dopamine uptake system sharing strong functional and molecular homology to that of the cloned neuronal dopamine transporter.

Protein kinase-mediated bidirectional trafficking and functional regulation of the human dopamine transporter.

Modification of the transport velocity of both the native neuronal and cloned presynaptic dopamine transporter (DAT) has been reported following activation/inhibition of second messenger system pathways. In order to identify the mechanism by which the functional activity of human DAT (hDAT) is regulated, we assessed the [3H]dopamine uptake kinetics, [3H] CFT binding characteristics, and, via immunofluorescent confocal microscopy, the cellular localization profiles of the hDAT expressed in both Sf9 and COS-7 cells following modulation of protein kinase C (PKC)- and protein kinase A (PKA)-dependent pathways. As with both native neuronal and cloned DATs, acute exposure of hDAT expressing Sf9 cells to the PKC activator PMA (1 microM), but not alphaPDD, reduced the Vmax (approximately 1 pmol/min/10(5) cells) for [3H]DA uptake by approximately 40%, an effect which was blocked by the protein kinase inhibitor staurosporine. Pretreatment of cells with staurosporine (500 nM) alone, however, increased [3H]DA uptake velocity by approximately 30%, an effect mimicked by the potent PKA inhibitor Rp-cAMPS. Activation of PKA-dependent pathways with Sp-cAMPS did not significantly modify DA uptake. Neither the Km of [3H]DA uptake (approximately 200 nM) nor the affinity of various substrates and transport inhibitors was altered by either PMA or staurosporine treatment. Despite changes in functional dopamine uptake velocity by PKC/PKA-dependent mechanisms, the estimated density of hDAT as indexed by whole-cell [3H] CFT binding was unchanged. Immunofluorescent confocal microscopy demonstrated that the observed functional consequence of PKC activation on [3H]DA uptake is associated with the rapid sequestration/internalization of hDAT protein from the cell surface, while the increase in DA uptake following PKC/PKA inhibition is the result of the recruitment of internalized or intracellular transporters to the plasma membrane. Identical rapid translocation patterns were observed in similarly treated COS-7 cells transiently expressing hDAT. These data suggest that the differential regulation of DAT transport capacity by both PKC- and PKA-dependent pathways are not a result of modifications in DAT catalytic activity. Moreover, the rapid shuttling of DATs between the plasma membrane and intracellular compartments provides an efficient means by which native DAT function may be regulated by second messenger systems, possibly following activation of presynaptic dopaminergic receptors, and suggests a role for cytoskeletal components in the dynamic regulation of DAT function.

High affinity recognition of serotonin transporter antagonists defined by species-scanning mutagenesis. An aromatic residue in transmembrane domain I dictates species-selective recognition of citalopram and mazindol.

Human and Drosophila melanogaster serotonin (5-HT) transporters (SERTs) exhibit similar 5-HT transport kinetics and can be distinguished pharmacologically by many, but not all, biogenic amine transporter antagonists. By using human and Drosophila SERT chimeras, major determinants of potencies of two transporter antagonists, mazindol and citalopram, were tracked to the amino-terminal domains encompassing transmembrane domains I and II. Species-scanning mutagenesis, whereby amino acid substitutions are made switching residues from one species to another, was employed on the eight amino acids that differ between human and Drosophila SERTs in this region, and antagonist potencies were reassessed in 5-HT uptake assays. A single mutation in transmembrane domain I of human SERT, Y95F, shifted both citalopram and mazindol to Drosophila SERT-like potencies. Strikingly, these potency changes were in opposite directions suggesting Tyr95 contributes both positive and negative determinants of antagonist potency. To gain insight into how the Y95F mutant might influence mazindol potency, we determined how structural variants of mazindol responded to the mutation. Our studies demonstrate the importance of the hydroxyl group on the heterocyclic nucleus of mazindol for maintaining species-selective recognition of mazindol and suggest that transmembrane domain I participates in the formation of antagonist-binding sites for amine transporters.

Nitrogen-based drugs are not essential for blockade of monoamine transporters.

