Intracellular cGMP Concentration Research Articles

Nutraceutical COMP-4 confers protection against endothelial dysfunction through the eNOS/iNOS-NO-cGMP pathway.

The nutraceutical COMP-4 -consisting of L-citrulline, ginger extract, and herbal components Paullinia cupana and muira puama-has been shown previously to stimulate the production of nitric oxide (NO) in a variety of tissue types. We hypothesized that COMP-4 may have a protective, stimulatory effect on the vascular endothelial cell. Human umbilical arterial endothelial cells were incubated for 24 hours with or without COMP-4 and, to replicate impairment of endothelial function, co-incubated with or without H2O2. NO intracellular content, nitrite formation and cGMP content in culture media, nitric oxide synthase (NOS) isoforms and mRNA content, pro-inflammatory cytokines, and PAI-1 expression and activity were measured. COMP-4 increased endothelial cell production of NO and cGMP and the expression of both endothelial NOS (eNOS) and inducible NOS (iNOS), in tandem with a reduction in cytokine expression and activity of PAI-1. Co-incubation of COMP-4 with H2O2 reversed detrimental effects of H2O2 on endothelial function, evidenced by improvement in NO availability and abrogation of the pro-inflammatory milieu. These results suggest that COMP-4 exerts a stimulatory effect on endothelial cell eNOS and iNOS to increase NO bioavailability, leading to a reduction in pro-inflammatory cytokines, particularly the prothrombotic PAI-1.

The multidrug resistance protein 4 is expressed and functionally active in isolated bladder from pig.

Multidrug resistance proteins type 4 (MRP4) and 5 (MRP5) play pivotal roles in the transport of cyclic nucleotides in various tissues. However, their specific functions within the lower urinary tract remain relatively unexplored. This study aimed to investigate the effect of pharmacological inhibition of MRPs on cyclic nucleotide signaling in isolated pig bladder. The relaxation responses of the bladder were assessed in the presence of the MRP inhibitor, MK571. The temporal changes in intra- and extracellular levels of cAMP and cGMP in stimulated tissues were determined by mass spectrometry. The gene (ABCC4) and protein (MRP4) expression were also determined. MK571 administration resulted in a modest relaxation effect of approximately 26% in carbachol-precontracted bladders. The relaxation induced by phosphodiesterase inhibitors such as cilostazol, tadalafil, and sildenafil was significantly potentiated in the presence of MK571. In contrast, no significant potentiation was observed in the relaxation induced by substances elevating cAMP levels or stimulators of soluble guanylate cyclase. Following forskolin stimulation, both intracellular and extracellular cAMP concentrations increased by approximately 15.8-fold and 12-fold, respectively. Similarly, stimulation with tadalafil + BAY 41-2272 resulted in roughly 8.2-fold and 3.4-fold increases in intracellular and extracellular cGMP concentrations, respectively. The presence of MK571 reduced only the extracellular levels of cGMP. This study reveals the presence and function of MRP4 transporters within the porcine bladder and paves the way for future research exploring the role of this transporter in both underactive and overactive bladder disorders.NEW & NOTEWORTHY This study investigates the impact of pharmacological inhibition of MRP4 and MRP5 transporters on cyclic nucleotide signaling in isolated pig bladders. MK571 administration led to modest relaxation, with enhanced effects observed in the presence of phosphodiesterase inhibitors. However, substances elevating cAMP levels remained unaffected. MK571 selectively reduced extracellular cGMP levels. These findings shed light on the role of MRP4 transporters in the porcine bladder, opening avenues for further research into bladder disorders.

Effect of polymorphisms in the FAM151B gene on testis-related trait parameters in Hu sheep

The family with sequence similarity 151 member B (FAM151B) gene family is widely distributed in animals. This gene family encodes proteins belonging to the phosphodiesterase superfamily which plays a role in hydrolyzing intracellular second messengers, and affects the proliferation of Sertoli cells in the testis by degrading intracellular cAMP or cGMP content, thereby affecting the sperm production capacity of the testis. In this study, mutations in the FAM151B gene were screened in 491 Hu sheep to analyze the association among FAM151B gene polymorphisms testicular traits, and sperm-related parameters. A synonymous mutation (5: g.77976636 C>T), was detected in FAM151B, resulting in three genotypes, namely, CC, TT, and TC, with genotypic frequencies of 0.33, 0.05, and 0.62, respectively. This synonymous mutation was found to be significantly correlated with testicular weight, long testicular circumference, short testicular circumference, epididymal weight, and sperm count (p <0.05). In addition, individuals with CC genotypes had significant reproductive performance advantages compared with those with TC and TT genotypes. The current study shows that the FAM151B gene may play important roles in testicular development, and its SNPs are associated with testicular parameters, and sperm count, which provides important indicators for ram selection at early stage.

