RESUMO
Extracellular vesicle (EV) are tiny membranous vesicles usually <500nm in size that recently emerged as a new paradigm in human intercellular signaling. EVs have shown a promising role in development of diagnostic markers in many pathophysiological disorders. The presence of chemosensory and therapeutically relevant G protein-coupled receptors (GPCRs) on EV membranes is poorly characterized. Here, we compare different methods including ultracentrifugation and polymer-charge-based separation to isolate EVs from cell culture media and human saliva. The presence of bitter taste GPCRs (T2R4 and T2R38) and a class A GPCR angiotensin II type 1 receptor on these EVs was characterized by qPCR, ELISA, and immunotransmission electron microscopy.
Assuntos
Vesículas Extracelulares/metabolismo , Receptor Tipo 1 de Angiotensina/isolamento & purificação , Receptores Acoplados a Proteínas G/isolamento & purificação , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Microscopia Eletrônica de Transmissão/métodos , Microscopia Imunoeletrônica/métodos , Oligopeptídeos/química , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/ultraestrutura , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestrutura , Ultracentrifugação/métodosRESUMO
It was already found that Ang II AT1 receptors are involved in the neuroadaptative changes induced by a single exposure to amphetamine, and such changes are related to the development of behavioral and neurochemical sensitization. The induction of the immediately early gene c-fos has been used to define brain activated areas by amphetamine. Our aim was to evaluate the participation of AT1 receptors in the neuronal activation induced by amphetamine sensitization. The study examined the c-fos expression in mesocorticolimbic areas induced by amphetamine challenge (0.5 mg/kg i.p) in animals pretreated with candesartan, a selective AT1 receptor blocker (3 mg/kg p.o × 5 days), and amphetamine (5 mg/kg i.p) 3 weeks before the challenge. Increased c-fos immunoreactivity was found in response to the amphetamine challenge in the dorsomedial caudate-putamen and nucleus accumbens, and both responses were blunted by the AT1 receptor blocker pretreatment. In the infralimbic prefrontal cortex, increased c-fos immunoreactivity was found in response to amphetamine and saline challenge, and both were prevented by the AT1 receptor blocker. No differences were found neither in ventral tegmental area nor prelimbic cortex between groups. Our results indicate an important role for brain Ang II in the behavioral and neuronal sensitization induced by amphetamine.
