Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor β (TGF-β) signaling. However, in vivo , natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice ( n =10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT 1) -antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (∼55% reduction, P <0.05) and α-smooth muscle actin (α-SMA; ∼40% reduction, P <0.05). Moreover, the number of α-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a ‘deep serial analysis of gene expression (Deep SAGE)’ transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation.

In the present study, we investigated the vasodilator properties of A-type, B-type and C-type natriuretic peptides (ANP, BNP and CNP respectively) and the NO (nitric oxide) donor sin-1 (3-morpholino-sydnonimine) in human by-pass grafts. In contrast with previous studies, the same vessel was used to demonstrate a direct link between cGMP production and functional relaxation. Remnants of the IMA (internal mammary artery) and SV (saphenous vein) were obtained from 82 patients undergoing coronary artery by-pass grafting. The responses to cumulative concentrations of ANP, BNP, CNP and sin-1 in vessel rings pre-contracted with a thromboxane A 2 agonist (U46619) were measured in an organ bath. Additionally, intracellular cGMP production after single submaximal dose application of these drugs to vessel rings was determined by a RIA. ANP ( P =0.001) and sin-1 ( P <0.001) caused significant concentration-dependent relaxation of the IMA. In the SV, only sin-1 ( P <0.001) induced marked concentration-dependent relaxation. At a single submaximal concentration, significant relaxation as well as intracellular cGMP production were found in response to ANP, BNP and sin-1 in the IMA. In contrast, in the SV, only sin-1 significantly induced cGMP production and relaxation. There was a moderate, but significant, correlation between intracellular cGMP net production and net relaxation in the IMA. In conclusion, ANP, as the most powerful relaxant of all the natriuretic peptides tested on the IMA, may be a possible alternative vasorelaxant to overcome peri-operative vasospasm in this artery. In contrast with sin-1, ANP and BNP were not effective vasorelaxants of the SV. Net relaxation in response to natriuretic peptides correlated with cGMP net concentrations in the IMA.

Endothelin-1 (ET-1) and atrial natriuretic peptide (ANP) play important roles in the regulation of body fluid balance in congestive heart failure (CHF). Renal production of ET-1 increases in CHF and it is a significant independent predictor of sodium excretion. ANP inhibits the ET system through cGMP, a second messenger of ANP. However, in severe CHF, plasma cGMP levels reached a plateau despite the activation of ANP secretion. Thus, ANP does not seem to sufficiently oppose exaggerated ET-1 actions in severe CHF, partially due to the accelerated degradation of cGMP, through phosphodiesterase type 5 (PDE5). We examined the chronic effects of a PDE5 inhibitor, T-1032 (1mg/kg per day, n = 5), on renal function and renal production of ET-1 in dogs with CHF induced by rapid ventricular pacing (270beats/min). Vehicle dogs were given a placebo ( n = 5) and normal dogs ( n = 5) served as normal controls without pacing. In this experimentally produced CHF, plasma levels of ET-1, ANP and cGMP were elevated and renal production of cGMP was increased compared with the normal group, associated with increases in renal expression of preproET-1 mRNA and the number of ET-1-positive cells in glomeruli. In the T-1032 group, systemic and renal production of cGMP were further increased compared with the vehicle group despite no significant difference in plasma ANP levels between the two groups. Subsequently, the agent significantly improved urine flow rate, sodium excretion rate and glomerular filtration rate (GFR) associated with reductions in renal expression of preproET-1 mRNA and the number of ET-1-positive cells compared with the vehicle group. Moreover, there was a significant negative correlation between the number of ET-1-positive cells and GFR ( r =-0.802 and P <0.001 respectively). Our results indicate that chronic PDE5 inhibition ameliorates the antagonistic relationship between renal ANP and ET-1 through the cGMP pathway, subsequently preventing renal dysfunction during the progression of CHF.

