RESUMO
We investigated the subcellular distribution of dopamine D(1), D(2) and D(5) receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D(1) receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D(2) receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D(5) receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D(5) receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D(1) receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D(5) and D(2) receptor subtypes were not significantly altered by cocaine treatment. These data imply that D(5) receptors are predominantly cytoplasmic, D(2) receptors are diffusely distributed within the cell, whereas D(1) receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation.
Assuntos
Cocaína/farmacologia , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Isoformas de Proteínas/metabolismo , Receptores Dopaminérgicos/metabolismo , Frações Subcelulares/metabolismo , Animais , Membrana Celular/metabolismo , Citoplasma/metabolismo , Detergentes/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Lobo Frontal/citologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The recent discovery of novel high-affinity and selective dopamine D3 receptor (DA D3R) antagonists and partial agonists has provided tools with which to further elucidate the role DA D3R plays in substance abuse. The present study was conducted to evaluate the transport, metabolism, pharmacokinetics, and brain uptake of the DA D3R-selective fluorenyl amides, NGB 2904 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide] fumarate) and JJC 4-077 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-9H-fluorene-2-carboxamide hydrochloride], and the 2-pyridylphenyl amides, CJB 090 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridine-2-yl)benzamide hydrochloride] and PG 01037 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-trans-but-2-enyl)-4-(pyridine-2-yl)benzamide hydrochloride], all of which have been studied in animal models of psychostimulant abuse. Additional screening with a panel of human and rat Supersomes was performed for NGB 2904 and PG 01037. Drug-stimulated ATPase activation assays and bidirectional transport and efflux assays were used to test for substrate specificity of NGB 2904 and PG 01037 for human and rat efflux transporters. All compounds exhibited moderate elimination half-lives, ranging from 1.49 to 3.27 h, and large volumes of distribution (5.95-14.19 l/kg). The brain-to-plasma ratios ranged from 2.93 to 11.81 and were higher than those previously reported for cocaine. Brain exposure levels of NGB 2904 and PG 01037 were significantly reduced after intraperitoneal administration compared with intravenous administration. The metabolism of these compounds was mediated primarily by CYP3A subfamilies. PG 01037 was a P-glycoprotein-transported substrate. Higher doses of these compounds are often required for in vivo action, suggesting decreased bioavailability via extravascular administration that may be attributed to high drug efflux and hepatic metabolism. These studies provide important preclinical information for optimization of next-generation D3R selective agents for the treatment of drug addiction.
Assuntos
Benzamidas/uso terapêutico , Estimulantes do Sistema Nervoso Central , Dopaminérgicos/uso terapêutico , Antagonistas de Dopamina/uso terapêutico , Fluorenos/uso terapêutico , Piperazinas/uso terapêutico , Receptores de Dopamina D3/agonistas , Receptores de Dopamina D3/antagonistas & inibidores , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Benzamidas/química , Benzamidas/farmacocinética , Encéfalo/metabolismo , Linhagem Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Inibidores das Enzimas do Citocromo P-450 , Dopaminérgicos/química , Dopaminérgicos/farmacocinética , Antagonistas de Dopamina/química , Antagonistas de Dopamina/farmacocinética , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Fluorenos/química , Fluorenos/farmacocinética , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Piperazinas/química , Piperazinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Solubilidade , Espectrofotometria UltravioletaRESUMO
The effect of progesterone and estrogen treatment on the expression and function of P-glycoprotein (P-gp) was evaluated in JAR cells and a P-gp overexpressing cell line, NCI-ADR-RES. Western blot analysis and real-time Q-PCR were used to evaluate P-gp protein and MDR1 mRNA expression respectively in the cells following incubation with progesterone (P4) and/or beta-estradiol (E2). Cellular uptake studies of the P-gp substrates, saquinavir and paclitaxel, were performed to evaluate function. Treatment with either E2 or P4 resulted in a significant increase in P-gp protein levels in the NCI-ADR-RES cells at concentrations of or greater than 100 nM or 10 nM, respectively. JAR cells also had increased levels of P-gp with 100 nM of P4 but were much more sensitive to E2 showing increased P-gp at a concentration of 1 nM. Furthermore, E2 or P4 treatment resulted in a significant decrease in cellular uptake of the P-gp substrates tested in these cells lines. Based on mRNA quantitation, a transient increase (2-fold) in MDR1 levels was observed at 8 h postincubation with either E2 or P4, while MDR1 levels remained high in the JAR cells treated with E2 for 72 h postincubation. The addition of actinomycin D, a transcription inhibitor negated the increase in P-gp by P4 and E2. P4 and E2 increase P-gp expression and function in NCI-ADR-RES and JAR cells with the ERalpha-expressing cells (JAR) much more sensitive to E2. Furthermore, transcriptional regulation by E2 and P4 likely contributes to the modulation of P-gp levels.