Leung S, O'Donnell M E, Martinez A, Palfrey H C
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637.
J Biol Chem. 1994 Apr 8;269(14):10581-9.
PC12 cells possess a bumetanide-sensitive Na/K/2Cl cotransport system similar to that found in other cell types. Between 10-15% of the total 86Rb influx in these cells is mediated by this pathway under normal conditions. The cotransporter has affinities of 16.5 mM for Nao and 0.7 mM for Ko, is absolutely dependent on Clo and is loop diuretic inhibitable (benzmetanide > bumetanide > piretanide > furosemide). The cotransporter can be activated (up to 8-fold) by cell shrinkage or (up to 4-fold) by treatment with the protein phosphatase inhibitors okadaic acid (EC50 approximately 650 nM) or calyculin A (EC50 approximately 8 nM). Cell shrinkage is followed by a bumetanide-sensitive regulatory volume increase as determined in cell sizing experiments. Calyculin A rapidly elevates normal cell volume in a diuretic-inhibitable manner. Cotransport activity and cell volume are also increased by nerve growth factor (NGF) treatment. The effect of NGF on cotransport rate is biphasic, with an initial rapid approximately 2.5-fold increase followed by a prolonged plateau, and is blocked by pretreatment of the cells with K252a (IC50 approximately 30 nM). By contrast, agents that raise cAMP or phorbol esters lead to an inhibition of cotransport, indicating that the NGF effect is not mediated by stimulation of either cAMP-dependent protein kinase or protein kinase C. Long term NGF treatment (> 2 days) leads to neurite formation and a maintained approximately 2-fold increase in cotransport activity. Bumetanide treatment does not affect the ability of cells to extend neurites, nor is the growth rate of cells in normal medium affected by the diuretic. These results suggest that the cotransport system in PC12 cells is acutely regulated by protein phosphorylation and dephosphorylation as well as cell shrinkage and that cotransport activity may be up-regulated during neuronotypic differentiation.
PC12细胞拥有一种对布美他尼敏感的Na/K/2Cl共转运系统，类似于在其他细胞类型中发现的系统。在正常条件下，这些细胞中86Rb总内流的10%-15%是由该途径介导的。该共转运体对Na⁺的亲和力为16.5 mM，对K⁺的亲和力为0.7 mM，绝对依赖于Cl⁻，并且可被袢利尿剂抑制（苄甲噻嗪>布美他尼>吡咯他尼>呋塞米）。该共转运体可通过细胞收缩激活（高达8倍）或通过用蛋白磷酸酶抑制剂冈田酸（EC50约为650 nM）或花萼海绵诱癌素A（EC50约为8 nM）处理激活（高达4倍）。如在细胞大小测量实验中所确定的，细胞收缩后接着是布美他尼敏感的调节性容积增加。花萼海绵诱癌素A以利尿剂可抑制的方式迅速升高正常细胞体积。神经生长因子（NGF）处理也会增加共转运活性和细胞体积。NGF对共转运速率有双相作用，最初迅速增加约2.5倍，随后是长时间的平台期，并且可被用K252a预处理细胞（IC50约为30 nM）阻断。相比之下，提高cAMP的试剂或佛波酯会导致共转运抑制，表明NGF的作用不是由cAMP依赖性蛋白激酶或蛋白激酶C的刺激介导的。长期NGF处理（>2天）会导致神经突形成，并且共转运活性维持增加约2倍。布美他尼处理不影响细胞延伸神经突的能力，利尿剂也不影响正常培养基中细胞的生长速率。这些结果表明，PC12细胞中的共转运系统受蛋白磷酸化和去磷酸化以及细胞收缩的急性调节，并且在神经元样分化过程中共转运活性可能上调。
