Intranigral substance P modulation of striatal dopamine: interaction with N-terminal and C-terminal substance P fragments.

The effects of unilateral injections of two substance P fragments, the N-terminal substance P (1-7) (SP1-7) and the C-terminal substance P (6-11) (SP6-11) into the substantia nigra, pars reticulata on dopamine (DA) release in the ipsilateral striatum of halothane-anaesthetized rats were studied using microdialysis. SP1-7 and SP6-11 were also tested for their ability to modify the DA stimulation produced by intranigral injections of SP or neurokinin A (NKA). In addition, the SP antagonist Spantide I was tested for its ability to modify the DA stimulation produced by an intranigral injection of SP1-7. Intranigral injections of SP1-7 (0.001-5.0 nmol) inhibited DA release after low doses (0.001-0.01 nmol), but stimulated DA release after high doses (0.1-5.0 nmol). Striatal dihydroxyphenylacetic acid (DOPAC) levels increased moderately after high doses of SP1-7 (1.0-5.0 nmol). Intranigral injections of SP6-11 (0.01-5.0 nmol) inhibited DA release, but enhanced striatal DOPAC levels, dose-dependently. SP1-7 (0.01-0.1 nmol), but not SP6-11 (0.1 nmol), blocked the stimulation of striatal DA release produced by intranigral SP (0.07 nmol). Neither SP1-7 (0.1 nmol) nor SP6-11 (0.1 nmol) could modify the stimulation of striatal DA release produced by intranigral NKA (0.09 nmol). The increase in DA release after a high dose of SP1-7 (1.0 nmol) was not modified by co-administration with Spantide I (0.07 nmol).(ABSTRACT TRUNCATED AT 250 WORDS)     

N- and C-terminal fragments of substance P: spinal effects in the rat tail flick test

Evidence exists to suggest that within the CNS, substance P may be enzymatically cleaved into fragments which may mediate some of the effects of substance P. As we have previously reported on the spinal effects of substance P, the present study examines the effects of selected substance P fragments on reaction time in the tail flick test. Peptides were administered via a chronically implanted intrathecal catheter to the L5 vertebral level in the rat. Administration of 6.5 nmoles of SP(1-7) produced a transient decrease in reaction time at 1 min after injection with a return to above control values by 5 min. Similar administration of SP(7-11) produced a smaller decrease in reaction time at 6 min which lasted until 16 min. Administration of 6.5 nmoles of SP(1-9), SP(8-11) and of CSF were without effect. As the effects of SP(1-7) on reaction time resembled those of similar administration of substance P in the earlier experiments, these results suggest that this fragment may be the active component involved in facilitating the tail flick reflex. Substance P may be degraded to the active fragment prior to receptor activation or alternatively, substance P and SP(1-7) may act on the same receptor.     

Specific labeling of rat brain substance P receptor with [3H]physalaemin

The binding of [3H]physalaemin [( 3H]PHY) to rat brain membranes is specific, saturable and reversible in the presence of monovalent cations and peptidase inhibitors. Monovalent cations increase the binding of [3H]PHY in an ionic strength (mu)-dependent manner with an optimal effect at mu higher than 0.3. Addition of 2.5 mM MnCl2 results in a 2-fold increase in the affinity (KD) and a 40% increase in the maximal receptor density (Bmax). Scatchard analysis under these conditions indicates the existence of a single population of noninteracting sites with KD of 3.6 nM and a Bmax of 76 fmol/mg of protein. Substance P (SP) and physalaemin are equipotent in inhibiting the binding of [3H]PHY, whereas the potency of SP(2-11), SP(3-11), and SP(4-11) decreased in inverse proportion to their length. The relative affinity of the different tachykinins, SP, and SP fragments in competing with [3H]PHY correlates with their potency to stimulate several bioassay systems, indicating that [3H]PHY labels a physiologically relevant binding site that correspond to the SP-P tachykinin receptor. Guanine nucleotides completely abolish the increase in the binding of [3H]PHY produced by 2.5 mM MnCl2, but in its absence, the nucleotides reduce binding only by 15%. Guanine nucleotides reduce binding to the same level regardless of the presence or absence of the divalent cation. Regional distribution studies confirm that the density of SP receptors is maximal in the olfactory bulb, followed by the hypothalamus, striatum, hippocampus, cortex, and cerebellum.     

