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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2584.)
© 1999 American Heart Association, Inc.

Vascular Biology

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

Petra M. Schwarz; Hartmut Kleinert; Ulrich Förstermann

From the Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany.

Correspondence to Dr Petra M. Schwarz, Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, 55101 Mainz, Germany. E-mail petra.schwarz{at}uni-mainz.de

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 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 µNOS I. Under certain conditions, this NOS I/µNOS I expression could serve as a backup system to the functionally predominant NOS III.

Key Words: smooth muscle • N^G-nitro-L-arginine • potassium chloride • RNase protection analysis • Western blot • immunohistochemistry

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