Bafilomycin A1 Molecular Effect on ATPase Activity of Subcellular Fraction of Human Colorectal Cancer and Rat Liver.

Bafilomycin A1 inhibits V-type H+ ATPases on the molecular level, which acidifies endo-lysosomes. 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.

ATPase Activity of the Subcellular Fractions of Colorectal Cancer Samples under the Action of Nicotinic Acid Adenine Dinucleotide Phosphate.

In tumor cells with defects in apoptosis, autophagy allows prolonged survival. Autophagy leads to an accumulation of damaged mitochondria by autophagosomes. An acidic environment is maintained in compartments of cells, such as autophagosomes, late endosomes, and lysosomes; these organelles belong to the "acid store" of the cells. Nicotinic acid adenine dinucleotide phosphate (NAADP) may affect the release of Ca2+ from these organelles and affect the activity of Ca2+ ATPases and other ion transport proteins. Recently, a growing amount of evidence has shown that the variations in the expression of calcium channels or pumps are associated with the occurrence, disease-presentation, and the prognosis of colorectal cancer. We hypothesized that activity of ATPases in cancer tissue is higher because of intensive energy metabolism of tumor cells. The aim of our study was to ascertain the effect of NAADP on ATPase activity on tissue samples of colorectal cancer patients' and healthy individuals. We tested the effect of NAADP on the activity of Na+/K+ ATPase; Ca2+ ATPase of endoplasmic reticulum (EPR) and plasma membrane (PM) and basal ATPase activity. Patients' colon mucus cancer samples were obtained during endoscopy from cancer and healthy areas (control) of colorectal mucosa of the same patients. Results. The mean activity of Na+/K+ pump in samples of colorectal cancer patients (n = 5) was 4.66 ± 1.20 µmol Pi/mg of protein per hour, while in control samples from healthy tissues of the same patient (n = 5) this value was 3.88 ± 2.03 µmol Pi/mg of protein per hour. The activity of Ca2+ ATPase PM in control samples was 6.42 ± 0.63 µmol Pi/mg of protein per hour and in cancer -8.50 ± 1.40 µmol Pi/mg of protein per hour (n = 5 pts). The mean activity of Ca2+ ATPase of EPR in control samples was 7.59 ± 1.21 µmol Pi/mg versus 7.76 ± 0.24 µmol Pi/mg in cancer (n = 5 pts). Basal ATPase activity was 3.19 ± 0.87 in control samples versus 4.79 ± 1.86 µmol Pi/mg in cancer (n = 5 pts). In cancer samples, NAADP reduced the activity of Na+/K+ ATPase by 9-times (p < 0.01) and the activity of Ca2+ ATPase EPR about 2-times (p < 0.05). NAADP caused a tendency to decrease the activity of Ca2+ ATPase of PM, but increased basal ATPase activity by 2-fold vs. the mean of this index in cancer samples without the addition of NAADP. In control samples NAADP caused only a tendency to decrease the activities of Na+/K+ ATPase and Ca2+ ATPase EPR, but statistically decreased the activity of Ca2+ ATPase of PM (p < 0.05). In addition, NAADP caused a strong increase in basal ATPase activity in control samples (p < 0.01). Conclusions: We found that the activity of Na+/K+ pump, Ca2+ ATPase of PM and basal ATPase activity in cancer tissues had a strong tendency to be higher than in the controls. NAADP caused a decrease in the activities of Na+/K+ ATPase and Ca2+ ATPase EPR in cancer samples and increased basal ATPase activity. In control samples, NAADP decreased Ca2+ ATPase of PM and increased basal ATPase activity. These data confirmed different roles of NAADP-sensitive "acidic store" (autophagosomes, late endosomes, and lysosomes) in control and cancer tissue, which hypothetically may be connected with autophagy role in cancer development. The effect of NAADP on decreasing the activity of Na+/K+ pump in cancer samples was the most pronounced, both numerically and statistically. Our data shows promising possibilities for the modulation of ion-transport through the membrane of cancer cells by influence on the "acidic store" (autophagosomes, late endosomes and lysosomes) as a new approach to the treatment of colorectal cancer.

Peculiarities of the effects of bile acids on atpase activity of the colon mucosa in patients with overweight and irritable bowel syndrome.

OBJECTIVE: The aim 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). PATIENTS AND METHODS: Materials and methods: Completely examined 12 patients with IBS and overweight.We estimated the ATPase activity of colon mucous of the patients with IBSspectrophotometrically 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. RESULTS: Results: We establishedthe specific activities of Na+/K+-ATPase, Ca2+-ATPase of EPR,Ca2+-ATPase of PM and basal Mg2+-ATPase. Therewere(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 causedany change of Na+/K+-ATPase , as well as Ca2+-ATPasesactivities, but statistically significant increased activity of Mg2+-ATPase of postmitochondrial subcellular fraction of colon mucous of the patients with IBS by 4 fold. CONCLUSION: 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.

A new method of diagnosis of the hiatal hernia associated with gastroesophageal reflux disease.

