Desensitization of the soluble guanylyl cyclase/cGMP pathway by lipopolysaccharide in rat isolated pulmonary artery but not aorta

Abstract

Background and purpose: To investigate the function of soluble guanylyl cyclase (sGC)/3',5'-cyclic guanosine monophosphate (cGMP) pathway in lipopolysaccharide (LPS)-induced changes in vascular reactivity of rat isolated pulmonary artery and aorta.

Experimental approach: Nitric oxide (NO) production, contraction responses to endothelin-1 (ET-1), relaxation responses to sodium nitroprusside (SNP), 8-pCPT-cGMP, BAY412272 and T-0156, SNP-induced cGMP production and expression of sGC(alpha1), sGC(beta1) and 3',5'-cyclic nucleotide phosphodiesterase-5 (PDE5) proteins were measured in LPS-treated pulmonary and aortic rings from male Wistar rats.

Key results: In both vessels, LPS (10 microg mL(-1), 20 h) increased NO production, which was inhibited by the selective inducible NOS (iNOS) inhibitor 1400W (1 microM). In the aorta, LPS decreased ET-1-induced contractility and this decrease was inhibited by the selective sGC inhibitor ODQ (10 microM) but not by removal of endothelium, or inhibitors of cyclooxygenase (indomethacin, 10 microM) or iNOS (1400W, 1 microM). Furthermore, aortic relaxation responses to the direct sGC activator BAY412272 were enhanced. In the pulmonary artery, SNP (1 nM to 30 microM)-induced relaxation and cGMP production, BAY412272-induced relaxation and sGC(beta1) protein expression were decreased, whereas relaxation responses to the PDE5-specific inhibitor T-0156 (0.1-100 nM) were enhanced. Relaxation responses to the phosphodiesterase-resistant cGMP analogue, 8-pCPT-cGMP, and protein expression levels of sGC(alpha1) and PDE5 were not altered in either vessel.

Conclusion and implications: LPS caused a selective hypocontractility of rat aorta to ET-1 mediated mainly through NO-independent sGC activation, whereas in the pulmonary artery, the effect of sGC activation was reduced by a decreased protein expression of sGC(beta1) together with increased PDE5 activity.

Figure 1

Lipopolysaccharide (LPS) treatment increased nitric oxide (NO) release from isolated rat pulmonary artery and aorta. NO release (total amount of nitrite plus nitrate, NOx) was determined in the supernatant spectrophotometrically using Greiss reagent after reduction of nitrate to nitrite. LPS-induced NO overproduction was inhibited by co-incubation with either nitro-L-arginine methyl ester (L-NAME, 100 μM) or 1400W (1 μM) (n=5). ^**P<0.01 compared with control group, ^##P<0.01 compared with LPS group using one-way ANOVA followed by Dunnett's post hoc test.

Figure 2

The specific soluble guanylyl cyclase (sGC) inhibitor ODQ prevented the lipopolysaccharide -induced decrease in ET-1-mediated contraction in the aorta. (a) Contraction to ET-1 (0.3–100 nM) was measured after incubation with LPS in the pulmonary and aortic arterial rings (n=9). (b) Pulmonary and aortic arterial rings were incubated with 10 μM ODQ before measuring contraction to ET-1 (0.3–100 nM) (n=6). ^**P<0.01 for E_max and pEC₅₀ values compared with control group using paired Student's t-test.

Figure 3

Sodium nitroprusside (SNP), BAY412272 and 8-pCPT-cGMP-induced relaxation responses were differentially modulated by lipopolysaccharide in pulmonary and aortic vessels. Pulmonary and aortic arterial rings were preconstricted with 30 nM ET-1 before measuring relaxation to (a) SNP (1 nM to 30 μM, n=6), (b) the nitric oxide-independent direct sGC activator BAY412272 (1 nM to 10 μM, n=4) or (c) the non-hydrolysable cGMP analogue 8-pCPT-cGMP (0.1–100 μM, n=5). ^##P<0.01 for E_max values, ^**P<0.01 for E_max and pEC₅₀ values compared with control group using paired Student's t-test.

Figure 4

Lipopolysaccharide (LPS) selectively decreased sodium nitroprusside (SNP)-induced cGMP production in the pulmonary artery. Pulmonary and aortic rings from LPS-treated and control groups were stimulated by SNP (100 μM for 10 min) in the presence of the non-selective phosphodiesterase inhibitor IBMX (100 μM) and cGMP levels were determined by ELISA (n=4). ^*P<0.05 compared with control group using paired Student's t-test.

Figure 5

Lipopolysaccharide (LPS) decreased protein expression levels of soluble guanylyl cyclase-β1 (sGC_β1), but not sGC_α1, in the pulmonary artery. (a) Representative immunoblots for sGC_α1 and sGC_β1 in the pulmonary artery (left) and the aorta (right). (b) Densitometric ratio of sGC protein subunits normalized to β-actin in the pulmonary artery (left) and the aorta (right) (n=4). ^**P<0.01 compared with control group using paired Student's t-test.

Figure 6

Selective increases in T-0156-induced relaxation by lipopolysaccharide (LPS) treatment in the pulmonary artery without changes in PDE5 protein expression levels. (a) Pulmonary and aortic arterial rings were preconstricted with 30 nM ET-1 before measuring relaxation to T-0156 (0.1–100 nM) (n=6). (b) Representative immunoblots for PDE5 in the pulmonary artery (left) and the aorta (right). (c) Densitometric ratio of PDE5 protein normalized to β-actin in the pulmonary artery (left) and the aorta (right) (n=4). ^**P<0.01 for E_max and pEC₅₀ values compared with control group using paired Student's t-test.