Increased cGMP Accumulation Research Articles

Long-term inhibition of PDE5 ameliorates aging-induced changes in rat testis.

NO-cGMP signaling pathway has been implicated in reduction of testicular steroidogenesis during aging. Here we analyzed the effect of PDE5 inhibition on old testicular phenotype formation. The old phenotype exhibited low testosterone and increased nitrite levels in circulation, increased cGMP accumulation in testicular interstitial fluid (TIF), progressive atrophy of testicular seminiferous tubules and enlargement of interstitial area followed by rise in blood vessel density and slight increase in the number of Leydig cells and macrophages. Leydig cells have reduced steroidogenic capacity, increased MAP kinases expression (MEK, ERK1/2, JNK) and antiapoptotic PRKG1 and AKT, suggesting increased proliferation/survival and accumulation of senescent Leydig cells in testis. In 12 month-old rats, a long-term treatment with sildenafil (PDE5 inhibitor) normalized testosterone/nitrite levels in circulation and cGMP accumulation in TIF; improved Leydig cell steroidogenic capacity; decreased MEK, ERK1/2 and PRKG1 expression; prevented an increase in the Leydig cells number and atrophy of seminiferous tubules leading to histological appearance of young rat testes. In 18 month-old rats, long-term PDE5 inhibition partially recovered testosterone and nitrite levels in serum; normalized PRKG1 expression without effect on MEK and ERK1/2; and slowed down Leydig cell and macrophage accumulation and regressive tubular changes. Culturing of primary Leydig cells from aged rats in presence of PDE5-inhibitor stimulated steroidogenic and MAPK gene expression. Taking together, results indicate that cGMP targeting alter both steroidogenesis and signaling pathways associated with cell proliferation/survival. The long-term PDE5 inhibition improves testicular steroidogenesis and slows-down regressive changes in testes during aging.

Apelin and Acetylcholine Activate Distinct Nitric Oxide Signaling Pathways to Cause Endothelium‐Dependent Relaxation of Isolated Coronary Arteries

The peptide, apelin, is expressed in fat cells, endothelial cells, and CNS neurons. Increasing evidence (e.g. inotropic and vasomotor effects) supports a role for apelin in the regulation of the cardiovascular system. Apelin is reported to cause vasodilation in peripheral arteries but the underlying molecular mechanisms are not well understood. In the present study, we investigated the role of nitric oxide (NO) in apelin‐induced relaxation of isolated rat coronary arteries, and compared it with the classic endothelium‐dependent vasodilator, acetylcholine. Receptors for apelin (APJ receptors) were expressed in coronary arteries, as determined by Western blot and RT‐PCR analysis (n=3). Immunofluorescence staining and confocal microscopy demonstrated the presence of APJ receptors on endothelial and smooth muscle cells in the coronary arterial wall. In freshly isolated coronary endothelial cells, apelin (10−6 M) caused an increase in DAF‐2 fluorescence that was abolished by nitro‐l‐arginine (3 × 10−5 M; NLA) and F13A (10−7 M), an APJ receptor antagonist, consistent with an increase in NO production. In coronary arterial rings contracted with 5‐HT (10−7 M), apelin caused endothelium‐dependent relaxations (pD2=6.90 ± 0.1; Emax=45 ± 6%; n=9) that were abolished in the presence of NLA, F13A, or iberiotoxin (10−7 M; IBTx), as well as in rings contracted with a depolarizing concentration of K+ (40 mM). Neither ODQ (10−5 M) nor DT‐2 (10−6 M), a protein kinase G (PKG) inhibitor, had any effect on apelin‐induced relaxations, and apelin (10−6 M) itself had no effect on intracellular cGMP accumulation in coronary arteries (0.09 ± 0.03 vs 0.05 ± 0.01 pmol/mg; n=4; p>0.05). By contrast, endothelium‐dependent relaxations to acetylcholine (pD2=6.89 ± 0.1; Emax=97 ± 2%; n=8) were inhibited by NLA, ODQ, and DT‐2. Acetylcholine (10−6 M) increased DAF‐2 fluorescence in isolated endothelial cells, and increased cGMP accumulation (0.08 ± 0.04 vs 0.26 ± 0.02 pmol/mg; n=4; p<0.05) in coronary arteries in an NLA‐ and ODQ‐sensitive manner. Patch clamp studies in freshly isolated coronary smooth muscle cells demonstrated that the NO donor, DEA NONOate (10−6 M) caused an increase in large conductance, calcium‐activated K (BKCa) current (peak current density (+80mV) = 53.02 ± 10.4 vs. 90.42 ± 8.6 pA/pF without and with DEA NONOate, respectively; n=6; p<0.05), that was inhibited by IBTx (peak current density = 33.8 ± 5.3 pA/pF; n=6; p<0.05 vs DEA NONOate alone) but not by ODQ (peak current density = 90.2 ± 7.9 pA/pF; n=6; p>0.05 vs DEA NONOate alone). Taken together, the data demonstrate that, in coronary arteries, endothelium‐derived NO causes arterial relaxation via two distinct signaling pathways: 1) which is activated by apelin, is sensitive to changes in membrane potential and requires activation of BKCa channels; this pathway is independent of cGMP formation and may be due to direct activation of BKCa channels by NO; and 2) which is activated by acetylcholine, is cGMP‐dependent and is mediated by activation of PKG. Differential regulation of distinct NO‐signaling pathways may provide opportunities for developing novel therapeutic strategies for the treatment of the coronary arterial dysfunction that often occurs in cardiovascular disease.Support or Funding InformationSupported by a grant from the NIH National Heart, Lung and Blood Institute (HL124338)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ML290 is a biased allosteric agonist at the relaxin receptor RXFP1

