Cyclase Activity Research Articles

The follicle-stimulating hormone receptor (FSHR) is expressed in human sperm and it may be considered as molecular marker of the detrimental effects related to the physiopathology of testicular varicocele

Localization of the follicle-stimulating hormone receptor (FSHR), has been always closely related to the testis and ovary. FSH/FSHR role in Sertoli cell, has been known, however, the sites of FSH action within the male reproductive system are not resolved yet. Few studies have raised the intriguing possibility that germ cells may exhibit FSHR, all the reports point to Sertoli cells as the exclusive FSH target cells in testis. Besides, the attention has been always paid on the FSHR several polymorphisms which affect receptor sensitivity and expression. The presence of FSHR in germinal cells from spermatogonia to spermatocytes, including round spermatids is controversial or excluded. The mechanisms by which testicular varicocele affects fertility remain undetermined. Recently, our studies showed that the disease causes damage in sperm at the molecular level opening a new chapter in the already multifaceted physiopathology of varicocele. Samples used in this study were from normozoospermic and from diagnosed varicocele of grade III on the left testis patients. To date four FSHR isoforms were discovered, FSHR1, FSHR2, FSHR3 and FSHR4. The activity of FSHR1 is mediated by G proteins, which activate adenylate cyclase. FSHR2 and FSHR3 also bind FSH, but this does not result in activation of adenylate cyclase. FSHR4 does not bind FSH. By western blot analysis, we showed that healthy sperm express FSHR1, FSHR2 and FSHR3 while FSHR4 is almost absent. Varicocele does not express FSHR2. Immunofluorescence assay evidences FSHR localization prevalently at the midpiece level, which was strongly reduced in varicocele sperm. Responses to different FSH concentrations on motility and survival were significantly reduced in varicocele respect to the normal sperm, probably due to the lower FSHR1 expression and FSHR2 absence. The FSHR significance in human male gamete also emerged from the acrosome reaction histochemical studies, during FSH treatment which significantly induced the process. Our data showed for the first time that human sperm express the FSHR and constrain the need of further studies on the molecular anatomy of human male gamete both in healthy and in pathological conditions related to the male genital apparatus, considering the high couple infertility linked to the male. The translation of these new researches in the clinic surgery of testicular varicocele needs to be taken into account since molecular alterations in sperm imply a decline in the acquisition of fertilizing ability, and to date controversies exist on the opportunity to intervene surgically.

Type IV Pili-Dependent Motility as a Tool to Determine the Activity of c-di-GMP Modulating Enzymes in Myxococcus xanthus.

The nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates multiple processes in bacteria including cellular motility. The rod-shaped Myxococcus xanthus cells move in the direction of their long axis using two distinct motility systems: type IV pili (T4P)-dependent motility and gliding motility. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase causes defects in T4P-dependent motility without affecting gliding motility. As both an increased and a decreased level of c-di-GMP affect T4P-dependent motility, M. xanthus represents a good model system to assess enzyme activity of diguanylate cyclases and phosphodiesterases using T4P-dependent motility as a readout. Here, we describe the assay, which allows correlating diguanylate cyclase and phosphodiesterase activity with T4P-dependent motility in M. xanthus.

Intracellular Molecular Mechanisms of Adrenergic Regulation of Pulmonary Vein Myocardium Membrane Potential

Pulmonary vein (PV) myocardium is characterized by numerous electrophysiological properties which make this tissue highly prone to spontaneous, ectopic activity partially due to resting potential (RP) instability. PV derived ectopy frequently underlies supraventricular arrhythmias, including atrial fibrillation. It has also been demonstrated that adrenergic stimulation causes proarrhythmic alterations in PV. Selective α1- and β-adrenoreceptors stimulation causes RP depolarization and hyperpolarization, respectively, at least in rats. The intracellular mechanisms of α1- and β-adrenoreceptors-dependent RP drifts are not investigated. Adenylate cyclase (AC) activator forscolin similarly to selective β-adrenoreceptors agonist isoproterenol (ISO) induced strong hyperpolarization in quiescent isolated perfused multicellular preparations of rat PV. Maximal value of hyperpolarization in PV was equal after application of both compounds. Proteinkinase A (PKA) inhibitors КТ5720, H-89 and Rp-adenosine-cAMP suppressed ISO-induced hyperpolarization in PV. Inhibitors of phospholipase C (U73122) or D (FIPI), similarly to proteinkinase C (PKC) inhibitor chelerythrine, failed to suppress α-adrenoreceptors-dependent phenylephrine-induced depolarization in rat PV myocardium. These results allow us to suggest that β-adrenoreceptors-dependent RP hyperpolarization in quiescent rat PV myocardium is only partially mediated by cAMP-dependent signal transduction pathway and by PKA. Besides, PKA-independent mechanisms also contribute to β-agonists effects in PV. In addition, α-adrenoreceptors-dependent depolarization in rat PV myocardium could be independent on PLC and PKC.

