H(+)-ATPases of renal cortical and medullary endosomes are differentially sensitive to Sch-28080 and omeprazole

Abstract

Adenosinetriphosphatase (ATPase) activity stimulated by K+ and inhibited by Sch-28080 (SCH), omeprazole (OME), and vanadate has been measured in microsomes from mammalian renal medulla and attributed to a kidney isoform of the H(+)-K(+)-ATPase. To determine whether the H(+)-K(+)-ATPase inhibitors could also inhibit the vacuolar (V)-type H(+)-adenosinetriphosphatase (H(+)-ATPase, i.e., H+ pump) in mammalian intracellular vesicles, we examined their effects on bafilomycin-sensitive acidification in renal cortical vesicles (CEV) and medullary endocytic vesicles (MEV). Rats were injected with fluorescein isothiocyanate-labeled dextran, and labeled endosomes were enriched from kidney tissue homogenates by differential and Percoll density gradient centrifugation. In the CEV, the V-type H+ pump was inhibited 25% by SCH and 30% by OME (100 microM each). Whereas the inhibition by OME was concentration and time dependent, the inhibition by SCH was only concentration dependent. Inhibition by these compounds was similar in the presence of 50 mM K+ (in = out) and in the complete absence of K+, thus ruling out a significant involvement of H(+)-K(+)-ATPase-mediated acidification. Inhibition, however, was not observed with 10 microM SCH and OME. The sensitivity of the V-type H+ pump to 100 microM SCH and OME in CEV was confirmed by the comparable inhibitions of intravesicular acidification observed in acridine orange fluorescence quench studies and by inhibition of Pi liberation in an ATPase assay. We also found that the V-type H+ pump in isolated rat liver endosomes is sensitive to 100 microM SCH and OME to a similar degree. In the MEV, acidification was only weakly affected by 100 microM SCH and OME, thus suggesting that H(+)-ATPases in endosomes from cortical and medullary tubules are different, possibly due to a previously described selective expression of subunit isoforms. Our finding indicates the importance of using low concentrations (< 10 microM) of OME and SCH in studies of H(+)-K(+)-ATPase in nongastric tissues to avoid misinterpretation of the data due to nonspecific inhibition of V-type H(+)-ATPases.

Transcriptomes of purified gastric ECL and parietal cells: identification of a novel pathway regulating acid secretion

“…ECL cells accumulate acridine orange [acidic dissociation constant (pK a) ϳ 9.0] in their histamine-containing vesicles due to the acidic pH of 5-5.5 generated by the activity of a V-type ATPase that is insensitive to inhibition by SCH28080 at concentrations Ͻ100 M (44). At this pH and dye concentration, the ECL cell histaminecontaining vacuoles show combined red and green fluorescent emission resulting in a yellow fluorescent appearance of ECL cells at an excitation wavelength of 488 nm.…”

Omeprazole Induces Apoptosis in Jurkat Cells

“…We therefore investigated whether lysosomal enzymes playa role in OM-induced apoptosis. A number of studies have evidenced the lysosomotropic properties of OM and have indicated that following activation by protonation, it can also affect the activity of proton pumps, such as the vacuolar H+-ATPase (5,6,8,49) in part responsible for the internal acid milieu of! ysosomes (9-10).…”

Proton Pump Inhibitors Induce Apoptosis of Human B-Cell Tumors through a Caspase-Independent Mechanism Involving Reactive Oxygen Species

“…They act as potent inhibitors of the gastric acid pump and have been used for short-or long-term treatments also with very high doses (150 mg/d) without major side effects (19,20). Besides targeting the gastric acid pump, PPI inhibit the V-ATPase activity (21)(22)(23). We reported that PPI sensitize chemoresistant human tumors to cytotoxic drugs (24), suggesting that proton pump activity may affect cancer cells homeostasis and particularly acidic vesicles trafficking (5,24).…”