RESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuroprotective peptide expressed in the central nervous system. To date, changes in the expression and effect of endogenous PACAP have not been clarified with respect to spinal cord injury (SCI). The aim of this study was to elucidate the expression pattern and function of endogenous PACAP on the contusion model of SCI using heterozygous PACAP knockout (PACAP(+/-)) and wild-type mice. Real-time polymerase chain reaction methods revealed that the level of PACAP mRNA increased gradually for 14 days after SCI and that PAC1R mRNA levels also increased for 7 days compared with intact control mice. PACAP and PAC1R immunoreactivities colabeled with a neuronal marker in the intact spinal cord. Seven days after SCI, PAC1R immunoreactivity was additionally co-expressed with an astrocyte marker. Wild-type mice gradually recovered motor function after 14 days, but PACAP(+/-) mice showed significantly impaired recovery from 3 days compared with wild-type mice. The injury volume at day 7 in PACAP(+/-) mice, and the number of single-stranded DNA-immunopositive cells as a marker of neuronal cell death at day 3 were significantly higher than values measured in wild-type mice. These data suggest that endogenous PACAP is upregulated by SCI and has a neuroprotective effect on the damaged spinal cord.
Asunto(s)
Proteínas del Tejido Nervioso/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Astrocitos/metabolismo , Muerte Celular , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/biosíntesis , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/deficiencia , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/biosíntesis , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/fisiología , Recuperación de la Función , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
INTRODUCTION: We evaluated the effects of diabetes on nitric oxide-mediated relaxations and nitric oxide synthase activity in male rat corpus cavernosum smooth muscles. METHODS: Eight-week-old male rats were assigned to three groups: control (injected with the vehicle), DM (diabetes mellitus, induced by injection with 65 mg/kg streptozotocin), and TES (testosterone, testosterone supplemented after induction of diabetes). After 8 weeks, corpus cavernosum smooth muscle strips were mounted in an organ bath for isometric tension recordings. Electrical field stimulation (EFS, 2-ms pulse duration, 0.3-20 Hz and 3 s train) was applied to the strips precontracted with 30 microM phenylephrine. The microdialysis probe was inserted into the strip, and Krebs-Henseleit solution was perfused into the probe. The dialysate during EFS was collected, and the amount of NO(-)(2)/NO(-)(3) (NOx) released in the dialysate was measured by the Greiss method. Sodium nitroprusside (0.1 nM to 10 mM) and carbachol (1 nM to 10 mM) were cumulatively added to the strips precontracted with 30 microM phenylephrine. RESULTS: EFS caused frequency-dependent relaxations and NOx releases of the strips. Pretreatment with N(omega)-nitro-L-arginine (100 microM) and tetrodotoxin (1 microM) completely inhibited the relaxations and NOx releases. The maximum relaxation was significantly greater in the DM group than in the control or TES group. The release of NOx was significantly greater in the DM group than in the control or TES group. Sodium nitroprusside, the endothelium-independent vasodilator, relaxed the tissues in all three groups. There were no significant differences among control, DM and TES groups in the maximum relaxation to sodium nitroprusside. CONCLUSION: The present data suggest that diabetes enhances nitric oxide synthase activity and nitric oxide-mediated relaxations in the male rat corpus cavernosum by the reduced testosterone level in the diabetic animals.