Cloning, characterization, and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte.

Abstract

Incubation of human articular chondrocytes with interleukin 113 results in the time-dependent expression of nitric oxide (NO) synthase. We report here the isolation of a cDNA clone which encodes a protein of 1153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. CHO cells transfected with a plasmid harboring this cDNA clone expressed NO synthase activity that was inhibited by some L-arginine analogues. The deduced amino acid sequence of the human chondrocyte inducible NO synthase shows 51% identity and 68% similarity with the endothelial NO synthase and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species.The synthesis of nitric oxide (NO) from L-arginine is now recognized as an important pathway for regulating the function of a wide variety of cells and tissues. NO exerts many of these effects through activation of the soluble guanylate cyclase (1). In the vessel wall, NO is synthesized by the vascular endothelium, to regulate smooth muscle tone and thus blood pressure (2-7). NO synthase is also present in the central nervous system, where NO is a neurotransmitter/ neuromodulator mediating the action of glutamate on N-methyl-D-aspartate receptors (8-11) and mediating/ modulating transmission in nerves previously recognized as nonadrenergic and noncholinergic (12). NO can also act as an autocrine regulator of some cells, including platelets, where it modulates their activation (13).NO generated by activated macrophages is also an important effector molecule in host defense, through a mechanism involving its interaction with iron-sulfur-centered enzymes (14) and/or superoxide anions (15). In this role, NO has been shown to possess antitumor (16) and antimicrobial activity against various parasites in vitro (17) and in vivo (18).NO is synthesized from L-arginine by the action of NO synthase(s), generating citrulline as a co-product. These enzymes are all NADPH-, FAD-, FMN-, and tetrahydrobiopterin-dependent (19). Both the neuronal and inducible NO synthases are P450-type heme proteins (20). In addition to control at the enzyme level, NO synthesis can also be regulated through the expression of different enzymes in various cell types. In endothelial cells (21,22) and neuronal (9, 10) and other (13, 23, 24) tissues, NO synthase activity is constitutively expressed and has a requirement for Ca2+ and calmodulin (22). In contrast, NO synthase is synthesized de novo in macrophages, hepatocytes, Kupffer cells, vascular smooth muscle, and vascular endothelium following activation with endotoxin and/or cytokines (1,19). The inducibleThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.NO sy...

Signalling and cellular specificity of airway nitric oxide production in pertussis

“…Chondrocytes are known to express a macrophage-like iNOS (Charles et al, 1993) in response to cytokines, such as IL-1 and tumour necrosis factor a (TNF-a), or LPS (Stadler et al, 1991). Previous reports have suggested that peptidoglycan may also mediate NO?…”

Human colon cancer cell lines show a diverse pattern of nitric oxide synthase gene expression and nitric oxide generation

“…USA). Positive control poly(A)+ mRNA samples were isolated from induced human chondrocytes as described previously (Charles et al, 1993) and the human skeletal muscle and placental mRNA was supplied by Clontech. Cambridge.…”

“…hepatocytes (Nussler et al, 1992), vascular smooth muscle (Scott-Burden et al, 1992), megakaryoblasts (Lelchuk et al, 1992), chondrocytes (Charles et al, 1993) and the human colonic adenocarcinoma cell line, DLD-1 (Sherman et al. 1993).…”

Nitric oxide and proteoglycan biosynthesis by human articular chondrocytes in alginate culture

“…The ability of articular chondrocytes to regenerate lost matrix may play a key role in preventing harmful pathological changes in the cartilage surface which lead to osteoarthritis. The mechanism through which IL-l inhibits PG synthesis by articular chondrocytes is unknown, IL-l also up-regulates expression of the inducible form of nitric oxide synthase (iNOS) within articular chondrocytes [5], leading to the synthesis of large amounts of nitric oxide (NO) [&9]. Indeed, synthesis of NO under these conditions is so great that it is unlikely to be irrelevant to the physiology of articular cartilage.…”