Lin C-S, Chow S, Lau A, Tu R, Lue T F
Knuppe Molecular Urology Laboratory, School of Medicine, University of California, San Francisco, CA 94143-1695, USA.
Int J Impot Res. 2002 Feb;14(1):15-24. doi: 10.1038/sj.ijir.3900802.
Sildenafil improves erectile function by inhibiting the cGMP-catalytic activity of phosphodiesterase type V (PDE5). We used rapid amplification of cDNA Ends-polymerase chain reaction (RACE-PCR) to isolate three PDE5 isoforms from human corpus cavernosum. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis on eight human cavernous tissue samples showed that all samples expressed the PDE5A1 at a lower level than the PDE5A2 isoform. Five samples expressed the PDE5A3 isoform at various levels while the other three did not. Analysis on non-penile tissues showed that all tissues expressed the A1 and A2 isoforms while only those that have substantial amounts of smooth muscle expressed the A3 isoform. Cloning and sequencing of the PDE5A gene showed that the isoform-specific 5'-ends of the PDE5 mRNAs are encoded from three alternative first exons arranged in the order of A1-A3-A2. Promoter activities were detected upstream from the A1-specific exon and in the intron preceding the A2-specific exon. The upstream PDE5A promoter is expected to direct the expression of all three PDE5 isoforms while the intronic PDE5A2 promoter only the A2 isoform. Both promoters were upregulated by increasing concentrations of either cAMP or cGMP. Several transcription factor AP2 and Sp1-binding sequences identified in the promoters are likely to be the mediators of cAMP/cGMP-responsiveness.
西地那非通过抑制5型磷酸二酯酶（PDE5）的cGMP催化活性来改善勃起功能。我们使用cDNA末端快速扩增-聚合酶链反应（RACE-PCR）从人海绵体中分离出三种PDE5亚型。对八份人海绵体组织样本进行的半定量逆转录-聚合酶链反应（RT-PCR）分析表明，所有样本中PDE5A1的表达水平均低于PDE5A2亚型。五个样本以不同水平表达PDE5A3亚型，而其他三个样本则不表达。对非阴茎组织的分析表明，所有组织均表达A1和A2亚型，而只有那些含有大量平滑肌的组织表达A3亚型。PDE5A基因的克隆和测序表明，PDE5 mRNA的亚型特异性5'末端由三个交替的第一外显子编码，其排列顺序为A1-A3-A2。在A1特异性外显子上游和A2特异性外显子之前的内含子中检测到启动子活性。预计上游PDE5A启动子可指导所有三种PDE5亚型的表达，而内含子PDE5A2启动子仅指导A2亚型的表达。两种启动子均通过增加cAMP或cGMP的浓度而上调。在启动子中鉴定出的几个转录因子AP2和Sp1结合序列可能是cAMP/cGMP反应性的介质。
