MPTP-induced model of Parkinson's disease in cytochrome P450 2E1 knockout mice.

Evidence for involvement of cytochrome P450 2E1 in the MPTP-induced mouse model of PD has been reported [Vaglini, F., Pardini, C., Viaggi, C., Bartoli, C., Dinucci, D., Corsini, G.U., 2004. Involvement of cytochrome P450 2E1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease. J. Neurochem. 91, 285-298]. We studied the sensitivity of Cyp2e1(-/-) mice to the acute administration of MPTP in comparison with their wild-type counterparts. In Cyp2e1(-/-) mice, the reduction of striatal DA content was less pronounced 7 days after MPTP treatment compared to treated wild-type mice. Similarly, TH immunoreactivity analysis of the substantia nigra of Cyp2e1(-/-) mice did not show any neuronal lesions after MPTP treatment. In contrast to this, wild-type animals showed a minimal but significant lesioning by the toxin as evaluated also by means of non-stereologic computerized assisted analysis of this brain area. Striatal levels of DA metabolites after 7 days were variably affected by the toxin, but consistent differences between the two animal strains were not observed. We evaluated short-term changes in the levels of striatal DA and its metabolites, and we monitored striatal MPP(+) levels. Striatal MPP(+) was cleared more rapidly in Cyp2e1(-/-) mice than in wild-type animals and, consistently, striatal DA content decreased faster in Cyp2e1(-/-) mice than in wild-type animals, and 3-methoxytyramine and HVA levels showed an early and sharp rise. Our findings suggest that Cyp2e1(-/-) mice are weakly sensitive to MPTP-induced brain lesions, markedly in contrast with a protective role of the enzyme as suggested previously. The differences observed between the knockout mice and their wild-type counterparts are modest and may be due to an efficient compensatory mechanism or genetic drift in the colonies.

Increased susceptibility to kainic acid-induced seizures in Engrailed-2 knockout mice.

The En2 gene, coding for the homeobox-containing transcription factor Engrailed-2 (EN2), has been associated to autism spectrum disorder (ASD). Due to neuroanatomical and behavioral abnormalities, which partly resemble those observed in ASD patients, En2 knockout (En2(-/-)) mice have been proposed as a model for ASD. In the mouse embryo, En2 is involved in the specification of midbrain/hindbrain regions, being predominantly expressed in the developing cerebellum and ventral midbrain, and its expression is maintained in these structures until adulthood. Here we show that in the adult mouse brain, En2 mRNA is expressed also in the hippocampus and cerebral cortex. Hippocampal En2 mRNA content decreased after seizures induced by kainic acid (KA). This suggests that En2 might also influence the functioning of forebrain areas during adulthood and in response to seizures. Indeed, a reduced expression of parvalbumin and somatostatin was detected in the hippocampus of En2(-/-) mice as compared to wild-type (WT) mice, indicating an altered GABAergic innervation of limbic circuits in En2(-/-) mice. In keeping with these results, En2(-/-) mice displayed an increased susceptibility to KA-induced seizures. KA (20 mg/kg) determined more severe and prolonged generalized seizures in En2(-/-) mice, when compared to WT animals. Seizures were accompanied by a widespread c-fos and c-jun mRNA induction in the brain of En2(-/-) but not WT mice. Long-term histopathological changes (CA1 cell loss, upregulation of neuropeptide Y) also occurred in the hippocampus of KA-treated En2(-/-) but not WT mice. These findings suggest that En2(-/-) mice might be used as a novel tool to study the link between epilepsy and ASD.

CYP 2E1 mutant mice are resistant to DDC-induced enhancement of MPTP toxicity.

In order to reach a deeper insight into the mechanism of diethyldithiocarbamate (DDC)-induced enhancement of MPTP toxicity in mice, we showed that CYP450 (2E1) inhibitors, such as diallyl sulfide (DAS) or phenylethylisothiocyanate (PIC), also potentiate the selective DA neuron degeneration in C57/bl mice. Furthermore we showed that CYP 2E1 is present in the brain and in the basal ganglia of mice (Vaglini et al., 2004). However, because DAS and PIC are not selective CYP 2E1 inhibitors and in order to provide direct evidence for CYP 2E1 involvement in the enhancement of MPTP toxicity, CYP 2E1 knockout mice (GONZ) and wild type animals (SVI) of the same genetic background were treated with MPTP or the combined DDC + MPTP treatment. In CYP 2E1 knockout mice, DDC pretreatment completely fails to enhance MPTP toxicity, although enhancement of MPTP toxicity was regularly present in the SVI control animals. The immunohistochemical study confirms our results and suggests that CYP 2E1 may have a detoxifying role.

