Andrea Vaccari

Selective MPP+ uptake into synaptic dopamine vesicles: possible involvement in MPTP neurotoxicity.

1 In the present study we provide evidence for a saturable, Mg2+/ATP‐ and temperature‐dependent, tetrabenazine‐, dopamine‐, and amphetamine‐sensitive uptake of 1‐methyl‐4‐phenylpyridinium ion (MPP+) in synaptic vesicles from mouse striatum. 2 Similarity in the properties of the vesicular uptake suggests that in the striatum dopamine and MPP+ share the vesicular carrier. 3 The presence of MPP+ vesicular uptake in dopamine‐rich regions such as striatum, olfactory, tubercles and hypothalamus, as well as its absence in cerebellum, cortex and pons‐medulla, suggest that monoamine vesicular carriers differ between highly and poorly dopamine‐innervated regions. 4 The restriction of active MPP+ uptake to the dopaminergic regions, which reflects the previously shown distribution of [3H]‐MPP+ binding sites in mouse brain membranes, indicates MPP+ as a marker of the vesicular carrier for dopamine in dopaminergic neurones. 5 A role in MPP+ neurotoxicity is suggested for this region‐specific, vesicular storage of the toxin.

High affinity binding of [3H]-tyramine in the central nervous system

1 Optimum assay conditions for the association of [3H]‐para‐tyramine ([3H]‐pTA) with rat brain membranes were characterized, and a saturable, reversible, drug‐specific, and high affinity binding mechanism for this trace amine was revealed. 2 The binding capacity (Bmax) for [3H]‐pTA in the corpus striatum was approximately 30 times higher than that in the cerebellum, with similar dissociation constants (KD). 3 The binding process of [3H]‐pTA involved the dopamine system, inasmuch as (a) highest binding capacity was associated with dopamine‐rich regions; (b) dopamine and pTA equally displaced specifically bound [3H]‐pTA; (c) there was a severe loss in striatal binding capacity for [3H]‐pTA and, reportedly, for [3H]‐dopamine, following unilateral nigrostriatal lesion; (d) acute in vivo reserpine treatment markedly decreased the density of [3H]‐pTA and, reportedly, of [3H]‐dopamine binding sites. 4 In competition experiments [3H]‐pTA binding sites, though displaying nanomolar affinity for dopamine, revealed micromolar affinities for the dopamine agonists apomorphine and pergolide, and for several dopamine antagonists, while having very high affinity for reserpine, a marker for the catecholamine transporter in synaptic vesicles. 5 The binding process of [3H]‐pTA was both energy‐dependent (ouabain‐sensitive), and ATP‐Mg2+‐insensitive; furthermore, the potencies of various drugs in competing for [3H]‐pTA binding to rat striatal membranes correlated well (r = 0.96) with their reported potencies in inhibiting [3H]‐dopamine uptake into striatal synaptosomes. 6 It is concluded that [3H]‐pTA binds at a site located on/within synaptic vesicles where it is involved in the transport mechanism of dopamine.

The tyramine-labelled vesicular transporter for dopamine: a putative target of pesticides and neurotoxins

This study defined the ability of a large sample of heterogeneous pesticides and neurotoxins to interact with the [3H]tyramine-labelled vesicular transporter of dopamine in rat striatum. Botanical (with rotenone as the most potent), and organochlorine (Kepone) insecticides, as well as fungicides (Zineb), as a whole, consistently inhibited [3H]tyramine binding, with Ki values ranging from 5 nM to 10 microM. ATP/Mg(2+)-dependent [3H]tyramine uptake to purified striatal synaptic vesicles was also inhibited by rotenone. Organophosphate and carbamate insecticides, and miscellaneous herbicides poorly antagonized [3H]tyramine binding, yielding Ki values exceeding 10 microM. Several, though not all, of the best recognized central neurotoxins tested were major binding antagonists. Their rank order of potency was 1-methyl-4-phenylpyridinium ion (MPP+) > trimethyltin > or = 6-hydroxydopamine > N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) > 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), with Ki values ranging from 35 nM to 3 microM. Overall, the potent interaction of selected pesticides and chemicals with the vesicular transporter for dopamine, although, by itself, not synonymous with neurotoxicity, would argue for a likely impairment of transmitter homeostasis, or the putative formation of neurodegenerative toxin pools.

