Decrease In cGMP Levels Research Articles

IMPAIRED NO SIGNALLING, ER STRESS AND RHO KINASE MEDIATE PERIPHERAL VASCULAR DYSFUNCTION IN A MODEL OF SMALL VESSEL DISEASE OF THE BRAIN (CADASIL)

Objective: CADASIL, a monogenic condition due to Notch3 mutations, is a very aggressive small vessel disease of the brain resulting in premature vascular dementia and stroke. Changes in cerebral vessels include vascular dysfunction and narrowing, and accumulation of granular osmiophilic material (GOM). It is not clear whether small peripheral arteries undergo similar damage. Thus our aim is to assess vascular dysfunction and associated mechanisms in mesenteric resistance arteries from CADASIL mice. Design and method: Mesenteric arteries (MA) from male CADASIL-causing Notch3 mutation (TgNotch3R169C) and wildtype (TgNotch3WT) mice (6 months old) were investigated. Blood pressure (BP) was assessed by tail-cuff and vascular function by myography. Results: BP and cardiac function were unchanged in CADASIL mice. By electron microscopy GOM deposits were identified in MA from CADASIL mice. Notch3 (WT: 2.0 ± 0.5 vs. 6.0 ± 1.3) and HeyL (WT: 1.1 ± 0.4 vs. 2.9 ± 0.6) mRNA were augmented in CADASIL mice (p < 0.01). CADASIL mice exhibited endothelial (Emax 109.9 ± 7.4 vs. 81.3 ± 5.4) and VSMC-dysfunction (pD2 7.8 ± 0.1 vs 6.8 ± 0.3); effects associated with increased eNOS inhibition (p-Thr495) (1.8-fold increase) and decreased cGMP levels (1.2 ± 0.2 vs 0.59 ± 0.2) (p < 0.05). Contraction to Ang II (Emax 33.7 ± 6.8 vs. 72.8 ± 4.4), phenylephrine (Emax: 70.6 ± 7.2 vs. 92.1 ± 4.2) and U46619 (Emax: 123.4 ± 4.4 vs. 215.1 ± 24.4) (p < 0.05) was increased in CADASIL; effects attenuated by fasudil (Rho-kinase inhibitor) and 4-PBA (ER stress inhibitor) (p < 0.05). MA structure and mechanics from CADASIL mice remain unchanged at this stage. U46619-induced calcium influx was increased in TgNotch3R169C VSMCs (AUC: 1.3-fold increase) as well as SERCA (1.5 ± 0.7 vs. 8.1 ± 1.4) and TRPM2 (0.7 ± 0.3 vs. 4.5 ± 1.6) mRNA levels (p < 0.05). BiP protein expression (ER stress marker) (1.7-fold increase) and gene expression of Rho GEFs (LARG: 1.1 ± 0.1 vs. 2.1 ± 0.3; PDZ: 0.9 ± 0.1 vs. 2.9 ± 0.4) were increased in MA from TgNotch3R169C vs. TgNotch3WT mice (p < 0.05). These effects may be associated with the increase in oxidative stress observed in VSMCs from peripheral arteries and brain vessels from CADASIL patients (p < 0.05). Conclusions: Our findings demonstrate that the cerebral vasculopathy associated with Notch3 mutations is also present in peripheral small vessels and identify ER stress and Rho kinase as potential new therapeutic targets in CADASIL, for which there are no disease-specific treatments.

TNFα induces endothelial dysfunction in rheumatoid arthritis via LOX-1 and arginase 2: reversal by monoclonal TNFα antibodies.

Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting joints and blood vessels. Despite low levels of low-density lipoprotein cholesterol (LDL-C), RA patients exhibit endothelial dysfunction and are at increased risk of death from cardiovascular complications, but the molecular mechanism of action is unknown. We aimed in the present study to identify the molecular mechanism of endothelial dysfunction in a mouse model of RA and in patients with RA. Endothelium-dependent relaxations to acetylcholine were reduced in aortae of two tumour necrosis factor alpha (TNFα) transgenic mouse lines with either mild (Tg3647) or severe (Tg197) forms of RA in a time- and severity-dependent fashion as assessed by organ chamber myograph. In Tg197, TNFα plasma levels were associated with severe endothelial dysfunction. LOX-1 receptor was markedly up-regulated leading to increased vascular oxLDL uptake and NFκB-mediated enhanced Arg2 expression via direct binding to its promoter resulting in reduced NO bioavailability and vascular cGMP levels as shown by ELISA and chromatin immunoprecipitation. Anti-TNFα treatment with infliximab normalized endothelial function together with LOX-1 and Arg2 serum levels in mice. In RA patients, soluble LOX-1 serum levels were also markedly increased and closely related to serum levels of C-reactive protein. Similarly, ARG2 serum levels were increased. Similarly, anti-TNFα treatment restored LOX-1 and ARG2 serum levels in RA patients. Increased TNFα levels not only contribute to RA, but also to endothelial dysfunction by increasing vascular oxLDL content and activation of the LOX-1/NFκB/Arg2 pathway leading to reduced NO bioavailability and decreased cGMP levels. Anti-TNFα treatment improved both articular symptoms and endothelial function by reducing LOX-1, vascular oxLDL, and Arg2 levels.