The effect of γ-irradiation at a dose of 1 Gy on the adenylate- and guanylate cyclase systems of rat platelets at different times of the post-radiation period

Objective. To study the effect of total γ-irradiation of the organism of rats at a dose of 1 Gy on the adenylate- and guanylate cyclase systems and their relationship with Ca2+ levels in the platelet cytoplasm.Materials and methods. The studies were carried out on outbred white male rats of mature age (6-7 months) weighing 250±30 g. Animals were irradiated (once and evenly) on the IGUR unit with 137Сs γ-quanta at a dose of 1 Gy (dose rate 0.62 Gy/min, for 1.61 min). Animals of the corresponding age served as controls. To determine the content of cyclic nucleotides, RIO cAMP/cGMP-iodine125-M-IBOX reagent kits were used. The amount of calcium in platelets was determined using a Fura-2/AM fluorescent probe using spectrofluorimeter SM 2203 “SOLAR” (Minsk, Belarus).Results. In the short period after irradiation, an increase in the activity of the adenylate cyclase system is observed. The cAMP level rises 1.8-1.5 times during 3-30 days of the post-radiation period. However, the increase in [Ca2+]cyt on the 3rd day after irradiation leads to its predominance by 1.8 times in relation to cAMP, which explains the appearance of an increased aggregation activity of the blood platelets in the immediate post-radiation period. An increase in the activity of the guanylate cyclase system and an increase in the intracellular content of cGMP are observed on the 90th day, which leads to a decrease in the Ca2+/cGMP ratio by 1.83 times and corresponds to a reduced platelet aggregation activity and the probability of bleeding in the long term of the rehabilitation period.Conclusion. The effects of radiation on the platelets of rats irradiated at a dose of 1 Gy are due to systemic disorders of the main intracellular regulatory mechanisms: Ca2+, cAMP, cGMP. Based on the data obtained, it can be concluded that pharmacological correction in the short-term after irradiation should be aimed at inhibiting the effects of [Ca2+]cyt and adenylate cyclase system activity, and in the long-term, at inhibition of guanylate cyclase system activity.

#3650 LINACLOTIDE IS PROTECTIVE AGAINST RENAL ISCHEMIA-REPERFUSION INJURY

Abstract Background and Aims Renal ischemia-reperfusion injury is associated with delayed graft function and poor long-term graft survival following transplantation. Linaclotide is a guanylate cyclase C (GC-C) receptor agonist that increases intracellular cGMP concentrations and promotes intestinal transport capacity by activating the GC-C receptor. Since CKD is a deficiency of cGMP in the kidney, increasing cGMP in the kidney by linaclotide would suppress fibrosis, protecting the kidney. Method An AKI rat model of unilateral nephrectomy plus contralateral ischaemia (30 min) and impaired reperfusion (up to 14 days) was used. Linaclotide was administered before and after surgery for 2 weeks, and blood and renal tissue samples were collected to determine its effect on renal function and whether it was protective against the progression of CKD. Results In the I/R injury+ group, creatinine levels were lower in the linaclotide-treated group than in the non-treated group. Sirius red staining showed significant improvement in fibrosis. The hydroxyproline content of kidneys was determined by the basic hydroxyproline method. RNA seq showed that genes down-regulated in I/R injury+ were the oxidative stress markers SOD3 and Gpx1. The expression of genes down-regulated in I/R injury+ was up-regulated to a degree similar to that in I/R injury- by treatment with linaclotide. These data suggest that linaclotide may ameliorate renal fibrosis by suppressing renal dysfunction through its antioxidant effects. Conclusion The antioxidant effect of linaclotide was found to suppress the decrease in renal function caused by I/R. We would like to apply this finding to human renal transplantation in the future to prevent renal function deterioration due to I/R.

The potential involvement of MRP5 pump in urethral dysfunction in streptozotocin-induced diabetic rats.

