Challenging the Norm: The Unrecognized Impact of Soluble Guanylyl Cyclase Subunits in Cancer.

Since the discovery of nitric oxide (NO), a long journey has led us to the present, during which much knowledge has been gained about its pathway members and their roles in physiological and various pathophysiological conditions. Soluble guanylyl cyclase (sGC), the main NO receptor composed of the sGCα1 and sGCß1 subunits, has been one of the central figures in this narrative. However, the sGCα1 and sGCß1 subunits remained obscured by the focus on sGC's enzymatic activity for many years. In this review, we restore the significance of the sGCα1 and sGCß1 subunits by compiling and analyzing available but previously overlooked information regarding their roles beyond enzymatic activity. We delve into the basics of sGC expression regulation, from its transcriptional regulation to its interaction with proteins, placing particular emphasis on evidence thus far demonstrating the actions of each sGC subunit in different tumor models. Exploring the roles of sGC subunits in cancer offers a valuable opportunity to enhance our understanding of tumor biology and discover new therapeutic avenues.

The Effect of Chronic Treatment with the Inhibitor of Phosphodiesterase 5 (PDE5), Sildenafil, in Combination with L-DOPA on Asymmetric Behavior and Monoamine Catabolism in the Striatum and Substantia Nigra of Unilaterally 6-OHDA-Lesioned Rats.

The use of phosphodiesterase inhibitors in the treatment of Parkinson's disease is currently widely discussed. The study aimed to investigate the impact of acute and chronic treatment with the phosphodiesterase 5 inhibitor, sildenafil, at low and moderate doses of 2 mg/kg and 6 mg/kg, and L-DOPA (12.5 mg/kg), alone or in combination, on asymmetric behavior and dopamine (DA) and serotonin metabolism in the striatum and substantia nigra of unilaterally 6-OHDA-lesioned rats. Acute administration of sildenafil at both tested doses jointly with L-DOPA significantly increased the number of contralateral rotations during a 2 h measurement compared to L-DOPA alone. The effect of a lower dose of sildenafil combined with L-DOPA was much greater in the second hour of measurement. However, the acute combined administration of a higher dose of sildenafil with L-DOPA resulted in an immediate and much stronger increase in the number of contralateral rotations compared to L-DOPA alone, already visible in the first hour of measurement. Interestingly, the chronic combined administration of 2 mg/kg of sildenafil and L-DOPA significantly reduced the number of contralateral rotations, especially during the first hour of measurement, compared to the long-term treatment with L-DOPA alone. Such an effect was not observed after the long-term combined treatment of a higher dose of sildenafil and L-DOPA compared to L-DOPA alone. The concentration of DA in the ipsilateral striatum and substantia nigra after the last combined chronic dose of sildenafil (2 or 6 mg/kg) and L-DOPA (12.5 mg/kg) was significantly higher than after L-DOPA alone. In spite of much stronger increases in the DA concentration in the ipsilateral striatum and substantia nigra, the number of contralateral rotations was reduced in the group of rats treated with the combination of 2 mg/kg sildenafil and L-DOPA compared to the group receiving L-DOPA alone. Moreover, the combined treatment with a low dose of sildenafil and L-DOPA had an opposite effect on DA catabolism, as assessed by DOPAC/DA and HVA/DA indexes, and these indexes were reduced in the ipsilateral striatum but increased in the contralateral striatum and substantia nigra compared to the treatment with L-DOPA alone. The results of the present study show that the addition of a low dose of a PDE5 inhibitor to the standard L-DOPA therapy differently modulates rotational behavior, the tissue DA concentration and its catabolism in the striatum and substantia nigra.

Hydrogen sulfide dysfunction in metabolic syndrome-associated vascular complications involves cGMP regulation through soluble guanylyl cyclase persulfidation.

