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 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 eventua...
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Published in | Cellular physiology and biochemistry Vol. 43; no. 3; pp. 1037 - 1051 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Basel, Switzerland
S. Karger AG
01.01.2017
Cell Physiol Biochem Press GmbH & Co KG |
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1015-8987 1421-9778 |
DOI: | 10.1159/000481701 |