Activation of the relaxin receptor RXFP1 has been associated with improved survival in acute heart failure. ML290 is a small molecule RXFP1 agonist with simple structure, long half-life and high stability. Here we demonstrate that ML290 is a biased agonist in human cells expressing RXFP1 with long-term beneficial actions on markers of fibrosis in human cardiac fibroblasts (HCFs). ML290 did not directly compete with orthosteric relaxin binding and did not affect binding kinetics, but did increase binding to RXFP1. In HEK-RXFP1 cells, ML290 stimulated cAMP accumulation and p38MAPK phosphorylation but not cGMP accumulation or ERK1/2 phosphorylation although prior addition of ML290 increased p-ERK1/2 responses to relaxin. In human primary vascular endothelial and smooth muscle cells that endogenously express RXFP1, ML290 increased both cAMP and cGMP accumulation but not p-ERK1/2. In HCFs, ML290 increased cGMP accumulation but did not affect p-ERK1/2 and given chronically activated MMP-2 expression and inhibited TGF-β1-induced Smad2 and Smad3 phosphorylation. In vascular cells, ML290 was 10x more potent for cGMP accumulation and p-p38MAPK than for cAMP accumulation. ML290 caused strong coupling of RXFP1 to Gαs and GαoB but weak coupling to Gαi3. ML290 exhibited signalling bias at RXFP1 possessing a signalling profile indicative of vasodilator and anti-fibrotic properties.

Enhanced serelaxin signalling in co-cultures of human primary endothelial and smooth muscle cells.

In the phase III clinical trial, RELAX-AHF, serelaxin caused rapid and long-lasting haemodynamic changes. However, the cellular mechanisms involved are unclear in humans. This study examined the effects of serelaxin in co-cultures of human primary endothelial cells (ECs) and smooth muscle cells (SMCs) on cAMP and cGMP signalling. Stimulation of HUVECs or human coronary artery endothelial cells (HCAECs) with serelaxin, concentration-dependently increased cGMP accumulation in co-cultured SMCs to a greater extent than in monocultures of either cell type. This was not observed in human umbilical artery endothelial cells (HUAECs) that do not express the relaxin receptor, RXFP1. Treatment of ECs with l-N(G) -nitro arginine (NOARG; 30μM, 30min) inhibited serelaxin-mediated (30nM) cGMP accumulation in HUVECs, HCAECs and co-cultured SMCs. In HCAECs, but not HUVECs, pre-incubation with indomethacin (30μM, 30min) also inhibited cGMP accumulation in SMCs. Pre-incubation of SMCs with the guanylate cyclase inhibitor ODQ (1μM, 30min) had no effect on serelaxin-mediated (30nM) cGMP accumulation in HUVECs and HCAECs but inhibited cGMP accumulation in SMCs. Serelaxin stimulation of HCAECs, but not HUVECs, increased cAMP accumulation concentration-dependently in SMCs. Pre-incubation of HCAECs with indomethacin, but not l-NOARG, abolished cAMP accumulation in co-cultured SMCs, suggesting involvement of prostanoids. In co-cultures, treatment of ECs with serelaxin caused marked cGMP accumulation in SMCs and with HCAEC also cAMP accumulation. Responses involved EC-derived NO and with HCAEC prostanoid production. Thus, serelaxin differentially modulates vascular tone in different vascular beds.

