Benning N, Leipziger J, Greger R, Nitschke R
Physiologisches Institut der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
Pflugers Arch. 1996 May;432(1):126-33. doi: 10.1007/s004240050114.
The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pHi) and intracellular Ca2+ activity ([Ca2+]i) of HT29 cells was investigated microspectrofluorimetrically using pH- and Ca2+- sensitive fluorescent indicators, [i.e. 2', 7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (Vm) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pKa value. Trimethylamine (20 mmol/l) increased pHi by 0.78 +/- 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pHi and a slow and incomplete recovery: pHi stayed 0.26 +/- 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pHi. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca2+]i transient which reached a peak value within 10-25 s and then slowly declined to a [Ca2+]i plateau. The initial Delta[Ca2+]i induced by trimethylamine (20 mmol/l) was 160 +/- 15 nmol/l (n = 17). The [Ca2+]i peak was independent of the Ca2+ activity in the bath solution, but the [Ca2+]i plateau was significantly lower under Ca2+-free conditions and could be immediately interrupted by application of CO2 (10%; n = 6), a manoeuvre to acidify pHi in HT29 cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca2+ stores completely abolished this [Ca2+]i transient. Tetramethylamine led to higher [Ca2+]i changes than the other amines tested and only this transient could be completely blocked by atropine (10(-6) mol/l). Trimethylamine (20 mmol/l) hyperpolarized Vm by 22.5 +/- 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 +/- 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pHi changes, release Ca2+ from intracellular, inositol-1,4, 5-trisphosphate-sensitive Ca2+ stores and increase Ca2+ influx into HT29 cells. The latter may be related to both the store depletion and the hyperpolarization.
利用pH和Ca²⁺敏感荧光指示剂[即分别为2',7'-双（羧乙基）-5（6）-羧基荧光素（BCECF）和fura-2]，采用显微分光荧光测定法研究了仲胺、叔胺和季胺甲基胺及乙胺对HT29细胞内pH（pHi）和细胞内Ca²⁺活性（[Ca²⁺]i）的影响。通过膜片钳技术研究膜电压（Vm）。仲胺和叔胺导致快速且稳定的浓度依赖性碱化，这与其pKa值无关。三甲胺（20 mmol/L）使pHi升高0.78±0.03 pH单位（n = 9），且在施加期间pH保持稳定。去除后导致pHi出现下冲且恢复缓慢且不完全：pHi比静息值酸性高0.26±0.06 pH单位。季胺四甲胺和四乙胺对pHi无影响。所有测试的仲胺和叔胺（二甲胺、二乙胺、三甲胺和三乙胺）均诱导[Ca²⁺]i瞬变，该瞬变在10 - 25秒内达到峰值，然后缓慢下降至[Ca²⁺]i平台期。三甲胺（20 mmol/L）诱导的初始Δ[Ca²⁺]i为160±15 nmol/L（n = 17）。[Ca²⁺]i峰值与浴液中的Ca²⁺活性无关，但在无Ca²⁺条件下[Ca²⁺]i平台期显著降低，并且可通过施加CO₂（10%；n = 6）立即中断，这是使HT29细胞内pHi酸化的一种操作。排空对卡巴胆碱或神经降压素敏感的细胞内Ca²⁺储存完全消除了这种[Ca²⁺]i瞬变。四甲胺导致的[Ca²⁺]i变化比其他测试胺更高，并且只有这种瞬变可被阿托品（10⁻⁶ mol/L）完全阻断。三甲胺（20 mmol/L）使Vm超极化22.5±3.7 mV（n = 16），并使全细胞电导增加2.3±0.5 nS（n = 16）。我们得出结论，仲胺和叔胺诱导稳定的碱性pHi变化，从细胞内对肌醇-1,4,5-三磷酸敏感的Ca²⁺储存中释放Ca²⁺，并增加Ca²⁺流入HT29细胞。后者可能与储存耗竭和超极化均有关。
