Increase In cGMP Content Research Articles

Dose-dependent beneficial hemodynamic effects of BAY 41-2272 in a canine model of acute pulmonary thromboembolism

The current therapy of acute pulmonary embolism is focused on removing the mechanical obstruction of the pulmonary vessels. However, accumulating evidence suggests that pulmonary vasoconstriction drives many of the hemodynamic changes found in this condition. We examined the effects of stimulation of soluble guanylate cyclase with BAY 41-2272 (5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrim idin-4-ylamine) in an anesthetized dog model of acute pulmonary embolism. Hemodynamic and arterial blood gas evaluations were performed in non-embolized dogs treated with vehicle ( N = 5), and in embolized dogs (intravenous injections of microspheres) that received BAY 41-2272 intravenously in doses of 0.03, 0.1, 0.3, and 1 mg/kg/h or vehicle (1 ml/kg/h of 1.13% ethanol in saline, volume/volume). Plasma cGMP and thiobarbituric acid reactive substances concentrations were determined using a commercial enzyme immunoassay and a fluorometric method, respectively. The infusion of BAY 41-2272 resulted in a decrease in pulmonary artery pressure by ~ 29%, and in pulmonary vascular resistance by ~ 46% of the respective increases induced by lung embolization (both P < 0.05). While the higher doses of BAY 41-2272 produced no additional effects on the pulmonary circulation, they caused significant arterial hypotension and reduction in systemic vascular resistance (both P < 0.05). Although BAY 41-2272 increased cGMP concentrations ( P < 0.05), it did not affect the hypoxemia and the increased oxidative stress caused by lung embolization. These results suggest that stimulation of soluble guanylate cyclase with low (but not high) doses of BAY 41-2272 produces selective pulmonary vasodilation during acute pulmonary embolism. The dose-dependent systemic effects produced by BAY 41-2272, however, may limit its usefulness in larger doses.

NO/cGMP/PKG pathway in platelets: inhibitory but not stimulatory

Platelets are specialized anucleate cells that play a key role in hemostasis through their ability to rapidly adhere to subendothelial matrix proteins and endothelial cells (platelet adhesion) and to other activated platelets (platelet aggregation). The importance of cyclic nucleotides and especially the NO-cGMP-PKG pathway as potent inhibitors of platelet activation has been well established by many investigators in human and animal platelets. However, recently a new mechanism of platelet activation by vWF, mediated by PKG (that sequentially activates p38 and ERK MAP kinases), was proposed [1,2]. Here we present data that activation of PKG by cGMP analogs or NO donors does not stimulate, but rather inhibits, p38 and ERK MAP kinases [3]. However, some PKG stimulators and inhibitors do affect platelets independently of PKG activity [4]. Our data also show that human and mouse platelets do not express functionally active eNOS. However, activation of the vWF receptor in human and mouse platelets stimulates basal guanylyl cyclase activity independently of NOS activation. Furthermore, a PDE5 inhibitor (sildenafil) increases cGMP content in both eNOS KO and WT mouse platelets, whereas in mouse aorta (in which sGC activity is strongly eNOS-dependent), sildenafil increased cGMP only in WT but not eNOS KO mice. In summary, our data do not provide any evidence for a "stimulatory role" of PKG in platelets. Our data suggest that vWF-induced increase in platelet cGMP is independent of platelet eNOS activity and may involve regulation of basal sGC activity by tyrosine phosphorylation. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB 688).