OBJECTIVE: Introduction: Gastroesophageal reflux disease (GERD) is one of the most common gastroduodenal diseases. The relationship between the hiatal hernia and the GERD is established. It is advisable to develop an accessible non-invasive diagnostic method for this combined pathology. The aim of the research was to estimate measuring of calcium in patients' saliva samples as simple non-invasive diagnostic method of GERD associated with the hiatal hernia. PATIENTS AND METHODS: Materials and methods: The samples of saliva were obtained from 37 patients with hiatal hernia associated with GERD and 22 healthy volunteers. The content of calcium in saliva was measured using calcium-sensitive dye Arsenazo III by photometrical method at a wavelength of 590-650 nm. RESULTS: Results: It has been established that in the saliva of patients with hiatal hernia, the calcium content was increased by 100.9% compared to the control group. Such a significant increase in the level of calcium in the saliva of patients with hiatal hernia may be due to the fact that the development of this pathology is a disorder of calcium homeostasis. CONCLUSION: Conclusions: It has been found that the calcium content in the saliva of patients with hiatal hernia exceeded the norm almost twice. Thus, the determination of calcium content in saliva can be used as a simple non-invasive diagnostic marker of hiatal hernia associated with GERD.

[Features of exchange of calcium saliva in patients with gastroesophageal reflux disease].

OBJECTIVE: Introduction: The pathogenesis of gastroesophageal reflux disease (GERD) is a topical issue of modern gastroenterology. There are a number of scientific papers on changes in the qualitative and quantitative composition of saliva, which triggers a cascade of biochemical reactions, the consequence of which is the destruction of the resistance of the mucous membrane of the esophagus. Calcium is a macro element that provides the normal functioning of cells. Parathormone also regulates the metabolism of calcium in the body. The aim: To study the level of calcium of saliva in patients with GERD before and after 6 months after treatment, to investigate the correlation with the concentration of parathyroid hormone. PATIENTS AND METHODS: Materials and methods: The samples of saliva of patients with GERD before and after treatment are analyzed. The content of calcium in saliva was determined by photometric method. To test the level of parathyroid hormone, the Intact-PTH ELISA test was used. The treatment was carried out with pantoprazole doses of 40 mg per day. Normal values of electrolyte levels in human saliva are set on 10 virtually healthy volunteers. Data processing was carried out using Microsoft Excel. Correlations were calculated using the Pearson method. RESULTS: Results: 25 samples of saliva have been analyzed: 15 patients with GERD and 10 healthy individuals. Concentration of parathormonone in plasma was consistent with normal values. The concentration of calcium saliva for practically healthy individuals was 2.48 ± 0.07 mmol / L, in patients with GERD before treatment 1.92 ± 0.16 mmol / L, after treatment 2.04 ± 0.07 mmol / L. The correlation coefficient between the level of calcium salivation in patients with GERD and the plasma parathyroid hormone concentration before treatment was 0.21, after treatment 0.73. CONCLUSION: Conclusions: The concentration of calcium is statistically significantly reduced in patients with GERD by 22.5% and increased by 10% after treatment. There is a high correlation between the concentration of calcium after treatment and the level of plasma parathyroid hormone.

[Features of the course of irritable bowel syndrome in patients with excess body weight].

OBJECTIVE: Introduction: Currently, the diagnosis of irritable bowel syndrome (IBS) is only based on clinical symptoms without clear interpretation and can be interpreted differently, complicating the diagnosis. Therefore, to study features of the prevalence and the course of IBS in patients with excessive body weight / obesity is of particular significance nowadays. The aim of the research is to evaluate the prevalence and the course of IBS in patients with obesity. PATIENTS AND METHODS: Materials and methods: Observation results for 120 obese persons who underwent in-patient treatment at various hospital departments in Western Ukraine have been analyzed to evaluate the prevalence of IBS. A thorough collection of complaints, anamnesis of the disease and lifestyle were conducted due to the survey; objective survey data were analyzed. The diagnosis of IBS was made according to Rome IV diagnostic criteria (2016). RESULTS: Results: Patients of all groups were gender and age-matched participants. The average body mass index was 33.9 ± 0.83 kg / m². Excessive body weight was revealed in 6 patients (5.0%). Degrees of obesity: (stage I) was diagnosed in 74 patients (61.6%), stage II was determined in 29 patients (24.2%), and stage III - in 11 patients (9,2%). The vast majority of patients (82.5%) complained of fullness or discomfort in the abdomen. Every second obese patient (52.5%) noted periodic bloating. Periodic nausea was recorded in every third patient with obesity (32.5%). Recurrent abdominal pain was determined in 62 (51.7%) of patients during the last 3 months: in females - 38 (61.3%), in males - 24 (38.7%) respectively. The prevalence of diarrhea with IBS compared to IBS with constipation was also noted: 36 (58.1%) versus 26 (41.9%) of cases. CONCLUSION: Conclusions: Irritable bowel syndrome was revealed in 51.7% of obese patients. Significant prevalence of combined pathology was noted in females. The prevalence of irritable bowel syndrome with diarrhea was determined.