Dopamine vesicular transporter

It is now possible to image not only postsynaptic, but pre-synaptic and intrasynaptic neurotransmission (Fig. 58-5). Presynaptic sites, such as the dopamine transporter and the serotonin transporter the presynaptic dopamine vesicular transporter (VMAT-2) and the acetylcholine transporter extrasynaptic sites such as the enzymes which break down neurotransmitters, e.g. MAO A and MAO B with radioligands that bind to post or pre-synaptic sites, i.e. dopamine competing with radioligands such as UC raclopride (see Fig. 58-9) (PET (Fig. 58-10) can be measured under basal conditions or following drugs which either decrease (e.g. AMPT) or increase (e.g. intravenous amphetamine) intrasynaptic dopamine. 

Together with dopamine, adrenaline and noradrenaline belong to the endogenous catecholamines that are synthesized from the precursor amino acid tyrosine (Fig. 1). In the first biosynthetic step, tyrosine hydroxylase generates l-DOPA which is further converted to dopamine by the aromatic L-amino acid decarboxylase ( Dopa decarboxylase). Dopamine is transported from the cytosol into synaptic vesicles by a vesicular monoamine transporter. In sympathetic nerves, vesicular dopamine (3-hydroxylase generates the neurotransmitter noradrenaline. In chromaffin cells of the adrenal medulla, approximately 80% of the noradrenaline is further converted into adrenaline by the enzyme phenylethanolamine-A-methyltransferase. 

Rostene, W., Boja, J.W., Scherman, D., Carroll, F.I., and Kuhar, M.J., Dopamine transport pharmacological distinction between the synaptic membrane and vesicular transporter in rat striatum, Eur. J. Pharmacol., 281, 175, 1992. 

Dopamine depleting agents. Reserpine, a natural alkaloid that blocks vesicular transport of monoamines, depletes stored monoamines, including DA. DA depletion is associated with the emergence of parkinsonism. This effect of reserpine was among the first clues that PD is the result of DA deficiency (see above). Generally, the parkinsonism resulting from reserpine is reversible. 

Since the enzyme that converts dopamine to norepinephrine (dopamine (3-hydroxylase) is located only within the vesicles, the transport of dopamine into the vesicle is an essential step in the synthesis of norepinephrine. This same transport system is essential for the storage of norepinephrine. There is a tendency for norepinephrine to leak from the vesicles into the cytosol. If norepinephrine remains in the cytosol, much of it will be destroyed by a mitochondrial enzyme, monoamine oxidase MAO). However, most of the norepinephrine that leaks out of the vesicle is rapidly returned to the storage vesicles by the same transport system that carries dopamine into the storage vesicles. It is important for a proper understanding of drug action to remember that this single transport system, called vesicular transport, is an essential element of both synthesis and storage of norepinephrine. 

FIGURE 23.7 Dopamine (DA) is synthesized within neuronal terminals from the precursor tyrosine by the sequential actions of the enzymes tyrosine hydroxylase, producing the intermediary L-dihydroxyphenylalanine (Dopa), and aromatic L-amino acid decarboxylase. In the terminal, dopamine is transported into storage vesicles by a transporter protein (T) associated with the vesicular membrane. Release, triggered by depolarization and entry of Ca2+, allows dopamine to act on postsynaptic dopamine receptors (DAR). Several distinct types of dopamine receptors are present in the brain, and the differential actions of dopamine on postsynaptic targets bearing different types of dopamine receptors have important implications for the function of neural circuits. The actions of dopamine are terminated by the sequential actions of the enzymes catechol-O-methyl-transferase (COMT) and monoamine oxidase (MAO), or by reuptake of dopamine into the terminal. 

Uptake of amine NTs from the neuronal cytosol into synaptic vesicles is achieved by vesicular monoamine transporters (VMAT1 and VMAT2) that sequester dopamine, epinephrine, norepinephrine and serotonin. A similar vesicle transporter (VGAT) sequesters GABA and glycine and a vesicular transporter (VAChT) sequesters acetylcholine into synaptic vesicles. 

Amphetamine acts on both transmitter transporters - the plasmalemmal one, which is the site of action of cocaine, and the vesicular transporter, which is targeted by reserpine. It is imported into the cell by the plasmalemmal transporter. This will result in inhibited reuptake of the physiological transmitter, not so much apparently by direct competition (as is the case with cocaine) but by subsequent endocytosis of the receptor. This is clearly shown in Figure 10.16. In the experiment shown, the dopamine reuptake transporter was recombinantly expressed in cultured cells and visualized by immimofluorescence . Initially, the fluorescence is confined to the surface of the cells expressing the transporter (Figure 10.16b, left panel). After exposure of the cells to amphetamine, the stain gradually disappears from the surface and is translocated into the cell interior, indicating endocytosis of the transporter". Simultaneously, the... 

