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Momohydroxylated bile acids, including taurolitocholate (TLC) and its 3-sulphate (TLC-S), have been shown to increase [Ca]i in cytosol of rat hepatocytes [1, 2]. These bile acids mobilize Ca2+ from the internal pool which is sensitive to inositol trisphosphate (IP3). However, bile-acid mediated Ca2+ release is independent of IP3 production. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a nucleotide which can to release calcium from specific type of intracellular store defined as endo-lysosomal system or acidic store. The aim of this study was to examine influence of NED-19 (antagonist of NAADP) on TLC-S-induced change of calcium content in cytosol of and endoplasmic reticulum of isolated mice hepatocytes in order to elucidate the role of acidic store in bile-acid mediated Ca2+ release. Isolated hepatocytes of mice were loaded with fluo-4 (2.5 μM). Fluorescent images were obtained using Leica SP2 MP dual two-photon confocal microscope. Isolated hepatocytes were permeabilized in suspension with saponine (0.1 mg/mL). Next the permeabilized suspension of hepatocytes was loaded with Mag-Fura-2 AM (5 μM). Measurement of Ca2+ content in store of permeabilized cells was conducted using spectrofluorimetric method. We confirmed that TLC-S (50, 100 and 200 μM) elicited cytosolic Ca2+-signals, which were not inhibited by the IP3-receptors (IP3Rs) antagonist 2-APB (100 μM). In suspensions of permeabilized murine hepatocytes TLC-S (100 μM) mobilized 66.10 ± 8.87 % of the total stored calcium as detected by ionomycin-induced release (10 μM). After application of TLC-S thapsigargin could release only 47.94 ± 3.05 %. Previous addition of NED-19 (100 nM) decreased fraction of calcium that is released by TLC-S and equals 33.25 ± 2.15 % of the total calcium. In this case, the following use of thapsigargin mobilized only 21.75 ± 10.68 %. Thus, previous application of NED-19 significantly (n = 6; P ≤ 0.01) reduced the proportion of calcium released by TLC-S 2-fold. It was observed that the rate of TLC-S-induced decrease of calcium content in the intracellular store was 1.8 times slower than after application of NED-19 (n = 6; Р ≤ 0.05). Previous application of NED-19 increased the rate of thapsigargin-evoked calcium content reduction by a factor of 2.5 (n = 6; Р ≤ 0.01). We suggest the impact of acid store in TLC-Selicited cytosolic Ca2+-signals in mice hepatocytes. Thus, the mechanism of TLC-S-induced calcium release is also NAADP-mediated.

Influence of taurolithocholate 3-sulphate on calcium content in cytosol and store of isolated mice hepatocytes