To examine the effects of i.v. administration of MK-571, a MRP4/5 pump inhibitor, on urethral function in the urethane-anesthetized rat, and the changes of urethral multidrug resistance protein 5 (MRP5) pump in streptozotocin (STZ)-induced diabetes mellitus (DM) rats. Isovolumetric cystometry and urethral perfusion pressure (UPP) measurements were carried out in normal control (NC) group and 8week DM groups under urethane anesthesia. When stable rhythmic bladder contractions were showed, UPP parameters were recorded after successive administration of various dose of MK-571. Additionally, urethral cyclic guanosine monophosphate (cGMP) protein level was evaluated by ELISA, and changes of MRP5 pump and neurogenic nitric oxide synthase (nNOs) in the urethra were examined with immunohistochemical staining and Western blot analysis. In NC group, UPPnadir was significantly decreased but UPP change increased after administration of MK-571, while no significant differences in UPP parameters were observed in 8-week DM group. Furthermore, urethral MRP5 protein level was up-regulated, whereas urethral cGMP and nNOS protein levels were down-regulated in 8-week DM group. MK-571 could not restore NO-mediated urethral relaxation dysfunction in DM rats, which may be attributed to the up-regulation of urethral MRP5 pump, and thus decrease of intracellular cGMP concentration in the urethra. These novel results would be useful for a better understanding of DM-related lower urinary tract dysfunction LUT (LUTD). Also, they could be helpful to study the importance of MRP pumps in the control of urethral relaxation mechanisms under physiological and pathological states.

Reproducibility of the Rod Photoreceptor Response Depends Critically on the Concentration of the Phosphodiesterase Effector Enzyme.

The high sensitivity of night vision requires that rod photoreceptors reliably and reproducibly signal the absorption of single photons, a process that depends on tight regulation of intracellular cGMP concentration through the phototransduction cascade. Here in the mouse (Mus musculus), we studied a single-site D167A mutation of the gene for the α subunit of rod photoreceptor phosphodiesterase (PDEA), made with the aim of removing a noncatalytic binding site for cGMP. This mutation unexpectedly eliminated nearly all PDEA expression and reduced expression of the β subunit (PDEB) to ∼5%-10% of WT. The remaining PDE had nearly normal specific activity; degeneration was slow, with 50%-60% of rods remaining after 6 months. Responses were larger and more sensitive than normal but slower in rise and decay, probably from slower dark turnover of cGMP. Remarkably, responses became much less reproducible than WT, with response variance increasing for amplitude by over 10-fold, and for latency and time-to-peak by >100-fold. We hypothesize that the increase in variance is the result of greater variability in the dark-resting concentration of cGMP, produced by spatial and temporal nonuniformity in spontaneous PDE activity. This variability decreased as stimuli were made brighter, presumably because of greater spatial uniformity of phototransduction and the approach to saturation. We conclude that the constancy of the rod response depends critically on PDE expression to maintain adequate spontaneous PDE activity, so that the concentration of second messenger is relatively uniform throughout the outer segment.SIGNIFICANCE STATEMENT Rod photoreceptors in the vertebrate retina reliably signal the absorption of single photons of light by generating responses that are remarkably reproducible in amplitude and waveform. We show that this reproducibility depends critically on the concentration of the effector enzyme phosphodiesterase (PDE), which metabolizes the second messenger cGMP and generates rod light responses. In rods with the D167A mutation of the α subunit of PDE, only 5%-10% of PDE is expressed. Single-photon responses then become much more variable than in WT rods. We think this variability is caused by spatial and temporal inhomogeneity in the concentration of cGMP in darkness, so that photons absorbed in different parts of the cell produce responses of greatly varying amplitude and waveform.

Abstract 11024: Interferon-Gamma Impairs Human Coronary Artery Endothelial Glucose Metabolism via Tryptophan Catabolism and Activates Fatty Acid Oxidization

