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 microNOS 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, microNOS 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 microNOS I. RNase protection analyses with an antisense RNA probe overlapping the microNOS I insert detected significant amounts of NOS I mRNA and lesser amounts of microNOS I mRNA in endothelium-denuded aorta. Western blots using a specific polyclonal antibody recognizing NOS I and microNOS 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/microNOS 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 microNOS 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 microNOS I. Under certain conditions, this NOS I/microNOS I expression could serve as a backup system to the functionally predominant NOS III.
Schwarz
P M
PM
Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany. petra.schwartz@uni-mainz.de
Kleinert
H
H
Förstermann
U
U
D000818:Animals; D016375:Antisense Elements (Genetics); D001012:Aorta, Abdominal / Q000201:enzymology*; D001013:Aorta, Thoracic / Q000201:enzymology*; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D018076:DNA, Complementary; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D007124:Immunoenzyme Techniques; D008564:Membrane Potentials / Q000502:physiology; D018482:Muscle, Skeletal / Q000201:enzymology; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D019335:Nitroarginine / Q000494:pharmacology; D009638:Norepinephrine / Q000494:pharmacology; D011189:Potassium Chloride; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017540:Tunica Media / Q000201:enzymology; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D016375:Antisense Elements (Genetics); D018076:DNA, Complementary; D012333:RNA, Messenger; D014662:Vasoconstrictor Agents; D019335:Nitroarginine; D011189:Potassium Chloride; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; C496322:Nos1 protein, rat; C496316:Nos3 protein, rat; D002118:Calcium; D009638:Norepinephrine