Regulation of membrane transport by cardiolipin

心磷脂（CL）是产生ATP所需的关键线粒体膜磷脂。在其合成时，CL通过tafazzin酶用亚油酸重塑以形成四氢萘油酰基-心磷脂（L4-CL）。CL的减少和/或其脂肪酸组成的改变导致产生ATP的能力降低。我们推测，适当的含量和脂肪酸组成的CL是需要最佳的溶质转运和跨细胞转运的生物膜。 具体目标1：确定心磷脂是否在体外调节溶质和药物跨细胞膜转运。hCMEC/D3细胞中的CL水平将使用CL合酶（hCLS 1）的siRNA敲低来降低。将测定对照和CL降低细胞中的线粒体能量代谢，并检查溶质、药物外排转运蛋白、脂肪酸转运蛋白以及紧密连接蛋白和炎症标志物的水平和转运能力。将检查放射性标记的脱氧葡萄糖、放射性标记的脂肪酸和罗丹明800的膜渗透性和摄取。我们预计，在CL水平降低的hCMEC/D3细胞中，线粒体功能（包括基础耗氧量、糖酵解速率、呼吸能力和ATP周转率）将降低，并且hCMEC/D3细胞中CL的降低将导致脂肪酸、葡萄糖、肌酸和罗丹明800转运到hCMEC/D3细胞中的减少。我们将在Transwell®平板上培养hCMEC/D3细胞，并测量放射性标记的葡萄糖、肌酸、油酸盐的顶侧至基底侧转运以及罗丹明800的转运。预期在Transwell®板上培养的hCMEC/D3细胞中CL的降低将导致线粒体功能降低，脂肪酸、葡萄糖、肌酸和罗丹明800转运到细胞中减少。CL水平将通过在细胞中表达hCLS 1来恢复，其中CL水平通过hCLS 1敲低降低，并检查所有上述参数。此外，作为推论实验，CL水平将通过在hCMEC/D3细胞中表达hCLS 1而升高，并检查上述参数。我们预计，适当含量的CL是必需的溶质和药物转运过程的正常功能。 具体目标2：确定心磷脂是否在体内调节溶质和药物穿过血脑屏障（BBB）的转运。通过敲低小鼠中的他法菌素降低全身L4-CL水平，并暂时测定这些动物和对照动物中放射性标记甘露醇的转运和渗透性以及伊文思蓝染料和罗丹明800的渗透性。将从脑中分离微血管毛细血管段，并测定线粒体功能。此外，Gad-DTPA对比增强的磁共振成像将用于检查体内穿过BBB的运输。L4-CL水平将在tafazzin被敲低的小鼠中恢复，并检查上述参数。我们预计，线粒体功能的微血管毛细血管段，运输和放射性标记的甘露醇和罗丹明800和Gad-DTPA渗透性的渗透性将减少tafazzin敲低，这可以逆转恢复正常的L4-CL水平。 拟议的研究将确定重要的运输/调节途径，可以通过CL的变化由线粒体功能调节。这些研究将创建一个新的研究计划，其长期愿景是表征溶质和药物膜转运，并确定通过CL介导的线粒体功能如何影响这一点，并可能有助于调节膜功能的药物开发。这些生物学研究在药物开发中具有根本的重要性。

Cardiolipin (CL) is a key mitochondrial membrane phospholipid required for the generation of ATP. Upon its synthesis CL is remodeled with linoleic acid to form tetralinoleoyl-cardiolipin (L4-CL) by the enzyme tafazzin. Reduction in CL and/or alteration in its fatty acid composition results in reduced ability to generate ATP. We hypothesize that the appropriate content and fatty acid composition of CL is required for optimal solute transport and transcellular transport across biological membranes. Specific Objective 1: Determine if cardiolipin regulates solute and drug transport across cell membranes in vitro. CL levels in hCMEC/D3 cells will be lowered using siRNA knock down of CL synthase (hCLS1). Mitochondrial energy metabolism in control and cells with reduced CL will be determined and the level and transport ability of solute, drug efflux transporters, fatty acid transporters as well as tight junction proteins and inflammatory markers will be examined. The membrane permeability and uptake of radiolabeled deoxyglucose, radiolabeled fatty acid and rhodamine 800 will be examined. We anticipate that mitochondrial function including basal oxygen consumption, glycolysis rate, respiratory capacity and ATP turnover will be lower in hCMEC/D3 cells with reduced CL levels and that reduction in CL in hCMEC/D3 cells will result in reduced fatty acid, glucose, creatine and rhodamine 800 transport into hCMEC/D3 cells. We will culture hCMEC/D3 cells on Transwell® plates and measure apical to basolateral transport of radiolabeled glucose, creatine, oleate, and transport of rhodamine 800. It is anticipated that reduction in CL in hCMEC/D3 cells cultured on Transwell® plates will result in reduced mitochondrial function, reduced fatty acid, glucose, creatine and rhodamine 800 transport into cells. CL levels will be restored by expressing hCLS1 in cells in which CL levels were reduced by hCLS1 knock down and all of the above parameters examined. In addition, as corollary experiments, CL levels will be elevated by expressing hCLS1 in hCMEC/D3 cells and the above parameters examined. We anticipate that the appropriate content of CL is required for proper function of solute and drug transport processes. Specific Objective 2: Determine if cardiolipin regulates solute and drug transport across the blood brain barrier (BBB) in vivo. Whole body L4-CL levels will be reduced by knock down of tafazzin in mice and the transport and permeability of radiolabeled mannitol, and Evan’s blue dye and rhodamine 800 permeability will be temporally determined from these and control animals. Microvessel capillary segments will be isolated from brains and mitochondrial function determined. In addition, magnetic resonance imaging with Gad-DTPA contrast enhancement will be used to examine transport across the BBB in vivo. L4-CL levels will be restored in mice in which tafazzin was knocked down and the above parameters examined. We anticipate that mitochondrial function in microvessel capillary segments, transport and permeability of radiolabeled mannitol and rhodamine 800 and Gad-DTPA permeability will be reduced by tafazzin knock down and that this can be reversed by restoration of normal L4-CL levels. The proposed studies will identify important transport/modulation pathways that can be regulated by mitochondrial function through changes in CL. These studies will create a new research program with the long-term vision of characterizing solute and drug membrane transport and determining how mitochondrial function mediated through CL can influence this and potentially aid in drug development for regulation of membrane function. These biological studies are of fundamental importance in drug development.