Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase.

Abstract

The constitutive endothelial cell nitric oxide synthase (NOS) importantly regulates vascular homeostasis. To gain understanding of this enzyme, a pEF BOS cDNA library of 5 X 105 clones was prepared from bovine aortic endothelial cells (BAEC) and screened with a 2.8-kb cDNA BamHI fragment of rat brain NOS. Clone pBOS13 was found to express NO synthase activity when transfected into COS-7 cells. Sequence analysis revealed sequences compatible with binding domains for calcium/calmodulin, flavin mononucleotide, flavin adenine nucleotide and NADPH. The deduced amino acid sequence revealed a protein with a relative mol mass of 133,286, which is 58% homologous to the rat cerebellar NOS and 51% homologous to the mouse macrophage NOS. The amino-terminal portion of the protein exhibits several characteristics peculiar to the endothelial cell NOS. These include a proline-rich region and several potential sites for proline-directed phosphorylation as well as a potential substrate site for acyl transferase. Northern hybridization to mRNA from cultured BAEC revealed an abundant 4.8-kb message, which was not increased by coincubation with tumor necrosis factor a, but was markedly increased by exposure to shear stress for 24 h. The unique features of the endothelial cell NO synthase, particularly in the amino terminal portion of the molecule, may provide for novel regulatory influences of enzyme activity and localization. (J.

Ventilation and oxygenation induce endothelial nitric oxide synthase gene expression in the lungs of fetal lambs.

“…The sequences of the oligonucleotides were 5 Ј -CCTCCG-GAGGGGCCCAAGTTCCCTCGC-3 Ј for oligonucleotide 1 and 5 Ј -CACGTCGAAGCGCCGTTTCCGGGGGT-3 Ј for oligonucleotide 2. The region amplified corresponds to amino acids 62-288 of the eNOS protein (24,(33)(34)(35). Total RNA, prepared from ovine fetal lung, was used in RT-PCR reactions (kit from Perkin-Elmer, Foster City, CA).…”

“…Initially, NOS activity and NO production are increased when endothelial cells are exposed to increased shear stress (12,14,15,(20)(21)(22)(23). If the shear is maintained there are then subsequent increases in eNOS mRNA and protein expression (24)(25)(26). The immediate increase in pulmonary blood flow at birth may increase shear forces on the pulmonary vascular endothelium inducing eNOS mRNA and protein expression.…”

Reversal of NO-induced nociceptive hypersensitivity by St. John’s wort and hypericin: NF-κB, CREB and STAT1 as molecular targets

“…Nitric oxide (NO) is a free radical that is synthesized by three isoforms of NOS: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) (Bredt et al 1991;Lamas et al 1992;Nishida et al 1992). NO produces a variety of biological actions under physiological and pathological conditions (Harrison 1997;Brown and Bal-Price 2003;Tieu et al 2003).…”

Up‐regulation of endothelial nitric oxide synthase expression by cyclic guanosine 3′,5′‐monophosphate

“…shear stress [7,9], chronic exercise training [10], protein kinase C [11] and lysophosphatidylcholine [12] were reported to regulate the molecular expression of ecNOS. Thus, there is now sufficient evidence to suggest that the expression of ecNOS, though it is a constitutive isoform, is subject to regulation by a variety of stimuli.…”

“…In human umbilical vein endothelial cells (HUVEC), tumor necrosis factor-c~ was shown to decrease the ecNOS mRNA levels in a dose-dependent manner by acting at the posttranscriptional level, thereby decreasing its half-life [8]. There are in vitro data showing that prolonged mechanical deformation of the endothelium by defined shear or cyclic stretching increases ecNOS gene expression, protein and activity [9,24]. In dogs, chronic exercise training was reported to increase ecNOS mRNA and coronary vascular NO production [10].…”