Sparing of striatal neurons coexpressing calretinin and substance P (NK1) receptor in Huntington''s disease

Immunohistochemical studies of the striatum in normal human subjects with a double-antigen localization method have revealed the presence of large and medium-sized aspiny neurons displaying immunoreactivity for both the calcium-binding protein calretinin and substance P (neurokinin-1) receptor. These large and medium-sized cells form two distinct classes of striatal interneurons, which together represent less than 3% of the total neuronal population of the human striatum. Observations made in four cases of Huntington's disease revealed that such doubly labeled interneurons are still present in the striatum of these patients, despite the marked atrophy of the structure. This study provides the first evidence for the existence of interneurons containing calretinin and expressing tachykinin receptors in the human striatum. It also demonstrates the selective sparing of these chemospecific striatal neurons in Huntington's disease.     

Reduction of [3H]substance P binding in the intermediolateral cell column after sympathectomy

Guanethidine-induced destruction of sympathetic postganglionic neurons in neonatal rats leads to transneuronal degeneration of the sympathetic preganglionic neurons. Using this model, we have been able to show a approximately 35% decrease in [3H]substance P ([3H]SP) binding in the intermediolateral cell column--suggesting that sympathetic preganglionic neurons possess substance P receptors. Our results show that [3H]substance P binding in the intermediolateral cell column is dependent on the integrity of sympathetic postganglionic neurons.     

Substance P fragments and striatal endogenous dopamine outflow: interaction with substance P

Accumulating evidence shows that N- and C-terminal substance P fragments have significant biological activity. Substance P(1–9) and substance P(6–11) have been reported to be major substance P metabolites in rat striatum. We investigated the effects of these fragments on endogenous dopamine outflow in rat striatal slices. Substance P-(1–9) and substance P-(6–11) induced a significant increase in dopamine outflow at 0.1 and 1 nM. The effects of substance P-(6–11) (1 nM) were reversed by the tachykinin NK₁ antagonist WIN 51,708 (17β-hydroxy-17α-ethynyl-5α-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) (2.5 nM), whereas the effects of substance P-(1–9) were not modified by the antagonist. Substance P-(1–9) and substance P-(6–11) (1 nM) did not increase the dopamine overflow induced by 25 mM KCl. The effects of the two fragments were reversed by the muscarinic antagonist atropine (1 μM) but not by nicotinic antagonists dihydro-β-erythroidine (0.5 μM) and pempidine (10 μM). The co-incubation of tissue with substance P and each fragment in a 1/1 or 10/1 ratio of substance P to metabolite revealed a negative interaction between parent and fragments. A similar pattern was observed when substance P was co-administered with the active fragments substance P(1–4), substance P(1–7), substance P(5–11) and substance P(8–11). The data show that substance P-(1–9) and substance P-(6–11) have modulatory effects similar to substance P. However, the presence of active substance P metabolites does not appear to amplify the signal mediated by the parent peptide.     

Modulation of [3H]DAGO binding by substance P (SP) and SP fragments in the mouse brain and spinal cord via MU1 interactions

Binding of [3H]DAGO to fresh, frozen or beta-funaltrexamine (beta-FNA) pretreated membranes of mouse brain and spinal cord was extensively studied using substance P (SP) or SP fragments as potential competitors and/or modulators. The objective was to determine whether SP exerts its analgesic effect by interacting with mu opioid receptors. The affinity of DAGO was reduced and binding capacity was increased in the presence of SP or the N-terminal SP fragments SP(1-9) and SP(1-4) but not the C-terminal SP fragment SP(5-11). Because sub-nanomolar concentrations of SP or N-terminal SP fragments displaced [3H] DAGO binding to a minor but detectable degree, it is suggested that SP interacts with mu 1 sites through its N-terminus portion. The effect of SP on DAGO binding was less in the spinal cord compared to the rest of the brain. Modulation of DAGO binding by SP was enhanced in the brain after pretreatment of membranes with the narcotic antagonist beta-FNA. These results suggest a novel mechanism for the analgesic action of SP.     

