cGMP Signaling Research Articles

Nitric oxide: an old drug but with new horizons in ophthalmology-a narrative review.

Based on basic knowledge and prior research on nitric oxide (NO), the potential of NO for treating eye diseases is reviewed, and the possibility of NO-based eye drops in clinical practice and the future potential of NO in ophthalmology are discussed. A PubMed search was performed for English-language original reports and reviews using the following key words: nitric oxide, eye, ocular, and drug. NO is synthesized in the human body by NO synthase (NOS) from L-arginine or through enzyme-dependent reduction of dietary nitrate. Three types of NOS (eNOS, nNOS, and iNOS) are abundantly expressed in the eye under normal physiologic or pathologic conditions. The biological effect of NO in the eye is dose dependent. Low intraocular NO concentrations, produced by eNOS or nNOS, have various cellular effects, including vasodilation, intraocular pressure (IOP) regulation, and neuroprotection. iNOS induced under pathologic ocular conditions produces high NO concentrations in the local environment and mediates tissue inflammation, ocular cell apoptosis, and neurodegeneration. In particular, increased iNOS has been reported in glaucoma and retinal ischemic or degenerative diseases. NO plays a vital role in ocular injury. NO can facilitate ocular surface wound healing while eradicating pathogens such as bacteria and Acanthamoeba in chemical burns or infectious keratitis. Furthermore, NO has antifibrotic activity via the cyclic guanosine monophosphate (cGMP) signaling pathway. NO causes smooth muscle relaxation, which can be used to inhibit myopia progression in children. NO can be a stem cell modulator and may help in treating ocular stem cell disorders. Because of its diverse biologic effects, NO can be a key player in regulating ocular inflammation in various ocular diseases, aiding ocular surface wound healing, controlling IOP in glaucoma, alleviating retinal disease, and suppressing myopia progression. Although there remain limitations to the effective use of highly unstable state, gaseous NO, the role of NO in the field of ophthalmology can be greatly expanded through the development of novel NO donors and effective delivery platforms.

Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor

The cardiac natriuretic peptides (NPs) control pivotal physiological actions such as fluid and electrolyte balance, cardiovascular homeostasis, and adipose tissue metabolism by activating their receptor enzymes [natriuretic peptide receptor-A (NPRA) and natriuretic peptide receptor-B (NPRB)]. These receptors are homodimers that generate intracellular cyclic guanosine monophosphate (cGMP). The natriuretic peptide receptor-C (NPRC), nicknamed the clearance receptor, lacks a guanylyl cyclase domain; instead, it can bind the NPs to internalize and degrade them. The conventional paradigm is that by competing for and internalizing NPs, NPRC blunts the ability of NPs to signal through NPRA and NPRB. Here we show another previously unknown mechanism by which NPRC can interfere with the cGMP signaling function of the NP receptors. By forming a heterodimer with monomeric NPRA or NPRB, NPRC can prevent the formation of a functional guanylyl cyclase domain and thereby suppress cGMP production in a cell-autonomous manner.

Nicorandil/ morphine crosstalk accounts for antinociception and hepatoprotection in hepatic fibrosis in rats: Distinct roles of opioid/cGMP and NO/KATP pathways