Introduction: Endothelial cells depend on glycolysis for much of energy production. Deranged endothelial glycolysis has been associated with various vascular pathobiologies. Interferon-gamma (IFN-γ) producing T-cells have been identified as the predominant pathologic cell subset in human atherosclerotic plaque. While the immunological consequences of these cells have been evaluated, their metabolic effects on endothelial cells remain unknown. The objective of this study was to determine the metabolic consequences of IFN-γ on human coronary artery endothelial cells (HCAEC). Hypothesis: We hypothesized that IFN-γ inhibits endothelial glucose metabolism and results in pathologic phenotypic changes in HCAEC. Methods: The metabolic effects of IFN-γ on primary HCAEC were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux and molecular mechanistic studies. Relevant cellular phenotype was assessed by measuring endothelial intracellular cGMP content and wound healing capacity. Results: IFN-γ inhibited glycolysis of HCAEC by 20 % ( p = 0.006) via global transcriptional suppression of glycolytic enzymes resulting from decreased basal nuclear hypoxia inducible factor 1α (HIF1α) availability. HIF1α destabilization (FC 0.72; p = 0.0039) was mediated by the sequestration of its binding partner HIF1β by aryl hydrocarbon receptor via a kynurenine surge (FC 6.24; p &lt; 0.0001) resulting from IFN-γ-induced tryptophan degradation. Endothelial fatty acid oxidation (FAO) increased by 19% ( p = 0.014) with its inhibition leading to a 23% ATP deficit ( p &lt; 0.0001). These metabolic derangements were associated with pathologic endothelial phenotypic changes, including decreased intracellular cGMP by 29 % ( p = 0.025) and impaired wound healing capacity by 38 % ( p = 0.0098). Conclusions: IFN-γ impairs endothelial glucose metabolism via tryptophan catabolism destabilizing HIF1α and results in a metabolic shift toward increased FAO. These findings suggest a novel mechanistic basis for pathologic T-lymphocyte-endothelial interaction in coronary artery disease mediated by IFN-γ, linking endothelial glucose, amino acid, and fatty acid metabolism and their adverse endothelial functional consequences.

Interferon-γ Impairs Human Coronary Artery Endothelial Glucose Metabolism by Tryptophan Catabolism and Activates Fatty Acid Oxidation.

Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. IFN-γ (interferon-γ)-producing CD4+ and CD8+ T lymphocytes have been identified as the predominant pathological cell subsets in human atherosclerotic plaques. Although the immunologic consequences of these cells have been extensively evaluated, their IFN-γ-mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN-γ, on human coronary artery endothelial cells. The metabolic effects of IFN-γ on primary human coronary artery endothelial cells were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cGMP content, wound-healing capacity, and adhesion molecule expression. IFN-γ exposure inhibited basal glycolysis of quiescent primary human coronary artery endothelial cells by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal HIF1α (hypoxia-inducible factor 1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN-γ-induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1β sequestration by the kynurenine-activated AHR (aryl hydrocarbon receptor). In addition, IFN-γ resulted in a 23% depletion of intracellular nicotinamide adenine dinucleotide in human coronary artery endothelial cells. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by >20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a proinflammatory state. IFN-γ impairs endothelial glucose metabolism by altered tryptophan catabolism destabilizing HIF1, depletes nicotinamide adenine dinucleotide, and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathological T lymphocyte-endothelial interactions in atherosclerosis mediated by IFN-γ, linking endothelial glucose, tryptophan, and fatty acid metabolism with the nicotinamide adenine dinucleotide balance and ATP generation and their adverse endothelial functional consequences.

Advances in Cyclic Nucleotide Phosphodiesterase-Targeted PET Imaging and Drug Discovery.

Cyclic nucleotide phosphodiesterases (PDEs) control the intracellular concentrations of cAMP and cGMP in virtually all mammalian cells. Accordingly, the PDE family regulates a myriad of physiological functions, including cell proliferation, differentiation and apoptosis, gene expression, central nervous system function, and muscle contraction. Along this line, dysfunction of PDEs has been implicated in neurodegenerative disorders, coronary artery diseases, chronic obstructive pulmonary disease, and cancer development. To date, 11 PDE families have been identified; however, their distinct roles in the various pathologies are largely unexplored and subject to contemporary research efforts. Indeed, there is growing interest for the development of isoform-selective PDE inhibitors as potential therapeutic agents. Similarly, the evolving knowledge on the various PDE isoforms has channeled the identification of new PET probes, allowing isoform-selective imaging. This review highlights recent advances in PDE-targeted PET tracer development, thereby focusing on efforts to assess disease-related PDE pathophysiology and to support isoform-selective drug discovery.

Nitric oxide impacts bovine sperm capacitation in a cGMP-dependent and cGMP-independent manner.

