Increases in calmodulin abundance and stabilization of activated inducible nitric oxide synthase mediate bacterial killing in RAW 264.7 macrophages

Heather Smallwood, Liang Shi, Thomas C. Squier

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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 2-fold increase in CaM abundance during the first 30 min that is blocked by inhibition of either NFκB nuclear translocation or protein synthesis. A similar 2-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 2 h; it remains at a very low abundance during the initial phase of macrophage activation. Increasing cellular CaM levels in stably transfected macrophages results in a corresponding increase in the abundance of the CaM/iNOS complex that promotes effective bacterial killing following infection by Salmonella typhimurium. Thus, LPS-dependent increases in CaM abundance 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 min following bacterial infection to upregulate the innate immune system involving macrophage activation.

Original languageEnglish (US)
Pages (from-to)9717-9726
Number of pages10
JournalBiochemistry
Volume45
Issue number32
DOIs
StatePublished - Aug 15 2006

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Macrophages
Nitric Oxide Synthase Type II
Calmodulin
Stabilization
Macrophage Activation
Chemical activation
Immune system
Lipopolysaccharides
Immune System
Bacterial Antigens
Salmonella
Pathogens
Protein Transport
Cellular Structures
Salmonella typhimurium
Infection
Bacterial Infections
Cell Wall
Nitric Oxide
Up-Regulation

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Increases in calmodulin abundance and stabilization of activated inducible nitric oxide synthase mediate bacterial killing in RAW 264.7 macrophages. / Smallwood, Heather; Shi, Liang; Squier, Thomas C.

In: Biochemistry, Vol. 45, No. 32, 15.08.2006, p. 9717-9726.

Research output: Contribution to journalArticle

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