Abstract
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 Ca(2+) content, NAADP-induced Ca(2+) 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 Ca(2+) and prevented the effect of NAADP on stored Ca(2+). This effect was dependent on EGTA-Ca(2+) buffers in the medium. Bafilomycin A1 significantly increased the activity of Ca(2+) ATPases of endoplasmic reticulum (EPR), but not plasma membrane (PM) Ca(2+) 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 Mg(2+) 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 Ca(2+) ATPase of EPR in the subcellular fraction of normal human colon mucosa and also in colon cancer tissue samples. In contrast, it decreased Ca(2+) 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 Mg(2+) ATPase activity in normal colon mucosa samples and in human colon cancer samples. It can be concluded that bafilomycin A1 targets NAADP-sensitive acidic Ca(2+) stores, effectively modulates ATPase activity, and assumes the link between acidic stores and EPR. Bafilomycin A1 may be useful for cancer therapy.