8-bromocyclic-gmp and Hypertension--Pulmonary

To investigate the downstream target genes regulated by the nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signal pathway and their possible roles in the pathogenesis of pulmonary hypertension (PH).. Digital gene expression tag profiling was performed to identify genes that are differentially expressed after activation of the NO-sGC-cGMP signal pathway in human pulmonary artery smooth muscles cells using 8-bromo-cyclic guanosine monophosphate, BAY 41-2272 and BAY 60-2770. Results were confirmed using real-time polymerase chain reaction. Gene ontology and signal transduction network analyses were also performed.. A number of genes were differentially expressed, including MMP1, SERPINB2, GREM1 and IL8. A total of 68 gene ontology terms and seven pathways were found to be associated with these genes. Most of these genes are involved in cell proliferation, cell migration and apoptosis, which may contribute to the pathological pulmonary vascular remodelling in PH.. These results may provide new insights into the molecular mechanisms of PH.

Topics: Benzoates; Biphenyl Compounds; Cyclic GMP; Down-Regulation; Gene Expression Profiling; Gene Ontology; Guanosine Monophosphate; Humans; Hydrocarbons, Fluorinated; Hypertension, Pulmonary; Myocytes, Smooth Muscle; Nitric Oxide; Pulmonary Artery; Pyrazoles; Pyridines; Reproducibility of Results; Signal Transduction; Soluble Guanylyl Cyclase; Up-Regulation

Mutations in the bone morphogenetic protein type II receptor (BMPR-II) are responsible for the majority of cases of heritable pulmonary arterial hypertension (PAH), and BMPR-II deficiency contributes to idiopathic and experimental forms of PAH. Sildenafil, a potent type-5 nucleotide-dependent phosphodiesterase inhibitor, is an established treatment for PAH, but whether sildenafil affects bone morphogenetic protein (BMP) signaling in the pulmonary circulation remains unknown.. Studies were undertaken in human pulmonary arterial smooth muscle cells (PASMCs) and in vivo in the monocrotaline rat model of PAH. In PASMCs, sildenafil enhanced BMP4-induced phosphorylation of Smad1/5, Smad nuclear localization, and Inhibitor of DNA binding protein 1 gene and protein expression. This effect was mimicked by 8-bromo-cyclic GMP. Pharmacological inhibition or small interfering RNA knockdown of cyclic GMP-dependent protein kinase I inhibited the effect of sildenafil on BMP signaling. In functional studies, we observed that sildenafil potentiated the antiproliferative effects of BMP4 on PASMC proliferation. Furthermore, sildenafil restored the antiproliferative response to BMP4 in PASMCs harboring mutations in BMPR-II. In the monocrotaline rat model of PAH, which is characterized by BMPR-II deficiency, sildenafil prevented the development of pulmonary hypertension and vascular remodeling, and partly restored Smad1/5 phosphorylation and Inhibitor of DNA binding protein 1 gene expression in vivo in monocrotaline exposed rat lungs.. Sildenafil enhances canonical BMP signaling via cyclic GMP and cyclic GMP-dependent protein kinase I in vitro and in vivo, and partly restores deficient BMP signaling in BMPR-II mutant PASMCs. Our findings demonstrate a novel mechanism of action of sildenafil in the treatment of PAH and suggest that targeting BMP signaling may be beneficial in this disease.

Topics: Animals; Antihypertensive Agents; Binding Sites; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Proliferation; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Disease Models, Animal; Dose-Response Relationship, Drug; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; Inhibitor of Differentiation Protein 1; Male; Monocrotaline; Muscle, Smooth, Vascular; Mutation; Myocytes, Smooth Muscle; Phosphodiesterase 5 Inhibitors; Phosphorylation; Piperazines; Promoter Regions, Genetic; Pulmonary Artery; Purines; Rats; Rats, Sprague-Dawley; RNA Interference; Signal Transduction; Sildenafil Citrate; Smad1 Protein; Smad5 Protein; Sulfones; Transfection; Vasodilator Agents

