Xu X, Zeng W, Diaz J, Muallem S
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
J Biol Chem. 1996 Oct 4;271(40):24684-90. doi: 10.1074/jbc.271.40.24684.
Imaging [Ca2+]i at high temporal resolution and measuring the properties of Ca2+ signaling in streptolysin O (SLO)-permeabilized cells were used to study the spacial organization of signaling complexes. Sequential stimulation of single cells within pancreatic acini with several Ca2+-mobilizing agonists revealed an agonist-specific pattern and propagation rate of Ca2+ waves in the same cells, with CCK8 stimulating the fastest and bombesin the slowest waves. More importantly, each agonist initiated the wave in a different region of the same cell. On the other hand, repetitive stimulation with the same agonist induced Ca2+ waves of the same pattern that were initiated from the same region of the cell. The agonist-specific Ca2+ signaling does not appear to be the result of coupling to different G proteins as infusion of an anti-Galphaq antibody into the cells through a patch pipette equally inhibited Ca2+ signaling by all agonists. Further evidence for compartmentalization of signaling complexes was developed in permeabilized cells. The time-dependent loss of Ca2+ signaling due to SLO permeabilization occurred in an agonist-specific manner in the sequence cabachol > bombesin > cholecystokinin. Signaling by all agonists could be completely restored with as low as 2 micro guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). At this low concentration GTPgammaS recoupled inositol 1,4,5-trisphosphate production and Ca2+ release, rather than enhancing phospholipase C activity. Priming of Ca2+ signaling by GTPgammaS was agonist-specific. Guanosine 5'-O-(thio)diphosphate (GDPbetaS) uncoupled the ability of signaling complexes to release Ca2+ much better than stimulating inositol 1,4,5-trisphosphate production. The uncoupling of Ca2+ signaling by GDPbetaS was also agonist-specific. The combined findings of agonist-specific initiation sites of the Ca2+ wave and differential access of guanine nucleotides to signaling complexes suggest spacial compartmentalization of Ca2+ signaling complexes. Each complex must include a receptor, G protein, and phospholipase C that are coupled to a specific portion of the Ca2+ pool.
采用高时间分辨率成像细胞内钙离子浓度（[Ca2+]i）以及测量链球菌溶血素O（SLO）通透细胞中钙离子信号转导特性的方法，研究信号复合物的空间组织。用几种钙离子动员激动剂依次刺激胰腺腺泡内的单个细胞，结果显示同一细胞内钙离子波呈现激动剂特异性模式和传播速率，其中胆囊收缩素八肽（CCK8）刺激产生的钙离子波最快，蛙皮素刺激产生的最慢。更重要的是，每种激动剂在同一细胞的不同区域引发钙离子波。另一方面，用同一激动剂重复刺激会诱导出相同模式的钙离子波，且均从细胞的同一区域起始。激动剂特异性钙离子信号转导似乎并非与不同G蛋白偶联的结果，因为通过膜片吸管向细胞内注入抗Gαq抗体可同等程度地抑制所有激动剂引起的钙离子信号转导。在通透细胞中还获得了信号复合物分隔的进一步证据。由于SLO通透导致的钙离子信号转导随时间的丧失以激动剂特异性方式发生，顺序为卡巴胆碱＞蛙皮素＞胆囊收缩素。所有激动剂的信号转导均可通过低至2微摩尔鸟苷5'-3-O-（硫代）三磷酸（GTPγS）完全恢复。在此低浓度下，GTPγS重新偶联肌醇1,4,5-三磷酸生成和钙离子释放，而非增强磷脂酶C活性。GTPγS对钙离子信号转导的引发具有激动剂特异性。鸟苷5'-O-（硫代）二磷酸（GDPβS）解偶联信号复合物释放钙离子的能力比刺激肌醇1,4,5-三磷酸生成的能力强得多。GDPβS对钙离子信号转导的解偶联也具有激动剂特异性。钙离子波激动剂特异性起始位点以及鸟嘌呤核苷酸对信号复合物的不同作用的综合研究结果表明钙离子信号复合物存在空间分隔。每个复合物必须包含一个受体、G蛋白和磷脂酶C，它们与钙离子池的特定部分偶联。
