Ji Guangju, Feldman Morris, Doran Robert, Zipfel Warren, Kotlikoff Michael I
Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850, USA.
J Gen Physiol. 2006 Mar;127(3):225-35. doi: 10.1085/jgp.200509422.
Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca(2+) release or Ca(2+) sparks and, in some spiking tissues, as Ca(2+) release that is triggered by the activation of sarcolemmal Ca(2+) channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca(2+) (DMNP-EDTA) in Fluo-4-loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca(2+) activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca(2+) release in the form of Ca(2+) sparks and Ca(2+) waves that were distinguishable from increases in Ca(2+) associated with Ca(2+) uncaging, unequivocally demonstrating that Ca(2+) release occurs subsequent to a localized rise in Ca(2+). TPFP-triggered Ca(2+) release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca(2+) sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca(2+) release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local Ca(2+) through inositol trisphosphate (InsP(3)) receptors (InsP(3)Rs). We conclude that CICR activated by localized Ca(2+) release bears essential similarities to those observed by the activation of I(Ca) (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca(2+) release through InsP(3)R can occur at high local Ca(2+).
肌浆网（SR）的钙诱导钙释放（CICR）在平滑肌中以自发的SR钙释放或钙火花的形式发生，而在一些产生动作电位的组织中，则以由肌膜钙通道激活触发的钙释放的形式发生。这两种过程都表现出空间定位，即释放发生在特定亚细胞区域的频率更高。我们利用在装载Fluo-4的膀胱平滑肌细胞中对笼锁钙（DMNP-EDTA）进行双光子闪光光解（TPFP），来确定局部钙浓度升高激活SR释放的程度，并进一步了解CICR背后的分子和生物物理过程。TPFP导致以钙火花和钙波形式的局部钙释放，这些与钙笼锁解除相关的钙浓度升高不同，明确表明钙释放发生在局部[Ca²⁺]i升高之后。TPFP触发的钙释放并不局限于少数几个释放区域，而是可以在细胞的所有区域被激活，释放通常发生在光解部位或其几微米范围内。正如预期的那样，CICR过程主要由兰尼碱受体（RYR）活性主导，因为兰尼碱消除了单个钙火花，并在FKBP12.6基因敲除细胞中以不同的阈值和动力学诱发释放。然而，TPFP CICR并未被兰尼碱完全抑制；在存在兰尼碱的情况下，具有不同动力学特征的钙释放以更高的TPFP阈值发生。这种高阈值释放被西司他汀C阻断，其药理学敏感性和动力学与通过肌醇三磷酸（InsP₃）受体（InsP₃Rs）在高局部[Ca²⁺]i时的CICR释放一致。我们得出结论，由局部钙释放激活的CICR与通过I（Ca）激活所观察到的情况具有本质相似性（即主要依赖于2型RYR），释放并不在空间上局限于少数几个特定亚细胞区域，并且通过InsP₃R的钙释放在高局部[Ca²⁺]i时可以发生。
