RESUMEN
Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg(-1), ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loaded with fluorescent calcium indicator were used to measure intracellular Ca(2+) transients. Sibutramine 5 and 20 mg kg(-1) reduced ejaculation latency whereas 50 mg kg(-1) increased ejaculation latency. Sibutramine 3 to 30 microM greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 microM there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl(2). In HEK293 cells, sibutramine 10 to 100 microM inhibited intracellular Ca(2+) transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca(2+) may be involved in the delayed ejaculation observed with high doses of sibutramine.
Asunto(s)
Depresores del Apetito/efectos adversos , Ciclobutanos/efectos adversos , Eyaculación/efectos de los fármacos , Contracción Isométrica/efectos de los fármacos , Vesículas Seminales/efectos de los fármacos , Conducto Deferente/efectos de los fármacos , Agonistas de Receptores Adrenérgicos alfa 1 , Animales , Calcio/metabolismo , Cloruro de Calcio/farmacología , Carbacol/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas In Vitro , Masculino , Neurotransmisores/farmacología , Norepinefrina/farmacología , Ratas , Ratas Wistar , Receptores Muscarínicos/metabolismo , Vesículas Seminales/metabolismo , Conducto Deferente/metabolismoRESUMEN
L-arginine is a basic amino acid that has versatile metabolic roles, being involved in the generation of a wide range of biologically active intermediates such as nitric oxide (NO), polyamines, creatine and L-amino acids [1]. Because the levels of L-arginine reflect a metabolic crossroads, the mechanisms of its synthesis and degradation in peripheral tissues are very well described. However, there is an increasing amount of data also implicating this amino acid as a mediator of central nervous system activities and those are not yet fully understood. Here we shall summarize the tissue-specific pathways controlling L-arginine intracellular and blood levels and also the emerging evidence pointing to a role of this metabolite in the central regulation of diverse physiological processes, as blood pressure control and inflammatory response. As a conclusion we shall discuss the advantages of targeting L-arginine metabolism over other NO donors, as a general strategy to correct NO deficiency related diseases and discuss a few new patents recently deposited which take into account the rational perspective outlined in the present review.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Arginina/metabolismo , Encéfalo/metabolismo , Transducción de Señal/fisiología , Animales , Arginina/farmacología , Encéfalo/efectos de los fármacos , Metabolismo Energético , Humanos , Modelos Biológicos , Óxido Nítrico/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Aiming to extend the knowledge about the diversity of bradykinin-potentiating peptides (BPPs) and their precursor proteins, a venom gland cDNA library from the South American rattlesnake (Crotalus dursissus terrificus, Cdt) was screened. Two novel homologous cDNAs encoding the BPPs precursor protein were cloned. Their sequence contain only one single longer BPP sequence with the typical IPP-tripeptide, and two short potential BPP-like molecules, revealing a unique structural organization. Several peptide sequences structurally similar to the BPPs identified in the precursor protein from Cdt and also from others snakes, were chemically synthesized and were bioassayed both in vitro and in vivo, by means of isolated smooth muscle preparations and by measurements of blood pressure in anaesthetized rats, respectively. We demonstrate here that a pyroglutamyl residue at the N-terminus with a high content of proline residues, even with the presence of a IPP moiety characteristic of typical BPPs, are not enough to determine a bradykinin-potentiating activity to these peptides. Taken together, our results indicate that the characterization of the BPPs precursor proteins and identification of characteristic glutamine residues followed by proline-rich peptide sequences are not enough to predict if these peptides, even with a pyroglutamyl residue at the N-terminus, will present the typical pharmacological activities described for the BPPs.
