The effects of intracellular pH changes on resting cytosolic calcium in voltage-clamped snail neurones
The effects of intracellular pH changes on resting cytosolic calcium in voltage-clamped snail neurones
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DOI：
10.1111/j.1469-7793.2001.0405k.x
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发表时间：
2001-02-01
影响因子：
5.5
通讯作者：
Schwiening, CJ
中科院分区：
文献类型：
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作者：
Willoughby, D;Thomas, RC;Schwiening, CJ
1.我们研究了改变细胞内pH值对电压钳位的蜗牛神经元细胞内游离钙浓度（[Ca ~（2+）]（i））的影响。使用荧光染料8-羟基芘-1，3，6-三磺酸（HPTS）测量细胞内pH（pH（i）），并使用弱酸和弱碱改变。使用fura-2或钙敏感微电极记录[Ca 2 +]（i）的变化。用5 mM或20 mM丙酸盐酸化神经元（分别类似于0.2或0.3 pH单位酸化）分别导致静息[Ca 2 +]（i）小幅降低5 +/- 2 nM（n = 4）和7 +/- 16 nM（n = 4）。在类似于40分钟灌流后去除20 mill丙酸盐导致类似于0.35 pH单位的碱化和伴随的静息[Ca 2 +]（i）31 +/- 9 nM的升高（n = 4，P < 0.05）。去除5 mM丙酸盐对[Ca 2 +]（i）没有显著影响。用3 mM浓度的弱碱性三甲胺（TMA）、氯化铵（NH 4Cl）和普鲁卡因灌流诱导的0.2-0.4 pH单位的受阻神经元的碱化伴随着显著的（P < 0.05）静息[Ca 2 +]（i）分别增加42 +/- 4 nM（n = 26）、30 +/- 7 nM（n = 5）和36 +/- 4 nh（n = 3）。TMA（0.5-6 mM）对[Ca 2 +]（i）的作用是剂量依赖性的，在pH（i）增加小于0.1 pH单位（0.5 mM TMA）期间[Ca 2 +]（i）增加。灌流的神经元与零钙（1 mM EGTA）林格氏溶液抑制去极化诱导的钙增加，但不是由第一次暴露于TMA（3 mM）产生的钙增加。在细胞外钙长时间缺乏（50分钟）TMA诱导的钙升高降低了64 +/- 10%相比，在外部钙的存在下观察到的（P < 0.05）.5，在用咖啡因（10 mM）灌注10分钟以耗尽内质网（ER）钙库（P < 0.05）。与对照相比，En钙泵抑制剂Clyclopiazonic acid（10-30 μ M CPA）抑制由TMA（3 mw）和NH，Cl（3 mM）产生的钙升高61 ± 4%（P < 0.05）。这些数据与刺激钙释放或抑制细胞内储存的钙再摄取的生理性细胞内碱性变化一致。钙的增加大大减少后应用咖啡因，治疗与CPA或长期去除外部钙。因此，ER可能是动员钙的来源。
1. We have investigated the effects of changing intracellular pH on intracellular free calcium concentration ([Ca2+](i)) in voltage-clamped neurones of the snail Helix aspersa. Intracellular pH (pH(i)) was measured using the fluorescent dye 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) and changed using weak acids and weak bases. Changes in [Ca2+](i) were recorded using either fura-2 or calcium-sensitive microelectrodes.2. Acidification of the neurones with 5 mM or 20 mM propionate ( similar to0.2 or 0.3 pH units acidification, respectively) caused a small reduction in resting [Ca2+](i) of 5 +/- 2 nM (n = 4) and 7 +/- 16 nM (n = 4), respectively. The removal of the 20 mill propionate after similar to 40 min superfusion resulted in an alkalinization of similar to0.35 pH units and an accompanying rise in resting [Ca2+](i) of 31 +/- 9 nM (n = 4, P < 0.05). The removal of 5 mM propionate did not significantly affect [Ca2+](i).3. Alkalinizations of 0.2-0.4 pH units of Helix neurones induced by superfusion with 3 mM concentrations of the weak bases trimethylamine (TMA), ammonium chloride (NH4Cl) and procaine were accompanied by significant (P < 0.05) increases in resting [Ca2+](i) of 42 +/- 4 nM (n = 26), 30 +/- 7 nM (n = 5) and 36 +/- 4 nh (n = 3), respectively. The effect, of TMA (0.5-6 mM) on [Ca2+](i) was dose dependent with an increase in [Ca2+](i) during pH(i) increases of less than 0.1 pH units (0.5 mM TMA).4. Superfusion of neurones with zero calcium (1 mM EGTA) Ringer solution inhibited depolarization-induced calcium increases but not the calcium increase produced by the first exposure to TMA (3 mM). In the prolonged absence of extracellular calcium (50 min) TMA-induced calcium rises were decreased by 64 +/- 10% compared to those seen in the presence of external calcium (P < 0.05).5, The calcium rise induced by TMA (3 mM) was reduced by 60 +/- 6% following a 10 min period of superfusion with caffeine (10 mM) to deplete the endoplasmic reticulum (ER) stores of calcium (P < 0.05).6. Clyclopiazonic acid (10-30 muM CPA), an inhibitor of the En calcium pump, inhibited the calcium rise produced by TMA (3 mw) and NH,CI (3 mM) by 61 +/- 4% compared to controls (P < 0.05). These data are consistent with physiological intracellular alkaline shifts stimulating release of calcium, or inhibiting re-uptake of calcium by an intracellular store. The calcium increase was much reduced following application of caffeine, treatment with CPA or prolonged removal of external calcium. Hence the ER was likely to he the source of mobilized calcium.