Interaction of 1-methyl-4-phenylpyridinium ion and tyramine with a site putatively involved in the striatal vesicular release of dopamine.

The neurotoxin MPP+ potently inhibited the striatal binding of [3H]‐tyramine, a putative marker for the vesicular transporter of dopamine, and provoked a massive in vivo release of striatal dopamine. Tetrabenazine, an established ligand for the vesicular catecholamine carrier, potently inhibited [3H]‐tyramine binding, tyramine‐provoked striatal efflux of dopamine and the fast component of MPP+‐induced dopamine release. It is concluded that MPP+ in the striatum, besides interacting with additional intracellular targets, avidly binds at a vesicular site functionally involved with the outward transport of dopamine.

The tyramine binding site in the central nervous system: An overview

The [3H]Tyramine (TY) binding site is proposed as a high affinity marker of the membrane carrier for dopamine (DA) in synaptic vesicles from DA-rich brain regions. Under precise assay conditions, there is neither a consistent association of TY with the neuronal, cocaine-sensitive DA transporter, nor with mitochondrial or microsomal targets. TY-labeled sites have a high affinity for selected toxins such as the Parkinsonian agent MPP+ (1-methyl-4-phenylpyridinium ion), or drugs such as diphenylalkylamine Ca2+-channel antagonists. The MPP+/TY site interaction, which in the striatum leads to depletion of vesicular DA, occurs in dopaminergic as well as in noradrenergic regions, though with different kinetic profiles. TY-labeled carriers for DA and noradrenaline (NA) in respective vesicles seem to be different entities, which might result in a region-specific rate of toxin sequestration and/or release from heterogeneous vesicles. Whereas MPP+ is a potent competitive-type inhibitor of [3H]TY binding, prenylamine-like Ca2+-channel antagonists can compete with TY for the vesicle site, in a tetrabenazine- or reserpine-like manner, and also inhibit TY binding thanks to the extra-channel directed impairment of membrane bioenergetics they are proposed to provoke. This follows from the generally-accepted assumption that similar mechanisms are operational for secretory organelles in adrenals and CNS, and from the marked sensitivity of TY binding to miscellaneous energy-disrupting agents. A model is therefore proposed, depicting the TY-, DA- or MPP+-labeled, vesicle carrier, as a dimeric protein which may switch from the cytoplasm-oriented, “recognition” state, to the vesicle-oriented, “transport” state, thanks to the establishment of an H+-ATPase-supported, membrane protein electrochemical gradient.

Selective effects of thiol reagents on the binding sites for imipramine and neurotransmitter amines in the rat brain.

1 The action of the antithyroid drugs methimazole (MMI) and propylthiouracyl (PTU) on the binding of [3H]‐imipramine, [3H]‐5‐hydroxytryptamine ([3H]‐5‐HT) (to 5‐HT1‐receptors) and [3H]‐spiperone (to 5‐HT2‐, D2‐receptors) of rat brain membranes has been examined. The synaptosomal uptake of [3H]‐5‐HT was also studied. 2 Micromolar concentrations of the disulphide bond reducing agents MMI, PTU, dithiothreitol (DTT) and mercaptoethanol increased both the binding of [3H]‐imipramine and the uptake of [3H]‐5‐HT. In contrast, they decreased the number of 5‐HT1‐receptors, and did not affect 5‐HT2‐ and D2‐sites. 3 Reaction with membrane‐bound sulphydryl (SH) groups by micromolar concentrations of N‐ethylmaleimide (NEM), hydroxymercuribenzoic acid (PCMB), or Ellmans reagent (DTNB) decreased the binding of [3H]‐imipramine, the number of 5‐HT1‐receptors, and the uptake of [3H]‐5‐HT. Millimolar concentrations of NEM were necessary in order to decrease partially 5‐HT2‐ and D2‐receptors. 4 The effects of NEM on imipramine recognition sites and on the uptake of 5‐HT could be prevented by DTT; protection was not obtained in other receptor systems. 5 Three groups of receptors have been, thus, postulated, based upon their different sensitivity towards alterations in membrane [disulphide bridges SH] equilibrium: Group I, including imipramine recognition sites and the uptake system for 5‐HT; Group II, including 5‐HT]‐receptors; Group III, including 5‐HT2‐ and D2‐receptors.