Cardiovascular disease is characterized by impaired exercise capacity and endothelial dysfunction, i.e. reduced bioavailability of nitric oxide (NO). Phosphodiesterase-5 (PDE5) inhibition is a promising vasodilator therapy, but its effects on pulmonary and systemic hemodynamic responses to exercise in the absence, and particularly in the presence, of endothelial dysfunction have not been studied. We investigated the effects of PDE5 inhibitor EMD360527 in chronically instrumented swine at rest and during exercise with and without NO synthase inhibition (N(ω)-nitro-l-arginine; NLA). PDE5 inhibition caused a 19 ± 3% decrease in systemic vascular resistance (SVR) and a 24 ± 4% decrease in pulmonary vascular resistance (PVR) at rest. At maximal exercise, PDE5 inhibition caused a 13 ± 1% decrease in SVR and a 29 ± 3% decrease in PVR. NLA enhanced PDE5-inhibition-induced pulmonary (decrease in PVR 32 ± 12% at rest and 41 ± 3% during exercise) and systemic (decrease in SVR 24 ± 5% at rest and 18 ± 3% during exercise) vasodilation. Similarly, NLA increased the pulmonary and systemic vasodilation to nitroprusside and 8-bromo-cyclic guanosine monophosphate (cGMP), indicating that inhibition of NO synthase increases responsiveness to stimulation of the NO/cGMP pathway. Thus, PDE5 inhibition causes pulmonary and systemic vasodilation that is, respectively, maintained and slightly blunted during exercise. The degree of dilation in both the pulmonary and systemic beds were paradoxically enhanced in the presence of reduced bioavailability of NO, suggesting that this vasodilator therapy is most effective in patients with cardiovascular disease.

Topics: Animals; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Endothelial Cells; Humans; Hypertension, Pulmonary; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Phosphodiesterase 5 Inhibitors; Physical Conditioning, Animal; Pulmonary Circulation; Swine; Vascular Resistance

Many infants with congenital diaphragmatic hernias (CDHs) experience persistent pulmonary hypertension that is refractory to treatment with inhaled nitric oxide (NO). We have examined the responses of isolated pulmonary arterioles from prenatal and postnatal rats with and without nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether)-induced CDH to a variety of activators of the NO-cyclic guanosine monophosphate (cGMP) pathway.. Right-sided CDH was induced in fetal rats by feeding nitrofen to pregnant rats on day 12 of gestation. Control rats were fed olive oil (vehicle). Third-generation pulmonary arterioles were isolated from the right lung of prenatal rats at term and from newborn rats within 8 hours after birth. Responses to increasing concentrations of sodium nitroprusside (SNP), atrial natriuretic peptide, or 8-bromo-cGMP were measured in pulmonary arterioles from control rats and from rats with nitrofen-induced CDH. Postnatal responses to 8-bromo-cGMP were also recorded in the presence of zaprinast, a type V phosphodiesterase inhibitor.. Pulmonary arterioles from prenatal rats did not dilate in response to SNP, atrial natriuretic peptide, or 8-bromo-cGMP. Vasodilatory responses of postnatal pulmonary arterioles from control rats to SNP and 8-bromo-cGMP were significantly greater than for arterioles from rats with CDH. Zaprinast pretreatment resulted in similar responses for postnatal CDH and control arterioles to 8-bromo-cGMP.. Postnatal pulmonary arterioles from CDH rats exhibit altered nitrovasodilator responsiveness, which may be due to rapid degradation of cGMP.

Topics: Animals; Arterioles; Atrial Natriuretic Factor; Cyclic GMP; Disease Models, Animal; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Hypertension, Pulmonary; Lung; Nitric Oxide; Nitroprusside; Pesticides; Phenyl Ethers; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Sprague-Dawley; Vasodilation; Vasodilator Agents

