Jean C. Shih

Human monoamine oxidase A and B genes map to xp11.23 and are deleted in a patient with norrie disease

Monoamine oxidase A and B (MAO A and B) are the central enzymes that catalyze oxidative deamination of biogenic amines throughout the body. The regional locations of genes encoding MAO A and B on the X chromosome were determined by using full-length cDNA clones for human MAO A and B, respectively. Using somatic cell hybrids, in situ hybridization, and field-inversion gel electrophoresis as well as deletion mapping in a patient with Norrie disease, we concluded that these two genes are close to each other and to the DXS7 locus (Xp 11.3).

5-HT-la and 5-HT-lb selectivity of two phenylpiperazine derivatives: Evidence for 5-HT-lb heterogeneity

The ability of m-trifluoromethylphenylpiperazine (TFMPP) and an N-substituted derivative, LY 165163 (p-NH2-PE-TFMPP), to discriminate 5-HT-1 binding sites labelled by [3H]5-HT is compared in rat corpus striatum and rat cortex. TFMPP displays at least a 30 fold selectivity for the 5-HT-1b subtype. Furthermore, TFMPP reveals heterogeneity within the 5-HT-1b binding sites. TFMPP displays a Kd of 6 nM for approximately two-thirds of the 5-HT-1b binding sites and a Kd of 273 nM for the remaining one third of the 5-HT-1b sites. p-NH2-PE-TFMPP, on the other hand, discriminates the 5-HT-1 sites in a manner similar to spiperone, displaying a 110 fold selectivity for the 5-HT-1a sites. p-NH2-PE-TFMPP displays a Kd of about 3 nM for the 5-HT-1a sites. p-NH2-PE-TFMPP does not discriminate subtypes within the 5-HT-1b binding sites. The significance of the selectivity of these two compounds as well as structurally related compounds is discussed.

Regulation of MAO-A and MAO-B Gene Expression

MAO A and B genes are made of 15 exons with identical exon-intron organization. They are located on X-chromosome organized in opposite direction, tail to tail with 24 kb apart. Both promoters are GC-rich and regulated by transcription factor Sp1. However, they have distinctly different features. MAO B gene, but not MAO A gene, has TATA box. MAO B promoter contains two clusters of overlapping Sp1 sites, the CACCC repressor element. Transcription factors Sp1 and Sp4 can activate MAO B promoter activity through the proximal cluster of Sp1 sites and its activation can be repressed by the over-expression of Sp3 and a related family member, BTEB2. Decreased methylation and transcription repressor Sp3 upregulate human MAO B, but not MAO A, gene expression during Caco-2 differentiation. MAO B, but not MAO A gene, could be activated by PMA (phorbol 12-myristate 13-acetate) by protein kinase C, MAPkinase signal transduction pathway involves cJun and Egr-1. The differences in MAO A and B gene regulation may explain the different tissue-specific expression and functions of these two important isoenzymes.

[3H]1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine: a selective radioligand for 5-HT1A receptors in rat brain

Abstract: 1‐[2‐(4‐Aminophenyl)ethyl]‐4‐(3‐trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5‐hydroxytryptamine (5‐HT, serotonin) binding to 5‐HT1A and 5‐HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5‐HT1A receptor radioligand was evaluated. With either 10 μM 5‐HT or 1 μM 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d‐Lysergic acid diethylamide and 5‐HT, two nonselective inhibitors of [3H]5‐HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5‐HT1 receptors. Spiperone and 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, two compounds that discriminate [3H]5‐HT binding to 5‐HT1A and 5‐HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5‐HT1A receptor subtype. Furthermore, the ability of N‐(m‐trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5‐HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin‐sensitive [3H]PAPP sites correlated with the densities of 5‐HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5‐HT1A receptor sites in rat brain.

Expression of Functional Human Monoamine Oxidase A and B cDNAs in Mammalian Cells

Abstract: Monoamine oxidase (MAO) A and B are important enzymes that metabolize biogenic amines throughout the body. Previous studies had suggested that both MAO A and B consist of two subunits of molecular masses of 63 and 60 kilodaltons, respectively. The cDNAs encoding one subunit of human liver MAO A and B have been expressed in mammalian cells by transfection of the individual clones. The proteins expressed from these cDNAs are shown to be catalytically active. Similar to the endogenous enzymes, the expressed MAO A prefers serotonin as a substrate and is sensitive to the inhibitor clorgyline. In contrast, the expressed MAO B prefers phenylethyl‐amine as a substrate and is sensitive to the inhibitor deprenyl. These results suggest that a single polypeptide of MAO A (or B), existing as either a monomer or homodimer, is enzymatically active. The ability to obtain functional MAO A and B from their respective cDNA clones allows us to study further the structure and function relationships of these important enzymes.

MAO-B knockout mice exhibit deficient habituation of locomotor activity but normal nicotine intake.

Low levels of monoamine oxidase‐B (MAO‐B) activity, such as those observed in smokers, are also associated with behavioral traits such as a heightened responsiveness to novelty. However, the exact mechanism by which low MAO‐B activity influences smoking and heightened responsiveness to novelty is still poorly understood. We used MAO‐B knockout (KO) mice to test the hypothesis that MAO‐B concomitantly affects locomotor responses in a novel inescapable open field and nicotine intake. Male wild‐type (WT) and MAO‐B KO mice were placed in an inescapable open field and their horizontal locomotor activity was measured for 30 min per day for 5 days. MAO‐B KO mice exhibited impaired within‐session habituation of locomotor activity, as compared to WT mice. Separate groups of male WT and MAO‐B KO mice were individually housed in their home cages with two water bottles. One of the bottles contained tap water and the other contained nicotine (0, 3.125, 6.25, 12.5, 25, 50 or 100 µg/ml). The total amount of water and nicotine solution consumed was measured every three days for 16 days. MAO‐B KO mice and WT mice consumed equal amounts of nicotine and exhibited comparable concentration‐dependent nicotine preference and aversion over a period of 16 days. The data suggest that the absence of MAO‐B impairs the ability of mice to habituate in the inescapable environment, but does not alter their nicotine intake.