Cytochrome P450 and Parkinson's disease: protective role of neuronal CYP 2E1 from MPTP toxicity.

Elucidation of the biochemical steps leading to the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP)-induced degeneration of the nigro-striatal dopamine (DA) pathway has provided new clues to the pathophysiology of Parkinson's Disease (PD). In line with the enhancement of MPTP toxicity by diethyldithiocarbamate (DDC), here we demonstrate how other CYP450 (2E1) inhibitors, such as diallyl sulfide (DAS) or phenylethylisothiocyanate (PIC), also potentiate the selective DA neuron degeneration in C57/bl mice. In order to provide direct evidence for this isozyme involvement, CYP 2E1 knockout mice were challenged with MPTP or the combined treatment. Here we show that these transgenic mice have a low sensitivity to MPTP alone, similarly to the wild type SVI, suggesting that it is likely that transgenic mice compensate for the missing enzyme. However, in these CYP 2E1 knockout mice, DDC pretreatment completely fails to enhance MPTP toxicity; this enhancement is instead regularly present in the SVI control animals. This study indicates that the occurrence of CYP 2E1 in C57/bl mouse brain is relevant for MPTP toxicity, and suggests that this isozyme may have a detoxificant role related to the efflux transporter of the toxin.

Assessing neuroprotection in Parkinson's disease: from the animal models to molecular neuroimaging in vivo.

An important goal in Parkinson's Disease research is to identify neuroprotective therapy, and the interaction between basic science and clinical research is needed to discover drugs that can slow or halt the disorder progression. At present there is not a perfect animal model of PD to test neuroprotective strategies, however the models that portray the basic characteristics needed are toxin-induced and gene-based models. The first group comprehends 6-OHDA e MPTP and recently rotenone, paraquat and epoxomicin treated animals that shows some of human disease characteristics. Gene-based models are various and, even if with limits, they seem suitable models to test neuroprotection in PD since they present replicable lesions, a predictable pattern of neurodegeneration and a well-characterized behavior, biochemistry and morphology to assist in the understanding of induced changes. In clinical trials researchers have first used as marker of disease progression clinical scores and motor tasks which are limited by the potential symptomatic effect of tested drugs and are not useful in the pre-clinical phases of PD. Recently has emerged the important role of neuroimaging (Dopamine Transporter SPECT, 18FDopa-PET) as surrogate biomarker of PD progression. Even if there are still concerns about the influence of regulatory effects of tested drugs, neuroimaging features could represent a good outcome measure to evaluate PD progression and putative neuroprotective effect of pharmacological and non-pharmacological manipulations.

D2/D3 dopamine receptor heterodimers exhibit unique functional properties.

Evidence for heterodimerization has recently been provided for dopamine D(1) and adenosine A(1) receptors as well as for dopamine D(2) and somatostatin SSTR(5) receptors. In this paper, we have studied the possibility that D(2) and D(3) receptors interact functionally by forming receptor heterodimers. Initially, we split the two receptors at the level of the third cytoplasmic loop into two fragments. The first, containing transmembrane domains (TM) I to V and the N-terminal part of the third cytoplasmic loop, was named D(2trunk) or D(3trunk), and the second, containing the C-terminal part of the third cytoplasmic loop, TMVI and TMVII, and the C-terminal tail, was named D(2tail) or D(3tail). Then we defined the pharmacological profiles of the homologous (D(2trunk)/D(2tail) and D(3trunk)/D(3tail)) as well as of the heterologous (D(2trunk)/D(3tail) and D(3trunk)/D(2tail)) cotransfected receptor fragments. The pharmacological profile of the cross-cotransfected fragments was different from that of the native D(2) or D(3) receptors. In most cases, the D(3trunk)/D(2tail) was the one with the highest affinity for most agonists and antagonists. Moreover, we observed that all of these receptor fragments reduced the expression of the wild type dopamine D(2) and D(3) receptors, suggesting that D(2) and D(3) receptors can form complexes with these fragments and that these complexes bind [(3)H]nemonapride less efficiently or are not correctly targeted to the membrane. In a second set of experiments, we tested the ability of the split and the wild type receptors to inhibit adenylyl cyclase (AC) types V and VI. All of the native and split receptors inhibited AC-V and AC-VI, with the exception of D(3), which was unable to inhibit AC-VI. We therefore studied the ability of D(2) and D(3) to interact functionally with one another to inhibit AC-VI. We found that with D(2) alone, R-(+)-7-hydroxydypropylaminotetralin hydrobromide inhibited AC-VI with an IC(50) of 2.05 +/- 0.15 nm, while in the presence of D(2) and D(3) it inhibited AC-VI with an IC(50) of 0.083 +/- 0.011 nm. Similar results were obtained with a chimeric cyclase made from AC-V and AC-VI. Coimmunoprecipitation experiments indicate that D(2) and D(3) receptors are capable of physical interaction.

