Formation Of Cyclic Guanosine Monophosphate Research Articles

Multiple-Ascending-Dose Study of the Soluble Guanylate Cyclase Stimulator, IW-1701, in Healthy Subjects

▪Background: Nitric oxide (NO), a key regulator of vascular health, modulates smooth muscle tone and blood pressure, inhibits vascular smooth muscle proliferation, and suppresses inflammation and platelet activation. These protective functions are mediated by soluble guanylate cyclase (sGC), a signaling enzyme that catalyzes the formation of cyclic guanosine monophosphate (cGMP) in response to NO binding. The compromised vascular health characteristic of the hemoglobinopathies may in part be due to deficient NO bioavailability and inadequate sGC-cGMP signaling. IW-1701 is an orally available sGC stimulator that binds to sGC and enhances NO-sGC-cGMP signaling.Methods: A Phase 1b placebo-controlled, randomized, multiple-ascending-dose study was conducted to assess safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (blood pressure, heart rate, platelet function, and plasma cGMP) of IW-1701 in healthy subjects. Five successive cohorts of 11 subjects each (8 active and 3 placebo) were randomized. The first 3 cohorts received 7 days of repeated once-daily (QD) doses of 1-, 2- or 5-mg IW-1701 respectively, and the last 2 cohorts received 7 days of lead-in QD doses of 3 mg followed by 7 days of up-titrated QD doses of 5 or 6 mg, respectively.Results: IW-1701 doses ranging from 1 to 6 mg were tolerated and no safety concerns were identified. There were no serious adverse events (SAEs), severe adverse events (AEs), or withdrawals due to AEs. All AEs were mild or moderate, most resolved on the day of onset. Among the 40 subjects who received IW-1701 for 7 consecutive days, the most common AEs were tachycardia and headache (each reported in 7 subjects), and hypotension and nausea (each reported in 3 subjects). Among the 16 subjects who received an up-titrated dose of IW-1701 for 7 additional days, headache was the only AE reported in >1 subject (2 subjects). IW-1701 was rapidly absorbed, with median Tmax achieved from 2 to 4 hours postdose. Systemic exposure to IW-1701 was less than dose proportional after a single dose (Cmax slope, 0.701 [90% CI, 0.578-0.824] and AUCtau slope, 0.833 [90% CI, 0.726-0.939]) and was dose proportional after 7 days of QD dosing (Cmax slope, 1.01 [90% CI, 0.873-1.14] and AUCtau slope, 1.03 [90% CI, 0.892-1.17]). Intersubject variability in PK was low to moderate (AUCtau %CV range, 10.6-39.1%). The accumulation ratio (based on RAUC) ranged from 1.95 to 2.63 across doses of 1-, 2-, 3-, and 5-mg IW-1701 after 7 days of QD dosing. Mean CL/F (apparent total body clearance) and Vz/F (apparent volume of distribution) ranged from 1080 to 1520 mL/h and 49.4 to 58.9 L, respectively. Mean terminal t1/2 was 30 hours, which is consistent with a QD dosing regimen. After 7 days of QD dosing, least squares mean changes from baseline in 24-hour ambulatory systolic blood pressure (±SE) (in cohort order) were -1.17±1.24 (placebo), -2.98±1.80 (1 mg), -5.37±1.71 (2 mg), -6.67±1.69 (5 mg), -6.55±1.95 (3 mg), and -4.98±1.68 mmHg (3 mg). After 14 days of QD dosing in the 2 cohorts that received up-titrated doses, the least squares mean changes from baseline in 24-hour ambulatory systolic blood pressure (±SE) were -1.51±2.42 (placebo), -6.85±2.03 (5 mg), and -8.64±2.08 mmHg (6 mg). Plasma cGMP levels were highly variable; in general, mean plasma cGMP levels were increased from baseline at dose levels ≥3 mg IW-1701. There were no clinically meaningful changes from baseline in platelet function assessment by PFA-100® at any dose level.Conclusion: IW-1701 was tolerated in healthy subjects with only mild and moderate AEs. Common AEs were consistent with the effects of sGC stimulation. Lead-in dosing with a lower dose (3 mg) appeared to improve the tolerability of higher doses (5 and 6 mg). The PK of repeat IW-1701 dosing was dose proportional with low-to-moderate intersubject variability and half-life consistent with QD dosing. Pharmacodynamic results, including blood pressure and plasma cGMP levels, demonstrated target engagement. Clinical evaluation of IW-1701 in diseases associated with NO deficiency, including sickle cell disease and achalasia, are planned or ongoing.– The authors gratefully acknowledge the early contributions of Bernard J. Lavins. – DisclosuresMittleman:Ironwood Pharmaceuticals: Employment, Equity Ownership. Wilson:Ironwood Pharmaceuticals: Employment, Equity Ownership. Sykes:Ironwood Pharmaceuticals: Employment, Equity Ownership. Mihova:Ironwood Pharmaceuticals: Employment, Equity Ownership. Chickering:Ironwood Pharmaceuticals: Employment, Equity Ownership. Hall:Ironwood Pharmaceuticals: Employment, Equity Ownership. Milne:Ironwood Pharmaceuticals: Employment, Equity Ownership. Currie:Ironwood Pharmaceuticals: Employment, Equity Ownership. Chien:Ironwood Pharmaceuticals: Employment, Equity Ownership.