Diabetes mellitus as a prothrombotic condition

Diabetes mellitus (DM) is characterized by fasting hyperglycaemia and a high risk of atherothrombotic disorders affecting the coronary, cerebral and peripheral arterial trees. The risk of myocardial infarction (MI) is 3-5 fold higher in Type 2 DM and a DM subject with no history of MI has the same risk as a non-DM subject with a past history of MI. In total around 70% of deaths are vascular with poorer outcomes to both acute events and cardiological interventions. It was proposed that clustering of vascular risk factors (hyperinsulinaemia, dysglycaemia, dyslipidaemia and hypertension) around insulin resistance (IR) accounted for the increase in risk with Type 2 DM. The importance of this became apparent with the recognition that risk clustering occurs in normoglycaemic and impaired glucose tolerance (IGT) subjects with IR, in total around 25% of the population in addition to long-standing Type 1 subjects with renal disease. Evidence indicates that thrombotic risk clustering also occurs in association with IR, suppression of fibrinolysis due to elevated concentrations of the fibrinolytic inhibitor, plasminogen activator inhibitor-1 (PAI-1) is invariable with IR and there is evidence that this is regulated by the effects of triglyceride on the PAI-1 gene promoter. Other studies indicated that prothrombotic risk (coagulation factors VII, XII and fibrinogen) also associates with the IR syndrome. The development of endothelial cell dysfunction with suppression of nitric oxide and prostacyclin synthesis, combined with platelet resistance to the anti-aggregatory effects of these hormones leads to loss of control over platelet activation. In addition, hyperglycaemia and glycation have marked effects on fibrin structure function, generating a clot which has a denser structure, resistant to fibrinolysis. The combination of increased circulating coagulation zymogens, inhibition of fibrinolysis, changes in fibrin structure/function and alterations in platelet reactivity creates a thrombotic risk clustering which underpins the development of cardiovascular disease.

Antisense and Short Hairpin RNA (shRNA) Constructs Targeting PIN (Protein Inhibitor of NOS) Ameliorate Aging-Related Erectile Dysfunction in the Rat

Over-expression of penile neuronal nitric oxide synthase (PnNOS) from a plasmid ameliorates aging-related erectile dysfunction (ED), whereas over-expression of the protein inhibitor of NOS (PIN), that binds to nNOS, increases ED. To improve this form of gene therapy for ED by comparing the electrical field response of short hairpin RNA (shRNA) for PIN with that of antisense PIN RNA. Both shRNA and antisense RNA gene therapy vectors increased intracavernosal pressure in aged rats. PIN small interfering RNA (siRNA), and plasmid constructs for cytomegalovirus promoter plasmid vector (pCMV-PIN), pCMV-PIN antisense RNA, pSilencer2.1-U6-PIN-shRNA; and pSilencer2.1-U6-randomer-shRNA were prepared and validated by transfection into HEK293 cells, determining the effects on PIN expression by Western blot. Plasmid constructs were then injected, followed by electroporation, into the penile corpora cavernosa of aged (20-month-old) Fisher 344 rats and, 1 month later, the erectile response was measured by intracavernosal pressure increase following electrical field stimulation (EFS) of the cavernosal nerve. PIN was estimated in penile tissue by Western blot and real-time reverse transcriptase-polymerase chain reaction. Cyclic guanosine monophosphate (cGMP) measurements were conducted by competitive enzyme immunoassay (EIA). Immunohistofluorescence detected PIN in corporal tissue sections. In cell culture, PIN siRNA and plasmid-expressed pU6-PIN-shRNA effectively reduced PIN expression from pCMV-PIN. pSilencer2.1-U6-PIN-shRNA corrected the impaired erectile response to EFS in aged rats and raised it above the value for young rats, more efficiently than pCMV-PIN antisense RNA. PIN mRNA expression in the penis was decreased by >70% by the shRNA but remained unaffected by the antisense RNA, whereas PIN protein expression was reduced in both cases, particularly in the dorsal nerve. PIN antisense increased cGMP concentration in treated tissue by twofold. pSilencer2.1-U6-PIN-shRNA gene therapy was more effective than the antisense PIN mRNA in ameliorating ED in the aged rat, thereby suggesting that PIN is indeed a physiological inhibitor of nNOS and nitrergic neurotransmission in the penis.

Contribution of captopril thiol group to the prevention of spontaneous hypertension

We aimed to compare the effect of angiotensin converting enzyme (ACE) inhibitors captopril (containing thiol group) and enalapril (without thiol group) on the development of spontaneous hypertension and to analyze mechanisms of their actions, particularly effects on oxidative stress and NO production. Six-week-old SHR were divided into three groups: control, group receiving captopril (50 mg/kg/day) or enalapril (50 mg/kg/day) for 6 weeks. At the end of experiment, systolic blood pressure (SBP) increased by 41 % in controls. Both captopril and enalapril prevented blood pressure increase, however, SBP in the captopril group (121+/-5 mmHg) was significantly lower than that in the enalapril group (140+/-5 mmHg). Concentration of conjugated dienes in the aorta was significantly lower in the captopril group compared to the enalapril group. Captopril and enalapril increased NO synthase activity in the heart and aorta to the similar level. Neither captopril nor enalapril was, however, able to increase the expression of eNOS. Both ACE inhibitors increased the level of cGMP. However, cGMP level was significantly higher in the aorta of captopril group. We conclude that captopril, beside inhibition of ACE, prevented hypertension by increasing NO synthase activity and by simultaneous decrease of oxidative stress which resulted in increase of cGMP concentration.

