MYOCARDIAL CA2+ TRANSPORT AND METABOLISM

这项建议的长期目标是了解细胞内 心肌细胞内钙离子的稳态。用荧光数字技术研究 成像显微镜(FDIM)检测细胞内钙离子浓度([Ca~(2+)]i) 纸巾。此外，细胞内钙离子的动态性质被放大 通过激活与磷脂相连的肌膜受体 崩溃了。尽管受体介导的钙信号是 尽管研究兴趣浓厚，但人们对其在心脏中的作用知之甚少。 因此，我们将利用几种技术来完成五个具体的 目标：1)完成已在进行的实验工作。2)至 心肌细胞内钙离子浓度空间分布的定量测定 细胞。3)提高FDIM的时间分辨率，以记录 [Ca~(2+)]的时间分布研究α~(2+)的影响 肾上腺素能受体、低亲和力M受体和嘌呤能 受体激活[Ca~(2+)]I)以评估蛋白激酶的作用 C(PKC)参与L型钙通道的调节。大多数实验都将 使用来自豚鼠和大鼠脑室的单个细胞。[Ca~(2+)]i 用FDIM确定。L类钙通道将被分离 全细胞膜片钳的电生理学研究。至相关 [Ca~(2+)]i测量心脏的功能方面， 乳头状肌的细胞内Na+活性和收缩能力将被测量 肌肉。[Ca~(2+)]i的量化将通过测量 Fura-2在两个波长的荧光比值和参考 在“体外”和“体内”条件下获得的校准。这个 计算机将提高成像[Ca2+]i的时间分辨率-- 受控双光束照明系统。要研究两者之间的联系 与磷脂周转和[Ca~(2+)]i偶联的受体 低亲和力的α1肾上腺素能受体激动剂(甲氧胺) M受体(卡巴胆碱)和嘌呤能受体(ATP)将 使用。确定与受体介导有关的钙离子来源 [Ca~(2+)]i变化，药物(如尼群地平对L型钙通道的作用) 抑制细胞内钙离子转运系统将被使用。信号的调制 用佛波醇酯研究PKC介导的L型钙通道， 合成的二酰甘油和纯化的PKC同工酶。这些建议 研究将提供有关细胞内钙稳态的信息 心肌。因为钙离子是心脏功能的关键调节因子。 生理和病理状态，这些研究将拓宽我们的 对正常心脏与异常心脏基本原理的认识 兴奋和收缩。

The long-term objective of this proposal is to understand intracellular Ca2+ homeostasis in cardiac muscle. Studies with fluorescence digital imaging microscope (FDIM) cellular Ca2+ concentration ([Ca2+]i) in numerous tissues. Moreover, the dynamic nature of intracellular Ca2+ is amplified by the activation of sarcolemmal receptors that are linked to phospholipid breakdown. Although receptor-mediated Ca2+ signal is the subject of intense research interest, little is known about its role in heart. Therefore, we will exploit several techniques to accomplish five specific aims: 1) to complete the experimental work already in progress. 2) To determine quantitatively the spatial distribution of [Ca2+]i in cardiac cells. 3) To improve the time resolution of our FDIM for recording the temporal distribution of [Ca2+]i. 4) To investigate the effects of alpha1- adrenergic receptor, low affinity muscarinic receptor and purinergic receptor activation of [Ca2+]i. 5) To assess the role of protein kinase C (PKC) in modulating L-type Ca2+ channels. Most of the experiments will use single cells from guinea pig and rat ventricles. The [Ca2+]i will be determined with FDIM. The L-type Ca2+ channels will be isolated electrophysiologically by the whole-cell patch-clamp. To correlated [Ca2+]i measurements with the functional aspects of the heart, intracellular Na+ activity and contractility will be measured in papillary muscles. The quantification of [Ca2+]i will be achieved by measuring the ratio values of fura-2 fluorescence at two wavelengths and referring to calibrations obtained from "in vitro" and "in vivo" conditions. The temporal resolution of imaging [Ca2+]i will be improved by a computer- controlled dual beam illumination system. To study the link between the receptors that are coupled to phosphoinostitide turnover and [Ca2+]i, agonists for the alpha1-adrenergic receptor (methoxamine), the low affinity muscarinic receptor (carbachol) and the purinergic receptor (ATP) will be used. To identify the sources of Ca2+ responsible for receptor-mediated [Ca2+]i changes, drugs (e.g. nitrendipine for L-type Ca2+ channels) that inhibit cellular Ca2+ - transport systems will be used. The modulation of L-type Ca2+ channels by PKC will be studied by using phorbol esters, synthetic diacylglycerols, and purified PKC isozymes. These proposed studies will provide information about intracellular Ca2+ homeostasis in cardiac muscle. Because Ca2+ is a key regulator for cardiac function in physiological and pathological states, these studies will broaden our understanding on the fundamental principles of normal and abnormal cardiac excitation and contraction.