Previous report indicated that nicorandil potentiated morphine antinociception and attenuated hepatic injury in liver fibrotic rats. Herein, the underlying mechanisms of nicorandil/morphine interaction were investigated using pharmacological, biochemical, histopathological, and molecular docking studies. Male Wistar rats were injected intraperitoneally (i.p.) with carbon tetrachloride (CCl4, 40%, 2 ml/kg) twice weekly for 5 weeks to induce hepatic fibrosis. Nicorandil (15 mg/kg/day) was administered per os (p.o.) for 14 days in presence of the blockers; glibenclamide (KATP channel blocker, 5 mg/kg, p.o.), L-NG-nitro-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor, 15 mg/kg, p.o.), methylene blue (MB, guanylyl cyclase inhibitor, 2 mg/kg, i.p.) and naltrexone (opioid antagonist, 20 mg/kg, i.p.). At the end of the 5th week, analgesia was evaluated using tail flick and formalin tests along with biochemical determinations of liver function tests, oxidative stress markers and histopathological examination of liver tissues. Naltrexone and MB inhibited the antinociceptive activity of the combination. Furthermore, combined nicorandil/morphine regimen attenuated the release of endogenous peptides. Docking studies revealed a possible interaction of nicorandil on µ, κ and δ opioid receptors. Nicorandil/morphine combination protected against liver damage as evident by decreased liver enzymes, liver index, hyaluronic acid, lipid peroxidation, fibrotic insults, and increased superoxide dismutase activity. Nicorandil/morphine hepatoprotection and antioxidant activity were inhibited by glibenclamide and L-NAME but not by naltrexone or MB. These findings implicate opioid activation/cGMP versus NO/KATP channels in the augmented antinociception, and hepatoprotection, respectively, of the combined therapy and implicate provoked cross talk by nicorandil and morphine on opioid receptors and cGMP signaling pathway. That said, nicorandil/morphine combination provides a potential multitargeted therapy to alleviate pain and preserve liver function.

Characterizing the Protein Isoforms of foraging (for), the PKGI Ortholog in Drosophila melanogaster.

The foraging (for) gene of Drosophila melanogaster encodes a cGMP-dependent protein kinase (PKG), which is a major effector of the cGMP signaling pathway involved in the regulation of behaviour and metabolic traits. Despite being well studied at the transcript level, little is known about the for gene at the protein level. Here, we provide a detailed characterization of the for gene protein (FOR) products and present new tools for their study, including five isoform-specific antibodies and a transgenic strain that carries an HA-labelled for allele (forBAC::HA). Our results showed that multiple FOR isoforms were expressed in the larval and adult stages of D. melanogaster and that the majority of whole-body FOR expression arises from three (P1, P1α, and P3) of eight putative protein isoforms. We found that FOR expression differed between the larval and adult stages and between the dissected larval organs we analyzed, which included the central nervous system (CNS), fat body, carcass, and intestine. Moreover, we showed that the FOR expression differed between two allelic variants of the for gene, namely, fors (sitter) and forR (rover), that are known to differ in many food-related traits. Together, our in vivo identification of FOR isoforms and the existence of temporal, spatial, and genetic differences in their expression lay the groundwork for determining their functional significance.

Silencing of PKG1 Gene Mimics Effect of Aging and Sensitizes Rat Vascular Smooth Muscle Cells to Cardiotonic Steroids: Impact on Fibrosis and Salt Sensitivity.

Background Marinobufagenin, NKA (Na/K-ATPase) inhibitor, causes vasoconstriction and induces fibrosis via inhibition of Fli1 (Friend leukemia integration-1), a negative regulator of collagen synthesis. In vascular smooth muscle cells (VSMC), ANP (atrial natriuretic peptide), via a cGMP/PKG1 (protein kinase G1)-dependent mechanism, reduces NKA sensitivity to marinobufagenin. We hypothesized that VSMCfrom old rats, due to downregulation of ANP/cGMP/PKG-dependent signaling, would exhibit heightened sensitivity to the profibrotic effect of marinobufagenin. Methods and Results Cultured VSMCfrom the young (3-month-old) and old (24-month-old) male Sprague-Dawley rats and young VSMCwith silenced PKG1 gene were treated with 1 nmol/L ANP, or with 1 nmol/L marinobufagenin, or with a combination of ANP and marinobufagenin. Collagen-1, Fli1, and PKG1 levels were assessed by Western blotting analyses. Vascular PKG1 and Fli1 levels in the old rats were reduced compared with their young counterparts. ANP prevented inhibition of vascular NKA by marinobufagenin in young VSMC but not in old VSMC. In VSMC from the young rats, marinobufagenin induced downregulation of Fli1 and an increase in collagen-1 level, whereas ANP blocked this effect. Silencing of the PKG1 gene in young VSMC resulted in a reduction in levels of PKG1 and Fli1; marinobufagenin additionally reduced Fli1 and increased collagen-1 level, and ANP failed to oppose these marinobufagenin effects, similar to VSMC from the old rats with the age-associated reduction in PKG1. Conclusions Age-associated reduction in vascular PKG1 and the resultant decline in cGMP signaling lead to the loss of the ability of ANP to oppose marinobufagenin-induced inhibition of NKA and fibrosis development. Silencing of the PKG1 gene mimicked these effects of aging.

