CELLULAR MECHANISMS OF HORMONAL MODULATION OF MUSCLE

描述：过去的研究表明，成年龙虾有横纹肌 表现出生化、生理和药理学上的相似性 脊椎动物的条纹肌肉和平滑肌肉。此外，正如 对于脊椎动物的肌肉和神经组织来说，龙虾的功能 横纹肌受到血液中荷尔蒙的调节。具体来说， 龙虾肌肉的静息张力、收缩和松弛动力学 都受到体内合成的胺和多肽激素的影响 神经分泌腺并通过全身传递到肌肉 发行量。这项研究提案的总体目标是 确定这些荷尔蒙的作用机制。 为了达到这个目标，申请者将研究荷尔蒙。 对兴奋收缩过程中若干步骤的影响 龙虾幼体指骨开口肌的偶联。仔鱼肌肉 之所以选择是因为单根纤维很容易分离和研究 与允许评估肌膜离子的技术组合 电导、细胞内钙水平的控制和监测 肌肉机械活动。 申请者将检验提出的两个解释这些影响的假设。 下列激素对龙虾肌肉功能的影响： “...5-羟色胺、章鱼胺、原胡萝卜素，两种阿片样多肽(龙虾 多肽F1和F2)和一种新发现的具有特异性的多肽 刺激cGMP代谢(肽G1)“；所有这些激素都是 由成年龙虾神经分泌腺合成并释放到 发行量。第一个要检验的假设是这些荷尔蒙 对兴奋-收缩耦合所涉及的各个步骤采取行动。这个 第二个假设是，它们导致了药物-机械耦合； 激素作用于膜上的受体以激活第二信使 启动收缩和调节肌肉张力的途径 兴奋-收缩耦合的激活……“。因此， 申请者将评估应用第二信使(GTP， CAMP和IP3)和在细胞内改变其水平的物质(例如， 百日咳毒素、磷酸二酯酶抑制剂)。 为了检验两个提出的假设，以下测量 将在接触化学品之前、期间和之后进行 如上所述： 1.记录整个细胞的钙、钾电流 膜片钳技术； 2.胞浆内钙浓度用Fura-2测定。 成像； 3.测量收缩蛋白对钙的敏感性。 对于用β-七叶皂苷渗透并暴露在不同环境中的肌肉纤维 胞外钙离子浓度； 4.肌肉的收缩能力将根据纤维的视频图像进行测量。 单个肌肉纤维缩短、收缩或吸光度改变 在Fura-2的等压点(260 Nm)。 幼虫肌肉制剂的一个明显优势是 测量1、2和4可以同时对单个 纤维。

DESCRIPTION: Past work has shown that adult lobster striated muscle exhibits biochemical, physiological, and pharmacological similarities to both striated and smooth muscle of vertebrates. Furthermore, just as for vertebrate smooth muscle and nervous tissue, the function of lobster striated muscle is modulated by blood borne hormones. Specifically, resting tone, contractility, and relaxation kinetics of lobster muscle are all influenced by amine and peptide hormones synthesized in neurosecretory glands and delivered to muscle via the general circulation. The overall objective of this research proposal is to determine the mechanism whereby these hormones act. In order to achieve this goal, the applicant will investigate hormonal effects on a number of the steps involved in excitation-contraction coupling of the dactyl opener muscle of larval lobsters. Larval muscle is selected because single fibers are readily isolated and investigated with a combination of techniques allowing evaluation of sarcolemmal ion conductances, control of intracellular calcium levels, and monitoring of muscle mechanical activity. The applicant will test two hypotheses proposed to explain the effects of the following hormones on the function of lobster muscle: "...serotonin, octopamine, proctolin, two opioid-like peptides (lobster peptides F1 and F2) and a newly identified peptide which specifically stimulates cGMP metabolism (peptide G1)"; all of these hormones are synthesized by adult lobster neurosecretory glands and released into the circulation. The first hypothesis to be tested is that these hormones act on various steps involved in excitation-contraction coupling. The second hypothesis is that they cause pharmacomechanical coupling; that is, the hormones act on membrane receptors to activate second messenger pathways which "...initiate contraction and modulate muscle tone without activation of excitation-contraction coupling...". Therefore, the applicant will evaluate the effects of applied second messengers (GTP, cAMP, and IP3) and substances altering their levels within cells (e.g., pertussis toxin, phosphodiesterase inhibitors). In order to test the two proposed hypotheses, the following measurements will be performed before, during, and after exposure to the chemicals noted above: 1. calcium and potassium currents will be recorded with the whole cell patch clamp technique; 2. cytoplasmic concentrations of calcium will be measured with Fura-2 imaging; 3. the sensitivity of contractile proteins to calcium will be measured for muscle fibers permeabilized with Beta-escin and exposed to varying concentrations of extracellular calcium; 4. muscle contractility will be measured from video images of fiber shortening, contractions of single muscle fibers, or absorbance changes at the isobestic point of Fura-2 (260 nm). A distinct advantage of the larval muscle preparation is that measurements 1, 2, and 4 can be performed simultaneously for a single fiber.