RESUMEN
We utilized rat fetal lung fibroblasts (RFL-6) to evaluate our PDE5 inhibitors at cellular level and observed a decrease in cGMP accumulation induced by sodium nitroprusside (SNP) and PDE5 inhibitors with passage. To further investigate this observation, we examined cGMP synthesis via soluble guanylyl cyclase (sGC) and degradation via phosphodiesterases (PDEs) at different passages. At passage (p)4, p9, p14, major cGMP and cAMP degradation activities were contributed by PDE5 and PDE4, respectively. The PDE5 activity decreased 50% from p4 to p14, while PDE4 activity doubled. The cGMP accumulation was evaluated in the presence of sodium nitroprusside (SNP) and/or PDE inhibitors in p4 and p14 cells. SNP together with sildenafil, a PDE5 inhibitor, induced dose-dependent increase in cGMP levels in cells at p4, but showed little effect on cells at p14. The possible down regulation of sGC at mRNA level was explored using real-time RT-PCR. The result showed the mRNA level of the alpha1 subunit of sGC decreased about 98% by p9, while the change on beta1 mRNA was minimal. Consistently, sGC activities in cell lysate decreased by 94% at p9. Forskolin stimulated a dramatic increase in cAMP levels in cells at all passages examined. Our results show that sGC activity decreased significantly and rapidly with passage due to a down regulation of the alpha1 subunit mRNA, yet the adenylyl cyclase activity was not compromised. This study further emphasized the importance of considering passage number when using cell culture as a model system to study NO/cGMP pathway.
Asunto(s)
Guanilato Ciclasa/metabolismo , Péptidos Cíclicos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Receptores de Superficie Celular/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , Técnicas de Cultivo de Célula , Línea Celular , Colforsina/farmacología , AMP Cíclico/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Expresión Génica , Guanilato Ciclasa/química , Pulmón , Óxido Nítrico , Ratas , Receptores de Superficie Celular/química , Rolipram/farmacología , SolubilidadRESUMEN
The synthesis of the fused tetracyclic pyrroloquinolones 9a-i in four steps is described. The PDE5 inhibitory activities of these compounds, their selectivities against PDE1, PDE2, PDE3, PDE4 and PDE6, the preclinical pharmacokinetic assessments and the in vivo efficacy in increasing intracavernosal pressure are presented and discussed.
