Pb(II) Induces Scramblase Activation and Ceramide-Domain Generation in Red Blood Cells

Abstract

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling.

Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells

“…Lipid extraction was performed as in Ahyayauch et al [27]. Briefly, cell pellets were first resuspended in perchloric acid (60% v/v), samples were then centrifuged at 14,000g for 15 min and supernatants were discarded.…”

CHO/LY-B cell growth under limiting sphingolipid supply: correlation between lipid composition and biophysical properties of sphingolipid-restricted cell membranes+

“…Fluorescence-activated cell sorting (FACS) was performed to evaluate how the decreased FBS concentration in the medium affected cell viability 38 . Cells were stained with Annexin-V-FITC and propidium iodide as indicated in the manual of the annexin V-FITC detection kit (CalbioChem, Darmstadt, Germany) and fluorescence was measured using a FACS Calibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ) as in Ahyayauch et al 39 . Annexin V-FITC fluorescence intensity was measured in fluorescence channel FL-1 with λ ex = 488 nm and λ em = 530 nm, while FL-3 was used for propidium iodide detection, with λ ex = 532 nm and λ em = 561 nm.…”

“…Whole cell lipid extract. Lipid extraction was performed following the method used in Ahyayauch et al 39 . Briefly, cell pellets were first dispersed in aqueous perchloric acid (60% v/v), then centrifuged at 14,000g for 15 min, and the supernatant was discarded.…”

Understanding Graphene Response to Neutral and Charged Lead Species: Theory and Experiment

“…Lead, belonging to the category of heavy metals, is one of the most dangerous substances that adversely affects intracellular biochemical processes in living creatures [ 1 , 2 , 3 , 4 , 5 ]. The negative impact on human health is originating from its significant toxicity [ 6 ] and ability to accumulate in the body [ 7 ].…”