The β subunit of soluble guanylyl cyclase GUCY1B3 exerts cardioprotective effects against ischemic injury via the PKCε/Akt pathway

The soluble form of guanylyl cyclase (sGC) is the main receptor for the signaling agent nitric oxide (NO), which regulates cardiomyocyte contractile function and attenuates cardiomyocyte hypertrophy. sGC catalyzes the formation of cyclic guanosine monophosphate (cGMP), a regulator of vascular tone, and cardiac NO-sGC-cGMP signaling modulates cardiac stress responses, including ischemia and reperfusion (IR) injury. Here, we investigated the role of GUCY1B3 (the β subunit of sGC) in cardiomyocyte IR injury and myocardial infarction (MI) in vitro and in vivo. GUCY1B3 was upregulated in neonatal rat ventricular myocytes in response to IR injury, and GUCY1B3 overexpression restored IR-induced cell death and apoptosis. Treatment with specific inhibitors of PKCδ, PKCε, and Akt suggested that the protective effects of GUCY1B3 were mediated by PKCε/Akt signaling. In a mouse model of coronary artery ligation-induced MI, GUCY1B3 silencing aggravated MI-induced cardiac dysfunction and increased infarct size and exacerbated cardiomyocyte apoptosis in association with the inactivation of PKCε and Akt. Our results suggest that GUCY1B3 exerts cardioprotective effects through the modulation of the PKCε/Akt activity and identify a potential mechanism involved in NO-sGC-cGMP signaling in the heart.

Renoguanylin stimulates apical CFTR translocation and decreases HCO3- secretion through PKA activity in the Gulf toadfish (Opsanus beta).

The guanylin peptides - guanylin, uroguanylin and renoguanylin (RGN) - are endogenously produced hormones in teleost fish enterocytes that are activators of guanylyl cyclase-C (GC-C) and are potent modulators of intestinal physiology, particularly in seawater teleosts. Most notably, they reverse normal net ion-absorbing mechanisms that are vital to water absorption, an important process for seawater teleost survival. The role of guanylin-peptide stimulation of the intestine remains unclear, but it is hypothesized to facilitate the removal of solids from the intestine by providing fluid to enable their removal by peristalsis. The present study used one member of this group of peptides - RGN - to provide evidence for the prominent role that protein kinase A (PKA) plays in mediating the effects of guanylin-peptide stimulation in the posterior intestine of the Gulf toadfish (Opsanus beta). Protein kinase G was found to not mediate the intracellular effects of RGN, despite previous evidence showing that GC-C activation leads to higher cyclic guanosine monophosphate formation. RGN reversed the absorptive short-circuit current and increased conductance in the Gulf toadfish intestine. These effects are correlated to increased trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel to the apical membrane, which is negated by PKA inhibition. Moreover, RGN decreased HCO3- secretion, likely by limiting apical HCO3-/Cl- exchange (possibly by reducing SLC26a6 activity), a reduction that was enhanced by PKA inhibition. RGN seems to alter PKA activity in the posterior intestine to recruit CFTR to the apical membrane and reduce HCO3- secretion.

