Liu H, Maurice D H
Department of Pharmacology & Toxicology, Queen's University, Kingston, Ontario, Canada.
Br J Pharmacol. 1998 Dec;125(7):1501-10. doi: 10.1038/sj.bjp.0702227.
A combination of pharmacological, molecular biological and biochemical approaches were used to investigate the differential expression of two cyclic GMP-inhibited cyclic nucleotide phosphodiesterase genes (PDE3A and PDE3B) in the rat. RT PCR using PDE3A- or PDE3B-specific oligonucleotide primers allowed amplification of products encoding PDE3A (508 bp) or PDE3B (499 bp) sequences from several rat tissues (heart, aorta, liver, kidney and epididymal fat), from primary cultures of aortic vascular smooth muscle cells (VSMC) as well as from an SV40 large T-antigen immortalized aortic VSMC line. Immunoblotting experiments with PDE3-selective antisera allowed the detection of both PDE3A and PDE3B immunoreactive proteins in several rat tissues, including tissues of the cardiovascular system, in primary cultures of aortic VSMC and in an SV40 large T-antigen immortalized aortic VSMC line. In all cases, PDE3A was expressed as a 120 kDa protein which was only detected in the cytosolic fraction. PDE3B was expressed as a 135 kDa protein and its expression was limited to the particulate fraction of all tissues and cells studied. Prolonged incubation of cultured aortic VSMC with agents that increase VSMC cyclic AMP (forskolin or 8-bromo-cyclic AMP) produced marked time-dependent increases in PDE3 activity which correlated with increases in PDE3A and PDE3B RT PCR signals and a marked increase in particulate PDE3 activity and PDE3B protein. The physiological, pharmacological and biochemical implications of these findings are discussed based on previous reports of the effects of PDE3 inhibitors in the cardiovascular system and the relevance of our findings are presented in the context of the development of PDE3A and/or PDE3B-selective pharmacological agents.
采用药理学、分子生物学和生物化学相结合的方法，研究大鼠体内两种环磷酸鸟苷抑制性环核苷酸磷酸二酯酶基因（PDE3A和PDE3B）的差异表达。使用PDE3A或PDE3B特异性寡核苷酸引物进行逆转录聚合酶链反应（RT-PCR），能够从大鼠的多种组织（心脏、主动脉、肝脏、肾脏和附睾脂肪）、主动脉血管平滑肌细胞（VSMC）原代培养物以及SV40大T抗原永生化主动脉VSMC系中扩增出编码PDE3A（508 bp）或PDE3B（499 bp）序列的产物。用PDE3选择性抗血清进行免疫印迹实验，可在大鼠的多种组织（包括心血管系统组织）、主动脉VSMC原代培养物以及SV40大T抗原永生化主动脉VSMC系中检测到PDE3A和PDE3B免疫反应蛋白。在所有情况下，PDE3A均以120 kDa的蛋白形式表达，且仅在胞质部分检测到。PDE3B以135 kDa的蛋白形式表达，其表达仅限于所有研究组织和细胞的颗粒部分。用能增加VSMC环磷酸腺苷（福斯可林或8-溴环磷酸腺苷）的试剂对培养的主动脉VSMC进行长时间孵育，可使PDE3活性出现明显的时间依赖性增加，这与PDE3A和PDE3B的RT-PCR信号增加以及颗粒性PDE3活性和PDE3B蛋白的显著增加相关。基于先前关于PDE3抑制剂在心血管系统中作用的报道，讨论了这些发现的生理、药理和生化意义，并在PDE3A和/或PDE3B选择性药理剂的开发背景下阐述了我们研究结果的相关性。