The neurotransmitter phenotype, (i.e., what type of neurotransmitter is stored and ultimately will be released from the synaptic bouton) is determined by the identity of the neurotransmitter transporter that resides on the synaptic vesicle membrane. Although some exceptions to the rule may exist all synaptic vesicles of a given neuron normally will express only one transporter type and thus will have a dehned neurotransmitter phenotype (this concept is enveloped in what is known as Dale s principle see also Reference 19). To date, four major vesicular transporter systems have been characterized that support synaptic vesicle uptake of glutamate (VGLUT 1-3), GABA and glycine (VGAT), acetylcholine (VAChT), and monoamines such as dopamine, norepinephrine, and serotonin (VMAT 1 and 2). Vesicles that store and release neuropeptides do not have specific transporters to load and concentrate the peptides but, instead, are formed with the peptides already contained within. 

The widespread presence of nicotinic receptors in the central nervous system coupled with the relative rarity of a classical synaptic role means that it is still difficult to describe a clear physiological role for these receptors. This is particularly true for receptors in presynaptic locations, for which the level and temporal pattern of exposure to the neurotransmitter are unknown. Approaches that are casting light on this problem include mouse knockout models and transmitter depletion by blockers of vesicular transport processes such as vesamicol the combination of these techniques has recently shown that normal evoked dopamine release in striatal slices is strongly dependent on endogenous cholinergic mechanisms that involve the activation of j32-containing nicotinic receptors (240). 

The pharmacology of amphetamine is considerably more complex. It does not only block monoamine reuptake, but also directly inhibits the vesicular monoamine transporter, causing an increase in cytosolic but not vesicular dopamine concentration. This may lead to reverse transport of the amines via the membrane-bound transporters. Further mechanisms of amphetamine action are direct MAO inhibition and indirect release of both dopamine and serotonin in the striatum. 

The vesicular monoamine transporters (VMATs) were identified in a screen for genes that confer resistance to the parkinsonian neurotoxin MPP+ [2]. The resistance apparently results from sequestration of the toxin inside vesicles, away from its primary site of action in mitochondria. In addition to recognizing MPP+, the transporter s mediate the uptake of dopamine, ser otonin, epinephrine, and norepinephrine by neurons and endocrine cells. Structurally, the VMATs show no relationship to plasma membrane monoamine transporters. 

Once returned to the presynaptic terminal, dopamine is repackaged into synaptic vesicles via the vesicular monoamine transporter (VMAT) or metabolized to dihydroxyphenylacetic acid (DOPAC) by monoamine oxidase (MAO). Two alternative pathways are available for dopamine catabolism in the synapse, depending on whether the first step is catalyzed by MAO or catechol-O-methyltransferase (COMT). Thus, dopamine can be either deaminated to 3,4-dihydroxyphenylacetic acid (DOPAC) or methylated to 3-methoxytyramine (3-MT). In turn, deamination of 3-MT and methylation of DOPAC leads to homovanillic acid (HVA). In humans, cerebrospinal fluid levels of HVA have been used as a proxy for levels of dopaminergic activity within the brain (Stanley et al. 1985). 

Pill, C., Drobny, H., Reither, H., Homykiewicz, O., and Singer, E.A., Mechanism of the dopaminereleasing actions of amphetamine and cocaine plasmalemmal dopamine transporter versus vesicular monoamine transporter, Mol. Pharmacol., 47, 368, 1995. 

Wilson J., Levey A., Bergeron C. et al. Striatal dopamine, dopamine transporter, and vesicular monoamine transporter in chronic cocaine users. Ann. Neurol. 40 428, 1996. 

The neuronal membrane norepinephrine transporter (NET), the dopamine transporter (DAT) and the vesicular membrane transporter (VMAT-2), which is the same in all catecholamine-containing neurons, have similar numbers of predicted transmembrane segments. They have different numbers of amino acids, pharmacological properties and chromosomal localizations. 

Pharmacologic targeting of monoamine transporters. Commonly used drugs such as antidepressants, amphetamines, and cocaine target monoamine (norepinephrine, dopamine and serotonin) transporters with different potencies. A shows the mechanism of reuptake of norepinephrine (NE) back into the noradrenergic neuron via the norepinephrine transporter (NET), where a proportion is sequestered in presynaptic vesicles through the vesicular monoamine transporter (VMAT). and C show the effects of amphetamine and cocaine on these pathways. See text for details. 

HTxR, serotonin receptor CB1R, cannabinoid-1 DAT, dopamine transporter GABA, y-aminobutyric acid Kir3 channels, G protein-coupled inwardly rectifying potassium channels LSD, lysergic acid diethylamide i -OR, H-opioid receptor nAChR, nicotinic acetylcholine receptor NET, norepinephrine transporter NMDAR, N -methyl-D-aspartate receptor SERT, serotonin transporter VMAT, vesicular monoamine transporter indicates data not available. 

Mechanism of action of cocaine and amphetamine on synaptic terminal of dopamine (DA) neurons. Left Cocaine inhibits the dopamine transporter (DAT), decreasing DA clearance from the synaptic cleft and causing an increase in extracellular DA concentration. Right Since amphetamine (Amph) is a substrate of the DAT, it competitively inhibits DA transport. In addition, once in the cell, amphetamine interferes with the vesicular monoamine transporter (VMAT) and impedes the filling of synaptic vesicles. As a consequence, vesicles are depleted and cytoplasmic DA increases. This leads to a reversal of DAT direction, strongly increasing nonvesicular release of DA, and further increasing extracellular DA concentrations. 

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