Increase In Cyclic GMP Research Articles

Protective Effects of Thymoquinone on Endothelial Cell Dysfunction in Hypercholesterolemia

Background: Increased reactive oxygen species (ROS) have been implicated as important mechanisms that contribute to endothelial dysfunction (ED). The administration of thymoquinone in animal models significantly inhibits ROS production. Purpose: The protective effects of thymoquinone on endothelial cell dysfunction were studied in cholesterol-fed rabbits. Thirty rabbits were randomly divided into five groups. Methods: The negative control group was fed a standard diet, the positive control group was fed the same diet with 2 % cholesterol, the Thymoquinone group was fed the same diet with 2 % cholesterol and Thymoquinone 100 mg/Kg BW/day, 200 mg/Kg BW/day or 400 mg/Kg BW/day. Results: The cholesterol-rich diet significantly increased Malondialdehyde (MDA) in the aortic blood vessels, as reflected by Thiobarbituric Acid-Reactive Substances (TBARS), inhibited endothelium-dependent vascular relaxations to acetylcholine and decrease cyclic GMP were compared with vessels from normal rabbits (negative control). In cholesterol-fed rabbits, Thymoquinone treatment decreased MDA in plasma production, improved endothelium-dependent relaxations to acetylcholine and increase cyclic GMP production. Conclusion: These results suggest that dietary treatment of rabbits with thymoquinone may prevent superoxide anion (O2-) induced inactivation of endothelium-dependent relaxing factor (EDRF), improve the endothelium-dependent relaxation to acetylcholine in the aortic blood vessels, and increase cyclic GMP content in aortic of cholesterol-fed rabbits.

The role of tadalafil in treated hypertensive patients with erectile dysfunction.

In this issue, Kloner and colleagues showed in a pooled safety analysis that tadalafil—a phosphodiesterase type 5 (PDE-5) inhibitor—is safe in treated hypertensive patients and is not associated with increased hypotension-related treatment adverse events and major cardiovascular (CV) events.1 The safety data were extracted from 72 Phase II–IV clinical studies of tadalafil used for erectile dysfunction (ED) or benign prostate hypertrophy. PDE-5 inhibitors are vasodilating drugs that work by blocking the degradation of cyclic GMP by PDE-5 in the smooth muscle cells lining the blood vessels supplying various tissues. The increase of the intracellular cyclic GMP prolongs its duration of action resulting in increase in nitric oxide bioavailability, smooth muscle relaxation, vasodilation and increased blood flow in the corpora cavernosa, and penile erection. The main PDE-5 inhibitors are sildenafil, vardenafil, avanafil, and tadalafil. They differ in their pharmacodynamic and pharmacokinetic properties allowing targeting the treatment to the patients' needs. Avanafil has the fastest onset of action and tadalafil has the longest duration of action (half-life of 17.5 h and duration of action up to 36 h).2 ED impairs the quality of life of affected individuals and may be a prognostic marker for the development of CV disease.3 The prevalence of ED in the general population is around 15–20% and it is significantly higher in patients with hypertension,2, 4 probably due to vascular changes. Hypertension is a major modifiable risk factor for CV disease and lowering blood pressure is beneficial. Lowering blood pressure may cause or aggravate ED and it is not clear whether it is a side effect of the antihypertensive treatment or a result of the blood pressure decrease. The development of ED is one of the major reasons to withdraw antihypertensive treatment leading to inadequate blood pressure control. PDE-5 inhibitors improve ED and improve adherence to antihypertensive therapy, improve blood pressure control, and thereby may reduce CV risk in hypertensive patients with ED. The effect of PDE-5 inhibitors is not limited to the corpora cavernosa and they also vasodilate other vascular beds thereby reducing blood pressure. There was a concern that blood pressure decrease may cause CV side effects particularly in patients with CV risk factors. Kloner and colleagues in this issue showed that in subjects not receiving antihypertensive medications the incidence of hypotension related treatment emergent adverse events was low, but it was significantly higher in the tadalafil group than in the placebo group.1 Most subjects (>90%) not receiving antihypertensive medications were normotensives, and in this group, lowering blood pressure by tadalafil may increase the incidence of hypotension-related events. However, in hypertensive patients on antihypertensive medications tadalafil does not increase the hypotension related events. The co-administration of tadalafil with doxazosin, a blood pressure lowering alpha blocker is associated with a remarkable blood pressure decrease that maybe deleterious.5 However, with other antihypertensive agents tadalafil is not associated with hypotensive related side effects. The co-administration of PDE-5 inhibitors with various antihypertensive drugs, except alpha blockers, is safe. This was observed even in those who used several antihypertensive agents.1 It has been shown that all PDE-5 inhibitors are safe in treated hypertensive patients.2 The hypotensive effect of PDE-5 inhibitors should be exploit to treat resistant hypertension, particularly in those with ED. Since tadalafil has the longest duration of action it will probably be the best PDE-5 inhibitor to use for lowering blood pressure. Low dose tadalafil (5 mg daily) can improve blood pressure control and erectile function. The fact that PDE-5 inhibitors can lower blood pressure and improve erectile function suggests that lowering blood pressure by itself does not cause ED and it is probably related to the antihypertensive medications. The safety profile of tadalafil presented by Kloner in this issue may justify wider use of the drug in hypertensive patients. Wide use of tadalafil in hypertensive patients may improve erectile function and blood pressure control leading to a decrease in CV events. Further long term studies should be done to prove this concept. The author has no competing interests.

