Messenger cGMP Research Articles

Safety, tolerability and blood pressure lowering of the innovative guanylyl cyclase a-receptor activator MANP in an ethnically diverse resistant hypertension population: a phase 1B clinical trial

Abstract Background/Introduction MANP is a novel atrial natriuretic peptide (ANP) analog bioengineered as a particulate guanylyl cyclase A receptor (GC-A) activator with superior receptor binding, resistance to neprilysin degradation, natriuretic, aldosterone suppressing and blood pressure (BP) lowering properties than native ANP. MANP is now under clinical development for resistant hypertension (RH). The rationale for its design was the fundamental role of the ANP/GC-A system in BP homeostasis through which GC-A activation with MANP leads to BP reduction through renal, vascular, and hormonal mechanisms mediated by the second messenger cGMP. Purpose We investigated the safety, tolerability, cGMP activation and BP reductions of MANP compared to placebo in subjects with RH. Methods We performed a double-blind, placebo controlled randomized trial in 36 RH patients comprised of approximately 50% non-African Americans (NAA) and 50% AA. MANP was administered in 6 cohorts receiving ascending subcutaneous (SQ) doses of MANP once daily from 3 ug/kg to a maximal dose of 7 ug/kg or placebo for 5 days and then followed for 21 days after dosing. Endpoints included safety, cGMP activation and changes in systolic BP (SBP), aldosterone (Aldo) levels and glomerular filtration rate (GFR). Results MANP was safe and well tolerated. One patient in the 7 ug/kg cohort was discontinued due to symptomatic hypotension. Compared to baseline, plasma cGMP increased in all MANP groups on Day 1 and Day 5 with no change in the placebo group. The maximal SBP reduction in the MANP group on Days 1 (D1) and 5 (D5) was -17.5 (D1) and -16 mmHg (D5) with 3 ug/kg (lowest dose) and -39.5 (D1) and -36.3 mmHg (D5) with 7 ug/kg (highest dose); Changes in the Placebo group were -6.3 (D1) and -16.7 mmHg (D5). Notably, the SBP reductions in AA were greater than NAA in the 4 and 7 ug/kg cohorts. After the 1-hour timepoint on both the first and fifth days, Aldo and percentage of Aldo reduction generally remained suppressed compared to baseline. Twenty-one days after MANP dosing, SBP remained lower in three groups receiving SQ MANP (4, 4.5 and 7 ug/kg) compared to placebo in association with a preservation of GFR. There were no reported drug related serious adverse events. Conclusions This clinical trial demonstrates that once a day SQ administration of MANP in RH subjects activates cGMP, without attenuation over 5 days, and reduces SBP and preserves GFR. At higher doses of MANP, SBP reductions were greater in AA compared to NAA. MANP also demonstrated Aldo inhibiting actions compared to placebo. Importantly, MANP was overall safe and well-tolerated. Our findings support the continued clinical development of MANP as a novel GC-A activating peptide for the treatment of RH.

Discovery and optimization of 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine derivatives as novel phosphodiesterase 4 inhibitors.

Phosphodiesterases (PDEs) are important intracellular enzymes that hydrolyze the second messengers cAMP and/or cGMP. Now several studies have shown that PDE4 received particular attention due to which it represents the most prominent cAMP-metabolizing enzyme involved in many diseases. In this study, we performed prescreening of our internal compound library and discovered the compound (PTC-209) with moderate PDE4 inhibitory activity (IC50 of 4.78 ± 0.08μM). And a series of 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine derivatives as novel PDE4 inhibitors starting from PTC-209 were successfully designed and synthesized using a structure-based discovery strategy. L19, the most potent inhibitor, exhibited good inhibitory activity (IC50 of 0.48 ± 0.02μM) and remarkable metabolic stability in rat liver microsomes. Our study presents an example of discovery novel PDE4 inhibitors, which would be helpful for design and optimization of novel inhibitors in future.