Acetamiprid inhibits testosterone synthesis by affecting the mitochondrial function and cytoplasmic adenosine triphosphate production in rat Leydig cells.

The insecticide acetamiprid is used to control noxious agricultural pests. However, it can cause mammalian toxicity. We evaluated the reproductive toxicity of acetamiprid in adult male Sprague Dawley rats. Rats were given oral acetamiprid alone or with vitamin E for 35 days. Rat plasma testosterone concentration and sperm quality decreased significantly as the levels of luteinizing hormone (LH) increased after exposure. At the same time, acetamiprid increased malondialdehyde and nitric oxide (NO) levels of Leydig cells. Further analysis showed that acetamiprid reduced the adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) production of Leydig cells, but the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and the activity of adenylyl cyclase were not changed. Acetamiprid exposure also significantly diminished protein levels of steroidogenic acute regulatory protein (STAR), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B), and cytochrome P450, family 11, subfamily a, polypeptide 1 (CYP11A1), and testicular mRNA levels, which are cAMP-dependent proteins that are essential for steroidogenesis. Electron microscopy indicated mitochondrial membrane damage in the Leydig cells of the testes of exposed rats. Vitamin E ameliorated the impairment of acetamiprid on Leydig cells. Our results indicate that acetamiprid causes oxidative stress and mitochondrial damage in Leydig cells and inhibits the synthesis of testicular ATP and cAMP. Acetamiprid disrupts subsequent testosterone biosynthesis by decreasing the rate of conversion of cholesterol to testosterone and by preventing cholesterol from entering the mitochondria within the Leydig cells. These effects caused reproductive damage to the rats.

Oxygen and Bis(3',5')-cyclic Dimeric Guanosine Monophosphate Binding Control Oligomerization State Equilibria of Diguanylate Cyclase-Containing Globin Coupled Sensors.

Bacteria sense their environment to alter phenotypes, including biofilm formation, to survive changing conditions. Heme proteins play important roles in sensing the bacterial gaseous environment and controlling the switch between motile and sessile (biofilm) states. Globin coupled sensors (GCS), a family of heme proteins consisting of a globin domain linked by a central domain to an output domain, are often found with diguanylate cyclase output domains that synthesize c-di-GMP, a major regulator of biofilm formation. Characterization of diguanylate cyclase-containing GCS proteins from Bordetella pertussis and Pectobacterium carotovorum demonstrated that cyclase activity is controlled by ligand binding to the heme within the globin domain. Both O2 binding to the heme within the globin domain and c-di-GMP binding to a product-binding inhibitory site (I-site) within the cyclase domain control oligomerization states of the enzymes. Changes in oligomerization state caused by c-di-GMP binding to the I-site also affect O2 kinetics within the globin domain, suggesting that shifting the oligomer equilibrium leads to broad rearrangements throughout the protein. In addition, mutations within the I-site that eliminate product inhibition result in changes to the accessible oligomerization states and decreased catalytic activity. These studies provide insight into the mechanism by which ligand binding to the heme and I-site controls activity of GCS proteins and suggests a role for oligomerization-dependent activity in vivo.

Formation of nitric oxide by aldehyde dehydrogenase-2 is essential for nitroglycerin-induced activation of soluble guanylate cyclase

Formation of nitric oxide by aldehyde dehydrogenase-2 is essential for nitroglycerin-induced activation of soluble guanylate cyclase

Modulation of growth cone filopodial length by carbon monoxide.

