Keeton R A, Runge S W, Moran W M
Department of Biology, University of Central Arkansas, Conway, AR 72035-5003, USA.
J Comp Physiol B. 2004 Mar;174(2):129-38. doi: 10.1007/s00360-003-0397-x. Epub 2003 Nov 25.
We used the Aplysia californica intestinal epithelium to investigate the effect of alanine-stimulated Na+ absorption on apical membrane exocytosis and whether stimulated exocytosis requires intact actin filaments. The fluid-phase marker fluorescein dextran was used to determine rates of apical membrane exocytosis. L-alanine significantly increased apical exocytosis by approximately 30% compared to controls, and there is a modest, positive correlation between alanine-stimulated exocytosis and short-circuit current (ISC). Thus, apical exocytosis is modulated to some extent by the magnitude of Na+ and alanine entry across the apical membrane. Apical exocytosis is also responsive to virtually any increase in Na+ and alanine entry because increments in alanine-stimulated ISC as small as 1 microA/cm2 stimulated exocytosis. We used D-alanine to determine which parameter (sensitivity to transport vs. magnitude of transport) was most important in activation of apical exocytosis. D-alanine-stimulated ISC was one-sixth that of L-alanine, but stimulated exocytosis was only 29% less than that of L-alanine. Therefore, the apical exocytic system is more responsive to small increases in transport than to the magnitude of transport. Latrunculin A (Lat-A) disrupts the actin cytoskeleton and reduced constitutive apical exocytosis by approximately 65% and completely abolished alanine-stimulated exocytosis. Hence, constitutive exocytosis and alanine-stimulated exocytosis require actin filaments for recruitment of vesicles to the apical membrane. During nutrient absorption, actin filament-regulated apical exocytosis may represent a negative feedback system that modulates apical membrane tension.
我们利用加州海兔肠道上皮来研究丙氨酸刺激的Na⁺吸收对顶端膜胞吐作用的影响，以及刺激的胞吐作用是否需要完整的肌动蛋白丝。使用液相标记物荧光素葡聚糖来测定顶端膜胞吐作用的速率。与对照组相比，L-丙氨酸使顶端胞吐作用显著增加了约30%，并且丙氨酸刺激的胞吐作用与短路电流（ISC）之间存在适度的正相关。因此，顶端胞吐作用在一定程度上受到Na⁺和丙氨酸跨顶端膜进入量的调节。顶端胞吐作用实际上对Na⁺和丙氨酸进入量的任何增加都有反应，因为丙氨酸刺激的ISC只要增加小至1微安/平方厘米就能刺激胞吐作用。我们使用D-丙氨酸来确定在激活顶端胞吐作用中哪个参数（对转运的敏感性与转运量）最为重要。D-丙氨酸刺激的ISC是L-丙氨酸的六分之一，但刺激的胞吐作用仅比L-丙氨酸少29%。因此，顶端胞吐系统对转运的小幅增加比对转运量更敏感。拉特罗毒素A（Lat-A）破坏肌动蛋白细胞骨架，使组成型顶端胞吐作用减少约65%，并完全消除丙氨酸刺激的胞吐作用。因此，组成型胞吐作用和丙氨酸刺激的胞吐作用需要肌动蛋白丝将囊泡募集到顶端膜。在营养物质吸收过程中，肌动蛋白丝调节的顶端胞吐作用可能代表一种调节顶端膜张力的负反馈系统。