Discovery of novel selective phosphodiesterase‑1 inhibitors for the treatment of acute myelogenous leukemia

Acute myelogenous leukemia (AML) is the most common form of acute leukemia in adults. PDE1 (Phosphodiesterase 1) is a subfamily of the PDE super-enzyme families that can hydrolyze the second messengers cAMP and cGMP simultaneously. Previous research has shown that suppressing the gene expression of PDE1 can trigger apoptosis of human leukemia cells. However, no selective PDE1 inhibitors have been used to explore whether PDE1 is a potential target for treating AML. Based on our previously reported PDE9/PDE1 dual inhibitor 11a, a series of novel pyrazolopyrimidinone derivatives were designed in this study. The lead compound 6c showed an IC50 of 7.5 nM against PDE1, excellent selectivity over other PDEs and good metabolic stability. In AML cells, compound 6c significantly inhibited the proliferation and induced apoptosis. Further experiments indicated that the apoptosis induced by 6c was through a mitochondria-dependent pathway by decreasing the ratio of Bcl-2/Bax and increasing the cleavage of caspase-3, 7, 9, and PARP. All these results suggested that PDE1 might be a novel target for AML.

Analysis of cGMP Signaling in Dictyostelium.

Biochemical assays are described to analyze signal transduction by the second messenger cGMP in Dictyostelium. The methods include enzyme assays to measure the activity and regulation of cGMP synthesizing guanylyl cyclases and cGMP-degrading phosphodiesterases. In addition, several methods are described to quantify cGMP levels. The target of cGMP in Dictyostelium is the large protein GbpC that has multiple domains including a Roc domain, a kinase domain, and a cGMP-stimulated Ras-GEF domain. A cGMP-binding assay is described to detect and quantify GbpC.

The Functionality of the DC Pair in a Rhodopsin Guanylyl Cyclase from Catenaria anguillulae

Rhodopsin guanylyl cyclases (RGCs) belong to the class of enzymerhodopsins catalyzing the transition from GTP into the second messenger cGMP, whereas light-regulation of enzyme activity is mediated by a membrane-bound microbial rhodopsin domain, that holds the catalytic center inactive in the dark. Structural determinants for activation of the rhodopsin moiety eventually leading to catalytic activity are largely unknown. Here, we investigate the mechanistic role of the D283-C259 (DC) pair that is hydrogen bonded via a water molecule as a crucial functional motif in the homodimeric C. anguillulae RGC. Based on a structural model of the DC pair in the retinal binding pocket obtained by MD simulation, we analyzed formation and kinetics of early and late photocycle intermediates of the rhodopsin domain wild type and specific DC pair mutants by combined UV–Vis and FTIR spectroscopy at ambient and cryo-temperatures. By assigning specific infrared bands to S-H vibrations of C259 we are able to show that the DC pair residues are tightly coupled. We show that deprotonation of D283 occurs already in the inactive L state as a prerequisite for M state formation, whereas structural changes of C259 occur in the active M state and early cryo-trapped intermediates. We propose a comprehensive molecular model for formation of the M state that activates the catalytic moiety. It involves light induced changes in bond strength and hydrogen bonding of the DC pair residues from the early J state to the active M state and explains the retarding effect of C259 mutants.

Recombinant protein delivery enables modulation of the phototransduction cascade in mouse retina

Inherited retinal dystrophies are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca2+. Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein 1 (GCAP1) were compared in biological systems of increasing complexity (in cyto, ex vivo, and in vivo). The analysis of protein biodistribution and the direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful complementary tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases.

Nitric oxide signaling pathways in the normal and pathological bladder: Do they provide new pharmacological pathways?-ICI-RS 2023.