Carbon monoxide (CO) is physiologically produced via heme degradation by heme oxygenase enzymes. Whereas CO has been identified as an important physiological signaling molecule, the roles it plays in neuronal development and regeneration are poorly understood. During these events, growth cones guide axons through a rich cellular environment to locate target cells and establish synaptic connections. Previously, we have shown that another gaseous signaling molecule, nitric oxide (NO), has potent effects on growth cone motility. With NO and CO sharing similar cellular targets, we wanted to determine whether CO affected growth cone motility as well. We assessed how CO affected growth cone filopodial length and determined the signaling pathway by which this effect was mediated. Using two well-characterized neurons from the freshwater snail, Helisoma trivolvis, it was found that the CO donor, carbon monoxide releasing molecule-2 (CORM-2), increased filopodial length. CO utilized a signaling pathway that involved the activation of soluble guanylyl cyclase, protein kinase G, and ryanodine receptors. While increases in filopodial length often occur from robust increases in intracellular calcium levels, the timing in which CO increased filopodial length corresponded with low basal calcium levels in growth cones. Taken together with findings of a heme oxygenase-like protein in the Helisoma nervous system, these results provide evidence for CO as a modulator of growth cone motility and implicate CO as a neuromodulatory signal during neuronal development and/or regeneration. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 677-690, 2017.

Abstract 128: Impaired Beta Adrenergic Receptor Resensitization Contributes to Human Heart Failture

Beta adrenergic receptors (βARs) are one of the most powerful regulators of cardiac function, and βAR downregulation and desensitization are hallmarks of heart failure. Agonist occupied βARs undergo desensitization by G-protein coupled receptor kinase phosphorylation leading to βAR internalization. Desensitized βARs are resensitized by dephosphorylation in the endosomes by PP2A; however, less is known about role of resensitization in heart failure. Although we have previously shown that resensitization is regulated by inhibition of PP2A by I2PP2A via PI3Kγ (Vasudevan et. al., 2011), the underlying mechanisms of I2PP2A binding to PP2A are not well understood. We used PyMOL software to find the binding interaction between PP2A and I2PP2A. Based on in silico predictions, we generated a mutant PP2A that when expressed would compete out I2PP2A and inhibit I2PP2A from binding to endogenous PP2A. Expression of PP2A mutant in β2AR expressing cells showed preservation of β2AR function following stimulation as measured by reduced β2AR phosphorylation, increased cAMP generation and increased phosphatase function. We also tested whether resensitization as a mechanism is altered in endstage human heart failure samples. Since resensitization occurs in endosomes, plasma membranes and endosomes were isolated from human heart samples and assessed for PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase (AC) activity as a measure of βAR function. Significant PI3Kγ activity was observed to increase in heart failure samples while AC activity was reduced in plasma membranes. Consistent with the reduced recovery in βAR function in heart failure samples, significant β2AR phosphorylation was observed in the endosomes and plasma membranes of human heart failure samples compared to non-failing. Correspondingly, we observed significant reduction in endosomal PP2A activity in heart failure samples versus controls suggesting inhibited resensitization occurs in human heart failure. Together our studies suggest that resensitization is inhibited in human heart failure and targeting I2PP2A may provide preservation of receptor function and beneficial cardiac remodeling.

Abstract 4662: Beta blockers abrogate EGFR TKI resistance induced by adrenergic receptor-mediated upregulation of IL-6 and modulation of the LKB1/AMPK/mTOR axis

Abstract Therapeutic strategies to target EGFR tyrosine kinase inhibitor (TKI) resistance mediated by mechanisms other than T790M are a major clinical challenge. Studies have implicated IL-6 as a mediator of EGFR TKI resistance, and IL-6 is known to be regulated by adrenergic receptors (AR) in some cancers. We investigated whether adrenergic pathways can promote T790M-independent EGFR TKI resistance in preclinical models and in clinical studies. We found that β2-AR was highly expressed in our panel of 119 cell lines and in NSCLC clinical specimens. Activation of β-ARs by stress hormones such as norepinephrine (NE) induced a dramatic rise in IL-6, and this occurred through β2-ARs. β-AR inhibitors (i.e. propranolol), but not α-AR inhibitors, blocked IL-6 induction. Analysis of downstream signaling pathways revealed that β2-ARs induced IL-6 expression through activation of adenylyl cyclase, p90RSK and CREB. To identify novel signaling pathways modulated by ARs, we stimulated NSCLC cell lines with NE and analyzed protein lysates by RPPA to detect expression and activation of >100 proteins. β-AR signaling inactivated the tumor suppressor LKB1 through phosphorylation of S428 and subsequently increased mTOR activity. LKB1 inactivation was critical for IL-6 induction. This finding is important as LKB1 loss is known to be a driver of NSCLC resistance and metastasis. Moreover, we found that β2-AR activation promoted EGFR TKI resistance in cell lines and in mouse models of EGFR mutant NSCLC. The effect of β-AR on EGFR TKI resistance was blocked by the addition of the beta blocker propranolol or IL-6 antibodies, in vitro and in vivo. Consistent with our preclinical studies, in the phase III ZEST clinical study testing erlotinib vs vandetanib, we found that high plasma levels of IL-6 was associated with a worse PFS and OS in the erlotinib arm. In addition, we found that circulating levels of IL-6 were significantly lower in NSCLC patients incidentally receiving beta blockers in the BATTLE trial. Finally, we analyzed the influence of incidental beta blocker use in the LUX-Lung3 study testing afatinib vs chemotherapy in EGFR mutant NSCLC patients. In patients not receiving beta blockers, the median PFS was 11.1 and 6.9 months for afatinib and chemotherapy, respectively, and afatinib improved PFS with a hazard ratio (HR) of 0.60. In patients receiving beta blockers, the median PFS was 13.6 and 2.5 for afatinib and chemotherapy, respectively, and afatinib improved PFS with a HR of 0.25. In conclusion, our preclinical and clinical data provide evidence that β2-AR activation can upregulate IL-6 in EGFR mutant+ NSCLC, modulate the LKB1/AMPK/mTOR axis, and promote EGFR TKI resistance. Moreover, EGFR mutant+ patients using beta blockers had greater relative PFS benefit from afatinib vs chemotherapy compared with those not using beta blockers, supporting future clinical testing of EGFR TKIs in combination with beta blockers. Citation Format: Monique B. Nilsson, Huiying Sun, Lixia Diao, Pan Tong, Youhong Fan, Hai Tran, Diane Liu, Guillermo Armaiz Pena, Jing Wang, Phil Rowe, Alan Webster, Jack Lee, Daniel Gomez, Waun Ki Hong, Ignacio Wistuba, Anil Sood, John Heymach. Beta blockers abrogate EGFR TKI resistance induced by adrenergic receptor-mediated upregulation of IL-6 and modulation of the LKB1/AMPK/mTOR axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4662.

Effect of N-phenyl-2-naphthylamine on activity of adenylate cyclase signal system components and virulence of bacterial phytopathogens and mutualists

The effect of N-phenyl-2-naphthylamine, negative allelochemical isolated from the exudates of roots of pea (Pisum sativum L.), on the growth and activity of the adenylate cyclase signal system and virulence factors of the bacteria Rhizobium leguminosarum bv. viciae and Pseudomonas siringae pv. pisi was studied. It was demonstrated that N-phenyl-2-naphthylamine at a physiological concentration nonspecifically inhibited the growth of these bacteria in both planktonic cultures and biofilms. One of the reasons for this phenomenon is the reduction of intra- and extracellular concentrations of cAMP due to greater activation of phosphodiesterase, which disrupts cAMP, in comparison to soluble adenylyl cyclase, which synthesizes it. At the same time, N-phenyl-2-naphthylamine did not affect activity of either membrane-bound adenylyl cyclase or bacterial virulence factors.

The synthetic peptide octarphin activates soluble guanylate cyclase in macrophages

The effect of the peptide octarphin (TPLVTLFK), a selective agonist of the nonopioid β-endorphin receptor, on the activity of soluble (sGC) and membrane-bound guanylate cyclase (mGC) of peritoneal macrophages from mouse, rat, and guinea pig has been studied. It has been found that, in the concentration range of 10–1000 nM, octarphin increases the activity of sGC and has no effect on the activity of mGC in macrophages of these species. The activating effect of the peptide toward sGC was dose-dependent and was maximal at a concentration of 100 nM. These results indicate that the increase in the concentration of guanosine 3',5'-cyclophosphate in macrophages in the presence of octarphin occurs through the activation of sGC. It can be stated that the activating effect of octarphin on peritoneal macrophages is accomplished in the following way: an increase in the activity of inducible NO synthase → an increase in NO production → activation of sGC → an increase in the intracellular cGMP level.

Riociguat and sildenafil for pulmonary hypertension: similarity and difference

Last decades, several drugs for treatment of pulmonary hypertension have been implemented in clinical practice. Sildenafil selectively inhibits phosphodiesterase-5 (PDE-5), increases cyclic guanosine monophosphate (cGMP) level in the pulmonary vascular wall leading to vasodilation, inhibition of proliferation and other specific effects. Sildenafil is indicated as single therapy and in sequential combination therapy of pulmonary arterial hypertension (PAH) (high-level evidence, high-class recommendation). However, therapeutic response on PDE-5 inhibitors is insufficient in a certain number of patients. Soluble guanylate cyclase (sGC) activators were proposed as a good alternative for such patients. Riociguat has dual mechanism of action: firstly, it is designed to increase cGMP biosynthesis via direct activation of sGC; this pathway resembles that of nitric oxide (NO) but is independent on NO. Secondly, riociguat can sensitise sGC to NO. Riociguat is indicated as single therapy and in sequential combination therapy of PAH and to treatment of chronic thromboembolic pulmonary hypertension (high-level evidence, high-class recommendation). A clinical trial RESPITE has been initiated in February, 2014, which is intended to evaluate efficacy of switching PAH patients from PDE-5 inhibitors to riociguat. Potential advantage of this drug is the mechanism independent of NO and of other PDE enzyme isoforms. Whether this advantage is clinically significant, remains to be seen, probably, in the RESPITE trial.

Evolutionarily conserved phosphorylation and palmitoylation-dependent regulation of dopamine D1-like receptors in vertebrates

In the vertebrate central nervous system, dopamine is a neurotransmitter, acting as a modulator of variety of neuronal functions, including cognition, emotion, locomotor activity, hunger and satiety, and endocrine system regulation. Dopamine receptors are composed of D 1 , D 2 , D 3 , D 4 and D 5 receptors, which are classified into two major subtypes, D 1 -like (D 1 and D 5 ) and D 2 -like (D 2 , D 3 and D 4 ) dopamine receptors. D 1 -like receptors stimulate adenylyl cyclase (AC) activity, whereas D 2 -like receptors inhibit AC activity and these receptors modulate many signaling pathways in dopaminergic synapses. Previous studies showed that post-translational protein phosphorylation and palmitoylation of mammalian D 1 receptors regulate functional properties, stability, cellular localization and membrane trafficking of the receptors. Here, I further focus on conservation of phosphorylation and palmitoylation sites found in vertebrate and invertebrate D 1 -like receptor homologs. Analysis of databases provides evidence to suggest that PKA- and GRKs-catalyzed phosphorylation sites of D 1 -like receptors have been completely conserved in the vertebrate lineage, in spite of the divergence of D 1 -like receptors full-length amino acid sequences during molecular evolution. These intracellular phosphorylated serine and threonine residues of D 1 -like receptors are evolutionarily conserved against mutation pressure throughout vertebrate species with a couple of exceptions. Furthermore, a PKC-mediated phosphorylation site of D 1 -like receptors is conserved in gnathostomes (jawed vertebrates) and not exist in agnathans (jawless vertebrates). Another phosphorylation site for PKC has been additionally acquired only in mammalian D 1 receptor orthologs. These findings suggest that the multiple kinases-dependent regulation of dopaminergic synapses by phosphorylation is so critical for complex vertebrate higher brain function. Namely, dynamic regulation of dopaminergic synapses made possible by reversible phosphorylation of D1-like receptors may ensure the specific refined functions of vertebrate nervous systems. In contrast, broadly conserved palmitoylation sites in both vertebrate and invertebrate D 1 -like receptor homologs indicate that palmitoylated cysteines play crucial roles for sustaining the structural and functional regulations of D 1 -like receptors.

