Nitric oxide (NO) and natriuretic peptides (NPs) cooperatively regulate vascular tone through activation of soluble (sGC) and particulate (pGC) guanylate cyclases, respectively (Hussain et al., 2001; Madhani et al 2003). Expression of inducible nitric oxide synthase (iNOS) in vascular tissue following exposure to lipopolysaccharide (LPS) results in the production of 'high-output' NO and altered responsiveness to vasoconstrictors and vasodilators (Chauhan et al. 2003). In this study, we examined the effect of LPS on the sensitivity of cGMP- and cAMP- vasorelaxant pathways in rat aorta in vitro.

Rat (male; Harlan-Sprague Dawley; 200-250g) thoracic aortic rings were set up under 1g tension for isometric recording in Krebs solution gassed with 95%O₂/5%CO₂ at 37ºC. All tissues were contracted to an approximate EC₈₀ with U46619 (mean g tension: 2.1±0.07 and 2.2±0.06, with and without LPS respectively; n=64; P>0.05) and cumulative concentration-response curves to ANP (0.01nM-0.1µM), CNP (0.1nM-1µM), SPER-NO (0.001-1µM), ACh (0.001-1µM), forskolin (0.001-1µM), 8-Br-cGMP (1-300µM) and 8-Br-cAMP (1-300µM) were constructed. The effect of endotoxin on the sensitivity of sGC and pGC pathways was investigated by treating vessels with LPS (0.3µg/ml; 4hrs) alone or co-incubated with the iNOS, cyclooxygenase or sGC inhibitors 1400W (10µM), indomethacin (10µM) and ODQ (5µM), respectively. pEC₅₀ values were used to compare the relaxant effects of drugs. Statistical analysis was by two-way ANOVA with P<0.05 taken to be significantly different.

LPS reduced the potency of ACh (pEC₅₀: 7.92±0.15 before and 7.18±0.23 after LPS; n=7; P<0.05) and SPER-NO (pEC50: 7.09±0.41 before and 6.47±0.68 after LPS; n=9; P<0.05). 1400W, but not indomethacin, preserved ACh- and SPER-NO- relaxations (pEC₅₀: ACh 7.80±0.357 and 7.35±0.39 in the presence of 1400W and indomethacin, respectively; SPER-NO 6.94±0.17 and 6.13±1.3 in the presence of 1400W and indomethacin, respectively; n5, P<0.05 versus control for both). LPS treatment also reduced the potency of 8-Br-cGMP (pEC₅₀: 3.99±0.22 before and 3.39±0.39 after LPS; n=7; P<0.05), but did not alter responses to forskolin (pEC₅₀: 6.15±0.74 before and 6.52±0.36 after LPS, respectively; n=6; P>0.05) or 8-Br-cAMP (pEC₅₀: 3.43±0.26 before and 3.58±0.21 after LPS; n=4; P>0.05). In LPS-treated vessels, the potency of CNP (pEC₅₀: 7.38±0.4 before and 6.54±0.43 after LPS n=4; P<0.05) and ANP (pEC₅₀: 8.95±0.17 before and 8.05±0.26 after LPS; n=10; P<0.05) was also significantly reduced. 1400W (pEC₅₀: 8.44± 0.29; n=6), ODQ (pEC₅₀: 8.59±0.37; n=7), or both inhibitors (8.89±0.38; n=5; all P<0.05 versus LPS alone), preserved relaxations to ANP in LPS-treated vessels. 1400W and/or ODQ also reversed established desensitisation to ANP after 4 hr incubation with LPS (pEC₅₀: 1400W alone, 8.26±0.25, n=7; ODQ alone, 8.83±0.18, n=7; 1400W + ODQ, 9.02±0.38, n=5; all P<0.05 versus LPS alone).

These results indicate that sGC and pGC signalling are desensitised following exposure to LPS, consistent with a compensatory mechanism offsetting high-output NO production by iNOS. This effect is specific to cGMP-dependent pathways since the cAMP pathway was unaltered. The mechanism of desensitisation is likely to involve a reversible biochemical change, since the responsiveness was restored immediately following removal of excess NO (by 1400W) or cGMP (by ODQ). These observations suggest that inflammatory cardiovascular disorders associated with excessive NO production (ie. septic shock) are characterised by specific impairment of GC-cGMP-mediated vasorelaxation that is readily reversible.

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