The nitric oxide (NO•)/soluble guanylate cyclase/cyclic-GMP (cGMP) signaling pathway is ubiquitous and regulates several functions in physiological systems as diverse as the vascular, nervous, and renal systems. However, its roles in determining normal and abnormal lower urinary tract functions are unclear. The aim was to identify potential therapeutic targets associated with this pathway to manage lower urinary tract functional disorders. This review summarizes a workshop held under the auspices of ICI-RS with a view to address these questions. Four areas were addressed: NO• signaling to regulate neurotransmitter release to detrusor smooth muscle; its potential dual roles in alleviating and exacerbating inflammatory pathways; its ability to act as an antifibrotic mediator; and the control by nitrergic nerves of lower urinary tract vascular dynamics and the contractile performance of muscular regions of the bladder wall. Central to much of the discussion was the role of the NO• receptor, soluble guanylate cyclase (sGC) in regulating the generation of the enzyme product, the second messenger cGMP. The redox state of sGC is crucial in determining its enzymic activity and the role of a class of novel agents, sGC activators, to optimize activity and to potentially alleviate the consequences of lower urinary tract disorders was highlighted. In addition, the consequences of a functional relationship between nitrergic and sympathetic nerves to regulate vascular dynamics was discussed. Several potential NO•-dependent drug targets in the lower urinary tract were identified that provide the basis for future research and translation to clinical trials.

Buxu Tongyu Granule Alleviates Myocardial Ischemia by Activating Vascular Smooth Muscle Cell Soluble Guanylate Cyclase to Inhibit Abnormal Vasomotion

Myocardial ischemia is a serious threat to human health, and vascular dysfunction is its main cause. Buxu Tongyu (BXTY) Granule is an effective traditional Chinese medicine (TCM) for treating myocardial ischemia. However, the underlying mechanism of BXTY is still unclear. In this study, we demonstrate that BXTY ameliorates myocardial ischemia by activating the soluble guanylate cyclase (sGC)–3′,5′-cyclic guanosine monophosphate (cGMP)–protein kinase G (PKG) signaling pathway in vascular smooth muscle cells (VSMCs) to dilate the arteries. BXTY was given by gavage for ten consecutive days before establishing an animal model of acute myocardial ischemia in mice via the intraperitoneal injection of pituitrin. The results showed that BXTY alleviated the symptoms of myocardial ischemia induced by pituitrin in mice, including electrocardiogram abnormalities and changes in plasma enzymes. In addition, BXTY dilated pre-constricted blood vessels and inhibited the vasoconstriction of the superior mesenteric artery in a dose-dependent but endothelial-independent manner. These effects were eliminated by pre-incubating vascular rings with the sGC inhibitors NS 2028 or ODQ, or with the PKG inhibitor KT 5823. Moreover, BXTY increased the protein expression of sGC-β1 and the intracellular second messenger cGMP level in mouse aortic vascular smooth muscle cells (MOVAs). NS 2028 or ODQ reversed these effects of BXTY. The expression level of the cGMP downstream effector protein PKG-1 increased after treating MOVAs with BXTY. NS 2028, ODQ, or KT 5823 also reversed this effect of BXTY. In conclusion, BXTY can improve the symptoms of acute myocardial ischemia in mice, and activating the sGC–cGMP–PKG pathway in VSMCs to induce vasodilation is its key pharmacodynamic mechanism.

Molecular tuning of calcium dependent processes by neuronal calcium sensor proteins in the retina

Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination mediated by phototransduction, which is under control of the two secondary messengers cGMP and Ca2+. Feedback mechanisms enable photoreceptor cells to regain their responsiveness after light stimulation and involve neuronal Ca2+-sensor proteins, named GCAPs (guanylate cyclase-activating proteins) and recoverins. This review compares the diversity in Ca2+-related signaling mediated by GCAP and recoverin variants that exhibit differences in Ca2+-sensing, protein conformational changes, myristoyl switch mechanisms, diversity in divalent cation binding and dimer formation. In summary, both subclasses of neuronal Ca2+-sensor proteins contribute to a complex signaling network in rod and cone cells, which is perfectly suited to match the requirements for sensitive cell responses and maintaining this responsiveness in the presence of different background light intensities.

