HORMONE REGULATION OF CA++ I- IN PANCREATIC ACINAR CELLS

胰腺腺泡分泌消化酶和富含盐分的液体。两者都有 活动受神经激素刺激的钙离子直接调节 震荡。因此，对钙离子调节的一般了解和 尤其是Ca~(2+)振荡是理解 这些细胞中的刺激-分泌耦合。在过去的十年里，我们 用多种方法研究了胰腺腺泡内的钙信号 跟踪钙离子和钙离子通量的实验系统和技术 穿过特定的细胞膜。其中一个核心发现就是 研究表明，钙离子本身就是该通路的主要调节者 负责钙离子调节。在这方面，我们最重要的是 最近的发现是：a)激动剂依赖的刺激 质膜Ca~(2+)泵，b)Ca~(2+)从 内部存储由蛋白激酶磷酸酶对调节， C)一氧化氮途径之间的可能关系， 增加缝隙连接(GJ)的通透性和钙离子内流。要建造 根据这些发现，我们开发出了一种激动剂 拮抗剂反应性溶链菌素O-通透性细胞系统。在……里面 此外，为了选择性地影响腺泡内的细胞，我们喷雾或 将所需成分的溶液注入单个细胞。我们建议 使用这些技术进行以下工作：a)研究 激动剂激活质膜钙泵的机制。 这需要一个激动剂反应系统，允许自由访问 细胞质。因此，分离出具有SLO通透性的细胞和膜 将被用来评估磷脂酶C的作用 激活、钙调素与细胞骨架结构 激动剂介导的泵激活；B)探讨调节机制 钙离子释放钙离子的能力。钙离子释放的双相调节 钙离子参与钙依赖蛋白的顺序激活 激酶和磷酸酶。激动剂刺激对此有深刻的影响 监管。为了明确地确定激酶磷酸酶对，我们 将使用SLO渗透系统来激活钙释放通道 用IP3或激动剂使通道失活，而用抗激动剂使通道失活 调节特定的蛋白激酶和磷酸酶的活性， C)建立钙离子进入、一氧化氮和差距之间的关系 交汇点。我们最近用FRAP和FRAP测量GJ渗透性 局域和全局诱发的钙振荡模式 刺激暗示了这些活动之间的关系。我们 建议使用FRAP和钙离子成像来研究NO的作用 钙离子内流和神经节细胞通透性的调节途径；D)研究 在追求目标a-c过程中发现的钙机制的调节 激动剂或血管紧张素转换酶刺激的钙振荡的整体信号 将通过注射特异性药物来研究非激动剂依赖的方式 调节蛋白质和化合物。我们希望拟议的研究 将发现钙离子调节的新方面，并导致更好的 钙离子信号转导及其在生理学中的作用 胰腺腺泡功能。

Pancreatic acini secrete digestive enzymes and NaCl-rich fluid. both activities are directly regulated by neurohormonal-stimulated Ca2+ oscillations. Therefore, understanding Ca2+ regulation in general and Ca2+ oscillations in particular hold the key to understanding stimulus-secretion coupling in these cells. In the last ten years, we have studied the Ca2+ signal in pancreatic acini using a variety of experimental systems and techniques to follow Ca2+ and Ca2+ fluxes across specific cellular membranes. A central finding of these studies is that Ca2+ itself is the chief regulator of the pathways responsible for Ca2+ regulation. In this respect, our most important recent findings are: a) the agonist-dependent stimulation of the plasma membrane Ca2+ pump, b) the realization that Ca2+ release from internal stores is regulated by a protein kinase phosphatase pair and, c) the possible relationship between the nitric oxide pathway, increased gap junction (GJ) permeability and Ca2+ entry. To build upon and extend these findings, we have developed an agonist antagonist responsive streptolysin O-permeable cell system. In addition, to selectively affect cells within an acinus we puff or inject single cells with solutions of desired composition. We propose to use these techniques to pursue the following: A) Study the Mechanism of Activation of the Plasma membrane Ca2+ Pump by Agonist. This requires an agonist-responsive system that allows free access to the cytosol. Therefore, SLO-permeable cells and membranes isolated from them will be used to evaluate the role of phospholipase C activation, Ca2+ calmodulin and the cytoskeleton architecture in agonist-mediated pump activation; B) Probe the Mechanism of Regulation of Ca2+ Release by Ca2+. The biphasic regulation of Ca2+ release by Ca2+ involves the sequential activation of Ca2+ -dependent protein kinase and phosphatase. Agonist stimulation profoundly affects this regulation. To unequivocally identify the kinase phosphatase pair, we will use the SLO-permeable system to activate the Ca2+ release channel by IP3 or agonist and inactivate the channel by ant-agonist while modulating the activity of specific protein kinases and phosphatases, C) Establish a Relationship Between Ca2+ Entry, Nitric Oxide and Gap Junctions. Our recent measurement of GJ permeability with FRAP and the patterns of Ca2+ oscillations evoked by focal and global stimulation suggest a relationship between these activities. We propose to use FRAP and imaging of Ca2+ to study the role of the NO pathway in regulating Ca2+ entry and GJ permeability; D) Study Regulation of Ca2+ mechanisms discovered in pursuing aims a-c in the overall signal of Ca2+ oscillations stimulated by agonists or an agonist-independent manner will be studied by injection of specific modulatory proteins and compounds. We hope that the proposed studies will uncover novel aspects of Ca2+ regulating and lead to better understanding of Ca2+ signaling and their role in the physiological function of pancreatic acini.