We have investigated the ionic events elicited in Balb-c 3T3 fibroblasts by basic fibroblast growth factor (bFGF), a peptide that binds to membrane receptors with tyrosine kinase activity and has a mitogenic action on many cell types. The peptide (0.2-100 ng ml-1) caused the appearance of an inward current, as observed in whole-cell patch-clamp experiments at a holding potential of -50 mV, that could last for tens of minutes and had a peak density of 4.6 +/- 2.6 pA pF-1. The reversal potential was 18.8 +/- 16.7 mV. 2. The current was reversibly abolished by removal of bFGF from the external bath. Inhibition of low-affinity FGF receptors had no effect on the activation of the inward current; it was completely abolished when cells were pre-incubated with tyrphostin or 5'-methylthioadenosine (MTA), two inhibitors of the tyrosine kinase activity of the high-affinity FGF receptors. The inward current was not activated by the emptying of internal calcium stores, as tested with 200 nM thapsigargin. 3. Values of peak current density comparable to control ones were obtained when either all Na+ ions or all Ca2+ ions were removed from the external solution; when both ions were completely removed, no inward current could be observed. The inward current was not affected by 2 microM nifedipine, and was reversibly blocked by the imidazole derivative SK&F 96365-A. 4. Measurements of free intracellular calcium concentration ([Ca2+]i) with the dye fura-2 showed that bFGF elicited sustained increases in [Ca2+]i that were completely dependent on external calcium and on the presence of the agonist and could last more than 1 h. 5. Single channel currents (conductance 7.9 pS) in response to bFGF stimulation could be recorded in the cell-attached configuration with 100 mM CaCl2 in the pipette. When the resting potential was brought near to 0 mV by external perfusion in a high-K+ solution, Vrev was about 0 mV. 6. We conclude that in Balb-c 3T3 cells bFGF induces an inward current that is carried at least partially by Ca2+ ions; this current in turn causes a long-lasting increase in intracellular Ca2+ concentration. The amplitude and time course of these bFGF-activated ionic events are compatible with their involvement in the control of cell proliferation.
摘要
我们研究了碱性成纤维细胞生长因子（bFGF）在Balb - c 3T3成纤维细胞中引发的离子事件。bFGF是一种能与具有酪氨酸激酶活性的膜受体结合的肽，对多种细胞类型具有促有丝分裂作用。在 - 50 mV的钳制电位下进行的全细胞膜片钳实验中观察到，该肽（0.2 - 100 ng/ml）引起内向电流的出现，此电流可持续数十分钟，峰值密度为4.6±2.6 pA pF⁻¹。反转电位为18.8±16.7 mV。2. 从外部浴液中去除bFGF后，电流可逆性消失。抑制低亲和力FGF受体对内向电流的激活没有影响；当细胞用 tyrphostin 或5'-甲基硫代腺苷（MTA）（两种高亲和力FGF受体酪氨酸激酶活性的抑制剂）预孵育时，内向电流完全消失。用200 nM毒胡萝卜素测试发现，内向电流不会因内部钙库排空而被激活。3. 当从外部溶液中去除所有Na⁺离子或所有Ca²⁺离子时，可获得与对照相当的峰值电流密度值；当两种离子都完全去除时，则观察不到内向电流。内向电流不受2 μM硝苯地平的影响，并被咪唑衍生物SK&F 96365 - A可逆性阻断。4. 用fura - 2染料测量细胞内游离钙浓度（[Ca²⁺]i）表明，bFGF引起[Ca²⁺]i持续升高，这完全依赖于外部钙以及激动剂的存在，且可持续超过1小时。5. 在吸管中含有100 mM CaCl₂的细胞贴附模式下，可记录到对bFGF刺激的单通道电流（电导为7.9 pS）。当通过在高钾溶液中外部灌流使静息电位接近0 mV时，Vrev约为0 mV。6. 我们得出结论，在Balb - c 3T3细胞中，bFGF诱导至少部分由Ca²⁺离子携带的内向电流；该电流进而导致细胞内Ca²⁺浓度的长期升高。这些bFGF激活的离子事件的幅度和时间进程与其参与细胞增殖控制相一致。