Apomorphine, dopamine and phenylethylamine reduce the proportion of phosphorylated insulin receptor substrate 1.

We tested the ability of dopamine, apomorphine, phenylethylamine and pergolide to inhibit the proliferation of fetal calf serum-stimulated human breast cancer (MCF)-7 cells. While the first three compounds were able to block the proliferation of MCF-7 cells, pergolide failed to do so (up to 100 microM). The inhibitory effect of dopamine, apomorphine and phenylethylamine was also evident in serum-starved insulin-stimulated MCF-7 cells. Apomorphine also inhibited the proliferation of the human oestrogen receptor-negative breast cancer (MDA-MB231) and prostate carcinoma (LNCaP) cell lines. In a second set of experiments, we measured the ability of dopamine, apomorphine, phenylethylamine and pergolide to inhibit the phosphorylation (or increase the dephosphorylation) of the insulin receptor substrate (IRS)-1, a major intracellular substrate of the insulin-like growth factor (IGF)-1 receptor. Dopamine, apomorphine and phenylethylamine all reduced to zero the level of phosphorylated IRS-1 with potencies ranging between 0.01 and 1 microM. Finally, we found that fibroblasts from IRS-1 null (-/-) mice were less sensitive to the anti-proliferative effect of apomorphine compared to fibroblasts from wild type-mice, suggesting that the inhibition of IRS-1 phosphorylation by apomorphine is an important aspect of the activity of this compound.

Reconstitution of functional dopamine D(2s) receptor by co-expression of amino- and carboxyl-terminal receptor fragments.

An N-terminal dopamine D(2s) receptor clone was constructed and coexpressed in COS-7 cells together with a separate gene fragment coding for the C-terminal sequence of the dopamine D(2s) receptor. The truncated receptor (referred to as D(2trunc)) contained transmembrane domains I-V and the N-terminal portion of the third cytoplasmic loop, whereas the C-terminal receptor fragment (referred to as D(2tail)) contained transmembrane domains VI and VII and the adjacent intra- and extracellular sequences of the dopamine D(2s) receptor. Expression in COS-7 cells of either of these two polypeptides alone did not result in any detectable [3H]methylspiperone binding activity. However, specific [3H]methylspiperone binding could be observed after coexpression of the D(2trunc) and D(2tail) gene constructs; the number of receptors present on the plasma membrane was about 10% with respect to that of the wild type. The binding properties of the coexpressed fragments were similar to those of the wild-type dopamine D(2s) receptor for agonists and antagonists. Functional stimulation of the cotransfected D(2trunc) and D(2tail) fragments with quinpirole resulted in the inhibition of adenylate cyclase activity. Maximal inhibition corresponds to a 28% decrease in forskolin-stimulated adenylate cyclase. The apparent IC(50) of quinpirole was 5.1+/-0.3 mcM. These findings confirm and extend analogous data for other G protein-coupled receptors and indicate that this phenomenon is of general importance for the entire family of these proteins.

Modulation of dihydroxyphenylacetaldehyde extracellular levels in vivo in the rat striatum after different kinds of pharmacological treatment.

We recently identified the direct product of dopamine (DA) by monoamine-oxidase (MAO) activity, dihydroxyphenylacetaldehyde (DOPALD) in the trans-striatal dialysate. Based on these findings, in this work, we directly measured the variations in DOPALD levels after various kinds of pharmacological treatment in rat striatal extracellular fluid. Using both reversible and irreversible MAO inhibitors, we found that MAO-A inhibition suppressed, whereas MAO-B inhibition did not modify DOPALD levels in the dialysate. The vesicular DA uptake blocker Ro 4-1284 led to an increase in extracellular DA and DOPALD, whereas the increase in extracellular DA obtained after administration of the plasma membrane DA uptake blocker GBR-12909 occurred without concomitant changes in DOPALD extracellular levels. Microinfusions of DA through the dialysis probe or systemic administration of L-DOPA increased striatal DOPALD to a greater extent compared with other DA metabolites, both in intact and in 6-hydroxydopamine (6-OHDA)-lesioned striatum. This study indicates that the direct product of MAO activity within the rat striatum derives from the activity of the isoenzyme MAO-A. The assay of DOPALD, together with DOPAC, represents a reliable tool to measure directly, in freely moving animals, DA oxidative metabolism. As recent studies have shown that microinfusions of exogenous DOPALD might induce cell death, pharmacological modulation of DOPALD levels might also be relevant for an understanding of the mechanisms involved in DA neurotoxicity.