Nitric oxide (NO) has diverse physiological and pathophysiological effects. The roles of NO in the renal and cardiac dysfunction found in cirrhosis are reviewed. In the kidneys of experimental animals with cirrhosis, several lines of evidence speak in favour of an enhanced production of NO, through the activation of both endothelial constitutive and inducible isoforms of NO synthase. In contrast with the situation in normal animals, inhibition of NO synthesis in rats with cirrhosis improves sodium and water excretion via blood pressure-dependent and -independent mechanisms, which indicates that the renal sodium and water retention of cirrhosis is related to an excess of NO production. The deleterious effect of excessive NO on the kidney may be mediated by peroxynitrite, a potent oxidant that is readily formed whenever superoxide anions and the ·NO radical are produced together. The peroxidation of arachidonic acid by peroxynitrite leads to the formation of F 2a -isoprostanes, which are powerful renal vasoconstrictors. F 2a -isoprostane levels are correlated with the severity of liver injury during cirrhosis. However, whether peroxynitrite or F 2a -isoprostanes are the elusive mediator of the NO-induced renal alterations in cirrhosis remains to be firmly established. NO is also involved in cardiac contractility, probably in the normal heart as well as in disease conditions such as non-cirrhotic and cirrhotic cardiomyopathy. In the latter state, evidence suggests that inducible NO synthase attenuates ventricular contractility, mediated by cGMP. Another gas that transduces its signal through cGMP, carbon monoxide, is also likely to play a role in cirrhotic cardiomyopathy, but the nature of the interaction between NO and carbon monoxide in this syndrome remains unclear.

Although gender and oestrogen treatment influence production of the vasorelaxant, NO, their influence on factors downstream in the NO signal-transduction pathway, specifically protein kinase G (PKG), remains unknown. We aimed to study the influence of sex hormones on PKG, along with the endothelial modulation of these effects, in rat thoracic aortic rings in two separate groups, control male and female rats and ovariectomized female rats after treatment with oestrogen or vehicle. Vessel preparations were preconstricted with phenylephrine (0.1 μ M). Constrictions were greater in male than female aortas. This differential effect was attenuated by endothelium removal, addition of the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3- a ]quinoxalin-1-one (ODQ, 1 μ M) and the nitric oxide synthase (NOS) inhibitor N G -monomethyl- l -arginine ( l -NMMA, 100 μ M), supporting the role of NO in maintenance of basal relaxation and vascular tone in females. We have examined the relative activity of the specific PKG subtypes 1α and 1β in vascular smooth muscle, based on relaxation of rat aortas by two cGMP analogues with different selectivity, β-phenyl-l- N 2 -ethano-8-bromo-cGMP (8-Br-PET-cGMP) and 8-(2-aminophenylthio)cGMP (8-APT-cGMP). 8-Br-PET-cGMP was more potent than 8-APT-cGMP in both sexes, suggesting that PKG 1α is the primary subtype involved in vasorelaxation. The gender differences in PKG activity were examined based on relaxation responses in male and female rat aortas. Both 8-Br-PET-cGMP and 8-APT-cGMP were more potent in aortas from male than female rats. In further studies on the endothelial modulation of relaxation with 8-APT-cGMP, the differential gender-vasorelaxation response was negated by endothelium removal and addition of the guanylate cyclase inhibitor ODQ (1 μ M), but not by the NOS inhibitor l -NMMA (100 μ M), suggesting that an endothelial-dependent factor other than NO may be responsible for the observed differential PKG-mediated vasorelaxation between the sexes. To further investigate oestrogen influence on PKG, treated female rats were studied. Contrary to our hypothesis, in the presence of 1 μ M ODQ, there were no differences in either the phenylephrine constriction, or the relaxation with 8-APT-cGMP from either sham-operated, vehicle-treated or oestrogen-treated ovariectomized rats. In conclusion, female rat aortas have greater basal NO production compared with males. Relaxant responses to PKG activation are greater in aortas from male compared with female rats. These findings suggest hormonal regulation of PKG; however, oestrogen treatment of ovariectomized rats did not affect PKG activity, suggesting factors other than oestrogen may be responsible for the gender differences noted in this study.

The research programme that started in 1985 led to the approval of Sildenafil (Viagra ® ), in 1998, as the first oral treatment for male erectile dysfunction. The initial project objective was the design and synthesis of novel inhibitors of phosphodiesterase that would increase tissue levels of cGMP, and that could be beneficial for the treatment of cardiovascular conditions. Starting from zaprinast, a weak phosphodiesterase inhibitor, computer modelling guided rational medicinal chemistry to achieve significant increases in potency and selectivity for the 5-isoenzyme within a novel series of pyrazolopyrimidinones. Optimization of structure–activity relationships and pharmacokinetic properties led to sildenafil, which proved essentially devoid of cardiovascular activity in clinical trials. However, the emerging role of nitric oxide and cGMP in controlling blood flow in the penis suggested that sildenafil would have a beneficial effect on erectile function. This hypothesis was confirmed by extensive clinical trials in nearly 5000 patients and the Food and Drug Administration approved sildenafil in March 1998 for male erectile dysfunction. Sildenafil is now available in over 100 countries and more than 150 million tablets have been dispensed worldwide. The sildenafil research programme reflects a traditional approach to drug discovery, but pressures to improve productivity have prompted major investments in genome sciences and new technologies. The impact of these initiatives on the drug discovery paradigm will be discussed, particularly with respect to shortening time scales between identifying gene sequences and submitting innovative products for regulatory approval.

This study was conducted to evaluate the influence of proinsulin C-peptide on erythrocyte Na + ,K + -ATPase and endothelial nitric oxide synthase activities in patients with type I diabetes. In a randomized double-blind study design, ten patients with type I diabetes received intravenous infusions of either human C-peptide or physiological saline on two different occasions. C-peptide was infused at a rate of 3 pmol·min -1 ·kg -1 for 60 min, and thereafter at 10 pmol·min -1 ·kg -1 for 60 min. At baseline and after 60 and 120 min, laser Doppler flow (LDF) was measured following acetylcholine iontophoresis or mild thermal stimulation (44 °C), and venous blood samples were collected to determine plasma cGMP levels and erythrocyte membrane Na + ,K + -ATPase activity. The LDF response to acetylcholine increased during C-peptide infusion and decreased during saline infusion [18.6±19.2 and -13.2±9.4 arbitrary units respectively; mean±S.E.M.; P < 0.05). No significant change in LDF was observed after thermal stimulation. The baseline plasma concentration of cGMP was 5.5±0.6 nmol·l -1 ; this rose to 6.8±0.9 nmol·l -1 during C-peptide infusion ( P < 0.05). Erythrocyte Na + ,K + -ATPase activity increased from 140±29 nmol of P i ·h -1 ·mg -1 in the basal state to 287±5 nmol of P i ·h -1 ·mg -1 during C-peptide infusion ( P < 0.01). There was a significant linear relationship between plasma C-peptide levels and erythrocyte Na + ,K + -ATPase activity during the C-peptide infusion ( r = 0.46, P < 0.01). No significant changes in plasma cGMP levels or Na + ,K + -ATPase activity were observed during saline infusion. This study demonstrates an effect of human proinsulin C-peptide on microvascular function, which might be mediated by an increase in NO production and an activation of the erythrocyte Na + ,K + -ATPase. These mechanisms are compatible with the previous observed microvascular effects of C-peptide in patients with type I diabetes.

We compared 5-hydroxytryptamine (5-HT)- and U46619-mediated contractions in bovine pulmonary conventional arteries (CA) and supernumerary arteries (SA). The effects of the NO synthase inhibitor N G -nitro- l -arginine methyl ester ( l -NAME) (100 μ M) and the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 μ M) on the responses of CA and SA to 5-HT and U46619 were also examined. In addition, the effects of the 5-HT 2B receptor antagonist SB 200646 (1 nM–1 μ M) on the responses to 5-HT in SA and CA were studied. Tissue cGMP levels were measured in the absence and presence of l -NAME, ODQ, 5-HT and U46619. 5-HT was approximately 30 times more potent in SA {-log [EC 50 (M)] (pEC 50 ) 6.32±0.13} than in CA (5.05±0.14). U46619 displayed a similar potency in both CA (pEC 50 7.80±0.07) and SA (7.75±0.12). l -NAME did not significantly alter the resting tone of CA or SA. In contrast, ODQ produced a transient increase in the tone of both CA and SA. Neither l -NAME nor ODQ altered the responses to 5-HT or U46619 in CA. In addition, neither l -NAME nor ODQ altered the responses to U46619 in SA, but both l -NAME and ODQ increased the magnitude of the response to 5-HT in SA without changing the sensitivity. Inhibition of the 5-HT 2B receptor with SB 200646 did not alter the response to 5-HT in SA or CA. Basal levels of cGMP (pmol/mg of protein) were similar in CA (1.16±0.33) and SA (0.8±0.51), and were not significantly changed in the presence of 5-HT or U46619. l -NAME and ODQ reduced the basal levels of cGMP in both SA and CA. The results suggest that endogenous NO selectively attenuates the vasoconstrictor response to 5-HT in SA, but not in CA. These results also suggest that the NO/cGMP pathway may have a role in maintaining low vascular tone, but that other mechanisms are able to compensate for the absence of this pathway.

1. Nitric oxide has platelet-stabilizing effects. Inhaled nitric oxide is used to treat pulmonary disorders, and may prolong bleeding times, suggesting that it has effects on haemostasis. We therefore examined if inhaled nitric oxide influences platelet function in vivo in healthy subjects. 2. Platelet aggregability (filtragometry ex vivo , which reflects aggregability in vivo ), bleeding time and platelet secretion products and cGMP in plasma were studied during inhalation of two different doses of nitric oxide (30 and 80 p.p.m.; 15 min at each dose level; n = 19) and during prolonged (55 min; n = 18) inhalation of 30p.p.m. nitric oxide. For comparison, studies were also performed before and after ingestion of 640 mg aspirin in 13 of the healthy subjects. 3. Plasma cGMP increased dose dependently during nitric oxide inhalation, suggesting guanylate cyclase activation in vivo. Platelet aggregability was, however, little affected and platelet secretion was not attenuated by nitric oxide inhalation. Bleeding time tended to increase (by 16–33%), but was significantly increased only after prolonged inhalation of nitric oxide at 30 p.p.m. 4. Aspirin (640 mg orally) caused pronounced and significant prolongations of filtragometry readings and bleeding time. Thus, the methods used were able to reveal platelet stabilization. 5. We conclude that nitric oxide inhalation causes only mild, if any, attenuation of platelet function in healthy subjects with a normal endogenous nitric oxide production. The effects may be different in disease states.

1. In order to determine whether atrial natriuretic peptide might play a role in the development of glomerular hyperfiltration in diabetes mellitus, we examined the effects of administration of glucose, albumin, atrial natriuretic peptide and an atrial natriuretic peptide receptor antagonist on renal function in rats with streptozotocin-induced diabetes mellitus and vehicle-treated control rats. 2. Four weeks after treatment, rats with diabetes mellitus had a higher mean plasma atrial natriuretic peptide concentration than controls [152 ± 5 (SE) versus 115 ± 6 pg/ml, P < 0.01] and a higher glomerular filtration rate (3.3 ± 0.1 versus 2.7 ± 0.2 ml min −1 kg −1 , P < 0.05). 3. Infusion of albumin or glucose caused significant increases in atrial pressure, plasma atrial natriuretic peptide concentration and urinary excretion of sodium and protein in both groups of rats. 4. Increasing plasma atrial natriuretic peptide concentration by 60% via atrial natriuretic peptide infusion increased urinary excretion of sodium and protein in both control rats and rats with diabetic mellitus. 5. Administration of the atrial natriuretic peptide receptor antagonist HS-142-1 to diabetic rats resulted in diminished urinary excretion of both sodium (−61 ± 14%, P < 0.02) and protein (−51 ± 17%, P < 0.05). These changes were associated with a significant reduction in glomerular filtration rate (−32 ± 11%, P < 0.05) and urinary cGMP excretion (−40 ± 14%, P < 0.05). No significant effects of HS-42-1 on renal function were observed in control rats. 6. These results suggest that elevated plasma glucose may lead to an increase in atrial natriuretic peptide secretion via a rise in atrial pressure, which in turn results in increased urinary sodium and protein excretion. Elevated plasma atrial natriuretic peptide appears to contribute to the proteinuria in diabetic rats.