Regulation of membrane transport by cardiolipin

心磷脂(CL)是一种重要的线粒体膜磷脂，是产生ATP所必需的。在其合成后，CL被亚油酸重塑，在他法津的作用下形成四氢呋喃油酰心磷脂(L4-CL)。CL的减少和/或其脂肪酸组成的改变会导致产生ATP的能力降低。我们假设，合适的CL含量和脂肪酸组成是通过生物膜进行最佳溶质转运和跨细胞转运所必需的。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的转运。通过siRNA敲除CL合成酶(HCLS1)可以降低hCMEC/D3细胞中的CL水平。测定对照组和CL降低细胞的线粒体能量代谢，检测溶质、药物外排转运体、脂肪酸转运体以及紧密连接蛋白和炎症标志物的水平和转运能力。将检测放射性标记脱氧葡萄糖、放射性标记脂肪酸和罗丹明800的膜通透性和摄取率。我们预计，CL水平降低的hCMEC/D3细胞的线粒体功能，包括基础氧耗、糖酵解速率、呼吸能力和ATP转换率将降低，而hCMEC/D3细胞CL水平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、油酸盐和罗丹明800的运输。预计在Transwell®平板上培养的hCMEC/D3细胞CL的减少将导致线粒体功能降低，脂肪酸、葡萄糖、肌酸和罗丹明800转运到细胞内的减少。在被hCLS1基因敲除后CL水平降低的细胞中表达hCLS1，并检测上述所有参数，可恢复CL水平。此外，作为推论，在hCMEC/D3细胞中表达hCLS1并检测上述参数后，CL水平将升高。我们预计，适当的CL含量是溶质和药物运输过程正常发挥作用所必需的。具体目标2：确定心磷脂在体内是否调节溶质和药物跨血脑屏障(BBB)的转运。通过在小鼠体内击倒他法津和放射性标记甘露醇的转运和渗透性，全身L4-CL水平将降低，伊万的蓝色染料和罗丹明800的渗透性将从这些动物和对照动物的时间上确定。从脑中分离出微血管段，并测定线粒体功能。此外，具有Gad-DTPA对比度增强的磁共振成像将用于检查体内跨血脑屏障的运输。Tafazzin被击倒的小鼠的L4-CL水平将恢复，并检测上述参数。我们预计Tafazzin将降低微血管毛细血管段线粒体的功能、放射性标记甘露醇和罗丹明800的转运和通透性以及Gad-DTPA的通透性，并可通过恢复正常的L4-CL水平来逆转这种情况。建议的研究将确定重要的转运/调制途径，这些途径可以通过CL的变化来调节线粒体的功能。这些研究将创建一个新的研究计划，着眼于描述溶质和药物膜运输的特征，并确定通过CL介导的线粒体功能如何影响这一点，并有可能帮助药物开发调节膜功能。这些生物学研究在药物开发中具有重要意义。

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.