BILE ACIDS AND HEPATOCELLULAR SIGNAL TRANSDUCTION

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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.