Photoreceptor Phosphodiesterase (PDE6): Structure, Regulatory Mechanisms, and Implications for Treatment of Retinal Diseases.

The photoreceptor phosphodiesterase (PDE6) is a member of large family of Class I phosphodiesterases responsible for hydrolyzing the second messengers cAMP and cGMP. PDE6 consists of two catalytic subunits and two inhibitory subunits that form a tetrameric protein. PDE6 is a peripheral membrane protein that is localized to the signal-transducing compartment of rod and cone photoreceptors. As the central effector enzyme of the G-protein coupled visual transduction pathway, activation of PDE6 catalysis causes a rapid decrease in cGMP levels that results in closure of cGMP-gated ion channels in the photoreceptor plasma membrane. Because of its importance in the phototransduction pathway, mutations in PDE6 genes result in various retinal diseases that currently lack therapeutic treatment strategies due to inadequate knowledge of the structure, function, and regulation of this enzyme. This review focuses on recent progress in understanding the structure of the regulatory and catalytic domains of the PDE6 holoenzyme, the central role of the multi-functional inhibitory γ-subunit, the mechanism of activation by the heterotrimeric G protein, transducin, and future directions for pharmacological interventions to treat retinal degenerative diseases arising from mutations in the PDE6 genes.

Abstract 12567: Circulating Factors Induce Cardiomyopathy After Burn Injury

Introduction: Our previous results in vivo indicated PDE5-cGMP-PKG was involved in burn-induced heart dysfunction and PDE5A inhibitor restored the dysfunction. It’s unknown if circulating factors after burn would injure cardiomyocytes. Hypothesis: Circulating factors released after burn induce cardiomyopathy. Methods: Human cardiomyocytes (AC16) were treated with sham-serum, burn-serum (24 hpb-serum) and burn/sildenafil-serum (24 hpb/SIL). We performed cut-edged biochemistry technologies and Illumina RNA sequencing (RNA-seq) in this study. GraphPad Prism 8.4.2 was used for statistics. Results: We found a significant decrease of cGMP level and an increase of cTN1 in 24 hpb-serum group. Treatment with the PDE5A inhibitor Sildenafil completely reversed this change similar to our in vivo work. To understand what bioactive molecules would be involved in the alterations by burn injury, human cardiomyocytes (Ac16) were employed to test the cardiomyocyte response to burn-induced circulating factors. We observed that 24 hpb-serum significantly 1) decreased cell viability and cell proliferation; as well as 2) increased cell cytotoxicity, cell apoptosis and cell ROS production. We also found 24 hpb-serum resulted in cell mitochondrial dysfunction by decreasing ATP production and mitochondrial membrane integrity/potential and increasing mitochondrial ROS. Seahorse and O2K approaches confirmed 24 hpb-serum-induced cardiomyocyte mitochondrial dysfunction as evidenced by decreases of mitochondrial basal respiration, proton leak, ATP production, and maximal respiration. 24 hpb/SIL serum rescued 24 hpb serum-induced Ac 16 cell response, at least partially. Advanced bioinformatic analyses identified 1415 upregulated genes and 1091 downregulated genes in 24 hpb-serum group and 776 upregulated genes and 113 downregulated genes restored in 24 hpb/SIL-serum group. We also analyzed and validated the differentially expressed genes. Conclusions: Our study not only confirmed burn induced heart dysfunction, but also provided evidence for understanding the pathogenic mechanism of circulating factors released after burn injury and preliminary genomic evidence for the mechanism for cardiomyopathy after burn injury.

Abstract MP31: Peripheral Vascular Dysfunction In A Model Of Small Vessel Disease Of The Brain (CADASIL) Involves Impaired Redox-sensitive Cyclic GMP/PKG Signaling

