Gish R G, Garcia C, Reedy T, Kaplowitz N, Langer G A
Liver Research Laboratory, Wadsworth V.A. Hospital, Los Angeles, California.
Anal Biochem. 1990 May 15;187(1):187-96. doi: 10.1016/0003-2697(90)90439-g.
We utilized a technique, previously used to study myocardial cells (G. A. Langer, J. S. Frank, and L. M. Nudd, 1979, Amer. J. Physiol. 237, H239-H246), to study 45Ca2+ isotope exchange kinetics in hepatocyte monolayers, cultured on scintillation disks, and perfused in a flow-through chamber. Isolated rat hepatocytes were plated directly on Primaria-coated disks impregnated with scintillation fluors which made up the walls of the perfusion chamber. Following the labeling of the cells with radioactive calcium (45Ca2+), to apparent asymptote, the washout of 45Ca2+ from the cells was measured. A large very fast turnover compartment, as well as small fast and slow turnover compartments, were identified in each experiment. Surface calcium (Ca2+) was determined by its displacement with 1 mM La3+ after asymptote had been reached during 45Ca2+ labeling (1.59 mmol Ca2+/kg dry wt). The rate constant for this compartment was faster than the washout of the chamber (greater than 3.4 min-1 with a t1/2 less than 12 s). The rate constants for the fast and slow exchangeable compartments were 0.11 min-1 (t1/2 = 6.5 min) and 0.013 min-1 (t1/2 = 56 min), respectively. The fast compartment contained 0.40 mmol Ca2+/kg dry wt and the slow compartment contained 0.27 mmol Ca2+/kg dry wt. Neither the fast nor the slow compartment was lanthanum displaceable. Release of 45Ca2+ in response to 100 microM phenylephrine, 10 nM angiotensin II, and 100-microM 2,5-ditert-butyl hydroquinone was measured during the washout phase. Ca2+ released by these compounds was determined to be 0.50 mmol 0.44, and 0.43 mmol Ca2+/kg dry cell wt, respectively. These agents had an effect only during the washout of the fast compartment. In conclusion, this novel technique of on-line measurement of 45Ca2+ exchange in hepatocyte monolayers identified three exchangeable compartments: (1) a very rapidly exchangeable surface compartment, (2) a fast "microsomal" hormone-releasable compartment, and (3) a slow, non-hormone-releasable compartment.
我们采用了一种先前用于研究心肌细胞的技术（G. A. 兰格、J. S. 弗兰克和L. M. 努德，1979年，《美国生理学杂志》237卷，H239 - H246页），来研究闪烁盘上培养并在流通室中灌注的肝细胞单层中的45Ca2 + 同位素交换动力学。将分离的大鼠肝细胞直接接种在涂有Primaria的圆盘上，圆盘浸渍有构成灌注室壁的闪烁荧光剂。在用放射性钙（45Ca2 +）标记细胞至明显渐近线后，测量45Ca2 + 从细胞中的洗脱情况。在每个实验中都鉴定出一个大的非常快速周转的区室，以及小的快速和慢速周转区室。在45Ca2 + 标记（1.59 mmol Ca2 + / kg干重）达到渐近线后，通过用1 mM La3 + 置换来测定表面钙（Ca2 +）。该区室的速率常数比室的洗脱速率快（大于3.4 min-1，t1/2小于12秒）。快速和慢速可交换区室的速率常数分别为0.11 min-1（t1/2 = 6.5分钟）和0.013 min-1（t1/2 = 56分钟）。快速区室含有0.40 mmol Ca2 + / kg干重，慢速区室含有0.27 mmol Ca2 + / kg干重。快速和慢速区室均不可被镧置换。在洗脱阶段测量了对100 microM去氧肾上腺素、10 nM血管紧张素II和100 - microM 2,5 - 二叔丁基对苯二酚的45Ca2 + 释放。这些化合物释放的Ca2 + 分别测定为0.50 mmol、0.44和0.43 mmol Ca2 + / kg干细胞重。这些试剂仅在快速区室的洗脱过程中有作用。总之，这种用于在线测量肝细胞单层中45Ca2 + 交换的新技术鉴定出三个可交换区室：（1）一个非常快速可交换的表面区室，（2）一个快速的“微粒体”激素可释放区室，以及（3）一个缓慢的、非激素可释放区室。
