The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells

Background/Aims: Glucose-stimulated insulin secretion (GSIS) of pancreatic beta-cells involves glucose uptake and metabolism, closure of K-ATP channels and depolarization of the cell membrane potential (V-mem), activation of voltage-activated Ca2+ currents (ICav) and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, K-ATP-independent mechanisms such as changes in intracellular pH (pH(i)) or cell volume, which also affect beta-cell viability, can elicit or modify insulin release. In beta-cells the regulation of pH(i) is mainly accomplished by Na+/H+ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the nongastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. Methods: In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi) recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. V-mem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (Delta psi(m)) were measured by flow cytometry. Results: We found that the a-subunit of the non-gastric H+/K+ ATPase (HK alpha 2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pH(i) recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 mu M SCH-28080 inhibited GSIS by similar to 50%, while basal release was unaffected. The L-type ICav blocker nifedipine caused a full inhibition of GSIS at 10 and 20 mu M. At 20 mu M, SCH-28080 inhibited ICav comparable to 20 mu M nifedipine and in addition augmented IKATP recorded at -60 mV and hyperpolarized V-mem by similar to 15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC50 values of 22.9 mu M and 15.3 mu M, respectively. At 20 mu M the percentages of Annexin-V+, caspase+ and propidium iodide+ cells were significantly increased after 24 and 48 h. Concurrently, the MCV was significantly decreased (apoptotic volume decrease, AVD) and the SSC signal was increased. At concentrations >40-50 mu M, SCH-28080 became progressively cytotoxic causing a steep increase in necrotic cells already 2 h post treatment and a breakdown of Delta psi(m) within 4 h under 50 and 100 mu M while 10 and 20 mu M had no effect on Delta psi(m) within 24 h. Conclusion: We demonstrate expression of HK alpha 2 in rat INS-1E cells. However, the pump is apparently non-functional under the given conditions. Nonetheless the H+/K+ ATPase blocker SCH-28080 inhibits insulin secretion and induces cell death. Importantly, we show that SCH-28080 inhibits ICav-and activates K-ATP channels identifying them as novel "off-targets" of the inhibitor, causing hyperpolarization of V-mem and inhibition of insulin secretion. (C) 2017 The Author(s) Published by S. Karger AG, Basel