Marszalek P E, Farrell B, Verdugo P, Fernandez J M
Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905, USA.
Biophys J. 1997 Sep;73(3):1160-8. doi: 10.1016/S0006-3495(97)78148-7.
We developed a method for measuring the efflux of 5-hydroxytryptamine (5-HT, serotonin) from isolated intact granules of the mast cell of the beige mouse. This method combines electroporation of the vesicle membrane with amperometric detection of 5-HT. A single secretory granule is placed between two platinum electrodes (distance approximately 100 microm) and positioned adjacent (<1 microm) to a carbon fiber microelectrode. A short (approximately 30 micros) high-intensity voltage pulse (electric field of approximately 5 kV/cm) is delivered to the electrodes to trigger the mechanical breakdown of the granule membrane, which activates the release of 5-HT. We observed concurrent swelling of the granule matrix with the oxidation of 5-HT at the carbon fiber electrode (overpotential + 650 mV). Similar to the release of secretory products during exocytosis, the oxidation current exhibits a spike-like time course with a noninstantaneous rising phase (time between onset of current and maximum flux, t(max)) with approximately 25% of the molecules released during this period. When the current reaches its maximum, the granule matrix attains its maximum swollen state. We found that the rising phase depends on the initial cross-sectional area of the granule (t(max) approximately 21r2) and reflects the time required for membrane rupture. The average t(1/2)spike of the amperometric spikes was found to be approximately 150 ms, which is 3-7 times faster than the t(1/2) measured during cellular exocytosis.
我们开发了一种用于测量米色小鼠肥大细胞分离完整颗粒中5-羟色胺（5-HT，血清素）流出的方法。该方法将囊泡膜的电穿孔与5-HT的安培检测相结合。将单个分泌颗粒置于两个铂电极之间（距离约100微米），并与碳纤维微电极相邻放置（<1微米）。向电极施加一个短的（约30微秒）高强度电压脉冲（电场约5 kV/cm），以触发颗粒膜的机械破裂，从而激活5-HT的释放。我们观察到颗粒基质同时肿胀，且5-HT在碳纤维电极上发生氧化（过电位+650 mV）。与胞吐过程中分泌产物的释放类似，氧化电流呈现出尖峰状的时间进程，其上升阶段并非瞬间完成（从电流开始到最大通量的时间，t(max)），在此期间约25%的分子被释放。当电流达到最大值时，颗粒基质达到最大肿胀状态。我们发现上升阶段取决于颗粒的初始横截面积（t(max)约为21r2），并反映了膜破裂所需的时间。安培尖峰的平均t(1/2)spike约为150毫秒，这比细胞胞吐过程中测得的t(1/2)快3 - 7倍。