We aimed to elucidate whether NO acts in in vitro sperm capacitation in bovine via cGMP/PKG1 pathway. For this, cryopreserved bovine sperm were capacitated in vitro with 20µg/ml heparin (Control) plus treatments: 1mM L-arginine (L-arg, NO precursor), 50µM Rp-8-Bromo-β-phenyl-1,N2 -ethenoguanosine-3',5'-cyclic monophosphorothioate (Rp-8-Br-cGMPS, selective inhibitor of the binding site for cGMP in PKG1), 1mM 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, NO scavenger), and the combinations of L-arg+RP-8-Br-cGMPS and L-arg+PTIO. Sperm motility and vigour were determined by phase-contrast microscopy, capacitation status by chlortetracycline staining, and the intracellular concentration of cGMP was measured by ELISA. Data were subjected to analysis of variance and means compared with SNK test at 5% probability. Motility and vigour were lower in sperm treated with PTIO when compared to Control and other treatments (p<.05). The L-arg treatment showed the highest percentage of capacitated sperm when compared to the Control and other treatments (Rp-8-Br-cGMPS, L-arg+Rp-8-Br-cGMPS and PTIO) (69.8±3.4%, 51.2±3.0, 51.1±2.1, 51.2±3.0 and 45.5±2.7, respectively) (p<.05). The capacitation ratio (%) was lower in treatments with Rp-8-Br-cGMPS, L-arg+Rp-8-Br-cGMPS and PTIO, respectively (p<.05). Lastly, cGMP concentration (pmol/ml) was lower in PTIO and L-arg+PTIO (1.3±0.3 and 1.6±0.4) and was higher in Rp-8-Br-cGMPS and L-arg+Rp-8-Br-cGMPS (3.7±0.4 and 4.0±0.5) treatments. We showed that during in vitro capacitation of cattle: (a) NO influences sperm motility and vigour; (b) NO is associated with cGMP synthesis through two independent pathways and (c) the cGMP/PKG1 pathway has a partial role in sperm capacitation and does not involve the L-arg/NO.

Cyclic nucleotide signalling compartmentation by PDEs in cultured vascular smooth muscle cells.

Up-regulation of phosphodiesterases (PDEs) is associated with several vascular diseases, and better understanding of the roles of each PDE isoform in controlling subcellular pools of cyclic nucleotides in vascular cells is needed. We investigated the respective role of PDE1, PDE5, and PDE9 in controlling intracellular cAMP and/or cGMP concentrations ([cAMP]i , [cGMP]i ) in cultured rat aortic smooth muscle cells (RASMCs). We used selective inhibitors of PDE1 (PF-04471141), PDE5 (sildenafil), and PDE9 (PF-04447943) to measure cAMP- and cGMP-PDE activities with a radioenzymatic assay, in RASMC extracts. Real-time [cAMP]i and [cGMP]i were recorded by Förster resonance energy transfer-imaging in single living cells, and cell proliferation was assessed in FBS-stimulated cells. PDE1, PDE5, and PDE9 represented the major cGMP-hydrolyzing activity in RASMCs. Basal PDE1 exerted a functional role in degrading in situ the cGMP produced in response to activation of particulate GC by C-type natriuretic peptide. In high intracellular Ca2+ concentrations, PDE1 also regulated the NO/soluble GC-dependent cGMP response, as well as the β-adrenoceptor-mediated cAMP response. PDE5 exerted a major role in degrading cGMP produced by NO and the natriuretic peptides. PDE9 only regulated the NO-induced [cGMP]i increase. All three PDEs contributed differently to regulate cell proliferation under basal conditions and upon cGMP-elevating stimuli. Our data emphasize the distinct roles of PDE1, PDE5, and PDE9 in local regulation of [cAMP]i and [cGMP]i , in vascular smooth muscle cells, strengthening the concept of PDEs as key actors in the subcellular compartmentation of cyclic nucleotides.

