N-oxygenation of amphetamine and methamphetamine by the human flavin-containing monooxygenase (form 3): role in bioactivation and detoxication.
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(+)- And (-)-amphetamine and methamphetamine were N-oxygenated by the cDNA expressed adult human flavin-containing monooxygenase form 3 (FMO3), their corresponding hydroxylamines. Two major polymorphic forms of human FMO3 were studied, and the results suggested preferential N-oxygenation by only one of the two enzymes. Chemically synthesized (+/-)-amphetamine hydroxylamine was also a substrate for the human FMO3 and it was converted to phenylpropanone oxime with a stereoselectivity ratio of trans/cis of 5:1. Human FMO3 also N-oxygenated methamphetamine to produce methamphetamine hydroxylamine. Methamphetamine hydroxylamine was also N-oxygenated by human FMO3, and the ultimate product observed was phenylpropanone. For amphetamine hydroxylamine, studies of the biochemical mechanism of product formation were consistent with the production of an N, N-dioxygenated intermediate that lead to phenylpropanone oxime. This was supported by the observation that alpha-deutero (+/-)-amphetamine hydroxylamine gave an inverse kinetic isotope effect on product formation in the presence of human FMO3. For methamphetamine, the data were consistent with a mechanism of human FMO3-mediated N,N-dioxygenation but the immediate product, a nitrone, rapidly hydrolyzed to phenylpropanone. The pharmacological activity of amphetamine hydroxylamine, phenylpropanone oxime, and methamphetamine hydroxylamine were examined for effects at the human dopamine, serotonin, and norepinephrine transporters. Amphetamine hydroxylamine and methamphetamine hydroxylamine were apparent substrates for the human biogenic amine transporters but phenylpropanone oxime was not. Presumably, phenylpropanone oxime or nitrone formation from amphetamine and methamphetamine, respectively, represents a detoxication process. Because of the potential toxic nature of amphetamine hydroxylamine and methamphetamine hydroxylamine metabolites and the polymorphic nature of N-oxygenation, human FMO3-mediated metabolism of amphetamine or methamphetamine may have clinical consequences. (+info)
Polyamine transport in bacteria and yeast.
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The polyamine content of cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) were ABC transporters, each consisting of a periplasmic substrate-binding protein, two transmembrane proteins and a membrane-associated ATPase. The crystal structures of the substrate-binding proteins (PotD and PotF) have been solved. They consist of two domains with an alternating beta-alpha-beta topology, similar to other periplasmic binding proteins. The polyamine-binding site is in a cleft between the two domains, as determined by crystallography and site-directed mutagenesis. Polyamines are mainly recognized by aspartic acid and glutamic acid residues, which interact with the NH(2)- (or NH-) groups, and by tryptophan and tyrosine residues that have hydrophobic interactions with the methylene groups of polyamines. The precursor of one of the substrate binding proteins, PotD, negatively regulates transcription of the operon for the spermidine-preferential uptake system, thus providing another level of regulation of cellular polyamines. The third transport system, catalysed by PotE, mediates both uptake and excretion of putrescine. Uptake of putrescine is dependent on membrane potential, whereas excretion involves an exchange reaction between putrescine and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport protein (TPO1) was identified. The properties of this protein are similar to those of PotE, and TPO1 is located on the vacuolar membrane. (+info)
Activation of a heterologously expressed octopamine receptor coupled only to adenylyl cyclase produces all the features of presynaptic facilitation in aplysia sensory neurons.
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Short-term behavioral sensitization of the gill-withdrawal reflex after tail stimuli in Aplysia leads to an enhancement of the connections between sensory and motor neurons of this reflex. Both behavioral sensitization and enhancement of the connection between sensory and motor neurons are importantly mediated by serotonin. Serotonin activates two types of receptors in the sensory neurons, one of which is coupled to the cAMP/protein kinase A (PKA) pathway and the other to the inositol triphosphate/protein kinase C (PKC) pathway. Here we describe a genetic approach to assessing the isolated contribution of the PKA pathway to short-term facilitation. We have cloned from Aplysia an octopamine receptor gene, Ap oa(1), that couples selectively to the cAMP/PKA pathway. We have ectopically expressed this receptor in Aplysia sensory neurons of the pleural ganglia, where it is not normally expressed. Activation of this receptor by octopamine stimulates all four presynaptic events involved in short-term synaptic facilitation that are normally produced by serotonin: (i) membrane depolarization; (ii) increased membrane excitability; (iii) increased spike duration; and (iv) presynaptic facilitation. These results indicate that the cAMP/PKA pathway alone is sufficient to produce all the features of presynaptic facilitation. (+info)
Octopamine receptors in the honey bee and locust nervous system: pharmacological similarities between homologous receptors of distantly related species.
