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1.
Peptides ; 29(6): 919-32, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18353507

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid neuropeptide with promising therapeutic applications for the treatment of several pathophysiological states related to neurodegenerative diseases. However, its use for therapeutic applications is actually limited by its restricted bioavailability and rapid degradation. Therefore, metabolically stable PACAP analogs represent promising tools to further investigate the physiological roles of PACAP and ascertain its usefulness in some clinical conditions. In this study, derivatives of PACAP27 and PACAP38 have been rationally designed to develop PAC1 receptor agonists resistant to peptidase action. Results showed that N-terminal modifications confer resistance to dipeptidyl peptidase IV, a major proteolytic process involved in PACAP degradation. Moreover, in vitro incubation of both PACAP isoforms in human plasma revealed that PACAP38 is rapidly metabolized, with a half-life of less than 5 min, while PACAP27 was stable in these experimental conditions. Hence, following the elucidation of its plasmatic metabolites, PACAP38 was modified at its putative endopeptidase and carboxypeptidase sites of cleavage. All peptide analogs were tested for their ability to bind the PAC1 receptor, as well as for their potency to induce calcium mobilization and inhibit PC12 cell proliferation through the PAC1 receptor. This approach revealed two leading compounds, i.e. acetyl-[Ala15, Ala20]PACAP38-propylamide and acetyl-PACAP27-propylamide, which exhibited improved metabolic stability and potent biological activity. This study describes innovative data related to PACAP metabolism in human plasma and depicts the development of a metabolically stable PACAP38 analog, acetyl-[Ala15, Ala20]PACAP38-propylamide, which behaves as a super-agonist towards the PAC1 receptor.


Assuntos
Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Células CHO , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Cricetinae , Cricetulus , DNA Complementar , Relação Dose-Resposta a Droga , Eletroporação , Humanos , Dados de Sequência Molecular , Células PC12 , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/sangue , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/síntese química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/isolamento & purificação , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Fatores de Tempo
2.
Diabetes ; 56(10): 2494-500, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17626888

RESUMO

OBJECTIVE: Orexins are neuropeptides involved in energy homeostasis. We investigated the effect of orexin A (OxA) and orexin B (OxB) on intestinal glucose transport in the rat. RESEARCH DESIGN AND METHODS AND RESULTS: Injection of orexins led to a decrease in the blood glucose level in oral glucose tolerance tests (OGTTs). Effects of orexins on glucose entry were analyzed in Ussing chambers using the Na(+)-dependent increase in short-circuit current (Isc) to quantify jejunal glucose transport. The rapid and marked increase in Isc induced by luminal glucose was inhibited by 10 nmol/l OxA or OxB (53 and 59%, respectively). Response curves to OxA and OxB were not significantly different with half-maximal inhibitory concentrations at 0.9 and 0.4 nmol/l, respectively. On the one hand, OxA-induced inhibition of Isc was reduced by the neuronal blocker tetrodotoxin (TTX) and by a cholecystokinin (CCK) 2R antagonist, indicating involvement of neuronal and endocrine CCK-releasing cells. The OX(1)R antagonist SB334867 had no effect on OxA-induced inhibition, which is likely to occur via a neuronal and/or endocrine OX(2)R. On the other hand, SB334867 induced a significant right shift of the concentration-effect curve for OxB. This OxB-preferring OX(1)R pathway was not sensitive to TTX or to CCKR antagonists, suggesting that OxB may act directly on enterocytic OX(1)R. These distinct effects of OxA and OxB are consistent with the expression of OX(1)R and OX(2)R mRNA in the epithelial and nonepithelial tissues, respectively. CONCLUSIONS: Our data delineate a new function for orexins as inhibitors of intestinal glucose absorption and provide a new basis for orexin-induced short-term control of energy homeostasis.


Assuntos
Glucose/metabolismo , Absorção Intestinal , Mucosa Intestinal/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neurônios/fisiologia , Neuropeptídeos/fisiologia , Animais , Trânsito Gastrointestinal , Teste de Tolerância a Glucose , Hipotálamo/fisiologia , Mucosa Intestinal/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Jejuno/efeitos dos fármacos , Jejuno/inervação , Jejuno/fisiologia , Masculino , Neuropeptídeos/genética , Orexinas , RNA/genética , RNA/isolamento & purificação , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetrodotoxina/farmacologia
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