Notch3/Hes5 Induces Vascular Dysfunction in Hypoxia-Induced Pulmonary Hypertension Through ER Stress and Redox-Sensitive Pathways.

Notch3 (neurogenic locus notch homolog protein 3) is implicated in vascular diseases, including pulmonary hypertension (PH)/pulmonary arterial hypertension. However, molecular mechanisms remain elusive. We hypothesized increased Notch3 activation induces oxidative and endoplasmic reticulum (ER) stress and downstream redox signaling, associated with procontractile pulmonary artery state, pulmonary vascular dysfunction, and PH development. Studies were performed in TgNotch3R169C mice (harboring gain-of-function [GOF] Notch3 mutation) exposed to chronic hypoxia to induce PH, and examined by hemodynamics. Molecular and cellular studies were performed in pulmonary artery smooth muscle cells from pulmonary arterial hypertension patients and in mouse lung. Notch3-regulated genes/proteins, ER stress, ROCK (Rho-associated kinase) expression/activity, Ca2+ transients and generation of reactive oxygen species, and nitric oxide were measured. Pulmonary vascular reactivity was assessed in the presence of fasudil (ROCK inhibitor) and 4-phenylbutyric acid (ER stress inhibitor). Hypoxia induced a more severe PH phenotype in TgNotch3R169C mice versus controls. TgNotch3R169C mice exhibited enhanced Notch3 activation and expression of Notch3 targets Hes Family BHLH Transcription Factor 5 (Hes5), with increased vascular contraction and impaired vasorelaxation that improved with fasudil/4-phenylbutyric acid. Notch3 mutation was associated with increased pulmonary vessel Ca2+ transients, ROCK activation, ER stress, and increased reactive oxygen species generation, with reduced NO generation and blunted sGC (soluble guanylyl cyclase)/cGMP signaling. These effects were ameliorated by N-acetylcysteine. pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension recapitulated Notch3/Hes5 signaling, ER stress and redox changes observed in PH mice. Notch3 GOF amplifies vascular dysfunction in hypoxic PH. This involves oxidative and ER stress, and ROCK. We highlight a novel role for Notch3/Hes5-redox signaling and important interplay between ER and oxidative stress in PH.

Characterization of 2 Novel Phosphodiesterase 2 Inhibitors Hcyb1 and PF-05180999 on Depression- and Anxiety-Like Behavior.

Phosphodiesterase 2A (PDE2A) represents a novel target for new therapies addressing psychiatric disorders. To date, the development of PDE2A inhibitors suitable for human clinical evaluation has been hampered by the poor brain accessibility and metabolic stability of the available compounds. Corticosterone (CORT)-induced neuronal cell lesion and restraint stress mouse model were used to measure the neuroprotective effect in cells and antidepressant-like behavior in mice. The cell-based assay showed that both Hcyb1 and PF were potent in protecting cells against stress hormone CORT insults by stimulating cAMP and cGMP signaling in hippocampal cells (HT-22). Administration of both compounds before treatment of CORT to cells increased cAMP/cGMP, VASP phosphorylation at Ser239 and Ser157, cAMP response element binding protein phosphorylation at Ser133, and brain derived neurotrophic factor BDNF expression. Further in vivo study showed that both Hcyb1 and PF displayed -antidepressant- and anxiolytic-like effects against restraint stress as indicated by reduced immobility time in the forced swimming and tail suspension tasks as well as increased open arm entries and time spent in open arms and holes visit in elevated plus maze and hole-board tests, respectively. The biochemical study confirmed that these antidepressant- and anxiolytic-like effects of Hcyb1 and PF were related to cAMP and cGMP signaling in the hippocampus. The results extend the previous studies and validate that PDE2A is a tractable target for drug development in the treatment of emotional disorders such as depression and anxiety.

