Ketamine inhibits LPS-induced calcium elevation and NF-kappa B activation in monocytes<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

J.Sun, Z.Q.Zhou, R.Lv,W.Y.Li and J.G.Xu

Department of Anesthesiology, jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, China

Introduction

Bacterial lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, is one of the most potent activators of innate immune cells, and induce fatal endotoxic shock syndrome with exaggerated production of inflammatory cytokines in experimental animals and humans^[1]. Monocytes and macrophages are accepted as the major origin of many inflammatory cytokines induced by LPS^[2,3]. LPS stimulation of monocytes activates various transcription factors, including the NF-kappa B family, which coordinates the induction of genes encoding inflammatory mediators, such as tumor necrosis factor alpha (TNF-α) and interleukin 1(IL-1)^[4].

Ketamine, an intravenous anesthetic, has been advocated for anesthesia in septic or severely ill patients because of its cardiovascular stimulating effects^[5,6].However several previous studies reported that ketamine could suppress LPSinduced TNF-a production and mortality in carrageenansensitized endotoxin shock mice ^{[7, 8]}. In vitro studies, ketamine is also proved to have the ability to suppress the production of proinflammatory cytokines following the administration of LPS ^[9－12].<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

The mechanism for the suppressive effect of ketamine on cytokine production is not clear. Our laboratory has reported that ketamine can prevent NF-kappa B translocation in the peripheral blood mononuclear cells (PBMC) after stimulating with LPS ^[10]. However NF-kappa B translocation is prominent about one hour after the beginning of LPS incubation. There it is not known whether ketamine has any effects on the early events in LPS-induced signal transduction in monocytes. Intracellular calcium seems to be a very important early second message in LPS signal transduction, and ketamine has the ability to prevent the elevation of intracellular calcium in myocardial cells through inhibition of transmembrane influx of Ca²⁺ by L-type voltage-dependent Ca²⁺ channels and release of Ca²⁺ from sarcoplasmic reticulum ^[13－15]. Our study was therefore intended to determine whether ketamine could attenuate calcium elevation in the monocytes exposed to LPS, and whether this attenuation was consistent with its anti-inflammatory effect.

Materials and methods

Experiment protocol

Isolated monocytes were treated with LPS (10 μg/ml final concentration) (Escherichia coli O111: B4, Sigma Chemical Co., St. Louis, Mo. USA) in the presence or absence of various concentrations of ketamine (10, 100, 1000 μM final concentration) (Hengrui, China). Intracellular calcium was monitored with laser confocal microscopy. NF-kappa B translocation was investigated with Electrophoretic mobility shift assay (EMSA).

Isolation of monocytes<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

Whole heparinized blood was obtained from healthy Wistar rats (220～250 g). PBMC were separated by density gradient centrifugation on Ficoll-Histopaque 1077 (Sigma Chemical Co., St. Louis. MO). The PBMC were enriched for monocytes by adherence on petri dish at 37°C for 1 h. Then, plates were vortexed and nonadherent cells were removed by vigorous washing with RPMI 1640 (Gibco, BRL). Adherent cells were then cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FCS overnight and monolayer was washed again before infection with LPS. More than 85% of cells purified by this technique were determined to be monocytes. Cell viability determined by trypan blue exclusion was 94%. Cells were exposed to LPS (10 mg/ml) and ketamine of various concentrations. We used cells from six individual animals.

Cytosolic Ca²⁺ measurements

Fluo-3/AM (10 μM final concentration) (Sigma Chemical Co., St.Louis. MO) was added into incubation medium and incubated for 30 min at 37°C before LPS infection. Images were acquired using the linescan mode of a confocal BioRad MRC1000 microscope (BioRad, USA) connected to a Nikon Diaphot microscope. Fluo-3 was excited at 488 nm and fluorescence emission was filtered and measured at 540 ±30 nm. Images were acquired every 20 s. Scanned lines were plotted vertically and each line was added to the right of the preceding line to form the linescan image. Fluorescence intensity and the curve of time course were analyzed by the computer automatically.

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