Na+ - and Cl- -coupled active transport of nitric oxide synthase inhibitors via amino acid transport system B(0,+).
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Nitric oxide synthase (NOS) inhibitors have therapeutic potential in the management of numerous conditions in which NO overproduction plays a critical role. Identification of transport systems in the intestine that can mediate the uptake of NOS inhibitors is important to assess the oral bioavailability and therapeutic efficacy of these potential drugs. Here, we have cloned the Na+ - and Cl- -coupled amino acid transport system B(0,+) (ATB(0,+)) from the mouse colon and investigated its ability to transport NOS inhibitors. When expressed in mammalian cells, ATB(0,+) can transport a variety of zwitterionic and cationic amino acids in a Na+ - and Cl- -coupled manner. Each of the NOS inhibitors tested compete with glycine for uptake through this transport system. Furthermore, using a tritiated analog of the NOS inhibitor N(G)-nitro-L-arginine, we showed that Na+ - and Cl- -coupled transport occurs via ATB(0,+). We then studied transport of a wide variety of NOS inhibitors in Xenopus laevis oocytes expressing the cloned ATB(0,+) and found that ATB(0,+) can transport a broad range of zwitterionic or cationic NOS inhibitors. These data represent the first identification of an ion gradient-driven transport system for NOS inhibitors in the intestinal tract. (+info)
Neurotransmitter transporters and their impact on the development of psychopharmacology.
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The synaptic actions of most neurotransmitters are inactivated by reuptake into the nerve terminals from which they are released, or by uptake into adjacent cells. A family of more than 20 transporter proteins is involved. In addition to the plasma membrane transporters, vesicular transporters in the membranes of neurotransmitter storage vesicles are responsible for maintaining vesicle stores and facilitating exocytotic neurotransmitter release. The cell membrane monoamine transporters are important targets for CNS drugs. The transporters for noradrenaline and serotonin are key targets for antidepressant drugs. Both noradrenaline-selective and serotonin-selective reuptake inhibitors are effective against major depression and a range of other psychiatric illnesses. As the newer drugs are safer in overdose than the first-generation tricyclic antidepressants, their use has greatly expanded. The dopamine transporter (DAT) is a key target for amphetamine and methylphenidate, used in the treatment of attention deficit hyperactivity disorder. Psychostimulant drugs of abuse (amphetamines and cocaine) also target DAT. The amino-acid neurotransmitters are inactivated by other families of neurotransmitter transporters, mainly located on astrocytes and other non-neural cells. Although there are many different transporters involved (four for GABA; two for glycine/D-serine; five for L-glutamate), pharmacology is less well developed in this area. So far, only one new amino-acid transporter-related drug has become available: the GABA uptake inhibitor tiagabine as a novel antiepileptic agent. (+info)
Experimental colitis: decreased Octn2 and Atb0+ expression in rat colonocytes induces carnitine depletion that is reversible by carnitine-loaded liposomes.
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Carnitine transporters have recently been implicated in susceptibility to inflammatory bowel disease (IBD). Because carnitine is required for beta-oxidation, it was suggested that decreased carnitine transporters, and hence reduced carnitine uptake, could lead to impaired fatty acid oxidation in intestinal epithelial cells, and to cell injury. We investigated this issue by examining the expression of the carnitine transporters OCTN2 and ATB0+, and butyrate metabolism in colonocytes in a rat model of IBD induced by trinitrobenzene sulfonic acid (TNBS). We found that Octn2 and Atb0+ expression was decreased in inflammatory samples at translational and functional level. Butyrate oxidation, evaluated based on CO2 production and acetyl-coenzyme A synthesis, was deranged in colonocytes from TNBS-treated rats. Treatment with carnitine-loaded liposomes corrected the butyrate metabolic alterations in vitro and reduced the severity of colitis in vivo. These results suggest that carnitine depletion in colonocytes is associated with the inability of mitochondria to maintain normal butyrate beta-oxidation. Our data indicate that carnitine is a rate-limiting factor for the maintenance of physiological butyrate oxidation in colonic cells. This hypothesis could also explain the contradictory therapeutic efficacy of butyrate supplementation observed in clinical trials of IBD. (+info)
Ancestry of neuronal monoamine transporters in the Metazoa.
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Selective Na(+)-dependent re-uptake of biogenic monoamines at mammalian nerve synapses is accomplished by three types of solute-linked carrier family 6 (SLC6) membrane transporter with high affinity for serotonin (SERTs), dopamine (DATs) and norepinephrine (NETs). An additional SLC6 monoamine transporter (OAT), is responsible for the selective uptake of the phenolamines octopamine and tyramine by insect neurons. We have characterized a similar high-affinity phenoloamine transporter expressed in the CNS of the earthworm Lumbricus terrestris. Phylogenetic analysis of its protein sequence clusters it with both arthropod phenolamine and chordate catecholamine transporters. To clarify the relationships among metazoan monoamine transporters we identified representatives in the major branches of metazoan evolution by polymerase chain reaction (PCR)-amplifying conserved cDNA fragments from isolated nervous tissue and by analyzing available genomic data. Analysis of conserved motifs in the sequence data suggest that the presumed common ancestor of modern-day Bilateria expressed at least three functionally distinct monoamine transporters in its nervous system: a SERT currently found throughout bilaterian phyla, a DAT now restricted in distribution to protostome invertebrates and echinoderms and a third monoamine transporter (MAT), widely represented in contemporary Bilateria, that is selective for catecholamines and/or phenolamines. Chordate DATs, NETs, epinephrine transporters (ETs) and arthropod and annelid OATs all belong to the MAT clade. Contemporary invertebrate and chordate DATs belong to different SLC6 clades. Furthermore, the genes for dopamine and norepinephrine transporters of vertebrates are paralogous, apparently having arisen through duplication of an invertebrate MAT gene after the loss of an invertebrate-type DAT gene in a basal protochordate. (+info)
Molecular targets for antiepileptic drug development.
