Endothelium-dependent relaxation is markedly reduced in atherosclerotic arteries. Recently, the endothelium-dependent relaxing factor has been identified as nitric oxide (NO). We used RNase protection assay and immunoblotting to elucidate the effect of atherogenic lipoprotein on the expression of constitutive NO synthase (cNOS) mRNA and protein levels in bovine aortic endothelial cells. Twenty-four-hour exposure to a low concentration of oxidized low-density lipoprotein (10 micrograms protein/mL) upregulated cNOS mRNA levels (2.4 +/- 0.4-fold, P < .01). However, native low-density lipoprotein and high-density lipoprotein did not have any effect on cNOS mRNA levels. Furthermore, 5 micrograms/mL of lysophosphatidylcholine (LPC) also upregulated cNOS mRNA levels (2.6 +/- 0.5-fold, P < .01) at 8 hours. This action of LPC was abolished with cycloheximide but not with staurosporine. We concluded that atherogenic lipoproteins upregulate cNOS mRNA and protein levels in bovine aortic endothelial cells. This observation supports the hypothesis that an impairment of endothelium-dependent vasodilatation in atherosclerotic vessels may not be due to a decrease in cNOS expression. Moreover, the LPC action on cNOS mRNA levels requires new protein synthesis.

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Endothelial cell nitric oxide synthase (NOS) is known to have a N-myristoylation consensus sequence. Such a consensus sequence is not evident in the macrophage, smooth muscle and neuronal NOS. A functional role for this N-terminal myristoylation is not clear yet. In the present study, we examined the effect of N-terminal myristoylation on the NOS activity determined by the conversion of L-[3H]arginine to L-[3H]citrulline and extracellular NO release determined by nitrite production in the conditioned medium from the COS-7 cells transfected with wild-type bovine aortic endothelial cell (BAEC) NOS cDNA or non-myristoylated BAEC-NOS mutant cDNA. NOS activity of wild type BAEC-NOS in COS-7 cells was localized in the particulate fraction and that of mutant NOS was in the cytosolic fraction. In contrast, nitrite production from COS-7 cells transfected with wild type BAEC-NOS cDNA was greater than that of mutant cDNA in a time dependent and a concentration dependent manner. These results suggest that membrane localization of NOS with myristoylation facilitates extracellular transport of NO and leads to enhanced NO signaling on the vascular smooth muscle cells and the intravascular blood cells including neutrophils, macrophages and platelets.

A reconstituted system containing a form of cytochrome P-450, cytochrome b5, NADPH-cytochrome P-450 reductase, and NADH-cytochrome b5 reductase, all purified from rabbit liver microsomes, could catalyze O-demethylation of p-nitroanisole in the presence of both NADPH and NADH. Omission of either cytochrome P-450 or cytochrome b5 from the system led to complete loss of the activity. The reconstituted activity was sensitive to carbon monoxide, metyrapone, phenyl isocyanide, and cyanide, indicating that the cytochrome P-450 used is cyanide-sensitive and is involved in the catalytic process. The maximal demethylase activity was attained when the system contained cytochrome P-450 and cytochrome b5 at a 1 : 1 molar ratio. Trypsin digestion of cytochrome b5 abolished the capacity of this cytochrome to reconstitute the demethylase activity. These results suggest that O-demethylation of p-nitroanisole by this particular form of cytochrome P-450 absolutely requires the intact form of cytochrome b5 and that the second electron needed for the demethylation may be donated to the cytochrome P-450 only by way of cytochrome b5.