Wiley J S, Chen R, Wiley M J, Jamieson G P
Haematology Department, Austin Hospital, Heidelberg, Victoria, Australia.
Arch Biochem Biophys. 1992 Feb 1;292(2):411-8. doi: 10.1016/0003-9861(92)90010-t.
Extracellular ATP is known to increase the membrane permeability of a variety of cells. Addition of ATP to human leukemic lymphocytes loaded with the Ca2+ indicator, fura-2, induced a rise in cytosolic Ca2+ concentration which was attenuated or absent in NaCl media compared with KCl, choline Cl, or NMG Cl media. In contrast, anti-immunoglobulin antibody gave similar Ca2+ transients in NaCl and KCl media. A half-maximal inhibition of peak ATP-induced Ca2+ response was observed at 10-16 mM extracellular Na+. Basal 45Ca2+ influx into lymphocytes was stimulated 9.6-fold by ATP added to cells in KCl media, but the effect of ATP was greatly reduced for cells in NaCl media. Hexamethylene amiloride blocked 74% of the ATP-stimulated Ca45 uptake of cells in KCl media. Flow cytometry measurements of fluo-3-loaded cells confirmed that the ATP-induced rise in cytosolic Ca2+ was inhibited either by extracellular Na+ or by addition of hexamethylene amiloride. Extracellular ATP stimulated 86Rb efflux from lymphocytes 10-fold and this increment was inhibited by the amiloride analogs in a rank order of potency 5-(N-methyl-N-isobutyl)amiloride greater than 5-(N,N-hexamethylene)amiloride greater than 5-(N-ethyl-N-isopropyl)amiloride greater than amiloride. ATP-induced 86Rb efflux showed a sigmoid dependence on the concentration of ATP and Hill analysis gave K1/2 of 90 and 130 microM and n values of 2.5 and 2.5 for KCl and NaCl media, respectively. However, the maximal ATP-induced 86Rb efflux was 3-fold greater in KCl than in NaCl media. Raising extracellular Na+ from 10 to 100 mM increased ATP-induced Na+ influx from a mean of 2.0 to 3.7 nEq/10(7) cells/min, suggesting either saturability or self-inhibition by Na+ of its own influx. These data suggest that ATP opens a receptor-operated ion channel which allows increased Ca2+ and Na+ influx and Rb+ efflux and these fluxes are inhibited by extracellular Na+ ions as well as by the amiloride analogs.
已知细胞外ATP可增加多种细胞的膜通透性。将ATP添加到负载Ca2+指示剂fura-2的人白血病淋巴细胞中，可诱导胞质Ca2+浓度升高，与KCl、胆碱氯或NMG Cl培养基相比，在NaCl培养基中这种升高减弱或不存在。相反，抗免疫球蛋白抗体在NaCl和KCl培养基中产生相似的Ca2+瞬变。在细胞外Na+浓度为10-16 mM时，观察到ATP诱导的Ca2+峰值反应受到半数抑制。在KCl培养基中添加ATP可使淋巴细胞的基础45Ca2+内流增加9.6倍，但在NaCl培养基中，ATP对细胞的作用大大降低。六甲铵阻断了KCl培养基中细胞ATP刺激的Ca45摄取的74%。对负载fluo-3的细胞进行流式细胞术测量证实，细胞外Na+或添加六甲铵均可抑制ATP诱导的胞质Ca2+升高。细胞外ATP刺激淋巴细胞的86Rb外流增加了10倍，这种增加被氨氯地平类似物以5-(N-甲基-N-异丁基)氨氯地平＞5-(N,N-六甲撑)氨氯地平＞5-(N-乙基-N-异丙基)氨氯地平＞氨氯地平的效力顺序抑制。ATP诱导的86Rb外流对ATP浓度呈S形依赖性，Hill分析得出KCl和NaCl培养基中K1/2分别为90和130 microM，n值分别为2.5和2.5。然而，ATP诱导的最大86Rb外流在KCl培养基中比在NaCl培养基中高3倍。将细胞外Na+从10 mM提高到100 mM，可使ATP诱导的Na+内流从平均2.0 nEq/10(7)细胞/分钟增加到3.7 nEq/10(7)细胞/分钟，这表明Na+自身内流存在饱和性或自抑制作用。这些数据表明，ATP打开了一个受体操纵的离子通道，使Ca2+和Na+内流增加以及Rb+外流增加，并且这些通量受到细胞外Na+离子以及氨氯地平类似物的抑制。
