cGMP Generation Research Articles

The Impact of Natriuretic Peptides on Heart Development, Homeostasis, and Disease.

During mammalian heart development, the clustered genes encoding peptide hormones, Natriuretic Peptide A (NPPA; ANP) and B (NPPB; BNP), are transcriptionally co-regulated and co-expressed predominately in the atrial and ventricular trabecular cardiomyocytes. After birth, expression of NPPA and a natural antisense transcript NPPA-AS1 becomes restricted to the atrial cardiomyocytes. Both NPPA and NPPB are induced by cardiac stress and serve as markers for cardiovascular dysfunction or injury. NPPB gene products are extensively used as diagnostic and prognostic biomarkers for various cardiovascular disorders. Membrane-localized guanylyl cyclase receptors on many cell types throughout the body mediate the signaling of the natriuretic peptide ligands through the generation of intracellular cGMP, which interacts with and modulates the activity of cGMP-activated kinase and other enzymes and ion channels. The natriuretic peptide system plays a fundamental role in cardio-renal homeostasis, and its potent diuretic and vasodilatory effects provide compensatory mechanisms in cardiac pathophysiological conditions and heart failure. In addition, both peptides, but also CNP, have important intracardiac actions during heart development and homeostasis independent of the systemic functions. Exploration of the intracardiac functions may provide new leads for the therapeutic utility of natriuretic peptide-mediated signaling in heart diseases and rhythm disorders. Here, we review recent insights into the regulation of expression and intracardiac functions of NPPA and NPPB during heart development, homeostasis, and disease.

Isthmin-1 alleviates cardiac ischaemia/reperfusion injury through cGMP-PKG signalling pathway.

Ischaemia/reperfusion (I/R) injury is an important complication of reperfusion therapy for acute myocardial infarction, extremely compromising the cardiac benefits of revascularization; however, specific and efficient treatment for cardiac I/R injury is still lacking. Isthmin-1 (ISM1) is a novel adipokine and plays indispensable roles in regulating glycolipid metabolism and cell survival. The present study aims to investigate the potential role and molecular mechanism of ISM1 in cardiac I/R injury using gain- and loss-of-function approaches. Cardiac-specific ISM1 overexpression and silence were achieved using an adeno-associated virus serotype 9 system, and then these mice were subjected to I/R surgery, followed by biochemical test, echocardiography and histopathologic examinations, etc. Meanwhile, neonatal rat cardiomyocytes (NRCMs) with ISM1 silence or overexpression also received simulated I/R (sI/R) injury to further verify its role in vitro. The potential downstream pathways and molecular targets of ISM1 were screened by RNA sequencing. We also treated injured mice and NRCMs with recombinant ISM1 (rISM1) to explore whether supplementation with ISM1 was sufficient to protect against I/R injury. Furthermore, acute myocardial infarction patients with percutaneous coronary intervention (PCI) and paired healthy controls were included to reveal the clinical relevance of circulating ISM1. Cardiac-specific ISM1 silencing aggravated while ISM1 overexpression alleviated I/R-induced acute cardiac injury and cardiac remodelling and dysfunction. Mechanistically, ISM1 targeted αvβ5 integrin to facilitate the nuclear accumulation of nuclear transcription factor Y subunit alpha, transcriptionally increased soluble guanylyl cyclase beta subunit expression, and eventually enhanced cGMP generation. Besides, we confirmed that treatment with rISM1 before or after reperfusion could confer cardioprotective effects in mice. Clinically, lower ISM1 levels post-PCI was associated with worse outcome in patients. ISM1 can protect against cardiac I/R injury through cGMP-PKG signalling pathway, and it is a promising therapeutic and predictive target of cardiac I/R injury.

PKC Inhibition Improves Human Penile Vascular Function and the NO/cGMP Pathway in Diabetic Erectile Dysfunction: The Role of NADPH Oxidase.

Erectile dysfunction (ED) is a frequent and difficult-to-treat condition in diabetic men. Protein kinase C (PKC) is involved in diabetes-related vascular and cavernosal alterations. We aimed to evaluate the role of PKC in endothelial dysfunction and NO/cGMP impairment associated with diabetic ED in the human corpus cavernosum (CC) and penile resistance arteries (PRAs) and the potential mechanisms involved. Functional responses were determined in the CC and PRAs in patients with non-diabetic ED and diabetic ED undergoing penile prosthesis insertion. PKC activator 12,13-phorbol-dibutyrate (PDBu) impaired endothelial relaxations and cGMP generation in response to acetylcholine in the CC from non-diabetic ED. PDBu also impaired responses to a PDE5 inhibitor, sildenafil, in non-diabetic ED patients. Conversely, a PKC inhibitor, GF109203X, improved endothelial, neurogenic, and PDE5-inhibitor-induced relaxations and cGMP generation only in the CC in diabetic ED patients. Endothelial and PDE5-inhibitor-induced vasodilations of PRAs were potentiated only in diabetes. Improvements in endothelial function in diabetes were also achieved with a specific inhibitor of the PKCβ2 isoform or an NADPH-oxidase inhibitor, apocynin, which prevented PDBu-induced impairment in non-diabetic patients. PKC inhibition counteracted NO/cGMP impairment and endothelial dysfunction in diabetes-related ED, potentially improving response to PDE5 inhibition.

Protein kinase PfPK2 mediated signalling is critical for host erythrocyte invasion by malaria parasite.

Signalling pathways in malaria parasite remain poorly defined and major reason for this is the lack of understanding of the function of majority of parasite protein kinases and phosphatases in parasite signalling and its biology. In the present study, we have elucidated the function of Protein Kinase 2 (PfPK2), which is known to be indispensable for the survival of human malaria parasite Plasmodium falciparum. We demonstrate that it is involved in the invasion of host erythrocytes, which is critical for establishing infection. In addition, PfPK2 may also be involved in the maturation of the parasite post-invasion. PfPK2 regulates the release of microneme proteins like Apical Membrane Antigen 1 (AMA1), which facilitates the formation of Tight Junction between the merozoite and host erythrocyte- a key step in the process of invasion. Comparative phosphoproteomics studies revealed that PfPK2 may be involved in regulation of several key proteins involved in invasion and signalling. Furthermore, PfPK2 regulates the generation of cGMP and the release of calcium in the parasite, which are key second messengers for the process of invasion. These and other studies have shed light on a novel signalling pathway in which PfPK2 acts as an upstream regulator of important cGMP-calcium signalling, which plays an important role in parasite invasion.

Evidence for Angiotensin II as a Naturally Existing Suppressor for the Guanylyl Cyclase A Receptor and Cyclic GMP Generation.

The natriuretic peptide system (NPS) and renin-angiotensin-aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date supports this notion. This study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro. Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in vivo to determine the influence of ANGII on ANP actions. The underlying mechanisms were further explored via in vitro approaches. In humans, ANGII demonstrated an inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and the interaction term between ANGII and natriuretic peptides increased the predictive accuracy of the base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed a positive association between cGMP and ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at a physiological dose attenuated cGMP generation mediated by ANP infusion. In vitro, we found the suppressive effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), as this suppression can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using surface plasmon resonance (SPR), we showed ANGII has low binding affinity to the guanylyl cyclase A (GC-A) receptor compared to ANP or BNP. Our study reveals ANGII is a natural suppressor for the cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights the importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular protection.

Abstract 13473: Impaired Atrial Natriuretic Peptide Potency in Human Acute Decompensated Heart Failure and Therapeutic Potential of a Novel Designer Peptide (MANP) Using an Innovative Ex-Vivo Precision Medicine Assay