Effect of icariin on cyclic GMP levels and on the mRNA expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in penile cavernosum

To further investigate the mechanisms of action of icariin (ICA), we assessed the effects of ICA on the in vitro formation of cGMP and cAMP in isolated rabbit corpus cavernosum. Isolated segments of rabbit corpus cavernosum were exposed to increasing concentrations of ICA and the dose-dependent accumulation of cGMP and cAMP was determined in the tissues samples by means of 125I radioimmunoassay. Responses of the isolated tissues preparations to ICA were compared with those obtained with the reference compounds sildenafil (Sild). Furthermore, the effects of ICA on the mRNA expression of specific cGMP-binding phosphodiesterase type V (PDE5) in rat penis were also observed. After incubation with ICA for 6 h or 14 h respectively, the levels of PDE5 mRNA were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that ICA increased cGMP concentrations directly (P < 0.05), but there was no significant effect on cAMP concentrations (P > 0.05). In the presence of sodium nitroprusside (SNP), a stimulatory agent of cGMP, both ICA and Sild increased cGMP concentrations with increasing dose (P < 0.01). Their EC50 was 4.62 (ICA) and 0.42 (Sild) micromol/L respectively. Under the same condition, ICA and Sild unaltered cAMP level significantly (P > 0.05). There were PDE5A1 and PDE5A2 mRNA expressions in rat corpus cavernosum with PDE5A2 being the dominant isoform. ICA could obviously inhibit these two isoforms mRNA expression in rat penis, and decrease PDE5A1 more pronouncedly (P < 0.01). The present study indicated that the aphrodisiac mechanisms of icariin involved the NO-cGMP signal transduction pathway, with increasing cGMP levels in the corpus cavernosum smooth muscle. The inhibitory effect of icariin on PDE5 mRNA expression, especially on PDE5A1, might account for its molecular mechanisms for its long-term activity.

Role of the nitric oxide (NO)/cGMP signaling in the relaxations of mouse corpus cavernosum (CC) induced by the soluble guanylyl cyclase stimulator (sGC) BAY 41‐2272.

OBJECTIVE To characterize the in vitro effects of the NO-independent sGC stimulator BAY 41-2272 on CC from wild-type (WT), nNOS −/− and eNOS −/− mice. METHODS CC strips were mounted in 4-ml myographs and isometric force was recorded. cGMP was measured using EIA kits. RESULTS BAY 41-2272 (0.01–10 μM) relaxed CC with pEC50 values of 6.34±0.04, 6.36±0.06 and 5.70±0.07 in WT, nNOS μ/μ and eNOS μ/μ mice, respectively. L-NAME (100 μM) rightward shifted the curves to BAY 41-2272 by 3-fold in CC from WT and nNOS μ/μ, but not eNOS μ/μ. ODQ (10 μM) reduced BAY 41-2272-evoked relaxations as evidenced by rightward shifts of 4-fold. Sildenafil (0.1 μM) potentiated the relaxations induced by BAY 41-2272 in all groups. BAY 41-2272 (0.1 μM) enhanced SNP-induced relaxations of CC from WT (CTL: 6.58±0.05; BAY: 7.06±0.01), nNOS μ/μ (CTL: 6.47±0.06; BAY: 6.84±0.03) and eNOS μ/μ (CTL: 6.74±0.04; BAY: 7.16±0.03). BAY 41-2272 potentiated acetylcholine (ACh, 0.01–1 μM)- and electrical field stimulation (EFS, 1–16 Hz)-induced relaxations. BAY 41-2272 (1 μM) caused a 30-fold increase in cGMP concentration in WT and nNOS −/− samples versus an 18-fold increase in eNOS −/− CC. Co-incubation with SNP (1 μM) resulted in a synergistic increase in cGMP levels in an ODQ-sensitive manner. CONCLUSION sGC activation in the penis by BAY 41-2272 directly or via enhancement of NO effects may provide a novel treatment for erectile dysfunction. Support: HL74167, AHA.

