DIFFERENTIAL INDUCTION OF CONSTITUTIVE AND INDUCIBLE NITRIC-OXIDE SYNTHASES BY DISTINCT INFLAMMATORY STIMULI IN BOVINE AORTIC ENDOTHELIAL-CELLS

Exposure to various combinations of cytokines and lipopolysaccharide ( LPS) has been reported to increase NO production in vascular endotheli al cells. The molecular entity of the newly expressed nitric oxide syn thase (NOS) in endothelial cells, however, has not yet been examined i n detail. In this report, we carried out biochemical characterizations and molecular identification of NOS isoform(s) expressed in cytokine/ LPS-treated bovine aortic endothelial cells (BAEC). The increased NOS activity in tumor necrosis factor-alpha(TNF-alpha)/LPS-treated BAEC wa s localized mainly in the cytosolic fraction and Ca2+-independent, whe reas that in interferon-alpha,beta(IFN-alpha,beta)/LPS-treated BAEC wa s preferentially in the membrane fraction and Ca2+-dependent, suggesti ng that TNF-alpha/LPS increased an inducible NOS (iNOS)-like activity, and IFN-alpha,beta/LPS increased an endothelial constitutive NOS (ecN OS)-like activity. Correspondingly, the different responses to the cyt okine/LPS pretreatment were demonstrated in semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using primers specifi c for NOS or ecNOS, that is, TNF-alpha/LPS elicited the expression of iNOS mRNA whereas IFN-alpha,beta/LPS increased that of ecNOS mRNA. A n uclear run-on transcription assay and an inhibition experiment by acti nomycin D indicated that the apparent increase of ecNOS in the IFN-alp ha,beta/LPS-treated BAEC was at least in part ascribed to the transcri ptional activation. The nucleotide sequences of the amplified PCR prod ucts in TNF-alpha/LPS- and IFN-alpha,beta/LPS-treated BAEC were 93% an d 99% identical to the corresponding regions of human hepatocyte iNOS and bovine ecNOS, respectively. These findings indicated that, in cyto kine/LPS-treated BAEC, two NOS isoforms whose molecular natures were c losely homologous to the conventional isoforms of NOS and ecNOS were d ifferently induced in response to distinct inflammatory stimuli.