Visualization of ceramide channels in lysosomes following endogenous palmitoyl-ceramide accumulation as an initial step in the induction of necrosis

• Published in: Biochemistry and biophysics reports Vol. 11; no. C; pp. 174 - 181
• Main Authors: Yamane, Mototeru, Moriya, Shota, Kokuba, Hiroko
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier 01.09.2017
• Subjects: MS, mass spectrometry; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; D-NMAPPD, N-[(1R,2R)-2-hydroxy-1-(hydroxy-methyl)-2-(4-nitrophenyl)ethyl]tetradecanamide; LC3B, autophagosomal membrane bound-microtubule-associated protein 1 light chain 3B; CA-74Me, [(2S,3S)-3-Propylcarbamoyloxirane-2-carbonyl]-L-isoleucyl-L-proline methyl ester; DTT, dithiothreitol; ECL, enhanced chemiluminescence reagent; DMEM, Dulbecco's modified Eagle's medium; SIM, selected ion monitoring; Bax, BCL2-associated X protein; DL-PDMP, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; [D7]d18:1, D-erythro-sphingosine-D7; d18:1, sphingosine; LMP, lysosomal membrane permeability; NAC, N-acetyl-L-cysteine; FBS, fetal bovine serum; CerS, ceramide synthase; PBS, phosphate buffered saline; d18:0, sphinganine; C16:0-Cer, palmitoyl-Cer; GlcCer, glucosyl ceramide; DAPI, 4′,6-diamidino-2-phenylindole; d18:1-[D31]C16:0-Cer, N-palmitoyl [D31]-D-erythro-sphingosine; acridine orange, 3,6-Bis(dimethylamino) acridine hydrochloride; Cer, ceramide; ISs, internal standards; LAMP-2, lysosome-associated membrane protein 2; APCI, atmospheric pressure chemical ionization; DMSO, dimethyl sulfoxide; [D7]d18:0, D-erythro-sphinganine-D7

In this study, we showed that the dual addition of glucosyl ceramide synthase and ceramidase inhibitors to A549 cell culture led to the possibility of ceramide channel formation via endogenous palmitoyl-ceramide accumulation with an increase in cholesterol contents in the lysosome membrane as an ini...