Sildenafil prevents indomethacin‐induced gastropathy in rats: role of leukocyte adherence and gastric blood flow

Nitric oxide (NO) is an important mediator of gastric mucosal defense. Sildenafil (SILD), a cyclic GMP-specific phosphodiesterase inhibitor, promotes an increase in cGMP concentrations in the gastrointestinal tract. cGMP mediates many of the biological actions of NO. We tested the hypothesis that SILD could increase mucosal defense against indomethacin-induced gastropathy in rats. SILD (1, 4 or 10 mg kg(-1), p.o.) pretreatment significantly reduced (P < 0.01) the gastric damage and the increase in gastric myeloperoxidase (MPO) activity elicited by indomethacin (20 mg kg(-1) p.o.), with the maximal effect at the dose of 10 mg kg(-1). L-NAME (3, 10 or 20 mg kg(-1), i.p.) dose dependently reversed the protective effects of SILD, an effect not seen when L-arginine (L-ARG) (200 mg kg(-1), i.p.) was co-administered with L-NAME. Indomethacin-induced leukocyte adhesion, assessed by intravital microscopy, was decreased (P < 0.01) by SILD, and this effect was reversed by L-NAME cotreatment. Indomethacin elicited a decrease in gastric blood flow and in gastric PGE2 levels. SILD was able to prevent the decrease in gastric blood flow (P < 0.01), without diminishing the inhibitory effect of indomethacin on prostaglandin synthesis. These results indicate that SILD, acting via NO-dependent mechanisms, prevents indomethacin-induced gastropathy, possibly through a reduction of leukocyte adhesion and maintenance of gastric blood flow.

Excessive activation of cyclic nucleotide‐gated channels contributes to neuronal degeneration of photoreceptors

In different animal models, photoreceptor degeneration was correlated to an abnormal increase in cGMP concentration. The cGMP-induced photoreceptor toxicity was demonstrated by applying the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine on retinal explants. To assess the role of cGMP-gated channels in this cGMP toxicity, the Ca(2+) channel blockers verapamil and L- and D-diltiazem, which block cGMP-gated channels with different efficacies, were applied to in vitro animal models of photoreceptor degeneration. These models included: (i) adult rat retinal explants incubated with zaprinast, a more specific inhibitor of the rod phosphodiesterase than 3-isobutyl-1-methylxanthine and (ii) rd mouse retinal explants. Photoreceptor apoptosis was assessed by terminal dUTP nick end labelling and caspase 3 activation. Effects of the blockers on the synaptic rod Ca(2+) channels were measured by patch-clamp recording. In the zaprinast-induced photoreceptor degeneration model, both diltiazem isomers rescued photoreceptors whereas verapamil had no influence. Their neuroprotective efficacy was correlated to their inhibition of cGMP-gated channels (l-diltiazem>d-diltiazem>verapamil=0). In contrast, all three Ca(2+) channel blockers suppressed rod Ca(2+) channel currents similarly. This suppression of the currents by the diltiazem isomers was very weak (16.5%) at the neuroprotective concentration (10 microm). In rd retinal explants, both diltiazem isomers also slowed down rod degeneration in contrast to verapamil. L-diltiazem exhibited this effect at concentrations ranging from 1 to 20 microm. This study further supports the photoreceptor neuroprotection by diltiazem particularly in the rd mouse retina, whereas the absence of neuroprotection by verapamil further suggests the role of cGMP-gated channel activation in the induction of photoreceptor degeneration.

MECHANISMS UNDERLYING RELAXATION OF RABBIT AORTA BY BAY 41‐2272, A NITRIC OXIDE‐INDEPENDENT SOLUBLE GUANYLATE CYCLASE ACTIVATOR