Abstract P493: Multiple-ascending-dose Study of IW-1973, a Soluble Guanylate Cyclase Stimulator

Background: Soluble guanylate cyclase (sGC), an enzyme that catalyzes the formation of cyclic guanosine monophosphate (cGMP) in response to nitric oxide (NO) binding, is a key mediator of local blood flow, inflammation, and fibrosis. IW-1973 is an orally available sGC stimulator that enhances NOsGC-cGMP signaling and reduces blood pressure (BP) in animal models of hypertension, both alone and in combination with other antihypertensive agents. Methods: A Phase 1b placebo-controlled, randomized, multiple-ascending-dose study was conducted to assess safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (BP, heart rate, platelet function, and plasma biomarkers) of IW-1973 in healthy subjects. Four successive cohorts of 11 subjects each (8 active and 3 placebo) were enrolled. Subjects received a starting dose once daily (QD) for 14 days followed by an up-titrated QD dose for 7 days. Results: IW-1973 doses ranging from 15 to 40 mg were tolerated. There were no serious adverse events (AEs), severe AEs, or discontinuations due to AEs. Among the 32 subjects who received IW-1973, the most common AEs (which occurred mainly at doses ≥30 mg) were headache (15 subjects), dizziness/postural dizziness (6) and orthostatic hypotension/BP decreased (4). AEs tended to resolve with continued dosing. The AE profile of higher doses was not clearly improved by up-titration from a lower starting dose. PK was dose proportional, both for C max and AUC, with a T max of 2-4 hours and an effective half-life of 24-37 hours. After 14 days of treatment, least squares mean change from baseline in 24-h ambulatory systolic BP (±SE) was -0.85±1.32 (placebo), -7.29±1.62 (15 mg), -3.27±1.61 (20 mg), -6.75±1.62 (30 mg), and -5.23±1.61 mmHg (≤40 mg, ≤0.5 mg/kg). After 21 days, the change from baseline was -4.81±1.19 (placebo), -8.21±1.46 (15 to 30 mg), -6.29±1.45 (20 to 40 mg), -9.05±1.56 (30 to 40 mg), and -6.58±1.45 mmHg (≤40 to ≥40 mg). IW-1973 produced a dose-related increase in plasma cGMP indicating target engagement. There was no clear effect of IW-1973 administration on platelet function as assessed by the PFA-100 ® system. Conclusion: Further clinical investigation of IW-1973 is ongoing or planned in multiple indications, including resistant hypertension.

Experimental and clinical aspects of melatonin and clock genes in diabetes.

The pineal hormone melatonin influences insulin secretion, as well as glucagon and somatostatin secretion, both invivo and invitro. These effects are mediated by two specific, high-affinity, seven transmembrane, pertussis toxin-sensitive, Gi-protein-coupled melatonin receptors, MT1 and MT2. Both isoforms are expressed in the β-cells, α-cells as well as δ-cells of the pancreatic islets of Langerhans and are involved in the modulation of insulin secretion, leading to inhibition of the adenylate cyclase-dependent cyclic adenosine monophosphate as well as cyclic guanosine monophosphate formation in pancreatic β-cells by inhibiting the soluble guanylate cyclase, probably via MT2 receptors. In this way, melatonin also likely inhibits insulin secretion, whereas using the inositol triphosphate pathway after previous blocking of Gi-proteins by pertussis toxin, melatonin increases insulin secretion. Desynchrony of receptor signaling may lead to the development of type 2 diabetes. This notion has recently been supported by genomewide association studies pinpointing variances of the MT2 receptor as a risk factor for this rapidly spreading metabolic disturbance. As melatonin is secreted in a clearly diurnal fashion, it is safe to assume that it also has a diurnal impact on the blood-glucose-regulating function of the islet. Observations of the circadian expression of clock genes (Clock, Bmal1, Per1,2,3, and Cry1,2) in pancreatic islets, as well as in INS1 rat insulinoma cells, may indicate that circadian rhythms are generated in the β-cells themselves. The circadian secretion of insulin from pancreatic islets is clock-driven. Disruption of circadian rhythms and clock function leads to metabolic disturbances, for example, type 2 diabetes. The study of melatonin-insulin interactions in diabetic rat models has revealed an inverse relationship between these two hormones. Both type 2 diabetic rats and patients exhibit decreased melatonin levels and slightly increased insulin levels, whereas type 1 diabetic rats show extremely reduced levels or the absence of insulin, but statistically significant increases in melatonin levels. Briefly, an increase in melatonin levels leads to a decrease in stimulated insulin secretion and vice versa. Melatonin levels in blood plasma, as well as the activity of the key enzyme of melatonin synthesis, AA-NAT (arylalkylamine-N-acetyltransferase) in pineal, are lower in type 2 diabetic rats compared to controls. In contrast, melatonin and pineal AA-NAT mRNA are increased and insulin receptor mRNA is decreased in type 1 diabetic rats, which also indicates a close relationship between insulin and melatonin. As an explanation, it was hypothesized that catecholamines, which reduce insulin levels and stimulate melatonin synthesis, control insulin-melatonin interactions. This conviction stems from the observation that catecholamines are increased in type 1 but are diminished in type 2 diabetes. In this context, another important line of inquiry involves the fact that melatonin protects β-cells against functional overcharge and, consequently, hinders the development of type 2 diabetes.

Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes

Nebivolol is a third-generation β-adrenergic receptor (β-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-γ coactivator-1α (PGC-1α), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-l-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and β3-AR blocker SR59230A markedly attenuated PGC-1α, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of β3-AR receptors.

Preserved pulmonary vasodilative properties of aerosolized brain natriuretic peptide

Introduction Inhalation of vasoactive substances is an effective treatment of pulmonary hypertension. The B-type natriuretic peptide (BNP) leads to relaxation of smooth muscle cells, caused by an increased formation of cyclic guanosine monophosphate (cGMP). The biologic activity of BNP using an inhalative approach has not been addressed. Methods In order to assess the vasorelaxing capacity of exogenous BNP in the isolated ventilated and buffer perfused rabbit lung model, a stable pulmonary vasoconstriction was established by either the application of endothelin-1 or the thromboxane A 2 mimetic U46619. This was followed by an intravascular or aerosol application of BNP. CGMP was measured in the recirculating buffer fluid using a radioimmunoassay technique. Results During a stable plateau of U46619 induced pulmonary vasoconstriction (mean pulmonary artery pressure, PAP 25.5 ± 0.23 mmHg), the intravascular administration of BNP induced a rapid vasodilation (mean PAP 18.13 ± 0.95 mmHg, p < 0.001). This vasodilation was dose dependent and was paralleled by a 6-fold increase of cGMP. When BNP was aerosolized, pulmonary vasoconstriction was also significantly alleviated in the U46619 model (mean PAP 22 ± 2.1 mmHg) and during endothelin-1 induced vasoconstriction (mean PAP 17.1 ± 2.47 mmHg). Correspondingly, inhalation caused a significant augmentation of cGMP levels was. Conclusion The vasodilative capability of BNP as an indicator of the biologic activity of this peptide is preserved during its aerosolization. Presumably these vascular actions are caused at least in part by an increased availability of cGMP.

Nitric oxide signaling in vascular biology

Nitric oxide (NO) research has expanded rapidly in the past 20 years, and the role of NO in physiology and pathology has been extensively studied. This review focuses on the pathways of NO synthesis and metabolism in vascular biological systems. Healthy vascular homeostasis is dependent on the integrity of the endothelium, which is a very large dynamic autocrine and paracrine organ with vasodilator, anti-inflammatory, and antithrombotic properties. The importance and relevance of NO signaling is stressed in this review. The potential role of nitrotyrosine formation with vascular pathological conditions is discussed. The use of pharmacologic, biochemical, and molecular biological approaches to characterize, purify, and reconstitute these regulatory pathways should lead to the development of new therapies for various pathological conditions that are characterized by an insufficient production of NO. With more than 77,000 publications in the field of NO signaling, this brief review can only focus on some aspects of the field as it applies to vascular biology. Many molecular targets have been identified for drug development dealing with NO and cyclic guanosine monophosphate formation, metabolism, and function. Many agents have been identified that are in pre-clinical evaluation or in clinical trials. Certainly, many should prove to be important therapeutic additions during the next decade.