Inhibition of Cyclic Nucleotide Efflux by Placental MRP Transporters Enhances Trophoblast Syncytialization

In the placenta, multinucleated syncytiotrophoblasts arise from the fusion of cytotrophoblasts, a process regulated by cyclic nucleotide (cAMP and cGMP) signaling. Syncytiotrophoblasts secrete hormones and express efflux transporters, including the multidrug resistance‐associated proteins (MRP) 1 and 5. Understanding the interplay of cyclic nucleotides, efflux transporters, and syncytialization is important because aberrant cytotrophoblast functioning contributes to the pathogenesis of placental diseases and toxicities. In the present study, we sought to determine whether MRP transporters regulate the syncytialization of human placental cells. BeWo b30 trophoblasts were treated with vehicle, MK‐571 (25 μM), 8‐Bromo‐cAMP (100 μM), forskolin (25 μM), IBMX (200 μM), or their combination for up to 24 hr. Markers of trophoblast cell fusion (glial cells missing homolog 1, GCM1; syncytin 2) and hormone secretion (human chorionic gonadotropin, hCGa and hCGb) were quantified by qPCR. Treatment of BeWo cells with MK‐571, a pharmacological inhibitor of MRPs, increased intracellular concentrations of cAMP and cGMP by 15‐ and 19‐fold, respectively. Moreover, MK‐571 treatment up‐regulated cell fusion and hormone secretion markers (GCM1, hCGa, hCGb, syncytin 2) by 20–200% under basal conditions. Similarly, MRP inhibition with MK‐571 further enhanced syncytialization and hormone markers following stimulation with the permeable cAMP analog 8‐Bromo‐cAMP (2‐ to 6‐fold), activation of cAMP synthesis with forskolin (20–160%), or inhibition of cAMP/cGMP breakdown by IBMX (50–130%). In addition, shRNAs were used to generate a stable BeWo B30 trophoblast cell line with reduced MRP5 expression. Control and MRP5‐knockdown cells were treated with vehicle or 8‐Bromo‐cAMP for 24 hr. In response to treatment with 8‐Bromo‐cAMP, the induction of GCM1, hCGa, and hCGb mRNA expression was higher in MRP5 knockdown cells as compared to control cells. Furthermore, MRP5‐knockdown cells were larger in size (including greater numbers of multinucleated cells) and exhibited decreased staining of the tight junction protein E‐cadherin, pointing to enhanced syncytialization. In conclusion, MRP transporters participate in trophoblast cell fusion, which likely represents a novel mechanism regulating placentation and placental toxicities.Support or Funding InformationSupported by F31ES029794, R01ES029275, T32ES007148, P30ES005022.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Molecular Organization of Human cGMP Specific Phosphodiesterase 6 (PDE6): Structural Implications of Somatic Mutations in Cancer and Retinitis Pigmentosa

In the cyclic guanosine monophosphate (cGMP) signaling pathway, phosphodiesterase 6 (PDE6) maintains a critical balance of the intracellular concentration of cGMP by catalyzing it to 5′ guanosine monophosphate (5′-GMP). To gain insight into the mechanistic impacts of the PDE6 somatic mutations that are implicated in cancer and retinitis pigmentosa, we first defined the structure and organization of the human PDE6 heterodimer using computational comparative modelling. Each subunit of PDE6αβ possesses three domains connected through long α-helices. The heterodimer model indicates that the two chains are likely related by a pseudo two-fold axis. The N-terminal region of each subunit is comprised of two allosteric cGMP-binding domains (Gaf-A & Gaf-B), oriented in the same way and interacting with the catalytic domain present at the C-terminal in a way that would allow the allosteric cGMP-binding domains to influence catalytic activity. Subsequently, we applied an integrated knowledge-driven in silico mutation analysis approach to understand the structural and functional implications of experimentally identified mutations that cause various cancers and retinitis pigmentosa, as well as computational saturation mutagenesis of the dimer interface and cGMP-binding residues of both Gaf-A, and the catalytic domains. We studied the impact of mutations on the stability of PDE6αβ structure, subunit-interfaces and Gaf-cGMP interactions. Further, we discussed the changes in interatomic interactions of mutations that are destabilizing in Gaf-A (R93L, V141 M, F162 L), catalytic domain (D600N, F742 L, F776 L) and at the dimer interface (F426A, F248G, F424 N). This study establishes a possible link of change in PDE6αβ structural stability to the experimentally observed disease phenotypes.

Buffalo oocyte-secreted factors promote cumulus cells apoptosis and the rate of cGMP production but not steroidogenesis.