1. The compound BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine) has been described as a potent, nitric oxide (NO)-independent, stimulator of soluble guanylate cyclase. In the present study, the mechanisms underlying the relaxant effect of BAY 41-2272 in endothelium-intact and -denuded precontracted rabbit aortic rings were investigated. 2. Male New Zealand white rabbits were anaesthetized with pentobarbital sodium. Aortic rings were transferred to 10 mL organ baths containing oxygenated and warmed Krebs' solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. Aortic rings were precontracted submaximally with phenylephrine (1 micromol/L). 3. The addition of BAY 41-2272 (0.01-10 micromol/L) to the organ bath produced concentration-dependent relaxations of the aortic rings with a higher potency in endothelium-intact (pEC50 6.59 +/- 0.05) compared with endothelium-denuded (pEC50 6.19 +/- 0.04; P < 0.05) preparations. No differences in maximal responses were observed in either preparation. The NO synthesis inhibitor NG-nitro-L-arginine methyl ester (100 micromol/L) produced a 2.1-fold rightward shift in endothelium-intact (P < 0.01) rings, but had no effect in endothelium-denuded rings. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 micromol/L) caused significant rightward shifts of the concentration-response curves to BAY 41-2272 of 4.9- and 2.6-fold in endothelium-intact and -denuded rings, respectively. The phosphodiesterase-5 inhibitor sildenafil (0.1 micromol/L) significantly potentiated the relaxant effects of BAY 41-2272 in both endothelium-intact and -denuded rings. 4. At 1 micromol/L, BAY 41-2272 significantly elevated the aortic cGMP content above basal levels in both endothelium-intact and -denuded rings. Furthermore, ODQ reduced BAY 41-2272-elicited increases in cGMP content by 17 and 90% in endothelium-intact and -denuded rings, respectively (P < 0.01). 5. In conclusion, BAY 41-2272 potently relaxes endothelium-intact and -denuded rabbit aortic rings. The basal release of endothelium-derived NO enhances BAY 41-2272-induced relaxations, suggesting a synergistic effect of BAY 41-2272 and NO on soluble guanylate cyclase. In addition, the endothelium-independent relaxation involves both GMP-dependent and -independent mechanisms.

Inhibition of carbachol‐evoked oscillatory currents by the NO donor sodium nitroprusside in guinea‐pig ileal myocytes

The effect of sodium nitroprusside (SNP) on carbachol (CCh)-evoked inward cationic current (Icat) oscillations in guinea-pig ileal longitudinal myocytes was investigated using the whole-cell patch-clamp technique and permeabilized longitudinal muscle strips. SNP (10 microm) completely inhibited I(cat) oscillations evoked by 1 microm CCh. 1H-(1,2,4) Oxadiazole [4,3-a] quinoxaline-1-one (ODQ; 1 microm) almost completely prevented the inhibitory effect of SNP on Icat oscillations. 8-Bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cGMP; 30 microm) in the pipette solution completely abolished Icat oscillations. However, a pipette solution containing Rp-8-Br-cGMP (30 microm) almost completely abolished the inhibitory effect of SNP on Icat oscillations. When the intracellular calcium concentration ([Ca2+]i) was held at a resting level using BAPTA (10 mm) and Ca2+ (4.6 microm) in the pipette solution, CCh (1 microm) evoked only the sustained component of Icat without any oscillations and SNP did not affect the current. A high concentration of inositol 1,4,5-trisphosphate (IP3; 30 microm) in the patch pipette solutions significantly reduced the inhibitory effect of SNP (10 microm) on Icat oscillations. SNP significantly inhibited the Ca2+ release evoked by either CCh or IP3 but not by caffeine in permeabilized preparations of longitudinal muscle strips. These results suggest that the inhibitory effects of SNP on Icat oscillations are mediated, in part, by functional modulation of the IP3 receptor, and not by the inhibition of cationic channels themselves or by muscarinic receptors in the plasma membrane. This inhibition seems to be mediated by an increased cGMP concentration in a protein kinase G-dependent manner.

Adrenomedullin induces heme oxygenase-1 gene expression and cGMP formation in rat vascular smooth muscle cells

Adrenomedullin (ADM) is a potent vasodilatory peptide. It regulates blood pressure by increasing cyclic adenosine monophosphate (cAMP) and guanosine-3′,5′-monophosphate (cGMP). We sought to investigate the effect of ADM on heme oxygenase-1 (HO-1) gene expression and cGMP formation in cultured rat vascular smooth muscle cells (VSMCs). ADM treatment, 10 −9 and 10 −8 mol/L, increased cGMP production, and it increased the intracellular cGMP content of platelets coincubated with VSMCs. It increased cGMP content by 158.8% and 273.5%, respectively; increased HO-1 activity by 49.5% and 87%, respectively; augmented HO-1 protein levels by 66% and 126%, respectively; upregulated the steady-state level of HO-1 mRNA by 73% and 159%, respectively, and increased HO-1 mRNA transcription synthesis by four- and seven-fold, respectively. These results suggest that ADM induces HO-1 gene expression and cGMP formation in rat VSMCs.