Dopamine agonists and analogues have an antiproliferative effect on CHO-K1 cells.

Epidemiological studies have shown a reduced incidence of cancer in Parkinson's disease. Since nearly all parkinsonian patients with clinical impairment are treated with L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine (DA)ergic agonists, a possibility exists that these therapeutic agents can influence the risk of cancer. We studied the antiproliferative effect of these therapeutic agents (and substances structurally correlated) on Chinese hamster ovary (CHO)-K1 cell growth. Among the compounds tested, apomorphine proved to be the most potent inhibitor of CHO-K1 cell growth, with an EC(50) of 3.35 +/- 0.12 micro M. The apomorphine analogues, apocodeine and hydroxyethylnorapomorphine, were less active as inhibitors of CHO-K1 cell growth. The activity of DA, 6-hydroxydopamine (6-OHDA), phenylethylamine (PEA), L-DOPA and bromocriptine as antiproliferative was one order of magnitude lower than that of apomorphine while pergolide was ineffective. To test whether or not the oxidative potential of these compounds was important for their antiproliferative effect, several antioxidants were assayed. Among them glutathione (GSH) and dithiothreitol (DTT) were effective in reversing the anti-proliferative effect of apomorphine, DA, 6-OHDA and PEA, conversely they did not work with bromocriptine. GSH and DTT are sulphydryl-reducing agents; while their effect could explain the efficacy against apomorphine, DA and 6-OHDA, it is difficult to understand why they should have any effect on PEA as this substance does not react with sulphydryl groups. The oxidative potential as a mechanism of action was also questioned by the results obtained with dihydrorhodamine 123, a probe that changes its fluorescent emission wave when oxidized. None of the compounds, with the exception of 6-OHDA, had any effect on the fluorescent emission wave of the probe at the maximal concentrations used to inhibit CHO-K1 cell growth. At concentrations five times higher, apomorphine and DA generated reactive oxygen species but PEA and bromocriptine did not. These data demonstrate that the antiproliferative effect of these compounds is not due to their oxidative potential, but another mechanism must be postulated.

Localization of a glutathione-dependent dehydroascorbate reductase within the central nervous system of the rat.

In this study, we describe for the first time the occurrence, within the central nervous system of the rat, of a dehydroascorbate reductase analogous to the one we recently described in the liver. Dehydroascorbate reductase plays a pivotal role in regenerating ascorbic acid from its oxidation product, dehydroascorbate. In a first set of experiments, we showed that a dehydroascorbate reductase activity is present in brain cytosol; immunoblotting analysis confirmed the presence of an immunoreactive cytosolic protein in selected brain areas. Immunotitration showed that approximately 65% of dehydroascorbate reductase activity of brain cytosol which was recovered in the ammonium sulphate fraction can be attributed to this enzyme. Using immunohistochemistry, we found that a variety of brain areas expresses the enzyme. Immunoreactivity was confined to the gray matter. Amongst the several brain regions, the cerebellum appears to be the most densely stained. The enzyme was also abundant in the hippocampus and the olfactory cortex. The lesion of norepinephrine terminals following systemic administration of DSP-4 markedly decreased immunoreactivity in the cerebellum. Apart from the possible co-localization of the enzyme with norepinephrine, the relative content of dehydroascorbate reductase in different brain regions might be crucial in conditioning regional sensitivity to free radical-induced brain damage. Given the scarcity of protective mechanisms demonstrated in the brain, the discovery of a new enzyme with antioxidant properties might represent a starting-point to increase our knowledge about the antioxidant mechanisms operating in several central nervous system disorders.

G protein-linked receptors: pharmacological evidence for the formation of heterodimers.