Introduction: Atrial natriuretic peptide (ANP) is the most potent endogenous activator of the guanylyl cyclase A receptor (GC-A) mediating cardiorenal protective actions by increasing cGMP production. To test the hypothesis that acute decompensated heart failure (ADHF) is associated with increased circulating ANP but with reduced GC-A potency, we developed an ex-vivo precision medicine assay to quantify the potency of circulating ANP in plasma from healthy and ADHF subjects. Further, we assessed the ex-vivo potency of MANP, a novel ANP analog with greater cGMP activation than ANP, also using plasma of healthy and ADHF subjects. Methods: For the potency assay, we engineered HEK293 cells to overexpress human GC-A. Plasma from individual healthy (n=4) and ADHF (n=4) subjects was incubated in the assay and cGMP was assessed. The endogenous ANP derived cGMP was evaluated against the cGMP response of equimolar synthetic ANP (S ANP ) to assess potency of ANP from each cohort. MANP mediated cGMP generation was also assessed in the assay using healthy and ADHF plasma. Results: Healthy plasma, in which plasma ANP was 26±5 pg/mL, generated 10.3±0.7 pmol/mL of cGMP. S ANP added to the assay to mimic endogenous ANP levels in healthy subjects produced 7.8±0.5 pmol/mL of cGMP. ADHF plasma with markedly elevated ANP (350±57pg/mL) generated 23.5±3.1 pmol/mL of cGMP. S ANP mimicking plasma ANP in ADHF produced 59.7±13.8 pmol/mL of cGMP, which was greater than cGMP generated from ADHF patient-derived plasma (p<0.05), consistent with reduced potency of endogenous ANP in ADHF. Low treatment dose of MANP (LD MANP ; 10 -8 M) in healthy plasma increased cGMP to 45±5.5 pmol/ml while high treatment dose (HD MANP ; 10 -6 M) increased cGMP to 167.1±14.1 pmol/mL. In markedly elevated ANP ADHF plasma, LD MANP increased cGMP to 60.4±9.7 pmol/mL while HD MANP increased cGMP to 200.8±5.4 pmol/mL (p<0.03 for both) demonstrating similar cGMP production by MANP in healthy and ADHF plasma. Conclusions: Employing a novel ex-vivo precision medicine ANP/GC-A/cGMP assay, we conclude that ADHF is characterized by elevated ANP yet with impaired potency in GC-A activation and cGMP generation. MANP overcomes this impairment and serves as a potential efficacious GC-A activating therapeutic in heart failure.

S511 Oral Nitroglycerin Solution to Treat Esophageal Food Impaction (NEFI): A Multicenter Retrospective Study

Introduction: Esophageal food impaction (EFI) is a relatively common occurrence, with estimated 13 episodes/100,000 people/year. It is initially treated with a trial of medications, such as glucagon and benzodiazepines; however, with limited success. Patients usually require an urgent endoscopy if these drugs fail. Recently, anecdotal reports of successful use of oral nitroglycerin (NTG) in EFI have been reported. The possible mechanism is NTG-induced smooth muscle relaxation by cGMP generation. However, a single-arm study found that oral NTG alleviated EFI symptoms in only 2 of the 17 subjects, and EGD showed the presence of food bolus in 12 out remaining 15 patients. This multicenter retrospective study aims to identify the effectiveness of oral NTG in EFI patients. Methods: This is a retrospective chart audit of patients who visited ER of 3 community hospitals with EFI symptoms from 1Jan 2020 to 1Nov 2021. Patients were identified by the discharge ICD-10 codes T18.128A and T18.2XXA. Inclusion criteria were adult and non-pregnant patients. Patients were excluded if they had swallowed non-food items such as metallic objects or batteries. The study was approved by IRB (1690712-1). Results: A total of 71 pts (mean age 62.52yrs) (Figure) received a maximum of 2 doses of oral NTG solution (prepared by dissolving 0.4 mg of NTG in 5-10cc of water) orally. Seventeen patients (24%) had improvement in their symptoms. The oldest patient was 102yrs old who had improvement in their symptoms and did not need urgent EGD. Twelve patients did not undergo EGD and were discharged from the ER with instructions to get elective EGD in 4-6 weeks as outpatients. Five patients who underwent EGD even after improvement in their symptoms showed the absence of food bolus in 3 patients, and the other two patients had esophagitis. A total of 13 patients (13/54 patients) who did not improve with NTG showed features of eosinophilic esophagitis. None of these patients had malignant appearing stricture on EGD. Predictors of non-response appeared to be a history of EoE and proximal impaction. Conclusion: Oral NTG solution worked in about 24% of pts presenting to ER with EFI. The efficacy of oral NTG appears to be similar to other currently used medications such as glucagon, and it can be used as an adjunct therapy when other conservative managements fail. The purpose of using NTG is not to avoid EGD but to avoid emergent EGD. Therefore, these patients should undergo EGD on the follow-up to determine the cause of food impaction.Figure 1.: Identification of included patients with esophageal food impaction

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes.

