BILE ACIDS AND HEPATOCELLULAR SIGNAL TRANSDUCTION

主要研究者的拨款建议是基于以下方面的重要作用 肝细胞钙稳态在维持肝功能。 静息状态下细胞溶质钙浓度约为100 nM 肝细胞，而血清，胆汁和细胞器钙 浓度分别为约2- 3 mM、2- 7 mM和2- 3 mM。增加 细胞质钙离子已被证明参与调节 蛋白质和DNA合成、糖原分解和细胞生长。最近， 已经提出钙参与胆汁酸代谢， 包括胆汁酸的摄取和分泌。此外，几种胆汁酸 已显示增加肝细胞胞质钙。的目标 本研究拟研究不同胆汁酸对大鼠肝组织的影响， 对肝细胞钙稳态的影响以及 不同钙池的钙流量对肝细胞的影响 代谢，特别是钙和胆汁酸的释放，以及对 胆汁酸摄取PI提出的第一个目标的目标是 在分离的金黄叙利亚仓鼠肝细胞模型中， 不同的途径参与调节钙稳态， 胆汁酸更确切地说：1）所示化合物的各自效力 胆汁酸对钙的动员，以及机制， 将研究哪种胆汁酸诱导细胞内钙流; 2)钙被动员的细胞内位点将是 特别强调联合国系统发挥的作用 内质网和线粒体; 3）胆汁分泌的机制 将检查酸诱导钙内流或/和钙外流; 最后，4）通过测试以下物质的作用来评估细胞特异性： 胆汁酸对培养的人成纤维细胞钙稳态的影响， 没有胆汁酸转运体第二个目标的目标是 在灌注的肝脏中测定胆汁酸对钙的影响， 胆汁酸通量，以及胆汁流量：1）胆汁酸对 将研究钙释放到胆汁和血流中; 2)将胆汁分泌钙与胆汁分泌钙进行比较 酸和胆汁流速。最后一个目标的目标是定义 钙对离体大鼠胆汁酸摄取的调节作用 肝细胞：1）钙动员激素和钙的能力 将确认改变肝细胞胆汁酸摄取的离子载体; 2） 细胞内外钙离子对胆汁作用 酸吸收将被确定; 3）钙对两者的具体影响 将探索钠依赖性和非钠依赖性胆汁酸摄取; （4）胆汁酸摄取抑制的调节机制 钙将被调查。预计，通过更好的 了解不同胆汁酸对 肝细胞钙稳态，我们将获得一个更清晰的认识， 代谢后果的机制， 胆汁酸对肝细胞钙稳态的影响， 酸分泌和胆结石形成有关。

The grant proposal of the PI is based on the significant role of hepatocellular calcium homeostasis in the maintenance of liver function. The cytosolic calcium concentration is around 100 nM in resting hepatocytes, while the serum, biliary and intracellular organelle calcium concentrations are around 2-3mM, 2-7mM and 2-3mM, respectively. Increased cytosolic calcium has been shown to be involved in the regulation of protein and DNA synthesis, glycogen breakdown and cell growth. Recently, calcium has been proposed to he involved in bile acid metabolism, including bile acid uptake and secretion. In addition, several bile acids have been shown to increase hepatocellular cytosolic calcium. The goal of this research proposal is to study the effects different bile acids exert on hepatocellular calcium homeostasis and the consequent effect that the flux of calcium from the different calcium pools has on hepatocellular metabolism, in particular, on calcium and bile acid release, as well as on bile acid uptake. The goal of the first aim proposed by the PI is to define, in the isolated Golden Syrian hamster hepatocyte model, the different pathways involved in the regulation of calcium homeostasis by bile acids. More precisely: 1) the respective potency of the indicated bile acids on the mobilization of calcium, as well as the mechanism by which bile acids induce intracellular calcium fluxes will be investigated; 2) the intracellular site from which the calcium is mobilized will be explored, with special emphasis given to the role played by the endoplasmic reticulum and the mitochondria; 3) the mechanism by which bile acids induce calcium influx or/and calcium efflux will be examined; finally 4) the cell specificity will be assessed by testing the effect of bile acids on calcium homeostasis in cultured human fibroblasts, which do not possess a bile acid transporter. The goal of the second aim is to determine, in the perfused liver, the effect of bile acids on calcium and bile acid fluxes, as well as on bile flow: 1) the effect of bile acids on the release of calcium into bile and the bloodstream will be investigated; 2) the biliary secretion of calcium will be compared to that of bile acids, and to the bile flow rate. The goal of the last aim is to define the effect of calcium on the regulation of bile acid uptake in isolated hepatocytes: 1) the ability of calcium-mobilizing hormones and calcium ionophores to alter hepatocellular bile acid uptake will be confirmed; 2) the respective effect of intracellular and extracellular calcium on bile acid uptake will be determined; 3) the specific effect of calcium on both sodium-dependent and sodium-independent bile acid uptake will be explored; finally, 4) the mechanism(s) regulating the inhibition of bile acid uptake by calcium will be investigated. It is expected that, through a better understanding of the differential effects of different bile acids on hepatocellular calcium homeostasis, we will gain a clearer perception of the mechanism of the metabolic consequences that the modification of hepatocellular calcium homeostasis by bile acids may have, as far as bile acid secretion and gallstone formation are concerned.