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-κB (NF-κB). 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-κB.

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 μg/site) produced a dose-related increase in plasma extravasation (18.2±3.2, 27.2±2.9, 40.4±9.6 μl/site) as compared to saline control (11.4±2.2 μl/site). This increase was maximal after 2 h; therefore this time point and the dose of LPS 10 μg/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 N^Gnitro-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 μmol/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 μl/site; SMT: 19.5±1.5, 17.0±1.6, 15.0±2.6 μl/site) as compared to LPS alone (27.2±2.9 μl/site). At the lowest concentration used (0.01 μmol/site), SMT significantly reduced plasma leakage by 30%±0.7 while L-NAME (0.01 μmol/site) was not effective.

4. Treatment with increasing concentrations of pyrrolidinedithyocarbamate (PDTC) (0.01, 0.1, 1 μmol/site), an inhibitor of NF-κB 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 μmol/site) suppressed LPS-induced NF-κB 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 μmol/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-κB activation, is correlated to the inhibition of iNOS protein expression.

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