Effect of Volatile Anesthetics on the Induction of Inducible Nitric Oxide Synthase by Lipopolysaccharide Stimulation.

2001 Fiscal Year Final Research Report Summary

• Project/Area Number: 11671477
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Anesthesiology/Resuscitation studies
• Research Institution: Akita University
• Principal Investigator: MASAKI Yoko Akita University, School of Medicine, Research Associate, 医学部, 助手 (30125744)
• Co-Investigator(Kenkyū-buntansha): ABE Kyoko Akita University, School of Medicine, Research Associate, 医学部, 助手 (30311575)
• Project Period (FY): 1999 – 2001
• Keywords: Isoflurane / Sevoflurane / LPS / iNOS / PKC

Research Abstract

Over production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) plays an important role in systemic vasodilation in septic shock. Although volatile anesthetics have been reported to attenuate NO release by iNOS, it is unclear whether inhalational anesthetics affect on iNOS gene expression. In this study, we have investigated the effect of isoflurane and sevoflurane on iNOS protein expression and protein kinase C (PKC) activity in lipopolysaccharide (LPS)-treated murine peritoneal macrophages.
Methods : Macrophages were obtained from mice (female C3H/HeN, 6W) with thioglycolate i.p. injection. Cells (10^6/ml) were plated in dishes (O.D. 60mm) and incubated with LPS (10μg/ml) in the box containing 0, 1 or 3MAC isoflurane or sevoflurane with 5% CO_2 for 24h at 37℃. NO amount in 10μl supernatants were measured by NO analyzer (SIEVERS). Cell lysates with 2% SDS Tris Buffer were subjected to SDS-PAGE and iNOS protein was detected at 130 KDa by Western blot analysis with the ant i-iNOS antibody. PKC activity was measured with PKC Assay System^<TM> (Amersham Pharmacia Biotech) and ^<32>P ATP after 2h incubation with 0 or 3MAC of each anesthetics. INOS expression and PKC activity were shown as percent of levels expressed in OMAC. Data were expressed as mean±SD.
Results : Both isoflurane and sevoflurane reduced NO accumulation by LPS (465.5±64.0, 604.8±17.0pmol) at 3MAC (158.4±28.8, 533.5±20.30pmol). iNOS protein expression was also decreased at 3MAC of isoflurane and sevoflurane (28.7±1.5, 57.2±4.4%). At 1MAC, both volatile anesthetics did not affect NO accumulation, whereas, iNOS protein expression was stimulated (112.3±1.2,135.1±1.8%). PKC was activated by isoflurane and sevoflurane at 3MAC (160.5±14.8, 135.0±7.1%).
Conclusions : Isoflurane and sevoflurane significantly reduced iNOS protein levels and NO release in LPS-treated macrophages at 3MAC, partly by PKC activation, Whereas, this inhibitory effect could not be observed at 1MAC concentrations, volatile anesthetics differentially modulate iNOS gene expression at concentrations.