IONIC CHANNELS IN DISSOCIATED SMOOTH MUSCLE CELLS

本申请是离子通道及其 对单个新鲜分离的平滑肌细胞的调节。这些离子 控制膜电位的通道，是 神经递质作用，是动作电位的基础，并与 负责细胞内钙离子释放的通道 调节胞浆 Ca(2+)浓度。因此，这些渠道在 平滑肌收缩的控制，这是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 和胃肠道的收缩能力。大多数研究将在一个 中华大蟾蜍胃分离细胞的制备 它们已经被描述得相当详细 从电生理、生化和形态上看，已经证明 可以很好地预测血管平滑肌的一般特性 来源多种多样。 将有三个主要领域进行研究。1.神经递质和第二 离子通道的信使调节。毒鼠碱、胆碱能调节 负责M-电流的Ca(2+)通道和K+通道 研究确定胞浆第二信使的作用，如 蛋白激酶C与G蛋白等“膜分隔”机制 渠道互动。以这项研究为原型，机制 潜在的各种其他兴奋和抑制的行为 将对神经肽等试剂进行检查。2.离子型脂肪酸 频道。最近，脂肪酸被证明对人体健康有直接影响。 在平滑肌和其他类型的细胞中有许多离子通道。这个 这种脂肪酸激活的机制及其生理作用将是 检查过了。对机械门控通道的监管。平滑的肌肉 细胞有相当多种类的机械门控通道，包括 拉伸激活的通道、拉伸停用的通道、通道 被拉伸和膜超极化激活，而流- 激活的通道。这些通道的特性及其调节 将会被研究。 这些研究将强调使用膜片钳技术来记录 通过单个离子通道的电流，也记录宏观电流 在细胞质可以改变的条件下。此外，物理上 核磁共振等技术将用于研究脂肪酸之间的相互作用 由膜蛋白和脂类组成。最后，Ca(2+)指示染料 Fura-2将被用来测定单个电压下的胞浆{Ca(2+)}。 夹闭细胞测定不同药物对细胞内钙释放的影响 (2+)来自细胞内储藏与跨膜的关系 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+)}.