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This review considers how recent advances in the physiology of ion channels and other potential molecular targets, in conjunction with new information on the genetics of idiopathic epilepsies, can be applied to the search for improved antiepileptic drugs (AEDs). Marketed AEDs predominantly target voltage-gated cation channels (the alpha subunits of voltage-gated Na+ channels and also T-type voltage-gated Ca2+ channels) or influence GABA-mediated inhibition. Recently, alpha2-delta voltage-gated Ca2+ channel subunits and the SV2A synaptic vesicle protein have been recognized as likely targets. Genetic studies of familial idiopathic epilepsies have identified numerous genes associated with diverse epilepsy syndromes, including genes encoding Na+ channels and GABA(A) receptors, which are known AED targets. A strategy based on genes associated with epilepsy in animal models and humans suggests other potential AED targets, including various voltage-gated Ca2+ channel subunits and auxiliary proteins, A- or M-type voltage-gated K+ channels, and ionotropic glutamate receptors. Recent progress in ion channel research brought about by molecular cloning of the channel subunit proteins and studies in epilepsy models suggest additional targets, including G-protein-coupled receptors, such as GABA(B) and metabotropic glutamate receptors; hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits, responsible for hyperpolarization-activated current Ih; connexins, which make up gap junctions; and neurotransmitter transporters, particularly plasma membrane and vesicular transporters for GABA and glutamate. New information from the structural characterization of ion channels, along with better understanding of ion channel function, may allow for more selective targeting. For example, Na+ channels underlying persistent Na+ currents or GABA(A) receptor isoforms responsible for tonic (extrasynaptic) currents represent attractive targets. The growing understanding of the pathophysiology of epilepsy and the structural and functional characterization of the molecular targets provide many opportunities to create improved epilepsy therapies. (+info)
Characterization of the antinociceptive actions of bicifadine in models of acute, persistent, and chronic pain.
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Bicifadine (1-p-tolyl-3-azabicyclo[3.1.0]hexane) inhibits monoamine neurotransmitter uptake by recombinant human transporters in vitro with a relative potency of norepinephrine > serotonin > dopamine (approximately 1:2:17). This in vitro profile is supported by microdialysis studies in freely moving rats, where bicifadine (20 mg/kg i.p.) increased extrasynaptic norepinephrine and serotonin levels in the prefrontal cortex, norepinephrine levels in the locus coeruleus, and dopamine levels in the striatum. Orally administered bicifadine is an effective antinociceptive in several models of acute, persistent, and chronic pain. Bicifadine potently suppressed pain responses in both the Randall-Selitto and kaolin models of acute inflammatory pain and in the phenyl-p-quinone-induced and colonic distension models of persistent visceral pain. Unlike many transport inhibitors, bicifadine was potent and completely efficacious in both phases of the formalin test in both rats and mice. Bicifadine also normalized the nociceptive threshold in the complete Freund's adjuvant model of persistent inflammatory pain and suppressed mechanical and thermal hyperalgesia and mechanical allodynia in the spinal nerve ligation model of chronic neuropathic pain. Mechanical hyperalgesia was also reduced by bicifadine in the streptozotocin model of neuropathic pain. Administration of the D(2) receptor antagonist (-)-sulpiride reduced the effects of bicifadine in the mechanical hyperalgesia assessment in rats with spinal nerve ligations. These results indicate that bicifadine is a functional triple reuptake inhibitor with antinociceptive and antiallodynic activity in acute, persistent, and chronic pain models, with activation of dopaminergic pathways contributing to its antihyperalgesic actions. (+info)
All aglow about presynaptic receptor regulation of neurotransmitter transporters.
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Mounting evidence supports the idea that neurotransmitter transporters are subject to many forms of post-translational regulation typically associated with receptors and ion channels, including receptor and kinase-mediated changes in transporter phosphorylation, cell surface trafficking, and/or catalytic activation. Although hints of this regulation can be achieved with traditional radiolabeled substrate flux techniques, higher resolution methods are needed that can localize transporter function in situ as well as permit real-time monitoring of transport function without confounds associated with coincident receptor activation. The elegant study by Bolan et al. (p. 1222) capitalizes on the fluorescent properties of a recently introduced substrate for the dopamine (DA) transporter (DAT), termed 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+), to illuminate a pertussis toxin-sensitive, extracellular signal-regulated kinase (ERK1/2)-dependent pathway by which presynaptic DA D(2) receptors regulate DATs. (+info)
The neurotransmitter cycle and quantal size.
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Changes in the response to release of a single synaptic vesicle have generally been attributed to postsynaptic modification of receptor sensitivity, but considerable evidence now demonstrates that alterations in vesicle filling also contribute to changes in quantal size. Receptors are not saturated at many synapses, and changes in the amount of transmitter per vesicle contribute to the physiological regulation of release. On the other hand, the presynaptic factors that determine quantal size remain poorly understood. Aside from regulation of the fusion pore, these mechanisms fall into two general categories: those that affect the accumulation of transmitter inside a vesicle and those that affect vesicle size. This review will summarize current understanding of the neurotransmitter cycle and indicate basic, unanswered questions about the presynaptic regulation of quantal size. (+info)