By means of the expression of two chimeric receptors, alpha(2)/M(3) and M(3)/alpha(2), in which the carboxy-terminal receptor portions, containing transmembrane domains VI and VII, were exchanged between the alpha(2C)-adrenergic and the M(3) muscarinic receptor, it has been shown that G protein-coupled receptors are able to interact functionally with each other at the molecular level to form (hetero)dimers. In the present study, we tested the hypothesis that interaction between two different muscarinic receptor subtypes can lead to the formation of a heterodimeric muscarinic receptor with a new pharmacological profile. Initially, muscarinic M(2) or M(3) wild-type receptors were expressed together with gene fragments originating from M(3) or M(2) receptors, respectively. Antagonist binding, performed with pirenzepine and tripitramine, revealed the presence of two populations of binding sites: one represents the wild-type M(2) or M(3) receptors, the other the heterodimeric M(2)/M(3) receptor. In another set of experiments, we constructed a point mutant M(2) receptor M(2) (Asn404-->Ser), in which asparagine 404 was replaced by serine. Although this receptor alone did not show any binding for N-[(3)H]methylscopolamine (up to 2 nM), when cotransfected with M(3), it resulted in the rescue of a high-affinity binding for tripitramine. These findings demonstrate that M(2) and M(3) muscarinic receptor subtypes can cross-interact with each other and form a new pharmacological heterodimeric receptor.

Is lithium able to reverse neurological damage induced by vinca alkaloids? (Short communication).

Lithium (Li) actively antagonises the inhibiting action of vinca alkaloids on human leukocyte chemotaxis; it proved to be related to the activation of microtubular system, possibly mediated by its inhibiting effect on cyclic AMP. Vinca alkaloids induce peripheral neuropathy and muscle damage. The molecular basis of this neurotoxicity has not been fully explained, but a possible role of neurofibrillary degeneration has been reported. We studied both in animals and in humans, whether Li is able to antagonise vinca alkaloid neurotoxicity.

Apomorphine has a potent antiproliferative effect on Chinese hamster ovary cells.

Apomorphine is a potent non selective agonist at the D1 and D2 dopamine receptors acting both pre- and post-synaptically. In this report we describe a novel function of apomorphine, independent from its dopaminergic activity. Apomorphine inhibits Chinese hamster ovary (CHO)-K1 cell proliferation in a dose-dependent manner. The EC50 of apomorphine-induced inhibition of CHO-K1 cell proliferation determined by cell counting was 3.24 +/- 0.07 microM. Remarkably, the dose-response curve obtained by measuring the incorporation of [3H]thymidine was practically identical to the previous one giving an EC50 of 3.52 +/- 0.04 microM. The dopaminergic antagonists SCH23390 and spiperone at a concentration of 10 microM (well beyond their Kd values for the dopamine D1- and D2-like receptors respectively) were not able to antagonize the effect of apomorphine on CHO-K1 cell proliferation. Apomorphine exerts its effect early during incubation; CHO-K1 cells exposed to apomorphine for a period as short as 1 h and then allowed to grow for three days were significantly reduced in number with respect to untreated control cells. After four hours of exposition to apomorphine (10 microM) the antiproliferative effect was similar to that seen when this compound was present in the bath for all three days. Concentrations of apomorphine higher than 10 microM induced cell death, and the colony was completely destroyed at 50 microM. Cytometric analyses showed a significant accumulation of CHO-K1 cells in the G2/M phase.

Effects of pretreatment with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) on methamphetamine pharmacokinetics and striatal dopamine losses.

We recently demonstrated that pretreatment with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) exacerbates experimental parkinsonism induced by methamphetamine. The mechanism responsible for this effect remains to be elucidated. In this study, we investigated whether the exacerbation of chronic dopamine loss in DSP-4-pretreated animals is due to an impairment in the recovery of dopamine levels once the neurotoxic insult is generated or to an increased efficacy of the effects induced by methamphetamine. We administered different doses of methamphetamine either to DSP-4-pretreated or to intact Swiss-Webster mice and evaluated the methamphetamine-induced striatal dopamine loss at early and prolonged intervals. As a further step, we evaluated the striatal pharmacokinetics of methamphetamine, together with its early biochemical effects. We found that previous damage to norepinephrine terminals produced by DSP-4 did not modify the recovery of striatal dopamine levels occurring during several weeks after methamphetamine. By contrast, pretreatment with DSP-4 exacerbated early biochemical effects of methamphetamine, which were already detectable 1 h after methamphetamine administration. In addition, in norepinephrine-depleted animals, the clearance of striatal methamphetamine is prolonged, although the striatal concentration peak observed at 1 h is unmodified. These findings, together with the lack of a methamphetamine enhancement when DSP-4 was injected 12 h after methamphetamine administration, suggest that in norepinephrine-depleted animals, a more pronounced acute neuronal sensitivity to methamphetamine occurs.

