The Putative Role of the Non-Gastric H+/K+-ATPase ATP12A (ATP1AL1) as Anti-Apoptotic Ion Transporter: Effect of the H+/K+ ATPase Inhibitor SCH28080 on Butyrate-Stimulated Myelomonocytic HL-60 Cells

摘要

biBackground/Aims /i/bThe ATP12A gene codes for a non-gastric Hsup+/sup/Ksup+/sup ATPase, which is expressed in a wide variety of tissues. The aim of this study was to test for the molecular and functional expression of the non-gastric Hsup+/sup/Ksup+/sup ATPase ATP12A/ATP1AL1 in unstimulated and butyrate-stimulated (1 and 10 mM) human myelomonocytic HL-60 cells, to unravel its potential role as putative apoptosis-counteracting ion transporter as well as to test for the effect of the Hsup+/sup/Ksup+/sup ATPase inhibitor SCH28080 in apoptosis. biMethods /i/bReal-time reverse-transcription PCR (qRT-PCR) was used for amplification and cloning of ATP12A transcripts and to assess transcriptional regulation. BCECF microfluorimetry was used to assess changes of intracellular pH (pHsubi/sub) after acute intracellular acid load (NHsub4/subCl prepulsing). Mean cell volumes (MCV) and MCV-recovery after osmotic cell shrinkage (Regulatory Volume Increase, RVI) were assessed by Coulter counting. Flow-cytometry was used to measure MCV (Coulter principle), to assess apoptosis (phosphatidylserine exposure to the outer leaflet of the cell membrane, caspase activity, 7AAD staining) and differentiation (CD86 expression). biResults /i/bWe found by RT-PCR, intracellular pH measurements, MCV measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL-60 cells. Treatment of HL-60 cells with 1 mM butyrate leads to monocyte-directed differentiation whereas higher concentrations (10 mM) induce apoptosis as assessed by flow-cytometric determination of CD86 expression, caspase activity, phosphatidylserine exposure on the outer leaflet of the cell membrane and MCV measurements. Transcriptional up-regulation of ATP12A and CD86 is evident in 1 mM butyrate-treated HL-60 cells. The Hsup+/sup/Ksup+/sup ATPase inhibitor SCH28080 (100 µM) diminishes Ksup+/sup-dependent pHsubi/sub recovery after intracellular acid load and blocks RVI after osmotic cell shrinkage. After seeding, HL-60 cells increase their MCV within the first 24 h in culture, and subsequently decrease it over the course of the next 48 h. This effect can be observed in the overall- and non-apoptotic fraction of both untreated and 1 mM butyrate-treated HL-60 cells, but not in 1 mM butyrate-stimulated phosphatidylserine-positive cells. These cells do not shrink from 24 h to 72 h and have finally a higher MCV than untreated cells unless they are exposed to SCH28080. 10 mM butyrate induces apoptosis within 24 h. biConclusion /i/bIn summary we show that in HL-60 cells ATP12A is a functionally active Hsup+/sup/Ksup+/sup ATPase that may counteract events during early apoptosis like intracellular acidosis, loss of intracellular Ksup+/sup ions and apoptotic volume decrease. Its expression and/or susceptibility to the Hsup+/sup/Ksup+/sup ATPase inhibitor SCH28080 becomes most evident in cells exposing phosphatidylserine on the outer leaflet of the cell membrane and therefore during early apoptosis.