In brain, monoamine transporters are principal targets of widely used therapeutic drugs including antidepressants, methylphenidate (Ritalin), and the addictive drug cocaine. Without exception, these transport blocking agents contain an amine nitrogen. A prevalent view and untested premise is that an amine nitrogen is needed to bind to the same counterion on the transporter as does the amine nitrogen of the monoamine neurotransmitter. We report that several compounds without nitrogen (8-oxa-bicyclo-3-aryl-[3.2.1] octanes, or aryloxatropanes) are active at monoamine transporters. One of these, tropoxane (0-914), bound with high affinity to the dopamine (IC50: 3.35 +/- 0.39 nM), serotonin (IC50: 6.52 +/- 2.05 nM), and norepinephrine (IC50: 20.0 +/- 0.3 nM) transporters in monkey brain, the human striatal dopamine transporter (IC50: 5.01 +/- 1.74 nM), and blocked dopamine transport (IC50: 7.2 +/- 3.0 nM) in COS-7 cells transfected with the human dopamine transporter. These unique compounds require a revision of current concepts of the drug binding domains on monoamine transporters, open avenues for discovery of a new generation of drugs and raise the issue of whether mammalian transporters and receptors may respond to, as yet, undiscovered non-amine bearing neurotransmitters or drugs.

The dopamine transporter carboxyl-terminal tail. Truncation/substitution mutants selectively confer high affinity dopamine uptake while attenuating recognition of the ligand binding domain.

In order to delineate structural motifs regulating substrate affinity and recognition for the human dopamine transporter (DAT), we assessed [3H]dopamine uptake kinetics and [3H]CFT binding characteristics of COS-7 cells transiently expressing mutant DATs in which the COOH terminus was truncated or substituted. Complete truncation of the carboxyl tail from Ser582 allowed for the expression of biphasic [3H]dopamine uptake kinetics displaying both a low capacity (Vmax approximately 0.4 pmol/10(5) cells/min) high affinity (Km approximately 300 nM) component and one exhibiting low affinity (Km approximately 15 microM] and high capacity (Vmax approximately 5 pmol/10(5)cells/min) with a concomitant 40% decrease in overall apparent Vmax relative to wild type (WT) DAT. Truncation of the last 22 amino acids or substitution of the DAT-COOH tail with sequences encoding the intracellular carboxyl-terminal of either dopamine D1 or D5 receptors produced results that were identical to those with the fully truncated DAT, suggesting that the induction of biphasic dopamine uptake kinetics is likely conferred by removal of DAT-specific sequence motifs distal to Pro597. The attenuation of WT transport activity, either by lowering levels of DAT expression or by pretreatment of cells with phorbol 12-myristate 13-acetate (1 microM), did not affect the kinetics of [3H]dopamine transport. The estimated affinity of dopamine (Ki approximately 180 nM) for all truncated/substituted DAT mutants was 10-fold lower than that of WT DAT (approximately 2000 nM) and appears selective for the endogenous substrate, since the estimated inhibitory constants for numerous putative substrates or uptake inhibitors were virtually identical to those obtained for WT DATs. In marked contrast, DAT truncation/substitution mutants displayed significantly reduced high affinity [3H]CFT binding interactions with estimated Ki values for dopamine and numerous other substrates and inhibitors tested from 10-100-fold lower than that observed for WT DAT. Moreover, co-expression of truncated and/or substituted DATs with WT transporter failed to reconstitute functional or pharmacological activities associated with both transporters. Instead, complete restoration of uniphasic low affinity [3H]dopamine uptake kinetics (Km approximately 2000 nM) and high affinity substrate and inhibitor [3H]CFT binding interactions attributable to WT DATs were evident. These data clearly suggest the functional independence and differential regulation of the dopamine translocation process from the characteristics exhibited by its ligand binding domain. The lack of functional phenotypic expression of mutant DAT activities in cells co-expressing WT transporter is consistent with the contention that native DATs may exist as multisubunit complexes, the formation and maintenance of which is dependent upon sequences encoded within the carboxyl tail.

Molecular and functional characterization of a partial cDNA encoding a novel chicken brain melatonin receptor.

