title: Induction of nitric oxide synthase II does not account for excess vascular nitric oxide production in experimental cirrhosis.
abstract: OBJECTIVE
  Excess production of nitric oxide (NO) reduces vasoconstriction of cirrhotic rat aorta. Expression of the inducible nitric oxide synthase (NOS II) in endothelial cells or vascular smooth muscle could account for this, as described with endotoxin or lipopolysaccharide (LPS) treatment. Alternatively, the endothelial NOS enzyme (NOS III) could be activated by an as-yet undescribed mechanism. Here we describe a combined study of the basal release of NO and quantitative measurement of the mRNA for NOS II and NOS III from thoracic aorta of an animal model of cirrhosis.
  
  
  METHODS
  Thoracic aortas of six normal, six cirrhotic (secondary biliary cirrhosis) and six intra-peritoneal LPS-treated (15 mg/kg) rats were removed. Dissected aortic rings were precontracted with norepinephrine (NE; 10(-6) M) and relaxed with acetylcholine (Ach; 10(-6) M) with or without pre-incubation with the specific NOS inhibitor (L-NNA, 10(-5) M). Total RNA was extracted from aorta, reverse transcribed (RT) and used in polymerase chain reaction (PCR) amplification with primers specific for NOS II, NOS III and beta-actin. PCR products were hybridized with fluorescein labelled cDNA probes and the relative intensity on film was analyzed by densitometry.
  
  
  RESULTS
  Compared to normal, NE caused 32% less contraction in cirrhotic and 43% less contraction in LPS-treated aortic rings. This response was corrected to normal by L-NNA pre-incubation. All the contracted rings relaxed with Ach. NOS II mRNA, was expressed only in LPS-treated aorta and not in aorta from normal and cirrhotic rats. NOS III mRNA levels were the same in normal, cirrhotic and LPS-treated rats: 1176 +/- 170, 1233 +/- 626 and 979 +/- 423, in arbitrary densitometry units, respectively.
  
  
  CONCLUSIONS
  NO is overproduced by aorta from cirrhotic and LPS-treated rats. In LPS-treated rats this could result from expression of NOS II, but this was not the case in the cirrhotic rat aorta. Comparable amounts of NOS III mRNA suggest that, in cirrhosis, increased activity of this enzyme and not increased NOS III expression is responsible for the overproduction of NO.
authors.affiliation: Dept. Medicine and Pharmacology, University of Sheffield, UK.
authors.forename: P
authors.initials: P
authors.lastname: Sogni
authors.forename: A P
authors.initials: AP
authors.lastname: Smith
authors.forename: A
authors.initials: A
authors.lastname: Gadano
authors.forename: D
authors.initials: D
authors.lastname: Lebrec
authors.forename: T W
authors.initials: TW
authors.lastname: Higenbottam
mesh_terms: D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism*; D004790:Enzyme Induction; D007527:Isoenzymes / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008106:Liver Cirrhosis, Experimental / Q000378:metabolism*; D008297:Male; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic; D014661:Vasoconstriction; D014664:Vasodilation
chemical_list: D007527:Isoenzymes; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase