[Show abstract][Hide abstract] ABSTRACT: We studied the influence of soluble guanylate (sGC) on renal blood flow (RBF), glomerular filtration rate (GFR), and RBF autoregulation and its role in mediating the hemodynamic effects of endogenous nitric oxide (NO). Arterial pressure (AP), heart rate (HR), RBF, GFR, urine flow (UV), and the efficiency and mechanisms of RBF autoregulation were studied in anesthetized rats during iv. infusion of sGC activator cinaciguat before and (except GFR) also after inhibition of NO-synthase (NOS) by L-NAME. Cinaciguat (0.1, 0.3, 1, 3, 10 µg*kg-1*min-1, n=7) reduced AP and increased HR, but did not significantly alter RBF. In clearance experiments (FITC-sinistrin, n=7) GFR was not significantly altered by cinaciguat (0.1 and 1 µg*kg(-1)*min(-1)), but RBF slightly rose (+12%) and filtration fraction (FF) fell (-23%). RBF autoregulatory efficiency (67 vs. 104%) and myogenic response (33 vs. 44 units) were slightly depressed (n=9). NOS inhibition (n=7) increased AP (+38 mmHg), reduced RBF (-53%), and greatly augmented the myogenic response in RBF autoregulation (97 vs. 35 units) attenuating the other regulatory mechanisms. These changes were reversed by 77%, 78%, and 90% by 1 µg*kg(-1)*min(-1) cinaciguat. In vehicle controls (n=3), in which cinaciguat-induced hypotension was mimicked by aortic compression, the NOS inhibition-induced changes were not affected. We conclude that sGC activation leaves RBF and GFR well maintained despite hypotension and only slightly impairs autoregulation. The ability to largely normalize AP, RBF, RBF autoregulation and renovascular myogenic response after NOS inhibition indicates that these hemodynamic effects of NO are predominantly mediated via sGC.
American journal of physiology. Renal physiology 09/2014; 307(9). DOI:10.1152/ajprenal.00229.2014 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The chaperone heat shock protein 90 (hsp90) associates with signaling proteins in cells including soluble guanylate cyclase (sGC). Hsp90 associates with the heme-free (apo) sGC-β1 subunit and helps to drive heme insertion as required for maturation of sGC to its nitric oxide (NO)-responsive active form. Here we found that NO caused apo-sGC-β1 to rapidly and transiently dissociate from hsp90 and associate with sGC-α1 in cells. This NO response: (i) Required that hsp90 be active and that cellular heme be available and be capable of inserting into apo-sGC-β1; (ii) was associated with an increase in sGC-β1 heme content; (iii) could be mimicked by the heme-independent sGC activator BAY 60-2770; (iv) was followed by desensitization of sGC toward NO, sGC-α1 disassociation, and reassociation with hsp90. Thus, NO promoted a rapid, transient, and hsp90-dependent heme insertion into the apo-sGC-β1 subpopulation in cells, which enabled it to combine with the sGC-α1 subunit to form the mature enzyme. The driving mechanism likely involves conformational changes near the heme site in sGC-β1 that can be mimicked by the pharmacologic sGC activator. Such dynamic interplay between hsp90, apo-sGC-β1, and sGC-α1 in response to NO is unprecedented, and represent new steps by which cells can modulate heme content and activity of sGC for signaling cascades.
[Show abstract][Hide abstract] ABSTRACT: Soluble guanylyl cyclase (sGC) plays an important role in cardiovascular function and catalyzes formation of cGMP. sGC is activated by nitric oxide and allosteric stimulators and activators. However, despite its therapeutic relevance, the regulatory mechanisms of sGC are still incompletely understood. A major reason for this situation is that no crystal structures of active sGC have been resolved so far. An important step towards this goal is the identification of high-affinity ligands that stabilize a sGC conformation resembling the active, "fully closed" state. Therefore, we examined inhibition of rat sGC α1β1 by 38 purine and pyrimidine nucleotides with 2,4,6,-trinitrophenyl- and (N-methyl)anthraniloyl substitutions at the ribosyl moiety and compared the data with those for the structurally related membranous ACs (mACs) 1, 2 and 5 and the purified mAC catalytic subunits VC1:IIC2. 2',3'-O-(2,4,6-Trinitrophenyl)-guanosine 5'-triphosphate (TNP-GTP) was the most potent sGC α1β1 inhibitor (Ki, 10.7 nM), followed by 2'-O-(N-methylanthraniloyl)-3'-deoxy-adenosine 5'-triphosphate (2'-MANT-3'-dATP) (Ki, 16.7 nM). Docking studies on a sGCαcat/sGCβcat model derived from the inactive heterodimeric crystal structure of the catalytic domains point to similar interactions of MANT- and TNP-nucleotides with sGC α1β1 and mAC VC1:IIC2. Reasonable binding modes of 2'-MANT-3'-dATP and bis-(M)ANT-nucleotides at sGC α1β1 require a 3'-endo ribosyl conformation (vs. 3'-exo in 3'-MANT-2'-dATP). Overall, inhibitory potencies of nucleotides at sGC α1β1 vs. mACs 1, 2 and 5 correlated poorly. Collectively, we identified highly potent sGC α1β1 inhibitors that may be useful for future crystallographic and fluorescence spectroscopy studies. Moreover, it may become possible to develop mAC inhibitors with selectivity relative to sGC.