Nitric oxide directly inhibits ghrelin-activated neurons of the arcuate nucleus

The hypothalamic arcuate nucleus (Arc) is a target site for signals regulating energy homeostasis. The orexigenic hormone ghrelin directly activates neurons of the medial arcuate nucleus (ArcM) in rats. Nitric oxide (NO) is a neuromodulator implicated in the control of food intake and body weight. NO is produced by nitric oxide synthase (NOS) and induces the formation of cyclic guanosine monophosphate (cGMP) via a stimulation of soluble guanylate cyclase (sGC). Both enzymes NOS and sGC have been identified in the Arc. Using extracellular recordings we characterized the effects of NO signaling on ArcM neurons and their co-sensitivity to ghrelin. The artificial NO donor sodium nitroprusside (10 − 4 M) reversibly inhibited 91% of all ArcM neurons by a direct postsynaptic mechanism. 52% of ArcM neurons were excited by ghrelin. In all but one of these neurons SNP caused inhibitory responses. The SNP-induced inhibitions were mediated by cGMP since they were blocked by the specific sGC inhibitor ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, 10 − 4 M). Furthermore, the membrane permeating cGMP analogue 8-Br-cGMP (10 − 4 M) mimicked the inhibitory responses of SNP. In immunohistological in vitro studies SNP induced a cGMP formation, which could also be blocked by ODQ. The current studies demonstrate that NO/cGMP signaling inhibits a large population of ArcM neurons including ghrelin-excited cells. Since an activation of the latter neurons is regarded as a correlate of negative energy balance, NO may represent an anorectic neuromodulator in the Arc and/or restrain the action of signals promoting energy intake. NO signaling in the Arc is also induced following inflammation suggesting a possible role of Arc-intrinsic NO in disease-related anorexia.

Mitochondria and NO for Longer Life

Calorie restriction extends life span in organisms ranging from yeast to mammals. Nisoli et al. find that when mice are subjected to calorie restriction, endothelial nitric oxide synthase (eNOS) expression and 3′,5′-cyclic guanosine monophosphate formation are increased. This change is accompanied by mitochondrial biogenesis, increased oxygen consumption, adenosine triphosphate production, and expression of sirtuin 1, a protein previously implicated in mediating the effects of calorie restriction on life span. These effects are strongly attenuated in mutant mice lacking eNOS. Thus, NO may play a role in the processes induced by calorie restriction and in life-span expansion in mammals. E. Nisoli, C. Tonello, A. Cardile, V. Cozzi, R. Bracale, L. Tedesco, S. Falcone, A. Valerio, O. Cantoni, E. Clementi, S. Moncada, M. O. Carruba, Calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS. Science 310 , 314-317 (2005). [Abstract] [Full Text]

Calorie Restriction Promotes Mitochondrial Biogenesis by Inducing the Expression of eNOS

Calorie restriction extends life span in organisms ranging from yeast to mammals. Here, we report that calorie restriction for either 3 or 12 months induced endothelial nitric oxide synthase (eNOS) expression and 3',5'-cyclic guanosine monophosphate formation in various tissues of male mice. This was accompanied by mitochondrial biogenesis, with increased oxygen consumption and adenosine triphosphate production, and an enhanced expression of sirtuin 1. These effects were strongly attenuated in eNOS null-mutant mice. Thus, nitric oxide plays a fundamental role in the processes induced by calorie restriction and may be involved in the extension of life span in mammals.

Expression of soluble guanylyl cyclase in rat cerebral cortex during postnatal development

Soluble guanylyl cyclase (sGC), the principle "receptor" for nitric oxide (NO), catalyzes the formation of cyclic guanosine monophosphate (cGMP), an intracellular second messenger. Studies in invertebrates have shown that the NO/cGMP pathway is involved in several aspects of neural development, including neuronal migration, dendritic and axonal outgrowth, and synaptogenesis. In vitro studies suggest a developmental role also in mammals. To investigate whether the NO/cGMP pathway might mediate these processes in vivo, we performed immunohistochemistry for sGC on sections from postnatal rat cerebral cortex. Early in postnatal development, migrating neurons in the cortical plate were immunonegative, whereas neurons deeper in the cortex that had completed migration were immunopositive. At the subcellular level, sGC preferentially stained dendrites rather than axons, but, at postnatal day 1 (PND1), sGC was found in a large fraction of axonal growth cones, especially those oriented toward the pial surface. At PND10-20 (the period of maximal synaptogenesis), sGC immunostaining was located mainly in dendritic shafts and was only occasionally associated with spines or axon terminals. These results support a role for the NO/cGMP pathway in dendritic development but argue against a major role in neuronal migration and synaptogenesis.

Red wine polyphenols‐induced, endothelium‐dependent NO‐mediated relaxation is due to the redox‐sensitive PI3‐kinase/Akt‐dependent phosphorylation of endothelial NO‐synthase in the isolated porcine coronary artery

An enhanced endothelial formation of nitric oxide (NO) by red wine polyphenolic compounds (RWPs) has been involved in the protective effect of chronic intake of red wine on coronary diseases. However, the mechanism underlying the activation of endothelial NO synthase (eNOS) remains unclear. In the presence of indomethacin and charybdotoxin plus apamin to prevent the formation of prostanoids and endothelium-derived hyperpolarizing factor, respectively, RWPs caused pronounced endothelium-dependent relaxations in porcine coronary arteries. Relaxations to RWPs were abolished by N(omega)-nitro-L-arginine (L-NA, a competitive inhibitor of NO synthase) and the membrane permeant analog of superoxide dismutase (SOD), MnTMPyP, and reduced by polyethylene glycol-SOD (PEG-SOD), PEG-catalase and inhibitors of PI3-kinase (wortmannin and LY294002). RWPs caused the L-NA-sensitive formation of NO, as assessed by electron spin resonance spectroscopy and the formation of cyclic guanosine monophosphate in coronary artery endothelial cells; these responses were reduced by MnTMPyP, PEG-catalase, and inhibitors of PI3-kinase. RWPs caused the sustained phosphorylation of Akt and eNOS at Ser1177 in endothelial cells, which were abolished by MnTMPyP and inhibitors of PI3-kinase. These data demonstrate that RWPs induce the redox-sensitive activation of the PI3-kinase/Akt pathway in endothelial cells which, in turn, causes phosphorylation of eNOS, resulting in an increased formation of NO.

Low micromolar concentrations of copper augment the impairment of endothelium-dependent relaxation of aortae from diabetic rabbits

Both diabetes mellitus (DM) and elevated plasma copper concentrations are risk factors for cardiovascular disease (CVD). DM is associated with impaired endothelial nitric oxide (NO) and with excess superoxide (O 2 .−) formation. Copper is also elevated in DM and is also associated with the generation of O 2 .−. To explore possible interactions between DM and copper, the effect of exogenous copper (CuCl 2) on endothelium-dependent relaxation and cyclic guanosine monophosphate (GMP) formation was investigated in aortae from diabetic rabbits. Rabbits were rendered diabetic by intravenous injection of alloxan. Six months after induction of DM, the aortae were excised, cut into rings, and mounted in an organ bath for isometric measurement of acetylcholine (Ach)-evoked relaxation in rings precontracted with phenylephrine (PE). In parallel studies, cyclic (c)GMP formation by aortic rings following stimulation with Ach, calcium ionophore A23187 (A23187) and sodium nitroprusside (SNP) was assessed using radiommunoassay. The effect of copper on these parameters was then studied using the same methods. Ach-evoked relaxation and Ach- and A23187-evoked cGMP formation were significantly impaired in aortae from diabetic rabbits compared to controls, effects that were reversed with superoxide dimutase (SOD) and catalase (CAT). In contrast, there were no significant differences in SNP-stimulated relaxation or cGMP formation in aortae from diabetic rabbits compared to controls. Copper (1 to 10 μmol/L) promoted a further significant inhibition of Ach-stimulated relaxation in aortae from diabetic but not control rabbits. This reduction by copper was again reversed by SOD and CAT. We conclude that copper augments the reduction of NO bioavailability, which is already impaired in aortae from diabetic rabbits due to excess production of O 2 .− and H 2O 2. These results indicate that patients with DM may be susceptible to copper-mediated vasculopathy at much lower concentrations than those that promote vasculopathy in nondiabetic patients.

Regulatory role of nitric oxide over extracellular taurine in the hippocampus of freely moving rats