3H]-etorphine and [3H]-diprenorphine receptor binding in vitro and in vivo: differential effect of Na+ and guanylyl imidodiphosphate

The in vivo and in vitro cerebral receptor binding kinetics of the opiate agonist etorphine and the antagonist diprenorphine were investigated in the rat. Although of similar receptor affinity in vitro in Tris buffer brain homogenates, etorphine exhibited considerably less affinity than diprenorphine in vivo. The hypothesis was tested whether the opiate receptor regulators, Na⁺ and GTP, are responsible for the low in vivo receptor affinity of the agonist. [³H]Etorphine and [³H]diprenorphine dissociation curves were similarly affected by Na⁺ and guanylyl imidodiphosphate (GPP(NH)P), a hydrolysis resistant GTP analog when added separately in vitro. However, the combination of Na⁺ and GPP(NH)P greatly accelerated only the [³H]etorphine off-rate over that with Na⁺ alone and reproduced the rapid dissociation half-life observed in vivo (t_1/2 < 1 min). In contrast, the receptor dissociation rate of the antagonist was not further accelerated by Na⁺ plus GPP(NH)P over that with Na⁺ alone. Moreover, Na⁺ plus GPP(NH)P decreased [³H]etorphine, but not [³H]diprenorphine, equilibrium binding in vitro. These results suggested that the lower in vivo affinity of etorphine than of diprenorphine is predominantly caused by the combined action of Na⁺ and GTP. Furthermore, the data are consistent with the hypothesis that both etorphine and diprenorphine bind to the opiate receptor in its high affinity form, but that only the agonist etorphine is capable of converting the high affinity form to one of low affinity in the presence of Na⁺ and guanine nucleotide. Assuming that production of the low affinity form reflects biological activation of the opiate receptor system, then this hypothesis is consistent with the high pharmacological potency of etorphine (agonistic ED₅₀ ～ 1μg/kg) relative to its low apparent in vivo receptor affinity, as well as with the low fractional receptor occupancy of etorphine (～ 2%) at its analgesic ED₅₀. Finally, in vivo [³H]etorphine and [³H]diprenorphine displacement curves obtained with unlabeled diprenorphine and etorphine showed that [³H]etorphine labels only a subpopulation of the total [³H]diprenorphine binding sites. It remains to be determined which subsites of the opiate receptor system mediate the agonistic actions of etorphine.     

[H]-etorphine and [H]-diprenorphine receptor binding in vitro and in vivo: differential effect of Na and guanylyl imidodiphosphate

The in vivo and in vitro cerebral receptor binding kinetics of the opiate agonist etorphine and the antagonist diprenorphine were investigated in the rat. Although of similar receptor affinity in vitro in Tris buffer brain homogenates, etorphine exhibited considerably less affinity than diprenorphine in vivo. The hypothesis was tested whether the opiate receptor regulators, Na⁺ and GTP, are responsible for the low in vivo receptor affinity of the agonist. [³H]Etorphine and [³H]diprenorphine dissociation curves were similarly affected by Na⁺ and guanylyl imidodiphosphate (GPP(NH)P), a hydrolysis resistant GTP analog when added separately in vitro. However, the combination of Na⁺ and GPP(NH)P greatly accelerated only the [³H]etorphine off-rate over that with Na⁺ alone and reproduced the rapid dissociation half-life observed in vivo (t_1/2 < 1 min). In contrast, the receptor dissociation rate of the antagonist was not further accelerated by Na⁺ plus GPP(NH)P over that with Na⁺ alone. Moreover, Na⁺ plus GPP(NH)P decreased [³H]etorphine, but not [³H]diprenorphine, equilibrium binding in vitro. These results suggested that the lower in vivo affinity of etorphine than of diprenorphine is predominantly caused by the combined action of Na⁺ and GTP. Furthermore, the data are consistent with the hypothesis that both etorphine and diprenorphine bind to the opiate receptor in its high affinity form, but that only the agonist etorphine is capable of converting the high affinity form to one of low affinity in the presence of Na⁺ and guanine nucleotide. Assuming that production of the low affinity form reflects biological activation of the opiate receptor system, then this hypothesis is consistent with the high pharmacological potency of etorphine (agonistic ED₅₀ 1μg/kg) relative to its low apparent in vivo receptor affinity, as well as with the low fractional receptor occupancy of etorphine ( 2%) at its analgesic ED₅₀. Finally, in vivo [³H]etorphine and [³H]diprenorphine displacement curves obtained with unlabeled diprenorphine and etorphine showed that [³H]etorphine labels only a subpopulation of the total [³H]diprenorphine binding sites. It remains to be determined which subsites of the opiate receptor system mediate the agonistic actions of etorphine.     