CADASIL, a monogenic condition due to Notch3 mutations, is a very aggressive small vessel disease of the brain resulting in premature vascular dementia and stroke. Changes in cerebral vessels include vascular dysfunction and narrowing, and accumulation of granular osmiophilic material (GOM). It is not clear whether small peripheral arteries undergo similar damage. Therefore, our aim is to assess vascular dysfunction and associated mechanisms in mesenteric resistance arteries from CADASIL mice. Mesenteric arteries (MA) from male CADASIL-causing Notch3 mutation (TgNotch3 R169C ) and wildtype (TgNotch3 WT ) mice (6 months old) were investigated. GOM deposits in MA from CADASIL mice were identified by electron microscopy. mRNA expression of Notch3 (WT: 2.0±0.5 vs. 6.0±1.3) and its downstream target HeyL (WT: 1.1±0.4 vs. 2.9±0.6) was augmented in CADASIL mice (p<0.01), suggesting increased Notch3 activation. CADASIL mice exhibited endothelial-dependent (Emax 109.9±7.4 vs. 81.3±5.4) and -independent dysfunction (pD 2 7.8±0.1 vs. 6.8±0.3); effects associated with increased eNOS inhibition (p-Thr 495 ) (1.8-fold increase) and decreased cGMP levels (1.2±0.2 vs. 0.59±0.2) (p<0.05). Plasma lipid peroxidation (0.8±0.1 vs. 2.0±0.3; p<0.05) and vascular reactive oxygen species (ROS) production (7.2±1.9 vs. 75.4±35.0; p<0.05) were increased in TgNotch3 R169C mice; processes associated with upregulation of soluble guanylate cyclase (sGC) oxidation and decreased sGC activity. H 2 O 2 levels were decreased in TgNotch3 R169C mice (1.9±0.2 vs. 1.1±1.9; p<0.05), which was associated with reduced activation of protein kinase G (PKG). Observations in TgNotch3 R169C mice were recapitulated in human CADASIL, where ROS levels (0.8±0.1 vs. 4.1±2.7; p<0.05) and sGC oxidation were also increased. Our findings demonstrate that the vasculopathy associated with a CADASIL Notch3 gain-of-function mutation in peripheral small vessels involves reduction in eNOS activation and redox-sensitive processes leading to impaired sGC/cGMP signalling pathway. We identify a potential new therapeutic target in CADASIL, for which there are no disease-specific treatments.

Is Innervation of the Neuromuscular Junction at the Diaphragm Modulated by sGC/cGMP Signaling?

We previously reported NO/sGC signaling in the upper respiratory pathway, receiving input from the respiratory neurons of the brainstem to phrenic motoneurons in the C3–C6 spinal cord. In order to assess whether innervation of the neuromuscular junction (NMJ) at the diaphragm is modulated by sGC/cGMP signaling, we performed unilateral 8-day continuous ligation of the phrenic nerve in rats. We examined sGCβ1 within the lower bulbospinal pathway (phrenic motoneurons, phrenic nerves and NMJs at the diaphragm) and the cGMP level in the contra- and ipsilateral hemidiaphragm. Additionally, we characterized the extent of phrenic nerve axonal degeneration and denervation at diaphragm NMJs. The results of our study show that continuous 8-day phrenic nerve ligation caused a marked increase in sGCβ1 (immunoreactivity and the protein level) in the ipsilateral phrenic motor pool. However, the protein sGCβ1 level in the phrenic nerve below its ligation and the cGMP level in the ipsilateral hemidiaphragm were evidently decreased. Using confocal analysis we discovered a reduction in sGCβ1-IR boutons/synaptic vesicles at the ipsilateral MNJs. These findings are consistent with the marked axonal loss (∼47%) and significant NMJs degeneration in the ipsilateral diaphragm muscle. The remarkable unilateral decrease in cGMP level in the diaphragm and the failure of EMG recordings in the ipsilateral hemidiaphragm muscle can be attributed to the fact that sGC is involved in transmitter release at the diaphragm NMJs via the sGC-cGMP pathway.

Linolenic Acid Attenuates the Vasodilation Induced by Acetylcholine in Isolated Rat Aortae.

This study aims to examine the effect of linolenic acid on the vasodilation or vasoconstriction induced by acetylcholine and bupivacaine in isolated rat aortae and its underlying mechanism. The effect of linolenic acid on the vasodilation induced by acetylcholine, the calcium ionophore A23187, sodium nitroprusside, and 8-bromoguanosine 3′,5′-cyclic monophosphate sodium salt (bromo-cyclic guanosine monophosphate [bromo-cGMP]) in endothelium-intact and endothelium-denuded aortae was examined. Linolenic acid inhibited vasodilation induced by acetylcholine, calcium ionophore A23187, and sodium nitroprusside. However, this fatty acid increased bromo-cGMP-induced vasodilation in endothelium-denuded aortae. Linolenic acid increased bupivacaine-induced contraction in endothelium-intact aortae, whereas it decreased bupivacaine-induced contraction in endothelium-intact aortae with Nω-nitro-l-arginine methyl ester and endothelium-denuded aortae. Linolenic acid inhibited acetylcholine- and bupivacaine-induced phosphorylation of endothelial nitric oxide synthase. Sodium nitroprusside increased cGMP in endothelium-denuded aortic strips, whereas bupivacaine decreased cGMP in endothelium-intact aortic strips. Linolenic acid decreased cGMP levels produced by bupivacaine and sodium nitroprusside. Together, these results suggest that linolenic acid inhibits acetylcholine-induced relaxation by inhibiting a step just prior to nitric oxide-induced cGMP formation. In addition, linolenic acid-mediated inhibition of vasodilation induced by a toxic concentration (3 × 10−4 M) of bupivacaine seems to be partially associated with inhibition of the nitric oxide–cGMP pathway.

Profound Decrease in Glomerular Arginine Transport by CAT (Cationic Amino Acid Transporter)-1 Contributes to the FLT-1 (FMS-Like Tyrosine Kinase 1) Induced Preeclampsia in the Pregnant Mice.

