RESUMO
Lewy body diseases such as Parkinson's disease involve intraneuronal deposition of the protein α-synuclein (AS) and depletion of nigrostriatal dopamine (DA). Interactions of AS with DA oxidation products may link these neurohistopathologic and neurochemical abnormalities via two potential pathways: spontaneous oxidation of DA to dopamine-quinone and enzymatic oxidation of DA catalyzed by monoamine oxidase to form 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is then oxidized to DOPAL-Q. We compared these two pathways in terms of the ability of DA and DOPAL to modify AS. DOPAL was far more potent than DA both in oligomerizing and forming quinone-protein adducts with (quinonizing) AS. The DOPAL-induced protein modifications were enhanced similarly by pro-oxidation with Cu(II) or tyrosinase and inhibited similarly by antioxidation with N-acetylcysteine. Dopamine oxidation evoked by Cu(II) or tyrosinase did not quinonize AS. In cultured MO3.13 human oligodendrocytes DOPAL resulted in the formation of numerous intracellular quinoproteins that were visualized by near-infrared spectroscopy. We conclude that of the two routes by which oxidation of DA modifies AS and other proteins the route via DOPAL is more prominent. The results support developing experimental therapeutic strategies that might mitigate deleterious modifications of proteins such as AS in Lewy body diseases by targeting DOPAL formation and oxidation. SIGNIFICANCE STATEMENT: Interactions of the protein α-synuclein with products of dopamine oxidation in the neuronal cytoplasm may link two hallmark abnormalities of Parkinson disease: Lewy bodies (which contain abundant AS) and nigrostriatal DA depletion (which produces the characteristic movement disorder). Of the two potential routes by which DA oxidation may alter AS and other proteins, the route via the autotoxic catecholaldehyde 3,4-dihydroxyphenylacetaldehyde is more prominent; the results support experimental therapeutic strategies targeting DOPAL formation and DOPAL-induced protein modifications.
Assuntos
Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Dopamina/análogos & derivados , Dopamina/química , Doença de Parkinson/metabolismo , alfa-Sinucleína/química , Ácido 3,4-Di-Hidroxifenilacético/efeitos adversos , Ácido 3,4-Di-Hidroxifenilacético/química , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Acetilcisteína/química , Antioxidantes/química , Linhagem Celular , Cobre/química , Cobre/metabolismo , Dopamina/efeitos adversos , Dopamina/metabolismo , Humanos , Monoaminoxidase/metabolismo , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Oligodendroglia/citologia , Oxirredução , Ligação Proteica , Conformação Proteica , Tolcapona/metabolismo , alfa-Sinucleína/metabolismoRESUMO
The catecholaldehyde hypothesis posits that 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediary metabolite of dopamine, is an autotoxin that challenges neuronal homeostasis in catecholaminergic neurons. DOPAL toxicity may involve protein modifications, such as oligomerization of α-synuclein (AS). Potential interactions between DOPAL and other proteins related to catecholaminergic neurodegeneration, however, have not been systemically explored. This study examined DOPAL-induced protein-quinone adduct formation ("quinonization") and protein oligomerization, ubiquitination, and aggregation in cultured MO3.13 human oligodendrocytes and PC12 rat pheochromocytoma cells and in test tube experiments. Using near-infrared fluorescence spectroscopy, we detected spontaneous DOPAL oxidation to DOPAL-quinone, DOPAL-induced quinonization of intracellular proteins in both cell lines, and DOPAL-induced quinonization of several proteins related to catecholaminergic neurodegeneration, including AS, the type 2 vesicular monoamine transporter, glucocerebrosidase, ubiquitin, and l-aromatic-amino-acid decarboxylase (LAAAD). DOPAL also oligomerized AS, ubiquitin, and LAAAD; inactivated LAAAD (IC50 54 µM); evoked substantial intracellular protein ubiquitination; and aggregated intracellular AS. Remarkably, N-acetylcysteine, which decreases DOPAL-quinone formation, attenuated or prevented all of these protein modifications and functional changes. The results fit with the proposal that treatments based on decreasing the formation and oxidation of DOPAL may slow or prevent catecholaminergic neurodegeneration.