DNA methylation inhibitor regulates guanylyl cyclase/natriuretic peptide receptor-A gene expression via 5'-untranslated region in mesangial and smooth muscle cells

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is activated by cardiac hormones atrial and brain natriuretic peptides (ANP, BNP) and produces intracellular second messenger cGMP. The physiological actions of ANP/NPRA/cGMP signaling cascade largely include diuresis, natriuresis, vasorelaxation, and organ protection, which maintains blood pressure and opposed renal, cardiac, and vascular injury and remodeling. However, the mechanisms that contribute to the activation of Npr1 (coding for GC-A/NPRA) expression is not well understood. The present study was aimed at understanding the epigenetic mechanisms of DNA methyltransferase (DNMT) inhibitor, 5-Azacytidine (5-Aza)-mediated Npr1 gene transcription. The studies were carried out in mouse mesangial cells (MMCs) and rat thoracic aortic smooth muscle (RTASM) cells, cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and ITS (insulin, transferrin, and sodium selenite) and treated with 5-Aza for 24 h. Western blot analysis showed that 5-Aza enhanced NPRA protein levels by 5-fold and 7-fold in a dose-dependent manner in MMCs and RTASM cells, respectively, and greatly stimulated intracellular accumulation of cGMP in ANP-treated cells. Treatment with 5-Aza attenuated DNMT activity and repressed DNMT1 protein levels in cells. The MethPrimer search result showed the presence of three cytosine-phosphate-guanine (CpG) islands in the Npr1 full length promoter and 5'-untranslated region (UTR), namely island 1 (-886 to -752, 134 bp), island 2 (-310 to -158, 152 bp), and island 3 (-154 to +310, 464 bp) from the transcription start site. Addition of 5'-UTR encompassing +56 to +359 bp region to the Npr1 promoter constructs, (-1982/+55) and proximal promoter (-356/+55), was inhibitory to Npr1 transcriptional activity, independent of the cell type. Furthermore, treatment with 5-Aza distinctly induced Npr1 promoter activity of the construct -356/5'-UTR by 10-fold in MMCs suggesting that the repressive effect of DNA methylation on Npr1 gene transcription might be via 5'-UTR. The results demonstrate that 5-Aza acts as a potent inducer of Npr1 expression and signaling and provides new insights in the epigenetic control of Npr1, an important player in the control of hypertension and cardiorenal axis. Supported by the NIH grants (HL062147 and DK133833). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Phosphodiesterase 7 as a therapeutic target – Where are we now?

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes that hydrolyse the intracellular second messengers cAMP and cGMP to their inactive forms 5’AMP and 5’GMP. Some members of the PDE family display specificity towards a single cyclic nucleotide messenger, and PDE4, PDE7, and PDE8 specifically hydrolyse cAMP. While the role of PDE4 and its use as a therapeutic target have been well studied, less is known about PDE7 and PDE8. This review aims to collate the present knowledge on human PDE7 and outline its potential use as a therapeutic target. Human PDE7 exists as two isoforms PDE7A and PDE7B that display different expression patterns but are predominantly found in the central nervous system, immune cells, and lymphoid tissue. As a result, PDE7 is thought to play a role in T cell activation and proliferation, inflammation, and regulate several physiological processes in the central nervous system, such as neurogenesis, synaptogenesis, and long-term memory formation. Increased expression and activity of PDE7 has been detected in several disease states, including neurodegenerative diseases such as Parkinson's, Alzheimer's and Huntington's disease, autoimmune diseases such as multiple sclerosis and COPD, and several types of cancer. Early studies have shown that administration of PDE7 inhibitors may ameliorate the clinical state of these diseases. Targeting PDE7 may therefore provide a novel therapeutic strategy for targeting a broad range of disease and possibly provide a complementary alternative to inhibitors of other cAMP-selective PDEs, such as PDE4, which are severely limited by their side-effects.