Endothelial dysfunction because of nitric oxide inactivation has been suggested to play a role in the pathogenesis of preeclampsia. During pregnancy, L-arginine transport by CAT-1 (cationic amino acid transporter 1), the only transporter for eNOS (endothelial nitric oxide synthase) is inhibited. We hypothesize that maternal arginine deficiency contributes to the development of preeclampsia. Adenovirus-mediated overexpression of sFlt-1 (soluble fms-like tyrosine kinase 1) in virgin and pregnant mice resulted in glomerular endotheliosis, hypertension, and albuminuria. L-arginine prevented the increase in blood pressure and albuminuria in Flt-1 pregnant but not in Flt-1 virgin mice. Flt-1 augmented arginine transport in pregnant but not in virgin dames. Ex vivo inhibition of CAT-2 leaving exclusively CAT-1 activity, decreased arginine transport velocities in Flt-1 animals more prominently in pregnant dames. Phosphorylated CAT-1/CAT-1 increased in pregnant, sFlt-1-pregnant, and sFlt-1 virgin mice. CAT-2 increased in Flt-1-pregnant and Flt-1-virgin dames. L-arginine augmented arginine transport in pregnant and Flt-pregnant mice and prevented the increase in pCAT-1 and CAT-2 expression. Glomerular cGMP (cyclic guanosine monophosphate) generation as a measure of eNOS activity was decreased in all Flt-1 treated animals. L-arginine abolished the decrease in cGMP levels only in Flt-1-pregnant mice. In conclusion, glomerular endothelial NO generation is compromised in Flt-1-pregnant mice because of CAT-1 inhibition induced by a combined effect of pregnancy and preeclampsia which involves: phosphorylation of CAT-1 and induction of CAT-2. These processes contribute to the clinical syndrome of preeclampsia in mice and are prevented by L-arginine.

Hypoxia/Reoxygenation of Rat Renal Arteries Impairs Vasorelaxation via Modulation of Endothelium-Independent sGC/cGMP/PKG Signaling.

Ischemia/reperfusion injury holds a key position in many pathological conditions such as acute kidney injury and in the transition to chronic stages of renal damage. We hypothesized that besides a reported disproportional activation of vasoconstrictor response, hypoxia/reoxygenation (H/R) adversely affects endothelial dilatory systems and impairs relaxation in renal arteries. Rat renal interlobar arteries were studied under isometric conditions. Hypoxia was induced by application of 95% N2, 5% CO2 for 60 min to the bath solution, followed by a 10 min period of reoxygenation (95% O2, 5% CO2). The effect of H/R on relaxation was assessed using various inhibitors of endothelial dilatory systems. mRNA expression of phosphodiesterase 5 (PDE5), NADPH oxidases (NOX), and nitric oxide synthase (NOS) isoforms were determined using qRT-PCR; cGMP was assayed with direct cGMP ELISA. Acetylcholine induced relaxation was impaired after H/R. Inhibition of the NOS isoforms with L-NAME, and cyclooxygenases (COXs) by indomethacin did not abolish the H/R effect. Moreover, blocking the calcium activated potassium channels KCa3.1 and KCa2.1, the main mediators of the endothelium-derived hyperpolarizing factor, with TRAM34 and UCL1684, respectively, showed similar effects in H/R and control. Arterial stiffness did not differ comparing H/R with controls, indicating no impact of H/R on passive vessel properties. Moreover, superoxide was not responsible for the observed H/R effect. Remarkably, H/R attenuated the endothelium-independent relaxation by sodium nitroprusside, suggesting endothelium-independent mechanisms of H/R action. Investigating the signaling downstream of NO revealed significantly decreased cGMP and impaired relaxation during PDE5 inhibition with sildenafil after H/R. Inhibition of PKG, the target of cGMP, did not normalize SNP-induced relaxation following H/R. However, the soluble guanylyl cyclase (sGC) inhibitor ODQ abolished the H/R effect on relaxation. The mRNA expressions of the endothelial and the inducible NOS were reduced. NOX and PDE5 mRNA were similarly expressed in H/R and control. Our results provide new evidence that impaired renal artery relaxation after H/R is due to a dysregulation of sGC leading to decreased cGMP levels. The presented mechanism might contribute to an insufficient renal reperfusion after ischemia and should be considered in its pathophysiology.

MicroRNA-425 and microRNA-155 cooperatively regulate atrial natriuretic peptide expression and cGMP production.

