Effects of lowering extracellular and cytosolic pH on calcium fluxes, cytosolic calcium levels, and transmitter release in presynaptic nerve terminals isolated from rat brain.
Effects of lowering extracellular and cytosolic pH on calcium fluxes, cytosolic calcium levels, and transmitter release in presynaptic nerve terminals isolated from rat brain.
复制标题
DOI：
10.1085/jgp.91.2.305
复制
发表时间：
1988-02
期刊：
影响因子：
--
通讯作者：
Nachshen DA
中科院分区：
文献类型：
--
作者：
Drapeau P;Nachshen DA
我们研究了细胞外和细胞内pH变化对放射性45Ca内流的影响，用钙敏感荧光指示剂fura-2监测的电离Ca （pCai）浓度，以及从大鼠脑纹状体分离并预先加载[3H]多巴胺的突触前神经末梢（突触体）多巴胺的外排。胞浆pH （pHi）通过在突触体上负载H+敏感荧光指示剂2′，7′-二（羧乙基）-5,6-羧荧光素（BCECF）来监测（见Nachshen， D. A.和P. Drapeau, 1988, Journal of General Physiology, 91:289-303）。外部培养基的pH从7.4突然下降到5.5，导致pHi从初始值7.2缓慢下降（在5分钟内）到大约5.8的稳态水平。当20 mM醋酸盐存在于酸性介质中时，pHi的下降速度最快（在2秒内），达到与不含醋酸盐时相似的水平（速度更慢）。因此，根据细胞外酸化过程中是否存在醋酸盐，可以在短时间内将pHi降低到不同的水平。细胞外酸化至pH 5.5（不含乙酸）在30秒内对pCai和多巴胺释放无显著影响（pHi = 6.4）。醋酸盐存在下的酸化将pHi降至5.8，而不影响pCai，但多巴胺外排增加了约20倍。在缺乏细胞外钙的情况下，也观察到这种基础多巴胺释放的增加。因此，终端内而非细胞外酸化可以以不依赖钙的方式刺激多巴胺的外排。在诺米芬（阻断多巴胺载体）存在的情况下，酸刺激多巴胺外排的高Q10（3.6）与H+激活囊泡多巴胺释放一致。当突触体在高钾（77.5 mM）溶液中去极化2秒并酸化（不含醋酸盐）时，45Ca的进入被平行阻断并诱发多巴胺释放（在pH 6.0和0.2 mM外部Ca时阻断50%）。当在酸性介质中加入乙酸进一步降低pHi时，Ca的进入仍然受阻，但引起多巴胺释放增加。因此，胞外酸化通过阻断电压门控的Ca通道抑制多巴胺的释放，而不是胞内酸化。胞质酸化对基础多巴胺和诱发多巴胺释放的刺激表明，静息和去极化突触体的囊泡释放是由胞质H+直接激活的。
We examined the effects of extracellular and intracellular pH changes on the influx of radioactive 45Ca, the concentration of ionized Ca (pCai) as monitored with the Ca-sensitive fluorescent indicator fura-2, and the efflux of dopamine in presynaptic nerve endings (synaptosomes) isolated from rat brain corpora striata and preloaded with [3H]dopamine. Cytosolic pH (pHi) was monitored by loading the synaptosomes with the H+-sensitive fluorescent indicator 2',7'- bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) (see Nachshen, D. A., and P. Drapeau, 1988, Journal of General Physiology, 91:289-303). An abrupt decrease of the pH of the external medium, from 7.4 to 5.5, produced a slow decrease of pHi (over a 5-min period) from an initial value of 7.2 to a steady state level of approximately 5.8. When 20 mM acetate was present in acidic media, pHi dropped as fast as could be measured (within 2 s) to a level similar to that reached (more slowly) in the absence of acetate. It was therefore possible to lower pHi over short time periods to different levels depending on whether or not acetate was present upon extracellular acidification. Extracellular acidification to pH 5.5 (in the absence of acetate) had no significant effect on pCai and dopamine release over a 30-s period (pHi = 6.4). Acidification in the presence of acetate lowered pHi to 5.8 without affecting pCai, but dopamine efflux increased approximately 20-fold. This increase in basal dopamine release was also observed in the absence of extracellular Ca. Thus, intraterminal, but not extracellular, acidification could stimulate the efflux of dopamine in a Ca-independent manner. The high Q10 (3.6) of acid-stimulated dopamine efflux in the presence of nomifensine (which blocks the dopamine carrier) was consistent with an activation of vesicular dopamine release by H+. When synaptosomes were both depolarized for 2 s in high- K (77.5 mM) solutions and acidified (in the absence of acetate), there was a parallel block of 45Ca entry and evoked dopamine release (50% block at pH 6.0 with 0.2 mM external Ca). When acetate was included in the acidic media to further reduce pHi, Ca entry remained blocked, but evoked dopamine release was increased. Therefore, extracellular, but not cytosolic, acidification inhibited the release of dopamine by blocking voltage-gated Ca channels. The stimulation by cytosolic acidification of both basal and evoked dopamine release suggests that vesicular release in resting and depolarized synaptosomes was directly activated by cytoplasmic H+.