Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform.

Abstract

Nitric oxide (NO) is a ubiquitous intercellular messenger molecule synthesized from the amino acid L-arginine by NO synthases in diverse cells and tissues. NO is synthesized in vascular endothelial cells and appears to play an important role in the control of blood pressure and platelet aggregation. A detailed understanding of the regulation of NO synthesis by endothelial cells has been hampered by the lack of molecular clones for endothelial NO synthase; the isolation and characterization ofsuch clones is reported herein. The constitutive NO synthases present in endothelial cells and in brain share common biochemical and pharmacologic features. We purified NO synthase from bovine brain and determined the amino acid sequence of several tryptic peptides. The sequence ofthe bovine brain peptides is nearly identical to the deduced amino acid sequence previously determined for the rat brain NO synthase.These sequence data were utilized to design PCR-generated NO synthase cDNA probes, which were used to isolate clones encoding NO synthase from a bovine aortic endothelial cell (BAEC) cDNA library. A full-length NO synthase cDNA clone was isolated, representing a protein of 1205 amino acids with a molecular mass of 133 kDa; transfection of this clone in a heterologous expression system demonstrated the expected enzymatic activity. The deduced amino acid sequence of the BAEC NO synthase cDNA differs at numerous residues from the sequence determined for the purified bovine brain protein and shows 50-60% sequence identity with recently isolated molecular clones for murine macrophage and rat brain NO synthase isoforms. Bovine genomic Southern blots probed with bovine brain and BAEC NO synthase cDNA probes identify distinct bands, indicating that these cDNAs are the products of different genes. Prolonged treatment of BAECs with the cytokine tumor necrosis factor a, which we have previously shown to result in a marked increase in NO synthase activity, is associated with a decrease in the abundance of the 4.8-kilobase BAEC NO synthase transcript. The increase in BAEC NO synthase activity induced by tumor necrosis factor a is thus likely to involve posttranscriptional mechanisms or the induction of a distinct endothelial NO synthase isoform.The vascular endothelium forms the lining of the circulatory system in all vertebrates and comprises a dynamic monocellular interface between the surrounding vascular cells and the soluble and cellular components of the blood. In response to diverse stimuli, the vascular endothelium synthesizes and secretes vasoactive agents that control vascular tone (1, 2). In 1980, a labile endothelium-derived compound was discovered to play an important role in relaxing vascular smooth muscle and was termed endothelium-derived relaxing factor (EDRF) (3). EDRF was subsequently shown to be equivalent to nitric oxide (NO) or a NO-containing compound (for review, see ref. 4). NO synthesis is being discovered in a striking diversity of tissues and cultured cells (5).Catalysis by NO synthase, in all e...

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