Sodium nitrite attenuates MMP-9 production by endothelial cells and may explain similar effects of atorvastatin.

Imbalanced matrix metalloproteinase (MMP) activity promotes cardiovascular alterations that are attenuated by statins. These drugs exert pleiotropic effects independent of cholesterol concentrations, including upregulation of nitric oxide (NO) formation and MMP downregulation. However, statins also increase tissue concentrations of nitrites, which activate new signaling pathways independent of NO. We examined whether atorvastatin attenuates MMP-9 production by human umbilical vein endothelial cells (HUVEC) stimulated with phorbol 12-myristate 13-acetate (PMA) by mechanisms possibly involving increased nitrite, and whether this effect results of NO formation. We also examined whether such an effect is improved by sildenafil, an inhibitor of phosphodiesterase-5 which potentiates NO-induced increases in cyclic GMP. MMP activity and nitrite concentrations were measured by gelatin zymography and ozone-based reductive chemiluminescence, respectively, in the conditioned medium of HUVECs incubated for 24 h with these drugs. Phospho-NFκB p65 concentrations were measured in cell lysate to assess NFκB activation. Atorvastatin attenuated PMA-induced MMP-9 gelatinolytic activity by mechanisms not involving NO, although it increased nitrite concentrations, whereas sildenafil had no effects. Combining both drugs showed no improved responses compared to atorvastatin alone. While sodium nitrite attenuated MMP-9 production by HUVECs, adding hemoglobin (NO scavenger) did not affect the responses to nitrite. Neither atorvastatin nor nitrite inhibited PMA-induced increases in phospho-NFκB p65 concentrations. These findings show that sodium nitrite attenuates MMP-9 production by endothelial cells and may explain similar effects exerted by atorvastatin. With both drugs, the inhibitory effects on MMP-9 production are not dependent on NO formation or on inhibition of NFκB activation. Our findings may help to elucidate important new nitrite-mediated mechanisms by which statins affect imbalanced MMP activity in a variety of cardiovascular disease.

S39 The Role Of Soluble Guanylate Cyclase Stimulator Bay 41-2272 On Remodelling Processes Relevant To The Pathogenesis Of Pulmonary Arterial Hypertension

Introduction and objectives Pulmonary arterial hypertension (PAH) is characterised by remodelling of small, muscular pre-capillary blood vessels. The subsequent rise in pulmonary vascular resistance leads to right ventricular failure and death. The aetiology of the remodelling process is largely unknown although defects in the bone morphogenetic protein receptor II (BMPR II) pathway are likely to be involved. Most of the therapies used thus far are aimed at pulmonary vasodilation. However it is unclear how much of the benefit seen with these medications is related to reverse remodelling. Riociguat is a “first in class” drug that stimulates soluble guanylate cyclase, with a consequent increase in cyclic GMP (and vasodilation). Riociguat has recently been shown to improve haemodynamics and exercise capacity in patients with idiopathic PAH and chronic thromboembolic PH (PATENT and CHEST). Here we sought to determine the effects of Bay 41–2272, the tool compound for riociguat, on remodelling processes in pulmonary vascular cells. Methods We used primary human endothelial (HPAECs) and smooth muscle cells (HPASMCs) as our target cells. Proliferation was measured using the CyQUANT proliferation kit after cells were treated with various concentration of Bay 41–2272 (kind gifted by Bayer Pharmaceuticals Ltd) for 72 h in the presence of 15% serum. Apoptosis was measured using Cell death ELISA kit and DAPI staining after cells were treated with various concentration of Bay 41–2272 for 24 and 48 h in the absence of serum. Results Bay41–2272 treatment increased HPASMC apoptosis after 24 and 48 h (Figure 1) by Cell death ELISA; this was further confirmed by DNA condensation assay (DAPI staining). Bay 41–2272 treatment also increased HPAEC apoptosis at 24 h. It was not clear at 48 h treatment whether HPAEC cell death was significant in the absence of serum. Bay 41–2272 treatment reduced HPASMC proliferation (Figure 2), but had no effect on HPAECs. Conclusions Our preliminary indicate that Bay 41–2272 increases apoptosis and inhibits proliferation, at least in HPASMCs, in vitro . Further studies are needed to fully characterise these effects on remodelling processes and to compare sGC stimulators, such as Bay 41–2272 to Type V phosphodiesterases.

Asterosap, an Egg Jelly Peptide, Elevate Intracellular Ca<sup>2+</sup> and Activate the Motility of Spermatozoa

Components from the outer envelopes of the egg that influence the flagellar beating and acrosome reaction of spermatozoa are regulated by ion flux across the plasma membrane. Asterosap, a sperm-activating peptide from the starfish egg jelly layer, causes a transient increase in intracellular cyclic GMP (cGMP) through the activation of the asterosap receptor, a guanylyl cyclase (GC), and causes an increase in intracellular Ca2+. Here we describe the pathway of asterosap-induced Ca2+ elevation using different Ca2+ channel antagonists. Fluo-4 AM, a cell permeable Ca2+ sensitive dye was used to determine the channel caused by the asterosap-induced Ca2+ elevation in spermatozoa. Different L-type Ca2+ channel antagonists, a non specific Ca2+ channel antagonist (nickel chloride), and a store-operated Ca2+ channel (SOC) antagonist do not show any significant response on asterosap-induced Ca2+ elevation, whereas KB-R7943, a selective inhibitor against Na+/Ca2+ exchanger (NCX) inhibited effectively. We also analyzed the flagellar movement of spermatozoa in artificial seawater (ASW) containing the asterosap at 100 nM ml?1. We found that spermatozoa swam vigorously with more symmetrical flagellar movement in asterosap than in ASW and KB-R7943 significantly inhibited the flagellar movement.DOI: http://dx.doi.org/10.3329/pa.v19i1.17358 Progress. Agric. 19(1): 79 - 88, 2008

