Multiple bombesin-like peptides with opposite functions from skin of Odorrana grahami

AbstractBombesin-like peptides (BLPs) are a family of neuroendocrinic peptides that mediate a variety of biological activities. Three mature BLPs from the skin secretions of the frog Odorrana grahami were purified. Several bombesin-like peptide cDNA sequences encoding precursors of BLPs were identified from the skin cDNA library of O. grahami. This is the maximal diversity of BLPs ever found in animals. Five mature BLPs (B1–B5) based on the amino acid sequences derived from the cDNA cloning were synthesized. In the in vitro myotropic contraction experiment, all synthesized BLPs displayed a stimulating effect toward rat stomach strips, except B4 and B5 which showed the opposite effect, suggesting that certain BLPs may act as antagonists of bombesin receptors while most other BLPs act as agonists. This finding will facilitate the finding of novel bombesin receptors and novel ligands of bombesin receptors. The diversity of amphibian BLPs and their precursors were also analyzed and results suggest that amphibian BLPs and corresponding precursors of various sizes and processing patterns can be used as markers of taxonomic and molecular phylogenetics. The remarkable similarity of preproregions gives rise to very different BLPs and 3′-terminal regions in distantly related frog species, suggesting that the corresponding genes form a multigene family originating from a common ancestor. The diversification of BLP loci could thus be part of an evolutionary strategy developed by amphibian species as a result of shifts to novel ecological niches when environmental factors change rapidly

Efeitos comportamentais do tratamento neonatal com um antagonista do receptor do peptídeo liberador de gastrina: perspectivas para um modelo animal de transtorno do desenvolvimento neurológico

O receptor do peptídeo liberador de gastrina (GRPR) vem sendo relacionado a doenças do sistema nervoso central, incluindo desordens do desenvolvimento neurológico associadas ao autismo. No presente estudo, analisamos os efeitos do bloqueio do GRPR durante o período neonatal em medidas comportamentais relevantes em modelos animais de desordens do desenvolvimento neurológico. Ratos Wistar machos receberam injeções intraperitoneais (i. p) de salina (SAL) ou do antagonista do GRPR [D-Tpi6, Leu13 psi(CH2NH)-Leu14] bombesina (6-14) (RC-3095; 1 ou 10 mg/kg) duas vezes ao dia, do primeiro ao décimo dias de vida. Os animais tratados com RC-3095 demonstraram déficits pronunciados em interação social quando testados no período de 30-35 dias de idade. Prejuízos na retenção da memória 24h após o treino em ambas as tarefas; de reconhecimento do objeto novo (RON) e de esquiva inibitória, foram demonstrados quando os animais foram testados aos 60-71 dias de idade. O bloqueio neonatal do GRPR não afetou o comportamento em teste de memória de curta duração, 1,5h após o treino, tampouco o comportamento em campo aberto. As implicações desses achados em modelos animais de desordens do desenvolvimento neurológico são discutidas.The gastrin-releasing peptide receptor (GRPR) has been implicated in central nervous system diseases, including neurodevelopmental disorders associated with autism. In the present study we examined the effects of GRPR blockade during the neonatal period on behavioral measures relevant to animal models of neurodevelopmental disorders. Male Wistar rats were given an intraperitoneal (i. p. ) injection of either saline (SAL) or the GRPR antagonist [D-Tpi6, Leu13 psi(CH2NH)-Leu14] bombesin (6-14) (RC-3095; 1 or 10 mg/kg) twice daily for 10 days from postnatal days (PN) 1 to 10. Animals treated with RC-3095 showed pronounced deficits in social interaction when tested at PN 30-35 and impaired 24-h retention of memory for both novel object recognition (NOR) and inhibitory avoidance tasks tested at PN 60-71. Neither short-term memory tested 1. 5 h posttraining nor open field behavior were affected by neonatal GRPR blockade. The implications of the findings for animal models of neurodevelopmental disorders are discussed