Melatonin delays leaf senescence in pak choi (Brassica rapa subsp. chinensis) by regulating biosynthesis of the second messenger cGMP

Melatonin (MT) is a low molecular weight compound with multiple biological functions in plants. It is known to delay leaf senescence in various species. However, no data are available on the MT signaling pathway in postharvest vegetables. This study demonstrates that MT increases cGMP concentration and the expression of the cGMP synthesis gene BcGC1 in pak choi. The cGMP inhibitor LY83583 destroys effect of MT delaying the leaf senescence. LY83583 also prevents MT treatment from reducing the expression of chlorophyll metabolism-related genes (BcNYC1, BcNOL, BcPPH1/2, BcSGR1/2, and BcPAO) and senescence genes (BcSAG12 and BcSAG21). It also inhibits MT from reducing the activity of the key chlorophyll catabolism enzymes Mg-dechelatase, pheophytinase, and pheide a oxygenase. Thus, the ability of MT to maintain high levels of chlorophyll metabolites is also destroyed. The Arabidopsis cGMP synthetic gene mutant atgc1 was used to confirm that delayed leaf senescence caused by MT is mediated, at least in part, by the second messenger cGMP.

Ubiquitination of NKCC2 by the cullin-RING E3 ubiquitin ligase family in the thick ascending limb of the loop of Henle.

The Na+/K+/2Cl- cotransporter (NKCC2) in the thick ascending limb of the loop of Henle (TAL) mediates NaCl reabsorption. cGMP, the second messenger of nitric oxide and atrial natriuretic peptide, inhibits NKCC2 activity by stimulating NKCC2 ubiquitination and decreasing surface NKCC2 levels. Among the E3 ubiquitin ligase families, the cullin-RING E3 ubiquitin ligase (CRL) family is the largest. Cullins are molecular scaffold proteins that recruit multiple subunits to form the CRL complex. We hypothesized that a CRL complex mediates the cGMP-dependent increase in NKCC2 ubiquitination in TALs. Cullin-1, cullin-2, cullin-3, cullin-4A, and cullin-5 were expressed at the protein level, whereas the other members of the cullin family were expressed at the mRNA level, in rat TALs. CRL complex activity is regulated by neuronal precursor cell-expressed developmentally downregulated protein 8 (Nedd8) to cullins, a process called neddylation. Inhibition of cullin neddylation blunted the cGMP-dependent increase in ubiquitinated NKCC2 while increasing the expression of cullin-1 by threefold, but this effect was not seen with other cullins. CRL complex activity is also regulated by cullin-associated Nedd8-dissociated 1 (CAND1). CAND1 binds to cullins and promotes the exchange of substrate-recognition proteins to target different proteins for ubiquitination. CAND1 inhibition exacerbated the cGMP-dependent increase in NKCC2 ubiquitination and decreased surface NKCC2 expression. Finally, cGMP increased neddylation of cullins. We conclude that the cGMP-dependent increase in NKCC2 ubiquitination is mediated by a CRL complex. To the best of our knowledge, this is the first evidence that a CRL complex mediates NKCC2 ubiquitination in native TALs.NEW & NOTEWORTHY The Na+/K+/2Cl- cotransporter (NKCC2) reabsorbs NaCl by the thick ascending limb. Nitric oxide and atrial natriuretic peptide decrease NaCl reabsorption in thick ascending limbs by increasing the second messenger cGMP. The present findings indicate that cGMP increases NKCC2 ubiquitination via a cullin-RING ligase complex and regulates in part surface NKCC2 levels. Identifying the E3 ubiquitin ligases that regulate NKCC2 expression and activity may provide new targets for the development of specific loop diuretics.

Discrimination between cyclic-nucleotides by cyclic-nucleotide gated ion channels.

Cyclic-nucleotide-gated (CNG) ion channels play important roles throughout the entire nervous system, particularly in the signal transduction of the retina and olfactory system. They are regulated by the cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). These two chemically very similar molecules bind to a specialized intracellular domain called the cyclic nucleotide-binding domain (CNBD). CNG channels are key in translating the chemical signal of the second messenger molecules cAMP and cGMP into an electrical response by inducing a conformational change in the CNBD that modulates the opening of the channel gate in the pore domain allowing the flow of ions across cell membranes. Functional measurements have shown that, in SthK (a prokaryotic homolog with high sequence and structural similarities to CNG channels), cGMP barely leads to pore opening while cAMP activates the channel. Here we address the question of how the CNBD can finely discriminate between the two chemically and structurally very similar ligands. Using a combination of atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), and force probe molecular dynamics simulations, we elucidate, quantitatively and at the atomic level, the mechanism of CNBD ligand discrimination in SthK. We find that cAMP binds to the SthK CNBD with a slightly stronger affinity than cGMP, and can access a deep-bound state that is inaccessible to cGMP. We propose that the deep binding of cAMP is the discriminatory state that is essential for cAMP-dependent channel activation. We identify crucial residues and dynamic hydrogen bonding patterns within the binding pocket that give rise to this discrimination.