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) synthesized by cardiomyocytes plays an important role in the regulation of cardiac function. Here we studied the impact of NO signalling on calcium influx in human right atrial myocytes and its relation to atrial fibrillation (AF).
Right atrial appendages (RAA) were obtained from patients in sinus rhythm (SR) and AF. The biotin-switch technique was used to evaluate endogenous S-nitrosylation of the α1C subunit of L-type calcium channels. Comparing SR to AF, S-nitrosylation of Ca(2+) channels was similar.Direct effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on L-type calcium current (ICa,L) were studied in cardiomyocytes with standard voltage-clamp techniques. In SR, ICa,L increased with SNAP (100 µM) by 48%, n/N=117/56, p<0.001). The SNAP effect on ICa,L involved activation of soluble guanylate cyclase and protein kinase A. Specific inhibition of PDE3 with cilostamide (1 µM) enhanced ICa,L to a similar extent as SNAP. However, when cAMP was elevated by PDE3 inhibition or β-adrenoceptor stimulation, SNAP reduced ICa,L, pointing to cGMP-cAMP cross-regulation. In AF, the stimulatory effect of SNAP on ICa,L was attenuated, while its inhibitory effect on isoprenaline- or cilostamide-stimulated current was preserved. cGMP elevation with SNAP was comparable between SR and AF group. Moreover, expression of PDE3 and sGC was not reduced in AF.
NO exerts dual effects on ICa,L in SR with increase of basal and inhibition of cAMP-stimulated current, and in AF NO only inhibits stimulated ICa,L. We conclude that in AF, cGMP regulation of PDE2 is preserved, but regulation of PDE3 is lost.
Cardiovascular Research 12/2013; 101(3). DOI:10.1093/cvr/cvt334 · 5.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Approved therapies for pulmonary arterial hypertension can induce oxygen desaturation when administered to patients with secondary forms of pulmonary hypertension (PH), probably due to an increase in ventilation/perfusion mismatch. Thus, so far these treatments have largely failed in secondary forms of PH.
We established an animal model of heterogeneous lung ventilation to evaluate the desaturation potential of mechanistically distinct vasoactive drugs launched or currently in clinical development for the treatment of PH. Single-lung ventilation was induced in five groups (N = 6) of anesthetized minipigs (7 weeks, 4 to 5 kg BW), and their hemodynamic parameters were monitored before and after intravenous injection of control (vehicle only), endothelin antagonist (bosentan; 0.3, 1, 3, 10 mg/kg), phosphodiesterase type 5 inhibitor (sildenafil; 3, 10, 30, 100 µg/kg), and soluble guanylate cyclase stimulators (BAY 41-8543 and riociguat; 1, 3, 10, 30 µg/kg). Cumulative doses were administered before successive unilateral ventilation cycles. The doses were chosen to achieve equal effect on blood pressure by the different pharmacologic principles.
Single-lung ventilation resulted in transient increases in mean pulmonary artery pressure (mPAP) and desaturation. In contrast to control, all drugs dose-dependently decreased hypoxic mPAP (a positive treatment effect) and increased area under the arterial hemoglobin saturation curve (unwanted desaturation effect). Riociguat and bosentan reduced hypoxic mPAP to the greatest extent, while the soluble guanylate cyclase stimulators riociguat and BAY 41-8543 lowered arterial oxygen saturation of hemoglobin the least.
Future investigations will be required to confirm these findings in clinical settings.