Prenatal low-level exposure to CO alters postnatal development of hippocampal nitric oxide synthase and haem-oxygenase activities in rats.

The effects of prenatal CO exposure (150 ppm from days 0 to 20 of pregnancy) on the postnatal development of hippocampal neuronal NO synthase (nNOS) and haem-oxygenase (HO-2) isoform activities in 15-, 30- and 90-d-old rats were investigated. Unlike HO-2, hippocampal nNOS activity increased from postnatal days 15-90 in controls. Prenatal CO produced a long-lasting decrease in either nNOS or HO-2. The results suggest that the altered developmental profile of hippocampal nNOS and HO-2 activities could be involved in cognitive deficits and long-term potentiation dysfunction exhibited by rats prenatally exposed to CO levels resulting in carboxyhaemoglobin (HbCO) levels equivalent to those observed in human cigarette smokers.

[3H]Tyramine binding: A comparison with neuronal [3H]-dopamine uptake and [3H]mazindol binding processes

Abstract[3H]Spiperone ([3H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [3H]tyramine ([3H]PTA), thus indicating that [3H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [3H]PTA binding and [3H]DA uptake processes were markedly decreased, with no effect on [3H]mazindol ([3H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [3H]PTA binding, did not affect the [3H]MAZ binding process. This further supported the suggestion that while [3H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [3H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.

Effects of neonatal antithyroid treatment on brain [3H]-imipramine binding sites

1 The action of the antithyroid, sulphydryl reagent methimazole (MMI) on the specific binding of [3H]‐imipramine in the cerebral cortex and corpus striatum of immature and mature rats has been examined. 2 Chronic administration of MMI through the first 30 days of life decreased the number of imipramine binding sites in cortical but not striatal membranes, as assessed 48 h after the last injection of goitrogen. 3 A similar treatment did not affect the binding profile of [3H]‐imipramine in mature rats. 4 Acute administration of MMI to 30 day‐old rats increased the number of imipramine binding sites shortly after the injection, an effect no longer evident 48 h later. 5 MMI in vitro increased the binding of [3H]‐imipramine. 6 It is concluded that maturational impairment of the hypothyroid cortex, rather than any alteration of membrane bound thiol groups, was a major cause for the diminished binding of [3H]‐imipramine in MMI‐treated, immature rats.

High Affinity Binding of p-Tyramine: A Process in Search of a Function

The presence of trace amines in the CNS of different animal species, their interactions with monoaminergic pathways, and their intrinsic pharmacological activities have justified recent efforts to ascertain whether they may play a neurotransmitter role. The interaction with specific binding sites is a major requisite for a neurotransmitter, though “binding site” does not mean “receptor”, i.e. a physiologically-relevant entity (Laduron, 1984). Additionally, the candidate transmitter must be taken up in the synapse with an energy-dependent process, and must also be released from storage organelles. Para-tyramine (pTA) is actively taken up by nerve tissue preparations such as brain slices (Dyck, 1978), synaptosomes (Ungar et al., 1977) and synaptic vesicles (Lentzen and Philippu, 1977), from where it also under goes both spontaneous and stimulus-evoked release (Lentzen and Philippu, 1977; Dyck, 1984). Furthermore, pTA displays electrophysiologic (Jones, 1984), behavioral (Stoof et al., 1976) and neuroendocrine (Becu-Villalobos et al., 1985) activities.