Oral administration of sildenafil restores learning ability in rats with hyperammonemia and with portacaval shunts†

Patients with liver disease with overt or minimal hepatic encephalopathy show impaired intellectual capacity. The underlying molecular mechanism remains unknown. Rats with portacaval anastomosis or with hyperammonemia without liver failure also show impaired learning ability and impaired function of the glutamate-nitric oxide-cyclic guanine monophosphate (glutamate-NO-cGMP) pathway in brain. We hypothesized that pharmacological manipulation of the pathway in order to increase cGMP content could restore learning ability. We show by in vivo brain microdialysis that chronic oral administration of sildenafil, an inhibitor of the phosphodiesterase that degrades cGMP, normalizes the function of the glutamate-NO-cGMP pathway and extracellular cGMP in brain in vivo in rats with portacaval anastomosis or with hyperammonemia. Moreover, sildenafil restored the ability of rats with hyperammonemia or with portacaval shunts to learn a conditional discrimination task. In conclusion, impairment of learning ability in rats with chronic liver failure or with hyperammonemia is the result of impairment of the glutamate-NO-cGMP pathway. Moreover, chronic treatment with sildenafil normalizes the function of the pathway and restores learning ability in rats with portacaval shunts or with hyperammonemia. Pharmacological manipulation of the pathway may be useful for the clinical treatment of patients with overt or minimal hepatic encephalopathy.

GSA: behavioral, histological, electrophysiological and neurochemical effects

Renal insufficient patients suffer from a variety of complications as direct and indirect consequence of accumulation of retention solutes. Guanidinosuccinic acid (GSA) is an important probable uremic toxin, increased in plasma, urine, cerebrospinal fluid and brain of patients with uremia and supposed to play a role in the pathogenesis of some neurological symptoms. GSA, an NMDA-receptor agonist and GABA-receptor antagonist, is suggested to act as an excitotoxin and shown to be convulsive. The effect of hippocampal (i.h.) GSA injection on behavior and hippocampal volume in mice is presented here. In addition, hippocampal cGMP concentration after systemic injection of GSA was measured. The effect of co-application of NMDA-receptor antagonist CGP37849 with GSA was tested, in vivo, after hippocampal GSA injection and, in vitro, on GSA evoked currents in spinal cord neurons. A significant dose-dependent effect of i.h. injection of GSA on cognitive performance, activity and social exploratory behavior was observed. There was a protective effect of CGP37849 on GSA induced behavioral alterations. Volume of hippocampal cornu ammonis region decreased significantly and dose-dependently after GSA injection. Systemic GSA injection increased cGMP concentration in hippocampal formation. It can be concluded that GSA is an important neurotoxin. As GSA is increased in patients with uremia, it probably contributes to their neurological symptoms. Knowledge of neurotoxic effects and mechanisms of action of GSA and other uremic retention solutes could help in the development of more efficient treatment of uremic patients. Animal models like the ‘GSA mouse model’ are useful tools for research in this context.

Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture.

The soybean phytoestrogen, genistein (Gen), has anabolic effects on bone through mechanisms that remain to be elucidated. We examined the role of nitric oxide (NO) and its downstream effector guanylyl cyclase (GC) in mediating the effects of Gen on the proliferation and osteoblastic maturation of primary mouse bone marrow-derived mesenchymal stem cells (BMSCs). Gen (10(-8) approximately 10(-6) M) resulted in a dose-dependent increase in cell proliferation as measured by increased [3H]thymidine incorporation, and stimulated osteoblastic maturation as assessed by culture duration-dependent increments in alkaline phosphatase (ALP) activity, calcium deposition into extracellular matrix and Runx2/Cbfa1 gene expression in BMSCs cultures. Gen also resulted in a dose-dependent increase in NO synthase (NOS) activity, NO formation, and cGMP production in BMSCs cultures. The effects of Gen were mimicked by 17beta-estradiol (E2, 10(-8) M). Concurrent treatment with the estrogen receptor (ER) antagonist ICI182,780 (10(-7) M) or the NOS inhibitor L-NAME (3 x 10(-3) M) diminished the Gen (10(-6) M)-mediated increase in NOS activity, NO production, and cGMP content. In contrast, a soluble GC inhibitor 1H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ, 10(-6) M) selectively blocked the Gen (10(-6) M)-mediated increase in cGMP content but not in NO production and NOS activity. Moreover, inhibition of ER, NOS activity or cGMP blocked Gen-induced proliferation and osteoblastic differentiation of BMSCs and Runx2/Cbfa1 gene expression in culture. Gen has estrogen-like activity and stimulates the proliferation and osteoblastic differentiation of mouse BMSCs at least in part through NO/cGMP pathway.