Acidic pH increases cGMP accumulation through the OGR1/phospholipase C/Ca2 +/neuronal NOS pathway in N1E-115 neuronal cells

Neuronal NO synthase (nNOS)-mediated cGMP accumulation has been shown to affect a variety of neuronal cell activities, regardless of whether they are detrimental or beneficial, depending on the amount of their levels, under the physiological and pathological situations. In the present study, we examined the role of proton-sensing G protein-coupled receptors (GPCRs), which have been identified as new pH sensors, in the acidic pH-induced nNOS/cGMP activity in N1E-115 neuronal cells. In this cell line, ovarian cancer G protein-coupled receptor 1 (OGR1) and G protein-coupled receptor 4 (GPR4) mRNAs are expressed. An extracellular acidic pH increased cGMP accumulation, which was inhibited by nNOS-specific inhibitors. Acidic pH also activated phospholipase C/Ca2+ pathways and Akt-induced phosphorylation of nNOS at S1412, both of which have been shown to be critical regulatory mechanisms for nNOS activation. The acidic pH-induced activation of the phospholipase C/Ca2+ pathway, but not Akt/nNOS phosphorylation, was inhibited by small interfering RNA specific to OGR1 and YM-254890, an inhibitor of Gq/11 proteins, in association with the inhibition of cGMP accumulation. Moreover cGMP accumulation was inhibited by 2-aminoethoxydiphenyl borate, an inhibitor of inositol 1,4,5-trisphosphate channel; however, it was not by wortmannin, a phosphatidylinositol 3-kinase inhibitor, which inhibited Akt/nNOS phosphorylation. In conclusion, acidic pH stimulates cGMP accumulation preferentially through the OGR1/Gq/11 proteins/phospholipase C/Ca2+/nNOS in N1E-115 neuronal cells. Akt-mediated phosphorylation of nNOS, however, does not appreciably contribute to the acidification-induced accumulation of cGMP.

Effect of overexpression of human aldehyde dehydrogenase 2 in LLC‐PK1 cells on glyceryl trinitrate biotransformation and cGMP accumulation

Recent studies suggest a primary role for aldehyde dehydrogenase 2 (ALDH2) in mediating the biotransformation of organic nitrates, such as glyceryl trinitrate (GTN), to the proximal activator of soluble guanylyl cyclase (sGC), resulting in increased cGMP accumulation and vasodilation. Our objective was to assess the role of ALDH2 in organic nitrate action using a cell culture model. Porcine renal epithelial (LLC-PK1) cells possess an intact NO-sGC-cGMP signaling system, and can be used as a biochemical model of organic nitrate action. We used a pcDNA3.1-human ALDH2 expression vector to establish a stably transfected cell line (PK1(ALDH2)) that overexpressed ALDH2, or small interfering RNA (siRNA) to deplete endogenous ALDH2, and assessed GTN biotransformation and GTN-induced cGMP formation. ALDH2 activity in the stably transfected cells was approximately sevenfold higher than wild-type cells or cells stably transfected with empty vector (PK1(vector)); and protein expression, as assessed by immunoblot analysis, was markedly increased. In PK1(ALDH2), GTN biotransformation was significantly increased as a result of increased glyceryl-1,2-dinitrate formation compared to wild-type or PK1(vector). However, the incubation of PK1(ALDH2) with 1 or 10 μM GTN did not alter GTN-induced cGMP accumulation compared with wild-type or PK1(vector) cells. Furthermore, siRNA-mediated depletion of ALDH2 had no effect on GTN-induced cGMP formation. In an intact cell system, neither overexpression nor depletion of ALDH2 affects GTN-induced cGMP formation, indicating that ALDH2 does not mediate the mechanism-based biotransformation of GTN to an activator of sGC.

Transient Rise of Serum Testosterone Level after Single Sildenafil Treatment of Adult Male Rats