The characterization of [3H]sulpiride binding sites in rat striatal membranes

The characteristics of [3H]sulpiride binding to the D2 dopamine receptor in rat striatal membranes were examined under several conditions. In the absence of sodium ions, the specific binding of [3H]sulpiride could not be detected. In the direct binding experiment, at 25 degrees C, the affinity of [3H]sulpiride for D2 receptors was increased in a dose-dependent manner of sodium ions whereas magnesium ions have opposite effects on an affinity of [3H]sulpiride binding. The lowering incubation temperature (4 degrees C) also produced a further increase in affinity of the ligand. Under all conditions, [3H]sulpiride labeled a single homogenous site of the receptor. On the other hand, the result from quantitative analysis of agonist/[3H]sulpiride competition curves indicated an existence of high (RH) and low (RL) affinity states for agonists and the proportion of two-affinity states was modulated by guanosine triphosphate (GTP), magnesium ions and lowering temperatures. GTP, together with sodium ions, caused a full conversion of RH to RL, while an increase in the affinity for agonists with a partial conversion of RL to RH could be induced by magnesium ions at 25 degrees C. At a lower (4 degrees C) temperature, the agonist competition curve indicated an existence of a single agonist low-affinity state (RL) and then, the effects of GTP and magnesium ions in the agonist affinity observed at 25 degrees C were abolished. These results can be incorporated into a two-step, ternary complex model involving an inhibitory guanine nucleotide binding protein for the agonist and antagonist interaction with D2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased [3H]glutamate receptor binding in aged rats

Na-independent [3H]glutamate binding to rat hippocampal membranes increases progressively as a function of age. The increased binding represents an increased number of binding sites without changes in their apparent affinity for glutamate. However, [3H]glutamate binding, measured with a saturating concentration of calcium does not change at various ages. This does not reflect a change in the apparent affinity of calcium ions to stimulate [3H]glutamate binding, but a decrease in their maximal stimulatory effect. These results are discussed in relationship to age-related changes in certain physiological and behavioral functions.     

Distinct kinetic binding propeties of N-[3H]-methylscopolamine afford differential labeling and localization of M1, M2, and M3 muscarinic receptor subtypes in primate brain

Three classes of muscarinic receptors in mammalian brain have been postulated on the basis of equilibrium and kinetic binding data. However, equilibrium binding assays alone have not permitted a clear demonstration of the localization of putative M1, M2, and M3 receptor subtypes in the brain because of the overlaping affinities of virtually all muscarinic antagonists. In the present study, the conditions for selective occupancy of the M1, M2, and M3 receptor subtypes in the brain of the rhesus monkey were based on the distinct kinetic and equilibrium binding properties of N-[³H]-methylscopolamine (NMS) at cloned m1–m4 muscarinic receptor subtypes expressed in A9L transfected cells. Quantitative autoradiography of the M1, M2, and M3 muscarinic receptor subtypes in the primate brain was performed according to the following strategy. The M1 (m1) receptor subtype was labeled directly with a non-saturating concentration of [³H]-pirenzepine. The M2 (m2) subtype was labeled by incubations consisting of short, two minute pulses of [³H]-NMS after a preincubation with 0.3 μM pirenzepine to occlude m1, m3, and m4 sites. Selective occupancy of the M3 (m3) receptor (subtype) was achieved by pre-incubation with 0.5 nM unlabeled NMS to partially occlude the m1, m2, and m4 sites, equilibrium with 0.5 nM [³H]-NMS, followed by a 60 minute tracer dissociation in the presence of 1 μM atropine. In vitro autoradiography demonstrated that the M1 receptor subtype was confined to forebrain structures. M1 receptors were prevalent throughout the cerebral cortical mantle, amygdala, hippocampus, and the striatum. Low to background levels of the M1 receptor subtype were measured over the thalamus, hypothalamus, and brainstem. The M2 subtype was widely distributed with elevated densities of binding sites seen over all primary sensory cortical areas, and within discrete thalamic, hypothalamic, and brainstem nuclei. The distribution of the M3 receptor subtype was largely coincident with the pattern of the M1 sites labeled by non-saturating concentrations of [³H]-pirenzepine with some notable exceptions. Within the cerebral cortical mantle, the M3 receptor exhibited an elevated gradient over the orbitofrontal gyrus and the temporal lobe. Within the striatum, the M3 subtype was elevated over the anterior and dorsal part of the caudate nucleus, while the M1 receptors were most prevalent over the ventromedial sector. Selective labeling of M3 receptors was seen over the medial division of the globus pallidus and within the substantia nigra pars reticulata. In contrast to the pattern of the M1 receptor subtype, M3 receptors were prevalent also over midline nuclei of the hypothalamus. These results demonstrate that the distinct kinetic and equilibrium binding profiles of N-methylscopolamine and pirenzepine for cloned muscarinic receptors provide a viable ligand autoradiographic strategy for mapping the distribution of M1, M2, and M3 receptors in brain. © 1993 Wiley-Liss, Inc.     

