Zorec R, Sikdar S K, Mason W T
Department of Neuroendocrinology, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, United Kingdom.
J Gen Physiol. 1991 Mar;97(3):473-97. doi: 10.1085/jgp.97.3.473.
The patch-clamp technique has been used to measure changes in membrane capacitance (Cm) of bovine lactotrophs in order to monitor fluctuations in cell surface area associated with exo- and endocytosis. Cells were prepared by an enrichment procedure and cultured for up to 14 d before use. Under whole-cell recording, cell cytoplasm was dialyzed with various Ca2(+)-containing solutions. The resting Cm of 6.05 +/- 1.68 pF was found to correlate well with squared cell radius, suggesting a specific Cm of 0.8 microF/cm2. Discrete Cm steps of 2-10 fF were recorded, which most likely reflect single fusion and retrieval events of prolactin-containing granules (0.2-0.6 microns in diameter). High Ca2+ resulted in a Cm increase of 20-50% from the resting value, demonstrating a role for [Ca2+]i in stimulus-secretion coupling. Spontaneous Cm changes have also been recorded, which presumably reflect prolactin secretion supported by a tonic influx of Ca2+ through the membrane. This is supported by the following findings: addition of Co2+ diminished or reversed the spontaneous Cm changes and decreased resting [Ca2+]i; and membrane depolarization increased Cm, indicating the role of voltage-activated channels in stimulus-secretion coupling. As bovine lactotrophs have been found to be largely devoid of spontaneous electrical activity, a mechanism involving modulation of a tonic Ca2+ influx is proposed; this is shown to provide adequate control of basal and triggered secretion monitored by Cm.
膜片钳技术已被用于测量牛催乳素细胞的膜电容（Cm）变化，以监测与胞吐作用和胞吞作用相关的细胞表面积波动。通过富集程序制备细胞，并在使用前培养长达14天。在全细胞记录下，用各种含Ca2+的溶液透析细胞胞质。发现静息Cm为6.05±1.68 pF，与细胞半径的平方密切相关，表明比Cm为0.8 μF/cm2。记录到2-10 fF的离散Cm阶跃，这很可能反映了含催乳素颗粒（直径0.2-0.6微米）的单个融合和回收事件。高Ca2+导致Cm从静息值增加20-50%，表明[Ca2+]i在刺激-分泌偶联中起作用。也记录到了自发的Cm变化，这可能反映了通过膜的Ca2+持续内流支持的催乳素分泌。以下发现支持了这一点：添加Co2+减少或逆转了自发的Cm变化，并降低了静息[Ca2+]i；膜去极化增加了Cm，表明电压激活通道在刺激-分泌偶联中的作用。由于已发现牛催乳素细胞在很大程度上缺乏自发电活动，因此提出了一种涉及调节Ca2+持续内流的机制；结果表明，这为通过Cm监测的基础分泌和触发分泌提供了充分的控制。