Assuntos
Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Acetilcisteína/farmacologia , Proteínas/química , Proteínas/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/farmacologia , Animais , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Oxirredução/efeitos dos fármacos , Células PC12 , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Quinonas/metabolismo , RatosRESUMO
OBJECTIVE: Lewy body forms of primary chronic autonomic failure (CAF) such as incidental Lewy body disease (ILBD), Parkinson's disease (PD), and pure autonomic failure evolving into dementia with Lewy bodies (PAF+DLB) feature cardiac sympathetic denervation, whereas multiple system atrophy (MSA) in most cases does not. What links Lewy bodies with cardiac sympathetic denervation in CAF? In familial PD, abnormalities of the alpha-synuclein (AS) gene cause CAF and cardiac sympathetic denervation; and in sporadic PD, brainstem Lewy bodies contain AS co-localized with tyrosine hydroxylase (TH), a marker of catecholaminergic neurons. Cytotoxicity from AS deposition within sympathetic neurons might explain noradrenergic denervation in Lewy body forms of CAF. We used immunofluorescence microscopy (IM) to explore this possibility in sympathetic ganglia obtained at autopsy from CAF patients. METHODS: Immunoreactive AS and TH were imaged in sympathetic ganglion tissue from 6 control subjects (2 with ILBD), 5 PD patients (1 with concurrent PSP), and 3 patients with CAF (2 PAF + DLB, 1 MSA). RESULTS: MSA involved normal ganglionic TH and no AS deposition. In ILBD TH was variably decreased, and TH and AS were co-localized in Lewy bodies. In PD TH was substantially decreased, and TH and AS were co-localized in Lewy bodies. In PAF + DLB TH was virtually absent, but AS was present in Lewy bodies. The PD + PSP patient had AS co-localized with tau but not TH. CONCLUSIONS: Sympathetic denervation and intraneuronal AS deposition are correlated across CAF syndromes, consistent with a pathogenic contribution of synucleinopathy to cardiac noradrenergic deficiency in Lewy body diseases.
Assuntos
Gânglios Simpáticos/metabolismo , Doença por Corpos de Lewy/metabolismo , Doença de Parkinson/metabolismo , Insuficiência Autonômica Pura/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/metabolismo , Doença Crônica , Gânglios Simpáticos/química , Gânglios Simpáticos/patologia , Humanos , Doença por Corpos de Lewy/patologia , Doença de Parkinson/patologia , Insuficiência Autonômica Pura/patologia , Tirosina 3-Mono-Oxigenase/análise , alfa-Sinucleína/análiseRESUMO
The catecholaldehyde hypothesis for the pathogenesis of Parkinson's disease proposes that the deaminated dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is toxic to nigrostriatal dopaminergic neurons. Inhibiting monoamine oxidase (MAO) should therefore slow the disease progression; however, MAO inhibition increases spontaneous oxidation of dopamine, as indicated by increased 5-S-cysteinyl-dopamine (Cys-DA) levels, and the oxidation products may also be toxic. This study examined whether N-acetylcysteine (NAC), a precursor of the anti-oxidant glutathione, attenuates the increase in Cys-DA production during MAO inhibition. Rat pheochromocytoma PC12 cells were incubated with NAC, the MAO-B inhibitor selegiline, or both. Selegiline decreased DOPAL and increased Cys-DA levels (p < 0.0001 each). Co-incubation of NAC at pharmacologically relevant concentrations (1-10 µM) with selegiline (1 µM) attenuated or prevented the Cys-DA response to selegiline, without interfering with the selegiline-induced decrease in DOPAL production or inhibiting tyrosine hydroxylation. NAC therefore mitigates the increase in spontaneous oxidation of dopamine during MAO inhibition.