Effects of Various Selective Phosphodiesterase Inhibitors on Carbachol-Induced Contraction and Cyclic Nucleotide Contents in the Guinea Pig Gall Bladder

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in the guinea pig gall bladder were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), and zaprinast (type 5), inhibited CCh-induced contractions in a concentration-dependent manner. The rank order of potency for the gall bladder was Ro20-1724 > vinpocetine > EHNA > milrinone > zaprinast, which was different from that of the trachea, taenia coli, and aorta. In the presence of CCh (0.3 muM), vinpocetine, milrinone, and Ro20-1724 each increased cAMP content, but not cGMP. By contrast, zaprinast increased cGMP content, but not cAMP, and EHNA increased both cAMP and cGMP contents. These results suggest that vinpocetine-, milrinone-, and Ro20-1724-induced relaxation was correlated with cAMP, zaprinast-induced relaxation was correlated with cGMP, and that EHNA-induced relaxation was correlated with cAMP and cGMP in the guinea pig gall bladder. In conclusion, the effect of PDE inhibitors in the guinea pig gall bladder was different from those in smooth muscles, such as the trachea, taenia coli, and aorta.

Antiproliferative effect of sildenafil on human pulmonary artery smooth muscle cells

Pulmonary arterial hypertension (PAH) is characterized by vasoconstriction and by obstructive changes of the pulmonary vasculature including smooth muscle cell proliferation which leads to medial hypertrophy and subsequent luminal narrowing. Sildenafil, an orally active inhibitor of cGMP phosphodiesterase-type-5, exerts pulmonary vasodilator activity in PAH patients. We evaluated the effects of sildenafil on growth of cultured human pulmonary artery smooth muscle cells (PASMC). The results indicate that sildenafil reduced DNA synthesis stimulated by PDGF and dose dependently inhibited PASMC proliferation. These effects were paralleled by a progressive increase in cGMP content, followed by an accumulation of cAMP. The treatment with 8-bromo-cGMP or dibutyryl-cAMP mimicked all the effects of sildenafil. On the other hand, treatment of PASMC with inhibitors of cGMP-dependent protein kinase (PKG) or cAMP-dependent protein kinase (PKA) reversed the antiproliferative effect of sildenafil. In addition, sildenafil inhibited the phosphorylation of ERK, a converging point for several pathways leading to cell proliferation. This effect was partially reduced by PKG inhibition and completely abolished by PKA inhibition.We conclude that sildenafil exerts an antiproliferative effect on human PASMC that is mediated by an interaction between the cGMP-PKG and the cAMP-PKA activated pathways, leading to inhibition of PDGF-mediated activation of the ERK.

Conservation of capa peptide-induced nitric oxide signalling in Diptera

In D. melanogaster Malpighian (renal) tubules, the capa peptides stimulate production of nitric oxide (NO) and guanosine 3', 5'-cyclic monophosphate (cGMP), resulting in increased fluid transport. The roles of NO synthase (NOS), NO and cGMP in capa peptide signalling were tested in several other insect species of medical relevance within the Diptera (Aedes aegypti, Anopheles stephensi and Glossina morsitans) and in one orthopteran out-group, Schistocerca gregaria. NOS immunoreactivity was detectable by immunocytochemistry in tubules from all species studied. D. melanogaster, A. aegypti and A. stephensi express NOS in only principal cells, whereas G. morsitans and S. gregaria show more general NOS expression in the tubule. Measurement of associated NOS activity (NADPH diaphorase) shows that both D. melanogaster capa-1 and the two capa peptides encoded in the A. gambiae genome, QGLVPFPRVamide (AngCAPA-QGL) and GPTVGLFAFPRVamide (AngCAPA-GPT), all stimulate NOS activity in D. melanogaster, A. aegypti, A. stephensi and G. morsitans tubules but not in S. gregaria. Furthermore, capa-stimulated NOS activity in all the Diptera was inhibited by the NOS inhibitor l-NAME. All capa peptides stimulate an increase in cGMP content across the dipteran species, but not in the orthopteran S. gregaria. Similarly, all capa peptides tested stimulate fluid secretion in D. melanogaster, A. aegypti, A. stephensi and G. morsitans tubules but are either without effect or are inhibitory on S. gregaria. Consistent with these results, the Drosophila capa receptor was shown to be expressed in Drosophila tubules, and its closest Anopheles homologue was shown to be expressed in Anopheles tubules. Thus, we provide the first demonstration of physiological roles for two putative A. gambiae neuropeptides. We also demonstrate neuropeptide modulation of fluid secretion in tsetse tubule for the first time. Finally, we show the generality of capa peptide action, to stimulate NO/cGMP signalling and increase fluid transport, across the Diptera, but not in the more primitive Orthoptera.