The objectives of this study were to investigate the effect of buffalo oocyte-secreted factors (OSFs) on cumulus cells (CCs) functions, apoptosis and cGMP generation, and whether the direct contact between oocyte and CCs is essential for oocyte-mediated regulation of CCs functions. Buffalo CCs were cultured during IVM within three groups: (a) intact cumulus-oocyte complexes (COCs), (b) CCs cocultured with denuded oocytes (DOs) (CCs+DOs) and (c) CCs monolayer cultured alone (CCsM). After 24hr of IVM, CCs were harvested for evaluation of the relative mRNA abundance of the genes encoding gap junction (GJA1), glycolysis (PFKP and LDHA), apoptosis (CASPASE-3 and BCL-2) and steroidogenesis (ER-β and PGR) by QRT-PCR, and CASPASE-3 proteins, using western blot. Intracellular cGMP content was also assessed by ELISA. Results showed that the relative abundance of LDHA, PFKP and BCL-2 significantly increased (p<0.05) in COCs, whereas GJA1 and CASPASE-3 exhibited lower expression (p<0.05) compared to CCs+DOs and CCsM groups. However, the expression levels of CASPASE-3, both mRNA and protein, were significantly (p<0.05) downregulated in CCs+DOs compared to CCsM. There was no significant difference in the expression level of PGR and ER-β between the groups. The intracellular content of cGMP was notably (p<0.05) higher in COCs compared to CCs+DOs and CCsM groups. In conclusion, this study demonstrated, for the first time, that buffalo OSFs protect CCs against apoptosis and stimulate their cGMP production; however, the regulation of cumulus glycolysis and gap junction is confined to those in close contact with the oocyte. Neither OSFs from COCs nor those from DOs have any effect on CCs steroidogenesis.

Developmental competence of buffalo (Bubalus bubalis) denuded oocytes cocultured with cumulus cells: Protective role of cumulus cells

The objectives of the present study were to evaluate the developmental competence of buffalo denuded oocytes (DOs) cocultured with cumulus cells (CCs) during in vitro maturation, and to investigate the mechanisms by which CCs promote oocyte maturation and development. Buffalo oocytes were matured in vitro for 24 h in three groups: (1) intact cumulus–oocyte complexes (COCs) (2) DOs cocultured with CCs (DOsCC), and (3) DOs cultured alone (DOs). Matured oocytes were used to determine the relative mRNA abundance of Gdf-9, Bmp15, Zar1, Caspase-3, Bcl-2, Zp2, Zp3, Cd9 and Pde3a by Rt-qPCR and CASPASE-3 protein expression by immunofluorescence. The intracellular content of cGMP, cAMP and MPF activity and the rate of embryonic development were also assessed. Results of the present study showed that in DOs, the relative mRNA abundance of Gdf-9, Bmp15, and Cd9 significantly (P < 0.05) decreased, whereas Caspase-3 (mRNA and protein levels), Bcl-2, and Pde3a exhibited higher expression than DOsCC and COCs. However, there was no significant difference among the groups in the expression level of Zar-1, Zp2, and Zp3. The intracellular content of cAMP and MPF activity was notably higher (P < 0.05) in DOs compared to COCs and DOsCC. There was no significant difference between COCs and DOsCC in cGMP content, which was significantly lower (P < 0.05) in DOs. Moreover, the cleavage and blastocyst rates were 58.4 ± 1.8%, 43.7 ± 1.1%, 18.4 ± 0.9% and 18.0 ± 1.3%, 11.0 ± 0.9% and 4.5 ± 0.6% in COCs, DOsCC and DOs groups, respectively. In conclusion, the presence of CCs protects buffalo DOs from apoptosis and promotes maturation through regulation of the intracellular content of cAMP and MPF activity and improves the fertilizing capacity of oocytes through modulation of the gamete fusion gene, Cd9.

Real-Time Imaging Reveals Augmentation of Glutamate-Induced Ca2+ Transients by the NO-cGMP Pathway in Cerebellar Granule Neurons.