To investigate the effects of long-term nitric oxide (NO) inhalation on the recovery process of right ventricular hypertrophy (RVH) and functional alterations in the NO-cyclic guanosine monophosphate (cGMP) relaxation pathway in rat conduit pulmonary arteries (PAs) in established chronic hypoxic pulmonary hypertension.. A total of 35 rats were exposed to chronic hypobaric hypoxia (380 mm Hg, 10% oxygen), and 39 rats were exposed to air for 10 days. Both groups were then exposed to 3 or 10 days of NO 10 ppm, NO 40 ppm, or air (control groups for each NO concentration), resulting in a total of 16 groups. Acetylcholine- and sodium nitroprusside (SNP)-induced relaxation were evaluated in precontracted PA rings. RVH was assessed by heart weight ratio of right ventricle to left ventricle plus septum.. NO inhalation had no effect on either the regression of RVH or the recovery process of impaired relaxation induced by acetylcholine or SNP in a endothelium-intact hypertensive conduit extrapulmonary artery or intrapulmonary artery (IPA). In a normal endothelium-intact conduit IPA, 40 ppm NO inhalation for 10 days partially augmented SNP-induced relaxation, but not that induced by acetylcholine.. Continuous NO inhalation did not affect the regression process of either established RVH or the impaired endogenous NO-cGMP relaxation cascade in a conduit PA in rats during the recovery period after chronic hypoxia.

Topics: Acetylcholine; Administration, Inhalation; Animals; Chronic Disease; Cyclic GMP; Endothelium-Dependent Relaxing Factors; Endothelium, Vascular; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Nitroprusside; Pulmonary Artery; Rats; Rats, Wistar; Vasodilation; Vasodilator Agents

Nitric oxide (NO) is thought to play an important role in the regulation of neonatal pulmonary vasculature. It has been suggested that neonates with pulmonary hypertension have a defective NO pathway. Therefore, we measured in 1-day-old piglets exposed to hypoxia (fraction of inspired O(2) = 0.10) for 3 or 14 days to induce pulmonary hypertension 1) the activity of NO synthase (NOS) via conversion of L-arginine to L-citrulline and the concentration of the NO precursor L-arginine in isolated pulmonary vessels, 2) the vasodilator response to the NO donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) and the cGMP analog 8-bromo-cGMP in isolated perfused lungs, and 3) the production of cGMP in response to SIN-1 in isolated perfused lungs. After 3 days of exposure to hypoxia, endothelial NOS (eNOS) activity was unaffected, whereas, after 14 days of hypoxia, eNOS activity was decreased in the cytosolic fraction of pulmonary artery (P < 0.05) but not of pulmonary vein homogenates. Inducible NOS activity was decreased in the cytosolic fraction of pulmonary artery homogenates after both 3 (P < 0.05) and 14 (P < 0.05) days of hypoxia but was unchanged in pulmonary veins. Pulmonary artery levels of L-arginine were unaffected by hypoxic exposure. After 3 days of exposure to hypoxia, the reduction in the dilator response to SIN-1 (P < 0.05) coincided with a decrease in cGMP production (P < 0.005), suggesting that soluble guanylate cyclase activity may be altered. When the exposure was prolonged to 14 days, dilation to SIN-1 remained decreased (P < 0.05) and, although cGMP production normalized, the dilator response to 8-bromo-cGMP decreased (P < 0.05), suggesting that, after prolonged exposure to hypoxia, cGMP-dependent mechanisms may also be impaired. In conclusion, neonatal hypoxia-induced pulmonary hypertension is associated with multiple disruptions in the NO pathway.

Topics: Animals; Animals, Newborn; Arginine; Blood Vessels; Cyclic GMP; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Pulmonary Circulation; Swine; Vasodilation; Vasodilator Agents

To determine whether acute pulmonary hypertension in utero alters fetal pulmonary vascular reactivity, we compared pulmonary vasodilation with an endothelium-dependent agonist, acetylcholine, with that of an endothelium-independent agonist, 8-bromo-guanosine 3',5'-cylic monophosphate. Acute pulmonary hypertension was produced in chronically prepared, late-gestation fetal lambs by 3 repeated 30-minute partial occlusions of the ductus arteriosus (DA). The first DA compression increased LPA blood flow from 80 +/- 10 to 180 +/- 21 mL/min (p < 0.01) and decreased pulmonary vascular resistance. In contrast, LPA blood flow did not change and pulmonary vascular resistance increased by 25% during the third period of DA compression. Pulmonary vasodilation during acetylcholine infusion after serial DA compressions was decreased in comparison with the acetylcholine-induced vasodilator response achieved during the baseline period (fall in pulmonary vascular resistance = -49 +/- 7% (baseline) versus -25 +/- 5% after repeated DA compressions; p < 0.05). In contrast, the vasodilator response to 8-bromo-guanosine 3',5'-cylic monophosphate remained intact. To determine whether decreased nitric oxide (NO) production may contribute to altered vasoreactivity after acute pulmonary hypertension, repeated DA compressions were performed after treatment with a nonspecific NO synthase inhibitor (nitro-L-arginine). NO synthase inhibition blocked the pulmonary vasodilation during the first DA compression period, and repeated DA compressions after NO synthase inhibition did not further alter the hemodynamic response to DA compression. These findings support the hypothesis that brief hypertension due to DA compression impairs endothelium-dependent pulmonary vasodilation in the fetus, and that this may be due to decreased NO production.

Topics: Acetylcholine; Animals; Carbon Dioxide; Cyclic GMP; Ductus Arteriosus; Endothelium, Vascular; Female; Gestational Age; Heart Rate, Fetal; Hemodynamics; Hypertension, Pulmonary; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxygen; Pregnancy; Pulmonary Circulation; Sheep; Vasodilation

Ligation of the ductus arteriosus of the fetal sheep produces severe pulmonary hypertension at birth. Standard tissue bath techniques were used to study third- and fourth-generation pulmonary arteries and veins isolated from fetal sheep with pulmonary hypertension created by ligation of the ductus arteriosus 11-12 days before birth as well as from age-matched control sheep. Vessels pretreated with indomethacin and propranolol were submaximally preconstricted with norepinephrine before exposure to A-23187 (10(-8) to 3 x 10(-7) M), sodium nitroprusside (SNP; 10(-9) to 10(-5) M), and nitric oxide (NO) gas (1-973 ppm). Pulmonary veins in both control and ligated animals relaxed similarly and completely to A-23187, SNP, and NO. Control pulmonary arteries relaxed by 16 +/- 2% to A-23187 and relaxed completely to SNP and NO, with concentration-response curves shifted rightward of those observed in pulmonary veins. Pulmonary arteries from ligated animals did not relax at all to A-23187. SNP relaxations in ligated arteries were shifted rightward of control. Ligated arteries relaxed by only 11 +/- 5% to the highest dose of NO. However, control and ligated pulmonary arteries relaxed similarly to 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP; 10(-5) to 10(-3) M) and atrial natriuretic peptide (10(-9) to 10(-7) M). These data are most simply explained by decreased arterial vascular smooth muscle sensitivity to NO at the level of soluble guanylate cyclase.

Topics: Animals; Atrial Natriuretic Factor; Cyclic GMP; Ductus Arteriosus; Fetus; Hypertension, Pulmonary; Ligation; Nitric Oxide; Nitroprusside; Pulmonary Artery; Sheep; Vasoconstriction; Vasodilator Agents

Selective reduction of pulmonary vascular resistance (PVR) remains a therapeutic goal for the treatment of pulmonary hypertension, but current therapeutic options remain limited. Although the gas nitric oxide (NO) selectively dilates the pulmonary vascular bed, it requires special equipment for administration, has a short biologic half-life, and is potentially toxic. We hypothesized that stimulation of the NO pathway at the level of its second messenger, guanosine 3',5'-cyclic monophosphate (cGMP), by targeted pulmonary delivery of a membrane-permeable nonhydrolyzable cGMP analogue would cause selective pulmonary vasodilation. Pulmonary hypertension was induced in 21 pigs by the intravenous infusion of a thromboxane A2 analogue (9,11-dideoxy-9 alpha,11 alpha-epoxymethanoprostaglandin F2 alpha). Inhaled 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) lowered PVR in a time- and dose-dependent manner, with maximal effect achieved after 20 min. Compared with physiological saline control, 8-BrcGMP inhalation (3.0 micrograms/kg) lowered PVR by 25 +/- 3% (P < 0.01), whereas there was no significant decline in systemic vascular resistance (4 +/- 6%); mean pulmonary arterial pressure declined 13 +/- 3% (P < 0.01), whereas there was little change in mean arterial pressure; cardiac output increased 10 +/- 4% (P < 0.05). PVR did not decrease after inhalation of noncyclic 8-bromoguanosine 5'-monophosphate, indicating that stimulation of the NO-cGMP pathway beyond the level of NO results in pulmonary vasodilation independent of stimulation of purinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Administration, Inhalation; Animals; Blood Pressure; Cyclic GMP; Dose-Response Relationship, Drug; Female; Hypertension, Pulmonary; Myocardial Contraction; Pulmonary Circulation; Swine; Vascular Resistance

