Streb H, Bayerdörffer E, Haase W, Irvine R F, Schulz I
J Membr Biol. 1984;81(3):241-53. doi: 10.1007/BF01868717.
We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983, Nature (London) 306:67-69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000 X g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of Ip3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r = 0.96, 1.00, 0.91, respectively) and NADPH cytochrome c reductase (r = 0.63, 0.98, 0.90, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochrome c oxidase (r = -0.64) and glutamate dehydrogenase (r = -0.75) and with the plasma membrane markers (Na+ + K+)-ATPase (r = -0.81) and alkaline phosphatase (r = -0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.
我们之前已经表明，肌醇-1,4,5-三磷酸（IP3）可从大鼠胰腺通透腺泡细胞的细胞内钙库中释放Ca2+（H. 斯特雷布等人，1983年，《自然》（伦敦）306:67 - 69）。这一观察结果表明，IP3可能是毒蕈碱受体激活与刺激过程中细胞内钙库释放Ca2+之间缺失的环节。为了定位细胞内对IP3敏感的钙池，在分离的亚细胞组分中测量了IP3诱导的Ca2+释放。通过胶原酶消化法分离得到的腺泡细胞制备了总匀浆。通过差速离心将内质网与线粒体、酶原颗粒和细胞核分离。通过在蔗糖阶梯梯度上离心或用高浓度MgCl2沉淀来分离质膜和内质网。总匀浆中每毫克蛋白质的IP3诱导的Ca2+释放与渗漏细胞中的相同，并且足够稳定，使得短时间的分离程序成为可能。在通过7000×g差速离心、蔗糖密度离心或MgCl2沉淀获得的组分中，IP3诱导的Ca2+释放与内质网标志物核糖核酸（r分别为0.96、1.00、0.91）和NADPH细胞色素c还原酶（r分别为0.63、0.98、0.90）密切相关。相比之下，在所有分析的组分中，与线粒体标志物细胞色素c氧化酶（r = -0.64）和谷氨酸脱氢酶（r = -0.75）以及质膜标志物（Na+ + K+）-ATP酶（r = -0.81）和碱性磷酸酶（r = -0.77）存在明显的负相关。通过电子显微镜评估，IP3诱导的Ca2+释放与组分中的酶原颗粒或细胞核含量无关。数据表明，肌醇-1,4,5-三磷酸从胰腺腺泡细胞的内质网中释放Ca2+。
