Bittner M A, Holz R W
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
J Biol Chem. 1992 Aug 15;267(23):16226-9.
We have examined the temperature sensitivity of exocytosis in digitonin-permeabilized chromaffin cells. The time course of secretion is markedly slowed by incubating the cells at 18 degrees C rather than 27 degrees C. We have previously shown that secretion has both ATP-dependent and ATP-independent components (Holz, R. W., Bittner, M. A., Peppers, S. C., Senter, R. A., and Eberhard, D. A. (1989) J. Biol. Chem. 264, 5412-5419). Reducing the temperature has no effect on ATP-independent secretion. However, cold (18 degrees C) greatly slows the ability of ATP to stimulate secretion. The ATP-requiring priming step itself is not affected by reducing the temperature since an effect of ATP can be seen after permeabilization at 18 degrees C if the cells are subsequently stimulated to secrete at 27 degrees C. When cells are permeabilized at 27 degrees C with ATP and then stimulated by Ca2+ in the absence of ATP, the secretion which was primed by ATP during the permeabilization step is inhibited 75% at 18 degrees C. Similar results are seen when ATP-dependent priming is enhanced by low concentrations of Ca2+. Thus, the temperature-sensitive step occurs after ATP and Ca2+ act to prime the cell. The temperature-sensitive step is likely to be overall rate-limiting step during the later phase of secretion, when the ATP-dependent priming process is limiting.
我们研究了洋地黄皂苷通透化嗜铬细胞中胞吐作用的温度敏感性。将细胞在18℃而非27℃孵育时，分泌的时间进程显著减慢。我们之前已经表明，分泌具有ATP依赖性和ATP非依赖性成分（霍尔兹，R.W.，比特纳，M.A.，佩珀斯，S.C.，森特，R.A.，和埃伯哈德，D.A.（1989年）《生物化学杂志》264卷，5412 - 5419页）。降低温度对ATP非依赖性分泌没有影响。然而，低温（18℃）极大地减慢了ATP刺激分泌的能力。需要ATP的引发步骤本身不受温度降低的影响，因为如果随后在27℃刺激细胞分泌，那么在18℃通透化后仍能看到ATP的作用。当细胞在27℃用ATP通透化，然后在无ATP的情况下用Ca²⁺刺激时，在通透化步骤中由ATP引发的分泌在18℃时受到75%的抑制。当低浓度Ca²⁺增强ATP依赖性引发时，也能看到类似结果。因此，温度敏感步骤发生在ATP和Ca²⁺作用使细胞引发之后。当ATP依赖性引发过程成为限制因素时，温度敏感步骤可能是分泌后期的总体限速步骤。