AimsAtrial natriuretic peptide (ANP), secreted primarily by atrial cardiomyocytes, decreases blood pressure by raising cyclic 3’,5’-guanosine monophosphate (cGMP) levels and inducing vasorelaxation, natriuresis, and diuresis. Raising the level of ANP has been shown to be an effective treatment for hypertension. To advance the future development of an anti-microRNA (miR) approach to increasing expression of ANP, we investigated the regulation of NPPA expression by two miRs: miR-425 and miR-155. We examined whether miR-425 and miR-155 have an additive effect on the expression and function of ANP.Methods and resultsHuman embryonic stem cell-derived cardiomyocytes (hESC-CMs) were transfected with miR-425, miR-155, or a combination of the two miRs. Two days later, NPPA expression was measured using real time qPCR. Each of the miRs decreased NPPA expression over a wide range of concentrations, with a significant reduction at concentrations as low as 1 nM. The combination of miR-425 and miR-155 reduced NPPA expression to a greater extent than either miR-425 or miR-155 alone. An in vitro assay was developed to study the potential biological significance of the miR-induced decrease in NPPA expression. The cooperative effect of miR-425 and miR-155 on NPPA expression was associated with a significant decrease in cGMP levels.ConclusionsThese data demonstrate that miR-425 and miR-155 regulate NPPA expression in a cooperative manner. Targeting both miRNAs with anti-miRs (possibly at submaximal concentrations) might prove to be a more effective strategy to modulate ANP levels, and thus blood pressure, than targeting either miRNA alone.

The dual role of cGMP in oocyte maturation of zebrafish

The roles of cyclic guanosine monophosphate (cGMP) signaling in oocyte maturation attracts much attention in mammals, but its roles in fish are still largely unknown. Using zebrafish as a model, we demonstrated for the first time in fish that cGMP is involved in oocyte maturation, and its functional model in oocyte maturation is different from that of mammals. The intracellular cGMP could be regulated by nitric oxide (NO), we found that all three NO synthase enzymes and four soluble guanylyl cyclases (sGC) are expressed in the zebrafish ovary. Intriguingly, either the activation or inhibition of the NO/sGC/cGMP pathway in fully grown follicles could lead to oocyte maturation. During oocyte maturation, cGMP levels increased in the follicular cell layer but decreased in oocytes, while NO levels increased in follicular cells but remained constant in oocytes. Based on these findings in zebrafish, we propose a hypothetical model on the dual role of cGMP in oocyte maturation: in follicular cells the LH signal could increase the level of NO and cGMP which induces oocyte maturation, while in the oocyte the decreased cGMP level can also induce oocyte maturation. These findings help us to understand the molecular mechanism of fish oocyte maturation.

Effects of fetal calf serum on cGMP pathway and oocyte lipid metabolism in vitro.

Intracellular levels of cyclic nucleotides, such as cGMP, are involved in the regulation of adipocyte lipolysis. Cumulus-oocyte complexes (COCs) express enzymes that both synthesise (guanylate cyclase) and degrade (phosphodiesterase (PDE) 5A) cGMP. Because serum interferes with lipid metabolism, its effects on the cGMP pathway and lipid content in bovine COCs were examined. COCs were matured in medium containing fetal calf serum (FCS; 2% or 10%) or 0.4% bovine serum albumin (BSA; control). At both 2% and 10%, FCS decreased cGMP levels in COCs compared with BSA (0.64 and 1.04 vs 9.46 fmol per COC respectively; P<0.05) and decreased transcript levels of guanylate cyclase 1, soluble, beta 3 (GUCY1B3), whereas PDE5A levels were increased. FCS also affected the expression of genes related to lipolysis, increasing relative expression of perilipin 2 (PLIN2) and carnitine palmitoyltransferase 1B (CPT1B) in cumulus cells. Effects of FCS and cGMP on the lipid content of oocytes and embryos were evaluated by Nile red staining. COCs were matured with 10% FCS, FCS+10-5 M sildenafil (SDF), a PDE5 inhibitor, or 0.4% BSA. The lipid content was increased in oocytes matured in FCS compared with BSA (fluorescence intensity 20.1 vs 17.61 respectively; P<0.05), whereas the lipid content in oocytes matured in FCS+SDF (fluorescence intensity 16.33) was similar to that in the BSA-treated group (P>0.05). In addition, lipid content was higher in embryos from oocytes matured with FCS than BSA (fluorescence intensity 31.12 vs 22.31 respectively; P<0.05), but was increased even further in the FCS+SDF-treated group (fluorescence intensity 40.35; P<0.05), possibly due to a compensatory mechanism during embryo culture without SDF for the reduction in lipid content during IVM. The present study provides, for the first time, evidence that the cGMP pathway may be involved in lipid metabolism in bovine COCs and that this pathway is affected by FCS.

Epidermal Growth Factor-Mobilized Intracellular Calcium of Cumulus Cells Decreases Natriuretic Peptide Receptor 2 Affinity for Natriuretic Peptide Type C and Induces Oocyte Meiotic Resumption in the Mouse.

