Potential Functional Significance of Brain-Type and Muscle-Type Nitric Oxide Synthase I Expressed in Adventitia and Media of Rat Aorta

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Abstract
—Skeletal muscle and myocardium express μNOS I, an elongated splice variant of neuronal-type nitric oxide (NO) synthase (NOS I), and NOS III, endothelial-type NO synthase, respectively. This study was designed to elucidate whether vascular smooth muscle also contains a constitutively expressed NO synthase isoform. In the rat, μNOS I contains an insert of 102 nucleotides after nucleotide 2865 of the cDNA, yielding a protein of 164 kd. Reverse transcription-polymerase chain reaction with primers flanking this insert and with insert-specific primers indicated that endothelium-denuded rat aorta expresses both brain-type NOS I and μNOS I. RNase protection analyses with an antisense RNA probe overlapping the μNOS I insert detected significant amounts of NOS I mRNA and lesser amounts of μNOS I mRNA in endothelium-denuded aorta. Western blots using a specific polyclonal antibody recognizing NOS I and μNOS I showed a major band of the 160-kd NOS I and a lesser band of a slightly larger protein in endothelium-denuded aorta. Immunohistochemistry demonstrated low levels of NOS I/μNOS I immunoreactivity in the medial layer of rat aorta, whereas the endothelium expressed only NOS III immunoreactivity. When the adventitia also was removed, NOS I and μNOS I mRNA decreased markedly but remained detectable in the medial layer. In functional experiments with endothelium-denuded rat aortic rings (that contained no NOS III), contractions induced by KCl were markedly increased in the presence of the NOS inhibitor NG-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 μNOS I. Under certain conditions, this NOS I/μNOS I expression could serve as a backup system to the functionally predominant NOS III.