Induction and cDNA sequence of inducible nitric oxide synthase from canine aortic smooth muscle cells.
An inducible isoform of nitric oxide synthase (type II, iNOS) is expressed in cardiac and vascular smooth muscle in response to inflammatory cytokines. The dog is an important large animal used for cardiovascular research including effects of exercise, heart failure, and allograft rejection. However, molecular probes for iNOS developed in other mammals have not been reliable for the study of iNOS induction in canine vascular smooth muscle. Experiments were designed to develop a molecular probe for canine iNOS. Smooth muscle cells were isolated from canine aortas. The cells (passages 3-10) were incubated for 1, 3, 6, 12, 24, 48, or 72 h in the absence and presence of Escherichia coli lipopolysaccharide (LPS) to induce iNOS. Total RNA was isolated from the cells using standard techniques. RT-PCR with primers against conserved regions of all known iNOS enzyme was used to clone the iNOS cDNA. RT-PCR showed a single band only from cells treated with LPS. Cloned cDNA from cultured canine aortic smooth muscle cells has 84% homology to human, 81% to rat, and 81% to mouse iNOS gene. Identification of the cDNA for canine iNOS will be useful in the study of differential, transcriptional regulation of inducible (type II) compared with constitutive endothelial (type III) NOS in canine studies of allograft rejection and cardiovascular disease.
Wang
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Division of Cardiothoracic Surgery, Departments of Surgery and Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
McGregor
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Miller
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D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000201:enzymology*; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D003907:Dexamethasone / Q000494:pharmacology; D004285:Dogs; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D006801:Humans; D008297:Male; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D016133:Polymerase Chain Reaction; D051381:Rats; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D018076:DNA, Complementary; D003907:Dexamethasone; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat