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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2008 Sep 22;155(8):1164–1173. doi: 10.1038/bjp.2008.358

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

M S H El-Awady 1, S V Smirnov 1, M L Watson 1,*
PMCID: PMC2607199  PMID: 18806822

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 sGCα1, sGCβ1 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 μg mL−1, 20 h) increased NO production, which was inhibited by the selective inducible NOS (iNOS) inhibitor 1400W (1 μM). In the aorta, LPS decreased ET-1-induced contractility and this decrease was inhibited by the selective sGC inhibitor ODQ (10 μM) but not by removal of endothelium, or inhibitors of cyclooxygenase (indomethacin, 10 μM) or iNOS (1400W, 1 μM). Furthermore, aortic relaxation responses to the direct sGC activator BAY412272 were enhanced. In the pulmonary artery, SNP (1 nM to 30 μM)-induced relaxation and cGMP production, BAY412272-induced relaxation and sGCβ1 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 sGCα1 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 sGCβ1 together with increased PDE5 activity.

Keywords: LPS, NO, ET-1, cGMP, sGC, PDE5, pulmonary artery, aorta

Introduction

Sepsis is a major cause of morbidity and mortality in hospitalized patients worldwide, with two main vasomotor complications, septic shock and pulmonary hypertension (Parsons et al., 1989; Lorente et al., 1993; Manthous et al., 1993). Excessive nitric oxide (NO) production, derived mainly from inducible NOS (iNOS), has been shown to contribute to the development of acute lung injury, delayed hypotension and vasoplegia in patients with septic shock (Feihl et al., 2001; Lopez et al., 2004) as well as in animals injected with bacterial lipopolysaccharide (LPS) (Szabo et al., 1995; Bishop-Bailey et al., 1997). The most important receptor molecule for NO is NO-sensitive soluble guanylyl cyclase (sGC), which catalyses the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). The amplitude and duration of a cGMP signal are determined by the activity of sGC, a heterodimeric cGMP-forming enzyme, and of the phosphodiesterases that degrade cGMP (Juilfs et al., 1999). Only two sGC isoforms, α1/β1 and α2/β1, exist, with the α1/β1 sGC heterodimer being the predominant isoform in most tissues including lung (Friebe and Koesling, 2003). The major cGMP-degrading phosphodiesterase isoform in vascular smooth muscle is the cGMP-binding 3′,5′-cyclic nucleotide phosphodiesterase-5 (PDE5) (Maurice et al., 2003).

Different strategies have been used to control excessive NO production in sepsis, such as inhibiting NO production preferentially with selective iNOS inhibitors or by inhibiting NO-dependent pathways such as sGC; however, results have been conflicting. For example, the potent and highly selective iNOS inhibitor 1400W (Garvey et al., 1997) has been shown to prevent LPS-induced hypotension (Bachetti et al., 2003) and delay hypotension and circulatory failure in rats in vivo (Cuzzocrea et al., 2006), but it only partially reverses LPS-induced hyporeactivity to phenylephrine in rat superior mesenteric artery in vitro (O'Brien et al., 2001) and is not effective in rabbits (Bachetti et al., 2003). ODQ, an irreversible and highly selective haem-site inhibitor of sGC (Garthwaite et al., 1995), prevents aortic hypocontractility to norepinephrine ex vivo in rat (Chen et al., 2005) and both in vitro and in vivo in rat and mouse (Zingarelli et al., 1999). Conversely, ODQ is only partially effective in restoring vascular reactivity to phenylephrine in rat superior mesenteric artery in vitro (O'Brien et al., 2001) and ex vivo in rat aorta (Wu et al., 1998).

Previous studies have shown that LPS decreases sGC activity in rat lung and aorta (Fernandes et al., 2006), in cultured rat aortic (Papapetropoulos et al., 1996) and pulmonary arterial smooth muscle cells (Scott and Nakayama, 1998). PDE5 activity is increased in rat lung after LPS injection (Holzmann et al., 1996) and 1 h after LPS inhalation in guinea pig lungs (Toward et al., 2005) but decreased after 48 h in the same model. These studies have examined whole lung tissue or cultured pulmonary arterial smooth muscle cells, but there are no direct measurements published using whole pulmonary arteries. Furthermore, these changes in NO signalling pathways have not been related to vascular reactivity in the same experimental model.

Together with NO, plasma levels of the powerful vasoconstrictor endothelin-1 (ET-1) are elevated in animal models of sepsis (Curzen et al., 1997; Fujii et al., 2000) and in patients with sepsis (Pittet et al., 1991) and its level correlates with the severity of illness (Pittet et al., 1991; Piechota et al., 2007). Furthermore, endothelin antagonism attenuates pulmonary hypertension in porcine endotoxic shock (Wanecek et al., 1999) and lung injury in sheep (Kuklin et al., 2004), suggesting an important function of ET-1 in sepsis. However, the function of ET-1 and its relation to the NO/cGMP pathway in endotoxic shock remain unclear.

This study investigated the function of the sGC/cGMP pathway in LPS-induced changes in vascular reactivity to ET-1 in rat isolated pulmonary artery and aorta as representatives of pulmonary and systemic vascular beds, respectively. Our results suggest that, in the pulmonary artery, LPS treatment impaired sGC activation by decreasing protein expression of sGCβ1 and by increasing PDE5 activity, whereas, in the aorta, LPS caused hypocontractility to ET-1 mainly through NO-independent activation of sGC.

Materials and methods

Tissue preparation

Animal housing and care was carried out in accordance with UK Home Office legislation and guidelines. In-house-bred male Wistar rats (250–300 g) were killed by cervical dislocation and the side branches of the pulmonary artery and the descending thoracic aorta were separated and placed in a cold oxygenated physiological solution composed of (mM): NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4·7H2O 1.2, NaHCO3 25 and glucose 11.1. The vessels were dissected free of fat and connective tissue and cut into rings (2–4 mm in length). Under sterile tissue culture conditions, vascular rings were then placed into 48-well plates, with each well containing one ring in 1 mL Dulbecco's modified Eagle medium supplemented with penicillin (100 unit mL−1), streptomycin (100 μg mL−1) and 10% foetal bovine serum. After 1 h stabilization, vascular rings were transferred to a fresh medium of the same composition (control group) or supplemented with LPS 10 μg mL−1 (LPS-treated group) and incubated for 20 h at 37 °C in humidified atmosphere of 5% CO2 in air. In some experiments, endothelium was removed mechanically by gentle rubbing before incubation. In other experiments, vascular rings were co-incubated with indomethacin (10 μM), 1400W (1 μM) or ODQ (10 μM). For NO measurement, phenol red-free Dulbecco's modified Eagle medium was used.

Measurement of NO release

In aqueous solutions, NO has a very short half-life and is oxidized to nitrite and nitrate, therefore NO production was determined by measuring the accumulation of these breakdown products in the culture medium (Bishop-Bailey et al., 1997). Nitrate was first reduced to nitrite and the total nitrite was measured spectrophotometrically by the Greiss reaction.

Pulmonary and aortic rings were divided into four groups: control, LPS, LPS+nitro-L-arginine methyl ester (100 μM) or LPS+1400W (1 μM), with each ring incubated in 1 mL medium as described above. After 20 h incubation, 60 μL supernatant was taken from each well and samples were incubated for 60 min at 37 °C with 0.2 U mL−1 nitrite reductase, 50 μM NADPH and 5 μM flavin adenine dinucleotide disodium to reduce nitrate to nitrite. Any remaining NADPH, which absorbs at 550 nm, was oxidized by the addition of 10 U mL−1 lactate dehydrogenase and 10 mM sodium pyruvate for a further 10 min at 37 °C. A final sample volume of 100 μL was then mixed with an equal volume of modified Greiss reagent for 5 min in 96-well plates. Absorbance was measured at 550 nm by Versamax tunable microplate reader with Softmax Pro software (Molecular Devices, Wokingham, Berks, UK) and total nitrite concentration was calculated using the standard curves of sodium nitrate (0–128 nmol mL−1) in culture medium run simultaneously in parallel.

In vitro vascular reactivity studies

Following 20 h of incubation, arterial rings from the control and the LPS-treated groups were mounted in an organ bath filled with 18 mL of the physiological salt solution at a temperature of 37 °C and bubbled with a mixture of 95% O2 and 5% CO2. Rings were allowed to equilibrate under 12 (aorta) and 7 (pulmonary artery) mN resting tension for 60 min, during which time the bath solution was replaced every 15 min and the resting tension was readjusted when necessary. Isometric tension generated by the vascular smooth muscle was measured using a force displacement transducer (K30, Hugosachs Elektronik, March, Germany) and recorded with a MacLab 4S unit linked to a PC running Chart v4.2 software (ADInstruments Ltd, Chalgrove, Oxfordshire, UK).

At the beginning of each experiment, arterial ring responsiveness was assessed by measuring contraction to 80 mM KCl, and this procedure was repeated until consistent responses were obtained, and then rings were washed until tension returned to the baseline. To measure tissue contractility to ET-1, the vasoconstrictor was added cumulatively to the organ bath and concentration (0.3–100 nM)–response curves were constructed. To measure vasorelaxation, rings were first preconstricted with 30 nM ET-1, and after reaching a steady-state contraction (plateau), cumulative concentration–response curves to sodium nitroprusside (SNP) (1 nM to 30 μM), 8-pCPT-cGMP (0.1–100 μM), BAY412272 (1 nM to 10 μM) or T-0156 (0.1–100 nM) were constructed. In de-endothelialized preparations, endothelium removal was confirmed by the absence of relaxation to 1 μM acetylcholine. Appropriate vehicle control experiments were also conducted, where vehicle effects were not observed.

Assay of SNP-induced cGMP production

To assess changes in cGMP production, a NO donor (100 μM SNP) was used to stimulate cGMP production in the presence of a non-selective phosphodiesterase inhibitor (100 μM IBMX) to prevent cGMP degradation (Toward et al., 2005; Fernandes et al., 2006). Rings from control and LPS groups were transferred to a HEPES-buffered physiological solution composed of (mM): NaCl 118, KCl 4.7, CaCl2 1.5, MgCl2 1.2, HEPES 10 and glucose 11.1) and incubated for 1 h at 37 °C. Rings were then incubated with the non-selective phosphodiesterase inhibitor 100 μM IBMX for 30 min followed by incubation with 100 μM SNP for an additional 10 min. Rings were then quickly frozen and homogenized in ice-cold 6% trichloroacetic acid to give a 10% (w/v) homogenate. Homogenates were centrifuged at 2000 g for 15 min at 4 °C, the supernatant was recovered and the pellet was discarded. The supernatant was washed four times with 5 volumes of water-saturated diethyl ether, and the upper ether layer was discarded after each wash. The remaining aqueous extract was heated at 60 °C for 10 min to remove any traces of ether, then lyophilized, and the dried extract was dissolved in a suitable volume of assay buffer. cGMP was measured in duplicate by ELISA, using a commercially available enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, UK) according to the manufacturer's instructions. Results were expressed as picomoles of cGMP per milligram of tissue weight.

Immunoblotting

After 20 h of incubation with either control or LPS, pulmonary and aortic rings were rapidly frozen in liquid nitrogen and stored at −80 °C until being used. Tissue was mechanically homogenized in 10 volumes of an ice-cold lysis buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl pH 7.5, 1% Nonidet P40, 10% glycerol, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM phenylmethanesulphonyl fluoride and 1% protease inhibitor cocktail). Homogenates were centrifuged (18 000 g for 15 min at 4 °C) and supernatant protein concentration was measured by the Bradford method using BSA as a standard. Supernatant samples were mixed with 5 × SDS sample buffer (10% SDS, 200 mM Tris-HCl, pH 6.8, 50% glycerol, 5% 2-mercaptoethanol and 0.01% bromophenol blue) and heated at 100 °C for 5 min. Protein samples (40 μg per lane) were subjected to SDS-PAGE on a 7.5% gel and transferred to nitrocellulose membrane (Whatman, Maidstone, UK) by semidry transfer blot (Transblot SD cell, Bio-Rad, CA, USA). Blots were blocked by incubation for 1 h with Tris-buffered saline-Nonidet P40 (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Nonidet P40) containing 5% non-fat milk with gentle shaking at room temperature to reduce non-specific binding. After blocking, membranes were subsequently incubated overnight at 4 °C with rabbit polyclonal anti-soluble guanylyl cyclase α1 antibody (1: 10 000 dilution, Abcam, Cambridge, UK), rabbit polyclonal anti-soluble guanylyl cyclase β1 antibody (1:4000 dilution, Abcam) or mouse monoclonal anti-phosphodiesterase 5 antibody (1:500 dilution, BD Biosciences, Oxford, UK) with gentle shaking. After washing with Tris-buffered saline-Nonidet P40 on the next day, blots were incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:10 000 dilution in Tris-buffered saline-Nonidet P40 with 2% milk; Dako Cytomation, Glostrup, Denmark). Immunoreactive protein bands were detected by ECL kit (Amersham Biosciences, Little Chalfont, Bucks, UK) and visualized on an X-ray film (Fujifilm Corporation, Tokyo, Japan). The intensity of the specific bands was quantified by densitometric analysis using Labimage software (Kapelan Bio-imaging Solutions, Halle, Germany). Membranes were reprobed with rabbit polyclonal anti-β-actin antibody (1:1000 dilution, Cell Signaling Technologies, Danvers, MA, USA) to confirm equal loading of proteins.

Data analysis

Data are expressed as mean±s.e.mean, where n equals the number of rats. Vascular relaxation was calculated as % of maximal steady state contraction induced by 30 nM ET-1. The highest response obtained was considered as the maximum response (Emax). Non-linear regression analysis (4-parameter curve fit) was carried out using Graphpad Prism software (Graphpad Software Inc., San Diego, CA, USA) and significant differences between groups were determined with paired Student's t-test or one-way ANOVA with Dunnett's post hoc test as appropriate.

Materials

Dulbecco's modified Eagle medium, penicillin–streptomycin solution and foetal bovine serum were purchased from Invitrogen (Paisley, UK), endothelin-1 (ET-1) from American Peptide Company (Sunnyvale, CA, USA), 1,2-dihydro-2-[(2-methyl-4-pyridinyl)methyl]-1-oxo-8-(2 -pyrimidinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and N-(3-aminomethyl)benzyl)acetamidine (1400W) from Tocris Bioscience (Avonmouth, UK), 3-isobutyl-1-methylxanthine (IBMX), 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY412272) and reduced nicotinamide adenosine dinucleotide phosphate (NADPH) from Calbiochem (Merck Chemicals Ltd, Nottingham, UK). Nonidet P-40, LPS (Escherichia coli O55:B5), 8-(-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate sodium salt (8-pCPT-cGMP), protease inhibitor cocktail, modified Greiss reagent (G4410), lactate dehydrogenase from rabbit muscle, nitrate reductase (Aspergillus), flavin adenine dinucleotide disodium, indomethacin and Nω-nitro-L-arginine methyl ester were purchased from Sigma-Aldrich Co. (Gillingham, Dorset, UK). Nomenclature of all receptors, drugs, enzymes and ion channels is according to the Guide to Receptors and Channels (Alexander et al., 2008).

Results

Effect of LPS treatment on NO release

Incubation of the pulmonary artery and the aorta with LPS caused a significant (5- to 10-fold) increase (P<0.01, n=5) in NO release (Figure 1). The increased levels of NO production after LPS exposure were not significantly different between the two preparations. Co-incubation of pulmonary and aortic rings with LPS together with either the non-specific NOS inhibitor nitro-L-arginine methyl ester (100 μM) or the iNOS-specific inhibitor 1400W (1 μM) prevented the LPS-induced overproduction of NO in both vessels (Figure 1).

Figure 1.

Figure 1

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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.

Effect of LPS treatment on vascular contractility

Incubation of arterial rings with LPS had a differential effect on ET-1-mediated contraction in the two types of blood vessels (Figure 2a). In the aorta, a significant decrease (P<0.01, n=9) in tissue contractility to ET-1 was observed in the LPS-treated group compared with the control group. This hyporeactivity to ET-1 was manifested both by a 26±4% decrease in Emax and an increase in EC50 in the LPS-treated group, relative to control values. In the pulmonary artery, however, no significant changes in contraction induced by ET-1 were found between control and LPS-treated groups. Similar aortic hyporeactivity to KCl and phenylephrine was also observed, without the pulmonary artery responses being affected (data not shown). In control experiments, there were no significant differences between freshly isolated and 20-h-incubated vascular rings, indicating that tissue responsiveness was not adversely influenced by prolonged incubation.

Figure 2.

Figure 2

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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 Emax and pEC50 values compared with control group using paired Student's t-test.

A decreased contractility to ET-1 in LPS-treated aorta was not affected by tissue preincubation with the iNOS-specific inhibitor 1400W (1 μM), the COX inhibitor indomethacin (10 μM) or by removal of endothelium (Table 1). Also, these treatments did not significantly alter ET-1-mediated contractility in the pulmonary artery (Table 1). In contrast, inhibition of sGC by incubation of tissues with 10 μM ODQ significantly prevented the LPS-induced decrease in contraction to ET-1 in the aorta, whereas the pulmonary artery was not affected (Figure 2b).

Table 1.

LPS-induced changes in vascular reactivity of isolated rat pulmonary artery and the aorta

Treatment Pulmonary artery
 Aorta
  Control LPS Control LPS
1400W
 pEC50 8.94±0.04 8.95±0.05 8.19±0.03 7.90±0.04**
 Emax 4.77±0.65 4.97±0.59 10.28±0.69 8.62±0.41**
         
Indomethacin
 pEC50 8.56±0.03 8.53±0.02 8.01±0.05 7.70±0.04**
 Emax 5.83±0.51 6.00±0.81 10.77±0.32 8.70±0.44**
         
Removal of endothelium
 pEC50 8.86±0.06 8.99±0.06 8.34±0.07 7.98±0.03**
 Emax 4.00±0.10 4.22±0.14 9.20±0.60 6.20±0.60**
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Data are expressed as mean±s.e.mean, n=6. Emax values are expressed in mN.

**P<0.01, significantly different compared with the corresponding control group using paired Student's t-test.

Effect of LPS treatment on vascular relaxation

Vascular relaxation to SNP was significantly decreased (P<0.01, n=6) by LPS in the pulmonary artery, as manifested by a 36±6% decrease in Emax in the LPS-treated group, relative to the control value, whereas no significant differences in SNP-induced vasorelaxation was found in the aorta (Figure 3a). In addition, vascular relaxation to the NO-independent direct sGC activator BAY412272 was significantly decreased (P<0.01, n=4) by LPS in the pulmonary artery, as manifested by a 32±8% decrease in Emax, whereas this BAY412272-induced vasorelaxation was significantly increased (P<0.01, n=4) in the aorta as manifested by a 15±7% increase in Emax (Figure 3b). To assess whether LPS-mediated pulmonary hyporeactivity to SNP was on account of decreased cGMP or impaired downstream cGMP-effector signalling molecules (such as protein kinase (PK)G); the vasorelaxation response to the phosphodiesterase-resistant cGMP analogue 8-pCPT-cGMP was studied. LPS did not significantly affect relaxation to 8-pCPT-cGMP either in the pulmonary artery or in the aorta (Figure 3c), suggesting that pulmonary hyporeactivity to SNP is likely to be because of either decreased cGMP synthesis by sGC or increased cGMP degradation (mainly by PDE5), or both.

Figure 3.

Figure 3

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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 Emax values, **P<0.01 for Emax and pEC50 values compared with control group using paired Student's t-test.

Effect of LPS treatment on SNP-induced cGMP production

Incubation with LPS significantly decreased (P<0.05, n=4) the SNP-stimulated cGMP production in the pulmonary artery by 24±4%, relative to the control value, whereas cGMP levels observed in the aorta were not significantly affected (Figure 4).

Figure 4.

Figure 4

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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.

Effect of LPS on protein expression levels of sGC subunits

In the pulmonary artery, LPS pretreatment caused a significant decrease (P<0.01, n=4) in protein expression levels of sGCβ1 subunit by 36±7% (protein density ratio to β-actin), whereas the expression of the sGCα1 subunit was not significantly affected (Figure 5). In contrast, LPS did not significantly change the protein expression levels of either sGC subunits in the aorta (Figure 5). As both subunits are required for sGC function, the decrease in the protein expression level of one subunit (sGCβ1 in the pulmonary artery) would impair sGC activity.

