PHOTOCHEMICAL REGULATION OF CALCIUM IN CELL PHYSIOLOGY

细胞内钙离子浓度的变化控制着无数的 生理过程包括肌肉收缩、分泌、有丝分裂、 通道门控、趋化性和气孔关闭。在过去 十年来，两种技术彻底改变了对钙离子的研究 细胞生理学的研究进展：用 比率、钙离子特定的荧光指示剂，如Fura-2；以及 细胞底物如ATP、cGMP、 等通过闪光光解从生物惰性或笼状的形式 技巧。这项提议的目标是使用快速光化学 控制二价阳离子水平以表征机理和 细胞生理过程的调节。特定于Ca2+ 耐光性螯合剂将被开发并用于操纵 细胞内钙离子浓度独立于其他效应器，如 如MG2、ATP、GTP等，因此这些物种的调节作用 对钙离子有依赖的细胞生理学可以定义。此外，一个 将合成镁离子特异的耐光性螯合剂。由于镁离子是 核苷酸依赖过程中的一个必要的辅助因素，即快速 阳离子的光化学释放将被用来启动这些 处理和表征镁离子/核苷酸复合体在 例如，肌肉收缩和分泌的机制。这些 研究将扩展那些已经用DM-硝酚进行的研究， 基于EDTA的耐光性螯合剂，由申请人开发， 结合高亲和力的钙和镁离子。这项建议的基础是 是一种名为硝基苯乙二胺四乙酸乙二酯的新型钙离子耐光络合剂， 它是最近合成的。这个笼子里的钙离子将被用在 结合钙离子荧光指示剂，研究其动力学和 黑素营养细胞分泌事件和收缩的调节 心肌和平滑肌，因为钙离子是关键的细胞内第二 这些系统中的信使。这些进程中的许多都分布在 病态。在对这些疾病进行充分描述之前 可以给予国家，对非疾病状态的更全面的了解 应该做到的。拟议的研究将有助于 更好地了解这些过程的正常运作。

Changes in intracellular Ca2+ concentration control a myriad of physiological processes including muscle contraction, secretion, mitosis, channel gating, chemotaxis and stomatal pore closure. During the past ten years two techniques have revolutionized the study of Ca2+ involvement in cell physiology: measurement of Ca2+ concentrations using ratiometric, Ca2+ specific fluorescent indicators such as fura-2; and the rapid increase in concentration of cellular substrates such as ATP, cGMP, etc. from a biologically inert or 'caged' form by flash-photolysis techniques. The objective of this proposal is to use rapid photochemical control of divalent cation levels to characterize the mechanism and regulation of cellular physiological processes. Ca2+-specific photolabile chelators will be developed and used to manipulate intracellular Ca2+ concentrations independently of other effectors, such as Mg2, ATP, GTP, etc. so that the regulatory roles that these species have on Ca2+-dependent cell physiology can be defined. Additionally, a Mg2+-specific photolabile chelator will be synthesized. Since Mg2+ is a necessary co-factor in nucleotide-dependent processes, the rapid photochemical release of the cation will be used to initiate these processes and characterize the role of Mg2+/nucleotide complexes in, for example, the mechanisms of muscle contraction and secretion. These studies will extend those already performed with DM-nitrophen, a photolabile chelator based on EDTA, developed by the applicant, which binds both Ca2+ and Mg2+ with high affinity. The basis of this proposal is a new Ca2+-specific photolabile chelator called nitrophenyl-EGTA, which has been recently synthesized. This caged Ca2+ will be used, in combination with fluorescent Ca2+ indicators, to study the kinetics and regulation of secretion events in melanotrophs and of contraction in cardiac, and smooth muscle because Ca2+ is the key intracellular second messenger in these systems. Many of these processes are distributed in pathological states. Before an adequate description of these disease states can be given, a more complete understanding of non-disease states should be accomplished. The proposed studies will contribute to a greater understanding of the normal functioning of these processes.