Assuntos
Acetilcisteína/farmacologia , Dopamina/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Selegilina/farmacologia , Animais , Relação Dose-Resposta a Droga , Oxirredução/efeitos dos fármacos , Células PC12 , RatosRESUMO
According to the catecholaldehyde hypothesis, the toxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to the loss of nigrostriatal dopaminergic neurons in Parkinson's disease. Monoamine oxidase-A (MAO-A) catalyzes the conversion of intraneuronal dopamine to DOPAL and may serve as a therapeutic target. The "cheese effect"-paroxysmal hypertension evoked by tyramine-containing foodstuffs-limits clinical use of irreversible MAO-A inhibitors. Combined MAO-A/B inhibition decreases DOPAL production in rat pheochromocytoma PC12 cells, but whether reversible MAO-A inhibitors or MAO-B inhibitors decrease endogenous DOPAL production is unknown. We compared the potencies of MAO inhibitors in attenuating DOPAL production and examined possible secondary effects on dopamine storage, constitutive release, synthesis, and auto-oxidation. Catechol concentrations were measured in cells and medium after incubation with the irreversible MAO-A inhibitor clorgyline, three reversible MAO-A inhibitors, or the MAO-B inhibitors selegiline or rasagiline for 180 minutes. Reversible MAO-A inhibitors were generally ineffective, whereas clorgyline (1 nM), rasagiline (500 nM), and selegiline (500 nM) decreased DOPAL levels in the cells and medium. All three drugs also increased dopamine and norepinephrine, decreased 3,4-dihydroxyphenylalanine, and increased cysteinyl-dopamine concentrations in the medium, suggesting increased vesicular uptake and constitutive release, decreased dopamine synthesis, and increased dopamine spontaneous oxidation. In conclusion, clorgyline, rasagiline, and selegiline decrease production of endogenous DOPAL. At relatively high concentrations, the latter drugs probably lose their selectivity for MAO-B. Possibly offsetting increased formation of potentially toxic oxidation products and decreased formation of DOPAL might account for the failure of large clinical trials of MAO-B inhibitors to demonstrate slowing of neurodegeneration in Parkinson's disease.
Assuntos
Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Ácido 3,4-Di-Hidroxifenilacético/antagonistas & inibidores , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Neurônios Dopaminérgicos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células PC12 , Doença de Parkinson/metabolismo , RatosRESUMO
The catecholaldehyde hypothesis predicts that monoamine oxidase (MAO) inhibition should slow the progression of Parkinson's disease, by decreasing production of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL). Inhibiting MAO, however, diverts the fate of cytoplasmic dopamine toward potentially harmful spontaneous oxidation products, indicated by increased 5-S-cysteinyl-dopamine (Cys-DA) levels. 3,4-Dihydroxyphenylethanol (hydroxytyrosol) is an abundant anti-oxidant phenol in constituents of the Mediterranean diet. Whether hydroxytyrosol alters enzymatic or spontaneous oxidation of dopamine has been unknown. Rat pheochromocytoma PC12 cells were incubated with hydroxytyrosol (10 µM, 180 min) alone or with the MAO-A inhibitor clorgyline (1 nM) or the MAO-B inhibitors rasagiline or selegiline (0.5 µM). Hydroxytyrosol decreased levels of DOPAL by 30 % and Cys-DA by 49 % (p < 0.0001 each). Co-incubation with hydroxytyrosol prevented the increases in Cys-DA seen with all 3 MAO inhibitors. Hydroxytyrosol therefore inhibits both enzymatic and spontaneous oxidation of endogenous dopamine and mitigates the increase in spontaneous oxidation during MAO inhibition.
Assuntos
Dopaminérgicos/farmacologia , Dopamina/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Álcool Feniletílico/análogos & derivados , Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Oxirredução/efeitos dos fármacos , Células PC12 , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Álcool Feniletílico/farmacologia , RatosRESUMO
Repeated systemic administration of the mitochondrial complex I inhibitor rotenone produces a rodent model of Parkinson's disease (PD). Mechanisms of relatively selective rotenone-induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the 'catecholaldehyde hypothesis,' buildup of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F-labeled catechols were measured in PC12 cells incubated with rotenone (0-1000 nM, 180 min), without or with F-dopamine (2 µM) to track vesicular uptake and catecholamine metabolism. Rotenone dose dependently increased DOPAL, F-DOPAL, and 3,4-dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD(+) was supplied, whereas the direct-acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective rotenone-induced toxicity in dopaminergic neurons. We report that rotenone, a mitochondrial complex I inhibitor that produces an animal model of Parkinson's disease, increases intracellular levels of the toxic dopamine metabolite 3,4-dihydroxyphenyl-acetaldehyde (DOPAL), via decreased DOPAL metabolism by aldehyde dehydrogenase (ALDH) and decreased vesicular sequestration of cytoplasmic dopamine by the vesicular monoamine transporter (VMAT). The results provide a novel mechanism for rotenone-induced toxicity in dopaminergic neurons.