Phosphodiesterase type 5 (PDE5) inhibitors have been established in therapy for a variety of physiological disorders including erectile dysfunction. Despite its popularity and wide usage in erectile dysfunction treatment, the short-term effect of PDE5 inhibition on Leydig cell functionality and testosterone dynamics is missing. This study was designed to assess the acute in vivo effects of sildenafil citrate (Viagra) treatment on testosterone production. Male adult rats were given sildenafil (1.25 mg/kg BW) per os, and testosterone production were analyzed 30, 60, 120, and 180 minutes after treatment. Additionally, in vitro effect of sildenafil extract on Leydig cell steroidogenesis was estimated. The formation of testicular interstitial fluid (TIF), and testosterone, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP) content was followed. Occurrence and phosphorylation of mature steroidogenic acute regulatory protein (StAR) and interaction with protein kinase G 1 (PRKG1) were assessed by immunoprecipitation and Western blot. Serum testosterone was increased 60 and 120 minutes after sildenafil treatment. In 60 minutes, TIF volume was doubled and stayed increased till the end of the experimental period. cGMP and testosterone content in TIF were increased 30 minutes after treatment, and cAMP decreased in 60 minutes. Further, sildenafil-induced stimulation of testosterone production was abolished by ex vivo addition of PRKG1 inhibitor but not by protein kinase A inhibitor. Sildenafil treatment increased the level of phosphorylated and total StAR protein. Moreover, co-immunoprecipitation of StAR and PRKG1 was increased following sildenafil treatment suggesting the active role of this kinase in initiation of testosterone synthesis. Additionally, sildenafil extract applied in vitro on primary Leydig cell culture increased cGMP accumulation and testosterone production in time- and dose-dependent manner without effect on cAMP level. Acute sildenafil treatment enlarged TIF volume but also stimulated testosterone production which may be significant considering the positive testosterone effect in regulation of sexual activity.

Angiotensin II Stimulates Thick Ascending Limb NO Production via AT2 Receptors and Akt1-dependent Nitric-oxide Synthase 3 (NOS3) Activation

Angiotensin II (Ang II) acutely stimulates thick ascending limb (TAL) NO via an unknown mechanism. In endothelial cells, activation of Ang II type 2 receptor (AT(2)) stimulates NO. Akt1 activates NOS3 by direct phosphorylation. We hypothesized that Ang II stimulates TAL NO production via AT(2)-mediated Akt1 activation, which phosphorylates NOS3 at serine 1177. We measured NO production by fluorescence microscopy. In isolated TALs, Ang II (100 nm) increased NO production by 1.1 +/- 0.2 fluorescence units/min (p < 0.01). Ang II increased cGMP accumulation by 4.9 +/- 1.3 fmol/microg (p < 0.01). Upon adding the AT(2) antagonist PD123319 (1 microm), Ang II failed to stimulate NO (0.1 +/- 0.1 fluorescence units/min; p < 0.001 versus Ang II); adding the AT(1) antagonist losartan (1 microm) resulted in Ang II stimulating NO by 0.9 +/- 0.1 fluorescence units/min. Akt inhibitor (5 microm) blocked Ang II-stimulated NO (-0.1 +/- 0.2 fluorescence units/min versus inhibitor alone). Phospho-Akt1 increased by 72% after 5 min (p < 0.006), returning to basal after 10 min. Phospho-Akt2 did not change after 5 min but increased by 115 and 163% after 10 and 15 min (p < 0.02). Phospho-Akt3 did not change. An AT(2) agonist increased pAkt1 by 78% (p < 0.02), PI3K inhibition blocked this effect. In TALs transduced with dominant negative Akt1, Ang II failed to stimulate NO (0.1 +/- 0.2 fluorescence units/min versus 1.2 +/- 0.2 for controls; p < 0.001). Ang II increased phospho-NOS3 at serine 1177 by 130% (p < 0.01) and 150% after 5 and 10 min (p < 0.02). Ang II increased phosphoNOS3 at serine 633 by 50% after 5 min (p < 0.01). Akt inhibition prevented NOS3 phosphorylation. We concluded that Ang II enhances TAL NO production via activation of AT(2) and Akt1-dependent phosphorylation of NOS3 at serines 1177 and 633.

Abstract: P172 THE INFLUENCE OF SILDENAFIL ON OXIDATIVE STRESS PARAMETERS DURING CORONARY AUTOREGULATION ON ISOLATED RAT HEART

Phosphodiesterase type 5 (PDE5) inhibitors have been established in therapy for a variety of physiological disorders including erectile dysfunction. Despite its popularity and wide usage in erectile dysfunction treatment, the short‐term effect of PDE5 inhibition on Leydig cell functionality and testosterone dynamics is missing.This study was designed to assess the acute in vivo effects of sildenafil citrate (Viagra) treatment on testosterone production.Male adult rats were given sildenafil (1.25 mg/kg BW) per os, and testosterone production were analyzed 30, 60, 120, and 180 minutes after treatment. Additionally, in vitro effect of sildenafil extract on Leydig cell steroidogenesis was estimated.The formation of testicular interstitial fluid (TIF), and testosterone, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP) content was followed. Occurrence and phosphorylation of mature steroidogenic acute regulatory protein (StAR) and interaction with protein kinase G 1 (PRKG1) were assessed by immunoprecipitation and Western blot.Serum testosterone was increased 60 and 120 minutes after sildenafil treatment. In 60 minutes, TIF volume was doubled and stayed increased till the end of the experimental period. cGMP and testosterone content in TIF were increased 30 minutes after treatment, and cAMP decreased in 60 minutes. Further, sildenafil‐induced stimulation of testosterone production was abolished by ex vivo addition of PRKG1 inhibitor but not by protein kinase A inhibitor. Sildenafil treatment increased the level of phosphorylated and total StAR protein. Moreover, co‐immunoprecipitation of StAR and PRKG1 was increased following sildenafil treatment suggesting the active role of this kinase in initiation of testosterone synthesis. Additionally, sildenafil extract applied in vitro on primary Leydig cell culture increased cGMP accumulation and testosterone production in time‐ and dose‐dependent manner without effect on cAMP level.Acute sildenafil treatment enlarged TIF volume but also stimulated testosterone production which may be significant considering the positive testosterone effect in regulation of sexual activity. Janjic MM, Stojkov NJ, Bjelic MM, Mihajlovic AI, Andric SA, and Kostic TS. Transient rise of serum testosterone level after single sildenafil treatment of adult male rats. J Sex Med **;**:**–**.

CGMP‐independent anti‐tumour actions of the inhibitor of soluble guanylyl cyclase, ODQ, in prostate cancer cell lines

Soluble guanylyl cyclase (sGC) is a receptor for nitric oxide that generates cGMP. This second messenger molecule has established roles in cellular physiology; however, less is known about its effects in tumour cells. The effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS2028), both selective sGC inhibitors on proliferation, death and migration were determined in prostate cancer cell lines. Western blot analysis confirmed the presence of alpha1 and beta1 subunits of sGC in LNCaP and PC-3 cells. Sodium nitroprusside (SNP) increased cGMP accumulation in LNCaP and PC-3, but not DU-145 cells. SNP-stimulated cGMP production in LNCaP cells was dose-dependently reduced by ODQ, with more than 90% inhibition being observed at 0.1 microM. ODQ activated caspase-3 in all three cell lines, but not in normal prostate epithelial cells, at concentrations over 10 muM. High concentrations of ODQ also promoted DNA fragmentation and nucleosome accumulation in the cytosol of LNCaP cells. Interestingly, the chemically related inhibitor, NS2028 was without effect on caspase-3. In addition, ODQ inhibited LNCaP, Du145 and PC-3 cell growth. Finally, although fibroblast growth factor-2 did not enhance cGMP levels in LNCaP cells, its ability to stimulate LNCaP motility was abolished by ODQ. These observations taken together suggest that the action of ODQ in LNCaP cells did not reflect sGC inhibition. We conclude that ODQ promotes cell death and inhibits growth and migration of prostate cancer cells and that these actions are independent of its effects on GMP levels.

Activation of soluble guanylyl cyclase (sGC) by the NO/heme-independent activator HMR1766 in cells

Most vascular diseases are characterized by an increase in the production of reactive oxygen species (ROS). The resulting oxidative stress reduces the biological activity of endothelial-derived nitric oxide (NO). In the present study we have evaluated the responsiveness of sGC in cells exposed to HMR1766, a NO-independent sGC activator that preferentially stimulates the oxidized/heme-free enzyme. Pre-treatment of rat aortic smooth muscle cells (RASMC) with 500 μM of H2O2 reduced the ability of both sodium nirtroprusside (SNP; a nitric oxide donor) and BAY 41–2272 (a NO-independent, heme-dependent sGC activator) to increase intracellular cGMP levels. In contrast, pre-treatment of cells with H2O2 significantly increased the HMR1766-stimulated cGMP accumulation. Similarly, pre-treatment of RASMC with SIN-1 (500 μM), a peroxynitrite donor, enhanced sGC activation by HMR1766. Adenovirus-mediated overepxression of catalase or superoxide dismutase in RASMC did not alter the responsiveness to HMR1766. To study if HMR1766 stimulates cGMP accumulation in cells expressing heme-deficient sGC we generated histidine 105 to phenylalanine (H105F) or cycteine (H105C) mutants of the sGC β1 subunit. In wild-type sGC-expressing cells NO-stimulated cGMP accumulation was blocked by ODQ, while HMR1766-stimulated cGMP was enhanced by ODQ. The H105F mutant was not sensitive to NO, as it is heme-deficient, but exhibited a 10-fold increase in cGMP accumulation after stimulation with HMR1766; this effect of HMR1766 was not affected by ODQ pretreatment. The α1/H105C enzyme was basally much more active than wt sGC and could be further stimulated with HMR1766, but not SNP. The above data indicate that HMR1766 is capable of activating oxidized and heme-free sGC in vivo.

