INFLUENCE OF TAUROLITHOCHOLATE 3-SULPHATE ON CALCIUM CONTENT IN CYTOSOL AND STORE..

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.


INTRODUCTION
Bile salts are synthesized from cholesterol in liver and represent the main driving force of the bile flow. Bile is crucial for intestinal absorption of fats and fat-soluble vitamins, as well as the elimination of excess cholesterol and waste products from body [3]. Previous work has shown that application of bile acids can cause the increase in the levels of cytosolic [Ca 2+ ] i in hepatocytes [1,4].
Specifically, bile acids activate calcium entry into the cells and cause depletion of internal calcium store [5]. Other effects, not linked to calcium signaling, have also been observed, including the increase in intracellular Na + concentration [6] and depolarization of inner mitochondrial membrane. [7].
In acinar pancreatic cells, it was also shown that bile acids can release calcium from both ER and acidic stores in secretory granular areas. In both stores TLC-S interacts with both the IP 3 Rs and the RyRs. TLC-S opens the RyRs through activation of NAADP [8]. In hepatocytes, it is still unclear if NAADP-sensitive acidic store is involved in TLC-S-induced Ca 2+ -signals. Therefore, the main purpose of this study was to examine such possibility.

MATERIALS AND METHODS
Isolation of hepatocytes. CD-1 male mice were humanely sacrificed in compliance with the provisions of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 1985) and in accordance with International Convention for the Protection of Animals. The protocol for hepatocyte isolation was as described in [9]. Isolated liver was perfused with buffer I without Ca 2+ : 140 mM NaCl; 4.7 mM KCl; 10 mM HEPES; 10 mM D-glucose; 100 µM EGTA; pH 7.4; the rate of perfusion was 5 mL/min at 37 °C. Next the liver was perfused with buffer I in the presence of 1.3 mM CaCl 2 and collagenase I (Worthington) for 10 min at 37 °C. Dissociated hepatocytes were centrifuged at 50 × g for 1 min and then transferred into buffer I containing 1 mM MgCl 2 and 1.3 mM CaCl 2 , pH 7.4.
Fluorescent [Ca 2+ ] measurement. After isolation, the cells were loaded with low affinity Ca 2+ -sensitive dye fluo-4 (2.5 µM) for 30-45 minutes at 36.5 °C. Cells were attached to poly-L-lysine-coated coverslips in flow chamber. All experiments were performed at room temperature.
Fluorescent images were obtained using Leica SP2 MP dual two-photon confocal microscope with a × 63 1.2 NA objective. For fluo-4 excitation and emission wavelengths were 488 nm (argon ion laser, 3 % power) and 510-590 nm, respectively. Fluorescent images were collected with frequency of 0.6-1.0 frame/second. Fluorescence signals were plotted as F/F 0 , with F as fluorescence during the experiment and F 0 as the initial level of fluorescence.
Measurement of [Ca 2+ ] content in store of permeabilized cells. Suspension of permeabilized hepatocytes (2 × 10 6 ) was used to load with fluorescent dye Mag-Fura-2 AM (5 µM). The dye was washed out before permeabilization. Isolated hepatocytes were permeabilized with saponine (0.1 mg/mL) in intracellular solution for 10 min. Cells were later washed with an intracellular solution based on K-HEPES, containing 20 mM NaCl; 127 mM KCl; 1.13 mM MgCl 2 ; 0.05 mM CaCl 2 ; 0.1 mM EGTA; 10.0 mM HEPES (KOH); 5 µg/mL oligomycine; 1 µg/mL rotenone; 2.0 mM АТP; рН 7.0. 2 mL of cell suspension were transferred to the spectrofluorometer cuvette. The fluorescence of Mag-Fura-2 AM was monitored using excitation wavelength 340-380 nm with emission at 500 nm. Cellular calcium content that was mobilized by 10 µM ionomycin was accepted as 100 % and represents the total amount of Ca 2+ within the internal pool.

TLC-S-induced Responses in the Intact Hepatocytes.
We have shown that TLC-S (50, 100 and 200 µM) elicited cytosolic Ca 2+ -signals, consistent with the previous findings described early [1,2]. A typical trace with repeated application of different concentration of TLC-S is shown in Fig.1. A and B. TLC-S (200 µM) induced calcium elevation in the cytosol of intact hepatocytes comparably half the size of 10 µM ATP effect ( Fig.1, B). After TLC-S-elicited Ca 2+ -signal takes place hepatocytes can answer to ATP but this signal has smaller amplitude yet longer plateau phase (Fig.1, B).
So, we investigated whether inhibitors of these channels influence the TLC-S-eli cited Ca 2+ release. We tested 2-aminoethyldiphenyl borate (2-APB) as the inhibitor of the IP 3 Rs. It was revealed that TLC-S-induced Ca 2+ -signals were not inhibited by the IP 3 Rs antagonist 2-APB (100 µM) (Fig. 1, C).
Monitoring of TLC-S action on the Ca 2+ storage organelles in suspension of permeabilized hepatocytes. In suspensions of murine hepatocytes, TLC-S (100 µM) mobilizes 66.10 ± 8.87 % of the total stored calcium released by ionomycin (10 µM). In this experiment, after exposure to TLC-S thapsigargin can release 47.94 ± 13.05 % of the total stored calcium. A typical trace showing the effect of bile acid on Mag-Fura-2 (5 µM) (F/F 0 ) in intracellular store of hepatocytes is shown on Fig.2

, A.
NAADP is the most potent Ca 2+ -mobilizing agent identified to date that acts in various cell types across phyla. It was shown to selectively target the lysosome-related organelles rich in Ca 2+ and H + and therefore called acidic Ca 2+ -stores. In hepatocytes they are presented as endo-lysosomal system of the cell [16]. There are many hypotheses about the mechanisms of NAADP action. Much evidence suggests that NAADP induces small yet localized cytoplasmic Ca 2+ -signals subsequently amplified into regenerative global Ca 2+ -signals by recruitment of endoplasmic reticulum via calcium-induced calcium release (CICR) [17]. The actual data collected on the NAADP-receptors remain disputable. The potential NAADP-sensitive Ca 2+ -channels candidates include TRPML1, TRPM2, TPCs and even RyRs [16,17]. In order to investigate the effects of NAADP in the cell, there was synthesized the selective antagonist of NAADP -NED-19. This small molecule is cell-permeable and fluorescent derivative of tryptophan. NED-19 is a powerful noncompetitive inhibitor of NAADP-binding process. It is also able to label the NAADP-receptors in intact cells and effectively block the NAADP-induced Ca 2+ -release. Thus, NED-19 is commonly used for studies of NAADP-mediated events [18]. We have found previously that NAADP triggered changes in stored Ca 2+ were completely abolished by NED-19 as antagonist of NAADP in permeabilized rat hepatocytes [15]. Time, s C After application of NED-19 (100 nM) fraction of calcium that was released by TLC-S decreases and made up only 33.25 ± 2.15 % of the total calcium released by ionomycin. In this case, the following use of thapsigargin mobilizes only 21.75 ± 10.68 % of the stored calcium in suspension of mice hepatocytes (Fig. 2, B). Thus, the previous application NED-19 significantly (n = 6; Р ≤ 0.01) reduced the proportion of calcium released by TLC-S 2-fold. We also calculated the velocity of calcium store emptying by TLC-S and thapsigargin in control and after previous application of NED-19. It was established that the rate of TLC-S-induced reduction of calcium level in the intracellular stores was 2-fold slower than after application of NED-19 (n = 6; Р ≤ 0.05). The same results were observed on thapsigargin-elicited calcium content decrease in endoplasmic reticulum -previous 100 µM TLC-S induce Ca 2+ release from intracellular store, the subsequent application of thapsigargin leads to depletion of Ca 2+ -store and the next adding of ionomycin releases residual Ca 2+ ; (B) NED-19 substantially reduces TLC-S-evoked Ca 2+ release and increases the rate of store emptying Рис. 2. Вплив TLC-S на вміст депонованого Са 2+ у пермеабілізованих гепатоцитах, навантажених magfura-2: (А) TLC-S у концентрації 100 мкмоль/л спричиняє вивільнення Са 2+ з депо, подальше застосування тапсигаргіну зумовлює спустошення останнього, а подальше використання іономіцину вивільняє залишковий Са 2+ ; (В) NED-19 суттєво пригнічує TLC-S-індуковане вивільнення Са 2+ з депо та підвищує швидкість його спустошення application of NED-19 increased its rate by 2.5 fold (n = 6; Р ≤ 0.01). We speculate that the rise in velocity of TLC-S-and thapsigargin-induced calcium release from endoplasmic reticulum after previous application of NED-19 is caused by destruction of the contact sites between NAADP-sensitive acidic stores and endoplasmic reticulum. We assume that the acidic Ca 2+ -store is important for refilling endoplasmic reticulum with calcium.