Expression of inducible nitric oxide (iNOS) in blood vessels during endotoxaemia, leads to an increase in vascular nitric oxide (NO) that plays a major role in the pathogenesis of bacterial sepsis. Our previous studies have shown that vascular iNOS expression is dependent upon an intact endothelium and iNOS independent NO production (Vo et al., 2003). Therefore we used eNOS knockout (KO) mice to examine the role of eNOS in regulating iNOS expression/function in a lipopolysaccharide (LPS)-induced model of sepsis.

Male (20-25g) WT (C57BL/6J) and eNOS KO mice were pretreated with saline or LPS (12.5mg/kg, i.v , 15h). Mice were sacrificed, the thoracic aorta removed, cut into rings and mounted under 0.3g tension in Krebs aerated with carbogen at 37 °C. Following equilibration concentration-response curves were constructed to either the thromboxane A₂ mimetic, U-46619 (U19; 0.001-3 µM), or phenylephrine (PE; 0.001-30 µM). Arteries were then precontracted with PE (~EC₈₀), and responses to acetylcholine (ACh; 0.001-10 µM) or spermine-NONOate (SPER-NO; 0.001-3 µM) determined. Some aortae were collected for iNOS/eNOS protein determination and blood samples collected by intra cardiac puncture for total plasma NOx measurements chemiluminescence NO analysis.

LPS treatment resulted in an elevation (p<0.0001, n>9) of the phenylephrine maximum response (Emax; 0.72±0.03g) compared to saline (Emax; 0.46±0.02g) that was absent in aortae of eNOS KO animals (n>9). In contrast LPS had no significant effect on responses to U-19 in aortae of WT (saline treated pEC₅₀=-7.4±0.06, n=12) or eNOS KO mice (saline treated pEC₅₀=-7.7±0.08, n=9). LPS treatment significantly suppressed responses to ACh (saline treated Emax=79± 5%, n=14; LPS treated Emax=53±4%, n=7) in WT aortae, whilst ACh had no effect in aortae of saline (n=7) or LPS (n=6) treated eNOS KO animals. In contrast LPS treatment decreased the potency of SPER-NO (saline treated pEC₅₀=-6.4±0.06, n=8; LPS treated pEC₅₀=-6.1±0.06, n=7, p<0.001), a suppression that was absent in aortae of eNOS KO mice (saline treated pEC₅₀=-6.4±0.11, n=10; LPS treated pEC₅₀=-6.8±0.14, n=9). Densitometric analysis of western blots of aortic tissue from WT and eNOS KO mice showed elevation of iNOS expression in LPS treated WT animals which was (P<0.05) reduced ~75% in eNOS KO mice (n=5-6). In addition plasma [NOx] analysis demonstrated a profound elevation in LPS-treated WT animals that was suppressed ~50% (p<0.05, n=5) in eNOS KO mice.

In conclusion, our data show an essential role for eNOS in regulating iNOS expression and consequent NO activity in conduit arteries during LPS-induced sepsis. In addition our study identifies eNOS as the mediator of the previously reported hyperreactivity to PE prevalent in conduit arteries following LPS treatment (Chauhan et al., 2002).

Chauhan, S.D et al. (2003) FASEB J, 17: 773-775.
Vo, P.A. et al. (2003) British Journal of Pharmacology, in Press.
SF is supported by an MRC PhD studentship .