Neuropeptide growth factors and small cell lung cancer

Quiescent cultures of Swiss 3T3 cells are a useful model system for elucidating the mechanisms of mitogenesis. Here, Swiss 3T3 cells were used to test novel mitogens and characterise bombesin antagonists. Bradykinin was identified as a potent mitogen, acting in synergy with insulin. Two groups of antagonists were characterised: the substance P analogues [DArg1, DPro2, DTrp7,9, Leu11]- substance P (antagonist A), [DArg1, DPhe5, DTrp7,9, Leu11]- substance P (antagonist D) and [Arg6, DTrp7,9, Leu11] substance P(6-11) (antagonist G); and the bombesin-specific antagonists (Leu13-psi(CH2NH)Leu14]bombesin and N- acetyl-GRP(20-26). The substance P antagonists appear to interact with a common domain on the receptors for three mitogenic neuropeptides in Swiss 3T3 cells: bombesin, vasopressin and bradykinin. The bombesin-specific antagonists did not interact with other receptors. Small cell lung cancer (SCLC) is an aggressive form of lung cancer. The bombesin-like peptides, including gastrin- releasing peptide (GRP), have been postulated to act as autocrine growth factors for these tumours. In SCLC cell lines, all the antagonists were shown to block the early effect of bombesin, mobilisation of cytosolic Ca2+. The substance P antagonists also inhibited SCLC growth in liquid and semisolid media, in a reversible, dose-dependent fashion. The bombesin-specific antagonists did not inhibit SCLC growth, suggesting that other growth factors blocked by the substance P antagonists may be more important for regulating SCLC growth. A variety of peptides and hormones were tested for their ability to mobilise cytosolic Ca2+ in SCLG cell lines. Ca2+ signals were induced by GRP, vasopressin, bradykinin, cholecystokinin, galanin and neurotensin, and all were blocked by antagonists D and G. These neuropeptides are suggested as possible growth factors for SCLC

Estrogen Regulation of the Pregnant Baboon Placental 11β-Hydroxysteroid Dehydrogenase (11β-HSD)-Catalyzed Metabolism of Cortisol and Cortisone and Its Effect on the Development of the Fetal Hypothalamic-Pituitary-Adrenal Axis

Pepe and Albrecht previously demonstrated that the estrogen-regulated change in transuteroplacental metabolism of cortisol (F) and cortisone (E) from preferential reduction (E to F) at midgestation to oxidation (F to E) near term results in a decline of bioactive cortisol crossing the placenta and reaching the fetus culminating in activation of the hypothalamic-pituitary adrenal axis of the baboon and the ontogenesis of rate-limiting steroidogenic enzymes culminating in de novo F secretion. Protein kinase A (PK-A) activity in the baboon fetal adrenal gland cytosolic fraction was increased 2-fold both at term [day 165 of gestation (term = 184 days)] and following treatment with estrogen at midgestation (day 100), compared to levels at mid-gestation. Protein kinase C (PK-C) activity in both cytosolic and membrane-bound fractions was similar at mid- and late gestation and not altered by treatment with estradiol. Secondly, we determined whether maturation of the fetal adrenal reflects enhanced expression of the mRNA for the ACTH precursor proopiomelanocortin (POMC). Pituitary POMC mRNA, was greater in baboon fetuses at term than at mid-gestation and increased on day 100 in estrogen-treated animals. Immunohistochemical studies confirmed that ACTH protein paralleled changes in POMC mRNA. Next, we determined whether the ontogenetic increase in POMC/ACTH in fetal baboon pituitaries reflected an increase in hypothalamic corticotropin-releasing hormone (CRH). CRH protein and CRH mRNA at mid-gestation were similar to concentrations in fetal baboons of late gestation and were not altered in fetuses in which the mother was treated with estradiol. In conclusion, the onset of fetal adrenal steroidogenic maturation normally near term and prematurely at mid-gestation following estrogen administration reflects increased expression of fetal pituitary POMC mRNA/ACTH protein and are not associated with a concomitant increase in hypothalamic CRH peptide or CRH mRNA. Thirdly, since the 11β-hydroxysteroid dehydrogenase (11β-HSD)-catalyzed metabolism of maternal cortisol and cortisone by the placenta is an important component in the sequence of events regulating the function of the baboon fetal pituitary-adrenocortical axis, both baboon 11β-HSD-1 and -2 gene promoters were isolated and sequenced and shown to exhibit extensive homology to their respective human sequences. Both genes were subcloned into luciferase reporter vectors and transiently-transfected into human placental JEG-3 cells. In the presence of 17β-estradiol and estrogen receptor α, basal promoter activities of both 11β-HSD-1 and -2 increased 8-fold. Finally, RT-PCR analysis demonstrated a significant reduction of both 11β-HSD-1 and -2 mRNAs in baboon placental syncytiotrophoblast-enriched fractions following reduction of maternal serum estrogen in vivo by a highly-specific P450-aromatase enzyme inhibitor, CGS 20267

