Cloning and Characterization of Inducible Nitric Oxide Synthase from Mouse Macrophages

TLDR

Nitric oxide serves as a signaling molecule, with constitutive NOS in endothelial cells and neurons activated by calcium/calmodulin, whereas macrophage NOS is induced by cytokines, remains active, and operates independently of calcium. A monospecific antibody was used to clone cDNA encoding two macrophage NO synthase isoforms, and liquid chromatography–mass spectrometry confirmed most of the amino acid sequence. Macrophage NO synthase differs markedly from cerebellar NOS, is transcriptionally induced by immune signals, and closely resembles the enzyme found in cytokine‑treated tumor cells and inflammatory neutrophils.

Abstract

Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.