Effect of nitroxyl on the hamster retinal nitridergic pathway

There is a growing body of evidence on the role of nitric oxide (NO) in retinal physiology. Recently, interest has developed in the functional role of an alternative redox form of NO, namely nitroxyl (HNO/NO −), because it is formed by a number of diverse biochemical reactions. The aim of the present report was to comparatively analyze the effect of HNO and NO on the retinal nitridergic pathway in the golden hamster. For this purpose, sodium trioxodinitrate (Angeli's salt) and diethylammonium ( Z)-1-( N, N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) were used as HNO and NO releasers, respectively. Angeli's salt and DEA/NO significantly decreased nitric oxide synthase activity. In addition, Angeli's salt (but not DEA/NO) significantly decreased l-arginine uptake. DEA/NO significantly increased cGMP accumulation at low micromolar concentrations, while Angeli's salt affected this parameter with a threshold concentration of 200 μM. Although Angeli's salt and DEA/NO significantly diminished reduced glutathione and protein thiol levels in a similar way, DEA/NO was significantly more effective than AS in increasing S-nitrosothiol levels. None of these compounds increased retinal lipid peroxidation. These results suggest that HNO could regulate the hamster retinal nitridergic pathway by acting through a mechanism that only partly overlaps with that involved in NO response.

Hydrogen peroxide restrains endothelium-derived nitric oxide bioactivity—Role for iron-dependent oxidative stress

Vascular diseases are characterized by impairment of endothelial-derived nitric oxide (NO) bioactivity and increased vascular levels of hydrogen peroxide (H 2O 2). Here we examined the implications of H 2O 2 for agonist-stimulated endothelial NO bioactivity in rabbit aortic rings and cultured porcine aortic endothelial cells (PAEC). Vessels pre-treated with H 2O 2 exhibited impaired endothelial-dependent relaxation induced by acetylcholine or calcium ionophore. In contrast, H 2O 2 had no effect on endothelium-independent relaxation induced by a NO donor, indicating a defect in endothelium-derived NO. This defect was not related to eNOS catalytic activity; treatment of PAEC with H 2O 2 enhanced agonist-stimulated eNOS activity indicated by increased eNOS phosphorylation at Ser-1177 and de-phosphorylation at Thr-495 and enhanced conversion of [ 3H]-L-arginine to [ 3H]-L-citrulline that was prevented by inhibitors of Src and phosphatidylinositol-3 kinases. Despite activating eNOS, H 2O 2 impaired endothelial NO bioactivity indicated by attenuation of the increase in intracellular cGMP in PAEC stimulated with calcium ionophore or NO. The decrease in cGMP was not due to impaired guanylyl cyclase as H 2O 2 treatment increased cGMP accumulation in response to BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase. At concentrations that impaired endothelial NO bioactivity H 2O 2 increased intracellular oxidative stress and size of the labile iron pool in PAEC. The increase in oxidative stress was prevented by the free radical scavenger's tempol or tiron and the iron chelator desferrioxamine and these antioxidants reversed the H 2O 2-induced impairment of NO bioactivity in PAEC. This study shows that despite promoting eNOS activity, H 2O 2 impairs endothelial NO bioactivity by promoting oxidative inactivation of synthesized NO. The study highlights another way in which oxidative stress may impair NO bioactivity during vascular disease.

Ammonia affects the activity and expression of soluble and particulate GC in cultured rat astrocytes

