IONIC CHANNELS IN DISSOCIATED SMOOTH MUSCLE CELLS

本申请是离子通道及其组合的延续。 调节单个新鲜分离的平滑肌细胞。 这些离子 通道控制膜电位，是靶点， 神经递质作用，构成动作电位的基础， 负责从细胞内储存释放Ca（2+）的通道 调节胞质 Ca（2+）浓度。 因此，这些渠道发挥着核心作用， 平滑肌收缩的控制，一个相当重要的话题， 治疗重要性，例如，在高血压、血管痉挛和子宫 和胃肠道收缩力。 大多数研究将在一个 制备来自蟾蜍，Bufo marinus， 这些都已经得到了相当详细的描述 电生理学、生物化学和形态学，并且已经证明 是平滑肌一般特性的良好预测因子， 各种来源。 将有三个主要的研究领域。 1.神经递质和第二 离子通道的信使调节。 毒蕈碱，胆碱能调节 负责M-电流的Ca（2+）通道和K+通道将被 研究以确定胞质第二信使的作用， 蛋白激酶C和“膜界定”机制，如G蛋白 渠道互动。 以这项研究为原型， 潜在的各种其他兴奋和抑制的行动， 将检查诸如神经肽的药剂。 2.离子型脂肪酸 渠道 最近，脂肪酸已被证明对 平滑肌和其他细胞类型中的许多离子通道。 的 这种脂肪酸活化的机制及其生理作用将是 考察 机械门控通道的调节。 平滑肌 细胞具有多种机械门控通道，包括 拉伸激活通道，拉伸失活通道，通道 由拉伸和膜超极化激活，以及流动- 激活渠道 这些通道的特性及其调节 将被研究。 这些研究将强调使用膜片钳技术记录 通过单个离子通道的电流，也记录宏观电流 在胞质溶胶可以改变的条件下。 此外，物理 核磁共振等技术将用于研究脂肪酸的相互作用 与膜蛋白和脂质结合。 最后，Ca（2+）指示染料 fura-2将被用来测量单个电压-电流信号中的胞浆{Ca（2+）}。 钳夹细胞以确定各种试剂对Ca释放的影响 （2+）从细胞内存储和跨膜之间的关系 Ca（2+）电流和{Ca（2+）}。

The present application is a continuation of ionic channels and their regulation in single, freshly dissociated smooth muscle cells. These ionic channels control the membrane potential, are the targets of neurotransmitter action, underlie the action potential and together with the channels responsible for Ca (2+) release from intracellular stores regulate the cytosolic Ca (2+) concentration. Consequently, these channels play a central role in the control of smooth muscle contraction, a topic of considerable therapeutic importance, for example, in hypertension, vasospasm and uterine and gastrointestinal contractility. Most studies will be carried out on a preparation of isolated cells from the stomach of the toad, Bufo marinus, which have been characterized in considerable detail electrophysiologically, biochemically, and morphologically, and have proven to be excellent predictors of general properties of smooth muscle from a variety of sources. There will be three major areas of study. 1. Neurotransmitter and second messenger regulation of ion channels. Muscarinic, cholinergic regulation of Ca (2+) channels and K+ channels responsible for M-current will be studied to determine the role of cytosolic second messengers such as protein kinase C and "membrane delimited" mechanisms such as G-protein channel interactions. Using this study as a prototype, the mechanisms underlying the actions of a variety of other excitatory and inhibitory agents such as neuropeptides will be examined. 2. Fatty acid of ionic channels. Recently fatty acids have been shown to exert a direct effect on a number of ionic channels in smooth muscle and other cell types. The mechanisms of this fatty acid activation and its physiological role will be examined. Regulation of mechanically gated channels. The smooth muscle cells have a considerable variety of mechanically gated channels, including stretch-activated channels, stretch inactivated channels, channels activated both by stretch and membrane hyperpolarization, and flow- activated channels. The properties of these channels and their regulation will be studied. The studies will emphasize the use of patch-clamp technology to record currents through single ionic channels and also record macroscopic currents under conditions where the cytosol can be altered. In addition, physical techniques such as NMR will be used in studying fatty acid interactions with membrane proteins and lipids. Finally, the Ca (2+) indicator dye fura-2 will be employed to measure cytosolic {Ca (2+)} in single, voltage- clamped cells to determine the effect of various agents on release of Ca (2+) from intracellular stores and the relationship between transmembrane Ca (2+) current and {Ca (2+)}.