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Title: Increases in Calmodulin Abundance and Stabilization of Activated iNOS Mediate Bacterial Killing in RAW 264.7 Macrophages

The rapid activation of macrophages in response to bacterial antigens is central to the innate immune system that permits the recognition and killing of pathogens to limit infection. To understand regulatory mechanisms underlying macrophage activation, we have investigated changes in the abundance of calmodulin (CaM) and iNOS in response to the bacterial cell wall component lipopolysaccharide (LPS) using RAW 264.7 macrophages. Critical to these measurements was the ability to differentiate free iNOS from the CaM-bound (active) form of iNOS associated with nitric oxide generation. We observe a rapid two-fold increase in CaM abundance during the first 30 minutes that is blocked by inhibition of NF?B nuclear translocation or protein synthesis. A similar two-fold increase in the abundance of the complex between CaM and iNOS is observed with the same time dependence. In contrast, there are no detectable increases in the CaM-free (i.e., inactive) form of iNOS within the first hour; it remains at a very low abundance during the initial phase of macrophage activation. Increasing cellular CaM levels in stably transfected cells results in a corresponding increase in the abundance of the CaM/iNOS complex that promotes effective bacterial killing following challenge by Salmonella typhimurium. Thus, LPS-dependent increases in CaM abundancemore » function in the stabilization and activation of iNOS on the rapid time-scale associated with macrophage activation and bacterial killing. These results explain how CaM and iNOS coordinately function to form a stable complex that is part of a rapid host-response that functions within the first 30 minutes following bacterial infection to up-regulate the innate immune system involving macrophage activation.« less
Authors:
; ;
Publication Date:
OSTI Identifier:
891139
Report Number(s):
PNWD-SA-7421
TRN: US200621%%583
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry, 45(32):9717-9726
Research Org:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; ABUNDANCE; ANTIGENS; CALMODULIN; CELL WALL; LIPOPOLYSACCHARIDES; MACROPHAGES; NITRIC OXIDE; PATHOGENS; PROTEINS; SALMONELLA TYPHIMURIUM; STABILIZATION; SYNTHESIS; TIME DEPENDENCE; TRANSLOCATION signal transduction; macrophage; pathogen; sensors