Effectiveness of homeopathic remedies in severe laminitic horses

Background : Equine laminitis is a severe inflammation of the internal lamellar of the horse foot due to an alteration of the periferic vascular flood flow. Marked vascular disturbances occur and microtrombi form in the digital vasculature, which is primed for neutrophil invasion and activation. Endotoxin exarcebate endothelial cell dysfunction, increasing endothelin-I and reducing Endothelial Nitrix Oxide Synthase (eNOS) activity. Ischaemic and inflammatory stimuli could also activate MMPs, leading to digital failure: destruction of the basement membrane bonding the interlocking lamellar leaflets, leads to detachment of phalanx bone from the hoof wall. Ischemia (I) triggers an inflammatory response that precipitates cell death during reperfusion (R). Nitric Oxide (NO) is an inflammatory mediator produced by macrophages and neutrophils during the inflammatory response. It is synthesised via oxidation of L-Arginine by Nitric Oxide Synthetase and plays an ambiguous role during I/R. Additionally, NO mediates the activation of Soluble Guanylyl Cyclase, which can also be activated by endogenous Carbon Monoxide (CO). Metabolism of heme and lipid peroxidation, which generate CO, are both enhanced during inflammation. Methods : 5 horses (4 mares and 1 stallion) diagnosed with acute severe laminitis were treated with homeopathic remedies; 10 healthy horses were selected to determine whether homeopathic remedies are useful and safe as a treatment for equine laminitis, if the level of NO can be re-established and the role of CO. Laminitic horses were administered oral homeopathic treatment only: Aconitum 30ch, Apis 15ch, Arnica 7ch, Belladonna 9ch, Bryonia 9ch and Nux vomica 9ch – 2 granules of each remedy every hour, 10 times per day for 10 days. Variables evaluated included signs of pain, grade of lameness, digital pulse, NO, Nitric Oxide Synthetase Expression, CO and Hemeoxygenase Expression in plasma levels. Results : Homeopathy-treated horses showed an obvious improvement after one day of treatment. Clinical signs of the disease (pain, severe lameness) had completely disappeared at the third day without side effects. NO diminished in laminitis has significant recovery levels in mares. CO and HO-1 levels were higher than in healthy horses.

Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel Fluorescence Resonance Energy Transfer Sensor.

Adenylyl cyclase (AC) activity relies on multiple effectors acting through distinct binding sites. Crystal structures have revealed the location of these sites, and biochemical studies have explored the kinetics of ACs, but the interplay between conformation and activity remains incompletely understood. Here, we describe a novel fluorescence resonance energy transfer (FRET) sensor that functions both as a soluble cyclase and a reporter of complementation within the catalytic domain. We report a strong linear correlation between catalytic domain complementation and cyclase activity upon stimulation with forskolin and Gαs. Exploiting this, we dissect the mechanism of action of a series of forskolin analogs and a P-site inhibitor, 2'-d3'-AMP. Finally, we demonstrate that this sensor is functional in live cells, wherein it reports forskolin-stimulated activity of AC.