Monoamine Oxidases Are Mediators of Endothelial Dysfunction in the Mouse Aorta

Monoamine oxidases (MAOs) generate H(2)O(2) as a by-product of their catalytic cycle. Whether MAOs are mediators of endothelial dysfunction is unknown and was determined here in the angiotensin II and lipopolysaccharide-models of vascular dysfunction in mice. Quantitative real-time polymerase chain reaction revealed that mouse aortas contain enzymes involved in catecholamine generation and MAO-A and MAO-B mRNA. MAO-A and -B proteins could be detected by Western blot not only in mouse aortas but also in human umbilical vein endothelial cells. Ex vivo incubation of mouse aorta with recombinant MAO-A increased H(2)O(2) formation and induced endothelial dysfunction that was attenuated by polyethylene glycol-catalase and MAO inhibitors. In vivo lipopolysaccharide (8 mg/kg IP overnight) or angiotensin II (1 mg/kg per day, 2 weeks, minipump) treatment induced vascular MAO-A and -B expressions and resulted in attenuated endothelium-dependent relaxation of the aorta in response to acetylcholine. MAO inhibitors reduced the lipopolysaccharide- and angiotensin II-induced aortic reactive oxygen species formation by 50% (ferrous oxidation xylenol orange assay) and partially normalized endothelium-dependent relaxation. MAO-A and MAO-B inhibitors had an additive effect; combined application completely restored endothelium-dependent relaxation. To determine how MAO-dependent H(2)O(2) formation induces endothelial dysfunction, cyclic GMP was measured. Histamine stimulation of human umbilical vein endothelial cells to activate endothelial NO synthase resulted in an increase in cyclic GMP, which was almost abrogated by MAO-A exposure. MAO inhibition prevented this effect, suggesting that MAO-induced H(2)O(2) formation is sufficient to attenuate endothelial NO release. Thus, MAO-A and MAO-B are both expressed in the mouse aorta, induced by in vivo lipopolysaccharide and angiotensin II treatment and contribute via the generation of H(2)O(2) to endothelial dysfunction in vascular disease models.

Pro-apoptotic effect of anti-β1-adrenergic receptor antibodies in periodontitis patients

An anti-β1-adrenergic antibody from the sera of periodontitis patients (anti-β1-AR IgG) against the second extracellular loop of the human β1-adrenoceptor (β1-AR) has been shown to cause rat atria apoptosis. The anti-β1-AR IgG binds and activates atria β1-AR, increasing the intracellular calcium concentration, which, in turn, activates caspases-3, -8, and -9. The β1-AR and the post-receptor activation of calcium/calmodulin (CaM) lead to increased inducible nitric oxide synthase (iNOS) activity, with an increase in cyclic GMP (cGMP) accumulation as well as increased JNK phosphorylation and cyclic AMP (cAMP) production. We also observed an apoptotic effect of anti-β1-AR IgG, with increased generation of PGE2. Comparatively, xamoterol, an authentic β1-AR agonist, mimicked the autoantibody effect on rat atria β1-AR apoptosis. Our results suggest that autoantibodies from the sera of periodontitis patients bind and interact with rat atria β1-AR, provoking apoptosis. This implicates a series of modulatory cardiac signaling events that could alter normal heart function and may occur with chronic stimulation of the atria β1-AR, which could lead to heart failure. These results suggest an important link between periodontitis and cardiovascular disease.

Increase of cyclic GMP in blood platelets by biogenic amines--a receptor-mediated effect?

Increase of cyclic GMP in blood platelets by biogenic amines--a receptor-mediated effect?

Role of Presynaptic Purinoceptors and Cyclic AMP on the Noradrenaline Release in Cat Cerebral Arteries

Field electrical stimulation (ES), K+ (50 mM) or ionophore X-537A (0.01 mM) induced tritium release from cat cerebral arteries preincubated with [3H]noradrenaline (NA). Adenosine and AMP (0.5 mM) did not modify tritium release caused by ionophore X-537A, but these agents and ATP (0.5 mM) significantly reduced that elicited by ES and K+; this reduction was antagonized by 1-methyl-3-isobutylxanthine (MIX; 0.05 mM). Inosine (0.5 mM) and the agonist of purinergic A2-receptors, 5'N-ethyl-carboxamide adenosine (NECA; 0.5 mM) had no effect, but the agonist of purinergic A2-receptors L-N6-phenylisopropyl adenosine (L-PIA; 0.1 mM) diminished tritium efflux caused by ES and K+. The adenosine inhibition of ES-induced radioactivity release was not affected by indomethacin (0.05 mM). MIX (0.05 mM) increased tritium release evoked by ES and K+. Agents that increase intracellular cyclic (c)AMP levels, such as dibutyryl cAMP (0.5 mM), the phosphodiesterase inhibitor Ro 20-1724 (0.1 mM), and the activators of adenylate cyclase, forskolin (0.005 mM) and NaF (2 mM) reduced tritium secretion elicited by ES and K+. However, the intracellular increase of cyclic GMP (cGMP) caused by 8-Br-cGMP did not affect this secretion. Dipyridamole (0.05 mM) and the adenosine deaminase inhibitor erythro-9-2-hydroxy-3 nonyl adenosine (EHNA; 0.1 mM) also produced inhibition of tritium secretion elicited by ES and K+. Dipyridamole reduced both the uptake of [3H]NA and [3H]adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

MT2 Receptors Mediate the Inhibitory Effects of Melatonin on Nitric Oxide-Induced Relaxation of Porcine Isolated Coronary Arteries

Previous studies from our laboratory demonstrated that melatonin inhibits nitric oxide (NO)-induced relaxation in porcine coronary arteries. The present study was designed to further characterize the mechanisms underlying this inhibitory effect of melatonin. Western immunoblot studies identified the presence of melatonin type 2 (MT(2)) receptors, but not MT(1) or MT(3) receptors, in porcine coronary arteries. Immunohistochemical analysis revealed that MT(2) receptors colocalized with α-actin in the smooth muscle cell layer. In coronary arterial rings suspended in organ chambers for isometric tension recording, melatonin (10(-7) M) inhibited relaxations induced by the exogenous NO donor sodium nitroprusside (SNP; 10(-9) to 10(-5) M) and by the α(2)-adrenoceptor agonist 5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline (UK14,304; 10(-9) to 10(-5) M), an endothelium-dependent vasodilator. The inhibitory effect of melatonin on SNP- and UK14,304-induced relaxations was abolished in the presence of the selective MT(2) receptor antagonists 4-phenyl-2-propionamidotetralin (4P-PDOT; 10(-7) M) and luzindole (10(-7) M). In contrast to melatonin, the selective MT(3) receptor agonist 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT; 10(-7) M) had no effect on the concentration-response curves to either SNP or UK14,304. Melatonin (10(-7) M) had no effect on coronary artery relaxation induced by 8-bromoguanosine 3',5'-cyclic monophosphate, but it significantly attenuated the increase in intracellular cyclic GMP levels in response to SNP (10(-5) M). This effect of melatonin was abolished in the presence of 4P-PDOT (10(-7) M). Taken together, these data support the view that melatonin acts on MT(2) receptors in coronary vascular smooth muscle cells to inhibit NO-induced increases in cyclic GMP and coronary arterial relaxation, thus demonstrating a novel function for MT(2) receptors in the vasculature.

Dipyridamole inhibits lipopolysaccharide-induced cyclooxygenase-2 and monocyte chemoattractant protein-1 via heme oxygenase-1-mediated reactive oxygen species reduction in rat mesangial cells

Dipyridamole contributes to its beneficial effects on inflammatory responses in many cell types. The anti-inflammatory mechanisms of dipyridamole on glomerular mesangial cells are mostly uncharacterized. In this study, we monitored the influence of dipyridamole on the expression levels of cyclooxygenase-2 (COX-2) and monocyte chemoattractant protein-1 (MCP-1) in rat mesangial cells stimulated with lipopolysaccharide. Dipyridamole was found to inhibit lipopolysaccharide-induced COX-2 and MCP-1 expression, and reduced lipopolysaccharide-induced reactive oxygen species generation in rat mesangial cells. This inhibitory effect of dipyridamole is independent on cyclic AMP and cyclic GMP increase. Tin protoporphyrin IX (SnPP), a heme oxygenase-1(HO-1) inhibitor, blocked the inhibitory effect of dipyridamole on lipopolysaccharide-induced COX-2 and MCP-1 expression. By applying specific inhibitors in rat mesangial cells, ERK1/2 and p38 MAPK signaling pathways were demonstrated to be involved in the lipopolysaccharide-induced inflammatory responses, and were inhibited by SnPP and N-acetylcysteine treatment. Additionally, dipyridamole was also found to upregulate HO-1 in rat mesangial cells. Therefore, our data suggest that dipyridamole inhibits the expression of COX-2 and MCP-1 in lipopolysaccharide-treated rat mesangial cells via HO-1-mediated reactive oxygen species reduction.

PHOSPHODIESTERASE-5 INHIBITORS USE IN PATIENTS WITH ERECTILE DYSFUNCTION AND CARDIOVASCULAR DISEASE IN CLINICAL PRACTICE

About 150 million men worldwide and about 50% of men aged 40-88 y.o. in outpatient practice suffer from erectile dysfunction (ED). There is a linear relation between the age and ED rate. The main reason of ED in the majority of men (about 80% of patients) is cardiovascular diseases (atherosclerosis, hypertension, diabetes mellitus), as well as certain risk factors (smoking, alcohol abuse, physical inactivity etc.). The problem of ED in cardiac outpatients and modern pharmacotherapy is discussed. The phosphodiesterase-5 (PDE5) inhibitors increase the relaxing effect of nitric oxide and increase cyclic GMP levels during sexual arousal. It results in increase of cavernosum blood flow, contributing to the physiological erection. Three PDE5 inhibitors (sildenafil, tadalafil, vardenafil) are used in clinical practice nowadays.

Inhibition of superoxide anion generation by CHS-111 via blockade of the p21-activated kinase, protein kinase B/Akt and protein kinase C signaling pathways in rat neutrophils

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111) inhibited superoxide anion (O 2 − ) generation, which was not mediated by scavenging the generated O 2 − or by a cytotoxic effect, and attenuated migration. CHS-111 had no effect on the arachidonic acid-induced NADPH oxidase activation or the GTPγS-stimulated Rac2 membrane translocation in cell-free systems, whereas it effectively attenuated the membrane recruitment of p40 phox, p47 phox and p67 phox, phosphorylation of Ser residues in p47 phox, association between p47 phox and p22 phox, and Rac activation in fMLP-stimulated neutrophils. Moreover, the phosphorylation and membrane recruitment of p21-activated kinase (PAK), PAK kinase activity and the interaction of PAK with p47 phox were inhibited by CHS-111. CHS-111 effectively reduced Akt kinase activity and the association between Akt and p47 phox, moderately inhibited the membrane recruitment of Akt and phospho-PDK1, and slightly attenuated Akt (Thr308) phosphorylation, whereas it had no effect on Akt (Ser473) phosphorylation or p110γ membrane translocation. The membrane recruitment of protein kinase C (PKC)-α, -βI, -βII, -δ and -ζ, PKC phosphorylation and PKC kinase activity was attenuated by CHS-111, whereas CHS-111 did not affect the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or downstream MAPK-activated protein kinase-2. Higher concentrations of CHS-111 were required to decrease fMLP-stimulated intracellular free Ca 2+ concentration ([Ca 2+] i) elevation in the presence but not in the absence of extracellular Ca 2+, and to reduce cellular cyclic AMP but slightly increase cyclic GMP levels. Taken together, these results suggest that CHS-111 inhibits fMLP-stimulated O 2 − generation in rat neutrophils through the blockade of PAK, Akt and PKC signaling pathways.

Platelet aggregation is inhibited by a nitric oxide-like factor released from human neutrophils in vitro

Thrombin-induced platelet aggregation was inhibited in vitro by washed human neutrophils. Aggregation was inhibited in a neutrophil concentration dependent manner but glutaraldehyde fixed neutrophils had no significant effect on platelet aggregation. The neutrophil-derived inhibitory factor had the pharmacological profile of nitric oxide. Its action was potentiated by both superoxide dismutase and M&B22, 948, a selective cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase inhibitor. Haemoglobin lessened this inhibitory action of neutrophils. L-Arginine, the substrate for nitric oxide formation, enhanced inhibition, whereas, L-canavanine, a structural analogue of L-arginine, prevented it. Nitric oxide release by neutrophils antagonized platelet ATP secretion and thromboxane B2 release. Inhibition was mediated by nitric oxide activation of guanylate cyclase with a subsequent rise in cyclic GMP. When neutrophils were stimulated with formyl-met-leu-phe, there was a further increase in platelet cyclic GMP. This was enhanced by superoxide dismutase, but lessened by haemoglobin. Leukotriene B4 stimulation of neutrophils promoted inhibition of platelet aggregation. Leukotriene B4 alone had no direct effect on thrombin-induced aggregation of platelets. Platelets, when incubated with neutrophils and stimulated with calcium ionophore A23187, increased leukotriene B4 production by neutrophils in a platelet concentration dependent manner. Platelets alone were unable to release leukotriene B4. The action of platelets in haemostasis is modified as they come into contact with neutrophils. This may be an important physiological mechanism.

Functional Interaction of a β-Adrenergic Agonist and Cyclic GMP Phosphodiesterase Inhibitor in Control and Hypertrophic Cardiomyocytes

This study tested the hypothesis that the positive inotropic effect of β-adrenoceptor stimulation would be inhibited by increases in cyclic GMP in control cardiomyocytes and that this response would be modified in hypertrophic cardiomyocytes. Cell functional data as well as GMP and cyclic AMP data were collected from 7 control and 7 1K1C (one-kidney-one-clip) renal hypertensive hypertrophic rabbits. Using isolated control and IKIC ventricular myocytes, data were obtained at baseline and after treatment with the β-adrenoceptor agonist isoproterenol (10^{–8, –6} mol/l) or the cyclic GMP-phosphodiesterase inhibitor zaprinast (10^–5 mol/l) followed by isoproterenol (10^{–8, –6} mol/l). We found that in control rabbits, isoproterenol (10^–6 mol/l) increased percent shortening (4.8 ± 0.2 to 6.4 ± 0.3%) and cyclic AMP (2.3 ± 0.3 to 5.0 ± 0.7 pmol/10⁵ cells). Zaprinast 10^–5 mol/l increased cyclic GMP (150 ± 20 to 209 ± 14 fmol/10⁵ cells) and decreased percent shortening (6.2 ± 0.4 to 5.2 ± 0.3). Zaprinast 10^–5 mol/l prevented the functional response to isoproterenol in control (5.2 ± 0.3 to 4.7 ± 0.3), without changing cyclic AMP levels. In 1K1C rabbits, isoproterenol (10^–6 mol/l) increased cyclic AMP (4.9 ± 0.8 to 7.6 ± 1.4 pmol/10⁵ cells) without changing function. Zaprinast 10^–5 mol/l increased cyclic GMP (182 ± 23 to 233 ± 24 fmol/10⁵ cells) and decreased percent shortening (6.6 ± 0.9 to 4.7 ± 0.5), but did not alter the lack of effect of isoproterenol in 1K1C. In control cardiomyocytes, cyclic GMP blunted the isoproterenol contraction response without changing cyclic AMP levels, but isoproterenol’s functional effect was not seen in 1K1C cardiomyocytes.

Nitric oxide from the laterodorsal tegmental neurons: Its possible retrograde modulation on norepinephrine release from the axon terminal of the locus coeruleus neurons

Nitric oxide released from the cholinergic neurons in the pons may play important roles in sleep–wake regulation. However, there are few reports demonstrating the mechanisms of nitric oxide release in the cholinergic neurons in the pons. The present study investigated the effects of drug delivery of N-methyl- d-aspartic acid on nitric oxide and the neurotransmitters released in the laterodorsal tegmental nucleus (LDT), one of the major cholinergic cell groups in the pons, in rats by in vivo microdialysis with a view to clarifying nitric oxide functions in the cholinergic system. The application of N-methyl- d-aspartic acid (1mM) into the LDT induced a significant increase in NO 2and NO 3 for 40 min ( P<0.001). Furthermore the same dose of N-methyl- d-aspartic acid induced a significant increase in cyclic GMP for 30 min ( P<0.05), as well as in acetylcholine ( P<0.001) and norepinephrine for 15 min ( P<0.001). 3-(4-Morpholinyl)-sydonone imine hydrochloride (a nitric oxide donor, 5mM) also induced significant increase in norepinephrine ( P<0.05). Pretreatment with 1mM 2-amino-5-phosphonopentanoic acid (an antagonist of N-methyl- d-aspartic acid receptor) prevented the N-methyl- d-aspartic acid-induced increase in cyclic GMP ( P<0.01), acetylcholine and norepinephrine ( P<0.01), while that with 1mM N G-nitro- l-arginine (an inhibitor of nitric oxide synthase) prevented the increase in cyclic GMP ( P<0.01) and norepinephrine ( P<0.01) but not in acetylcholine. These results suggested that nitric oxide release in the LDT induced by activation of the N-methyl- d-aspartic acid receptor on the cholinergic neurons of the LDT, then through the cyclic GMP system, facilitates norepinephrine release from the terminals of noradrenergic neurons in the locus coeruleus. Based on these findings, we propose a possible role of nitric oxide in the LDT is as a retrograde regulator of norepinephrine release from the locus coeruleus.

