Intracellular calcium mediates the cytotoxicity of lipid A in LLC-PK1 cells

PR Mayeux and SV Shah

Department of Pharmacology, University of Arkansas for Medical Sciences, Little Rock.

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.

Volume 266, Issue 1, pp. 47-51, 07/01/1993
Copyright © 1993 by American Society for Pharmacology and Experimental Therapeutics

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