RESUMO
Axon guidance molecules, critical for neurodevelopment, are also implicated in morphological and other neurodaptative changes mediated by physiological or pharmacological events in adult brain. As an example, the psychostimulant cocaine markedly alters axon guidance molecules in adult brain of cocaine-treated rats. To decipher a potential link between drug-induced activation of G-protein-coupled receptors (GPCRs) and modulation of axon guidance molecules, we investigated whether GPCR activity in a SK-N-MC human neuroepithelioma cell line (which expresses low levels of D1 dopamine receptors) affects gene expression of axon guidance molecules (semaphorins, ephrins, netrins, and their receptors). Using real-time polymerase chain reaction, we identified 17 of 26 axon guidance molecules in these cells, with varying levels of expression. Forskolin, which raised intracellular cAMP levels 340%, increased EphA5, EphB2, and Neuropilin1 expression, paralleling reported changes in the rat hippocampus after cocaine treatment. The dopamine receptor agonist dihydrexidine, which raised cAMP levels 22%, promoted regulatory changes in EphrinA1, EphrinA5, EphB1, DCC, and Semaphorin3C, whereas (+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF81297) altered EphA5, EphrinA1, EphrinA5, and neuropilin1. cAMP and other signal transduction pathways may regulate gene expression of axon guidance molecules, potentially linking monoamine receptor activation to signal transduction cascades, transcriptional regulation of axon guidance molecules, and alterations in neural networks.
Assuntos
Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Receptores Acoplados a Proteínas G/fisiologia , Adaptação Fisiológica , Benzazepinas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Colforsina/farmacologia , AMP Cíclico/metabolismo , Agonistas de Dopamina/farmacologia , Efrinas/genética , Efrinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptores de Netrina , Neuropilina-1/genética , Neuropilina-1/metabolismo , Fenantridinas/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Semaforinas/genética , Semaforinas/metabolismoRESUMO
The vesicular monoamine transporter-2 (VMAT2) sequesters monoamine neurotransmitters into vesicles and prevents neurotoxicity. Human or monkey striatum generated three VMAT2 immunoreactive proteins of approximately 75 kDa, approximately 52-55 kDa, and approximately 45 kDa. The approximately 55-kDa band is considered the unglycosylated native protein. Deglycosylation of the VMAT2 from striatum or human VMAT2 expressed in HEK293 cells yielded a approximately 45-kDa, but not a 55-kDa immunoreactive band. We investigated this apparent mismatch between observed molecular size and predicted size.
Assuntos
Corpo Estriado/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Análise de Variância , Animais , Sequência de Bases , Western Blotting/métodos , Clonagem Molecular/métodos , Corpo Estriado/efeitos dos fármacos , Dopamina/farmacocinética , Glicosilação , Humanos , Macaca mulatta , Substâncias Macromoleculares/metabolismo , Modelos Moleculares , Peso Molecular , Mutagênese , Fosforilação , Transfecção , Trítio/farmacocinética , Proteínas Vesiculares de Transporte de Monoamina/química , Proteínas Vesiculares de Transporte de Monoamina/genéticaRESUMO
The synthesis and preliminary pharmacological evaluation of 8,9-dihydroxy-1,2,3,11b-tetrahydrochromeno[4,3,2,-de]isoquinoline (5, now named dinoxyline) is described. This molecule was designed as a potential bioisostere that would conserve the essential elements of our beta-phenyldopamine D(1) pharmacophore (i.e., position and orientation of the nitrogen, hydroxyls, and phenyl rings). Previously, we have rigidified these elements using alkyl bridges, as exemplified in the dopamine D(1) full agonist molecules dihydrexidine (1) and dinapsoline (2). This approach has been modified and we now show that it is possible to tether these elements using an ether linkage. Preliminary pharmacology has revealed that 5 is a potent full D(1) agonist (K(0.5) <10 nM; EC(50)=30 nM), but also has high affinity for brain D(2)-like and cloned D(2) and D(3) receptors. Interestingly, whereas 1 and 2 and their analogues have only moderate affinity for the human D(4) receptor, 5 also has high affinity for this isoform. Moreover, although N-alkylation of 1 and 2 increases D(2) affinity, the N-allyl (15) and N-n-propyl (17) derivatives of 5 had decreased D(2) affinity. Therefore, 5 may be engaging different amino acid residues than do 1 and 2 when they bind to the D(2) receptor. This is the first example of a ligand with high affinity at all dopamine receptors, yet with functional characteristics similar to dopamine. These rigid ligands also will be useful tools to determine specific residues of the receptor transmembrane domains that are critical for agonist ligand selectivity for the D(4) receptor.