165 CRYOPRESERVATION OF IN VITRO PRODUCED BOVINE EMBRYOS AFTER LIPID DECREASE WITH FORSKOLIN

Forskolin® (F-6886) is being used to induce lipolysis and increase cryotolerance, to be an activator of adenylate cyclase, and elevating the cyclic adenosine monophosphate (cAMP) levels. The objective of this experiment was to induce the chemical lipolysis of embryos to improve vitrification and the hypothesis would be that Forskolin decrease the amount of lipid droplets, improve the production of blastocysts, and increase the survival rate after vitrification and warming. Eight random effect were performed which oocytes (N = 1172) were matured in TCM 199® supplemented with 10% of fetal bovine serum (FBS), under 5% CO2 atmosphere, at a temperature of 38.5°C and absolute humidity for 24 h. Semen was selected by Percoll gradient with a final concentration of the 2 × 106 sperm mL–1. Presumptive zygotes were cultured in SOFaa and 2.5% of FBS and were kept in an incubator with 5% CO2, 5% O2 and 90% N2 at 38.5°C and absolute humidity until Day 6, when Forskolin was added and remained until Day 7; control (group without Forskolin); F 2.5 µM (group with 2.5 µM Forskolin); F 5 µM (group with 5 µM Forskolin). On Day 7 (Day 0 = IVF) the rate of blastocyst formation was observed then they were vitrified. Apoptosis was analysed using the TUNEL technique, and the lipid content analysis was performed with Sudan Black B® (S-0395). To estimate the lipid content of embryos, 1 photo at a blastocyst group was performed and submitted to the program ImageJ 1.14 (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). The embryos were limited to obtain the area (μm2), and gray intensity mean (arbitrary units), and gray intensity per area was calculated (arbitrary units/μm2). Data were analysed by ANOVA with PROC GLM of SAS (SAS Institute, Cary, NC, USA). Sources of variation in the model including treatment and replicas were regarded as fixed and random effects, respectively. Data are presented as mean and standard least-squares error. For all analyzes was adopted the significance level of 5%. There was no difference in blastocyst rate: control (37.0 ± 4.0%), F 2.5 μM (38.6 ± 4.0%), F 5 μM (40.7 ± 4.0%). There were difference in lipids content between all groups: control (136.8 ± 2.2ab); F 2.5 μM (128.5 ± 2.2b), F 5 μM (135.6 ± 2.3c; P < 0.05). The F 2.5 μM group showed the higher rate of apoptotic cells compared to other groups: control (12.1 ± 3.5%a), F 2.5 μM (16.7 ± 4.1%b), F 5 μM (11.1 ± 6.5%a; P < 0.05). After vitrification, there was no difference in re-expansion: control (71.3 ± 8.9%), F 2.5 μM (73.1 ± 8.9%); F 5 μM (66.1 ± 8.9%) and apoptosis rate: control (22.3 ± 3.1%); F 2.5 μM (37.3 ± 3.8%); F 5 μM (33.2 ± 6.5%) between the groups. The Forskolin was effective at lower concentration to diminish lipids concentrations in embryos. But when we analysed the apoptotic cell, the lower concentration of Forskolin damaged embryos, but this effect could be diminished after vitrification and warming, when the drug did not increase the apoptotic cells. However, we need to study other concentrations of Forskolin. FAPESP (2010/50410–2/2014/21289–1) is acknowledged for support.

STEM-05GPR133 IS ENRICHED IN GLIOBLASTOMA STEM CELLS AND REGULATES THE RESPONSE TO HYPOXIA

Intratumoral heterogeneity in glioblastoma (GBM) is exemplified by the diversity of tumor microenvironments, which include normoxic hypervascular areas and necrotic regions, which are considered hypoxic. GBM stem cells (GSCs) play a central role in tumor growth and therapy resistance. How GSCs adapt to diverse GBM microenvironments remains an important and unanswered question. We recently discovered that CD133-expressing GSCs are metabolically adept at expanding in hypoxic conditions and do not require Notch signaling for their self-renewal. Transcriptional analysis indicated that CD133+ GSCs have 17.8 ± 8.8-fold enriched expression of GPR133 (n = 3 biospecimens), a member of the adhesion family of G protein-coupled receptors. Immunostaining with GPR133 antibody revealed that GPR133 expression is restricted to hypoxic regions within GBM tumors (12/12 GBM biospecimens) and not present in normal brain. We observed that GPR133 mRNA expression correlates with hypoxia-induced transcripts, such as CA9 and VEFGA (n = 3 primary cultures). To test the hypothesis that GPR133 expression is regulated by oxygen tension, we subjected GBM cultures to 1% O2 in vitro and found that GPR133 transcript was consistently upregulated (n = 5 cultures). To examine whether GPR133 is important for GSC self-renewal, we tested the effect of shRNA-mediated knockdown on in vitro tumorsphere formation ability. GPR133 knockdown depleted CD133+ GSCs and inhibited tumorsphere formation under both normoxic and hypoxic conditions (p < 0.05). GPR133 knockdown also reduced in vivo tumorigenicity and increased survival of implanted mice (n = 3). Using colorimetric assays, we found that CD133+ GSCs have 26.2 ± 12.53% higher cAMP levels compared to CD133- GBM cells (n = 3) and that GPR133 knockdown downregulated cAMP levels to 47.25 ± 27.27% of scramble control (n = 3), suggesting that GPR133 signals through activation of adenylate cyclase and cAMP elevation.