An approach based on homology probing was used to clone a partial cDNA encoding a novel melatonin (ML) receptor (MLR) from chicken (Gallus domesticus) brain. Based on available deduced amino-acid sequence, the chicken MLR (cMLR) displayed greater sequence homology to the frog (Xenopus) MLR than cloned human/mammalian receptors, with overall identities of 73% and 66%, respectively. In order to gain functional expression, a chimeric frog/chicken (flc)MLR was constructed in which the 5' end of the cMLR, including the N-terminus, TM1 and part of the first intracellular loop was substituted by fMLR sequence. [125I]Iodo-ML bound with high affinity (Kd of approximately 35 pM) to COS-7 cells transiently expressing the flcMLR in a saturable and guanine nucleotide-sensitive manner with the following rank order of potency: 2-iodo-ML > ML > 6-Cl-ML > S20750 > 6-OH-ML > S20642 > S20753 > N-acetyl-5HT >> 5-HT. Estimated Ki values for these compounds at the flcMLR correlated well to those obtained in native chicken brain membranes. In line with the observed structural similarity to the fMLR, the flcMLR exhibited affinities for ML, 6-Cl-ML and 6-OH-ML approximately 10-fold lower than mammalian receptors. Functionally, opposing interactions between ML and dopamine receptor signal transduction pathways were observed with ML potently inhibiting dopamine D1A-receptor-mediated cAMP accumulation in cells (HEK-293) transiently co-expressing these receptors. cMLR mRNAs were found expressed in chicken brain and kidney with trace levels observed in the lung. The availability of cloned vertebrate MLRs distinct at both the amino acid and pharmacological level from their mammalian counterparts may now allow for the identification of those amino-acid residues and structural motifs that regulate ML-binding specificity and affinity.

The dopamine transporter: immunochemical characterization and localization in brain.

Antibodies specific for the dopamine transporter (DAT) was developed and characterized by immunoblot analysis, immunoprecipitation, and immunocytochemistry, and used for immunolocalization of transporter protein in rat brain at the light microscopic level. Antibodies targeting the N-terminus, the second extracellular loop, and the C-terminus were generated from fusion proteins containing amino acid sequences from these respective regions. Immunoblot analysis demonstrated that N-terminus and loop antibodies were specific for expressed cloned DAT, recognized transporter protein in rat and human striatal membranes, and were sensitive to preabsorption with excess homologous fusion protein. Immunoprecipitation studies demonstrated that anti-DAT antisera recognized solubilized, radiolabeled DAT protein in a concentration-dependent manner. DAT immunocytochemistry with these antibodies were also sensitive to preabsorption with fusion protein and to lesions of dopaminergic mesostriatal and mesocorticolimbic pathways. Regional distribution of DAT coincided with established dopaminergic innervation of several regions, including ventral mesencephalon, medial forebrain bundle, and dorsal and ventral striatum. However, certain mismatches between immunocytochemical distributions of DAT and tyrosine hydroxylase were apparent, indicating that dopaminergic systems are heterogeneous and may use independent mechanisms for the regulation of dopamine levels in brain. The generation of specific DAT antibodies will permit further characterization of the cellular and subcellular localization of DAT protein, and of dopaminergic circuits in neurological and psychiatric disorders.

The dopamine transporter and dopamine D2 receptor messenger RNAs are differentially expressed in limbic- and motor-related subpopulations of human mesencephalic neurons.

Dysfunction of dopamine neural systems is hypothesized to underlie neuropsychiatric disorders and psychostimulant drug abuse. At least three dopamine systems have been characterized in the brain-nigrostriatal, mesolimbic, and mesocortical. Abnormalities of nigrostriatal dopamine neurons cause motor impairment leading to Parkinson's disease, whereas dysfunction of mesolimbic and mesocortical dopamine neurons are most implicated in psychotic disorders such as schizophrenia and in drug addition. One of the primary neural sites of action of potent antipsychotic agents and psychostimulant drugs of abuse are dopamine receptors and dopamine transporters which, respectively, mediate the induction and termination of dopamine's actions. Very limited information is, however, available about which particular set of dopaminergic cells in the human brain actually express the genes for these dopamine-specific proteins. In this study, we observed that the dopamine transporter and D2 receptor messenger RNAs are differentially expressed within the human mesencephalon: highest expression in ventral subpopulations of the substantia nigra pars compacta neurons with lowest expression in the mesolimbic/mesocortical ventral tegmental area and retrorubral cell groups. These findings suggest that motor- and limbic-related mesencephalic neurons in the human brain differ in the degree of dopamine transporter and D2 receptor gene expression.

