Evidence that inducible nitric oxide synthase is involved in LPS‐induced plasma leakage in rat skin through the activation of nuclear factor‐κB

Abstract

1. Rats challenged with lipopolysaccharide (LPS) produce large amounts of nitric oxide (NO) following the induction of the inducible NO-synthase (iNOS) in several tissues and organs. Recent studies have shown that the expression of iNOS is regulated at the transcriptional level by a transcription nuclear factor-kappaB (NF-kappaB). In this study we investigated the role of NO in a model of LPS-induced plasma-leakage in rat skin and the involvement of NF-kappaB. 2. Plasma leakage in the rat skin was measured over a period of 30 min to 2 h as the local accumulation of intravenous (i.v.) injection of [125I]-human serum albumin ([125I]-HSA) in response to intradermal (i.d.) injection of LPS. LPS (1, 10, 100 microg/site) produced a dose-related increase in plasma extravasation (18.2+/-3.2, 27.2+/-2.9, 40.4+/-9.6 microl/site) as compared to saline control (11.4+/-2.2 microl/site). This increase was maximal after 2 h; therefore this time point and the dose of LPS 10 microg/site was used in all the successive experiments. 3. To investigate the role of NO in LPS-induced plasma leakage in rat skin, the non-selective NOS inhibitor NG-nitro-L-arginine-methyl ester (L-NAME) or the more selective iNOS inhibitor S-methyl-isothiourea (SMT) was injected i.d. with LPS. L-NAME and SMT (0.01, 0.1 and 1 micromol/site) inhibited LPS-induced plasma leakage in a dose-related fashion (L-NAME: 26.0+/-5.5, 20.2+/-1.6, 18.0+/-2.0 microl/site; SMT: 19.5+/-1.5, 17.0+/-1.6, 15.0+/-2.6 microl/site) as compared to LPS alone (27.2+/-2.9 microl/site). At the lowest concentration used (0.01 micromol/site), SMT significantly reduced plasma leakage by 30\%+/-0.7 while L-NAME (0.01 micromol/site) was not effective. 4. Treatment with increasing concentrations of pyrrolidinedithyocarbamate (PDTC) (0.01, 0.1, 1 micromol/site), an inhibitor of NF-kappaB activation, injected i.d. 30 min before LPS challenge, inhibited in a concentration-dependent fashion LPS-induced plasma leakage by 9.0+/-0.6, 33+/-4.0, 51+/-2.0\% respectively. Moreover, PDTC (0.1, 1 micromol/site) suppressed LPS-induced NF-kappaB DNA-binding. 5. Western blot analysis showed significant levels of iNOS proteins in the skin samples of LPS-treated rats, as compared to basal levels present in saline-injected rat skin. PDTC (0.1, 1.0 micromol/site) dose-dependently decreased the amount of iNOS protein expression induced by LPS. 6. Our results indicate that LPS-induced plasma leakage in rat skin is modulated by NO mainly produced by the inducible isoform of NOS. Furthermore, the suppression of plasma leakage by PDTC, an inhibitor of NF-kappaB activation, is correlated to the inhibition of iNOS protein expression