To investigate the role of endothelium-derived relaxing factor (EDRF) in the regulation of resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation, we studied the hemodynamic effects of N omega-nitro-L-arginine (a new stereospecific EDRF inhibitor) in 10 spontaneously breathing lambs and then compared the hemodynamic responses to five vasodilators during pulmonary hypertension induced by the infusion of U-46619 (a thromboxane A2 mimetic) or N omega-nitro-L-arginine. N omega-nitro-L-arginine caused a significant dose-dependent increase in pulmonary arterial pressure. Pretreatment with L-arginine blocked this increase, but pretreatment with D-arginine did not, suggesting that N omega-nitro-L-arginine is a competitive inhibitor of L-arginine for EDRF production. During U-46619 infusions, acetylcholine, ATP-MgCl2, isoproterenol, sodium nitroprusside, and 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP) decreased pulmonary arterial pressure. During N omega-nitro-L-arginine infusions, the decrease in pulmonary arterial pressure caused by acetylcholine and ATP-MgCl2 (endothelium-dependent vasodilators) was significantly attenuated, but the decrease caused by isoproterenol, sodium nitroprusside, and 8-bromo-cGMP (endothelium-independent vasodilators) was unchanged. This study supports the hypothesis that EDRF in part mediates resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation. N omega-nitro-L-arginine is useful for studying EDRF inhibition in intact animals.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Adenosine Triphosphate; Animals; Arginine; Binding, Competitive; Blood Pressure; Cyclic GMP; Endothelium, Vascular; Hemodynamics; Hypertension, Pulmonary; Isoproterenol; Lung; Nitric Oxide; Nitroarginine; Nitroprusside; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Sheep; Vasodilation

The effects of endothelium-dependent vasodilation on pulmonary vascular hemodynamics were evaluated in a variety of in vivo and in vitro models to determine 1) the comparability of the hemodynamic effects of acetylcholine (ACh), bradykinin (BK), nitric oxide (NO), and 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP), 2) whether methylene blue is a useful inhibitor of endothelium-dependent relaxing factor (EDRF) activity in vivo, and 3) the effect of monocrotaline-induced pulmonary hypertension on the responsiveness of the pulmonary vasculature to ACh. In isolated rat lungs, which were preconstricted with hypoxia, ACh, BK, NO, and 8-bromo-cGMP caused pulmonary vasodilation, which was not inhibited by maximum tolerable doses of methylene blue. Methylene blue did not inhibit EDRF activity in any model, despite causing increased pulmonary vascular tone and responsiveness to various constrictor agents. There were significant differences in the hemodynamic characteristics of ACh, BK, and NO. In the isolated lung, BK and NO caused transient decreases of hypoxic vasoconstriction, whereas ACh caused more prolonged vasodilation. Pretreatment of these lungs with NO did not significantly inhibit ACh-induced vasodilation but caused BK to produce vasoconstriction. Tachyphylaxis, which was agonist specific, developed with repeated administration of ACh or BK but not NO. Tachyphylaxis probably resulted from inhibition of the endothelium-dependent vasodilation pathway proximal to NO synthesis, because it could be overcome by exogenous NO. Pretreatment with 8-bromo-cGMP decreased hypoxic pulmonary vasoconstriction and, even when the hypoxic pressor response had largely recovered, subsequent doses of ACh and NO failed to cause vasodilation, although BK produced vasoconstriction. These findings are compatible with the existence of feedback inhibition of the endothelium-dependent relaxation by elevation of cGMP levels. Responsiveness to ACh was retained in lungs with severe monocrotaline-induced pulmonary hypertension. Many of these findings would not have been predicted based on in vitro studies and illustrate the importance for expanding studies of EDRF to in vivo and ex vivo models.

Topics: Acetylcholine; Animals; Bradykinin; Cyclic GMP; Endothelium, Vascular; Hemodynamics; Hypertension, Pulmonary; Hypoxia; Lung; Male; Methylene Blue; Monocrotaline; Nitric Oxide; Pulmonary Circulation; Pyrrolizidine Alkaloids; Rats; Rats, Inbred Strains; Vasodilation