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 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.
摘要:
巴弗洛霉素A1在分子水平上抑制V型H+ATP酶,酸化内溶酶体。该研究的主要目的是评估巴弗洛霉素A1对Ca2含量的影响,NAADP诱导的Ca2+释放,大鼠肝细胞和人结肠癌样品中的ATP酶活性。金霉素(CTC)用于定量测量透化大鼠肝细胞中储存的钙。ATP酶活性是通过ATP水解后释放的正磷酸盐含量来确定的,该含量来自大鼠肝脏以及患者的结肠粘膜样品和结直肠癌样品。在大鼠肝细胞中,巴弗洛霉素A1降低了储存的Ca2,并阻止了NAADP对储存的Ca2的影响。这种作用取决于培养基中的EGTA-Ca2+缓冲液。巴弗洛霉素A1显著增加内质网Ca2+ATP酶(EPR)的活性,而不是大鼠肝脏中的质膜(PM)Ca2ATPases。巴弗洛霉素A1也阻止了NAADP对这些泵的影响。此外,巴弗洛霉素A1降低了大鼠肝脏亚细胞部分的Na/KATPase活性,并增加了基础Mg2ATPase活性。同时施用巴弗洛霉素A1和NAADP增强了这些作用。巴弗洛霉素A1增加了正常人结肠粘膜亚细胞部分以及结肠癌组织样品中EPR的Ca2ATPase的活性。相比之下,它降低了正常人结肠粘膜样品中的Ca2ATPasePM活性,并且没有引起结肠癌的变化。巴弗洛霉素A1降低了正常结肠粘膜样品和人结肠癌样品中的Na/KATPase活性,并增加了基础Mg2ATPase活性。可以得出结论,巴弗洛霉素A1靶向NAADP敏感的酸性Ca2存储,有效调节ATP酶活性,并假设酸性存储和EPR之间的联系。巴弗洛霉素A1可用于癌症治疗。