CGMP signaling: a potential therapeutic target for neurodegeneration in glaucoma?

cGMP signaling: a potential therapeutic target for neurodegeneration in glaucoma?

Histopathological and transcriptomic analyses reveal the reproductive endocrine-disrupting effects of decabromodiphenyl ethane (DBDPE) in mussel Mytilus galloprovincialis

The novel brominated flame retardant DBDPE has become a widespread environmental contaminant and could affect reproductive endocrine system in vertebrates. However, information about reproductive endocrine-disrupting effects of DBDPE on invertebrates is totally unknown. In this study, mussels Mytilus galloprovincialis were exposed to 1, 10, 50, 200 and 500 μg/L DBDPE for 30 days. Histopathological and transcriptomic analyses were performed to assess the reproductive endocrine-disrupting effects of DBDPE in mussels and the potential mechanisms. DBDPE promoted the gametogenesis in mussels of both sexes according to histological observation, gender-specific gene expression (VERL and VCL) and histological morphometric parameter analysis. Transcriptomic analysis demonstrated that DBDPE suppressed the genes related to cholesterol homeostasis and transport in both sexes via different LRPs- and ABCs-mediated pathways. DBDPE also disturbed nongenomic signaling pathway including signaling cascades (GPR157-IP3-Ca2+) in males and secondary messengers (cGMP) in females, and subsequently altered the expression levels of reproductive genes (VMO1, ZAN, Banf1 and Hook1). Additionally, dysregulation of energy metabolism in male mussels induced by DBDPE might interfere with the reproductive endocrine system. Overall, this is the first report that DBDPE evoked reproductive endocrine-disruptions in marine mussels. These findings will provide important references for ecological risk assessment of DBDPE pollution in marine environment.

Radiosynthesis and Preclinical Evaluation of an 18F-Labeled Triazolopyridopyrazine-Based Inhibitor for Neuroimaging of the Phosphodiesterase 2A (PDE2A).

The cyclic nucleotide phosphodiesterase 2A is an intracellular enzyme which hydrolyzes the secondary messengers cAMP and cGMP and therefore plays an important role in signaling cascades. A high expression in distinct brain areas as well as in cancer cells makes PDE2A an interesting therapeutic and diagnostic target for neurodegenerative and neuropsychiatric diseases as well as for cancer. Aiming at specific imaging of this enzyme in the brain with positron emission tomography (PET), a new triazolopyridopyrazine-based derivative (11) was identified as a potent PDE2A inhibitor (IC50, PDE2A = 1.99 nM; IC50, PDE10A ~2000 nM) and has been radiofluorinated for biological evaluation. In vitro autoradiographic studies revealed that [18F]11 binds with high affinity and excellent specificity towards PDE2A in the rat brain. For the PDE2A-rich region nucleus caudate and putamen an apparent KD value of 0.24 nM and an apparent Bmax value of 16 pmol/mg protein were estimated. In vivo PET-MR studies in rats showed a moderate brain uptake of [18F]11 with a highest standardized uptake value (SUV) of 0.97. However, no considerable enrichment in PDE2A-specific regions in comparison to a reference region was detectable (SUVcaudate putamen = 0.51 vs. SUVcerebellum = 0.40 at 15 min p.i.). Furthermore, metabolism studies revealed a considerable uptake of radiometabolites of [18F]11 in the brain (66% parent fraction at 30 min p.i.). Altogether, despite the low specificity and the blood–brain barrier crossing of radiometabolites observed in vivo, [18F]11 is a valuable imaging probe for the in vitro investigation of PDE2A in the brain and has potential as a lead compound for further development of a PDE2A-specific PET ligand for neuroimaging.