Nitrite is a cGMP generator in isolated platelets

cGMP is generated in blood platelets following activation of soluble guanylate cyclase (sGC) by nitric oxide (NO). Once synthesised, cGMP activates cGMP-dependent protein kinase (PKG) and causes an increase in intracellular cAMP concentration by inhibiting phosphodiesterase 3 (PDE3), responsible for its degradation. PKG and the cAMP-dependent protein kinase (PKA) phosphorylate a number of substrates with inhibitory effects on platelet function [1]. While NO is mainly generated by endothelial NO synthase (eNOS) in the endothelium, reduction of nitrite constitutes an alternative vascular source of NO during hypoxic and acidic conditions. Nitrite originates from reduction of dietary nitrate, found in foods such as beetroot and green leafy vegetables. Nitrite in the circulation can be converted to NO by several nitrite reductases in blood and tissues [2], and triggers vasodilatation [3] thus lowering blood pressure [4]. It has also been demonstrated that when eNOS activity is impaired, NO generated through the nitrate/nitrite pathway exerts negative effects on platelet function [5]. Whilst deoxyhaemoglobin is a candidate nitrite-reductase in blood [6], we aimed to characterise the effects of nitrite in isolated platelets. As such, we used a preparation of washed platelets and supraphysiological concentrations of nitrite in vitro and characterised nitrite-dependent cGMP generation and inhibition of platelet function. Our data show that nitrite at high concentrations (1mM) is a powerful generator of cGMP in platelets in the absence of extracellular reductases. While cGMP accumulation is inhibited by sGC inhibitor ODQ, NO scavengers have a partial effect. This seems to indicate that nitrite acts through both NO-dependent and independent mechanisms. Accordingly, phosphorylation of the the cAMP and cGMP-dependent substrate VASP is strongly increased and, similarly to cGMP generation, is completely dependent on sGC activity but not NO. The increase of cGMP induced by high concentrations of nitrite inhibits platelet function as measured by platelet aggregation and secretion. However, whilst being dependent on sGC activity, the effect of nitrite on aggregation is not dependent on NO.generation. Lower concentrations of nitrite synergise with inhibition of PDEs, in particular PDE5 responsible for cGMP degradation, to trigger detectable VASP phosphorylation and inhibition of aggregation. Nitrite triggers generation of cGMP in platelets and exerts inhibitory effects independently of extracellular reductases and other blood cells. Nitrite acts partly through reduction to NO and partly through an uncharacterised direct effect on sGC.

Age, noise and cGMP: Pharmacological activation of soluble guanylyl cyclase (sGC) interacts with the progression of age related and noise induced hearing loss

Age, noise and cGMP: Pharmacological activation of soluble guanylyl cyclase (sGC) interacts with the progression of age related and noise induced hearing loss

Functions of NO-GC1 and NO-GC2 in pain processing

Background Chronic pain in response to tissue inflammation (inflammatory pain) or nerve injury (neuropathic pain) is often unresponsive to currently available treatments. A large body of evidence indicates that production of nitric oxide (NO) and activation of NO-sensitive guanylyl cyclase (NO-GC) essentially contributes to the processing of chronic pain. NO-GC is a heterodimer consisting of one a subunit (a1 or a2) and one b1 subunit and exists in two isoforms (NO-GC1 and NOGC2). However, the functional role of NO-GC1 and NO-GC2 in pain processing remains poorly understood. Here, we investigated the expression of NO-GC isoforms in pain-relevant tissues (dorsal root ganglia and the spinal cord) and characterized the nociceptive behavior of mice lacking a1 or a2 in models of acute nociceptive, inflammatory and neuropathic pain.