Stimulating Role of Calcium and Cyclic GMP in Mediating the Effect of Magnetopriming for Alleviation of Salt Stress in Soybean Seedlings

This current study examined the role of calcium (Ca) and Cyclic GMP (cGMP) in mitigating the adverse effect of salt stress through magnetopriming of soybean cultivar JS-335 seeds with a static magnetic field (SMF, 200 mT for 1 h). The salt stress (50 mMNaCl) extensively reduced the early seedling growth (64%), vigour Index-I (71%), vigour Index-II (39%), total amylase (59%), protease (63%), and nitrate reductase (NR, 19%) activities in un-primed soybean seedlings. However, magnetopriming and Ca treatment enhanced all of these measured parameters along with remarkable increase in reactive oxygen species (ROS) and nitric oxide (NO) content. The exogenous application of Ca2+, cGMP and ROS regulators such as nifedipine (Ca2+ channel blocker), EGTA, ethylene glycol-β-amino ethyl ether tetra acetic acid (Ca2+chelators), genistein (cGMP blocker), and dimethyl thiourea (DMTU, H2O2 inhibitor) negatively affects the SMF-induced seedling length, seedling vigour, ROS, NO, and enzyme activities such as protease, total amylase, and NR in soybean seedlings. Results presented by using specific various biochemical inhibitors of Ca, cGMP, or ROS signalling in vivo indicated that Ca and cGMP are also involved with ROS and NO in the signal transduction of magnetic field enthused soybean seed germination and seedling growth under salt stress.

It takes two to tango: cardiac fibroblast-derived NO-induced cGMP enters cardiac myocytes and increases cAMP by inhibiting PDE3

The occurrence of NO/cGMP signalling in cardiac cells is a matter of debate. Recent measurements with a FRET-based cGMP indicator in isolated cardiac cells revealed NO-induced cGMP signals in cardiac fibroblasts while cardiomyocytes were devoid of these signals. In a fibroblast/myocyte co-culture model though, cGMP formed in fibroblasts in response to NO entered cardiomyocytes via gap junctions. Here, we demonstrate gap junction-mediated cGMP transfer from cardiac fibroblasts to myocytes in intact tissue. In living cardiac slices of mice with cardiomyocyte-specific expression of a FRET-based cGMP indicator (αMHC/cGi-500), NO-dependent cGMP signals were shown to occur in myocytes, to depend on gap junctions and to be degraded mainly by PDE3. Stimulation of NO-sensitive guanylyl cyclase enhanced Forskolin- and Isoproterenol-induced cAMP and phospholamban phosphorylation. Genetic inactivation of NO-GC in Tcf21-expressing cardiac fibroblasts abrogated the synergistic action of NO-GC stimulation on Iso-induced phospholamban phosphorylation, identifying fibroblasts as cGMP source and substantiating the necessity of cGMP-transfer to myocytes. In sum, NO-stimulated cGMP formed in cardiac fibroblasts enters cardiomyocytes in native tissue where it exerts an inhibitory effect on cAMP degradation by PDE3, thereby increasing cAMP and downstream effects in cardiomyocytes. Hence, enhancing β-receptor-induced contractile responses appears as one of NO/cGMP’s functions in the non-failing heart.

Functional Axis of PDE5/cGMP Mediates Timosaponin-AIII-Elicited Growth Suppression of Glioblastoma U87MG Cells.

Glioblastoma (GBM) is the most aggressive brain tumor, with high mortality. Timosaponin AIII (TIA), a steroidal saponin isolated from the medicinal plant Anemarrhena asphodeloides Bge., has been shown to possess anticancer properties in various cancer types. However, the effect of TIA on GBM is unknown. In this study, we reveal that TIA not only inhibited U87MG in vitro cell growth but also in vivo tumor development. Moreover, we found that the cause of TIA-induced cell growth suppression was apoptosis. When seeking to uncover antitumor mechanisms of TIA, we found that TIA diminished the expression of cGMP-specific phosphodiesterase 5(PDE5) while elevating the levels of guanylate cyclases (sGCβ), cellular cGMP, and phosphorylation of VASPser239. Following the knockdown of PDE5, PDE5 inhibitor tadalafil and cGMP analog 8-Bro-cGMP both inhibited cell growth and inactivated β-catenin; we reason that TIA elicited an antitumor effect by suppressing PDE5, leading to the activation of the cGMP signaling pathway, which, in turn, impeded β-catenin expression. As β-catenin is key for cell growth and survival in GBM, this study suggests that TIA elicits its anti-tumorigenic effect by interfering with β-catenin function through the activation of a PDE5/cGMP functional axis.

