Agulló L, García A
Instituto de Biología Fundamental V. Villar Palasi, Universidad Autónoma de Barcelona, Bellaterra, Spain.
Eur J Pharmacol. 1997 Mar 26;323(1):119-25. doi: 10.1016/s0014-2999(97)00019-8.
Cyclic GMP (cGMP) formation induced by agonist stimulation of Ca2+/calmodulin-dependent nitric oxide (NO) synthase type I is known to occur in both granule cell and astrocyte cultures from rat cerebellum. Here we show that in these same cells cGMP is predominantly hydrolyzed by a Ca2+/calmodulin-dependent phosphodiesterase. At 10 microM cGMP, Ca2+ (25 microM) stimulated basal (Ca(2+)-independent) phosphodiesterase activity about 6 times in granular neurons and 15 times in astrocytes. The calmodulin antagonist calmidazolium blocked the Ca(2+)-dependent phosphodiesterase activity and exogenous calmodulin increased 3-4-fold the stimulatory potency of Ca2+ in both cell types (EC50 values 1.26 +/- 0.20 and 1.50 +/- 0.42 microM in the absence and 0.38 +/- 0.11 and 0.39 +/- 0.14 microM in the presence of 1 microM calmodulin, for neurons and astrocytes, respectively). In both cell types K(m) values for cGMP at 25 microM Ca2+ were similar (1.72 +/- 0.20 and 1.92 +/- 0.09 microM) and phosphodiesterase activities were inhibited by isozyme-selective phosphodiesterase inhibitors with potencies analogous to those described for Ca2+/calmodulin-phosphodiesterases or phosphodiesterase type 1 isoforms in other preparations. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) effectively blocked the Ca2+/calmodulin-phosphodiesterase activity in granule cell and astrocyte extracts (IC50 values at 1 microM cGMP: 31 +/- 10 microM and 46 +/- 6 microM, respectively), in contrast to the apparent inability of this compound to inhibit the Ca(2+)-dependent activity reported in whole brain extracts. These results demonstrate that comparable phosphodiesterase type 1 activities are found in the cytosols of cerebellar granule cells and astrocytes and suggest that these activities may play an important role in controlling cGMP levels in cells where the Ca(2+)-dependent NO synthase type I is stimulated.
由激动剂刺激I型钙/钙调蛋白依赖性一氧化氮（NO）合酶诱导的环鸟苷酸（cGMP）生成，已知在大鼠小脑的颗粒细胞和星形胶质细胞培养物中均会发生。在此我们表明，在这些相同的细胞中，cGMP主要由一种钙/钙调蛋白依赖性磷酸二酯酶水解。在10微摩尔cGMP浓度下，钙离子（25微摩尔）使颗粒神经元中的基础（非钙依赖性）磷酸二酯酶活性提高约6倍，使星形胶质细胞中的该活性提高15倍。钙调蛋白拮抗剂氯米达唑可阻断钙依赖性磷酸二酯酶活性，而外源性钙调蛋白使钙离子在两种细胞类型中的刺激效力提高3至4倍（在无1微摩尔钙调蛋白时，颗粒神经元和星形胶质细胞的半数有效浓度（EC50）值分别为1.26±0.20和1.50±0.42微摩尔；在有1微摩尔钙调蛋白时，分别为0.38±0.11和0.39±0.14微摩尔）。在两种细胞类型中，25微摩尔钙离子浓度下cGMP的米氏常数（Km）值相似（分别为1.72±0.20和1.92±0.09微摩尔），磷酸二酯酶活性被同工酶选择性磷酸二酯酶抑制剂抑制，其效力类似于其他制剂中描述的钙/钙调蛋白磷酸二酯酶或1型磷酸二酯酶同工型。非选择性磷酸二酯酶抑制剂3 - 异丁基 - 1 - 甲基黄嘌呤（IBMX）有效阻断了颗粒细胞和星形胶质细胞提取物中的钙/钙调蛋白磷酸二酯酶活性（在1微摩尔cGMP浓度下的半数抑制浓度（IC50）值分别为3l±10微摩尔和46±6微摩尔），这与该化合物在全脑提取物中明显无法抑制钙依赖性活性形成对比。这些结果表明，在小脑颗粒细胞和星形胶质细胞的胞质溶胶中发现了相当的1型磷酸二酯酶活性，并表明这些活性可能在控制I型钙依赖性NO合酶被刺激的细胞中的cGMP水平方面发挥重要作用。
