Ruttner Z, Ligeti L, Reinlib L, Hines K, McLaughlin A C
Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland.
Cell Calcium. 1993 Jun;14(6):465-72. doi: 10.1016/0143-4160(93)90005-q.
Fluorescent calcium indicators have been widely used to assess cytoplasmic calcium concentration in cells. To examine the role of calcium ions on different physiological functions (e.g. in case of liver; bile secretion, glucose metabolism, etc.) there is a need for whole organ studies. We have developed a technique to estimate intracellular free calcium changes in perfused rat liver. Krebs-Henseleit perfused livers were loaded with 7 microM or 35 microM Indo-1/AM. An area 3 mm in diameter and approximately 300 microns in depth was illuminated at 340 nm. Fluorescence was monitored with photomultiplier tubes at 3 wavelengths (400 nm for Ca-bound dye, 504 nm for free dye and 464 nm for NADH). The viability of liver preparations was assessed by measurement of the concentrations of lactate dehydrogenase and alanine aminotransferase in the effluent. Loading of the livers with 7 microM Indo-1/AM via the portal vein resulted in a 5-fold increase of fluorescence at 400 nm. However the dye 'leaked' out of the liver with a half-time of 18 min. Probenecid (a specific anion carrier blocker) inhibited loss of dye in a dose dependent fashion (2.5-10 mM). Transient calcium elevations were observed in response to vasopressin (5-50 nM) at physiological levels, ethanol (0.3-0.8 M) and the calcium ionophore, ionomycin. Certain limitations were apparent with this approach: (1) it was necessary to use an anion carrier blocker to maintain a relatively steady dye concentration; (2) endogenous NADH fluorescence interfered with the calcium signal; and (3) absolute values of calcium concentration could not be determined.
荧光钙指示剂已被广泛用于评估细胞内的细胞质钙浓度。为了研究钙离子在不同生理功能中的作用（例如在肝脏中；胆汁分泌、葡萄糖代谢等），需要进行全器官研究。我们开发了一种技术来估计灌注大鼠肝脏中的细胞内游离钙变化。用Krebs-Henseleit溶液灌注的肝脏加载了7微摩尔或35微摩尔的Indo-1/AM。在340纳米波长下照射直径3毫米、深度约300微米的区域。用光电倍增管在3个波长下监测荧光（钙结合染料为400纳米、游离染料为504纳米、NADH为464纳米）。通过测量流出液中乳酸脱氢酶和丙氨酸转氨酶的浓度来评估肝脏制剂的活力。通过门静脉向肝脏加载7微摩尔的Indo-1/AM导致400纳米处的荧光增加了5倍。然而，染料以18分钟的半衰期从肝脏中“泄漏”出来。丙磺舒（一种特异性阴离子载体阻滞剂）以剂量依赖方式（2.5 - 10毫摩尔）抑制染料的流失。在生理水平下，观察到血管加压素（5 - 50纳摩尔）、乙醇（0.3 - 0.8摩尔）和钙离子载体离子霉素引起的瞬时钙升高。这种方法存在一些明显的局限性：（1）必须使用阴离子载体阻滞剂来维持相对稳定的染料浓度；（2）内源性NADH荧光干扰钙信号；（3）无法确定钙浓度的绝对值。