Abstract P006: Systematic Identification Of Metabolites Associated With Manp, A Novel Anp Analog, In Human Hypertension With Metabolic Syndrome

Introduction: The atrial natriuretic peptide (ANP) regulates blood pressure (BP) and cardiorenal homeostasis via activation of guanylyl cyclase A (GC-A) receptor and second messenger cGMP. We recently demonstrated the safety, tolerability, and BP-lowering properties of MANP, an ANP analog and best-in-class GC-A activator, in subjects with essential hypertension (HTN). However, the effects of MANP on human metabolome during HTN remains undefined. Hypothesis: A single subcutaneous (SQ) administration of MANP can trigger metabolomic changes in humans. Methods: Twenty-two patients with HTN and metabolic syndrome (MetS) were randomized to receive placebo (N=5) or MANP (N=17, 2.5 μg/kg). Untargeted liquid chromatography mass spectrometry-based metabolomics and informatics were conducted on plasma of all subjects at baseline, 1 h post injection (hpi), and 6 hpi. Top circulating metabolites associated with MANP treatment were identified via a comparison of abundance between before and after MANP administration. Results: Compared to placebo, MANP resulted in the most prominent cGMP increase at 1 hpi and BP lowering effect at 6 hpi. With untargeted approach, we identified totally 1384 circulating metabolites. Principal component analysis supported distinct metabolic signatures at baseline, 1 hpi and 6 hpi in subjects receiving MANP. In MANP group, we found 31 metabolites at 1 hpi and 148 metabolites at 6 hpi showing significant change in abundance (adjusted P value < 0.05) compared to baseline. By contrast, <10 metabolites with difference in circulating abundance at either 1 hpi or 6 hpi compared to baseline were found in placebo group. Specifically, 8 annotated metabolites were identified with significant and progressive changes at both 1 and 6 hpi after treatment of MANP but not placebo, which included spisulosine, paecilaminol, quinine ethyl carbonate, thyrotropin releasing hormone, 3-epideoxycholic acid, hellicoside, pentanorvitamin D3, and tetracosanal. Conclusion: SQ administration of MANP triggers changes in a wide range of circulating metabolites beyond cGMP activation and BP reduction in humans. The biological outcome and clinical impact of these MANP-associated metabolites may help guide a precision medicine approach in HTN with MetS.

Roles of Natriuretic Peptides and the Significance of Neprilysin in Cardiovascular Diseases.

Simple SummaryThe endocrine effects of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in the vasculature, and the autocrine effects of ANP and BNP in cardiomyocytes are mediated by the common guanylyl cyclase A receptor (GC-A) expressed in various tissues and cell types. C-type natriuretic peptide (CNP) has paracrine actions that regulate vascular resistance and moderate myocardial stiffness via guanylyl cyclase B receptor (GC-B). Genetically modified mice have revealed the physiological roles of ANP and BNP in blood pressure, cardiac remodeling, and acute myocardial infarction. Molecular pathways in GC-A signaling specifically in cardiomyocytes were also investigated. ANP and BNP via the GC-A signaling phosphorylate regulator of G-protein signaling subtype 4 (RGS4) result in the inhibition of Gαq signaling coupled with angiotensin II type 1A receptor, inhibit the activation of transient receptor potential C6 (TRPC6), and attenuate genomic actions of the cardiac mineralocorticoid receptor (MR). Moreover, recent studies showed the physiological roles of CNP via GC-B in blood pressure and cardiac stiffness. Since natriuretic peptides are degraded by neprilysin (NEP), inhibiting NEP activity is expected to enhance the actions of natriuretic peptides. Experimental studies and clinical trials have shown the effect of NEP inhibition on cardiac remodeling, acute myocardial infarction, and hypertension.Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) activate the guanylyl cyclase A receptor (GC-A), which synthesizes the second messenger cGMP in a wide variety of tissues and cells. C-type natriuretic peptide (CNP) activates the cGMP-producing guanylyl cyclase B receptor (GC-B) in chondrocytes, endothelial cells, and possibly smooth muscle cells, cardiomyocytes, and cardiac fibroblasts. The development of genetically modified mice has helped elucidate the physiological roles of natriuretic peptides via GC-A or GC-B. These include the hormonal effects of ANP/BNP in the vasculature, autocrine effects of ANP/BNP in cardiomyocytes, and paracrine effects of CNP in the vasculature and cardiomyocytes. Neprilysin (NEP) is a transmembrane neutral endopeptidase that degrades the three natriuretic peptides. Recently, mice overexpressing NEP, specifically in cardiomyocytes, revealed that local cardiac NEP plays a vital role in regulating natriuretic peptides in the heart tissue. Since NEP inhibition is a clinically accepted approach for heart failure treatment, the physiological roles of natriuretic peptides have regained attention. This article focuses on the physiological roles of natriuretic peptides elucidated in mice with GC-A or GC-B deletion, the significance of NEP in natriuretic peptide metabolism, and the long-term effects of angiotensin receptor-neprilysin inhibitor (ARNI) on cardiovascular diseases.