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Honey bees are perhaps the most versatile models to study the cellular and pharmacological basis underlying behaviours ranging from learning and memory to sociobiology. For both aspects octopamine (OA) is known to play a vital role. The neuronal octopamine receptor of the honey bee shares pharmacological similarities with the neuronal octopamine receptor of the locust. Both, agonists and antagonists known to have high affinities for the locust neuronal octopamine receptor have also high affinities for the bee neuronal octopamine receptor. The distribution of receptors is more or less congruent between locusts and bees. Optic lobes and especially the mushroom bodies are areas of greatest octopamine receptor expression in both species, which mirrors the physiological significance of octopamine in the insect nervous system. The neuronal octopamine receptor of insects served as a model to study the pharmacological similarity of homologous receptors from distantly related species, because bees and locusts are separated by at least 330 million years of evolution. (+info)
Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor.
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The trace amine para-tyramine is structurally and functionally related to the amphetamines and the biogenic amine neurotransmitters. It is currently thought that the biological activities elicited by trace amines such as p-tyramine and the psychostimulant amphetamines are manifestations of their ability to inhibit the clearance of extracellular transmitter and/or stimulate the efflux of transmitter from intracellular stores. Here we report the discovery and pharmacological characterization of a rat G protein-coupled receptor that stimulates the production of cAMP when exposed to the trace amines p-tyramine, beta-phenethylamine, tryptamine, and octopamine. An extensive pharmacological survey revealed that psychostimulant and hallucinogenic amphetamines, numerous ergoline derivatives, adrenergic ligands, and 3-methylated metabolites of the catecholamine neurotransmitters are also good agonists at the rat trace amine receptor 1 (rTAR1). These results suggest that the trace amines and catecholamine metabolites may serve as the endogenous ligands of a novel intercellular signaling system found widely throughout the vertebrate brain and periphery. Furthermore, the discovery that amphetamines, including 3,4-methylenedioxymethamphetamine (MDMA; "ecstasy"), are potent rTAR1 agonists suggests that the effects of these widely used drugs may be mediated in part by this receptor as well as their previously characterized targets, the neurotransmitter transporter proteins. (+info)
Phylogenetic analysis of 277 human G-protein-coupled receptors as a tool for the prediction of orphan receptor ligands.
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BACKGROUND: G-protein-coupled receptors (GPCRs) are the largest and most diverse family of transmembrane receptors. They respond to a wide range of stimuli, including small peptides, lipid analogs, amino-acid derivatives, and sensory stimuli such as light, taste and odor, and transmit signals to the interior of the cell through interaction with heterotrimeric G proteins. A large number of putative GPCRs have no identified natural ligand. We hypothesized that a more complete knowledge of the phylogenetic relationship of these orphan receptors to receptors with known ligands could facilitate ligand identification, as related receptors often have ligands with similar structural features. RESULTS: A database search excluding olfactory and gustatory receptors was used to compile a list of accession numbers and synonyms of 81 orphan and 196 human GPCRs with known ligands. Of these, 241 sequences belonging to the rhodopsin receptor-like family A were aligned and a tentative phylogenetic tree constructed by neighbor joining. This tree and local alignment tools were used to define 19 subgroups of family A small enough for more accurate maximum-likelihood analyses. The secretin receptor-like family B and metabotropic glutamate receptor-like family C were directly subjected to these methods. CONCLUSIONS: Our trees show the overall relationship of 277 GPCRs with emphasis on orphan receptors. Support values are given for each branch. This approach may prove valuable for identification of the natural ligands of orphan receptors as their relation to receptors with known ligands becomes more evident. (+info)
Regulation of chloride permeability by endogenously produced tyramine in the Drosophila Malpighian tubule.
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The Malpighian (renal) tubule of Drosophila melanogaster is a useful model for studying epithelial transport. The purpose of this study was to identify factors responsible for modulating transepithelial chloride conductance in isolated tubules. I have found that tyrosine and several of its metabolites cause an increase in chloride conductance. The most potent of these agonists is tyramine, which is active at low nanomolar concentrations; the pharmacology of this response matches that of the previously published cloned insect tyramine receptor. In addition, the tubule appears capable of synthesizing tyramine from applied tyrosine, as shown by direct measurement of tyrosine decarboxylase activity. Immunohistochemical staining of tubules with an antibody against tyramine indicates that the principal cells are the sites of tyramine production, whereas previous characterization of the regulation of chloride conductance suggests that tyramine acts on the stellate cells. This is the first demonstration of a physiological role for an insect tyramine receptor. (+info)
Modulation of early olfactory processing by an octopaminergic reinforcement pathway in the honeybee.
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Processing of olfactory information in the antennal lobes of insects and olfactory bulbs of vertebrates is modulated by centrifugal inputs that represent reinforcing events. Octopamine release by one such pathway in the honeybee antennal lobe modulates olfactory processing in relation to nectar (sucrose) reinforcement. To test more specifically what role octopamine plays in the antennal lobe, we used two treatments to disrupt an octopamine receptor from Apis mellifera brain (AmOAR) function: (1) an OAR antagonist, mianserin, was used to block receptor function, and (2) AmOAR double-stranded RNA was used to silence receptor expression. Both treatments inhibited olfactory acquisition and recall, but they did not disrupt odor discrimination. These results suggest that octopamine mediates consolidation of a component of olfactory memory at this early processing stage in the antennal lobe. Furthermore, after consolidation, octopamine release becomes essential for recall, which suggests that the modulatory circuits become incorporated as essential components of neural representations that activate odor memory. (+info)