Gilchrist J S, Palahniuk C, Bose R
Department of Oral Biology, University of Manitoba, Winnipeg, Canada.
Mol Cell Biochem. 1997 Jul;172(1-2):159-70.
In this report we describe the application of spectroscopic methods to the study of Ca2+ release by isolated native sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle. To date, dual-wavelength spectroscopy of arsenazo III and antipyrylazo III difference absorbance have been the most common spectroscopic methods for the assay of SR Ca2+ transport. The utility of these methods is the ability to manipulate intraluminal Ca2+ loading of SR vesicles. These methods have also been useful for studying the effect of both agonists and antagonists upon SR Ca2+ release and Ca2+ uptake. In this study, we have developed the application of Calcium Green-2, a long-wavelength excitable fluorescent indicator, for the study of SR Ca2+ uptake and release. With this method we demonstrate how ryanodine receptor Ca2+ channel opening and closing is regulated in a complex manner by the relative distribution of Ca2+ between extraluminal and intraluminal Ca2+ compartments. Intraluminal Ca2+ is shown to be a key regulator of Ca2+ channel opening. However, these methods also reveal that the intraluminal Ca2+ threshold for Ca2+-induced Ca2+ release varies as a function of extraluminal Ca2+ concentration. The ability to study how the relative distribution of a finite pool of Ca2+ across the SR membrane influences Ca2+ uptake and Ca2+ release may be useful for understanding how the ryanodine receptor is regulated, in vivo.
在本报告中，我们描述了光谱方法在研究兔骨骼肌分离的天然肌浆网（SR）膜释放Ca2+中的应用。迄今为止，偶氮胂III和安替比拉宗III差示吸光度的双波长光谱法一直是测定SR Ca2+转运最常用的光谱方法。这些方法的实用性在于能够控制SR囊泡腔内Ca2+的负载。这些方法对于研究激动剂和拮抗剂对SR Ca2+释放和Ca2+摄取的影响也很有用。在本研究中，我们开发了应用钙绿-2（一种长波长可激发荧光指示剂）来研究SR Ca2+摄取和释放。通过这种方法，我们证明了兰尼碱受体Ca2+通道的开闭如何通过胞外和胞内Ca2+区室之间Ca2+的相对分布以复杂的方式受到调节。腔内Ca2+被证明是Ca2+通道开放的关键调节因子。然而，这些方法也揭示了Ca2+诱导的Ca2+释放的腔内Ca2+阈值随胞外Ca2+浓度而变化。研究有限的Ca2+池在SR膜上的相对分布如何影响Ca2+摄取和Ca2+释放，这对于理解体内兰尼碱受体如何被调节可能是有用的。