Solubilization of Serotonin1a and Serotonin1b Binding Sites from Bovine Brain

Serotonin, (5‐hydroxytryptamine1, 5‐HT1) binding sites have been solubilized from bovine brain cortex using a mixture of 0.1 % Nonidet P‐40 and 0.3% digitonin in a low‐salt buffer containing 0.1 % ascorbic acid. The affinity of [3H]5‐HT for the soluble cortical binding sites (2.1 nM) is identical to its affinity at membrane‐bound binding sites (2.1 nM). [3H]8‐Hydroxy‐2‐(di‐n‐propylamino)tetraIin ([3H]DPAT), a selective 5‐HT1a, radioligand, also binds to soluble cortical binding sites with high affinity (1.8 nM) comparable with its affinity in the crude membranes (1.7 nM). A significant correlation exists in the rank order potency of serotonergic agents for [3H]5‐HT binding and for [3H]DPAT binding to crude and soluble membranes. The density of [3H]DPAT binding sites relative to the [3H]5‐HT sites in the solubilized cortical membranes (35%) corresponds well with the proportion of 5‐HT1a sites in the crude membranes determined by spiperone displacement (33%), suggesting that both the 5‐HT1a and 5‐HT1b binding sites have been cosolubilized. [3H]5‐HT binding in the soluble preparations was inhibited by GTP, suggesting that a receptor complex may have been solubilized. [3H]Spiperone‐specific binding was not detectable in this preparation, suggesting that 5‐HT2 sites were not cosolubilized.

Peptide displacement of [3H]5-hydroxytryptamine binding to bovine cortical membranes

Chemical studies have demonstrated that peptides such as the encephalitogenic (EAE) peptide of myelin basic protein (MBP) and luteinizing hormone-releasing hormone (LHRH) can bind serotonin (5-hydroxytryptamine, 5-HT) in vitro. The present research was undertaken to determine whether such binding interferes with 5-HT binding to its 5-HT1 receptors on bovine cerebral cortical membranes. EAE peptide and LHRH displaced [3H]5-HT with IC50s of 4.0 x 10(-4) and 1.8 x 10(-3) M respectively. MBP itself also showed apparent displacing ability with an IC50 of 6.0 x 10(-5) M, though it also caused aggregation of cortical membranes that might have interfered with normal receptor binding. These results support previous suggestions that the tryptophan peptide region of MBP may act as a 5-HT receptor in the neural system. We also tested the effects of muramyl dipeptide (N-acetyl-muramyl-L-Ala-D-isoGln, MD), a bacterial cell-wall breakdown product that acts as a slow-wave sleep promoter, binds to LHRH and EAE peptide, and competes for 5-HT binding sites on macrophages. It showed no significant displacement of 5-HT binding to cortical membranes (IC50 greater than 10(-1) M), but its D-Ala analogue did (IC50 = 1.7 x 10(-3) M). Thus, it seems likely that the 5-HT-related effects of naturally occurring muramyl peptides are physiologically limited by receptor types.

Separation of L-phenylalanine: Pyruvate transaminase and L-phenylalanine: α-ketoglutarate transaminase by Sephadex-electrophoresis

Abstract By means of a Sephadex-electrophoresis column, L-phenylalanine: pyruvate transaminase (PPT) was separated from L-phenylalanine: α-ketoglutarate transaminase (PKT) from rat liver. These enzymes differed in heat lability in vitro and in their inducibility by glucagon in vivo . PPT was heat-stable and was induced by chronic glucagon injection. On the other hand, PKT was heat-labile and was not induced by glucagon under the experimental conditions used. These studies provide evidence that distinct enzymes catalyze the transamination of phenylalanine with pyruvate or with α-ketoglutarate as the amino acceptor.

Identification of 5-hydroxytryptamine1A receptor proteins in bovine frontal cortex

Abstract: 5‐Hydroxytryptamine1A (5‐HT1A) receptor proteins were identified by a novel approach in which photoaffinity labeling technique was used in conjunction with affinity column chromatography. 5‐HT1A receptors were solubilized from bovine frontal cortical membranes with 0.3% digitonin and 0.1% Nonidet P‐40, and bound effectively to l‐[2‐(4‐aminophenyl)ethyl]‐4‐(3‐trifluoromethylphenyl)piperazine (PAPP)‐coupled Affi‐Gel 10 in a time‐dependent manner. PAPP was shown previously to be a selective ligand for the 5‐HT1A receptor. Two protein bands with molecular masses of approximately 55,000 and 38,000 daltons revealed on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis were eluted from the affinity column with either 1 mM 5‐HT or 1 μM [3H]l‐[2‐(4‐azidophenyl)ethyl]‐4‐(3‐trifluoromethyI‐phenyl)piperazine ([3H]p‐azido‐PAPP). [3H]p‐Azido‐PAPP is a selective photoaffinity labeling probe for the 5‐HT1A receptor. The intensity of these two protein bands and the incorporation of [3H]p‐azido‐PAPP into these two proteins decreased significantly when the solubilized fraction was prein‐cubated with excess 5‐HT or PAPP (saturating all 5‐HT1A receptors) prior to affinity column chromatography. These results suggest strongly that these two proteins are related to the 5‐HT1A receptor protein. The isoelectric points of the photolabeled 5‐HT1A receptor proteins were 6.0 and 6.5.