Heterogeneous subregional binding patterns of 3H-WIN 35,428 and 3H-GBR 12,935 are differentially regulated by chronic cocaine self-administration.

We examined the influence of chronic cocaine exposure, in an unlimited access self-administration paradigm, on density of the dopamine transporter (3H-WIN 35,428 and 3H-GBR 12,935 binding) and concentration of monoamine (dopamine, serotonin, noradrenaline and metabolites) neurotransmitters in rat brain. In normal rodent striatum 3H-WIN 35,428 and 3H-GBR 12,935 binding to the dopamine transporter, although generally similar, showed different subregional rostrocaudal and mediolateral gradients, suggesting that the two ligands might bind to different subtypes or states of the dopamine transporter. Following chronic, unlimited access cocaine self-administration, binding of 3H-WIN 35,428 was significantly elevated in whole nucleus accumbens (+69%, p < 0.001) and striatum (+65%, p < 0.001) on the last day of cocaine exposure ("on-cocaine group"); whereas in the 3 week withdrawn animals ("cocaine-withdrawn group"), levels were either normal (striatum) or reduced (-30%, p < 0.05, nucleus accumbens). Although similar changes in 3H-GBR 12,935 binding were observed, this dopamine transporter ligand showed a smaller and highly subregionally dependent increase in binding in striatal subdivision of the on-cocaine group, but a more marked binding reduction in the cocaine-withdrawn animals. As compared with the controls, mean dopamine levels were reduced in striatum (-15%, p < 0.05) of the on-cocaine group and in nucleus accumbens (-40%, p < 0.05) of the cocaine-withdrawn group. These data provide additional support to the hypothesis that some of the long-term effects of cocaine exposure (drug craving, depression) could be consequent to reduced nucleus accumbens dopamine function. Our data also suggest that dopamine transporter concentration, and perhaps function, might undergo up- or downregulation, either as a direct effect of cocaine, or indirectly as part of a homeostatic response to altered synaptic dopamine levels, and therefore might participate in the neuronal events underlying cocaine-induced behavioral changes.

Cloning, expression, and localization of a chloride-facilitated, cocaine-sensitive serotonin transporter from Drosophila melanogaster.

We report here on the isolation and characterization of a serotonin (5HT) transporter from Drosophila melanogaster. A 3.1-kb complementary DNA clone (dSERT) was found to encode a protein of 622 amino acid residues with a predicted molecular mass of approximately 69 kDa and a putative transmembrane topology characteristic of cloned members of the mammalian Na+/Cl- neurotransmitter cotransporter gene family. dSERT displays highest overall amino acid sequence identity with the mammalian 5HT (51%), norepinephrine (47%), and dopamine (47%) transporters and shares with all transporters 104 absolutely conserved amino acid residues. Upon transient expression in HeLa cells, dSERT exhibited saturable, high-affinity, and sodium-dependent [3H]5HT uptake with estimated Km and Vmax values of approximately 500 nM and 5.2 x 10(-18) mol per cell per min, respectively. In marked contrast to the human SERT (hSERT), 5HT-mediated transport by dSERT was not absolutely dependent on extracellular Cl-, while the sodium-dependent uptake of 5HT was facilitated by increased extracellular Cl- concentrations. dSERT displays a pharmacological profile and rank order of potency consistent with, but not identical to, mammalian 5HT transporters. Comparison of the affinities of various compounds for the inhibition of 5HT transport by both dSERT and hSERT revealed that antidepressants were 3- to 300-fold less potent on dSERT than on hSERT, while mazindol displayed approximately 30-fold greater potency for dSERT. Both cocaine and RTI-55 inhibited 5HT uptake by dSERT with estimated inhibition constants of approximately 500 nM, while high concentrations (> 10 microM) of dopamine, norepinephrine, octopamine, tyramine, and histamine failed to inhibit transport. In situ hybridization reveals the selective expression of dSERT mRNA to specific cell bodies in the ventral ganglion of the embryonic and larval Drosophila nervous system with a distribution pattern virtually identical to that of 5HT-containing neurons. The dSERT gene was mapped to position 60C on chromosome 2. The availability of the gene encoding the unique ion dependence and pharmacological characteristics of dSERT may allow for identification of those amino acid residues and structural motifs that confer the pharmacologic specificity and genetic regulation of the 5HT transport process.