[H]substance P binding in the intermediolateral cell column and striatum of the rat

[³H]substance P binding was studied in the intermediolateral cell column and striatum in the rat using slide-mounted sections. The intermediolateral cell column had a single high affinity binding component with a dissociation constant,K_d = 1.45 nM and the number of sites,B_max= 18.1fmol per mg protein. The striatum had aK_d = 0.77nM and aB_max= 23.5fmol per mg protein. The relative potency of various substance P-like tachykinins in displacing [³H]substance P suggested that both these areas may contain a substance P-P (for physalaemin) receptor subtype. (d-Pro⁴,d-Trp^7,9)substance P(4–11), a substance P antagonist, has a relatively low affinity (micromolar range) in both these areas.     

Difference in in vivo receptor binding between [3 H]N-methylspiperone and [3 H]raclopride in reserpine-treated mouse brain

Summary The in vivo binding of [³ H]N-methylspiperone (NMSP) and [³ H]raclopride was compared in mice treated with reserpine (5 mg/kg, 24 hr prior to the tracer injection). With both radioligands, selective accumulation of radioactivity in the striatum following intravenous injection was observed, whereas a relatively low accumulation and a rapid decline in radioactivity in the cerebellum was seen. Reserpine significantly decreased [³ H]NMSP binding in vivo, however it increased [³ H]raclopride binding. By compartment model analysis, it was found that the decrease in [³ H]NMSP binding was primarily due to the decrease in the association rate (K3) and the increase in [³ H]raclopride was due to the decrease in the dissociation rate (K4) in vivo. As both Kd and Bmax of dopamine D2 receptors have been reported to be unaltered by reserpine, these results suggested that some unknown factors except Kd and Bmax which influence on in vivo binding of receptors might be changed by reserpine. These results revealed that it is of importance to measure kinetics of ligand-receptor binding in vivo rather than static analysis. These two different types of radioligands can be combined to reveal functional roles of dopamine receptor in vivo, especially in the study of the human brain with positron emission tomography (PET).     

Ontogeny of adenosine uptake sites in guinea pig brain: differential profile of [3H]nitrobenzylthioinosine and [3H]dipyridamole binding sites

The ontogenetic profile of adenosine uptake sites was investigated in guinea pig cerebral cortex and cerebellum using as ligand probes the uptake inhibitors, [3H]nitrobenzylthioinosine ([3H]NBI) and [3H]dipyridamole ([3H]DPR). In cerebral cortex [3H]NBI binding was highest at E50 and decreased subsequently until P28 while in cerebellum after a first peak at E50 and a subsequent decline it increased again until P28. [3H]DPR binding increased by 25% from E40 to P28 in cerebral cortex while in cerebellum hardly any binding could be detected before E50 and it afterwards increased by more than 250% until P28. Scatchard analysis demonstrated that [3H]NBI labeled approximately as many sites as [3H]DPR in cerebral cortex at E44 while at P28 [3H]DPR labeled more than double as many sites. Accordingly, NBI was more potent in displacing [3H]DPR binding at E44 than at P28. These findings suggest that part of the [3H]DPR binding sites, i.e. the NBI-insensitive one develops later than [3H]NBI binding sites during ontogeny in guinea pig cerebral cortex and cerebellum.     

Effects of dopaminergic agonists and antagonists on [H]apomorphine binding to striatal membranes: sulpiride lack of interactions with positive cooperative [H]apomorphine binding