Asunto(s)
Antihipertensivos/aislamiento & purificación , Venenos de Crotálidos/química , Crotalus/metabolismo , Oligopéptidos/aislamiento & purificación , Precursores de Proteínas/aislamiento & purificación , Glándulas Salivales/metabolismo , Secuencia de Aminoácidos , Animales , Antihipertensivos/síntesis química , Antihipertensivos/farmacología , Secuencia de Bases , Presión Sanguínea/efectos de los fármacos , Clonación Molecular , Cobayas , Íleon/efectos de los fármacos , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Oligopéptidos/síntesis química , Oligopéptidos/genética , Oligopéptidos/farmacología , Precursores de Proteínas/síntesis química , Precursores de Proteínas/genética , Precursores de Proteínas/farmacología , Ratas , Ratas Wistar , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Relación Estructura-ActividadRESUMEN
This work describes chemical properties and anti-hypertensive activity of an oral pharmaceutical formulation obtained from the complexation of beta-cyclodextrin (beta-CD) with bradykinin potentiating penta peptide (BPP-5a) founded in the Bothrops jararaca poison. Physical chemistry characterizations were recorded in order to investigate the intermolecular interactions between species in complex. Circular dichroism data indicated conformational changes of BPP-5a upon complexation with beta-CD. ROESY and theoretical calculations showed a selective approximation of triptophan moiety into cavity of beta-CD. Isothermal titration calorimetry data indicated an exothermic formation of the complex, which is accomplished by reduction of entropy. The anti-hypertensive activity of the BPP-5a/beta-CD complex has been evaluated in spontaneous hypertensive rats, showing better results than pure BPP-5a.
Asunto(s)
Química Farmacéutica/métodos , Oligopéptidos/química , beta-Ciclodextrinas/química , Inhibidores de la Enzima Convertidora de Angiotensina/química , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Antihipertensivos/química , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Bothrops , Calorimetría , Dicroismo Circular , Frecuencia Cardíaca/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Masculino , Modelos Moleculares , Estructura Molecular , Oligopéptidos/farmacología , Ratas , Ratas Endogámicas SHR , Termodinámica , Venenos de Víboras/químicaRESUMEN
NUDEL-oligopeptidase is a cytosolic cysteine peptidase, active towards oligopeptides and involved in the conversion and inactivation of a number of bioactive peptides. This protein interacts with neuronal proteins and is essential for brain development and cortical organization during embryogenesis. In this study, 5'-flanking sequences of the human and rabbit NUDEL-oligopeptidase gene were cloned into the pGL3 reporter gene vector and the promoter activity of the full-length fragment and deletions series was measured in transient transfection assays using two different cell lines, namely, C6 rat glioma and NH15 human neuroblastoma. Overall, a very similar pattern of promoter activity was obtained for both rabbit and human NUDEL-oligopeptidase promoter sequences, and their respective serial deletion constructs upon transient transfection into these cell lines. The only exception was for the longest rabbit upstream sequence that displayed about 1.8-fold higher luciferase expression upon transfection into NH15 neuronal cells than that observed upon transfection into C6 glioma cells. On the other hand, no significant difference was observed for the human longest sequence. These results are in good agreement with the expression pattern of NUDEL-oligopeptidase in human and rabbit tissues.
Asunto(s)
Proteínas Portadoras/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Animales , Secuencia de Bases , Northern Blotting , Línea Celular Tumoral , Sistema Nervioso Central/metabolismo , Clonación Molecular , Vectores Genéticos/genética , Humanos , Luciferasas , Datos de Secuencia Molecular , Conejos , Ratas , Análisis de Secuencia de ADNRESUMEN
Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr(613) (EP24.16) or Tyr(612) (EP24.15) to either Phe or Ala promoted a strong reduction of k(cat)/K(M) for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k(cat)/K(M) of 2x10(5) M(-1) s(-1) for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k(cat)/K(M)=3x10(5) M(-1) s(-1)) which contains Gly at the corresponding position; the wild type EP24.15 has a k(cat)/K(M) of 2.5x10(4) M(-1) s(-1) for this substrate.
Asunto(s)
Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Alanina/genética , Alanina/fisiología , Animales , Sitios de Unión , Catálisis , Cinética , Metaloendopeptidasas/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad , Especificidad por Sustrato , Tirosina/genética , Tirosina/fisiologíaRESUMEN
The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (beta-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P(1), although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli HslVU protease suggested two putative threonine catalytic groups, one in the N-domain (T(136), K(168), and Y(264)) and the other in the C-domain (T(375), K(409), and S(502)). Mutagenesis studies showed that the replacement of K(409) by A caused a complete loss of the proteolytic activity, whereas the mutation of K(168) to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T(375), K(409), and S(502) at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.