Melmed R N, Karanian P J, Berlin R D
J Cell Biol. 1981 Sep;90(3):761-8. doi: 10.1083/jcb.90.3.761.
We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4'acetamido, 4-isothiocyano 2,2' disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.
我们已经探究了细胞体积受微管组装状态和环磷酸腺苷（cAMP）代谢调控且可能与形状变化相协调的可能性。用秋水仙碱处理J774.2小鼠巨噬细胞会导致微管迅速解体，并且与一极有大突起的细胞比例显著增加（从15%-20%增加到90%以上）相关。这提供了一个简单的实验系统，其中悬浮的几乎整个细胞群体都发生了形状变化。然后可以通过同位素稀释技术对细胞体积的平行变化进行定量。我们发现秋水仙碱引起的形状变化伴随着细胞体积约20%的减小。诺考达唑而非光秋水仙碱会引起细胞形状和细胞体积的相同变化。体积减小并非由于细胞裂解或胞饮作用受到抑制。我们还研究了体积减小的机制。秋水仙碱诱导哇巴因敏感的钠钾依赖性ATP酶活性出现小幅但可重复的增加。然而，用哇巴因抑制该酶/转运系统既未改变细胞体积，也未阻止秋水仙碱诱导的体积减小。另一方面，阴离子转运抑制剂4-乙酰氨基-4'-异硫氰酸-2,2'-二磺酸芪（SITS）抑制了秋水仙碱的作用，因此表明阴离子转运系统在细胞体积调节中发挥作用。由于已知秋水仙碱在多个系统中可激活腺苷酸环化酶，且细胞形状变化通常由提高cAMP的激素诱导，我们还研究了cAMP对细胞体积的影响。作用于增加cAMP的试剂（激活腺苷酸环化酶的霍乱毒素、磷酸二酯酶抑制剂异丁基甲基黄嘌呤（IBMX）以及二丁酰环磷酸腺苷）均导致与秋水仙碱相当的体积减小。为了确定有效的代谢途径，我们研究了J774.2的两个突变体，一个缺乏腺苷酸环化酶，另一个环磷酸腺苷依赖性蛋白激酶活性显著降低。霍乱毒素在这两个突变体中均未产生体积变化。环磷酸腺苷在缺乏环化酶的细胞系中引起的体积减小与野生型相当，但在缺乏激酶的细胞系中未引起体积变化。该分析确定了环磷酸腺苷和秋水仙碱的不同作用。环磷酸腺苷诱导的体积减小需要环磷酸腺苷依赖性蛋白激酶的作用。另一方面，秋水仙碱在突变体和野生型中均诱导了相当的体积变化，因此不需要该激酶。
