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

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.