Assuntos
Aldeído Desidrogenase/metabolismo , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/enzimologia , Rotenona/farmacologia , Desacopladores/farmacologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Neoplasias Encefálicas/metabolismo , Dopamina/metabolismo , Complexo I de Transporte de Elétrons/efeitos dos fármacos , Glioblastoma/metabolismo , Glioma/metabolismo , Humanos , NAD/metabolismo , Células PC12 , RatosRESUMO
The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is detoxified mainly by aldehyde dehydrogenase (ALDH). We find that the fungicide benomyl potently and rapidly inhibits ALDH and builds up DOPAL in vivo in mouse striatum and in vitro in PC12 cells and human cultured fibroblasts and glial cells. The in vivo results resemble those noted previously with knockouts of the genes encoding ALDH1A1 and 2, a mouse model of aging-related Parkinson's disease (PD). Exposure to pesticides that inhibit ALDH may therefore increase PD risk via DOPAL buildup. This study lends support to the "catecholaldehyde hypothesis" that the autotoxic dopamine metabolite DOPAL plays a pathogenic role in PD.
Assuntos
Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Aldeído Desidrogenase/metabolismo , Antifúngicos/metabolismo , Benomilo/metabolismo , Doença de Parkinson/etiologia , Ácido 3,4-Di-Hidroxifenilacético/química , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Desidrogenase/genética , Aldeídos/química , Aldeídos/toxicidade , Animais , Antifúngicos/química , Antifúngicos/toxicidade , Benomilo/química , Benomilo/toxicidade , Linhagem Celular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Células PC12 , RatosRESUMO
The incidence of Parkinson's disease (PD) correlates with environmental exposure to pesticides, such as the organochlorine insecticide, dieldrin. Previous studies found an increased concentration of the pesticide in the striatal region of the brains of PD patients and also that dieldrin adversely affects cellular processes associated with PD. These processes include mitochondrial function and reactive oxygen species production. However, the mechanism and specific cellular targets responsible for dieldrin-mediated cellular dysfunction and the structural components of dieldrin contributing to its toxicity (toxicophore) have not been fully defined. In order to identify the toxicophore of dieldrin, a structure-activity approach was used, with the toxicity profiles of numerous analogues of dieldrin (including aldrin, endrin, and cis-aldrin diol) assessed in PC6-3 cells. The MTT and lactate dehydrogenase (LDH) assays were used to monitor cell viability and membrane permeability after treatment with each compound. Cellular assays monitoring ROS production and extracellular dopamine metabolite levels were also used. Structure and stereochemistry for dieldrin were found to be very important for toxicity and other end points measured. Small changes in structure for dieldrin (e.g., comparison to the stereoisomer endrin) yielded significant differences in toxicity. Interestingly, the cis-diol metabolite of dieldrin was found to be significantly more toxic than the parent compound. Disruption of dopamine catabolism yielded elevated levels of the neurotoxin, 3,4-dihydroxyphenylacetaldehyde, for many organochlorines. Comparisons of the toxicity profiles for each dieldrin analogue indicated a structure-specific effect important for elucidating the mechanisms of dieldrin neurotoxicity.
Assuntos
Dieldrin/análogos & derivados , Dieldrin/toxicidade , Neurônios Dopaminérgicos/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dieldrin/química , Dieldrin/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Espécies Reativas de Oxigênio/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Parkinson's disease entails profound loss of nigrostriatal dopaminergic terminals, decreased vesicular uptake of intraneuronal catecholamines, and relatively increased putamen tissue concentrations of the toxic dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde (DOPAL). The objective of this study was to test whether vesicular uptake blockade augments endogenous DOPAL production. We also examined whether intracellular DOPAL contributes to apoptosis and, as α-synuclein oligomers may be pathogenetic in Parkinson's disease, oligomerizes α-synuclein. Catechols were assayed in PC12 cells after reserpine to block vesicular uptake, with or without inhibition of enzymes metabolizing DOPAL-daidzein for aldehyde dehydrogenase and AL1576 for aldehyde reductase. Vesicular uptake was quantified by a method based on 6F- or (13) C-dopamine incubation; DOPAL toxicity by apoptosis responses to exogenous dopamine, with or without daidzein+AL1576; and DOPAL--induced synuclein oligomerization by synuclein dimer production during DOPA incubation, with or without inhibition of L-aromatic-amino-acid decarboxylase or monoamine oxidase. Reserpine inhibited vesicular uptake by 95-97% and rapidly increased cell DOPAL content (p = 0.0008). Daidzein+AL1576 augmented DOPAL responses to reserpine (p = 0.004). Intracellular DOPAL contributed to dopamine-evoked apoptosis and DOPA-evoked synuclein dimerization. The findings fit with the 'catecholaldehyde hypothesis,' according to which decreased vesicular sequestration of cytosolic catecholamines and impaired catecholaldehyde detoxification contribute to the catecholaminergic denervation that characterizes Parkinson's disease.
