MYOCARDIAL CA2+ TRANSPORT AND METABOLISM

这项提案的长期目标是了解细胞内的 心肌中的Ca 2+稳态。 荧光数字化研究 图像显微镜（FDIM）细胞Ca 2+浓度（[Ca 2 +]i）在许多 组织中 此外，细胞内Ca 2+的动态性质被放大， 通过激活与磷脂连接的肌膜受体 崩溃 虽然受体介导的Ca 2+信号是研究的主题， 研究兴趣浓厚，但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实现五个具体的 目的：1）完成已在进行的实验工作。 2)到 定量测定心肌[Ca ~（2+）]i的空间分布 细胞 3)为了提高FDIM的时间分辨率， [Ca 2 +]i的时间分布 4)为了研究alpha 1- 肾上腺素能受体、低亲和力毒蕈碱受体和嘌呤能受体 [Ca2+]i. 5)为了评估蛋白激酶的作用 C（PKC）在调节L-型钙通道中的作用。 大多数实验将 使用豚鼠和大鼠心室的单细胞。 [Ca 2 +]i将是 用FDIM确定。 将分离L型Ca 2+通道 通过全细胞膜片钳电生理学。 相关的 [Ca2+]i心脏功能方面的测量， 细胞内Na+活性和收缩性将在乳头状肌中测量。 肌肉. [Ca 2 +]i的定量将通过测量 Fura-2荧光在两个波长处的比值， 从“体外”和“体内”条件获得的校准。 的 将通过计算机提高成像[Ca 2 +]i的时间分辨率- 受控双光束照明系统。 为了研究 与磷酸肌醇周转和[Ca 2 +]i偶联的受体， α 1-肾上腺素能受体激动剂（甲氧胺），低亲和力 毒蕈碱受体（卡巴胆碱）和嘌呤能受体（ATP）将被 采用 为了确定负责受体介导的Ca 2+的来源， [Ca2+]i变化，药物（如尼群地平用于L型钙通道）， 将使用抑制细胞Ca 2 + -转运系统。 的调制 PKC介导的L-型Ca 2+通道将通过使用佛波醇酯进行研究， 合成的二酰基甘油和纯化的PKC同工酶。 这些拟议 研究将提供有关细胞内Ca 2+稳态的信息， 心肌 由于Ca 2+是心脏功能的关键调节因子， 生理和病理状态，这些研究将扩大我们的 了解正常和异常心脏的基本原理 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.