In silico elucidation of potential drug targets against oxygenase domain of Human eNOS Dysfunction.

Nitric Oxide (NO) signaling pathway plays a vital role in various physiological and pathophysiological processes including vasodilation, neurogenesis, inflammation, translation and protein regulation. NO signaling pathway is associated with various diseases such as cardiovascular diseases, vision impairment, hypertension and Alzheimer's disease. Human Endothelial Nitric Oxide Synthase (eNOS) bound with calcium regulatory protein (calmodulin (CaM)) to produce NO which initiates cGMP pathway. The current study employs to screen the novel compounds against human eNOS independent of calcium regulatory protein (CaM). The current effort emphasized that the deficiency of CaM leads to dysfunction of cGMP signaling pathway. In this work, a hybrid approach of high-throughput virtual screening and comparative molecular docking studies followed by molecular dynamic simulation analyses were applied. The screening of top ranked two novel compounds against eNOS were reported that showed effective binding affinity, retrieved through the DrugBank and ZINC database libraries. Comparative molecular docking analyses revealed that Val-104, Phe-105, Gln-247, Arg-250, Ala-266, Trp-330, Tyr-331, Pro-334, Ala-335, Val-336, Tyr-357, Met-358, Thr-360, Glu-361, Ile-362, Arg-365, Asn-366, Asp-369, Arg-372, Trp-447 and Tyr-475 are potent residues for interactional studies. High-throughput virtual screening approach coupled with molecular dynamic simulation and drug likeness rules depicted that ZINC59677432 and DB00456 are potent compounds to target eNOS. In conclusion, the proposed compounds are potent against eNOS based on extensive in silico analyses. Overall, the findings of this study may be helpful to design therapeutic targets against eNOS.

Myokine Musclin Is Critical for Exercise-Induced Cardiac Conditioning.

This study investigates the role and mechanisms by which the myokine musclin promotes exercise-induced cardiac conditioning. Exercise is one of the most powerful triggers of cardiac conditioning with proven benefits for healthy and diseased hearts. There is an emerging understanding that muscles produce and secrete myokines, which mediate local and systemic "crosstalk" to promote exercise tolerance and overall health, including cardiac conditioning. The myokine musclin, highly conserved across animal species, has been shown to be upregulated in response to physical activity. However, musclin effects on exercise-induced cardiac conditioning are not established. Following completion of a treadmill exercise protocol, wild type (WT) mice and mice with disruption of the musclin-encoding gene, Ostn, had their hearts extracted and exposed to an ex vivo ischemia-reperfusion protocol or biochemical studies. Disruption of musclin signaling abolished the ability of exercise to mitigate cardiac ischemic injury. This impaired cardioprotection was associated with reduced mitochondrial content and function linked to blunted cyclic guanosine monophosphate (cGMP) signaling. Genetic deletion of musclin reduced the nuclear abundance of protein kinase G (PKGI) and cyclic adenosine monophosphate (cAMP) response element binding (CREB), resulting in suppression of the master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), and its downstream targets in response to physical activity. Synthetic musclin peptide pharmacokinetic parameters were defined and used to calculate the infusion rate necessary to maintain its plasma level comparable to that observed after exercise. This infusion was found to reproduce the cardioprotective benefits of exercise in sedentary WT and Ostn-KO mice. Musclin is essential for exercise-induced cardiac protection. Boosting musclin signaling might serve as a novel therapeutic strategy for cardioprotection.

Acute deletion of the central MR/GR steroid receptor correlates with changes in LTP, auditory neural gain, and GC-A cGMP signaling.

