Colorectal cancer is influenced by genetic mutations, lifestyle factors, and diet, particularly high fat intake, which raises bile acid levels in the intestinal lumen. This study hypothesized that bile acids contribute to tumorigenesis by disrupting ion transport and ATPase activity in the intestinal mucosa. The effects of 3-sulfo-taurolithocholic acid (TLC–S) on ATPase activity were investigated in colorectal cancer samples from 10 patients, using adjacent healthy tissue as controls, and in rodent liver function. ATPase activity was measured spectrophotometrically by determining inorganic phosphorus (Pi) in postmitochondrial fractions. Ca2+ dynamics were assessed in isolated mouse hepatocytes with fluorescence imaging, and rat liver mitochondria were studied using polarographic methods to evaluate respiration and oxidative phosphorylation. TLC–S increased Na+/K+ ATPase activity by 1.5 times in colorectal cancer samples compared to controls (p ≤ 0.05). In healthy mucosa, TLC–S decreased Mg2+ ATPase activity by 3.6 times (p ≤ 0.05), while Mg2+ ATPase activity in cancer tissue remained unchanged. TLC–S had no significant effect on Ca2+ ATPase activity in healthy colon mucosa but showed a trend toward decreased activity in cancer tissue. In rat liver, TLC–S decreased Ca2+ ATPase and Na+/K+ ATPase activities while increasing basal Mg2+ ATPase activity (p ≤ 0.05). Additionally, TLC–S induced cytosolic Ca2+ signals in mouse hepatocytes, partially attenuated by NED-19, an NAADP antagonist (p ≤ 0.05). TLC–S also reduced the V3 respiration rate of isolated rat liver mitochondria during α-ketoglutarate oxidation. These findings suggest that TLC–S modulates ATPase activity differently in cancerous and healthy colon tissues, playing a role in colorectal cancer development. In rat liver, TLC–S affects mitochondrial activity and ATPase function, contributing to altered cytosolic calcium levels, providing insight into the mechanistic effects of bile acids on colorectal cancer and liver function.

Bafilomycin A1 inhibits V-type H+ ATPases on the molecular level, which acidifies endolysosomes. The main objective of the study was to assess the effect of bafilomycin A1 on Ca2+content, NAADP-induced Ca2+ release, and ATPase activity in rat hepatocytes and human colon
cancer samples. Chlortetracycline (CTC) was used for a quantitative measure of stored calcium in permeabilized rat hepatocytes. ATPase activity was determined by orthophosphate content released after ATP hydrolysis in subcellular post-mitochondrial fraction obtained from rat liver as well as from patients’ samples of colon mucosa and colorectal cancer samples. In rat hepatocytes, bafilomycin A1 decreased stored Ca2+ and prevented the effect of NAADP on stored Ca2+. This effect was dependent on EGTA–Ca2+ buffers in the medium. Bafilomycin A1 significantly increased the activity of Ca2+ATPases of endoplasmic reticulum (EPR), but not plasma membrane (PM) Ca2+ ATPases in rat liver.
Bafilomycin A1 also prevented the effect of NAADP on these pumps. In addition, bafilomycin A1 reduced Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in the subcellular fraction of rat liver. Concomitant administration of bafilomycin A1 and NAADP enhanced these effects. Bafilomycin A1 increased the activity of the Ca2+ ATPase of EPR in the subcellular fraction of normal human colon mucosa and also in colon cancer tissue samples. In contrast, it decreased Ca2+ ATPase PM activity in samples of normal human colon mucosa and caused no changes in colon cancer. Bafilomycin A1 decreased Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity
in normal colon mucosa samples and in human colon cancer samples. It can be concluded that bafilomycin A1 targets NAADP-sensitive acidic Ca2+ stores, effectively modulates ATPase activity, and assumes the link between acidic stores and EPR. Bafilomycin A1 may be useful for cancer therapy.
Keywords: molecular mechanisms; colon cancer; ATPase; autophagy; hepatocytes; liver; NAADP; biomarkers; bafilomycin A1; Ca2+ store 