Here, by using in vitro and ex vivo approaches, we elucidate the impairment of the hydrogen sulfide (H2S) pathway in vascular complications associated with metabolic syndrome (MetS). In the in vitro model simulating hyperlipidemic/hyperglycemic conditions, we observe significant hallmarks of endothelial dysfunction, including eNOS/NO signaling impairment, ROS overproduction, and a reduction in CSE-derived H2S. Transitioning to an ex vivo model using db/db mice, a genetic MetS model, we identify a downregulation of CBS and CSE expression in aorta, coupled with a diminished L-cysteine-induced vasorelaxation. Molecular mechanisms of eNOS/NO signaling impairment, dissected using pharmacological and molecular approaches, indicate an altered eNOS/Cav-1 ratio, along with reduced Ach- and Iso-induced vasorelaxation and increased L-NIO-induced contraction. In vivo treatment with the H2S donor Erucin ameliorates vascular dysfunction observed in db/db mice without impacting eNOS, further highlighting a specific action on smooth muscle component rather than the endothelium. Analyzing the NO signaling pathway in db/db mice aortas, reduced cGMP levels were detected, implicating a defective sGC/cGMP signaling. In vivo Erucin administration restores cGMP content. This beneficial effect involves an increased sGC activity, due to enzyme persulfidation observed in sGC overexpressed cells, coupled with PDE5 inhibition. In conclusion, our study demonstrates a pivotal role of reduced cGMP levels in impaired vasorelaxation in a murine model of MetS involving an impairment of both H2S and NO signaling. Exogenous H2S supplementation through Erucin represents a promising alternative in MetS therapy, targeting smooth muscle cells and supporting the importance of lifestyle and nutrition in managing MetS.

Identification of new multi-substituted 1H-pyrazolo[3,4-c]pyridin-7(6H)-ones as soluble guanylyl cyclase (sGC) stimulators with vasoprotective and anti-inflammatory activities.

Herein, we describe the rational design, synthesis and in vitro functional characterization of new heme-dependent, direct soluble guanylyl cyclase (sGC) agonists. These new compounds bear a 1H-pyrazolo[3,4-c]pyridin-7(6H)-one skeleton, modified to enable efficient sGC binding and stimulation. To gain insights into structure-activity relationships, the N6-alkylation of the skeleton was explored, while a pyrimidine ring, substituted with various C5'-polar groups, was installed at position C3. Among the newly synthesized 1H-pyrazolo[3,4-c]pyridin-7(6H)-ones, derivatives 14b, 15b and 16a display characteristic features of sGC "stimulators" in A7r5 vascular smooth muscle cells in vitro. They strongly synergize with the NO donor, sodium nitroprusside (SNP) in inducing cGMP generation in a manner that requires the presence of a reduced heme moiety associated with sGC, and elevate the cGMP-responsive phosphorylation of the protein VASP at Ser239. In line with their sGC stimulating capacity, docking calculations of derivatives 16a, 15(a-c) on a cryo-EM structure of human sGC (hsGC) in an ΝΟ-activated state indicated the implication of 1H-pyrazolo[3,4-c]pyridin-7(6H)-one skeleton in efficient bonding interactions with the recently identified region that binds known sGC stimulators, while the presence of either a N6-H or N6-methyl group pointed to enhanced binding affinity. Moreover, the in vitro functional effects of our newly identified sGC stimulators were compatible with a beneficial role in vascular homeostasis. Specifically, derivative 14b reduced A7r5 cell proliferation, while 16a dampened the expression of adhesion molecules ICAM-1 and P/E-Selectin in Human Umbilical Vein Endothelial Cells (HUVECs), as well as the subsequent adhesion of U937 leukocytes to the HUVECs, triggered by tumor necrosis factor alpha (TNF-α) or interleukin-1 beta (IL-1ß). The fact that these compounds elevate cGMP only in the presence of NO may indicate a novel way of interaction with the enzyme and may make them less prone than other direct sGC agonists to induce characteristic hypotension in vivo.

Soluble guanylyl cyclase beta1 subunit targets epithelial-to-mesenchymal transition and downregulates Akt pathway in human endometrial and cervical cancer cells.

Endometrial and cervical cancer are among the most frequently diagnosed malignancies globally. Nitric oxide receptor-soluble guanylyl cyclase (sGC) is a heterodimeric enzyme composed of two subunits, α1 and ß1. Previously we showed that sGCα1 subunit promotes cell survival, proliferation, and migration, but the role of sGCß1 subunit has not been addressed. The aim of the present work was to study the impact of sGCß1 restoration in proliferation, survival, migration, and cell signaling in endometrial and cervical cancer cells. We found that sGCß1 transcript levels are reduced in endometrial and cervical tumors vs normal tissues. We confirmed nuclear enrichment of sGCß1, unlike sGCα1. Overexpression of sGCß1 reduced cell viability and augmented apoptotic index. Cell migration and invasion were also negatively affected. All these sGCß1-driven effects were independent of sGC enzymatic activity. sGCß1 reduced the expression of epithelial-to-mesenchymal transition factors such as N-cadherin and ß-catenin and increased the expression of E-cadherin. sGCß1 impacted signaling in endometrial and cervical cancer cells through significant downregulation of Akt pathway affecting some of its main targets such as GSK-3ß and c-Raf. Our results show for the first time that sGCß1 exerts several antiproliferative actions in ECC-1 and HeLa cell lines by targeting key regulatory pathways.

