Stimulation of Suicidal Erythrocyte Death by Rottlerin

Abstract

Background/Aims: The phytochemical polyphenol rottlerin is a potent activator of diverse Ca²⁺ -sensitive K⁺ channels. Those channels play a decisive role in the execution of eryptosis, the suicidal death of erythrocytes, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) and ceramide. The present study explored, whether rottlerin induces eryptosis and, if so, to test for the involvement of Ca²⁺ entry and ceramide. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to rottlerin (1 - 5 µM) significantly increased the percentage of annexin-V-binding cells, an effect paralleled by significant decrease of forward scatter. Up to 5 µM rottlerin failed to significantly increase average Fluo3-fluorescence. Rottlerin (5 µM) did, however, significantly increase the ceramide abundance. Rottlerin (5 µM) further significantly increased hemolysis. The effect of rottlerin (5 µM) on annexin-V-binding was virtually abolished by removal of extracellular Ca²⁺. Conclusions: Rottlerin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca²⁺ entry and ceramide.

Oxidative stress, eryptosis and anemia: a pivotal mechanistic nexus in systemic diseases

“…The process of eryptosis is characterized by cell membrane scrambling leading to breakdown of the cell membrane asymmetry and translocation of phosphatidylserine from the interior to the cell membrane surface . The cell membrane scrambling is typically paralleled by erythrocyte shrinkage and by the formation of blebs on the erythrocyte surface.…”

Rottlerin Stimulates Exosome/Microvesicle Release Via the Increase of Ceramide Levels Mediated by Ampk in an In Vitro Model of Intracellular Lipid Accumulation

“…To elucidate the mechanism responsible for the effects of rottlerin, we characterized several pathways. Based on previous studies by Kosaka et al showing that rottlerin increased ceramide concentrations after long treatments [ 17 ], and our results on the action of other drugs and compounds on intracellular lipid trafficking [ 10 , 40 ], we first focused on sphingolipid metabolism. We showed that rottlerin treatment increased ceramide content in C6 cells, together with the degradation of hexosylceramides.…”

“…Rottlerin has been used for decades as a selective protein kinase C-δ inhibitor [ 15 ], although some controversy on this aspect still exists [ 16 ]. Moreover, extended treatment of rottlerin increases cellular ceramide concentrations in erythrocytes [ 17 ]. However, the potential effects of rottlerin on exosomes/microvesicle release have not been examined.…”

Inhibition of Suicidal Erythrocyte Death by Chronic Hypoxia

“…Ceramide could enhance Ca 2+ sensitivity of cell membrane scrambling (Mischitelli et al, 2016). Ceramide abundance at rat RBC surface was quantified using specific antibodies.…”