Peripheral regulatory mechanisms in erection

The most important pathway underlying the penile erection is the nonadrenergic/noncholinergic signalling, which through the release of nitric oxide (NO), leads to an intracellular increase of cyclic GMP (cGMP), the main secondary messenger mediating tumescence in the penis. Interestingly, both cGMP formation and degradation are affected by testosterone (T). In fact, beyond the well-known role of T in regulating sexual desire and NO release, recent experimental evidences from our group showed that T also regulates the expression of phosphodiesterase type 5 (PDE5), the hydrolytic enzyme involved in cGMP breakdown. This antithetic role of T seems to be the main way through which the peripheral hormonal regulation of penile erections occurs, allowing an important synchronization between erectile processes and sexual desire. Hence, erections are still possible in hypogonadal conditions where a decreased cGMP formation, because of impaired NO production, is counterbalanced by a reduced cGMP hydrolysis. The purpose of this review is to describe evidences about the peripheral role of T in regulating penile erection and to justify the importance to test T plasma levels in those patients with erectile dysfunction who do not respond to PDE5 inhibitors.

Role of the Adventitia in the Cyclic GMP-Mediated Relaxant Effect of Nω-Hydroxy-L-Arginine in Rat Aorta

N^ω-hydroxy-L-arginine (L-NOHA), the stable intermediate of the nitric oxide synthase (NOS)-catalyzed reaction, can induce NO/cyclic GMP-dependent relaxation in the rat aorta, in an endothelium- and NOS-independent manner. In this study, the role of the adventitia in the endothelium-independent effect of L-NOHA was investigated. Despite a decrease in norepinephrine (NE)-induced precontraction, adventitia removal in the rat aorta did not markedly alter the relaxant effect of forskolin, S-nitroso-N-acetylpenicillamine or glyceryl trinitrate. In contrast, both inhibition of NE-induced contraction and relaxation of NE-precontracted rings produced by L-NOHA were diminished in the absence of adventitia. Moreover, exposure to L-NOHA significantly enhanced the cyclic GMP level in the media of the aorta with, but not without adventitia. These findings demonstrate the role of the adventitia in the L-NOHA-induced decrease in tone and increase in cyclic GMP in the endothelium-denuded rat aorta. They suggest that NO or an NO-related compound formed from L-NOHA in the adventitia may produce paracrine effects.

Chronic hyperammonemia in vivo impairs long‐term potentiation in hippocampus by altering activation of cyclic GMP‐dependent‐protein kinase and of phosphodiesterase 5

Long-term potentiation (LTP) is impaired in the CA1 area of hippocampal slices from rats with chronic moderate hyperammonemia. We studied the mechanisms by which hyperammonemia in vivo impairs LTP. This process requires sequential activation of soluble guanylate cyclase, cyclic GMP-dependent protein kinase (PKG) and cyclic GMP-degrading phosphodiesterase. Application of the tetanus induced a rapid increase of cyclic GMP in slices from control or hyperammonemic rats, which is followed in control slices by a sustained decrease in cyclic GMP due to sustained activation of cyclic GMP-degrading phosphodiesterase, which in turn is due to sustained activation of PKG. In slices from rats with chronic hyperammonemia tetanus-induced decrease in cyclic GMP was delayed and transient due to lower and transient activation of PKG and of the phosphodiesterase. Hyperammonemia-induced impairment of LTP may be involved in the alterations of cognitive function in patients with hepatic encephalopathy.