Dysfunctions of NO-cGMP signaling have been implicated in various neurological disorders. We have studied the potential crosstalk of cGMP and Ca2+ signaling in cerebellar granule neurons (CGNs) by simultaneous real-time imaging of these second messengers in living cells. The NO donor DEA/NO evoked cGMP signals in the granule cell layer of acute cerebellar slices from transgenic mice expressing a cGMP sensor protein. cGMP and Ca2+ dynamics were visualized in individual CGNs in primary cultures prepared from 7-day-old cGMP sensor mice. DEA/NO increased the intracellular cGMP concentration and augmented glutamate-induced Ca2+ transients. These effects of DEA/NO were absent in CGNs isolated from knockout mice lacking NO-sensitive guanylyl cyclase. Furthermore, application of the cGMP analogues 8-Br-cGMP and 8-pCPT-cGMP, which activate cGMP effector proteins such as cyclic nucleotide-gated cation channels and cGMP-dependent protein kinases (cGKs), also potentiated glutamate-induced Ca2+ transients. Western blot analysis failed to detect cGK type I or II in our primary CGNs. The addition of phosphodiesterase (PDE) inhibitors during cGMP imaging showed that CGNs degrade cGMP mainly via Zaprinast-sensitive PDEs, most likely PDE5 and/or PDE10, but not via PDE1, 2, or 3. In sum, these data delineate a cGK-independent NO-cGMP signaling cascade that increases glutamate-induced Ca2+ signaling in CGNs. This cGMP–Ca2+ crosstalk likely affects neurotransmitter-stimulated functions of CGNs.

Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy.

The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG) channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP). Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and voltage-gated potassium channels (KCN). In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.

A novel epididymal quiescence factor inhibits sperm motility by modulating NOS activity and intracellular NO-cGMP pathway.

Mature and potentially motile spermatozoa stored in cauda epididymis in an inactive state for approximately 30 days; however, during ejaculation they regain motility. To understand the actual molecular mechanism of the sperm quiescence during caudal stay, a proteinaceous quiescence factor (QF) has been purified from caprine epididymal plasma to apparent homogeneity. In the present study complete purification, detailed characterization as well as mechanistic pathway of QF has been described. QF is purified to 215-fold with 45% activity recovery. It is a 59 kDa monomeric protein with isoelectric point 5.8 and optimally active at pH 7.5. Circular dichroism spectroscopy and atomic force microscopy study confirm its α-helical secondary structure and globular tertiary conformation. QF is a thermo-stable protein as higher temperature does not alter its helical structure. N-terminal amino acid sequencing and MALDI analysis of QF did not find 100% similarity with any available protein of the database, proved its novelty. QF at 2 μM dose inhibits sperm progressive forward motility within 10 min. This motility inhibitory activity of QF is mediated by reducing NOS enzyme activity and subsequently decreasing the intracellular NO and cGMP concentration. It does not modulate intracellular Ca++ and cAMP concentration. QF has no adverse effect on DNA integrity and morphology of spermatozoa. Motility inhibitory action of QF is reversible. Thus, the role of QF in maintaining energy saving quiescence state of mature cauda spermatozoa and its reactive nitrogen species reducing activity may lead to a new direction for storage of spermatozoa and idiopathic male infertility.

Cyclic guanosine monophosphate modulates accumulation of phosphodiesterase 5 inhibitors in human platelets

Sildenafil and tadalafil are widely-used phosphodiesterase 5 (PDE5) inhibitors for which no clear dose-response relationship could be established. Using isolated and/or recombinant PDE5, it has been demonstrated that cGMP can increase the affinity of this enzyme for sildenafil and tadalafil. We thus hypothesized that in cells expressing the nitric oxide – soluble guanylyl cyclase – cyclic guanosine monophosphate – PDE5 (NO-sGC-cGMP-PDE5) pathway such as platelets, the presence of NO increases the intracellular cGMP content and thus promotes the intracellular accumulation of sildenafil or tadalafil. As a cell model, isolated and washed human platelets were used. Platelet suspensions were incubated with sildenafil or tadalafil at different concentrations and for various time intervals with or without an NO donor to increase intraplatelet cGMP concentrations. Intracellular sildenafil or tadalafil was quantified by ultra-performance liquid chromatography tandem mass spectrometry and intracellular cGMP by an enzyme-linked immunosorbent assay. Sildenafil accumulated in platelets with an up to 4-fold higher accumulation when platelets were pretreated with an NO donor (p < .0001). Accumulation of tadalafil in platelets was even higher, whereas the increase was 2-fold when an NO donor was present (p < .001). This accumulation was time-dependent and happened concomitantly with a rise in intracellular cGMP. Our data demonstrate that intracellular cGMP increases intracellular PDE5 inhibitor concentrations most likely by raising the affinity of these compounds for PDE5. These findings suggest that PDE5 inhibitor action in humans is critically influenced by modulators of the activity of the NO pathway.