Triggering of Suicidal Erythrocyte Death by the Antibiotic Ionophore Nigericin

Abstract

The K + ,H + ionophore and antibiotic nigericin has been shown to trigger apoptosis and is thus considered for the treatment of malignancy. Cellular mechanisms involved include induction of oxidative stress, which is known to activate erythrocytic Ca 2+ -permeable unselective cation channels leading to Ca 2+ entry, increase in cytosolic Ca 2+ activity ([Ca 2+ ] i ) and subsequent stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. This study explored whether and how nigericin induces eryptosis. Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca 2+ ] i from Fluo3 fluorescence, pH i from BCECF fluorescence, ceramide abundance utilizing antibodies and reactive oxygen species (ROS) formation from DCFDA-dependent fluorescence. A 48-hr exposure of human erythrocytes to nigericin significantly increased the percentage of annexin-V-binding cells (0.1-10 nM), significantly decreased forward scatter (0.1-1 nM), significantly decreased cytosolic pH (0.1-1 nM) and significantly increased Fluo3 fluorescence (0.1-10 nM). Nigericin (1 nM) slightly, but significantly, increased ROS, but did not significantly modify ceramide abundance. Materials and MethodsErythrocytes, solutions and chemicals. Li-heparin-anticoagulated fresh blood samples were kindly provided by the blood bank of the University of T€ ubingen. The study was approved by the ethics committee of the University of T€ ubingen (184/2003 V). The blood was centrifuged at 120 9 g for 20 min. at 21°C and the platelet-and leucocyte-containing supernatant were disposed. Erythrocytes were incubated in vitro at a haematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO 4 , 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; pH 7.4), 5 glucose and 1 CaCl 2 , at 37°C for 48 hr. Where indicated, erythrocytes were exposed to nigericin (Enzo Life Sciences, L€ orrach, Germany) at the indicated concentrations.Annexin V binding and forward scatter. After incubation under the respective experimental condition, a 150-ll cell suspension was centrifuged at 350 rcf for 3 min. and, after trashing the supernatant,

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“…Unfortunately, most ROS/inflammasome-related research uses nigericin to stimulate cells (Heid et al 2013; Nomura et al 2015; Antonopoulos et al 2015; Guey et al 2014; Yaron et al 2016). Nigericin acts as a H + , K + ionophore (Bissinger et al 2016) and anti-porter of H + and K + ions (Bissinger et al 2016; Budunova and Mittelman 1992). This is important for 3 reasons.…”