Dosing Challenges and Benefits of Vericiguat in a Patient With Decompensated Heart Failure: A Case Report.

Vericiguat has emerged as a promising add-on therapy for decompensated heart failure with reduced ejection fraction (HFrEF) patients requiring hospitalization or IV diuretic administration. In the VICTORIA trial (Vericiguat Global Study in Subjects with Heart Failure with Reduced Ejection Fraction), vericiguat was demonstrated to significantly reduce mortality and hospitalization rates. However, the effect of vericiguat on patients receiving SGLT2 inhibitors remains uncertain. In this report, we present a complicated case of dilated heart failure receiving low doses of foundational therapy due to a patient's intolerance but still experiencing recurrent hospital readmissions. Following six months of low-dose vericiguat as an add-on therapy, the patient exhibited important improvements in various clinical parameters, including cardiac and renal function. Nonetheless, further investigation is crucial to substantiate the additional benefits of combination therapy. These findings provide further evidence for the potential benefits of vericiguat when treating HFrEF.

New Therapeutics for Heart Failure: Focusing on cGMP Signaling.

Current drugs for treating heart failure (HF), for example, angiotensin II receptor blockers and ß-blockers, possess specific target molecules involved in the regulation of the cardiac circulatory system. However, most clinically approved drugs are effective in the treatment of HF with reduced ejection fraction (HFrEF). Novel drug classes, including angiotensin receptor blocker/neprilysin inhibitor (ARNI), sodium-glucose co-transporter-2 (SGLT2) inhibitor, hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, soluble guanylyl cyclase (sGC) stimulator/activator, and cardiac myosin activator, have recently been introduced for HF intervention based on their proposed novel mechanisms. SGLT2 inhibitors have been shown to be effective not only for HFrEF but also for HF with preserved ejection fraction (HFpEF). In the myocardium, excess cyclic adenosine monophosphate (cAMP) stimulation has detrimental effects on HFrEF, whereas cyclic guanosine monophosphate (cGMP) signaling inhibits cAMP-mediated responses. Thus, molecules participating in cGMP signaling are promising targets of novel drugs for HF. In this review, we summarize molecular pathways of cGMP signaling and clinical trials of emerging drug classes targeting cGMP signaling in the treatment of HF.

Targeting Soluble Guanylyl Cyclase during Ischemia and Reperfusion.

Ischemia and reperfusion (IR) damage organs and contribute to many disease states. Few effective treatments exist that attenuate IR injury. The augmentation of nitric oxide (NO) signaling remains a promising therapeutic target for IR injury. NO binds to soluble guanylyl cyclase (sGC) to regulate vasodilation, maintain endothelial barrier integrity, and modulate inflammation through the production of cyclic-GMP in vascular smooth muscle. Pharmacologic sGC stimulators and activators have recently been developed. In preclinical studies, sGC stimulators, which augment the reduced form of sGC, and activators, which activate the oxidized non-NO binding form of sGC, increase vasodilation and decrease cardiac, cerebral, renal, pulmonary, and hepatic injury following IR. These effects may be a result of the improved regulation of perfusion and decreased oxidative injury during IR. sGC stimulators are now used clinically to treat some chronic conditions such as heart failure and pulmonary hypertension. Clinical trials of sGC activators have been terminated secondary to adverse side effects including hypotension. Additional clinical studies to investigate the effects of sGC stimulation and activation during acute conditions, such as IR, are warranted.

From End-of-Life Care to Improved Quality of Life and Better Prognosis by Using Vericiguat: A Case Report From Costa Rica.