Untersuchungen zur Rolle der Glutaminyl-Cyclase bei der pathologischen Bildung von Pyroglutamyl-Peptiden - [kumulative Dissertation]

Posttranslationale Modifikationen spielen eine wichtige Rolle bei der Reifung von Proteinen und beeinflussen deren Funktion und Stabilität im Organismus. Die Bildung von Pyroglutamat (pGlu) ist für eine Reihe von Peptidhormonen, wie z.B. Thyreoliberin (TRH) und Gonadoliberin (GnRH) sowie sezernierten Proteinen beschrieben worden. Die pGlu-Modifikation entsteht durch intramolekulare Zyklisierung eines Glutaminylrestes. Die Reaktion wird in Tieren und Pflanzen durch Glutaminyl-Cyclasen (QC; EC 2.3.2.5) katalysiert. Im menschlichen Organismus geht offensichtlich eine Reihe von pathophysiologischen Veränderungen mit einer Bildung von Pyroglutamylpeptiden einher. Dies betrifft u.a. neurodegenerative Erkrankungen wie die Alzheimersche Krankheit oder entzündliche Prozesse. In der vorliegenden Arbeit wurde die QC-vermittelte Bildung von pGlu am N-Terminus der Peptide Aβ und CCL2 untersucht, welchen offenbar Schlüsselfunktionen in diesen Erkrankungen zukommen. Es konnte gezeigt werden, dass QC auch die Zyklisierung von Glutamat am N-Terminus von Aβ-Peptiden nach amyloidogener und Prohormonkonvertase-vermittelter Prozessierung in Säugerzellkultur katalysiert. Die Applikation eines spezifischen QC-Inhibitors verringerte sowohl die Glutaminyl- als auch die Glutamylzyklisierung. Die QC-katalysierte Bildung von pGlu-Aβ verläuft in den zellulären Modellsystemen vorwiegend intrazellulär. Der N-terminale pGlu-Rest von CCL2 beeinflusst die Stabilität und somit die chemotaktische Potenz dieses Chemokins. Durch eine Sezernierung von nicht vollständig gereiftem CCL2 kommt es zu einem Abbau durch Aminopeptidasen, wie z.B. DP4. Die Applikation eines QCInhibitors beeinflusste die arteriosklerotischen Veränderungen im Modell der „cuff“-induzierten Arteriosklerose. In einem weiteren Teil der Arbeit konnte ein Isoenzym der QC isoliert und charakterisiert werden. Humane QC und isoQC weisen eine Sequenzidentität von ca. 50% auf. Im Unterschied zur QC ist die isoQC ein Typ II-Transmembranprotein, welches im Golgi-Apparat zurückgehalten wird. Diesbezüglich ähnelt das Protein Glykosyltransferasen. Aufgrund der stabilisierenden Wirkung des pGlu-Restes für den N-Terminus verschiedener Proteine hat die Inhibierung von QC eine proteolysefördernde und dadurch aktivitätsmodulierende Wirkung. Dies könnte zur Behandlung verschiedener Krankheitssymptome dienen, welche durch das Auftreten von stabilen pGlu-Peptiden verursacht werden. Insofern sind die im Rahmen dieser Arbeit gewonnenen Erkenntnisse für die Entwicklung spezifischer QC-Inhibitoren zur Behandlung einer Reihe von pGlu-Proteinabhängigen Erkrankungen bedeutsam.von Holger Cyni

Somatostatin, cortistatin, ghrelin and their receptors: From an endocrine system to a pleiotropic system of pathophysiologic relevance