Neurotoxic effects of ammonia are mediated by increased accumulation of nitric oxide (NO), which combines with free radicals to form a highly toxic compound, peroxynitrite. Previous experiments in vivo and in vitro have suggested that this phenomenon engages neuron-derived NO and is coupled to changes in the accumulation of cGMP. The present study accounted for the facts that: (i) astrocytes, not neurons are the morphological target of ammonia, and (ii) both NO-dependent, soluble (sGC) and NO-independent, particulate guanylate cyclase (pGC) mediate cGMP production in the cells. Neocortical rat astrocytes were treated for 1 or 24 h with 5 mM ammonium chloride (“ammonia”) and then subjected to: (i) cGMP measurement, and (ii) mRNA and/or protein expression analysis of α1 and β1 subunits of sGC and two pGC forms: pGC-A and pGC-B. Treatment with ammonia for 1 h increased accumulation of cGMP and sGCβ1 mRNA expression, without producing significant changes in the protein expression. This was followed by a decrease of cGMP level at 24 h treatment, associated with a decreased expression of sGCβ1 and sGCα1 mRNA and sGCβ1 protein. Expression of pGC-A and pGC-B mRNA was elevated in ammonia-treated astrocytes after 24 h. Accordingly, increased cGMP accumulation was noted in the presence of a specific sGC inhibitor (ODQ). The results show that ammonia affects cGMP production in astrocytes, and that this may involve not only sGC but also pGC.

Endothelin-1 stimulates NO production and inhibits cAMP accumulation in rat inner medullary collecting duct through independent pathways

Endothelin-1 (ET-1) inhibition of vasopressin (AVP)-stimulated cAMP accumulation in the collecting duct has been hypothesized to be mediated, at least in part, by nitric oxide (NO). To examine this, the effect of ET-1 on NO production by acutely isolated rat inner medullary collecting duct (IMCD) cell suspensions and the role of NO in mediating ET-1 effects on AVP-stimulated cAMP accumulation were studied. ET-1 dose dependently (first evident at 100 pM ET-1) increased IMCD NO production as determined by DAF-FM fluorescence. ET(B) receptor (BQ-788), but not ET(A) receptor (BQ-123), antagonism blocked this effect. Nonspecific NO synthase (NOS) inhibitors [N(G)-nitro-L-arginine methyl ester (L-NAME) or N(G)-monomethyl-L-arginine] or NOS-1 inhibitors (SMTC or VNIO) inhibited the ET-1 response, whereas NOS-2 or NOS-3 inhibitors (L-NAA or 1400W) were ineffective. ET-1 also increased cGMP accumulation. ET-1 caused a 35% reduction in AVP-stimulated cAMP levels; however, this response was not affected by L-NAME or SMTC. The addition of L-arginine, NADPH, tetrahydrobiopterin, or tempol (to reduce superoxide-dependent conversion of NO to peroxynitrate) did not affect the response. NO donors (SNAP or spermine NONOate), at concentrations that stimulated DAF-FM fluorescence and increased cGMP levels, did not alter AVP-stimulated cAMP accumulation in the IMCD cell suspensions. In conclusion, ET-1 stimulates IMCD NO production through activation of the ET(B) receptor and NOS-1. However, neither ET-1-mediated NO production nor NO donors inhibit AVP-stimulated cAMP accumulation, indicating that NO does not mediate ET-1 inhibition of cAMP production by the IMCD.

Endothelial Nitric Oxide Production Stimulated by the Bioflavonoid Chrysin in Rat Isolated Aorta

In the present study, the effects of the bioflavonoid chrysin (5,7-dihydroxyflavone) were analysed on nitric oxide (NO) production from vascular endothelium. In aortic rings, incubation with chrysin or acetylcholine (both at 10 microM) increased L-NAME-sensitive endothelial NO release as measured using the fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA). Moreover, chrysin increased cGMP accumulation only in aortic rings with endothelium. However, at this concentration, chrysin had no effect either on basal or on NADPH-stimulated vascular superoxide production. Moreover, at this low concentration, chrysin, similar to acetylcholine, induced aortic relaxation, which was abolished by both endothelial deprivation and NO synthase inhibition. Endothelium-dependent relaxation induced by chrysin was unaltered by removal of extracellular calcium and incubation with the intracellular calcium chelator BAPTA, while the phosphatidylinositol (PI)-3 kinase inhibitor wortmannin suppressed the endothelial dependence. In conclusion, chrysin stimulated NO release from endothelial cells leading to vascular cGMP accumulation and subsequent endothelium dependent aortic relaxation. Chrysin-stimulated NO release is calcium independent and possibly mediated via PI3-kinase.