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.

PKGIα is activated by metal-dependent oxidation in vitro but not in intact cells

Type I cGMP-dependent protein kinases (PKGIs) are important components of various signaling pathways and are canonically activated by nitric oxide– and natriuretic peptide–induced cGMP generation. However, some reports have shown that PKGIα can also be activated in vitro by oxidizing agents. Using in vitro kinase assays, here, we found that purified PKGIα stored in PBS with Flag peptide became oxidized and activated even in the absence of oxidizing agent; furthermore, once established, this activation could not be reversed by reduction with DTT. We demonstrate that activation was enhanced by addition of Cu2+ before storage, indicating it was driven by oxidation and mediated by trace metals present during storage. Previous reports suggested that PKGIα Cys43, Cys118, and Cys196 play key roles in oxidation-induced kinase activation; we show that activation was reduced by C118A or C196V mutations, although C43S PKGIα activation was not reduced. In contrast, under the same conditions, purified PKGIβ activity only slightly increased with storage. Using PKGIα/PKGIβ chimeras, we found that residues throughout the PKGIα-specific autoinhibitory loop were responsible for this activation. To explore whether oxidants activate PKGIα in H9c2 and C2C12 cells, we monitored vasodilator-stimulated phosphoprotein phosphorylation downstream of PKGIα. While we observed PKGIα Cys43 crosslinking in response to H2O2 (indicating an oxidizing environment in the cells), we were unable to detect increased vasodilator-stimulated phosphoprotein phosphorylation under these conditions. Taken together, we conclude that while PKGIα can be readily activated by oxidation in vitro, there is currently no direct evidence of oxidation-induced PKGIα activation in vivo.

Cardiac Natriuretic Peptide And Cgmp Impairment In Human Decompensated Heart Failure: Employing A Novel Ex Vivo Natriuretic Peptide/GC-A/cGMP Potency Assay To Evaluate Natriuretic Peptide Therapeutic Capacity

Cardiac Natriuretic Peptide And Cgmp Impairment In Human Decompensated Heart Failure: Employing A Novel Ex Vivo Natriuretic Peptide/GC-A/cGMP Potency Assay To Evaluate Natriuretic Peptide Therapeutic Capacity

Angiotensin II As A Natural Suppressor On Cardiac Natriuretic Peptide System: Molecular Insights And Novel Therapeutic Design

<h3>Introduction</h3> Therapeutic strategies harnessing both renin-angiotensin system (RAS) and natriuretic peptide system (NPS) mediated effects, such as Sacubitril/Valsartan, has been proven to be promising in treating human heart failure (HF). However, molecular understanding of the interaction between RAS and NPS remains to be fully elucidated. While angiotensin II (ANGII), the central RAS hormone, is thought to interact with NPS at multiple levels, whether and how it may influence the actions of particulate guanylyl cyclase A (pGC-A), the only functional receptor for both atrial and b-type natriuretic peptide (ANP and BNP), has not been investigated. <h3>Hypothesis</h3> We hypothesize that ANGII naturally suppresses pGC-A and the inhibition of this crosstalk would be a novel therapeutic strategy to combat cardiovascular disease (CVD) of which RAS hyperactivation is a hallmark feature. <h3>Methods</h3> Plasma ANP, BNP, cGMP, and ANGII were measured in 128 healthy humans to define the balance between ANGII and NPS. cGMP responses to acute infusion of ANP alone and in the presence of ANGII was characterized in normal rats. The molecular mechanisms including involvement of ANGII type I receptor (AT1R) and its downstream target protein kinase C (PKC) were investigated in HEK293 cell overexpressing pGC-A and together with AT1R. CRRL-58, an innovative dual-functional peptide was designed to activate pGC-A and inhibit PKC, and tested for cGMP generation in HEK293 cells. <h3>Results</h3> In healthy human, plasma cGMP was positively associated with ANP or BNP only in low (≤ 4.5 pg/mL) but not those with high (> 4.5 pg/mL) ANGII levels (see Figure). Infusion of ANP (300 pmol/kg/min) in normal rats increased plasma and urinary cGMP, however ANP mediated cGMP generation was reduced in the presence of ANGII (50 pmol/kg/min). Notably, the suppressive effect of ANGII on pGC-A was recapitulated in HEK293 cell overexpressing both pGC-A and AT1R, but not in HEK293 cell overexpressing pGC-A alone. Meanwhile, we found PKC, a well-established downstream target of AT1R, was a key mediator for this observed crosstalk, as the suppression effect of ANGII was largely ablated by Go6983, a pan PKC inhibitor. Notably, CRRL-58 had superior potency in cGMP generation compared to the currently best-in-class ANP-analog, MANP, in vitro. <h3>Conclusions</h3> Our human and experimental data demonstrate that ANGII is a natural suppressor of pGC-A activation via AT1R and PKC signaling, but not via pGC-A per se. Moreover, we report for the first time that CRRL-58, an innovative bioengineered peptide that activates pGC-A receptor while inhibiting PKC signaling, potently generates cGMP. Together, our study provides new insights into the interaction between RAS and NPS, and highlights a novel and mechanism-based therapeutic avenue for treating HF and other CVDs.

