Intracellular calcium mediates the cytotoxicity of lipid A in LLC-PK1 cells.
Acute renal failure is a frequent and serious complication of endotoxemia. However, the biochemical events that might ultimately lead to renal cell injury have not been examined previously. The renal tubular epithelial cell line LLC-PK1 was used to evaluate the role of intracellular-free calcium ([Ca++]i) in lipid A-induced cytotoxicity. Lipid A, considered to be the active component of bacterial endotoxin, produced a time- and concentration-dependent cytotoxicity as assessed by loss of trypan blue exclusion (38 +/- 3% cell deaths in 120 min with 50 micrograms/ml, n = 4). Chelation of [Ca++]i by quin 2 and inhibition of Ca++ release by 8-(N,N-dimethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride each protected against lipid A (50 micrograms/ml) cytotoxicity whereas removal of extracellular Ca++ was without effect. Fura-2 fluorescence was used to demonstrate that lipid A induces a rise in [Ca++]i in a concentration-dependent manner. Extracellular Ca++ was not required for the rise in [Ca++]i. Lipid A (50 micrograms/ml) produced a similar rise in [Ca++]i in the presence of 1 mM Ca++ (144 +/- 3 nM above basal) or in the absence of added Ca++ (155 +/- 28 nM, n = 4). The peak rise in [Ca++]i occurred rapidly (within 30 sec), clearly preceding cell death. The observations that lipid A induces a rise in [Ca++]i that precedes cell death, and that quin 2 and 8-(N,N-dimethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride protect against cell death suggest release of intracellular Ca++ mediates the cytotoxicity of lipid A in this renal tubular epithelial cell line.
Mayeux
P R
PR
Department of Pharmacology, University of Arkansas for Medical Sciences, Little Rock.
Shah
S V
SV
D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured / Q000187:drug effects; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006706:Homeostasis / Q000187:drug effects; D007424:Intracellular Fluid / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007700:Kinetics; D008050:Lipid A / Q000633:toxicity*; D008954:Models, Biological; D013552:Swine; D014343:Trypan Blue / Q000493:pharmacokinetics
D000634:Aminoquinolines; D002021:Buffers; D005456:Fluorescent Dyes; D008050:Lipid A; D014343:Trypan Blue; C035739:Quin2; D002118:Calcium