We have studied the effects of drugs which manipulate nitric oxide (NO) levels as well the effect of N-methyl- d-aspartate (NMDA) infusion on extracellular taurine in rat hippocampus using in vivo microdialysis. The NO donor S-nitroso- N-acetylpenicillamine (SNAP) increased dialysate taurine in a concentration-dependent manner, and this effect was blocked by the inhibitor of soluble guanylate cyclase1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ). NMDA (100 μM) increased hippocampal taurine release, an effect that was reversed by the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5; 10 μM). The non-selective nitric oxide synthase (NOS) inhibitor N-nitro- l-arginine methyl ester ( l-NAME; 100 μM and 1.0 mM) increased extracellular taurine in a concentration-dependent manner while 7-nitroindazole (7-NI), a relatively selective neuronal NOS (nNOS) inhibitor, at the same concentrations decreased extracellular taurine. l-NAME (1.0 mM) infused prior to NMDA did not alter the effect of NMDA on extracellular taurine having an effect essentially identical to that seen with l-NAME infused alone. In contrast, when 7-NI was infused for 30 min prior to NMDA, taurine levels were no longer increased above basal. This suggests to us that taurine efflux is mediated by two different mechanisms: an NMDA-evoked, 7-NI-sensitive pathway which may be dependent on cyclic guanosine monophosphate formation, and an l-NAME-modulated mechanism which presumably involves other members of the NOS group of enzymes than nNOS alone.

The role of protein nitration in the inhibition of platelet activation by peroxynitrite

Peroxynitrite at low concentrations (3–10 μM) inhibited agonist-induced platelet aggregation by a mechanism not dependent on the formation of cyclic guanosine monophosphate. Platelets recovered completely from peroxynitrite-induced inhibition within 30 min. Peroxynitrite induced nitration of cytosolic proteins, but this diminished to near basal levels within 60 min of exposure to the oxidant. During this period there was a reduction in tyrosine phosphorylation of specific proteins such as syk, but this was not due to direct nitration of these same proteins. The inhibition of phosphorylation was reversible with platelet proteins recovering the ability to be phosphorylated within 15 min of exposure to peroxynitrite. Conversely, peroxynitrite increased phosphorylation of other proteins, but again these events were not directly linked to nitration. Nitration may affect the phosphorylation of tyrosine residues in a number of proteins, but by an indirect route, possibly by acting on proteins upstream in the signalling cascades. We suggest that low concentrations of peroxynitrite reversibly inhibit platelet aggregation by preventing the phosphorylation of key signalling proteins.

Effects of Short- and Long-Term Exercise on Urinary cGMP Excretion in Healthy Subjects and in Patients with Coronary Artery Disease

The possibility that systemic formation of cyclic guanosine monophosphate (cGMP) could reflect the level of cardiovascular fitness was investigated. The relations between physical activity and systemic formation of cGMP were evaluated in healthy volunteers and in patients with coronary artery disease (CAD). No significant differences were observed in the basal urinary excretion of cGMP in highly trained runners, sedentary subjects, and in patients with CAD, despite the large differences in aerobic exercise training between groups. In addition, the basal levels of cGMP in CAD patients failed to increase after a 12-week cardiac rehabilitation program. Short-term exercise, on the other hand, was associated with significant increases in urinary cGMP excretion. A 42-km marathon increased urinary cGMP excretion by 272%. The 15-km race increased urinary cGMP excretion by 330%. In CAD patients, 30 min of supervised exercise on a treadmill, at 80% of patient's maximal heart rate, induced a 60% increase in urinary cGMP, which returned to preexercise levels 90 min after termination of the exercise. Completion of the 12-week cardiac rehabilitation program improved exercise capacity and the magnitude of increase in cGMP levels induced by short-term treadmill exercise. Our findings suggest that cGMP increases during and shortly after short-term exercise and that the magnitude of the increase seems dependent on the intensity of the exercise and on physical fitness. Exercise training in healthy subjects and in CAD patients enhanced the amount of cGMP produced during short-term exercise, which might be responsible for some of the protective cardiovascular actions of exercise. The short half-life of cGMP may explain why the basal resting levels of cGMP are not appropriate predictors of a subject's physical fitness.

Does Diabetes Mellitus Affect the Progress of Tolerance to Isosorbide Dinitrate (ISDN) in Corporal Tissue?

