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

• Published in: Cellular physiology and biochemistry Vol. 43; no. 3; pp. 1037 - 1051
• Main Authors: Jakab, Martin, Ketterl, Nina, Fürst, Johannes, Beyreis, Marlena, Kittl, Michael, Kiesslich, Tobias, Hauser-Kronberger, Cornelia, Gaisberger, Martin, Ritter, Markus
• Format: Journal Article
• Language: English
• Published: Basel, Switzerland S. Karger AG 01.01.2017; Cell Physiol Biochem Press GmbH & Co KG
• Subjects: Animals; Apoptosis; Apoptosis - drug effects; ATP12A; ATP1AL1; Calcium - metabolism; Cell adhesion & migration; Cell death; Cell Line, Tumor; Cell Size - drug effects; Cell Survival - drug effects; Cell volume; Colon - metabolism; Diabetes; Diabetes mellitus; Enzyme-Linked Immunosorbent Assay; Gene expression; Glucose; Glucose - pharmacology; H(+)-K(+)-Exchanging ATPase - chemistry; H(+)-K(+)-Exchanging ATPase - genetics; H(+)-K(+)-Exchanging ATPase - metabolism; H+/K+ ATPase; Hydrogen-Ion Concentration; Imidazoles - toxicity; INS-1E cells; Insulin; Insulin - analysis; Insulin - metabolism; Insulin Secretion; Insulinoma - metabolism; Insulinoma - pathology; KATP Channels - metabolism; Membrane Potentials - drug effects; Metabolism; Neuroendocrine tumors; Nifedipine - toxicity; Original Paper; Patch-Clamp Techniques; Phosphatidylserines - pharmacology; Prostate; Proton Pump Inhibitors - toxicity; Rats; RNA, Messenger - metabolism; Diabetes mellitus; INS-1E cells; ATP1AL1; H+/K+ ATPase; Cell volume; ATP12A; Insulin; Apoptosis
• ISSN: 1015-8987; 1421-9778
• DOI: 10.1159/000481701

Background/Aims: Glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells involves glucose uptake and metabolism, closure of K ATP channels and depolarization of the cell membrane potential (V mem ), activation of voltage-activated Ca 2+ currents (ICa v ) and influx of Ca 2+ , 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 β-cell viability, can elicit or modify insulin release. In β-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 non-gastric 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 (pH i ) recovery after acute acidic load was measured by NH 4 Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. V mem , K + and Ca 2+ 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 (ΔΨ m ) were measured by flow cytometry. Results: We found that the α-subunit of the non-gastric H + /K + ATPase (HKα2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH 4 Cl prepulsing experiments no K + -dependent pH i recovery was observed under Na + -free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release was unaffected. The L-type ICa v blocker nifedipine caused a full inhibition of GSIS at 10 and 20 µM. At 20 µM, SCH-28080 inhibited ICa v comparable to 20 µM nifedipine and in addition augmented IK ATP recorded at -60 mV and hyperpolarized V mem by ∼15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC 50 values of 22.9 µM and 15.3 µM, respectively. At 20 µM 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 µM, SCH-28080 became progressively cytotoxic causing a steep increase in necrotic cells already 2 h post treatment and a breakdown of ΔΨ m within 4 h under 50 and 100 µM while 10 and 20 µM had no effect on ΔΨ m within 24 h. Conclusion: We demonstrate expression of HKα2 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 ICa v - and activates K ATP channels identifying them as novel “off-targets” of the inhibitor, causing hyperpolarization of V mem and inhibition of insulin secretion.