Effects of dopaminergic agonists and antagonists on [³H]apomorphine binding to striatal membranes of rat brain was examined. Haloperidol and spiroperidol exhibited biphasic inhibition of [³H]apomorphine binding; one of which had the Hill coefficient of 0.9, whereas the other had that of 0.4. The former accounted for 65% of [³H]apomorphine binding while the latter consisted of 35% of the binding. Furthermore, the latter disappeared after kainic acid lesions. On the other hand, sulpiride and metoclopramide reduced [³H]apomorphine binding to 31% with the Hill coefficient of 0.9. The inhibition of [³H]apomorphine binding with the Hill coefficient of 0.4 which was shown by haloperidol and spiroperidol was not observed for sulpiride and metoclopramide.Previously, we demonstrated non- and positive-cooperative [³H]apomorphine binding to striatal membranes. In the present study, it has been also shown that sulpiride inhibits non-cooperative [³H]apomorphine binding leaving that with allosteric properties unaffected. No inhibition of dopamine-sensitive adenylate cyclase was observed by 10⁻⁴ M sulpiride while 90% inhibition was obtained with 10⁻⁵ M haloperidol. From those results, it is suggested that non-cooperative [³H]apomorphine binding is not coupled with dopamine-sensitive adenylate cyclase.     

Folates: epileptogenic effects and enhancing effects on [3H]TBOB binding to the GABAA-receptor complex

The biochemical mechanism responsible for the convulsive effects of folates was investigated. The epileptogenic effects of folates were determined in vivo by quantification of the seizures following intracortical application in rats. The rank order of epileptogenic effects is: folic acid greater than or equal to 5-HCO-H4 folate greater than H2 folate greater than 5-CH3-H4 folate. This sequence of epileptogenicity in vivo is compared to the rank order of the effects of folates on radioligand binding to the GABAA-receptor complex in vitro. The inhibitory potencies of folates on [3H]muscimol and [3H]diazepam bindings did not correlate with their epileptogenic effects. However, folates reverse the inhibiting effect of GABA on the binding of the cage convulsant [3H]TBOB [( 3H]t-butylbicycloorthobenzoate). The rank order of this in vitro effect (folic acid greater than 5-HCO-H4 folate greater than H2 folate = 5-CH3-H4 folate) resembles the rank order of epileptogenicity determined in vivo. A relationship between the in vivo and in vitro effects is therefore suggested.     

Antipsychotic drug administration differentially affects [3H]muscimol and [3H]flunitrazepam GABA(A) receptor binding sites

Post-mortem studies of the human brain indicate that certain GABA(A) receptor subtypes may be differentially altered in schizophrenia. Increased binding to the total population of GABA(A) receptors using [3H]muscimol is observed in the post-mortem schizophrenic brain, yet a proportion of these receptors which bind benzodiazepines and are labelled with [3H]flunitrazepam, show decreased or unaltered expression. Data from animal studies suggest that antipsychotic drugs alter GABA(A) receptor expression in a subtype selective manner, but in the opposite direction to that observed in schizophrenia. To broaden our understanding of the effects of antipsychotic drugs on GABA(A) receptors, we examined the saturation binding maximum (B(max)) and binding affinity (K(D)) of [3H]muscimol and [3H]flunitrazepam in the prefrontal cortex (PFC), hippocampus and thalamus of male SD rats that received a sucrose solution containing either haloperidol (1.5 mg/kg), olanzapine (6.5 mg/kg) or no drug daily for up to 28 days using quantitative receptor autoradiography. [3H]Muscimol binding density was increased most prominently in the PFC after 7 days, with larger and more prolonged effects being induced by the atypical antipsychotic drug olanzapine in subcortical regions. While no changes were observed in [3H]muscimol binding in any region after 28 days of drug administration, [3H]flunitrazepam binding density (B(max)) was increased for both antipsychotic treatments in the PFC only. These findings confirm that the subset of GABA(A) receptors sensitive to benzodiazepines are regulated differently from other GABA(A) receptor subtypes following antipsychotic drug administration, in a time- and region-dependent manner.     

Characterization of a putatively vesicular binding site for [3H]MPP+ in mouse striatal membranes.

[3H] N-Methyl-4-phenylpyridinium ion (MPP+) binds with a fully reversible, high affinity process to a population of sites mainly localized in the mouse striatum (Bmax = 168 +/- 15 fmol/mg protein, KD = 1.4 +/- 0.4 nM). The majority of specifically-bound radioactivity was localized in the synaptosomal fraction. Unilateral, striatal denervation with 6-hydroxydopamine (6-OHDA) markedly (by 65-70%) decreased the number of [3H]MPP+ sites. Besides dopamine, the vesicular markers tyramine, tetrabenazine and reserpine inhibited [3H]MPP+, while mazindol was a poor displacer. Adenosine triphosphate (ATP) and Mg(2+)-ions did not affect [3H]MPP+ binding. It is concluded that these sites may represent a marker of striatal storage vesicles for dopamine.