UDC 577.352:616-006.44:542.978

Endo-lysosomal system through the process of autophagy 
is involved in the pathogenesis of many diseases. Acidification of these organelles is carried out by V-type H+-ATPases, which is inhibited by bafilomycin A1. Endosomes and lysosomes are also important Ca2+-storage in a cell. Nіcotіnіc acіd adenіne dіnucleotіde phosphate (NAADP) releases Cа2+ from endo-lysosomes. The main purpose of the study was to found out the effect of bafilomycin A1 and NAADP on stored Ca2+ and on the ATPase activity of rat hepatocytes. The stored Ca2+ was estimated using chlorotetracycline in permeabilized hepatocytes of rats. ATPase activity was determined by level of orthophosphate spectrophotometrically. It was found that bafilomycin A1 reduces stored Ca2+ in permeabilized hepatocytes of rats in the micromolar range of concentration (20 and 0.04 mkM) and averted the effect of NAADP on calcium content. Lower concentrations of bafilomycin A1 (0.001 mkM) did not alter the content of stored calcium, but prevented the influence of NAADP in permeabilized hepatocytes of rats. In the subcellular fraction of rat liver bafilomycin A1 (0.001 mkM) increased Ca2+-ATPase and basal Mg2+-ATPase activities and reduced Na+/K+-ATPase activity. Preincubation of the subcellular fraction with bafilomycin A1 completely averts any changes in the activity of estimated ATPases by means of NAADP. It was concluded that the bafilomycin-sensitive store in hepatocytes of rats is NAADP-sensitive endo-lysosomal Ca2+-store. Using of bafilomycin A1 may be useful in treating autophagy-depended diseases. 

Метою є дослідження впливу жовчних кислот на активність АТФаз слизової оболонки товстої кишки у пацієнтів із надмірною вагою та СПК.

Матеріали та методи. Комплексно обстежено 12 пацієнтів із СПК та надмірною вагою. Діагноз СПК встановлювали згідно Римських критеріїв ІV (2016). Для виключення запальної патології кишечника проводили CITO TEST Calprotectin-Lactoferrin. Індекс маси тіла розраховували за формулою Кеттле. Визначали активність АТФази слизової оболонки товстої кишки хворих на СПК спектрофотометрично, визначивши вміст ортофосфату (Фн), який вивільнився після гідролізу АТФ. Ми вивчали вплив 3-сульфату тауролітохолату (TLC-S) на специфічну активність Na+/K+-АТФази, Ca2+-АТФази ендоплазматичного ретикулума (ЕПР), Ca2+-АТФази плазматичної мембрани (ПМ) та базальної Mg2+-АТФази постмітохондріальної субклітинної фракції слизової ободової кишки хворих на СПК.

Результати досліджень. Ми виміряли специфічну активність Na+/K+-АТФази, Ca2+-АТФази ЕПР та ПМ і базальну Mg2+ -АТФазу, які становили (6,06 ± 1,61), (5,88 ± 1,19), (8,86 ± 1,56) (6,44 ± 2,02) мкмоль Фн/мг білка на годину, відповідно. TLC-S (50 мкМ) не спричиняв будь-якої зміни Na+/K+- та Ca2+ -АТФазної активності, але у 4 рази статистично значущо підвищував активність Mg2+-АТФази постмітохондріальної субклітинної фракції слизової оболонки товстої кишки хворих на СПК.

Висновки. TLC-S збільшував активність базальної Mg2+-АТФази у постмітохондріальній фракції слизової ободової кишки пацієнтів із надмірною вагою та СПК, але не впливав на активність Na+/K+-АТФази та Ca2+-АТФази. Висловлено припущення, що активація базальної Mg2+-АТФази за дії TLC-S може вказувати на роль ендо-лізосомальної системи епітеліоцитів слизової оболонки товстої кишки у розвитку патології СПК.

The purpose of the work is to investigate the effect of bile acids on the ATPase activity of the colon mucosa in patients with overweight and irritable bowel syndrome (IBS).

Materials and methods. Completely examined 12 patients with IBS and overweight. We estimated the ATPase activity of colon mucous of the patients with IBS spectrophotometrically by determined the content of orthophosphate that was released after ATP hydrolysis. We studied the effect of 3-sulphate of taurolitocholate (TLC-S) on specific activities of Na+/K+-ATPase, Ca2+-ATPase of endoplasmatic reticulum (EPR), Ca2+-ATPase of plasmatic membrane (PM) and basal Mg2+-ATPase of postmitochondrial subcellular fraction of colon mucous of the patients with IBS.

Research results. We established the specific activities of Na+/K+-ATPase, Ca2+-ATPase of EPR, Ca2+-ATPase of PM and basal Mg2+-ATPase. There were (6.06 ± 1.61), (5.88 ± 1.19), (8.86 ± 1.56) (6.44 ± 2.02) μmol Pi/ mg protein per hour, respectively. TLC-S (50 μM) did not caused any change of Na+/K+-ATPase , as well as Ca2+-ATPases activities, but statistically significant increased activity of Mg2+-ATPase of postmitochondrial subcellular fraction of colon mucous of the patients with IBS by 4 fold.

Conclusions. TLC-S increased basal Mg2+-ATPase in the postmitochondrial fraction of colon mucous of the patients with overweight and IBS, but had no effect on Na+/K+-ATPase and Ca2+-ATPases activities. It has been suggested that activation of basal Mg2+-ATPase under by TLC-S may indicates the role of the endo-lysosomal system of epitheliocytes of colon mucous in developing of pathology IBS.

Key words: irritable bowel syndrome, overweight, ATPase, bile acids.