Plant Nitrate Reductase Gene Fragments Enhance Nitrite Production in Activated Murine Macrophage Cell Lines

Abstract

Nitrate reductase (NR) gene fragments (1.1 kb and 800 bp) from the barley plant were incorporated into pSV 2neo and transfected by electroporation into a variety of cell lines of different functionality. Only transfected murine macrophage cell lines demonstrated appreciably enhanced NO − 2 production (i.e., NR activity) both in the presence and absence of exogenous nitrate (NO − 3). Addition of NO − 3 caused the greatest increase in NO − 2 production when macrophages were primed with interferon-gamma (INF-γ) and lipopolysaccharide (LPS). Transfection of RAW 264.7 murine macrophages led to isolation of several novel neomycin-resistant subpopulations designated NR10 1, NR10 2 (both containing the 1.1 kb NR fragment) and NR800 5 (containing the 800 bp NR fragment). Similarly transfected nonleukocytic and leukocytic stem cell lines showed no significant NO − 2 production. Outside of the macrophage cell lines, only the murine T cell line EL-4 showed evidence of mild nitrite production enhancement. The mechanism of enhanced NO − 2 formation in NR transfected murine macrophages is unknown. However, study of these novel cells may lead to greater understanding of the expression of a plant NR in mammalian cells and highly controlled production of a cytotoxic molecule (NO − 2) in macrophages.