For erection to take place, the penile arteries and sinusoids have to dilate, thereby increasing the blood flow into the penis. There is increasing evidence that release of l-arginine derived nitric oxide (NO) from nonadrenergic–noncholinergic (NANC) nerves and from the sinusoidal endothelium is a major event in penile smooth muscle relaxation and promotes the endogenous formation of cyclic guanosine monophosphate (cGMP). Nitrovasodilators can be attributed to the activation of soluble guanylate cyclase, resulting in an increase in intracellular level of cyclic guanosine monophosphate, but prolonged exposure to high levels of nitroglycerine and other organic nitroesters induces tolerance against the cardiovascular effect. In this study, the aim was to determine the effect of diabetes on the corporal smooth muscle relaxant effect of ISDN and the effect of diabetes on the process of tolerance to the drug. For this purpose, alloxan-induced diabetic rabbits were used to form diabetes group. The responses of the corpus cavernous strips obtained from control and alloxan-induced diabetic rabbit were studied in organ chamber. In conclusion, prolonged in vitro exposure of corpus cavernosum strips obtained from control and diabetic groups to high concentrations of ISDN caused significant desensitization to the relaxant effect the drug. So, prolonged exposure of corporal tissue to the agents like nitroglycerine, used for treatment of impotence, may render ineffective the therapy in diabetic erectile impotence. However, intolerance to nitric oxide provides a rationale for the concept of using nitro oxide agents (like SNP) in the treatment of diabetic erectile dysfunction.

Trilinolein potentiates the pro-aggregating effect of phorbol-12-myristate 13-acetate in human polymorphonuclear leukocytes

Trilinolein, a triacylglycerol with linoleic acid as the only type of fatty acid residue in all three of the glycerol esterified positions, was recently reported to have an antiplatelet effect, mediated through stimulating nitric oxide and cyclic guanosine monophosphate (GMP) formation. In our study, trilinolein induced aggregation of human polymorphonuclear neutrophils (PMNs) and, pretreatment with 0.1 nM trilinolein enhanced phorbol-12-myristate 13-acetate (PMA) induced aggregation. Further investigation showed that trilinolein at concentrations ranging from 0.1 nM to 10 μM increased cyclic GMP formation after 10 min of incubation with PMNs. Pretreatment of trilinolein with 10 μM d-sphingosine, before being incubated with PMNs, attenuated the stimulatory effect of trilinolein on cyclic GMP formation, and pretreatment of 10 μM d-sphingosine also attenuated the aggregation induced by PMA and trilinolein. We conclude that trilinolein can induce the aggregation of human PMNs, and enhance the aggregation induced by PMA.

Hydrogen peroxide-induced endothelium-dependent

In phenylephrine-precontracted rings, H 2O 2 produced an endothelium-dependent relaxation at concentrations of 4.4 × 10 −7 to ∼4.4 × 10 −5 M. Removal of extracellular Ca 2+ ([Ca 2+] 0) markedly attenuated the relaxant effects of H 2O 2. Complete inhibition of the H 2O 2 relaxant action was obtained after buffering intracellular Ca 2+ ([Ca 2+] i) in endothelial cells, with 10 μM acetyl methyl ester of bis (o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM). These relaxant effects of H 2O 2 were nearly abolished by 15 × 10 −5 M N G-monomethyl-arginine (L-NMMA) or 5 × 10 −5 M N G-nitro-L-arginine (L-NAME) and were attenuated markedly by the presence of either 10 −6 M Fe 2+, 10 −6 M Fe 3+, or 5 × 10 −6 M methylene blue. These inhibitory effects of L-NMMA or L-NAME could be reversed partly by 5 × 10 −5 M L-arginine. These Fe 2+- and Fe 3+-induced inhibitions of H 2O 2-stimulated relaxation were reduced significantly by either 1.0 mM deferoxamine (a Fe 2+ chelator) or 100 μM dimethyl sulfoxide (DMSO). In addition, 17-octadecynoic acid (2.5 μM) or proadifen (10 μM) (both antagonists of cytochrome P450 metabolism of fatty acids) markedly decreased the H 2O 2 relaxant effects. Proadifen (10 μM) produced concentration-dependent impairment of vasorelaxation to acetylcholine. A variety of amine antagonists and a cyclo-oxygenase inhibitor all fail to interfere with or attenuate the H 2O 2-induced relaxations. Our observations suggest that, at suitable pathophysiologic concentrations, H 2O 2 could induce release of an endothelium-derived relaxing factor, probably nitric oxide, from endothelial cells. The H 2O 2 relaxant effects are clearly Ca 2+-dependent and require formation of cyclic guanosine monophosphate (cGMP). These vasorelaxing effects of H 2O 2 appear to be induced by H 2O 2 itself. Hydrogen peroxide may stimulate production of some unknown metabolites metabolized by cytochrome P450–dependent enzymes.