Assuntos
Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Dopamina/metabolismo , Neurônios/metabolismo , Transtornos Parkinsonianos/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/toxicidade , Animais , Dopamina/deficiência , Neurônios/efeitos dos fármacos , Neurônios/patologia , Células PC12 , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/etiologia , RatosRESUMO
Dopamine (DA) has been implicated as an endogenous neurotoxin to explain the selective neurodegeneration as observed for Parkinson's disease (PD). In addition, oxidative stress and lipid peroxidation are hypothesized culprits in PD pathogenesis. DA undergoes catabolism by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) via aldehyde dehydrogenase (ALDH). As a minor and compensatory metabolic pathway, DOPAL can be reduced to 3,4-dihydroxyphenylethanol (DOPET) via cytosolic aldehyde or aldose reductase (AR). Previous studies have found DOPAL to be significantly more toxic to DA cells than DA and that the major lipid peroxidation products, that is, 4-hydroxynonenal (4HNE) and malondialdehyde (MDA), potently inhibit DOPAL oxidation via ALDH. The hypothesis of this work is that lipid peroxidation products inhibit DOPAL oxidation, yielding aberrant levels of the toxic aldehyde intermediate. To test this hypothesis, nerve growth factor-differentiated PC6-3 cells were used as a model for DA neurons. Cell viability in the presence of 4HNE and MDA (2-100 microM) was measured by MTT assay, and it was found that only 100 microM 4HNE exhibited significant cytotoxicity. Treatment of cells with varying concentrations of 4HNE and MDA resulted in reduced DOPAC production and significant elevation of DOPAL levels, suggesting inhibition of ALDH. In cells treated with 4HNE that exhibited elevated DOPAL, there was a significant increase in DOPET. However, elevated DOPET was not observed for the cells treated with MDA, suggesting MDA to be an inhibitor of AR. Using isolated cytosolic AR, it was found that MDA but not 4HNE inhibited reductase activity toward DOPAL, surprisingly. These data demonstrate that the oxidative stress products 4HNE and MDA inhibit the aldehyde biotransformation step of DA catabolism yielding elevated levels of the endogenous neurotoxin DOPAL, which may link oxidative stress to selective neurodegeneration as seen in PD.
Assuntos
Aldeídos/metabolismo , Dopamina/análise , Dopamina/metabolismo , Estresse Oxidativo , Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Ácido 3,4-Di-Hidroxifenilacético/análise , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Aldeído Redutase/metabolismo , Aldeídos/farmacologia , Aldeídos/toxicidade , Animais , Peroxidação de Lipídeos , Malondialdeído/farmacologia , Malondialdeído/toxicidade , Oxirredução , Células PC12 , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/análise , Álcool Feniletílico/metabolismo , RatosRESUMO
Three analogues of the dual mu-/delta-antagonist, H-Dmt-Tic-R-NH-CH2-Ph (R = 1, Lys-Z; 2, Lys-Ac; 3, Lys) were examined in vivo: 1 and 2 exhibited weak bioactivity, while 3 injected intracerebroventricularly was a potent dual antagonist for morphine- and deltorphin C-induced antinociception comparable to naltrindole (delta-antagonist), but 93% as effective as naloxone (nonspecific opioid receptor antagonist) and 4% as active as CTOP, a mu antagonist. Subcutaneous or oral administration of 3 antagonized morphine-induced antinociception indicating passage across epithelial and blood-brain barriers. Mice pretreated with 3 before morphine did not develop morphine tolerance indicative of a potential clinical role to inhibit development of drug tolerance.