Figure 5.

Figure 5

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Lipopolysaccharide (LPS) decreased protein expression levels of soluble guanylyl cyclase-β1 (sGCβ1), but not sGCα1, in the pulmonary artery. (a) Representative immunoblots for sGCα1 and sGCβ1 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.

Function of PDE5 in LPS-induced changes in vasorelaxation

The function of PDE5 activity in LPS-induced impairment in vasorelaxation in the pulmonary artery was investigated using the potent and highly selective PDE5 inhibitor T-0156. Vasorelaxation in response to T-0156 was significantly increased (P<0.01, n=6) by LPS in the pulmonary artery, as manifested by a 24±4% increase in Emax in the LPS-treated group, relative to control values, but not in the aorta (Figure 6a).

Figure 6.

Figure 6

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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 Emax and pEC50 values compared with control group using paired Student's t-test.

For the protein expression levels of PDE5, two bands were obtained at 95 and 85 kDa as PDE5 is dimeric (Lin et al., 2006). LPS treatment of the pulmonary artery and the aorta did not significantly alter PDE5 protein expression levels in either vessel (Figures 6b and c). These results therefore suggest that the LPS-induced increase in the effects of T-0156 in the pulmonary artery was likely to be due to changes in PDE5 activity.

Discussion and conclusion

The results presented here show that LPS activates sGC through a NO-independent pathway in the aorta. The effect of this activation is impaired in the pulmonary artery by decreasing protein expression of sGCβ1 and by increasing PDE5 activity. As a consequence, LPS causes selective hypocontractility to ET-1 only in the aorta, whereas the pulmonary artery is not affected.

In both vessels, LPS significantly increased NO production through iNOS, as it was blocked by the selective iNOS inhibitor 1400W (1 μM). This NO overproduction by LPS through iNOS was reported previously in the aorta (Griffiths et al., 1995) and in the pulmonary artery (Bishop-Bailey et al., 1997). We found, however, that ET-1-mediated contraction was significantly impaired only in the aorta but not in the pulmonary artery, a finding similar to observation in clinical sepsis where systemic vasodilation and pulmonary hypertension occur at the same time (Lorente et al., 1993; Manthous et al., 1993) and to an ex vivo rat endotoxemic model using phenylephrine (McIntyre et al., 1997). We have examined LPS-induced hypocontractility at the 20 h time point only, but the expression levels of several relevant genes (ET-1, ETA, ETB, eNOS and iNOS) at 4, 8 and 20 h in the aorta and pulmonary artery appear to change in parallel (data not shown). This does not exclude the possibility of other LPS effects at different time points.

Neither endothelial removal nor inhibition of COX affected vascular changes induced by LPS, consistent with previous reports (McKenna, 1990; O'Brien et al., 2001), suggesting that these are not involved in LPS-induced aortic hypocontractility to ET-1. Moreover, inhibition of iNOS by 1400W was unable to prevent this aortic hypocontractility to ET-1, although the same concentration of 1400W suppressed the NO overproduction in both vessels. On the other hand, incubation with the sGC inhibitor ODQ prevented aortic LPS-induced hypocontractility to ET-1, leading us to conclude that the hypocontractility is mediated through NO-independent regulators of sGC activity. In addition, the aortic relaxation responses to the new non-NO-based haem-dependent sGC activator BAY412272 (Stasch et al., 2001; Boerrigter and Burnett, 2007) were enhanced in the aorta, confirming that LPS treatment enhances NO-independent sGC activation in the aorta. Our experiments show that inhibition of sGC could be more effective than NOS inhibition in preventing vascular hyporeactivity induced by LPS.

Lipopolysaccharide treatment in our study decreased pulmonary artery relaxation responses to both the NO donor SNP and the sGC activator BAY412272, suggesting that LPS treatment impaired the activation (both NO-dependent and NO-independent) of the sGC/cGMP pathway in the pulmonary artery. This impairment in sGC/cGMP pathway could be either due to an altered cGMP synthesis and/or metabolism or due to downstream changes in cGMP-effector signalling molecules such as PKG. Because the relaxation to the non-hydrolysable cGMP analogue 8-pCPT-cGMP was not affected in the LPS-treated vessels, cGMP-effector signalling molecules are not involved; leaving the possibility that LPS modified cGMP synthesis and/or degradation.