PLoS ONE 08/2013; 8(8):e73502. DOI:10.1371/journal.pone.0073502 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aims:
Hemoglobin-based oxygen carriers (HBOC) provide a potential alternative to red blood cell (RBC) transfusion. Their clinical application has been limited by adverse effects, in large part thought to be mediated by the intravascular scavenging of the vasodilator nitric oxide (NO) by cell-free plasma oxy-hemoglobin. Free hemoglobin may also cause endothelial dysfunction and platelet activation in hemolytic diseases and after transfusion of aged stored RBCs. The new soluble guanylate cyclase (sGC) stimulator Bay 41-8543 and sGC activator Bay 60-2770 directly modulate sGC, independent of NO bioavailability, providing a potential therapeutic mechanism to bypass hemoglobin-mediated NO inactivation.
Infusions of human hemoglobin solutions and the HBOC Oxyglobin into rats produced a severe hypertensive response, even at low plasma heme concentrations approaching 10 μM. These reactions were only observed for ferrous oxy-hemoglobin and not analogs that do not rapidly scavenge NO. Infusions of L-NG-Nitroarginine methyl ester (L-NAME), a competitive NO synthase inhibitor, after hemoglobin infusion did not produce additive vasoconstriction, suggesting that vasoconstriction is related to scavenging of vascular NO. Open-chest hemodynamic studies confirmed that hypertension occurred secondary to direct effects on increasing vascular resistance, with limited negative cardiac inotropic effects. Intravascular hemoglobin reduced the vasodilatory potency of sodium nitroprusside (SNP) and sildenafil, but had no effect on vasodilatation by direct NO-independent activation of sGC by BAY 41-8543 and BAY 60-2770.
Innovation and conclusion:
These data suggest that both sGC stimulators and sGC activators could be used to restore cyclic guanosine monophosphate-dependent vasodilation in conditions where cell-free plasma hemoglobin is sufficient to inhibit endogenous NO signaling.
[Show abstract][Hide abstract] ABSTRACT: The soluble guanylyl cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC's salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (β1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58-2667 bound structure reveals that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has about 10% higher fold maximal stimulation compared to BAY 58-2667. S-nitrosylation of the BAY 60-2770 substituted Nostoc H-NOX domain causes subtle changes in the vicinity of the S-nitrosylated C122 residue. These shifts could impact the adjacent YxSxR motif and αF helix and as such potentially inhibit either heme incorporation or NO-activation of sGC and thus provide a structural basis for desensitization.
[Show abstract][Hide abstract] ABSTRACT: To investigate the effects of the selective Rho-associated protein kinase (ROCK) inhibitor azaindole-1 on erectile function under physiologic and pathophysiologic conditions in the rat.
The effect of intracavernosal (i.c.) injections of azaindole-1 on change in intracavernous pressure (ICP), ICP/mean arterial pressure (MAP), area under the curve (AUC), and response duration were investigated in the anesthetized rat under control conditions and when nonadrenergic noncholinergic neurotransmission and cholinergic function or soluble guanylyl cyclase (sGC) were inhibited or after cavernosal nerve crush injury.
The i.c. injections of azaindole-1 produced dose-related increases in ICP/MAP and AUC that were long-lasting at the highest doses studied compared with the prototypical ROCK inhibitor fasudil. Erectile responses were not altered by 7-nitroindazole and atropine in doses that reduced the response to cavernosal nerve stimulation by 86%, indicating that they were independent of NO release by cavernosal nerves or activation of muscarinic receptors in the corpora cavernosa. Erectile responses to azaindole-1 were not altered by the sGC inhibitor ODQ in a dose that attenuated responses to the NO donor sodium nitroprusside, indicating that they were independent of an action on sGC. The erectile response to i.c. injections of azaindole-1 or Y-27632, which was reported to be NO/cyclic guanosine monophosphate-dependent, was not attenuated after cavernosal nerve crush injury.
The present studies indicate that azaindole-1 has long-lasting erectile activity that is independent of NO release, muscarinic receptor, or sGC activation or the integrity of the cavernosal nerves.
[Show abstract][Hide abstract] ABSTRACT: Background
Although evidence now suggests cGMP is a negative regulator of cardiac hypertrophy, the direct consequences of the soluble guanylyl cyclase (sGC) activator BAY 58-2667 on cardiac remodeling, independent of changes in hemodynamic load, has not been investigated. In the present study, we tested the hypothesis that the NO•-independent sGC activator BAY 58-2667 inhibits cardiomyocyte hypertrophy in vitro. Concomitant impact of BAY 58-2667 on cardiac fibroblast proliferation, and insights into potential mechanisms of action, were also sought. Results were compared to the sGC stimulator BAY 41-2272.
