de Diego A M G, Arnáiz-Cot J J, Hernández-Guijo J M, Gandía L, García A G
Facultad de Medicina, Instituto Teófilo Hernando, Universidad Autónoma de Madrid, Madrid, Spain.
Acta Physiol (Oxf). 2008 Oct;194(2):97-109. doi: 10.1111/j.1748-1716.2008.01871.x. Epub 2008 Jun 28.
This study looks into the physiology of the exocytosis of catecholamines released by adrenal medullary chromaffin cells. We have comparatively explored the exocytotic responses elicited by two different patterns of depolarizing stimulation: the widely employed square depolarizing pulses (DPs) and trains of acetylcholine-like action potentials (APs), likely the physiological mode of stimulation in the intact innervated adrenal medulla. APs were applied at 30 Hz, a frequency similar to that produced in a stressful situation.
Patch-clamp, cell membrane capacitance, single cell amperometry and fluorescence were the techniques used. The variations of calcium entry measured as the integral of the calcium current, cytosolic calcium (measured with the calcium-sensitive fluorescent probe fluo-4) and exo-endocytosis (membrane capacitance variations) were the parameters measured.
Trains of AP depolarizations produced distinct responses compared to those of square depolarizations: (1) Calcium current amplitude decreased to a lesser extent along the AP train; (2) calcium entry and capacitance increments raised linearly with stimulation time whereas they deviated from linearity when square depolarizations were used; (3) slower activation and faster delayed decay phase of cytosolic calcium transients; (4) capacitance increments varied linearly with calcium entry with APs and deviated from linearity with longer depolarizations; (5) little endocytosis after stimulation with longer trains of APs and pronounced endocytosis with longer square depolarizations.
Stimulation of chromaffin cells with trains of APs produced patterns of cytosolic calcium transients, exocytotic and endocytotic responses quite different from those elicited by the widely employed DPs. Our study is relevant from the methodological and physiological points of view.
本研究探讨肾上腺髓质嗜铬细胞释放儿茶酚胺的胞吐作用的生理学机制。我们比较研究了两种不同模式的去极化刺激引发的胞吐反应：广泛应用的方形去极化脉冲（DPs）和类似乙酰胆碱的动作电位序列（APs），后者可能是完整的神经支配肾上腺髓质中的生理性刺激模式。以30Hz的频率施加APs，该频率与应激状态下产生的频率相似。
采用膜片钳、细胞膜电容、单细胞安培法和荧光技术。测量的参数包括：以钙电流积分衡量的钙内流变化、胞质钙（用钙敏荧光探针fluo-4测量）以及胞吐-内吞作用（膜电容变化）。
与方形去极化相比，APs序列产生了不同的反应：（1）在APs序列中，钙电流幅度下降幅度较小；（2）钙内流和电容增加随刺激时间呈线性增加，而使用方形去极化时则偏离线性；（3）胞质钙瞬变的激活较慢且延迟衰减阶段较快；（4）APs刺激时，电容增加与钙内流呈线性关系，较长时间去极化时则偏离线性；（5）较长序列的APs刺激后内吞作用较弱，较长时间的方形去极化刺激后内吞作用明显。
用APs序列刺激嗜铬细胞产生的胞质钙瞬变、胞吐和内吞反应模式与广泛应用的DPs引发的反应截然不同。我们的研究从方法学和生理学角度来看都具有重要意义。