The complex mechanism by which stress can affect sensory processes such as hearing is still poorly understood. In a previous study, the mineralocorticoid (MR) and/or glucocorticoid receptor (GR) were deleted in frontal brain regions but not cochlear regions using a CaMKIIα-based tamoxifen-inducible Cre ERT2/loxP approach. These mice exhibit either a diminished (MRTMXcKO) or disinhibited (GRTMXcKO) auditory nerve activity. In the present study, we observed that mice differentially were (MRTMXcKO) or were not (GRTMXcKO) able to compensate for altered auditory nerve activity in the central auditory pathway. As previous findings demonstrated a link between central auditory compensation and memory-dependent adaptation processes, we analyzed hippocampal paired-pulse facilitation (PPF) and long-term potentiation (LTP). To determine which molecular mechanisms may impact differences in synaptic plasticity, we analyzed Arc/Arg3.1, known to control AMPA receptor trafficking, as well as regulators of tissue perfusion and energy consumption (NO-GC and GC-A). We observed that the changes in PPF of MRTMXcKOs mirrored the changes in their auditory nerve activity, whereas changes in the LTP of MRTMXcKOs and GRTMXcKOs mirrored instead the changes in their central compensation capacity. Enhanced GR expression levels in MRTMXcKOs suggest that MRs typically suppress GR expression. We observed that hippocampal LTP, GC-A mRNA expression levels, and ABR wave IV/I ratio were all enhanced in animals with elevated GR (MRTMXcKOs) but were all lower or not mobilized in animals with impaired GR expression levels (GRTMXcKOs and MRGRTMXcKOs). This suggests that GC-A may link LTP and auditory neural gain through GR-dependent processes. In addition, enhanced NO-GC expression levels in MR, GR, and MRGRTMXcKOs suggest that both receptors suppress NO-GC; on the other hand, elevated Arc/Arg3.1 levels in MRTMXcKOs and MRGRTMXcKOs but not GRTMXcKOs suggest that MR suppresses Arc/Arg3.1 expression levels. Conclusively, MR through GR inhibition may define the threshold for hemodynamic responses for LTP and auditory neural gain associated with GC-A.

A label-free LC/MS-based enzymatic activity assay for the detection of PDE5A inhibitors.

Phosphodiesterase type 5 (PDE5), a cyclic nucleotide phosphodiesterase, controls the duration of the cyclic guanosine monophosphate (cGMP) signal by hydrolyzing cGMP to GMP. Inhibiting the activity of PDE5A has proven to be an effective strategy for treating pulmonary arterial hypertension and erectile dysfunction. Current enzymatic activity assay methods for PDE5A mainly use fluorescent or isotope-labeled substrates, which are expensive and inconvenient. Here, we developed an LC/MS-based enzymatic activity assay for PDE5A without labeling, which detects the enzymatic activity of PDE5A by quantifying the substrate cGMP and product GMP at a concentration of 100nM. The accuracy of this method was verified by a fluorescently labeled substrate. Moreover, a new inhibitor of PDE5A was identified by this method and virtual screening. It inhibited PDE5A with an IC50 value of 870nM. Overall, the proposed strategy provides a new method for screening PDE5A inhibitors.

Inhibition of phosphodiesterase 5A by tadalafil improves SIRT1 expression and activity in insulin-resistant podocytes

A decrease in intracellular levels of 3′,5′-cyclic guanosine monophosphate (cGMP) has been implicated in the progression of diabetic nephropathy. Hyperglycemia significantly inhibits cGMP-dependent pathway activity in the kidney, leading to glomerular damage and proteinuria. The enhancement of activity of this pathway that is associated with an elevation of cGMP levels may be achieved by inhibition of the cGMP specific phosphodiesterase 5A (PDE5A) using selective inhibitors, such as tadalafil. Hyperglycemia decreased the insulin responsiveness of podocytes and impaired podocyte function. These effects were associated with lower protein amounts and activity of the protein deacetylase sirtuin 1 (SIRT1) and a decrease in the phosphorylation of adenosine monophosphate-dependent protein kinase (AMPK). We found that PDE5A protein levels increased in hyperglycemia, and PDE5A downregulation improved the insulin responsiveness of podocytes with reestablished SIRT1 expression and activity. PDE5A inhibitors potentiate nitric oxide (NO)/cGMP signaling, and NO modulates the activity and expression of SIRT1. Therefore, we investigated the effects of tadalafil on SIRT1 and AMPK in the context of improving the insulin sensitivity in podocytes and podocyte function in hyperglycemia. Our study revealed that tadalafil restored SIRT1 expression and activity and activated AMPK by increasing its phosphorylation. Tadalafil also restored stimulating effect of insulin on glucose transport in podocytes with high glucose-induced insulin resistance. Additionally, tadalafil improved the function of podocytes that were exposed to high glucose concentrations. Our results display novel mechanisms involved in the pathogenesis of glomerulopathies in diabetes, which may contribute to the development of more effective treatment strategies for diabetic nephropathy.