Neonatal rat cardiomyocytes were incubated with endothelin-1 (ET1, 60nmol/L) in the presence and absence of BAY 41-2272 and BAY 58-2667 (0.01–0.3 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. The impact of both sGC ligands on basal and stimulated cardiac fibroblast proliferation in vitro was also determined.
We now demonstrate that BAY 58-2667 (0.01–0.3 µmol/L) elicited concentration-dependent antihypertrophic actions, inhibiting ET1-mediated increases in cardiomyocyte 2D area and de novo protein synthesis, as well as suppressing ET1-induced cardiomyocyte superoxide generation. This was accompanied by potent increases in cardiomyocyte cGMP accumulation and activity of its downstream signal, vasodilator-stimulated phosphoprotein (VASP), without elevating cardiomyocyte cAMP. In contrast, submicromolar concentrations of BAY 58-2667 had no effect on basal or stimulated cardiac fibroblast proliferation. Indeed, only at concentrations ≥10 µmol/L was inhibition of cardiac fibrosis seen in vitro. The effects of BAY 58-2667 in both cell types were mimicked by BAY 41-2272.
Our results demonstrate that BAY 58-2667 elicits protective, cardiomyocyte-selective effects in vitro. These actions are associated with sGC activation and are evident in the absence of confounding hemodynamic factors, at low (submicromolar) concentrations. Thus this distinctive sGC ligand may potentially represent an alternative therapeutic approach for limiting myocardial hypertrophy.
PLoS ONE 11/2012; 7(11):e44481. DOI:10.1371/journal.pone.0044481 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction. Soluble guanylate cyclase (sGC) is the receptor for nitric oxide (NO) and in pathophysiologic conditions where NO formation or bioavailability is impaired, erectile dysfunction (ED) occurs.
Aim. The aim of this study was to investigate erectile responses to the sGC stimulator BAY 41-8543 in physiologic and pathophysiologic conditions.
Methods. Increases in intracavernosal pressure (ICP) in response to intracavernosal (ic) injections of BAY 41-8543 were investigated in the anesthetized rat.
Main Outcome Measures. Increases in ICP/MAP in response to ic injections of BAY 41-8543 and the interaction of BAY 41-8543 with exogenous and endogenously released NO were investigated and the effect of the sGC stimulator on cavernosal nerve injury was assessed. The mechanism of the increase in ICP/MAP in response to ic injection of acetylcholine was investigated.
Results. The ic injections of BAY 41-8543 increased ICP/MAP and the duration of the response. BAY 41-8543 was less potent than sodium nitroprusside (SNP) and ic injections of BAY 41-8543 and SNP produced a larger response than the algebraic sum of responses to either agent alone. Simultaneous ic injection of BAY 41-8543 and cavernosal nerve stimulation produced a greater response than either intervention alone. Atropine and cavernosal nerve crush injury decreased the response to nerve stimulation and ic injection of BAY 41-8543 restored the response.
Conclusion. These data show that BAY 41-8543 has significant erectile activity and can synergize with exogenous and endogenously released NO. This study shows that atropine and nerve crush attenuate the response to cavernosal nerve stimulation and that BAY 41-8543 can restore the response. The results with atropine, L-NAME and hexamethonium indicate that the response to ic injection of acetylcholine is mediated by muscarinic receptors and the release of NO with no significant role for nicotinic receptors. These results suggest that BAY 41-8543 would be useful in the treatment of ED. Lasker GF, Pankey EA, Allain AV, Dhaliwal JS, Stasch J-P, Murthy SN, and Kadowitz PJ. Analysis of erectile responses to BAY 41-8543 and muscarinic receptor stimulation in the rat. J Sex Med 2013;10:704–718.