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Estradiol/farmacologia , Estrogênios/farmacologia , Placenta/citologia , Progesterona/farmacologia , Progestinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Dactinomicina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Leupeptinas/farmacologia , Gravidez , Inibidores da Síntese de Proteínas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Neuropathic pain resulting from spinal cord injury is often accompanied by maladaptive plasticity of the central nervous system, including the opioid receptor-rich periaqueductal gray (PAG). Evidence suggests that sensory signaling via the PAG is robustly modulated by dopamine D1- and D2-like receptors, but the effect of damage to the spinal cord on D1 and D2 receptor protein expression and function in the PAG has not been examined. Here we show that 21days after a T10 or C6 spinothalamic tract lesion, both mice and rats display a remarkable decline in the expression of D1 receptors in the PAG, revealed by western blot analysis. These changes were associated with a significant reduction in hindpaw withdrawal thresholds in lesioned animals compared to sham-operated controls. We investigated the consequences of diminished D1 receptor levels by quantifying D1-like receptor-mediated phosphorylation of ERK1,2 and CREB, events that have been observed in numerous brain structures. In naïve animals, western blot analysis revealed that ERK1,2, but not CREB phosphorylation was significantly increased in the PAG by the D1-like agonist SKF 81297. Using immunohistochemistry, we found that SKF 81297 increased ERK1,2 phosphorylation in the PAG of sham animals. However, in lesioned animals, basal pERK1,2 levels were elevated and did not significantly increase after exposure to SKF 81297. Our findings provide support for the hypothesis that molecular adaptations resulting in a decrease in D1 receptor expression and signaling in the PAG are a consequence of SCL.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Substância Cinzenta Periaquedutal/metabolismo , Receptores de Dopamina D1/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Benzazepinas/farmacologia , Proteína de Ligação a CREB/metabolismo , Modelos Animais de Doenças , Agonistas de Dopamina/farmacologia , Feminino , Masculino , Camundongos Transgênicos , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Substância Cinzenta Periaquedutal/patologia , Fosforilação , Ratos Long-Evans , Ratos Sprague-Dawley , Ratos Transgênicos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/genética , Traumatismos da Medula Espinal/patologiaRESUMO
Striatal medium spiny neurons are an important site of convergence for signaling mediated by the neurotransmitters dopamine and glutamate. We report that in striatal neurons in primary culture, signaling through group I metabotropic glutamate receptors (mGluRs) 1/5 and the D1 class of dopamine receptors (DRs) 1/5 converges to increase phosphorylation of the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2). Induction of mitogen-activated protein kinase kinase-dependent signaling cascades by either mGluR1/5 or DR1/5 gave rise to increases in phosphorylation of ERK2. Coactivation of mGluR1/5 and DR1/5 with (S)-3,5-dihydroxyphenylglycine and (+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride enhanced the phosphorylation of ERK2. This interaction between mGluR1/5 and DR1/5 required protein kinase C (PKC), because the PKC inhibitors calphostin C, bisindolylmaleimide I, and Gö6976 blocked DR1/5-enhanced phosphorylation of ERK2. Use of the phosphatase inhibitors calyculin and okadaic acid indicated that inhibition of protein phosphatases 1 and 2A dramatically enhanced ERK2 phosphorylation by mGluR1/5. Coactivation of mGluR1/5 and DR1/5 also enhanced cAMP-response element binding protein (CREB) phosphorylation (compared with each receptor agonist alone) but did not enhance CREB-mediated transcriptional activity. Thus, signal transduction pathways activated by DR1/5 and mGluR5 interact to modify downstream events in striatal neurons while retaining numerous regulatory checkpoints.
Assuntos
Corpo Estriado/citologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Neurônios/metabolismo , Receptores de Dopamina D1/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Análise de Variância , Animais , Benzazepinas/farmacologia , Benzoatos/farmacologia , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Ácidos Dicarboxílicos/farmacologia , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Interações Medicamentosas , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Imunofluorescência , Glicina/análogos & derivados , Glicina/farmacologia , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Modelos Biológicos , Neurônios/efeitos dos fármacos , Fenilacetatos/farmacologia , Fosforilação , Gravidez , Proteína Quinase C/farmacologia , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
BACKGROUND: Motor cortex stimulation (MCS) is a potentially effective treatment for chronic neuropathic pain. The neural mechanisms underlying the reduction of hyperalgesia and allodynia after MCS are not completely understood. OBJECTIVE: To investigate the neural mechanisms responsible for analgesic effects after MCS. We test the hypothesis that MCS attenuates evoked blood oxygen-level dependent signals in cortical areas involved in nociceptive processing in an animal model of chronic neuropathic pain. METHODS: We used adult female Sprague-Dawley rats (n = 10) that received unilateral electrolytic lesions of the right spinal cord at the level of C6 (SCL animals). In these animals, we performed magnetic resonance imaging (fMRI) experiments to study the analgesic effects of MCS. On the day of fMRI experiment, 14 days after spinal cord lesion, the animals were anesthetized and epidural bipolar platinum electrodes were placed above the left primary motor cortex. Two 10-min sessions of fMRI were performed before and after a session of MCS (50 µA, 50 Hz, 300 µs, for 30 min). During each fMRI session, the right hindpaw was electrically stimulated (noxious stimulation: 5 mA, 5 Hz, 3 ms) using a block design of 20 s stimulation off and 20 s stimulation on. A general linear model-based statistical parametric analysis was used to analyze whole brain activation maps. Region of interest (ROI) analysis and paired t-test were used to compare changes in activation before and after MCS in these ROI. RESULTS: MCS suppressed evoked blood oxygen dependent signals significantly (Family-wise error corrected P < 0.05) and bilaterally in 2 areas heavily implicated in nociceptive processing. These areas consisted of the primary somatosensory cortex and the prefrontal cortex. CONCLUSIONS: These findings suggest that, in animals with SCL, MCS attenuates hypersensitivity by suppressing activity in the primary somatosensory cortex and prefrontal cortex.