Dynamics of intracellular cGMP during chemotaxis in Dictyostelium cells.

Cyclic guanosine 3',5'-monophosphate (cGMP) is a ubiquitous important second messenger involved in various physiological functions. Here, intracellular cGMP (cGMPi) was visualized in chemotactic Dictyostelium cells using the fluorescent probe, D-Green cGull. When wild-type cells were stimulated with a chemoattractant, fluorescence transiently increased, but guanylate cyclase-null cells did not show a change in fluorescence, suggesting that D-Green cGull is a reliable indicator of cGMPi. In the aggregation stage, the responses of cGMPi propagated in a wave-like fashion from the aggregation center. The oscillation of the cGMPi wave was synchronized almost in phase with those of other second messengers, such as the intracellular cAMP and Ca2+. The phases of these waves preceded those of the oscillations of actomyosin and cell velocity, suggesting that these second messengers are upstream of the actomyosin and chemotactic migration. An acute increase in cGMPi concentration released from membrane-permeable caged cGMP induced a transient shuttle of myosin II between the cytosol and cell cortex, suggesting a direct link between cGMP signaling and myosin II dynamics.

CNP Promotes Antiarrhythmic Effects via Phosphodiesterase 2.

Ventricular arrhythmia and sudden cardiac death are the most common lethal complications after myocardial infarction. Antiarrhythmic pharmacotherapy remains a clinical challenge and novel concepts are highly desired. Here, we focus on the cardioprotective CNP (C-type natriuretic peptide) as a novel antiarrhythmic principle. We hypothesize that antiarrhythmic effects of CNP are mediated by PDE2 (phosphodiesterase 2), which has the unique property to be stimulated by cGMP to primarily hydrolyze cAMP. Thus, CNP might promote beneficial effects of PDE2-mediated negative crosstalk between cAMP and cGMP signaling pathways. To determine antiarrhythmic effects of cGMP-mediated PDE2 stimulation by CNP, we analyzed arrhythmic events and intracellular trigger mechanisms in mice in vivo, at organ level and in isolated cardiomyocytes as well as in human-induced pluripotent stem cell-derived cardiomyocytes. In ex vivo perfused mouse hearts, CNP abrogated arrhythmia after ischemia/reperfusion injury. Upon high-dose catecholamine injections in mice, PDE2 inhibition prevented the antiarrhythmic effect of CNP. In mouse ventricular cardiomyocytes, CNP blunted the catecholamine-mediated increase in arrhythmogenic events as well as in ICaL, INaL, and Ca2+ spark frequency. Mechanistically, this was driven by reduced cellular cAMP levels and decreased phosphorylation of Ca2+ handling proteins. Key experiments were confirmed in human iPSC-derived cardiomyocytes. Accordingly, the protective CNP effects were reversed by either specific pharmacological PDE2 inhibition or cardiomyocyte-specific PDE2 deletion. CNP shows strong PDE2-dependent antiarrhythmic effects. Consequently, the CNP-PDE2 axis represents a novel and attractive target for future antiarrhythmic strategies.

Characterizing the Specific Recognition of Xanthurenic Acid by GEP1 and GEP1-GCα Interactions in cGMP Signaling Pathway in Gametogenesis of Malaria Parasites.

