Lipopolysaccharide-induced changes in resistance artery vasoactivity: interaction between eNOS and iNOS

S.A. Francis, & A. Ahluwalia. Clinical Pharmacology, William Harvey Research Institute, Barts and The London, London EC1M 6BQ.

Expression of inducible NO synthase (iNOS) in blood vessels, during endotoxaemia, elevates vascular NO production, a response thought to play a major role in the associated altered vascular reactivity. Our previous studies indicated an essential role for endothelial NOS (eNOS) in the regulation of iNOS expression and activity during lipopolysaccaride (LPS)-induced sepsis in conduit arteries (Vo et al., 2005; Francis et al., 2005). Here we used eNOS knockout (KO) animals to investigate a possible role for eNOS in LPS-induced changes in vascular reactivity within small resistance arteries.

Male (25-30g) WT (C57BL/6J) and eNOS KO mice were treated with saline or LPS (12.5mg/kg, i.v , 4 or 15h). Mice were sacrificed; and the mesenteries removed for iNOS protein determination. For functional studies 3rd order arteries were dissected, cleaned of surrounding fat and mounted in tension myographs, bathed in Krebs solution aerated with carbogen at 37 oC. Following equilibration, arteries were normalised using standard procedures and concentration-response curves constructed to either the thromboxane A 2 mimetic, U-46619 (U19; 0.001-3μM), or phenylephrine (PE; 0.001-30μM). Arteries were then precontracted with U19 (~EC80), and responses to acetylcholine (ACh; 0.001-10μM) or the NO donor spermine-NONOate (SPER-NO; 0.001-3µM) determined.

Densitometric analysis of western blots of mesenteric tissue showed that LPS-induced vascular iNOS expression was substantially reduced in eNOS KO animals (analysis indicates ~20 fold decrease, p<0.05, n=3) vs WT animals (WT LPS; 24±9 KO LPS; 3±1). LPS treatment of WT animals suppressed (P<0.01, n>6) PE responses at 15 but not 4h (LPS 15h; pEC50=5.5±0.07, n=6 vs saline; pEC50=5.8±0.05, n>10). However this effect of LPS was profoundly enhanced in eNOS KO animals (PE Emax; 4.2±0.6mN; pEC50=5.6±0.17, n=13; p<0.001) compared to saline treatment (PE Emax; 9.0±0.2mN; pEC50=6.2±0.03, n=13). Responses to U-19 were unaffected by LPS treatment in WT arteries but significantly reduced in eNOS KO arteries (P<0.001; saline pEC50=8.6±0.4; LPS pEC50=7.8±0.08, n=13). In WT arteries responses to ACh were significantly suppressed following LPS, at 4 (P<0.001) but not 15 h (saline Emax=77±5%; LPS 4h Emax=55±14%, n>5), whilst responses to SPER-NO were suppressed at both time points (saline pEC50=6.4±0.07; LPS 4h pEC50=6.2±0.08; LPS 15h pEC 50=5.9±0.09, n>5). Responses to SPER-NO were enhanced in arteries of saline treated eNOS KO animals compared to WT (pEC50=7.0±0.05, n=14), and the potency of SPER-NO was significantly reduced after 15h LPS treatment (pEC50=6.3±0.08, n=12).

Together, our data show an essential role for eNOS in regulating iNOS expression and therefore vascular reactivity of resistance arteries during LPS-induced sepsis. However, in the absence of eNOS, additional compensatory mediators may be upregulated to produce the endotoxaemia-induced hyporesponsiveness, independently of iNOS.

Francis, S.A et al (2005) British Journal of Pharmacology, in Press. Vo, P.A. et al. (2005) The Journal Of Biological Chemistry, 280: 7239-7243.

SF is supported by an MRC PhD studentship