Vitamin D Regulates Natriuretic Peptide Receptor‐A Gene Expression via Modulation of Histone Deacetylases and DNA Methyltransferase Activity

Cardiac hormones atrial and brain natriuretic peptides (ANP and BNP) activate guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and produces the second messenger cGMP. cGMP activates downstream signaling and biological consequences of ANP/NPRA such as natriuresis, diuresis, vasorelaxation, antimitogenic responses, and cardiac and renal anti‐hypertrophic effects. The activity and expression of Npr1 gene (coding for GC‐A/NPRA) assessed primarily through ANP‐stimulated cGMP production, are mediated by agents, including autoregulation involving natriuretic peptides, osmotic sensors, and other external and internal stimuli, but the hormonal and epigenetic mechanisms mediating Npr1 regulation are not well understood. The objective of present study was to study the effect of Vitamin D (vitD) in the regulation of Npr1 gene transcription and expression via modulation of epigenetic factors. Our bioinformatics study with the murine Npr1promoter has shown presence of 4 vitD response elements (VDRE) in the region ‐583 to ‐495 from the transcription start site having perfect and VDRE‐like consensus sequence. Npr1promoter activity was determined by transient transfection of Npr1 promoter deletion constructs in cultured rat thoracic aortic smooth muscle cells (RTASMCs) and mouse mesangial cells (MMCs) treated with Vitamin D₃ (1α, 25‐dihydroxy; VD3) using dual luciferase assay kit. Luciferase assay demonstrated that VD3 enhances Npr1 promoter activity by more than 5‐6‐fold in RTASMCs and MMCs in a dose‐dependent manner. Western blot and densitometry analysis showed increasing concentrations of VD3 significantly induced NPRA protein levels by 3.5‐fold in MMCs and 4.5‐fold in RTASMCs and maximum effect was observed at 100 nM. There was 45%‐50% inhibition of histone deacetylase (HDAC) activity in presence of VD3 in both cell lines as measured by HDAC activity/inhibition ELISA kit. Moreover, treatment with VD3 reduced class I HDAC enzymes, HDAC1 and HDAC3 protein levels and dose‐dependently enhanced acetylation level of histones, H3 at lysine residues 9 and 14 (H3‐K9/14 ac) and H4 at lysine residue 12 (H4‐K14ac). Treatment of RTASMCs and MMCs with VD3 attenuated DNA methyltransferase (DNMT) activity by 40%‐55% as measured by DNMT activity/inhibition ELISA kit. The results demonstrate that VD3 regulate Npr1 gene expression by modulation of histone deacetylases and DNMT activity. The identification of epigenetic targets of vitamin D signaling as a regulator of Npr1gene transcription and protein expression will have important implications in hypertension and cardiovascular regulation.