Assuntos
Analgésicos Opioides/farmacologia , Morfina/farmacologia , Oligopeptídeos/farmacologia , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides mu/antagonistas & inibidores , Animais , Área Sob a Curva , Relação Dose-Resposta a Droga , Tolerância a Medicamentos , Injeções Intraventriculares , Injeções Subcutâneas , Ligantes , Masculino , Camundongos , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Tempo de Reação/efeitos dos fármacos , Somatostatina/análogos & derivados , Somatostatina/farmacologiaRESUMO
Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) and [Dmt1]EM-2 (Dmt = 2',6'-dimethyl-l-tyrosine) analogues, containing alkylated Phe3 derivatives, 2'-monomethyl (2, 2'), 3',5'- and 2',6'-dimethyl (3, 3', and 4', respectively), 2',4',6'-trimethyl (6, 6'), 2'-ethyl-6'-methyl (7, 7'), and 2'-isopropyl-6'-methyl (8, 8') groups or Dmt (5, 5'), had the following characteristics: (i) [Xaa3]EM-2 analogues exhibited improved mu- and delta-opioid receptor affinities. The latter, however, were inconsequential (Kidelta = 491-3451 nM). (ii) [Dmt1,Xaa3]EM-2 analogues enhanced mu- and delta-opioid receptor affinities (Kimu = 0.069-0.32 nM; Kidelta = 1.83-99.8 nM) without kappa-opioid receptor interaction. (iii) There were elevated mu-bioactivity (IC50 = 0.12-14.4 nM) and abolished delta-agonism (IC50 > 10 muM in 2', 3', 4', 5', 6'), although 4' and 6' demonstrated a potent mixed mu-agonism/delta-antagonism (for 4', IC50mu = 0.12 and pA2 = 8.15; for 6', IC50mu = 0.21 nM and pA2 = 9.05) and 7' was a dual mu-agonist/delta-agonist (IC50mu = 0.17 nM; IC50delta = 0.51 nM).
Assuntos
Oligopeptídeos/síntese química , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides mu/agonistas , Tirosina/análogos & derivados , Tirosina/síntese química , Animais , Ligação Competitiva , Encéfalo/metabolismo , Cobaias , Técnicas In Vitro , Ligantes , Masculino , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/fisiologia , Plexo Mientérico/fisiologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/fisiologia , Oligopeptídeos/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sinaptossomos/metabolismo , Tirosina/farmacologia , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/fisiologiaRESUMO
Opioidmimetics containing 3-[H-Dmt-NH-(CH(2))(m)]-6-[H-Dmt-NH-(CH(2))(n)]-2(1H)-pyrazinone symmetric (m = n, 1-4) (1 - 4) and asymmetric (m, n = 1 - 4) aliphatic chains (5 - 16) were synthesized using dipeptidyl chloromethylketone intermediates. They had high mu-affinity (K(i)mu = 0.021 - 2.94 nM), delta-affinity (K(i)delta = 1.06 - 152.6 nM), and mu selectivity (K(i)delta/K(i)mu = 14 - 3,126). The opioidmimetics (1 - 16) exhibited mu agonism in proportion to their mu-receptor affinity. delta-Agonism was essentially lacking in the compounds except (4) and (16), and (1) and (2) indicated weak delta antagonism (pA(2) = 6.47 and 6.56, respectively). The data verify that a specific length of aliphatic linker is required between the Dmt pharmacophore and the pyrazinone ring to produce unique mu-opioid receptor ligands.
Assuntos
Analgésicos Opioides/química , Pirazinas/síntese química , Receptores Opioides mu/agonistas , Humanos , Mimetismo Molecular , Ligação Proteica , Pirazinas/farmacologia , Receptores Opioides mu/metabolismo , Relação Estrutura-AtividadeRESUMO
Substitution of Gly with side-chain-protected or unprotected Lys in lead compounds containing the opioid pharmacophore Dmt-Tic [H-Dmt-Tic-Gly-NH-CH(2)-Ph, mu agonist/delta antagonist; H-Dmt-Tic-Gly-NH-Ph, mu agonist/delta agonist; and H-Dmt-Tic-NH-CH(2)-Bid, delta agonist (Bid = 1H-benzimidazole-2-yl)] yielded a new series of compounds endowed with distinct pharmacological activities. Compounds (1-10) included high delta- (Ki(delta) = 0.068-0.64 nM) and mu-opioid affinities (Ki(mu) = 0.13-5.50 nM), with a bioactivity that ranged from mu-opioid agonism {10, H-Dmt-Tic-NH-CH[(CH2)4-NH2]-Bid (IC50 GPI = 39.7 nM)} to a selective mu-opioid antagonist [3, H-Dmt-Tic-Lys-NH-CH2-Ph (pA2(mu) = 7.96)] and a selective delta-opioid antagonist [5, H-Dmt-Tic-Lys(Ac)-NH-Ph (pA2(delta) = 12.0)]. The presence of a Lys linker provides new lead compounds in the formation of opioid peptidomimetics containing the Dmt-Tic pharmacophore with distinct agonist and/or antagonist properties.