Natriuretic peptide type C (NPPC) activation of the guanylyl cyclase-linked natriuretic peptide receptor (NPR) 2 maintains oocyte meiotic arrest. Luteinizing hormone (LH)-dependent epidermal growth factor (EGF) receptor signaling elevates calcium of cumulus cells to inactivate NPR2, resulting in meiotic resumption. This study investigated the regulatory mechanism of calcium on NPR2 inactivation. In mouse ovarian follicles, LH, through the activation of EGF receptor, significantly elevated calcium levels in cumulus cells, but decreased the binding affinity of NPR2 for NPPC. In cultured cumulus-oocyte complexes, the activation of EGF receptor by EGF mobilized intracellular calcium of cumulus cells to decrease NPR2 affinity and cGMP levels, resulting in meiotic resumption. However, hormone treatments had not changed NPR2 protein levels. In addition, the removal of magnesium ions from the medium decreased the binding affinity of NPR2 for NPPC, resulting in a decrease in cGMP levels and meiotic resumption. It is concluded that magnesium ions are required to maintain functional NPR2, and that LH-dependent EGF receptor signaling mobilizes intracellular calcium of cumulus cells to reduce NPPC-NPR2 interaction that is required for meiotic resumption.

Suppressive effect of exogenous carbon monoxide on endotoxin-stimulated platelet over-activation via the glycoprotein-mediated PI3K-Akt-GSK3β pathway

Platelet activation is an important event involved in the pathophysiological processes of the coagulation system. Clinical evidence has shown that platelets undergo distinctive pathological processes during sepsis. Unfortunately, how platelets physiologically respond to inflammation or sepsis is not well understood. In this study, we used a lipopolysaccharide (LPS)-stimulated platelet model to systemically investigate alterations in membrane glycoprotein expression, molecular signaling, morphology and critical functions of platelets. We found that platelet adhesion, aggregation, secretion, and spreading on immobilized fibrinogen and the expression of platelet membrane glycoproteins were significantly increased by LPS stimulation, and these changes were accompanied by a significant decrease in cGMP levels and an abnormal distribution of platelet α-granules. Exogenous CO reversed these alterations. Profound morphological changes in LPS-stimulated platelets were observed using atomic force microscopy and phase microscopy. Furthermore, the elevated activities of PI3Ks, AKt and GSK-3β were effectively suppressed by exogenous CO, leading to the improvement of platelet function. Together, these results provide evidence that platelet over-activation persists under LPS-stimulation and that exogenous CO plays an important role in suppressing platelet activation via the glycoprotein-mediated PI3K-Akt-GSK3β pathway.

PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis

Sepsis severity has been positively correlated with platelet dysfunction, which may be due to elevations in nitric oxide (NO) and cGMP levels. Protein kinase C, Src kinases, PI3K and AKT modulate platelet activity in physiological conditions, but no studies evaluated the role of these enzymes in platelet aggregation in sepsis. In the present study we tested the hypothesis that in sepsis these enzymes positively modulate upstream the NO-cGMP pathway resulting in platelet inhibition. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg, i.p.) and blood was collected after 6 h. Platelet aggregation was induced by ADP (10 μM). Western blotting assays were carried out to analyze c-Src and AKT activation in platelets. Intraplatelet cGMP levels were determined by enzyme immunoassay kit. Phosphorylation of c-SRC at Tyr416 was the same magnitude in platelets of control and LPS group. Incubation of the non-selective Src inhibitor PP2 (10 μM) had no effect on platelet aggregation of LPS-treated rats. LPS increased intraplatelet cGMP levels by 5-fold compared with control group, which was accompanied by 76% of reduction in ADP-induced platelet aggregation. The guanylyl cyclase inhibitor ODQ (25 μM) and the PKG inhibitor Rp-8-Br-PET-cGMPS (25 μM) fully reversed the inhibitory effect of LPS on platelet aggregation. Likewise, the PKC inhibitor GF109203X (10 μM) reversed the inhibition by LPS of platelet aggregation and decreased cGMP levels in platelets. AKT phosphorylation at Thr308 was significantly higher in platelets of LPS compared with control group, which was not reduced by PI3K inhibition. The AKT inhibitor API-1 (20 μM) significantly increased aggregation and reduced cGMP levels in platelets of LPS group. However, the PI3K inhibitor wortmannin and LY29004 had no effect on platelet aggregation of LPS-treated rats. Therefore, inhibition of ADP-induced platelet aggregation after LPS injection is mediated by cGMP/PKG-dependent mechanisms, and PKC and AKT act upstream upregulating this pathway.

IWP-121: a novel sGC stimulator that reduces blood pressure and exhibits anti-fibrotic and anti-inflammatory activities in the Dahl Salt-Sensitive rat model