173 Effects of Phosphodiesterase 5 Inhibitor on Pulmonary Vascular Reactivity in The Fetal Lamb

Background: Nitric Oxide (NO) released by pulmonary vascular endothelium is a potent vasodilator related to increased cGMP content. Hydrolysis of cGMP is achieved predominantly by cGMP–specific phosphodiesterases (PDEs). Sildenafil (SILD) is a selective phosphodiesterase–5 (PDE–5) inhibitor. The purpose of the study was to assess the effects of sildenafil on pulmonary vascular circulation during the perinatal period.Methods: 12 pregnant ewes were operated on between 128 and 130 days gestation (term = 145 d). Catheters were placed into the ascending aorta, superior vena cava, main pulmonary artery and left atrium. An ultrasonic flow transducer was placed around left pulmonary artery (LPA). An inflatable vascular occluder was placed around ductus arteriosus (DA). Acute DA compression increases pulmonary artery pressure (PAP) causing an increase in pulmonary blood flow (Q) and a progressive decline in pulmonary vascular resistance (PVR). Fetal lambs were divided in two groups: 1) a SILD group infused continuously with SILD at a rate of 24 mg/day (n=6) and; 2/ control group (CONT) infused with saline (n=6). After 24 hrs of infusion, we compared the hemodynamic response of LPA to: 1) increased fetal PaO2 (maternal O2 inhalation for 30 min), and; 2) increase in vascular shear-stress (flow-induced pulmonary vasodilatation related to DA compression).Results: Mean PAP, Q and PVR were similar in both groups before and after 24 h of SILD or saline infusion. Despite similar baseline values, PVR during maternal O2 inhalation was lower in SILD than in CONT group (0.23±0.01 vs 0.27±0.01 mmHg/ml.min−1 respectively) (p<0.01). Furthermore, drop in PVR during acute DA compression was greater in the SILD group (from 0.54±0.03 to 0.26±0.02 mmHg/ml.min−1) than in the CONT group (from 0.55±0.04 to 0.39±0.02 mmHg/ml.min−1) (p<0.01).Conclusion: Although no difference was found in the basal pulmonary vascular tone, sildenafil increases pulmonary vascular reactivity in the ovine fetus. These data suggest that PDE–5 is involved in the regulation of pulmonary vascular reactivity during the perinatal period. We further speculate that specific inhibitor of PDE–5 may potentiate birth related pulmonary vasodilator stimuli and could improve conditions associated with failure to circulatory adaptation at birth.

Cobalt-60 gamma radiation increased the nitric oxide generation in cultured rat vascular smooth muscle cells

Radiation is a promising and new treatment for restenosis following angioplasty. Nitric oxide has been proposed as a potential “anti-restenotic” molecule. We radiated the cultured rat vascular smooth muscle cells with Cobalt-60 gamma radiation at doses of 14 and 25Gy and observed nitrite production, cGMP content, L-arginine uptake, inducible nitric oxide synthase (iNOS) activity, and the gene expression of iNOS. Results showed that radiation at doses of 14 and 25Gy increased cGMP content by 92.4% and 86.4%, respectively. Radiation at the dose of 25Gy increased the iNOS activity and nitrite content, but radiation at the dose of 14Gy had no significant effect on iNOS activity and NO production. Both doses of radiation significantly decreased the L-arginine transport. Radiation at the doses of 14 and 25Gy increased iNOS gene expression significantly, which was consistent with the effect of radiation on iNOS activity. In conclusion, radiation induces the NO generation by up-regulating the iNOS activity.