RESUMO
An electrochemical impedimetric immunosensor was developed for the detection of the neuropeptide Corticotropin Releasing Hormone (CRH) based on the immobilization of half-antibody fragments on gold nanoparticles (AuNp). Then, the optimal conditions for the obtainment of AuNp through electroplating on a bare gold electrode were studied. The results showed that the obtainment of AuNp at a fixed potential of -0.2â¯V for 330â¯s, at 80⯰C and 2·10-3 mol·L-1 of HAuCl4 generates an adequate nanostructured surface and is a highly reproducible method. Also, the optimal conditions for immobilizing the half-antibody on AuNp were studied. The interaction of the CRH with the recognition layer of the immobilized half-antibody on the nanostructured surface was carried out by incubation at 4⯰C for 2â¯h. A dissolution of [Fe(CN)6]4-/[Fe(CN)6]3- as a redox probe was used to study the electrochemical responses of the nanostructured surface and the immobilization processes of the half-antibody and detection of CRH, using cyclic voltammetry and electrochemical impedance spectroscopy. An immunosensor was obtained for the specific detection of CRH, within a range of 10.0-80.0⯵gâ¯mL-1, with a limit of detection of 2.7⯵gâ¯mL-1 and a limit of quantification of 9.2⯵gâ¯mL-1. Additionally, the association constant between the CRH and the immobilized half-antibody was calculated at 1.96·105 M-1.
Assuntos
Técnicas Biossensoriais , Hormônio Liberador da Corticotropina/isolamento & purificação , Espectroscopia Dielétrica , Fragmentos de Imunoglobulinas/química , Anticorpos/química , Anticorpos/imunologia , Anticorpos Imobilizados/química , Hormônio Liberador da Corticotropina/química , Hormônio Liberador da Corticotropina/imunologia , Técnicas Eletroquímicas , Humanos , Imunoensaio , Fragmentos de Imunoglobulinas/imunologia , Limite de Detecção , Nanopartículas Metálicas/química , NanoestruturasRESUMO
Clinical and experimental studies have shown that the activation of corticotropin-releasing hormone (CRH) and noradrenergic systems mediate stress-induced anxiety. Repeated immobilization stress (RIS) has been shown to induce long-lasting anxiety behavior and changes in noradrenaline turnover. The present work was aimed at studying the effect of RIS on the in situ expression of CRH-LI in the central extended amygdala and paraventricular nucleus of the hypothalamus (PVN). Our results showed that RIS for 15 days induces a significant increase of CRH-LI expression in the central extended amygdala. The increase in CRH-LI expression in the central extended amygdala was sustained even after a 25-day stress-free period. The concomitant administration of desipramine (DMI), a specific noradrenaline uptake inhibitor, fully prevented the RIS-induced increase in CRH expression. RIS also induced an increase of CRH-LI expression in the PVN that was prevented by the concomitant DMI administration. In contrast to the sustained effect observed in the central extended amygdala, the RIS-induced increase of CRH-LI expression in the PVN was nonlasting. DMI administration also prevented the RIS-induced increase of adrenal gland weight. The present findings showing that RIS induces a sustained increase of CRH expression in the central extended amygdala suggest that the repeated activation of CRH neurons and CRH receptors in the central extended amygdala may underlie the long-lasting anxiety behavior induced by RIS. Further studies should address the mechanisms involved in the effect of DMI and its eventual relevance in the therapeutic actions of DMI.
Assuntos
Tonsila do Cerebelo/metabolismo , Antidepressivos Tricíclicos/uso terapêutico , Hormônio Liberador da Corticotropina/metabolismo , Desipramina/uso terapêutico , Estresse Psicológico/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/fisiologia , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Ansiedade/psicologia , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Hormônio Liberador da Corticotropina/imunologia , Imuno-Histoquímica , Masculino , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/fisiologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Ratos , Ratos Sprague-Dawley , Restrição Física , Núcleos Septais/efeitos dos fármacos , Núcleos Septais/metabolismo , Estresse Psicológico/psicologiaRESUMO
In order to investigate the production and secretion of hypothalamic factors by the prolactin and proopiomelanocortin (POMC)-derived, peptide-producing, transplantable rat pituitary tumor 7315a, we determined the concentrations of corticotropin-releasing factor (CRF)- and vasopressin (AVP)-like immunoreactivities (IR) in the tumor extracts [14.0 +/- 1.6 (SE) and 4.2 +/- 0.9 pmol/g, respectively] and incubation media (0.26 +/- 0.01 and 0.07 +/- 0.01 pmol/10(7) cells/h, respectively). Total peptide content correlated well with tumor weight. Moreover, there is a very good correlation between the CRF and AVP IR, but not as good between CRF or AVP IR and POMC peptide IR tumor contents. Tumor extracts were chromatographed on Sephadex G-75 and compared with chromatograms of stalk median eminence (SME) extracts from normal Buffalo rats. CRF IR in tumor chromatograms gave an unusual pattern of peaks. About 31% of the total CRF IR was eluted in the high molecular weight region. The major portion of CRF IR was located in a wide region of lower molecular weight. The AVP radioimmunoassay revealed a similar pattern of peaks in tumor and SME chromatograms. A propressophysin-like peak and a smaller peak coeluting with synthetic AVP were detected. Immunohistochemical staining of consecutive sections of the tumor indicated that AVP and CRF are often found in the same cell, but the CRF and AVP-producing cells are clearly distinct from the POMC peptide-producing cells.