Effect of Ramipril on Endothelin-1 Expression in Myocardial Tissue at Wistar Rats Induced Myocardial Infarction

BACKGROUND: Acute myocardial infarction occurs due to a sudden decrease in coronary blood flow caused by coronary artery embolism, coronary dissection, or coronary vasospasm. The Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor; it is synthesized and released from vascular and endocardial endothelial cells and myocytes. The action of ET-1 induces endothelial dysfunction in the coronary circulation through several mechanisms, such as reduced NO pathway activity, increased oxidative stress and inflammation, and interference with glucose and lipid metabolism. Ramipril is one of the angiotensin-converting enzyme inhibitors (ACE-I) that can reduce the formation of ET-1 by enhancing the NO expression. NO can down-regulate the ET-1 secretion through soluble guanylate cyclase activation and increased cellular generation of cGMP. AIM: This study aimed to investigate the effect of Ramipril on ET-1 expression in rats-induced myocardial infarction. METHODS: Six-week-old male Wistar rats were randomly allocated into three groups: negative control, positive control was given NaCl 0.9% and treatment group treated with ramipril 3 mg/kg/day orally for 7 days. Myocardial infarction was induced in positive and treatment group by subcutaneous injection of isoproterenol, and 24 h after the last administration, rats were sacrificed to evaluate the relative expression of ET-1 using the real-time polymerase chain reaction and 2-ΔΔCt method. RESULTS: The average expression for the negative control was 0.0098, positive control was 0.0136 and treatment group was 0.0118, with p = 0.210 (p &gt; 0.05). CONCLUSION: Our data suggest that there is no difference between groups for the relative expression of ET-1.

Discovery of small molecule guanylyl cyclase A receptor positive allosteric modulators

The particulate guanylyl cyclase A receptor (GC-A), via activation by its endogenous ligands atrial natriuretic peptide (ANP) and b-type natriuretic peptide (BNP), possesses beneficial biological properties such as blood pressure regulation, natriuresis, suppression of adverse remodeling, inhibition of the renin-angiotensin-aldosterone system, and favorable metabolic actions through the generation of its second messenger cyclic guanosine monophosphate (cGMP). Thus, the GC-A represents an important molecular therapeutic target for cardiovascular disease and its associated risk factors. However, a small molecule that is orally bioavailable and directly targets the GC-A to potentiate cGMP has yet to be discovered. Here, we performed a cell-based high-throughput screening campaign of the NIH Molecular Libraries Small Molecule Repository, and we successfully identified small molecule GC-A positive allosteric modulator (PAM) scaffolds. Further medicinal chemistry structure-activity relationship efforts of the lead scaffold resulted in the development of a GC-A PAM, MCUF-651, which enhanced ANP-mediated cGMP generation in human cardiac, renal, and fat cells and inhibited cardiomyocyte hypertrophy invitro. Further, binding analysis confirmed MCUF-651 binds to GC-A and selectively enhances the binding of ANP to GC-A. Moreover, MCUF-651 is orally bioavailable in mice and enhances the ability of endogenous ANP and BNP, found in the plasma of normal subjects and patients with hypertension or heart failure, to generate GC-A-mediated cGMP ex vivo. In this work, we report the discovery and development of an oral, small molecule GC-A PAM that holds great potential as a therapeutic for cardiovascular, renal, and metabolic diseases.

Abstract 10303: CRRL290: A Novel and Mechanism-Based Therapeutic Peptide Targeting the Crosstalk Between Angiotensin II and the Natriuretic Peptide System for Cardiorenal Disease

Introduction: Therapeutic strategies dual-targeting renin-angiotensin system (RAS) and natriuretic peptide system (NPS), such as Sacubitril/Valsartan, have been proven promising in treating cardiorenal disease (CRD). However, mechanisms behind the crosstalk between RAS and NPS remain to be fully elucidated. While angiotensin II (ANGII), the central RAS hormone, is thought to interact with NPS at multiple levels, whether and how it may influence the actions of particulate guanylyl cyclase A (pGC-A), the receptor activated by ANP and BNP to increase its second messenger cGMP, has not been investigated. Hypothesis: We hypothesize ANGII naturally suppresses pGC-A activity and inhibition of this ANGII mediated action can be a new strategy to combat CRD. Methods: Studies consist of biomarker analyses in plasma from healthy humans, drug infusions in normal rats, molecular characterization in human HEK293 cells, and in vitro assessment of CRRL290, an innovative dual-functional peptide designed to activate pGC-A and inhibit protein kinase C (PKC). Results: In 128 healthy humans, plasma cGMP was positively associated with ANP or BNP in low (≤ 4.5 pg/mL) but not those with high (&gt; 4.5 pg/mL) ANGII levels. Infusion of ANP (300 pmol/kg/min) in normal rats increased plasma and urinary cGMP, however ANP mediated cGMP generation was reduced in the presence of ANGII. Interestingly, suppression effect of ANGII on pGC-A was only recapitulated in HEK293 cell overexpressing both pGC-A and AT1R, but not in HEK293 cell overexpressing pGC-A alone. We also found PKC, a known downstream target of AT1R, was a key mediator as the suppression effect of ANGII was largely ablated by Go6983, a pan PKC inhibitor. Accordingly, we designed and synthesized CRRL290. Notably, CRRL290 had superior potency in cGMP generation compared to the currently best-in-class ANP-analog, MANP, in HEK293 cells. Conclusions: We demonstrate that ANGII is a natural suppressor of pGC-A via AT1R and PKC signaling. We also report for the first time that CRRL290, a novel bioengineered peptide that activates pGC-A receptor while inhibiting PKC signaling, potently generates cGMP. Our study provides new insights into the RAS-NPS interaction, and highlights a novel and mechanism-based therapeutic avenue for treating CRD.

Blockade of the natriuretic peptide clearance receptor attenuates proteinuria in a mouse model of focal segmental glomerulosclerosis.

Glomerular podocytes play a key role in proteinuric diseases. Accumulating evidence suggests that cGMP signaling has podocyte protective effects. The major source of cGMP generation in podocytes is natriuretic peptides. The natriuretic peptide clearance receptor (NPRC) binds and degrades natriuretic peptides. As a result, NPRC inhibits natriuretic peptide‐induced cGMP generation. To enhance cGMP generation in podocytes, we blocked natriuretic peptide clearance using the specific NPRC ligand ANP(4‐23). We then studied the effects of NPRC blockade in both cultured podocytes and in a mouse transgenic (TG) model of focal segmental glomerulosclerosis (FSGS) created in our laboratory. In this model, a single dose of the podocyte toxin puromycin aminonucleoside (PAN) causes robust albuminuria in TG mice, but only mild disease in non‐TG animals. We found that natriuretic peptides protected cultured podocytes from PAN‐induced apoptosis, and that ANP(4‐23) enhanced natriuretic peptide‐induced cGMP generation in vivo. PAN‐induced heavy proteinuria in vehicle‐treated TG mice, and this increase in albuminuria was reduced by treatment with ANP(4‐23). Treatment with ANP(4‐23) also reduced the number of mice with glomerular injury and enhanced urinary cGMP excretion, but these differences were not statistically significant. Systolic BP was similar in vehicle and ANP(4‐23)‐treated mice. These data suggest that: 1. Pharmacologic blockade of NPRC may be useful for treating glomerular diseases such as FSGS, and 2. Treatment outcomes might be improved by optimizing NPRC blockade to inhibit natriuretic peptide clearance more effectively.

