Tortorici G, Zhang B X, Xu X, Muallem S
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235-9040.
J Biol Chem. 1994 Nov 25;269(47):29621-8.
Streptolysin O-permeabilized pancreatic acini were used to study compartmentalization of Ca2+ signaling and Ca2+ pools. In these cells, the inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ channels could be activated by a number of agonists (carbachol, cholecystokinin, or bombesin) or by activation of the entire cellular phospholipase C pool with GTP gamma S. Surprisingly, each of the antagonists interacting with acinar cells inactivated the channels after stimulation with GTP gamma S. In addition, when permeabilized cells were stimulated with more than one agonist, any antagonist to the specific agonists employed inactivated the channels. The aberrant behavior of the antagonists in permeable cells was not related to a loss of specificity since (a) when added before GTP gamma S, the antagonists had no effect on Ca2+ release and (b) when cells were stimulated with a single agonist, the antagonists prevented only the effect of their specific agonist. The differential behavior of the antagonists in intact and permeable cells suggests a compartmentalization of Ca2+ signaling into separate, agonist-specific units that is modified by cell permeabilization. Further evidence for compartmentalization of signaling was obtained by showing that the partial agonist (the CCK octapeptide analogue JMV-180) can access and release only 50% of the cholecystokinin- or IP3-mobilizable Ca2+ pool in intact and permeable cells. Kinetic measurements revealed a multiphasic time course of agonist-evoked Ca2+ release in permeable cells. At high agonist concentrations, all phases were fast and merged into an apparent single event of Ca2+ release. The phases were separated by three independent protocols: reduction in agonist concentrations, addition of heparin, or addition of guanosine-5'-O-(thio)diphosphate. Since all protocols that caused phase separation reduce IP3-mediated Ca2+ release, these findings demonstrate heterogeneity in the affinity for IP3 of channels present in compartmentalized Ca2+ pools of the same cells. Compartmentalization of signaling and the heterogeneity in the affinity for IP3 resulted in a quantal agonist-evoked Ca2+ release. The overall findings are discussed in the context of an integrated model of compartmentalization of signaling complexes, Ca2+ pools, and IP3-activated Ca2+ channels.
使用经链球菌溶血素O通透处理的胰腺腺泡来研究Ca2+信号传导和Ca2+池的区室化。在这些细胞中，肌醇1,4,5-三磷酸（IP3）依赖性Ca2+通道可被多种激动剂（卡巴胆碱、胆囊收缩素或蛙皮素）激活，或通过用GTPγS激活整个细胞磷脂酶C池来激活。令人惊讶的是，在与腺泡细胞相互作用的每种拮抗剂在GTPγS刺激后都会使通道失活。此外，当用多种激动剂刺激通透细胞时，针对所使用的特定激动剂的任何拮抗剂都会使通道失活。拮抗剂在可通透细胞中的异常行为与特异性丧失无关，因为（a）在GTPγS之前添加时，拮抗剂对Ca2+释放没有影响；（b）当用单一激动剂刺激细胞时，拮抗剂仅阻止其特定激动剂的作用。拮抗剂在完整细胞和通透细胞中的不同行为表明Ca2+信号传导区室化为单独的、激动剂特异性单元，而细胞通透化会对其进行修饰。通过显示部分激动剂（胆囊收缩素八肽类似物JMV-180）在完整细胞和通透细胞中只能进入并释放50%的胆囊收缩素或IP3可动员的Ca2+池，获得了信号传导区室化的进一步证据。动力学测量揭示了激动剂诱发的Ca2+释放在通透细胞中的多相时间进程。在高激动剂浓度下，所有阶段都很快，并合并为明显的单一Ca2+释放事件。这些阶段通过三种独立的方案分开：降低激动剂浓度、添加肝素或添加鸟苷-5'-O-（硫代）二磷酸。由于所有导致阶段分离的方案都会减少IP3介导的Ca2+释放，这些发现证明了同一细胞区室化Ca2+池中存在的通道对IP3的亲和力存在异质性。信号传导的区室化以及对IP3亲和力的异质性导致了量子化的激动剂诱发的Ca2+释放。在信号复合物、Ca2+池和IP3激活的Ca2+通道区室化的综合模型背景下讨论了总体发现。