Pharmacological heterogeneity of the cloned and native human dopamine transporter: disassociation of [3H]WIN 35,428 and [3H]GBR 12,935 binding.

Controversy exists as to whether the functional state of the dopamine (DA) transporter is identical to sites mediating the specific binding of selective DA transporter radioligands. Therefore, we compared the pharmacological profile of numerous dopamine transport substrates and inhibitors on [3H]DA uptake with the binding of [3H]WIN 35,428 and [3H]GBR 12,935 to COS-7 cells transiently expressing the cloned human DA transporter. [3H]DA uptake and [3H]WIN 35,428 binding was specific, saturable, and to a single class of binding sites with an estimated Km/Vmax of approximately 2 microM and 6 pmol/min/10(5) cells for DA uptake and Kd/Bmax values of approximately 10 nM and 113 fmol/10(5) cells for [3H]WIN 35,428. [3H]DA uptake was inhibited in a concentration-dependent and uniphasic manner by dopaminergic agents with an appropriate rank order of potency for the DA transporter. Although most uptake blockers inhibited [3H]WIN 35,428 binding in a uniphasic manner, WIN 35,428, Lu 19,005, D-amphetamine, and DA clearly displayed the presence of both high and low affinity components. Comparison of the Ki values for the inhibition of [3H]DA uptake with [3H]WIN 35,428 binding reveals that, for uptake blockers and D-amphetamine, it is the high affinity component that shares pharmacological identity with effects on DA uptake (r = 0.9985), whereas for DA it is the low affinity site. In striking contrast, however, [3H]GBR 12,935 binding to COS-7 cells could not be made to exhibit a pharmacological profile indicative of the DA transporter and suggests that the site regulating functional [3H]DA uptake may not be identical with sites labeled by [3H]GBR 12,935 in these cells. Moreover, these sites appear unrelated to those previously described in native membranes as "piperazine acceptor" or P450 proteins. Comparison of Ki values and rank order of potency for the inhibition of [3H]WIN 35,428 or [3H]GBR 12,935 binding to human caudate membranes reveals pharmacological homology, but not identity, with that of the cloned DA uptake process. Taken together, these data suggest that 1) [3H]WIN 35,428 recognizes two sites of the DA transporter, of which only one appears to represent the functional state of the protein, and 2) [3H]WIN 35,428 and [3H]GBR 12,935 do not appear to bind the same functional form/state of the DA transporter. Whether the nonidentity of binding sites is a manifestation of some post-translational regulatory event (e.g., phosphorylation/accessory binding protein) or caused by the existence of multiple molecular forms of the DA transporter is currently unknown.

Enhancement in antioxidant-based hepatoprotective activity of Trolox by its conjugation to lactosylphenylpyranoside.

When Trolox (a polar analog of vitamin E) is conjugated to p-aminophenyl-beta-D-lactopyranoside, the resulting lactosylphenyl Trolox becomes a markedly more stable and effective hepatoprotector than Trolox. In primary rat hepatocytes exposed to xanthine oxidase-hypoxanthine, lactosylphenyl Trolox prolonged cell survival better than did Trolox, mannitol or ascorbate. In rats that underwent 80-min partial hepatic ischemia, infusion of lactosylphenyl Trolox at 2.9 to 5.7 mumol/kg body wt just before reoxygenation salvaged the organ more extensively than did Trolox. Mechanistically, we showed (a) that lactosylphenyl Trolox does not inhibit xanthine oxidase; (b) that lactosylphenyl Trolox effectively scavenges oxyradicals generated with xanthine oxidase and the peroxyl radicals produced with 2,2'-azo-bis(2-amidinopropane) HCl; (c) that both in hepatocytes and in vivo, lactosylphenyl Trolox is distinctly more cytoprotective than either or both of its precursors; and (d) that lactosylphenyl Trolox is amphipathic (i.e., it has both hydrophilic and hydrophobic properties), which enable it to better access and protect the lipid and aqueous milieus of the cell than the lipophile vitamin E and the moderately polar Trolox. Thus there are strong fundamental reasons for lactosylphenyl Trolox being an effective antioxidant-based hepatoprotector.

