US-Poland Research on Hormonal Control of Calcium Transport Across the Plasma Membrane in Hepatocytes

这项由宾夕法尼亚州立大学好时医学中心的Kathryn F. Lanoue博士和波兰华沙Nencki研究所的Jerzy Duszinski博士共同开展的美国-波兰研究项目，旨在鉴定和表征催化细胞外钙依赖激素流入肝细胞的转运体。研究人员将在原代培养中研究装载fura-2的肝细胞，使用荧光数字成像系统与全细胞膜片钳显微注射技术相结合，以确定假定的钙通道的电流电压关系，以及钙内流的焓及其Ph依赖性。初步实验表明，钙转运到细胞内不依赖于激素在质膜中钙动员受体的占用，也不依赖于细胞内IP3水平，而是依赖于细胞内激素释放钙储存的消耗程度。这些储存的钙的消耗刺激钙以分级的方式进入细胞。研究人员将测试内质网中钙敏感酶的假设，并向细胞质溶胶释放一种代谢物，该代谢物调节质膜钙的通透性。他们将设计实验来证明这种可扩散代谢物的存在并对其进行表征。研究人员计划使用钙质膜转运蛋白抑制剂作为监测携带fura-2的肝细胞质膜囊泡中钙转运蛋白活性的手段。他们将使用囊泡分析系统来筛选可能的调节因子的影响，并作为后期溶解、重构和纯化的基础。这个生物学项目通过使美国和东欧的顶尖专家在共同感兴趣和能力强的领域结合互补的人才和汇集资源，实现了推进科学知识的计划目标。

This U.S.-Poland research project between Dr. Kathryn F. Lanoue of Pennsylvania State University Hershey Medical Center and Jerzy Duszinski of the Nencki Institute of Warsaw, Poland, is to identify and characterize the transporter which catalyzes hormone dependent influx of extracellular calcium into liver cells. The researchers will study fura-2 loaded hepatocytes in primary culture, using a fluorescence digital imaging system in conjunction with whole cell patch clamp microinjection techniques, to determine the current voltage relationships of the putative calcium channel, as well as the enthalpy of calcium influx and its Ph dependence. Preliminary experiments indicate the calcium transport into the cell is not dependent on hormone occupancy of calcium mobilizing receptors in the plasma membrane, or on IP3 levels in the cell but it is dependent on the extent of depletion of intracellular hormone releasable calcium stores. Calcium depletion of these stores stimulates calcium entry into the cell, in a graded manner. The researchers will test the hypothesis that a calcium sensitive enzyme in the endoplasmic reticulum elaborates and releases to the cytosol a metabolite which regulates plasma membrane calcium permeability. They will design experiments to demonstrate the existence of such a diffusible metabolite and to characterize it. The researchers plan to use an inhibitor of the calcium plasma membrane transporter as a means to monitor calcium transporter activity in hepatocyte plasma membrane vesicles loaded with fura-2. They will use the vesicular assay system to screen the influence of putative regulators and as a basis for later solubilization, reconstitution, and purification. %%% This project in biology fulfills the program objective of advancing scientific knowledge by enabling leading experts in the United States and Eastern Europe to combine complementary talents and pool resources in areas of strong mutual interest and competence.