Barnette M S, Manning C D, Price W J, Barone F C
Division of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania.
J Pharmacol Exp Ther. 1993 Feb;264(2):801-12.
Cyclic nucleotides mediate relaxation of gastrointestinal smooth muscle. The intracellular concentration of these second messengers is determined by a balance between their synthesis and metabolism. Because cyclic nucleotide phosphodiesterase enzymes (PDE) are the sole enzymes responsible for their degradation, it is essential to determine the role of the various PDE isozymes in regulating cyclic nucleotide content of gastrointestinal smooth muscle. To examine the role of different PDE isozymes in colonic smooth muscle motility, soluble PDE activity was measured in fractions obtained from homogenates of canine colon using DEAE sepharose chromatography. PDE activity was determined using [3H]cyclic AMP (cAMP) (1 microM) or [3H]cyclic GMP (cGMP) (1 microM) as a substrate. Results indicated that colonic smooth muscle contains at least two forms of PDE with a high affinity for cGMP. One form was stimulated by calmodulin (type I) and the other was inhibited by low concentrations of zaprinast (type V). In addition, colonic smooth muscle contains at least two isozymes that prefer cAMP as a substrate. One form was inhibited by SB 94120 and cGMP (type III) and the other by rolipram (type IV). An additional peak of PDE activity was identified. The hydrolysis of cAMP by this peak was greatly enhanced by the presence of cGMP, suggesting that this activity belonged to type II or cGMP-stimulated PDE. The functional role of these isozymes was evaluated by determining the ability of selective PDE inhibitors to antagonize a carbachol (0.3 microM)-induced contraction of isolated circular colonic muscle strips in the presence of forskolin (0.2 microM). Concentration-dependent decreases in contractile activity were observed with the following potency order: rolipram > Ro 20-1724 > isobutyl methylxanthine > SB 94120 > zaprinast. These results demonstrate that colonic smooth muscle contains several PDE isozymes and that selective inhibition of PDE isozymes can increase cyclic nucleotide content and antagonize contractile responses. Functionally, PDE IV appears to be very important in reducing contractile activity, suggesting that selective PDE IV inhibitors might be useful in the treatment of gut hypermotility disorders.
环核苷酸介导胃肠平滑肌的舒张。这些第二信使的细胞内浓度由其合成与代谢之间的平衡决定。由于环核苷酸磷酸二酯酶（PDE）是负责其降解的唯一酶类，因此确定各种PDE同工酶在调节胃肠平滑肌环核苷酸含量中的作用至关重要。为了研究不同PDE同工酶在结肠平滑肌运动中的作用，使用DEAE琼脂糖层析法测定了从犬结肠匀浆获得的各组分中的可溶性PDE活性。以[3H]环磷酸腺苷（cAMP）（1微摩尔）或[3H]环磷酸鸟苷（cGMP）（1微摩尔）作为底物测定PDE活性。结果表明，结肠平滑肌含有至少两种对cGMP具有高亲和力的PDE形式。一种形式受钙调蛋白刺激（I型），另一种受低浓度扎普司特抑制（V型）。此外，结肠平滑肌含有至少两种以cAMP为底物的同工酶。一种形式受SB 94120和cGMP抑制（III型），另一种受咯利普兰抑制（IV型）。还鉴定出一个额外的PDE活性峰。cGMP的存在极大地增强了该峰对cAMP的水解作用，表明该活性属于II型或cGMP刺激的PDE。通过测定选择性PDE抑制剂在福斯高林（0.2微摩尔）存在下拮抗卡巴胆碱（0.3微摩尔）诱导的离体结肠环形肌条收缩的能力，评估了这些同工酶的功能作用。观察到收缩活性呈浓度依赖性降低，其效力顺序如下：咯利普兰＞Ro 20-1724＞异丁基甲基黄嘌呤＞SB 94120＞扎普司特。这些结果表明，结肠平滑肌含有多种PDE同工酶，选择性抑制PDE同工酶可增加环核苷酸含量并拮抗收缩反应。在功能上，PDE IV在降低收缩活性方面似乎非常重要，这表明选择性PDE IV抑制剂可能对治疗肠道运动亢进性疾病有用。
