Pledger W J, Thompson W J, Epstein P M, Strada S J
J Cell Physiol. 1979 Sep;100(3):497-507. doi: 10.1002/jcp.1041000312.
A rapid reduction of cyclic nucleotide phosphodiesterase activity occurs after the replating of confluent cultures of BHK 21 c/13 fibroblasts into fresh medium. This reduction in activity depends on the density to which the cultures are reseeded and the concentration of serum in the medium. Enzyme activity in BHK cells is restored after 24 to 48 hours if cells are diluted into medium containing 10% fetal calf serum or 0.5% fetal calf serum supplemented with insulin (10(-6)M), but not into 0.5% serum alone. The restoration in enzyme activity is blocked by cycloheximide or Actinomycin D. When BHK cells become quiescent by maintanance in 0.5% serum conditions for 48 hours, a rapid (15--60 minutes) increase in cyclic AMP phosphodiesterase activity occurs when 10% serum is added to the cultures. Enzyme activity is increased even further after 24 to 48 hours in the 10% serum. Cycloheximide or Actinomycin D do not affect the rapid increase in enzyme activity in response to serum, but completely inhibit the long term increase. In contrast to serum, insulin (10(-8) to 10(-6)M) has no short term effect, but does increase enzyme activity after 24 to 48 hours to levels comparable to those seen with addition of 10% serum. As is the case with serum, this long term effect of insulin on enzyme activity is prevented by inhibitors of protein and RNA synthesis. Kinetic analyses of cyclic AMP phosphodiesterase activity in homogenates of quiescent BHK cells indicate the presence of only high Km (congruent to 20 muM) enzyme activity. Addition of serum or insulin to quiescent cells results in the appearance of apparent low Km enzyme activity in homogenates. Sucrose gradient analysis of BHK cells displays two forms of cyclic AMP phosphodiesterase enzyme activity: a 3--4 S form and 5--6 S form. In quiescent cells, the 5--6 S form greatly predominates relative to the 3--4 S form. Addition of serum to quiescent cells results in a rapid appearance of increased 3--4 S form enzyme activity. Insulin also increases the activity of this higher affinity 3--4 S enzyme form after 24 to 48 hours in culture. The functional significance of short and long term regulation of cyclic nucleotide phosphodiesterase(s) in cells is discussed.
将汇合的BHK 21 c/13成纤维细胞培养物重新接种到新鲜培养基后，环核苷酸磷酸二酯酶活性迅速降低。这种活性降低取决于重新接种时培养物的密度以及培养基中血清的浓度。如果将细胞稀释到含有10%胎牛血清或补充有胰岛素（10⁻⁶M）的0.5%胎牛血清的培养基中，BHK细胞中的酶活性在24至48小时后恢复，但单独稀释到0.5%血清中则不会恢复。酶活性的恢复被环己酰亚胺或放线菌素D阻断。当BHK细胞在0.5%血清条件下维持48小时而静止时，向培养物中添加10%血清会导致环磷酸腺苷磷酸二酯酶活性迅速（15 - 60分钟）增加。在10%血清中培养24至48小时后，酶活性进一步增加。环己酰亚胺或放线菌素D不影响酶活性对血清的快速增加，但完全抑制长期增加。与血清不同，胰岛素（10⁻⁸至10⁻⁶M）没有短期作用，但在24至48小时后确实会将酶活性增加到与添加10%血清时相当的水平。与血清情况一样，胰岛素对酶活性的这种长期作用被蛋白质和RNA合成抑制剂所阻止。对静止BHK细胞匀浆中环磷酸腺苷磷酸二酯酶活性的动力学分析表明仅存在高Km（约20μM）的酶活性。向静止细胞中添加血清或胰岛素会导致匀浆中出现明显的低Km酶活性。对BHK细胞的蔗糖梯度分析显示两种形式的环磷酸腺苷磷酸二酯酶活性：3 - 4 S形式和5 - 6 S形式。在静止细胞中，5 - 6 S形式相对于3 - 4 S形式占主导地位。向静止细胞中添加血清会导致3 - 4 S形式的酶活性迅速增加。在培养24至48小时后，胰岛素也会增加这种高亲和力3 - 4 S酶形式的活性。文中讨论了细胞中环核苷酸磷酸二酯酶短期和长期调节的功能意义。