In this case report, we present the evolution of a heart failure with reduced ejection fraction (HFrEF) patient who was set to receive end-of-life care but demonstrated improvement following treatment with vericiguat in combination with foundational therapy. Vericiguat is a novel soluble guanylate cyclase stimulant that has been proven helpful for treating decompensated heart failure with HFrEF, decreasing hospitalization rates and mortality of cardiovascular causes. This medication is currently indicated in patients who require IV diuretics administration or hospitalization due to decompensated heart failure. This is a case study of a 62-year-old woman with dilated heart failure and reduced left ventricular ejection fraction (LVEF), who was a wheelchair user due to severe cardiovascular symptoms and various comorbidities, who was referred to our heart failure program for treatment. Despite previous treatment, the patient experienced persistent cardiovascular symptoms and required palliative care. After optimizing the foundational therapy, the patient's condition improved but continued to require hospitalization. Vericiguat was initiated as an add-on. After six months, the patient's LVEF improved by 9%, and she is now asymptomatic with a considerable decrease in pro-B-type natriuretic peptide levels and is wheelchair independent due to enhance exercise resistance. However, the echocardiogram revealed a progression in the dysfunction of both the mitral and aortic valves. The patient's renal function and quality of life scores also changed over time. Vericiguat therapy, as an adjunct to foundational therapy, improved exercise tolerance and symptom relief. However, further investigation is necessary to assess the effects of vericiguat on renal function and disease progression in individuals with HFrEF.

Oxidized soluble guanylyl cyclase causes erectile dysfunction in alcoholic mice.

BACKGROUND AND PURPOSE: Alcohol abuse has been associated with erectile dysfunction (ED), but the implicated molecular mechanisms are unresolved. This study analyses the role of alterations in soluble guanylyl cyclase (sGC) in ED. EXPERIMENTAL APPROACH: ED was analysed in adult male C57BL/6J mice subjected to the Chronic Intermittent Ethanol (CIE) paradigm. Erectile function was assessed in anaesthetised mice in vivo by evaluating intracavernosal pressure (ICP) and in vitro in isolated mice corpora cavernosa (CC) mounted in a myograph. Protein expression and reactive oxygen species were analysed by western blot and dihydroethidium staining, respectively. KEY RESULTS: In CIE mice, we observed a significant decrease in the relaxant response of the CC to stimulation of NO release from nitrergic nerves by electrical field stimulation, to NO release from endothelial cells by acetylcholine, to the PDE5 inhibitor sildenafil, and to the sGC stimulator riociguat. Conversely, the response to the sGC activator cinaciguat, whose action is independent of the oxidation state of sGC, was significantly enhanced in these CC. The responses to adenylyl cyclase stimulation with forskolin were unchanged. We found an increase in reactive oxygen species in the CC from CIE mice as well as an increase in CYP2E1 and NOX2 protein expression. In vivo pre-treatment with tempol prevented alcohol-induced erectile dysfunction. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that alcoholic mice show ED in vitro and in vivo due to an alteration in the redox state of sGC and suggest that sGC activators may be effective in ED associated with alcoholism.

Inhibitory Peptide of Soluble Guanylyl Cyclase/Trx1 Interface Blunts the Dual Redox Signaling Functions of the Complex.

Soluble guanylyl cyclase (GC1) and oxido-reductase thioredoxin (Trx1) form a complex that mediates two NO signaling pathways as a function of the redox state of cells. Under physiological conditions, reduced Trx1 (rTrx1) supports the canonical NO-GC1-cGMP pathway by protecting GC1 activity from thiol oxidation. Under oxidative stress, the NO-cGMP pathway is disrupted by the S-nitrosation of GC1 (addition of a NO group to a cysteine). In turn, SNO-GC1 initiates transnitrosation cascades, using oxidized thioredoxin (oTrx1) as a nitrosothiol relay. We designed an inhibitory peptide that blocked the interaction between GC1 and Trx1. This inhibition resulted in the loss of a) the rTrx1 enhancing effect of GC1 cGMP-forming activity in vitro and in cells and its ability to reduce the multimeric oxidized GC1 and b) GC1's ability to fully reduce oTrx1, thus identifying GC1 novel reductase activity. Moreover, an inhibitory peptide blocked the transfer of S-nitrosothiols from SNO-GC1 to oTrx1. In Jurkat T cells, oTrx1 transnitrosates procaspase-3, thereby inhibiting caspase-3 activity. Using the inhibitory peptide, we demonstrated that S-nitrosation of caspase-3 is the result of a transnitrosation cascade initiated by SNO-GC1 and mediated by oTrx1. Consequently, the peptide significantly increased caspase-3 activity in Jurkat cells, providing a promising therapy for some cancers.