Assuntos
Dipeptídeos/síntese química , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidores , Tetra-Hidroisoquinolinas/síntese química , Animais , Dipeptídeos/química , Dipeptídeos/farmacologia , Estimulação Elétrica , Cobaias , Íleo/efeitos dos fármacos , Íleo/fisiologia , Técnicas In Vitro , Ligantes , Masculino , Camundongos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Relação Estrutura-Atividade , Tetra-Hidroisoquinolinas/química , Tetra-Hidroisoquinolinas/farmacologia , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/fisiologiaRESUMO
The Aba-Gly scaffold, incorporated into Dmt-Tic ligands (H-Dmt-Tic-Gly-NH-CH2-Ph, H-Dmt-Tic-Gly-NH-Ph, H-Dmt-Tic-NH-CH2-Bid), exhibited mixed micro/delta or delta opioid receptor activities with micro agonism. Substitution of Tic by Aba-Gly coupled to -NH-CH2-Ph (1), -NH-Ph (2), or -Bid (Bid=1H-benzimidazole-2-yl) (3) shifted affinity (Ki(micro)=0.46, 1.48, and 19.9 nM, respectively), selectivity, and bioactivity to micro-opioid receptors. These compounds represent templates for a new class of lead opioid agonists that are easily synthesized and suitable for therapeutic pain relief.
Assuntos
Analgésicos Opioides/síntese química , Benzazepinas/síntese química , Glicina/análogos & derivados , Glicina/síntese química , Peptídeos/química , Receptores Opioides mu/agonistas , Analgésicos Opioides/química , Analgésicos Opioides/farmacologia , Animais , Benzazepinas/farmacologia , Glicina/farmacologia , Cobaias , Técnicas In Vitro , Ligantes , Mimetismo Molecular , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/fisiologia , Plexo Mientérico/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/metabolismo , Relação Estrutura-Atividade , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
A new environment-sensitive fluorophore, 6-N,N-(dimethylamino)-2,3-naphthalimide (6DMN) was introduced in the delta-selective opioid peptide agonist H-Dmt-Tic-Glu-NH(2) and in the mu-selective opioid peptide agonist endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH(2)). Environment-sensitive fluorophores are a special class of chromophores that generally exhibit a low quantum yield in aqueous solution but become highly fluorescent in nonpolar solvents or when bound to hydrophobic sites in proteins or membranes. New fluorescent delta-selective irreversible antagonists (H-Dmt-Tic-Glu-NH-(CH(2))(5)-CO-Dap(6DMN)-NH(2) (1) and H-Dmt-Tic-Glu-Dap(6DMN)-NH(2) (2)) were identified as potential fluorescent probes showing good properties for use in studies of distribution and internalization of delta receptors by confocal laser scanning microscopy.
Assuntos
Corantes Fluorescentes/síntese química , Imidas/química , Naftalenos/química , Oligopeptídeos/síntese química , Peptídeos Opioides/síntese química , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Animais , Ligação Competitiva , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Cobaias , Técnicas In Vitro , Masculino , Camundongos , Contração Muscular , Naftalimidas , Neuroblastoma , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/fisiologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Peptídeos Opioides/química , Peptídeos Opioides/farmacologia , Ensaio Radioligante , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Relação Estrutura-AtividadeRESUMO
[Dmt1]Endomorphin-1 is a novel analogue of the potent mu-opioid agonist endomorphin-1. Given the physiological role of endomorphin-1 in vivo, this compound was investigated to determine if the antinociception occurred through systemic, supraspinal or in a combination of both neuronal pathways. This compound exhibited a potent dose-dependent effect intracerebroventricularly in both spinal and supraspinal regions, and was blocked by opioid antagonist naloxone, which verified the involvement of opioid receptors. Specific opioid antagonists characterized the apparent receptor type: beta-funaltrexamine (mu1/mu2-irreversible antagonist) equally inhibited spinal- and central-mediated antinociception; on the other hand, naloxonazine (mu1-subtype) was ineffective in both neural pathways and naltrindole (delta-selective antagonist) partially (26%), though not significantly, blocked only the spinal-mediated antinociception. Therefore, spinal antinociception was primarily triggered by mu2-subtypes without involvement of mu1-opioid receptors; however, although a slight enhancement of antinociception by delta-receptors cannot be completely ruled out since functional bioactivity indicated mixed mu-agonism/delta-antagonism. In terms of the CNS action, [Dmt1]endomorphin-1 appears to act through mu2-opioid receptor subtypes.
