Studies on Intracellular Signal Transduction Pathway Involved in Human and Rabbit's Corpus Cavernosal Smooth Muscle Relaxation / 대한남성과학회지

PURPOSE: Nitric oxide (NO) and phosphodiesterases (PDEs) play key roles in mediating relaxation of corpus cavernosal smooth muscle by increasing intracellular cGMP level. Here, we investigated effects of NO-donor (sodium nitroprusside, SNP) and penile specific type-V PDE inhibitor (zaprinast) in human and rabbit corpus cavernosal cells and tissues in vitro. MATERIALS AND METHODS: The cultured smooth muscle cells and tissues of human and rabbit corpus cavernosum were treated with increasing concentrations of SNP or zaprinast for 5 and 20 minutes, respectively, and intracellular cGMP levels were measured by radioimmunoassay. Organ bath study was performed to measure the relaxation effects of drugs on precontracted corpus cavernosal muscle strips. RESULTS: Although both NO-donor and type-V PDE inhibitor effectively stimulated the accumulation of cGMP in a dose-dependent manner, magnitude of cGMP increase and specificity of drug were found to be species-dependent. In human corpus cavernosal tissues, cGMP was increased upto 10- and 5-folds by SNP and zaprinast, respectively. However, magnitude of increase was much less in cultured smooth muscle cells. In rabbit, SNP effect was most prominent in cultured cells and effects of SNP and zaprinast were modest in tissues. Both agents also resulted in effective relaxation of human and rabbit cavernosal tissue strips. Similar patterns of dose-response curves were shown between results from the organ bath studies and cGMP radioimmunoassay with cavernosal smooth muscle cells. CONCLUSIONS: Present results show that effects of SNP and zaprinast are not coincident in different species, suggesting possible species-specificities of these two agents. Measurement of cGMP changes in cultured cavernosal smooth muscles cells could be reflected to the relaxation effects of drugs on corpus cavernosal muscle strips.

Development of New Gene Therapy for Erectile Dysfunction Using Antisense cDNA for Type V Phosphodiesterase Gene / 대한남성과학회지

PURPOSE: We examined the feasibility of gene therapy for erectile dysfunction using cultured human corpus cavernosal smooth muscle cells. MATERIALS AND METHODS: The vector construct was designed to contain a fusion gene of enhanced green fluorescent protein (gfp) and beta-galactosidase (lacZ) which was under control of CMV promoter. Cells within the second passage were transfected with the vector DNA only and vector DNA containing a part of cDNA in an antisense orientation for human type V phosphodiesterase (PDE) gene using lipofection. Reporter gene expressions were investigated by fluorescence microscopy and X-gal staining at 24-hour interval. Effects of gene transfer of type V PDE antisense cDNA were investigated after 48 hours of gene transfection using the RT-PCR for type V PDE gene and measurement of intracellular cGMP level treated by sodium nitroprusside (SNP), NO-donor, of various concentrations. RESULTS: Expressions of gfp and lacZ were observed for upto 72 hours after gene transfection. Results from RT-PCR analysis also confirmed the gene expression at the transcriptional level. Type V PDE mRNA expression was significantly inhibited and magnitude of cGMP increase was significantly enhanced by gene transfer of antisense cDNA for type V PDE gene compared with non-transfectant control cells. CONCLUSIONS: Our results demonstrate that the liposome-mediated gene transfer was shown to be effective in corpus cavernosal smooth muscle cells. Gene transfer of antisense cDNA for type V PDE gene effectively inhibited the expression of type V PDE gene at the transcriptional and translational levels, suggesting that this newly developed gene transfer system may be a potential gene therapy in the treatment of erectile dysfunction.