Nitric Oxide Signaling in the Auditory Pathway.

Nitric oxide (NO) is of fundamental importance in regulating immune, cardiovascular, reproductive, neuromuscular, and nervous system function. It is rapidly synthesized and cannot be confined, it is highly reactive, so its lifetime is measured in seconds. These distinctive properties (contrasting with classical neurotransmitters and neuromodulators) give rise to the concept of NO as a “volume transmitter,” where it is generated from an active source, diffuses to interact with proteins and receptors within a sphere of influence or volume, but limited in distance and time by its short half-life. In the auditory system, the neuronal NO-synthetizing enzyme, nNOS, is highly expressed and tightly coupled to postsynaptic calcium influx at excitatory synapses. This provides a powerful activity-dependent control of postsynaptic intrinsic excitability via cGMP generation, protein kinase G activation and modulation of voltage-gated conductances. NO may also regulate vesicle mobility via retrograde signaling. This Mini Review focuses on the auditory system, but highlights general mechanisms by which NO mediates neuronal intrinsic plasticity and synaptic transmission. The dependence of NO generation on synaptic and sound-evoked activity has important local modulatory actions and NO serves as a “volume transmitter” in the auditory brainstem. It also has potentially destructive consequences during intense activity or on spill-over from other NO sources during pathological conditions, when aberrant signaling may interfere with the precisely timed and tonotopically organized auditory system.

Novel soluble guanylyl cyclase activators increase glomerular cGMP, induce vasodilation and improve blood flow in the murine kidney.

Background and PurposeGeneration of cGMP via NO‐sensitive soluble guanylyl cyclase (sGC) has been implicated in the regulation of renal functions. Chronic kidney disease (CKD) is associated with decreased NO bioavailability, increased oxidative stress and oxidation of sGC to its haem‐free form, apo‐sGC. Apo‐sGC cannot be activated by NO, resulting in impaired cGMP signalling that is associated with chronic kidney disease progression. We hypothesised that sGC activators, which activate apo‐sGC independently of NO, increase renal cGMP production under conditions of oxidative stress, thereby improving renal blood flow (RBF) and kidney function.Experimental ApproachTwo novel sGC activators, runcaciguat and BAY‐543, were tested on murine kidney. We measured cGMP levels in real time in kidney slices of cGMP sensor mice, vasodilation of pre‐constricted glomerular arterioles and RBF in isolated perfused kidneys. Experiments were performed at baseline conditions, under L‐NAME‐induced NO deficiency, and in the presence of oxidative stress induced by ODQ.Key ResultsMouse glomeruli showed NO‐induced cGMP increases. Under baseline conditions, sGC activator did not alter glomerular cGMP concentration or NO‐induced cGMP generation. In the presence of ODQ, NO‐induced glomerular cGMP signals were markedly reduced, whereas sGC activator induced strong cGMP increases. L‐NAME and ODQ pretreated isolated glomerular arterioles were strongly dilated by sGC activator. sGC activator also increased cGMP and RBF in ODQ‐perfused kidneys.Conclusion and ImplicationsGC activators increase glomerular cGMP, dilate glomerular arterioles and improve RBF under disease‐relevant oxidative stress conditions. Therefore, sGC activators represent a promising class of drugs for chronic kidney disease treatment.LINKED ARTICLESThis article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc

Activation of the iNOS/NO/cGMP pathway by Revactin® in human corporal smooth muscle cells