Dental Pulp Inflammation Initiates the Occurrence of Mast Cells Expressing the α1 and ß1 Subunits of Soluble Guanylyl Cyclase.

The binding of nitric oxide (NO) to heme in the ß1 subunit of soluble guanylyl cyclase (sGC) activates both the heterodimeric α1ß1 and α2ß1 isoforms of the enzyme, leading to the increased production of cGMP from GTP. In cultured human mast cells, exogenous NO is able to inhibit mast cell degranulation via NO-cGMP signaling. However, under inflammatory oxidative or nitrosative stress, sGC becomes insensitive to NO. The occurrence of mast cells in healthy and inflamed human tissues and the in vivo expression of the α1 and ß1 subunits of sGC in human mast cells during inflammation remain largely unresolved and were investigated here. Using peroxidase and double immunohistochemical incubations, no mast cells were found in healthy dental pulp, whereas the inflammation of dental pulp initiated the occurrence of several mast cells expressing the α1 and ß1 subunits of sGC. Since inflammation-induced oxidative and nitrosative stress oxidizes Fe2+ to Fe3+ in the ß1 subunit of sGC, leading to the desensitization of sGC to NO, we hypothesize that the NO- and heme-independent pharmacological activation of sGC in mast cells may be considered as a regulatory strategy for mast cell functions in inflamed human dental pulp.

Co-expression of soluble guanylyl cyclase subunits and PDE5A shRNA to elevate cellular cGMP level: A potential gene therapy for myocardial cell death.

BACKGROUND: Genetic manipulation on the NO-sGC-cGMP pathway has been rarely achieved, partially due to complexity of the soluble guanylyl cyclase (sGC) enzyme. OBJECTIVE: We aim to develop gene therapy directly targeting the pathway to circumvent cytotoxicity and tolerance after prolonged use of NO-donors and the insufficiency of PDE inhibitors. METHODS: In this study, we constructed lentivirus vectors expressing GUCY1A3 and GUCY1B3 genes, which encoded the α1 and ß1 subunits of soluble guanylyl cyclase (sGC), respectively, to enhance cGMP synthesis. We also constructed lentiviral vector harboring PDE5A shRNA to alleviate phosphodiesterase activity and cGMP degradation. RESULTS: Transductions of human HEK293 cells with the constructs were successful, as indicated by the fluorescent signal and altered gene expression produced by each vector. Overexpression of GUCY1A3 and GUCY1B3 resulted in increased sGC enzyme activity and elevated cGMP level in the cells. Expression of PDE5A shRNA resulted in decreased PDE5A expression and elevated cGMP level. Co-transduction of the three lentiviral vectors resulted in a more significant elevation of cGMP in HEK293 cells without obvious cytotoxicity. CONCLUSION: To the best of our knowledge, this is the first study to show that co-expression of exogenous subunits of the soluble guanylyl cyclase could form functional enzyme and increase cellular cGMP level in mammalian cells. Simultaneous expression of PDE5A shRNA could alleviate feedback up-regulation on PDE5A caused by cGMP elevation. Further studies are required to evaluate the effects of these constructs in vivo.

Complementary mechanisms of modulation of spontaneous phasic contractions by the gaseous signalling molecules NO, H2S, HNO and the polysulfide Na2S3 in the rat colon.

OBJECTIVES: Reactive oxygen and nitrogen species may be produced during inflammation leading to the formation of NO, H2S or HNO. Enzymes such as iNOS, CSE and CBS might also be responsible for polysulfide production. Since these signalling molecules might have an impact on colonic motility, the aim of this study was to compare their effect on rat colonic slow phasic contractions (SPC). METHODS: Organ bath measurements with strips obtained from rat proximal colon were performed using the polysulfide Na2S3, sodium nitroprusside (NaNP), sodium hydrogen sulfide (NaHS), Angeli's salt as NO, H2S, and HNO donors, respectively. TTX (1 µM) was used to block neuronal activity. RESULTS: All four molecules, concentration-dependently, inhibited the amplitude and frequency of SPC both in the circular and longitudinal muscle layer. The relative potency was NaNP>Angeli's salt>NaHS>Na2S3. The inhibitory response induced by NaNP (1 µM) and Angeli's salt (50 µM) was reversed by ODQ (10 µM) whereas the inhibitory effect of NaHS (1 mM) was reversed by apamin (1 µM) and glibenclamide (10 µM). Na2S3 (1 mM) response was partially reversed by apamin (1 µM) and glibenclamide (10 µM). High concentrations of Na2S3 caused an increase in tone. Low concentrations of NaHS or Na2S3 did not potentiate NaNP responses. CONCLUSIONS: All signalling molecules inhibit SPC in both muscle layers. The effect is independent of neural activity and involves guanylyl cyclase (NO and HNO) and SKCa and KATP channels (NaHS or Na2S3). Other pathways might also be involved in Na2S3 responses. Accordingly, complementary mechanisms of inhibition might be attributable to these signalling molecules.