Assuntos
Analgesia , Oligopeptídeos/farmacologia , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides mu/efeitos dos fármacos , Animais , Encéfalo/fisiologia , Cobaias , Íleo/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Camundongos , Naloxona , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Dor , Medição da Dor , Medula Espinal/fisiologia , Cauda , Ducto Deferente/efeitos dos fármacosRESUMO
INTRODUCTION: Glial cytoplasmic inclusions (GCIs) containing alpha-synuclein (AS) are a neuropathologic hallmark of multiple system atrophy (MSA). Oligomerized AS is thought to be the pathogenic form of the protein. Glial cells normally express little AS, but they can take up AS from the extracellular fluid. 3,4-Dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediate in the intra-neuronal metabolism of dopamine (DA), potently oligomerizes AS. In this study we tested whether DOPAL is taken up by human glial cells and augments intracellular oligomerization of AS. METHODS: DOPAL (exogenous or endogenous from co-incubation with PC12 cells) and AS (native or A53T mutant form) were added to the incubation medium of glial cells (glioblastoma or MO3.13 oligodendrocytes). Glial cellular contents of DOPAL and its intracellular metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at up to 180 min of incubation. Glial cellular AS oligomers were quantified by Western blotting. RESULTS: Neither glioblastoma nor MO3.13 cells contained endogenous catecholamines or AS. Co-incubation of the cells with DA-producing PC12 cells produced time-related increases in DOPAL and DOPAC contents. Similarly, glial cellular DOPAL and DOPAC contents increased rapidly after addition of DOPAL to the medium. After addition of native or A53T-AS, intracellular AS also increased. Incubation of glial cells with both DOPAL and AS enhanced the intracellular oligomerization of native and A53T-AS. CONCLUSIONS: DOPAL is transmissible to glial cells and enhances intracellular oligomerization of AS. An interaction of DOPAL with AS might help explain the formation of CGIs in MSA.
Assuntos
Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Neuroglia/metabolismo , alfa-Sinucleína/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/farmacologia , Animais , Catecolaminas/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Glioblastoma/patologia , Humanos , Mutação/genética , Neuroglia/efeitos dos fármacos , Células PC12 , Agregados Proteicos/efeitos dos fármacos , Agregados Proteicos/genética , Ratos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Fatores de Tempo , alfa-Sinucleína/genética , alfa-Sinucleína/farmacologiaRESUMO
INTRODUCTION: There is intense interest in identifying cerebrospinal fluid (CSF) biomarkers of Parkinson's disease (PD), both for early diagnosis and to track effects of putative treatments. Nigrostriatal dopamine depletion characterizes PD. Predictably, CSF levels of 3,4-dihydroxyphenylacetic acid (DOPAC), the main neuronal metabolite of dopamine, are decreased in PD, even in patients with recent onset of the movement disorder. Whether low CSF DOPAC is associated specifically with parkinsonism has been unclear. In the neuronal cytoplasm dopamine undergoes not only enzymatic oxidation to form DOPAC but also spontaneous oxidation to form 5-S-cysteinyl-dopamine (Cys-DA). Theoretically, oxidative stress or decreased activity of aldehyde dehydrogenase (ALDH) in the residual nigrostriatal dopaminergic neurons would increase CSF Cys-DA levels with respect to DOPAC levels. PD, parkinsonian multiple system atrophy (MSA-P), and pure autonomic failure (PAF) are synucleinopathies; however, PAF does not entail parkinsonism. We examined whether an elevated Cys-DA/DOPAC ratio provides a specific biomarker of parkinsonism in synucleinopathy patients. METHODS: CSF catechols were assayed in PD (n = 24), MSA-P (n = 32), PAF (n = 18), and control subjects (n = 32). RESULTS: Compared to controls, CSF DOPAC was decreased in PD and MSA-P (p < 0.0001 each). In both diseases Cys-DA/DOPAC ratios averaged more than twice control (0.14 ± 0.02 and 0.13 ± 0.02 vs. 0.05 ± 0.01, p < 0.0001 each), whereas in PAF the mean Cys-DA/DOPAC ratio was normal (0.05 ± 0.01). CONCLUSIONS: CSF Cys-DA/DOPAC ratios are substantially increased in PD and MSA-P and are normal in PAF. Thus, in synucleinopathies an elevated CSF Cys-DA/DOPAC ratio seems to provide a specific biomarker of parkinsonism.