Somatostatina (SST)/cortistatina (CST) y ghrelina son los más conocidos miembros de dos sistemas complejos formados por un gran número de péptidos y receptores (ssts y GHS-Rs, respectivamente), que, aunque exhiben claras diferencias desde el punto de vista estructural, evolutivo y funcional, presentan importantes similitudes y coincidencias. De hecho, además de los bien conocidos efectos opuestos de los sistemas SST/CST y ghrelina en la regulación de varios tipos celulares en la adenohipófisis, actuando directamente a nivel hipofisario o indirectamente a través del hipotálamo, ambos sistemas ejercen además un gran número de funciones biológicas diversas. Sin embargo, numerosas pruebas apuntan a que los sistemas de la SST/CST y de la ghrelina no son sistemas de neuro-péptidos y receptores aislados con funciones diversas y separadas, sino que componen un red de componentes interrelacionados fisiológicamente capaces de interaccionar funcionalmente a nivel molecular, celular y orgánico para regular multitud de funciones. Teniendo en cuenta estos antecedentes, el reto consiste en identificar, caracterizar y colocar en un contexto fisiológico y en una posición de posible aplicación terapéutica los elementos que componen el supra-sistema SST/CST/ghrelina y receptores, sus interacciones y los nuevos elementos. En este sentido, el objetivo general de la Tesis consiste en ampliar nuestros conocimientos acerca del papel de los sistemas de la SST/CST y la ghrelina en condiciones pato-fisiológicas, extendiendo las acciones más clásicas a nivel hipotálamo-hipofisario, e incorporando nuevas funciones e interacciones en la interfase del cáncer, el metabolismo y los desórdenes neurodegenerativos. Los resultados obtenidos en el presente estudio se resumen en los siguientes: 1)Los sistemas de la SST y la ghrelina están regulados diferencialmente y de manera tejido-dependiente en respuesta a insultos metabólicos en el eje hipotálamo-hipófisis-estómago, donde parece que estos sistemas se regulan de manera cruzada. De hecho, el ayuno altera, en ratones, la expresión de SST y ghrelina en dicho eje, así como los niveles de ghrelina plasmáticos. Interesantemente, los ratones deficientes en SST presentan elevados niveles de ghrelina en plasma y en estómago, lo que sugiere que los niveles basales de SST son importantes en la regulación de la ghrelina. Además, la regulación de la expresión de la enzima responsable de la acilación de la ghrelina, así como su actividad enzimática y la disponibilidad de sustrato, podrían actuar sinérgicamente para regular los niveles plasmáticos de ghrelina acilada bajo condiciones de estrés metabólico en ratones. 2)Los sistemas de la SST/CST y de la ghrelina están desregulados en cáncer de mama en mujeres, donde algunos de los nuevos componentes de estos sistemas (sst5TMD4, In2-ghrelina, GHS-R1b, GOAT) pueden tener una contribución especial en esta patología. Así, el receptor truncado sst5TMD4 está presente en cánceres de mama agresivos, donde se asocia con marcadores de mal pronóstico. Además, en líneas celulares derivadas de cáncer de mama, su expresión incrementa su malignidad, posiblemente a través de la disrupción de sistema "protector"compuesto por SST y sst2. Por otro lado, una nueva variante de la ghrelina, la In2-ghrelina, así como la GOAT o el GHS-R1b, están altamente expresados en muestras humanas de cáncer de mama y su sobreexpresión en líneas celulares de cáncer de mama provoca un incremento en su tasa de proliferación. 3)Los sistemas de la SST/CST y de la ghrelina, en pacientes con la enfermedad de Alzheimer, están profundamente alterados en el giro temporal, una de las regiones corticales más importantes en procesos cognitivos. Puesto que los estos sistemas parecen ejercer acciones protectoras influyendo directamente en los procesos relacionados con la memoria, la desregulación de estos sistemas en el lóbulo temporal de los pacientes con enfermedad de Alzheimer puede contribuir a las alteraciones de los procesos cognitivos observadas en esta patología. En conclusión, los sistemas de la SST/CST y de la ghrelina están presentes y regulados diferencialmente en ejes y en condiciones patológicas claramente diferentes y distantes de sus originales funciones neuroendocrinas a nivel hipotálamo-hipofisario. Por lo tanto, parece razonable proponer que la SST/CST, la ghrelina, los péptidos relacionados y sus receptores conforman dos sistemas pleiotrópicos profundamente interrelacionados, los cuales podrían ser considerados como nuevas dianas para la intervención terapéutica