Zhou L, Thompson W J, Potter D E
Department of Pharmacology and Toxicology, Morehouse School of Medicine, Atlanta, Georgia 30310, USA.
Invest Ophthalmol Vis Sci. 1999 Jul;40(8):1745-52.
To characterize cyclic nucleotide phosphodiesterase isozyme activities in human trabecular meshwork cells and primary cultures of porcine trabecular meshwork cells.
Radioimmunoassay of acetylated acid extracts was used to determine changes in cyclic adenosine monophosphate (cAMP) and cyclic quanosine monophosphate (cGMP) in human trabecular meshwork cells treated with phosphodiesterase isoform selective inhibitors. Cyclic nucleotide phosphodiesterase activities were measured using the two-step radioisotope procedure (Thompson). Enzyme activities in the supernatant of human cells were fractionated using anion-exchange chromatography. Additionally, human and porcine trabecular meshwork cell transcripts of phosphodiesterase family-specific isoforms were studied by reverse transcription-polymerase chain reaction and nucleotide sequencing.
In intact human cells, selective inhibitors for phosphodiesterase 4 (rolipram) and 5 (E4021) gene families were effective in augmenting cyclic nucleotide accumulation in response to isoproterenol or sodium nitroprusside, respectively. cAMP and cGMP hydrolytic activities, resolved using Trisacryl M anion-exchange chromatography, showed a cAMP phosphodiesterase peak that was minimally sensitivity to cGMP but modestly inhibited by rolipram and a cGMP phosphodiesterase peak that was sensitive to inhibition by E4021. Further evaluation of the cGMP phosphodiesterase demonstrated Michaelis-Menten kinetics and competitive inhibition by E4021. Messenger RNA transcripts for phosphodiesterase 4, 5, and 7 isozymes were isolated in human trabecular meshwork cells. However, in porcine trabecular meshwork cells only isozymes for phosphodiesterase 4 and 5 isozymes were detected.
Human trabecular meshwork cells express phosphodiesterase 4, 5, and 7 gene family isoforms and enzyme activities, suggesting that selective isoform inhibitors could be used to augment the actions of antiglaucoma drugs that use cyclic nucleotides as second messengers.
鉴定人小梁网细胞和猪小梁网细胞原代培养物中的环核苷酸磷酸二酯酶同工酶活性。
采用乙酰化酸提取物放射免疫分析法，测定用磷酸二酯酶同工型选择性抑制剂处理的人小梁网细胞中环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）的变化。使用两步放射性同位素法（汤普森法）测量环核苷酸磷酸二酯酶活性。用人细胞上清液中的酶活性通过阴离子交换色谱法进行分离。此外，通过逆转录-聚合酶链反应和核苷酸测序研究磷酸二酯酶家族特异性同工型的人及猪小梁网细胞转录本。
在完整的人细胞中，磷酸二酯酶4（咯利普兰）和5（E4021）基因家族的选择性抑制剂分别有效地增强了对异丙肾上腺素或硝普钠反应中环核苷酸的积累。使用Trisacryl M阴离子交换色谱法解析的cAMP和cGMP水解活性显示，一个对cGMP敏感性最低但被咯利普兰适度抑制的cAMP磷酸二酯酶峰，以及一个对E4021抑制敏感的cGMP磷酸二酯酶峰。对cGMP磷酸二酯酶的进一步评估显示其符合米氏动力学且被E4021竞争性抑制。在人小梁网细胞中分离出了磷酸二酯酶4、5和7同工酶的信使核糖核酸转录本。然而，在猪小梁网细胞中仅检测到磷酸二酯酶4和5同工酶。
人小梁网细胞表达磷酸二酯酶4、5和7基因家族同工型及酶活性，这表明选择性同工型抑制剂可用于增强以环核苷酸作为第二信使的抗青光眼药物的作用。
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