Journal of Sexual Medicine 09/2012; 10(3). DOI:10.1111/j.1743-6109.2012.02912.x · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Therefore, administration of BH4 is considered a promising therapy for cardiovascular diseases associated with endothelial dysfunction and oxidative stress. Here we report on a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. Treatment of cultured porcine aortic endothelial cells with nitroglycerin (GTN) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) resulted in heme oxidation of soluble guanylate cyclase (sGC), as evident from diminished NO-induced cGMP accumulation that was paralleled by increased cGMP response to a heme- and NO-independent activator of soluble guanylate cyclase [4-([(4-carboxybutyl)[2-(5-fluoro-2-([4'-(trifluoromethyl)biphenyl-4-yl]methoxy)phenyl)ethyl]amino]methyl)benzoic acid (BAY 60-2770)]. Whereas scavenging of superoxide and/or peroxynitrite with superoxide dismutase, tiron, Mn(III)tetrakis(4-benzoic acid)porphyrin, and urate had no protective effects, supplementation of the cells with BH4, either by application of BH4 directly or of its precursors dihydrobiopterin or sepiapterin, completely prevented the inhibition of NO-induced cGMP accumulation by GTN and ODQ. Tetrahydroneopterin had the same effect, and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of sGC against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress.
[Show abstract][Hide abstract] ABSTRACT: The number of annual hospitalizations for heart failure (HF) and the mortality rates among patients hospitalized for HF remains unacceptably high. The search continues for safe and effective agents that improve outcomes when added to standard therapy. The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway serves an important physiologic role in both vascular and non-vascular tissues, including regulation of myocardial and renal function, and is disrupted in the setting of HF, leading to decreased protection against myocardial injury, ventricular remodeling, and the cardio-renal syndrome. The impaired NO-sGC-cGMP pathway signaling in HF is secondary to reduced NO bioavailability and an alteration in the redox state of sGC, making it unresponsive to NO. Accordingly, increasing directly the activity of sGC is an attractive pharmacologic strategy. With the development of two novel classes of drugs, sGC stimulators and sGC activators, the hypothesis that restoration of NO-sGC-cGMP signaling is beneficial in HF patients can now be tested. Characterization of these agents in pre-clinical and clinical studies has begun with investigations suggesting both hemodynamic effects and organ-protective properties independent of hemodynamic changes. The latter could prove valuable in long-term low-dose therapy in HF patients. This review will explain the role of the NO-sGC-cGMP pathway in HF pathophysiology and outcomes, data obtained with sGC stimulators and sGC activators in pre-clinical and clinical studies, and a plan for the further clinical development to study these agents as HF therapy.
[Show abstract][Hide abstract] ABSTRACT: Responses to a selective azaindole-based Rho kinase (ROCK) inhibitor (azaindole-1) were investigated in the rat. Intravenous injections of azaindole-1 (10-300 µg/kg), produced small decreases in pulmonary arterial pressure and larger decreases in systemic arterial pressure without changing cardiac output. Responses to azaindole-1 were slow in onset and long in duration. When baseline pulmonary vascular tone was increased with U46619 or L-NAME, the decreases in pulmonary arterial pressure in response to the ROCK inhibitor were increased. The ROCK inhibitor attenuated the increase in pulmonary arterial pressure in response to ventilatory hypoxia. Azaindole-1 decreased pulmonary and systemic arterial pressures in rats with monocrotaline-induced pulmonary hypertension. These results show that azaindole-1 has significant vasodilator activity in the pulmonary and systemic vascular beds and that responses are larger, slower in onset, and longer in duration when compared with the prototypical agent fasudil. Azaindole-1 reversed hypoxic pulmonary vasoconstriction and decreased pulmonary and systemic arterial pressures in a similar manner in rats with monocrotaline-induced pulmonary hypertension. These data suggest that ROCK is involved in regulating baseline tone in the pulmonary and systemic vascular beds, and that ROCK inhibition will promote vasodilation when tone is increased by diverse stimuli including treatment with monocrotaline.