Gametogenesis is an essential step for malaria parasite transmission and is activated in mosquito by signals including temperature drop, pH change, and mosquito-derived xanthurenic acid (XA). Recently, a membrane protein gametogenesis essential protein 1 (GEP1) was found to be responsible for sensing these signals and interacting with a giant guanylate cyclase α (GCα) to activate the cGMP-PKG-Ca2+ signaling pathway for malaria parasite gametogenesis. However, the molecular mechanisms for this process remain unclear. In this study, we used AlphaFold2 to predict the structure of GEP1 and found that it consists of a conserved N-terminal helical domain and a transmembrane domain that adopts a structure similar to that of cationic amino acid transporters. Molecular docking results showed that XA binds to GEP1 via a pocket similar to the ligand binding sites of known amino acid transporters. In addition, truncations of this N-terminal sequence significantly enhanced the expression, solubility, and stability of GEP1. In addition, we found that GEP1 interacts with GCα via its C-terminal region, which is interrupted by mutations of a few conserved residues. These findings provide further insights into the molecular mechanism for the XA recognition by GEP1 and the activation of the gametogenesis of malaria parasites through GEP1-GCα interaction.

Soluble guanylate cyclase agonist, isoliquiritigenin attenuates renal damage and aortic calcification in a rat model of chronic kidney failure

AimsChronic kidney disease (CKD) is a growing fatal health problem worldwide associated with vascular calcification. Therapeutic approaches are limited with higher costs and poor outcomes. Adenine supplementation is one of the most relevant CKD models to human. Insufficient Nitric Oxide (NO)/ cyclic Guanosine Monophosphate (cGMP) signaling plays a key role in rapid development of renal fibrosis. Natural products display proven protection against CKD. Current study therefore explored isoliquiritigenin, a bioflavonoid extracted from licorice roots, potential as a natural activator for soluble Guanylate Cyclase (sGC) in a CKD rat model. Materials and methods60 male Wistar rats were grouped into Control group (n = 10) and the remaining rats received adenine (200 mg/kg, p.o) for 2 wk to induce CKD. They were equally sub-grouped into: Adenine untreated group and 4 groups orally treated by isoliquiritigenin low or high dose (20 or 40 mg/kg) with/without a selective sGC inhibitor, ODQ (1-H(1,2,4)oxadiazolo(4,3-a)-quinoxalin-1-one, 2 mg/kg, i.p) for 8 wk. Key findingsLong-term treatment with isoliquiritigenin dose-dependently and effectively amended adenine-induced chronic renal and endothelial dysfunction. It not only alleviated renal fibrosis and apoptosis markers but also aortic calcification. Additionally, this chalcone neutralized renal inflammatory response and oxidative stress. Isoliquiritigenin beneficial effects were associated with up-regulation of serum NO, renal and aortic sGC, cGMP and its dependent protein kinase (PKG). However, co-treatment with ODQ antagonized isoliquiritigenin therapeutic impact. SignificanceIsoliquiritigenin seems to exert protective effects against CKD and vascular calcification by activating sGC, increasing cGMP and its downstream PKG.

The Effects of Different Doses of Sildenafil on Coronary Blood Flow and Oxidative Stress in Isolated Rat Hearts.

The dose-response relationship of sildenafil effects on cardiac function is not completely elucidated. The aim of this study was to assess the effects of different doses of sildenafil on coronary flow and oxidative stress in isolated rat hearts. Coronary flow and markers of oxidative stress, including nitrite outflow, and superoxide anion production in coronary effluent, were determined for isolated rat hearts. The experiments were performed during control conditions and in the presence of sildenafil (10, 20, 50, 200 nM) alone or with Nω-nitro-L-arginine monomethyl ester (L-NAME) (30 μM). Sildenafil was shown to result in a significant increase in coronary flow at lower coronary perfusion pressure (CPP) values at all administered doses, whereas, with an increase in CPP, a reduction in coronary flow was observed. An increase in nitric oxide (NO) was most pronounced in the group treated with the lowest dose of sildenafil at the highest CPP value. After the inhibition of the NO-cyclic guanosine monophosphate (cGMP) signaling (NOS) system by L-NAME, only a dose of 200 nM sildenafil was high enough to overcome the inhibition and to boost release of O2-. That effect was CPP-dependent, with statistical significance reached at 80, 100 and 120 mmHg. Our findings indicate that sildenafil causes changes in heart vasculature in a dose-dependent manner, with a shift from a vasodilatation effect to vasoconstriction with a pressure increase. The highest dose administered is capable of producing superoxide anion radicals in terms of NOS system inhibition.