Folylpolyglutamate synthetase from beef liver: purification and some properties.

The polypeptide chain of folylpolyglutamate synthetase from beef liver has been isolated and partially characterized. This polypeptide has an apparent molecular weight of 73,000. Its amino-terminal residue is blocked. Amino acid analysis agrees with the hydrophobic properties and the pI (6.0) of this cytosolic enzyme. Polyclonal antibodies to the denatured enzyme have been prepared.

Activation of folypolyglutamate synthetase by pteroic acid.

The glutamylation of methotrexate catalyzed by beef liver folypolyglutamate synthetase (FPGS) is activated by addition of pteroic acid. Pteroic acid causes greater stimulation of FPGS, including glutamylation of tetrahydrofolate, at neutral pH values (i.e., below the pH optimum of 8.4). We have attributed this activation to a conformational change of FPGS induced by pteroic acid.

Folate analogues as substrates of mammalian folylpolyglutamate synthetase.

The antifolate drugs methotrexate (MTX) and aminopterin (AM) have been tested as substrates for folylpolyglutamate synthetase (FPGS) partially purified from beef liver. The Km for MTX is 100 microM, and that for AM is 25 microM. These values are considerably higher than those for either tetrahydrofolate or folinic acid. Based on their ratios of Vmax to Km, AM is a better substrate than is MTX for the beef liver FPGS. Both are poorer substrates than tetrahydrofolate. The 7-hydroxy metabolites of MTX and AM also are substrates for FPGS. The reactivity of 7-hydroxymethotrexate is similar to that of MTX, but 7-hydroxyaminopterin is a poorer substrate than AM. Folinic acid, often used as the rescue agent in high-dose MTX therapy, has a low Km with mammalian FPGS (7 microM). Its activity is comparable to that of the best substrate, tetrahydrofolate. Low concentrations of folinic acid prevent the formation of polyglutamates of MTX. This inhibition is competitive, presumably because folinic acid and MTX are competing substrates for FPGS. The activities of folate and antifolate substrates also have been determined with rat liver FPGS. With near-saturating concentrations of AM, MTX, or 7-hydroxymethotrexate, the reaction velocity exceeds that with an optimal concentration of tetrahydrofolate. However, the Km values of the folate analogues all are greater than those of the tetrahydrofolate coenzymes. In contrast to the formation of long-chain polyglutamates observed when tetrahydrofolate or folinic acid was the substrate, beef liver FPGS, under our reaction conditions, cannot catalyze the formation from MTX monoglutamate of polyglutamates longer than the triglutamate. MTX di- and triglutamates are poorer substrates than is MTX itself. Longer polyglutamates of MTX, while having no activity as substrates, must bind to the enzyme, because they are inhibitors. Our observations using MTX and AM with the enzymatic FPGS system help to rationalize the therapeutic use of antifolates.

Folypolyglutamate synthetase from beef liver: assay, stabilization, and characterization.

A reliable assay for folylpolyglutamate synthetase has been devised and tested. Conditions have been established for the complete separation of [3H]glutamate and the tritium-labelled products on columns of DEAE-cellulose. The availability of this assay has aided us in partially purifying and characterizing the synthetase from extracts of beef liver. Suitable conditions have been found for the stabilization of the activity of both crude and partially purified folylpolyglutamate synthetase. The apparent Km values for L-glutamate (0.82 mM), dl-L-tetrahydrofolate (9 microM), ATP (25 microM with 10 mM MgCl2), KCl (3 mM), and 2-mercaptoethanol (5 mM) have been estimated. Several oxidized pteridine substrates have been tested. Of the antifolates tested, aminopterin is the more active substrate. The chain lengths of folate polyglutamates have been measured by chromatography on columns of DEAE-cellulose, with elution by a gradient of sodium acetate. Conjugates as long as hexaglutamates have been detected. The identities of the polyglutamates of tetrahydrofolate have been verified by hydrolysis in the presence of conjugase and by double-labelling experiments.