Background Soluble guanylate cyclase (sGC) is an intracellular receptor that can be activated by nitric oxide (NO) and sGC stimulators to produce cyclic guanosine monophosphate (cGMP), thereby modulating a number of downstream cellular and physiological responses including phosphorylation of VASP and vasodilation. In the Dahl Salt-Sensitive (DSS) rat model of hypertension, cGMP production by sGC is decreased, most likely due to reactive oxygen species (ROS) converting NO to peroxynitrite, resulting in depleted pools of NO available to bind to sGC. In this study we evaluated the efficacy of a novel sGC stimulator (IWP-121) in the DSS model. Male DSS rats (230-270 grams) received high-salt diet (8% NaCl) for 2 weeks followed by high salt plus compound for 6 additional weeks. IWP-121 was administered at doses of 1, 3, and 10 mg/kg/ day in the chow (n=8/group). Losartan (30 mg/kg/day in the water) was used as a positive control, in addition to both High Salt (HS) and Normal Salt (NS). All groups were compared to HS control group for analyses. IWP-121 dose dependently decreased mean blood pressure (MAP) throughout the study. Additionally, IWP-121 (at all doses tested) and losartan had statistically significant effects on decreasing heart hypertrophy and plasma NT-proBNP but only IWP-121 had an effect on attenuating liver hypertrophy. IWP-121 decreased microalbuminuria (an indicator of kidney end organ damage) as well as attenuated serum biomarkers known to be involved in inflammatory and fibrotic processes. Conclusion In the rat DSS model of hypertension, there is a decrease in cGMP levels most likely due to the inactivation of endogenous NO by ROS. The sGC stimulator IWP-121, when administered in the diet exhibited sustained dosedependent reduction in blood pressure. Additionally, IWP-121 attenuated heart and liver hypertrophy and reduced NT-proBNP, a biomarker of heart failure. The compound reduced levels of biomarkers for inflammation and fibrosis, and demonstrated renal end organ protection. sGC stimulators, like IWP-121 may have broad therapeutic application by modulating multiple relevant therapeutic endpoints including blood pressure, hypertrophy, inflammation, and fibrosis.

Upregulation of RhoA/Rho kinase pathway and downregulation of cGMP/PKG pathway in smooth muscle impair peristaltic reflex and pellet propulsion in the colon of diabetic mice (1110.5)

Abnormalities in extrinsic and intrinsic innervation and in the distribution of interstitial cells of Cajal are known to contribute to altered gut motility in diabetes. Smooth muscle dysfunction in relation to diabetes has not been fully explored. Aim. To test the hypothesis that upregulation of RhoA/Rho kinase pathway and downregulation of cGMP/PKG pathway contribute to increased muscle tone in diabetes. Methods. Colonic smooth muscle cells from type 1 (NOD) or type 2 diabetes (db/db) mice were used to measure i) RhoA and PDE5 expression; 2) ACh‐induced Rho kinase activity and muscle contraction; 3) NO‐induced PDE5 activity, cGMP levels and muscle relaxation. Colonic segments were used to measure peristaltic reflex and pellet propulsion. Results. Smooth muscle cells isolated from the colon of NOD and db/db mice showed an increase in RhoA and PDE5 expression, Rho kinase and PDE5 activity, and muscle contraction, and a decrease in cGMP levels and muscle relaxation relative to control mice. Consistent with an increase in RhoA and PDE5 expression, orad contraction was increased by 20%, caudad relaxation decreased by 60%, and pellet propulsion decreased by 35% in db/db mice relative to control. Discussion. In diabetic colon upregulation of the RhoA/Rho kinase pathway and downregulation of the cGMP/PKG pathway causes an increase in muscle contraction and decrease in relaxation leading to a decrease in pellet propulsion.

165 FETAL CALF SERUM INFLUENCES CYCLIC GMP PATHWAY, WHICH IN TURN AFFECTS THE LIPID CONTENT IN IN VITRO-MATURED BOVINE OOCYTES

The sensitivity of IVP embryos to cryopreservation is often associated with lipid accumulation in the cytoplasm induced by the presence of fetal calf serum (FCS) during culture. Intracellular levels of cyclic (c)AMP and cGMP are involved in the regulation of lipolysis in adipocytes; high levels stimulate lipolysis whereas low levels lead to lipogenesis. Both nucleotides are present in bovine oocytes, together with the enzymes for their synthesis and degradation. The aim of this study was to analysis the effect of FCS on the cGMP pathway and the influence of cGMP on cytoplasmic lipids in bovine oocytes. In experiments 1 and 2, cumulus–oocyte complexes (COC) were cultured for 24 h in maturation medium with different proportions of FCS (2 and 10%) and a control group was matured with 0.4% BSA. After this period, transcripts for cGMP pathway were assessed by real-time PCR (GUCY1B3 and PDE5, cGMP synthesis and degradation enzymes, respectively; experiment 1) in oocytes and cumulus cells, and cGMP levels were measured in COC using commercial enzyme immunoassay kits (EIA; experiment 2). In experiments 3 and 4, COC were matured for 24 h with 0.4% BSA and different concentrations of the phosphodiesterase (PDE)5 inhibitor (0, 10–7, and 10–5 M sildenafil) to inhibit cGMP degradation and a control group was matured with 0.4% BSA. The nucleotide levels were measured in COC (experiment 3) and the oocytes were stained with Nile Red (1 μg mL–1) for evaluation of lipid content (experiment 4). Statistical analyses were performed by ANOVA followed by Tukey post hoc test using SAS software (SAS Institute Inc., Cary, NC, USA). Data for gene expression from 5 replicates and for cGMP measurements and lipid content from 3 replicates were log10-transformed into before analyses. The level of significance was 5%. The presence of FCS reduced GUCY1B3 expression in both cells and increased PDE5A in cumulus cells (P &lt; 0.05). In experiment 2, the groups treated with 2 (0.64 fmol/COC) and 10% FCS (1.04 fmol/COC) showed decreased cGMP levels compared with control (9.46 fmol/COC; P &lt; 0.05). In experiment 3, inhibition of PDE5A increased cGMP levels in the treated groups (36 and 56 fmol/COC for 10–7 and 10–5 M sildenafil, respectively) compared with control (9.5 fmol/COC; P &lt; 0.05). Therefore, sildenafil showed inverse effects compared with FCS (experiment 2). In experiment 4, oocytes treated with 10–7 and 10–5 M sildenafil showed a reduced lipid content compared with controls (11.6 ± 9.4 v. 13.9 μm2 fluorescence intensity, respectively; P &lt; 0.05). The results suggest that FCS in maturation medium affects the cGMP pathway, interfering with the transcription of genes that control its levels, which in turn results in nucleotide reduction. Inhibition of PDE5 increases cGMP levels and reduces the lipid content of oocytes, indicating that changes in this pathway caused by FCS may affect lipid metabolism of oocytes. More studies are underway to better understand this mechanism. The authors acknowledge FAPESP 2012/00170-0 for financial support.