BackgroundThe combination of the nutraceuticals, Paullinia cupana, ginger rhizome, muira puama, and the amino acid L-citrulline (COMP-4) has been shown to stimulate the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and cGMP in rat corpora cavernosa smooth muscle cells (CSMC). When administered to middle-aged rats, long-term treatment with COMP-4 resulted in both an increase in the number of CSMC and an improvement in erectile function. We, therefore, aimed to determine whether a commercial formulation of COMP-4, Revactin®, could have a similar stimulatory effect on human CSMC.MethodsPrimary human CSMC cultures (HCSMC) were grown and incubated with Revactin® for up to 24 hours. cGMP generation and nitrite formation were determined by ELISA and Griess reaction, respectively. IBMX (1 mM), sildenafil (0.4 mM), and L-NIL (4 µM) were utilized as modulators of the NO-cGMP pathway. iNOS, endothelial NOS (eNOS), and neuronal NOS (nNOS) expressions were determined by Western blot.ResultsRevactin® up-regulated both nitrite formation and cGMP expression, achieving the highest expression at 24 hours in the HCSMC. These effects were completely blocked by L-NIL. Revactin® up-regulated iNOS expression, but not that of eNOS or nNOS.ConclusionsThe results presented in this study confirmed that Revactin® activated the iNOS-NO-cGMP pathway intracellularly in HCSMC. It still needs to be determined whether the upregulation of this pathway would be an effective approach for counteracting the fibrosis and apoptosis of the corporal smooth muscle cells associated with aging.

Structural Perspectives on the Mechanism of Soluble Guanylate Cyclase Activation

The enzyme soluble guanylate cyclase (sGC) is the prototypical nitric oxide (NO) receptor in humans and other higher eukaryotes and is responsible for transducing the initial NO signal to the secondary messenger cyclic guanosine monophosphate (cGMP). Generation of cGMP in turn leads to diverse physiological effects in the cardiopulmonary, vascular, and neurological systems. Given these important downstream effects, sGC has been biochemically characterized in great detail in the four decades since its discovery. Structures of full-length sGC, however, have proven elusive until very recently. In 2019, advances in single particle cryo–electron microscopy (cryo-EM) enabled visualization of full-length sGC for the first time. This review will summarize insights revealed by the structures of sGC in the unactivated and activated states and discuss their implications in the mechanism of sGC activation.

Replacement of heme by soluble guanylate cyclase (sGC) activators abolishes heme-nitric oxide/oxygen (H-NOX) domain structural plasticity.

The gasotransmitter nitric oxide (NO) is a critical endogenous regulator of homeostasis, in major part via the generation of cGMP (cyclic guanosine monophosphate) from GTP (guanosine triphosphate) by NO's main physiological receptor, the soluble guanylate cyclase (sGC). sGC is a heterodimer, composed of an α1 and a β1 subunit, of which the latter contains the heme-nitric oxide/oxygen (H-NOX) domain, responsible for NO recognition, binding and signal initiation. The NO/sGC/cGMP axis is dysfunctional in a variety of diseases, including hypertension and heart failure, especially since oxidative stress results in heme oxidation, sGC unresponsiveness to NO and subsequent degradation. As a central player in this axis, sGC is the focus of intense research efforts aiming to develop therapeutic molecules that enhance its activity. A class of drugs named sGC “activators” aim to replace the oxidized heme of the H-NOX domain, thus stabilizing the enzyme and restoring its activity. Although numerous studies outline the pharmacology and binding behavior of these compounds, the static 3D models available so far do not allow a satisfactory understanding of the structural basis of sGC's activation mechanism by these drugs. Herein, application NMR describes different conformational states during the replacement of the heme by a sGC activators. We show that the two sGC activators (BAY 58-2667 and BAY 60-2770) significantly decrease the conformational plasticity of the recombinant H-NOX protein domain of Nostoc sp. cyanobacterium, rendering it a lot more rigid compared to the heme-occupied H-NOX. NMR methodology also reveals, for the first time, a surprising bi-directional competition between reduced heme and these compounds, pointing to a highly dynamic regulation of the H-NOX domain. This competitive, bi-directional mode of interaction is also confirmed by monitoring cGMP generation in A7r5 vascular smooth muscle cells by these activators. We show that, surprisingly, heme's redox state impacts differently the bioactivity of these two structurally similar compounds. In all, by NMR-based and functional approaches we contribute unique experimental insight into the dynamic interaction of sGC activators with the H-NOX domain and its dependence on the heme redox status, with the ultimate goal to permit a better design of such therapeutically important molecules.