Nicotine prevents experimental parkinsonism in rodents and induces striatal increase of neurotrophic factors.

The repeated finding of an apparent protective effect of cigarette smoking on the risk of Parkinson's disease is one of the few consistent results in the epidemiology of this disorder. Among the numerous substances that originate from tobacco smoke, nicotine is by far the most widely studied. Nicotine is a natural alkaloid that has considerable stimulatory effects on the CNS. Its effects on the CNS are mediated by the activation of neuronal heteromeric acetylcholine-gated ion channel receptors (nAChRs, also termed nicotinic acetylcholine receptors). In the present study, we describe the neuroprotective effects of (-)-nicotine in two animal models of parkinsonism: diethyldithiocarbamate-induced enhancement of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice and methamphetamine-induced neurotoxicity in rats and mice. The neuroprotective effect of (-)-nicotine was very similar to that of the noncompetitive NMDA receptor antagonist (+)-MK-801. In parallel experiments, we found that (-)-nicotine induces the basic fibroblast growth factor-2 (FGF-2) and the brain-derived neurotrophic factor in rat striatum. The effect of (-)-nicotine on the induction of FGF-2 was prevented by the nAChR antagonist mecamylamine. We also found that (+)-MK-801 was able to induce FGF-2 in the striatum. As trophic factors have been reported to be neuroprotective for dopaminergic cells, our data suggest that the increase in neurotrophic factors is a possible mechanism by which (-)-nicotine protects from experimental parkinsonisms.

Antagonist binding profile of the split chimeric muscarinic m2-trunc/m3-tail receptor.

Recent evidence suggests that G-protein-coupled receptors can behave as multiple subunit receptors, and can be split into parts, maintaining their binding ability. Transfection of a truncated muscarinic m2 receptor (containing transmembrane domains I-V, named m2-trunc) with a gene fragment coding for the carboxyl-terminal receptor portion of the muscarinic m3 receptor (containing transmembrane domains VI and VII, named m3-tail) results in the formation of a binding site with a high affinity for the muscarinic ligand N-[3H]methylscopolamine. In this paper we analyse the antagonist binding profile of this chimeric m2-trunc/m3-tail receptor in comparison with the wild-type muscarinic m2 and m3 receptors. While many of the substances tested had an intermediate affinity for the chimeric m2-trunc/m3-tail receptor compared with m2 and m3, some compounds were able to distinguish between the chimeric m2-trunc/m3-tail receptor on the one hand and the m2 or the m3 receptor on the other. Among them, tripitramine (a high-affinity M2 receptor antagonist) bound to the m2-trunc/m3-tail receptor with the same affinity as m2, but it bound to the m3 receptor with a 103-fold lower affinity; pirenzepine (a selective muscarinic M1 receptor antagonist) bound to the chimeric receptor with an affinity that was 12- and 3-fold higher than that of m2 and m3, respectively. The results of this study demonstrate that the chimeric m2-trunc/m3-tail receptor has a pharmacological profile distinct from that of the originating muscarinic m2 and m3 receptors.

Noradrenergic modulation of methamphetamine-induced striatal dopamine depletion.

Noradrenergic (NE) neurons belonging to the locus coeruleus (LC), much more than the A1 and A2 areas, are lost in Parkinson's disease (PD). In this study, we reproduced the selective pattern of NE loss involving axons arising from the LC using the selective neurotoxin N-(-2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) (50 mg/kg). In these experimental conditions, we investigated whether NE loss potentiates methamphetamine-induced striatal dopamine (DA) depletion in mice and rats. Administration of a moderate dose of methamphetamine to C57B1/6N mice or Sprague-Dawley rats produced only a partial striatal DA depletion 7 days after drug administration. Pre-treatment with DSP-4, in both animal species, significantly enhanced methamphetamine-induced striatal DA depletion. Administration of a lower dose of methamphetamine did not decrease striatal DA levels when injected alone, but produced a significant decrease in striatal DA when given to DSP-4-pretreated rodents. Moreover, we found that agents reducing the noradrenergic activity (i.e., the alpha-2 agonist clonidine) enhanced, whereas alpha-2 antagonists decreased, methamphetamine toxicity. Enhancement of methamphetamine toxicity did not occur if the noradrenergic lesion was produced 12 hr after methamphetamine administration. By contrast, exacerbation of methamphetamine toxicity in NE-depleted animals was accompanied by increased extracellular DA levels measured with brain dialysis and by a more severe acute DA depletion measured in striatal homogenates.