Increased PDE5 activity and decreased Rho kinase and PKC activities in colonic muscle from caveolin-1−/− mice impair the peristaltic reflex and propulsion

Caveolae are specialized regions of the plasma membrane that concentrate receptors and associated signaling molecules critical in regulation of cellular response to transmitters and hormones. We have determined the effects of caveolin-1 (Cav-1) deletion, caveolin-1 siRNA, and caveolar disruption in mice on the signaling pathways that mediate contraction and relaxation in colonic smooth muscle and on the components of the peristaltic reflex in isolated tissue and propulsion in intact colonic segments. In Cav-1-/- mice, both relaxation and contraction were decreased in smooth muscle cells and muscle strips, as well as during both phases of the peristaltic reflex and colonic propulsion. The decrease in relaxation in response to the nitric oxide (NO) donor was accompanied by a decrease in cGMP levels and an increase in phosphodiesterase 5 (PDE5) activity. Relaxation by a PDE5-resistant cGMP analog was not affected in smooth muscle of Cav-1-/- mice, suggesting that inhibition of relaxation was due to augmentation of PDE5 activity. Similar effects on relaxation, PDE5 and cGMP were obtained in muscle cells upon disruption of caveolae by methyl-β-cyclodextrin or suppression of Cav-1. Sustained contraction mediated via inhibition of myosin light chain phosphatase (MLCP) activity is regulated by Rho kinase and PKC via phosphorylation of two endogenous inhibitors of MLCP: myosin phosphatase-targeting subunit (MYPT1) and 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), respectively. The activity of both enzymes and phosphorylation of MYPT1 and CPI-17 were decreased in smooth muscle from Cav-1-/- mice. We conclude that the integrity of caveolae is essential for contractile and relaxant activity in colonic smooth muscle and the maintenance of neuromuscular function at organ level.

Heterozygous Mutations in Natriuretic Peptide Receptor-B (NPR2) Gene as a Cause of Short Stature in Patients Initially Classified as Idiopathic Short Stature

Based on the stature observed in relatives of patients with acromesomelic dysplasia, type Maroteaux, homozygous for mutations in natriuretic peptide receptor B gene (NPR2), it has been suggested that heterozygous mutations in this gene could be responsible for the growth impairment observed in some children with idiopathic short stature (ISS). The objective of the study was to investigate the presence of NPR2 mutations in a group of patients with ISS. The NPR2 coding region was directly sequenced in 47 independent patients with ISS. The functional consequences of NPR2 nonsynonymous variations were established using in vitro cell-based assays. Three novel heterozygous NPR2 mutations were identified: c.226T>C (p.Ser76Pro), c.788G>C (p.Arg263Pro), and c.2455C>T (p.Arg819Cys). These allelic variants were not found in our controls or in the 1000 Genomes database. In silico analysis suggested that the three missense mutations are probably damaging. All of them were selected for in vitro functional evaluation. Cells transfected with the three mutants failed to produce cyclic GMP after treatment with C-type natriuretic peptide. Cells cotransfected with mutant and wild-type-NPR-B (1:1) showed a significant decrease in cGMP levels after C-type natriuretic peptide stimulation in comparison with cells cotrasnfected with empty vector and wild type, suggesting a dominant-negative effect. These three mutations segregated with short stature phenotype in an autosomal dominant pattern (height SD score ranged from -4.5 to -1.7). One of these patients and two relatives have disproportionate short stature, whereas in another patient a nonspecific skeletal abnormality was observed. All three of these patients were treated with recombinant human GH (33-50 μg/kg · d) without significant height SD score change during therapy. We identified heterozygous NPR2 mutations in 6% of patients initially classified as ISS. Affected patients have mild and variable degrees of short stature without a distinct phenotype. Heterozygous mutations in NPR2 could be an important cause of nonsyndromic familial short stature.