title: Endothelial nitric oxide synthase plays a main role in producing nitric oxide in the superacute phase of hepatic ischemia prior to the upregulation of inducible nitric oxide synthase.
abstract: BACKGROUND
  The aim of this study was to determine the intrahepatic kinetics of different types of nitric oxide (NO) synthase, such as endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS), during repeated ischemia/reperfusion (I/R).
  
  
  METHODS
  Three different protocols of hepatic I/R in rats were designed as follows: 60 min of ischemia and 30 min of reperfusion (I/R 60/30); 5 min of ischemia and 5 min of reperfusion (I/R 5/5); and repeating 15 min of ischemia and 5 min of reperfusion for four cycles (I/R 15/5 × 4). Intrahepatic NO levels were measured using a selective NO sensor. Changes in hepatic microcirculation during I/R 5/5 were investigated using intravital microscopy. Hepatic expression of eNOS, phospho-eNOS, and iNOS were evaluated during repeated I/R by Western blot, reverse transcription polymerase chain reaction, and immunohistochemistry.
  
  
  RESULTS
  During I/R 60/30, intrahepatic NO levels gradually increased and then reached a plateau approximately 15 min after starting ischemia. During I/R 5/5, the sinusoids after 5 min reperfusion were dilated compared with the sinusoids before ischemia. The expression of phospho-eNOS during I/R 15/5 × 4 markedly increased during the first ischemia, and then the levels attenuated during the subsequent repeating I/R cycles; however, the expression of iNOS gradually increased, as observed by Western blot, reverse transcription polymerase chain reaction, and immunohistochemical analysis. An impact of NO production by phospho-eNOS activation during the superacute phase of I/R was also confirmed using pharmacologic NOS inhibitors.
  
  
  CONCLUSIONS
  Our results firstly demonstrated an altered activation of the phospho-eNOS system and iNOS over the course of repeated hepatic I/R.
authors.affiliation: Division of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Miyake
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Yokoyama
authors.forename: Toshio
authors.initials: T
authors.lastname: Kokuryo
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Mizutani
authors.forename: Atsushi
authors.initials: A
authors.lastname: Imamura
authors.forename: Masato
authors.initials: M
authors.lastname: Nagino
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D008099:Liver / Q000098:blood supply* / Q000378:metabolism* / Q000473:pathology; D008107:Liver Diseases / Q000378:metabolism / Q000473:pathology; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000378:metabolism*; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III
keywords: Ischemia; Nitric oxide; Reperfusion; eNOS; iNOS