NAADP-sensitive Сa(2+) stores in permeabilized rat hepatocytes

Sv, Bychkova; Ti, Chorna

doi:10.15407/ubj86.05.065

Cited by 5 publications

(14 citation statements)

References 23 publications

(34 reference statements)

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“…The Ca 2 + mobilizing properties of NAADP have been characterized in a variety of cell types from different organisms across phyla underscoring that NAADP is a ubiquitous messenger . Some NAADP‐sensitive pathways were indentified in breast carcinoma cells .…”

Section: Resultsmentioning

confidence: 99%

“…[23][24][25][26] In addition, previous research on permeabilized hepatocytes established that NAADP in concentration of 7 μM significantly decreases the stored calcium concentration. 27 Bafilomycin A1 (Sigma, USA) was added as selective inhibitor H + pumps V-type in concentrations 0.001, 1, and 2 μM. The total ATPase activity of postmitochondrial fraction was calculated by the difference of inorganic phosphorus in the media with different composition (supplemented with bafilomycin A1 or NAADP) expressed as micromoles of inorganic phosphorus equivalent to 1 mg of protein per 1 h. Specific Na + /K + -ATPase activity was calculated as difference of inorganic phosphorus content in medium with or without ouabain (1 mM).…”

Section: Isolation Of Nk/ly Mitochondria and Subcellular Postmitochmentioning

confidence: 99%

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Effect of bafilomycin and NAADP on membrane‐associated ATPases and respiration of isolated mitochondria of the murine Nemeth‐Kellner lymphoma

Hreniukh

Bychkova

Kulachkovsky

et al. 2016

Cell Biochemistry & Function

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Bafilomycin A1 potentiates the effect of NAADP by inhibiting the mitochondrial energetic process in lymphoma cells and activity of Na /K -ATPase. The obtained data show promising possibility to use bafilomycin A1 and NAADP as chemotherapeutic agents for lymphoma cells treatment. This is important because lymphomas are seventh most common form of cancer. Today the lymphoma mortality is 15% to 30%, whereas the effectiveness of malignant neoplasms treatment is less than 50%.

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Section: Resultsmentioning

confidence: 99%

Section: Isolation Of Nk/ly Mitochondria and Subcellular Postmitochmentioning

confidence: 99%

Effect of bafilomycin and NAADP on membrane‐associated ATPases and respiration of isolated mitochondria of the murine Nemeth‐Kellner lymphoma

Hreniukh

Bychkova

Kulachkovsky

et al. 2016

Cell Biochemistry & Function

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“…It was suggesed that the effect of NAADP is dependent on ER luminal calcium in hepatocytes [6]. Besides, it was also assumed that RyRs have been involved in indirect modulation of the NAADP effect via increasing the level of ER luminal calcium [6]. This suggestion predicts presence of the specific membrane contact sites between acidic orga nelles and the ER.…”

Section: Introductionmentioning

confidence: 99%

Influence of NAADP and bafilomycine A1 on activity of ATPase in liver postmitochondrial fraction

Bychkova¹

2015

Biol. Stud.

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Recycling of the endo-lysosomal organelles occur by permanent in the cell. This store is thought to be the acidic store because V-type of H +-ATPase is responsible for their acidification. Bafilomycine A1 is a selective inhibitor of vacuolar H + ATPase, and is often used as anticancer drug. However, it is not clear if applying of bafilomycine A1 is safe for normal cells. Endo-lysosomes are important cellular Ca 2+ storage, sensitive to nicotinic acid adenine dinucleotide phosphate (NAADP). Hepatocytes were shown to have NAADP-sensitive acidic store. But the correlation between acidic store and endoplasmatic reticulum or plasmatic membrane during endo-lysosomal organelles recycling and fusion is still unknown. The main goal of this study was to examine influence of bafilomycine A1 and NAADP on activity of Na + /K +-ATPase, basal Mg 2+-ATPase and Ca 2+-ATPase of plasmatic membrane (PM) and endoplasmatic reticulum (ER) for better understanding of the relationship between acidic organelles and PM/ER. Besides possibility of bafilomycine A1 applying as anticancer drug is examined. All experiments were conducted using rat liver postmitochondrial fraction. It was shown that SERCA activity is increasing under bafilomycine A1 presence, as well as NAADP. At the same time, changes in PMCA activity was not found. Preincubation of rat liver postmitochondrial fraction with bafilomycine A1 completely prevented NAADPinduced increasing of SERCA activity. Bafilomycine A1 decreased activity of Na + /K +-ATPase and amplificated NAADP-induced decreasing of this pump. Applying of NAADP caused more intensive decrease in Na + /K +-ATPase activity after preincubation of this fraction with bafilomycine A1. It was also shown that bafilomycine A1 caused increasing of basal Mg 2+-ATPase activity and NAADP intensify bafilomycine A1-induced changes of function of this pump. These effects are realized due to changes of pH inside the endolysosomal ogranels, acidification of incubation medium, as well as calcium concentration in local contact sites. A conclusion was made that NAADP-sensitive store is bafilomycine A1-sensitive, which also has direct contact sites to ER, but not to PM. Besides, existing of NAADPsensitive, but bafilomycine-insensitive store in this fraction is supposed, which is more likely represented by endosomes. So, applying bafilomycine A1 in anticancer therapy may cause a damage of endo-lysosomal organelles recycling in healthy cells.

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“…Additionally, Zhang's group has shown that NAADP-sensitive Ca 2+ release channel is present in the lysosome of native liver cells [14]. We have also observed NAADPevoked Ca 2+ -release in permeabilized hepatocytes [15]. So, we investigated whether inhibitors of these channels influence the TLC-S-eli cited Ca 2+ release.…”

Section: Resultsmentioning

confidence: 91%

“…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…”

Section: Resultsmentioning

confidence: 99%

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

Bychkova¹

2015

Biol. Stud.

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Momohydroxylated bile acids, including taurolitocholate (TLC) and its 3-sulphate (TLC-S), have been shown to increase [Ca 2+ ] i in cytosol of rat hepatocytes [1,2]. These bile acids mobilize Ca 2+ from the internal pool which is sensitive to inositol trisphosphate (IP 3 ). However, bile-acid mediated Ca 2+ release is independent of IP 3 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 Ca 2+ 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 Ca 2+ -signals, which were not inhibited by the IP 3 -receptors (IP 3 Rs) 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 Ca 2+ -signals in mice hepatocytes. Thus, the mechanism of TLC-S-induced calcium release is also NAADP-mediated.

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NAADP-sensitive Сa(2+) stores in permeabilized rat hepatocytes

Abstract: Nicotinic acid adenine dinucleotide phosphate (NaaDP) is a nucleotide that is potent to release cal-

Cited by 5 publications

References 23 publications

Effect of bafilomycin and NAADP on membrane‐associated ATPases and respiration of isolated mitochondria of the murine Nemeth‐Kellner lymphoma

Effect of bafilomycin and NAADP on membrane‐associated ATPases and respiration of isolated mitochondria of the murine Nemeth‐Kellner lymphoma

Influence of NAADP and bafilomycine A1 on activity of ATPase in liver postmitochondrial fraction

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

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