Design, synthesis and biological evaluation of new 3,4-dihydroquinoxalin-2(1H)-one derivatives as soluble guanylyl cyclase (sGC) activators.

Herein, we present the structure-based design, synthesis and biological evaluation of novel mono- and di-carboxylic 3,4-dihydroquinoxalin-2(1H)-one derivatives as potential heme-independent activators of soluble guanylate cyclase (sGC). Docking calculations of several known sGC agonists by utilizing both a homology model of human sGC ß1 Η-ΝΟΧ domain and a recent cryo-EM structure of the same domain guided the structural optimization of various designed compounds. Among these, mono- and di-carboxylic 3,4-dihydroquinoxalin-2(1H)-one derivatives arose as promising candidate sGC activators. A series of such compounds was synthesized and assessed for their effect on sGC activity. None of them was able to trigger any detectable activation of native sGC in prostate cancer (LnCaP) or rat aortic smooth muscle (A7r5) cells, even after loss of heme by treatment with the heme oxidant ODQ. Furthermore, selected derivatives did not exhibit any antagonistic effect against the known heme-independent sGC activator BAY 60-2770 nor any additive or synergistic effect with the heme-dependent NO donor sodium nitroprusside (SNP) on heme-associated sGC in A7r5 cells. However, when tested in vitro using purified recombinant sGC enzyme, the dicarboxylic 3,4-dihydroquinoxalin-2(1H)-one derivative 30d was able to increase the enzymatic activity of both the wild-type α1/ß1 sGC dimer (by 4.4-fold, EC50 = 0.77 µΜ) as well as the heme-free α1/ß1 His105Ala mutant sGC (by 4.8-fold, EC50 = 1.8 µΜ). Notably, the activity of compound 30d towards the mutant α1/ß1 Η105A enzyme was comparable with that previously reported by us for the bona fide activator BAY 60-2770, using the functionally equivalent wild-type sGC preparation treated with ODQ. These results indicate that compound 30d can indeed act as a promising sGC activator and may serve as a basic structure in the design of novel, optimized analogues with enhanced sGC agonistic activity and improved efficiency in cell-based and in vivo systems.

Association Between Copayment and Adherence to Medications for Pulmonary Arterial Hypertension.

Background Pharmacologic treatment for pulmonary arterial hypertension (PAH) improves exercise capacity, functional class, and hemodynamic indexes. However, monthly prescription costs often exceed $4000. We examined associations between (1) medication copayment and (2) annual household income with adherence to pulmonary vasodilator therapy among individuals with PAH. Methods and Results We used administrative claims data from an insured population in the United States to identify individuals diagnosed with PAH between 2015 and 2020. All individuals had ≥1 medication claim for endothelin receptor antagonists, phosphodiesterase type-5 inhibitors, prostanoids or prostacyclin receptor agonists, or the soluble guanylate cyclase stimulator riociguat. We defined copayments as low, medium, or high, as determined by their distributions for each medication class. Annual household income was categorized as <$40 000, $40 000 to $74 999, and ≥$75 000. The primary outcome was medication adherence, defined by proportion of days covered ≥80%. We studied 4025 adults (aged 65.9±13.3 years; 71.2% women). Compared with those with annual household income ≥$75 000, individuals in the <$40 000 and $40 000 to $74 999 categories had no significant differences in medication adherence. Compared with those with low copayments, individuals with high copayments had decreased adherence to prostanoids (odds ratio [OR], 0.36 [95% CI, 0.20-0.65]; P<0.001) and combination therapy with endothelin receptor antagonist and phosphodiesterase type-5 inhibitor (OR, 0.61 [95% CI, 0.38-0.97]; P=0.03). Conclusions We identified associations between copayment and adherence to prostanoids and combination therapy among individuals with PAH. Copayment may be a structural barrier to medication adherence and merits inclusion in studies examining access to pharmacotherapy among individuals with PAH.