Effect of nitroxyl on human platelets function

There is a growing body of evidence on the role of nitric oxide (NO) in human platelet physiology regulation. Recently, interest has developed in the functional role of an alternative redox form of NO, namely nitroxyl (HNO/NO-), because it is formed by a number of diverse biochemical reactions. The aim of the present study was to comparatively analyze the effect of HNO and NO on several functional parameters of human platelets. For this purpose, sodium trioxodinitrate (Angeli's salt,AS) and sodium nitroprusside (SNP) were used as HNO and NO releasers, respectively. BothAS and SNP significantly inhibited platelet aggregation and ATP release induced by different agonists and adrenaline. AS or SNP did not modify the expression of platelet glycoproteins (Ib, IIb-IIIa, la-IIa, IV), whereas they substantially decreased the levels of CD62P, CD63 and of PAC-1 (a platelet activated glycoprotein IIb/IIIa epitope) after the stimulation with ADP. AS and SNP significantly increased cGMP accumulation in a 1H-[1,2,4]oxadiazolo [4,3-a] quinoxalin-1-one (ODQ)-sensitive manner. However, while L-cysteine reduced the effect of AS, it increased the effect of SNP on this parameter. Accordingly, a differential effect of L-cysteine was observed on the antiaggregatory effect of both compounds. In summary, these results indicate that HNO is an effective inhibitor of human platelet aggregation.

Vanadate is a potent activator of endothelial nitric-oxide synthase: evidence for the role of the serine/threonine kinase Akt and the 90-kDa heat shock protein.

We investigated the molecular mechanisms of sodium vanadate (vanadate)-induced nitric oxide (NO) production. Exposure of bovine lung microvascular cells (BLMVEC) to vanadate increased the release of biologically active NO in endothelium/smooth muscle cocultures, as measured by the accumulation of its surrogate marker, cGMP. This release was sensitive to NO synthase (NOS) inhibition and was greater than that observed with ionomycin. Although calcium chelators (BAPTA, EGTA) inhibited basal and ionomycin-induced NO production, they failed to inhibit vanadate-induced NO release. Moreover, in the absence of calcium/calmodulin, cell lysates from vanadate-treated cells exhibited greater NOS activity compared with control cells. Vanadate activates the phosphoinositide3-kinase (PI3-K)/Akt pathway, which is known to increase endothelial NOS (eNOS) activity by direct phosphorylation of Ser-1179. Treatment of BLMVEC with vanadate resulted in phosphorylation of both Akt and endothelial NOS. In addition, wortmannin, a PI3-K inhibitor, blocked both the vanadate-induced phosphorylation of eNOS and the increase in cGMP accumulation. Similarly, adenovirus-mediated gene transfer of an activation deficient form of Akt (AA-Akt) blocked the release of NO brought about by vanadate. To further investigate the mechanism of action of vanadate, eNOS was immunoprecipitated and its association with proteins that alter eNOS activity was tested. Immunoblots demonstrated that the eNOS-caveolin interaction remained unaffected by vanadate, whereas vanadate promoted recruitment of the 90-kDa heat shock protein (hsp90) to eNOS. We conclude that vanadate causes NO release via a mechanism that involves Akt-induced eNOS phosphorylation and increased binding of the activator protein hsp90 to eNOS.

Relaxation and cGMP formation in response to sildenafil and sodium nitroprusside in saphenous veins from normotensive and hypertensive patients

Background This study was designed to measure cyclic guanosine 3′5′ monophosphate (cGMP) formation and relaxation response to sildenafil given either alone or in combination with sodium nitroprusside in saphenous veins obtained from normotensive and hypertensive patients. Methods Saphenous vein rings were obtained from 13 hypertensive and nine normotensive patients undergoing coronary artery bypass surgery. The vein rings were suspended in organ bath chambers for isometric recording of tension. The effect of sildenafil on sodium nitroprusside-induced cGMP formation was also assessed. Results Sildenafil (10 nmol/L to 100 μmol/L) and sodium nitroprusside (0.01 to 100 nmol/L) caused concentration-dependent relaxations that were of greater magnitude in veins from normotensive patients. Sildenafil (1 to 10 μmol/L) amplified the relaxation induced by sodium nitroprusside in both groups of veins, but this effect was more pronounced in veins from hypertensive patients. Levels of cGMP in response to sodium nitroprusside were significantly lower in veins from hypertensive subjects. However, in the presence of sildenafil, the increase in cGMP levels in response to sodium nitroprusside was significantly greater in the hypertensive as compared with the normotensive group. Conclusions Although the relaxant effects of sildenafil are less pronounced in veins from hypertensive patients, the synergistic interaction sildenafil–sodium nitroprusside is more effective in veins from hypertensive patients, mainly due to an increase in cGMP accumulation.

Calcium-independent and cAMP-dependent Modulation of Soluble Guanylyl Cyclase Activity by G Protein-coupled Receptors in Pituitary Cells

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.