Involvement of a reduced synthesis of cGMP in the pulmonary artery was indicated by the decreased sGC activity and was confirmed by a significant decrease in the expression of the sGCβ1 subunit. As both sGC subunits are required for the enzyme activity, reduced levels of either subunit lead to reduced sGC activity. Importantly, sGCβ1 contains the major haem-binding domain and seems to be more rapidly and robustly regulated after inflammatory stimuli (Takata et al., 2001; Friebe and Koesling, 2003). Although previous studies demonstrate the involvement of sGC modulation in LPS-induced effects, these studies detected sGC in whole lung tissue or in isolated vascular smooth muscle cells, but not in isolated pulmonary arteries. For example, LPS decreased sGC activity and sGCβ1 protein expression in rat lung (Fernandes et al., 2006) and the expression of sGCβ1 mRNA and both sGC protein subunits in mice lung (Glynos et al., 2007). It is noteworthy that LPS decreased sGCα1 but not sGCβ1 expression in cultured rat aortic (Papapetropoulos et al., 1996) or pulmonary (Scott and Nakayama, 1998) smooth muscle cells, raising the possibility that expression of sGC subunits may be influenced by cell isolation or culture.

Besides reduced cGMP synthesis by sGC, an increased degradation of cGMP by PDE5 could also contribute to NO hyporeactivity in the pulmonary artery. This was directly confirmed using T-0156; a potent and highly selective PDE5 inhibitor (Mochida et al., 2002). As the enhanced pulmonary artery relaxation to T-0156 in LPS-treated tissue was not associated with changes in protein expression of PDE5, the most likely explanation is that the activity of PDE5 was increased. An increased PDE5 activity was demonstrated in perfused rat lungs isolated from rats 18 h after LPS injection (Holzmann et al., 1996) and 1 h after LPS inhalation in guinea pig lungs (Toward et al., 2005) but decreased after 48 h in the same model. The mechanism for increased PDE5 activity could include PKG-mediated phosphorylation and allosteric cGMP binding (either of which upregulate PDE5 activity) and a decreased PP1 phosphatase activity and/or expression (Lin et al., 2006). The latter will lead to a decreased dephosphorylation of PDE5, leading to maintained or enhanced PDE5 activity (Lin et al., 2006). As our results with the non-hydrolysable cGMP analogue suggest that downstream cGMP-effector signalling molecules are not affected by pretreatment of vessels with LPS, downregulation of PP1 phosphatase activity and/or expression seems likely.

In sepsis, excessive vasodilation is counterbalanced by increased release of vasoconstrictors such as ET-1 and angiotensin II (Knotek et al., 2000), therefore reduced sensitivity to sGC activation and increased PDE5 activity in pulmonary arteries could contribute to the development of pulmonary hypertension during sepsis. These mechanisms could also explain, at least in part, why a significant fraction (up to 30%) of patients with pulmonary hypertension fail to respond to therapeutic doses of inhaled NO (Holzmann et al., 1996).

To summarize, we have demonstrated that LPS treatment in vitro causes a selective hypocontractility of rat aorta to ET-1, which is largely mediated by NO-independent activation of sGC. The pulmonary artery is not affected because LPS induces a desensitization of the sGC/cGMP-dependent pathway by decreasing protein expression levels of sGCβ1, and hence sGC activity, and increasing PDE5 activity. Therefore, sGC and/or PDE5-selective inhibitors could be important in controlling systemic and pulmonary vasomotor complications in sepsis.

Acknowledgments

This study was supported by grant from the Egyptian Government (PhD studentship MM31/04 to MSHEA). We gratefully acknowledge the technical assistance of Kevin Smith.

Abbreviations

cGMP

3′,5′-cyclic guanosine monophosphate

ET-1

endothelin-1

iNOS

inducible NOS

LPS

lipopolysaccharide

NO

nitric oxide

PDE5

3′,5′-cyclic nucleotide phosphodiesterase-5

sGC

soluble guanylyl cyclase

SNP

sodium nitroprusside

Conflict of interest

The authors state no conflict of interest.

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