Soluble guanylyl cyclase: Molecular basis for ligand selectivity and action in vitro and in vivo.

Nitric oxide (NO), carbon monoxide (CO), oxygen (O2), hydrogen sulfide (H2S) are gaseous molecules that play important roles in the physiology and pathophysiology of eukaryotes. Tissue concentrations of these physiologically relevant gases vary remarkable from nM range for NO to high µM range of O2. Various hemoproteins play a significant role in sensing and transducing cellular signals encoded by gaseous molecules or in transporting them. Soluble guanylyl cyclase (sGC) is a hemoprotein that plays vital roles in a wide range of physiological functions and combines the functions of gaseous sensor and signal transducer. sGC uniquely evolved to sense low non-toxic levels of NO and respond to elevated NO levels by increasing its catalytic ability to generate the secondary signaling messenger cyclic guanosine monophosphate (cGMP). This review discusses sGC's gaseous ligand selectivity and the molecular basis for sGC function as high-affinity and selectivity NO receptor. The effects of other gaseous molecules and small molecules of cellular origin on sGC's function are also discussed.

Backbone and side chain NMR assignment of the heme-nitric oxide/oxygen binding (H-NOX) domain from Nostoc punctiforme.

Soluble guanylate cyclase (sGC) is considered as the primary NO receptor across several known eukaryotes. The main interest regarding the biological role and its function, focuses on the H-NOX domain of the ß1 subunit. This domain in its active form bears a ferrous b type heme as prosthetic group, which facilitates the binding of NO and other diatomic gases. The key point that still needs to be answered is how the protein selectively binds the NO and how the redox state of heme and coordination determines H-NOX active state upon binding of diatomic gases. H-NOX domain is present in the genomes of both prokaryotes and eukaryotes, either as a stand-alone protein domain or as a partner of a larger polypeptide. The biological functions of these signaling modules for a wide range of genomes, diverge considerably along with their ligand binding properties. In this direction, we examine the prokaryotic H-NOX protein domain from Nostoc punctiforme (Npun H-NOX). Herein, we first report the almost complete NMR backbone and side-chain resonance assignment (1H, 13C, 15 N) of Npun H-NOX domain together with the NMR chemical shift-based prediction of the domain's secondary structure elements.

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection through the purinergic P2Y13 receptor and nitric oxide signaling.

Red blood cells (RBCs) are suggested to play a role in cardiovascular regulation by exporting nitric oxide (NO) bioactivity and ATP under hypoxia. It remains unknown whether such beneficial effects of RBCs are protective in patients with acute myocardial infarction. We investigated whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia-reperfusion injury and whether such effect involves NO and purinergic signaling in the RBCs. RBCs from patients with STEMI undergoing primary coronary intervention and healthy controls were administered to isolated rat hearts subjected to global ischemia and reperfusion. Compared to RBCs from healthy controls, RBCs from STEMI patients reduced myocardial infarct size (30 ± 12% RBC healthy vs. 11 ± 5% RBC STEMI patients, P < 0.001), improved recovery of left-ventricular developed pressure and dP/dt and reduced left-ventricular end-diastolic pressure in hearts subjected to ischemia-reperfusion. Inhibition of RBC NO synthase with L-NAME or soluble guanylyl cyclase (sGC) with ODQ, and inhibition of cardiac protein kinase G (PKG) abolished the cardioprotective effect. Furthermore, the non-selective purinergic P2 receptor antagonist PPADS but not the P1 receptor antagonist 8PT attenuated the cardioprotection induced by RBCs from STEMI patients. The P2Y13 receptor was expressed in RBCs and the cardioprotection was abolished by the P2Y13 receptor antagonist MRS2211. By contrast, perfusion with PPADS, L-NAME, or ODQ prior to RBCs administration failed to block the cardioprotection induced by RBCs from STEMI patients. Administration of RBCs from healthy subjects following pre-incubation with an ATP analog reduced infarct size from 20 ± 6 to 7 ± 2% (P < 0.001), and this effect was abolished by ODQ and MRS2211. This study demonstrates a novel function of RBCs in STEMI patients providing protection against myocardial ischemia-reperfusion injury through the P2Y13 receptor and the NO-sGC-PKG pathway.