Potential functional significance of brain-type and muscle-type nitric oxide synthase I expressed in adventitia and media of rat aorta

Skeletal muscle and myocardium express mu NOS I, an elongated splice varian t of neuronal-type nitric oxide (NO) synthase (NOS I), and NOS III, endothe lial-type NO synthase, respectively. This study was designed to elucidate w hether vascular smooth muscle also contains a constitutively expressed NO s ynthase isoform. In the rat, mu NOS I contains an insert of 102 nucleotides after nucleotide 2865 of the cDNA, yielding a protein of 164 kd. Reverse t ranscription-polymerase chain reaction with primers flanking this insert an d with insert-specific primers indicated that endothelium-denuded rat aorta expresses both brain-type NOS I and mu NOS I. RNase protection analyses wi th an antisense RNA probe overlapping the mu NOS I insert detected signific ant amounts of NOS I mRNA and lesser amounts of mu NOS I mRNA in endotheliu m-denuded aorta. Western blots using a specific polyclonal antibody recogni zing NOS I and mu NOS I showed a major band of the 160-kd NOS I and a lesse r band of a slightly larger protein in endothelium-denuded aorta. Immunohis tochemistry demonstrated low levels of NOS I/mu NOS I immunoreactivity in t he medial layer of rat aorta, whereas the endothelium expressed only NOS II I immunoreactivity. When the adventitia also was removed, NOS I and mu NOS I mRNA decreased markedly but remained detectable in the medial layer. In f unctional experiments with endothelium-denuded rat aortic rings (that conta ined no NOS III), contractions induced by KCl were markedly increased in th e presence of the NOS inhibitor N-G-nitro-L-arginine These data demonstrate that 2 subforms of NOS I are expressed in nonendothelial components of rat aorta: NOS I and lesser amounts of mu NOS I. Under certain conditions, thi s NOS I/mu NOS I expression could serve as a backup system to the functiona lly predominant NOS III.