Canadian Journal of Physiology and Pharmacology 05/2012; 90(7):825-35. DOI:10.1139/y2012-061 · 1.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cinaciguat (BAY 58-2667) is a novel nitric oxide (NO)-independent activator of soluble guanylate cyclase (sGC), which induces cGMP-generation and vasodilation in diseased vessels. We tested the hypothesis that cinaciguat might trigger protection against ischemia/reperfusion (I/R) in the heart and adult cardiomyocytes through cGMP/protein kinase G (PKG)-dependent generation of hydrogen sulfide (H(2)S). Adult New Zealand White rabbits were pretreated with 1 or 10 μg/kg cinaciguat (iv) or 10% DMSO (vehicle) 15 min before I/R or with 10 μg/kg cinaciguat (iv) at reperfusion. Additionally, adult male ICR mice were treated with either cinaciguat (10 μg/kg ip) or vehicle 30 min before I/R or at the onset of reperfusion (10 μg/kg iv). The PKG inhibitor KT5283 (KT; 1 mg/kg ip) or dl-propargylglycine (PAG; 50 mg/kg ip) the inhibitor of the H(2)S-producing enzyme cystathionine-γ-lyase (CSE) were given 10 and 30 min before cinaciguat. Cardiac function and infarct size were assessed by echocardiography and tetrazolium staining, respectively. Primary adult mouse cardiomyocytes were isolated and treated with cinaciguat before simulated ischemia/reoxygenation. Cinaciguat caused 63 and 41% reduction of infarct size when given before I/R and at reperfusion in rabbits, respectively. In mice, cinaciguat pretreatment caused a more robust 80% reduction in infarct size vs. 63% reduction when given at reperfusion and preserved cardiac function following I/R, which were blocked by KT and PAG. Cinaciguat also caused an increase in myocardial PKG activity and CSE expression. In cardiomyocytes, cinaciguat (50 nM) reduced necrosis and apoptosis and increased H(2)S levels, which was abrogated by KT. Cinaciguat is a novel molecule to induce H(2)S generation and a powerful protection against I/R injury in heart.
[Show abstract][Hide abstract] ABSTRACT: Although inhaled NO (iNO) therapy is often effective in treating infants with persistent pulmonary hypertension of the newborn (PPHN), up to 40% of patients fail to respond, which may be partly due to abnormal expression and function of soluble guanylate cyclase (sGC). To determine whether altered sGC expression or activity due to oxidized sGC contributes to high pulmonary vascular resistance (PVR) and poor NO responsiveness, we studied the effects of cinaciguat (BAY 58-2667), an sGC activator, on pulmonary artery smooth muscle cells (PASMC) from normal fetal sheep and sheep exposed to chronic intrauterine pulmonary hypertension (i.e., PPHN). We found increased sGC α(1)- and β(1)-subunit protein expression but lower basal cGMP levels in PPHN PASMC compared with normal PASMC. To determine the effects of cinaciguat and NO after sGC oxidation in vitro, we measured cGMP production by normal and PPHN PASMC treated with cinaciguat and the NO donor, sodium nitroprusside (SNP), before and after exposure to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an sGC oxidizer), hyperoxia (fraction of inspired oxygen 0.50), or hydrogen peroxide (H(2)O(2)). After treatment with ODQ, SNP-induced cGMP generation was markedly reduced but the effects of cinaciguat were increased by 14- and 64-fold in PPHN fetal PASMC, respectively (P < 0.01 vs. controls). Hyperoxia or H(2)O(2) enhanced cGMP production by cinaciguat but not SNP in PASMC. To determine the hemodynamic effects of cinaciguat in vivo, we compared serial responses to cinaciguat and ACh in fetal lambs after ductus arteriosus ligation. In contrast with the impaired vasodilator response to ACh, cinaciguat-induced pulmonary vasodilation was significantly increased. After birth, cinaciguat caused a significantly greater fall in PVR than either 100% oxygen, iNO, or ACh. We conclude that cinaciguat causes more potent pulmonary vasodilation than iNO in experimental PPHN. We speculate that increased NO-insensitive sGC may contribute to the pathogenesis of PPHN, and cinaciguat may provide a novel treatment of severe pulmonary hypertension.
[Show abstract][Hide abstract] ABSTRACT: The soluble guanylate cyclase (sGC) activator Cinaciguat (BAY 58-2667) represents a novel class of drugs that selectively activate oxidised sGC. The extent of oxidised sGC depends on the patient's oxidative burden. We here describe two platelet-based assays that allow determining the extent of oxidised sGC and thus provide a basis for an individualised pharmacotherapy.
Platelets obtained from patients with (n=12) and without (n=12) coronary artery disease (CAD) were examined by flow cytometry (P-selectin expression), and Western blots (vasodilator associated phosphoprotein, VASP-phosphorylation). Results were compared to maximal oxidation of sGC achieved by the oxidising agent ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one). Treatment of platelets with Cinaciguat resulted in differential activation of oxidised sGC. Platelet P-selectin expression and VASP-phosphorylation revealed significant differences (p=0.012, p=0.039, respectively) between CAD and non-CAD patients.
We describe platelet-based assays that allow the determination of patients' oxidative status and thus allow the prediction of pharmacological response to direct sGC activators.