Stimulation of Suicidal Erythrocyte Death by Ipratropium Bromide

Abstract

Background/Aims: Ipratropium bromide, an anticholinergic agent widely used in obstructive lung disease, has previously been shown to trigger suicidal death of nucleated cells or apoptosis. Despite their lack of mitochondria and nuclei, key organelles in the execution of apoptosis, erythrocytes may similarly undergo suicidal cell death, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). The present study explored whether ipratropium bromide triggers eryptosis. Methods: [Ca Ca²⁺]_i was estimated utilizing Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, and hemolysis from hemoglobin release. Results: A 48 h exposure to ipratropium bromide (1 nM) significantly increased [Ca²⁺]_i, decreased forward scatter and increased annexin-V-binding. Ipratropium bromide treatment was followed by slight but significant increase of hemolysis. Removal of extracellular Ca²⁺ or inhibition of Ca²⁺ permeable cation channels with amiloride (1 mM) virtually abolished cell membrane scrambling. Ca²⁺ ionophore ionomycin (1 µM, 30 min) increased the percentage of phosphatidylserine exposing erythrocytes to similarly high levels in the absence and presence of ipratropium bromide (1 nM). Conclusions: Ipratropium bromide triggers suicidal erythrocyte death or eryptosis, an effect mainly due to stimulation of Ca²⁺-entry.