Elsevier

Nitric Oxide

Volume 8, Issue 2, March 2003, Pages 127-132
Nitric Oxide

Regulation of nitric oxide production from macrophages by lipopolysaccharide and catecholamines

https://doi.org/10.1016/S1089-8603(02)00148-9Get rights and content

Abstract

Catecholamines are elaborated in stress responses to mediate vasoconstriction, and elevate systemic vascular resistance and blood pressure. They are elaborated in disorders such as sepsis, cocaine abuse, and cardiovascular disease. The aim of the study was to determine whether catecholamines affect nitric oxide (NO) production, as NO is a vasodilator and counteracts the harmful effects of catecholamines. RAW264.7 macrophage cells were cultured with lipopolysaccharide (LPS) ± epinephrine, norepinephrine, and dopamine at 5×10−6 M concentrations for 24 h. Supernatants were harvested for measuring NO by spectrophotometry using the Greiss reagent and cells were harvested for detecting inducible NO synthase (iNOS) by Western blot. NO production in RAW 264.7 macrophages was increased significantly by addition of LPS (0.5–10 ng/ml) in a dose-dependent fashion. The NO production induced by LPS was further enhanced by epinephrine and norepinephrine, and to a lesser extent by dopamine. These increases in NO correlated with expression of iNOS protein in these cells. The enhancing effect of iNOS synthesis by epinephrine and norepinephrine on LPS-induced macrophages was down regulated by β-adrenoceptor antagonist, propranolol, and dexamethasone. The results suggest that catecholamines have a synergic effect on LPS in induction of iNOS synthesis and NO production, and this may mediate some of the vascular effects of infection. These data support a novel role for catecholamines in disorders such as septic shock and cocaine use, and indicate that β-adrenoceptor antagonists and glucocorticoids may be used therapeutically for modulation of the catecholamine–NO axis in disease states.

Section snippets

Experimental procedures

Materials. Mouse Raw264.7 macrophage cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA). Raw264.7 cell line was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 μg/ml). LPS (Escherichia coli serotype 0111:B4), epinephrine, norepinephrine, and dopamine were purchased from Sigma (St. Louis, MO).

Effect of LPS and catecholamines on NO production in macrophages. Two milliliters of RAW264.7 murine

Results

Effect of catecholamines and LPS on nitrite production from macrophages. Two milliliters of RAW264.7 murine macrophage cells (1.8×106/ml) were cultured in 12-well tissue culture plates with medium or LPS at a concentration ranging from 0.5 to 10 ng/ml for 24 h. The nitrite production from macrophages treated with LPS was increased in a dose-dependent manner. LPS at concentrations of 5–10 ng/ml significantly (P<0.05) increased nitrite production. Epinephrine (Epi), norepinephrine (Nor), or dopamine

Discussion

Catecholamines are elaborated in stress responses to mediate vasoconstriction and elevate systemic vascular resistance and blood pressure. Sympathetic nervous system activation together with microbial infection results in a synergistic interaction that could modulate inflammatory processes [26]. In septic shock, NO has been shown to be involved in vascular collapse, which is a major contributor to mortality. Thus, it is important to understand the interaction between catecholamines and LPS with

Acknowledgements

This work was supported by NIH Grant HL63070, the Ruth R. Harris Endowment, and RDC and CVRI of ETSU.

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